epidermal-growth-factor and Carcinoma--Squamous-Cell

Head and neck squamous cell carcinoma (HNSCC) develops from the mucosal epithelium of the oral cavity, pharynx, and larynx, and is the most common malignancy of the head and neck, the incidence of which continues to rise. The epidermal growth factor receptor is thought to play a key role in the pathogenesis of HNSCC. Inhibition of epidermal growth factor receptor has been identified as an effective target for the treatment of HNSCC. Many phytochemicals have emerged as potential new drugs for the treatment of HNSCC. A systematic search was conducted for research articles published in PubMed, and Medline on relevant aspects. This review provides an overview of the available literature and reports highlighting the in vitro effects of phytochemicals on epidermal growth factor in various HNSCC cell models and in vivo in animal models and emphasizes the importance of epidermal growth factor as a current therapeutic target for HNSCC. Based on our review, we conclude that phytochemicals targeting the epidermal growth factor receptor are potentially effective candidates for the development of new drugs for the treatment of HNSCC. It provides an idea for further development and application of herbal medicines for cancer treatment.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Phytochemicals; Squamous Cell Carcinoma of Head and Neck

The field of cutaneous oncology is exploding with innovative treatment options, specifically in the field of targeted therapy. These advances offer new hope to select patients with high risk skin cancers. In part two of our series on targeted therapy for skin cancer, we focus our attention on squamous cell carcinoma. We begin with the epidermal growth factor receptor inhibitors and branch out into newer areas of active research.

Topics: Antineoplastic Agents; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Clinical Trials as Topic; Dermatology; Epidermal Growth Factor; Humans; Molecular Targeted Therapy; Skin Neoplasms

We have identified tyrosine-phosphorylated proteins on membrane from A-431 human epidermoid carcinoma cells by using detection with anti-phosphotyrosine antibody followed by PMF analysis. In there, on-membrane digestion for these protein spots was carried out on microscale region using chemical inkjet technology and the resulting tryptic digests were directly analyzed by MALDI-TOF MS. Proteins identified by a database search included phosphoproteins that are known to be markedly phosphorylated on tyrosine sites after the cells are treated with epidermal growth factor (EGF). This procedure is a rapid and easily handled approach that enables both detection and identification of phosphoproteins on a single blot membrane.

Topics: Antibodies, Phospho-Specific; Blotting, Western; Carcinoma, Squamous Cell; Cell Extracts; Cell Line, Tumor; Cell Membrane; Electrophoresis, Gel, Two-Dimensional; Epidermal Growth Factor; Humans; Molecular Weight; Peptide Mapping; Phosphoproteins; Phosphotyrosine; Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry

The role of dysregulated epidermal growth factor receptor-tyrosine kinase (EGFR-TK) activity in promoting tumor resistance to radiation therapy is discussed, and evidence supporting the rationale for the use of gefitinib (IRESSA, ZD1839) to enhance tumor radiosensitivity is reviewed.. A review of the literature regarding the role of EGFR-TK signaling in tumor response to radiation therapy was conducted, and results were summarized from preclinical and clinical studies of gefitinib in the treatment of solid tumors alone and in combination with radiation therapy.. Preclinical results indicate that EGFR-TK activity in tumors can block the cytotoxic effects of radiation therapy and enhance tumor repopulation, resulting in failure of local tumor control. In xenograft tumor models, gefitinib in combination with ionizing radiation resulted in additive to synergistic growth inhibition. In randomized clinical trials, gefitinib has demonstrated efficacy with favorable tolerability as monotherapy for patients with advanced non-small-cell lung cancer or head-and-neck carcinomas who had previously received standard therapies.. These results indicate that there is potential for improved responses by combining gefitinib with radiation therapy in non-small-cell lung cancer, head-and-neck cancers, and other solid tumors.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Combined Modality Therapy; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioblastoma; Head and Neck Neoplasms; Humans; Lung Neoplasms; Neoplasm Proteins; Neoplasms; Quinazolines; Radiation Tolerance; Radiation-Sensitizing Agents

The activation of the epidermal growth factor receptor (REGF) participates in oncogenesis by inducing cell proliferation, cell mobility and angiogenesis, and inhibiting apoptosis. This activation might be due to numerous abnormalities, including increased expression of its ligand. Although based on retrospective analyses with no standard technique of evaluation, the level of REGF expression is a prognosis factor for several tumors. It appears to be an indicator of poor prognosis which might influence treatments in head and neck tumors, cancers of the cervix, oesophagus, bladder and ovary. Its prognostic value is not observed in non-small cell lung cancer and remains to be demonstrated in adenocarcinomas such as colorectal tumors and beast cancer. Because of its role in oncogenesis and its prognostic value, REGF might in the future become a therapeutic target.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Male; Neoplasm Proteins; Neoplasms; Organ Specificity; Prognosis; Receptor, ErbB-2; Transforming Growth Factor alpha

Expression of squamous cell carcinoma (SCC) antigen emerged concurrently with squamous formation of the uterine cervix and increased during the neoplastic transformation of the cervical squamous epithelium. SCC antigen expression differed considerably among the histomorphologic cell types of cervical carcinoma. Large cell nonkeratinizing carcinoma contained high levels of the antigen. In contrast, no appreciable expression of SCC antigen was observed in small cell nonkeratinizing carcinoma. The pattern of SCC antigen expression closely coincided with EGF receptor (EGF-R) expression in cervical squamous neoplasia. This suggests that the expression of SCC and EGF-R in cervical carcinoma is related to the differentiation or dedifferentiation processes of the tumor cells. SCC production by CaSki cervical epidermoid carcinoma cells was stimulated by EGF. It seems likely that an autocrine system, in which EGF serves as the signal, may exist in cervical squamous carcinoma. 17beta-estradiol and L-triiodothyronine were found to upregulate EGF-R expression, proliferative potential and SCC production in the CaSki cervical carcinoma cells.

Topics: Antigens, Neoplasm; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cervix Uteri; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Serpins; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Squamous cell carcinoma of the head and neck is a disease predominantly of males and is due to a variety of known environmental irritants, notably cigarette smoke. Dietary, viral and immunological factors may also be relevant. Head and neck squamous cancers express epidermal growth factor receptors and some show weak levels of oestrogen receptor activity, but a reliable serum marker of tumour burden remains to be identified. The prognosis is found to be less favourable in females, in those with advanced T stage, in association with multiple node involvement, especially where extracapsular spread is present and where the T4/T8 ratio is elevated. Administration of heterologous blood during therapy may also have an adverse effect on prognosis. Interested clinicians must remember that most cases are preventable.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Male; Prognosis; Sex Factors; T-Lymphocytes

Topics: Animals; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Herpes Simplex; Humans; Male; Mouth Neoplasms; Oncogenes; Papillomaviridae; Tumor Virus Infections

The role of EGF receptor concentration in tumor growth was investigated in athymic mice by measuring the rate of growth of clonal human epidermoid carcinoma A431 cells containing different extents of EGF receptor gene amplification and protein expression. A direct correlation-between the rate of tumor growth and EFG receptor concentration was found, supporting previous cell culture studies that quantitated the relationship between activated EGF receptors and cell proliferation. Holo EGF receptor is activated by ligand binding to the extracellular domain to activate cytoplasmic tyrosine protein kinase activity. A model of single molecule transmembrane signaling is proposed. The function of two phosphorylation sites on the EGF receptor has been analyzed by use of site-directed mutagenesis. Comparison of normal and mutant hEGF receptors expressed in rodent cells lacking endogenous EGF receptors indicates that: 1) Thr654, located 10 amino acids carboxyl terminal to the inner membrane boundary, is a major site of heterologous regulation via protein kinase C catalyzed phosphorylation, and 2) Tyr1173, the major site of self-phosphorylation, located at the carboxyl terminus, provides a secondary level of regulation of receptor function by acting as a competitive inhibitor with exogenous substrates.

Topics: Animals; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation; Humans; Mice; Mice, Nude; Models, Genetic; Protein Sorting Signals

Ten cell lines established from surgical specimens of human squamous carcinomas of the tongue and larynx have been investigated with respect to their motility, ultrastructure, karyotypes, certain biochemical features, interaction with normal epithelial and stromal elements and capacity to infiltrate three-dimensional organoid systems. All the cell lines have maintained several morphological and biochemical characteristics indicating a common origin, although the extent to which each line displays this heritage is variable. The phenotypes of each of the individual cell lines are, however, notably stable. Data are provided for epithelial surface markers (including epidermal growth factor, EGF) and for the synthesis and release of prostaglandins and proteases which may be involved in invasive mechanisms. Encounters between the cell lines and organoid substrata (embryonic chick heart spheroids, human amnion, chick chorioallantoic membrane) are described: the results indicate a scale of invasiveness ranging from lack of penetration to full-thickness infiltration by cells showing various distinctive growth patterns. Correlation between in vitro and in vivo findings is discussed, and it is suggested that the biological heterogeneity of the lines may reflect inherent properties of the original carcinoma cell populations which are more distinctly expressed in vitro.

Topics: Aged; Animals; Bone and Bones; Calcium-Binding Proteins; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Cartilage; Cell Communication; Cell Line; Cell Movement; Chick Embryo; Epidermal Growth Factor; Female; Humans; Karyotyping; Keratins; Laryngeal Neoplasms; Male; Membrane Proteins; Microscopy, Electron; Middle Aged; Mucin-1; Tongue Neoplasms

Human exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and chlorinated analogs commonly results in pathological changes in the skin and its appendages characterized by thickening of the epidermis (acanthosis), hyperkeratosis and squamous metaplasia of the epithelial lining of the sebaceous glands. Acneform lesions (chloracne) develop as hair follicles dilate and fill with keratin and sebaceous glands become cystic. In animal models it has been found that the chloracneogenic potential of the halogenated aromatic compounds examined corresponds with the relative affinity of these same compounds for the cytosolic TCDD receptor. This receptor controls the coordinate expression of a number of inducible enzyme activities and in certain cell targets can alter normal programs of proliferation and differentiation. In this report we describe some of our ongoing studies on the mechanisms of action of TCDD in normal human epidermal cells and squamous cell carcinoma (SCC) lines. These systems permit detailed investigation of the molecular and biochemical events underlying pathologic changes in the skin and offer the potential of establishing a risk assessment model for halogenated aromatic compounds by using human target cells.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; Chromosome Mapping; Dioxins; Epidermal Growth Factor; Humans; Hyperplasia; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; Receptors, Drug; Risk; Skin

Most drugs available for cancer chemotherapy exert their effects through cytodestruction. Although significant advances have been attained with these cytotoxic agents in several malignant diseases, response is often accompanied by significant morbidity and many common malignant tumours respond poorly to existing cytotoxic therapy. Development of chemotherapeutic agents with non-cytodestructive actions appears desirable. Considerable evidence exists which indicates that (a) the malignant state is not irreversible and represents a disease of altered maturation, and (b) some experimental tumour systems can be induced by chemical agents to differentiate to mature end-stage cells with no proliferative potential. Thus, it is conceivable that therapeutic agents can be developed which convert cancer cells to benign forms. To study the phenomenon of blocked maturation, squamous carcinoma SqCC/Y1 cells were employed in culture. Using this system it was possible to demonstrate that physiological levels of retinoic acid and epidermal growth factor were capable of preventing the differentiation of these malignant keratinocytes into a mature tissue-like structure. The terminal differentiation caused by certain antineoplastic agents was investigated in HL-60 promyelocytic leukaemia cells to provide information on the mechanism by which chemotherapeutic agents induce cells to by-pass a maturation block. The anthracyclines aclacinomycin A and marcellomycin were potent inhibitors of N-glycosidically linked glycoprotein biosynthesis and transferrin receptor activity, and active inducers of maturation; temporal studies suggested that the biochemical effects were associated with the differentiation process. 6-Thioguanine produced cytotoxicity in parental cells by forming analog nucleotide. In hypoxanthine-guanine phosphoribosyltransferase negative HL-60 cells the 6-thiopurine initiated maturation; this action was due to the free base (and possibly the deoxyribonucleoside), a finding which separated termination of proliferation due to cytotoxicity from that caused by maturation.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Leukemia, Myeloid; Models, Biological; Naphthacenes; Thioguanine; Tretinoin

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; Epithelial Cells; Fetal Hemoglobin; Fibroblasts; Gene Expression Regulation; Glycopeptides; Growth Inhibitors; Growth Substances; Hemoglobins; Humans; Isoenzymes; Liver; Models, Biological; Mutation; Neoplasms; Peptides; Protein Kinases; RNA, Messenger; Thalassemia; Transcription, Genetic; Transforming Growth Factors

Oral mucositis (OM) is considered a painful and debilitating side effect in patients receiving head and neck cancer treatment. Low-level laser therapy (LLLT) proved to be effective to prevent and treat chemoradiotherapy-induced OM. The aim of this study was to evaluate the effect of LLLT in the severity of OM in patients with head and neck cancer and on the release of salivary inflammatory mediators. Clinical (score of OM severity) and biochemical parameters (concentration of inflammatory mediators, growth factors, and enzymes in saliva) were used.. Thirty patients were randomized into two groups: control and laser. LLLT was performed three times a week in the laser group, while control group received sham irradiation. OM severity was assessed according to the World Health Organization (WHO) and National Cancer Institute (NCI) scales. Pro-inflammatory and anti-inflammatory cytokines (TNF-α, IL-6, IL-1β, IL-10, TGF-β), growth factors (EGF, FGF, VEGF), and metalloproteinases (MMP2/TIMP2, MMP9/TIMP2) concentrations were assessed using ELISA test. Saliva samples were collected on admission, and at the 7th, 21st, and 35th sessions of radiotherapy.. The laser group showed a reduction in the severity of OM, which coursed with significantly diminished salivary concentration of EGF and VEGF in the 7th radiotherapy session and of IL-6 and FGF in the 35th. There was a trend for reduced levels of IL-1β, TNF-α, IL-10, TGF-β, MMP2/TIMP2, MMP9/TIMP2 in the laser group compared to the control, however, no statistically significant differences were found.. These findings demonstrated that LLLT was effective in reducing the severity of chemoradiotherapy-induced OM and was associated with the reduction of inflammation and repair.

Topics: Carcinoma, Squamous Cell; Chemoradiotherapy; Cytokines; Double-Blind Method; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Low-Level Light Therapy; Male; Metalloproteases; Middle Aged; Saliva; Severity of Illness Index; Stomatitis; Vascular Endothelial Growth Factor A

We show the result of a randomized phase II clinical trial with an epidermal growth factor (EGF)-based cancer vaccine in advanced non-small-cell lung cancer (NSCLC) patients, evaluating immunogenicity, safety, and effect on survival.. Eighty patients with stage IIIB/IV NSCLC after finishing first-line chemotherapy were randomly assigned to receive best supportive care or EGF vaccinations.. Vaccination was safe. Adverse events were observed in less than 25% of cases and were grade 1 or 2 according to National Cancer Institute Common Toxicity Criteria. Good anti-EGF antibody response (GAR) was obtained in 51.3% of vaccinated patients and in none of the control group. Serum EGF concentration showed a major decrease in 64.3% of vaccinated patients. GAR patients survived significantly more than those with poor antibody response (PAR). Also, patients whose serum EGF dropped below 168 pg/mL survived significantly more than the rest. There was a trend to an increased survival for vaccinated patients compared with controls. The survival advantage for vaccinated patients compared with controls was statistically significant in the subgroup of patients with age younger than 60 years.. Vaccination with EGF was safe and provoked an increase in anti-EGF antibody titers and a decrease in serum EGF. There was a direct correlation between antibody response and survival. There was a direct correlation between decrease in serum EGF and survival. In patients younger than 60 years, vaccination was associated with increased survival.

Topics: Adenocarcinoma; Adult; Aged; Cancer Vaccines; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunotherapy, Active; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Survival Analysis; Treatment Outcome

Regardless of the presence or absence of specific diagnostic mutations, many cancer patients fail to respond to EGFR-targeted therapeutics, and a personalized approach is needed to identify putative (non)responders. We found previously that human peripheral blood and EGF can modulate the activities of EGFR-specific drugs on inhibiting clonogenity in model EGFR-positive A431 squamous carcinoma cells. Here, we report that human serum can dramatically abolish the cell growth rate inhibition by EGFR-specific drugs cetuximab and erlotinib. We show that this phenomenon is linked with derepression of drug-induced G1S cell cycle transition arrest. Furthermore, A431 cell growth inhibition by cetuximab, erlotinib, and EGF correlates with a decreased activity of ERK1/2 proteins. In turn, the EGF- and human serum-mediated rescue of drug-treated A431 cells restores ERK1/2 activity in functional tests. RNA sequencing revealed 1271 and 1566 differentially expressed genes (DEGs) in the presence of cetuximab and erlotinib, respectively. Erlotinib- and cetuximab-specific DEGs significantly overlapped. Interestingly, the expression of 100% and 75% of these DEGs restores to the no-drug level when EGF or a mixed human serum sample, respectively, is added along with cetuximab. In the case of erlotinib, EGF and human serum restore the expression of 39% and 83% of DEGs, respectively. We further assessed differential molecular pathway activation levels and propose that EGF/human serum-mediated A431 resistance to EGFR drugs can be largely explained by reactivation of the MAPK signaling cascade.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Serum

Highly homologous E3 ubiquitin ligases, Cbl and Cbl-b, mediate ubiquitination of EGF receptor (EGFR), leading to its endocytosis and lysosomal degradation. Cbl and Cbl-b, are thought to function in a redundant manner by binding directly to phosphorylated Y1045 (pY1045) of EGFR and indirectly via the Grb2 adaptor. Unexpectedly, we found that inducible expression of Cbl or Cbl-b mutants lacking the E3 ligase activity but fully capable of EGFR binding does not significantly affect EGFR ubiquitination and endocytosis in human oral squamous cell carcinoma (HSC3) cells which endogenously express Cbl-b at a relatively high level. Each endogenous Cbl species remained associated with ligand-activated EGFR in the presence of an overexpressed counterpart species or its mutant, although Cbl-b overexpression partially decreased Cbl association with EGFR. Binding to pY1045 was the preferential mode for Cbl-b:EGFR interaction, whereas Cbl relied mainly on the Grb2-dependent mechanism. Overexpression of the E3-dead mutant of Cbl-b slowed down EGF-induced degradation of active EGFR, while this mutant and a similar mutant of Cbl did not significantly affect MAPK/ERK1/2 activity. EGF-guided chemotaxis migration of HSC3 cells was diminished by overexpression of the E3-dead Cbl-b mutant but was not significantly affected by the E3-dead Cbl mutant. By contrast, the inhibitory effect of the same Cbl mutant on the migration of OSC-19 cells expressing low Cbl-b levels was substantially stronger than that of the Cbl-b mutant. Altogether, our data demonstrate that Cbl and Cbl-b may operate independently through different modes of EGFR binding to jointly control receptor ubiquitination, endocytic trafficking, and signaling.

Topics: Carcinoma, Squamous Cell; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Mouth Neoplasms; Proto-Oncogene Proteins c-cbl; Ubiquitin-Protein Ligases

Head and neck squamose cell carcinoma (HNSCC) is an aggressive group of tumors that are generally heterogeneous. Despite treatment advances, disease-free survival has not significantly improved. Therefore, it is of great importance to understand the molecular etiology of HNSCC and genetic alterations in the signal pathways in order to develop new therapeutic approaches. In this study, firstly we used a cytokine array to analyze the secretomes of HNSCC patients and healthy controls. In the next step, the results from the cytokine sequence were validated by qRT-PCR and western blot, including genes in the associated signaling pathway. In array analysis, the levels of EGF, IGF-1, IGFBP-1, and PDGFBB were significantly higher in patients than in the controls. The results of qRT-PCR analyses showed that expression levels of

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cytokines; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor I; Placenta Growth Factor; Receptor, Platelet-Derived Growth Factor beta; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix proteolysis and other factors. Studies have shown a potential relationship between growth factors, metallothionein 2A (MT2A) and matrix metalloproteinase (MMP) activation in malignant tumours. The aim of this study was to downregulate MT2A in cells (Cal27) derived from human squamous cell carcinoma.. Cal27 cells with reduced MT2A were subjected to proliferation, migration and invasion assays. Immunofluorescence and western blot confirmed MT2A depletion by siRNA. Growth curve assays assessed cell proliferation. Indirect immunofluorescence analysed the expression of MT2A, MMP-2, MMP-9, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), tumour necrosis factor alpha (TNF-α) and Ki67. Zymography evaluated the effects of MT2A silencing on MMP-2 and -9 expression. Migration and invasion activities were evaluated using migration and invasion assays.. CAL27 cells displayed MT2A, MMP-2, MMP-9, EGF, TGF-α, TNF-α and Ki67. MT2A depletion decreased MMP-9, EGF, TGF-α and Ki67 protein levels, while increasing TNF-α.. MT2A downregulation reduced cell proliferation, migration and invasion activities. Therefore, MT2A has an important role in cell proliferation, migration and invasion in human oral SCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; Humans; Ki-67 Antigen; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metallothionein; Transforming Growth Factor alpha; Tumor Necrosis Factor-alpha

Macropinocytosis is a type of endocytosis accompanied by actin rearrangement-driven membrane deformation, such as lamellipodia formation and membrane ruffling, followed by the formation of large vesicles, macropinosomes. Ras-transformed cancer cells efficiently acquire exogenous amino acids for their survival through macropinocytosis. Thus, inhibition of macropinocytosis is a promising strategy for cancer therapy. To date, few specific agents that inhibit macropinocytosis have been developed. Here, focusing on the mechanosensitive ion channel Piezo1, we found that Yoda1, a Piezo1 agonist, potently inhibits macropinocytosis induced by epidermal growth factor (EGF). The inhibition of ruffle formation by Yoda1 was dependent on the extracellular Ca

Topics: Biological Transport; Calcium; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Humans; Ion Channels; Pinocytosis; Pyrazines; Thiadiazoles

The expression of programmed death ligand-1 (PD-L1) is controlled by complex mechanisms. The elucidation of the molecular mechanisms of PD-L1 expression is important for the exploration of new insights into PD-1 blockade therapy. Detailed mechanisms of the in situ expression of PD-L1 in tissues of oral squamous cell carcinomas (OSCCs) have not yet been clarified. We examined the mechanisms of PD-L1 expression focusing on the phosphorylation of downstream molecules of epidermal growth factor (EGF) and interferon gamma (IFN-γ) signaling in vitro and in vivo by immunoblotting and multi-fluorescence immunohistochemistry (MF-IHC), respectively. The in vitro experiments demonstrated that PD-L1 expression in OSCC cell lines is upregulated by EGF via the EGF receptor (EGFR)/PI3K/AKT pathway, the EGFR/STAT1 pathway, and the EGFR/MEK/ERK pathway, and by IFN-γ via the JAK2/STAT1 pathway. MF-IHC demonstrated that STAT1 and EGFR phosphorylation was frequently shown in PD-L1-positive cases and STAT1 phosphorylation was correlated with lymphocyte infiltration and EGFR phosphorylation. Moreover, the phosphorylation pattern of the related molecules in PD-L1-positive cells differed among the cases investigated. These findings indicate that PD-L1 expression mechanisms differ depending on the tissue environment and suggest that the examination of the tissue environment and molecular alterations of cancer cells affecting PD-L1 expression make it necessary for each patient to choose the appropriate combination drugs for PD-1 blockade cancer treatment.

Topics: B7-H1 Antigen; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Interferon-gamma; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Programmed Cell Death 1 Receptor; Squamous Cell Carcinoma of Head and Neck

Gastroesophageal reflux disease (GERD) may present as nonerosive reflux disease (NERD), erosive esophagitis (EE), or be complicated by Barrett's esophagus (BE). The explanation as to what determines the phenotype of GERD is awaited. Therefore, we assessed the correlation between the growth factors expression and endoscopic as histologic findings in GERD patients.. The squamous esophageal epithelium of 50 patients (20-NERD, 7-EE, 15-BE, 8 controls) was examined by: (1) magnification endoscopy with evaluation of minimal GERD changes such as: microerosions, white spots, palisade blood vessels visibility, and intrapapillary capillary loops (IPCLs) appearance, (2) histology, (3) immunohistochemistry with evaluation of the expression of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and their receptors (VEGFR and EGFR).. The expression of VEGF, but not VEGFR, EGF, and EGFR, was significantly increased in EE patients compared to NERD patients and controls. VEGF levels correlated significantly with the presence of white spots, but not with other minimal endoscopic and histologic features. The EGFR expression correlated positively with basal cell hyperplasia and enlarged IPCLs.. Our findings suggest a correlation between growth factors expression and findings in conventional endoscopy, formation of endoscopic minimal changes, and histologic lesions.

Topics: Barrett Esophagus; Carcinoma, Squamous Cell; Endoscopy, Gastrointestinal; Epidermal Growth Factor; ErbB Receptors; Esophageal Mucosa; Gastroesophageal Reflux; Humans; Phenotype; Vascular Endothelial Growth Factor A

Cr(VI) is broadly applied in industry. Cr(VI) exposure places a big burden on public health, thereby increasing the risk of lung squamous cell carcinoma (LUSC). The mechanisms underlying Cr(VI)-induced LUSC remain largely elusive. Here, we report that the cancer stem cell (CSC)/tumour-initiating cell (TIC)-like subgroup within Cr(VI)-transformed bronchial epithelial cells (CrT) promotes lung cancer tumourigenesis. Mechanistically, Cr(VI) exposure specifically increases the expression levels of aldehyde dehydrogenase 1A1 (ALDH1A1), a CSC marker, through KLF4-mediated transcription. ALDH1A1 maintains self-renewal of CrT/TICs and facilitates the expression and secretion of EGF from CrT/TICs, which subsequently promotes the activation of EGFR signalling in differentiated cancer cells and tumour growth of LUSC. In addition, the ALDH1A1 inhibitor A37 and gemcitabine synergistically suppress LUSC progression. Importantly, high ALDH1A1 expression levels are positively correlated with advanced clinical stages and predict poor survival in LUSC patients. These findings elucidate how ALDH1A1 modulates EGF secretion from TICs to facilitate LUSC tumourigenesis, highlighting new therapeutic strategies for malignant lung cancers.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Lung; Lung Neoplasms; Neoplastic Processes; Retinal Dehydrogenase; Tics

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Mouth Neoplasms; Squamous Cell Carcinoma of Head and Neck

Accumulating evidence suggests that specific non-coding RNAs exist in many types of malignant tissues, and are involved in cancer invasion and metastasis. However, little is known about the precise roles of non-coding RNAs in squamous cell carcinoma (SQCC) invasion and migration. Recently, the dentin matrix protein-1 (DMP-1) gene locus was identified as a transcriptionally active site in squamous cell carcinoma (SQCC) tissue and cells. However, it is unclear whether RNA associated with cell migration exist at the DMP-1 gene locus in SQCC cells. We identified a novel promoter-associated non-coding RNA in the antisense strand of DMP-1 gene locus, promoter-associated non-coding RNA (panRNA)-DMP-1, by the RACE method in SQCC cells and tissues, and characterized the functions of panRNA-DMP-1 in EGF-driven SQCC cell migration. The inhibition of endogenous panRNA-DMP-1 expression by specific siRNAs and exogenous over-expression of panRNA-DMP-1 resulted in increased and suppressed cellular migration toward EGF in SQCC cells, respectively, and nuclear expression of panRNA-DMP-1 was induced by EGF stimulation. Mechanistically, suppression of panRNA-DMP-1 expression increased EGFR nuclear localization upon EGF treatment and nuclear panRNA-DMP-1 physically interacted with EGFR, which was confirmed by RNA immunoprecipitation assay using a bacteriophage-delivered PP7 RNA labeling system. Furthermore, co-immunoprecipitation assay revealed that suppression of panRNA-DMP-1 stabilized EGFR interaction with STAT3, a known co-transcription factors of EGFR, to induce migratory properties in many cancer cells. Based on these findings, panRNA-DMP-1 is an EGFR-associating RNA that inhibits the EGF-induced migratory properties of SQCC possibly by regulating EGFR nuclear localization and EGFR binding to STAT3.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix Proteins; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Phosphoproteins; RNA, Antisense; RNA, Neoplasm

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Humans

p120-catenin (p120) serves as a stabilizer of the calcium-dependent cadherin-catenin complex and loss of p120 expression has been observed in several types of human cancers. The p120-dependent E-cadherin-β-catenin complex has been shown to mediate calcium-induced keratinocyte differentiation via inducing activation of plasma membrane phospholipase C-γ1 (PLC-γ1). On the other hand, PLC-γ1 has been shown to interact with phosphatidylinositol 3-kinase enhancer in the nucleus and plays a critical role in epidermal growth factor-induced proliferation of oral squamous cell carcinoma (OSCC) cells. To determine whether p120 suppresses OSCC proliferation and tumor growth via inhibiting PLC-γ1, we examined effects of p120 knockdown or p120 and PLC-γ1 double knockdown on proliferation of cultured OSCC cells and tumor growth in xenograft OSCC in mice. The results showed that knockdown of p120 reduced levels of PLC-γ1 in the plasma membrane and increased levels of PLC-γ1 and its signaling in the nucleus in OSCC cells and OSCC cell proliferation as well as xenograft OSCC tumor growth. However, double knockdown of p120 and PLC-γ1 or knockdown of PLC-γ1 alone did not have any effect. Immunohistochemical analysis of OSCC tissue from patients showed a lower expression level of p120 and a higher expression level of PLC-γ1 compared with that of adjacent noncancerous tissue. These data indicate that p120 suppresses OSCC cell proliferation and tumor growth by inhibiting signaling mediated by nuclear PLC-γ1.

Topics: Calcium, Dietary; Carcinoma, Squamous Cell; Catenins; Cell Differentiation; Cell Proliferation; Epidermal Growth Factor; Head and Neck Neoplasms; Mouth Neoplasms; Phospholipase C gamma; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have longterm effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model.. In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors.. Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis.. All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins.. These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis.

Topics: Amino Acid Motifs; Animals; Calcium-Binding Proteins; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Discoidin Domain; Epidermal Growth Factor; Genetic Therapy; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Tumor Cells, Cultured

Patients failing radiotherapy for laryngeal squamous cell carcinoma (LSCC) often require salvage total laryngectomy which has major functional consequences, highlighting a need for biomarkers of radiotherapy resistance. In other tumour types, radioresistance has been linked to epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF-1R). Here, we evaluated IGF-1R and EGFR as predictors and mediators of LSCC radioresistance.. We compared IGF-1R and EGFR immunohistochemical scores in patients with LSCC achieving long-term remission post-radiotherapy (n = 23), patients treated with primary laryngectomy (n = 22) or salvage laryngectomy following radiotherapy recurrence (n = 18). To model radioresistance in vitro, two LSCC cell lines underwent clinically relevant irradiation to 55 Gy in 2.75 Gy fractions.. Type 1 insulin-like growth factor receptor expression was higher in pre-treatment biopsies of radiotherapy failures compared with those in long-term remission and was upregulated post-radiotherapy. Patients undergoing primary laryngectomy had more advanced T/N stage and greater tumour IGF-1R content than those achieving long-term remission. Pre-treatment EGFR did not associate with radiotherapy outcomes but showed a trend to upregulation post-irradiation. In vitro, radiosensitivity was enhanced by inhibition of EGFR but not IGF. Repeated irradiation upregulated IGF-1R in BICR18 and SQ20B cells and EGFR in SQ20B, and enhanced SQ20B radioresistance. Repeatedly irradiated SQ20B_55 cells were not radiosensitised by inhibition of IGF and/or EGFR, but IGF-1R:EGFR co-inhibition suppressed baseline cell survival more effectively than blockade of either pathway alone, and more effectively than in parental cells.. Radiation upregulates IGF-1R and may enhance IGF/EGFR dependence, suggesting that IGF/EGFR blockade may have activity in LSCCs that recur post-radiotherapy.

Topics: Aged; Carcinoma, Squamous Cell; Cohort Studies; Epidermal Growth Factor; Female; Humans; Laryngeal Neoplasms; Laryngectomy; Male; Middle Aged; Predictive Value of Tests; Radiation Tolerance; Receptor, IGF Type 1; Signal Transduction; Somatomedins

The protein voltage-gated sodium channel Nav1.5 is highly upregulated in various types of cancer and, in general, promotes cancer cell invasiveness and metastatic progression. A previous study found that Nav1.5 was highly expressed in poorly differentiated oral squamous cell carcinoma (OSCC). However, whether Nav1.5 enhances invasiveness and metastasis of OSCC are still unknown. In this study, we found that Nav1.5 was highly expressed in OSCC cell lines compared with normal oral keratinocyte HOK cell line by using western blot analysis. CCK-8 assay results revealed that downregulation of Nav1.5 expression by its specific siRNA reduced proliferation of OSCC HSC-3 cells. Moreover, transwell assay results showed Nav1.5 knockdown significantly inhibited migration and invasion of HSC-3 cells. Meanwhile, qRT-PCR and western blot analysis results showed that epidermal growth factor (EGF) induced Nav1.5 expression in a time- and dose-dependent manner. In addition, EGF promoted proliferation, migration and invasion of HSC-3 cells. Importantly, the Nav1.5 inhibitor tetrodotoxin significantly inhibited the proliferation of HSC-3 cells and impeded the migration and invasion of HSC-3 cells. Furthermore, it was found that siRNA-mediated knockdown of Nav1.5 also lessened the proliferation of HSC-3 cells and blocked the migration and invasion of HSC-3 cells. Taken together, these results indicate that Nav1.5 is involved in the progression of OSCC and Nav1.5 promotes the proliferation, migration and invasion of OSCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; NAV1.5 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; RNA Interference; Sodium Channel Blockers; Tetrodotoxin

The aim of this study was to evaluate the expression of Akt, PTEN, Mdm2 and p53 proteins in three different head and neck squamous cell carcinoma (HNSCC) cell lines (HN6, HN19 and HN30), all of them treated with epidermal growth factor (EGF) and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of Hsp90 protein.. Immunofluorescence and western blot were performed in order to analyze the location and quantification, respectively, of proteins under the action 17-AAG and EGF.. Treatment with EGF resulted in increased levels of Akt, PTEN and p53 in all cell lineages. The expression of Mdm2 was constant in HN30 and HN6 lineages, while in HN19 showed slightly decreased expression. Under the action 17-AAG, in HN6 and HN19, the expression of PTEN and p53 proteins was suppressed, while Akt and Mdm2 expression was reduced. Finally, in the HN30 cell lineage were absolute absence of expression of Akt, Mdm2 and p53 and decreased expression of PTEN.. These data allow us to speculate on the particular utility of 17-AAG for HNSCC treatment through the inhibition of Akt protein expression, especially in the cases that retain the expression of PTEN protein.

Topics: Benzoquinones; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Lineage; Epidermal Growth Factor; Head and Neck Neoplasms; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-mdm2; PTEN Phosphohydrolase; Squamous Cell Carcinoma of Head and Neck; Tumor Suppressor Protein p53

Epidermal growth factor (EGF) promotes tumourigenesis and tissue repair of epithelial and mesenchymal cells and has a role in chemotaxis, mitogenesis, cell motility, and cytoprotection. It also enhances the growth of cancers. EGF may therefore have a role in the initiation or promotion of oral carcinogenesis. The cases of 152 patients with oral squamous cell carcinoma whose preoperative serum EGF level was determined by enzyme-linked immunosorbent assay were analyzed retrospectively, along with those of 40 age- and sex-matched controls. Patients with higher levels of EGF were more likely to have neck lymph node metastasis (P=0.026), advanced stage cancer (P=0.04), and a worse survival status (P=0.0019). Multivariate analysis using the Cox proportional hazards model indicated that the EGF level was an independent predictor of poor survival (hazard ratio 1.99, P=0.018). Patients with higher preoperative serum EGF levels had significantly poorer cancer-specific survival by Kaplan-Meier analysis (P=0.032). This study indicates that a higher preoperative serum EGF level is associated with neck lymph node metastasis, more advanced stage, and poor survival. EGF should be considered as a potential prognostic biomarker and a therapeutic target for patients with oral cancer.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Mouth Neoplasms; Neoplasm Staging; Prognosis; Retrospective Studies; Survival Rate

Migration and invasion are the most important characteristics of human malignancies which limit cancer drug therapies in the clinic. Tongue squamous cell carcinoma (TSCC) is one of the rarest types of cancer, although it is characterized by a higher incidence, rapid growth and greater potential for metastasis compared with other oral neoplasms worldwide. Studies have demonstrated that the phenolic compound obovatol exhibits anti‑tumor effects. However, the potential mechanisms underlying obovatol‑mediated signaling pathways have not been completely elucidated in TSCC. The present study investigated the anti‑tumor effects and potential molecular mechanisms mediated by obovatol in TSCC cells and tissues. The results of the present study demonstrated that obovatol exerted cytotoxicity in SCC9 TSCC cells, and inhibited their migration and invasion. In addition, obovatol induced apoptosis in SCC9 TSCC cells by increasing caspase 9/3 and apoptotic protease enhancing factor 1 expression levels. Western blot analysis demonstrated that obovatol inhibited the expression of pro‑epidermal growth factor (EGF), Janus kinase (JAK), and signal transducer and activator of transcription (STAT) in SCC9 TSCC cells. A study of the molecular mechanisms demonstrated that depletion of EGF reversed the obovatol‑mediated inhibition of SCC9 TSCC cell growth and aggressiveness. Animal experiments indicated that obovatol significantly inhibited TSCC tumor growth and increased the number of apoptotic cells in tumor tissues. In conclusion, the results of the present study provided scientific evidence that obovatol inhibited TSCC cell growth and aggressiveness through the EGF‑mediated JAK‑STAT signaling pathway, suggesting that obovatol may be a potential anti‑TSCC agent.

Topics: Animals; Antigens, CD; Antineoplastic Agents, Phytogenic; Biphenyl Compounds; Cadherins; Carcinoma, Squamous Cell; CDC2 Protein Kinase; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinase 2; Epidermal Growth Factor; Fibronectins; Gene Expression Regulation, Neoplastic; Humans; Janus Kinases; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Phenyl Ethers; Signal Transduction; STAT Transcription Factors; Tongue Neoplasms; Vimentin; Xenograft Model Antitumor Assays

Genetic amplification, overexpression, and increased signaling from the epidermal growth factor receptor (EGFR) are often found in oral squamous cell carcinoma (OSCC) and thus EGFR is frequently targeted molecularly by the therapeutic antibody cetuximab. We assessed effects of cetuximab in control of EGF-driven malignant traits of OSCC cells. EGF stimulation promoted progression level of mesenchymal traits in OSCC cells, which were attenuated by cetuximab but incompletely. We pursued a potential mechanism underlying such incomplete attenuation of OSCC malignant traits. Cetuximab promoted secretion of EGFR-EVs by OSCC cells and failed to inhibit EGF-driven secretion of EGFR-EVs. Cetuximab was also found to be robustly secreted with the EGFR-EVs by the OSCC cells. Thus, EGF promotes the level of mesenchymal traits of OSCC cells and secretion of EGFR-EVs, which involve cetuximab resistance.

Topics: Antibodies, Monoclonal, Humanized; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Extracellular Vesicles; Humans; Mouth Neoplasms

Activated platelets release functional, high m.w. epidermal growth factor (HMW-EGF). In this study, we show platelets also express epidermal growth factor (EGF) receptor (EGFR) protein, but not ErbB2 or ErbB4 coreceptors, and so might respond to HMW-EGF. We found HMW-EGF stimulated platelet EGFR autophosphorylation, PI3 kinase-dependent AKT phosphorylation, and a Ca

Topics: Autocrine Communication; B-Cell Lymphoma 3 Protein; Blood Platelets; Carcinogenesis; Carcinoma, Squamous Cell; Cell Movement; Cells, Cultured; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Interleukin-1beta; Mouth Neoplasms; Neoplasm Invasiveness; Platelet Activation; Proto-Oncogene Proteins; Transcription Factors

Cancer cells have been known to overexpress the epidermal growth factor receptor (EGFR) and hence relevant multiple‑targeted therapies have been developed, with a recent clinical application of the antibody‑mediated inhibition of the EGFR. However, this strategy is not useful in cancer cells with mutations in KRAS; a GTPase downstream of EGFR which constitutively activates the pathway without EGF stimulation. Furthermore, mutations in EGFR also reduce the binding of monoclonal antibodies and thereby render them ineffective. In the present study, we designed a chimeric EGF protein fused to the truncated N‑terminal domain fragment of Pseudomonas aeruginosa exotoxin A (EGF‑ETA), which has ADP‑ribosylation activity and induces apoptosis. The EGF‑ETA protein was expressed in E. coli as a His‑tagged fusion. Our results showed that EGF‑ETA significantly inhibited the proliferation of EGFR‑positive A431 epidermoid carcinoma (IC50 27 ng/ml) and HN5 head and neck squamous cell carcinoma (IC50 36 ng/ml) cells. However, its effect on cancer cells with little or no EGFR expression was limited (A549‑IC50 1,000 ng/ml; MCF‑7‑IC50 >10,000 ng/ml). Compared to cetuximab, EGF‑ETA was highly potent in its killing capacity of HN5 cancer cells at 1,000 ng/ml, while cetuximab had little effect at 1,000 ng/ml. Furthermore, EGF‑ETA was just as potent in HCT116 (KRAS G13D) and SW480 (KRAS G12V) colon cancer cell lines harbouring KRAS hyperactivating mutations when compared to KRAS wild‑type HT29 colon cancer cells. Finally, co‑incubation of EGF‑ETA with an anti‑EGF antibody abrogated its effect on the EGFR‑positive A431 cells. Our results show that the chimeric EGF‑ETA toxin is extremely effective against EGFR‑positive cancers and raises the potential to further develop this chimera for use in targeting EGFR‑positive tumours resistant to monoclonal antibodies.

Topics: ADP Ribose Transferases; Antibodies, Anti-Idiotypic; Apoptosis; Bacterial Toxins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Humans; Ligands; Proto-Oncogene Proteins p21(ras); Pseudomonas aeruginosa; Pseudomonas aeruginosa Exotoxin A; Recombinant Fusion Proteins; Squamous Cell Carcinoma of Head and Neck; Virulence Factors

Inhibiting BRD4 has emerged as a promising anticancer strategy, and inhibitors such as JQ1 can suppress cell growth in oral squamous cell carcinoma (OSCC). However, the mechanism through which JQ1 exerts its anticancer activity has not been reported. Moreover, JQ1 does not markedly inhibit proliferation and increase apoptosis in OSCC when used as a monotherapy. Herein, we explore the mechanism of JQ1 in OSCC and probe ways to increase its therapeutic potential. In this study, we used two cell lines, Cal27, and Scc25. We found that BRD4 was highly expressed in OSCC tissues when compared with adjacent non-tumor tissues, and JQ1 worked through the EGFR-mediated signaling pathway in tumor cells. Furthermore, we demonstrated that JQ1 induced an increased treatment effect in vitro and in vivo when combined with a PI3K inhibitor. Interestingly, subsequent mechanistic analyses indicated that further suppressing EGFR and BRD4 expression was instrumental to this functional synergism. Moreover, we found that upregulating EGFR expression by EGF stimulation protected cells treated with JQ1 from apoptosis, while knockdown of EGFR before addition of JQ1 successfully mimicked the combination treatment results. In summary, our findings revealed that JQ1 can act by inhibiting the EGFR-mediated signaling pathway, and EGFR expression influences the sensitivity of OSCC to JQ1. Regarding clinical use, this study demonstrates that BRD4 is a novel therapeutic target and EGFR can be used as a biomarker to identify the most appropriate anti-BRD4 treatment strategy in OSCC.

Topics: Aminopyridines; Analysis of Variance; Animals; Antineoplastic Agents; Apoptosis; Azepines; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Female; Gene Knockdown Techniques; Humans; Mice; Morpholines; Mouth Neoplasms; Nuclear Proteins; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; Transcription Factors; Triazoles; Xenograft Model Antitumor Assays

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. The majority of HNSCCs overexpress Epidermal Growth Factor Receptor (EGFR), an essential receptor tyrosine kinase (RTK) that promotes HNSCC growth and metastasis. Therefore, EGFR has been used as an important therapeutic target to treat HNSCC. Inhibition of EGFR stimulates autophagy in cancer cells. However, the role of autophagy in EGFR inhibitor-induced cancer suppression is still in a debate. Here, we reveal that the first- and the second-generation EGFR tyrosine kinase inhibitors (TKIs) differentially affect HNSCC autophagy. The second-generation EGFR TKIs have much stronger effects on autophagy than the first-generation TKIs. The second-generation EGFR TKIs not only promote autophagy initiation signaling but also block autophagic flux by disturbing the lysosomes function, indicating a novel mechanism by which EGFR TKIs modulate cancer cell autophagy. Blocking the initiation of autophagy does not affect the second-generation EGFR TKI-induced HNSCC growth suppression. This suggests that the anti-growth effect of the second-generation EGFR TKIs on HNSCC is not dependent on autophagy.

Topics: Autophagy; Carcinoma, Squamous Cell; Cell Proliferation; Dose-Response Relationship, Drug; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Squamous Cell Carcinoma of Head and Neck; Treatment Outcome

The activation of receptor tyrosine kinases (RTKs) results in cellular effects including cell proliferation, survival, migration and invasion; RTKs also play an important role in tumourigenesis. It has been reported that EGFR signalling controls the migration of oral squamous cell carcinoma (OSCC) SAS and HSC3 cells but not of HSC4 cells, although the proliferation of HSC4 cells is regulated by EGF/EGFR. In the present study, we investigated the roles of EGFR and the c-Met signalling pathway in cell migration via filopodia and lamellipodia formation, which may be prerequisites for migration. To explore the role of c-Met in cell migration, we inhibited c-Met RTK activity using the c-Met inhibitor SU11274 and activated c-Met using hepatocyte growth factor (HGF) in three OSCC cell lines HSC4, SAS and Ca9-22 and investigated migration potency using a wound healing assay. We showed that inhibition of c-Met significantly suppressed, and activation of c-Met significantly promoted, the migration of OSCC cells. Additionally, the migration of SAS and Ca9-22 cells was inhibited by the EGFR inhibitors AG1478 and cetuximab and promoted by EGF treatment. Moreover, migration potency was correlated with lamellipodia formation. Furthermore, western blot analyses demonstrated that SU11274 decreased and HGF increased lamellipodin protein levels as well as phosphorylated c-Met levels. Collectively, we demonstrated that c-Met signalling induced lamellipodia formation by upregulating lamellipodin, thereby promoting the migration of OSCC cells.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Indoles; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Piperazines; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-met; Pseudopodia; Quinazolines; Signal Transduction; Sulfonamides; Tyrphostins

Recent studies have revealed that the epidermal growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) are overexpressed in various types of human tumors and are attractive targets for anticancer drugs. In the present study, the expression of EGFR and IGF-1R in esophageal squamous cell carcinoma (ESCC) and adjacent normal tissues in a tissue microarray was firstly detected by immunohistochemical staining. In addition, their co-overexpression was observed in 48 out of 75 (64%) patients. Based on the findings, the antitumor activity of an EGFR/IGF-1R bispecific and enediyne-energized fusion protein EGF-LDP-IGF-AE, which we constructed recently by fusing two ligands (EGF and IGF-1) with an enediyne antibiotic lidamycin (LDM), on ESCC were evaluated. Binding assay indicated that the EGF-LDP-IGF protein bound to esophageal cancer cells, and then internalized into the cytoplasm. In vitro, the enediyne‑energized fusion protein EGF-LDP-IGF-AE exhibited extremely potent cytotoxicity to ESCC cells with IC50 values between 10-10 and 10-15 mol/l. In vivo, EGF-LDP‑IGF-AE also markedly suppressed the growth of human KYSE450 xenografts by 75.1% when administered at 0.3 mg/kg in a nude mouse model, and its efficacy was significantly higher than that of LDM (at maximum tolerated dosage) and mono-specific counterparts. In addition, EGF-LDP-IGF-AE arrested cell cycle progression and it concentration-dependently induced cell apoptosis as well as inhibited the activation of EGFR/IGF-1R and two major downstream signaling pathways (PI3K/AKT and RAS/MAPK). These data imply the potential clinical application of EGF-LDP-IGF-AE for ESCC patients with EGFR and/or IGF-1R overexpression.

Topics: Aminoglycosides; Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Proliferation; Enediynes; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Ligands; Male; Mice; Protein Binding; Receptor, IGF Type 1; Recombinant Fusion Proteins; Signal Transduction; Xenograft Model Antitumor Assays

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Skin Neoplasms

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in such cancers as head and neck squamous cell carcinoma (HNSCC); however, whether the metabolic enzyme, pyruvate dehydrogenase kinase 1 (PDK1), mediates EGF-enhanced HNSCC metastasis remains unclear. Of interest, we found that EGF induced PDK1 expression in HNSCC. Tumor cell transformation induced by EGF was repressed by PDK1 knockdown, and the down-regulation of PDK1 expression or inhibition of its activity significantly blocked EGF-enhanced cell migration and invasion. In addition, depletion of PDK1 impeded EGF-enhanced binding of HNSCC cells to endothelial cells as well as the metastatic seeding of tumor cells in lungs. PDK1 depletion inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Furthermore, PDK1 overexpression induced MMP-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Of interest, depletion of fibronectin inhibited PDK1-enhanced MMP-1-3 and MMP-9 expression as well as Rac1/cdc42 activation and tumor invasion. These results demonstrate that EGF-induced PDK1 expression enhances HNSCC metastasis

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Fibronectins; Head and Neck Neoplasms; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Protein Serine-Threonine Kinases; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Squamous Cell Carcinoma of Head and Neck; Transcriptional Activation; Up-Regulation

Epithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal-regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.

Topics: Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; Female; Follistatin-Related Proteins; Genes, Switch; Head and Neck Neoplasms; Immunoprecipitation; Mass Spectrometry; Mice, Inbred NOD; MicroRNAs; Wound Healing

SIP-SII, the sulfated Sepiella maindroni ink polysaccharide (SIP), has been manifested to possess anti-tumor and anti-metastasis activity in vivo and in vitro. In the present study, we evaluated its inhibitory effect on the epidermal growth factor (EGF)-induced migration and invasion of human epidermoid carcinoma cell (KB cell line) as well as the related signaling pathways. The results of MTT assay indicated that SIP-SII inhibited the proliferation of KB cells in a concentration and time dependent manner. Notably, the attenuation of cell growth by SIP-SII was enlarged in the presence of EGF. The wound healing assay and transwell invasion assay were used to evaluate the effect of SIP-SII on the EGF-induced migration and invasion of KB cells and the results showed that SIP-SII markedly attenuated the EGF-induced migration and invasion. Besides, the EGF-induced matrix metalloproteinase-2 (MMP-2) expression was also suppressed by SIP-SII. However, SIP-SII showed no significant inhibition of the EGF-induced matrix metalloproteinase-9 (MMP-9) expression. Further research revealed that SIP-SII decreased the EGF-induced phosphorylation of epidermal growth factor receptor (EGFR), Akt and p38, but no significant suppression on EGF-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) and c-Jun N-terminal kinases (JNK) by SIP-SII treatment was observed. The involvement of EGFR/Akt/p38 pathway was confirmed by evidence that SIP-SII would enlarge the inhibitory effect of the specific signal pathway inhibitors. These results indicate that SIP-SII has the potential to be used as the inhibitor of tumor metastasis especially for cancers characterized by over-activation of EGF/EGFR signaling.

Topics: Animals; Carcinoma, Squamous Cell; Cell Movement; Decapodiformes; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; p38 Mitogen-Activated Protein Kinases; Polysaccharides; Proto-Oncogene Proteins c-akt; Signal Transduction

Epidermal growth factor (EGF) is important for cancer cell proliferation, angiogenesis and metastasis in many types of cancer. However, the mechanisms involved in EGF-induced head and neck squamous cell carcinoma (HNSCC) metastasis remain largely unknown. In this study, we reveal that angiopoietin-like 4 (ANGPTL4) plays an important role in the regulation of EGF-induced cancer metastasis. We showed that EGF-induced ANGPTL4 expression promoted anoikis resistance and cancer cell migration and invasion in HNSCC. In addition, depletion of ANGPTL4 inhibited EGF-induced cancer cell invasion. Autocrine production of EGF-induced ANGPTL4 regulated the expression of matrix metalloproteinases (MMPs). The induction of MMP-1 gene expression by ANGPTL4-activated integrin β1 signalling occurred through the AP-1 binding site in the MMP-1 gene promoter. Furthermore, down-regulation of MMP-1 impeded EGF- and recombinant ANGPTL4-enhanced HNSCC cell migration and invasion. Depletion of ANGPTL4 significantly blocked EGF-primed extravasation and metastatic seeding of tumour cells and MMP-1 expression in lungs. However, no effect of ANGPTL4 on tumour growth was observed. These results suggest that EGF-induced expression and autocrine production of ANGPTL4 enhances HNSCC metastasis via the up-regulation of MMP-1 expression. Inhibition of ANGPTL4 expression may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.

Topics: Angiopoietin-Like Protein 4; Angiopoietins; Anoikis; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Genes, jun; Head and Neck Neoplasms; Humans; Integrin beta1; Matrix Metalloproteinase 1; Neoplasm Metastasis; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

"Warburg effect", the enhanced glycolysis or aerobic glycolysis, confers cancer cells the ability to survive and proliferate even under stressed conditions. In this study, we explored the role of epidermal growth factor (EGF) in orchestrating Warburg effect, the epithelial-mesenchymal transition (EMT) process, and the acquisition of cancer stem-like cell properties in human oral squamous cell carcinoma (OSCC) cells. Our results showed that EGF induces EMT process in OSCC cells, which correlates with the acquisition of cancer stem-like properties, including the enrichment of CD44+/CD24- population of cancer cells and an increased expression of CSC-related genes, aldehyde dehydrogenase-1 (ALDH1) and Bmi-1. We also showed that EGF concomitantly enhanced L-lactate production, while blocking glycolysis by 2-deoxy-D-glucose (2-DG) robustly reversed EGF-induced EMT process and CSC-like properties in OSCC cells. Mechanistically, we demonstrated that EGF promoted EMT process and CSC generation through EGFR/PI3K/HIF-1α axis-orchestrated glycolysis. Using an orthotopic tumor model of human OSCC (UM-SCC1) injected in the tongue of BALB/c nude mice, we showed that treatment with 2-DG in vivo significantly inhibited the metastasis of tumor cells to the regional cervical lymph nodes and reduced the expression of ALDH1 and vimentin in both in situ tumors and tumor cell-invaded regional lymph nodes. Taken together, these findings have unveiled a new mechanism that EGF drives OSCC metastasis through induction of EMT process and CSC generation, which is driven by an enhanced glycolytic metabolic program in OSCC cells.

Topics: Aldehyde Dehydrogenase 1 Family; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; CD24 Antigen; Cell Line, Tumor; Deoxyglucose; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Glycolysis; Head and Neck Neoplasms; Humans; Hyaluronan Receptors; Hypoxia-Inducible Factor 1, alpha Subunit; Isoenzymes; Lactic Acid; Mice, Inbred BALB C; Mice, Nude; Mouth Neoplasms; Neoplastic Stem Cells; Phenotype; Phosphatidylinositol 3-Kinase; Polycomb Repressive Complex 1; Retinal Dehydrogenase; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Time Factors; Xenograft Model Antitumor Assays

Gefitinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor which has been proven effective for cancer treatment. In this study, we sought to determine whether gefitinib could increase the in vivo tumor uptake of human

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Iodine Radioisotopes; Mice; Mice, Nude; Protein Transport; Quinazolines; Radiation Tolerance; Radiochemistry; Tissue Distribution

Head and neck squamous cell carcinoma is frequently associated with aberrant epidermal growth factor receptor (EGFR) signaling, which contributes to tumor growth. Here, the functional importance of EGFR ligands in relation to proliferation and sensitivity to the EGFR-targeted therapy cetuximab was investigated in three tongue cancer cell lines.. The influence of epidermal growth factor (EGF), amphiregulin (AR), and epiregulin (EPR) on tumor cell proliferation and cetuximab response was evaluated by the addition of recombinant human (rh) proteins or by siRNA-mediated downregulation of the endogenous ligand production. The expression, activation and cellular distribution of EGFR were assessed by Western blot analysis and immunocytochemistry.. EGF downregulation suppressed the proliferation of all investigated tumor cell lines, whereas the response to an increased level of EGF differed between EGFR-overexpressing and EGFR-non-overexpressing cell lines. Furthermore, tumor cells consistently displayed increased cetuximab resistance upon the addition of rhEGF, whereas EGF silencing was associated with an improved cetuximab response. The data regarding AR and EPR were inconclusive.. Our data suggest that the amount of EGF is a determinant of the tumor cell proliferation rate and the response to cetuximab treatment in tongue cancer. Thus, EGF is a potential predictive biomarker of poor cetuximab response and a possible treatment target.

Topics: Amphiregulin; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cetuximab; Down-Regulation; Drug Resistance, Neoplasm; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Head and Neck Neoplasms; Humans; Immunohistochemistry; Ligands; Molecular Targeted Therapy; Squamous Cell Carcinoma of Head and Neck; Tongue Neoplasms

Activation of β1 integrins in dormant tumor cells has been linked to metastatic progression, suggesting that therapies designed to maintain β1 integrins in an inactive state may be useful in the prevention of metastatic disease. Our earlier studies have demonstrated that EGF regulates the activation state of the α5β1 integrin in EGFR overexpressing tumor cells through an ERK/p90RSK signaling pathway. Activation of this pathway by EGF resulted in the filamin A dependent inactivation of the α5β1 integrin receptor for fibronectin. The current study was designed to address the role of EGFR overexpression in the regulation of α5β1 integrin activation state by EGF. Lentiviral knockdown of EGFR coupled with limited dilution cloning was used to develop A431 squamous carcinoma cell lines expressing high, moderate, and low levels of EGFR. Inactivation of α5β1 integrin by EGF was shown to correlate with both the level of EGFR expression and the extent of p90RSK phosphorylation, but not with the level of ERK phosphorylation, suggesting that high levels of EGFR promote α5β1 integrin inactivation through sustained activation of p90RSK. Treatment of cells with EGFR kinase inhibitor resulted in a reactivation of the integrin which could be reversed with the phosphatase inhibitor, menadione. Taken together, these findings indicate that p90RSK may function to maintain dormancy in tumor cells expressing high levels of EGFR. © 2015 Wiley Periodicals, Inc.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha5beta1; MAP Kinase Signaling System; Phosphorylation; RNA, Small Interfering; Signal Transduction; Up-Regulation

The initiation and progression of cancer is closely associated with the tumor microenvironment. The overexpression of oncogenes during tumor growth and progression by stromal stimuli can affect the aggressiveness of the cancer. In this study, in vitro and in vivo studies were performed to examine the role of stromal epidermal growth factor (EGF) in enhancing the invasive potential of in low-invasive cancer. EGF was tested in order to elucidate the specific molecules that participate in increasing the invasive potential of low-invasive cancer cells. EGF stimulation enhanced cancer invasion in an EGF receptor (EGFR)-dependent manner. EGF induced insulin-like growth factor-II mRNA-binding protein-3 (IMP-3) and podoplanin (PDPN) expression, which play an important role in oral squamous cell carcinoma (OSCC) cell invasion. An apparent tumor mass was not observed in the mouse xenograft; however, multiple tumor microfoci were seen in mice injected with IMP-3-overexpressing cells. These results show that EGF stimulates IMP-3 expression, thereby increasing cancer invasion and tumor progression.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Mice; Mice, Inbred BALB C; Mouth Neoplasms; Neoplasm Invasiveness; RNA-Binding Proteins; RNA, Messenger; Tumor Microenvironment

CD166 has been considered a relatively specific marker of stem cells and cancer stem cells, and the altered expression of CD166 has also been reported as a prognostic marker of several other types of cancer. However, the molecular functions of CD166 in these cancer cells are largely unknown. In this study, we found that CD166 significantly enhanced epidermal growth factor receptor (EGFR) phosphorylation and prolonged epidermal growth factor (EGF)/EGFR signalling activation. In addition, EGF stimulation in CD166-overexpressing oral squamous carcinoma cells led to enhanced colony formation, invasion capacity and cytoskeletal re-organization in vitro and elevated tumourigenesis in vivo. Taken together, the results of our study identify CD166 as an intriguing therapeutic target for patients suffering from oral squamous cell carcinoma (OSCC).

Topics: Antigens, CD; Carcinoma, Squamous Cell; Cell Adhesion Molecules, Neuronal; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Fetal Proteins; Humans; Mouth Neoplasms; Phosphorylation; Signal Transduction

Head and neck squamous cell carcinoma (HNSCC) represents the most frequent malignancy in the head and neck region, and the survival rate has not been improved significantly over the past three decades. It has been reported the infiltrated macrophages contribute to the malignant progression of HNSCC. However, the crosstalk between macrophages and cancer cells remains poorly understood. In the present study, we explored interactions between monocytes/macrophages and HNSCC cells by establishing the direct co-culture system, and found that the crosstalk promoted the migration and invasion of cancer cells by enhancing the invadopodia formation through a CCL2/EGF positive feedback loop. Our results demonstrated HNSCC cells educated monocytes into M2-like macrophages by releasing C-C motif chemokine ligand 2 (CCL2, or MCP-1). And the M2-like macrophages secreted epithelial growth factor (EGF), which increased the motility of HNSCC cells by enhancing the invadopodia formation. These subcellular pseudopodia degraded extracellular matrix (ECM), facilitating tumor local invasion and distant metastasis. Moreover, EGF up-regulated CCL2 expression in HNSCC cells, which recruited monocytes and turned them into M2-like macrophages, thus forming a positive feedback paracrine loop. Finally, we reported that curcumin, a powerful natural drug, suppressed the production of EGF and CCL2 in macrophages and cancer cells, respectively, blocking the feedback loop and suppressing the migration and invasion of HNSCC cells. These results shed light on the possibilities and approaches based on targeting the crosstalk between cancer cells and monocytes/macrophages in HNSCC for potential cancer therapy.

Topics: Carcinoma, Squamous Cell; Cell Communication; Cell Differentiation; Cell Movement; Cell Polarity; Chemokine CCL2; Curcumin; Epidermal Growth Factor; Feedback, Physiological; Head and Neck Neoplasms; Humans; Macrophages; Monocytes; Neoplasm Invasiveness; Squamous Cell Carcinoma of Head and Neck

The purpose of this study was to evaluate whether an exogenous epidermal growth factor (EGF) could induce anti-tumor and radiosensitizing effects in vivo.. BALB/c-nu mice that were inoculated with A431 (human squamous cell carcinoma) cells in the right hind legs were divided into five groups: I (no treatment), II (EGF for 6 days), III (EGF for 20 days), IV (radiotherapy [RT]), and V (RT plus concomitant EGF). EGF was administered intraperitoneally (5 mg/kg) once a day and the RT dose was 30 Gy in six fractions. Hematoxylin and eosin (H&E) stained sections of tumor, liver, lung, and kidney tissues were investigated. Additionally, tumors were subjected to immunohistochemistry staining with caspase-3.. EGF for 6 days decreased tumor volume, but it approached the level of the control group at the end of follow-up (p=0.550). The duration of tumor shrinkage was prolonged in group V while the slope of tumor re-growth phase was steeper in group IV (p=0.034). EGF for 20 days decreased tumor volume until the end of the observation period (p < 0.001). Immunohistochemistry revealed that mice in group V showed stronger intensity than those in group IV. There were no abnormal histological findings upon H&E staining of the normal organs.. EGF-induced anti-tumor effect was ascertained in the xenograft mouse models with A431 cells. Concomitant use of EGF has the potential role as a radiosensitizer in the design of fractionated irradiation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Humans; Mice; Mice, Nude; Neoplasms, Experimental; Radiotherapy

Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where it has been shown to promote tumor cell invasion upon phosphorylation. One mechanism by which EGFR promotes tumor progression is by activating signal cascades that lead to loss of E-cadherin, a transmembrane glycoprotein of the cell-cell adherence junctions; however mediators of these signaling cascades are not fully understood. One such mediator, RhoC, is activated upon a number of external stimuli, such as epidermal growth factor (EGF), but its role as a mediator of EGF-stimulated migration and invasion has not been elucidated in HNSCC. In the present study, we investigate the role of RhoC as a mediator of EGF-stimulated migration and invasion in HNSCC. We show that upon EGF stimulation, EGFR and RhoC were strongly activated in HNSCC. This resulted in activation of the phosphatidylinositol 3-Kinase Akt pathway (PI3K-Akt), phosphorylation of GSK-3β at the Ser(9) residue, and subsequent down regulation of E-cadherin cell surface expression resulting in increased tumor cell invasion. Knockdown of RhoC restored E-cadherin expression and inhibited EGF-stimulated migration and invasion. This is the first report in HNSCC demonstrating the role RhoC plays in mediating EGF-stimulated migration and invasion by down-regulating the PI3K-Akt pathway and E-cadherin expression. RhoC may serve as a treatment target for HNSCC.

Topics: Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Gene Knockdown Techniques; Head and Neck Neoplasms; Humans; Models, Biological; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; rho GTP-Binding Proteins; rhoC GTP-Binding Protein; Signal Transduction; Snail Family Transcription Factors; Squamous Cell Carcinoma of Head and Neck; Transcription Factors

The number of epidermal growth factor receptor (EGFR)-targeting drugs in the development for cancer treatment is continuously increasing. Currently used EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors have specific limitations related to toxicity and development of resistance, and there is a need for alternative treatment strategies to maximize the clinical potential of EGFR as a molecular target. This study describes the design and production of a novel EGFR-targeted fusion protein, rGel/EGF, composed of the recombinant plant toxin gelonin and EGF. rGel/EGF was custom-made for administration by photochemical internalization (PCI), a clinically tested modality for cytosolic release of macromolecular therapeutics. rGel/EGF lacks efficient mechanisms for endosomal escape and is therefore minimally toxic as monotherapy. However, PCI induces selective and efficient cytosolic release of rGel/EGF in EGFR-expressing target cells by light-directed activation of photosensitizers accumulated selectively in tumor tissue. PCI of rGel/EGF was shown to be highly effective against EGFR-expressing cell lines, including head and neck squamous cell carcinoma (HNSCC) cell lines resistant to cetuximab (Erbitux). Apoptosis, necrosis and autophagy were identified as mechanisms of action following PCI of rGel/EGF in vitro. PCI of rGel/EGF was further shown as a highly tumor-specific and potent modality in vivo, with growth inhibitory effects demonstrated on A-431 squamous cell carcinoma (SCC) xenografts and reduction of tumor perfusion and necrosis induction in SCC-026 HNSCC tumors. Considering the small amount of rGel/EGF injected per animal (0.1 mg/kg), the presented in vivo results are highly promising and warrant optimization and production of rGel/EGF for further preclinical evaluation with PCI.

Topics: Animals; Antibodies, Monoclonal, Humanized; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Photochemistry; Recombinant Fusion Proteins; Ribosome Inactivating Proteins, Type 1; Squamous Cell Carcinoma of Head and Neck; Toxins, Biological

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) is associated with enhanced invasion and metastasis in head and neck squamous cell carcinoma (HNSCC). Long Pentraxin PTX3 is involved in immune escape in cancer cells. Here, we identified PTX3 as a promoting factor that mediates EGF-induced HNSCC metastasis. EGF-induced PTX3 transcriptional activation is via the binding of c-Jun to the activator protein (AP)-1 binding site of the PTX3 promoter. PI3K/Akt and NF-κB were essential for the PTX3 activation. EGF-induced PTX3 expression was blocked in c-Jun- and NF-κB-knockdown cells. EGF-mediated PTX3 secretion resulted in the enhancement of cell migration and invasion, and interactions between cancer and endothelial cells. The tail-vein injection animal model revealed that depletion of PTX3 decreased EGF-primed tumor cell metastatic seeding of the lungs. In addition, fibronectin, matrix metalloproteinase-9 (MMP9) and E-cadherin were essential components in EGFR/PTX3-mediated cancer metastasis. In conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF. Autocrine production of EGF-induced PTX3 in turn induces metastatic molecules, activating inflammatory cascades and metastasis.

Topics: Animals; C-Reactive Protein; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Heterografts; Humans; JNK Mitogen-Activated Protein Kinases; Male; Mice; Mice, SCID; Neoplasm Metastasis; NF-kappa B; Phosphatidylinositol 3-Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Serum Amyloid P-Component; Signal Transduction; Squamous Cell Carcinoma of Head and Neck; Transcription Factor AP-1; Transfection

Cisplatin is a cytotoxic chemotherapeutic drug frequently used to treat many solid tumors, including head and neck squamous cell carcinoma (HNSCC). EGF receptor (EGFR) inhibitors have also shown efficacy as alternatives to cisplatin in some situations. However, large clinical trials have shown no added survival benefit from the use of these two drugs in combination. Possible explanations for this include overlapping downstream signaling cascades. Using in vitro studies, we tested the hypothesis that cisplatin and EGFR inhibitors rely on the activation of the tumor suppressor STAT1, characterized by its phosphorylation at serine (S727) or tyrosine (Y701) residues. Cisplatin consistently increased the levels of p-S727-STAT1, and STAT1 siRNA knockdown attenuated cisplatin-induced cell death. EGFR stimulation also activated p-S727-STAT1 and p-Y701-STAT1 in a subset of cell lines, whereas EGFR inhibitors alone decreased levels of p-S727-STAT1 and p-Y701-STAT1 in these cells. Contrary to our hypothesis, EGFR inhibitors added to cisplatin treatment caused variable effects among cell lines, with attenuation of p-S727-STAT1 and enhancement of cisplatin-induced cell death in some cells and minimal effect in other cells. Using HNSCC tumor specimens from a clinical trial of adjuvant cisplatin plus the anti-EGFR antibody panitumumab, higher intratumoral p-S727-STAT1 appeared to correlate with worse survival. Together, these results suggest that cisplatin-induced cell death is associated with STAT1 phosphorylation, and the addition of anti-EGFR therapy to cisplatin has variable effects on STAT1 and cell death in HNSCC.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Squamous Cell; Cell Death; Cell Line, Tumor; Cetuximab; Cisplatin; Epidermal Growth Factor; ErbB Receptors; Gene Knockdown Techniques; Head and Neck Neoplasms; Humans; p38 Mitogen-Activated Protein Kinases; Panitumumab; Phosphorylation; Prognosis; RNA, Small Interfering; Squamous Cell Carcinoma of Head and Neck; STAT1 Transcription Factor

Oral lichen planus (OLP) is classified as a potential malignant disorder, and epidermal growth factor (EGF) may play a key role in cancer development. The aim of this study was to compare serum and saliva EGF among patients with OLP and oral squamous cell carcinoma (OSCC). A cross-sectional study was performed on 27 patients with OLP (10 reticular and 17 atrophic-erosive forms), 27 patients with OSCC and 27 healthy control group. The study was conducted at the Cancer Department, Clinic of Oral Medicine, Tehran University of Medical Sciences. The serum and saliva EGF were assayed by ELISA method. Statistical analysis of ANOVA was used. The mean serum EGF in OLP and OSCC patients was significantly lower compared to healthy control group (P<0.05), but no significant difference was observed between OLP and OSCC patients. There was no significant difference in mean salivary EGF among groups. As serum EGF levels appear to be statistically similar in OLP and OSCC, it seems that EGF might play a role in the pathogenesis of OLP and its cancerization.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Case-Control Studies; Cross-Sectional Studies; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Humans; Iran; Lichen Planus, Oral; Male; Middle Aged; Mouth Neoplasms; Saliva

Lymphatic metastasis is an important determinant of aggressive malignant tumors. Identification of key genes that regulate carcinoma cell metastasis will aid in understanding progression of esophageal squamous cell carcinoma (ESCC). Guanylate-binding protein 1 (GBP1) is a GTP-binding protein family member with high GTPase activity. While the role of GBP1 has been demonstrated in colorectal cancer (CRC) and ovarian cancer, such a role has not been identified in ESCC. Herein, we assessed GBP1 expression in ESCC via immunohistochemistry (IHC) analysis of 215 clinical ESCC specimens and found that the expression of GBP1 was significantly upregulated in lymph node metastatic ESCCs compared with non-metastatic cases. We further demonstrated that GBP1 expression was increased with epidermal growth factor (EGF) stimulus in ESCC cell lines and had a positive correlation with EGFR expression in ESCC tissue samples. Finally, we found that overexpression of GBP1 in ESCC cells induced more lymph node metastasis in an animal model. In summary, our results demonstrate that GBP1 promotes lymph node metastasis and has a positive correlation with EGFR expression in ESCC.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Disease Models, Animal; Epidermal Growth Factor; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Lymph Nodes; Lymphatic Metastasis; Male; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; RNA, Messenger; Up-Regulation

Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the "aggressive" phenotype of these cells, which includes enhanced inflammatory signaling (e.g., IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG), and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA, and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene (BP) also elicited similar responses. In conclusion, this study identified AREG, EREG, and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy.

Topics: Azo Compounds; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; Epiregulin; Fibroblast Growth Factor 2; Gene Expression; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Platelet-Derived Growth Factor; Pyrazoles; Receptors, Aryl Hydrocarbon; Squamous Cell Carcinoma of Head and Neck

Epithelial-mesenchymal transition (EMT) is a crucial programme in cancer metastasis. Epidermal growth factor (EGF) is a key inducer of EMT, and Ezrin has an important role in this process. However, how Ezrin is activated and whether it mediates EGF-induced EMT in tongue squamous cell carcinomas (TSCCs) through activating NF-κB remains obscure.. We used two TSCC cell lines as a cell model to study invasion and EMT in vitro, and used nude mice xenografts model to evaluate metastasis of TSCC cells. Finally, we evaluated the level of pEzrin Tyr353, nuclear p65 and EMT markers in TSCC clinical samples.. Ezrin Tyr353 was phosphorylated through Akt (but not ERK1/2, ROCK1) pathway, and lead to the activation of NF-κB in EGF-treated TSCC cells. Akt and NF-κB inhibitors blocked EGF-induced EMT, and suppressed invasion and migration of TSCC cells. In vivo, silencing Ezrin significantly suppressed EGF-enhanced metastasis of TSCC xenografts. Finally, high levels of expression of pEzrin Tyr353, nuclear p65, vimentin and low level of expression of E-cadherin were correlated with cancer metastasis and poor patient prognosis.. Our data suggest that Akt/Ezrin Tyr353/NF-κB pathway regulates EGF-induced EMT and metastasis inTSCC, and Ezrin may serve as a therapeutic target to reverse EMT in tongue cancers and prevent TSCC progression.

Topics: Animals; Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cytoskeletal Proteins; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mice; Neoplasm Metastasis; NF-kappa B; Proto-Oncogene Proteins c-akt; Signal Transduction; Tongue Neoplasms; Xenograft Model Antitumor Assays

It has been reported that the epidermal growth factor receptor (EGFR) expression is associated with the extracellular matrix metalloproteinase inducer (EMMPRIN) in some solid tumors; however, the relationship of EMMPRIN with EGFR in head and neck cancers is not fully understood. To determine the relationship between EMMPRIN and EGFR in head and neck squamous cell carcinoma (HNSCC), HNSCC cells were stimulated with epidermal growth factor (EGF), a ligand of EGFR. EMMPRIN expression in HNSCC cells was upregulated by EGF. In addition, EGF stimulation induced HNSCC cell invasion and MMP-9 expression. This increase in invasion and MMP-9 expression was abrogated by downmodulation of EMMPRIN. Furthermore, to determine the effects of combined EMMPRIN and EGFR targeting in HNSCC, HNSCC cells were treated with an EMMPRIN function-blocking antibody and the EGFR inhibitor AG1478. This combined treatment resulted in greater inhibition of HNSCC cell proliferation and migration compared with the individual agents alone. These results suggest that EMMPRIN mediates EGFR-induced tumorigenicity and that combined targeting of EMMPRIN and EGFR may be an efficacious treatment approach.

Topics: Basigin; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Emerging evidence suggests that endothelial cell-secreted factors contribute to the pathobiology of squamous cell carcinoma (SCC) by enhancing invasive migration and resistance to anoikis. Here, we report that SCC cells within the perivascular niche have undergone epithelial to mesenchymal transition (EMT) in a primary human SCC of a patient that developed distant metastases. Endothelial cell-secreted EGF induced EMT of human SCC cells in vitro and also induced acquisition of a stem-like phenotype. In vivo, tumor xenografts vascularized with EGF-silenced endothelial cells exhibited a smaller fraction of cancer stem-like cells (ALDH(+)CD44(+)) and were less invasive than tumors vascularized with control endothelial cells. Collectively, these results demonstrated that endothelial cell-EGF induces EMT and acquisition of stem-like properties by head and neck tumor cells. On this basis, we suggest that vascular endothelial cells contribute to tumor dissemination by secreting factors that endow carcinoma cells with enhanced motility and stemness.

Topics: Aged; Animals; Carcinoma, Squamous Cell; Cell Communication; Cell Growth Processes; Cell Line, Tumor; Cell Movement; Endothelial Cells; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Head and Neck Neoplasms; Heterografts; Humans; Male; Mice; Mice, SCID; Neoplastic Stem Cells; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

To investigate the association between genetic polymorphisms of growth factor-related genes and prognosis in patients with advanced esophageal squamous cell carcinoma (ESCC).. A total of 334 ESCC patients with advanced tumor stages (stages IIB, III and IV) were enrolled in the study. The genotypes of 14 candidate single nucleotide polymorphisms (SNPs) involved in growth factor-related functions were analyzed using iPLEX Gold technology from the genomic DNA of peripheral leukocytes, and were correlated with the clinical outcome of patients. Serum levels of growth factors were examined by enzyme-linked immunosorbent assay (ELISA).. The genetic polymorphisms of EGF:rs4444903, EGF:rs2237051 and VEGF:rs2010963 showed significant associations with overall survival (OS) of advanced ESCC patients (A/A+ A/G vs. GG, [HR = 0.77, 95% CI = 0.60-0.99, P = 0.039 for rs4444903; A/G+ G/G vs. A/A, [HR = 0.74, 95% CI = 0.58-0.95, P = 0.019 for rs2237051; G/G+G/C vs. C/C, [HR] inves = 0.69, 95% CI = 0.50-0.95, P = 0.023 for rs2010963). EGFR:rs2227983 and 3 SNPs of PIK3CA also showed borderline significant correlation with OS of advanced ESCC patients (P = 0.058 for rs2227983; P = 0.069, 0.091 and 0.067 for rs6443624, rs7651265 and rs7621329 of PIK3CA respectively). According to cumulative effect analysis of multiple SNPs, patients carrying 4 unfavorable genotypes exhibited more than a 3-fold increased risk of mortality. Finally, both EGF and VEGF expression levels significantly associated with patient mortality.. The genetic variants and expression levels of EGF and VEGF can serve as prognostic predictors in patients with advanced ESCC, and thus provide more information for optimizing personalized therapies for patients with ESCC.

Topics: Aged; Carcinoma, Squamous Cell; Disease-Free Survival; Epidermal Growth Factor; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Female; Genetic Predisposition to Disease; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Neoplasm Recurrence, Local; Neoplasm Staging; Polymorphism, Single Nucleotide; Prognosis; Vascular Endothelial Growth Factor A

Epithelial-mesenchymal transition (EMT) is a developmental program that is associated with esophageal squamous cell carcinoma (ESCC) progression and metastasis. Recently, C/EBPβ has been reported to be an EMT inducer in cancer. However, the detailed molecular mechanisms remain unclear. Here, we report for the first time, that the truncated CCAAT-enhancer-binding protein β (C/EBPβ) LIP isoform is abnormally overexpressed and correlated with cancer metastasis in clinical specimens of human ESCC. Furthermore, we demonstrate that C/EBPβ LIP mediates epithelial growth factor (EGF)-induced EMT and increases migration and invasion of esophageal cancer cells in a manner that is dependent on miR-203 inactivation. Finally, we identified miR-203 as a direct target of C/EBPβ LIP. Disruption of C/EBPβ LIP attenuated the EGF-mediated decrease in miR-203, whereas overexpression of C/EBPβ LIP alone markedly suppressed miR-203. In addition, we demonstrated that C/EBPβ LIP inhibited miR-203 transcription by directly interacting with a conserved distal regulatory element upstream of the miR-203 locus, and in doing so, orchestrated chromatin remodeling. In conclusion, our results have revealed a new regulatory mechanism that involves C/EBPβ-LIP-mediated downregulation of miR-203, which plays a key role in EMT and metastasis.

Topics: Carcinoma, Squamous Cell; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs

Emerging data suggest that cancer stem cells (CSCs) exist in equilibrium with differentiated cells and that stochastic transitions between these states can account for tumor heterogeneity and drug resistance. The aim of this study was to establish an in vitro system that recapitulates stem cell plasticity in head and neck squamous cell cancers (HNSCCs) and identify the factors that play a role in the maintenance and repopulation of CSCs. Tumor spheres were established using patient-derived cell lines via anchorage-independent cell culture techniques. These tumor spheres were found to have higher aldehyde dehydrogenase (ALD) cell fractions and increased expression of Kruppel-like factor 4, SRY (sex determining region Y)-box 2, and Nanog and were resistant to γ-radiation, 5-fluorouracil, cisplatin, and etoposide treatment compared with monolayer culture cells. Monolayer cultures were subject to single cell cloning to generate clones with high and low ALD fractions. ALDHigh clones showed higher expression of stem cell and epithelial-mesenchymal transition markers compared with ALDLow clones. ALD fractions, representing stem cell fractions, fluctuated with serial passaging, equilibrating at a level specific to each cell line, and could be augmented by the addition of epidermal growth factor (EGF) and/or insulin. ALDHigh clones showed increased EGF receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-1R) phosphorylation, with increased activation of downstream pathways compared with ALDLow clones. Importantly, blocking these pathways using specific inhibitors against EGFR and IGF-1R reduced stem cell fractions drastically. Taken together, these results show that HNSCC CSCs exhibit plasticity, with the maintenance of the stem cell fraction dependent on the EGFR and IGF-1R pathways and potentially amenable to targeted therapeutics.

Topics: Adult; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Head and Neck Neoplasms; Humans; Insulin-Like Growth Factor I; Kruppel-Like Factor 4; Male; Middle Aged; Neoplastic Stem Cells; Receptor, IGF Type 1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Oral squamous cell carcinoma (OSCC) is one of the most prevalent carcinomas worldwide. MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression and modulate physiological or pathological processes including OSCC carcinogenesis. miR-31 has been found to be up-regulated in OSCC and to act as an oncogenic miRNA. However, the molecular mechanism underlying miR-31 up-regulation in OSCC is still obscure. The activation of epidermal growth factor receptor (EGFR) signaling axis plays key roles in driving oral carcinogenesis. Our screening identified that there is up-regulation of miR-31, miR-181b and miR-222 in OSCC cells following EGF treatment. Subsequent analysis showed that EGF treatment led to AKT activation, which then resulted in miR-31 up-regulation. Moreover, EGF treatment and the AKT activation induced by exogenous expression up-regulated C/EBPβ expression. The miR-31 up-regulation induced by EGF was abrogated by AKT inhibition or by the knockdown of C/EBPβ expression. In OSCC cell subclones stably overexpressing the functional isoform of C/EBPβ, miR-31 expression was up-regulated. Curcumin is a natural ingredient exhibiting anti-cancer potential. It was found that curcumin attenuated AKT activation and the up-regulation of C/EBPβ and miR-31 caused by EGF stimulation in OSCC cells. Lastly, concordance across the expression of EGFR, the expression of C/EBPβ and the expression of miR-31 in OSCC tissues was found. This study describes a novel scenario where the up-regulation of miR-31 expression in OSCC is, at least in part, a consequence of EGFR oncogenic activation. Although the AKT activation and C/EBPβ expression after EGF treatment might not be directly linked, both events are the crucial mediators underlying miR-31 up-regulation in the EGFR signaling axis.

Topics: Carcinoma, Squamous Cell; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Curcumin; Epidermal Growth Factor; Humans; MicroRNAs; Mouth Mucosa; Mouth Neoplasms; Proto-Oncogene Proteins c-akt; Signal Transduction; Up-Regulation

The epithelial-mesenchymal transition (EMT) is suggested to be a crucial factor for the development of an invasive and metastatic cell phenotype, which is characterized by down-regulation of epithelial adhesive proteins (e.g. E-cadherin) and induction of mesenchymal proteins (e.g. vimentin). Therefore, there is a great clinical interest to specify this phenotype. Different growth factors induce EMT, such as epithelial growth factor (EGF) and transforming growth factor beta 1 (TGFβ1). The role of EMT in human papilloma virus (HPV)-positive squamous cell carcinoma (SCC) is still not understood. The aim of this study was to investigate the expression pattern in p16-positive and -negative SCC cells of vimentin, β-catenin and E-cadherin after stimulation with growth factors.. We incubated the p16-positive CERV196 and p16-negative HNSCC22B SCC cell lines with EGF and EGF/TGFβ1 (10 ng/ml) and detected E-cadherin, vimentin and β-catenin by immunocytochemistry and enzyme-linked immunosorbent assay after 5, 24 and 96 h.. We found a low expression of vimentin in all studied tumor cell lines. The negative control of HNSCC22B cells showed a higher intrinsic level of membranous E-cadherin and β-catenin. We found statistically significant EGF/TGFβ1-induced expression of vimentin dependent on incubation time in p16-negative HNSCC22B cells. Particularly in the presence of EGF, we detected an increase of β-catenin and vimentin expression in p16-positive SCC tumor cell lines in addition to induced cell scattering and unexpected expression of E-cadherin.. In conclusion, E-cadherin, β-catenin and vimentin expression are important features to characterize EMT-like events. We were able to show incomplete EGF-induced EMT with β-catenin expression in p16-positive SCC. Extended studies are required to investigate the mechanistic role of EMT markers, especially in p16-positive SCC, in order to develop new anti-SCC therapies to block EMT progression.

Topics: beta Catenin; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Immunohistochemistry; Neoplasm Proteins; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor beta1; Vimentin

Girdin, an actin-binding Akt substrate, regulates actin reconstruction and Akt-dependent cell motility in fibroblasts and in a human breast cancer cell line. We examined whether Girdin is also involved in the motility of esophageal squamous cell carcinoma (ESCC) cells. Immunofluorescent staining and migration assays were performed, using KYSE cell lines, to examine whether Girdin is involved in the motility of ESCC cells. Upon EGF stimulation, Girdin colocalized with filamentous actin (F-actin) in the lamellipodia as determined by immunofluorescent staining. In migration assays, cell motility was significantly reduced in KYSE cell lines transfected with Girdin siRNA compared with the negative control. In addition, we examined the relationship between Girdin expression and clinical data, using specimens resected from ESCC patients. In immunohistochemical (IHC) analyses using specimens resected from ESCC patients, overall survival was significantly longer in cases showing lower Girdin expression compared to cases with higher Girdin expression. Collectively, Girdin appears to be involved in the motility of ESCC cells. The levels of Girdin expression correlated inversely with the survival of ESCC patients. Therefore, in ESCC, Girdin may be a prognostic marker and may serve as a therapeutic target as well.

Topics: Aged; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Esophageal Neoplasms; Esophagus; Female; Gene Expression; Gene Knockdown Techniques; Humans; Intestinal Mucosa; Kaplan-Meier Estimate; Lymphatic Metastasis; Male; Microfilament Proteins; Middle Aged; Prognosis; Pseudopodia; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Vesicular Transport Proteins

Phenethyl isothiocyanate (PEITC) is a natural compound that is involved in chemoprevention as well as inhibition of cell growth and induction of apoptosis in several types of cancer cells. Previous studies have revealed that PEITC suppresses the invasion of AGS gastric and HT-29 colorectal cancer cells. However, the effects of PEITC on the metastasis of SAS oral cancer cells remain to be determined. Our results showed that PEITC treatment inhibited the invasion of EGF-stimulated SAS cells in a concentration-dependent manner, but appeared not to affect the cell viability. The expression and enzymatic activities of matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) were suppressed by PEITC. Concomitantly, we observed an increase in the protein expression of both tissue inhibitor of metalloproteinase-1 (TIMP-1) and -2 (TIMP-2) in treated cells. Furthermore, PEITC treatments decreased the protein phosphorylation of epidermal growth factor receptor (EGFR) and downstream signaling proteins including PDK1, PI3K (p85), AKT, phosphorylated IKK and IκB to inactivate NF-κB for the suppression of MMP-2 and MMP-9 expression. In addition, PEITC can trigger the MAPK signaling pathway through the increase in phosphorylated p38, JNK and ERK in treated cells. Our data indicate that PEITC is able to inhibit the invasion of EGF-stimulated SAS oral cancer cells by targeting EGFR and its downstream signaling molecules and finally lead to the reduced expression and enzymatic activities of both MMP-2 and MMP-9. These results suggest that PEITC is promising for the therapy of oral cancer metastasis.

Topics: Anticarcinogenic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Humans; I-kappa B Kinase; Isothiocyanates; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mouth Neoplasms; Neoplasm Invasiveness; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.

Topics: Allosteric Regulation; Animals; Binding Sites; Carcinoma, Squamous Cell; Cell Line, Tumor; Crystallography, X-Ray; Cyclin-Dependent Kinases; Epidermal Growth Factor; Flavonoids; G1 Phase; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Neoplasm Transplantation; Protein Kinase Inhibitors; Retinoblastoma Protein; S Phase; Skin Neoplasms

Combination treatment (chemoradiotherapy) is the standard treatment for locally advanced head and neck squamous cell carcinoma (HNSCC); however, treatment resistance and local recurrence are significant problems. A high level of epidermal growth factor receptor (EGFR) has been associated with a more aggressive phenotype as well as decreased responsiveness to radio- or chemotherapy. We examined the role of EGFR status and EGFR ligand expression for the treatment response.. Intrinsic sensitivity to radiotherapy, cisplatin, and cetuximab treatments was investigated in 25 HNSCC cell lines. EGFR gene copy number, mRNA and protein expression, EGFR and Akt phosphorylation status, and mRNA expression of the EGFR ligands were analyzed using quantitative PCR and ELISA and assessed for their impact on treatment sensitivity.. Different treatment modalities yielded great diversity in outcome; of note, cetuximab treatment stimulated growth in one cell line. When treatments were combined primarily additive effects were observed. While radioresistance tended to be associated with a high level of phosphorylated EGFR (pEGFR; P = 0.09), cetuximab-resistant cells had low levels of pEGFR (P = 0.13). The three most cetuximab-sensitive cell lines had high EGFR gene copy numbers. Furthermore, cetuximab treatment response was significantly correlated with epiregulin mRNA expression (r = -0.408, P = 0.043). Cisplatin-resistant tumor cells expressed significantly lower levels of EGFR protein (P = 0.04) compared to cisplatin-sensitive cells and tended to have lower levels of phosphorylated Akt (pAkt; P = 0.13) and lower expression levels of amphiregulin (P = 0.18).. Epidermal growth factor receptor status and ligand expression influence the treatment sensitivity of HNSCC cells and may be useful as predictive markers.

Topics: Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Chemoradiotherapy; Cisplatin; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Dosage; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Phosphorylation; Proto-Oncogene Proteins c-akt; Radiation Tolerance; Statistics, Nonparametric

The epidermal growth factor receptor (EGFR) plays an important role in the pathogenesis of head and neck squamous cell carcinoma (HNSCC). Despite the high expression of EGFR in HNSCC, EGFR inhibitors have only limited success as monotherapy. The Grb2-associated binder (GAB) family of adaptor proteins acts as docking/scaffolding molecules downstream of tyrosine kinase receptors. We hypothesized that GAB1 may amplify EGFR-induced signaling in HNSCCs and therefore could play a role in the reduced sensitivity of HNSCC to EGFR inhibitors. We used representative human HNSCC cell lines overexpressing wild type EGFR, and expressing GAB1 but not GAB2. We demonstrated that baseline Akt and MAPK signaling were reduced in HNSCC cells in which GAB1 expression was reduced. Furthermore, the maximal EGF-induced activation of the Akt and MAPK pathway was reduced and delayed, and the duration of the EGF-induced activation of these pathways was reduced in cells with GAB1 knock-down. In agreement with this, HNSCC cells in which GAB1 levels were reduced showed an increased sensitivity to the EGFR inhibitor gefitinib. Our work demonstrates that GAB1 plays an important role as part of the mechanism of by which EGFR induces induced activation of the MAPK and AKT pathway. Our results identify GAB1 as an amplifier of the EGFR-initiated signaling, which may also interfere with EGFR degradation. These findings support the emerging notion that reducing GAB1 function may sensitize HNSCC to EGFR inhibitors, hence representing a new therapeutic target HNSCC treatment in combination with EGFR targeting agents.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; GRB2 Adaptor Protein; Head and Neck Neoplasms; Humans; Mitogen-Activated Protein Kinase Kinases; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

Arming antibody with toxins is a new approach in cancer therapy. We evaluated the efficacy of cetuximab-ZZ-PE38 immunocomplex in killing cancer cells in vitro and inhibiting tumor growth in nude mice.. Several cancer cell lines and human foreskin fibroblasts were tested for epidermal growth factor receptor (EGFR) expression and cetuximab binding using Western blot assay, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. Cell survival in vitro was estimated by XTT assay. Tumor size was measured twice a week.. Cetuximab-ZZ-PE38 immunocomplex was significantly more effective in killing head and neck cancer cells than nonspecific IgG-ZZ-PE38 complex or free ZZ-PE38, whereas normal cells were not affected. Tumor treatment with immunocomplex resulted in tumor shrinkage. The immunocomplex was safe to mice at a therapeutic dosage of 0.25 mg/mL, whereas the dosage of 0.50 mg/mL induced liver toxicity.. Cetuximab-ZZ-PE38 immunocomplex is a highly effective agent in killing EGFR-positive cancer cells and in tumor shrinkage.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Culture Techniques; Cell Line, Tumor; Cetuximab; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Head and Neck Neoplasms; Humans; Male; Mice; Mice, Nude; Prostatic Neoplasms

The migratory ability of tumor cells requires cytoskeletal rearrangement processes. Epidermal growth factor receptor (EGFR)-signaling tightly correlates with tumor progression in head and neck squamous cell carcinomas (HNSCCs), and has previously been implicated in the regulation of cytokeratin (CK) expression. In this study, HNSCC cell lines were treated with EGF, and CK expression levels were monitored by Western blot analysis. Changes in cellular morphology were documented by fluorescence- and atomic force microscopy. Some of the cell lines demonstrated an EGF-dependent modulation of CK expression levels. Interestingly, regression of some CK subtypes or initial up-regulation followed by downregulation at higher EGF-levels could also be observed in the tested cell lines. Overall, the influence of EGF on CK expression levels appeared variable and cell-type-dependent. Real-time cellular analysis of EGF-treated and -untreated HNSCC cell lines demonstrated a rise over time in cellular impedance. In three of the EGF-treated HNSCC cell lines, this rise was markedly higher than in untreated controls, whereas in one of the cell lines the gain of cellular impedance was paradoxically reduced after EGF treatment, which was found to correlate with changes in cellular morphology rather than with relevant changes in cellular viability or proliferation. After treating HNSCC cells with EGF, CK filaments frequently appeared diffusely distributed throughout the cytoplasm, and in some cases were found in a perinuclear localization, the latter being reminiscent to observations by other groups. In summary, the data points to a possible role of EGFR in modulating HNSCC cell morphology.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Shape; Cell Survival; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Keratinocytes; Keratins; Microscopy, Atomic Force; Phenotype; Plakophilins; Squamous Cell Carcinoma of Head and Neck

Caveolin-1 (Cav1) is a scaffolding protein that serves to regulate the activity of several signaling molecules. Its loss has been implicated in the pathogenesis of several types of cancer, but its role in the development and progression of cutaneous squamous cell carcinoma (cSCC) remains largely unexplored. Herein, we use the keratinocyte cell line PAM212, a murine model of cSCC, to determine the function of Cav1 in skin tumor biology. We first show that Cav1 overexpression decreases cell and tumor growth, whereas Cav1 knockdown increases these attributes in PAM212 cells. In addition, Cav1 knockdown increases the invasive ability and incidence of spontaneous lymph node metastasis. Finally, we demonstrate that Cav1 knockdown increases extracellular signaling-related kinase 1/2 mitogen-activated protein kinase/activator protein-1 pathway activation. We attribute the growth and invasive advantage conferred by Cav1 knockdown to increased expression of activator protein-1 transcriptional targets, including cyclin D1 and keratin 18, which show inverse expression in PAM212 based on the expression level of Cav1. In summary, we demonstrate that loss of Cav1 affects several characteristics associated with aggressive human skin tumors and that this protein may be an important modulator of tumor growth and invasion in cSCC.

Topics: Animals; Carcinoma, Squamous Cell; Caveolin 1; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Models, Animal; Enzyme Activation; Epidermal Growth Factor; Gene Knockdown Techniques; Humans; Keratin-18; Keratinocytes; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Serum; Skin Neoplasms; Transcription Factor AP-1

In the course of screening for an inhibitor of farnesyl transferase (FTase), we identified two compounds, N-benzyl-aclacinomycin A (ACM) and N-allyl-ACM, which are new derivatives of ACM. N-benzyl-ACM and N-allyl-ACM inhibited FTase activity with IC50 values of 0.86 and 2.93 μM, respectively. Not only ACM but also C-10 epimers of each ACM derivative failed to inhibit FTase. The inhibition of FTase by N-benzyl-ACM and N-allyl-ACM seems to be specific, because these two compounds did not inhibit geranylgeranyltransferase or geranylgeranyl pyrophosphate (GGPP) synthase up to 100 μM. In cultured A431 cells, N-benzyl-ACM and N-allyl-ACM also blocked both the membrane localization of H-Ras and activation of the H-Ras-dependent PI3K/Akt pathway. In addition, they inhibited epidermal growth factor (EGF)-induced migration of A431 cells. Thus, N-benzyl-ACM and N-allyl-ACM inhibited EGF-induced migration of A431 cells by inhibiting the farnesylation of H-Ras and subsequent H-Ras-dependent activation of the PI3K/Akt pathway.

Topics: Aclarubicin; Alkyl and Aryl Transferases; Antibiotics, Antineoplastic; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Membrane; Cell Movement; Epidermal Growth Factor; Farnesyltranstransferase; Genes, ras; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Humans; Inhibitory Concentration 50; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt

The expression of cytokines, such as IL-1β, and the activation of the epidermal growth factor receptor (EGFR) are crucial regulators in the process of carcinogenesis. The correlation between growth factor and activated cytokine signals in the control of tumor development is a critical issue to be clarified. In our study, we found that the IL-1β gene and protein expression were induced by EGF in squamous cell carcinoma. To clarify the mechanism involved in EGF-regulated IL-1β expression, we examined the transcriptional activity and mRNA stability of IL-1β in EGF-treated cells. We found that EGF induced the expression of IL-1β and was mediated through transcriptional activation, but not through mRNA stability. The involvement of Akt and NF-κB signaling pathways in the EGF-induced IL-1β gene expression was confirmed by knockdown of RelA and Akt in cells or treating cells with Akt and NF-κB inhibitors, LY294002 and parthenolide, respectively. The expression of dominant negative IκB also repressed the activation of NF-κB and inhibited EGF-induced IL-1β expression. Using immunofluorescence staining assay, the EGF-stimulated nuclear translocation of NF-κB (p65) was inhibited by pre-treating cells with LY294002 and parthenolide. Furthermore, EGF increased the binding of NF-κB to the NF-κB binding site of the IL-1β promoter through the activation of the Akt/NF-κB pathway, which resulted in activating IL-1β promoter activity. The expression and secretion of IL-1β induced by EGF considerably reduced chemotherapeutic drug cisplatin-induced cell death. These results showed that EGF enhanced the expression of IL-1β, which was mediated by the Akt/NF-κB pathway. The activation of EGF signaling and increase of IL-1β contributed to chemotherapeutic resistance of cancer cells, suggesting that the expression of IL-1β may be used as a biomarker to evaluate successful cancer treatment.

Topics: Biomarkers, Tumor; Blotting, Western; Carcinoma, Squamous Cell; Chromones; Cisplatin; Cytotoxins; DNA Primers; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1beta; Morpholines; NF-kappa B; Oncogene Protein v-akt; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Sesquiterpenes; Signal Transduction

Molecular mechanisms other than activating KRAS mutations should underlie the occurrence of weaker versus stronger responses to cetuximab (CTX) in EGFR-dependent carcinomas with either an intact KRAS signaling or in which KRAS mutations do not predict CTX efficacy. We hypothesized that KRAS wild-type (WT) tumor cell-line models chronically adapted to grow in the presence of CTX could be interrogated to establish if the positive predictive value of the mRNAs coding for the EGFR ligands amphiregulin (AR) and epiregulin (EPI) could be significantly altered during and/or after treatment with CTX. Gene expression analyses using real-time (kinetic) RT-PCR were performed to monitor the transcriptional evolution of EGFR ligands EGF, TGFα, AR, BTC, EPI, NRG and HB-EGF in experimental modes induced to exhibit acquired resistance to the mono-HER1 inhibitor CTX, the mono-HER2 inhibitor trastuzumab (Tzb) or the dual HER1/HER2 inhibitor lapatinib (LPT). Gene expression signatures for EGFR ligands distinctively related to the occurrence of unresponsiveness to CTX, Tzb or LPT, with minimal overlap between them. CTX's molecular functioning largely depended on the overproduction of the mRNAs coding for the EGFR ligands AR and EPI. Thus, a dramatic down-regulation of AR/EPI mRNA expression occurred upon loss of CTX efficacy in EGFR-positive tumor cells with an intact regulation of RAS signaling. Unlike KRAS mutations, which are informative of unresponsiveness to CTX solely in mCRC, our hypothesis-generating data suggest that expression status of AR and EPI mRNAs might be evaluated as dynamic predictors of response in KRAS WT patients receiving any CTX-based therapy.

Topics: Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Drug Resistance, Neoplasm; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Real-Time Polymerase Chain Reaction; Receptor, ErbB-2; RNA, Messenger; Time Factors; Up-Regulation; Vulvar Neoplasms

Epidermal growth factor (EGF) stimulates cell proliferation by binding to its receptor (epidermal growth factor receptor), and the overexpression of this receptor is associated with poorer prognosis. The EGF gene presents a polymorphism at position 61 (A/G), associated with higher EGF production. We examined the association between this polymorphism and cervical cancer through a case-control study.. This study used the PCR-restriction fragment length polymorphism method on a sample of 384 women with cervical lesions and 500 controls of white ethnicity.. In cases of cervical cancer, we found an increased risk of progression to advanced disease (The International Federation of Gynecology and Obstetrics stage IIb/IV) (odds ratios=2.05; 95% confidence intervals=1.11 to 3.79; P=0.021), and this risk was particularly evident in G carriers for younger women (odds ratios=2.96; 95% confidence intervals=1.41-6.20, P=0.003).. We hypothesize the onset of an advanced disease-driven selective pressure due to the effect of oncogenic human papillomavirus types in a favorable genetic background observed in G carrier women. These results suggest that EGF functional polymorphism may influence cervical cancer prognosis through an EGF/epidermal growth factor receptor pathway.

Topics: Adenocarcinoma; Adult; Carcinoma, Squamous Cell; Case-Control Studies; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Follow-Up Studies; Genetic Predisposition to Disease; Humans; Middle Aged; Neoplasm Invasiveness; Neoplasm Staging; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Prognosis; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Mtss1 is located within chromosomal region 8q23-24, which is one of the three most commonly amplified regions in head and neck squamous cell carcinoma (HNSCC). Mtss1 is lost in metastatic cells, but confusingly is commonly overexpressed in primary tumors. Here we address possible reasons why Mtss1 is positively selected for in primary tumors. We find that Mtss1 enhances the localization of the epidermal growth factor (EGF) receptor to the plasma membrane, prolonging EGF signaling and resulting in enhanced proliferation in HNSCC. Depletion of Mtss1 results in decreased EGF receptor levels and decreased phosphorylation of Erk1/2 and Akt. However, when cells are at high density and adherent to each other, analogous to conditions in a solid tumor, Mtss1 does not confer any growth advantage, either in basal conditions or following EGF stimulation. This could indicate why Mtss1 might be lost in metastases, but preserved in early primary tumors. This is supported by an organotypic assay showing that Mtss1-expressing cells display a less proliferative more epithelial-like morphology on top of a collagen matrix. Furthermore, xenograft tumors expressing Mtss1 initially grow more rapidly, but later show less proliferation and more differentiation. Mtss1 positively modulates EGF signaling at low cell densities to promote proliferation and, therefore, may be beneficial for the early stages of primary HNSCC tumor growth. However, at high cell densities, Mtss1 impacts negatively on EGF signaling and this suggests why it inhibits metastasis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Mice; Mice, Nude; Microfilament Proteins; Neoplasm Proteins; Neoplasm Transplantation; Signal Transduction; Squamous Cell Carcinoma of Head and Neck

In this report we describe the contribution of prostaglandin E(2) (PGE(2)) derived from the inducible microsomal PGE-synthase type-1 (mPGES-1) to the epidermal growth factor receptor (EGFR) oncogenic drive in tumor epithelial cells and in tumor-bearing mice. EGFR stimulation upregulated expression of mPGES-1 in HT-29, A431 and A549 cancer cells. Egr-1, a transcription factor induced by EGF, mediated this response. The Egr-1 rise provoked the overexpression of mPGES-1 messenger and protein, and enhanced PGE(2) formation. These changes were suppressed either by silencing Egr-1, or by upstream blockade of EGFR or ERK1/2 signals. Further, in a clonogenic assay on tumor cells, EGF induced a florid tumorigenic phenotype, which regressed when mPGES-1 was silenced or knocked down. EGF-induced mPGES-1 overexpression in epithelial cell reduced E-cadherin expression, whereas enhancing that of vimentin, suggesting an incipient mesenchymal phenotype. Additionally, inhibiting the EGFR in mice bearing the A431 tumor, the mPGES-1 expression and the tumor growth, exhibited a parallel decline. In conclusion, these findings provide novel evidence that a tight cooperation between the EGF/EGFR and mPGES-1 leads to a significant tumorigenic gain in epithelial cells, and provide clues for controlling the vicious association.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Transformation, Neoplastic; Early Growth Response Protein 1; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Silencing; Humans; Intramolecular Oxidoreductases; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Prostaglandin-E Synthases; Signal Transduction; Up-Regulation

Predictive strategies for the treatment efficacy of cetuximab are currently not available for head and neck cancer. We investigated the correlation between the expression of epidermal growth factor receptor (EGFR) ligands and EGFR expression, and the growth inhibitory activity of cetuximab in a panel of head and neck squamous cell carcinoma (HNSCC) cell lines.. The growth inhibiting effect of cetuximab was measured for eight HNSCC cell lines and correlated with the autocrine production of five EGFR ligands as measured by ELISA, and the mRNA expression of two ligands, as measured by quantitative RT-PCR. EGFR expression was assessed by western blot analysis.. There was a good correlation between the expression of four of the EGFR ligands (TGF-α, amphiregulin, epiregulin and epigen) and the growth inhibiting effect of cetuximab. TGF-α had the highest predictive potential but had to be combined with epigen for full prediction. EGFR expression also correlated with cetuximab sensitivity but less clearly.. The results indicate that the expression of several EGFR ligands has to be used to predict sensitivity to cetuximab in HNSCC. This has to be further evaluated in clinical samples.

Topics: Amphiregulin; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epigen; Epiregulin; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glycoproteins; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Predictive Value of Tests; Reverse Transcriptase Polymerase Chain Reaction; Squamous Cell Carcinoma of Head and Neck; Transforming Growth Factor alpha

The presence of lymph node metastases is one of the most important prognostic indicators in head and neck squamous cell carcinomas (HNSCCs). An alteration of the E-cadherin-catenins complex and EGFR is essential for the invasiveness of cancer cells. Caveolin-1, the major structural protein of the caveolae, represents a scaffolding molecule for several signaling proteins including EGFR. Although caveolin-1 has been shown to play a role in inducing the invasive phenotype of cancer cells, its role appears to be cell-type specific and for some tumors it has not been defined yet. In this study we used 57 HNSCC specimens to investigate whether the abnormal expression of caveolin-1 was associated with the derangement of the E-cadherin-catenins complex and with the overexpression of ErbB receptors. We demonstrate that in HNSCCs caveolin-1 overexpression is associated with the simultaneous abnormal expression of at least one member of the E-cadherin/α-β catenins complex and multiple ErbB receptors as well as with lymph node metastases. We also demonstrate that chronic stimulation of a human hypopharyngeal carcinoma cell line (FaDu) with EGF induced the internalization of β-catenin and caveolin-1 and their co-localization with EGFR. Moreover, EGF treatment induced an increased physical interaction between EGFR/β-catenin/caveolin-1 and between E-cadherin/β-catenin/caveolin-1. These molecular events were associated with an increased directional motility of FaDu cells in vitro. These findings may provide new insight into the biology of HNSCC progression and help to identify subgroups of primary HNSCCs with a more aggressive behavior.

Topics: alpha Catenin; beta Catenin; Cadherins; Carcinoma, Squamous Cell; Caveolin 1; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Hypopharyngeal Neoplasms; Lymph Nodes; Neoplasm Invasiveness; Neoplasm Metastasis; Receptor, ErbB-2

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Kaplan-Meier Estimate; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Prognosis; Prospective Studies; Squamous Cell Carcinoma of Head and Neck

One of the key drivers for squamous cell carcinoma (SCC) proliferation is activation of the epidermal growth factor receptor (EGFR), a known proto-oncogene. However, the mechanism of EGFR-dependent SCC proliferation remains unclear. Our previous studies indicate that epidermal growth factor (EGF)-induced SCC cell proliferation requires the SH3 domain of phospholipase C-γ1 (PLC-γ1), but not its catalytic activity. The SH3 domain of PLC-γ1 is known to activate the short form of nuclear phosphatidylinositol 3-kinase enhancer (PIKE) that enhances the activity of nuclear class Ia phosphatidylinositol 3-kinase (PI3K) required for proliferation. However, PIKE has been described for more than a decade to be present exclusively in neuronal cells. In the present study, we found that PIKE was highly expressed in malignant human keratinocytes (SCC4 and SCC12B2) but had low expression in normal human keratinocytes. Immunohistochemical analysis showed strong nuclear staining of PIKE in human epidermal and tongue SCC specimens but little staining in the adjacent non-cancerous epithelium. Treatment of SCC4 cells with EGF-induced translocation of PLC-γ1 to the nucleus and binding of PLC-γ1 to the nuclear PIKE. Knockdown of PLC-γ1 or PIKE blocked EGF-induced activation of class Ia PI3K and protein kinase C-ζ and phosphorylation of nucleolin in the nucleus as well as EGF-induced SCC cell proliferation. However, inhibition of the catalytic activity of PLC-γ1 had little effect. These data suggest that PIKE has a critical role in EGF-induced SCC cell proliferation and may function as a proto-oncogene in SCC.

Topics: Carcinoma, Squamous Cell; Cell Nucleus; Cell Proliferation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Gene Knockdown Techniques; GTP-Binding Proteins; GTPase-Activating Proteins; Humans; Keratinocytes; Nucleolin; Phosphatidylinositol 3-Kinase; Phospholipase C gamma; Phosphoproteins; Phosphorylation; Protein Kinase C; Proto-Oncogene Mas; Reference Values; RNA-Binding Proteins; Signal Transduction; src Homology Domains; Tongue Neoplasms

Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti-tumour signal inhibitors on the migratory activity.. Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time-lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting.. In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose-dependent activation of cell migration. Addition of the EGFR-specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF-induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI-3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF-induced migration was particularly sensitive to PI-3 K-inhibition, while in C12 cells, both HGF- and EGF-induced migration were highly sensitive to p38-blockade.. The results demonstrate that the MEK/ERK, p38 and PI-3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.

Topics: Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; MAP Kinase Signaling System; Mouth Neoplasms; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Signal Transduction; Tumor Cells, Cultured

Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2',3'-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner.

Topics: Animals; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cyclic AMP; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Mice; Neurocalcin; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Skin Neoplasms; Snail Family Transcription Factors; Transcription Factors; Tumor Suppressor Proteins

Fucosyltransferase IV (FUT4) has been implicated in cell adhesion, motility, and tumor progression in human epidermoid carcinoma A431 cells. We previously reported that it promotes cell proliferation through the ERK/MAPK and PI3K/Akt signaling pathways; however, the molecular mechanisms underlying FUT4- induced cell invasion remain unknown. In this study we determined the effect of FUT4 on expression of matrix metalloproteinase (MMP)-12 induced by EGF in A431 cells. Treatment with EGF resulted in an alteration of cell morphology and induced an increase in the expression of MMP-12. EGF induced nuclear translocation of nuclear factor κB (NF-κB) and resulted in phosphorylation of IκBα in a time-dependent manner. In addition, ERK1/2 and p38 MAPK were shown to play a crucial role in mediating EGF-induced NF-κB translocation and phosphorylation of IκBα when treated with the MAPK inhibitors, PD98059 and SB203580, which resulted in increased MMP-12 expression. Importantly, we showed that FUT4 up-regulated EGF-induced MMP-12 expression by promoting the phosphorylation of ERK1/2 and p38 MAPK, thereby inducing phosphorylation/ degradation of IκBα, NF-κB activation. Base on our data, we propose that FUT4 up-regulates expression of MMP-12 via a MAPK-NF-κB-dependent mechanism.

Topics: Analysis of Variance; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cytosol; Epidermal Growth Factor; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Proteins; Lewis X Antigen; MAP Kinase Signaling System; Matrix Metalloproteinase 12; NF-kappa B; NF-kappa B p50 Subunit; Protein Transport; Transcription Factor RelA; Transfection

One challenge of squamous cell carcinoma of the head and neck (SCCHN) chemotherapy is a small percentage of tumor cells that arrest in the G0 phase of the cell cycle and are thus not affected by chemotherapy. This could be one reason for tumor recurrence at a later date. The recruitment of these G0-arresting cells into the active cell cycle and thus, proliferation, may increase the efficacy of chemotherapeutic agents. The aim of this study was to investigate whether stimulation with recombinant epidermal growth factor (EGF) or serotonin leads to an increased tumor cell proliferation in xenografts. Detroit 562 cells were injected into NMRI-Foxn1nu mice. Treatment was performed with 15 µg murine or human EGF, or 200 µg serotonin. The control mice were treated with Lactated Ringer's solution (5 mice/group). Tumor size was measured on days 4, 8 and 12 after tumor cell injection. The EGF stimulated mice showed a significantly higher tumor growth compared to the serotonin-stimulated mice and the untreated controls. In the present study, we show that it is possible to stimulate tumor cells in xenografts by EGF and thus, enhance cell proliferation, resulting in a higher tumor growth compared to the untreated control group. In our future investigations, we plan to include a higher number of mice, an adjustment of the EGF dosage and cell subanalysis, considering the heterogeneity of SCCHN tumors.

Topics: Amino Acid Sequence; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Ki-67 Antigen; Mice; Molecular Sequence Data; Neoplasm Transplantation; Platelet Endothelial Cell Adhesion Molecule-1; Recombinant Proteins; Serotonin; Tumor Burden

The effectiveness of near-infrared imaging (NIR) interrogation of epidermal growth factor receptor (EGFR) expression as a sensitive biomarker of oral squamous cell carcinoma (OSCC) response to arsenic trioxide therapy was studied in mice.. A431 OSCC in vitro were exposed to 0 µM, 0.5 µM, 2.5 µM, or 5 µM of As(2)O(3) for 0 h, 24 h, 48 h and 72 h. Confocal microscopy and flow cytometry confirmed EGFR expression and demonstrated a sensitivity dose-related signal decline with As(2)O(3) treatment. Next, mice with pharynx-implanted A431 cells received As(2)O(3) i.p. every 48 h at 0.0, 0.5, 2.5, or 5 mg/kg/day (n = 6/group) from day 0 to 10. An intravenous NIR probe, EGF-Cy5.5, was injected at baseline and on days 4, 8, and 12 for dynamic NIR imaging. Tumor volume and body weights were measured three times weekly.. In vitro, A431 EGFR expression was well appreciated in the controls and decreased (p<0.05) with increasing As(2)O(3) dose and treatment duration. In vivo EGFR NIR tumor signal intensity decreased (p<0.05) in As(2)O(3) treated groups versus controls from days 4 to 12, consistent with increasing dosage. Tumor volume diminished in a dose-related manner while body weight was unaffected. Immunohistochemical staining of excised tumors confirmed that EGFR expression was reduced by As(2)O(3) treatment in a dose responsive pattern.. This study demonstrates for the first time that OSCC can be interrogated in vivo by NIR molecular imaging of the EGFR and that this biomarker is effective for the longitudinal assessment of OSCC response to As(2)O(3) treatment.

Topics: Animals; Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Biomarkers, Tumor; Body Weight; Carbocyanines; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Injections, Intraperitoneal; Injections, Intravenous; Magnetic Resonance Imaging; Mice; Mice, Nude; Mouth Neoplasms; Neoplasms, Experimental; Oxides; Recombinant Fusion Proteins; Spectroscopy, Near-Infrared; Tumor Burden; Tumor Cells, Cultured

Human podoplanin is a type-1 transmembrane sialomucin-like glycoprotein that is involved in cell migration, tumor cell invasion and metastasis. Our recent study of oral squamous cell carcinoma (OSCC) demonstrated that the degree of immunohistochemical expression of podoplanin was correlated with the severity of epithelial dysplasia and significantly associated with a poor pathologic grade of differentiation. Furthermore, it has been reported that Src directly associates with the epidermal growth factor receptor (EGFR) in OSCC cells upon stimulation with EGF and phosphorylates Crk-associated substrate (Cas), podoplanin acting downstream of Src and Cas to promote cell migration. However, the molecular function of podoplanin remains unclear. In this study we performed real-time RT-PCR, Western blotting and scratch assay using OSCC cell lines in order to clarify the molecular biological function of podoplanin expression associated with various growth factors including EGF and with the Src-Cas signaling pathway. Podoplanin was found to have a marked influence on cancer cell migration and the expression of matrix metalloprotease-9 (MMP-9) in the oral cavity upon stimulation with EGF. Podoplanin promotes oral cancer cell migration, and the EGF-Src-Cas pathway is one of the possible mechanisms responsible for progression of cancer in the oral cavity.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Crk-Associated Substrate Protein; Epidermal Growth Factor; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Membrane Glycoproteins; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; Tumor Microenvironment

Topics: Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cetuximab; Epidermal Growth Factor; Erlotinib Hydrochloride; Female; Humans; Male; Orbital Neoplasms; Protein Kinase Inhibitors; Quinazolines

Chemotherapy drug resistance is a major obstacle in the treatment of cancer. It can result from an increase in levels of cellular drug efflux pumps, such as P-glycoprotein (P-gp). Lapatinib, a growth factor receptor tyrosine kinase inhibitor, is currently in clinical trials for treatment of breast cancer. We examined the impact of co-incubation of chemotherapy drugs in combination with lapatinib in P-gp over-expressing drug resistant cells. Unexpectedly, lapatinib treatment, at clinically relevant concentrations, increased levels of the P-gp drug transporter in a dose- and time-responsive manner. Conversely, exposure to the epidermal growth factor (EGF), an endogenous growth factor receptor ligand, resulted in a decrease in P-gp expression. Despite the lapatinib-induced alteration in P-gp expression, use of accumulation, efflux and toxicity assays demonstrated that the induced alteration in P-gp expression by lapatinib had little direct impact on drug resistance.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antineoplastic Combined Chemotherapy Protocols; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Squamous Cell; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lapatinib; Lung Neoplasms; Male; Middle Aged; Protein Kinase Inhibitors; Quinazolines; Time Factors

Epithelial-mesenchymal transition (EMT) is suggested to be crucial for the development of an invasive and metastatic carcinoma cell phenotype. Therefore, the definition of this phenotype is of great clinical interest. We recently evidenced vimentin positive cells in oral squamous cell carcinoma (OSCC) invasive front expressing laminin γ2 chain mRNA implicating an EMT origin of these cells. To further elucidate the nature of these cells, we have investigated the relation between EMT criteria and laminin-332 expression in a cell culture model of transforming growth factor beta-1 (TGFβ1)/epithelial growth factor (EGF) long time co-stimulation. We demonstrate that in contrast to TGFβ1 or EGF alone, co-stimulation induces phenotype transition in OSCC cells which fulfils the criteria of EMT in terms of vimentin up-regulation and E-cadherin down-regulation on protein level as well as cell scattering. Furthermore, cells displayed a strongly enhanced invasiveness and adhesion to type I-IV collagens. Phenotype transition is accompanied by an enhanced expression of laminin-332, especially of its γ2 chain. We further analyse the expression of extracellular matrix related genes by RT-PCR profiling. With respect to strongly enhanced proteins, data confirm the EMT phenotype of co-stimulated OSCC cells and expression of laminin-332. Furthermore, alpha catenin, collagen type 16, the integrin α7 and β1 chains, and MMP11 are suggested as candidates with potential role in EMT in OSCC. In summary we are able to show that EMT in OSCC is mediated by multiple growth factors and is accompanied by laminin γ2 chain up-regulation evidencing the existence of an intermediate Vim(+) /Ln332(+) EMT phenotype as seen in situ.

Topics: Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Extracellular Matrix Proteins; Humans; Kalinin; Laminin; Mouth Neoplasms; Neoplasm Invasiveness; Transforming Growth Factor beta1; Vimentin

Epidermal growth factor receptor (EGFR) overexpression correlates with recurrence and with treatment resistance in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to evaluate the relationship of EGFR gene copy number utilizing FISH and protein expression with automated quantitative analysis (AQUA) and to correlate those with patient outcome.. A tissue microarray composed of 102 HNSCC treated with (chemo)radiation was constructed and analyzed for EGFR copy number by FISH (Vysis; Abbott Laboratories) and EGFR protein expression using AQUA analysis of EGFR staining scored on a scale of 0 to 255. We evaluated associations of EGFR FISH status and AQUA score with clinicopathologic parameters and survival prognosis.. Eleven (17.2%) of 64 tumors with FISH results showed EGFR high polysomy and/or gene amplification (FISH positive). Protein levels assessed by AQUA in FISH-positive cases were significantly higher (P = 0.04) than in FISH-negative cases. Using the continuous AQUA scores for EGFR expression, AQUA and FISH showed significant agreement (Pearson's ρ = 0.353, P = 0.04). Patients with high tumor EGFR protein expression had inferior 5-year overall survival (27.7%) compared with those with low tumor EGFR expression (54%; P = 0.029). There was no significant association between EGFR FISH status and overall survival (P = 0.201). In the multivariate model, high tumor EGFR protein expression status remained an independent prognostic factor for overall survival (P = 0.047).. EGFR protein content correlates with gene copy number if protein content is quantitated and automatically analyzed, as with AQUA. EGFR protein levels assessed by AQUA strongly predict for patient outcome in HNSCC, whereas EGFR FISH status does not provide prognostic information.

Topics: Carcinoma; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Gene Dosage; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; In Situ Hybridization, Fluorescence; Male; Neoplasms, Squamous Cell; Predictive Value of Tests; Prognosis; Proteins; Squamous Cell Carcinoma of Head and Neck; Survival Analysis; Tissue Array Analysis

Kirsten Ras (K-Ras) mutations have been implicated as a key predictive marker of resistance to therapies targeting the epidermal growth factor receptor (EGFR). To determine whether Harvey Ras (H-Ras) mutations also can confer resistance to EGFR-targeted therapy, we expressed a constitutively active H-Ras (Ras G12V) in A431 human vulvar squamous carcinoma cells. Compared with corresponding control cells, A431-Ras cells exhibited marked resistance to the EGFR inhibitors cetuximab and gefitinib, reducing inhibition of Akt and Erk phosphorylation, inhibition of HIF-1α expression and transcriptional activity, and antitumor effects in vitro and in vivo. Our data indicate that constitutively active H-Ras can also confer resistance to anti-EGFR therapy in cancer cells.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cetuximab; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gefitinib; Genes, ras; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Models, Biological; Phosphorylation; Quinazolines; ras Proteins; Vulvar Neoplasms

The objective of this research was to investigate the salivary levels of epidermal growth factor (EGF) in patients with oral squamous cell carcinoma (OSCC) in comparison with clinically healthy individuals and to verify the immunoexpression of EGF in tumor samples. In addition, the relationship between salivary levels and tumoral EGF expression with clinicopathologic features was investigated. We carried out an investigation on EGF expression in lesion samples and in saliva of OSCC patients through immunohistochemistry and enzyme-linked immunosorbent assay, respectively. EGF salivary levels of OSCC patients were also compared with levels in saliva of healthy controls. EGF levels were significantly lower in OSCC patients in comparison with the control group. Smoking, tumor location, and alcohol consumption affected salivary levels of EGF. Strong immunoexpression of EGF was associated with a more aggressive histologic pattern of the lesion. There was no significant association among salivary levels and immunohistochemical expression of EGF. Although EGF expression is frequently observed in tumors, salivary levels of EGF are reduced in patients with OSCC samples. Tobacco and alcohol may decrease EGF in saliva, which may contribute to oral carcinogenesis. Indeed, further investigations are needed to elucidate the EGF pathways.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alcohol Drinking; Biomarkers; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Mouth; Mouth Neoplasms; Prognosis; Risk Factors; Saliva; Smoking

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.

Topics: Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phenotype; Phosphatidylinositol 3-Kinases; Proteolysis

Invasion of tumor cells into the local stroma is an important component in cancer progression. Here we report studies of the in vivo invasion of head and neck squamous cell carcinoma (HNSCC) cells in response to applied gradients of a growth factor [epidermal growth factor (EGF)] and a chemokine (CXCL12), using orthotopic floor-of-mouth models. Analysis of the invading cells indicated that >75% of them were tumor cells, about 15% macrophages, and <10% were unidentified. Surprisingly, although macrophages invaded together with tumor cells, macrophage contributions were not required for HNSCC invasion. CXCL12-induced in vivo invasion of HNSCC cells was also observed and found to occur via a unidirectional transactivation of epidermal growth factor receptor (EGFR) through CXCR4. Inhibition of tumor necrosis factor-α-converting enzyme using TNF-α protease inhibitor-2 selectively inhibited CXCL12-induced invasion but not EGF-induced invasion, consistent with CXCL12 activation of EGFR via release of EGFR ligands.

Topics: ADAM Proteins; ADAM17 Protein; Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemokine CXCL12; Disease Models, Animal; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Macrophages; Mice; Mice, Nude; Neoplasm Invasiveness; Signal Transduction

To investigate the mechanisms responsible for epidermal growth factor (EGF)-induced proliferation of human esophageal squamous cell carcinoma cells.. (3)H-thymidine incorporation assay was used to assess the proliferation of HKESC-1 cells exposed to EGF stimulation. Enzyme immunoassay was used to measure PGE(2) release from HKESC-1 cells, and the protein levels of cyclooxygenase 1 (COX-1), COX-2, EP1 and EP2 in EGF-stimulated cells were determined by Western blotting.. EGF upregulated COX-2 protein expression but produced no obvious effect on COX-1 protein expression in HKESC-1 cells. As a consequence of increased COX-2, EGF further enhanced cellular PGE(2) release. EGF stimulation also resulted in increased protein expression of EP2, a subtype of PGE(2) receptors. Both the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor SC-236 completely abolished EGF-induced PGE(2) release, and suppressed the mitogenic effect of EGF.. EGF stimulates the proliferation of HKESC-1 cells by increasing COX-2 protein expression and PGE(2) release. Upregulated EP2 protein expression may further amplify the mitogenic action of PGE(2).

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Dinoprostone; Epidermal Growth Factor; Esophageal Neoplasms; Humans; Pyrazoles; Receptors, Prostaglandin E, EP2 Subtype; Sulfonamides; Up-Regulation

Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro.. Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis.. In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis.. Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Dose-Response Relationship, Radiation; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Gamma Rays; Head and Neck Neoplasms; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Quinazolines; Signal Transduction; Sirolimus; Squamous Cell Carcinoma of Head and Neck; TOR Serine-Threonine Kinases; Tyrphostins

Genetic factors are known to be important in the development of esophageal squamous cell carcinoma (ESCC). Epidermal growth factor (EGF) can activate several signaling pathways leading to proliferation, differentiation, and tumorigenesis of epithelial tissues by binding with its receptor. Interindividual variations in EGF production were genetically contributed to EGF +61 G/A polymorphism. The purpose of this study is to investigate the potential association between EGF gene polymorphism and ESCC in a Chinese population. In this study, we analyzed single nucleotide polymorphism of EGF +61 G/A in 158 patients with ESCC and 212 age- and sex-matched controls in a Chinese population using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy and DNA sequencing. The variant genotypes of GA/AA were associated with a significantly decreased risk of ESCC compared with the wild-type homozygote GG (OR = 0.657, 95% CI: 0.434-0.996). However, no significant difference was observed between the EGF +61 G/A polymorphism and the risk of ESCC when the analyses were stratified in terms of age, gender, smoking status, different clinical stage, and lymph node status. The EGF +61 G/A polymorphism is associated with ESCC in a Chinese population. Our data suggests that the EGF gene may play a role in the development of ESCC.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Esophageal Neoplasms; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Middle Aged; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide

To determine the expression of nucleostemin (NS), epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) mRNA in human esophageal squamous cell carcinoma (ESCC) tissues and their association in a human ESCC cell line.. The expression of NS, EGF and EGFR mRNA was determined in paired normal esophageal and ESCC tissues of 62 patients using in situ hybridization. The association between NS and EGF or EGFR was examined using immunoblotting and real time polymerase chain reaction in a human ESCC cell line transfected with NS siRNA or treated with a selective EGFR inhibitor.. In normal esophageal and ESCC tissues, the positive detection rates were 21.0% (13/62) and 69.4% (43/62) for NS mRNA staining, 40.3% (25/62) and 77.4% (48/62) for EGF mRNA staining, and 30.6% (19/62) and 75.8% (41/62) for EGFR mRNA staining, respectively. These results indicated that NS, EGF and EGFR mRNA expression was upregulated mostly in ESCC tissues. Moreover, the expression of NS, EGF and EGFR mRNA was positively correlated with tumor grade, invasion and lymphatic metastasis of ESCC cells. NS mRNA was co-expressed with EGF and EGFR mRNA in ESCC tissues. The in vitro studies using a human ESCC cell line showed that knockdown of NS with NS siRNA significantly reduced EGF and EGFR expression. However, inhibition of the EGFR kinase activity with a specific EGFR kinase inhibitor had minimal effect on NS expression.. The upregulation of NS, EGF and EGFR mRNA frequently occurs in ESCC tissues and is associated with malignancy of human esophageal squamous tumors. NS is required for EGF and EGFR expression.

Topics: Aged; Carcinoma, Squamous Cell; Carrier Proteins; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Male; Middle Aged; Nuclear Proteins; RNA, Messenger; Up-Regulation

The full clinical relevance of the expression pattern of HER family of type I receptor tyrosine kinases in laryngeal squamous cell carcinoma remains to be elucidated. We evaluated the clinical relevance of such parameter in our population.. This study examined the expression pattern of HER family receptor members by quantitative immunohistochemistry and the amount of the EGF binding sites by a radioligand binding assay, in the same group of 67 LSCC patients, analysing the correlation between the expression of the four HER receptors and the clinical and prognostic parameters.. HER1 levels inversely correlated with that of HER2-4, while HER2-4 directly correlated among them. Cox univariate analysis using HER1-4 values as continuous covariates indicated that HER1 expression was directly associated with the risk of death and relapse while that of HER2-4 was inversely associated with the risk of death. Among the patients with high HER1 expressing tumours, those with tumours co-expressing HER2-4 showed a lower risk of death and relapse (in particular regional relapse) than those with tumours displaying a negative HER2-4 status.. The evaluation of HER2-4 status adds more power to the prognostic role of HER1 detection. In the era of molecularly targeted therapy, the expression of HER family of receptor tyrosine kinases in LSCC may hold relevant clinical significance and turn out to be a key factor in prognostic assessment and in treatment planning.

Topics: Binding Sites; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Middle Aged; Prognosis; Radioligand Assay; Receptor Protein-Tyrosine Kinases; Retrospective Studies

Factors that promote the aggressiveness of squamous cell carcinoma of the breast are not well understood. To examine the involvement of cell motility and the mechanism of this behavior, a squamous cell carcinoma cell line of the breast (HBC9) was established from a metastatic lymph node of a Japanese woman. HBC9 expressed epidermal growth factor receptor (EGFR), but was negative for Her2 or Her3.The invasive ability of HBC9 was compared with that of four breast ductal carcinoma cell lines by Matrigel invasion assay. EGF stimulation induced the formation of surface protrusions and cell migration in HBC9 cells, and significantly increased the number of cells migrating through the Matrigel. The invasive ability of HBC9 was compared with other cell lines of breast carcinoma; it was much greater than that of MCF-7, BT474, or HBC5, but did not differ significantly from that of MDA-MB-231. Observation of the surface protrusions of HBC9 by confocal laser microscopy revealed co-localization of Arp2 and N-WASP with actin polymerization, detected by visualization with phalloidin, indicating that the protrusions induced by EGF were invadopodia. In HBC9 cells, cortactin also co-localized with the N-WASP/Arp2/3 complex in the protrusions. Immunohistochemistry of 12 cases of squamous cell carcinoma of the breast revealed expression of cortactin and EGFR in all of them, and this was confirmed by western blotting in two cases. These results suggest that EGF-dependent enhancement of cell motility by formation of invadopodia associated with cortactin is a cause of the clinical aggressiveness of squamous cell carcinoma of the breast.

Topics: Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cortactin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Neoplasm Invasiveness

Overexpression of epidermal growth factor receptor (EGFR) in several types of malignant tumors correlates with disease progression. EGFR could, therefore, be an excellent candidate for targeted radionuclide diagnostics. However, the high natural expression of EGFR in the liver may be problematic. The aim of this study was to improve the tumor-to-liver ratio of radiolabeled epidermal growth factor (EGF) by blocking its uptake by the liver with a nonradiolabeled EGFR-targeting molecule in tumor-bearing mice. Intraperitoneally injected nonradiolabeled EGF was first evaluated as a blocking agent, preadministered at various time intervals before intravenous injection of (125)I-labeled EGF. The anti-EGFR Affibody molecule (Z(EGFR:955))(2) was then assessed as a blocking agent of (111)In-labeled EGF in a dual isotope study (50, 100, and 200 microg, preadministered 30 or 60 min before (111)In-EGF). The 30-min preadministration of nonradiolabeled EGF significantly decreased (125)I-EGF uptake in the liver, whereas uptake in the tumor remained unchanged. Furthermore, preadministration of only 50 microg (Z(EGFR:955))(2) as a blocking agent 30 min before the (111)In-EGF decreased the uptake of (111)In-EGF by the liver and increased its uptake by the tumor, thereby increasing the tumor-to-liver ratio sixfold. We conclude that the Affibody molecule (Z(EGFR:955))(2) shows promise as a blocking agent that could enhance the outcome of radionuclide-based EGFR-expressing tumor diagnostics and imaging.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Indium Radioisotopes; Iodine Radioisotopes; Liver; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Tissue Distribution; Transplantation, Heterologous

Connexin43 (Cx43) is known to have tumor-suppressive effects, but the underlying mechanisms are still poorly understood. In keratinocytes, we previously showed that the COOH-terminal domain of Cx43 directly interacts with the tumor suppressor Cav-1. We now show that rat epidermal keratinocytes (REK) that are reduced in Cx43 present features of epithelial-to-mesenchymal transition and are more invasive than their control counterparts, whereas overexpression of Cx43 inhibited the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)- and epidermal growth factor (EGF)-induced invasive properties. Carbenoxolone did not alter the inhibitory effect of Cx43 against TPA- and EGF-induced cell invasion, indicating the involvement of a gap junctional intercellular communication-independent mechanism. Interestingly, the association of Cx43 with Cav-1 was found to be reduced after TPA and EGF treatment. Accordingly, the colocalization of Cx43 with Cav-1 was diminished in cells from a human epidermal squamous cell carcinoma, as well as in sections from human keratinocyte tumors, suggesting that Cx43/Cav-1 interaction plays a protective role against keratinocyte transformation. As opposed to cells that overexpress Cx43-GFP, invasion could be induced in rat epidermal keratinocytes that overexpressed a GFP-tagged truncated mutant of Cx43 (Delta244-GFP) that we previously showed not to interact with Cav-1, as well as in cells that overexpressed Cx43-GFP but were reduced in Cav-1. Our data show that Cx43 possesses tumor-suppressive properties in keratinocytes and provide the first evidence that the Cx43/Cav-1 interaction is altered in keratinocyte transformation processes, as well as in human keratinocyte tumors, and that this association might play a role in Cx43-mediated tumor suppression.

Topics: Animals; Anti-Ulcer Agents; Blotting, Western; Carbenoxolone; Carcinogens; Carcinoma, Squamous Cell; Caveolin 1; Cell Communication; Cell Proliferation; Connexin 43; Epidermal Cells; Epidermal Growth Factor; Epidermis; Epithelial Cells; Fluorescent Antibody Technique; Gap Junctions; Green Fluorescent Proteins; Humans; Immunoprecipitation; Keratinocytes; Mesoderm; Neoplasm Invasiveness; Rats; RNA, Small Interfering; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta1

The erbB receptors and their ligands are involved in the pathogenesis and progression of oral squamous cell carcinoma (OSCC). Although EGFR and Her-2 are frequently overexpressed in OSCC, few studies evaluated these proteins in saliva and their association with the tumor, which may represent potential usefulness in a clinical setting.. The levels of EGFR, Her-2, and EGF were evaluated in saliva of 46 patients with OSCC before and after the surgical removal of the lesion, as well as in matched healthy controls. The relationship of salivary levels and EGFR and Her-2 immunoexpression in tumor samples with clinicopathological features was analyzed.. EGFR and Her-2 salivary levels did not show difference between to pre-surgery and control groups, however, both demonstrated an increase after surgical removal of the tumor. No association was detectable among receptor salivary levels, tissue expression and clinicopathological features. EGF levels in pre-surgery group were significantly lower when compared to the control group.. EGFR and Her-2 were not considered to be valuable salivary tumor markers in OSCC, however, lower levels of EGF in saliva may suggest a higher susceptibility for OSCC development.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Case-Control Studies; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Mouth Neoplasms; Receptor, ErbB-2; Saliva

The overexpression of fascin in human carcinomas is associated with aggressive clinical phenotypes and poor prognosis. However, the molecular mechanism underlying the increased expression of fascin in cancer cells is largely unknown. Here, we identified a Sp1 binding element located at -70 to -60 nts of the FSCN1 promoter and validated that Sp1 specifically bound to this element in esophageal carcinoma cells. Fascin expression was enhanced by Sp1 overexpression and blocked by Sp1 RNAi knockdown. Specific inhibition of ERK1/2 decreased phosphorylation levels of Sp1, and thus suppressed the transcription of the FSCN1, resulting in the down-regulation of fascin. Stimulation with EGF could enhance fascin expression via activating the ERK1/2 pathway and increasing phosphorylation levels of Sp1. These data suggest that FSCN1 transcription may be subjected to the regulation of the EGF/EGFR signaling pathway and can be used as a viable biomarker to predict the efficacy of EGFR inhibitors in cancer therapies.

Topics: Carcinoma, Squamous Cell; Carrier Proteins; Down-Regulation; Epidermal Growth Factor; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Humans; Microfilament Proteins; Mitogen-Activated Protein Kinases; Phosphorylation; Proteins; Signal Transduction

Overexpression and/or amplification of the epidermal growth factor receptor (EGFR) is present in 35-45% of primary glioblastoma multiforme tumors and has been correlated with a poor prognosis. In this study, we investigated the effect of cetuximab and intracellular signaling pathways downstream of EGFR, important for cell survival and proliferation. We show insufficient EGFR downregulation and competition with endogenous EGFR ligands upon cetuximab treatment. Dose-response experiments showed inhibition of EGFR phosphorylation without affecting two of the prominent downstream signaling pathways. Our results indicate that amplification and/or overexpression of EGFR is an unsatisfactory predictor for response to cetuximab.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Cell Survival; Cetuximab; Cycloheximide; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Gene Amplification; Gene Expression Regulation, Neoplastic; Glioma; Head and Neck Neoplasms; Humans; Phosphorylation; Signal Transduction

The epidermal growth factor receptor (EGFR) is a key driver in the process of squamous cell carcinoma (SCC) cell mitogenesis. Phospholipase C-gamma1 (PLC-gamma1) is a downstream target of EGFR signaling, but the role and necessity of PLC-gamma1 in EGFR-induced cell mitogenesis remain unclear. In the present study, we report an elevated expression of PLC-gamma1 in human SCC biopsies relative to adjacent normal epidermis, and in human SCC cell lines compared to normal human keratinocytes. EGFR-induced SCC cell mitogenesis was blocked by small interfering RNA knockdown of PLC-gamma1. However, inhibition of the catalytic activity of phospholipase C had no effect on EGFR-induced SCC cell mitogenesis. In response to the EGFR ligand epidermal growth factor (EGF), PLC-gamma1 was translocated not only to the plasma membrane but also to the nucleus. These data suggest that PLC-gamma1 is required for EGFR-induced SCC cell mitogenesis and the mitogenic function of PLC-gamma1 is independent of its lipase activity.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Epidermal Growth Factor; ErbB Receptors; Gene Knockdown Techniques; Humans; Keratinocytes; Mitosis; Phospholipase C gamma; Protein Transport; RNA, Small Interfering

Phospholipase C-gamma1 (PLC-gamma1) is a multiple-domain protein and plays an important role in epidermal growth factor (EGF)-induced cell mitogenesis, but the underlying mechanism is unclear. We have previously demonstrated that PLC-gamma1 is required for EGF-induced mitogenesis of squamous cell carcinoma (SCC) cells, but the mitogenic function of PLC-gamma1 is independent of its lipase activity. Earlier studies suggest that the Src homology 3 (SH3) domain of PLC-gamma1 possesses mitogenic activity. In the present study, we sought to determine the role of the SH3 domain of PLC-gamma1 in EGF-induced SCC cell mitogenesis. We examined the effect of overexpression of PLC-gamma1, a catalytically active PLC-gamma1 mutant lacking the SH3 domain or a catalytically inactive PLC-gamma1 mutant lacking the X domain on EGF-induced SCC4 (tongue squamous cell carcinoma) cell mitogenesis. We found that overexpression of PLC-gamma1 enhanced EGF-induced SCC4 cell mitogenesis. This enhancement was abolished by deletion of the SH3 domain but not by deletion of the X catalytic domain. These data suggest that the SH3 domain, but not the catalytic domain, is required for PLC-gamma1 to mediate EGF-induced SCC4 cell mitogenesis.

Topics: Carcinoma, Squamous Cell; Catalytic Domain; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Humans; Mitosis; Phospholipase C gamma; src Homology Domains

The anti-apoptotic protein BCL-x(L) and the cell cycle inhibitor p21(CIP1/WAF1) were previously implicated in head and neck cancer. Several reports point to a role of the epidermal growth factor receptor (EGFR, ErbB-1, HER1) in regulating their expression. In the present study, we investigated the influence of EGFR on these tumor-associated factors. HNSCC cell lines were incubated with EGF or with the EGFR-specific kinase inhibitor AG1478. Western blot analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were deployed to measure BCL-x(L) and p21(CIP1/WAF1) protein and mRNA levels. A dose-dependent rise of BCL-x(L) as well as p21(CIP1/WAF1) protein was noted after incubation with EGF, whereas inhibition with AG1478 reduced basal expression levels. No influence on BCL-2 was seen. Interestingly, qRT-PCR revealed that p21(CIP1/WAF1) but not BCL-x(L) transcript levels were induced after EGF treatment. Taken together, it can be stated that p21(CIP1/WAF1) and BCL-x(L) but not BCL-2 levels are tightly regulated by EGFR in HNSCC cell lines. BCL-x(L) induction appears to be due to protein stabilization rather than transcriptional activation, which is the likely cause of p21(CIP1/WAF1) induction. The noted variability in EGF response of HNSCC cells could reflect frequently observed variations in clinical response rates after implementation of anti-EGFR therapies.

Topics: bcl-X Protein; Blotting, Western; Carcinoma, Squamous Cell; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Phosphorylation; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tyrphostins

Topics: beta-Defensins; Biomarkers; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Erythroplasia; Humans; Leukoplakia, Oral; Mouth Neoplasms; Precancerous Conditions; Prognosis; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

To report toxicity data from the first 13 consecutive patients with locally advanced head and neck squamous cell carcinoma (LAHNSCC), ineligible for cisplatin, treated with concurrent cetuximab and radiotherapy (RT) at our institution.. Data were collected prospectively between August 2007 and May 2008. Planned treatment consisted of a cetuximab loading dose (400mg/m(2)) via intravenous infusion 1 week prior and then weekly (250mg/m(2)) with 70Gy in 35 daily fractions over 7 weeks.. Median age was 68 years (range 52-82 years). The predominant primary sites were hypopharyngeal (5) and oropharyngeal (5). Ineligibility for cisplatin consisted of renal impairment (5), hearing impairment (4) and of other major co-morbidities (4). Of the 13 patients, 10 (77%) had grade 3/4 skin reactions and 10 (77%) grade 3/4 mucositis. Six (46%) patients required admission for management of severe skin reactions and/or mucositis with 4 (31%) requiring a treatment break, median 10 days (9-15days). Only 4 (31%) patients managed to complete the planned 8 cycles of cetuximab. Of the 9 patients with 12-week post-therapy data, 7 (78%) achieved a complete response.. Our early experience with cetuximab/RT has demonstrated a higher rate of toxicity compared with the recently reported randomised trial, resulting in low treatment compliance and delays in completing RT.

Topics: Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cetuximab; Combined Modality Therapy; Drug Eruptions; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Otorhinolaryngologic Neoplasms; Radiodermatitis; Radiotherapy Dosage

Human beta-defensins (hBDs) are small, cationic antimicrobial peptides produced by oral and other mucosal epithelia. More recently, hBDs have been shown to regulate adaptive immunity. In this study, we provide new information about the potential role of hBD-3 in the progression of oral cancer. In normal human oral epithelia, hBD-3 is produced by mitotically active cells in the basal layers of oral epithelium, whereas hBD-1 and -2 are coexpressed in the differentiated spinosum and granulosum layers. Interestingly, premalignant cells in carcinoma in situ lesions overexpress hBD-3, but not hBD-1 and hBD-2, correlating with specific recruitment and infiltration of macrophages. Our in vitro studies demonstrate that hBD-3 chemoattracts THP-1 monocytic cells and that epidermal growth factor (EGF) significantly induces hBD-3 expression in oral epithelial cells via mitogen-activated protein kinase (MAPK) kinase MEK1/2, p38 MAPK, protein kinase C (PKC), and phosphoinositide 3 kinase (PI3K), but not via Janus kinase (JAK) and signal transducer and activator of transcription (STATs). These results suggest that hBD-3 serves as a mitogen responsive gene in the initiation of oral cancer and may act as a motility signal to recruit tumor-associated macrophages.

Topics: beta-Defensins; Carcinoma in Situ; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Humans; Macrophages; Microscopy, Fluorescence; Mouth Neoplasms; Phosphotransferases; Precancerous Conditions; STAT Transcription Factors

Snail is a regulator of epithelial-mesenchymal transition (EMT) and considered crucial to carcinoma metastasis, myofibroblast transdifferentiation, and fibroblast activation. To investigate the role of Snail in oral squamous cell carcinoma (OSCC), its immunohistochemical expression was analysed in 129 OSCC samples and correlated to nodal metastasis, histological grade, E-cadherin, and alpha smooth-muscle-actin (alpha SMA). The results were compared to findings in 23 basal cell carcinomas (BCC). Additionally, the influence of TGF beta 1 and EGF on Snail, E-cadherin, vimentin, and alpha SMA expression was analysed in two OSCC cell lines. As a result, Snail-positive cells were mainly found in the stroma of the OSCC invasive front without statistically significant correlation to histological grade or nodal metastasis. Snail was co-localised to alpha SMA but not to E-cadherin or cytokeratin and showed a significant correlation to the loss of membranous E-cadherin. All BCCs were Snail negative. In OSCC culture, the growth-factor-mediated EMT-like phenomenon was accompanied by alpha SMA down-regulation. In summary, Snail expression in OSCC is a stromal phenomenon associated with the myofibroblast phenotype and not related to growth-factor-mediated transdifferentiation of the carcinoma cells themselves. Consequently, Snail immunohistochemistry cannot contribute to the prediction of the metastatic potential. Furthermore, stromal Snail expression is suggested to be the result of mutual paracrine interaction of fibro-/myofibroblasts and dedifferentiated carcinoma cells leading to the generation of a special type of carcinoma-associated fibroblasts.

Topics: Actins; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Cadherins; Carcinoma, Squamous Cell; Epidermal Growth Factor; Fibroblasts; Humans; Keratins; Middle Aged; Mouth Neoplasms; Myoblasts; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta1; Vimentin

Hypoxia-inducible factor (HIF) accumulates when tumors grow under hypoxic conditions. The genesis of tumors, however, usually involves normoxic conditions. In this study, we were interested in examining the potential role of aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF-1beta in tumor growth under normoxic conditions, specifically when cells are treated with epidermal growth factor (EGF), which is known to affect the gene expression of tumor growth-related protein COX-2 (cyclooxygenase-2). The results showed that EGF receptor inhibitor, AG1478, abolished EGF-induced nuclear accumulation of ARNT as well as the expression of COX-2. ARNT small interfering RNA inhibited the promoter activity, mRNA level, and protein expression of COX-2 in cells treated with EGF. In contrast, CoCl(2)-induced HIF-1alpha exhibited no effect on COX-2 expression. EGF also stimulated the formation of the ARNT.c-Jun complex as well as the complex binding to the COX-2 promoter. ARNT small interfering RNAs blocked EGF-activated cell migration. Moreover, COX-2 and ARNT were cohorts present distinctively in clinical specimens of human cervical squamous cell carcinoma and were almost nondetectable in adjacent normal or noncancerous cervical tissues. Our results revealed that ARNT plays an important role in EGF-regulated COX-2 gene expression and may thus be related to either a cause or a consequence of tumorigenesis in cervical cancer.

Topics: Active Transport, Cell Nucleus; Aryl Hydrocarbon Receptor Nuclear Translocator; Carcinoma, Squamous Cell; Cell Line, Tumor; Cobalt; Cyclooxygenase 2; Epidermal Growth Factor; Female; Humans; Microscopy, Fluorescence; Models, Biological; Promoter Regions, Genetic; Quinazolines; RNA, Messenger; Tyrphostins; Uterine Cervical Neoplasms

Carbon nanotube-based drug delivery holds great promise for cancer therapy. Herein we report the first targeted, in vivo killing of cancer cells using a drug-single wall carbon nanotube (SWNT) bioconjugate, and demonstrate efficacy superior to nontargeted bioconjugates. First line anticancer agent cisplatin and epidermal growth factor (EGF) were attached to SWNTs to specifically target squamous cancer, and the nontargeted control was SWNT-cisplatin without EGF. Initial in vitro imaging studies with head and neck squamous carcinoma cells (HNSCC) overexpressing EGF receptors (EGFR) using Qdot luminescence and confocal microscopy showed that SWNT-Qdot-EGF bioconjugates internalized rapidly into the cancer cells. Limited uptake occurred for control cells without EGF, and uptake was blocked by siRNA knockdown of EGFR in cancer cells, revealing the importance of EGF-EGFR binding. Three color, two-photon intravital video imaging in vivo showed that SWNT-Qdot-EGF injected into live mice was selectively taken up by HNSCC tumors, but SWNT-Qdot controls with no EGF were cleared from the tumor region in <20 min. HNSCC cells treated with SWNT-cisplatin-EGF were also killed selectively, while control systems that did not feature EGF-EGFR binding did not influence cell proliferation. Most significantly, regression of tumor growth was rapid in mice treated with targeted SWNT-cisplatin-EGF relative to nontargeted SWNT-cisplatin.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cisplatin; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Mice; Nanotubes, Carbon; Substrate Specificity; Tissue Distribution

nm23-H1 was found to diminish metastatic potential of carcinoma cell lines and therefore was placed in the group of metastatic suppressor genes. Its protein product has a function of a nucleoside diphosphate kinase (NDPK) as well as protein kinase and nuclease. Though it was found that Nm23-H1 is involved in many cellular processes, it is still not known how it promotes metastatic suppressor activity. Since the process of metastasis is dependent on adhesion properties of cells, the goal of our work was to describe the adhesion properties of CAL 27 cells (oral squamous cell carcinoma of the tongue) overexpressing FLAG/nm23-H1. In our experiments, cells overexpressing nm23-H1 show reduced migratory and invasive potential. Additionally, cells overexpressing nm23-H1 adhere stronger on substrates (collagen IV and fibronectin) and show more spread morphology than the control cells. Results obtained by EGF induction of migration revealed that the adhesion strength predetermined cell response to chemoattractant and that Nm23-H1, in this cell type, does not interfere with, EGF induced, Ras signaling pathway. These data contribute to the overall knowledge about nm23-H1 and its role in cell adhesion, migration, and invasion, especially in oral squamous cell carcinoma.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Flow Cytometry; Humans; Immunohistochemistry; Immunoprecipitation; Integrin beta1; Microscopy, Confocal; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; Neoplasm Invasiveness; NM23 Nucleoside Diphosphate Kinases; Oligopeptides; Peptides; Protein Binding; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transfection

It has been suggested that epithelial cyclooxygenase-2 (COX-2) promotes oral carcinogenesis and carcinoma malignancy through increased prostaglandin E(2) (PGE(2)) production. Although oral squamous cell carcinomas (OSCC) often express COX-2, they may also produce PGE(2) in a COX-1-dependent manner. We used 6 isolated cell lines to investigate which COX isoforms OSCC may use for PGE(2) production. COX-1 and -2 expression patterns divided the 6 OSCC cell lines into 3 distinct groups: both COX isoforms low, only COX-1 high, or both COX isoforms high. Multicolor immunohistofluorescence staining confirmed the COX-expression profiles in organotypic 3D cultures and the COX-2 dominance in OSCC tumors. Epidermal growth factor (EGF) stimulation induced COX-2 (but not COX-1) expression and increased PGE(2) production, which was attenuated by COX-2 (but not COX-1) specific inhibition or siRNA-mediated COX-2 gene knockdown. Thus, PGE(2) production in OSCC cell lines was COX-2-dependent.

Topics: Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Dinoprostone; Epidermal Growth Factor; Female; Gene Expression; Gene Knockdown Techniques; Humans; Intramolecular Oxidoreductases; Isoenzymes; Male; Middle Aged; Mouth Neoplasms; Prostaglandin-E Synthases; RNA, Small Interfering

Gefitinib targeting of the epidermal growth factor receptor (EGFR) has shown limited activity in clinical trials of head and neck squamous cell carcinoma (HNSCC). To investigate the underlying molecular mechanism, the proteomic signatures and responses of EGFR and downstream signals have been studied in a panel of HNSCC cell lines and tumor specimens pre- and post-gefitinib treatment.. The IC(50) of gefitinib for HNSCC cell lines were determined using 3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay. The effects of gefitinib on activation of EGFR and downstream signaling molecules were determined by Western blot, ELISA, and reverse-phase protein microarray (RPMA). The biomarkers involved in the signaling pathways were examined in HNSCC tumor specimens from patients in a phase I gefitinib trial.. In vitro, gefitinib inhibited cell proliferation with differing IC(50), and suppressed activation of EGFR and downstream signaling molecules protein kinase B (AKT), extracellular signal-regulated kinase 1/2, signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappaB. The drug sensitivity was statistically correlated with activation of phosphorylated AKT (p-AKT) and phosphorylated STAT3 (p-STAT3) detected by ELISA, and consistent with results measured by RPMA. In patient samples, a broad suppression of activation of EGFR and downstream signaling molecules was observed in a molecular responder patient, in contrast to a lack of inhibition or increased activation of biomarkers in different pathways in nonresponder patients.. Gefitinib sensitivity is correlated with p-AKT and p-STAT3 activation in HNSCC cell lines and tumor specimens. p-AKT and p-STAT3 could serve as potentially useful biomarkers and drug targets for further development of novel therapeutic agents for HNSCC.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Clinical Trials, Phase I as Topic; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Head and Neck Neoplasms; Humans; Inhibitory Concentration 50; NF-kappa B; Phosphorylation; Protein Array Analysis; Protein Kinase Inhibitors; Proteomics; Quinazolines; Signal Transduction

Coexistence of pulmonary tuberculosis (TB) and lung cancer in clinic poses significant challenges for the diagnostic and treatment of both diseases. Although association of chronic inflammation and cancer is well-documented, causal relationship between TB infection and lung cancer are not understood. We present experimental evidence that chronic TB infection induces cell dysplasia and squamous cell carcinoma (SCC) in a lung-specific manner. First, squamous cell aggregates consistently appeared within the lung tissue associated with chronic TB lesions, and in some cases resembled SCCs. A transplantable tumor was established after the transfer of cells isolated from TB lung lesions into syngeneic recipients. Second, the (Mycobacterium tuberculosis) MTB-infected macrophages play a pivotal role in TB-induced carcinogenesis by inducing DNA damage in their vicinity and by the production of a potent epidermal growth factor epiregulin, which may serve as a paracrine survival and growth factor responsible for squamous metaplasia and tumorigenesis. Third, lung carcinogenesis during the course of chronic TB infection was more pronounced in animals with severe lung tissue damage mediated by TB-susceptibility locus sst1. Together, our experimental findings showed a causal link between pulmonary TB and lung tumorigenesis and established a genetic model for further analysis of carcinogenic mechanisms activated by TB infection.

Topics: Animals; Antitubercular Agents; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Chronic Disease; Disease Models, Animal; Epidermal Growth Factor; Epiregulin; Female; Gene Expression; Genetic Predisposition to Disease; Host-Pathogen Interactions; Isoniazid; Lung; Lung Neoplasms; Macrophages; Male; Mice; Mice, Congenic; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred Strains; Mycobacterium tuberculosis; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Tuberculosis, Pulmonary

We examined the anti-angiogenic effects of Ganoderma tsugae methanol extract (GTME) on human epidermoid carcinoma A-431 cells. Our data indicate that GTME inhibits the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in vitro and in vivo, and also inhibits the capillary tube formation of human umbilical vein endothelial cells (HUVECs). We also show that the suppression of VEGF expression by GTME can be restored by treatment with EGF. These results suggest that GTME inhibits VEGF expression via the suppression of EGFR expression, resulting in the downregulation of VEGF secretion from epidermoid carcinoma A-431 cells. These findings reveal a novel role for G. tsugae in inhibiting EGFR and VEGF expression, which are important for tumor angiogenesis and growth. Thus, GTME may provide a potential therapeutic approach for anti-tumor treatment.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents, Phytogenic; Carcinoma, Squamous Cell; Cell Line, Tumor; Cells, Cultured; Drug Resistance, Neoplasm; Drugs, Chinese Herbal; Endothelium, Vascular; Epidermal Growth Factor; ErbB Receptors; Ganoderma; Gene Expression Regulation, Neoplastic; Humans; Methanol; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Neovascularization, Pathologic; Neovascularization, Physiologic; Phytotherapy; Plant Extracts; Reishi; Skin Neoplasms; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

The phenotypic switching called epithelial-to-mesenchymal transition is frequently associated with epithelial tumor cell progression from a comparatively benign to an aggressive, invasive malignancy. Coincident with the emergence of such cellular plasticity is an altered response to transforming growth factor-beta (TGF-beta) as well as epidermal growth factor (EGF) receptor amplification. TGF-beta in the tumor microenvironment promotes invasive traits largely through reprogramming gene expression, which paradoxically supports matrix-disruptive as well as stabilizing processes. ras-transformed HaCaT II-4 keratinocytes undergo phenotypic changes typical of epithelial-to-mesenchymal transition, acquire a collagenolytic phenotype, and effectively invade collagen type 1 gels as a consequence of TGF-beta1 + EGF stimulation in a three-dimensional physiologically relevant model system that monitors collagen remodeling. Enhanced collagen degradation was coupled to a significant increase in matrix metalloproteinase (MMP)-10 expression and involved a proteolytic axis composed of plasmin, MMP-10, and MMP-1. Neutralization of any one component in this cascade inhibited collagen gel lysis. Similarly, addition of plasminogen activator inhibitor type 1 (SERPINE1) blocked collagen degradation as well as the conversion of both proMMP-10 and proMMP-1 to their catalytically active forms. This study therefore identifies an important mechanism in TGF-beta1 + EGF-initiated collagen remodeling by transformed human keratinocytes and proposes a crucial upstream role for plasminogen activator inhibitor type 1-dependent regulation in this event.

Topics: Carcinoma, Squamous Cell; Cell Line, Transformed; Collagen Type I; Epidermal Growth Factor; Fibrinolysin; Humans; Keratinocytes; Matrix Metalloproteinase 1; Matrix Metalloproteinase 10; Plasminogen Activator Inhibitor 1; Skin Neoplasms; Transforming Growth Factor beta1

The epidermal growth factor receptor (EGFR, ErbB1, HER1) is frequently overexpressed in head and neck squamous cell carcinomas (HNSCCs) and correlates with disease progression and reduced survival of the patient. The aim of this study was to investigate the influence of EGFR stimulation on cisplatin sensitivity in 3 previously well characterized HNSCC cell lines. The HNSCC cell lines UMB-SCC-745, -864 and UT-SCC-26A were incubated for 13 h in the presence of 0.1, 1, 10, 100 or 1000 ng/mL EGF. Cell cycle checkpoint distribution was determined by FACS analysis. Effects of cisplatin on cell viability were evaluated with the MTT assay. UT-SCC-26A cells were resistant to the highest cisplatin concentrations (100 microM). However, during treatment with rising EGF levels UT-SCC-26A tumor cells became susceptible to cisplatin. Cell cycle analysis revealed a very low level of UT-SCC-26A cells in G(2)/M phase that rose after stimulation with EGF. Also, after EGFR stimulation, cyclin D1 and CHK2 levels increased most prominently in UT-SCC-26A, whereas CHK2 levels dropped again at higher EGF concentrations. Overall, cellular proliferation and cisplatin IC(50) exhibited inverse behaviour. Taken together the data suggest that EGFR stimulation in some cases could be an important prerequisite for cisplatin sensitivity. Therefore, future studies should investigate potential opposing effects of such combination therapies that consist of cell proliferation inhibitors in conjunction with cisplatin treatment. In addition, studies should also include investigations regarding possible therapeutic benefits of tumor cell proliferation-activating agents, that could render resistant HNSCC tumor cells sensitive to chemotherapy treatment.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Cisplatin; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Tumor Cells, Cultured

It is well known that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effect of endothelial cell-secreted factors on the phenotype and behavior of tumor cells. The hypothesis underlying this study is that endothelial cells initiate signaling pathways that enhance tumor cell survival and migration. Here, we observed that soluble mediators from primary human dermal microvascular endothelial cells induce phosphorylation of signal transducer and activator of transcription 3 (STAT3), Akt, and extracellular signal-regulated kinase (ERK) in a panel of head and neck squamous cell carcinoma (HNSCC) cells (OSCC-3, UM-SCC-1, UM-SCC-17B, UM-SCC-74A). Gene expression analysis demonstrated that interleukin-6 (IL- 6), interleukin-8 (CXCL8), and epidermal growth factor (EGF) are upregulated in endothelial cells cocultured with HNSCC. Blockade of endothelial cell-derived IL-6, CXCL8, or EGF by gene silencing or neutralizing antibodies inhibited phosphorylation of STAT3, Akt, and ERK in tumor cells, respectively. Notably, activation of STAT3, Akt, and ERK by endothelial cells enhanced migration and inhibited anoikis of tumor cells. We have previously demonstrated that Bcl-2 is upregulated in tumor microvessels in patients with HNSCC. Here, we observed that Bcl-2 signaling induces expression of IL-6, CXCL8, and EGF, providing a mechanism for the upregulation of these cytokines in tumor-associated endothelial cells. This study expands the contribution of endothelial cells to the pathobiology of tumor cells. It unveils a new mechanism in which endothelial cells function as initiators of molecular crosstalks that enhance survival and migration of tumor cells.

Topics: Anoikis; Blotting, Western; Carcinoma, Squamous Cell; Cell Communication; Cell Line, Tumor; Cell Movement; Cell Survival; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Endothelial Cells; Epidermal Growth Factor; Gene Expression; Humans; Interleukin-6; Interleukin-8; Mitogen-Activated Protein Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Signal Transduction; STAT3 Transcription Factor

EGF is known to affect adherens junctions and disrupt cell-cell adhesion in a variety of carcinomas but the underlying mechanisms are not completely understood. Using human tumor epithelial cells overexpressing EGFR we demonstrated that EGF-induced cell scattering was mediated by protein kinase C-delta (PKC-delta). PKC-delta knockdown by siRNA significantly inhibited EGF-induced internalization of E-cadherin into the cytoplasm and blocked cell scattering. EGF phosphorylated PKC-delta at Y311 and ectopic expression of the mutant Y311F prevented PKC-delta binding to E-cadherin and EGF-induced cell scattering. Moreover, depletion of Src using siRNA decreased EGF-induced phosphorylation of PKC-delta at Y311 and blocked scattering. Finally, EGF reduced expression of the tight junction protein, occludin, and this effect was also mediated by PKC-delta through Src. In summary, PKC-delta mediated the effects of EGF on adherens and tight junctions thereby playing an important role in cell-cell adhesion with possible wider implications in tumor metastasis or epithelial-to-mesenchymal transition.

Topics: Amino Acid Substitution; Cadherins; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Foreskin; Humans; Imidazoles; Infant, Newborn; Keratinocytes; Kidney; Male; Protein Kinase C-delta; Pyridines; RNA, Small Interfering; Tight Junctions

Clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase, has been shown in non-small-cell lung carcinoma patients with EGF receptor mutations, so these mutations are useful marker(s) to find a responder for the drug. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. We previously reported that the expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells was down-regulated by EGF treatment, and that forced expression of BRAK in tumor cells decreased the tumorigenicity of the cells in xenografts. Thus, we investigated the relationship between restoration of BRAK expression by gefitinib and the efficacy of the drug for tumor suppression. We found that EGF down-regulated BRAK expression through the MEK-extracellular signal regulated kinase pathway and that this down-regulated expression was restored by gefitinib in vitro. Oral administration of gefitinib significantly (P < 0.001) reduced tumor growth of xenografts of three HNSCC cell lines (HSC-2, HSC-3, and HSC-4), in female athymic nude mice, accompanied by an increase in BRAK expression specifically in tumor tissue. This tumor-suppressing effect of the drug was not observed in the case of BRAK non-expressing cells. Furthermore introduction of BRAK shRNA vector reduced both the expression levels of BRAK in HSC-3 cells and the antitumor efficacy of gefitinib in vivo. Our data showing an inverse relationship between BRAK expression levels in tumor cells and the tumor growth rate indicate that the gefitinib-induced increase in BRAK expression is beneficial for tumor suppression in vivo.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemokines, CXC; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Head and Neck Neoplasms; Humans; Mutation; Quinazolines; RNA, Messenger

The intracellular signalling cascade(s) mediating epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression is poorly defined in oral carcinomas. Investigation of two different oral squamous cell carcinoma (OSCC) cell lines with high EGF-induced COX-2 expression revealed, however, that this expression was dependent on two mitogen-activated protein kinase (MAPK) pathways [extracellular signal-regulated kinase 1/2 (ERK1/2) and p38] because combined inhibition of these pathways was needed to abolish EGF-induced COX-2 expression. Surprisingly, inhibition of phosphoinositide-3 kinase (PI3K) increased EGF-induced COX-2 expression in the basaloid OSCC cell line (C12), suggesting a PI3K-controlled, inhibitory COX-2-regulating pathway. Neither the transcription factor nuclear factor-kappaB (NF-kappaB), nor Src, was involved in EGF-induced COX-2 expression. The results suggest that EGF-induced COX-2 expression is regulated by several pathways, and emphasizes that individual tumors use different strategies for intracellular signalling.

Topics: Aged; Carcinoma, Basosquamous; Carcinoma, Squamous Cell; Carcinoma, Verrucous; Cell Line, Tumor; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Enzymologic; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Signal Transduction; src-Family Kinases; Tumor Cells, Cultured

This study aimed to characterize estrogen receptor expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues, and to evaluate estrogen receptor and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC.. Estrogen receptor expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to estrogen receptor and EGFR ligands or inhibitors. Estrogen receptor and EGFR protein expression in patient tissues was assessed by immunohistochemical staining.. Phospho-mitogen-activated protein kinase (P-MAPK) levels were significantly increased following combined estrogen and EGF treatment. Treatment of HNSCC cells with estrogen and EGF significantly increased cell invasion compared with either treatment alone, whereas inhibiting these two pathways resulted in reduced invasion compared with inhibiting either pathway alone. EGFR (P = 0.008) and nuclear estrogen receptor alpha (ER alpha(nuc); P < 0.001) levels were significantly increased in HNSCC tumors (n = 56) compared with adjacent mucosa (n = 30), whereas nuclear estrogen receptor beta (ER beta(nuc)) levels did not differ (P = 0.67). Patients with high ER alpha(nuc) and EGFR tumor levels had significantly reduced progression-free survival compared with patients with low tumor ER alpha(nuc) and EGFR levels (hazards ratio, 4.09; P = 0.01; Cox proportional hazards). In contrast, high ER beta(nuc) tumor levels were not associated with reduced progression-free survival alone or when combined with EGFR.. ER alpha and ER beta were expressed in HNSCC, and stimulation with estrogen receptor ligands resulted in both cytoplasmic signal transduction and transcriptional activation. Estrogen receptor and EGFR cross-talk was observed. Collectively, these studies indicate that estrogen receptor and EGFR together may contribute to HNSCC development and disease progression.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estrogens; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinases; Mucous Membrane; Neoplasm Invasiveness; Receptor Cross-Talk; Receptors, Estrogen; Signal Transduction

Epidermal growth factor (EGF)-induced cyclooxygenase-2 (COX-2) expression in squamous cell carcinomas is mediated through the extracellular signal-regulated kinase 1/2 and p38 pathways. Examination of a basaloid and a conventional oral squamous cell carcinoma cell line revealed that inhibition of c-Jun N-terminal kinase (JNK) with SP600125 increased EGF-induced (but not basal) COX-2 transcription 1.5-1.9-fold in extracellular signal-regulated kinase 1/2 and p38 pathway-dependent manners. Although JNK may phosphorylate the cyclosporine A-sensitive transcription factor, nuclear factor of activated T cells c3, it was seemingly not involved because cyclosporine A did not reduce EGF-induced COX-2 expression. Thus, JNK negatively regulated EGF-induced extracellular signal-regulated kinase 1/2 and/or p38-mediated COX-2 transcription, presumably through activating an unidentified phosphatase.

Topics: Anthracenes; Calcineurin Inhibitors; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclooxygenase 2; Cyclosporine; Dactinomycin; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mouth Neoplasms; NFATC Transcription Factors; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Synthesis Inhibitors; Transcription, Genetic

Aberrant expression of N-cadherin is associated with tumor progression in squamous cell carcinomas (SCCs). Consequently, we examined the regulation of N-cadherin by TGFbeta1, an important mediator of keratinocyte and SCC function. N-cadherin expression was increased in oral SCC (OSCC) cell lines, regulating motility and correlating with TGFbeta1 production. Moreover, in normal keratinocytes TGFbeta1 increased expression of N-cadherin to regulate motility. TGFbeta1-mediated N-cadherin expression in the oral keratinocytes was blocked using siRNA targeting Smads. Unexpectedly, we found that EGF blocked TGFbeta1-mediated N-cadherin expression in oral keratinocytes and not in OSCC cells. Mechanistically, EGF enhanced Smad phosphorylation in the linker region, and attenuated TGFbeta1-mediated phosphorylation of Smad at the C-terminus, localization of Smad to the nucleus as well as Smad-driven promoter activity exclusively in oral keratinocytes but not in OSCC cells. The effect of EGF on TGFbeta1-mediated Smad-driven promoter activity and N-cadherin expression was reversed when activation of ERK1/2 was blocked. Although EGF and TGFbeta1 independently promoted migration of both oral keratinocytes and OSCC cells, EGF decreased TGFbeta1-mediated migration of oral keratinocytes but enhanced migration of OSCC cells. Together, these data support a model wherein EGF signaling has an important negative regulatory role on TGFbeta1-mediated N-cadherin expression and motility in normal oral keratinocytes, and in which loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.

Topics: Cadherins; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Epithelium; Humans; Keratinocytes; Mitogen-Activated Protein Kinase 3; Mouth; Mouth Neoplasms; Signal Transduction; Transforming Growth Factor beta1; Tumor Cells, Cultured

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Drug Combinations; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotoxins; Models, Biological; Mouth Neoplasms; Saponins

Acquisition of epithelial-mesenchymal transition (EMT) by primary carcinoma cells is associated with disrupted epithelial integrity, local invasion, and ultimately metastasis. Little is known about the existence and function of EMT in cervical cancer. This study aims to investigate the regulation of EMT in cervical squamous cell carcinoma.. We investigated the molecular events of EMT in surgical specimens, which present the progression of cervical carcinoma. Two cervical cancer cell lines and the primary culture of normal cervical epithelia were used to study the regulatory mechanisms of EMT.. The chronic epidermal growth factor (EGF) treatment induces the elongation of cell shape, increases cell scattering, and enhances cell invasion. EGF treatment down-regulates E-cadherin and up-regulates vimentin in cervical cancer cells. These characteristics are consistent with the morphologic changes, molecular events, and functional significance of EMT. EGF receptor (EGFR) signaling inactivates glycogen synthase kinase-3beta, which results in the nuclear accumulation of up-regulated Snail and then leads to EMT program. alpha(5)beta(1) integrin signaling and extracellular matrix fibronectin can modulate EGF-induced EMT. Importantly, the immunofluorescent stainings of surgical specimens indicate that cervical carcinoma progression is accompanied by EGFR overexpression, which is in parallel with decreased E-cadherin and increased vimentin. Up-regulation and nuclear accumulation of Snail correlate with EMT program in tumor tissues.. EGF cooperates with alpha(5)beta(1) integrin signaling to induce EMT in cervical cancer cells via up-regulated Snail. Blockade of EGFR activity or expression may provide a potential target for the treatment of cervical cancer progression.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Nucleus; Disease Progression; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Integrin alpha5beta1; Mesoderm; Models, Biological; Snail Family Transcription Factors; Transcription Factors; Uterine Cervical Neoplasms

The quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine (2) and (3-bromophenyl)quinazoline-4,6-diamine (3) were labelled with (99m)Tc using the "4 + 1" mixed-ligand system [Tc(NS3)(CN-R)] and the tricarbonyl moiety fac-[Tc(CO)3]+. In the "4 + 1" approach the technetium(iii) is stabilized by a monodentate isocyanide bearing a quinazoline fragment (L1,L2 ) and by the tetradentate tripodal ligand tris(2-mercaptoethyl)-amine (NS3). In the "4 + 1" approach, 99mTc-labelling was performed in a two-step procedure, the complexes [Tc(NS3)(L1)] (7a) and [Tc(NS3)(L2)] (8a) being obtained in about 50-70% yield. In the tricarbonyl approach, the fac-[Tc(CO)3]+ unit is anchored by two different monoanionic chelators bearing the quinazoline derivatives (3-chloro-4-fluorophenyl)quinazoline-4,6-diamine (2) and (3-bromophenyl)quinazoline-4,6-diamine (3). Both chelators have a N2O donor atom set, but one contains a pyrazolyl ring (L5H) and the other contains a pyridine unit (L6H). In both cases the conjugation of the quinazoline to the chelator was done through the secondary amine of the potentially tridentate and monoanionic chelators, the corresponding 99mTc-complexes (10a, 11a) being obtained in quantitative yield. The identities of the 99mTc-labelled quinazolines (7a, 8a, 10a, 11a) were confirmed by comparison with the HPLC profiles of the analogous Re compounds (7, 8, 10, 11). All these Re complexes were characterized by NMR and IR spectroscopy, elemental analysis and in some cases by MS and X-ray diffraction analysis. In vitro studies indicate that the quinazoline fragments, after conjugation to the cyano group (L1, L2) or to the pyrazolyl containing chelator (L5H), as well as the corresponding Re complexes (7, 8, 10) inhibit significantly the EGFR autophosphorylation and also inhibit A431 cell growth. These two effects were also found for the pyridine-containing chelator (L6H) and corresponding Re complex (11), although to a lesser extent.

Topics: Biomarkers; Carcinoma, Squamous Cell; Cell Proliferation; Chelating Agents; Chromatography, High Pressure Liquid; Crystallography, X-Ray; Epidermal Growth Factor; ErbB Receptors; Humans; Magnetic Resonance Spectroscopy; Models, Molecular; Organotechnetium Compounds; Phosphorylation; Quinazolines; Rhenium; Tumor Cells, Cultured

To generate and evaluate a modular recombinant transporter (MRT) for targeting 211 At to cancer cells overexpressing the epidermal growth factor receptor (EGFR).. The MRT was produced with four functional modules: (1) human epidermal growth factor as the internalizable ligand, (2) the optimized nuclear localization sequence of simian vacuolating virus 40 (SV40) large T-antigen, (3) a translocation domain of diphtheria toxin as an endosomolytic module, and (4) the Escherichia coli hemoglobin-like protein (HMP) as a carrier module. MRT was labeled using N-succinimidyl 3-[211 At]astato-5-guanidinomethylbenzoate (SAGMB), its 125 I analogue SGMIB, or with 131 I using Iodogen. Binding, internalization, and clonogenic assays were performed with EGFR-expressing A431, D247 MG, and U87MG.wtEGFR human cancer cell lines.. The affinity of SGMIB-MRT binding to A431 cells, determined by Scatchard analysis, was 22 nM, comparable to that measured before labeling. The binding of SGMIB-MRT and its internalization by A431 cancer cells was 96% and 99% EGFR specific, respectively. Paired label assays demonstrated that compared with Iodogen-labeled MRT, SGMIB-MRT and SAGMB-MRT exhibited more than threefold greater peak levels and durations of intracellular retention of activity. SAGMB-MRT was 10-20 times more cytotoxic than [211 At]astatide for all three cell lines.. The results of this study have demonstrated the initial proof of principle for the MRT approach for designing targeted alpha-particle emitting radiotherapeutic agents. The high cytotoxicity of SAGMB-MRT for cancer cells overexpressing EGFR suggests that this 211 At-labeled conjugate has promise for the treatment of malignancies, such as glioma, which overexpress this receptor.

Topics: Alpha Particles; Antigens, Polyomavirus Transforming; Astatine; Benzoates; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Nucleus; Confidence Intervals; Dihydropteridine Reductase; Diphtheria Toxin; Drug Carriers; Endosomes; Epidermal Growth Factor; ErbB Receptors; Escherichia coli Proteins; Glioblastoma; Guanidine; Guanidines; Hemeproteins; Humans; NADH, NADPH Oxidoreductases; Radioimmunotherapy

Numerous candidate genes have been proposed as susceptibility factors for the development of nasopharyngeal carcinoma (NPC). Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) interaction plays a pivotal role in cell proliferation, differentiation, and tumourigenesis of epithelial tissues. To our knowledge, however, no study has examined the relationship between the EGF/EGFR and NPC. The aim of this study is to investigate the potential association between single-nucleotide polymorphisms of EGF +61 G/A and EGFR +2073 A/T and NPC. A total of 173 patients with NPC and 206 age- and sex-matched controls were the participants. Genotypes were determined using a polymerase chain reaction-restriction fragment length polymorphism strategy and DNA sequencing. There were no significant differences in the genotype and allele frequencies of EGF +61 G/A and EGFR +2073 A/T polymorphisms between the group of patients with NPC and the control group in a Chinese population (for EGF +61 G/A: OR=1.29, 95% CI: 0.95-1.74; for EGFR +2073 A/T: OR=0.91, 95% CI: 0.67-1.23). Further studies are still needed to explore the complicated interaction between environmental factors and EGF +61 G/A and EGFR +2073 A/T polymorphisms in the risk of NPC, particularly in ethnically different populations.

Topics: Adult; Alleles; Base Sequence; Carcinoma, Squamous Cell; Case-Control Studies; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Female; Gene Frequency; Humans; Male; Middle Aged; Nasopharyngeal Neoplasms; Polymorphism, Genetic

In studies on lipid metabolomics, liquid chromatography/ mass spectrometry (LC/MS) is a robust and popular technique. Although effective reversed-phase (RP) LC/ MS methods enabling the separation of phospholipid molecular species have been developed, RPLC methods to analyze phosphatidylinositol phosphates (PIPs) have not been reported. In this study, we developed conditions suitable for PIP analysis. Coupled with (diethylamino)ethyl (DEAE)-cellulose pretreatment, at least 1 pmol each of phosphatidylinositol monophosphates (PIP1), bisphosphates (PIP2), and triphosphates standards per approximately 6 x 10(6) cultured cells could be measured. Using these methods, we detected elevated concentrations of more than 12 PIP1 species in epidermal growth factor (EGF)-stimulated A431 cells, a human epidermoid carcinoma cell line. The PIP2 species detected were not elevated after stimulation. We also detected EGF-induced increases in the levels of several phosphatidic acid species using another set of methods. Our method sensitively determined PIPs within a biological sample and is thus suitable for analysis of phoisphoiniositide metabolism.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Line, Tumor; Cells; Chromatography, Liquid; DEAE-Cellulose; Epidermal Growth Factor; Humans; Mass Spectrometry; Molecular Structure; Phosphatidic Acids; Phosphatidylinositol Phosphates; Sensitivity and Specificity

We previously revealed a novel signal pathway involving S100A11 for inhibition of the growth of normal human keratinocytes (NHK) caused by high Ca(++) or transforming growth factor beta. Exposure to either agent resulted in transfer of S100A11 to nuclei, where it induced p21(WAF1). In contrast, S100A11 has been shown to be overexpressed in many human cancers. To address this apparent discrepancy, we analyzed possible new functions of S100A11, and we provide herein evidence that 1) S100A11 is actively secreted by NHK; 2) extracellular S100A11 acts on NHK to enhance the production of epidermal growth factor family proteins, resulting in growth stimulation; 3) receptor for advanced glycation end products, nuclear factor-kappaB, Akt, and cAMP response element-binding protein are involved in the S100A11-triggered signal transduction; and 4) production and secretion of S100A11 are markedly enhanced in human squamous cancer cells. These findings indicate that S100A11 plays a dual role in growth regulation of epithelial cells.

Topics: Carcinoma, Squamous Cell; Cell Proliferation; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; Humans; Keratinocytes; Proto-Oncogene Proteins c-akt; Receptor for Advanced Glycation End Products; Receptors, Immunologic; S100 Proteins; Signal Transduction; Transcriptional Activation

Treatment of oral squamous cell carcinoma (OSCC) is currently based on surgery and radiotherapy. Prolongation of the survival time of patients with progressing tumors is infrequently achieved. To improve the therapeutic options, targeted therapies are a favorable alternative. Therefore, we analyzed the effect of a chimeric toxin (CT) named SE consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis. A second construct (SA2E) additionally contains a peptidic adapter designed to enhance efficacy of the CT in vivo and to reduce side effects. The IC(50) values for an OSCC cell line (BHY) were 0.27 nM and 0.73 nM for SE and SA2E, respectively, while fibroblasts remained unaffected. To investigate primary tumor cells, we developed a technique to analyze freshly prepared OSCC cells of 28 patients in a stem cell assay directly after surgery. Cells were treated for 1 h with the CTs, subsequently seeded into soft agar and colony growth determined after 1-2 weeks In spite of the short time of CT incubation, the amount of colonies was reduced to about 78% by 10 nM and to 69% by 100 nM of either toxin. A combined application of 10 nM SA2E with a saponin from Gypsophila paniculata reduced the amount of surviving cells to 68%. The results demonstrate the impact of the CTs on OSCC cells and depict that the stem cell assay is suitable to determine the potential of anti-tumor drugs before studies in vivo will be initiated.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Epidermal Growth Factor; Escherichia coli; Humans; Immunotoxins; Inhibitory Concentration 50; Mouth Neoplasms; Saponins; Time Factors

To evaluate the prognostic value of the expressions of epidermal growth factor receptor (EGFR) and cyclin D1 in early glottic cancer treated with radiotherapy only.. One hundred fifty-one patients with T1-2, N0 glottic cancer who had been treated with radiotherapy at Seoul National University Hospital since 1992 through 2004. Immunohistochemical staining for EGFR and cyclin D1 were performed on the formalin-fixed paraffin-embedded tissues of 25 patients who developed local recurrence and on the tissues of 25 matched patients free from disease. Patterns and degrees of expression were compared between these 2 groups.. High EGFR (p = .047) and high cyclin D1 (p = .040) expressions were both found to be significantly associated with a poor prognosis. No association was found between EGFR and cyclin D1 status (p = .158), but EGFR and cyclin D1 status in combination were found to be significantly associated with local control. The patients with both high EGFR and high cyclin D1 expression had the poorest outcome compared with the others (14 months vs 29 months of median time to progression). Patterns of EGFR and cyclin D1 expression changed after recurrence, but these changes were not found to alter the ultimate prognosis.. The molecular biomarkers, EGFR and cyclin D1 have a prognostic significance in early glottic cancer. These markers in combination seem to play an important role in tumor relapse and may be useful for selecting patients with a poor outcome after radiotherapy.

Topics: Biomarkers, Tumor; Biopsy, Needle; Carcinoma, Squamous Cell; Case-Control Studies; Cyclin D1; Early Diagnosis; Epidermal Growth Factor; Female; Follow-Up Studies; Glottis; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Neoadjuvant Therapy; Neoplasm Recurrence, Local; Neoplasm Staging; Probability; Reference Values; Retrospective Studies; Risk Assessment; Survival Analysis; Treatment Outcome

Cripto-1 is an epidermal growth factor-Cripto/FRL1/Cryptic family member that plays a role in early embryogenesis as a coreceptor for Nodal and is overexpressed in human tumors. Here we report that in the two-stage mouse skin carcinogenesis model, Cripto-1 is highly up-regulated in tumor promoter-treated normal skin and in benign papillomas. Treatment of primary mouse keratinocytes with Cripto-1 stimulated proliferation and induced expression of keratin 8 but blocked induction of the normal epidermal differentiation marker keratin 1, changes that are hallmarks of tumor progression in squamous cancer. Chemical or genetic blockade of the transforming growth factor (TGF)-beta1 signaling pathway using the ALK5 kinase inhibitor SB431542 and dominant negative TGF-beta type II receptor, respectively, had similar effects on keratinocyte differentiation. Our results show that Cripto-1 could block TGF-beta1 receptor binding, phosphorylation of Smad2 and Smad3, TGF-beta-responsive luciferase reporter activity, and TGF-beta1-mediated senescence of keratinocytes. We suggest that inhibition of TGF-beta1 by Cripto-1 may play an important role in altering the differentiation state of keratinocytes and promoting outgrowth of squamous tumors in the mouse epidermis.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Differentiation; DNA Replication; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Reporter; Keratinocytes; Membrane Glycoproteins; Mice; Neoplasm Proteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Skin Neoplasms; Transforming Growth Factor beta1

Epidermal growth factor (EGF) is excreted in a high concentration in human saliva and modulates the growth and differentiation of various cancer cells. To elucidate the molecular mechanisms by which EGF affects oral cancer growth and invasion, we analyzed the Matrigel invasion activity of the cultured oral cancer cell line. Cells grown under the influence of EGF were subjected to Matrigel invasion assays and cells grown in the absence of EGF were used as controls. Gelatin-zymography and Northern blot analyses quantified the invasiveness and tumorigenicity. Chloramphenicol acetyltransferase assay (CAT assay) determined the EGF stimulation of matrix metalloproteinase (MMP) expression. EGF increased the number of cells penetrating a Matrigel membrane. Gelatin-zymography and Northern blot analysis revealed that MMP9 and Ets1 expressions correlated with EGF but MMP2 was not changed. a transient transfection assay revealed that EGF increased the promoter activities of the MMP9 genes in HSC3 and SAS cells. These results suggest that EGF increases the invasion activity of oral cancer cells partly by increasing MMP9.

Topics: Biocompatible Materials; Blotting, Northern; Carcinoma, Squamous Cell; Chloramphenicol O-Acetyltransferase; Collagen; Drug Combinations; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Laminin; Matrix Metalloproteinase 9; Mouth Neoplasms; Neoplasm Invasiveness; Proteoglycans; Saliva; Tumor Cells, Cultured

We examined the expression of epiregulin and amphiregulin mRNA in 39 oral SCCs, 2 epithelial dysplasias and 7 normal gingivae by real-time RT-PCR. The mean expression level of epiregulin mRNA was higher in oral SCCs (0.29+/-0.50) than normal gingivae (0.01+/-0.007) and epithelial dysplasias (0.01+/-0.001). The expression level of epiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.023). Epiregulin mRNA was higher in stage III/IV than in stage I/II oral SCCs. However, a significant association was not found. The mean expression level of amphiregulin mRNA was higher in oral SCCs (0.18+/-0.24) than normal gingivae (0.002+/-0.003) and epithelial dysplasias (0.01+/-0.001). Amphiregulin mRNA was significantly higher in oral SCCs than normal gingivae (Mann-Whitney U test, P=0.001). We then examined the expression of four EGF receptor mRNA in oral SCCs. The expression levels of HER1, HER2, HER3 and HER4 mRNA in oral oral SCCs were increased compared to those in normal gingivae. A significant correlation was found between the mRNA expression levels of epiregulin and HER2, HER3 and HER4 (Spearman's correlation coefficient by rank test, P=0.031, P=0.004 and P=0.027, respectively). Patients with oral SCC that have a high expression of epiregulin had a significantly shorter survival than those with low a expression (log-rank test, P<0.05). These results indicate that human epiregulin is closely linked to the increased or abnormal cell proliferation in human oral SCC.

Topics: Amphiregulin; Carcinoma, Squamous Cell; Cell Proliferation; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Ligands; Male; Mouth Neoplasms; Prognosis; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate

To evaluate the possibility of treatment with antiepidermal growth factor receptor (EGFR) mAb, Cetuximab against esophageal squamous cell carcinoma (SCC), we performed detail analysis of the antibody-dependent cellular cytotoxicity (ADCC) mediated by Cetuximab against esophageal SCC. Esophageal SCC cell lines with various levels of EGFR (n = 8) were evaluated for their Cetuximab-mediated ADCC by (51)Cr-release assay. As a result, Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC and the activities reflected the degree of EGFR expression on the esophageal SCC. The activities of Cetuximab-mediated ADCC by patients' PBMC were impaired in comparison with those by healthy donors' PBMC. Moreover, the inhibition of transforming growth factor (TGF)-beta could enhance Cetuximab-mediated ADCC against TGF-beta-producing SCC. In conclusion, Cetuximab was able to induce ADCC against EGFR-expressing esophageal SCC. Some modalities aiming at enhancing the Cetuximab-mediated ADCC may be necessary for successful Cetuximab treatment of patients with esophageal SCC.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibody-Dependent Cell Cytotoxicity; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Proliferation; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; Interleukin-2; Leukocytes, Mononuclear; Transforming Growth Factor alpha; Transforming Growth Factor beta2; Tumor Cells, Cultured

Receptor targeting is an effective method of enhancing fluorescence signal in tumors for optical imaging. We previously used epidermal growth factor (EGF) conjugated to IRDye 800CW to detect and track orthotopic prostate tumors in mice. In this study, our goal was to identify a reliable assay for targeting agent integrity in vitro that correlated with signal strength in vivo. Binding of IRDye 800CW EGF to intact A431 human epidermoid carcinoma cells was quantified in a microplate assay. Specificity was confirmed by competition with unlabeled EGF or monoclonal antibody blocking. Biological activity of intact and damaged targeting agents relative to unlabeled EGF was determined by binding and stimulation of extracellular signal-regulated kinase (ERK) phosphorylation. Both assays indicated a reduction of up to 60% of the fluorescence intensity with damaged agents. Using a research prototype imaging system optimized for IRDye 800CW detection, we compared the efficacy of intact and damaged targeting agents for imaging subcutaneous tumors in mice. In live animal images and in sections of the excised tumors, damaged targeting agents consistently yielded diminished fluorescence signals corresponding to the reduction observed in microplate assays. This is the first study to directly correlate targeting agent signal strength in whole cell binding, In-Cell Western, and in vivo near-infrared imaging.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Chromatography, Gel; Chromatography, High Pressure Liquid; Coloring Agents; Epidermal Growth Factor; ErbB Receptors; Humans; Indicators and Reagents; Mice; Mice, Nude; Mice, SCID; Sensitivity and Specificity; Spectrometry, Fluorescence; Transplantation, Heterologous

The aim of the present study was to investigate if treatment with lysosomotropic weak bases could increase the intracellular retention of radiohalogens and thereby increase the therapeutic effect of radionuclide tumor targeting.. Four different lysosomotropic bases, chloroquine, ammonium chloride, amantadine, and thioridazine, were investigated for their ability to increase radiohalogen retention in vitro. The two most promising substances, chloroquine and ammonium chloride, were studied in several cell lines (A431, U343MGaCl2:6, SKOV-3, and SKBR-3) in combination with radiolabeled epidermal growth factor (EGF) or the HER2 binding affibody (Z(HER2:4))(2).. The uptake and retention of radionuclides was found to be substantially increased by simultaneous treatment with the lysosomotropic bases. The effect was, however, more pronounced in the epidermal growth factor:epidermal growth factor receptor (EGF:EGFR) system than in the (Z(HER2:4))(2):HER2 system. The therapeutic effect of ammonium chloride treatment combined with (211)At-EGF was also studied. The effect obtained after combined treatment was found to be much better than after (211)At-EGF treatment alone.. The encouraging results from the present study indicate that the use of lysosomotropic weak bases is a promising approach for increasing the therapeutic effect of radionuclide targeting with radiohalogens.

Topics: Amantadine; Ammonium Chloride; Antimalarials; Antipsychotic Agents; Antiviral Agents; Astatine; Carcinoma, Squamous Cell; Cell Line, Tumor; Chloroquine; Epidermal Growth Factor; Glioma; Humans; Iodine Radioisotopes; Radioimmunotherapy; Radioisotopes; Recombinant Fusion Proteins; Thioridazine

The induction of apoptosis might be a promising treatment for cancers refractory to conventional therapies, such as esophageal cancer. In this study, we examined whether epidermal growth factor-induced growth inhibition results from apoptosis of esophageal squamous cell carcinoma (SCC) cells as a result of STAT1 activation and evaluated whether interferon gamma (IFN-gamma) can induce apoptosis of cancer cells in vivo.. To assess the function of STAT1, we established stable transfectants expressing dominant-negative STAT1. Apoptosis was assessed by several experimental techniques, including flow cytometry. Differentiation was evaluated by Western blot test with involucrin used as a marker. In vivo, cancer cells were injected into male BALB/c nu/nu mice. Two weeks later, the mice started to receive injections of IFN-gamma or saline into a tail vein four times per week. Concentrations of IFN-gamma in the tumors were analyzed by enzyme-linked immunosorbent assay. Apoptosis was evaluated by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining.. Epidermal growth factor inhibited the growth of esophageal SCC cells by causing apoptosis through several pathways involving STAT1 activation. IFN-gamma induced the apoptosis of cancer cells, but it also promoted the differentiation (not apoptosis) of primary cultured cells derived from normal esophageal epithelium. IFN-gamma also inhibited the growth of xenograft tumors of esophageal SCC cells in vivo.. Our results suggest that IFN-gamma is one candidate for cytokine-based therapy of cancer. IFN-gamma-induced STAT1 activation might be involved in the apoptosis of esophageal SCC cells and in the terminal differentiation of normal squamous cells. Further studies of STAT1 signaling pathways may provide the basis for new targeted therapeutic strategies for esophageal SCC.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Caspases; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Esophageal Neoplasms; Flow Cytometry; Genes, Dominant; Humans; Immunoprecipitation; In Situ Nick-End Labeling; Interferon-gamma; Male; Mice; Mice, Inbred BALB C; Recombinant Proteins; Signal Transduction; STAT1 Transcription Factor; Tumor Cells, Cultured

Locally advanced skin cancers including squamous cell carcinoma (SCC) of the skin are increasing in incidence. Patients are often elderly with significant comorbidities and therapy can be difficult. New targeted therapies, such as treatment directed at the epidermal growth factor receptor (EGFR), may be effective and less toxic in these patients. However, before designing appropriate clinical trials it is necessary to characterize the expression and activation of targets such as the EGFR to evaluate the rationale of using EGFR inhibitors (EGFRIs) in the treatment of this type of cancer.. To characterize the expression and activation by phosphorylation of EGFR in SCC of the skin by quantitative Western blotting using the LiCor immunofluorescence detection system with validation by immunohistochemistry. Secondary objectives were to evaluate downstream targets of EGFR expression and activation in SCC of the skin and to examine the associations between EGFR, pathological features and clinical behaviour of these tumours.. Twenty-one mainly locally advanced skin SCCs collected in our institution and stored in our tissue bank over a 4-year period were used for the study.. Nine of 21 (43%) tumours expressed EGFR above background. Of those nine, five expressed phosphorylated EGFR. There was no correlation with downstream activation of canonical signalling pathways, pathological features or clinical behaviour.. EGFR is expressed in a minority of tumours and then is not always activated. These results show that, before designing a trial with a targeted agent such as an EGFRI in SCC of the skin, it is important to verify the presence of the appropriate target to maximize the best outcome.

Topics: Adolescent; Adult; Carcinoma, Squamous Cell; Child; Child, Preschool; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Infant; Male; Neoplasm Proteins; Phosphorylation; Retrospective Studies; Skin Neoplasms; Treatment Outcome

Topics: Adenine; Age Factors; Aged; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Follow-Up Studies; Gene Frequency; Genotype; Guanine; Heart Transplantation; Humans; Italy; Kidney Transplantation; Liver Transplantation; Male; Middle Aged; Organ Transplantation; Polymorphism, Single Nucleotide; Prognosis; Proportional Hazards Models; Risk Assessment; Risk Factors; Skin Neoplasms; Time Factors

Oridonin, an active component isolated from the plant Rabdosia rubescens, has been reported to exhibit antitumor effects, but little is known about its molecular mechanism of action. In this study, we first investigated the mechanism involved in oridonin-induced cell death in human epidermoid carcinoma A431 cells, which overexpress epidermal growth factor receptor (EGFR). After treatment with various doses of oridonin for 24 h, the majority of A431 cells underwent apoptosis in a time- and dose-dependent manner as measured by an LDH activity-based assay. Treatment with oridonin at various concentrations for 24 h caused significant inhibition on the total tyrosine kinase activities and downregulation of EGFR expression or EGFR phosphorylation. Oridonin significantly affected the localization of EGFR and phosphorylated EGFR on the cell membrane. However, genistein (a well-known tyrosine kinase inhibitor) did not induce apoptotic A431 cell death. Importantly, oridonin exhibited much stronger inhibitory effect on the total tyrosine kinase activities or EGFR tyrosine phosphorylation as well as much stronger suppression on EGFR and phosphorylated EGFR localization than genistein in A431 cells. Taken together, oridonin exerted a potential inhibitory effect on the tyrosine kinase activity of A431 cells. The decrease in the tyrosine kinase activity and the blockage of EGFR tyrosine phosphorylation might be one of the causes of oridonin-induced A431 cell death.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; Diterpenes; Diterpenes, Kaurane; DNA Fragmentation; Epidermal Growth Factor; ErbB Receptors; Humans; L-Lactate Dehydrogenase; Necrosis; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases

Loss of E-cadherin-mediated cell-cell junctions has been correlated with cancer cell invasion and poor patient survival. p120-catenin has emerged as a key player in promoting E-cadherin stability and adherens junction integrity and has been proposed as a potential invasion suppressor by preventing release of cells from the constraints imposed by cadherin-mediated cell-cell adhesion. However, it has been proposed that tyrosine phosphorylation of p120 may contribute to cadherin-dependent junction disassembly during invasion. Here, we use small interfering RNA (siRNA) in A431 cells to show that knockdown of p120 promotes two-dimensional migration of cells. In contrast, p120 knockdown impairs epidermal growth factor-induced A431 invasion into three-dimensional matrix gels or in organotypic culture, whereas re-expression of siRNA-resistant p120, or a p120 isoform that cannot be phosphorylated on tyrosine, restores the collective mode of invasion employed by A431 cells in vitro. Thus, p120 promotes A431 cell invasion in a phosphorylation-independent manner. We show that the collective invasion of A431 cells depends on the presence of cadherin-mediated (P- and E-cadherin) cell-cell contacts, which are lost in cells where p120 expression is knocked down. Furthermore, membranous p120 is maintained in invasive squamous cell carcinomas in tumours suggesting that p120 may be important for the collective invasion of tumours cells in vivo.

Topics: Base Sequence; Carcinoma, Squamous Cell; Catenins; Cell Adhesion Molecules; Cell Line, Tumor; Delta Catenin; DNA Primers; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; Phosphoproteins; Phosphorylation; Recombinant Proteins; RNA, Small Interfering; Tyrosine

To determine whether fluorescently labeled anti-epidermal growth factor (EGFR) antibody could be used to detect residual disease and to guide surgical resections by comparing the sensitivity and specificity of optical fluorescence imaging with the sensitivity and specificity of histopathologic evaluation.. A preclinical model of head and neck squamous cell carcinoma.. Mice xenografted with SCC-1 tumor cells.. The mice underwent systemic injection with anti-EGFR antibody (cetuximab) conjugated to an optically active fluorophore (Cy5.5). Both a subcutaneous flank model (n = 18) and an orthotopic murine model (n = 15) were used to assess for the presence of residual disease by fluorescent stereomicroscopy after subtotal resections of tumors. Histologic analysis was performed to confirm the presence or absence of disease.. In the subcutaneous flank model, a diagnostic dose (50 microg) and therapeutic dose (250 microg) of fluorescent-labeled anti-EGFR were administered. When a diagnostic dose was given, the sensitivity was 86%, which was less than the 91% sensitivity when the higher dose was given. Tumor biopsy specimens in which disease was detected by histologic analysis but not by fluorescence (false-negative result) averaged 166 cells (range, 50-350 cells). The specificity of optical fluorescence to predict the presence of tumor in both groups was 100%. In the floor of the mouth model, we demonstrated a sensitivity of 81% and a specificity of 100%. False-negative results were obtained in a tumor fragment measuring less than 0.5 mm in diameter.. These data support further investigation of fluorescently labeled anti-EGFR antibody to detect disease in the surgical setting.

Topics: Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line, Tumor; Diagnosis, Differential; Disease Models, Animal; Epidermal Growth Factor; Fluorescent Antibody Technique; Head and Neck Neoplasms; Humans; Immunohistochemistry; Mice; Mouth Floor; Neoplasm, Residual; Sensitivity and Specificity; Staining and Labeling; Transplantation, Heterologous

In this study, we examined ErbB1 signaling in human basal and squamous cell carcinomas (BCC and SCC) of the skin in vivo. We used enzyme-linked immunosorbent assay, laser capture microdissection-coupled real-time reverse transcriptase-polymerase chain reaction, and immunohistochemistry to assess expression and activation levels of ErbB1 protein, ligands, and potential downstream effectors, in BCC and SCC tumors, stroma, and adjacent epidermis. Although total ErbB1 protein and mRNA were similar in cancerous and normal skin, we found that ErbB1 activation (phospho-Tyr(1068)) was greater in bulk SCC versus BCC or normal skin. In addition, three ErbB1 ligand transcripts (amphiregulin, heparin-binding epidermal growth factor-like growth factor, and transforming growth factor-alpha) were up-regulated in tumor cells of SCC but not BCC. Expression of these ligands was also increased in asymptomatic epidermis adjacent to both SCC and BCC, relative to normal skin. Interestingly, betacellulin transcript levels were inversely regulated compared with the other ligands. Consistently, downstream ErbB1 effectors (Erk1/2 and Akt) were activated in tumor cells of SCC but not of BCC and in adjacent epidermis of both BCC and SCC. These results demonstrate that ErbB1 signaling is hyperactive in tumor cells of SCC but not of BCC and in nearby asymptomatic epidermis of both tumor types. Our results suggest that targeting ErbB1 signaling might be of benefit in the treatment of SCC.

Topics: Amphiregulin; Animals; Betacellulin; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glycoproteins; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Proto-Oncogene Proteins c-akt; RNA, Messenger; Signal Transduction; Skin; Skin Neoplasms; Transforming Growth Factor alpha

Squamous cell carcinoma of the skin (SCCS) is rarely encountered by medical oncologists owing to success of local therapies. When advanced SCCS requires systemic palliation, treatment with conventional chemotherapy, such as cisplatin, is often precluded by a patient's age or medical comorbidities. Cetuximab is a human and mouse chimeric antibody against epidermal growth factor receptor, a tyrosine kinase receptor richly expressed by SCCS cells, including lymph node metastases. This drug, approved for treatment of squamous cell carcinoma of the upper aerodigestive tract as well as colorectal cancer, is well tolerated. Toxic effects include acneiform rash and diarrhea. Preclinical data suggest that epidermal growth factor receptor is important in SCCS carcinogenesis.. Herein, we report 2 cases of elderly patients with extensive, in-transit recurrence of SCCS who have been treated with palliative cetuximab. The drug was well tolerated, with the exception of acneiform rash requiring dose reduction in 1 patient. Both patients had excellent responses to cetuximab: the first patient had complete response by week 16 of treatment and the second a near-complete response by week 12. In both cases, initial response to cetuximab was evident by week 4 of therapy.. To our knowledge, these are the first reported cases of cetuximab use in patients with SCCS. The encouraging responses justify the prospective study of cetuximab in SCCS.

Topics: Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cetuximab; Combined Modality Therapy; Epidermal Growth Factor; Face; Female; Humans; Male; Neoplasm Recurrence, Local; Nose; Scalp; Skin Neoplasms

EGF in high concentrations has a growth-inhibitory effect on human epidermoid carcinoma cells A431. The transcription factor STAT1 is the most probable candidate for mediating this effect. In the present study, we demonstrated a strong reduction of the expression level of STAT1 in EGF-resistant sub-clones of A431 cells. EGF resistance was reversed by introducing wild-type STAT1, but not its Y701F mutant. Moreover, blocking the activity of Src family kinases reduced tyrosine phosphorylation of STAT1 and STAT3 and protected A431 cells from the EGF-induced growth inhibition. To further elucidate roles of STATs in A431 cell growth and survival, clones of A431 cells expressing short hairpin RNA (shRNA) against STAT1 or STAT3 were generated. Neither STAT1 nor STAT3 knockdown exerted any effect on growth rate or apoptotic death of A431 cells in the absence of EGF. However, upon EGF treatment A431 cells with knocked down STAT1 continued to grow and demonstrated a significantly lower level of apoptosis as compared to A431 cells. The knockdown of STAT3 did not alter cell growth or apoptosis. Taken together, our experiments prove the essential role of tyrosine phosphorylated STAT1, but not of STAT3, in EGF-induced apoptosis in A431 cells.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; DNA Fragmentation; Electrophoresis, Agar Gel; Epidermal Growth Factor; Humans; Phosphorylation; RNA, Small Interfering; Signal Transduction; src-Family Kinases; STAT1 Transcription Factor; STAT3 Transcription Factor; Tyrosine

Glycogen synthase kinase 3beta (GSK3beta) is a multifunctional serine/threonine kinase. We showed that the expression of GSK3beta was drastically down-regulated in human cutaneous squamous cell carcinomas and basal cell carcinomas. Due to its negative regulation of many oncogenic proteins, we hypothesized that GSK3beta may function as a tumor suppressor during the neoplastic transformation of epidermal cells. We tested this hypothesis using an in vitro model system, JB6 mouse epidermal cells. In response to epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), the promotion-sensitive JB6 P+ cells initiate neoplastic transformation, whereas the promotion-resistant JB6 P- cells do not. JB6 P- cells expressed much higher levels of GSK3beta than JB6 P+ cells; JB7 cells, the transformed derivatives of JB6, had the least amount of GSK3beta. The activity of GSK3beta is negatively regulated by its phosphorylation at Ser9. EGF and TPA induced strong Ser9 phoshorylation in JB6 P+ cells, but phosphorylation was seen at a much lesser extent in JB6 P- cells. EGF and TPA-stimulated Ser9 phosphorylation was mediated by phosphoinositide-3-kinase (PI3K)/Akt and protein kinase C (PKC) pathways. Inhibition of GSK3beta activation significantly stimulated activator protein-1 (AP-1) activity. Overexpression of wild-type (WT) and S9A mutant GSK3beta in JB6 P+ cells suppressed EGF and TPA-mediated anchorage-independent growth in soft agar and tumorigenicity in nude mice. Overexpression of a kinase-deficient (K85R) GSK3beta, in contrast, potentiated anchorage-independent growth and drastically enhanced in vivo tumorigenicity. Together, these results indicate that GSK3beta plays an important role in skin tumorigenesis.

Topics: Animals; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Enzyme Activation; Epidermal Growth Factor; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Male; Mice; Mice, Inbred BALB C; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Protein Kinase C; Signal Transduction; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transfection

Aberrant epidermal growth factor receptor (EGFR) signaling is a major cause of tumor progression and metastasis; the underlying mechanisms, however, are not well understood. In particular, it remains elusive whether deregulated EGFR pathway is involved in epithelial-mesenchymal transition (EMT), an early event that occurs during metastasis of cancers of an epithelial origin. Here, we show that EGF induces EGFR-expressing cancer cells to undergo a transition from the epithelial to the spindle-like mesenchymal morphology. EGF reduced E-cadherin expression and increased that of mesenchymal proteins. In search of a downstream mediator that may account for EGF-induced EMT, we focused on transcription repressors of E-cadherin, TWIST, SLUG, and Snail and found that cancer cells express high levels of TWIST and that EGF enhances its expression. EGF significantly increases TWIST transcripts and protein in EGFR-expressing lines. Forced expression of EGFR reactivates TWIST expression in EGFR-null cells. TWIST expression is suppressed by EGFR and Janus-activated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) inhibitors, but not significantly by those targeting phosphoinositide-3 kinase and MEK/ERK. Furthermore, constitutively active STAT3 significantly activates the TWIST promoter, whereas the JAK/STAT3 inhibitor and dominant-negative STAT3 suppressed TWIST promoter. Deletion/mutation studies further show that a 26-bp promoter region contains putative STAT3 elements required for the EGF-responsiveness of the TWIST promoter. Chromatin immunoprecipitation assays further show that EGF induces binding of nuclear STAT3 to the TWIST promoter. Immunohistochemical analysis of 130 primary breast carcinomas indicates positive correlations between non-nuclear EGFR and TWIST and between phosphorylated STAT3 and TWIST. Together, we report here that EGF/EGFR signaling pathways induce cancer cell EMT via STAT3-mediated TWIST gene expression.

Topics: Animals; Base Sequence; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Dogs; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Mesoderm; Mice; Molecular Sequence Data; Promoter Regions, Genetic; STAT3 Transcription Factor; Swiss 3T3 Cells; Twist-Related Protein 1; Up-Regulation

The study aimed at investigating whether genetic polymorphisms in BCL2, FAS, CCND1, EGF and EGFR genes influence the outcome of patients of esophageal squamous cell cancer treated with radiotherapy, with or without chemotherapy. Sixty nine histologically confirmed, previously untreated, patients with a squamous cell esophageal cancer were inducted into this study. Genotyping of BCL2 (ala43thr), FAS (A-670G), CCND1 (G870A), EGF (+61A/G) and EGFR (G497A) polymorphisms were determined using the polymerase chain reaction followed by restriction fragment length polymorphism methodology. Genotyped data was analyzed using univariate and multivariate logistic regression statistical tests for predicting the survival outcome. Genotypes of BCL2, FAS, CCND1 and EGFR polymorphisms independently did not influence outcome significantly. However, patients with EGF +61AG genotype had median survival of 25.5 months (95% CI = 5.2-45.5), whereas those with EGF +61GG genotype had survival of only 3.7 months (95% CI = 0.0-9.8, p = 0.006). In univariate cox-regression analysis, interaction of genotypes EGF+61GG*radiotherapy tumor dose (< or =50 Gy) and EGF +61GG *upper third tumor location showed high hazard of death, 6.6 (95% CI = 2.0-21.5, p = 0.002) and 26.8 (95% CI = 3.7-194.2, p = 0.001) while EGF+61AG*middle third tumor location had reduced hazard 0.20 (95%CI = 0.06-0.60, p = 0.004). The pilot study suggests that EGF +61AG and +61GG genotypes may predict clinical outcome in esophageal cancer patients treated with radiotherapy with or without chemotherapy. EGF +61AG genotype was associated with improved survival, however +61GG genotype adversely affected the outcome in patients particularly with upper third location of tumor and lower dose (< or =50) of radiotherapy.

Topics: Adult; Aged; Aged, 80 and over; Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; fas Receptor; Female; Humans; Male; Middle Aged; Polymorphism, Genetic; Proto-Oncogene Proteins c-bcl-2; Survival Analysis; Treatment Outcome

Regulation of cell growth and apoptosis is one of the pleiotropic functions of annexin A1 (ANXA1). Although previous reports on the overexpression of ANXA1 in many human cancers and on growth suppression and/or induction of apoptosis by ANXA1 may indicate the tumor-suppressive nature of ANXA1, molecular mechanisms of the function of ANXA1 remain largely unknown. Here we provide evidence that ANXA1 mechanistically links the epidermal growth factor-triggered growth signal pathway with cytosolic phospholipase A(2) (cPLA(2)), an initiator enzyme of the arachidonic acid cascade, through interaction with S100A11 in normal human keratinocytes (NHK). Ca(2+)-dependent binding of S100A11 to ANXA1 facilitated the binding of the latter to cPLA(2), resulting in inhibition of cPLA(2) activity, which is essential for the growth of NHK. On exposure of NHK to epidermal growth factor, ANXA1 was cleaved solely at Trp(12), and this cleavage was executed by cathepsin D. In squamous cancer cells, this pathway was shown to be constitutively activated. The newly found mechanistic intersection may be a promising target for establishing new measures against human cancer and other cell growth disorders.

Topics: Annexin A1; Apoptosis; Calcium; Carcinoma, Squamous Cell; Cathepsin D; Cell Line, Tumor; Epidermal Growth Factor; Humans; Keratinocytes; Phospholipases A2, Cytosolic; Protein Binding; S100 Proteins; Signal Transduction; Tumor Suppressor Proteins

Epidermal growth factor (EGF) plays an important role in tumourigenesis by binding with its receptor, EGFR. Variations in the DNA sequence in the EGF gene can lead to an alteration in EGF production and/or activity, which can affect an individual's susceptibility to lung cancer. To test this hypothesis, this study examined the association between the +61 A>G polymorphism in the 5'-untranslated region of the EGF gene and the risk of lung cancer in a Korean population.. The EGF+61 A>G genotype was determined in 432 lung cancer patients and 432 healthy age- and gender-matched control subjects.. The +61 AA and +61 AG genotypes were not significantly associated with the risk of lung cancer compared with the +61 GG genotype (adjusted OR = 1.02, 95% CI: 0.77-1.37; and adjusted OR = 0.81, 95% CI: 0.51-1.29, respectively). In addition to the reference model, the EGF+61 A>G polymorphism had no significant association with the risk of lung cancer under both dominant and recessive models for the +61A allele (adjusted OR = 0.98, 95% CI: 0.74-1.29; and adjusted OR = 0.80, 95% CI: 0.51-1.24, respectively).. These results suggest that the EGF+61 A>G polymorphism may not significantly affect the susceptibility to lung cancer in the Korean population.

Topics: Adenocarcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Staging; Polymorphism, Single Nucleotide; Risk Factors

Arsenic has been effectively used to treat acute promyelocytic leukemia, and can induce cell cycle arrest or apoptosis in human solid tumors. Previously, we have demonstrated that As2O3 can induce p21WAF1/CIP1 (p21) expression in A431 cells and then due to cellular cytotoxicity. Presently, we have clarified these signaling events and compared them with EGF. Using reporter assay, RT-PCR and Western blotting, we show that c-Src activation might be a prerequisite for As2O3-induced EGFR/Ras/Raf/ERK signaling. Furthermore, with the aids of 5'-deletion and site-directed mutagenesis, we demonstrate that Sp1 binding sites, ranging from -64 to -84 bp, are essential for As2O3- or EGF-regulated p21 expression. Finally, our experiments utilizing cycloheximide prompt the suggestion that the stability of mRNA or protein also contributes to As2O3- or EGF-induced p21 expression. Taken together, we conclude that the Sp1 binding sites are required for As2O3-induced p21 gene transcription through c-Src/EGFR/Ras/Raf/ERK pathway. Furthermore, post-transcriptional or post-translational stabilization mechanism is also essential for As2O3-induced p21 expression. EGF-induced p21 expression may involve similar mechanisms as those that operate in the As2O3-mediated reactions in A431 cells.

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Binding Sites; Carcinoma, Squamous Cell; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase 8; Oxides; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Proto-Oncogene Proteins pp60(c-src); Sp1 Transcription Factor; Transcriptional Activation

Polypeptide growth factors play key roles in the processes of cell migration and invasion. In this study, we have used cDNA microarrays to identify target genes whose expression is differentially modulated by the growth factors TGFbeta and EGF. HN4 and HN12 cell lines, established from primary tumor and a lymph node metastasis arising in one patient with head and neck squamous cell carcinoma, were treated with 2nM EGF or 50pM TGFbeta for 24h and extracted RNA was used to prepare labeled cDNAs which were hybridized to NCI UniGem 2.0 cDNA microarrays containing 9128 features. Results revealed constitutive overexpression of 41 genes and underexpression of 109 genes in metastatic HN12 compared to HN4 under conditions of serum withdrawal. Furthermore, TGFbeta treatment resulted in relative upregulation of 53 genes and downregulation of 91 genes in HN12 compared with HN4, whereas cells treated with EGF showed relative upregulation of 67 genes and downregulation of 113 genes. Partial overlap was found between TGFbeta and EGF-modulated gene sets. Results were verified for a subset of each category using quantitative PCR, western blotting and zymography. The data indicate that TGFbeta and EGF differentially affect gene expression in primary and metastatic HNSCC cells, and likely contribute to the invasive properties of metastatic cells through regulation of both common and specific mediators for each growth factor.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Flow Cytometry; Gene Expression; Head and Neck Neoplasms; Humans; Microarray Analysis; Neoplasm Invasiveness; Phenotype; Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta

Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.

Topics: beta-Cyclodextrins; Biosensing Techniques; Calcium; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line, Tumor; Cholesterol; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Humans; Mitogen-Activated Protein Kinase 3; Optics and Photonics; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinase C; ras Proteins; Transcriptional Activation

Epidermal growth factor receptor (EGFR) inhibitors can decrease vascular endothelial growth factor (VEGF) expression and tumor angiogenesis. In the current study, we investigate the molecular pathways by which this occurs using two drugs that have been used in the clinic, gefitinib (Iressa) and erlotinib (Tarceva). The decrease in VEGF expression by gefitinib in SQ20B squamous cell carcinoma cells was opposed by adenoviral expression of Akt in these cells. The hypoxia-inducible factor-1 (HIF-1) binding site located at approximately -1 kbp in the VEGF promoter was not required for down-regulation of promoter activity by gefitinib under normoxia. Furthermore, the drug decreased activity of a reporter containing the -88/+54 region. In a gel shift assay, gefitinib led to decreased retardation of a labeled DNA oligonucleotide probe corresponding to the -88/-66 region of the VEGF promoter, which contains Sp1 binding sites. These effects of gefitinib on VEGF promoter activity and DNA binding were both reversed by Akt expression. Phosphorylation of Sp1 was decreased in the presence of gefitinib. Gefitinib also decreases VEGF expression by decreasing HIF-1alpha expression. This occurs due to decreased protein translation without any change in the level of HIF-1alpha mRNA. Together, these results suggest that gefitinib decreases VEGF expression both by decreasing Sp1 binding to the proximal core VEGF promoter and by down-regulating HIF-1alpha expression. Similar results were obtained with erlotinib in SQ20B and gefitinib in HSC3 squamous carcinoma cells. These results indicate that there are at least two separate mechanisms by which EGFR inhibitors decrease VEGF expression.

Topics: Binding Sites; Carcinoma, Squamous Cell; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Head and Neck Neoplasms; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Promoter Regions, Genetic; Protein Kinase Inhibitors; Quinazolines; Sp1 Transcription Factor; Vascular Endothelial Growth Factor A

We constructed photo-reactive epidermal growth factor (EGF) bearing p-azido phenylalanine at the C-terminal (HEGFP) by genetic engineering to investigate the possibility of immobilized EGF as a novel artificial extracellular matrix (ECM). The constructed recombinant protein was immobilized to glass surface by ultraviolet irradiation. A431 cells adhered both to HEGFP-immobilized and collagen-coated surfaces. Interaction between immobilized HEGFP and EGF receptors in the A431 cells was independent of Mg(2+) although integrin-mediated cell adhesion to natural ECMs is dependent on Mg(2+). Phosphorylation of EGF receptors in A431 cells was induced by immobilized HEGFP as same as soluble EGF. DNA uptake of hepatocytes decreased by immobilized HEGFP whereas it increased by soluble EGF. Liver-specific functions of hepatocytes were maintained for 3 days by immobilized HEGFP whereas they were not maintained by soluble EGF, indicating that immobilized HEGFP follows different signal transduction pathway from soluble EGF.

Topics: Adsorption; Carcinoma, Squamous Cell; Cell Adhesion; Cell Culture Techniques; Cell Line, Tumor; Epidermal Growth Factor; Extracellular Matrix; Humans; Light; Photochemistry; Protein Binding; Protein Engineering; Recombinant Proteins; Tissue Engineering

Grb2-associated binder 1 (GAB1) is a scaffold protein involved in numerous interactions that propagate signaling by growth factor and cytokine receptors. Here we explore in silico and validate in vivo the role of GAB1 in the control of mitogenic (Ras/MAPK) and survival (phosphatidylinositol 3-kinase (PI3K)/Akt) signaling stimulated by epidermal growth factor (EGF). We built a comprehensive mechanistic model that allows for reliable predictions of temporal patterns of cellular responses to EGF under diverse perturbations, including different EGF doses, GAB1 suppression, expression of mutant proteins, and pharmacological inhibitors. We show that the temporal dynamics of GAB1 tyrosine phosphorylation is significantly controlled by positive GAB1-PI3K feedback and negative MAPK-GAB1 feedback. Our experimental and computational results demonstrate that the essential function of GAB1 is to enhance PI3K/Akt activation and extend the duration of Ras/MAPK signaling. By amplifying positive interactions between survival and mitogenic pathways, GAB1 plays the critical role in cell proliferation and tumorigenesis.

Topics: Adaptor Proteins, Signal Transducing; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; Feedback; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Models, Biological; Transfection

Protein kinase C (PKC) zeta has been implicated as a mediator of epidermal growth factor (EGF) receptor (EGFR) signaling in certain cell types. Because EGFR is ubiquitously expressed in squamous cell carcinomas of the head and neck (SCCHN) and plays a key role in tumor progression, we determined whether PKCzeta is required for tumor cell proliferation and viability. Examination of total and phosphorylated PKCzeta expression in normal oral mucosa, dysplasia, and carcinoma as well as SCCHN tumor cell lines revealed a significant increase in activated PKCzeta expression from normal to malignant tissue. PKCzeta activity is required for EGF-induced extracellular signal-regulated kinase (ERK) activation in both normal human adult epidermal keratinocytes and five of seven SCCHN cell lines. SCCHN cells express constitutively activated EGFR family receptors, and inhibition of either EGFR or mitogen-activated protein kinase (MAPK) activity suppressed DNA synthesis. Consistent with this observation, inhibition of PKCzeta using either kinase-dead PKCzeta mutant or peptide inhibitor suppressed autocrine and EGF-induced DNA synthesis. Finally, PKCzeta inhibition enhanced the effects of both MAPK/ERK kinase (U0126) and broad spectrum PKC inhibitor (chelerythrine chloride) and decreased cell proliferation in SCCHN cell lines. The results indicate that (a) PKCzeta is associated with SCCHN progression, (b) PKCzeta mediates EGF-stimulated MAPK activation in keratinocytes and SCCHN cell lines, (c) PKCzeta mediates EGFR and MAPK-dependent proliferation in SCCHN cell lines; and (d) PKCzeta inhibitors function additively with other inhibitors that target similar or complementary signaling pathways.

Topics: Alkaloids; Amino Acid Sequence; Benzophenanthridines; Butadienes; Carcinoma, Squamous Cell; Cell Growth Processes; Cell Line, Tumor; Cell Transformation, Neoplastic; DNA, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Keratinocytes; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mouth Mucosa; Mouth Neoplasms; Nitriles; Phenanthridines; Protein Kinase C; Protein Kinase Inhibitors

In a recent report, we introduced Extended Range Proteomic Analysis (ERPA), an intermediate approach between top-down and bottom-up proteomics, for the comprehensive characterization at the trace level (fmol level) of large and complex proteins. In this study, we extended ERPA to determine quantitatively the temporal changes that occur in the tyrosine kinase receptor, epidermal growth factor receptor (EGFR), upon stimulation. Specifically A 431 cells were stimulated with epidermal growth factor after which EGFR was immunoprecipitated at stimulation times of 0, 0.5, 2, and 10 min as well as 4 h. High sequence coverage was obtained (96%), and methods were developed for label-free quantitation of phosphorylation and glycosylation. A total of 13 phosphorylation sites were identified, and the estimated stoichiometry was determined over the stimulation time points, including Thr(P) and Ser(P) sites in addition to Tyr(P) sites. A total of 10 extracellular domain N-glycan sites were also identified, and major glycoforms at each site were quantitated. No change in the extent of glycosylation with stimulation was observed as expected. Finally potential binding partners to EGFR were identified based on changes in the amount of protein pulled down with EGFR as a function of time of stimulation. Many of the 19 proteins identified are known binding partners of EGFR. This work demonstrates that comprehensive characterization provides a powerful tool to aid in the study of important therapeutic targets. The detailed molecular information will prove useful in future studies in tissue.

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Line, Tumor; Chromatography, Liquid; Epidermal Growth Factor; ErbB Receptors; Glycosylation; Humans; Mass Spectrometry; Molecular Sequence Data; Peptides; Phosphorylation; Protein Interaction Mapping; Protein Processing, Post-Translational; Proteome; Proteomics; Signal Transduction

In order to find a suppressor(s) of tumor progression in vivo for oral carcinoma (OC), we searched for molecules down-regulated in OC cells when the cells were treated with epidermal growth factor (EGF), whose receptor is frequently over-activated in OC. The expression of BRAK, which is also known as CXC chemokine ligand14 (CXCL14), was down-regulated significantly by the treatment of OC cells with EGF as observed by cDNA microarray analysis followed by reverse-transcriptase polymerase chain reaction analysis. The EGF effect was attenuated by the co-presence of a MEK inhibitor. The rate of tumor formation in vivo of BRAK-expressing vector-transfected tumor cells in athymic nude mice was significantly lower than that of mock vector-transfected ones. In addition tumors formed in vivo by the BRAK-expressing cells were significantly smaller than those of the mock-transfected ones. These results indicate that BRAK/CXCL14 is a chemokine, having suppressive activity toward tumor progression of OC in vivo.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Chemokines, CXC; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; Mice; Neoplasm Transplantation; Signal Transduction; Tongue Neoplasms; Transfection; Tumor Suppressor Proteins

Overexpression of the epidermal growth factor receptor (EGFR, ErbB1, HER1) is frequent in head and neck squamous cell carcinomas (HNSCCs) and correlates with disease progression. Inhibition of EGFR with the kinase inhibitor AG1478 abolished receptor phosphorylation and reduced cell proliferation. However, treatment of HNSCC cells with cetuximab (Erbitux), a monoclonal antibody designed to block the EGFR ligand binding site, led to paradox EGFR activation due to hyperphosphorylation of tyrosine 1173, however, with a concomitant reduction in Erk1/2 phosphorylation levels. No pronounced influence on cell proliferation levels could be observed after treatment with this antibody. Since cetuximab appears able to activate EGFR in HNSCC cell lines, it is necessary to rethink the exact mechanisms by which cetuximab that recently was approved for the treatment of advanced head and neck cancer, inhibits tumor growth.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Survival; Cetuximab; Cisplatin; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Head and Neck Neoplasms; Humans; Phosphorylation; Quinazolines; Tyrosine; Tyrphostins

Alteration of the epidermal growth factor (EGF) signaling pathway occurs frequently in human cancer cells and may subsequently affect the cell survival towards anti-cancer agents. To elucidate the effect of long-term EGF treatment on the chemo-sensitivity of human cancer cells, human squamous carcinoma A431 cells (AP) were incubated continuously with 50 ng/ml EGF for 30 weeks and these cells were designated as the AC cells. The long-term EGF treatment did not alter the EGFR level and the EGF-induced protein tyrosine phosphorylation pattern in the AC cells. By MTT assay, the AC cells were shown to be more resistant than the AP cells to doxorubicin, etoposide and amsacrine but not to cisplatin. Among the drug-resistant proteins, topoisomerase IIalpha (topoII) was downregulated in the AC cells while there was no apparent change in the levels of P-glycoprotein, MRP-1 or glutathione- S-transferase-pi as compared to the AP cells. Furthermore, knockdown of topoII by antisense topoII oligonucleotide transfection decreased the sensitivity to doxorubicin, etoposide and amsacrine in the A431 cells. Results from the present study support an idea that long-term treatment with EGF may induce drug resistance in cells through the downregulation of topoII.

Topics: Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Survival; DNA Topoisomerases, Type II; Down-Regulation; Drug Resistance, Neoplasm; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Oligonucleotides; Phosphorylation; Tetrazolium Salts; Thiazoles; Tyrosine

Salivary epidermal growth factor (EGF) effect on oral cancer biology is unknown. We examined changes in minute volumes of whole resting and stimulated saliva, EGF concentration and its output (ELISA) in whole resting and stimulated saliva before and 2 weeks after surgical treatment in oral carcinoma patients compared to the control group. After stimulation salivary flow increased both in the control (P=0.003) and in the patients group--before (P=0.007) and after surgery (P=0.005). Higher stimulated saliva volume levels were observed before surgery than in post-treatment patients (P=0.032). A trend was seen with increasing EGF salivary concentrations after tumour excision both in resting (P=0.508) and stimulated (P=0.647) saliva. A similar ascending tendency of EGF output in stimulated saliva of post-treatment patients was observed (P=0.878). Decreased levels of EGF concentration in saliva before and its contrary tendency after surgical treatment may suggest an important role of EGF in oral cancer tumourogenesis.

Topics: Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Male; Middle Aged; Mouth Neoplasms; Postoperative Period; Saliva; Salivation

Epidermal growth factor is a potent growth-promoting factor for a variety of tissue cells in vivo and in vitro. Epidermal growth factor binds, phosphorylates, and activates epidermal growth factor receptors on the cell surface. In this study, we attempted to design functional peptide mimics by panning a phage display library on the anti-epidermal growth factor monoclonal antibody. By using anti-epidermal growth factor monoclonal antibody as a mold of the structure of the binding site of epidermal growth factor, high-efficiency probing was expected. From a random peptide phage display library, phage clones that bind to the anti-epidermal growth factor monoclonal antibody were isolated. One of the phage clones also exhibited binding activity to the epidermal growth factor receptor. The amino acid sequence of this phage clone showed slight similarity to the primary sequence of epidermal growth factor. We synthesized this motif to a 9-amino-acid intramolecularly disulfide-linked peptide. This synthetic peptide inhibited mitogenesis as well as epidermal growth factor receptor tyrosine phosphorylation, which is induced by epidermal growth factor. The present results suggest that the peptide synthesized in this study may mimic the epidermal growth factor receptor-binding region in epidermal growth factor.

Topics: Animals; BALB 3T3 Cells; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; Humans; Mice; Molecular Mimicry; Peptide Library; Peptides; Protein Binding

The epidermal growth factor receptor (EGFR) is a key regulator of growth, differentiation, and survival of epithelial cancers. In a small subset of tumors, the presence of activating mutations within the ATP binding site confers increased susceptibility to gefitinib, a potent tyrosine kinase inhibitor of EGFR. Agents that can inhibit EGFR function through different mechanisms may enhance gefitinib activity in patients lacking these mutations. Mevalonate metabolites play significant roles in the function of the EGFR; therefore, mevalonate pathway inhibitors may potentiate EGFR-targeted therapies.. In this study, we evaluated the effect of lovastatin on EGFR function and on gefitinib activity. Effects on EGFR function were analyzed by Western blot analysis using phosphospecific antibodies to EGFR, AKT, and extracellular signal-regulated kinase. Cytotoxic effects of lovastatin and/or gefitinib were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry.. Lovastatin treatment inhibited EGF-induced EGFR autophosphorylation by 24 hours that was reversed by the coadministration of mevalonate. Combining lovastatin and gefitinib treatments showed enhanced inhibition of AKT activation by EGF in SCC9 cells. The combination of 10 mumol/L lovastatin and 10 mumol/L gefitinib treatments showed cooperative cytotoxicity in all 8 squamous cell carcinomas, 4 of 4 non-small cell lung carcinoma and 4 of 4 colon carcinoma cell lines tested. Isobologram and flow cytometric analyses of three representative cell lines with wild-type EGFR ATP binding sites confirmed that this combination was synergistic inducing a potent apoptotic response.. Taken together, these results show that targeting the mevalonate pathway can inhibit EGFR function. They also suggest the potential utility of combining these clinically relevant therapeutic approaches.

Topics: Adenosine Triphosphate; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Binding Sites; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Colonic Neoplasms; Drug Synergism; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Lovastatin; Lung Neoplasms; Mevalonic Acid; Mitogen-Activated Protein Kinases; Mutation; Phosphorylation; Quinazolines; Signal Transduction; Tumor Cells, Cultured

A suitable model analyzing the behavior of well-differentiated squamous cell carcinoma has not yet been established. We tried to establish such a system using a reconstructed oral mucosa, in which T3M-1 squamous cell carcinoma cells were cultured on 3T3 fibroblast-containing collagen gel. Fibroblasts promoted the stratification and keratinization of T3M-1 cells. During growth, the Ki-67 index of T3M-1 cells with fibroblasts was higher than that of T3M-1 cells alone. Fibroblasts increased the expression of involucrin, a differentiating marker of keratinocytes, in T3M-1 cells. They also promoted the invasion of T3M-1 cells into the gel. When T3M-1 cells alone were cultured in a fibroblast-conditioned (FC) medium, the fibroblast-induced phenomena mentioned above were almost replicated. In addition, epidermal growth factqr (EGF) promoted T3M-1 cells growth, but not the invasion. cDNA microarray analysis showed that FC medium increased the expression of EGF receptor and several other mRNAs of T3M-1 cells. The data suggest that T3M-1 cells, under cancer-stromal fibroblast interaction, undergo invasive growth with their well-differentiated squamous phenotype, and that this interaction may be mediated partly by soluble molecules (e.g., EGF) in an autocrine or paracrine pathway. Our system will probably provide a useful model for analyzing the biological behavior of well-differentiated squamous cell carcinoma.

Topics: 3T3 Cells; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Coculture Techniques; Collagen; Culture Media, Conditioned; Epidermal Growth Factor; Fibroblasts; Gels; Hepatocyte Growth Factor; Humans; Ki-67 Antigen; Mice; Mouth Neoplasms; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Protein Precursors; Receptors, Interleukin-1; Stromal Cells

Imatinib mesylate is a tyrosine kinase inhibitor of the ABL, platelet-derived growth factor receptor (PDGFR), and c-kit kinases. Inhibition of BCR-ABL and c-kit accounts for its clinical activity in leukemia and sarcoma, respectively. In this report, we describe other cellular targets for imatinib. Treatment of head and neck squamous carcinoma cells with clinically relevant concentrations of imatinib-induced changes in cell morphology and growth similar to changes associated with epidermal growth factor receptor (EGFR) activation. Imatinib-induced changes were blocked with the EGFR antagonist cetuximab, which suggested direct involvement of EGFR in this process. Western blot analysis of cells incubated with imatinib demonstrated activation of EGFR and downstream signaling that was reduced by inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase 1 (MEK1) and EGFR, but not Her2/ErbB2. An in vitro kinase assay showed that imatinib did not directly affect EGFR kinase activity, suggesting involvement of EGFR-activating molecules. Inhibitors and neutralizing antibodies against heparin-binding epidermal growth factor-like growth factor (HB-EGF), and to a lesser extent transforming growth factor-alpha, reduced imatinib-mediated mitogen activated protein kinase (MAPK) activation. Imatinib stimulated the rapid release of soluble HB-EGF and the subsequent induction of membrane-bound HB-EGF, which correlated with biphasic MAPK activation. Together, these results suggested that imatinib affects EGFR activation and signaling pathways through rapid release and increased expression of endogenous EGFR-activating ligands. Although, imatinib primarily inhibits tyrosine kinases, it also stimulates the activity of EGFR tyrosine kinase in head and neck squamous tumors. This finding demonstrates the need for careful use of this drug in cancer patients.

Topics: Antineoplastic Agents; Benzamides; Blotting, Western; Carcinoma, Squamous Cell; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Heparin-binding EGF-like Growth Factor; Humans; Imatinib Mesylate; Inhibitory Concentration 50; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Piperazines; Platelet-Derived Growth Factor; Pyrimidines; Stem Cell Factor

Cell lines pairs were established from a primary squamous carcinoma of tongue and a lymph node metastasis and their biological behavior characterized. HN12 cells, derived from metastatic SCC, formed tumors upon subcutaneous transplantation to athymic mice, whereas HN4, derived from a primary lesion in the same individual, were non-tumorigenic in this assay. EGF stimulated proliferation of HN4 cells; in comparison, not only were metastatic HN12 cells refractory to the stimulatory effects of this growth factor but showed inhibition at higher growth factor concentrations. However, in contrast to the effects on proliferation, EGF (10 ng/ml) readily induced HN12 cells to invade in Boyden chamber assays whereas HN4 were non-invasive under these conditions. The invasive properties of HN12 cells were apparently independent of MMP-2 activity, as levels of active MMP-2 were higher in the non-invasive cells. However, EGF stimulated MMP-9 activity in invasive cells. Additionally, HN12 cells expressed constitutively high levels of active MMP-7 and MMP-3/10. The pharmacological agents LY294002, PD098059, SP600125, or SB202190 inhibited invasion of HN12 cells, suggesting requirement for phosphoinositide 3-OH kinase- and mitogen activated protein kinase-dependent pathways in the process. The data indicate that distinct biochemical differences distinguish metastatic squamous carcinoma cells from those derived from corresponding primary tumors, resulting in their contrasting biological properties.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Epidermal Growth Factor; Lymphatic Metastasis; Mice; Mice, SCID; Neoplasm Invasiveness; Neoplasm Metastasis; Tongue Neoplasms

Hydrogen peroxide (H(2)O(2)) has been shown to be an important modulator of intracellular phosphatase activity involved in cell signaling pathways, including signaling by members of the receptor tyrosine kinase family of receptors such as the epidermal growth factor receptor (EGFR). Intracellular H(2)O(2) can be generated by mitochondria-dependent pathways, whereas we recently showed that H(2)O(2) could be generated extracellularly by receptor-ligand interaction. Here, we show that H(2)O(2) produced by EGF-EGFR interaction can modulate the activity of intracellular protein tyrosine phosphatases (PTPs). Using purified proteins, we found that EGFR-ligand interaction generates H(2)O(2) that is capable of inhibiting the activity of PTP1B in vitro. Furthermore, the addition of catalase rescued phosphatase inhibition consequent to EGF-EGFR interaction. Using cells that overexpress EGFR, we found that the addition of extracellular catalase prevented EGF-induced inhibition of EGFR-associated phosphatase activity. Our findings suggest that extracellular H(2)O(2) generated by EGFR-ligand interaction permeates the plasma membrane and inhibits EGFR-associated tyrosine phosphatase activity, thereby modulating downstream signal transduction pathways.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Extracellular Fluid; Humans; Hydrogen Peroxide; Kidney; Ligands; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases

Interleukin-18 (IL-18) is a proinflammatory cytokine. This protein has a role in regulating immune responses and exhibits significant anti-tumor activities. Epidermal growth factor (EGF) is an important growth factor that plays a central role in the regulation of cell cycle and differentiation. It was proposed that a targeted delivery of IL-18 by generation of IL-18-EGF fusion protein might decrease adverse effects and result in enhancing cytotoxic and antitumor activities. In the present study, a fusion protein, consisting of EGFR binding domain fused to human IL-18 mature peptide via a linker peptide of (Gly(4)Ser) 3, was constructed and expressed in the insect cell line Sf9 using Bac-to-Bac baculovirus expression system. We showed that the purified recombinant fusion protein induced similar levels of IFN-gamma to that of native IL-18 protein in human PBMC in the presence of ConA. Furthermore, EGF receptor competitive test in human epithelial cancer A431 cell line showed that EGF-IL18 fusion protein can specifically bind with EGFR by competing with native EGF protein. These suggest that this rationally designed protein can be further developed as novel tumor therapeutics.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Drug Delivery Systems; Drug Design; Epidermal Growth Factor; ErbB Receptors; Gene Targeting; Humans; Interleukin-18; Protein Engineering; Recombinant Fusion Proteins; Spodoptera; Transfection

Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer.. DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts.. A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors.. (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line, Tumor; Drug Evaluation, Preclinical; Epidermal Growth Factor; ErbB Receptors; Feasibility Studies; Female; Gene Expression Regulation, Neoplastic; Glioma; Humans; Isotope Labeling; Metabolic Clearance Rate; Mice; Mice, Inbred BALB C; Mice, Nude; Organ Specificity; Organometallic Compounds; Radionuclide Imaging; Radiopharmaceuticals; Tissue Distribution

Gefitinib (Iressa(TM), ZD1839) is an orally active, selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. Phase I studies showed that it is well tolerated, with evidence of tumor regression in patients with advanced non-small-cell lung cancer (NSCLC). Therefore, we aimed to assess the antitumor activity and tolerability of gefitinib in a series of patients with previously treated, advanced NSCLC, as a part of a compassionate use program.. To be eligible, all patients were required to have histologically or cytologically proven advanced or metastatic NSCLC, prior chemotherapy with at least one cisplatin-containing chemotherapy regimen or contraindication to cytotoxic drugs, Eastern Cooperative Oncology Group performance status < or =2, and adequate hematological, renal and hepatic parameters. All patients provided signed informed consent. Patient re-evaluation was performed every 4-6 weeks.. Seventy-three consecutive patients were enrolled. Response rate, including complete and partial response, was 9.6%; an additional 43.8% of patients achieved stable disease, for an overall disease control of 53.4%. EGFR1 status was evaluated by immunocytochemistry in 25 patients. According to EGFR1 immunoreactivity all responses were observed with medium/strong imunoreactivity while three out of four responses were observed in high expressive patients. Median survival for all patients was 4 months while it reached 6 months for patients with disease control. The 1-year survival rate was 13.1% for the entire series and 23.2% for patients with disease control. Non-hematological toxicity was generally mild.. Gefitinib has promising activity with a good toxicity profile in patients with progressive NSCLC who have received one or two prior chemotherapy regimens. A possible relationship within response and EGFR1 expression is suggested.

Topics: Adenocarcinoma; Adult; Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Neoplasm Recurrence, Local; Protein-Tyrosine Kinases; Quinazolines; Survival Analysis

Epidermal growth factor (EGF) has been shown to induce proliferation in cells, however, the role of prostaglandin E(2) (PGE(2)) plays in EGF-induced proliferation in still unclear. EGF and PGE(2) showed proliferation responses in epidermoid carcinoma cell A431 by MTT and [(3)H] thymidine incorporation assay. Activation of the EGF receptor and extracellular signal-regulated protein kinases (ERK1/2), but not p38 and JNK, appeared 10 min after EGF treatment, whereas total amounts of ERK1/2, p38 and JNK remained unchanged in A431 cells, accompanied by induction of COX-2 and PGE(2) production. PD98059, a specific ERK1/2 inhibitor, inhibited EGF-induced proliferation with concomitant decreases in ERK1/2 phosphorylation and COX-2/PGE(2) induction. Non-steroid anti-inflammatory drugs (NSAIDs) such as aspirin and diclofenac, a COX activity inhibitor, inhibited EGF-induced proliferation by blocking PGE(2) production. The addition of PGE(2) reversed the inhibitory effects of PD98059, aspirin, and diclofenac on EGF-induced proliferation. This suggests that COX-2/PGE(2) activation involves in EGF-induced proliferation and locates at the downstream of ERK1/2 activation. Furthermore, the natural product, 3-OH flavone, showed the most-potent inhibitory activity on EGF-induced proliferation among 9 structurally-related compounds, and suppression of EGF receptor phosphorylation, ERK1/2 phosphorylation, and COX-2/PGE(2) production by 3-OH flavone was identified. PGE(2) addition attenuates the inhibitory activity of 3-OH flavone on EGF-induced proliferation by MTT assay and colony formation by soft agar assay. Additionally, 3-OH flavone also showed more-specific inhibition on EGF- than on fetal bovine serum (FBS)-induced proliferation in A431 cells. Results of our present study provide evidence to demonstrate that PGE(2) is an important downstream molecule in EGF-induced proliferation, and 3-OH flavone, which inhibits PGE(2) production by blocking MAPK cascade, might reserve potential for development as an anti-cancer drug.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Carcinoma, Squamous Cell; Cell Division; Colony-Forming Units Assay; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Prostaglandin-Endoperoxide Synthases; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Abnormalities in the expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) contribute to the progression, invasion, and maintenance of the malignant phenotype in human cancers, including those of the head and neck and breast. Accordingly, agents such as the EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (Iressa) are promising, biologically based treatments that are in various stages of preclinical and clinical development. The process of tumor progression requires, among other steps, increased transformation, directional migration, and enhanced cell survival; this study explored the effect of ZD1839 on the stimulation of c-Src and p21-activated kinase 1 (Pak1), which are vital for transformation, directional motility, and cell survival of cancer cells.. We examined the effect of ZD1839 on biochemical and functional assays indicative of directional motility and cell survival, using human head and neck squamous cancer cells and breast cancer cells.. ZD1839 effectively inhibited c-Src activation and Pak1 activity in exponentially growing cancer cells. In addition, ZD1839 suppressed EGF-induced stimulation of EGFR autophosphorylation on Y1086 and Grb2-binding Y1068 sites, c-Src phosphorylation on Y215, and Pak1 activity. ZD1839 also blocked EGF-induced cytoskeleton remodeling, redistribution of activated EGFR, and in vitro invasiveness of cancer cells.. These studies suggest that the EGFR-TKI ZD1839 may cause potent inhibition of the Pak1 and c-Src pathways and, therefore, have potential to affect the invasiveness of human cancer cells deregulated in these growth factor receptor pathways.

Topics: Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cell Survival; CSK Tyrosine-Protein Kinase; Cytoskeleton; Disease Progression; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Head and Neck Neoplasms; Humans; Microscopy, Fluorescence; Neoplasm Invasiveness; p21-Activated Kinases; Phenotype; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Quinazolines; Signal Transduction; src-Family Kinases; Transfection; Vinculin

The majority of cancer cells derived from epithelial tissue express Lewis-Y (LeY) type difucosylated oligosaccharides on their plasma membrane. This results in the modification of cell surface receptors by the LeY antigen. We used the epidermal growth factor (EGF) receptor family members ErbB1 and ErbB2 as model systems to investigate whether the sugar moiety can be exploited to block signaling by growth factor receptors in human tumor cells (i.e., SKBR-3 and A431, derived from a breast cancer and a vulval carcinoma, respectively). The monoclonal anti-LeY antibody ABL364 and its humanized version IGN311 immunoprecipitated ErbB1 and ErbB2 from detergent lysates of A431 and SKBR-3, respectively. ABL364 and IGN311 blocked EGF- and heregulin-stimulated phosphorylation of mitogen-activated protein kinase [MAPK = extracellular signal-regulated kinase 1/2] in SKBR-3 and A431 cells. The effect was comparable in magnitude with that of trastuzumab (Herceptin) and apparently noncompetitive with respect to EGF. Stimulation of MAPK by ErbB was dynamin dependent and contingent on receptor internalization. ABL364 and IGN311 changed the intracellular localization of fluorescent EGF-containing endosomes and accelerated recycling of intracellular [(125)I]EGF to the plasma membrane. Taken together, these observations show that antibodies directed against carbohydrate side chains of ErbB receptors are capable of inhibiting ErbB-mediated signaling. The ability of these antibodies to reroute receptor trafficking provides a mechanistic explanation for their inhibitory action.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Iodine Radioisotopes; Kinetics; Lewis Blood Group Antigens; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphorylation; Precipitin Tests; Receptor, ErbB-2; Tunicamycin; Vulvar Neoplasms

Discovery of the common and ubiquitous molecular targets for the disruption of angiogenesis, that are independent of the characteristics of malignant tumors, is desired to develop the more effective antitumor drugs. In this study, we propose that the platelet-derived growth factor receptor-alpha (PDGFRalpha)-p70S6K signal transduction pathway in mesenchymal cells, which is required for functional angiogenesis induced by fibroblast growth factor-2, is the potent candidate. Using murine limb ischemia as a tumor-free assay system, we demonstrated that p70S6K inhibitor rapamycin (RAPA) targets mesenchymal cells to shut down the sustained expression of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), via silencing of the PDGFRalpha-p70S6K pathway. Irrespective of the varied expression profiles of angiogenic factors in each tumor tested, RAPA constantly led the tumors to dormancy and severe ischemia in the time course, even associated with upregulated expression of VEGF from tumors. Because RAPA showed only a minimal effect to hypoxia-related expression of VEGF in culture, these results suggest that RAPA targets the host-vasculature rather than tumor itself in vivo. Thus, our current study indicates that the PDGFRalpha-p70S6K pathway is an essential regulator for FGF-2-mediated therapeutic neovascularization, as well as for the host-derived vasculature but not tumors during tumor angiogenesis, via controlling continuity of expression of multiple angiogenic growth factors.

Topics: Animals; Carcinoma, Squamous Cell; Cell Hypoxia; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Hepatocyte Growth Factor; Hindlimb; Humans; Ischemia; Liver Neoplasms, Experimental; Male; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Nude; Mouth Neoplasms; Neoplasm Proteins; Neovascularization, Pathologic; Neovascularization, Physiologic; Platelet-Derived Growth Factor; Receptor, Platelet-Derived Growth Factor alpha; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Stromal Cells

Gefitinib (Iressa, ZD1839), a quinazoline tyrosine kinase inhibitor that targets the epidermal growth factor receptor (EGFR), is approved for patients with advanced non-small cell lung cancer (NSCLC) in several countries including Japan. However, the mechanism of drug sensitivity to gefitinib is not fully understood. In this study, we examined the molecular basis of sensitivity to gefitinib using nine human lung cancer cell lines derived from NSCLC. PC9 was the most sensitive to gefitinib of the nine NSCLC cell lines when assayed either by colony formation or MTS assays. The various cell lines expressed different levels of EGFR, HER2, HER3, and HER4, but there was no correlation between levels of EGFR and/or HER2 expression and drug sensitivity. Phosphorylation of EGFR, protein kinase B/AKT (Akt), and extracellular signal-regulated kinase (ERK) 1/2 was inhibited by much lower concentration of gefitinib in PC9 cells than in the other eight cell lines under exponential growing conditions. About 80% of cell surface EGFR in PC-9 was internalized within 10 min, whereas only about 30-50% of the cell surface EGFR was internalized in more drug-resistant cell lines in 15-60 min. The present study is the first to demonstrate that sensitivity to growth inhibition by gefitinib in NSCLC cell lines under basal growth condition is associated with dependence on Akt and ERK1/2 activation in response to EGFR signaling for survival and proliferation and also that drug sensitivity may be related to the extent of EGF-induced down-regulation of cell surface EGFR.

Topics: Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Cell Survival; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-cbl; Quinazolines; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Ubiquitin-Protein Ligases

Fatty acid synthase (FAS) is the enzyme responsible for the endogenous synthesis of saturated long-chain fatty acids from the precursors acetyl-CoA and malonyl-CoA. A growing body of evidence indicates that FAS is over expressed in several human cancers, such as prostate, breast, bladder, liver, lung, melanoma and oral squamous cell carcinoma (SCC). In the present study we used human oral SCC cell lines (SCC-4, -9, -15 and -25) as a model to investigate the role of FAS in the pathogenesis of oral cancer. RT-PCR and western blot experiments demonstrated that FAS is differentially expressed by the four oral SCC cell lines, with the highest production in SCC-9 followed by SCC-25. FAS expression in SCC-4 and -15 was similarly lower than the other cell lines. Proliferation curves and immunocytochemistry for PCNA and Ki-67 demonstrated that SCC-25 has the highest proliferative potential. In addition, the specific inhibitor of FAS activity cerulenin was able to significantly reduce the proliferation of oral SCC cells. Expression of androgen receptor was low in SCC-4, -9 and -15 and undetectable in SCC-25, whereas EGFR and c-erb-B2 were expressed in high amounts by the four cell lines. Immunocytochemical reactions showed that SCC-25 expresses higher levels of EGF compared to the other three cell lines. Finally, oral SCC cells exposed to nanomolar concentrations of exogenous EGF presented a reduction in the FAS protein levels concomitant with a decrease in their proliferation rates. Taken together, our results indicate that FAS is expressed in an apparently androgen-independent fashion in oral SCC cells and it is necessary for their proliferation.

Topics: Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Fatty Acid Synthases; Humans; Mouth Neoplasms; Receptor, ErbB-2; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Gefitinib ('Iressa', ZD1839) is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has demonstrated antitumour activity and favourable tolerability in Phase II studies. We investigated whether EGFR expression levels could predict for response to gefitinib in patients with advanced non-small-cell lung cancer (NSCLC), who received gefitinib (250 mg day(-1)) as part of a worldwide compassionate-use programme. Tissue samples were analysed by immunohistochemistry to assess membrane EGFR immunoreactivity. Of 147 patients enrolled in our institution, 50 patients were evaluable for assessment of both clinical response and EGFR expression. The objective tumour response rate was 10% and disease control was achieved in 50% of patients. Although high EGFR expression was more common in squamous-cell carcinomas than adenocarcinomas, all objective responses were observed in patients with adenocarcinoma. Response and disease control with gefitinib were not associated with high EGFR expression. Overall, median survival was 4 months, and the 1-year survival rate was 18%. Strong EGFR staining correlated with shorter survival time for all patients. Gefitinib demonstrated promising clinical activity in this group of patients with NSCLC. These results have also shown that EGFR expression is not a significant predictive factor for response to gefitinib.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Predictive Value of Tests; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Survival Rate

The mechanisms by which c-erbB-dependent signaling contribute to the invasive potential of HNSCC remain to be fully elucidated. We have previously shown that c-erbB autocrine and/or paracrine stimulation upregulates MMP-9 but has no effect on the related gelatinase, MMP-2. BTC, a major c-erbB ligand, has the ability to efficiently activate all c-erbB receptors and to bind directly to EGFR and c-erbB-4. BTC is commonly expressed in HNSCC cells and exerts the most potent effects in terms of MMP induction relative to other c-erbB ligands so far tested. In the present study, we explored the contribution of major downstream events triggered by BTC/c-erbB receptor signaling to the regulation of MMP-9 and in vitro invasiveness of HNSCC cells. In human HNSCC cell lines, SIHN-006 and Detroit-562, BTC treatment resulted in rapid tyrosine phosphorylation of all c-erbB receptors whereas both endogenous MMP-9 and BTC-stimulated MMP-9 were predominantly mediated via EGFR. BTC induced ERK1/2, JNK/SAPK and Akt phosphorylation with differing kinetics but not p38 kinase. The BTC-dependent activation of JNK and PI3K/Akt pathways occurred predominantly via EGFR, whereas activation of the MEK-1/ERK pathway occurred via all 4 c-erbB receptors, although again predominantly via EGFR. Selective inhibition of ERK/MAPK (by PD98059 or U0126) and PI3K (by LY294002 or wortmannin) led to marked reduction of both basal and BTC-induced MMP-9 activity and invasive ability of HNSCC cells. In contrast, inhibition of p38 kinase with SB203580 produced no such effects. A specific inhibitor of NF-kappa B, BAY 11-7085, also blocked the stimulatory effect of BTC. No remarkable inhibition of MMP-9 and invasion was observed on targeting other cellular activities, such as PKA, PKC and PLC-gamma. Taken together, our data show that BTC induces MMP-9 production and invasion primarily through activation of EGFR, MAPK and PI3K/Akt in HNSCC cells.

Topics: Betacellulin; Carcinoma, Squamous Cell; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Genes, erbB; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction

Cyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high-level prostaglandin production during inflammation and carcinogenesis. In this study, the transcriptional regulation of COX-2 expression induced by epidermal growth factor (EGF) in human epidermoid carcinoma A431 cells was studied. EGF treatment induced the expression of COX-2 mRNA, protein, promoter and enzyme activity in a time-dependent manner. EGF-induced COX-2 promoter activity was inhibited by overexpression of the dominant-negative forms of Ras and ERK2. Induction of COX-2 and c-Jun by EGF was completely suppressed by MEK inhibitor combined with JNK inhibitor. Analysis of the COX-2 promoter binding proteins by gel mobility shift assay and DNA affinity precipitation assay revealed that c-Jun and p300 binding to CRE/E-box site were responsible for the EGF-induced COX-2 gene transcription. Overexpression of p300 significantly enhanced COX-2 promoter activity in cells overexpressed of c-Jun or treated with EGF. EGF- and c-Jun-induced transcription of COX-2 promoter was repressed by cotransfection of E1A in a dose-dependent manner. All together, these results indicated that the EGF-induced expression of COX-2 in A431 cells was mediated through the Ras-ERK/JNK signaling pathway, and subsequent induction of c-Jun following MAPK activation, in cooperation with coactivator p300, was required for the EGF response.

Topics: Carcinoma, Squamous Cell; Cyclooxygenase 2; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; MAP Kinase Signaling System; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase Kinases; Nuclear Proteins; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins c-jun; rac GTP-Binding Proteins; Sequence Deletion; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured

We have previously reported that interferon-alpha (IFNalpha) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNalpha and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNalpha induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNalpha-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNalpha induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNalpha-treated transfected cells caused a recovery of the apoptosis. Notably, IFNalpha and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNalpha induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNalpha-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 microM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNalpha. In conclusion, our data are the first demonstration that IFNalpha can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Caspases; Cell Line, Tumor; Cytochromes c; Epidermal Growth Factor; Gelsolin; Gene Expression; Humans; Interferon-alpha; Oropharyngeal Neoplasms

Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor alpha (TGF-alpha) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein-coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src-transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRP-induced proliferation or invasion of HNSCC cells. In addition to TGF-alpha release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-alpha and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-alpha and amphiregulin in HNSCC.

Topics: Amphiregulin; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gastrin-Releasing Peptide; Glycoproteins; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphorylation; Receptors, Bombesin; src-Family Kinases; Transforming Growth Factor alpha

Retinoids have been shown the most powerful inducers of transglutaminase activity, a well known marker of differentiation. In this work, we tested the effects of all-trans retinoic acid and EGF, used alone or in combination, on transglutaminase activity in a squamous, epithelial carcinoma cell line, HEp-2. We demonstrated that nanomolar EGF further enhances transglutaminase activity previously induced by all-trans retinoic acid. Confocal laser scanning microscopy revealed functional changes in transglutaminase activity localisation, at first restricted to the outermost region of cytosol, then diffused both in the membrane region and extracellular space. RT-PCR showed the presence of mRNA transcripts of different transglutaminases (1, 2, 3). Transglutaminase 2 expression was increased by either all-trans retinoic acid or EGF, and further up-regulated by the simultaneous addition of both substances. These effects were confirmed by Western blotting with transglutaminase 2 specific antibody. The results obtained by combined use of retinoic acid and EGF suggest that transglutaminase activity and expression are differently regulated, and that EGF-signalling can be involved in differentiation of epithelial carcinoma cells induced by retinoids.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Line, Tumor; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; GTP-Binding Proteins; Humans; Protein Glutamine gamma Glutamyltransferase 2; Transglutaminases; Tretinoin; Up-Regulation

Hepatocyte growth factor (HGF), the ligand for the c-met proto-oncogene product, is a multifunctional protein that enhances tumor cell motility, extracellular matrix invasion, and mitogenic or morphogenic activities of various cell types. In this study we examined the expression of the c-Met receptor in human oral squamous cell carcinoma (SCC) in vivo and in vitro to explore its relationship to tumor progression and invasiveness. Biopsy specimens of human oral SCC were immunohistochemically stained for c-Met. Nearly all primary oral SCC lesions and lymph node metastases consistently showed intense staining for c-Met, whereas normal oral mucosa showed faint to negative staining only on basal cells. In a panel of human oral SCC cell lines, we found a strong correlation between the levels of c-Met expression and the cells' response to HGF in motility and invasion assays. Sensitivity to HGF also correlated with the expression of the c-Met 9-kb mRNA. When the non-invasive HOC-605 cell line, which expresses a low level of c-Met receptor, was transfected with an expression plasmid containing human c-met cDNA, the transfectant cells showed motile and invasive responses to HGF. Immunostaining and immunoprecipitation studies demonstrated that E-cadherin and c-Met were physically associated at SCC cell-cell junctions, suggesting a direct role for c-Met in induction of junctional integrity. Importantly, HGF caused a rapid elevation of unbound beta-catenin, suggesting its availability for nuclear signal transduction and triggering of cell motility and invasiveness. Thus, overexpression of c-Met may facilitate disruption of E-cadherin junctions. Collectively, these results suggest that HGF/c-Met signaling is a common event in oral SCC that may trigger phenotype modulation and enhanced invasion and metastasis.

Topics: beta Catenin; Blotting, Northern; Cadherins; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Intercellular Junctions; Mouth Neoplasms; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Mas; Proto-Oncogene Proteins c-met; Signal Transduction; Trans-Activators; Tyrosine

In an effort to elucidate the role of ErbB receptors in human head and neck squamous cell carcinoma (HNSCC), expression abnormalities and subcellular localization of epidermal growth factor receptor (EGFR), ErbB2, ErbB3, and ErbB4 were investigated along with EGF and tenascin by immunohistochemistry in 38 carcinomas as compared to adjacent normal mucosa of 24 cases. Although tumour-specific overexpression affected each ErbB receptor (EGFR 47%, ErbB2 29%, ErbB3 21%, ErbB4 26%), EGFR abnormalities were most prevalent. The latter, and overexpression of more than two ErbB receptors in the same tumour, which always included EGFR, correlated with metastatic disease. ErbB products were specifically detected on the cell membrane and in the cytoplasm. In contrast, ErbB4 was uniquely localized to the nucleus in 7 carcinomas and a tumour-derived cell line, indicating a role for regulated intramembrane proteolysis resulting in nuclear ErbB4 translocation in HNSCC. Expression of prototype ligand EGF or low-affinity stromal activator tenascin correlated significantly with EGFR overexpression, implying chronic EGFR activation. Simultaneous overexpression of additional ErbB receptors in most of these cases suggested recurrent involvement of receptor heterodimers. In spite of frequent ErbB receptor alterations, autologous ErbB serum antibodies were rare, with only 1 of 38 tumour patients exhibiting an ErbB2-specific immune response. Based on upregulation of several known immunosuppressive molecules, scarcity of ErbB-specific antibodies is consistent with attenuation of natural tumour-specific immune responses in HNSCC.

Topics: Antibody Formation; Carcinoma, Squamous Cell; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Genes, erbB; Genes, erbB-2; Head and Neck Neoplasms; Humans; Immunohistochemistry; Neoplasm Proteins; Receptor, ErbB-4; Receptors, Interleukin-2; Tenascin; Transforming Growth Factor beta; Translocation, Genetic

Synthesis and biological evaluation of a new class of 1-aryl-4-amino-1H-pyrazolo[3,4-d]pyrimidine derivatives are reported. A preliminary cellular assay system using the tumor cell line A431 responding to epidermal growth factor (EGF) for its growth, shows that the new compounds are potent inhibitors of cell growth. The inhibition of tumor cell proliferation is not associated with blockage of EGF receptor (EGFR), but substantially due to the interference with the signalling pathway at the level of Src tyrosine kinase and at the level of the downstream effector signal mitogen activated protein kinases (MAPKs), ERK1-2.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Humans; Mitogen-Activated Protein Kinases; Pyrazoles; Pyrimidines; Signal Transduction; src-Family Kinases; Tumor Cells, Cultured

Epidermal growth factor (EGF)-mediated Ca2+ signaling in multiple cell lines derived from human gliomas and in the A431 epidermoid carcinoma cell line was observed using fluorescence videomicroscopy. Bath application of EGF evoked an oscillatory increase in [Ca2+]i in 4 different human glioma cell lines as well as the A431 cell line. This effect was blocked by the EGF receptor tyrosine kinase inhibitors gefitinib and erlotinib, as well as by the EGFR antibody cetuximab. In addition to this acute Ca2+ signaling response, transient exposure to EGF also potentiated subsequent Ca2+ signaling responses to other stimuli. Tumor cells transiently exposed to EGF (5 minutes), showed a sustained increase in propagation of intercellular Ca2+ waves, which have been previously shown to involve release of ATP and activation of purinergic receptors. Cells transiently exposed to EGF also showed a sustained potentiation of the Ca2+ signaling response to ATP. In contrast to the acute Ca2+ signaling response to EGF, this sustained potentiation of purinergic intercellular signaling was not blocked by gefitinib or erlotinib, while it was blocked by cetuximab. These results indicate that while the acute Ca2+ signaling response requires tyrosine kinase activation, the sustained potentiation of intercellular signaling occurs via a distinct pathway. Distinct intra- and intercellular Ca2+ signaling pathways may be mechanisms by which EGF modulates the growth and migration of tumor cells.

Topics: Adenosine Triphosphate; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Calcium; Calcium Signaling; Carcinoma, Squamous Cell; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Gefitinib; Glioma; Humans; Protein Kinase Inhibitors; Quinazolines; Receptors, Purinergic; Tumor Cells, Cultured

Signaling pathways initiated by the epidermal growth factor receptor (EGFR) play important roles in the response to ionizing radiation. In this study the consequences of inhibiting the EGFR on the response of A431 cells (human vulvar squamous cell carcinoma cells that overexpress EGFR) to radiation, were investigated in vitro and in vivo, using the selective EGFR-tyrosine kinase inhibitor, ZD1839 ("Iressa").. The effect of ZD1839 on proliferation, apoptosis, and clonogenic survival after radiation was determined in vitro. For in vivo studies, athymic nude mice with established subcutaneous A431 xenografts (approximately 100 mm(3)) were treated with either a single 10 Gy fraction or 4 daily 2.5 Gy fractions of radiation with or without ZD1839 (75 mg/kg/day intraperitoneally for 10 days) to determine effects on tumor growth delay.. Treatment of A431 cells with ZD1839 in vitro reduced proliferation, increased apoptosis, and reduced clonogenic survival after radiation. Strikingly greater than additive effects of ZD1839 in combination with radiation on tumor growth delay were observed in vivo after either a single 10 Gy fraction (enhancement ratio: 1.5) or multiple 4 x 2.5 Gy fractions (enhancement ratio: 4). ZD1839 reduced tumor vascularity, as well as levels of vascular endothelial growth factor (VEGF) protein and mRNA induced by stimulation with epidermal growth factor (EGF), suggesting a possible role of inhibition of angiogenesis in the effect.. Inhibiting EGFR-mediated signal transduction cascades with ZD1839 potentiates the antitumor effect of single and multiple fractions of radiation. These data provide preclinical rationale for clinical trials of EGFR inhibitors including ZD1839 in combination with radiation.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Drug Screening Assays, Antitumor; Endothelial Growth Factors; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Mice; Mice, Nude; Phosphorylation; Quinazolines; Radiation-Sensitizing Agents; Radiotherapy Dosage; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Nitric oxide (NO*) strongly inhibits the proliferation of human A431 tumour cells. It also inhibits tyrosine phosphorylation of a 170-kDa band corresponding to the epidermal growth factor receptor (EGFR) and induces the phosphorylation at tyrosine residue(s) of a 58-kDa protein which we have denoted NOIPP-58 (nitric oxide-induced 58-kDa phosphoprotein). The NO*-induced phosphorylation of NOIPP-58 is strictly dependent on the presence of EGF. Phosphorylation of NOIPP-58 and inhibition of the phosphorylation of the band corresponding to EGFR are both cGMP-independent processes. We also demonstrate that the p38 mitogen-activated protein kinase (p38MAPK) pathway is activated by NO* in the absence and presence of EGF, whereas the activity of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and the c-Jun N-terminal kinase 1/2 (JNK1/2) pathways are not significantly affected or are slightly decreased, respectively, on addition of this agent. Moreover, we show that the p38MAPK inhibitor, SB202190, induces rapid vanadate/peroxovanadate-sensitive dephosphorylation of prephosphorylated EGFR and NOIPP-58. We propose that the dephosphorylation of both NOIPP-58 and EGFR are mediated by a p38MAPK-controlled phosphotyrosine-protein phosphatase (PYPP). Activation of the p38MAPK pathway during nitrosative stress probably prevents the operation of this PYPP, allowing NOIPP-58, and in part EGFR, to remain phosphorylated and therefore capable of generating signalling events.

Topics: Carcinoma, Squamous Cell; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrazines; Kinetics; Mitogen-Activated Protein Kinases; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Phosphotyrosine; Thymidine; Tumor Cells, Cultured

Phospholipase D (PLD) has been implicated in the signal transduction pathways initiated by several mitogenic protein tyrosine kinases. We demonstrate for the first time that most notably PLD2 and to a lesser extent the PLD1 isoform are tyrosine phosphorylated by c-Src tyrosine kinase via direct association. Moreover, epidermal growth factor induced tyrosine phosphorylation of PLD2 and its interaction with c-Src in A431 cells. Interaction between these proteins is via the pleckstrin homology domain of PLD2 and the catalytic domain of c-Src. Coexpression of PLD1 or PLD2 with c-Src synergistically enhances cellular proliferation compared with expression of either molecule. While PLD activity as a lipid-hydrolyzing enzyme is not affected by c-Src, wild-type PLDs but not catalytically inactive PLD mutants significantly increase c-Src kinase activity, up-regulating c-Src-mediated paxillin phosphorylation and extracellular signal-regulated kinase activity. These results demonstrate the critical role of PLD catalytic activity in the stimulation of Src signaling. In conclusion, we provide the first evidence that c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src. This transmodulation between c-Src and PLD may contribute to the promotion of cellular proliferation via amplification of mitogenic signaling pathways.

Topics: Animals; Carcinoma, Squamous Cell; Catalytic Domain; Cell Division; Cells, Cultured; CSK Tyrosine-Protein Kinase; Cytoskeletal Proteins; Enzyme Activation; Epidermal Growth Factor; Female; Humans; Mice; Mice, Inbred BALB C; Mitogen-Activated Protein Kinases; Mutation; Paxillin; Phospholipase D; Phosphoproteins; Phosphorylation; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Rats; Signal Transduction; src-Family Kinases; Tyrosine

We isolated human KB adenocarcinoma cisplatin-resistant (CP-r) cell lines with multidrug-resistance phenotypes because of reduced accumulation of cisplatin and other cytotoxic compounds such as methotrexate and heavy metals. The uptake of horseradish peroxidase (HRPO) and Texas Red dextran was decreased several-fold in KB-CP-r cells, indicating a general defect in fluid-phase endocytosis. In contrast, although EGF receptors were decreased in amount, the kinetics of EGF uptake, a marker of receptor-mediated endocytosis, was similar in sensitive and resistant cells. However, 40-60% of the (125)I-EGF released into the medium after uptake into lysosomes of KB-CP-r cells was TCA precipitable as compared to only 10% released by sensitive cells. These results indicate inefficient degradation of internalised (125)I-EGF in the lysosomes of KB-CP-r cells, consistent with slower processing of cathepsin L, a lysosomal cysteine protease. Treatment of KB cells by bafilomycin A(1), a known inhibitor of the vacuolar proton pump, mimicked the phenotype seen in KB-CP-r cells with reduced uptake of HRPO, (125)I-EGF, (14)C-carboplatin, and release of TCA precipitable (125)I-EGF. KB-CP-r cells also had less acidic lysosomes. KB-CP-r cells were crossresistant to Pseudomonas exotoxin, and Pseudomonas exotoxin-resistant KB cells were crossresistant to cisplatin. Since cells with endosomal acidification defects are known to be resistant to Pseudomonas exotoxin and blocking of endosomal acidification mimics the CP-r phenotype, we conclude that defective endosomal acidification may contribute to acquired cisplatin resistance.

Topics: Biological Transport; Carboplatin; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Endocytosis; Epidermal Growth Factor; Horseradish Peroxidase; Humans; Lysosomes

The cytotoxicity of a diphtheria toxin-human epidermal growth factor fusion protein (DAB(389)EGF) was tested against 14 human glioma cell lines. After cells were cultured for 48 h with various concentrations of DAB(389)EGF, the percentage reduction in thymidine incorporation was determined. For 13 of 14 cell lines, potent cytotoxicity was observed, with IC(50)s of 0.4-50 pM. The epidermal growth factor receptor (EGFR) density of these cell lines was determined by immunofluorescence microscopy, flow cytometry, and radioligand binding. These assays correlated well with each other and demonstrated EGFR levels of 15,000-230,000/cell for 13 of 14 cell lines. The cell line U138MG, which lacked EGFR, was the only cell line insensitive to DAB(389)EGF. Linear regression analysis showed a good correlation between EGFR density and DAB(389)EGF sensitivity (P < 0.001) and between results of flow cytometry and radiolabeled binding assays of EGFR density (P = 0.01). DAB(389)EGF may have potential for intracranial therapy of EGFR-positive glioblastomas.

Topics: Brain Neoplasms; Carcinoma, Squamous Cell; Diphtheria Toxin; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Glioblastoma; Humans; Inhibitory Concentration 50; Kinetics; Microscopy, Fluorescence; Recombinant Fusion Proteins; Tumor Cells, Cultured

Granular cell tumors (GCT) are uncommon benign neoplasms that have a predilection for the head and neck region. These tumors can frequently be associated with pseudoepitheliomatous hyperplasia (PEH), which in turn may be mistaken for squamous cell carcinoma. Although epidermal growth factors are overexpressed in squamous cell carcinomas of the head and neck, their presence in PEH, especially its relation to GCT, is unknown. We hypothesize that the expression of epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor alpha (TGFalpha) in GCT have a role in the development of PEH overlying some GCT. Sections from 13 cases of GCT (five with overlying PEH) were examined histologically and evaluated immunohistochemically using monoclonal antibodies for EGFR, EGF, and TGFalpha. These were compared with nine cases of PEH independent of GCT. Two of five GCT with overlying PEH and two of six GCT without overlying PEH stained positively for TGFalpha. None of the GCT stained with EGFR or EGF. All cases of PEH, whether or not associated with GCT, were reactive for EGFR and EGF. Four of the five cases of PEH overlying GCT stained with TGFalpha. The staining pattern and intensity of all three antibodies were comparable to that of the adjacent normal squamous mucosa. Among the three antibodies, only TGFalpha in GCT appears to be related to the development of PEH. Epidermal growth factor receptor and EGF do not seem to be directly involved. The reason of PEH formation associated with GCT in the absence of growth factors is unknown.

Topics: Adolescent; Adult; Aged; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Child; Diagnosis, Differential; Epidermal Growth Factor; ErbB Receptors; Female; Granular Cell Tumor; Head and Neck Neoplasms; Humans; Hyperplasia; Immunohistochemistry; Male; Middle Aged; Retrospective Studies; Transforming Growth Factor alpha

A431 epidermoid carcinoma cells have an increased expression of EGF receptor. In contrast to many other cell lines and primary cells, these respond to EGF in high (more than 1 ng/ml) concentrations by cell cycle arrest, apoptosis and detachment. Clonal variants of A431 (1a and 8a), able to grow in the presence of EGF in high concentration, were previously developed in our laboratory (Gudkova, Sorokin, 1989). Here we tested upper pathways of signal transduction from EGF receptor in the clonal variants, as compared to A431. We found no reasonable difference in the expression of EGF receptor, as well as in its EGF-induced phosphorylation in A431 and clonal variants. There were also no changes in the amount and activation of ERK MAP kinase in different cell lines. In contrast, the amount of STAT1 transcription factor, known to play pro-apoptotic and antiproliferative roles, was strictly diminished in both the clonal variants tested (1a and 8a), as compared to the parental line A431. However, EGF-induced tyrosine phosphorylation of STAT1 decreased only in 8a. Increased phosphorylation of Akt protein kinase, the key component of PI-3 kinase of the anti-apoptotic and proliferative signaling pathway, was also observed in clonal variants. The data obtained demonstrate that resistance to EGF can be acquired in cells having similar levels of EGF receptor expression and phosphorylation, but different in STAT1 or PI-3 kinase signal transduction pathways. These pathways may presumably represent two antagonistic key elements regulating A431 proliferation and apoptosis.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Division; Clone Cells; DNA-Binding Proteins; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Genetic Variation; Humans; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured; Tyrosine

The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ("Iressa", ZD1839), a reversible growth inhibitor of EGFR-expressing tumour cells, has been shown to enhance the antitumour effect of ionising radiation, and also to increase the uptake of radioiodinated EGF. Thus, combination of gefitinib treatment and radionuclide targeting is an interesting option for therapy of brain tumours that are difficult to treat with conventional methods. The aim of this study was to evaluate how pre-treatment with gefitinib affects binding of astatinated EGF ((211)At-EGF) to cultured glioma U343 cells, which express high levels of EGFR. The growth of U343 cells in the presence of gefitinib was investigated, and it was found that gefitinib does not significantly inhibit the growth of these cells. Nevertheless, the uptake of (211)At-EGF in U343 cells was markedly increased (up to 3.5 times) in cells pre-treated with gefitinib (1 microM). This indicates that a combination of gefitinib treatment and radionuclide targeting to EGFR might be a useful therapeutic modality, even for patients who do not respond to treatment with gefitinib alone.

Topics: Astatine; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Glioma; Humans; Metabolic Clearance Rate; Protein-Tyrosine Kinases; Quinazolines; Radioisotopes; Radiopharmaceuticals

Expression of MMP-7 in human esophageal squamous cell carcinoma lines (TE-9 and -10) was investigated. Under normal culture conditions, immunocytochemical staining and enzymography demonstrated the production of MMP-7 in their cytoplasm and its gelatinolytic activity in the medium of TE-9 and -10 cell cultures. When EGF was added to the cultures, Western blotting and RT-PCR analysis showed a dose-dependent increase in the amount of MMP-7 synthesized in the TE-9 cells, whereas in TE-10 cells, EGF failed to stimulate MMP-7 production. For luciferase reporter analysis of MMP-7 transcription, pMMP7LucWI (1132-bp) and pMMP7LucWII (334-bp) were cloned in the luciferase pGL3-basic vector. The promoter activity was enhanced from 1.5- to 2-fold by the addition of EGF to TE-9 cells transfected with pMMP7LucWI or WII, even though the response was low as compared with that of 12-O-tetra-decanoyl-phorbol-13-acetate (TPA); and in TE-10 cells, only TPA enhanced the promoter activity of MMP-7. Luciferase promoter analysis using a pMMP7WII mutant series revealed that the AP-1 site was essential for transcription of the MMP-7 gene in TE-9 cells, and Tcf-I was also an important site, and that to a lesser degree, Tcf-II and PEA3s participated in the transcription of the MMP-7 gene in these cells. Unlike the results with TE-9 cells, in TE-10 cells, EGF failed to stimulate transcription of the MMP-7 gene; and up-regulation of the promoter activity by TPA was dependent on the AP-1 site and to lesser degree, on Tcf-I and Tcf-II. These results suggest that EGF plays also an important role in MMP-7 production of TE-9 cells and that there is a difference not only in EGF-intracellular signaling system but also in regulation mechanisms of MMP-7 transcription by beta-catenin-Tcf and/or PEA3 system between these 2 carcinoma lines.

Topics: Carcinogens; Carcinoma, Squamous Cell; DNA-Binding Proteins; Epidermal Growth Factor; Esophageal Neoplasms; Gene Expression Regulation, Enzymologic; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 1-alpha; Hepatocyte Nuclear Factor 1-beta; Humans; Luciferases; Lymphoid Enhancer-Binding Factor 1; Matrix Metalloproteinase 7; Mutagenesis, Site-Directed; Mutation; Nuclear Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Sequence Deletion; Tetradecanoylphorbol Acetate; Transcription Factor AP-1; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Oncolytic virotherapy with conditionally replicating viruses is a promising approach for treating advanced cancers. Promiscuous tropism and low tumor transduction have represented limiting issues, which targeting approaches seek to overcome. An approach utilizing a secretory targeting molecule for the epidermal growth factor pathway (sCAR-EGF) has previously been shown to be compatible with replicating adenoviruses, when an E1-deleted vector was used in a dual-virus system in conjunction with a replication-competent agent. Here, we constructed a virus that replicates in cancer cells and codes for sCAR-EGF. Interestingly, the oncolytic potency of the novel agent was not improved over nontargeted controls in vitro or in vivo. These results suggest that the expression of biologically active proteins can be counterproductive to virus replication.

Topics: Adenoviridae; Animals; Carcinoma, Squamous Cell; Cell Line; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Epidermal Growth Factor; ErbB Receptors; Gene Targeting; Genetic Therapy; Genetic Vectors; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Receptors, Virus; Recombinant Fusion Proteins; Transduction, Genetic; Tumor Cells, Cultured; Virus Replication

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Dose Fractionation, Radiation; Endothelial Growth Factors; Epidermal Growth Factor; Female; Mice; Mice, Nude; Quinazolines; Radiation-Sensitizing Agents; Radiotherapy Dosage; Tumor Cells, Cultured

The EGFR-TKI (epidermal growth factor receptor tyrosine kinase inhibitor) gefitinib ['Iressa' (trademark of the AstraZeneca group of companies), ZD1839] increases the cellular uptake of radiolabelled epidermal growth factor (EGF). We investigated gefitinib treatment combined with astatine-211 EGF targeting in vitro using two cell lines expressing high levels of EGFR: A431 (sensitive to gefitinib) and U343MGaCl2:1 (resistant to gefitinib). In both cell lines, the uptake of 211At-EGF was markedly increased by concomitant treatment with gefitinib. Survival was investigated using both a clonogenic survival assay and a cell growth assay. Combined gefitinib and 211At-EGF treatment reduced the survival of U343 cells 3.5-fold compared with 211At-EGF alone. In A431 cells, 211At-EGF treatment resulted in very low survival, but combined treatment with gefitinib increased the survival by about 20-fold. These results indicate that combined treatment with gefitinib might increase the effect of ligand-mediated radionuclide therapy in gefitinib-resistant tumours and decrease the effect of such therapy in gefitinib-sensitive tumours.

Topics: Antineoplastic Agents; Astatine; Astrocytoma; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Cell Survival; Combined Modality Therapy; Epidermal Growth Factor; Gefitinib; Humans; Quinazolines; Radiopharmaceuticals; Treatment Outcome

The epidermal growth factor (EGF) receptor plays a pivotal role in growth regulation of epidermal keratinocytes. Its expression and function can be markedly altered during malignant transformation in squamous cell carcinoma. The present study investigated the potential of growth inhibition by signal-transduction inhibitors in EGF-dependent epithelial cell lines in vitro. Benign HaCaT keratinocytes and malignant A431 cells were grown in vitro and exposed to various concentrations of a panel of eleven kinase and phosphodiesterase inhibitors. Cell growth was measured after 24 h and 48 h using fluorescence labeling with Hoechst 33342 and propidium iodide. Significant growth inhibition was achieved with all inhibitors when applied to HaCaT cells. The strongest growth inhibition was achieved with inhibitors H-7, A3 and diacylglycerol kinase inhibitors I and II. A431 cells were inhibited significantly by H-7, A3 and H-9. Selected signal-transduction inhibitors such as A3, H-7 and H-9 acting on intracellular kinases are capable of suppressing growth of EGF-dependent benign and malignant epithelial cell lines in vitro. They might be of future potential in the treatment of epithelial cancer but further studies are necessary.

Topics: Benzimidazoles; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Line, Tumor; Coloring Agents; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Humans; Keratinocytes; Propidium; Signal Transduction

We previously demonstrated that epidermal growth factor (EGF) induces a decrease in dihydropyrimidine dehydrogenase (DPD), which is the first and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), in EGF receptor (EGFR)-positive human SKG-IIIb uterine cervical carcinoma cells, and thereby increased the sensitivity of the cells to 5-FU. In the present study, we examined the individual and combined effects of 5-FU and EGF on growth and DPD activity in SKG-IIIb cells, and also investigated the mode of regulation of DPD activity. The cells showed sensitivity to 5-FU, and growth was stimulated by EGF. When the agents were used in combination, the sensitivity of SKG-IIIb cells to 5-FU was increased roughly sixfold at maximum, as judged in terms of the 50% growth-inhibitory concentration. We then examined the effects of 5-FU and EGF on DPD. Either agent inhibited DPD activity and protein expression in a concentration-dependent manner. Expression of DPD mRNA was concentration-dependently inhibited by treatment with 5-FU and by EGF at a concentration that strongly stimulated cell growth. Further, combination treatment inhibited DPD activity, as well as DPD protein and mRNA expression, more strongly than did treatment with 5-FU or EGF alone. These results suggest that inhibition of DPD activity by EGF or 5-FU is regulated at least at the level of protein expression and that regulation via mRNA is also involved. The above findings indicate that 5-FU and EGF act synergistically in suppressing DPD activity and that the use of 5-FU against tumors in which EGF plays an important role would maximize the potential of 5-FU as an anticancer drug.

Topics: Antimetabolites, Antineoplastic; Carcinoma, Squamous Cell; Cell Division; Dihydrouracil Dehydrogenase (NADP); DNA Primers; Dose-Response Relationship, Drug; Drug Synergism; Drug Therapy, Combination; Epidermal Growth Factor; Female; Fluorouracil; Humans; Immunoblotting; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Lymph node metastasis and local invasion of head and neck squamous cell carcinoma (HNSCC) is associated with a poor prognosis. However, little is known about the factors governing tumor cell invasion in HNSCC. Phospholipase Cgamma-1 (PLCgamma-1) contributes to tumor cell invasion in experimental systems when activated by the epidermal growth factor receptor (EGFR). We hypothesized that EGFR overexpression in HNSCC mediates invasion via PLCgamma-1. On EGFR ligand stimulation, phosphorylation of PLCgamma-1 increased in all of the HNSCC cell lines tested (4 of 4). In the presence of EGFR-specific tyrosine kinase inhibitor (PD153035) or an anti-EGFR antibody (C225), PLCgamma-1 activation was abrogated indicating that PLCgamma-1 was downstream of EGFR. Blocking cellular PLC with an inhibitor (U73122) reduced inositol phosphate turnover in all of the HNSCC cell lines examined, and treatment with the PLC inhibitor or antisense oligonucleotides targeting PLCgamma-1 significantly reduced in vitro invasiveness of HNSCC cell lines through Matrigel. To determine the clinical relevance of these findings, we compared levels of PLCgamma-1 in tumor and paired normal tissue from 33 patients with HNSCC. PLCgamma-1 levels were significantly higher (P < 0.0001) in the tumors compared with the normal mucosa of HNSCC patients. Levels of activated PLCgamma-1 were analyzed in 20 patients. Tumors expressed higher levels of phosphorylated PLCgamma-1 compared with normal adjacent mucosa (P = 0.05). Thus, PLCgamma-1 may mediate invasion and metastasis downstream of EGFR in HNSCC.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Invasiveness; Phospholipase C gamma; Phosphorylation; Recombinant Proteins; Signal Transduction; Type C Phospholipases

Epidermal growth factor receptor (EGFR) plays a role in laryngeal squamous cell carcinoma (SCC) development and progression. The flavonoid quercetin (Q) and the antiestrogen tamoxifen (TAM) inhibit proliferation of both primary laryngeal SCC and laryngeal carcinoma cell lines, through still uncharacterized mechanisms. We studied Q and TAM inhibitory effect on epidermal growth factor (EGF)-stimulated Hep2 and CO-K3 laryngeal squamous cell lines. Q and TAM (0.1-1.0 microM) induced more apoptosis in EGF growth-stimulated than in unstimulated Hep2 cells. EGF neither stimulated CO-K3 cell growth nor enhanced Q and TAM-induced apoptosis. Mitogen-activated protein kinase (MAPK) analysis revealed that in Hep2 cells, but not in CO-K3 cells, EGF induced a time-dependent phosphorylation of p42, p44, p38, and p46. In Hep2 cells, but not in CO-K3 cells, Q and TAM produced, upon EGF treatment, a twofold increase of p38 and p46 and an enhancement of p42 and p44 dephosphorylation, suggesting a requirement of EGFR. The enhancing effect was due to a p38 and p46 dephosphorylation delayed kinetics. An antiphosphorylated p38 antibody prevented Q and TAM inhibitory effect on p42 and p44 phosphorylations, suggesting that the EGF-dependent increase in Q and TAM apoptotic effect on Hep2 cells could depend on the p38 inhibition of the survival kinases p42 and p44. In SCC, EGFR overexpression is an early event from dysplasia to neoplasia. We conclude that the capacity of Q and TAM to increase apoptosis in EGFR-activated cells makes these compounds possible chemopreventive drugs in subjects at risk of developing laryngeal cancer.

Topics: Anticarcinogenic Agents; Apoptosis; Carcinoma, Squamous Cell; Cell Division; Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Laryngeal Neoplasms; Male; Middle Aged; Mitogen-Activated Protein Kinases; Phosphorylation; Quercetin; Tamoxifen; Time Factors

Activation of telomerase, which stabilizes the telomere length of chromosomes, is crucial for the continued growth or progression of cancer cells. In a previous study, we showed that telomerase is frequently activated in skin tumors. Because epidermal growth factor plays an important role during the tumorigenesis of epithelial tissue, we have now examined the role of epidermal growth factor signaling in regulating telomerase activity using HSC-1 human cutaneous squamous cell carcinoma cells. Treatment of HSC-1 cells with AG 1478, an inhibitor of the epidermal growth factor receptor, or with a neutralizing antibody to the epidermal growth factor receptor, significantly suppressed their telomerase activity, in association with inhibiting their growth. The suppression of telomerase activity was obvious at day 3 and was maximal at day 5 after treatment with AG 1478. The suppression of telomerase activity correlated with the decreased expression of human telomerase catalytic subunit (hTERT) mRNA, the rate-limiting determinant of its enzyme activity. The expression of c-Myc and of Sp1 proteins, transcription factors for hTERT, were also suppressed by AG 1478 in HSC-1 cells, but the expression of Ets-2 protein, another transcription factor, was not affected. The expression of Mad-1, a competitor of c-Myc, was increased. Inhibition of ERK, Src, or Akt suppressed telomerase activity in HSC-1 cells, but to a lesser extent than did treatment with AG 1478. Serum starvation suppressed telomerase activity, but addition of epidermal growth factor or transforming growth factor alpha did not increase it, indicating the involvement of other epidermal growth factor receptor ligands in the activation of telomerase in HSC-1 cells. These data indicate that blockade of the epidermal growth factor receptor might be effective in inhibiting telomerase activity of squamous cell carcinomas, which would lead to the suppression of tumor growth.

Topics: Carcinoma, Squamous Cell; Cell Cycle Proteins; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Quinazolines; Repressor Proteins; RNA, Messenger; Skin Neoplasms; Telomerase; Transforming Growth Factor alpha; Tyrphostins

Phospholipase C gamma 1 (PLC gamma 1), an enzyme participating in phosphoinositide turnover, is one of the key elements in cell signaling. Here it is shown that treatment of A431 carcinoma cells with proteasome inhibitors Mg132 and lactacystin results in increasing the PLC gamma 1 intracellular level. Simultaneously, several additional bands with lower electrophoretic mobilities were detected on immunoblots, using anti-PLC gamma 1 antibodies. PLC gamma 1 ubiquitinilation was shown using immunoprecepitation. In control A 431 cells, PLC gamma 1 is ubiquitinilated, but the addition of EGF greatly induces the ubiquitinilation of the protein. Association of PLC gamma 1 with ubiquitin-ligase c-Cb1 was shown. Dynamics of ubiquitinilation under EGF treatment is in a close agreement with that of association of PLC gamma 1 and c-Cb1. It is concluded that PLC gamma 1 is ubiquitinilated and degraded by proteasomes. PLC gamma 1 ubiquitinilation is an EGF-dependent process.

Topics: Acetylcysteine; Carcinoma, Squamous Cell; Cell Line, Tumor; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epidermal Growth Factor; Epithelial Cells; Humans; Leupeptins; Multienzyme Complexes; Phospholipase C gamma; Proteasome Endopeptidase Complex; Type C Phospholipases; Ubiquitins

The intracellular distribution of hsp70 and hdj1 was studied using immunofluorescent method. In nonstimulated cells hsp70 and hdj1 were observed in the cytoplasm of A431 cells. When 100 ng/ml EGF was added for 15 min, both hsp70 and hdj1 were accumulated in the nuclei. Later on (up to 1 h) hsp70 was exported from the nuclei to be observed mainly in the cytoplasm, whereas hdj1 remained in the nuclei. In cells exposed to tyrphostin AG1478, this inhibitor of tyrosine kinase activity of EGF receptor prevented EGF-dependent accumulation of hsp70 and hdj1 in the nuclei. U73122, an inhibitor of phospholipase C activity, induced tyrosine phosphorylation of EGF receptor without EGF stimulation. In cells treated with U73122, both hsp70 and hdj1 were detected in the nuclei of non-stimulated cells. It is concluded that the intracellular distribution of heat shock proteins in A431 cells depends on tyrosine kinase activity of EGF receptor. Here we report for the first time the influence of EGF on the intracellular redistribution of heat shock proteins.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Estrenes; Fluorescent Antibody Technique; Heat-Shock Proteins; Hot Temperature; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Phosphorylation; Protein-Tyrosine Kinases; Pyrrolidinones; Quinazolines; Time Factors; Type C Phospholipases; Tyrphostins; Vanadates

With ongoing efforts to target the epidermal growth factor receptor (EGFR)-mediated tumour growth in the treatment of selected human malignancies, there is a need to determine the expression levels of EGFR and to evaluate its prognostic value in various malignancies in the Asia-Pacific region.. A total of 172 patients with head and neck squamous cell carcinomas from Australia, Hong Kong, Singapore, and Taiwan were selected for EGFR detection. Immunohistochemical staining was performed to evaluate EGFR expression. EGFR expression was present in 88.4% (152/172) of all cases tested. Specifically, EGFR expression was found in 91.3% (42/46), 84.6% (22/26), 84.1% (37/44), 96.0% (24/25), and 87.1% (27/31) cases of head and neck squamous cell carcinomas from the oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx, respectively. The results demonstrate a stronger EGFR expression in T4 tumours (P=0.017) and later clinical stages (P=0.016). No significant correlation was seen with risk factors, primary tumour site and ethnicity.. The majority of head and neck squamous cell carcinomas express EGFR, indicating the importance of studying the efficacy of anti-cancer therapy through this pathway. The results also show similar rates of receptor expression in head and neck squamous cell carcinoma patients from our region compared with other parts of the world.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Australia; Carcinoma, Squamous Cell; Disease Progression; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Hong Kong; Humans; Immunohistochemistry; Male; Middle Aged; Prognosis; Sex Factors; Singapore; Taiwan

Epidermal growth factor receptor (EGFR) is up-regulated and contributes to the loss of growth control in squamous cell carcinoma of the head and neck (SCCHN). Previously, we reported an association between autocrine stimulation of EGFR and constitutive signal transducers and activators of transcription (STAT) 3 activation in SCCHN cells in vitro and in vivo. Here, we evaluated the role of activated STAT3 in tumor progression and EGFR-independent mitogenic signaling. We found that SCCHN cells stably transfected with a dominant active STAT3 construct expressed elevated levels of STAT3 target genes, including Bcl-X(L) and cyclin D1, and demonstrated increased proliferation in vitro and more rapid tumor growth rates in vivo. Cell cycle analysis demonstrated an increased proportion of STAT3 construct transfectants in G(2)-M. These findings provide evidence that constitutive STAT3 activation contributes to tumor growth in SCCHN, independent of the EGFR autocrine axis.

Topics: Autocrine Communication; bcl-X Protein; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cyclin D1; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Proto-Oncogene Proteins c-bcl-2; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

Previous studies have shown that EGF can induce the tyrosine phosphorylation of caveolin-1 in murine fibroblasts following ErbB1 (EGF receptor) mutation or overexpression, but the cell signaling events linking EGF action with caveolin phosphorylation are not fully established. In this regard, we examined multiple human carcinoma cell lines that express various ErbB family members, including A431 epidermoid carcinoma cells and several squamous carcinoma cell lines. In all cases, EGF treatment induced the tyrosine phosphorylation of caveolin-1 in a time- and EGF dose-dependent manner, and immunoblotting analysis revealed that this phosphorylation occurred at tyrosine-14. The EGF-dependent phosphorylation of caveolin-1 was observed at low temperatures (4 degrees C) and was enhanced by caveolae-disrupting agents (cyclodextrin), suggesting that this EGF-dependent system is in a low temperature-stable arrangement that allows for their interaction under conditions where mobility in the membrane is altered. To further assess the events linking EGF action with caveolin phosphorylation, we evaluated the ligand specificity of these responses and their dependence on known effectors of EGF receptor function. We observed that EGF and HB-EGF, but not heregulin, promoted caveolin-1 phosphorylation in A431 cells, suggesting that these responses are linked to EGF receptor activation and not solely occurring via the activation of other endogenous ErbB family members. In addition, the EGF-induced phosphorylation of caveolin-1 in A431 cells was blocked by the Src kinase antagonists PP1 and PP2, but not by the MEK inhibitor PD98059, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin, or cytoskeleton-disrupting agents, such as cytochalasin D, colchicine, and nocadazole. Altogether, these data indicate that multiple human carcinoma cells exhibit an EGF receptor-dependent tyrosine phosphorylation of caveolin-1 and that this process is sensitive to Src family kinase inhibitors. These observations support a role for caveolin tyrosine phosphorylation in the profile of cellular responses by which Src potentiates cancer progression following EGF receptor overexpression.

Topics: Androstadienes; Carcinoma, Squamous Cell; Caveolae; Caveolin 1; Caveolins; Cell Line; Chromones; Colchicine; Cyclodextrins; Cytochalasin D; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Humans; Kinetics; Morpholines; Nucleic Acid Synthesis Inhibitors; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; src-Family Kinases; Tumor Cells, Cultured; Tyrosine; Wortmannin

Invasive squamous cell carcinoma (SCC) cells degrade extracellular matrix (ECM) via an extracellular protease cascade that includes urokinase-type plasminogen activator (uPA), plasmin, and the metalloprotease (MMP) family of collagenases. In this study, treatment of oral SCC cells with epidermal growth factor (EGF) stimulated the cells to invade Matrigel (constructive basement membrane (BM) protein). EGF-induced cell invasion was inhibited by antibodies to uPA and by synthetic uPA inhibitors. EGF also induced increased expression of uPA and uPA receptor (uPAR) proteins and mRNA, as well as transcription factor activator protein-1 (AP-1)-DNA binding. These EGF-induced changes were inhibited by treatment with dexamethasone (DEX). DEX treatment also stimulated the production of plasminogen activator inhibitor type 1. Moreover, transfection of SCC cells with AP-1 decoy oligodeoxynucleotides (ODNs) resulted in the suppression of EGF-induced uPA and uPAR expression and Matrigel invasion. These results suggest that oral SCC cells invade Matrigel mainly through the uPA-plasmin cascade, which is mediated by the transcription factor AP-1. The uPA-uPAR interaction is essential for augmenting proteolytic activity and uPAR-mediated signaling, which ultimately induce motility and invasion. Since DEX inhibits the expression of both uPA and uPAR, it may be a useful treatment for oral SCC.

Topics: Carcinoma, Squamous Cell; Cell Movement; Dexamethasone; DNA; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Mouth Neoplasms; Neoplasm Invasiveness; Oligodeoxyribonucleotides; Plasminogen Activator Inhibitor 1; RNA, Messenger; Transcription Factor AP-1; Transfection; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

We investigated the interactions between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) pathways in head and neck squamous cell carcinomas (HNSCCs) and in two carcinoma cell lines. HNSCCs showed an up-regulation of both pathways which were strongly correlated with each other (p=0.02) and with tumor vascularization (p=0.0001 and p=0.008, respectively). In carcinoma cells, Escherichia coli lipopolysaccharide (LPS) and EGF treatment up-regulated both pathways. NOS inhibitor N(G)-monomethyl-L-arginine methyl ester (L-NAME) inhibited this up-regulation. LPS or EGF induced iNOS expression that was not altered by NOS or COX-2 inhibitors. Conversely, LPS or EGF promoted COX-2 expression that was decreased by L-NAME. The NO donor S-nitroso-acetyl-penicillamine (SNAP) up-regulated COX-2 pathway and this effect was reduced by the guanylate cyclase inhibitor methylene blue. Thus, in squamous carcinoma cells, NO increases the activity of COX-2 pathway and this effect is probably mediated by endocellular cGMP level, with potential implications on tumor growth, angiogenesis, and therapy.

Topics: Carcinoma, Squamous Cell; Cyclic GMP; Cyclooxygenase 2; Enzyme Activation; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Isoenzymes; Lipopolysaccharides; Membrane Proteins; Neovascularization, Pathologic; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin-Endoperoxide Synthases; S-Nitroso-N-Acetylpenicillamine; Signal Transduction; Statistics as Topic; Tumor Cells, Cultured; Up-Regulation

Tumor metastasis represents a complex multistep process that requires migration, invasion, and angiogenesis. In this study, we examined the impact of molecular blockade of the epidermal growth factor receptor on the invasive and metastatic capacity of human squamous cell carcinoma (SCC) of the head and neck using in vitro and in vivo model systems. Treatment with the anti-epidermal growth factor receptor antibody C225 attenuated the migration of SCC-1 tumor cells through a chemotaxis chamber in a dose-dependent manner. Incubation of SCC cells with 10-100 nM C225 for 4 h resulted in 40-60% inhibition of cell migration. Furthermore, in the presence of C225, the capacity of SCC-1 to invade across a layer of extracellular matrix (Matrigel) was significantly inhibited. Using an in vivo orthotopic floor-of-mouth xenograft model, locoregional tumor invasion of SCC-1 into muscle, vessel, bone, and perineural tissues was inhibited in C225-treated mice. This inhibition was additionally characterized by down-regulation in the expression of matrix metalloproteinase-9. These data suggest that inhibition of metastatic potential by C225 may be mediated via decreased migration and invasion of SCC cells. Regarding angiogenesis in vitro, we first studied human umbilical vascular endothelial cells, which established a capillary-like network structure (tube formation) in the presence of reconstituted Matrigel. Treatment with C225 reduced cell-to-cell interaction of human umbilical vascular endothelial cells, resulting in disruption of tube formation. The effect of C225 was additionally examined using an in vivo tumor xenograft neovascularization model of angiogenesis. Systemic treatment with C225 not only reduced tumor growth and the number of blood capillaries but also hindered the growth of established vessels toward the tumor. Taken together, these results provide evidence that C225 can suppress tumor-induced neovascularization and metastasis in SCC of the head and neck.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Carcinoma, Squamous Cell; Cell Movement; Cells, Cultured; Cetuximab; Chemotaxis; Collagen; Dose-Response Relationship, Drug; Down-Regulation; Drug Combinations; Endothelium, Vascular; Epidermal Growth Factor; Extracellular Matrix; Fibronectins; Head and Neck Neoplasms; Humans; Immunohistochemistry; Laminin; Matrix Metalloproteinase 9; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Proteoglycans; Time Factors; Tumor Cells, Cultured

Epidermoid carcinoma (PSCC) of the endometrium is a rare form of endometrial cancer that constitutes about 0.1% of all malignant epithelial tumors of the uterus. The diagnosis of PSCC is based on strict criteria and is made in the absence of a glandular component of the tumor. Squamous cell carcinoma of the endometrium should enter the differential diagnosis in postmenopausal patients in the presence of atypical squamous cells in the uterine curettage, while the cervical biopsies are negative for malignancy. The presence of HPV should be investigated as well, so that its pathogenetic relation is clarified. While no significant relation was found to p-53, C-NEU and EGFR expression this investigation must be continued because. HPV may interact with tumor suppressor genes.

Topics: Aged; Biomarkers, Tumor; Biopsy, Needle; Carcinoma, Squamous Cell; Endometrial Neoplasms; Epidermal Growth Factor; Fatal Outcome; Female; Genes, p53; Humans; Immunohistochemistry; Papillomavirus Infections; Proto-Oncogene Proteins; Sensitivity and Specificity; Tumor Virus Infections

Epidermal growth factor receptor (EGFR) tyrosine kinase is a potential target for anticancer therapy. ZD1839 (Iressa) is a selective inhibitor of EGFR tyrosine kinase. In this study, we investigated the question as to whether the antitumor effect of ZD1839 is partly attributable to antiangiogenic activity and the potential mechanisms involved. Both ZD1839 and SU5416 [a vascular endothelial growth factor (VEGF)-receptor tyrosine kinase inhibitor] inhibited the migration of human umbilical vein endothelial cell cocultivated with EGF-stimulated cancer cells. ZD1839 also inhibited EGF-induced migration and the formation of tube-like structures by human microvascular endothelial cells. Moreover, ZD1839 almost completely blocked EGF-induced neovascularization of mice cornea, and SU5416 partially blocked neovascularization. In contrast, ZD1839 did not inhibit VEGF-induced angiogenesis. However, EGF-induced up-regulation of the angiogenic factors, VEGF and IL-8, was almost completely blocked by ZD1839. The antitumor effects of ZD1839 could, therefore, be mediated in part by the inhibition of tumor angiogenesis through direct effects on microvascular endothelial cells that express EGFR and also through reduced production of proangiogenic factors by tumor cells.

Topics: Angiogenesis Inhibitors; Animals; Carcinoma, Squamous Cell; Cell Movement; Cells, Cultured; Cornea; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Humans; Interleukin-8; Lymphokines; Male; Mitogen-Activated Protein Kinase Kinases; Neovascularization, Physiologic; Quinazolines; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vulvar Neoplasms

The purpose of this study was to investigate the effects of a potent LHRH agonist, [D-Trp(6)]LHRH on the basal and EGF-induced cell proliferation and the metastasis-associated properties in A431 human epidermoid carcinoma. [D-Trp(6)]LHRH time-dependently inhibited the basal and EGF-stimulated growth of A431 cancer cells. It is assumed that phosphorylation/dephosphorylation of cellular proteins is highly related to cell growth. This study demonstrates that [D-Trp(6)]LHRH decreased the basal and EGF-induced total cellular kinase activity, particularly the tyrosine phosphorylation of several cellular proteins including the EGFR. In contrast, [D-Trp(6)]LHRH did not cause detectable changes in basal and EGF-stimulated serine/threonine phosphorylation of A431 cellular proteins. The inhibitory effect of [D-Trp(6)]LHRH on A431 cell proliferation was associated with apoptosis as evidenced by the cell morphology and DNA integrity (ladder pattern), the expression of interleukin 1beta-converting enzyme (ICE) and activation of caspase. Furthermore, EGF could rescue the remaining attached A431 cells following [D-Trp(6)]LHRH treatment for 48 hr, which suggests that limited exposure to [D-Trp(6)]LHRH did not channel all cells to irreversible apoptotic process. We also determined the effects of [D-Trp(6)]LHRH on metastasis-associated properties in A431 cells. [D-Trp(6)]LHRH reduced both basal and EGF-stimulated secretion of MMP-9 and MMP-2. In addition, [D-Trp(6)]LHRH suppressed the basal and EGF-induced invasive activity of A431 cells based on an in vitro invasion assay. In conclusion, this study indicates that [D-Trp(6)]LHRH may act partly through activating tyrosine phosphatase activity to inhibit cell proliferation and the metastasis-associated properties of A431 cancer cells. Our work suggests that [D-Trp(6)]LHRH may be therapeutically useful in limiting the tumor growth and metastasis of some neoplasms.

Topics: Antineoplastic Agents, Hormonal; Carcinoma, Squamous Cell; Caspases; Cell Division; DNA Fragmentation; Enzyme Activation; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Immunoblotting; Matrix Metalloproteinases; Neoplasm Invasiveness; Phosphorylation; Precipitin Tests; Protein Tyrosine Phosphatases; Serine; Threonine; Time Factors; Triptorelin Pamoate; Tumor Cells, Cultured

An oral epithelial cell line derived from buccal mucosa squamous cell carcinoma, HO-1-N-1, was used to elucidate the role of epidermal growth factor (EGF) in saliva on wound healing of the oral mucosa. The effects of EGF on DNA synthesis, and cell migration was studied and the related signal transduction pathways examined. DNA synthesis by HO-1-N-1 cells was stimulated dose-dependently by 1-10 ng ml(-1) EGF, but significantly inhibited by addition of a PI3-K inhibitor (wortmannin), a p38 MAPK inhibitor (SB203580) or an MEKs inhibitor (PD98059). Cell migration was also accelerated by addition of 1-10 ng ml(-1) EGF; however, the migration rate was decreased to 30% by adding PD98059, to 40% by adding a tyrosine kinase inhibitor (herbimycin A), and to 60% by adding wortmannin or dexamethasone. These results indicate that the physiologic concentration of EGF in saliva may stimulate proliferation and migration of oral epithelial cells for wound healing, when the oral mucosa has been injured. Furthermore, this study revealed that EGF-stimulated signal transduction pathways for epithelial cell proliferation and cell migration are different.

Topics: Androstadienes; Carcinoma, Squamous Cell; Cell Division; Cell Movement; Dexamethasone; DNA; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Glucocorticoids; Humans; Immunity, Mucosal; MAP Kinase Signaling System; Mouth Mucosa; Mouth Neoplasms; Saliva; Salivary Proteins and Peptides; Tumor Cells, Cultured; Wortmannin; Wound Healing

The 11p15 mucin genes (MUC2, MUC5AC, MUC5B and MUC6) possess a cell-specific pattern of expression in normal lung that is altered during carcinogenesis. Growth factors of the epidermal growth factor family are known to target key genes that in turn may affect the homeostasis of lung mucosae. Our aim was to study the regulation of the 11p15 mucin genes both at the promoter and protein levels to assess whether their altered expression may represent a key event during lung carcinogenesis. Studies were performed in the mucoepidermoid NCI-H292 lung cancer cell line. Cell treatment with epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), or tumor necrosis factor alpha (TNF-alpha) resulted in a dramatic increase of MUC2 and MUC5AC mRNAs levels, promoter activity, and apomucin expression, whereas those of MUC5B and MUC6 were unchanged. pGL3 deletion mutants of MUC2, MUC5AC, and MUC5B promoters were constructed and used in transient transfection assays to characterize EGF- and TGF-alpha-responsive regulatory regions within the promoters. They were located in the -2627/-2097 and -202/-1 regions of MUC2 and MUC5AC promoters, respectively. Finally, we demonstrate that transcription factor Sp1 not only binds and activates MUC2 and MUC5AC promoters but also participates to their EGF- and TGF-alpha-mediated up-regulation. We also show that Sp3 is a strong inhibitor of 11p15 mucin gene transcription. In conclusion, MUC2 and MUC5AC are two target genes of EGFR ligands in lung cancer cells, and up-regulation of these two genes goes through concomitant activation of the EGFR/Ras/Raf/Extracellular Signal-regulated Kinase-signaling pathway and Sp1 binding to their promoters.

Topics: Base Sequence; Carcinoma, Squamous Cell; DNA Primers; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Mucin 5AC; Mucin-2; Mucins; Polymerase Chain Reaction; ras Proteins; Sp1 Transcription Factor; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We report 6 cases of pseudoepitheliomatous hyperplasia (PEH) mimicking squamous cell carcinoma in association with an atypical CD30+ dermal infiltrate. Three patients had lymphomatoid papulosis type A, and 3 patients had cutaneous CD30+ lymphoma. All 6 cases showed histologic evidence of PEH with keratinocyte atypia. In 4 cases there was significant atypia to prompt a diagnosis of squamous cell carcinoma. Three of these received treatment with wide local excision and 2 had been engrafted. Immunohistochemical staining for epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) showed similar expression in lesional and perilesional skin. Epidermal growth factor receptor (EGFR) expression by the proliferating epithelium was similar to that of the suprabasal adjacent normal epidermis. There was no aberrant expression of EGF, TGF-alpha, and EGFR by atypical lymphocytes. These cases demonstrate that PEH associated with CD30+ lymphoproliferative disease may closely resemble squamous cell carcinoma, thereby leading to inappropriate diagnosis and treatment.

Topics: Adult; Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Humans; Immunohistochemistry; Ki-1 Antigen; Lymphoma, T-Cell, Cutaneous; Lymphomatoid Papulosis; Male; Middle Aged; Skin; Skin Neoplasms; Transforming Growth Factors

Despite recent advances in tumor surgery and multimodal treatment regimens the prognosis of squamous cell carcinomas of the head and neck is still relatively poor and has shown only slow progress. Additional clinical and biological factors are therefore urgently needed that aid tumor diagnosis, early detection of tumor recurrences, prediction of the results of therapeutic interventions, and identification of subsets of patients with unfavorable outcome during follow-up. A tumor marker should fulfill the criteria of specificity and clinical effectiveness. So far none of the known serological and biological markers fits all these criteria. Although some biological markers can be determined serologically without the need for tumor tissue, their low specificity limits their applicability to early detection of tumor recurrences during follow-up. A large number of molecular or genetic markers have been examined in squamous cell carcinomas of the head and neck. All of these are controversial regarding their prognostic value and clinical effectiveness. UICC tumor stage and patient's age and performance still remain the basis for therapeutic decisions. Further search for more sensitive and specific markers is needed to improve individual treatment and prognostic statements.

Topics: Autoantibodies; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Genes, erbB-2; Genes, p53; Genes, Tumor Suppressor; Head and Neck Neoplasms; Humans; Immunohistochemistry; Point Mutation; Prognosis; Proliferating Cell Nuclear Antigen

The regulatory mechanism through which the phospholipase D (PLD) isoforms PLD1 and PLD2 are activated is poorly understood. We investigated the possibility that the PLD isozymes are differentially regulated in response to pharmacologic stimulants in cells. In this report, we demonstrate for the first time that H2O2 and EGF differentially induce tyrosine phosphorylation of the PLD isozymes in A431 cells, which express both PLD1 and PLD2. H2O2 induced tyrosine phosphorylation of PLD1 and PLD2, whereas EGF only caused the tyrosine phosphorylation of PLD2. Both agents also induced phosphorylation of the EGF receptor. Interestingly, the PLD isozymes were associated with the EGF receptor and PKC-alpha in a ligand independent manner. Activation of PLD by H2O2 and EGF nearly correlated with tyrosine phosphorylation of the protein in PLD1 immune complexes. Activation of PLD by both agents was inhibited by the PKC inhibitor, Ro 31-8220, and by the down-regulation of PKC. Pretreatment of the cells with the tyrosine kinase inhibitor tyrphostin AG1478 resulted in inhibition of the H2O2 and EGF-induced tyrosine phosphorylation and PLD activation. These results indicate that H2O2 and EGF induce differential tyrosine phosphorylation of PLD isozymes. Also, the activation of PLD by these agonists involves tyrosine phosphorylation and PKC activation.

Topics: Carcinoma, Squamous Cell; Catalase; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrogen Peroxide; Isoenzymes; Oxidants; Phospholipase D; Phosphorylation; Protein Kinase C; Protein Kinase C-alpha; Tumor Cells, Cultured; Tyrosine

Several inhibitors of EGF receptor (EGFR) tyrosine kinase activity have been developed that compete with ATP at its binding site such as the quinazolines PD 153035 and ZD 1839 or the 4,5-dianilino-phthalimides DAPH1 and DAPH2. When tested on human A431 cells, the quinazolines completely blocked EGF-induced receptor phosphorylation at 100 nM, whereas it was inhibited by DAPH1 and DAPH2 by only 20% at 3 microM. Quinazoline-treated A431 as well as tumor cells expressing less EGFR (A549, MDA MB 231, and T47D) bound 3- to 6-fold more (125)I-labeled EGF than untreated intact control cells. Scatchard analysis revealed the disappearance of low- and high-affinity EGFR on A431 cells upon PD 153035 treatment. A single receptor class of intermediate ligand binding affinity emerged and its number corresponded to the sum of the two classes. DAPH1 and DAPH2 did not change ligand binding properties of EGFR. PD 153035 exerted the most potent effects on EGF binding to A431 or on inhibiting EGF-stimulated growth of rat MTLn3 cells at low ligand concentrations. Cross-linking of EGFR on PD 153035-treated A431 cells indicated the formation of inactive dimers that further increased upon addition of EGF. Chemical cross-linking of (125)I-labeled EGF to PD 153035-treated A431 cells revealed increased binding to monomeric and dimeric EGFR. Thus, the quinazolines sequestered EGFR plus the ligand into inactive receptor/ligand complexes. This novel mode of action of quinazoline tyrosine kinase inhibitors may be the basis for their extraordinary potency especially in conditions when the ligand is present in limiting amounts.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Kinetics; Ligands; Lung Neoplasms; Mammary Neoplasms, Experimental; Phthalimides; Quinazolines; Rats; Signal Transduction; Tumor Cells, Cultured

An antitumor glycoprotein [streptococcal acidic glycoprotein (SAGP)] purified from an extract of Streptococcus pyogenes inhibited the growth of human epidermoid carcinoma A431 cells overexpressing epidermal growth factor receptor (EGFR) in a time- and a concentration-dependent manner. The antiproliferative effect of SAGP was diminished by preincubating the cells with pertussis toxin and by coadministration of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases (PTPases). Western blot analysis showed that the immunoreactivity of a M(r) 170,000 band of cell lysate to antiphosphotyrosine antibody was reduced by SAGP, and the effect was abolished by sodium orthovanadate. The phosphotyrosine level of the precipitant with anti-EGFR antibody was reduced by SAGP, which was abolished by preincubation with pertussis toxin or by a coadministration with sodium orthovanadate. The PTPase activity transiently increased in the lysate of cells incubated with SAGP and was inhibitable by sodium orthovanadate. Additionally, preincubation of serum-starved A431 cells with SAGP decreased the epidermal growth factor-induced tyrosine phosphorylation of EGFR, and the effect of SAGP was sodium orthovanadate sensitive. These findings indicate that dephosphorylation of the M(r) 170,000 EGFR by activation of PTPase(s) may be responsible in part for the antiproliferative effect of SAGP on A431 cells.

Topics: Antibiotics, Antineoplastic; Bacterial Proteins; Carcinoma, Squamous Cell; Catalase; Cell Division; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Growth Inhibitors; GTP-Binding Proteins; Humans; MAP Kinase Signaling System; Pertussis Toxin; Phosphorylation; Precipitin Tests; Protein Tyrosine Phosphatases; Tumor Cells, Cultured; Tyrosine; Vanadates; Virulence Factors, Bordetella

We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.

Topics: Animals; Carcinoma, Squamous Cell; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Growth Substances; I-kappa B Proteins; Intercellular Signaling Peptides and Proteins; Interleukin-1; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; RNA, Messenger; Transforming Growth Factor beta

Growth factor receptors of the tyrosine kinase family regulate proliferation of a variety of cell types. In some human cancers, the epidermal growth factor receptor (EGFR) and its ligands often are overexpressed, leading to both constitutive and autocrine activation. Intracellular signaling via this receptor takes place through several mechanisms of action, including activation of ras and the mitogen-activated protein kinase (MAPK) pathway. Our previous studies have shown that human squamous cell carcinoma (SCC) lines overexpress EGFR and do not increase proliferation in response to exogenous epidermal growth factor (EGF). The vitamin A metabolite retinoic acid (RA) has been used as a chemotherapeutic drug in the treatment of SCC. RA decreases proliferation of SCC lines, in part owing to inhibition of EGFR expression. However, we previously found that treatment of SCC lines with inhibitory doses of RA sensitized cells to the proliferative effects of EGF. We now present a mechanism of action for this effect. RA inhibited expression of EGFR and proteins in the MAPK signaling pathway. Expression of these molecules returned to basal levels within 24 h after RA withdrawal. RA also inhibited autocrine secretion of EGF, which returned to basal levels with slower kinetics. During this time, addition of exogenous EGF stimulated mitosis in SCC lines. These data suggested that signaling proteins downstream of overexpressed EGFR may have limited the mitotic response in SCC lines. In support of this hypothesis, overexpression of the EGFR adaptor protein Grb2 increased cell proliferation and restored EGF-induced mitosis.

Topics: Adaptor Proteins, Signal Transducing; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; GRB2 Adaptor Protein; Humans; Proteins; Signal Transduction; Transfection; Tretinoin; Tumor Cells, Cultured

Epithelial growth factor receptor (EGFR) sends signals to the proliferation signal transduction system, receiving two ligands: epithelial growth factor (EGF) and transforming growth factor-alpha (TGF-alpha). This immunohistochemical study examined the roles of EGFR and its ligands in the proliferation of normal and neoplastic vulvar squamous cells in 25 patients with vulvar squamous cell carcinoma (VSCC), 10 patients with vulvar condyloma acuminata (VCA), 15 patients with vulvar intra-epithelial neoplasm I-II or III (VIN I-II or III), and 5 subjects with vulvar normal squamous cells (VNSC). EGFR was detected in a few basal cells in 40% of the VNSC, in highly dysplastic cells in 40% of the VIN III, in many neoplastic cells in 80% of the VCA, and in some malignant cells in 64% of the VSCC. EGF was seen in the cytoplasm in 20% of the VIN I-II, 100% of the VIN III, 100% of the VCA, and 100% of the VSCC. Diffuse TGF-alpha was weakly expressed in the cytoplasm in 100% of the VNSC, more intensely in 100% of the VIN and 100% of the VCA, and intensely in 100% of the VSCC. These findings led to the suggestion that the TGF-alpha-EGFR system maintains the growth of normal squamous cells and, in part, maintains the growth of dysplastic and neoplastic squamous cells in the vulva. EGF expression was an early sign of neoplasia. The expression of EGFR with overexpression of its two ligands contributed to the proliferation of dysplastic and neoplastic squamous cells in VIN III and VCA. EGFR expression appeared to contribute to essential neoplastic abnormalities in 64% of the VSCC.

Topics: Carcinoma in Situ; Carcinoma, Squamous Cell; Condylomata Acuminata; DNA, Viral; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Ligands; Papillomaviridae; Papillomavirus Infections; Transforming Growth Factor alpha; Tumor Virus Infections; Vulva; Vulvar Neoplasms

Head and neck squamous cell carcinomas (HNSCCs) are characterized by a marked propensity for local invasion and dissemination to cervical lymph nodes, with distant metastases developing in 30-40% of cases. Overexpression of the epidermal growth factor receptor (EGFR/c-erbB-1) and/or its ligands and high levels of certain matrix metalloproteinases (MMPs) have been associated with poor prognosis. The aim of this study was to examine the effects of EGFR ligands on gelatinase expression and invasion in HNSCC cell lines. We tested epidermal growth factor (EGF), transforming growth factor alpha, betacellulin, heparin-binding EGF, and amphiregulin and measured expression of gelatinases MMP-9 and MMP-2 in an established squamous carcinoma cell line (Detroit-562) and in two cell lines newly derived from patients with head and neck cancers (SIHN-005A and SIHN-006). Incubation of the cell lines with EGF-like ligands up-regulated MMP-9 (but not MMP-2) expression as measured by semiquantitative reverse transcription-PCR in a dose-dependent manner, with the effects being most marked in cells with high EGFR levels and undetectable in cells with low levels. Maximum stimulation was obtained in a concentration range of 10-100 nM. In addition, we confirmed by zymography that gelatinolytic activity consistent with MMP-9 (Mr 92,000) was up-regulated in parallel with increases in gene expression. Betacellulin (which binds both to EGFR and c-erbB-4 receptors) consistently increased MMP-9 expression and activation to a significantly greater degree than the other four ligands when tested at equimolar concentrations. In parallel with MMP-9 up-regulation, all EGF-like ligands increased tumor cell invasion through Matrigel in in vitro Transwell assays. These activities were independent of ligand effects on cell proliferation. Antagonist (ICR62) or agonist (ICR9) anti-EGFR monoclonal antibodies, respectively, inhibited or potentiated MMP-9 activity and tumor cell invasion induced by all ligands. Furthermore, a monoclonal antibody that neutralizes MMP-9 activity (Abl) also inhibited ligand-induced invasion of HNSCC. We confirmed that tumor cell lines used in these studies (and a larger series not reported here) generally expressed multiple c-erbB receptors and ligands. These results indicate that autocrine or paracrine signaling through EGFR potentiates the invasive potential of HNSCC via the selective up-regulation and activation of MMP-9. Furthermore, ligands such as betacellulin (w

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Ligands; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; RNA, Messenger; Tumor Cells, Cultured

In rat liver epithelial cells constitutively expressing transforming growth factor alpha (TGFalpha), c-Met is constitutively phosphorylated in the absence of its ligand, hepatocyte growth factor. We proposed that TGFalpha and the autocrine activation of its receptor, epidermal growth factor receptor (EGFR), leads to phosphorylation and activation of c-Met. We found that there is constitutive c-Met phosphorylation in human hepatoma cell lines and the human epidermoid carcinoma cell line, A431 which express TGFalpha, but not in normal human hepatocytes. Constitutive c-Met phosphorylation in A431, HepG2, AKN-1, and HuH6 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR. Exposure to exogenous TGFalpha or EGF increased the phosphorylation of c-Met in the human epidermoid carcinoma cell line, A431. The increase of c-Met phosphorylation by TGFalpha in A431 cells was inhibited by neutralizing antibodies against TGFalpha and/or EGFR and by the EGFR-specific inhibitor tyrphostin AG1478. These results indicate that constitutive c-Met phosphorylation, and the increase of c-Met phosphorylation by TGFalpha or EGF, in tumor cell lines is the result of the activation via EGFR. We found that c-Met in tumor cells co-immunoprecipitates with EGFR regardless of the existence of their ligands in tumor cells, but not in normal human hepatocytes. We conclude that c-Met associates with EGFR in tumor cells, and this association facilitates the phosphorylation of c-Met in the absence of hepatocyte growth factor. This cross-talk between c-Met and EGFR may have significant implications for altered growth control in tumorigenesis.

Topics: Animals; Carcinoma, Hepatocellular; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Hepatocyte Growth Factor; Humans; Liver Neoplasms; Phosphorylation; Protein Binding; Proto-Oncogene Proteins c-met; Quinazolines; Rats; Receptor Cross-Talk; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrphostins

Gangliosides have been described as modulators of growth factor receptors. For example, GM3 addition in cell culture medium inhibits epidermal growth factor (EGF)-stimulated receptor autophosphorylation. Furthermore, depletion of ganglioside by sialidase gene transfection appeared to increase EGF receptor (EGFR) autophosphorylation. These data suggested that changes in GM3 content may result in different responses to EGF. In this study, the ceramide analog d-threo-1-phenyl-2-decannoylamino-3-morpholino-1-propanol ([D]-PDMP), which inhibits UDP-glucose-ceramide glucosyltransferase, and addition of GM3 to the culture medium were used to study the effects of GM3 on the EGFR. Addition of 10 microM [D]-PDMP to A431 cells resulted in significant GM3 depletion. Additionally, EGFR autophosphorylation was increased after EGF stimulation. When exogenous GM3 was added in combination with [D]-PDMP, the enhanced EGFR autophosphorylation was returned to control levels. [D]-PDMP also increased EGF-induced cell proliferation, consistent with its effect on autophosphorylation. Once again, the addition of GM3 in combination with [D]-PDMP reversed these effects. These results indicate that growth factor receptor functions can be modulated by the level of ganglioside expression in cell lines. Addition of GM3 inhibits EGFR activity and decrease of GM3 levels using [D]-PDMP treatment enhances EGFR activity. Modulation of growth factor receptor function may provide an explanation for how transformation-dependent ganglioside changes contribute to the transformed phenotype.

Topics: Carcinoma, Squamous Cell; Cell Division; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; G(M3) Ganglioside; Gangliosides; Glucosyltransferases; Humans; Kinetics; Morpholines; Signal Transduction; Tumor Cells, Cultured

In Drosophila melanogaster and Caenorhabditis elegans, kinase suppressor of Ras (KSR) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf. Attempts to characterize the biochemical and biological properties of mammalian KSR, however, have had limited success. Although some studies demonstrated a requirement of KSR kinase activity for its action, others indicated the kinase function of KSR is dispensable and suggested that KSR acts primarily as a scaffold protein. Interpretations of KSR function are further hampered by the lack of a standardized assay for its kinase activity in vitro. To address this issue, we established a two-stage in vitro kinase assay in which KSR never comes in contact with any recombinant kinases other than c-Raf-1. Using this assay, we show that KSR immunoprecipitated from quiescent COS-7 cells overexpressing Flag-tagged KSR was inactive, but its activity was rapidly and markedly induced upon epidermal growth factor treatment. Moreover, KSR-reconstituted mitogen-activated protein kinase activation was detected in KSR immunoprecipitates depleted of all contaminating kinases (c-Raf-1, MEK1, ERK2) by multiple high salt washes. Only full-length kinase-active KSR was capable of signaling c-Raf-1-dependent activity as kinase inactive and C- and N-terminal deletion mutants were without effect. Furthermore, endogenous KSR isolated from A431 cells, which contain high levels of activated EGF receptor, displays constitutively enhanced kinase activity. Hence, KSR kinase activity is not an artifact of overexpression but a property intrinsic to this protein. The recognition of EGF as a potent activator of KSR kinase activity and the availability of a well defined in vitro kinase assay should facilitate the definition of the function of KSR as a Ras-effector molecule.

Topics: Animals; Carcinoma, Squamous Cell; COS Cells; Enzyme Activation; Epidermal Growth Factor; Humans; Mitogen-Activated Protein Kinases; Protein Kinases; Proto-Oncogene Proteins c-raf; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.

Topics: Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; CDC2-CDC28 Kinases; Cell Adhesion; Cell Cycle; Cell Division; Collagen; Cyclin A; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Dose-Response Relationship, Drug; Epidermal Growth Factor; Flow Cytometry; Humans; Neoplasms, Glandular and Epithelial; Protein Serine-Threonine Kinases; Retinoblastoma Protein; Time Factors; Tumor Cells, Cultured

It is known that EGF induces the cell-cycle arrest in A431 cells that possess high numbers of EGF receptors and it was previously suggested that p21/WAF1 protein was a major effector molecule of the EGF-mediated cell-cycle arrest of A431 cells. Here, we further investigate this phenomenon using the decoy double-strand oligonucleotides for STAT-binding sequence (STAT decoy) and IkappaB, an inhibitor of the nuclear factor kappa B (NFkappaB). Addition of STAT decoy restored EGF-induced A431 cell-growth arrest. Interestingly, infection of adenovirus vectors to express IkappaB (AxIkappaBalphaDeltaN) as the inhibitor of NFkappaB also reversed the A431 cell-growth inhibition. The individual treatment of two inhibitors partially inhibited the WAF1 gene expression, whereas simultaneous treatment of two inhibitors exhibited more efficient inhibition. These observations suggest the activation of NFkappaB via IkappaB degradation and STAT1 via specific receptor kinase activity synergistically induce WAF1 gene expression in A431 cells. Thus, NFkappaB and STAT1 pathways mutually interact to play an important role in the EGF-induced intracellular reaction.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Genes, Reporter; Humans; I-kappa B Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Transfection; Tumor Cells, Cultured

Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type IV collagen. Enhanced expression has been related to tumor progression both in vitro and in vivo. The control of MMP transcription is complex, but recently, epidermal growth factor receptor (EGFR) expression has been implicated in up-regulation of MMP-9 in tumor cells in vitro. Our objective was to evaluate the relationship between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on clinicopathological parameters and survival. This is a retrospective study of 169 patients who underwent resection for stage I-IIIa NSCLC with a postoperative survival >60 days. Minimum follow-up was 2 years. Standard avidin-biotin complex immunohistochemistry was performed on 4-microm paraffin-embedded sections from the tumor periphery using monoclonal antibodies to EGFR and MMP-9. MMP-9 was expressed in the tumor cells of 88 of 169 (52%) cases. EGFR expression was found in 94 of 169 (56%) cases [membranous, 55 of 169 (33%); cytoplasmic, 39 of 169 (23%)]. MMP-9 expression was associated with poor outcome in univariate (P = 0.0023) and multivariate (P = 0.027) analysis. Membranous, cytoplasmic, and overall EGFR expression were not associated with outcome (P = 0.13, 0.99, and 0.17, respectively). MMP-9 expression showed a strong correlation with EGFR expression (P < 0.0001) and EGFR membranous expression (P = 0.002) but not with cytoplasmic EGFR expression (P = 0.18). Co-expression of MMP-9 and EGFR (37%) conferred a worse prognosis (P = 0.0001). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (P = 0.0019). Our results show that a significant proportion of NSCLC tumors co-express MMP-9 and EGFR. The co-expression of these markers confers a poor prognosis. This finding suggests that EGFR signaling pathway may play an important role in the invasive behavior of NSCLC via specific up-regulation of MMP-9.

Topics: Adenocarcinoma; Adult; Age Factors; Aged; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Membrane; Cytoplasm; Disease Progression; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Matrix Metalloproteinase 9; Middle Aged; Multivariate Analysis; Prognosis; Retrospective Studies; Sex Factors; Signal Transduction; Time Factors; Up-Regulation

High epidermal growth factor (EGF) concentration (10(-8) M) induces inhibition of A431 cell proliferation, resulting in part from an apoptotic process. For some cells escaping this process, proliferation was associated with a decrease in apoptosis. Moreover, these surviving cells displayed marked morphological changes consisting of filopodia formation and cell aggregation. Disrupting cell-cell contacts by lowering extracellular calcium concentration reversed the resistance process, suggesting that apoptosis protection by aggregation may involve intercellular adhesion and cell-cell survival signals probably mediated by calcium-requiring molecules such as integrins. From a panel of integrins tested, only alpha 2 beta 1 integrin cell-surface expression was up-regulated after high apoptotic EGF treatment, and this up-regulation was not observed under a growth-stimulatory EGF concentration (10(-11) M). Double-labeling analysis (alpha 2 beta 1/DNA) implicated alpha 2 beta 1 integrin in the resistance process since 99% of cells that up-regulated alpha 2 beta 1 integrin survived a high dose of EGF. Moreover, the involvement of alpha 2 beta 1 integrin up-regulation in the survival of A431 cells that escape EGF-induced apoptosis was verified using the blocking anti-alpha 2 beta 1 integrin antibody, which was shown to decrease the survival of EGF-stimulated cells. Furthermore, under our culture conditions, alpha 2 beta 1 integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions, suggesting that alpha 2 beta 1 integrin is involved more directly in cell-cell interaction than in cell-substrate adhesion. Our results provide evidence that EGF-induced up-regulation of alpha 2 beta 1 integrin contributes to the enhancement of cell-cell adhesion, leading to cell aggregate formation, which permits the escape of A431 cells to EGF-induced death by alpha 2 beta 1 integrin signaling.

Topics: Antibodies, Monoclonal; Apoptosis; Calcium; Calcium Signaling; Carcinoma, Squamous Cell; Cell Adhesion; Cell Aggregation; Cell Size; Epidermal Growth Factor; Humans; Integrins; Neoplasm Proteins; Receptors, Collagen; Tumor Cells, Cultured; Up-Regulation

Expression of extracellular matrix-degrading proteases is required for tumor cell invasion. In the present study we examined the production of type I collagen-degrading matrix metalloproteinases (MMPs) in the invasive oral squamous cell carcinoma-derived cell line HSC-3. In the absence of serum or exogenous growth factors, HSC-3 cells displayed no collagen degradation activity. Addition of serum slightly increased collagen proteolysis. However, addition of epidermal growth factor (EGF) resulted in nearly complete degradation of the collagen matrix. Zymography showed that MMP-2 and -9 are secreted by HSC-3 cells. EGF stimulated secretion of an additional gelatinase with a molecular weight similar to that of MMP-1. Immunoblotting of conditioned medium confirmed that EGF and, to a lesser degree type I collagen, increased production of MMP-1. Finally, in situ hybridization revealed intense expression of MMP-1 in oral squamous cell carcinoma specimens. Together, these results indicate that MMP-1 is expressed, induced by EGF, and required for type I collagen degradation in oral squamous cell carcinoma.

Topics: Biopsy; Blotting, Western; Carcinoma, Squamous Cell; Collagen; Epidermal Growth Factor; Humans; In Situ Hybridization; Matrix Metalloproteinase 1; Mouth Neoplasms; Neoplasm Invasiveness; Neoplasm Proteins

The small leucine-rich proteoglycan decorin interacts with the epidermal growth factor receptor (EGFR) and triggers a signaling cascade that leads to elevation of endogenous p21 and growth suppression. We demonstrate that decorin causes a sustained down-regulation of the EGFR. Upon stable expression of decorin, the EGFR number is reduced by approximately 40%, without changes in EGFR expression. However, EGFR phosphorylation is nearly completely abolished. Concurrently, decorin attenuates the EGFR-mediated mobilization of intracellular calcium and blocks the growth of tumor xenografts by down-regulating the EGFR kinase in vivo. Thus, decorin acts as an autocrine and paracrine regulator of tumor growth and could be utilized as an effective anti-cancer agent.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Signaling; Carcinoma, Squamous Cell; Cell Division; Decorin; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix Proteins; Female; Humans; Mice; Mice, Nude; Phosphorylation; Proteoglycans; Recombinant Proteins; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Argos, the inhibitor of the Drosophila epidermal growth factor (EGF) receptor, remains the only known extracellular inhibitor of this family of receptors in any organism. The functional domain of Argos includes an atypical EGF domain and it is not clear whether it binds to the EGF receptor or if it acts via a distinct receptor to reduce Egfr activity indirectly. Here we present two lines of evidence that strongly suggest that Argos directly interacts with the EGF receptor. First, Argos is unable to inhibit a chimeric receptor that contains an extracellular domain from an unrelated RTK, indicating the need for the EGF receptor extracellular domain. Second, Argos can inhibit the Drosophila EGF receptor even when expressed in human cells, implying that no other Drosophila protein is necessary for inhibition. We also report that Argos and the Drosophila activating ligand, Spitz, can influence mammalian RTK activation, albeit in a cell-type specific manner. This includes the first evidence that Argos can inhibit signalling in mammalian cells, raising the possibility of engineering an effective human EGF receptor/ErbB antagonist. Oncogene (2000) 19, 3560 - 3562

Topics: Animals; Carcinoma, Squamous Cell; Culture Media, Conditioned; Drosophila melanogaster; Drosophila Proteins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Eye Proteins; Humans; Insect Proteins; KB Cells; Ligands; MAP Kinase Signaling System; Membrane Proteins; Nerve Tissue Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein Structure, Tertiary; Receptor Protein-Tyrosine Kinases; Recombinant Fusion Proteins; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase Cgamma1 and activity of protein kinase Calpha (PKCalpha). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nm) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Carcinoma, Squamous Cell; Enzyme Activation; Epidermal Growth Factor; Humans; Isoenzymes; Kinetics; Lipoxygenase Inhibitors; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phospholipase C gamma; Phosphorylation; Protein Kinase C; Protein Kinase C-alpha; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases

The aim of this study was to compare the profile of EGFr expression in transitional cell carcinoma of the bladder (TCC) and in oral squamous cell carcinoma (OSCC). In addition, to study the influence of EGF stimulation on the expression of major histocompatibility complex class I antigens, placental alkaline phosphatase (PLAP) as well as changes in tumour cell sensitivity to cisplatin using immunocytochemical staining, a colorimetric assay and SDS-gel electrophoresis. The results showed that: a) strong EGFr expression could be seen in 22/88 (27%) cases of TCCs. In oral tumours the values for non-invasive ameloblastoma and invasive OSCC were 4/25 (16%) and 30/41 (73%) respectively. b) EGFr expression in tumour cell lines paralleled that of tumour biopsies. The number of lines expressing high and low EGFr expression amongst TCCs were 4 and 4 and in OSCCs were 3 and 1 respectively. c) Exposure of tumour cell lines to EGF led to: i) an increase in EGFr expression (stimulatory indices SI, ranged from 1.06 to 2.58) for TCCs but a decrease in the case of OSCCs (SI ranged from 0.01 to 0.85). The corresponding SI values for class I antigens were 0.95-1.16 and 0.10-0.84. ii) A significant reduction in expression of PLAP by OSCC cell lines. iii) An increased susceptibility of OSCC cell lines to cisplatin by as much as 14% (p<0.001). These data demonstrated the overexpression of EGFr in a significant proportion of TCCs. As for oral tumours it depended on whether they were of an invasive or non-invasive type. In the invasive cases the majority overexpressed EGFr. The exposure of OSCC but not TCC tumour cells to EGF resulted in down regulation of EGFr and class I antigens. The expression of PLAP was also significantly reduced. Exposure of OSCC cells to EGF resulted in their increased susceptibility to cisplatin. The data supports the notion that the mitogenic activation of some tumour cells by EGF resulted in a reduction of their immune visibility, differentiation status and an increase in chemosensitivity.

Topics: Alkaline Phosphatase; Antineoplastic Agents; Biopsy; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Cisplatin; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Histocompatibility Antigens Class I; Humans; Isoenzymes; Mouth Neoplasms; Neoplasms; Placenta; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms

We recently reported that multiple c-erbB ligands differentially modulate in vitro proliferation, invasion and expression of matrix metalloproteinases in human head and neck squamous carcinoma cells (HNSCC). In order to evaluate further the importance of c-erbB ligands in tumor progression, the expression and regulation of this growth factor family in HNSCC cells was studied. We demonstrate that mRNAs for the 6 major c-erbB ligands, namely, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), betacellulin (BTC), heparin-binding epidermal growth factor-like growth factor (HB-EGF), amphiregulin (AR) and heregulin (HRG), are expressed in a large panel of HNSCC cell lines. In addition to TGF-alpha, other ligands (notably BTC and HRG-beta1) are involved in the autocrine growth regulation of these cells. Each c-erbB ligand when applied exogenously, induced mRNA expression of both itself and the remaining family members and a differential response in the kinetics of induction was found. HB-EGF and HRG mRNAs were induced rapidly (within 1 hr) and to a greater extent (3.2-6.2- and 4.8-7. 3-fold increase) than TGF-alpha, BTC and AR mRNAs (1.6-2.7, 1.8-3.6- and 1.6-4.2-fold, respectively). This pattern was observed for all inducing ligands tested. Analysis of mRNA stability, and concurrent treatment with BTC (as an inducing ligand) and cycloheximide (to inhibit protein synthesis) suggested both transcriptional and post-transcriptional regulatory mechanisms. These results support and extend previous observations of c-erbB receptor signaling as a critical element in the pathogenesis and progression of HNSCC, and emphasize the role of autocrine ligand production.

Topics: Amphiregulin; Antibodies; Betacellulin; Carcinoma, Squamous Cell; Cell Division; EGF Family of Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Head and Neck Neoplasms; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Neuregulin-1; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

Dihydropyrimidine dehydrogenase (DPD) and pyrimidine nucleoside phosphorylase (PyNPase) are the first and rate-limiting enzymes that regulate 5-fluorouracil (5-FU) metabolism, and tumoral DPD activity appears to be a promising predictor of 5-FU sensitivity. However, the regulatory mechanisms determining these enzyme activities have not been fully understood. We investigated the biological effects of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha on cell growth and tumoral DPD and PyNPase activities, and the subsequent effects on 5-FU sensitivity in uterine cervical carcinoma SKG-IIIb cells. The treatment of tumor cells with EGF or TGF-alpha resulted in a concentration-dependent increase in tumor cell growth and PyNPase activity, whereas tumoral DPD activity was inhibited. Their stimulatory effects on tumor cell growth correlated well with PyNPase activity, but were inversely related to DPD activity (P < 0.01). 5-FU sensitivity of tumor cells increased in the presence of EGF or TGF-alpha. These growth factors were shown to stimulate the first, rate-limiting enzyme activity in 5-FU anabolism and to inhibit that in 5-FU catabolism, leading to enhancement of the antiproliferative action of 5-FU at achievable therapeutic levels. The tumor environmental factors, EGF and TGF-alpha, may act as intrinsic regulators of DPD and PyNPase activities that affect the 5-FU sensitivity of individual tumors.

Topics: Antimetabolites, Antineoplastic; Carcinoma, Squamous Cell; Cell Division; Dihydrouracil Dehydrogenase (NADP); Drug Synergism; Epidermal Growth Factor; Female; Fluorouracil; Humans; Oxidoreductases; Pentosyltransferases; Pyrimidine Phosphorylases; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Aberrant expression of platelet-derived growth factor and its receptor (PDGFR) has been implicated in various human disorders, including cardiovascular disease and certain types of cancer. Inhibitors of the tyrosine kinase activity of PDGFR are leads in the development of novel agents to combat these diseases. We describe here a novel, potent inhibitor of PDGFR tyrosine kinase, 3-(4-dimethylamino-benzylidenyl)-2-indolinone (DMBI). The compound also inhibits signal transduction through fibroblast growth factor receptor 1 (FGFR1), but is not active towards epidermal growth factor receptor (EGFR) or c-Src tyrosine kinase. The activity of DMBI and other tyrosine kinase inhibitors was compared in a cell-based assay as well as in an assay based on purified recombinant platelet-derived growth factor beta-receptor (beta-PDGFR) lacking the transmembrane and ligand-binding domain. We showed that this truncated beta-PDGFR could dimerize, and that dimerization was required for tyrosine kinase activity. Tyrosine kinase activity was modulated by inhibitors of beta-PDGFR autophosphorylation in cells, but not by specific inhibitors of EGFR or c-Src tyrosine kinase. We conclude that beta-PDGFR lacking the transmembrane and ligand-binding domain retains the essential properties of the full-length receptor tyrosine kinase.

Topics: 3T3 Cells; Animals; Becaplermin; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cytoplasm; Enzyme Inhibitors; Epidermal Growth Factor; Fibroblast Growth Factor 2; Growth Substances; Humans; Indoles; Kinetics; Mice; Muscle, Smooth, Vascular; Phosphorylation; Phosphotyrosine; Platelet-Derived Growth Factor; Proto-Oncogene Proteins c-sis; Pulmonary Artery; Receptor Protein-Tyrosine Kinases; Receptor, Platelet-Derived Growth Factor beta; Receptors, Platelet-Derived Growth Factor; Recombinant Proteins; Transfection; Tumor Cells, Cultured

Previous studies in our laboratory have shown that the major green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), suppressed autophosphorylation of epidermal growth factor (EGF) receptor induced by EGF in human A431 epidermoid carcinoma cells. In this study, we examined the inhibitory effects of black tea polyphenols, including theaflavin (TF-1), a mixture (TF-2) of theaflavin-3-gallate (TF-2a) and theaflavin-3'-gallate (TF-2b), theaflavin-3,3'-digallate (TF-3) and the thearubigin fraction on the autophosphorylation of the EGF and PDGF receptors in A431 cells and mouse NIH3T3 fibroblast cells, respectively. First, we examined the effects of these polyphenols on the proliferation of A431 and NIH3T3 cells. Both EGCG and TF-3 strongly inhibited the proliferation of A431 and NIH3T3 cells more than the other theaflavins did. In cultured cells with pre-treatment of tea polyphenol, TF-3 was stronger than EGCG on the reduction of EGF receptor and PDGF receptor autophosphorylation induced by EGF and PDGF, respectively. Other theaflavins slightly reduced the autophosphorylation of the EGF and PDGF receptors; furthermore, TF-3 could reduce autophosphorylation of the EGF receptor (or PDGF receptor) even with co-treatment with EGF (or PDGF) and TF-3, but EGCG was inactive under these conditions. In addition, TF-3 was stronger than EGCG in blocking EGF binding to its receptor. These results suggest that not only the green tea polyphenol, EGCG, but also the black tea polyphenol, TF-3, have an antiproliferative activity on tumor cells, and the molecular mechanisms of antiproliferation may block the growth factor binding to its receptor and thus suppress mitogenic signal transduction.

Topics: 3T3 Cells; Animals; Biflavonoids; Carcinoma, Squamous Cell; Catechin; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gallic Acid; Growth Inhibitors; Humans; Laryngeal Neoplasms; Mice; Phenols; Phosphorylation; Platelet-Derived Growth Factor; Polyphenols; Protein Binding; Protein Processing, Post-Translational; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Tea; Tumor Cells, Cultured

Because regional spread to lymph nodes without systemic spread is a relatively common event in squamous cell cancer of the head and neck (SCCHN), it is possible that lymphoid-related receptors or cytokines might directly impact the growth of these tumors. In the present study, we have shown by flow cytometry and Western blotting that the central lymphoid regulatory molecule, CD40, is expressed on the surface of all seven SCCHN tumor cell lines studied. Tumor cell lines also expressed epidermal growth factor (EGF) receptor, MHC class I, and CD95 (Fas) but did not uniformly express other important lymphoid regulatory molecules such as CD80, CD86, or interleukin (IL) 2 receptor components. CD40 ligation by trimeric CD40 ligand (CD40L) resulted in a 20-45% inhibition of tumor cell growth in three of seven cell lines tested. The cytokines IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-11, and IL-15 neither inhibited nor stimulated growth in any of the cell lines tested. EGF had pleiotropic effects on cell growth; it inhibited growth in two cell lines, stimulated growth in one cell line, and had no effect in four cell lines. When coligation by EGF and CD40L was studied, additive or supra-additive growth inhibition was seen in four cell lines. Three cell lines were unaffected by EGF, CD40, or coligation with both reagents. Examination of tumor tissues from 12 previously untreated patients representing a broad spectrum of patients presenting with SCCHN demonstrated CD40 expression in all 12 tumor specimens. This study supports the notion that CD40 is a regulatory molecule for the growth of SCCHN. The important role of CD40-CD40L interactions in the regulation of immune cells in the lymph node and the unique high-level expression of CD40L by these immune cells lend support to the hypothesis that this ligand/receptor pair is an important mediator of cell growth in SCCHN.

Topics: Antibodies, Monoclonal; Blotting, Western; Carcinoma, Squamous Cell; CD40 Antigens; CD40 Ligand; Cell Division; Cell Membrane; Cytokines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Flow Cytometry; Head and Neck Neoplasms; Humans; Immunohistochemistry; Immunophenotyping; Membrane Glycoproteins; Receptors, Cell Surface; Transforming Growth Factor alpha; Tumor Cells, Cultured

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

Topics: Actins; Antigens, Surface; Carbazoles; Carcinoma, Squamous Cell; Cell Membrane; Cell Size; Chemotaxis; Desmosomes; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Indoles; Integrin alpha6beta4; Integrins; Keratins; Laminin; Phosphorylation; Phosphoserine; Phosphotyrosine; Protein Kinase C; Pseudopodia; Signal Transduction; Tumor Cells, Cultured

The proteolytic activity of gelatinase A, a member of the matrix metalloproteinase (MMP) family, is considered to be a critical factor in tumor cell penetration of the extracellular matrix. To express catalytic activity, however, gelatinase A requires activation by another MMP, membrane type 1-matrix metalloproteinase (MT1-MMP). The head and neck squamous cell carcinoma cell line, UM-SCC-1, forms a quiescent monolayer atop collagen unless stimulated with epidermal growth factor (EGF; 3.5 nmol/L), which induces single cell invasion within 48 hours. To determine the role of the MT1-MMP/gelatinase A protease system in an in vitro stromal invasion model, expression vectors for MT1-MMP and gelatinase A were transfected into UM-SCC-1 (SCC-1/MT and SCC-1/gelA, respectively). SCC-1/MT tumor cells were found to invade in the absence of growth factor stimulation. Additionally, these cells displayed shorter onset to invasion and penetrated deeper into the collagen gel with EGF stimulation than did control vector transfectants. SCC-1/gelA cells similarly demonstrated invasion in the absence of EGF and a heightened invasive potential under EGF-stimulated conditions. These results suggest that the MT1-MMP/gelatinase A protease system participates in squamous cell carcinoma invasion of collagenous matrices.

Topics: Awards and Prizes; Carcinoma, Squamous Cell; Enzyme Activation; Epidermal Growth Factor; Extracellular Matrix; Humans; In Vitro Techniques; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Matrix Metalloproteinases, Membrane-Associated; Metalloendopeptidases; Neoplasm Invasiveness; Otorhinolaryngologic Neoplasms; Tumor Cells, Cultured

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on migration, invasion and proteinase expression of gynecological cultured cancer cells (SKG-IIIb cervical squamous cell carcinoma, OMC-4 cervical adenocarcinoma, SNG-M endometrial adenocarcinoma and OMC-3 ovarian adenocarcinoma), and whether these growth factors affect thymidine phosphorylase/platelet-derived endothelial cell growth factor expression of tumor cells. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were stimulated by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. The zymography of tumor-conditioned medium showed that the treatment of tumor cells with EGF and TGF-alpha resulted in the increase of type IV collagenases, stromelysin and urokinase-type plasminogen activator which was partly confirmed by immunoblot analysis. The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor which has angiogenic activity, was also upregulated by these growth factors. These results suggest that EGF and TGF-alpha act as positive regulators on the invasion process of gynecological tumor cells which may be associated with their stimulatory action on the motility of tumor cells, the expression of proteinases secreted by tumor cells and the angiogenic phenotype.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Movement; Endometrial Neoplasms; Epidermal Growth Factor; Female; Genital Neoplasms, Female; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Ovarian Neoplasms; Thymidine Phosphorylase; Transforming Growth Factor alpha; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Uterine Cervical Neoplasms

Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC-3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC-3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA-induced migration, but only partly inhibited EGF-induced migration. Protein kinase A was also involved in the EGF-induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC-3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF- and PMA-stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF- and PMA-stimulated HSC-3 cells, whereas the fluorescence signal was hardly detected in non-stimulated cells. Talin and beta1 integrin were immunolocalized at focal contacts in non-stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC-3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non-stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin-associated focal contacts were regarded as migratory phenotypes.

Topics: Carcinogens; Carcinoma, Squamous Cell; Cell Adhesion; Cell Movement; Epidermal Growth Factor; Fibronectins; Humans; Intercellular Junctions; Pseudopodia; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vinculin

The growth factor-activated mitogenic pathways are often disregulated in tumour cells and, therefore, they can provide specific molecular targets for novel anti-tumour approaches. 8-Chloro-cAMP (8-Cl-cAMP), a synthetic cAMP analogue, is a novel anti-tumour agent that has recently undergone clinical evaluation. We investigated the effects of 8-Cl-cAMP on the epidermal growth factor (EGF)/EGF receptor (EGF-R) signalling in human epidermoid cancer KB cells, which are responsive to the mitogenic stimulus of EGF. We found that the growth-promoting activity of EGF was completely abolished when EGF treatment was performed in combination with 8-Cl-cAMP. The inhibition of the EGF-induced proliferation by 8-Cl-cAMP was paralleled by the blockade of the EGF-stimulated activation of mitogen-activated protein kinases (MAPK), ERK-1 and ERK-2. Conversely, we found an increase of EGF-R expression and EGF-R tyrosine phosphorylation when KB cells were growth inhibited by 8-Cl-cAMP. Moreover, the activity of Raf-1 and MEK-1 protein kinases, the activators upstream MAPK in the phosphorylation cascade induced by EGF, was not modified in 8-Cl-cAMP-treated cells. We concluded that the impairment of KB cell response to EGF, induced by 8-Cl-cAMP, resides in the specific inhibition of MAPK/ERKs activity while the function of the upstream elements in the EGF-R signalling is preserved.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Carcinoma, Squamous Cell; Cell Division; DNA, Neoplasm; Enzyme Activation; Epidermal Growth Factor; Humans; Mitogen-Activated Protein Kinases; Signal Transduction; Stimulation, Chemical; Tumor Cells, Cultured

Evidence suggests that there is an association between the abnormal expression of members of the c-erbB receptor tyrosine kinase family and poor prognosis in head and neck squamous cell carcinomas (HNSCC). Until now, the relative contributions of different c-erbB ligands to HNSCC progression have not been clearly defined. In this paper we examined the effects of ligands with different c-erbB receptor specificities in terms of their stimulation of HNSCC proliferation, expression of matrix metalloproteinases (MMPs) and invasion. Heregulin-beta1 (HRG-beta1; selective c-erbB3/B4 ligand) was found to stimulate proliferation in the majority of cell lines, whereas epidermal growth factor (EGF; EGFR ligand) and betacellulin (BTC; EGFR/B4 ligand) induced variable responses. All three ligands up-regulated multiple MMPs including collagenases, stromelysins, matrilysin and gelatinase B (MMP-9) but had minimal or no effects on gelatinase A (MMP-2), MT1-MMP and tissue inhibitors of MMPs (TIMPs). MMP-9 mRNA was induced to a higher level than other MMPs, although with slower kinetics. HRG-beta1 was less active than EGF and BTC at the optimal concentration (relative potency of EGF:BTC:HRG = 3:4:1). In vitro invasion through Matrigel was also increased by all three ligands in proportion to their MMP up-regulation. A specific anti-EGFR monoclonal antibody (mAb ICR62) inhibited MMP up-regulation, migration and invasion induced by all three ligands, whereas an anti-c-erbB-2 mAb ICR12 inhibited mitogenic and motogenic responses following ligand stimulation but had no effect on MMP expression. These results suggest that c-erbB ligands may differentially potentiate the invasive phenotype of HNSCC via co-operative induction of cell proliferation, migration and proteolysis. The EGFR signalling pathway appears to be the dominant component controlling the proteolytic and invasive phenotype in HNSCC, whereas the c-erbB-2 signalling pathway is responsible, in part, for the mitogenic and motogenic effects of ligands.

Topics: Antibodies, Monoclonal; Betacellulin; Carcinoma, Squamous Cell; Cell Division; Culture Media, Conditioned; Enzyme Induction; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Growth Substances; Head and Neck Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Proteins; Neuregulin-1; Pharyngeal Neoplasms; Phenotype; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Tumor Cells, Cultured

Keratinocyte growth factor (KGF) has potent mitogenic activity on normal epithelial cells and has been found to enhance intestinal crypt cell survival in irradiated mice and to prevent radiation and chemotherapy-induced mucositis in animal models. The purpose of the study reported here is to investigate the effect of recombinant human KGF on the proliferation and survival of human squamous carcinoma cell lines following irradiation.. The level of KGF receptor (KGFR) mRNA in normal Balb/Mk cell line and human head and neck squamous carcinoma cell lines was assessed using a RNase protection assay. The clonogenic assay and MTT assay were used to study the proliferative effects of KGF on human tumor cell lines and Balb/MK cell line in vitro. Effects of KGF on in vivo tumor growth and radiosensitivity were studied in three KGFR-positive human squamous cell carcinoma xenografts (FaDu, Detroit 562 and A431) in nude mice, and a murine KGFR-negative melanoma tumor (B16) in Balb/c mice.. Seven of 10 tumor cell lines studied expressed KGFR mRNA. None of these tumor cell lines showed enhanced proliferation when exposed to KGF for 2 days or less. Prolonged exposure to KGF for 7 days or longer resulted in low level stimulation of proliferation in both clonogenic and MTT assays in four of seven KGFR-positive cell lines. Two KGFR-negative cell lines also had a low proliferative response to KGF in a clonogenic assay, but not in the MTT assay. Normal keratinocyte Balb/MK cells, which expressed a moderate level of KGFR mRNA, had a strongly proliferative response to KGF. Its KGF enhancement ratio (KER) of plating efficiency was 24-70 times higher than that of the tumor cells studied (p < 0.001). The KGF-stimulated tumor cell growth was almost completely inhibited by heparin or epidermal growth factor (EGF). There were no significant differences (p > 0.05) in the survival of any of tumor cell lines in the presence or absence of KGF (100 ng/ml) irradiated with doses of 0-15 Gy, and no significant differences (p > 0.05) between the radiobiological parameters D0, Dq, and n number from the SHMT model, alpha, beta, and alpha/beta ratio from the LQ model and SF2 for radiation survival curves for cell lines irradiated in the presence or absence of KGF. Three KGFR-positive human squamous cell carcinoma xenografts in nude mice, and a murine KGFR-negative melanoma tumor in Balb/c mice treated with 1.0 mg/kg of KGF for 3 days grew at the same rate as in untreated mice.. The recombinant human KGF resulted in little or no stimulation of the proliferation of human head and neck squamous tumor cell lines and did not affect the radiosensitivity of these cell lines in vitro and in vivo. Therefore, KGF may be of clinical value in preventing radiation-induced mucositis and may have the potential to increase the therapeutic index of radiotherapy for treatment of cancers.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Survival; Colony-Forming Units Assay; Epidermal Growth Factor; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Growth Substances; Humans; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; Receptor, Fibroblast Growth Factor, Type 2; Receptors, Fibroblast Growth Factor; Receptors, Growth Factor; Recombinant Proteins; RNA, Messenger; Tumor Cells, Cultured

We set out to investigate the interactions between malignant transformation of keratinocytes, presence of oncoproteins and immunosurveillance in squamous cell carcinoma (SCC) and in a preneoplastic lesion, actinic keratosis (AK).. Samples of SCC, AK and normal skin (NS) were subjected to quantitative analysis using the following antibodies: anti-p53, Ki67, OKT6, OK-DR, B7/BB1, anti-CD54, anti-CD11, OKT3, OKT4, OKT8; positivity for ras-p21, EGFr and bcl-2 was evaluated by semiquantitative analysis.. Oncoprotein alterations and increased keratinocyte proliferative activity were observed both in AK and SCC. The number of Langerhans cells (CD1a+ cells) was similar in the two lesions but lower in SCC compared to AK. The proportion of CD1a(+)-B7/BB1+ cells was slightly higher in AK and SCC than in NS. The Langerhans cells expressed the HLA-DR antigen in all groups. Values were highest in AK and NS, and quite low in SCC. Cytotoxic T lymphocytes were more numerous in SCC than in AK and NS. Interestingly, the total CD4/CD8 ratio was much lower in SCC than in AK and NS, which indicates an increase in the CD8+ subpopulation in samples of SCC. In the epithelia of SCC samples there were a considerable number of B7/BB1+ keratinocytes.. We suggest that alterations in the immunodefence mechanisms have an important role in the transformation of AK into SCC, and that these changes affect not only lymphocytes, but also professional (i.e., Langerhans cells) and non-professional (i.e., keratinocytes) antigen presenting cells.

Topics: Aged; Analysis of Variance; Biopsy, Needle; Carcinoma, Squamous Cell; CD3 Complex; CD4 Antigens; CD8 Antigens; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Female; HLA-DR Antigens; Humans; Immunohistochemistry; Intercellular Adhesion Molecule-1; Keratinocytes; Keratosis; Male; Middle Aged; Oncogene Proteins; Precancerous Conditions; Skin Neoplasms

Mutations in receptors for the vitamin A metabolite retinoic acid (RAR) that repress retinoic acid (RA)-responsive gene expression have been identified and characterized. We previously reported an absence of target gene response to RA in all but one of a series of transformed human epithelial cell lines. To elucidate the mechanisms of this unresponsiveness, we created stable transfectants that expressed an RARalpha mutant (RARalpha403) previously shown to have dominant negative activity due to a C-terminal truncation. All clones exhibited repressed RA-responsive gene expression. These cells grew slowly and demonstrated greater growth inhibition by RA. Pretreatment of both control and experimental groups with RA enhanced epidermal growth factor-induced proliferation despite RA-dependent downregulation of epidermal growth factor receptor expression. In addition, clones expressing the mutant RARalpha were 60% less invasive in an in vitro assay. This reduced invasiveness correlated with decreased gelatinase activity in these cells. We showed for the first time that a dominant negative mutation in RARalpha can function as a tumor suppressor in transformed epithelial cells.

Topics: Carcinoma, Squamous Cell; Cell Division; Clone Cells; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; Receptors, Retinoic Acid; Recombinant Proteins; Retinoic Acid Receptor alpha; Sequence Deletion; Transfection; Tretinoin; Tumor Cells, Cultured

erbB2, a member of the epidermal growth factor (EGF) receptor-type tyrosine kinase receptor family, is overexpressed in breast carcinomas with poor prognosis. We examined the cell surface association of this receptor with itself and with other cell surface proteins by the Förster-type fluorescence resonance energy transfer using whole antibodies and Fab fragments. We found that erbB2 molecules homoassociate in unstimulated SK-BR-3, BT474 and BT474-M (a metastatic version of the parent BT474 line) breast tumor cells, and that the interaction was enhanced by EGF treatment in suspensions of SK-BR-3 and BT474-M cells. BT474 cells (with low EGF receptor expression) and attached SK-BR-3 cells do not respond to EGF. Image microscopic energy transfer measurements found considerable pixel-by-pixel heterogeneity in the homoassociation state of erbB2. In accordance with the EGF-induced redistribution of erbB2, EGF receptor was found to be in close proximity to erbB2 in FRET measurements. By labeling different epitopes on erbB2 and the lipid bilayer, we were able to prepare an epitope map of erbB2 molecule. Our data suggest the existence of dynamic cell surface patterns of erbB2 and point to functions fulfilled by these molecular complexes.

Topics: Breast Neoplasms; Carcinoma, Squamous Cell; Cell Membrane; Energy Transfer; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Humans; Models, Molecular; Receptor, ErbB-2; Receptors, Transferrin; Tumor Cells, Cultured

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor alpha (TGFalpha), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFalpha mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Amphiregulin; Animals; Betacellulin; Carcinogens; Carcinoma, Squamous Cell; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Ligands; Mice; Mice, Inbred SENCAR; Papilloma; RNA, Messenger; Skin; Skin Neoplasms; Transforming Growth Factor alpha; Up-Regulation

The known 2-aminoimidazole alkaloid naamidine A (1) was isolated from a Fijian Leucetta sp. sponge as an inhibitor of the epidermal growth factor (EGF) receptor. The compound exhibited potent ability to inhibit the EGF signaling pathway and is more specific for the EGF-mediated mitogenic response than for the insulin-mediated mitogenic response. Evaluation in an A431 xenograft tumor model in athymic mice indicated that naamidine A exhibited at least 85% growth inhibition at the maximal tolerated dose of 25 mg/kg. Preliminary mechanism of action studies indicate that the alkaloid fails to inhibit the binding of EGF to the receptor and has no effect on the catalytic activity of purified c-src tyrosine kinase.

Topics: 3T3 Cells; Alkaloids; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; CSK Tyrosine-Protein Kinase; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Imidazoles; Mice; Mice, Nude; Neoplasm Transplantation; Porifera; Protein-Tyrosine Kinases; src-Family Kinases; Transplantation, Heterologous

The regulation of parathyroid hormone-related protein expression by colchicine, vinblastine, nocodazole, taxol, transforming growth factor-beta1 (TGFbeta1), and epidermal growth factor (EGF) was investigated in a canine squamous carcinoma cell line (SCC 2/88 cells). SCC 2/88 cells were stably transfected with a human P2/P3 PTHrP promoter-luciferase reporter gene construct and gene expression was measured after chemical treatments. The greatest increase in reporter gene expression was observed after colchicine treatment and small increases occurred after treatment with vinblastine, taxol, TGFbeta1, or EGF. Nocodazole had no significant effect on reporter gene expression. Colchicine also increased PTHrP steady state mRNA expression and PTHrP secretion by SCC 2/88 cells. These results demonstrated that PTHrP production was increased in SCC 2/88 cells by colchicine and suggested that factors or events during mitosis are capable of stimulating PTHrP production. An increase in PTHrP production during mitosis of malignant epithelial cells may be important in the pathogenesis of humoral hypercalcemia of malignancy.

Topics: Animals; Blotting, Northern; Carcinoma, Squamous Cell; Colchicine; Dogs; Epidermal Growth Factor; Gene Expression Regulation; Luciferases; Lumicolchicines; Neoplasm Proteins; Nocodazole; Paclitaxel; Parathyroid Hormone-Related Protein; Proteins; Radioimmunoassay; RNA, Messenger; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Vinblastine

The roles of protein-tyrosine phosphatases (PTPs) in processes such as cell growth and adhesion are poorly understood. To explore the ability of specific PTPs to regulate cell signaling pathways initiated by stimulation of growth factor receptors, we expressed the receptor-like PTP, PTPalpha, in A431 epidermoid carcinoma cells. These cells express high levels of the epidermal growth factor (EGF) receptor and proliferate in response to the autocrine production of transforming growth factor-alpha. Conversely, EGF stimulation of A431 cells in vitro leads to growth inhibition and triggers the rapid detachment of these cells from the substratum. Although PTPalpha expression did not alter the growth characteristics of either unstimulated or EGF-stimulated cells, this phosphatase was associated with increased cell-substratum adhesion. Furthermore, PTPalpha-expressing A431 cells were strikingly resistant to EGF-induced cell rounding. Overexpression of PTPalpha in A431 cells was associated with the dephosphorylation/activation of specific Src family kinases, suggesting a potential mechanism for the observed alteration in A431 cell-substratum adhesion. Src kinase activation was dependent on the D1 catalytic subunit of PTPalpha, and there was evidence of association between PTPalpha and Src kinase(s). PTPalpha expression also led to increased association of Src kinase with the integrin-associated focal adhesion kinase, pp125(FAK). In addition, paxillin, a Src and/or pp125(FAK) substrate, displayed increased levels of tyrosine phosphorylation in PTPalpha-expressing cells and was associated with elevated amounts of Csk. In view of these alterations in focal adhesion-associated molecules in PTPalpha-expressing A431 cells, as well as the changes in adhesion demonstrated by these cells, we propose that PTPalpha may have a role in regulating cell-substratum adhesion.

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Adhesion; Cell Adhesion Molecules; Cell Division; Cell Size; Cloning, Organism; Cytoskeletal Proteins; Epidermal Growth Factor; ErbB Receptors; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Kinetics; Molecular Sequence Data; Paxillin; Peptide Fragments; Phosphopeptides; Phosphoproteins; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Receptor, Insulin; Recombinant Proteins; Rosaniline Dyes; src Homology Domains; Substrate Specificity; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Normal as well as neoplastic cells traverse extracellular matrix barriers by mobilizing proteolytic enzymes in response to epidermal growth factor (EGF)-EGF receptor (EGFR) or hepatocyte growth factor/scatter factor (SF)-c-Met interactions. The plasminogen activator-plasminogen axis has been proposed to play a key role during cell invasion, but the normal development of plasminogen activator- as well as that of plasminogen-deficient mice supports the existence of alternate proteolytic systems that permit cells to traverse extracellular matrix barriers. To characterize the role that matrix-degrading proteinases play in EGF- or SF-stimulated invasion, a human squamous carcinoma cell line (UM-SCC-1) was triggered atop the matrices of type I collagen or human dermal explants in a three-dimensional culture system. During EGF- or SF-induced invasion, UM-SCC-1 cells expressed urokinase-type plasminogen activator (uPA) and uPA receptor as well as the matrix metalloproteinases (MMPs), membrane-type MMP-1, collagenase 1, stromelysin 1, and gelatinase B. Despite the presence of a positive correlation between uPA receptor-uPA expression and growth factor-stimulated invasion, UM-SCC-1 invasion was not affected by inhibitors directed against the plasminogen activator-plasminogen axis. In contrast, both recombinant and synthetic MMP inhibitors completely suppressed invasion by either EGF- or SF-stimulated cells without affecting either proteinase expression or cell motility across collagen-coated surfaces. These data demonstrate that MMPs, but not the plasminogen activator-plasmin system, can directly regulate the ability of either EGF- or SF-stimulated tumor cells to invade interstitial matrix barriers.

Topics: Animals; Carcinoma, Squamous Cell; Cell Movement; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; Metalloendopeptidases; Neoplasm Invasiveness; Rats; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The epidermal growth factor receptor (EGF-R) and its major ligands EGF and transforming growth factor alpha (TGF alpha) play an important role in the development of multiple human tumors. However, little is known of the comparative effects of each ligand on the regulation of EGF-R expression. To investigate this issue we used two similar human epidermoid cancer cell lines that overexpress EGF-Rs (KB and A431). In KB cells, EGF and TGF alpha increased EGF-R mRNA and protein levels by 2-3 fold over 8 h, associated with a greater than 4-fold stabilization of EGF-R mRNA half-life. EGF and TGF alpha also increased transcription of EGF-R mRNA 2-3-fold in KB cells. In contrast, EGF and TGF alpha only minimally increased EGF-R mRNA and protein in A431 cells, without changing EGF-R mRNA half-life. Basal EGF-R mRNA half-life was 2 fold greater in A431 cells than in KB cells (6-7 h versus 2-3 h), whilst the half-life of a mutant 2.6 kb EGF-R mRNA present in A431 cells, which lacks the 3-untranslated region (3'-UTR), was 2 fold greater than the full-length EGF-R mRNA. RNA gel-shift studies demonstrated that KB and A431 cells contain cytoplasmic proteins that bind specifically to an AU-rich sequence from the 3'-UTR of EGF-R mRNA. Taken together, these results demonstrate that in KB cells EGF and TGF alpha upregulate EGF-R expression at both transcriptional and post-transcriptional levels. The identification of AU-rich EGF-R mRNA-specific RNA-binding proteins from epidermoid cancer cells that overexpress EGF-Rs suggests that regulated RNA-protein interactions involving this region may play a central role in modulating EGF-R mRNA stability.

Topics: 3' Untranslated Regions; Carcinoma, Squamous Cell; Cycloheximide; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

Protein phosphorylation/dephosphorylation on tyrosine residues are regulated by tyrosine kinases/phosphatases. The tyrosine phosphatase SH-PTP2 (PTP1D, PTP2C) interacts physically through its SH2 domain with phosphorylated epidermal growth factor receptor (EGFR). In KB cells, an oral epidermoid carcinoma, high epidermal growth factor (EGF) concentrations (10-9, 10-8 and 10-7 M) downregulate its receptor for the duration of the incubation with EGF. Thus, it was hypothesized that in KB cells, SH-PTP2 expression would also be downregulated by high EGF concentrations. This hypothesis was investigated by incubating the KB cells with increasing concentrations of EGF (0, 10-11, 10-10, 10-9, 10-8, 10-7 M) and by evaluating the expression of SH-PTP2 protein under these conditions. This study showed that EGF 10-7 and 10-8 M significantly decreased SH-PTP2 protein expression compared to controls. EGF 10-10 and 10-11 M did not change the expression of SH-PTP2 protein. We conclude that high EGF concentrations downregulate the expression of SH-PTP2 protein.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Growth Factor; Humans; Intracellular Signaling Peptides and Proteins; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; SH2 Domain-Containing Protein Tyrosine Phosphatases; src Homology Domains; Time Factors; Tumor Cells, Cultured

The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with a gene encoding human epidermal growth factor (hEGF). The hybrid human protein was isolated from Escherichia coli inclusion bodies, refolded and purified to homogeneity. The fusion protein competed with 125I-hEGF for binding to hEGF receptors (EGFR) and had ribonucleolytic activities approaching those of hpRNase1. Several conformations having different enzymatic activities could be detected after reversed-phase high-performance liquid chromatographic analysis, the less hydrophobic molecules being the most active. The hybrid protein was specifically cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line, with an IC50 of approximately 10(-7) M. In contrast, recombinant hpRNase1 had an IC50 higher than 10(-4) M. A mixture of free hEGF and free hpRNasel was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected in EGFR-deficient control cells. Taken together, these data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast cancers overexpressing EGFR, which correlate with a poor prognosis and cannot be cured by surgery alone. Engineering hybrid molecules with endogenous human proteins for targeted therapy may alleviate the dose-limiting immunogenicity and toxicity of conventional immunotoxins.

Topics: Binding, Competitive; Carcinoma, Squamous Cell; Cell Death; Chromatography, High Pressure Liquid; Enzyme Stability; Epidermal Growth Factor; ErbB Receptors; Escherichia coli; Gene Expression; Humans; Immunotoxins; Pancreas; Protein Folding; Recombinant Fusion Proteins; Ribonucleases; Tumor Cells, Cultured

Epidermal growth factor (EGF), determined by immunoprecipitation and Western blot analysis, increased both enzyme activity and protein level of 12-lipoxygenase in the solubilized microsomes of human epidermoid carcinoma A431 cells, respectively. The EGF-induced expression of 12-lipoxygenase mRNA was inhibited by transcription inhibitors such as actinomycin D and 5,6-dichlorobenzimidazole riboside. Promoters of different lengths for human 12-lipoxygenase gene were used to prepare the luciferase fusion vectors. These construct plasmids were transiently transfected into A431 cells, and the induction of luciferase expression by EGF was examined. A 4- to 6-fold increase in luciferase reporter activity stimulated by EGF for 18 h treatment was observed in plasmids with the 5'-flanking region length of -951 bp and that of -224 bp upstream from translation starting site. The time-dependent induction of luciferase activity by EGF paralleled the EGF-induced enzyme activity and expression of 12-lipoxygenase protein. Taken together, the results of this study indicate that EGF enhanced the transcription of the human 12-lipoxygenase gene, resulting in an increase in the amount and activity of 12-lipoxygenase.

Topics: Arachidonate 12-Lipoxygenase; beta-Galactosidase; Carcinoma, Squamous Cell; Cytosol; Dactinomycin; Dichlororibofuranosylbenzimidazole; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation; Genes, Reporter; Humans; Luciferases; Microsomes; Nucleic Acid Synthesis Inhibitors; Promoter Regions, Genetic; RNA, Messenger; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The functional 5' flanking region of the human 12-lipoxygenase in epidermoid carcinoma A431 cells was characterized. By a primer extension method, the transcription initiation sites were mapped at -47 adenosine, -48 guanosine and -55 guanosine upstream of the ATG translation start codon. Transient transfection with a series of 5' and 3' deletion constructs showed that the 5' flanking region spanning from -224 to -100 bp was important for the basal expression of 12-lipoxygenase gene. Gel mobility shift assays with antibodies of transcription factors showed that both Sp1 and Sp3 required highly GC-rich Sp1 sites within this region for binding. Disruption of two Sp1 recognition motifs residing at -158 to -150 bp and -123 to -114 bp by site-directed mutagenesis markedly reduced the basal 12-lipoxygenase promoter activity and abolished the retarded bands in a gel-shift assay, indicating that these two Sp1-binding sites were essential for gene expression. The same two Sp1-binding sites in this promoter region were also responsible for epidermal growth factor (EGF)-induced expression of 12-lipoxygenase gene. Moreover, EGF also induced the transcriptional activation of luciferase driven by SV40 early promoter, which contained rich Sp1-binding sites. Taken together, the results suggest that two specific Sp1 consensus sites are involved in the mediation of the basal promoter activity as well as EGF induction of the 12-lipoxygenase gene and that Sp1 and Sp3 transcription factors might have a role in their regulation.

Topics: Adenosine; Animals; Arachidonate 12-Lipoxygenase; Base Sequence; Binding Sites; Carcinoma, Squamous Cell; Codon; Consensus Sequence; DNA Primers; Epidermal Growth Factor; Gene Expression Regulation; Genes, Reporter; Guanosine; Humans; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Sequence Deletion; Sp1 Transcription Factor; Transcription, Genetic; Transfection; Tumor Cells, Cultured

We previously found that interferon alpha2 recombinant (IFNalpha) increases the expression of epidermal growth factor receptor (EGF-R) in the human epidermoid cancer KB cell line. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on KB cell cycle kinetics. IFNalpha (1000 i.u./ml) for 48 h decreased the S-phase fraction and diminished the expression of Ki67 and proliferating cell nuclear antigen on KB cells. Incubation of IFNalpha-treated KB cells with 10 nM EGF for 12 h reversed these effects. We then studied several biochemical markers of cell proliferation. Ornithine decarboxylase activity was decreased to about one-tenth by IFNalpha and partly restored by EGF. Hypusine is contained only in eukaryotic initiation factor 5A and its levels are correlated with cell proliferation. IFNalpha decreased hypusine synthesis by 75%; exposure of cells to EGF for 12 h restored hypusine synthesis almost completely. We also studied the effects of IFNalpha on the cytotoxicity of the recombinant toxin TP40, which inhibits elongation factor 2 through EGF-R binding and internalization. IFNalpha greatly enhanced the TP40-induced inhibition of protein synthesis in KB cells. In conclusion, IFNalpha, which affects protein synthesis machinery and increases EGF-R expression, enhances the tumoricidal activity of TP40 and hence could be useful in the setting of anti-cancer therapy.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Epidermal Growth Factor; Exotoxins; Humans; Interferon alpha-2; Interferon-alpha; Lysine; Ornithine Decarboxylase; Oropharyngeal Neoplasms; Recombinant Proteins; Transforming Growth Factor alpha; Tumor Cells, Cultured

Cyclooxygenase-2 (Cox-2), the inducible form of cyclooxygenase, is up-regulated in tumors and transformed cells. Because this enzyme catalyzes the formation of prostaglandins from arachidonic acid, chemopreventive strategies that suppress its expression could be useful for preventing cancer. We investigated whether retinoids suppressed basal expression of Cox-2 or EGF-mediated induction of Cox-2 in human oral squamous carcinoma cells. Treatment with retinoids [all-trans-retinoic acid (all-trans-RA), 9-cis-RA, 13-cis-RA, and retinyl acetate] suppressed both basal levels of Cox-2 and EGF-mediated induction of Cox-2 protein and synthesis of prostaglandin E2. Retinoids also suppressed the induction of Cox-2 mRNA by EGF. Transient transfection experiments showed that EGF caused about a 100% increase in Cox-2 promoter activity, an effect that was suppressed by retinoids. Levels of epidermal growth factor receptor were unaffected by retinoids. Epidermal growth factor caused a nearly 10-fold increase in mitogen-activated protein kinase activity; this effect was not blocked by retinoids.

Topics: Carcinoma, Squamous Cell; Cyclooxygenase 2; Epidermal Growth Factor; Humans; Isoenzymes; Membrane Proteins; Mouth Neoplasms; Prostaglandin-Endoperoxide Synthases; Retinoids; Transcription, Genetic; Tumor Cells, Cultured

We demonstrated the effect of epidermal growth factor (EGF) on the production of PGE2 in human squamous carcinoma A431 cells. The production of PGE2 was increased by stimulating the cells with EGF for 2 h and reached a maximum for 10 h. EGF was also found to augment the release of arachidonic acid (AA) following the increase in phospholipase A2 (PLA2) activity (1.7-fold). The induced PLA2 activity was diminished by 4-bromophenacyl bromide, but not by dithiothreitol, indicating that the EGF-induced release of AA was due to the increase in the activity of cytosolic PLA2 (cPLA2). On the other hand, cyclooxygenase (COX) activity was increased (1.6-fold) within 2 h after the EGF-treatment and the induced activity was inhibited by cycloheximide. In addition, Northern blot analysis showed that the level of COX-2 mRNA was increased by the EGF-treatment, whereas no COX-2 mRNA was detected in the untreated cells, indicating that the EGF-induced COX activity was resulted from the increase in the production of COX-2. These results suggest that EGF augments the production of PGE2 by increasing not only the activity of cPLA2 but also the production of COX-2 in A431 cells.

Topics: Acetophenones; Arachidonic Acid; Blotting, Northern; Carcinoma, Squamous Cell; Cloning, Molecular; Cycloheximide; Cyclooxygenase 2; Dinoprostone; Dithiothreitol; DNA, Complementary; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Membrane Proteins; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Tumor Cells, Cultured

Suramin disrupts several kinds of growth factor receptors. Since human esophageal squamous cell carcinoma expresses abundant epidermal growth factor receptors (EGFR) and proliferates in an autocrine and paracrine manner, it was expected that suramin inhibits tumor growth by disrupting EGFR.. We studied the effect of suramin on the human esophageal squamous cell carcinoma cell line KEsC-II in vitro and in an animal model.. Cell proliferation was stimulated at a low concentration and inhibited at a high concentration of suramin in vitro. Since autophosphorylation of EGFR was stronger at the low concentration and weaker at the high concentration of suramin compared with the control, the effect of suramin was thought to be via phosphorylation of receptors. In the animal model tumor growth was significantly stimulated in the suramin-treated group compared with the control group, and the BrdU labeling index was also higher in the suramin-treated group.. As it was impossible to increase the dose of suramin intravenously because of side effects, administration of suramin by another method, such as subcutaneous injection around the tumor, may increase the concentration of suramin in the tumor tissue and promote the anti-tumor effect of suramin.

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Disease Models, Animal; Dose-Response Relationship, Drug; Epidermal Growth Factor; Esophageal Neoplasms; Humans; Immunoblotting; Immunohistochemistry; Injections, Intravenous; Male; Mice; Mice, Nude; Neoplasm Transplantation; Reference Values; Suramin; Tumor Cells, Cultured

The overexpression of the human epidermal growth factor receptor (EGFR) has been demonstrated in many malignancies like squamous cell carcinoma of the head and neck, cervix, breast etc. which are most prevalent in India. This is often associated with poor prognosis and high mortality in these patients. Monoclonal antibodies generated against EGFR which inhibit binding of ligands like EGF to their receptor have anti-tumor activity and hence therapeutic application. One such monoclonal antibody designated as CIBCNSH3 generated in our laboratory has been found to recognize an epitope in the extracellular domain of EGFR by immunoprecipitation. By immunoperoxidase test this antibody exhibited strong reactivity to EGFR in head and neck cancers and breast cancers studied. It also inhibited the binding of Epidermal Growth Factor (EGF) to its receptor on MDA MB468 breast cancer cells rich in EGFR as revealed by competitive binding assay using 125I EGF, indicating its anti-tumor activity. The in vivo therapeutic efficacy has been demonstrated by injecting i.p. into tumor bearing mice 200 micrograms of the antibody for 4 consecutive days and then 100 micrograms twice a week resulting in complete regression of tumors of initial tumor size of 0.5-1.0 cm diameter. These results were compared with a control antibody against EGFR and also a nonspecific antibody which were administered to different groups of animals. In vivo studies performed using cell lines in culture like MDA MB468, MDA MB157 and HN5 with overexpression of EGFR revealed 98% cell death when incubated with different concentrations of the antibody. This monoclonal antibody seems to have a promising future application as therapeutic agent for tumors which overexpress EGFR.

Topics: Animals; Antibodies, Monoclonal; Binding, Competitive; Breast Neoplasms; Carcinoma; Carcinoma, Squamous Cell; Cell Division; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Epitopes; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Immunization, Passive; Mammary Neoplasms, Experimental; Mice; Multiple Myeloma; Neoplasm Proteins; Radioimmunodetection; Transplantation, Heterologous; Tumor Cells, Cultured

Dynamics of nuclear translocation of transcription factor Stat1 in human epidermoid carcinoma A431 cells in response to the epidermal growth factor (EGF) was examined by immunofluorescent microscopy and in cytoplasmic and nuclear extracts by Western blot. In has been shown that a prolonged presence of EGF induces a rapid tyrosine phosphorylation and nuclear translocation of Stat1 (within 5 min). The maximum amount of this protein in the nucleus was reached 30 min after the cell treatment to be maintained at the same level for 5 h. To study the dynamics of the export of Stat1 from the nucleus, a gentle treatment of cells with acetate buffer, pH 4.5, was used for extracting the surface-bound EGF. In this case, a complete dephosphorylation of Stat1 in the cytoplasm was observed in 30 min and the export of the Stat1 from the nucleus lasted for 1-6 h. These studies suggest the existence of an EGF-dependent dynamic equilibrium between the import and export of Stat1 in A431 cells.

Topics: Acetates; Binding Sites; Biological Transport; Blotting, Western; Buffers; Carcinoma, Squamous Cell; Cell Nucleus; Cytoplasm; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Humans; Hydrogen-Ion Concentration; Microscopy, Fluorescence; Phosphorylation; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Tyrosine

Topics: Arachidonate 12-Lipoxygenase; Biotin; Carcinoma, Squamous Cell; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Phalloidine; Phthalimides; Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Topics: Arachidonate 12-Lipoxygenase; Arachidonic Acid; Blotting, Western; Carcinoma, Squamous Cell; Cytosol; Enzyme Induction; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Microsomes; Neoplasm Proteins; RNA, Messenger; Tumor Cells, Cultured

Tea polyphenols are known to inhibit a wide variety of enzymatic activities associated with cell proliferation and tumor progression. The molecular mechanisms of antiproliferation are remained to be elucidated. In this study, we investigated the effects of the major tea polyphenol (-)-epigallocatechin gallate (EGCG) on the proliferation of human epidermoid carcinoma cell line, A431. Using a [3H]thymidine incorporation assay, EGCG could significantly inhibit the DNA synthesis of A431 cells. In vitro assay, EGCG strongly inhibited the protein tyrosine kinase (PTK) activities of EGF-R, PDGF-R, and FGF-R, and exhibited an IC50 value of 0.5-1 microgram/ml. But EGCG scarcely inhibited the protein kinase activities of pp60v-src, PKC, and PKA (IC50 > 10 micrograms/ml). In an in vivo assay, EGCG could reduce the autophosphorylation level of EGF-R by EGF. Phosphoamino acid analysis of the EGF-R revealed that EGCG inhibited the EGF-stimulated increase in phosphotyrosine level in A431 cells. In addition, we showed that EGCG blocked EGF binding to its receptor. The results of further studies suggested that the inhibition of proliferation and suppression of the EGF signaling by EGCG might mainly mediate dose-dependent blocking of ligand binding to its receptor, and subsequently through inhibition of EGF-R kinase activity.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Catechin; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Phosphotyrosine; Receptor Protein-Tyrosine Kinases; Receptors, Fibroblast Growth Factor; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Tea; Tumor Cells, Cultured

The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) results in the dimerization and self-phosphorylation of the receptor. Both of these responses were followed as a function of time and the concentration of EGF receptor. Dimerization of EGF receptor was monitored by immunoblotting the protein after it had been cross-linked with glutaraldehyde. The capacity for self-phosphorylation was followed by measuring the relative level of incorporation of [32P]phosphate into EGF receptor on autoradiograms of the same immunoblots used for the assay of its dimerization. When these two properties were followed as a function of time, it was found that dimerization preceded the appearance of the capacity for self-phosphorylation. Both dimeric and monomeric forms of EGF receptor were self-phosphorylated in the presence of EGF, but the dimeric form was phosphorylated preferentially to the monomeric form. When the dimerization and the capacity for self-phosphorylation were followed as a function of the concentration of dimeric EGF receptor, it was observed that the self-phosphorylation of dimeric EGF receptor increased as the concentration of dimeric EGF receptor increased. An equation including terms representing both intramolecular and intermolecular rates of self-phosphorylation was fit to the plots of self-phosphorylation as a function of concentration of EGF receptor. These fits demonstrate that intramolecular self-phosphorylation within dimers of EGF receptor is insignificant and that self-phosphorylation is an intermolecular process between dimers of EGF receptor.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cross-Linking Reagents; Dimerization; Epidermal Growth Factor; ErbB Receptors; Glutaral; Humans; Kinetics; Phosphorus Radioisotopes; Phosphorylation; Radioisotope Dilution Technique; Tumor Cells, Cultured

We investigated the effects of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha on migration, invasion and matrix metalloproteinase (MMP) expression of uterine cervical-carcinoma SKG-IIIb cells, and whether these growth factors affect pyrimidine-nucleoside-phosphorylase (PyNPase) activity and 5'-deoxy-5-fluorouridine (5'-dFUrd) sensitivity of tumor cells. Tumor-cell migration along a gradient of substratum-bound fibronectin and invasion into reconstituted basement membrane were stimulated by 0.1 to 100 ng/ml of EGF and TGF-alpha in a concentration-dependent manner. The zymography of tumor-conditioned medium showed that the treatment of tumor cells with EGF and TGF-alpha resulted in an increase of the 92-kDa type-IV collagenase (MMP-9), which was confirmed by immunoblot analysis. These growth factors also up-regulated the expression of PyNPase activity of tumor cells and consequently enhanced the anti-proliferative action of 5'-dFUrd, a cytostatic that is biotransformed to 5-fluorouracil (5-FUra) by PyNPase. However, EGF and TGF-alpha did not have significant effects on the 5-FUra sensitivity of tumor cells. These results suggest that EGF and TGF-alpha, tumor environmental factors, simultaneously up-regulate the potential of uterine cervical-carcinoma cells to invade extracellular matrices and their PyNPase activity, which are subsequently associated with the specific action of 5'-dFUrd selectively killing tumor cells of gynecological origin with high invasive and metastatic potential.

Topics: Basement Membrane; Carcinoma, Squamous Cell; Chemotaxis; Collagen; Collagenases; Dose-Response Relationship, Drug; Drug Combinations; Epidermal Growth Factor; Female; Fibronectins; Floxuridine; Gelatin; Humans; Laminin; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Pentosyltransferases; Proteoglycans; Pyrimidine Phosphorylases; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Cervical Neoplasms

The epidermal growth factor receptor (EGFR) is activated by a variety of ligands including EGF and transforming growth factor-alpha (TGFalpha), whereas no ligand for the homologous ErbB2 oncoprotein has yet been identified. Here we use both an ErbB2 phosphoantibody (aPY1222) and an activation-specific EGFR antibody to show that low concentrations of EGF induce more efficient tyrosine phosphorylation of ErbB2 in A431 cells than does equimolar TGFalpha, while EGFR is more potently activated by TGFalpha. Co-precipitation studies confirm that heterodimerization of activated EGFR and transphosphorylated ErbB2 is readily induced by EGF but not TGFalpha. EGFR downregulation is also more efficiently induced by EGF, suggesting that ligand-dependent modification of ErbB2 may be required to terminate EGFR signalling in cells expressing both receptor types. These findings indicate that EGF and TGFalpha differ in their abilities to induce tyrosine phosphorylation and heterodimerization of ErbB2, and raise the possibility that ErbB2 exerts its oncogenic effect in part by impairing TGFalpha-dependent EGFR downregulation.

Topics: Antibodies; Antibody Specificity; Carcinoma, Squamous Cell; Dimerization; Down-Regulation; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Ligands; Phosphorylation; Precipitin Tests; Receptor, ErbB-2; Transforming Growth Factor alpha; Tumor Cells, Cultured; Tyrosine

Lysophosphatidic acid (LPA) is a lipid mediator which acts on its putative G protein-coupled receptor (GPCR). Recently, activation of signal transducers and activators of transcription (STATs) mediated by GPCR has been reported. In this study, we examined the effect of LPA on STAT activation using the electrophoretic mobility shift assays and the heterologous promoter analysis in human epidermoid carcinoma A431 cells. We found that LPA inhibited epidermal growth factor (EGF)-induced Stat1 activation in a concentration-dependent manner. Other phospholipase C (PLC)-coupled GPCR agonists, bradykinin and ATP, also inhibited Stat1 activation. This inhibitory effect of LPA was completely mimicked by an activator of protein kinase C (PKC), a PLC-downstream effector. These findings suggest that the inhibitory effect on EGF-induced Stat1 activation may be a general characteristic of PLC-coupled GPCRs and PKC pathway may be mainly associated with this inhibitory effect. This is the first evidence showing that GPCR agonists inhibit the Janus kinase-independent Stat1 activation induced by receptor tyrosine kinase.

Topics: Bradykinin; Carcinoma, Squamous Cell; DNA-Binding Proteins; Epidermal Growth Factor; Epinephrine; Genes, Reporter; GTP-Binding Proteins; Humans; Lysophospholipids; Nuclear Proteins; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Protein Kinase C; Receptors, Cell Surface; Regulatory Sequences, Nucleic Acid; Signal Transduction; STAT1 Transcription Factor; Tetradecanoylphorbol Acetate; Trans-Activators; Transcription, Genetic; Tumor Cells, Cultured; Type C Phospholipases

We have shown previously that the epidermal growth factor peptide (EGF) may be radiolabeled with 99mTc at room temperature and neutral pH by using the N-hydroxysuccinimide ester of S-acetyl mercaptoacetyltriglycine (MAG3) as a bifunctional chelator. By a competition binding assay, we found that MAG3-conjugated EGF retained biological activity. Furthermore, the labeled peptide exhibited saturation binding to EGF receptor-positive tumor cell lines which could be inhibited by presaturation of the cells with unlabeled, native EGF. Biodistribution in normal mice at 3 h postadministration showed rapid clearance with minimal retention of the label in sampled organs. We have now investigated the tumor localization properties in mice of this labeled peptide. Nude mice implanted with the EGF receptor-positive tumors A431 and LS-174T were administered labeled EGF and a labeled control peptide (BPTI, aprotinin). Tumor uptake at 12 h postadministration was 0.44% injected dose/g for EGF/g vs. 0.09 for the control. Pretreatment of tumored mice with unlabeled EGF blocked about half the tumor uptake. Animals were also administered an anti-EGF receptor antibody labeled with 99mTc via MAG3. Relative to the antibody, tumor-to-muscle ratios were improved from 6 to 15 and tumor-to-blood ratios from 0.4 to 7 with EGF. These favorable results along with documented evidence of overexpression of the EGF receptor in many human tumors suggest that 99mTc-EGF should be considered further for tumor detection.

Topics: Animals; Autoradiography; Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Humans; Mice; Mice, Nude; Organotechnetium Compounds; Radionuclide Imaging; Tissue Distribution

The overexpression in tumor cells of (proto)-oncogenic receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or ErbB2/neu (also known as HER-2) is generally thought to contribute to the development of solid tumors primarily through their effects on promoting uncontrolled cell proliferation. However, agents that antagonize the function of the protein products encoded by these (proto)-oncogenes are known to behave in vivo in a cytotoxic-like manner. This implies that such oncogenes may regulate critical cell survival functions, including angiogenesis. The latter could occur as a consequence of regulation of relevant growth factors by such oncogenes. We therefore sought to determine whether EGFR or ErbB2/neu may contribute to tumor angiogenesis by examining their effects on the expression of vascular endothelial cell growth factor (VEGF)/vascular permeability factor (VPF), one of the most important of all known inducers of tumor angiogenesis. We found that in vitro treatment of EGFR-positive A431 human epidermoid carcinoma cells, which are known to be heavily dependent on VEGF/VPF in vivo as an angiogenesis growth factor, with the C225 anti-EGFR neutralizing antibody caused a dose-dependent inhibition of VEGF protein expression. Prominent suppression of VEGF/VPF expression in vivo, as well as a significant reduction in tumor blood vessel counts, were also observed in established A431 tumors shortly after injection of the antibody as few as four times into nude mice. Transformation of NIH 3T3 fibroblasts with mutant ErbB2/neu, another EGFR-like oncogenic tyrosine kinase, resulted in a significant induction of VEGF/VPF, and the magnitude of this effect was further elevated by hypoxia. Moreover, treatment of ErbB2/neu-positive SKBR-3 human breast cancer cells in vitro with a specific neutralizing anti-ErbB2/neu monoclonal antibody (4D5) resulted in a dose-dependent reduction of VEGF/VPF protein expression. Taken together, the results suggest that oncogenic properties of EGFR and ErbB2/neu may, at least in part, be mediated by stimulation of tumor angiogenesis by up-regulating potent angiogenesis growth factors such as VEGF/VPF. These genetic changes may cooperate with epigenetic/environmental effects such as hypoxia to maximally stimulate VEGF/VPF expression. Therapeutic disruption of EGFR or ErbB2/neu protein function in vivo may therefore result in partial suppression of angiogenesis, a feature that could enhance the therapeutic index of s

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Breast Neoplasms; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Growth Factors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lymphokines; Mice; Mice, Nude; Neoplasm Transplantation; Neovascularization, Pathologic; Neutralization Tests; Receptor, ErbB-2; Signal Transduction; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Phosphoinositide-specific phospholipase C gamma 1 is associated with EGF receptor in A-431 cells after EGF treatment. It is shown that phospholipase C gamma 1 is co-localized with internalized receptors as well as with membrane ones during receptor-mediated endocytosis. Nocodazole is known as an inhibitor of microtubule assembly, thus leading to a complete disappearance of microtubule network. Nocodazole had no effect on phospholipase C gamma 1--EGF receptor association and co-localization, but the intracellular distribution of both the proteins differed dramatically. Phospholipase C gamma 1 and EGF receptor were localized in endosomes in the periphery of the cell. Besides, pretreatment of A-431 cells with nocodazole resulted in decreasing tyrosine phosphorylation of some proteins. These data suggest that the internalized receptor may serve as an additional starting point for triggering cell signalling.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Isoenzymes; Microtubules; Nocodazole; Phosphatidylinositols; Phospholipase C gamma; Receptors, Mitogen; Signal Transduction; Substrate Specificity; Tumor Cells, Cultured; Type C Phospholipases

Transforming growth factor beta 1 (TGF beta 1) increases the phosphorylation of the epidermal growth factor (EGF) receptor and inhibits the growth of A431 cells, but the mechanism of TGF beta 1 signaling is unknown. Recent studies from this and other laboratories suggest a novel sphingomyelin signal transduction pathway (1-4). Ceramide, which is generated by sphingomyelinase action, can be deacylated to sphingoid bases, which are potential inhibitors of protein kinase C (PKC). Ceramide appears to have bioeffector properties. Cell-permeable ceramide analogs stimulate monocytic differentiation of human leukemia (HL60) cells (1), as well as the phosphorylation of the EGF receptor at Thr669 in A431 human epidermoid carcinoma cells (2). Further studies (3,4) demonstrate the existence of a ceramide-activated protein kinase (CAPK) that may mediate some of these aspects. The present studies aim to investigate the mechanism of TGF beta 1 signaling and to explore whether TGF beta 1's pathway involves activation of PKC by 1,2-Diacylglycerol (DAG) and/or stimulation of a CAPK by ceramide. Ceramide and DAG levels of A431 cells are determined by thin layer chromatography (TLC) after treatment with either TGF beta 1 or with EGF. 100 pM TGF beta 1 treatment for 1 hr increases the cellular contents of DAG 2-fold. 20 nM EGF treatment for 15 min decreases it 0.5-fold. Ceramide levels are reduced 2-fold by TGF beta 1 and almost unaffected by EGF. To evaluate the involvement of other components of signal transduction, the effects of TGF beta 1 and EGF on PKC activity are studied. 20 nM EGF decreases membrane PKC activity to 0.5-fold of controls, whereas 100 pM TGF beta 1 treatment of A431 cells increases this activity 4-fold. Modulation of PKC activity is paralled by translocation of the enzyme between the cytosol and the membrane as determined by Western immunoblot analysis. These studies suggest that TGF beta 1 and EGF may have regulatory effects on both sphingolipid and phospholipid metabolisms which could transmodulate both the CAPK and the PKC mediated signal tranduction pathways.

Topics: Carcinoma, Squamous Cell; Cell Division; Ceramides; Epidermal Growth Factor; ErbB Receptors; HL-60 Cells; Humans; Kinetics; Phosphorylation; Protein Kinase C; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-raf; Second Messenger Systems; Signal Transduction; Transforming Growth Factor beta; Triglycerides; Tumor Cells, Cultured

Topics: Arachidonate 12-Lipoxygenase; Arachidonic Acid; Carcinoma, Squamous Cell; Enzyme Induction; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Hydroquinones; Isoenzymes; Microsomes; Protein Kinase C; Protein-Tyrosine Kinases; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

In an effort to identify events initiating up-regulation of epidermal growth factor receptor after single and repeated radiation exposures, we investigated the role of epidermal growth factor receptor, a receptor protein tyrosine kinase, in radiation-induced signal transduction. Human malignant mammary, MCF-7, and squamous, A431, cells showed low baseline phospho-tyrosine levels of epidermal growth factor receptor, permitting reproducible dose-dependent stimulation of epidermal growth factor receptor autophosphorylation after exposure to epidermal growth factor. MCF-7 cells exhibited a mean 2.3-fold increase (95% confidence interval: 1.91, 2.65; P < 0.0001) in levels of epidermal growth factor phosphorylation in response to exposures of 2 Gy, which was substantially less than the epidermal growth factor receptor Y phosphorylation induced by epidermal growth factor. A quantitatively similar radiation response was seen in A431 cells. In the dose range of 1 to 4 Gy, no clear dose response was seen. There was a rapid induction of radiation-induced epidermal growth factor receptor Y phosphorylation, starting within 2 min, with maximum values between 0.5 and 5 min after radiation exposure followed by a slower decline to baseline levels after 20 min. The data presented identify the epidermal growth factor receptor protein tyrosine kinase associated with the plasma membrane as one target for ionizing radiation in the dose range used in radiotherapy.

Topics: Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cobalt Radioisotopes; Confidence Intervals; Dose-Response Relationship, Radiation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Phosphorylation; Phosphotyrosine; Signal Transduction; Tumor Cells, Cultured; Up-Regulation

Retinoic acid (RA) has been shown to be effective in eradicating premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck (SCCHN) in clinical trials. The basis for this effect is unclear. We have previously demonstrated that messenger RNA from tumor growth factor-alpha (TGF-alpha) and its receptor, the epidermal growth factor (EGFR), is unregulated in tumors and histologically normal mucosal samples from patients with SCCHN compared with control normal mucosa from patients without cancer, implicating this ligand-receptor pair in an autocrine growth pathway early in the molecular pathogenesis of this disease. In this report, we examined the hypothesis that the action of RA on the mucosa of the upper aerodigestive tract is mediated via downregulation of steady-state TGF-alpha and/or EGFR mRNA levels. Following exposure to all-trans-RA, a series of SCCHN cell lines demonstrated a 35.4% reduction in TGF-alpha mRNA expression (P = 0.022) and 58.5% reduction in EGFR mRNA (P = 0.0027). Nuclear run-on analysis indicated that the RA-mediated reduction of TGF-alpha and EGFR steady-state mRNA levels was a result of decreased gene transcription. These results suggest that the clinical effects of RA in SCCHN patients may be due to a downmodulation of TGF-alpha and EGFR mRNA production.

Topics: Carcinoma, Squamous Cell; Down-Regulation; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Keratolytic Agents; Mouth Mucosa; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor alpha; Tretinoin; Tumor Cells, Cultured

Here we report that epidermal growth factor (EGF) inhibits the replication of vesicular stomatitis virus (VSV) in human epidermoid carcinoma A-431 cells, as measured by reduction in infectious viral particle production, cell protection assay, and inhibition of viral protein synthesis. The observed antiviral effects of EGF were not apparently due to inhibitory effects on the initial events in VSV infection as postinfection treatment of cells with EGF also resulted in significant inhibition in the levels of viral proteins. The EGF-mediated inhibition of VSV protein synthesis was specific in nature as prior treatment of cells with an excess of anti-EGF receptor monoclonal antibody, which blocks EGF binding, reduced the inhibition of VSV proteins elicited by EGF. In summary, the observed antiviral action of EGF was an early effect of EGF and was mediated via an EGF-responsive cellular pathway(s) that could impair VSV replication.

Topics: Antiviral Agents; Carcinoma, Squamous Cell; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Interferon-alpha; Tumor Cells, Cultured; Vesicular stomatitis Indiana virus; Viral Proteins; Virus Replication

Epidermal growth factor (EGF) is a potent mitogen for keratinocytes. Although the role of the EGF receptor in cell proliferation has been extensively studied, the consequences of EGF receptor activation with respect to cell differentiation remain less well characterized. Our studies demonstrate that stimulation of the EGF receptor substantially suppresses cellular differentiation in squamous cell carcinoma lines that overexpress the EGF receptor, as assessed by an EGF-dependent reduction of cornified envelope formation. Only a modest ligand-dependent decrease in cornified envelope formation was observed in normal keratinocytes. The response is dependent on the concentration of EGF and is evident after 1-2 days of EGF treatment. With extended EGF treatment, the messenger RNA levels for involucrin, a major structural component of the cornified envelope, were unaltered by EGF. In contrast, membrane-associated transglutaminase enzyme activity, which predominantly represents type 1 (keratinocyte) transglutaminase, is markedly inhibited by EGF. The lost of type 1 transglutaminase activity is associated with reduced levels of the messenger RNA and protein. These studies suggest that the functional consequences of EGF receptor activation in squamous cell carcinomas involve not only aberrant growth regulation, but, additionally, reduction of terminal differentiation capacity.

Topics: Blotting, Western; Carcinoma, Squamous Cell; Cell Differentiation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Keratinocytes; Male; Protein Precursors; RNA, Messenger; Transglutaminases; Tumor Cells, Cultured

Following the discovery of 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (4; PD 153035) as an extremely potent (IC(50) 0.025 nM) inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), several fused tricyclic quinazoline analogues have been prepared and evaluated for their ability to inhibit the enzyme. The most potent compound was the linear imidazo[4,5-g]quinazoline (8), which exhibited an IC(50) of 0.008 nM for inhibition of phosphorylation of a fragment of phospholipase C-gamma-1 as substrate. While N-methyl analogues of 8 showed similar potency, analogous N-[2-(dimethylamino)ethyl] derivatives were less effective. The next most potent compounds were the linear pyrazoloquinazolines (19 and 20) (IC(50)s 0.34 and 0.44 nM) and pyrroloquinazoline (21) (IC(50) 0.44nM), while several other linear tricyclic ring systems of similar geometry to 8 (triazolo-, thiazolo-, and pyrazinoquinazolines) were less effective. In the imidazo[4,5-g]quinazoline and pyrroloquinazoline series, the corresponding angular isomers were also much less effective than the linear ones. These results are consistent with structure-activity relationship studies previously developed for the 4-[(3-bromophenyl)amino] quinazolines, which suggested that small electron-donating substituents at the 6- and 7-positions were desirable for high potency. Cellular studies of the linear imidazoloquinazoline 8 show that it can enter cells and rapidly and very selectively shut down EGF-stimulated signal transmission by binding competitively at the ATP site of the EGFR.

Topics: 3T3 Cells; Adenosine Triphosphate; Animals; Binding Sites; Carcinoma, Squamous Cell; Cell Cycle; Cell Line; DNA; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fibroblast Growth Factor 2; Growth Substances; Heterocyclic Compounds; Humans; Imidazoles; Indicators and Reagents; Magnetic Resonance Spectroscopy; Mice; Mitosis; Molecular Structure; Phosphorylation; Platelet-Derived Growth Factor; Protein-Tyrosine Kinases; Quinazolines; Structure-Activity Relationship; Thymidine

Various types of tumors show aberrant expression and overexpression of epidermal growth factor (EGF) receptor and the degree of receptor expression correlates with a malignant phenotype in many epithelial tumors. However, in vitro evidence supporting the advantageous role of receptor overexpression is deficient. In this study, we compared the effects of exogenous EGF on the cell colony morphology in monolayer and collagen gel culture between HSC-1 squamous carcinoma cells overexpressing EGF receptor and their revertant subline cells. These cells formed coherent cell colonies under routine culture conditions, but addition of EGF induced dissociation of cell colonies within 24 h in the parent HSC-1 cells, though not in the subline cells. Since the colony dissociation apparently involved loss of cell-cell adhesion, we also studied the effects of EGF on E-cadherin expression and its function. Cell aggregation assays showed that EGF reduced E-cadherin function dose-dependently in the parent cells, but not in the subline cells. However, immunoblotting analysis and ELISA showed the absence of downregulation or degradation of E-cadherin. Instead, EGF tyrosine phosphorylated cadherin/catenin complex components including beta-catenin and increased the detergent solubility of E-cadherin in the parent cells. These results suggest that EGF modified the functional association between E-cadherin and actin filament through tyrosine phosphorylation of the cadherin/catenin complex and thereby made the adhesion molecule incompetent. Our results indicate that the ligand activation of overexpressed EGF receptor impairs E-cadherin-mediated cell-cell adhesion and causes dissociation of the squamous carcinoma cell colonies, which facilitates tumor cell invasion in vivo. This might be relevant to the advantageous role of EGF receptor overexpression in malignant phenotype of epithelial tumor cells.

Topics: beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Cell Size; Collagen; Cytoskeletal Proteins; Detergents; Epidermal Growth Factor; ErbB Receptors; Humans; Octoxynol; Phosphorylation; Skin Neoplasms; Solubility; Trans-Activators; Tumor Cells, Cultured; Tyrosine

Recombinant human ribonuclease 1 (RNase 1) was chemically linked to recombinant human epidermal growth factor (EGF). The EGF-RNase conjugate showed dose-dependent cytotoxicity for EGF receptor-overexpressing A431 and TE-8 human squamous carcinoma cells with an IC50 of 2 x 10(-7)M and 10(-6)M, respectively, whereas the IC50 of RNase alone was almost 10(-4)M. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone. The conjugate showed no detectable cytotoxicity against EGF receptor-deficient small cell lung cancer cells (H69). Addition of excess EGF in the medium protected A431 cells from the EGF-RNase conjugate cytotoxicity. The cytotoxicity of the EGF-RNase conjugate was positively correlated with the EGF receptor numbers of each cell line. The chimeric toxin composed of only human proteins might be a more useful anti-cancer agent with less immunogenicity than the conventional chimeric toxins.

Topics: Carcinoma, Squamous Cell; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Humans; Ribonuclease, Pancreatic; Tumor Cells, Cultured

In order to ascertain the effects of epidermal growth factor (EGF) on human cancer invasion abilities, three cell lines of human oral squamous cell carcinoma were studied using a phagokinetic track assay and zymography. EGF (1-100 ng/ml) was found to inhibit the growth but enhance the random motility of all three cell lines in a concentration-dependent fashion. Exposure to EGF, dose-dependently, led to an increased production of urokinase-type plasminogen activator and M(r) 92 kD matrix metalloproteinase by the same cells. These results strongly suggest that EGF may promote human squamous cell carcinoma invasion and metastasis.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Movement; Epidermal Growth Factor; Extracellular Matrix; Humans; Metalloendopeptidases; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Because of its unique DNA-cleaving and strand-passing activities, topoisomerase II is involved in many aspects of DNA metabolism, including replication, transcription, recombination, and repair. The cytotoxic potential of topoisomerase II-targeted drugs, such as etoposide, is related to their ability to stabilize covalently linked enzyme-DNA complexes, which are intermediates in the enzyme's catalytic cycle. Epidermal growth factor receptor is expressed on the cell surface of the majority of squamous cell carcinomas, and epidermal growth factor binding is known to stimulate a number of cellular transduction pathways, including tyrosine kinase, protein kinase C, and phospholipase C. Because topoisomerase II is a proliferation-dependent protein and has been shown to be a high-affinity substrate for many of these cellular transduction pathways, the effects of epidermal growth factor on cellular regulation and sensitivity to etoposide were studied with the human oral cavity squamous cell line, KB. Topoisomerase II catalytic activity was rapidly and transiently inhibited after the addition of epidermal growth factor to the cellular growth media. Western blot on nuclear extracts did not demonstrate alterations in topoisomerase II polypeptide levels to account for changes in catalytic activity. Epidermal growth factor treatment also led to the formation of stabilized, covalently linked enzyme-DNA complexes. Furthermore, epidermal growth factor-induced, topoisomerase II-mediated DNA strand breaks were additive to those induced by etoposide. This study indicates that epidermal growth factor specifically regulates the catalytic and DNA-cleaving activities of topoisomerase II in KB cells. This may direct clinical strategies for circumventing the intrinsic cellular resistance to chemotherapy commonly observed in squamous cell carcinomas of the head and neck.

Topics: Antineoplastic Agents, Phytogenic; Blotting, Western; Carcinoma, Squamous Cell; Catalysis; Cell Division; DNA Damage; DNA Topoisomerases, Type II; DNA, Neoplasm; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Etoposide; Head and Neck Neoplasms; Humans; KB Cells; Tumor Cells, Cultured

The ATDC gene was originally identified by its ability to complement the radiosensitivity defect of an ataxia telangiectasia (AT) fibroblast cell line. Because hypersensitivity to ionizing radiation is an important feature of the AT phenotype, we reasoned that ATDC may function generally in the suppression of radiosensitivity. Previous work in our laboratory focused on radiosensitization mechanisms in human squamous carcinoma (SC) cells, especially A431 cells. To establish a basis for investigating the role of ATDC in radiation-responsive signaling pathways in human SC cells, we characterized ATDC message and protein expressions in A431 cells. ATDC message expression was also compared among human epidermoid cells (A431 cells, HaCaT spontaneously immortalized human keratinocytes and normal human epidermal keratinocytes) and a normal human fibroblast cell line (LM217). We made the following major observations: (i) the relative abundance of ATDC message is substantially higher in the epidermoid cells than in the fibroblast cell line, which has a message level comparable to those reported for other fibroblast lines; (ii) ATDC is constitutively phosphorylated on serine/threonine in A431 cells; (iii) in A431 cells, ATDC is a substrate for the serine/threonine protein kinase C (PKC) but not the epidermal growth factor (EGF) receptor tyrosine kinase; and (iv) EGF decreases ATDC message and protein expressions in A431 cells after a 24-hr exposure. The phosphorylation studies suggest that the ability of ATDC to modulate cellular radiosensitivity may be mediated in part through a PKC signaling pathway.

Topics: Ataxia Telangiectasia; Base Sequence; Carcinoma, Squamous Cell; Cell Line, Transformed; Cell Transformation, Viral; DNA-Binding Proteins; DNA, Complementary; Epidermal Growth Factor; Fibroblasts; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Molecular Sequence Data; Neoplasm Proteins; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Recombinant Fusion Proteins; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Simian virus 40; Skin; Transcription Factors; Tumor Cells, Cultured

Previous studies have reported inhibition of A431 squamous carcinoma cell growth by nanomolar concentrations of epidermal growth factor (EGF), a potent mitogen for cells of epithelial origin. In this study, we examined potential mechanisms through which inhibition of keratinocyte growth mediated by EGF might occur by analysing components of the cell cycle regulatory machinery in A431, HN6 and HN30 keratinocytes in the presence of growth inhibitory or growth stimulatory doses of EGF. Treatment of cells with 25 pM EGF produced an increase in [3H]thymidine incorporation in A431, HN6 and HN30 cells, with respect to control cultures. Exposure to 2.5 nM EGF reduced [3H]thymidine incorporation in A431 cells and HN6 cells to 11% and 70% of control levels, respectively, whereas HN30 cells continued to proliferate in the presence of EGF. [3H]thymidine incorporation assays carried out over 24 h revealed repression of DNA synthesis in A431 cells after 12 h exposure to 2.5 nM EGF compared to untreated cells. Flow cytometry studies demonstrated accumulation of cells in G0/G1 after addition of 2.5 nM, but not 25 pM EGF. Western blot analysis revealed elevation of p21 (WAF1/CIP1/SDI1) protein levels in A431 and HN6 cells under growth-inhibitory conditions. Stimulatory doses of EGF did not induce p21 in these cells. Northern blot hybridization demonstrated elevated levels of p21 mRNA within 4 h of exposure of A431 cells to 2.5 nM EGF, which remained elevated above basal levels at 24 h. In vitro kinase assays demonstrated temporal differences in CDK2 and CDK6 activities which were related to EGF concentration. Immunocomplex Western blotting demonstrated increased association of p21 with CDK2 and CDK6 in A431 cells treated with 2.5 nm EGF. Furthermore, temporal alterations in the association of PCNA with p21 and with CDK6 were observed. The data indicate that p21 is a likely mediator of EGF-induced growth-inhibition, probably through mechanisms involving sequestration of PCNA and inhibition of CDK activity.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; G1 Phase; Humans; Keratinocytes; Molecular Sequence Data; Mouth Neoplasms; Proliferating Cell Nuclear Antigen; Resting Phase, Cell Cycle; Time Factors; Tumor Cells, Cultured

A system has been developed for the efficient production of heterologous proteins using Bacillus brevis as a host that secretes large amounts of cell wall protein into the medium. The promoter region and signal peptide-encoding region of the cell wall protein gene were used to construct an expression-secretion vector. Bacterial proteins such as amylases can be produced in large amounts by this system (1 g/l or more), but mammalian proteins such as human alpha-amylase are produced at a low level (one or two orders of magnitude less than for bacterial proteins). The highly efficient secretion of human epidermal growth factor (h-EGF, more than 1 g/l) was obtained with B. brevis HPD31 as the host and plasmid pHY481, derived from B. brevis 481, as the vector. Recombinant hEGF was purified easily from the culture supernatant by two steps. Purified hEGF had the identical NH2-terminal amino acid sequence and COOH-terminal amino acid sequence with those of the authentic hEGF, and it was fully active in biological assays. This recombinant hEGF has been shown to be successful for biological wool harvesting (CSIRO, Australia). These results, in combination with previous results, indicate that foreign proteins of diverse origins can be produced efficiently as functional proteins in B. brevis.

Topics: Amino Acid Sequence; Animals; Bacillus; Biological Assay; Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; Cloning, Molecular; Epidermal Growth Factor; Humans; Liver; Plasmids; Rats; Recombinant Proteins; Restriction Mapping; Sequence Homology, Amino Acid; Tumor Cells, Cultured

This study was aimed at gaining an understanding of metabolic events responsible for the inhibition of cells in G2 phase, a known physiological restriction site in the cell cycle of multicellular organisms. In an earlier study, phosphatidic acid was proposed as an inhibitory mediator in the epidermal growth factor (EGF)-induced inhibition of A431 cells in G2 phase via the phospholipase C pathway [Kaszkin, Richards and Kinzel (1992) Cancer Res. 52, 5627-5634]. We show here that the phorbol ester phorbol 12-myristate 13-acetate (PMA) induces a reversible inhibition of the G2/M transition in A431 cells under conditions of phospholipase D-catalysed phosphatidic acid formation. Such PMA-induced inhibition in G2 phase is largely attenuated in the presence of 1-propanol (but not of 2-propanol). In this case the amount of phosphatidic acid is reduced to almost control levels, and instead phosphatidylpropanol is formed. In the case of EGF-induced activation of a phospholipase D the amount of phosphatidic acid is only slightly decreased in the presence of a primary alcohol. Under these conditions the EGF-induced G2 delay was not affected. The correlation between the formation of phosphatidic acid and the G2 delay induced by PMA, as well as by an exogenous bacterial phospholipase D (from Streptomyces chromofuscus), could be supported by using synchronized cells in order to increase the population of cells in G2 phase. This study indicates that the formation of substantial amounts of phosphatidic acid immediately before entry into mitosis seems to be important for establishing a delay in the cell cycle at the G2/M border by exogenous ligands.

Topics: 1-Propanol; Carcinoma, Squamous Cell; Diglycerides; Enzyme Activation; Epidermal Growth Factor; G2 Phase; Humans; Mitosis; Phosphatidic Acids; Phospholipase D; Prophase; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Mammalian pancreatic ribonuclease (RNase) was conjugated chemically via a disulfide bond to human or murine epidermal growth factor (EGF). The conjugation between EGF and RNase was ascertained by SDS-PAGE using reduced and nonreduced conjugates. The EGF-RNase conjugate retained potent RNase activity and competed with 125I-EGF for binding to EGFR to the same extent as unconjugated EGF. Both the human and murine EGF-RNase conjugates showed dose-dependent cytotoxicity against EGFR-overexpressing A431 human squamous carcinoma cells with IC50 values of 3 x 10(-7) M and 6 x 10(-7) M, respectively, whereas free RNase had an IC50 of 10(-4) M. Against the EGFR-deficient small-cell lung cancer cell line H69, the EGF-RNase conjugate had no cytotoxic effect. The Human EGF-RNase conjugate showed dose-dependent cytotoxicity against other squamous carcinoma cell lines (TE-5, TE-1) and breast cancer cell lines (BT-20, SK-BR-3, MCF-7) and the cytotoxicity of the conjugate correlated positively with the level of expression of EGFR by each cell line. An unconjugated mixture of EGF and RNase had no greater effect than RNase alone on any cell line. Excess free EGF blocked EGF-RNase conjugate cytotoxicity against A431 cells. These results suggest that the EGF-RNase conjugate may be a more effective anticancer agent with less immunogenicity than coventional chimeric toxins.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; Immunotoxins; Lung Neoplasms; Ribonucleases; Tumor Cells, Cultured

Interleukin-6 (IL-6) is a multifunctional cytokine that is produced not only by a variety of normal cells but also by cancer cells. IL-6 produced by cancer cells stimulates the proliferation of these cancer cells in an autocrine/ paracrine manner and causes paraneoplastic syndromes including hypercalcemia, cachexia, and leukocytosis. We have reported previously that a human oral squamous cancer associated with hypercalcemia produces large amounts of IL-6, that animals bearing this cancer exhibit elevated levels of plasma IL-6, and that neutralizing antibodies to human IL-6 reverse hypercalcemia in tumor-bearing animals, indicating an important role of IL-6 in the hypercalcemia in this model. Because these cancer cells overexpress epidermal growth factor receptors (EGFR) with intrinsic tyrosine kinase (TK) activity similar to many other squamous cancers, we examined the effects of herbimycin A, a tyrosine kinase inhibitor, on IL-6 production and hypercalcemia in animals bearing this cancer to develop a new approach to treat the hypercalcemia associated with malignancy. Intraperitoneal administration (once a day for 2 days) of herbimycin A to cancer-bearing hypercalcemic mice reduced the plasma levels of human IL-6 and impaired the hypercalcemia. During 2-day treatment with herbimycin A, no changes were observed in tumor size. Of interest, plasma levels of mouse, but not human, soluble IL-6 receptors were also elevated. However, herbimycin A showed no effects on plasma levels of mouse soluble IL-6 receptors. Herbimycin A suppressed the tyrosine autophosphorylation of EGFR and IL-6 mRNA expression and production, all of which were stimulated by EGF. The data raise the possibility that TK inhibitors may be potential mechanism-based therapeutic agents for the treatment of hypercalcemia associated with squamous cancers which overexpress EGFR.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, CD; Benzoquinones; Carcinoma, Squamous Cell; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Hypercalcemia; Interleukin-6; Lactams, Macrocyclic; Male; Mice; Mice, Nude; Neoplasm Transplantation; Protein-Tyrosine Kinases; Quinones; Receptors, Interleukin; Receptors, Interleukin-6; Rifabutin; Solubility; Tumor Cells, Cultured

Epidermal growth factor (EGF), amylase and haptocorrin are molecules produced in the salivary glands. The aim of the present study was to determine immunohistochemical and quantitative alterations in EGF as compared with haptocorrin and amylase following radiotherapy for oral cancer. Changes in the salivary secretion of EGF are of interest because of the importance of EGF in mucosal regeneration. Immunohistochemical studies on normal tissue from parotid and submandibular glands have demonstrated EGF in the serous acini with a tendency to single cell expression in the parotid gland. Amylase has been found in the serous acini of both the submandibular and parotid glands. Haptocorrin was localized in the duct system of both glands. In the submandibular glands with radiotherapy induced sialoadenitis only very few acini with weak or no staining for EGF and amylase were demonstrated, while no changes were observed in the staining for haptocorrin. Analysis on stimulated whole saliva samples collected from 20 healthy individuals and from 20 patients prior to, and 1, 2 and 3 weeks following radiotherapy showed significant reduction in salivary contents of EGF and amylase after treatment as expressed per g protein (p < 0.0002). The salivary content of haptocorrin increased significantly after treatment (p < 0.002). These alterations may be explained by the different cellular sites of the molecules studied, the serous acini being more sensitive to ionising radiation than the duct system. The concentration of EGF in saliva before treatment was significantly higher in patients than in the control group (p < 0.02), which may indicate that the tumors induce increased secretion of salivary EGF, or alternatively that the oral tumors contribute with EGF to the saliva. In conclusion we have demonstrated a reduction in the mitogenic peptide EGF both immunohistochemically and quantitatively following irradiation for oral cancer, results which may contribute to the understanding of the clinical signs of mucositis.

Topics: Aged; Amylases; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Middle Aged; Mouth; Mouth Neoplasms; Nasopharyngeal Neoplasms; Nasopharynx; Salivary Glands

A case of synchronous squamous cell carcinomas in the soft palate, larynx and esophagus is reported, along with findings of molecular-pathological analysis. A biopsy sample from the aryngeal carcinoma revealed well differentiated squamous cell carcinoma harboring two point mutations at codons 144 and 148 of the p53 gene but not at codon 299, and more than 50% of the cancer cells showed accumulation of p53 protein immunohistochemically. The esophageal tumor, which was moderately differentiated squamous cell carcinoma, showed immunoreactivity for p53 within the nuclei of 25-50% of cancer cells with a missense mutation at codon 299 but not at codon 144 or 148. This cancer also showed immunoreactivity for transforming growth factor alpha. On the other hand, the poorly differentiated squamous cell carcinoma in the soft palate showed negative immunoreactivity for p53 and no point mutation in exons 5 to 8 of the gene. These results suggest that the three synchronous squamous cell carcinomas arose as independent events.

Topics: Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Genes, p53; Humans; Immunohistochemistry; Laryngeal Neoplasms; Male; Mutation; Neoplasms, Multiple Primary; Palatal Neoplasms; Palate, Soft; Polymerase Chain Reaction; Stomach Neoplasms; Transforming Growth Factor alpha; Tumor Suppressor Protein p53

Human A431 epidermoid carcinoma cells express 12-lipoxygenase enzymatic activity. However, the isoform identity based on cDNA sequence data is not known. Further, the simultaneous characterization of the intracellular distribution of 12-lipoxygenase protein and activity is lacking. Here we report that the cDNA sequence from RT-PCR-amplified 12-lipoxygenase mRNA is identical with the platelet-type 12-lipoxygenase isoform, and the leukocyte-type isoform of 12-lipoxygenase is not expressed in A431 cells. The predominant amount (78%) of 12-lipoxygenase protein resides in the cytosol. In contrast, the predominant (98%) 12-lipoxygenase activity is localized in the membrane fraction. Western blot and immunofluorescence data demonstrate that epidermal growth factor increases total cellular 12-lipoxygenase protein and enhances the association of 12-lipoxygenase protein with perinuclear or nuclear membrane sites. In addition, epidermal growth factor stimulates 12-lipoxygenase activity resulting in generation of 12(S)-hydroxyeicosatetraenoic acid from cellular arachidonate. In contrast, both 12-lipoxygenase protein and activity decrease approximately 80% within 24 h during serum starvation. The recovery of 12-lipoxygenase expression in serum-deprived cells can be induced by readdition of epidermal growth factor or serum. Further, the basal expression of 12-lipoxygenase depends on signal pathways requiring protein tyrosine kinase activity, since genistein, herbimycin A, and tyrphostin 25 reduce the expression of 12-lipoxygenase protein in A431 cells.

Topics: Arachidonate 12-Lipoxygenase; Base Sequence; Blood Platelets; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Culture Media, Serum-Free; Cytosol; DNA Primers; DNA, Complementary; Epidermal Growth Factor; Exons; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Polymerase Chain Reaction; Protein-Tyrosine Kinases; RNA, Messenger; Sequence Homology, Nucleic Acid; Signal Transduction; Transcription, Genetic

Tissue-specific delivery of a variety of molecules has been a valuable technique for biological and medical research. Therefore, we have constructed a recombinant plasmid containing the coding regions for streptavidin core and mature human transforming growth factor-alpha (TGF-alpha). The recombinant plasmid has been expressed in Escherichia coli to produce a chimeric protein with both streptavidin and TGF-alpha activity. The streptavidin-TGF-alpha chimeric protein (ST-TGF-alpha) could efficiently transfer biotinylated beta-galactosidase into A431 cells via the epidermal growth factor receptor. More than 99% of the cells contained the enzyme transferred. Furthermore, ST-TGF-alpha complexed with biotinylated-glucose oxidase had a significant cytotoxic effect when incubated with A431 cells. These findings suggest that the ST-TGF-alpha chimeric protein could be used to deliver proteins of interest into target cells without the need for chemical linkage or genetic construction. Essentially, ST-TGF-alpha serves as a high-modular "molecular bridge" for the passage of a wide variety of effector molecules into target cells.

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Galactosidase; Biotin; Carcinoma, Squamous Cell; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Glucose Oxidase; Humans; Molecular Sequence Data; Radioligand Assay; Recombinant Fusion Proteins; Streptavidin; Transforming Growth Factor alpha; Tumor Cells, Cultured

Two types of EGF-mediated endocytosis have been identified in A431 cells by the method of subcellular fractionation in Percoll density gradients. One ("slow") type of endocytosis is characterized by 125I-EGF retention in the fraction of light endosomes, while the other ("fast") type demonstrates efficient 125I-EGF transition into heavy endosomes and lysosomes. 32P-ATP phosphorylation assay of Percoll fractions, followed by alkaline treatment on the gels, has demonstrated that "slow" cells reveal an increased basal level of EGF-receptor tyrosine kinase (TK) activity compared to the "fast" cells. Pretreatment of "fast" cells with Mn2+, which was shown to induce TK stimulation without EGF (Mohammadi [correction of Muhammedi et al., 1993), caused a dramatic decrease in 125I-EGF transition to heavy endosomes and lysosomes. Analysis of tyrosine phosphorylation of the receptor, being performed in Mn(2+)-pretreated A431 cells, has confirmed the significant increase of 32P-incorporation into unoccupied EGFR. Taken together, our data suggest that sorting of internalized EGF-receptor complexes depends on the basal TK activity level of EGFR.

Topics: Animals; Carcinoma, Squamous Cell; Cell Compartmentation; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Male; Mice; Organelles; Phosphorylation; Tumor Cells, Cultured

Transforming growth factor beta (TGF beta) was examined regarding its regulation of the mitogen EGF. A431 human epidermoid carcinoma cells were treated with TGF beta and epidermal growth factor (EGF) (10 ng/ml each) to determine if TGF beta modulates EGF-induced Ca2+ signaling and c-Fos oncoprotein levels. Changes in [Ca2+]i were determined by digital imaging analysis or photon counting. In HBSS + Ca2+ (1.37 mM), EGF treatment resulted in a transient increase in [Ca2+]i from 75 to 150 nM, which lasted approximately 3.5 min and re-equilibrated to 90 nM. In nominally Ca(2+)-free (2-5 muM) HBSS, EGF caused a [Ca2+]i elevation that peaked at 140 nM and returned to baseline. TGF beta in HBSS + Ca2+ did not elicit a [Ca2+]i increase, although affinity labeling revealed types I, II, and III TGF beta receptors. TGF beta added simultaneously with EGF in HBSS + Ca2+ caused a gradual rise in [Ca2+]i from 50 to 100 nM over 16 min. Pretreatment with TGF beta (3 h; 10 ng/ml) abolished the EGF-induced [Ca2+]i elevation. EGF or TGF beta treatments increased c-Fos immunoreactivity by around 1 h. In summary, EGF elevated [Ca2+]i in the presence or absence of [Ca2+]e, resulting in high [Ca2+]n, associated with tyrosine and threonine phosphorylation, and increased c-Fos oncoprotein immunoreactivity. TGF beta did not increase [Ca2+]i but did increase c-Fos; TGF beta + EGF added simultaneously altered the EGF-induced [Ca2+]i elevation, and TGF beta pretreatment eliminated EGF-induced [Ca2+]i elevation. This suggests that TGF beta can regulate EGF in A431 cells and that increased c-Fos may not be mediated by Ca2+.

Topics: Calcium; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique, Indirect; Humans; Immunohistochemistry; Phosphorylation; Proto-Oncogene Proteins c-fos; Transforming Growth Factor beta; Tumor Cells, Cultured

The [(3)H]inositol incorporation into the membrane fraction of A-431 human epidermoid carcinoma cells was markedly increased by stimulation of the cells with either epidermal growth factor (EGF), ATP, bradykinin, or a calcium ionophore A23187 in the presence of 1 mM extracellular calcium ions; most incorporated [(3)H]inositol was found to have accumulated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI synthesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Pretreatment of cells with pertussis toxin (IAP, islet-activating protein) inhibited the PI synthesis, [Ca(2+)]i elevation, and inositol trisphosphate (IP(3)) production by ATP, suggesting that the phospholipase C(PLC) system coupled with IAP-sensitive G protein is involved in the ATP-stimulated PI synthesis. On the other hand, the ATP stimulation increased the release of [(3)H]choline and [(32)P)phosphatidic acid (PA) from radiolabeled cells, and such release was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, the ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP(3) production and [Ca(2+)]i elevation, this fact suggests that the lAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P(2)-purinergic receptors appears to activate the IAP-sensitive PLC system and IAP-insensitive PLD system, both of which are essential for the stimulation of PI synthesis. The present results imply the general prospect that ligand stimulation, which mobilizes second messengers and consumes their precursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Bradykinin; Calcimycin; Calcium; Carcinoma, Squamous Cell; Chelating Agents; Choline; Diacylglycerol Kinase; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Ionophores; Neoplasm Proteins; Pertussis Toxin; Phosphatidic Acids; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositols; Phospholipase D; Phosphotransferases (Alcohol Group Acceptor); Pyrimidinones; Receptors, Purinergic P2; Signal Transduction; Staurosporine; Sulfonamides; Thiazoles; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

Overexpression of epidermal growth factor receptor (EGFr) in squamous carcinomas has been demonstrated extensively. Preliminary clinical studies have shown that radiolabeled anti-EGFr monoclonal antibodies can localize to these tumors. The aims of this study were to determine the tolerance, pharmacokinetics, and radiolocalization properties of 131I-labeled EGF in patients (n = 9) with advanced squamous lung cancer. Patients' vital signs and symptoms were monitored regularly for 3 days. Daily scintigrams and biological samples for pharmacokinetic analysis were obtained for 3-4 days. 99mTc-labeled human serum albumin was administered to patients with positive tumor scans. Six patients had positive tumor scans, and five of them had received >/=1.0 mg EGF. In all of these cases, tumors were visualized the same day of the infusion, although best tumor-background contrast was obtained at 50-74 h. There were no false-positive images. Whole-body radioactivity retention rose significantly with increasing EGF doses; most labeled EGF was eliminated by urinary excretion. Tumor:normal tissue uptake ratios increased during the course of the study. All patients presented self-limited, dose-related gastrointestinal adverse effects. In conclusion, recombinant 131I-labeled EGF administered i.v. can localize to squamous lung cancer efficiently, can be administered safely to patients, and has more advantageous pharmacokinetic properties than monoclonal antibodies. Further studies are warranted to determine more accurately the potential of EGF and EGF-related peptides in the imaging and/or therapy of EGFr-overexpressing human cancers.

Topics: Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Epidermal Growth Factor; Humans; Iodine Radioisotopes; Lung Neoplasms; Middle Aged; Radionuclide Imaging

To determine whether testosterone administration was capable of modifying salivary gland carcinogenesis, female mice were given 1 mg of 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland and then Group 1 mice received 5 mg of testosterone propionate and Group 2 mice received vehicle, olive oil, subcutaneously for 8 weeks. Twelve weeks after the start of the experiment, the weight of the left submandibular gland of the Group 2 mice was greater than that of the Group 1 mice. The incidences of submandibular gland carcinoma in Groups 1 and 2 were 41% (12/29) and 57% (17/30), respectively. Epidermal growth factor (EGF) levels of the left submandibular gland were significantly higher in Group 1 as compared with Group 2. These findings indicate that testosterone increases the production of EGF in the DMBA-injected submandibular gland, but does not promote the development of submandibular gland carcinoma.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adenocarcinoma; Animals; Carcinoma; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Mice; Organ Size; Submandibular Gland; Submandibular Gland Neoplasms; Testosterone

The discovery of EGFR gene amplification in glioblastoma multiforme has prompted interest in experimental therapies to target the receptor on brain tumor cells. To develop an animal model for in vivo study of such strategies, we transfected C6 glioma cells with a plasmid containing the neomycin resistance gene and the human EGFR gene under the control of the glucocorticoid-inducible MMTV promoter. Following selection with G418, individual clones that expressed EGFR at high levels were selected. Kinetics of EGF binding fit a dual site model indicating the presence of both high (KA = 2.5 x 10(9) M-1) and low (KA = 3.3 x 10(7) M-1) affinity receptors. To assess growth in vivo, graded numbers of either wild-type or transfected cells were implanted into the brains of CD Fischer 344 rats. No differences in survival were observed between groups of animals injected with either wild-type or transfected cells at inocula of 10(3) or 10(4) respectively. In addition, one-third of animals (7/21) challenged with 10(5) or 10(6) transfected cells survived > 50 days compared to 0% of animals (0/12) challenged with 10(5) or 10(6) wild-type cells. Such an effect suggests greater immunogenicity of transfected cells, but only at the larger inocula. Since C6 glioma cells will grow in both outbred and inbred strains, our model should have a number of applications including the in vivo study of EGFR targeting for glioma therapy.

Topics: Animals; Binding, Competitive; Carcinoma, Squamous Cell; Cell Division; Cell Line; DNA, Complementary; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Male; Rats; Rats, Inbred F344; Recombinant Proteins; Time Factors; Transfection; Tumor Cells, Cultured

Some human neoplasms show aberrant expression or overexpression of epidermal growth factor (EGF) receptor, and the degree of the receptor expression is correlated with the malignant phenotype in certain epithelial tumors including squamous carcinoma cells. Since phenotypic transformation of cells could involve quantitative and qualitative alteration of integrin function, the effects of EGF on cell-matrix interactions were studied using HSC-1 cells, a human squamous carcinoma cell line showing EGF receptor overexpression. The EGF-treated HSC-1 cells interacted with matrix proteins differently from the untreated cells, as shown by cell adhesion and phagokinetic track assays. Among fibronectin, laminin, fibrinogen, and type I collagen, fibronectin was the most efficient substratum to promote untreated HSC-1 cell adhesion and migration. Pretreatment of the cells with 50 ng/ml EGF for 18 h selectively increased the number of spread cells and the size of the individual cell migration area on type I collagen by 250 and 400%, respectively. The same pretreatment diminished cell adhesion and migration on other substrata so that the EGF treatment converted type I collagen as the most efficient substratum for cell adhesion and migration of the HSC-1 cells. ELISA and immunoprecipitation studies showed that EGF up-regulated the expression of alpha 2 beta 1 integrin collagen receptor in a time- and dose-dependent manner by stimulating biosynthesis of alpha 2 subunit, but did not up-regulate those of the alpha 3 beta 1, alpha 5 beta 1, or alpha v beta 3 integrins. These results suggest that EGF preferentially enhances HSC-1 cell interaction with type I collagen, leading to the enhanced cellular migratory activity on the substratum, as a result of selective up-regulation of alpha 2 beta 1 integrin expression.

Topics: Antigens, CD; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Adhesion; Cell Movement; Collagen; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix Proteins; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Integrin beta1; Integrins; Oligopeptides; Skin Neoplasms; Tumor Cells, Cultured; Up-Regulation

Epidermal growth factor (EGF) mediates many pleiotrophic biological effects, one of which is alteration of cellular morphology. In the present study, we examine the possibility that this alteration in cell morphology is caused in part by the dysfunction of cadherin-mediated cell-cell adhesion using the human oesophageal cancer cell line TE-2R, which expresses E-cadherin and EGF receptor. In the presence of EGF, TE-2R changed its shape from round to fibroblastic and its colony formation from compact to sparse. Vanadate, a tyrosine phosphatase inhibitor, further potentiated the EGF response, whereas herbimycin A, a tyrosine kinase inhibitor, interfered with it. Moreover, EGF enabled the cells to invade in organotypic raft culture. These phenomena were accompanied not by decreased expression of the E-cadherin molecule but by a change in its localisation from the lateral adhesion site to the whole cell surface. Both alpha- and beta-catenin, cadherin-binding proteins, were also expressed at the same level throughout these morphological changes. Finally, we examined tyrosine phosphorylation of E-cadherin and alpha- and beta-catenin, and observed tyrosine phosphorylation of beta-catenin induced by EGF. These results suggest that EGF counteracts E-cadherin-mediated junctional assembly through phosphorylation of beta-catenin and modulates tumour cell behaviour to a more aggressive phenotype.

Topics: alpha Catenin; Benzoquinones; beta Catenin; Cadherins; Carcinoma, Squamous Cell; Cell Adhesion; Cell Aggregation; Cell Line; Culture Techniques; Cytoskeletal Proteins; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Fibroblasts; Fluorescent Antibody Technique; Gels; Humans; Lactams, Macrocyclic; Neoplasm Invasiveness; Neoplasm Proteins; Phenotype; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Quinones; Rifabutin; Signal Transduction; Trans-Activators; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tyrosine; Vanadates

We have examined the interaction between c-Src and epidermal growth factor (EGF) receptor in A431 cells. c-Src was found exclusively in the Triton X-100-solubilized particulate fraction and activated up to 3-fold within 1 min after EGF treatment of the cells. Association between c-Src and EGF receptor was detected by immunoprecipitation of c-Src followed by immunoblotting with anti-EGF receptor antibody. The c-Src-EGF receptor complex was found in both EGF-treated and untreated cells, but an augmented complex formation was observed in EGF-treated cells. We have isolated the complex by DEAE-cellulose column chromatography and found that a site-specific anti-c-Src antibody, which was raised against a synthetic peptide corresponding to residues 413 to 431 of human c-Src, did not recognize the c-Src protein in the complex, while other c-Src-specific antibodies tested did. Incubation of the complex with this synthetic peptide resulted in a partial dissociation of the complex. These results suggest that the specific region of c-Src is involved in the association with EGF receptor.

Topics: Amino Acid Sequence; Animals; Antibodies; Binding Sites; Carcinoma, Squamous Cell; Cell Line; Chromatography, DEAE-Cellulose; Cytosol; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Mice; Molecular Sequence Data; Octoxynol; Peptides; Proto-Oncogene Proteins pp60(c-src); Tumor Cells, Cultured

Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells.

Topics: Actins; Blotting, Western; Carcinoma, Squamous Cell; Cell Line; Cytochalasin D; Epidermal Growth Factor; Humans; Intercellular Junctions; Membrane Proteins; Phosphoproteins; Phosphorylation; Phosphotyrosine; Tumor Cells, Cultured; Tyrosine; Zonula Occludens-1 Protein

The effect of epidermal growth factor (EGF) on the biological behaviour of human tumours in vivo is still controversial. We investigated the effect of EGF on the growth of an EGF receptor-hyperproducing human epidermoid carcinoma, A431 tumour, and on a human small-cell lung carcinoma, H69 tumour, without detectable EGF receptor by using sialoadenectomised (sialex) mice as an endogenous EGF-suppressed animal model. The plasma EGF concentration in the sialex athymic mice was significantly lower than that in the sham-operated mice (P < 0.05). After exogenous EGF replacement with an implanted minipump, the plasma EGF concentration was significantly increased in both groups (P < 0.05). There was no significant difference between the body weight growth curves of sialex and sham-operated mice with and without EGF treatment. The tumour weight of A431, both estimated and measured in sialex mice, was significantly lower than that in sham-operated control mice (P < 0.05), and the growth of A431 tumour was significantly increased by exogenous EGF treatment (P < 0.05). Mitotic activity of these tumours detected by immunohistochemical staining for incorporated bromodeoxyuridine indicated a mitosis-stimulatory effect of endogenous and exogenous EGF on A431 tumours. In contrast to these findings on A431 tumours, a growth-stimulatory effect of endogenous and exogenous EGF was not observed in the H69 tumour. These results suggest a growth-promoting effect of physiological levels of endogenous EGF on EGF receptor-hyperproducing human tumours in vivo.

Topics: Animals; Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mitosis; Neoplasm Transplantation; Transplantation, Heterologous

Proliferation of some cultured human tumor cell lines bearing high numbers of epidermal growth factor (EGF) receptors is paradoxically inhibited by EGF in nanomolar concentrations. In the present study, we have investigated the biochemical mechanism of growth inhibition in A431 human squamous carcinoma cells exposed to exogenous EGF. In parallel, we studied a selected subpopulation, A431-F, which is resistant to EGF-mediated growth inhibition. We observed a marked reduction in cyclin-dependent kinase-2 (CDK2) activity when A431 and A431-F cells were cultured with 20 nM EGF for 4 h. After further continuous exposure of A431 cells to EGF, the CDK2 activity remained at a low level and was accompanied by persistent G1 arrest. In contrast, the early reduced CDK2 activity and G1 accumulation in A431-F cells was only transient. We found that, at early time points (4-8 h), EGF induces p21Cip1/WAF1 mRNA and protein expression in both EGF-sensitive A431 cells and EGF-resistant A431-F cells. But only in A431 cells, was p21Cip1/WAF1 expression sustained at a significantly increased level for up to 5 d after addition of EGF. Induction of p21Cip1/WAF1 by EGF could be inhibited by a specific EGF receptor tyrosine kinase inhibitor, tyrphostin AG1478, suggesting that p21Cip1/WAF1 induction was a consequence of receptor tyrosine kinase activation by EGF. We also demonstrated that the increased p21Cip1/WAF1 was associated with both CDK2 and proliferating cell nuclear antigen (PCNA). Taken together, our results demonstrate that p21Cip1/WAF1 is an important mediator of EGF-induced G1 arrest and growth inhibition in A431 cells.

Topics: Carcinoma, Squamous Cell; CDC2-CDC28 Kinases; Cell Cycle; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Growth Inhibitors; Humans; Nitriles; Phenols; Proliferating Cell Nuclear Antigen; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

The expression of EGF/EGFR in 47 laryngeal surgical specimens from 44 patients was examined. PCNA analysis as an index of proliferating cells was also performed in 32 cases of laryngeal cancer, six cases of pre-cancerous lesions and nine cases of normal laryngeal mucosa. EGFR failed to show a significant correlation with tumour behaviour, but EGF expression was statistically significantly higher in malignant (SCC) than in non-malignant tissues (pre-cancerous and normal tissues) (p < 0.006), and PCNA also showed a statistically significant difference (p < 0.016) between the two. In malignant tissues when EGF/EGFR in 'double-positive' and 'double-negative' cases was compared, a statistically significant difference in PCNA was found (p < 0.029); but this was not seen in non-malignant tissues. Our results support the hypothesis that an autocrime mechanism exists in laryngeal cancer and in this mechanism EGF may play an important role in tumour progression, especially when EGFR is overexpressed.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Laryngeal Neoplasms; Larynx; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Precancerous Conditions; Proliferating Cell Nuclear Antigen

The effect of glucocorticoids on epidermal growth factor (EGF)-induced expression of 12-lipoxygenase in human epidermoid carcinoma A431 cells was studied. A significant suppression of the EGF-induced expression of 12-lipoxygenase was observed in cells pretreated with 1 microM dexamethasone for 2 hr. The same pretreatment for 8 hr resulted in 55 and 54% inhibition of EGF-induced 12-lipoxygenase activity and mRNA expression, respectively. Cortisol, but not sex and mineral steroids, had a similar inhibitory effect. The glucocorticoid antagonist RU486 completely blocked the inhibitory effect of dexamethasone, suggesting that the action of dexamethasone was mediated through the ligation of glucocorticoid receptors. The results indicated that pretreatment of A431 cells with glucocorticoids resulted in a down-regulation of the EGF-induced expression of 12-lipoxygenase at the mRNA and enzyme activity level, which was mediated through glucocorticoid receptor activation.

Topics: Arachidonate 12-Lipoxygenase; Carcinoma, Squamous Cell; Dexamethasone; Enzyme Induction; Epidermal Growth Factor; Glucocorticoids; Humans; Hydrocortisone; Mifepristone; RNA, Messenger; Tumor Cells, Cultured

Tenascin-C, a 6-armed extracellular matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during tumorigenesis in association with stromal-epithelial interactions. We have previously shown that de novo synthesis of tenascin-C is induced by the diffusible factor EGF in tenascin-C-non-producing human epidermoid carcinoma cells in stromal-epithelial interactions. We now demonstrate that the addition of human tenascin-C or tenascin-C peptides to the culture medium of these cells had little effect on the induction of tenascin-C. The physiological regulators of tenascin-C induction through the EGF receptor, however, have not yet been characterized. We show that steroid hormones down-regulate EGF-induced tenascin-C glycoprotein and its mRNA in these tenascin-C-non-producing carcinoma cells. Of the steroids examined, hydrocortisone most effectively inhibited the secretion of tenascin-C. These steroids did not affect EGF-induced autophosphorylation or de novo synthesis of EGF receptors, nor did they compete for the binding of EGF to its receptor. Our results indicate that the induction of tenascin-C by EGF and its down-regulation by steroids might proceed in these carcinoma cells through separate signal transduction pathways.

Topics: Amino Acid Sequence; Animals; Carcinoma, Squamous Cell; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Mice; Mice, Nude; Molecular Sequence Data; Steroids; Tenascin; Tumor Cells, Cultured

Cholera toxin and pertussis toxin were inhibitory to the incorporation of thymidine into A431 cells in serum-free culture. Cholera toxin enhanced the growth inhibitory effect of epidermal growth factor (EGF) on A431 cells, whereas pertussis toxin attenuated the effect. Cholera toxin increased the concentration of intracellular cyclic AMP (cAMP) to three-times the initial concentration at 120 minutes and it increased the concentration of intracellular inositol trisphosphate (IP3) rapidly but transiently. Pertussis toxin reduced the concentration of IP3 both in EGF treated and untreated A431 cells at 10 minutes. cAMP was not involved in pertussis toxin-mediated effects. In conclusion, the intracellular cAMP and IP3 concentrations in CT-treated A431 cells are compatible with previous reports regarding the growth inhibitory effects on A431 cells. The inhibitory effect of PTX on the EGF-induced increase of intracellular IP3 is thought to be compatible with the finding that PTX attenuated the EGF-induced growth inhibition.

Topics: Carcinoma, Squamous Cell; Cell Division; Cholera Toxin; Cyclic AMP; DNA; Epidermal Growth Factor; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Mitosis; Pertussis Toxin; Thymidine; Tumor Cells, Cultured; Virulence Factors, Bordetella

Epiregulin, a novel epidermal growth factor (EGF)-related growth regulating peptide, was purified from conditioned medium of the mouse fibroblast-derived tumor cell line NIH3T3/clone T7. It was a 46-amino-acid single chain polypeptide, and its amino acid sequence exhibited 24-50% amino acid sequence identity with sequences of other EGF-related growth factors. Epiregulin exhibited bifunctional regulatory properties: it inhibited the growth of several epithelial tumor cells and stimulated the growth of fibroblasts and various other types of cells. Epiregulin bound to the EGF receptors of epidermoid carcinoma A431 cells much more weakly than did EGF, but was nevertheless much more potent than EGF as a mitogen for rat primary hepatocytes and Balb/c 3T3 A31 fibroblasts. These findings suggest that epiregulin plays important roles in regulating the growth of epithelial cells and fibroblasts by binding to receptors for EGF-related ligands.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cells, Cultured; Chromatography; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Durapatite; Epidermal Growth Factor; Epiregulin; ErbB Receptors; HeLa Cells; Humans; Liver; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Rats; Sequence Homology, Amino Acid; Tumor Cells, Cultured

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Line, Transformed; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Humans; Kinetics; Mice; Mice, Inbred BALB C; Mice, Nude; Oncogene Proteins v-sis; Oncogenes; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyridines; Pyrimidines; Receptors, Platelet-Derived Growth Factor; Retroviridae Proteins, Oncogenic; Signal Transduction; Tetradecanoylphorbol Acetate; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured

Increased phosphorylation in cancers can stimulate growth and up-regulate certain receptors. To test whether the functional response of phosphatase receptors is up-regulated during carcinogenesis, we examined the effects of ligands on net phosphorylation in isolated membranes derived from hamster cheek-pouch tissues undergoing malignant transformation. The buccal mucosa of groups of Syrian golden hamsters was exposed thrice weekly to 0.5% dimethylbenzanthracene (DMBA) in acetone for 2-12 weeks to produce premalignant and malignant tissues. Homogenates of these tissues were then incubated with [32P]ATP in the presence of epidermal growth factor (EGF), agonist of somatostatin analogue RC-160, luteinizing-hormone-releasing hormone (LH-RH) [D-Trp6]LH-RH, or combinations of EGF, RC-160, and [D-Trp6]LH-RH. Changes compared to controls in phosphorylation in response to ligands provided estimates of kinase or phosphatase activity. Phosphorylation increased continuously, from the first application of DMBA in a linear fashion, and independently of EGF stimulation. RC-160 and [D-Trp6]LH-RH reduced phosphorylation in vitro. This response occurred in premalignant (weeks 6-10 after DMBA application) as well as malignant tissues (week 12 after DMBA application), but was not significant in normal tissues. The results show a continuous augmentation in phosphatase activity prior to the appearance of cancers, but with a delay in expression following the primary event of increased kinase activity. Significantly less phosphorylation of substrates was induced by both RC-160 and [D-Trp6]LH-RH after in vitro activation by EGF than in the absence of EGF. This suggests that EGF activates latent systems of hormonal receptors. Collectively, these results support the hypothesis that the enhancement of the hormonally stimulated phosphatase in cancers occurs secondarily to the increased kinase activity.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cricetinae; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Mesocricetus; Mouth Mucosa; Phosphorylation; Precancerous Conditions; Protein Tyrosine Phosphatases; Receptor Protein-Tyrosine Kinases; Somatostatin; Time Factors; Up-Regulation

The molecular mechanisms underlying the growth inhibition of human tumor cells induced by recombinant interferon-alpha (IFN alpha) are mostly unknown. It has been proposed that this effect could be related to down-regulation and/or impaired function of peptide growth factor receptors (PGF-Rs) in tumor cells exposed to IFN alpha. However, we have previously described that IFN alpha-induced growth inhibition of human epidermoid carcinoma cells is paralleled by up-regulation of epidermal growth factor receptor (EGF-R). Here we report that an increase in EGF-R synthesis is detectable after 3 hr of exposure to cytostatic concentration of IFN alpha in epidermoid KB tumor cells. In these experimental conditions IFN alpha does not depress and even potentiates EGF-R function. IFN alpha-treated KB cells retain sensitivity to the cytotoxic activity of the anti-EGF-R 225 monoclonal antibody (MAb), which acts through receptor blockade, and are sensitized to the growth-promoting effect of EGF. EGF-induced tyrosine (tyr) phosphorylation both of total cellular protein extracts and of the immunoprecipitated EGF-R is increased in IFN alpha-treated cells. We conclude that a cross-talk between IFN alpha and EGF occurs in KB cells since IFN alpha, at cytostatic concentration, potentiates the effects mediated by the EGF-R.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Interferon alpha-2; Interferon-alpha; KB Cells; Kinetics; Molecular Weight; Recombinant Proteins; Time Factors; Up-Regulation

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of A431 cells, leading to a approximately 30% rise in cellular filamentous actin levels. EGF-induced actin polymerization depends upon EGF receptor (EGFR) tyrosine kinase activity, since the selective tyrosine kinase inhibitor AG213 abolishes EGF-induced actin polymerization. In accordance, confocal laser scanning microscopy shows that newly assembled actin filaments localize selectively to the tyrosine-phosphorylated EGFR in the plasma membrane, since actin polymerization is not observed at the internalized tyrosine-phosphorylated EGFR. Actin binding proteins (ABP's) are generally believed to regulate actin filament assembly. Ca2+ is known as one of the important regulatory factors for the activity of ABP's in vitro [15]. Therefore, we investigated the importance of the EGF-induced transient rise in [Ca2+]i for the regulation of actin polymerization in vivo. Continuous high [Ca2+]i in the millimolar range induces a prominent rise in cellular filamentous actin levels to approximately 50% over control cells. However, actin polymerization is unimpaired under conditions which effectively block the EGF-induced [Ca2+]i transient. These data demonstrate that EGF-induced actin polymerization localizes to the activated EGFR in the membrane skeleton, independent of the cytosolic free calcium transient.

Topics: Actins; Adenosine Triphosphate; Calcimycin; Calcium; Carcinoma, Squamous Cell; Catechols; Cell Line; Cell Membrane; Cytosol; Egtazic Acid; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Kinetics; Macromolecular Substances; Microscopy, Confocal; Nitriles; Phosphorylation; Skin Neoplasms; Time Factors; Tumor Cells, Cultured; Tyrphostins

We previously reported that the octapeptide somatostatin (SS) analogue SMS 201-995 (SMS) unexpectedly stimulates the growth of A431 human epidermoid carcinoma cells in vitro. In the present study, we found that both SS-14 and SS-28 also stimulated the growth of A431 cells in vitro. The proliferative effect of SS-28 also stimulated the growth of A431 cells in vitro. The proliferative effect of SS-28 was greater than that of SS-14. In contrast, there was no difference in A431 cell tumor weight or area between the SMS-treated or untreated athymic, tumor-bearing mice. The serum epidermal growth factor (EGF) level of the SMS-treated mice was significantly lower than that of the untreated mice. Decreases in serum EGF may attenuate the proliferative effect of SMS in vivo.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; Humans; Male; Mice; Mice, Nude; Somatostatin; Tumor Cells, Cultured

Polyamines--putrescine, spermidine, and spermine--are ubiquitous cellular components that play an important role in cell growth and differentiation. Using A431 cells, a cell line that overexpresses the epidermal growth factor (EGF) receptor, we found that polyamines modulate EGF-mediated growth inhibition. The natural polyamine, putrescine, was the most effective, followed by diamines containing lower and higher methylene bridging between the amino groups. To understand the mechanism, we examined the effects of polyamines on EGF-mediated signal transduction in A431 cells. All three polyamines partially inhibited EGF-receptor tyrosine kinase activity in a dose-dependent manner. The maximal inhibition was 75% with spermidine. Polyamine effects were exerted 12-16 h after treatment, although HPLC analysis revealed uptake of polyamines within 1 h. Homologues of putrescine had no significant effect on tyrosine kinase activity, indicating structural specificity of naturally occurring polyamines in this process. Amine oxidase inhibitors did not alter spermidine and spermine-mediated effects, suggesting that the inhibition of tyrosine kinase activity was not a consequence of the oxidative metabolism of polyamines. Difluoromethylornithine, a specific inhibitor of polyamine biosynthesis, did not affect EGF receptor tyrosine kinase activity. Polyamines also had no effect on EGF receptor levels or EGF-EGF receptor high-affinity binding, indicating that they are not competitive inhibitors of the EGF receptor tyrosine kinase. Our results suggest that polyamine action in A431 cells involves modulation of EGF receptor signal transduction pathways.

Topics: Biogenic Polyamines; Carcinoma, Squamous Cell; Epidermal Growth Factor; Humans; Phosphorylation; Receptor Protein-Tyrosine Kinases; Tumor Cells, Cultured

Some types of malignant neoplasms are consequence of genetic changes. The starting of oncogenesis begins when some genetic alterations arises, or the genome acquires some peculiar and pathological traits owing to the action of physical, chemical or viral agents. These alterations set in motion an activatory phenomenon upon the proto-oncogene which is turned into an oncogene. The oncogene is responsible for fixed alterations of the genome inducing to an anomalous cellular growth. In squamous carcinomata of the head and neck has been observed mutations of the p-53 tumour-suppressor gene, alike amplifications of the ras-gene, of the C-myc proto-oncogene and also of the epidermal growth factor gene (EGFG). The viruses induce mutations on the genome. That would be the explanatory reason for the association carcinoma of the rhinopharynx-Epstein-Barr virus.

Topics: Carcinoma, Squamous Cell; DNA, Viral; Epidermal Growth Factor; Gene Amplification; Genes, ras; Genome, Human; Head and Neck Neoplasms; Herpesvirus 4, Human; Humans; Nasopharynx; Point Mutation; Proto-Oncogene Mas; Proto-Oncogenes; RNA, Viral

To investigate the contribution of the N-terminal sequence of h-EGF to its biological activity and the formation of three intramolecular disulfide bonds by oxidative refolding via air oxidation, five derivatives of h-EGF with a single N-terminal amino acid deletion were synthesized by solid-phase synthesis. The homogeneity of the synthetic peptides was confirmed by analytical reversed-phase HPLC, amino acid analysis, and FAB-MS. The pairing of the three disulfide bridges in synthetic peptides was determined by thermolytic digestion. All N-truncated derivatives of h-FGF formed the correct intramolecular three disulfide linkages during oxidative refolding and had equipotent activity in both EGF receptor binding on A-431 epidermoid carcinoma cells and mitogenesis on NIH-3T3 fibroblast cells, compared with authentic h-EGF. The results suggested that the five residues from N-terminal sequence of h-EGF have no effect on the formation of the correct disulfide linkages in h-EGF and do not exert a significant influence on its biological activity.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Binding, Competitive; Carcinoma, Squamous Cell; Cell Division; Chromatography, High Pressure Liquid; Cysteine; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Indicators and Reagents; Kinetics; Mice; Molecular Sequence Data; Peptide Fragments; Protein Conformation; Protein Folding; Tumor Cells, Cultured

The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.

Topics: Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Energy Transfer; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Kinetics; Macromolecular Substances; Microscopy, Fluorescence; Models, Structural; Models, Theoretical; Spectrometry, Fluorescence; Time Factors; Tumor Cells, Cultured

F.2a and B.2, cell clones of the human squamous cell carcinoma SCC-12, were examined to characterize their interactions through the expression of growth factors. F.2a was nontumorigenic yet B.2 was fully tumorigenic when injected into the flanks of athymic nude mice. Combination injections of F.2a and a subtumorigenic level of B.2 produced tumors. F.2a and B.2 overexpressed the 4.8-kb transcript for transforming growth factor-alpha (TGF-alpha) as well as the 10.5- and 5.8- kb transcripts for the epidermal growth factor receptor. Neither clone expressed the transcript for epidermal growth factor, while both expressed transcripts for insulin-like growth factor-I (IGF-I) of 8.15, 4.9, and 1.6 kb and transcripts for its receptor of 8.5 and 6.5 kb. Conditioned medium (CM) from either clone stimulated the growth of itself and the other clone in tissue culture. Both clones produced intracellular TGF-alpha detectable by immunohistochemical staining, but not detectable in CM by enzyme-linked immunosorption assay. IGF-I was detected at low levels in CM by radioimmunoassay. Neutralizing antibodies to TGF-alpha but not IGF-I partially inhibit the growth of both clones in tissue culture. These results suggest that (1) TGF-alpha is active in autocrine signaling (2) IGF-I is not directly stimulatory, and (3) additional factors, as yet unidentified, are present in CM and enhance tumor growth. It is concluded that human squamous cell carcinoma SCC-12 is composed of tumorigenic and nontumorigenic clones which interact to enhance growth.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin-Like Growth Factor I; Mice; Mice, Nude; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

To elucidate the effect of ionizing radiation on the membrane anchored signal transduction, the binding of 125I epidermal growth factor (EGF) to its receptor (EGF-R) and the EGF-dependent EGF-R tyrosine phosphorylation were examined in a human squamous cell carcinoma cell line, A431. The significant suppression of 125I EGF binding to A431 cells was observed from 3-5 h after 10 Gy irradiation, whereas this inhibition was not observed both in non-irradiated and in 5 Gy-irradiated cells. This phenomenon was mediated by the protein kinase C pathway, because the inhibition was not observed in cells which had been pretreated with phorbol ester and treated with an inhibitor of the enzyme, H7. Scatchard analysis showed that the receptor affinity was decreased. In contrast, the level of EGF-dependent EGF-R-tyrosine phosphorylation was not decreased, compared with non-irradiated cells. These results suggest that ionizing radiation may modulate the function of EGF/EGF-R interaction through the direct activation of protein kinase C.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Phosphorylation; Protein Kinase C; Signal Transduction; Tumor Cells, Cultured; Vulvar Neoplasms; X-Rays

Photodynamic therapy combines photosensitizers absorbing light in the red spectral region and irradiation with light of the corresponding wavelength. To analyse the influence of cell differentiation on susceptibility to photodynamic therapy, we compared the proliferation inhibition induced by photodynamic therapy on normal human keratinocytes, spontaneously transformed human keratinocyte cell line HaCat and on squamous cell carcinoma lines. Cells were irradiated with polychromatic methylene blue as well as the precursor of protoporphyrin, 5-aminolevulinic acid. When incubated with Photosan-3, normal human keratinocytes exhibited and ED50 about 10-fold lower than the other cell lines studied. When methylene blue and 5-aminolevulinic acid were used, photodynamic therapy had comparable effects on all cell types. Stimulation of normal human keratinocytes with either EGF-alpha or IFN-gamma resulted in an increase susceptibility to photodynamic therapy when 5-aminolevulinic acid was used. This effect was more pronounced in the case of EGF-alpha. Our experiments suggest that activation and differentiation are two important parameters determining susceptibility to photodynamic therapy.

Topics: Aminolevulinic Acid; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line, Transformed; Dose-Response Relationship, Drug; Epidermal Growth Factor; Hematoporphyrins; Humans; Interferon-gamma; Keratinocytes; Methylene Blue; Photochemotherapy; Photosensitizing Agents; Skin Neoplasms; Tumor Cells, Cultured

The interaction between epidermal growth factor (EGF) and specific membrane receptors in A-431 cells results in stimulation of its tyrosine kinase (TK) activity. The intrinsic tyrosine kinase phosphorylates both the receptor itself and different intracellular substrates. Upon EGF binding, clustering and internalization of EGF-receptor (EGF-R) complexes are observed. In the present study by incubating A-431 cells with EGF at 4 or at 37 degrees C, eluting surface-bound EGF we were able to compare the TK-activity of internalized EGF receptors to those localized on the plasma membrane. By the method of the phosphorylation of exogenic substrate poly (Glu/Tyr) 4:1 in vitro it was shown that the total TK-activity was equal to 3 microM PO4- incorporated/mg protein/min. Under conditions, when 40% EGF-R complexes were internalized their activity consisted of 46% of the total after inactivation 60% surface-bound receptors. We draw a conclusion that the TK-activity of EGF-receptors is remained during their internalization.

Topics: Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Ionizing radiation is believed to stimulate the repopulation of squamous carcinoma cells that survive the early portion of a fractionated course of radiotherapy. To characterize any intrinsic radiation-induced adaptive response and to examine whether epidermal growth factor (EGF) influences this response, A431 and 183A cells were irradiated with repeated daily exposures of 0.5-0.75 Gy and then grown in monolayer culture for 7 days with or without EGF at a 1 ng/ml concentration. Cell numbers were quantified using a microtiter dye-reduction assay. EGF alone caused approximately 70% and 30% growth inhibition of human SC A431 and 183A cells, respectively. Although radiation alone did not affect proliferative rates in these conditions, radiation eliminated the EGF-related growth inhibition in both cell lines. This effect was dose dependent in single radiation exposure experiments. Cell cycle analyses indicated that EGF initially promoted entry into S-phase 3 days after treatment but caused a G1-S block after 7 days. Treatment with radiation recruited cells into S-phase and G2-M, an effect which was sustained 7 days after treatment, overriding the influence of EGF. Radiation-induced modulation of the response of human squamous carcinoma cells to EGF in vitro after single and repeated radiation exposures suggests a proliferation response that may underlie enhanced repopulation of tumor clonogens in vivo.

Topics: Carcinoma, Squamous Cell; Cell Count; Cell Cycle; Cell Division; Dose-Response Relationship, Radiation; Epidermal Growth Factor; ErbB Receptors; Humans; Tumor Cells, Cultured

Certain tumor cells, such as squamous carcinomas and gliomas, can have an increased number of epidermal-growth-factor (EGF) receptors. The EGF receptors can in these cases be targets for toxic conjugates with specific binding. EGF-based toxic conjugates are potential targeting agents. We have analyzed the internalization and excretion of 125I administered in the form of 125I-EGF-dextran in squamous-carcinoma A431 cells. 125I-EGF without dextran was used for comparison. A431 cells have large numbers of EGF receptors and are capable both of recycling and of degradation of internalized receptor-ligand complexes. The binding of 125I-EGF-dextran and 125I-EGF was receptor-specific, since both ligands competed with non-radioactive EGF for binding. The amount of internalized 125I as a function of time increased continuously within 24 hr following administration of radioactivity as 125I-EGF-dextran. The time pattern was quite different when 125I-EGF without dextran was applied. In the latter case, the amount of internalized radioactivity decreased already after a few hours, probably depending on degradation of EGF. Pre-incubation of the cells with 125I-EGF-dextran or 125I-EGF and analysis of retained and released 125I activity at different times after washing showed that the 125I activity was retained for longer periods of time when EGF-dextran was used instead of EGF. About 30% of the internalized 125I activity was retained after 24 hr when EGF-dextran was used, compared with excretion of nearly all the radioactivity within 5 hr when EGF was used. In some experiments a high concentration of non-radioactive EGF, 5 micrograms/ml, was given to the cells after pre-incubation with 125I-EGF-dextran. This changed the retention and excretion patterns, so that a larger amount of 125I was excreted in the macromolecular fraction and a smaller amount of 125I activity was retained in the cells. Gel chromatography of the 125I activity released into the culture medium showed that the variations in molecular weight were larger after administration of a high concentration of non-radioactive EGF, most likely due to partial degradation of EGF-dextran. The results regarding excretion are in conformity with a model of competition between recycling of EGF-dextran-EGF-receptor complexes and "trapping" of EGF-dextran in the lysosomes followed by slow degradation. For targeting purposes, it is worth noting that the radioactivity administered in the form of 125I-EGF-dextran had a longer r

Topics: Binding, Competitive; Carcinoma, Squamous Cell; Cell Division; Culture Media; Dextrans; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Tumor Cells, Cultured

Epidermal growth factor (EGF) increases 12-lipoxygenase mRNA by about 2-fold with a lag period of 4 to 8 hr, which precedes the increase in 12-lipoxygenase activity by 2 to 4 hr in human epidermoid carcinoma A431 cells. Induction of 12-lipoxygenase expression in human erythroleukemia cells by phorbol 12-myristate 13-acetate (PMA) has been reported previously. The present report describes a study of the involvement of protein kinase C (PKC) in EGF-induced 12-lipoxygenase expression in A431 cells. EGF-induced 12-lipoxygenase expression was inhibited by methyl 2,5-dihydroxycinnamate, a tyrosine kinase inhibitor. Staurosporine and calphostin C, which are two PKC inhibitors, inhibited EGF-induced enzyme activity and mRNA expression of 12-lipoxygenase. 1,2-Dioctanoyl-sn-glycerol (a membrane-permeant diacylglycerol) and PMA significantly induced enzyme activity and mRNA expression. Simultaneous treatment of cells with EGF and PMA did not exhibit an additive effect, suggesting that EGF and PMA share a common biochemical pathway in 12-lipoxygenase induction. Expression of mRNA for PKC alpha, delta and zeta was detected in A431 cells, whereas no mRNA expression for PKC beta 1, gamma and epsilon was observed. Taken together, these results suggest that EGF-induced 12-lipoxygenase expression is at least in part mediated by the PKC signal transduction pathway.

Topics: Alkaloids; Arachidonate 12-Lipoxygenase; Carcinoma, Squamous Cell; Diglycerides; Enzyme Induction; Epidermal Growth Factor; Humans; Naphthalenes; Phosphatidylinositol Phosphates; Polycyclic Compounds; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We have characterized 24 hypopharyngeal squamous cell carcinomas and 5 normal hypopharyngeal tissues by immunostaining with antibodies against epidermal growth factor (EGF), EGF-Receptor (EGFR), p53, v-erb B, and ras p21. The Avidin-Biotin Complex (ABC) technique was employed. Overexpression of p53 appeared in 17 of 24 cases of squamous cell carcinoma of the hypopharynx (normal mucosa: none, well differentiated: 60%, moderately differentiated: 71.4%, poorly differentiated: 71.4%). Some dysplastic mucosae surrounding cancer lesions showed overexpression of p53. EGF and EGFR tended to be expressed more strongly in carcinoma [EGF: 29.1% (well differentiated: 30%, moderately differentiated: 28.6%, poorly differentiated: 28.6%); EGFR: 50% (well differentiated: 60%, moderately differentiated: 42.9%, poorly differentiated: 42.9%)] than in normal mucosa (EGF: 0%, EGFR: 20%). The v-erb B stained positively in carcinoma [62.5% (well differentiated: 70%, moderately differentiated: 71.4%, poorly differentiated: 42.9%)] but negatively in normal mucosa. These data suggest that genetic mutations of p53 probably play an important role at an early stage of tumorigenesis, and that the networks of EGF, EGFR and v-erb B probably are involved in the development of hypopharyngeal squamous cell carcinoma.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hypopharynx; Immunohistochemistry; Male; Middle Aged; Oncogene Proteins v-erbB; Pharyngeal Neoplasms; Proto-Oncogene Proteins p21(ras); Tumor Suppressor Protein p53

The ratio of tyrosine-phosphorylated EGF-receptors was estimated for surface and endosomal EGF-receptor complexes on A431 cells. The cells were incubated with 125I-EGF, and then 125I-EGF was covalently bound to the receptor by cross-linking reagent-suberate. Following the cell lysis, the tyrosine-phosphorylated receptors were immunoprecipitated by polyclonal antiphosphotyrosine antibody. The ratio between the whole pool of 125I-EGF-linked and tyrosine-phosphorylated receptors were determined by densitometry of gels. It is found that only 20-45% of EGF-linked receptors are tyrosine-phosphorylated. The same share of tyrosine-phosphorylation was found for internalized EGF-receptor complexes.

Topics: Carcinoma, Squamous Cell; Cell Membrane; Endosomes; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Ligands; Membrane Proteins; Phosphorylation; Precipitin Tests; Protein Binding; Tumor Cells, Cultured; Tyrosine

Many of the effects of growth factors or hormones are mediated through the activation of protein kinase cascades. In this regard, it is well established that the activity of several protein kinases can be dramatically increased when cells are treated with a variety of stimuli. Since 1987, there have been several reports demonstrating that the activity of casein kinase II (CKII) can be acutely increased by hormones or growth factors. However, these are a number of discrepancies regarding the activation of CKII. In this study, we have examined CKII activities in extracts prepared from cells following treatment with stimuli that had been previously shown to elicit dramatic increases in CKII activity. Human WI.38 diploid lung fibroblasts were stimulated with serum or a variety of other stimuli including insulin, platelet-derived growth factor, fibroblast growth factor, epidermal growth factor, or phorbol myristate acetate. Human A431 epidermal carcinoma cells were similarly treated with epidermal growth factor. No reproducible increases in CKII activity were observed in response to any of these treatments. By comparison, a dramatic increase in kinase activity towards a synthetic peptide based on phosphorylation sites within the ribosomal S6 protein was consistently measured. Our observations indicate that CKII is not regulated in a similar manner by growth factors as are the protein kinases of the MAP kinase cascade, e.g., MAP kinase itself or ribosomal protein S6 kinase.

Topics: Amino Acid Sequence; Animals; Bombesin; Carcinoma, Squamous Cell; Casein Kinase II; Cattle; Cell Line; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Induction; Epidermal Growth Factor; Fetal Blood; Fibroblast Growth Factors; Fibroblasts; Growth Substances; Humans; Insulin; Ionomycin; Lung; Molecular Sequence Data; Neoplasm Proteins; Peptide Fragments; Phosphorylation; Platelet-Derived Growth Factor; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Ribosomal Protein S6; Ribosomal Protein S6 Kinases; Ribosomal Proteins; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Although human squamous cell carcinoma (SCC) cell lines frequently contain an elevated number of epidermal growth factor (EGF) receptor accompanied with amplification of EGF receptor/c-erbB gene, it is well known that EGF inhibits the growth of these cells in culture at doses that stimulate the growth of epidermal keratinocytes and dermal fibroblasts. To study this growth inhibitory effect of EGF on the SCC cell lines, 3H-thymidine incorporation into DNA and cell cycle distribution were analyzed. In HSC-1 and NA cells, which contain the highest number of EGF receptor among these SCC cell lines, the inhibition of 3H-thymidine incorporation was apparent 2 to 4 hours after treatment with 100 ng/ml of EGF and reached more than 95% inhibition after 24 hours. Two-color cell cycle analysis using fluorescein isothiocyanate (FITC)-conjugated anti-Bromodeoxyuridine (BrdU) antibody and propidium iodide revealed that this inhibitory effect was due to cell cycle arrest not only in G1 but also in G2 phase. This effect was well correlated to the sensitivity to the growth inhibitory effect of EGF among the 4 SCC cell lines. These observations suggest that the SCC cells contain altered machineries which regulate the normal cell growth in both G1 and G2 phases, and the EGF affects these machineries via overexpressed its receptor.

Topics: Carcinoma, Squamous Cell; Cell Cycle; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Thymidine; Tumor Cells, Cultured

Specific small ribonucleoprotein (alpha-RNP) complexes have been identified and characterized in the human epidermal carcinoma A-431 cells. The alpha-RNP complexes contain Alu-homologous small RNA, along with other small antisense RNA species. The epidermal growth factor (EGF) has been shown to induce selective specific changes in the expression of the small alpha-RNAs, the expression of the Alu-like RNA being repressed. Specific changes in the protein composition of the alpha-RNP complexes have been detected under the influence of EGF.

Topics: Carcinoma, Squamous Cell; DNA, Neoplasm; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Nucleic Acid Hybridization; Ribonucleoproteins, Small Nuclear; RNA, Neoplasm; RNA, Small Nuclear; Tumor Cells, Cultured

Two subpopulations of epidermal growth factor (EGF) receptor with different affinity to EGF have been demonstrated for many cell types. There are reasons to assume a key role of high-affinity receptors in stimulation of cell response to EGF. Nevertheless, characteristics of its action remain obscure. In the present work an attempt has been made to study internalization and intracellular compartmentalization of high-affinity receptors. For this purpose endocytosis of 125I-EGF in A431 cells was stimulated by low (less than 1 nM) EGF concentrations as well as after blocking of low-affinity binding sites with specific monoclonal antibodies 2E9. By subcellular fractionation in 17% Percoll gradient it was demonstrated that in both cases internalized 125I-EGF was found first in light, and then in heavy endosomes staying there for a long time. Effectiveness of 125I-EGF delivery to prelysosomal heavy endosomes as well as initial internalization rate is maximal at low EGF concentrations and is reduced dramatically with increasing of receptor occupancy. Monoclonal antibody Mab108 specifically recognizing high-affinity receptors were capable of stimulation of receptor internalization with initial rate higher than that of high EGF concentrations, but Mab108-receptor complexes were localized in light endosomes. Preincubation of the cells with low concentrations of EGF led to redistribution of considerable portion of 125I-Mab108 into heavy endosomes, which may be a result of high-affinity receptor tyrosine kinase activation. Our data confirm a hypothesis of TK involvement in regulation of both internalization and sorting of EGF-receptor complexes. Structural organization of high-affinity receptors is not sufficient for receptor targeting to degradation pathway.

Topics: Antibodies, Monoclonal; Antibody Affinity; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Ligands; Tumor Cells, Cultured

The epidermal growth factor (EGF) receptor is overexpressed in squamous cell carcinoma and the EGF receptor has been proposed as a potential target for new therapeutic agents in this tumour type. We have utilized a tyrphostintype inhibitor of the EGF receptor tyrosine kinase domain (RG50864) to study EGF-dependent proliferation and phosphorylation in two human squamous cell carcinoma cell lines. There were selected on the basis that whereas both cell lines have a large number of EGF receptors, one is growth inhibited by EGF (A431) while the proliferation of the other cell line (B2A4) is stimulated by EGF. EGF induced receptor autophosphorylation in each of the two cell lines; however, the level of phosphorylation was greater in the A431 cells than in the B2A4 cells. The pattern of proteins phosphorylated in response to EGF was different in the two squamous cell lines. RG50864 antagonized the EGF-dependent proliferation of B2A4 cells, but was unable to reverse the inhibitory effect of EGF on A431 cell growth. RG50864 partially inhibited EGF receptor autophosphorylation in both cell lines and completely inhibited the EGF-dependent phosphorylation of other cellular proteins, one of which co-migrated with MAP2kinase in both cell lines. Moreover, different dose-response relationships for the inhibition of phosphorylation of various proteins were observed in A431 versus B2A4 cells. As a substrate competitive inhibitor of the EGF receptor tyrosine kinase, the primary mode of action of RG50864 may be to prevent the association and/or phosphorylation of multiple specific substrates of the receptor in a fashion which may be cell line dependent. The precise relationship of these phosphorylation events to tyrphostin sensitivity remains to be established.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Catechols; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Nitriles; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tyrphostins

The activation of A1 and A2 adenosine receptors was shown to modulate DNA synthesis. In A431 cells, low concentrations of adenosine (approximately 10 microM) inhibited while higher concentrations (approximately 100 microM) stimulated [14C]thymidine incorporation. Epidermal growth factor (EGF) (10 ng/ml) could partially alleviate the inhibitory effects and further enhance the stimulatory effects of adenosine. Treatment of the cells with adenosine analogues (R-PIA and NECA) and antagonists (theophylline and DPCPX), which are selective for either the A1 (inhibitory) or A2 (stimulatory) receptors, indicated the involvement of these two receptors in the biphasic response to adenosine.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Carcinoma, Squamous Cell; DNA, Neoplasm; Epidermal Growth Factor; Humans; Phenylisopropyladenosine; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline; Tumor Cells, Cultured; Xanthines

We have studied insulin-stimulated threonine phosphorylation of cellular proteins by immunoblotting and immunoprecipitation using antiphosphothreonine antibody (anti-P-Thr). A 50-kilodalton protein (p50) was found to be greatly phosphorylated on threonine residues upon insulin stimulation in intact rat hepatoma cells (Fao) and Chinese hamster ovary cells overexpressing human insulin receptor (CHO-HIR). Insulin induced threonine phosphorylation of this protein in a dose-dependent manner, with an ED50 of 3-6 x 10(-9) M. The 50-kilodalton phosphoprotein (pp50) was detectable 20 min after exposure of the cells to insulin, and phosphorylation reached a maximum after 90 min. Immunoprecipitation of pp50 with anti-P-Thr required extraction of the cellular proteins with sodium dodecyl sulfate and dithiothreitol, and subcellular fractionation of the cells revealed that pp50 is present in the membrane fraction, implying that pp50 is a protein integrated into the membrane component in the cells. Tryptic phosphopeptide mapping of the pp50 was distinct from that of the insulin receptor beta-subunit. Phosphoamino acid analysis of the pp50 demonstrated that insulin increased phosphorylation, mainly of threonine and moderately of serine, whereas pp50 did not contain phosphotyrosine. Cycloheximide, a protein synthesis inhibitor, did not affect the insulin-induced appearance of pp50 in the cells. pp50 was not detectable in A431 cells and KB cells stimulated by epidermal growth factor. These data suggest that p50 is a novel endogenous substrate for insulin-sensitive serine/threonine kinase in intact cells.

Topics: Animals; Antibodies; Carcinoma, Squamous Cell; CHO Cells; Cricetinae; Cycloheximide; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Insulin; Liver Neoplasms, Experimental; Membrane Proteins; Molecular Weight; Peptide Mapping; Phosphorylation; Precipitin Tests; Protein Serine-Threonine Kinases; Rats; Receptor, Insulin; Threonine; Tumor Cells, Cultured

The binding of epidermal growth factor (EGF) or an EGF-like growth factor to the EGF receptor is the initial event which leads to receptor activation, and consequently the induction of cell growth. In order to study this binding interaction in detail, we produced the extracellular domain of the EGF receptor (EGFR) using the baculovirus expression system. Affinity-labeling and Western-blot analyses revealed that the baculovirus-infected insect cells secrete active EGFR extracellular domain relatively efficiently, however a significant amount of inactive EGFR extracellular domain is retained within the cells. The apparent dissociation constant (Kd) of the secreted EGFR extracellular domain for EGF and transforming growth factor alpha (TGF-alpha), as determined using an immobilized receptor binding assay, was approximately 200 nM. Interestingly, this Kd value is 30-40-fold lower than that of the full-length EGFR derived from detergent-solubilized A431 cell membranes. The stoichiometry of binding of the EGFR extracellular domain to EGF and TGF-alpha was examined by band-shift analysis on non-denaturing PAGE and was estimated to be 1:1. We have also shown, using sedimentation equilibrium analysis, that ligand binding induces significant dimerization of the EGFR extracellular domain. Finally, we carried out site-specific mutagenesis on the EGFR extracellular domain in order to define the ligand-binding region. We identified amino acid residues which are close to the binding site since they are common to the epitopes of several ligand-competitive monoclonal antibodies. However, these residues do not contribute directly to ligand binding since the affinity of the mutated EGFR extracellular domain for EGF and TGF-alpha was unaffected.

Topics: Amino Acid Sequence; Animals; Baculoviridae; Base Sequence; Binding Sites; Blotting, Western; Carcinoma, Squamous Cell; Cell Line; Chickens; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Macromolecular Substances; Male; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Recombinant Proteins; Spodoptera; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Antibodies directed against the epidermal growth factor receptor may impede proliferation and induce differentiation of head and neck squamous cell carcinoma. To test this hypothesis, we examined the effect of monoclonal antibody 528 directed against epidermal growth factor receptor on the proliferation and differentiation of monolayer cells and multicenter tumor spheroids from three head and neck squamous cell carcinoma cell lines (1483, MDA 686Ln, and MDA 886Ln) and the epidermal growth factor-responsive vulvar carcinoma A431. All head and neck squamous cell carcinoma lines were shown to express high levels of epidermal growth factor receptor by Scatchard analyses. Epidermal growth factor inhibited the growth of monolayer cells but stimulated the growth of 886 and A431 multicellular tumor spheroids. Epidermal growth factor modulated the differentiation of A431 and 686 with respect to involucrin immunohistochemistry and cornified enveloped competency. Monoclonal antibody 528 directed against epidermal growth factor receptor inhibited cellular proliferation as measured by cell number, thymidine incorporation, and multicellular tumor spheroid volume. A mild promotion of differentiation was observed in the epidermal growth factor-responsive cells. In conclusion, monoclonal antibody 528 directed against epidermal growth factor receptor inhibits growth of head and neck squamous cell carcinoma cells bearing high levels of epidermal growth factor receptors and promotes differentiation in some tumors. The use of a multicellular tumor spheroid model to evaluate growth factor responsiveness and inhibition of proliferation may more accurately reflect in vivo tumor growth than monolayer cells. Antibodies against epidermal growth factor receptor may prove effective in modulating disease progression in patients with head and neck squamous cell carcinoma.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Mice; Tumor Cells, Cultured

The introduction of a bacterial aminoglycoside phosphotransferase gene (neo gene) into A-431 cells was found to result in disappearance of high-affinity binding sites of the epidermal growth factor receptor (EGFR), probably by affecting the phosphorylation level of the receptors. Using A-431 cells and their neo gene-transfectants, we studied the relation between "rounding" and the high-affinity sites for EGF; and we also examined the role of protein kinase C (PKC) and A (PKA) in the EGF-induced cell rounding. Pretreatment of A-431 and their transfectant cells with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 ng/ml), an activator of PKC, for 30 min inhibited both the EGF-induced cell rounding and expression of high-affinity binding sites for EGF. However, both of these responses were recovered when cells were pretreated with TPA for 20 h, which treatment is known to result in depletion of PKC by a process called "down regulation". A similar recovery was also observed when cells were pretreated with forskolin (100 microM), an activator of PKA, for 30 min. Both cell rounding and EGFR high-affinity binding sites disappeared by activation of PKC, and reappeared by activation of PKA. These results suggest that the rounding of A-431 cells by EGF was induced via the high-affinity binding sites of EGFR.

Topics: Binding Sites; Carcinoma, Squamous Cell; Colforsin; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Kanamycin Kinase; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

To characterize the presence and production of various phospholipid metabolites by head and neck squamous cell carcinoma (HNSCC) and squamous cell carcinoma cell lines in vitro and in vivo.. The HNSCC tumor homogenates and supernatants of HNSCC tumor cultures and established squamous cell carcinoma cell lines were assayed for prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and platelet activating factor (PAF). In vitro experiments were carried out under baseline conditions or with exposure to several known immunomodulators (epidermal growth factor, bacterial lipopolysaccharide, and interleukin 1).. The HNSCC tumor tissue was obtained from primary tumor or cervical lymph node metastasis of surgical resections.. Prostaglandin E2, LTB4, and PAF were measured in tumor homogenates and cell culture supernatants using standardized radioimmunoassay kits.. All tumor homogenates (eight of eight) contained detectable levels of PGE2 (range, 324 to 2258 pg/g of tumor tissue) and LTB4 (range, 790 to 41,900 pg/g of tumor tissue); PAF was detected in six of eight homogenates (range, 7362 to 40,788 pg/g of tumor tissue). All of the short-term primary HNSCC tumor cultures and squamous carcinoma lines produced PGE2 (range, 90 to 1160 pg/10(6) cells), and half of the cultures produced LTB4 (range, 100 to 1700 pg/10(6) cells); none of the cultures or cell lines produced detectable levels of PAF. Interleukin 1 significantly enhanced production of PGE2 by tumor cultures (P < .02). Characterization of tumor cultures with a fibroblast antibody marker, BR2, revealed that 26% to 64% of tumor culture cells were fibroblasts.. Prostaglandin E2, LTB4, and PAF are present in the tumor microenvironment, where they may be involved in the local immunosuppression phenomenon seen in HNSCC. Both PGE2 and LTB4 were produced in vitro by tumor cultures and squamous cell carcinoma cell lines; PAF was not produced by tumor cultures in vitro and therefore may be a product of local immune cells in HNSCC in vivo. Interleukin 1 and PGE2 may interact in immunoregulation in the HNSCC tumor microenvironment.

Topics: Aged; Analysis of Variance; Animals; Carcinoma, Squamous Cell; Dinoprostone; Epidermal Growth Factor; Evaluation Studies as Topic; Fibroblasts; Head and Neck Neoplasms; Humans; Immune Tolerance; Interleukin-1; Leukotriene B4; Lipopolysaccharides; Middle Aged; Neoplasm Staging; Phenotype; Platelet Activating Factor; Radioimmunoassay; Regression Analysis; Tumor Cells, Cultured

We examined effect of the tyrosine kinase inhibitor herbimycin A on A431 human epidermoid carcinoma cells which over-express epidermal growth factor (EGF) receptor. Herbimycin A inhibited the autophosphorylation of EGF-stimulated receptors in intact cells both time- and dose-dependently. The inhibition was found to be due to a decrease in the level of expression of the receptor amount, because herbimycin A equally decreased the receptor quantity and EGF-stimulated receptor kinase activity in intact cells, but did not exhibit a direct inhibitory effect on EGF receptor kinase activity in vitro. The decrease of the level of EGF receptor was also confirmed by 125I-EGF binding to herbimycin A-treated cells, and Scatchard analysis showed that the decrease in the receptor number occurred in the major population of the low-affinity binding ones, whereas the number with high-affinity binding was unaffected. Interestingly, although the proliferation of A431 cells was inhibited by EGF, herbimycin A converted EGF into a stimulatory ligand for cell growth, as determined by both cell number and DNA synthesis. These findings indicated that herbimycin A decreased the level of expression of EGF receptor by a mechanism other than inactivation of the receptor kinase and reversed the transformed phenotype of A431 cells to a normal one in the proliferative response to EGF.

Topics: Benzoquinones; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Lactams, Macrocyclic; Ligands; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; Tumor Cells, Cultured

Receptor-activated breakdown of complex sphingolipids has been proposed as a mechanism for generating sphingoid base-containing putative second messenger molecules whose actions may modulate responses to extracellular signals. In human epidermoid carcinoma A431 cells, sphingosine (1-10 microM) by itself had no effect on intracellular free calcium concentrations ([Ca2+]i), yet within seconds, markedly enhanced the epidermal growth factor (EGF)-evoked Ca2+ influx (by up to 2-fold), but failed to alter Ca2+ release from the intracellular stores. Ca2+ signals evoked by serum were not affected by sphingosine. The response to sphingosine was dose-dependent and saturable, exhibiting an EC50 of 2.3 microM. In contrast, a ceramide, N-acetylsphingosine (10 microM), sphingosine 1-phosphate (10 microM), and sphingosylphosphorylcholine (10 microM) inhibited EGF-evoked elevations in [Ca2+]i. The latter two compounds by themselves transiently increased [Ca2+]i. N-Octanoylsphingosine, N,N-dimethylsphingosine, sphingomyelin, and stearylamine were inactive. The potentiation of calcium signals by sphingosine occurred at all concentrations of EGF tested (0.15-15 nM) and did not alter the EGF receptor protein kinase activity as determined by antiphosphotyrosine immunoblotting. Antiphosphoserine immunoblotting revealed that sphingosine (10 microM for 3 min) increased the phosphoserine content of two proteins with approximate molecular masses of 40 and 70 kDa. Serine hyperphosphorylation of the 40-kDa protein was also observed in cells treated with EGF alone, whereas the intensity of the 70-kDa band was highest in cells treated with both sphingosine and EGF. The modulation of growth factor receptor-regulated signaling, including changes in [Ca2+]i, may constitute a mechanism by which elevations in cellular levels of specific sphingolipids, which occur transiently upon activation of certain receptors and chronically in sphingolipid storage diseases, exert their physiological and pathophysiological effects.

Topics: Calcium; Carcinoma, Squamous Cell; Epidermal Growth Factor; Humans; Phosphorylation; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Second Messenger Systems; Sphingolipids; Sphingosine; Tumor Cells, Cultured

To assess the functional activity of internalized epidermal growth factor (EGF) receptors in A-431 cells we investigated their ability to be both the target and activator of serine and threonine protein kinases. By incubating A-431 cells with EGF at 4 degrees C wr at 37 degrees C, eluting surface-bound EGF with an acid buffer, and immunoprecipitating the EGF receptor with different antibodies, we were able to compare the phosphorylation state of internalized EGF receptors to those found on the plasma membrane in intact cells. Tryptic phosphopeptide mapping and subsequent phosphoamino acid analysis revealed four tyrosine, one threonine, and seven serine phosphorylation sites in the molecule of plasma membrane receptor, while internalized EGF receptor contained one additional threonine and three additional serine phosphorylation sites. Because acid-mediated removal of EGF from its receptor demonstrated that the majority of EGF-induced phosphorylation required the continuous presence of activated receptor to be maintained, the conclusion was made that internalized EGF receptors may be not only the target of protein kinases whose activity was detected in our assay but also an activator of at least some of them. The interaction between internalized EGF receptors and nontyrosine protein kinase was also observed in vitro. Membrane-associated protein kinase was detected which phosphorylated a serine residue of the EGF receptor molecule in an EGF-dependent manner. Subcellular fractionation revealed the presence of the serine protein kinase both in the fractions of plasma membranes and high-density endosomes. These results demonstrate that at its prelysosomal stage, internalization does not impair the functional activity of EGF receptor.

Topics: Amino Acids; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Peptide Fragments; Peptide Mapping; Phosphopeptides; Phosphoproteins; Phosphorylation; Subcellular Fractions; Tumor Cells, Cultured

Prostaglandins may inhibit or promote tumor cell replication, depending on the cell system that is investigated. In our laboratory, we have established and characterized four different specific human cancer cell lines. The objectives of this study were to examine and compare the prostaglandin endoperoxide synthase (PG synthase, EC 1.14.99.1) activity of these cell lines by measuring the conversion of arachidonate to 3H-PGE2 and 3H-PGF2 alpha. We found that the oral epidermal carcinoma cell line (OEC-M1) had a moderate degree of PG synthase activity. Enzyme activity could be partially blocked (statistically significant) by the addition of epidermal growth factor (EGF) at 20 ng/mL and almost completely inhibited by platelet-derived growth factor at (PDGF) 20 mU/mL. By contrast, we discovered that the human breast adenocarcinoma cell line (BC-M1) did not contain significant PG synthase, and enzyme activity could be significantly activated by the addition of epidermal growth factor at 20 ng/mL and platelet-derived growth factor at 20 mU/mL. We also found that the human stomach adenocarcinoma cell line (SCM-1) had a significant amount of PG synthase activity, and these PG synthase activities were not activated or inhibited by EGF at 20 ng/mL or PDGF at 20 mU/mL. Furthermore, the human fibrosarcoma (FS-M1) cell line also contained a moderate degree of PG synthase activity, which could be significantly inhibited by PDGF at 20 mU/mL but was not inhibited by EGF at 20 ng/mL. The results suggest that EGF and PDGF may be involved in the regulation of the PG synthase activities of human oral, breast, stomach, and fibrosarcoma cancer cells.

Topics: Adenocarcinoma; Breast Neoplasms; Buttocks; Carcinoma, Squamous Cell; Chromatography, Thin Layer; Cyclooxygenase Inhibitors; Dinoprost; Dinoprostone; Enzyme Activation; Epidermal Growth Factor; Female; Fibrosarcoma; Gingival Neoplasms; Humans; Organ Specificity; Platelet-Derived Growth Factor; Prostaglandin-Endoperoxide Synthases; Stomach Neoplasms; Tumor Cells, Cultured

EGF is involved in the regulation of cell proliferation in normal as well as in neoplastic tissues. The A431 cells that over-express EGFR, display in vitro ambivalent growth properties in response to EGF, since stimulation induced by low concentrations (10(-12) M-10(-10) M) is reversed with increasing concentrations (10(-9) M-10(-8) M). To assess differential mechanisms of signal transduction that determine growth stimulatory and inhibitory activity, we have studied the MAP kinase activation induced by mitotic and antimitotic concentrations of EGF. We demonstrate that the presence of a growth stimulatory concentration of EGF (10(-12) M) leads to a moderate but persistent activation of p42 MAP kinase during all the time of the EGF treatment. Conversely, an early peak of kinase activation that rapidly falls down below the basal level, is observed when a growth inhibitory concentration of EGF (10(-8) M) is used. Moreover, the addition of Na-orthovanadate in 10(-8) M EGF-treated cells leads to the rescue of the MAP kinase activity, suggesting that the loss of kinase activity induced by growth inhibitory EGF concentrations involves the dephosphorylation of the MAP kinase. In conclusion, our data demonstrate that the dual effect (stimulator/inhibitor) of EGF on the proliferation of A431 cells is associated to differential mechanisms of p42 MAP kinase regulation.

Topics: Blood; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Squamous Cell; Cell Division; Enzyme Activation; Epidermal Growth Factor; Kinetics; Mitogen-Activated Protein Kinase 1; Protein Tyrosine Phosphatases; Tumor Cells, Cultured; Vanadates

The regulation of parathyroid hormone-related protein (PTH-rP) mRNA levels and immunoreactive (ir)PTH-rP formation by peptide growth factors, particularly transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF), in squamous cell carcinomas of gynecologic origin is largely unknown.. PTH-rP mRNA levels were evaluated by Northern analysis in A431 cells (derived from a human vulvar epidermoid carcinoma) and ME-180 cells (derived from a human papillomavirus-infected squamous cell carcinoma of the cervix). PTH-rP protein levels in cell culture media were evaluated using both radioimmunoassay and immunoradiometric assay techniques. These results were compared with those from a lung carcinoid cell line known to produce PTH-rP, namely, NCI-H727 cells.. TGF-beta 1 or EGF treatment caused an increase in the levels of PTH-rP mRNA in A431 cells; these increases in PTH-rP mRNA were detectable after 60 minutes of treatment, were maximal at approximately 4-8 hours, and were approximately additive. Immunoreactive PTH-rP was not detectable (using two different PTH-rP immunoassays) in the culture medium or cell sonicates of A431 cells before or after treatment with TGF-beta 1, EGF, or TGF-beta 1 plus EGF. ME-180 cells responded to EGF (but not to TGF-beta 1) with an increase in the level of PTH-rP mRNA as early as 2 hours; irPTH-rP was present (by use of either immunoassay) in the medium of these cells at 8 and 24 hours. In NCI-H727 (human lung carcinoid) cells, TGF-beta 1 and EGF acted alone and synergistically to effect increases in PTH-rP mRNA and the accumulation of irPTH-rP.. TGF-beta 1 and EGF regulation of PTH-rP gene expression in squamous cell carcinomas of gynecologic origin is unique for each cell line studied and different from that in human lung carcinoid cells.

Topics: Base Sequence; Carcinoid Tumor; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genital Neoplasms, Female; Humans; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vulvar Neoplasms

Epidermal growth factor (EGF) has been shown to radiosensitize A431 and other human squamous carcinoma cells with high numbers of surface EGF receptors. In this study of the mechanistic basis of EGF-induced radiosensitization, both EGF and ionizing radiation caused G1 phase arrests in cycling A431 cells, but only radiation caused a G2-M arrest. However, EGF enhanced the magnitude of this G2-M arrest, suggesting an interaction of signaling pathways involved in cellular responses to EGF and radiation damage. EGF and radiation also uniquely perturbed cyclin A and B1 mRNA levels during the time of maximum radiation-induced G2-M arrest. The effects of EGF on G2-M events probably originated in cells in G1. It is possible that aberrant EGF signal transduction in human squamous carcinoma cells may be exploited as a novel strategy for radiotherapy.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cyclins; Epidermal Growth Factor; ErbB Receptors; G2 Phase; Humans; Mitosis; Radiation, Ionizing; RNA, Messenger; Stimulation, Chemical; Tumor Cells, Cultured

The fungal metabolite BE-23372M is a structurally novel protein kinase inhibitor. Its IC50 for epidermal growth factor (EGF) receptor kinase was 0.03 microM. IC50 values of BE-23372M for other protein tyrosine kinases, erbB-2, p43v-abl, insulin receptor kinase, and p60c-src were 0.42, 1.0, 3.3, and 4.5 microM, respectively, and the IC50 for protein kinase C, a serine/threonine kinase, was 4.1 microM. Cdc2 kinase, casein kinases I and II and cAMP-dependent protein kinase were not inhibited by 20 microM BE-23372M. A kinetic study showed that BE-23372M was competitive with respect to the substrate peptide and to ATP. Autophosphorylation of solubilized EGF receptor kinase was clearly inhibited by 0.1 microM BE-23372M. Autophosphorylation of EGF receptor in A431 cells was also inhibited. These results show that BE-23372M is a potent and specific EGF receptor kinase inhibitor. It should be a valuable tool for EGF receptor kinase research.

Topics: Amino Acid Sequence; Animals; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Furans; Humans; Molecular Sequence Data; Moths; Phenols; Phosphorylation; Protein Kinase Inhibitors; Tumor Cells, Cultured

Cluster 13 was defined by 2 independently derived murine monoclonal antibodies (MAbs), RS7 (IgG1) and MR54 (IgG2a), which were raised against human squamous-cell carcinoma of the lung and a human ovarian-carcinoma cell line, respectively. Immunologic and biochemical evidence demonstrated that RS7 and MR54, as well as 2 additional MAbs, MR6 (IgG2a) and MR23 (IgG1), generated in the same fusion as MR54, recognize the same antigen, a 46- to 48-kDa glycoprotein. Evaluation of the expression of antigen on the surface of tumor cell lines, Western blotting analyses, competitive binding studies, and double-determinant ELISA assays, support this conclusion. Two distinct epitopes are defined by these MAbs. In order to further characterize this antigen, amino-acid-sequence analyses were performed on peptides derived from antigen purified by affinity chromatography with MAb RS7. The sequence data obtained from 2 peptides, which were independently generated by CNBr cleavage and trypsin digestion respectively indicated identity to GA733-1. The GA733-1 genomic DNA sequence predicted a type-1 membrane protein of 35 kDa, with 4 potential N-linked glycosylation sites. The GA733-1 protein product has not been identified previously, and MAbs to this tumor-associated antigen were not previously known.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Blotting, Western; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Line; Epidermal Growth Factor; Epithelial Cell Adhesion Molecule; Female; Humans; Immunoglobulin G; Lung Neoplasms; Mice; Molecular Sequence Data; Multigene Family; Neoplasms; Ovarian Neoplasms; Sequence Homology, Amino Acid; Tumor Cells, Cultured

This study was performed to characterize the interaction of epidermal growth factor and radiation in two human head and neck squamous cell cancer cell lines of vastly different radiosensitivities (UM-SCC-6 Radiosensitive; UM-SCC-1 radioresistant).. The two human head and neck squamous cell cancers (UM-SCC-1 and UM-SCC-6) were grown in medium and following the appropriate treatments, cell survival was assessed by a standard colony formation assay. Growth inhibition was assessed by monitoring cell counts following treatment and flow cytometry was used to assess cell cycle distributions.. It was determined that exposure to epidermal growth factor (10 ng/ml) for 24 h prior to radiation resulted in radiosensitization in both cell lines, however, the magnitude of radiosensitization was greater in the radiosensitive UM-SCC-6 cells compared to the radioresistant UM-SCC-1 cells. Treatment of the UM-SCC-6 cells with epidermal growth factor (EGF) (10 ng/ml) for 24 h resulted in a growth delay, however, cell growth returned to normal approximately 24 h following removal of EGF. Similar treatment of the UM-SCC-1 cells resulted in no growth inhibition. The 24 h pre-radiation exposures to EGF (10 ng/ml) did not affect the radiation-induced growth delay in either cell line. Additionally, the 24 h exposures to EGF (10 ng/ml) did not affect the radiation-induced growth delay in either cell line. Additionally, the 24 h exposures to EGF (10 ng/ml) did not cause the cells to enter a more radiosensitive cell cycle phase. Further work will be necessary to determine whether events associated with the EGF-induced growth delay in the UM-SCC-6 cells are associated with the enhanced EGF-induced radiosensitization in these cells compared to UM-SCC-1 cells.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Survival; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Radiation Tolerance; Tumor Cells, Cultured

This study examined the expression of epidermal growth factor (EGF) cell-surface receptors, the response to exogenous ligand and the autocrine production of transforming growth factor alpha (TGF-alpha) in normal and carcinoma-derived human oral keratinocytes. One of eight malignant cell lines overexpressed EGF receptors, while the remainder expressed receptor numbers similar to normal cells. Exogenous EGF stimulated incorporation of tritiated thymidine in a dose-dependent manner. In keratinocytes expressing normal numbers of EGF receptors, the cellular response to exogenous EGF correlated positively with total EGF receptor number. SCC-derived keratinocytes produced more TGF-alpha than normal cells. There was no statistical correlation between the autocrine production of TGF-alpha, EGF cell-surface receptor expression and cellular response to exogenous EGF. While the growth-stimulatory effects of exogenous TGF-alpha were inhibited by the addition of a neutralising antibody, the presence of this antibody in conditioned medium failed to produce a similar decrease in growth. The results indicate that overexpression of EGF receptors is not an invariable characteristic of human oral squamous carcinoma-derived cell lines. Further, the contribution of TGF-alpha to the growth of normal and carcinoma-derived human oral keratinocytes in vitro may be less significant than previously documented.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Keratinocytes; Male; Middle Aged; Mouth Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Integrin expression in squamous cell carcinomas have been reported to be down-regulated in vivo. Because integrins are molecules whose functions include the reorganization of cytoskeleton in response to the surrounding extracellular matrix, we have examined the expression of integrins in the transformed cell line A431 when grown as multicellular spheroids or as monolayers. The spheroids were grown to sizes of approximately 100 microns and approximately 600 microns. Since larger A431 spheroids require epidermal growth factor for growth, we also investigated the effect of this growth factor on the expression of integrins in cells grown as monolayers or as small spheroids. Immunostaining studies using monoclonal antibodies specific for alpha 6, beta 1, and beta 4 subunits revealed a strong staining pattern in the periphery of the spheroids. The interior cells of the spheroids showed a moderate, positive reaction with the beta 1 antibody but significantly reduced from that at the periphery. Anti-alpha 2 antibody, on the other hand, revealed a uniform staining around the cells throughout the spheroids. Western blot analyses confirmed an overall diminution of alpha 6 and beta 1 protein levels in the spheroids compared with monolayers. Northern blot analyses showed that the low expression of integrin subunits alpha 6, beta 1, and beta 4 in spheroids was due to a reduction in mRNA transcripts. Northern blot analyses, however, showed no significant change in the expressions of alpha 2, alpha 5, or beta 5 mRNA. Conversely, the expression of alpha v was slightly reduced in spheroids. Epidermal growth factor increased the mRNA expression of alpha 2, alpha 6, beta 1, and beta 4 integrin subunits in cells grown either as monolayers or as spheroids whereas epidermal growth factor had no detectable effect on the expression of alpha v or beta 5. These results mimic the pattern of expression found in vivo and indicate that cell-cell contact and the microenvironments of cells within a spheroid regulate the expression and distribution of a subset of integrin molecules.

Topics: Animals; Blotting, Northern; Blotting, Western; Carcinoma, Squamous Cell; Cell Communication; Cell Division; Down-Regulation; Epidermal Growth Factor; Humans; Immunohistochemistry; Integrins; Mice; Models, Biological; Receptors, Laminin; Tumor Cells, Cultured

Previous studies have demonstrated quantitation of epidermal growth factor receptors (EGFR) to be of prognostic significance in breast, bladder, esophageal and other neoplasms. However, the relatively large quantity of unfixed tissue required for epidermal growth factor radioligand binding assays (RLBA) has precluded its application to cytologic specimens and small biopsy specimens. For this reason we evaluated reverse transcription intron differential polymerase chain reaction (RTIDPCR) as an assay of EGFR gene expression. Squamous cell carcinoma (A431 and SiHa), transitional cell carcinoma (HT1376, T24, RT4), mammary (MCF7) and endocervical (HeLa) adenocarcinoma, and leukemia (K562) cell lines were used to compare RTIDPCR and RLBA. RTIDPCR involved reverse transcription of RNA and amplification of cDNA using primers for beta-actin and EGFR. Good agreement was observed between the RLBA and RTIDPCR results. RNA extracted from fresh cells, Diff-Quik-stained smears and formalin-fixed, paraffin-embedded cell pellet sections yielded similar results. These data suggest that RTIDPCR may be useful in evaluating gene expression by cells processed as cytologic specimens.

Topics: Adenocarcinoma; Base Sequence; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Genes, Neoplasm; Humans; Introns; Leukemia, Myeloid; Molecular Sequence Data; Neoplasms; Polymerase Chain Reaction; RNA, Neoplasm; Staining and Labeling; Tissue Embedding; Tissue Preservation; Tumor Cells, Cultured

A431 cells, a human epidermoid carcinoma, possess specific [3H]platelet-activating factor (PAF) and [3H]WEB 2086 binding sites indicating the presence of PAF receptors. PAF-stimulated PLC as determined by the increase in inositol phosphate levels. Pretreatment of A431 cells with genistein, a putative tyrosine kinase inhibitor, abolished the ability of PAF to activate PLC, whereas pretreatment with staurosporine, a protein kinase C inhibitor, potentiated the ability of PAF to activate PLC. Pretreatment of A431 cells with phorbol-12-myristate-13-acetate, a protein kinase C activator, blocked PAF-stimulated PLC. Overnight exposure of cells to pertussis toxin (PT) partially blocked the ability of PAF to stimulate PLC. Based on these observations the involvement of PT-sensitive and -insensitive guanine nucleotide-binding protein(s) (G-protein) as well as the role of tyrosine kinase in the activation of PLC by PAF was considered further. PT treatment of A431 cell membranes obliterated PAF-stimulated GTPase and indicated that PT-insensitive membrane-associated G-proteins were not involved in PAF actions. In alpha-toxin permeabilized cells, PT blocked GTP-gamma-S potentiation of PLC activation by PAF, thus suggesting that PT-insensitive G-proteins were not involved in PAF activation of PLC in A431 cells. PAF stimulated tyrosine kinase activity as observed with the increase in radioactivity associated with proteins immunoprecipitated with polyclonal antibodies to phosphotyrosine residues. This increase was blocked by PAF receptor antagonists, CV 6209 and TCV 309, and by pretreatment with genistein. PAF also activated the phosphorylation of pp60c-src and Src associated proteins in A431 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carcinoma, Squamous Cell; Enzyme Activation; Epidermal Growth Factor; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; Phosphorylation; Platelet Activating Factor; Platelet Membrane Glycoproteins; Protein-Tyrosine Kinases; Proto-Oncogene Proteins pp60(c-src); Receptors, Cell Surface; Receptors, G-Protein-Coupled; Tumor Cells, Cultured; Type C Phospholipases

Fifteen laryngeal squamous cell carcinomas were investigated for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) using immunohistochemical methods. In a recent study the same material was characterized for epidermal growth factor receptors (EGF receptors) which were confined predominantly to the undifferentiated cells. The expression of this growth factor system in malignant cells may play a role in carcinogenesis and/or tumour growth. All carcinomas were positive for TGF-alpha and 12 were positive for EGF. In moderately-to-well differentiated carcinomas, the immunoreactivity was mainly detected in the cytologically more differentiated cells. Nine sections included both laryngeal stratified squamous epithelium of normal appearance and carcinoma. The immunoreactivity was here again localized in the cytologically more differentiated cells above the basal cell layer. The present investigation and our previous results confirm the existence of EGF receptors, TGF-alpha and EGF in laryngeal carcinomas. In addition, we conclude that the conditions do exist for growth factors to act through an autocrine system in poorly differentiated tumours and through a paracrine system in the moderately-to-well differentiated tumours.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cytoplasm; Endothelium; Epidermal Growth Factor; Epithelium; Exocrine Glands; Female; Humans; Immunohistochemistry; Keratins; Laryngeal Muscles; Laryngeal Neoplasms; Macrophages; Male; Mucins; Neoplasm Staging; Transforming Growth Factor alpha

Autocrine neoplastic growth circuits are based on excess synthesis of growth factors and/or cognate membrane receptors. We analyzed by immunohistochemistry 19 typical lung carcinoids for the expression of the epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), EGF receptor (EGFr), and EGFr-related c-erbB-2 protein (p185). Thirteen tumors (68%) were positive for TGF alpha, 11 for EGFr (58%), three for EGF (16%), and four for p185 (21%). Six carcinoids (32%) were consistently negative for these gene products. The following patterns of coexpression could be documented: TGF alpha, EGFr, EGF, and p185: two cases (11%); TGF alpha, EGFr, and EGF: one case (5%); TGF alpha, EGFr, and p185: two cases (11%); TGF alpha and EGFr: six cases (32%); TGF alpha by itself: two cases (11%). Thus, EGFr was coexpressed with its ligands, TGF alpha and EGF, and the receptor encoded by c-erbB-2 was detected in carcinoids positive for EGFr and TGF alpha. Therefore, alterations of EGF/EGFr-related growth control pathways may be implicated in the pathogenesis of pulmonary carcinoids via the establishment of autocrine growth promoting circuits, as documented in adenocarcinomas and squamous cell carcinomas of the lung.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Proto-Oncogene Proteins; Receptor, ErbB-2; Transforming Growth Factor alpha

This study investigates the regulation of protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) activity by epidermal growth factor (EGF). Cytosol from EGF-treated A-431 human epidermoid carcinoma cells was used as a source of PTPase activity, and tyrosine-phosphorylated ErbB2, EGF receptor, phospholipase C-gamma 1, and the Ras GTPase-activating protein were used as substrates to monitor PTPase activity. EGF stimulated PTPase activity that was selective toward these substrates, as it dephosphorylated ErbB2 and the EGF receptor, but not phospholipase C-gamma 1 and the Ras GTPase-activating protein. EGF stimulated PTPase activity in several cell lines, regardless of EGF receptor number, and the activity was localized in the cytosol. The dephosphorylation activity in vitro was dependent on the presence of reducing agents and was inhibited by orthovanadate. Agonists such as phorbol 12-myristate 13-acetate, isoproterenol, or ATP were unable to stimulate PTPase activity. Physiological relevance is indicated by experiments showing that EGF treatment of a human mammary cancer cell line rapidly induced the dephosphorylation of ErbB2.

Topics: 3T3 Cells; Animals; Carcinoma, Squamous Cell; Cytosol; Epidermal Growth Factor; ErbB Receptors; GTP-Binding Proteins; Humans; Isoenzymes; Kinetics; Mice; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; rap GTP-Binding Proteins; Receptor, ErbB-2; Substrate Specificity; Tumor Cells, Cultured; Type C Phospholipases

Previous studies have reported that the proliferation of A431 cells, a human squamous cell carcinoma cell line, was stimulated by picomolar epidermal growth factor (EGF) but inhibited by nanomolar EGF. This biphasic dose-response phenomenon is not observed in normal human epithelial cells where nanomolar EGF is usually mitogenic. We have examined the effects of inhibitory and stimulatory concentrations of EGF on the growth and differentiation of A431 cells. In the presence of 100 pM EGF, A431 cells showed a mild increase in growth rate (129% of control) compared to cells grown in the absence of EGF. At 10 nM EGF, growth inhibition to 63% of control was observed. EGF at 10 nM stimulates a twofold increase both in cornified envelope formation and in epidermal transglutaminase activity, suggesting that high concentrations of EGF induce terminal differentiation in A431 cells. Mitogenic concentrations of EGF (100 pM) had no significant effect on these differentiation markers. Chronic exposure of A431 cells to 20 or 50 nM EGF resulted in EGF-resistant A431 variants that are neither growth arrested nor induced to terminally differentiate by 10 nM EGF. Removal of EGF from the growth medium of the EGF-resistant cells resulted in the reversion of these cells back to the wild-type A431 biphasic response pattern within 2 weeks. Our results suggest that A431 cells have the capacity to non-mutatively alter their response pattern to EGF in vitro to maintain themselves in a state of optimum proliferation and away from terminal differentiation.

Topics: Binding Sites; Carcinoma, Squamous Cell; Cell Differentiation; Epidermal Growth Factor; Humans; Transglutaminases; Tumor Cells, Cultured

The demonstration that alpha-sialic acid is the minimal determinant recognized by human reovirus is compatible with the finding that this virus binds to multiple sialoglycoproteins on the host cell surface. However, the identities of these proteins have remained unknown. By applying detergent-solubilized plasma membranes from the human epidermoid carcinoma A431 cell line to immobilized reovirions, we have identified the 150- to 170-kDa epidermal growth factor (EGF) receptor as one of the cell surface proteins recognized by reovirus. Direct interaction between the N-terminal extracellular domain of the EGF receptor and reovirus was confirmed by the demonstration that of a number of proteins secreted by A431 cells, the 105-kDa N-terminal cell surface domain of the EGF receptor was the major protein recognized by the virus. However, as expected, reovirus and EGF did not compete for the same binding site on the EGF receptor of intact A431 cells.

Topics: Autoradiography; Carcinoma, Squamous Cell; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Membrane Proteins; Molecular Weight; Receptors, Virus; Reoviridae; Sulfur Radioisotopes; Tumor Cells, Cultured

Suramin, a drug used for the treatment of African trypanosomiasis and onchoceriasis, has potential anti-cancer activity. As to the mechanism of action, it is considered to interfere with the action of tumor growth factor and specific growth enzymes. In the present study, the effect of suramin on proliferation of human head and neck squamous cell carcinoma cells was determined. A significant growth inhibition in all three cell lines tested was observed when the concentration of suramin was 100 micrograms/ml or more. The hypothesis was tested to ascertain whether growth inhibition may be due to selective interference with the action of epidermal growth factor. Suramin at growth inhibiting concentrations only partly antagonized the growth stimulating effect of EGF indicating that other mechanisms of action may also contribute to the growth inhibitory activity of suramin. Furthermore, evidence was found that serum proteins other than growth factors presented in the culture medium have a growth stimulating effect on cells and a strongly antagonistical effect on suramin activity.

Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Head and Neck Neoplasms; Humans; Laryngeal Neoplasms; Mouth Neoplasms; Pharyngeal Neoplasms; Serum Albumin, Bovine; Suramin; Tumor Cells, Cultured

From 1976 to 1991, 151 cases of small cell carcinoma of the lung were diagnosed by fiberoptic bronchoscopic biopsy at our institution. One hundred twenty-eight of 151 cases provided suitable material for the examination of the morphologic changes in the bronchial surface epithelium. Thirty-seven percent of the cases showed normal bronchial epithelium, 47% showed benign squamous metaplasia, 9% showed atypical squamous metaplasia, and 5% showed squamous cell carcinoma in situ. Immunohistochemical examination for bombesin and epidermal growth factor was performed on selected biopsy specimens. The biopsy specimens chosen for immunohistochemistry included 20 specimens that showed normal bronchial epithelium, 20 specimens with benign squamous metaplasia, 12 specimens with atypical squamous metaplasia, and seven specimens with squamous cell carcinoma in situ. All the specimens showed positive staining with anti-bombesin. With anti-epidermal growth factor 10% of biopsy specimens with normal epithelium showed positive staining. The positive reaction increased from 25% for biopsy specimens with benign squamous metaplasia to 58% for biopsy specimens with atypical squamous metaplasia and to 71% for biopsy specimens with carcinoma in situ. These findings suggest a connection between epidermal growth factor production by small cell carcinoma of the lung cells and changes in the bronchial surface epithelium.

Topics: Biopsy; Bombesin; Bronchi; Carcinoma in Situ; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; Epithelial Cells; Epithelium; Humans; Immunohistochemistry; Lung Neoplasms; Metaplasia

The Src homology (SH) region 2 binds to phosphorylated tyrosine residues and SH3 domains may interact with cytoskeletal molecules and GTPase-activating proteins for Rho/Rac proteins (the small GTP-binding proteins related to Ras). The recently cloned Ash/Grb-2 protein, a 25-28 kDa molecule composed entirely of SH2 and SH3 domains, is a mammalian homolog of the Caenorhabditis elegans Sem-5 protein, which communicates between a receptor protein tyrosine kinase and a Ras protein. In the present study the function of Ash/Grb-2 was investigated by microinjecting cells with an anti-Ash antibody. The antibody abolished both S phase entry and the reorganization of actin assembly to ruffle formation upon stimulation with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). On the other hand, anti-Ash antibody had no effect on S phase entry or actin stress fiber formation induced by either serum or lysophosphatidic acid. Since the induction of DNA synthesis, ruffle induction and stress fiber formation involve a function of Ras, Rac activation and Rho activation respectively, the findings strongly suggest that Ash plays a critical role in the signaling of both pathways downstream from growth factor receptors to Ras and Rac. Consistent with this, Ash co-precipitated with EGF receptor from EGF-stimulated cells. Other proteins of approximately 21, 29, 135 and 160 kDa were also detected in the anti-Ash antibody immunoprecipitates, suggesting a role of Ash as a linker molecule in signal transduction downstream of growth factor receptors.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibodies; Becaplermin; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cytoskeleton; Epidermal Growth Factor; GRB2 Adaptor Protein; GTP-Binding Proteins; Humans; Kidney; Microinjections; Platelet-Derived Growth Factor; Proteins; Proto-Oncogene Proteins c-sis; Rats; Recombinant Fusion Proteins; Recombinant Proteins; Signal Transduction; Tumor Cells, Cultured

The effects of removal of the submandibular gland (sialoadenectomy) and administration of human urinary epidermal growth factor on the 9,10-dimethyl-1,2-benzanthracene-induced tumor formation were investigated with the use of a hamster cheek pouch model. Syrian hamsters were treated with 0.5% 9,10-dimethyl-1,2-benzanthracene for 6 weeks. Thereafter hamsters in group 1 underwent a sham operation and those in groups 2 and 3 underwent a sialoadenectomy. Subsequently, hamsters in groups 1 and 2 were given 0.9% sodium chloride and group 3 received the human urinary epidermal growth factor at a dose of 0.25 mg/kg body weight subcutaneously three times a week for 8 weeks. Sixteen weeks after the start of the experiment, the mean number of tumors that were less than 3-mm in diameter in groups 1 and 3 was significantly greater than that in group 2 (p < 0.05). The overall incidence and mean number of all carcinomas irrespective of size showed no differences among the experimental groups. These results indicate that epidermal growth factor synthesized in the submandibular gland may enhance the induction of cheek pouch tumor.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cricetinae; Epidermal Growth Factor; Hyperplasia; Male; Mesocricetus; Mouth Neoplasms; Papilloma; Submandibular Gland

Using cultured A431 cells, a comparative analysis was performed of endocytosis stimulated by the epidermal growth factor (EGF) just following the ligand influence (early endocytosis) and after a 3 hour incubation of A431 cells with EGF (delay endocytosis). It is shown that at the early endocytosis the total amount of 125I-EGF, associated with cells, is decreased less than at the delay endocytosis. The decrease in total amount of cell-associated 125I-EGF was accompanied with the increase in the EGF concentration in the incubation medium. The data obtained suggested a lower rate of internalization for delay endocytosis. The Scatchard analysis of 125I-EGF-binding has shown that both high affinity EGF-receptor and low affinity EGF-receptor undergo the down-regulation. The Percoll gradient fractionation of EGF-loaded cells has shown that at the delay endocytosis 125I-EGF is displaced to the fraction of heavy endosomes and lysosomes slower than at the early endocytosis. It is suggested that a delay of EGF-receptor complexes transition from endosomes to lysosomes may arise during delay endocytosis.

Topics: Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Down-Regulation; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Ligands; Microsomes; Time Factors; Tumor Cells, Cultured

EGF-induced hydrolysis of phosphatidylinositol 4, 5, biphosphate was compared in A431 cells with respect to their growth response to EGF. A431 cells which express 4- to 5-fold more EGF receptors than A431-4 cells were growth inhibited, while A431-4 cells were growth stimulated by EGF within the same dose range. Treatment of A431 cells with EGF resulted in a 2-fold increase in cellular IP3 levels and the effect in A431-4 cells was not as obvious. In the presence of tyrosine kinase inhibitor coumaric acid (0.2 approximately 2 microM), A431 cell growth was stimulated, rather than inhibited, by EGF in a dose-dependent manner. In contrast, the stimulation of A431-4 cell growth by EGF was reduced under the same conditions. Furthermore, in the presence of coumaric acid (up to 0.5 microM), EGF-induced production of inositol phosphates in A431 cells was not obviously affected. Taken together, the results suggest that EGF-induced growth inhibition of A431 cells may be due to a quantitative changes of EGF-receptor tyrosine kinase activity in areas other than the recruitment and activation of phosphatidylinositol-specific phospholipase C gamma.

Topics: Carcinoma, Squamous Cell; Cell Division; Coumaric Acids; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Inositol Phosphates; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Type C Phospholipases

We investigated the growth-regulatory mechanism of 2 esophageal squamous-cancer cell lines, TE2-NS and TE3-OS cells, both of which can grow stably in protein-free conditions in vitro. Protein-free conditioned media from TE2-NS and TE3-OS cells stimulated the growth of these cells. Exogenous epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), insulin-like growth factor (IGF)-I and -II enhanced cell proliferation by 2.2- to 3.8-fold in protein-free conditions, as compared with an untreated control. Receptor-binding assays showed that both TE2-NS and TE3-OS cells possessed a single class of high-affinity binding sites for IGF-I and 2 classes of binding sites for TGF-alpha, as confirmed on the cell membrane by immunochemistry. These results suggest that EGF, TGF-alpha and IGFs are candidates for the autocrine growth factor in cancer cells. The addition of inhibitory monoclonal antibodies against TGF-alpha and EGFR, but not those against either EGF or IGF-IR, significantly inhibited growth of the cells. Immunocytochemical staining and ELISA of the conditioned media both confirmed the production of TGF-alpha protein, but not EGF protein, in these cell lines. The data for a protein-free culture system strongly suggested that TGF-alpha, but not EGF or IGF, is biologically important as an autocrine growth factor in the growth of these cell lines in vitro.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Receptor, IGF Type 1; Transforming Growth Factor alpha; Tumor Cells, Cultured

Humoral hypercalcemia of malignancy is a paraneoplastic syndrome associated with a variety of solid neoplasms including squamous cell carcinomas of various sites. Parathyroid hormone-related protein (PTHrP) is a newly recognized hormone that has been implicated as one of the major causative factors in the pathogenesis of this syndrome. A canine oral squamous carcinoma cell line (SCC 2/88) was used to investigate the regulation of production of PTHrP in response to agents that alter keratinocyte differentiation/proliferation in vitro.. SCC 2/88 cells grown in serum-free media were exposed to various factors and PTHrP production was measured by radioimmunoassay. This cell line spontaneously produced substantial amounts of PTHrP (up to 7,000 pg/ml) without the need for a fibroblast-feeder layer. Production of PTHrP decreased at cellular confluence, and with increasing passage number.. Epidermal growth factor, cholera toxin, calcium, 1,25-dihydroxyvitamin D, ionomycin, trans-retinoic acid, transforming growth factor-beta 1 and hydrocortisone stimulated production of PTHrP by SCC 2/88 cells to various degrees. Transforming growth factor-beta 1 was the most potent stimulator of PTHrP production, with a maximal stimulation of 25-fold over control. Monensin decreased PTHrP secretion as early as 6 hours post-treatment and by 48 hours, there was no detectable PTHrP in the conditioned cell culture medium. Calcium, cholera toxin, ionomycin, and transforming growth factor-beta 1 decreased keratinocyte proliferation as measured by cell counts at all doses tested.. The results of this study revealed that SCC 2/88 cells spontaneously produced substantial amounts of PTHrP under baseline conditions and that compounds known to affect keratinocyte differentiation/proliferation up-regulated production of PTHrP. These cells will be valuable to investigate the molecular regulation of PTHrP production by squamous cell carcinomas.

Topics: Animals; Calcitriol; Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; Cholera Toxin; Dog Diseases; Dogs; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Hydrocortisone; Ionomycin; Keratinocytes; Kinetics; Mouth Neoplasms; Neoplasm Proteins; Parathyroid Hormone-Related Protein; Protein Biosynthesis; Radioimmunoassay; Time Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Carcinoma, Squamous Cell; Cell Membrane Permeability; Coated Pits, Cell-Membrane; Cytosol; Egtazic Acid; Endocytosis; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Kinetics; Ligands; Microscopy, Immunoelectron; Receptors, Transferrin; Tumor Cells, Cultured

The binding of 125I-epidermal growth factor (EGF) to the plasma membranes of 54 samples of human lung tumors was determined. These included 34 squamous cell carcinomas and 20 adenocarcinomas. Twenty samples of histologically normal lung excised surgically along with the tumors were used as controls. Most of the plasma membranes showed an EGF receptor level higher than that of normal tissue. A moderate increase in the amount of 125I-EGF bound (2-5 fold) was observed in the majority of the tumors. Only a few cases (5-10% of the total) showed a large increase (> than 10 fold). The binding of 125I-EGF was compared with clinical stages and grades of differentiation. No correlation between the stage of the tumor and 125I-EGF binding was observed. However, the highest levels of EGF receptor (EGF-R) were found in poorly differentiated squamous cell carcinomas. The total amount and the distribution pattern of gangliosides and phospholipids were analyzed in individual tumors. A decrease in GD1b, GD1a and sphingomyelin and an increase in GM1 and GM3 was observed. No correlation was detected when tumors with the highest or lowest levels of gangliosides or phospholipids were compared with tumors exhibiting the highest binding of 125I-EGF.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Gangliosides; Humans; Iodine Radioisotopes; Lung Neoplasms; Membrane Lipids; Middle Aged; Phospholipids

The binding of the Epidermal Growth Factor (EGF) and the amount of EGF-like activity (EGF-A) was analysed in 75 cervical carcinomas and possible clinical implications were tested. EGF-A was significantly increased in patients with metastases to the pelvic and/or para-aortic lymph nodes. The EGF-receptor (EGF-R) capacity was inversely related to the histological grade (p < 0.05) and was reduced in highly differentiated tumours. In stage I and II disease, the clinical outcome was significantly reduced, if the receptor capacity or the level of EGF-A was increased (> 100 fmol/mg protein and > 0.5 ng/mg protein, respectively). The measurement of EGF-R capacity and EGF-A provides new and additional information for the prediction of the prognosis in cervical cancer.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cervix Uteri; Epidermal Growth Factor; ErbB Receptors; Female; Follow-Up Studies; Humans; Lymphatic Metastasis; Neoplasm Staging; Survival Rate; Uterine Cervical Neoplasms

We demonstrated that four human oesophageal squamous cell carcinoma cell lines (TE8, TE9, TE10 and TE11) produced matrix metalloproteinase-1 (proMMP-1/tissue collagenase), 2 (ProMMP-2/'type IV collagenase'), 3 (proMMP-3/stromelysin), and 9 (proMMP-9/92-kDa gelatinase) as members of a matrix metalloproteinase (MMP) family, which degrades extracellular matrix macromolecules. Under normal culture conditions, in immunoblot analysis, proMMP-1 of M(r) = 53,00 was detected in one cell line (TE8), proMMP-2 of M(r) = 72,000 in three cell lines (TE9, TE10, and TE11), and proMMP-3 of M(r) = 57,000 in all four cell lines. In addition to these enzymes, in enzymography, a gelatinolytic activity around M(r) = 92-kDa, likely to be proMMP-9, was detected in only one cell line (TE10) under normal culture conditions. When these cell lines were treated with epidermal growth factor (EGF), however, the agent stimulated three cell lines (TE8, TE10 and TE11) to produce proMMP-9 in a dose-dose dependent manner. Oesophageal carcinoma-conditioned medium stimulated oesophageal fibroblasts to produce proMMP-1, -2, and -3, suggesting that the interaction between oesophageal carcinoma and stromal fibroblasts also plays a role in the production of MMPs by the latter. Our present study illustrates that oesophageal squamous cell carcinoma produces a variety of MMPs including proMMP-1, -2, -3, and -9 in vitro, suggesting that the ability of MMP production of the tumour may play an important role in its malignant behaviour and that the production of proMMP-9 may be regulated by EGF via overexpression of EGF receptors.

Topics: Carcinoma, Squamous Cell; Collagenases; Culture Media; Epidermal Growth Factor; Esophageal Neoplasms; Humans; Matrix Metalloproteinase 9; Metalloendopeptidases; Tumor Cells, Cultured

Antisera against murine Son of sevenless (Sos) recognize a protein of M(r) 155,000 in rat-1 fibroblasts with specific guanine nucleotide exchange activity toward p21c-Ha-ras. Epidermal growth factor (EGF) receptor coimmunoprecipitates with Sos from EGF-stimulated, but not quiescent, cells. The SH2 and SH3 domain-containing "adapter" protein Grb2 is also found in Sos immunoprecipitates in an EGF-inducible manner. In vitro reconstitution shows that Grb2 is required for the binding of activated EGF receptor to Sos. A phosphopeptide corresponding to tyrosine 1068 of the EGF receptor blocks both the assembly of the complex and EGF stimulation of nucleotide exchange on p21ras in a permeabilized cell system. These results suggest that EGF-induced activation of nucleotide exchange on p21ras proceeds through the recruitment of cytosolic Sos to a complex with EGF receptor and Grb2 at the plasma membrane.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antibodies; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Glutathione Transferase; GRB2 Adaptor Protein; Humans; Immune Sera; Immunoblotting; Kinetics; Membrane Proteins; Proteins; Proto-Oncogene Proteins p21(ras); Rats; Recombinant Fusion Proteins; Son of Sevenless Proteins; Tumor Cells, Cultured

The expression of mRNAs for epidermal growth factor (EGF), transforming growth factor alpha(TGF alpha), EGFR, platelet-derived growth factor (PDGF) A and B chain, PDGF receptor (PDGFR), transforming growth factor beta (TGF beta), erbB-2 and estrogen receptor (ER) genes was first examined in 6 human esophageal carcinoma cell lines, 6 xenoplanted and 15 surgically resected esophageal carcinomas. Secondly, the effect of EGF and TGF alpha on the expression of these genes by the TE-1 esophageal carcinoma cell line was investigated. The expression of EGF mRNA was detected in 8 (29.6%) of 27 tumors including the cell lines, whereas the TGF alpha and EGFR genes were expressed in 21 (77.8%) and 24 (88.9%) tumors respectively. PDGF B chain and PDGFR were detected in 18 (66.7%) and 20 (74.1%), respectively, and ER mRNA was observed in 16 (59.3%) tumors. Genes for PDGF A chain and TGF beta and the erbB-2 gene were commonly expressed. On the other hand, exogenous EGF and TGF alpha stimulated the expressions of fos and myc genes by TE-1 cells. The expression of mRNAs for TGF alpha, PDGF A and B chain and the erbB-2 genes was also increased after treatment with EGF. TGF alpha increased the accumulation of mRNAs for EGF, TGF alpha, EGFR, PDGF A and B chain and the erbB-2 gene. Moreover, the expression of mRNAs for interstitial collagenase, stromelysin and type IV collagenase was increased after EGF or TGF alpha treatment. These results indicate that EGF and TGF alpha may regulate the multi-growth-factor receptor expression and may play a central role for tumor invasion and metastasis as autocrine modulators for human esophageal carcinoma.

Topics: Adenocarcinoma; Adult; Aged; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Metalloendopeptidases; Middle Aged; Platelet-Derived Growth Factor; Receptors, Estrogen; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected with a monoclonal mouse antibody and EGF with polyclonal rabbit antiserum. Thirty-five of the tumours were positive for TGF-alpha and 26 of the tumours for EGF. None of the poorly differentiated tumours was positive for EGF, but they all were for TGF-alpha. In sections including normal differentiated oral mucosa, the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus paralleling the situation observed in the normal differentiated oral mucosa. In four cases, material was available from both a primary tumour and a metastasis. Three of these were positive for TGF-alpha and EGF with the same staining pattern as that of the primary tumours. This investigation together with our previous results confirms the existence of TGF-alpha, EGF, and EGF-receptors in the majority of oral squamous cell carcinomas and their metastases.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Mouth Mucosa; Mouth Neoplasms; Transforming Growth Factor alpha

A retrospective study was performed on 52 advanced tongue cancer patients (stages III + IV) in order to assess the prognostic value of circulating prolactin, tissue polypeptide-specific antigen (TPS), insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF). These markers were correlated with short-term prognosis (2 years). The advanced tongue cancer patients had significantly elevated levels of prolactin (p < 0.02), TPS (p < 0.05) and EGF (p < 0.01) and low levels of IGF-1 when compared to controls. The patients were grouped according to the commonly used cut-off levels of these markers. A significant difference in survival was observed only between two subgroups of prolactin (< 15.0 and > 15.0 ng/ml; p < 0.001). Hence, prolactin may provide an independent predictor of short-term prognosis in patients with advanced tongue cancer.

Topics: Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; Growth Substances; Humans; Insulin-Like Growth Factor I; Life Tables; Male; Peptides; Prognosis; Prolactin; Retrospective Studies; Survival Analysis; Survival Rate; Tissue Polypeptide Antigen; Tongue Neoplasms

Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive beta-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate the EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

Topics: Calcimycin; Carcinogens; Carcinoma, Squamous Cell; Colforsin; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Gravitation; Humans; Ionophores; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rotation; Signal Transduction; Space Flight; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Weightlessness; Weightlessness Simulation

The absence of specific stimulators of cell proliferation, i.e. of growth factors, is one of the causes of failure in long-term cultivation of the marine invertebrate cells. In search for such stimulators in tissues of marine invertebrates, we succeeded in discovering in some tissues a stimulator of cell proliferation, similar to EGF, with a comparatively high content of it in tissues of Mytilus edulis. The similarity of the obtained factor with EGF was shown by the substitution of 125I-EGF, connected with EGF receptors on the surface of A431 cells, with analysed extracts, as well as by the ability of this extract to induce the internalization of EGF-receptor complexes. The fraction of acid/ethanol extract of M.edulis stimulating the cell proliferation in resting mouse fibroblasts Swiss 3T3, was obtained in the same conditions as EGF did, using reversed phase chromatography. Thus, the factor from tissues of M.edulis may belong to the family of EGF-like factors.

Topics: 3T3 Cells; Animals; Bivalvia; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Horseshoe Crabs; Humans; Iodine Radioisotopes; Mice; Sea Cucumbers; Sea Urchins; Starfish; Tumor Cells, Cultured

Using 3T3 and 3T6 mouse fibroblasts and A431 epidermoid carcinoma cells, we previously observed that extracellular ATP and ADP were mitogens and they synergized with other growth factors (Huang, N., Wang, D. and Heppel, L. A. (1989) Proc. Natl. Acad. Sci. USA 86, 7904-7908). We now report that ATP and ADP stimulated Na+ entry, intracellular alkalinization and Na+/K+ pump activity, which are early events that had been proposed to play a central role in DNA synthesis. In addition, ATP, ADP and AMPPNP stimulated uridine uptake by a pathway involving arachidonic acid metabolism. In A431 cells, activation of protein kinase C also contributed to ATP-dependent stimulation of uridine uptake. Concentrations of indomethacin and pertussis toxin which inhibited uridine uptake also blocked arachidonic acid metabolism and DNA synthesis. ATP acted as a competence factor. Interestingly, ATP did not have to be continuously present to stimulate uridine uptake. It was equally effective even when it was washed away after brief treatment of cells.

Topics: 3T3 Cells; Adenosine Diphosphate; Adenosine Triphosphate; Animals; Biological Transport; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Growth Substances; Humans; Hydrogen-Ion Concentration; Indomethacin; Insulin; Kinetics; Mice; Pertussis Toxin; Phorbol 12,13-Dibutyrate; Potassium; Protein Kinase C; Rubidium; Rubidium Radioisotopes; Sodium; Sodium-Potassium-Exchanging ATPase; Uridine; Virulence Factors, Bordetella

A431 cells have an abundance of Epidermal Growth Factor (EGF) receptors which possess intrinsic tyrosine kinase activity. Treatment of membranes isolated from A431 cells with EGF caused a 2-3 fold increase in phosphorylation of a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly) which is a substrate for tyrosine kinase. Treatment of these membranes with 0.1 to 100 mM ethanol altered basal tyrosine kinase activity in a biphasic manner; increase at 10 mM and decrease at 100 mM ethanol. The treatment of the membranes with the same concentrations of ethanol also altered EGF's ability to stimulate tyrosine kinase activity: increase at 0.1 mM ethanol and decrease at 10 mM. Strikingly, EGF-stimulated tyrosine kinase was more sensitive to ethanol than the basal activity. Experiments with other alcohols showed a relationship between chain length and the inhibitory ability of the alcohol. These data demonstrate a biochemical effect of low concentrations of ethanol on tyrosine kinase. Interestingly, ethanol treatment of A431 cells inhibited EGF-stimulated phosphorylation of PLC-gamma 1 which is a substrate for EGF receptor tyrosine kinase. It is concluded that ethanol at low concentrations has significant modulatory effect on basal and EGF-stimulated tyrosine kinase, as well as PLC-gamma 1 phosphorylation.

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Ethanol; Humans; Isoenzymes; Molecular Sequence Data; Oligopeptides; Phosphorylation; Protein-Tyrosine Kinases; Substrate Specificity; Tumor Cells, Cultured; Type C Phospholipases

Two peaks of mitogen-activated protein (MAP) kinase activator activity are resolved upon ion exchange chromatography of cytosolic extracts from epidermal growth factor-stimulated A431 cells. Two forms of the activator (1 and 2) have been purified from these peaks, using chromatography on Q-Sepharose, heparin-agarose, hydroxylapatite, ATP-agarose, Sephacryl S-300, Mono S, and Mono Q. The two preparations each contained one major protein band with an apparent molecular mass of 46 or 45 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Evidence identifying the MAP kinase activators as the 46- and 45-kDa proteins is presented. Using inactive mutants of MAP kinase as potential substrates, it was found that each preparation of MAP kinase activator catalyzes phosphorylation of the regulatory residues, threonine 188 and tyrosine 190, of Xenopus MAP kinase. These results support the concept that the MAP kinase activators are protein kinases. These MAP kinase kinases demonstrate an apparent high degree of specificity toward the native conformation of MAP kinase, although slow autophosphorylation on serine, threonine, and tyrosine residues and phosphorylation of myelin basic protein on serine and threonine residues is detected as well.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Squamous Cell; Cell Line; Chromatography; Chromatography, Affinity; Chromatography, Gel; Durapatite; Enzyme Activation; Epidermal Growth Factor; Humans; Hydroxyapatites; Mitogen-Activated Protein Kinase Kinases; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Proteins; Substrate Specificity; Xenopus

The tyrosine kinase activity of the epidermal growth factor receptor (EGFR-TK) was determined at varying poly-Glu6Ala3Tyr1 (GAT) or [Val5]-angiotensin II (AT) and constant ATP concentrations and vice versa. With GAT as substrate, double reciprocal plots intersected practically on the abscissa following EGFR-TK pre-activation with EGF, but below the abscissa without EGF pre-activation. The EGFR-TK inhibitors App(NH)p (5'-adenylyl-beta, gamma-imidodiphosphate) and ADP were competitive with ATP and noncompetitive with GAT. Four families of 1/v vs. 1/[ATP] plots, constructed at different fixed concentrations of ADP and a different constant concentration of GAT for each family, yielded Slope1/ATP replots which intersected to the left of the ordinate and below the abscissa. GAT and AT, as cosubstrates, were competitive with each other and noncompetitive with ATP; 1/v vs. 1/[GAT] or 1/[AT] plots were hyperbolic and reached horizontal asymptotes when v was expressed as the rate of common product formation. All data were subjected to computer best-fit analysis by a program written especially for this purpose. We conclude that (i) the EGFR-TK reaction follows a Sequential Bi-Bi Rapid Equilibrium Random mechanism, and (ii) EGF induces conformational changes in the EGFR-TK active center which lead to marked decreases in the apparent dissociation constants of both substrates of the kinase reaction and a concomitant increase in initial velocities and Vmax (apparent).

Topics: Carcinoma, Squamous Cell; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Mathematics; Models, Theoretical; Protein-Tyrosine Kinases; Substrate Specificity; Tumor Cells, Cultured

The A9 antigen is a basement membrane antigen of normal squamous epithelial cells that is strongly expressed in many squamous carcinomas. High expression of this antigen is associated with early relapse in squamous cell carcinomas of the head and neck. We now know that the A9 antigen is structurally, immunologically, and functionally similar to the alpha 6 beta 4 integrin that has been shown to be linked to metastatic behavior in murine tumor models. The alpha 6 and beta 4 genes have been cloned and sequenced, and a model has been constructed from the deduced amino acid composition. In this study we present a hypothetical model and use it to design experiments to assess the factors that influence the expression of the A9/alpha 6 beta 4 integrin in normal and malignant keratinocytes. High calcium induces down regulation of A9/alpha 6 beta 4 antigen in normal but not malignant keratinocytes within 24 hours. Although calcium can down-regulate beta 4 message in tumor cells in the absence of epidermal growth factor (EGF), transcription of beta 4 increased in the tumor cells under the conditions we used for assessing antigen expression (calcium plus EGF). Retinoic acid also stimulated transcription of beta 4 in tumor cells, but this was partially inhibited by the presence of high calcium. Phosphorylation of the beta 4 chain was stimulated by epidermal growth factor and calcium in normal keratinocytes, but in the malignant cells phosphorylation was constant regardless of the culture conditions. Our results indicate that high expression of the alpha 6 beta 4 integrin is associated with conditions that favor migration and undifferentiated proliferation of normal keratinocytes and that malignant keratinocytes differ from normal keratinocytes by constitutive phosphorylation of beta 4 and by failure to downregulate beta 4 transcription in response to calcium in the presence of EGF.

Topics: Antigens, Neoplasm; Blotting, Northern; Calcium; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; Humans; Integrins; Keratinocytes; Models, Molecular; Mouth Neoplasms; Phosphorylation; Proteolipids; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; RNA, Messenger; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

Transforming growth factor alpha (TGF-alpha) is a single chain polypeptide which exists in a variety of forms differing in molecular weight. These forms are variously present in normal and neoplastic cells. Of particular interest are TGF-alpha's well-known mitogenic properties. The transition from a normal to a neoplastic cellular state results from signalling defects that may depend upon, inter alia, abnormal levels of expression and secretion of TGF-alpha. It is known that the secretion of TGF-alpha may be enhanced appreciably by agents such as phorbol 12-myristate 13-acetate (PMA), serum factors and epidermal growth factor (EGF). Here, we compare the efficacy of these three agents in the elevation of TGF-alpha secretion in the well studied A431 cell line with their previously undocumented efficacy in certain interesting, but little known, human oesophageal squamous cell carcinoma (SCC) lines.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; In Vitro Techniques; Secretory Rate; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha; Tumor Cells, Cultured

Recovery of the cell cycle in cells A 431 and in human embryo fibroblasts (EFH) differs much. Unlike EFH, A 431 cells have: 1) synchronized exit of cells from G1 into S phase after 5 Gr irradiation; 2) G2-block; 3) much less manifestation of these two phenomena in the presence of EGF; 4) a lesser effectiveness of the repair of DNA single-strand breaks. EGF stimulation of the repair of radiation-induced DNA lesions, SSB in particular, may be of great importance for the postirradiation cell cycle recovery.

Topics: Carbon Radioisotopes; Carcinoma, Squamous Cell; Cell Cycle; Cell Line; Cells, Cultured; DNA Damage; DNA Repair; DNA, Neoplasm; DNA, Single-Stranded; Embryo, Mammalian; Epidermal Growth Factor; Fibroblasts; Humans; Thymidine; Time Factors; Tumor Cells, Cultured

Cytokines are known to play an important role in host defense by regulating the function, growth, and differentiation of the cells of the immune system. We hypothesize that, in the tumor microenvironment, tumor cells and resident tissue cells (e.g., fibroblasts) also produce cytokines that may regulate the local immune response to tumors. Initially, homogenates of eight head and neck squamous cell carcinomas (HNSCC) were assayed for the presence of interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to establish the presence of these cytokines in the tumors in vivo. We detected IL-1 in all tumor homogenates and IL-4, IL-6, and GM-CSF in some homogenates. To assess the ability of HNSCC to produce these cytokines, supernatants of short-term primary cultures of HNSCC were assayed for the same cytokines. No IL-1 was detected, although baseline levels of IL-4, IL-6, and GM-CSF were present. However, the stimulation of primary tumor cultures with exogenous IL-1 induced or significantly enhanced production of IL-4 (p < 0.01), IL-6 (p < 0.001), and GM-CSF (p < 0.02). These results support our hypothesis that HNSCC secrete cytokines that may influence the response of local immune cells. Our data also suggest that IL-1 may have a central role in regulating the local immune response through the enhancement or induction of cytokine production by tumor and/or resident tissue cells.

Topics: Carcinoma, Squamous Cell; Culture Techniques; Epidermal Growth Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Head and Neck Neoplasms; Humans; Interleukin-1; Interleukin-4; Interleukin-6; Lipopolysaccharides; Middle Aged; Phenotype; Tumor Cells, Cultured

I. Serial histopathologic study of esophageal squamous cell carcinoma. A review of 335 cases of squamous cell carcinoma disclosed 55 cases (16.4%) with glandular components in addition to the ordinary component of squamous cell carcinoma, suggesting that this type of esophageal tumor had originated not only from the covering squamous epithelium but from esophageal gland or ductal epithelium. Intra-epithelial carcinoma concomitant with squamous cell carcinoma was seen in 95 cases (28.4%). The incidences of coexistence in such lesion were higher in the groups of early stage esophageal cancer. These observations support the concept of field carcinogenesis of esophageal cancer. II. Histopathologic study of squamous epithelial dysplasia. Among 91 cases without preoperative treatment, there were 40 dysplastic lesions in 23 cases (25.3%). The continuity of dysplasia to the carcinoma was 48.3% and it was often encountered in severe dysplasia rather than in moderate or mild dysplasia, suggesting some relationship between the severity of dysplasia and carcinoma. III. Immunohistochemical study of EGF and c-myc. Among 27 cases, EGF was positive in 10 (37.0%). c-myc was positive in 18 (66.7%) not only cancer but normal epithelium suggesting that some change of products of oncogene occurred also in the normal epithelium of the patients of esophageal cancer.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Esophageal Neoplasms; Humans; Immunohistochemistry; Proto-Oncogene Proteins c-myc

It has been shown elsewhere that the epidermal growth factor (EGF) in A431 cells can recycle in receptor-bound state (Teslenko et al., 1987; Sorkin et al., 1989, 1991). Present study deals with the action of primaquine, a lysosomotropic agent, on EGF-receptor complexes (EGF-RC). By the method of indirect immunofluorescence with anti-EGF-R monoclonal antibody it is found that following a 1 h incubation of cells at 37 degrees C in the presence of EGF a bright staining of endosomes appears in the intranuclear region, while after incubation of the cells at 4 degrees only margins of cells are stained. Such a pattern of fluorescence is peculiar of endocytosis in A431 cells. When the cells were incubated in the presence of a 0.3 mM primaquine for 1 h, the immunostaining is changed: bright compact spot in the para-Golgi region appeared. The effect of primaquine is reversible. When the cells after preincubation with EGF were incubated in the absence of EGF for 3 h at 37 degrees C, the staining of cell margins could be observed again, demonstrating the recycling of EGF-RC. Under similar conditions of cell incubation, but in the presence of primaquine, the staining of the para-Golgi region was not changed. In the experiments with 125I-EGF it was shown that intracellular accumulations of 125I-EGF were maintained when the cells were incubated in the presence of 0.3 mM primaquine. It is concluded that primaquine inhibits the recycling of EGF-R in A431 cells.

Topics: Carcinoma, Squamous Cell; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Iodine Radioisotopes; Lysosomes; Primaquine; Time Factors; Tumor Cells, Cultured

Three morphologically distinct cell lines--F.2a, V, and B.2--were isolated from a single human squamous cell carcinoma. Although all three cell lines can grow indefinitely in culture, they differ in a number of important transformation-related phenotypes. Only B.2 is strongly tumorigenic when injected into the flanks of nude mice, and only V can efficiently grow in semisolid media. The dominance of these traits was investigated by generating somatic cell hybrids among the three cell lines. F.2a was able to suppress the tumorigenicity of B.2 cells, whereas B.2 inhibited the capacity for anchorage-independent growth of V, the latter trait being a function of the ability of these epithelial cells to differentiate when deprived of support. The influence of exogenously added growth factors was also evaluated. This study indicates that the particular tumor we examined consisted of a heterogeneous population of cells with distinct growth and differentiation capacities.

Topics: Animals; Carcinoma, Squamous Cell; Cell Adhesion; Cell Differentiation; Cell Division; Cell Fusion; Cell Line; Culture Media, Serum-Free; Epidermal Growth Factor; Genetic Markers; Humans; Keratinocytes; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; Transfection; Transforming Growth Factor alpha; Transplantation, Heterologous

Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-alpha in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFR1) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-1 protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-alpha at concentrations greater than 0.1 nM resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-alpha was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-alpha in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Lung Neoplasms; Protein Precursors; Tetradecanoylphorbol Acetate; Transforming Growth Factor alpha

Neutral red stains both normal and cancer mitotic cells, but uptake by living mitotic cancer cells is distinctly higher than in normal cells. This new approach to cancer cell identification is demonstrated in 4 established tumorigenic cancer cell lines: human skin epidermoid carcinoma A431, mouse Cloudman malignant melanoma, human oral epidermoid carcinoma and rat hepatoma. Human Chang liver cells served as normal controls. With epidermal growth factor (EGF) prepulse, neutral red uptake is dramatically enhanced. The possibility of a causal relationship with M-phase specific phosphorylation is discussed.

Topics: Animals; Carcinoma, Squamous Cell; Cell Nucleus; Epidermal Growth Factor; Humans; Interphase; Liver; Liver Neoplasms, Experimental; Melanoma, Experimental; Mitosis; Mouth Neoplasms; Neoplasms, Experimental; Neutral Red; Rats; Skin Neoplasms; Tumor Cells, Cultured

Several malignant neoplasms express high levels of epidermal growth factor receptors. The aim of this study was to assess whether 123I-labeled epidermal growth factor can concentrate in lymph node metastases of squamous cell carcinomas of the cervix. 14 patients with advanced cervical cancer were selected because of their high probability of lymph node metastases. Planar scintigrams were recorded from the lower and upper abdomen following subcutaneous injection of 123I-labeled epidermal growth factor into the web space of each foot. Scintigraphic images were interpreted without knowledge of computerized tomography scan (n = 13) and ultrasound (n = 9) results from the pelvic lymph nodes. In 2 patients, histological verification was performed by diagnostic biopsy of pelvic lymph nodes. Nodal involvement was confirmed by computerized tomography for 4 of the 11 positive scans and by ultrasound for 2. In 11 out of 14 patients an increased uptake of 123I-labeled epidermal growth factor could also be seen in the primary tumour. Our findings suggest that targeting of cervical cancer lymph node metastases can be achieved by in vivo binding of 123I-labeled epidermal growth factor with receptors on tumour cell surfaces.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Iodine Radioisotopes; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Radionuclide Imaging; Uterine Cervical Neoplasms

A431 cells were treated with epidermal growth factor (EGF) to study the mechanism by which this factor accelerates the glycolytic flux. After EGF treatment, fructose-2,6-bisphosphate (Fru-2,6-P2) levels rose up to 2-fold. This change correlated with an increase in phosphofructokinase-2 activity, which was not due to a change in the transcription or translation of the enzyme, neither in the amount of enzyme. PK-C does not appear to be involved in the signalling mechanism since EGF was equally potent in PK-C depleted cells than in control cells. The increase in Fru-2,6-P2 levels was lower and more transient in cells treated with EGF in a calcium-free medium than in the presence of the cation, and it was restored by the addition of calcium to the medium. These results suggest a possible role for calcium-mediated pathways in the control of Fru-2,6-P2 levels in A431 cells.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Fructosediphosphates; Glycolysis; Humans; Phosphofructokinase-1; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The concentration of epidermal growth factor in human and bovine milk was measured by radioreceptor assay. Both human placental plasma membranes and a human epidermoid carcinoma cell were used as the epidermal growth factor receptor source in the assay. The use of placental plasma membrane in the radioreceptor assay gave erroneous results for bovine milk and overestimated the concentration of epidermal growth factor in human milk. Intact cells appear to provide a more accurate measure of the concentration of epidermal growth factor in milk samples. Using A431 cells, we found very low concentrations of epidermal growth factor in bovine milk (less than 2 ng/ml) compared to human milk (30-40 ng/ml). No epidermal growth factor activity was found in several cows' milk-based infant formulas. These results highlight the caution which must be taken when measuring trace substances such as polypeptide growth factors in complex samples such as milk.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chemical Fractionation; Chromatography, Gel; Colostrum; Epidermal Growth Factor; ErbB Receptors; Evaluation Studies as Topic; Milk; Milk, Human; Placenta; Radioligand Assay

We have established three cell lines (IMC-2, IMC-3, and IMC-4) from a human maxillary tumor, which exhibited different sensitivities to epidermal growth factor (EGF). It was inhibitory to colony-forming abilities of IMC-3 and IMC-4 cells in culture, while it affected that of IMC-2 cells slightly if at all. The differential sensitivities to EGF among the three cell lines were reproducibly observed when several cell sublines were further established from tumors appearing in nude mice. Saturation-binding kinetics with 125I-EGF showed similar levels of EGF-binding activities among the three cell lines. However, IMC-2, IMC-3, and IMC-4 showed almost similar sensitivities to cisplatin. Autophosphorylation of EGF receptor in the presence of EGF proceeded at similar levels among the three cell lines. Tumor growth was followed in nude mice when IMC-2, IMC-3, and IMC-4 at 1 x 10(7) cells were inoculated. The IMC-2 tumors enlarged at much faster rates than the other two cell lines. The IMC-4 tumors showed very slow growth rates, and IMC-3 tumors enlarged at an intermediate rate. These data suggest that the maxillary tumor used comprised cell populations that differed in their growth behaviors in response to EGF.

Topics: Carcinoma, Squamous Cell; Cisplatin; Epidermal Growth Factor; ErbB Receptors; Humans; Maxillary Neoplasms; Phosphorylation; Tumor Cells, Cultured

We have used an antisense approach to investigate the role of overexpression of the normal human epidermal growth factor (EGF) receptor in the transformed phenotype of KB cells, which are a tumor derived human cell line. Initial experiments performed in vitro, showed that antisense RNA complementary to the entire coding region (AS-FL) or to parts of the EGF-R mRNA (AS-3', AS-5', and AS-K) effectively blocked translation of EGF-R mRNA. In addition, upon microinjection into KB cells, the in vitro synthesized antisense RNAs were able to inhibit transiently the synthesis of EGF-R. Inhibition was concentration-dependent, both in vitro and in cells, and the most effective constructs were those complementary to the entire coding region (AS-FL) or to the 3'-coding end of the mRNA (AS-3'). Transfection of the same EGF-R antisense RNA constructs into the human epidermoid carcinoma KB cell line gave rise to several clones stably expressing elevated levels of antisense RNA and resulting in low residual levels of EGF receptor. The most reduced clones exhibited a totally restored serum-dependent growth and were severely impaired in colony formation and growth in agar. In addition the severity of the phenotype was directly proportional to the residual amount of EGF-R expressed. We conclude that over-expression of normal EGF-R plays a direct primary role in the development of the transformed phenotype of this human cancer cell line.

Topics: Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Humans; Microscopy, Fluorescence; Phenotype; Plasmids; Precipitin Tests; Protein Biosynthesis; RNA, Antisense; RNA, Messenger; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The effect of epidermal growth factor on the radiation response of two human squamous carcinoma cell lines, A431 (from vulva) and SiHa (from cervix), was examined. In both lines, cells in S phase were more radioresistant than cells in other cell cycle phases. Epidermal growth factor present after irradiation enhanced the radiation response of A431 cells in different cell cycle phases, whereas no effect was seen for SiHa cells. The enhancement was maximum with 10 ng/ml epidermal growth factor and was associated mainly with a reduction in the shoulder region of the cell survival curve. The ratio between the n values of the control and epidermal growth factor treated total cell population, G1, S, and G2M cells is 2.2, 4.1, 1.7, and 2.2, respectively. Epidermal growth factor reduced plating efficiency by about 50% for A431 cells in different cell cycle phases whereas a slight increase in plating efficiency was seen for SiHa cells. The present results indicate that epidermal growth factor related radiosensitization is dependent on both cell line and cell cycle.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Survival; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Female; Humans; In Vitro Techniques; Radiation Tolerance; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vulvar Neoplasms

Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha (TGF-alpha) was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.

Topics: Blotting, Northern; Carcinoma, Squamous Cell; Cell Aggregation; Cell Communication; Cell Count; Epidermal Growth Factor; Humans; Radioimmunoassay; RNA, Messenger; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Activation of epidermal growth factor (EGF) receptors stimulates inositol phosphate production in rat hepatocytes via a pertussis toxin-sensitive mechanism, suggesting the involvement of a G protein in the process. Since the first event after receptor-G protein interaction is exchange of GTP for GDP on the G protein, the effect of EGF was measured on the initial rates of guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) association and [alpha-32P]GDP dissociation in rat hepatocyte membranes. The initial rate of [35S]GTP gamma S binding was stimulated by EGF, with a maximal effect observed at 8 nM EGF. EGF also increased the initial rate of [alpha-32P]GDP dissociation. The effect of EGF on [35S]GTP gamma S association was blocked by boiling the peptide for 5 min in 5 mM dithiothreitol or by incubation of the membranes with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). EGF-stimulated [35S]GTP gamma S binding was completely abolished in hepatocyte membranes prepared from pertussis toxin-treated rats and was inhibited in hepatocyte membranes that were treated directly with the resolved A-subunit of pertussis toxin. The amount of guanine nucleotide binding affected by occupation of the EGF receptor was approximately 6 pmol/mg of membrane protein. Occupation of angiotensin II receptors, which are known to couple to G proteins in hepatic membranes, also stimulated [35S]GTP gamma S association with and [alpha-32P]GDP dissociation from the membranes. The effect of angiotensin II on [alpha-32P]GDP dissociation was blocked by the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II, demonstrating that the guanine nucleotide binding was receptor-mediated. In A431 human epidermoid carcinoma cells, EGF stimulates inositol lipid breakdown, but the effect is not blocked by treatment of the cells with pertussis toxin. In these cells, EGF had no effect on [35S]GTP gamma S binding. Occupation of the beta-adrenergic receptor in A431 cell membranes with isoproterenol did stimulate [35S] GTP gamma S binding, and the effect could be completely blocked by l-propranolol. These results support the concept that in hepatocyte membranes, EGF receptors interact with a pertussis toxin-sensitive G protein via a mechanism similar to other hormone receptor-G protein interactions, but that in A431 human epidermoid carcinoma cells, EGF may activate phospholipase C via different mechanisms.

Topics: Animals; Binding Sites; Calcium; Carcinoma, Squamous Cell; Cell Membrane; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Isoproterenol; Kinetics; Liver; Male; Pertussis Toxin; Phosphorylation; Protein-Tyrosine Kinases; Rats; Rats, Inbred Strains; Virulence Factors, Bordetella

Topics: 4-Nitroquinoline-1-oxide; Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Keratinocytes; Mice; Mice, Nude; Mouth Neoplasms; Rats; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Transplantation, Heterologous

We have used 123I-labelled epidermal growth factor (EGF) scans to study 14 patients with advanced cervical cancer. Abnormal lymph node imaging was seen most clearly 6-8 h after the injection and revealed abnormal uptake by pelvic lymph nodes in 11 patients. 4 of these 11 had abnormal computerised tomographic and ultrasound scans; in the other 7 conventional radiology did not confirm the presence of disease.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Evaluation Studies as Topic; Female; Humans; Iodine Radioisotopes; Lymphatic Metastasis; Pelvis; Tomography, Emission-Computed; Uterine Cervical Neoplasms

By the use of rhodamine-phalloidin, the distribution of actin in A-431 cells during the action of epidermal growth factor (EGF) has been studied. Changes in the pattern of staining are observed in 30-60 s after addition of the EGF. Microvilli and wrinkles are created on the cell surface. Following a 5-10 min action of EGF, rhodamine-phalloidin stained intensely ruffles and cell borders. After 60 min, the ruffling of cell surface disappeared, and actin was seen concentrating on the cell borders only. Electron microscopy of the EGF-treated A-431 cells lysed by Triton X-100 also revealed some vigorous fibrillar bunches on the cell edges.

Topics: Actins; Carcinoma, Squamous Cell; Cell Line; Cytochalasin B; Cytoskeleton; Epidermal Growth Factor; Humans; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Staining and Labeling; Time Factors; Tumor Cells, Cultured; Vinblastine

Phosphorylation of the epidermal growth factor (EGF) receptors in endosomes isolated from A431 cells was studied using antiphosphotyrosin antibody (anti-P-Tyr). A431 cells were preincubated with EGF and then washed with acid buffer to remove surface-bound EGF. Endosomes were isolated from such cells by the method of subcellular fractionation on Percoll density gradient. Addition to isolated endosomes of anti-P-Tyr complexes with immunogold resulted in a significant shift of endosome peak to the high density region. This fact indicates that anti-P-Tyr interacts with phosphotyrosine residues of EGF receptors localized in endosomes.

Topics: Antibodies; Carcinoma, Squamous Cell; Cell Fractionation; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Organelles; Phosphorylation; Phosphotyrosine; Tumor Cells, Cultured; Tyrosine

Fourteen patients suffering from advanced inoperable cervical cancer were investigated by planar scintigraphy after subcutaneous administration of a radiolabelled (I-123) epidermal growth factor (EGF). The objective of the study was to test whether labelled EGF concentrates in lymph node metastases of squamous cell cancer of the cervix uteri. Scintigraphic results were correlated with the gynecological findings, computed tomography (CT), ultrasound (US) and in two patients with histology. A series of scintigrams were performed up to 24 hours post injection. Slight activity in liver and kidneys was found already 30 min after s.c. injection of EGF. Optimal imaging quality for the lymphatics was obtained at 6-8 hours post injection. Accumulation in the pelvic lymph nodes was documented by the region of interest technique (ROI). Lymph nodes of the inguinal and iliac communis region were marked in all cases. Due to this, accumulation of EGF could not be called selective. In 11/14 patients hot spots were correlated to other pelvic lymph nodes. In 4/11 patients with positive EGF-scanning metastatic disease was confirmed by CT scan and/or US examination. 2/11 patients underwent a Probatoria operation. The respective histological reports confirmed our scintigraphic results. In conclusion, labelled EGF did not fulfil our theoretical expectations of excellent accumulation in lymph node metastases and cannot at present be recommended for routine clinical use.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Radionuclide Imaging; Uterine Cervical Neoplasms

Using monoclonal antibody, G6K12, a novel differentiation marker of keratinocyte, we examined the effect of EGF and retinoic acid on the differentiation of SCC-25, a cell line established from human squamous cell carcinoma. EGF inhibited the expression of cell-surface differentiation marker in low cell-density, but not in high cell-density. Retinoic acid (more than 10(-8) M) inhibited the expression of the antigens. The terminal differentiation of SCC-25 cells occurred at the condition of high cell density. These results obtained demonstrate that EGF and retinoic acid inhibit the terminal differentiation of SCC-25 cells, and that the cell density is the important factor for the commitment of the terminal differentiation.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Depression, Chemical; Epidermal Growth Factor; Humans; Tretinoin; Tumor Cells, Cultured

Topics: Alkaline Phosphatase; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Female; Giant Cells; Glycolipids; Glycosylphosphatidylinositols; Growth Substances; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Insulin; Isoenzymes; Kinetics; Phosphatidylinositols; Placenta; Pregnancy; Thionucleotides; Trophoblasts

This study examined the characteristics of keratinocytes from 4-nitroquinoline N-oxide-treated rat oral epithelium that formed well differentiated carcinomas or spindle cell tumours when transplanted s.c. to athymic mice. Small polygonal-type cells in early culture passage gave rise to xenografts that were well differentiated carcinomas with keratin positivity and a differential reactivity with two anti-vimentin antibodies (positive Vim Dako, negative Vim 3B4). Progressive culture of the polygonal cells resulted in cells of a more stellate-like morphology which, when transplanted, formed spindle cell tumours that were keratin negative and vimentin positive (Vim Dako and Vim 3B4). The staining pattern of the xenografts was similar to that of the cultured cell derivatives. By contrast to normal oral keratinocytes which were stimulated by epidermal growth factor (EGF), the parental and xenograft-derived cells were markedly less responsive and at times refractory to EGF. Low affinity EGF receptors characterized normal keratinocytes whilst parental and xenograft-derived cells from well differentiated carcinomas and spindle cell tumours expressed diminished numbers of low and high affinity and high affinity EGF receptors respectively. Normal keratinocytes and parental tumour cells were inhibited by transforming growth factor (TGF-beta) whereas xenograft-derived cell lines from both well differentiated carcinomas and spindle cell tumours were refractory to TGF-beta. TGF-beta receptors were predominantly of high affinity although xenograft-derived cells, particularly from spindle cell tumours, expressed increased numbers of receptors which were of lower affinity. The results indicate that spindle cell tumours are a variant of well differentiated carcinomas and express an aberrant pattern of differentiation. Resistance to EGF-induced stimulation and TGF-beta-induced inhibition appears not to be an integral part of the tumour phenotype but the pattern of EGF and TGF-beta receptor expression closely follows the degree of tumour differentiation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Keratinocytes; Male; Mouth Neoplasms; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Thymidine; Transforming Growth Factor beta; Tritium

Treatment of A431 human epidermoid cells with epidermal growth factor (EGF; 20 nM) results in decreased proliferation. This is associated with blockage of the cells in the S and/or G2 phases of the cell cycle. We found that tyrphostin, a putative tyrosine kinase inhibitor, in the range of 50 to 100 microM, partially reversed the growth-inhibitory and cell cycle changes induced by EGF. By using high-pressure liquid chromatography with electrochemical detection, we found that tyrphostin was readily incorporated into A431 cells, reaching maximal levels within 1 h. Although tyrphostin (50 to 100 microM) had no effect on high-affinity binding of EGF to its receptor in A431 cells for up to 24 h, the compound partially inhibited EGF-stimulated EGF receptor tyrosine kinase activity. However, this effect was evident only after prolonged treatment of the cells (4 to 24 h) with the drug. When the peak intracellular concentration of tyrphostin occurred (1 h), no inhibition of tyrosine kinase activity was observed. After both 1 and 24 h, tyrphostin was a less effective inhibitor of tyrosine kinase activity than the potent tumor promoter 12-O-tetradecanoyl phorbol-13-acetate, which almost completely blocked EGF receptor autophosphorylation. On the basis of our data, we hypothesize that tyrphostin is not a competitive inhibitor of the EGF receptor tyrosine kinase in intact cells and that it functions by an indirect mechanism.

Topics: Biological Transport; Carcinoma, Squamous Cell; Catechols; Cell Division; Cell Line; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Structure; Nitriles; Protein-Tyrosine Kinases; Tyrphostins

We studied A431 epidermoid carcinomas xenografted into male nude mice either in the unperturbed state or after either surgical removal of the salivary glands or i.p. injection of exogenous epidermal growth factor (0.2 mg/kg daily for 7 days). The percentage of hypoxic cells in unperturbed tumors was 10.5% (95% confidence limits, 6.6-16.8%). In mice that received epidermal growth factor injections, hypoxic percentages decreased to 3.7% (1.7-7.8%), and tumor growth rates increased. In sialoadenectomized mice, hypoxic percentages increased to 35.2% (27.1-45.6%), and tumor growth rates decreased. These data indicate that the biology of solid tumors can be significantly modified by the host status.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Hypoxia; Epidermal Growth Factor; Humans; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Submandibular Gland

Topics: Animals; Carcinoma, Squamous Cell; Cell Adhesion; Cell Communication; Endocrine Glands; Endothelium; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Humans

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 microM C8-cer was effective, and 3 microM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 microM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 microM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol.10(6) cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 microM) also increased cellular ceramide levels to 227 pmol.10(6) cells-1, but did not increase its own cellular level of 12 pmol.10(6) cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at effective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine ma

Topics: Autoradiography; Blotting, Western; Carcinoma, Squamous Cell; Ceramides; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Humans; Peptide Mapping; Phosphorylation; Signal Transduction; Sphingosine; Tumor Cells, Cultured; Tyrosine

The presence of EGF-autocrine secretion was investigated in a cell line derived from a human squamous cell carcinoma (HSC-1). The cell line was found to secrete the epidermal growth factor (EGF)-like substance. Meanwhile, normal human and uninvolved and involved psoriatic epidermal cells did not show any evidence of EGF secretion in culture. Not was any evidence of EGF secretion observed in vitro in several malignant cell lines other than HSC-1 derived from human squamous cell carcinomas, adenocarcinoma and melanoma. The addition of EGF did not stimulate, but rather inhibited, the growth of HSC-1 cells in GIT medium as well as Dulbecco's modified essential medium with low concentrations of fetal calf serum (0.5-1%) in vitro. Overexpression of EGF receptors is known to occur in HSC-1. The results suggest that HSC-1 cells exhibit autocrine secretion of the EGF-like substance but not autostimulation in anchorage-dependent cell culture.

Topics: Carcinoma, Squamous Cell; Cell Division; Culture Media; Epidermal Growth Factor; Humans; Reference Values; Skin; Skin Neoplasms; Time Factors; Tumor Cells, Cultured

Topics: Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Radioligand Assay; Transforming Growth Factor alpha

Previous studies have shown that the presence of epidermal growth factor (EGF) after irradiation enhanced the radiosensitivity of CaSki cells. To examine the role of EGF receptor density and related growth response in EGF associated radiosensitisation, four human squamous carcinoma cell lines were used. The total number of EGF receptors for HN5, A431, CaSki, and SiHa cells is 5.2 x 10(6), 1.6 x 10(6), 7.9 x 10(5) and 1.1 x 10(5) respectively, and the dissociation constant (Kd) for low affinity EGF receptors is 11.8, 3.8, 1.7 and 0.8 nM respectively. The Kd for high affinity receptors differs slightly among the four cell lines, 0.09 to 0.21 nM. EGF inhibited the growth of A431, CaSki, and HN5 cells, but stimulated the growth of SiHa cells. Due to the presence of 10 ng ml-1 EGF after irradiation, radiosensitivity enhancement associated with reduced shoulder size of the survival curve was observed. The extent of sensitisation was similar for A431, CaSki, and HN5 cells, with no effect on SiHa cells. At this concentration, EGF present during the clonogenic assay period after irradiation also reduced the plating efficiency (PE) of the monolayer cultures of A431, CaSki, and HN5 cells, but increased that of SiHa cells. The radiation response of mouse 3T3 cells (less than 5,000 receptors) was not sensitised by EGF. A similar level of radiosensitivity enhancement by EGF was observed for parental and conditioned A431 cultures. The conditioned cells were grown in 50 ng ml-1 EGF for 10 weeks and did not demonstrate growth inhibition and PE reduction by treatment with EGF. The EGF receptor numbers and binding affinity of these cells were the same as for the parental cells. The results from the conditioned cells support the hypothesis that EGF related radiosensitisation is EGF receptor density dependent.

Topics: Animals; Binding Sites; Carcinoma, Squamous Cell; Cell Division; Dose-Response Relationship, Radiation; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Tumor Cells, Cultured; Tumor Stem Cell Assay

Epidermal growth factor (EGF) activates a well characterized signal transduction system in human A431 epidermoid carcinoma cells, which leads to rapid and transient expression of the c-fos proto-oncogene. In order to investigate the influence of altered gravity on EGF-induced signal transduction, we have studied the EGF-induced c-fos expression under simulated hypo- and hypergravity conditions. In this report we show that EGF-induced fos expression is decreased under simulated hypogravity conditions, while hypergravity has a stimulatory effect on EGF-induced fos expression. These results show that the EGF-activated signal transduction system is influenced by gravity, and that gravity exerts its effects already in the early phases of the signal transduction cascade.

Topics: Blotting, Northern; Carcinoma, Squamous Cell; Cycloheximide; DNA-Binding Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Gravitation; Humans; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

Exposure of RL95-2 human endometrial adenosquamous carcinoma cells of early passage (less than 30 passages) and late passage (greater than 250 passages) to epidermal growth factor (EGF) resulted in density- and concentration-dependent effects. At low seeding density, EGF (20 nM) inhibited the growth of early passage cells, whereas at high seeding density, 4.98 nM and 20 nM concentrations of EGF stimulated their growth. Furthermore, the growth of late passage cells was stimulated by 0.0166 nM EGF and inhibited by 4.98 nM and 20 nM EGF at both seeding densities. EGF (20 nM) caused marked morphological changes of both passages at the low seeding density. Inhibition of invasion of both passages through Matrigel-coated filters was seen at low seeding density, while at the high seeding density, EGF enhanced invasiveness. At high seeding density, EGF stimulated an increase in urokinase type plasminogen activator activity, which may have augmented the ability of cells to degrade the extracellular matrix. In addition, the ability of high seeding density cells of both passages to adhere to matrigel after EGF treatment correlated with invasiveness.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Neoplasm Invasiveness; Tissue Plasminogen Activator; Tumor Cells, Cultured; Uterine Neoplasms

A431 cells, a human epidermoid carcinoma cell line, express an unusually large number of cell surface receptors for the epidermal growth factor (EGF). The growth rate of A431 cells was estimated by measuring [3H]thymidine incorporation at the logarithmic growth phase. The growth of the cells in protein-free medium was partially inhibited by exposure of the cells to pertussis toxin, islet-activating protein (IAP). The growth in both serum-containing and protein-free medium was inhibited by high concentrations of EGF, and these inhibitions were partially reversed by treatment of the cells with IAP. The effects of IAP were well correlated with the degree of ADP-ribosylation of a membrane 40-kDa protein. Thus, IAP sensitive G-proteins appear to be involved in the signal transduction of both positive and negative regulation of A431 cell growth. The possibility is also discussed that phosphatidylinositol turnover may participate in growth regulation.

Topics: Carcinoma, Squamous Cell; Cell Division; DNA; Epidermal Growth Factor; GTP-Binding Proteins; Humans; Kinetics; Pertussis Toxin; Phosphatidylinositols; Signal Transduction; Tumor Cells, Cultured; Virulence Factors, Bordetella

Sertoli cell conditioned medium (SCCM) contains a potent mitogen, Sertoli cell secreted growth factor (SCSGF). A431 cells, derived from a human epidermoid carcinoma have provided an excellent model cell line for the study of this apparently unique activity secreted by rat Sertoli cells in vitro. Previously, it was shown that SCCM contained an epidermal growth factor (EGF)-like activity which was thought to be the mitogen for A431 cells. The present study showed that these two factors are distinct entities. The secretion of the EGF-like activity decreased with increasing number of culture days, while that of SCSGF and of another Sertoli cell specific protein, transferrin remained constant. The addition of SCCM stimulated whereas 2.5 ng/ml EGF inhibited the A431 cell growth. The proliferative response of A431 cells to a wide variety of growth factors and known Sertoli cell secretions was investigated. SCSGF was the only growth factor of known Sertoli cell secretions tested (transforming growth factors (TGF alpha, TGF beta), EGF, bombesin, fibroblast growth factor (FGF), platelet derived growth factor (PDGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), prostaglandins E-1 and E-2, insulin, transferrin and lactate) which stimulated A431 cell proliferation. SCSGF was mitogenic for A431 cells even in the presence of serum in the culture medium. The partially purified SCSGF was heat- and acid-stable, protease-sensitive with a molecular weight of 14,000. It did not bind to heparin or concanavalin A-Sepharose. The secretion of a mitogenic activity by the Sertoli cell which is different from other previously identified growth factors and which coincides with active spermatogenesis could have important implications in the regulation of spermatogenesis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cells, Cultured; Chromatography, Affinity; Chromatography, High Pressure Liquid; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Kinetics; Male; Rats; Rats, Inbred Strains; Sertoli Cells; Transforming Growth Factor beta

The intracellular sorting of EGF-receptor complexes (EGF-RC) has been studied in human epidermoid carcinoma A431 cells. Recycling of EGF was found to occur rapidly after internalization at 37 degrees C. The initial rate of EGF recycling was reduced at 18 degrees C. A significant pool of internalized EGF was incapable of recycling at 18 degrees C but began to recycle when cells were warmed to 37 degrees C. The relative rate of EGF outflow at 37 degrees C from cells exposed to an 18 degrees C temperature block was slower (t1/2 approximately 20 min) than the rate from cells not exposed to a temperature block (t1/2 approximately 5-7 min). These data suggest that there might be both short- and long-time cycles of EGF recycling in A431 cells. Examination of the intracellular EGF-RC dissociation and dynamics of short- and long-time recycling indicated that EGF recycled as EGF-RC. Moreover, EGF receptors that were covalently labeled with a photoactivatable derivative of 125I-EGF recycled via the long-time pathway at a rate similar to that of 125I-EGF. Since EGF-RC degradation was also blocked at 18 degrees C, we propose that sorting to the lysosomal and long-time recycling pathway may occur after a highly temperature-sensitive step, presumably in the late endosomes.

Topics: Carcinoma, Squamous Cell; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Temperature; Tumor Cells, Cultured

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.

Topics: Carcinoma, Squamous Cell; Cytoskeleton; Detergents; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Molecular Weight; Phosphorylation; Serine Endopeptidases; Tumor Cells, Cultured

The effect of epidermal growth factor on radiosensitivity of cells in spheroids and its relationship to radioresistance associated with cell-cell interactions was examined. A human squamous carcinoma cell lines, CaSki, was used. Epidermal growth factor present for 48 hr before irradiation reduced the plating efficiency but did not affect the radiosensitivity of cells. However, epidermal growth factor present after irradiation, that is, during the period of colony formation, reduced the plating efficiency and increased the radiosensitivity of cells from spheroids. Both effects were maximum at 10 ng/ml epidermal growth factor. The enhancement in radiation response was not related to epidermal growth factor effects on potentially lethal and sublethal damage repair. In the absence of cell-cell interactions, such as monolayer cultures and spheroids disaggregated for 15 hr before irradiation, radiosensitivity enhancement by epidermal growth factor was associated with reduced shoulder of the cell survival curve. However, in the presence of cell-cell interactions, such as intact spheroids and spheroids disaggregated immediately before irradiation, in addition to reduced shoulder, epidermal growth factor treatment increased the slope compared to that of the monolayer cultures. The results indicate that epidermal growth factor enhances cellular radiosensitivity and modifies effects of cell-cell interactions.

Topics: Carcinoma, Squamous Cell; Cell Communication; Clone Cells; Epidermal Growth Factor; Female; Humans; Radiation Tolerance; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Epidermal growth factor (EGF) induces rapid rounding of A431 human epidermoid carcinoma cells. This process is dependent upon temperature and EGF concentration. To investigate the possible influence of gravity variations on EGF-induced cell rounding of A431 cells, experiments were performed using a fast-rotating clinostat and centrifuge, thereby simulating microgravity and higher gravity values, respectively. We demonstrated that simulated microgravity conditions enhance EGF-induced cell rounding significantly, whereas hypergravity values do not show significant effects on this process. These results suggest that simulated microgravity modulates growth factor-induced signal transduction.

Topics: Adaptation, Physiological; Carcinoma, Squamous Cell; Cell Membrane; Cells, Cultured; Epidermal Growth Factor; Gravitation; Humans; Models, Biological; Temperature

Unregulated or increased expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation; modulation of such a receptor by physiological agents could be, therefore, of clinical interest. We have studied the binding ability, the availability at cell surface, and the synthesis of EGF-R in the A431 and KB human epidermoid cancer cell lines after treatment with recombinant alpha-interferon (IFN-alpha). After 48 h of treatment, IFN-alpha induces, in both cell lines, growth inhibition and enhances class I major histocompatibility HLA complex expression, which is a common marker of IFN action. [125I]EGF total binding assessed after 48 h of treatment with IFN-alpha shows a dose-dependent upregulation of EGF-R binding capacity. Saturation plots of the binding data show that IFN-alpha treatment does not dramatically alter the affinity of the EGF-R and indicate that IFN-alpha only increases the number of low affinity receptors. We show that this effect is due to a specific increase in the synthesis of the receptor protein, as assessed by immunoprecipitation of [35S]methionine-labeled cell extracts. Electron microscopy analysis has confirmed an increase of EGF-R proteins at cell surface without major changes in the morphology of the cells. Taken together, these results indicate that IFN-alpha consistently induces both the binding capacity and the synthesis of EGF-R in human epidermoid cancer cells and suggest the use of such a mechanism for new anticancer therapies.

Topics: Binding Sites; Carcinoma, Squamous Cell; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, MHC Class I; HLA Antigens; Humans; In Vitro Techniques; Interferon Type I; Microscopy, Immunoelectron; Tumor Cells, Cultured; Up-Regulation

Increased levels of epidermal growth factor receptor (EGFR) have been shown on squamous cell carcinomas. Recently, we described a squamous cell carcinoma (MH-85) derived from the oral cavity which was associated with several paraneoplastic syndromes including hypercalcemia and cachexia. This tumor induced the same paraneoplastic syndromes in nude mice (BALB/c, nu/nu, male, 4-6 weeks old). Scatchard analysis revealed that there are two classes of EGFR in MH-85. The dissociation constant and number of binding sites for the high affinity receptors were 38 pM and 5 x 10(4)/cell, respectively, and 2.2 nM and 6 x 10(5) cell, respectively, for the low affinity receptors. Growth of MH-85 in culture was stimulated by epidermal growth factor (EGF) and inhibited by monoclonal antibody 108 to human EGFR, which recognizes the extracellular domain of the EGF receptor. Surgical removal of submandibular glands from male nude mice resulted in a dramatic decrease in plasma EGF levels and a significant reduction of tumor growth, hypercalcemia, and cachexia. When EGF (5 micrograms/mouse, every 2 days for 6 weeks, i.p.) was administered to these sialoadenectomized animals, tumor growth increased, with a parallel increase in hypercalcemia. When monoclonal antibody 108 (1 mg/mouse, i.p.) was given 1, 5, and 10 days after MH-85 tumor implantation, tumor formation was retarded, which resulted in delayed onset of hypercalcemia and cachexia. Moreover, when the antibody was injected 6 times in nude mice exhibiting large tumors and profound hypercalcemia and cachexia, there was a striking decrease in tumor growth, which was accompanied with a reversal of hypercalcemia and cachexia. These results indicate that growth of the human squamous cell carcinoma MH-85 is dependent on the EGFR pathway and that subsequent development of hypercalcemia and cachexia is dependent on tumor growth. They also suggest that agents which interfere with the EGFR pathway may have therapeutic potential as anticancer agents in some human tumors.

Topics: Animals; Cachexia; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Hypercalcemia; Maxillary Sinus Neoplasms; Mice; Mice, Nude; Paraneoplastic Syndromes

In several cell types heparin exerts an antiproliferative action; here we report that heparin inhibited the growth of human epidermoid carcinoma A431 cells. Heparin binding to the cell surface was necessary for growth inhibition; binding was influenced by the molecular weight of heparin. Inhibition of A431 cell proliferation was evident in the presence and in the absence of serum, thus indicating that heparin did not act by binding and 'subtracting' nutrients or other serum factors. In confluent A431 cells, EGF induced DNA synthesis, but heparin did not inhibit this effect; consistently, it did not affect inositol lipid turnover triggered by EGF or bradykinin.

Topics: Bradykinin; Carcinoma, Squamous Cell; Cell Division; Cell Line; Culture Media; DNA Replication; Epidermal Growth Factor; Heparin; Humans; Kinetics

The immunohistochemical localizations of human epidermal growth factor (hEGF) and EGF receptor (EGFr) in oral tissues, including normal mucosa, leukoplakia and squamous cell carcinoma were examined by the use of monoclonal antibodies to hEGF and EGFr. In normal mucosa and leukoplakia, immunostaining of hEGF was limited to an underlying layer of connective tissue near the epithelium. The intensity of extracellular staining appeared to increase with the degree of epithelial malignancy and was eventually most striking in the stroma of invasive carcinoma. The epithelial cells in normal mucosa, leukoplakia, and squamous cell carcinoma showed negligible immunoreactivity for hEGF. Expression of EGFr appeared to be associated with the proliferative activity of cells and/or epithelial malignancy. In normal mucosa, anti-EGFr monoclonal antibody reacted only with the basal cell layer. In all sections of leukoplakia, the positive cells for EGFr were found in the prickle cell layer in addition to the basal cell layer. Most tumour cells in squamous cell carcinoma were strongly positive for EGFr. These findings indicate increased expression of hEGF and EGFr with malignancy. The characteristic localization of extracellular hEGF in the underlying connective tissue and in stroma of oral mucosal tumours suggests a possible epithelial-mesenchymal interaction in hEGF secretion.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Leukoplakia, Oral; Mouth Mucosa; Mouth Neoplasms

The epidermal growth factor receptor (EGFR) level in 56 esophageal cancer tissues was measured by 125I-EGF binding assay to elucidate its role in tumor progression. The survival rate of patients with high EGFR level (more than 50 fmol/mg protein) was significantly lower than that of patients with low EGFR level (less than 50 fmol/mg protein, P less than 0.01), although a correlation between EGFR level and the pathologic findings was not observed. The expression of EGF was examined immunohistochemically using anti-EGF monoclonal antibody in 100 esophageal cancer tissues; EGF-positive tumor cells were detected in 92.0%. The immunoreactivity of EGF was classified arbitrarily into four grades according to the number of stained tumor cells. The expression of EGF significantly correlated with the differentiation of esophageal squamous cell carcinoma (P less than 0.01, by chi-square test). The survival rate of patients with high EGF immunoreactivity (Grade 2 or 3) was much lower than in those with lower grade (0 or 1) tumors, (P less than 0.01). Patients with both high EGFR level and EGF immunoreactivity had a much worse prognosis than if both were low. Furthermore, the mitotic index was higher in groups with both high EGFR and EGF than if both were low (16.39 +/- 5.35 versus 6.90 +/- 3.31). These results suggest that EGF and EGFR in the autocrine system may play an important role in tumor progression in esophageal cancer and their expression could be of prognostic significance.

Topics: Antibodies, Monoclonal; Carcinoma; Carcinoma, Squamous Cell; Cell Differentiation; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Follow-Up Studies; Humans; Immunohistochemistry; Lymphatic Metastasis; Melanoma; Mitotic Index; Neoplasm Staging; Survival Rate

Neoplastic canine keratinocytes derived from a spontaneous oral squamous cell carcinoma were maintained in culture for more than 45 passages. The presence of desmosomes and keratin filaments was demonstrated by electron microscopy and immunohistochemistry. The keratinocytes were grown in two different culture conditions to induce variations in the stage of differentiation, i.e., in submerged cultures and at the air-liquid interface. For comparison, normal canine keratinocytes were grown under the same conditions. Anisocytosis was present in neoplastic cultures grown submerged in medium. Grown at the air-liquid interface, neoplastic keratinocytes differentiated into a well-organized, multilayered stratified squamous epithelium analogous to normal keratinocytes. Rare areas of irregular growth and formation of whorls were detected. Expression of lectin binding sites and specific cell surface antigens of neoplastic and normal keratinocytes demonstrated marked similarities between the two cell lines. Neoplastic cells lacked certain surface antigens that are present on normal cells. Squamous cell carcinoma cells grew faster than normal canine keratinocytes as demonstrated by growth curve evaluation. Neoplastic keratinocytes responded to growth stimulation by epidermal growth factor and cholera toxin as do normal keratinocytes. Neoplastic cells grown in medium lacking these factors proliferated faster than growth factor stimulated normal keratinocytes.

Topics: Animals; Carcinoma, Squamous Cell; Cell Count; Cell Differentiation; Cell Division; Cells, Cultured; Cholera Toxin; Culture Media; Dog Diseases; Dogs; Epidermal Growth Factor; Histocytochemistry; Immunohistochemistry; Keratinocytes; Microscopy, Electron; Microscopy, Electron, Scanning; Mouth; Mouth Neoplasms; Tumor Cells, Cultured

Epidermal growth factor (EGF) may either stimulate or inhibit cell growth. To elucidate the mechanism of these varied effects, we compared EGF action in parental A431 cells in which cell growth is inhibited, and clone 15, a mutant of these cells resistant to EGF growth inhibition. In both lines, EGF receptor was present in similar concentrations and underwent tyrosine phosphorylation to the same extent. Likewise, in both lines, acute exposure to EGF stimulated an increase in free cytoplasmic [Ca2+], as well as a similar increase in phosphorylation of lipocortin 1, a major substrate for the EGF receptor kinase whose phosphorylation is calcium-dependent. On the other hand, pretreatment of clone 15 cells with EGF for 72 h abolished EGF-induced phosphorylation of lipocortin 1 and led to a loss of the increase in cytoplasmic free [Ca2+], whereas no such desensitization was seen in the parental A431 cells. These data indicate a link between EGF-induced increase in cytoplasmic calcium, lipocortin phosphorylation, and cell growth and suggest that differences in mechanisms of desensitization to these immediate actions of EGF may lead to altered growth response to this hormone.

Topics: Annexins; Calcium; Calcium-Binding Proteins; Carcinoma, Squamous Cell; Cell Division; Cytoplasm; Drug Tolerance; Epidermal Growth Factor; ErbB Receptors; Humans; Mutation; Phosphorylation; Phosphotyrosine; Tumor Cells, Cultured; Tyrosine

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.

Topics: 1-Methyl-3-isobutylxanthine; Alprostadil; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cholera Toxin; Colforsin; Cyclic AMP; Diglycerides; Epidermal Growth Factor; ErbB Receptors; Glycerides; Humans; Kinetics; Peptide Mapping; Phosphopeptides; Phosphorylation; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.

Topics: 1-Methyl-3-isobutylxanthine; Bucladesine; Carcinoma, Squamous Cell; Cell Line; Cholera Toxin; Colforsin; Cyclic AMP; Drug Synergism; Epidermal Growth Factor; Humans; Isoproterenol; Kinetics; Tumor Cells, Cultured

Functional state of internalized epidermal growth factor (EGF) receptor in A-431 cells has been studied. The use of photoaffinity [125I]EGF derivative allowed us to establish that inside the cell the EGF retains its connection with the receptor. With the help of polyclonal antibodies to phosphotyrosine, it has been shown that EGF-receptor complexes maintain their phosphorylated state during internalization. The internalized EGF receptor kinase as well as that localized in the plasma membrane appeared to be able to phosphorylate synthetic peptide substrate introduced into the cell.

Topics: Biological Transport; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphotyrosine; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tyrosine

Phosphorylated receptors of the epidermal growth factor (EGF) were localized in the human epidermoid carcinoma cells using immunofluorescent staining with antibody to phosphotyrosine. The application of EGF at 4 degrees C was seen to induce a characteristic fluorescence of the cell margins, whereas no cell staining occurs in the absence of EGF. After a 1 hour incubation of cells at 37 degrees C, within which the internalized EGF receptor complexes are accumulated in the juxtanuclear compartment near the para-Golgi region, the staining with antiphosphotyrosine antibody reveals the receptors in this region. It is concluded that the internalized EGF-receptor complexes may remain in the phosphorylated state.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Hydrogen-Ion Concentration; Phosphorylation; Phosphotyrosine; Staining and Labeling; Temperature; Tumor Cells, Cultured; Tyrosine

Primary reactions on the addition of the epidermal growth factor (EGF) were investigated for the strains of A-431 cells, resistant to the antiproliferative effect of EGF. In spite of differences of EGF reception in the obtained strains, the EGF receptors in membrane preparations of these strains maintain the phosphorylating ability after addition of EGF. The rate of internalization of 125I-EGF in normal and resistant cells is the same. The production of a secreted fragment of the EGF receptor in resistant cells is lower than in normal ones. Questions of regulation of production of the normal receptor and of its shortened secreted fragment are discussed.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Ligands; Phosphorylation; Tumor Cells, Cultured

We observed that human epidermal growth factor (hEGF) alone prolonged the survival time of mice bearing various murine syngeneic tumors as well as athymic nude mice bearing human xenografts. No changes in the subcutaneous solid tumor mass volume were observed. Prolongation of survival time by hEGF was observed in mice bearing murine epidermoid carcinoma (BSC) and human gastric carcinoma (KATO III), but not in murine epidermoid carcinoma (KLN205) or human epidermoid carcinoma (A431). Human tumor cells such as A431, KATO III, and murine tumor cells, KLN205, BSC had roughly 2 X 10(6), 3 X 10(4), 1.3 X 10(3) and 1 X 10(3) EGF receptors/cell, respectively. Although KLN205 and BSC tumor cells maintained nearly the same number of EGF receptors, the effects of hEGF were very different. Although A431 tumor cells had nearly 100 times more receptors than KATO III cells, the prolongation of survival time of mice bearing A431 by hEGF was no better than that of mice bearing KATO III. Accordingly, it appears that this prolongation of survival time by hEGF is independent of the number of EGF receptors on tumor cells. In addition, hEGF was shown to inhibit experimental pulmonary metastasis of murine BSC tumor, but was ineffective with murine KLN205 tumor. These results suggest that prolongation of survival time by hEGF may result from the inhibition of tumor cell metastasis and EGF may play a role in preventing the metastasis of certain malignant neoplasms unrelated to its effects through the EGF receptor on tumor cells.

Topics: Adenocarcinoma; Animals; Carcinoma, Squamous Cell; Colonic Neoplasms; Epidermal Growth Factor; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Stomach Neoplasms; Time Factors

The present work was performed to clarify the effects of epidermal growth factor (EGF) on the growth of human breast cancer and carcinoma of the esophagus. Human breast cancer MX-1, UM-1, and esophageal cancer ES-4 were transplanted to the subcutaneous tissue of nude mice. Human EGF (2 micrograms) was injected locally into the subcutaneous tissue surrounding the tumor. The tumor growth was followed for 7 days after treatment, and the estimated tumor weight, tumor doubling time, tumor growth curve, tumor growth rate, and results from histologic examination were used to evaluate the effects of EGF on the growth of tumors. We investigated the dose effect on the growth of these tumors using various concentrations of EGF. We also studied the EGF receptor status of these transplanted tumors and its effect on the influence of EGF treatment. A growth-inhibitory effect was noted in these three tumors with 2 micrograms of EGF. EGF inhibited growth of ES-4 tumor in a dose-dependent manner. Treatment with 2 ng of EGF or saline did not inhibit growth. However treatment with 20 ng or 200 ng of EGF inhibited growth in proportion to the concentrations. All tumors were positive by the EGF receptor binding assay. The efficacy of EGF treatment correlated with EGF receptor levels. EGF receptor levels were also influenced by EGF treatment. These results suggest that human EGF showed an anti-tumor effect on MX-1 and UM-1 breast cancer and also on ES-4 esophageal cancer.

Topics: Animals; Breast Neoplasms; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Female; Humans; Male; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

Previous work identified a protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669. An assay for this protein kinase activity present in homogenates prepared from A431 human epidermoid carcinoma cells was developed using a synthetic peptide substrate corresponding to residues 663-681 of the EGF receptor (peptide T669). Here we report that a greater initial rate of T669 phosphorylation was observed in experiments using homogenates prepared from EGF- or phorbol ester-treated cells compared with control cells. EGF and 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a 6-fold and a 2-fold increase in protein kinase activity, respectively. A kinetic analysis of T669 phosphorylation demonstrated that the increase in protein kinase activity observed was accounted for by an increase in Vmax. To examine the interaction between protein kinase C and signal transduction by the EGF receptor, the effect of pretreatment of cells with PMA on the subsequent response to EGF was investigated. Treatment of cells with PMA caused greater than 90% inhibition of the EGF-stimulated tyrosine phosphorylation of the EGF receptor and abolished the EGF-stimulated formation of soluble inositol phosphates. In contrast, PMA was not observed to inhibit the stimulation of T669 protein kinase activity caused by EGF. Thus, the apparent functional desensitization of the EGF receptor caused by PMA does not inhibit signal transduction mediated by the T669 protein kinase. Our results demonstrate that EGF receptor transmodulation alters the pattern of signal-transduction pathways that are utilized by the EGF receptor.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Inositol Phosphates; Kinetics; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Phosphorylation; Protein-Tyrosine Kinases; Signal Transduction; Substrate Specificity; Tetradecanoylphorbol Acetate

UCVA-1 cells, derived from human pancreas adenocarcinoma, have a high number of epidermal growth factor (EGF) receptors (1.0 x 10(6) per cell) but their growth is not inhibited by EGF, unlike other EGF receptor-hyperproducing tumour cells. In UCVA-1 cells EGF activates neither the phosphatidylinositol turnover nor protein kinase C. EGF, however, enhances the phosphorylation of EGF receptors at specific tyrosine residues, indicating that the EGF receptor kinase is active and subject to autophosphorylation. Downmodulation of EGF receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) is also observed. Using an anti-phosphotyrosine antibody several phosphoproteins, including EGF receptors, were immunoprecipitated from UCVA-1 cell lysates, whereas more than 20 phosphoproteins were detected in other EGF receptor-hyperproducing tumour cells (NA), indicating that tyrosine-phosphorylation of endogenous substrates by EGF receptor kinase is significantly reduced in UCVA-1 cells. Thus, non-responsiveness of UCVA-1 cells to EGF is correlated with the reduced tyrosine phosphorylation.

Topics: Carcinoma, Squamous Cell; Cell Division; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Pancreatic Neoplasms; Peptide Mapping; Phosphatidylinositols; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Tyrosine

The amino terminal portion of urokinase type plasminogen activator (uPA) interacts with a cell surface binding protein/receptor that recognizes a region of the molecule autonomous from that of the catalytic domain of the enzyme, which mediates the conversion of plasminogen to plasmin. The expression of cell surface uPA receptors (uPA-Rs) and their association with uPA have been implicated in cellular invasion and tissue destruction. Treatment of A431 squamous carcinoma cells (SqCC) with epidermal growth factor (EGF) has previously been shown to result in an induction of the synthesis and extracellular accumulation of uPA and the plasminogen-dependent proteolysis of extracellular matrix (ECM). Regulation of cell membrane associated uPA activity by EGF and its influence on uPA-R expression in A431 cells, which possess an unusually large number of EGF-R (greater than 10(6)), and in an EGF-R expression variant (A431/A5), which contains 20-fold fewer cell surface EGF-R, were assessed. Exposure to 5-50 ng/mL of EGF for 24 hr enhanced uPA activity 2- to 3-fold in partially purified membrane preparations derived from A431 cells in a concentration dependent manner. In contrast, no changes in tissue type plasminogen activator (tPA) activity were detected under similar conditions. A431/A5 cell membrane preparations did not exhibit such an EGF mediated response. In accord with EGF enhanced uPA activity, a 2-fold increase in immunoreactive cell surface associated uPA was observed in A431 cells using indirect immunofluorescence staining. The increase in cell surface uPA produced by exposure to EGF required protein synthesis and could be blocked by cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; Fibrinolytic Agents; Humans; Plasminogen Activators; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Urokinase-Type Plasminogen Activator

Transforming Growth Factor beta is a potent autocrine inhibitor of the growth of untransformed keratinocytes. We found each of eight human squamous carcinoma cell lines to be refractory to the anti-proliferative action of Transforming Growth Factor beta. Although each of these carcinoma cell lines expressed the 53-65 kDa (type I) and the 280-300 (type III) Transforming Growth Factor beta-receptor proteins, the 73-85 kDa (type II) species was detectable in only one of these cell lines. Furthermore, although Transforming Growth Factor beta-sensitive non-neoplastic mouse keratinocytes expressed type II binding proteins, human keratinocytes did not. Our findings suggest that resistance to the growth-inhibitory actions of Transforming Growth Factor beta is a common feature of human squamous carcinoma cell lines but does not correlate with the expression of cell-surface receptors for this growth factor.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; DNA; Drug Resistance; Epidermal Growth Factor; Humans; Keratinocytes; Kinetics; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta; Tumor Cells, Cultured

The expression of human epidermal growth factor (hEGF) was examined immunohistochemically in 86 esophageal cancer lesions, comprising 67 primary tumors and 19 metastatic lymph nodes. In the normal esophagus, the parabasal and intermediate cell layers showed a weak expression of hEGF, however, hEGF-positive tumor cells were detected in 62 (92.5 per cent) of the 67 primary esophageal carcinomas and in 18 (94.7 per cent) of the 19 metastatic lymph nodes. In this study, the immunoreactivity of hEGF was classified into 4 grades according to the number of stained tumor cells. A significant correlation was observed between the histologic type and the grade of hEGF immunoreactivity (Chi-square test, p less than 0.01). hEGF immunoreactivity in well differentiated squamous cell carcinomas was significantly higher than in other squamous cell carcinomas, although there were no correlations between other pathological findings and hEGF immunoreactivity. Patients with hEGF immunoreactivities of grades II or III had much worse prognoses than those with grades 0 or I (p less than 0.05). In 22 esophageal carcinomas and 10 normal esophageal mucosae, EGF receptor (EGFR) contents were measured by the competitive binding assay. The average EGFR content (101.3 +/- 35.7 fmol/mg protein, mean +/- SE) of the esophageal carcinomas was significantly higher than that (5.3 +/- 1.2) of the normal esophageal mucosae (p less than 0.05). Moreover, in hEGF negative tumors, EGFR contents were lower than in hEGF positive tumors. These results suggest that hEGF and EGFR show increased production in squamous cell carcinomas and could to be useful prognostic factors in patients with esophageal cancer.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Humans; Immunoenzyme Techniques; Survival Rate

As a first step toward developing a structural map of key sites on the epidermal growth factor (EGF) receptor, we have used resonance energy transfer to measure the distance of closest approach between the receptor-bound growth factor molecule and lipid molecules at the surface of the plasma membrane. EGF, specifically labeled at its amino terminus with fluorescein 5-isothiocyanate, was used as an energy donor in these experiments, while either octadecylrhodamine B or octadecylrhodamine 101, inserted into plasma membranes isolated from human epidermoid carcinoma (A431) cells, served as the energy acceptors. The energy transfer measurements indicate that the amino terminus of the bound growth factor is about 67 A away from the plasma membrane. On the basis of the dimensions of the EGF molecule, this suggests that EGF binds to a site on its receptor that is a considerable distance (52-82 A) from the surface of these cells. Identical results were obtained under conditions where the receptor functions as an active tyrosine kinase, suggesting that the relative juxtaposition of the EGF binding domain to the membrane surface does not change with receptor autophosphorylation or with the activation of the receptor tyrosine kinase activity.

Topics: Binding Sites; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Membrane Proteins; Neoplasm Proteins; Protein Binding; Protein Conformation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

In this paper we demonstrate that isolated cytoskeletons of normal keratinocytes cultured under differentiation inducing conditions exhibit a high level of epidermal growth factor (EGF) binding. This binding is approximately 300% higher than the binding of intact cells. In contrast, various squamous carcinoma cell lines or normal keratinocytes cultured under differentiation retarding conditions exhibit EGF binding to isolated cytoskeletons which is around 10-20% of the binding to intact cells. Incubation of normal keratinocytes in the presence of arotinoid ethyl sulfone resulted in a marked decrease of the ability of the cells to differentiate, and a decrease of EGF binding to isolated cytoskeletons. These results suggest a close relationship between the differentiation capacity of the cells and the presence of cytoskeleton-associated EGF receptors. Similar results were obtained for low density lipoprotein (LDL) binding.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Cytoskeleton; Depression, Chemical; Epidermal Growth Factor; ErbB Receptors; Keratinocytes; Lipoproteins, LDL; Neoplasm Proteins; Receptors, LDL; Retinoids; Tumor Cells, Cultured

Retinoic acid (RA) increases epidermal growth factor (EGF) receptors in many cells; in ME180 cells, a human epidermoid carcinoma, RA resulted in a dose- and time-dependent reduction of EGF binding. In RA-treated ME180 cells, binding was 41% of the control. The reduction of EGF binding was due to a decrease in the number of receptors, from 8.7 x 10(4) to 3.6 x 10(4) per cell. The difference was present 8 h after the addition of RA and was reversible 3 days after its removal. Scatchard analysis indicated that RA did not change the binding affinity of EGF (Kd = 1 nM). Also, RA did not alter the rate of EGF internalization or the down-regulation induced by exogenous EGF. Flow-cytometric analysis revealed that RA did not alter the cell cycle. Soluble cell membrane extracts were prepared in a Tris buffer with protease inhibitors, immunoprecipitated, electrophoresed, and immunoblotted with an antiserum to EGF receptors. The EGF receptor band of Mr 170,000 was decreased in RA-treated cells. These results suggest that RA reduces the synthesis of EGF receptors in ME180 cells.

Topics: Carcinoma, Squamous Cell; Cell Division; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Time Factors; Tretinoin; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Epidermal growth factor (EGF) has been shown to inhibit the multiplication of the human epidermoid carcinoma cell line A-431. In the present report it is shown that, despite growth inhibition, EGF caused a marked synthesis of DNA and nonhistone proteins, without progression into mitosis. This event was associated with a retraction of the monolayer into colonies of cells. This suggests that the cell cycle of A-431 cells is controlled by two surface membrane signals: one generated by EGF stimulating the synthetic events of the G1 and S phases; a second signal, leading to progression into mitosis appears either not to be generated or to be inhibited by EGF.

Topics: Carcinoma, Squamous Cell; Cell Division; DNA; Epidermal Growth Factor; Humans; Interphase; Mitosis; Protein Biosynthesis; Tumor Cells, Cultured

In order to ascertain autocrine growth factors in esophageal carcinomas, we analysed expression of mRNAs and proteins for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in 6 esophageal carcinoma cell lines. Gene alterations were also examined. All of the esophageal carcinoma cell lines expressed mRNA for EGFR and TGF-alpha genes. Interestingly, EGF mRNA of about 5.0 kb was also detected in TE-1, TE-2, and TE-8 cells. Production of protein was also confirmed by binding assay and ELISA on 3 of the 6 cell lines. The cells had a relatively high number of EGFRs and produced TGF-alpha and EGF protein at the same time. Furthermore, anti-EGF (KEM-10) and anti-TGF-alpha (WA-3) monoclonal antibodies (MAbs) inhibited spontaneous uptake of tritiated thymidine (3H-TdR) by TE-1 cells which expressed EGF, TGF-alpha and EGFR mRNA and protein. These results strongly suggest that EGF and/or TGF-alpha produced by carcinoma cells function as autocrine growth factors for human esophageal carcinomas.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Blotting, Northern; Blotting, Southern; Carcinoma, Squamous Cell; Cell Line; DNA Probes; DNA, Neoplasm; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Radioligand Assay; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factors; Tumor Cells, Cultured

Stimulation of A-431 carcinoma cells with epidermal growth factor (EGF) causes dramatic morphologic responses including ruffling, rounding, and bulk-phase pinocytosis. In attempts to explore the mechanisms responsible for changes in plasmalemma topography, we have investigated the effects of exogenous sterols thought to alter membrane fluidity. Light and scanning electron microscopy revealed a time- and concentration-dependent inhibition of ruffling (greater than 90%) by cholesterol. This effect could be duplicated by preincubation of the cells with comparable levels of low-density lipoproteins (LDL). EGF-stimulated bulk-phase endocytosis also is inhibited by treatment with cholesterol. No alteration of EGF binding, kinase stimulation, or internalization was detected in cells incubated in cholesterol-enriched medium (175 micrograms/ml in 0.5% ethanol), nor did cholesterol or LDL have any effect on EGF-stimulated rounding. Morphometry of electron micrographs from cholesterol-treated cells revealed a selective depletion of interdigitating lateral surface membrane that normally appears to be recruited to generate apical ruffles. Thus, the sterol inhibition of ruffling may be due to redistribution of plasmalemma rather than to changes in membrane viscosity. Together with previous observations, these data suggest that EGF-stimulated ruffling and bulk-phase pinocytosis are related phenomena, whereas EGF-stimulated cell rounding is an independent process.

Topics: Carcinoma, Squamous Cell; Cell Membrane; Cholesterol; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Lipoproteins, HDL; Lipoproteins, LDL; Microscopy, Electron; Microscopy, Electron, Scanning; Phosphorylation; Structure-Activity Relationship; Tumor Cells, Cultured

We examined the effects of epidermal growth factor (EGF) on the anchorage-dependent and -independent growth of four human squamous carcinoma cell lines that overexpress EGF receptors. While EGF inhibited anchorage-dependent growth, it stimulated anchorage-independent growth of all four cell lines tested. The results suggest that the proliferative responses to EGF are characterized by a preference for anchorage-independent, rather than -dependent growth, in cells overexpressing EGF receptors. Moreover, as EGF has been shown to stimulate the in vivo growth of squamous carcinoma cells overexpressing EGF receptors, it is also suggested that the in vitro EGF responsiveness of these cells in soft agar, but not in monolayer, better correlates with the in vivo EGF responsiveness.

Topics: Carcinoma, Squamous Cell; Cell Adhesion; Cell Count; Cell Division; DNA; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasms; Tumor Cells, Cultured

Epidermal growth factor (EGF) treatment of A-431 epidermoid carcinoma cells elicited a redistribution of phospholipase C-gamma 1 (PLC-gamma 1) from a predominantly cytosolic localization to membrane fractions. The temporal coincidence of this redistribution with EGF stimulation of inositol phosphate formation and EGF increased phosphorylation of PLC-gamma 1 suggests that the membrane association of PLC-gamma 1 is a significant event in second messenger transduction.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cytosol; Epidermal Growth Factor; Humans; Isoenzymes; Kinetics; Phosphopeptides; Protein Binding; Trypsin; Type C Phospholipases

The potential significance of cell-cell interactions on EGF receptor (EGFR) activity was investigated in cultured adherent A431 cells seeded as single-cell suspensions with different initial cell densities. In dense cultures, EGFRs were mainly localised at cell boundaries and in microvilli as shown by immunofluorescence analysis with an EGFR-specific antibody while in sparse cultures the distribution of EGFRs was more diffuse. Scatchard analysis showed that as cell density decreased the number of high-affinity receptors increased considerably. Upon treatment of adherent intact cells with EGF all cells in sparse cultures contained activated EGFRs as demonstrated by immunofluorescence analysis with a phosphotyrosine-specific antibody, while in dense cultures mainly cells at the periphery of a cluster and especially at their expanding borders exhibited activated EGFRs. EGF-induced phosphorylation in intact cells was greatly enhanced in sparse compared with dense cultures as demonstrated by immunoprecipitation with a phosphotyrosine-specific antibody. In contrast to intact cells, in cytoskeleton preparations, obtained after mild detergent treatment of adherent cells, EGFRs were able to undergo EGF-independent phosphorylation. Pretreatment of cells with EGF led to enhanced tyrosine phosphorylation of cytoskeletal-associated proteins. Our observations suggest that cell density has a considerable effect on the subcellular localisation as well as biological activity of the EGFR. Thus, in intact A431 cells growing with extensive cell-cell interactions some negative control mechanisms preventing EGFR activation may be exerted by adjacent cells.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Communication; Cell Line; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Kinetics; Phosphorylation; Tumor Cells, Cultured

The biochemical properties of lung cancer cell lines were investigated. Bombesin-like peptides were present in three small cell lung cancer (SCLC) cell lines examined and three of four lung carcinoids but not in five non-small cell lung cancer (NSCLC) cell lines. Therefore SCLC and some lung carcinoids, but not NSCLC, are enriched in neuroendocrine properties. In contrast, 125I-EGF bound with high affinity to all five NSCLC cell lines and three of four lung carcinoids but not to the three SCLC cell lines examined. For lung carcinoid cell line NCI-H727, 125I-EGF bound with high affinity (Kd = 6 nM) to a single class of sites (Bmax = 110,000/cell). The 125I-EGF bound was rapidly internalized at 37 degrees C but not 4 degrees C. Using Western blot techniques and antiphosphotyrosine antibodies, EGF induced phosphorylation of a major 170 Kd protein. Using immunoprecipitation techniques and anti-EGF receptor antibodies a major 170 Kd protein was labeled. These data indicate that biologically active EGF receptors are present on NSCLC and lung carcinoid cell lines.

Topics: Adenocarcinoma; Bombesin; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms; Peptides; Phosphorylation; Tumor Cells, Cultured

An expression vector was constructed from part of pSV2neo with the 3'-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction in cell-surface EGF receptor in response to ZnSO4. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma, Squamous Cell; Cloning, Molecular; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Genetic Vectors; Humans; Metallothionein; RNA; RNA, Antisense; RNA, Neoplasm; Tumor Cells, Cultured; Zinc

Transforming growth factors have a wide range of biological activities related to cell proliferation and differentiation. In general TGF-alpha promotes cell proliferation while TGF-beta may stimulate or inhibit proliferation depending on the cell type and growth factor environment. Cultured human keratinocytes, skin and oral squamous cell carcinomas were analysed for the presence of transcripts and protein for the transforming growth factors alpha & beta. Both growth factors were detected in cultured keratinocytes (which have receptors for and respond to both ligands), and in medium conditioned by these cells. Additionally transcripts for TGF-alpha were found preferentially in the basal, proliferative compartment of cultured keratinocytes. Similarly both growth factors were detected in oral squamous cell carcinomas and a highly significant inverse correlation was found between the levels of TGF-alpha and the epidermal growth factor receptor in these tumours. The data for TGF-alpha are consistent with the existence of an autocrine growth control loop influencing cell proliferation in both a normal cell type and malignant epithelial tissues, a process that in keratinocytes and responsive squamous cell carcinomas could be modulated by TGF-beta.

Topics: Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epithelial Cells; Humans; Keratins; Mouth Neoplasms; Protein Precursors; Skin; Transforming Growth Factors; Tumor Cells, Cultured

Effects of epidermal growth factor (EGF) on growth and tumor antigen-4 (TA-4) production of newly established uterine cervical cancer cell line (OMC-1) are reported. OMC -1 was established from a metastatic lesion of Virchow's lymph node of a large cell non-keratinizing squamous cell carcinoma of the uterine cervix and successively subcultured for about 4 years. Scatchard analysis of EGF binding to OMC-1 cells indicated a single class of binding sites with a dissociation constant (Kd) of 360 pM. The theoretical maximum number of binding sites was 2.4 x 10(4) sites/cell. The growth of OMC-1 cells was stimulated by EGF at 0.01-0.1 nM and inhibited at higher concentrations. The TA-4 production of OMC-1 cells was slightly stimulated by EGF at 0.01-1 nM and significantly stimulated at 10 nM. OMC-1 may serve as one of the available model systems for studies of regulation of proliferation and tumor marker production by EGF, particularly in cervical squamous cell carcinomas.

Topics: Antigens, Neoplasm; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Tumor Cells, Cultured; Uterine Cervical Neoplasms

The role of epidermal growth factor in the regulation of the proteolytic and RBC cytolytic activity of the A431 cancer cell line has been evaluated using our previously described gelatin/polyacrylamide electrophoretic assay and tumor-induced RBC cytolysis assay, respectively. A431 cells maintained in 10% fetal bovine serum were actively cytolytic for RBC (release index, 53.0 +/- 2.9%), whereas serum-starved cells maintained in serum-free medium were not cytolytic for RBC. RBC cytotoxicity was restored by adding as little as 3.4 pM epidermal growth factor to the serum-deprived cells. The RBC cytolytic stimulating activity of epidermal growth factor could be mimicked by the metal chelating agent 1,10-phenanthroline, suggesting a possible role for calcium ions in the action of epidermal growth factor and proteases. An enriched cell membrane preparation of A431 cells was also cytolytic for RBC but was unaffected by metal chelating agents. RBC-induced cytotoxicity was inhibited by the protease inhibitor leupeptin. Gelatin substrate gels of enriched A431 cell membrane preparations and serum-free supernatants revealed a pattern of high- and low-molecular-weight proteases that were stimulated by metal chelators and inhibited by leupeptin. The activity of these proteases appears to be regulated by epidermal growth factor by a process that may involve divalent cations.

Topics: Carcinoma, Squamous Cell; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Erythrocytes; Humans; Molecular Weight; Peptide Hydrolases; Protein Biosynthesis; Tosyllysine Chloromethyl Ketone; Tumor Cells, Cultured

The interaction of epidermal growth factor (EGF) with specific cell surface receptors initiates biochemical events in target cells which result in cellular proliferation and differentiation. In this report the regulation of extracellular-associated plasminogen activator (PA) production by EGF in human squamous cell carcinomas and its influence on tumor cell-mediated degradation of extracellular matrix (ECM) is described. The studies utilized the vulvar carcinoma cell line A431, which possesses an unusually large number of EGF receptors (EGF-Rs), and two A431 EGF-R expression variants (A5 and A7), which contain up to 20-fold fewer cell surface EGF-Rs. EGF enhanced the production of urokinase (u) PA activity by two- to threefold in A431 tumor cells, in a concentration-dependent manner, following a 24-h treatment, as determined by substrate hydrolysis assays, while no changes in tissue-type PA occurred. In contrast, A5 and A7 tumor cells failed to demonstrate such a response. Time course studies of the EGF-mediated induction of uPA activity in A431 tumor cells indicated that within 8 h after exposure to EGF, a twofold increase above basal untreated control levels was observed using the substrate hydrolysis assay. EGF increased the steady state levels of uPA mRNA threefold in A431 tumor cells following a 24-h treatment, while in contrast, no such response was observed in EGF-R variant tumor cells. In accord with an EGF enhancement of uPA mRNA levels in A431 tumor cells, a similar increase of two- to threefold in the de novo synthesis of [35S]methionine-radiolabeled uPA was observed by immunoprecipitation following EGF treatment, while no measurable increase was observed in the EGF-R tumor variants. A431 tumor cells progressively degraded [3H]glucosamine-radiolabeled bovine corneal subendothelial ECM in the presence of EGF, resulting in 8.7-, 4.3-, and 1.7-fold increases above untreated control values, after a 48-h exposure to 100, 10, and 1 ng/ml of EGF, respectively. In contrast, A5 and A7 tumor cells did not demonstrate an increase in ECM degradation in the presence of EGF, even though these tumor cells possessed the ability to degrade ECM in the absence of the growth factor. The observed increase in ECM degradation mediated by EGF in A431 tumor cells was dependent upon the presence of plasminogen and could be inhibited by an anticatalytic uPA monoclonal antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: Carcinoma, Squamous Cell; Cell Line; Enzyme Induction; Enzyme Precursors; Epidermal Growth Factor; Extracellular Matrix; Genetic Variation; Humans; Immunoblotting; Kinetics; Nucleic Acid Hybridization; Plasminogen Activators; RNA, Messenger; Transcription, Genetic; Urokinase-Type Plasminogen Activator

A variant clone resistant to high doses of colchicine (KB-C1) derived from human cancer KB cell line is resistant to various anticancer agents. The KB-C1 cells were much more resistant to epidermal growth factor and a chimeric toxin, EGF-Pseudomonas exotoxin (PE), than the parental KB cells. KB-C1 cells have decreased numbers of EGF-receptors, though the affinity of the receptors is similar to that in the parental KB cells. A drug-sensitive revertant (C1-R2) partially recovered its EGF-receptor activity. Northern blot analysis showed a decreased level of EGF-receptor mRNA in KB-C1 cells, while the multidrug-resistance gene, mdr-1, was expressed at very high levels in KB-C1 cells, but not in KB or C1-R2 cells. The drug-resistant cells were less tumorigenic than the parental cells when injected into nude mice. A decreased expression of EGF-receptor in these cells may be one of the pleiotropic properties of multidrug-resistant cells and may perhaps represent the basis for their reduced tumorigenicity.

Topics: Animals; Blotting, Northern; Carcinoma, Squamous Cell; Cell Survival; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; KB Cells; Kinetics; Mice; Neoplasm Transplantation

Pseudomonas exotoxin is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The substitution of the cell recognition domain (domain Ia) with a growth factor such as transforming growth factor alpha (TGF alpha), creates a cell-specific cytotoxic agent, TGF alpha-PE40, which kills cells bearing epidermal growth factor (EGF) receptors. We have used TGF alpha-PE40 to define the role of sequences in domains II, Ib, and III. Various mutations were made in these domains and mutant forms of TGF alpha-PE40 expressed in Escherichia coli. Mutant proteins were then tested for their ADP-ribosylation, EGF receptor-binding, and cell-killing activities. Additionally, the amino boundary of domain III, which contains the ADP-ribosylation activity, was determined by deletion analysis. Data indicate that (i) the functional amino terminus of domain III is near amino acid 400; (ii) deletion of various regions in domain II or conversion of cysteines 265 and 268 to serines results in a loss of cytotoxicity which ranged from 10-fold to more than 150-fold, indicating that domain II is essential for full expression of cytotoxicity; (iii) deletion of the amino terminus of domain Ib results in a molecule with somewhat increased cytotoxic activity, indicating that domain Ib is not essential for the cytotoxic effect of TGF alpha-PE40; and (iv) TGF alpha-PE40, produced by denaturing and refolding of insoluble material from inclusion bodies, binds better to EGF receptors and is about 10-fold more cytotoxic to cells bearing EGF receptors than is the secreted form of soluble TGF alpha-PE40.

Topics: Adenosine Diphosphate Ribose; Amino Acid Sequence; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Exotoxins; Genes, Bacterial; Humans; Iodine Radioisotopes; Mutation; Protein Synthesis Inhibitors; Pseudomonas aeruginosa; Recombinant Fusion Proteins; Tumor Cells, Cultured

Morphometric assessment including epithelial indices which express morphological features of the epithelium, mitotic index, mean nuclear area, mean form factor of the nucleus and cellular infiltration in the stroma was performed in 14 cases with oral non-dysplastic epithelium and 66 cases with dysplastic epithelium. The results from morphometry showed a close relationship to the histological severity of dysplasia determined by the histological criteria of Bánóczy. Immunohistochemical localization of carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA) and epidermal growth factor (EGF) were investigated in 14 cases of non-dysplastic epithelium, 66 cases of dysplastic epithelium and 16 cases of squamous cell carcinoma, and compared with the morphometric results. Positive rates of CEA and EMA reaction in epithelial dysplasia increased with the advance of the dysplastic grade. Those of EGF reaction decreased with the advance of the dysplastic grade. It is suggested that morphometry and immunohistochemistry are useful in confirmation of the histological severity of oral epithelial dysplasia.

Topics: Antigens, Neoplasm; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Chi-Square Distribution; Epidermal Growth Factor; Humans; Immunohistochemistry; Leukoplakia, Oral; Membrane Glycoproteins; Mitotic Index; Mouth Neoplasms; Mucin-1

Lung cancer tissues from 68 patients were examined for epidermal growth factor (EGF) receptor levels and EGF receptor gene copy numbers. Histologic cell types of these lung cancer tissues included squamous-cell carcinoma (n = 30), adenocarcinoma (n = 28), large-cell carcinoma (n = 4), and small-cell carcinoma (n = 6). Tissues of squamous-cell carcinoma exhibited exceptionally high 125I-EGF binding activity, and those of small-cell carcinoma showed no EGF binding activity. Southern blot hybridization analysis revealed EGF receptor gene amplification in the squamous-cell carcinomas with high EGF binding activity. The EGF receptor levels in squamous-cell carcinomas and adenocarcinomas were compared with their pathological staging grouping and pathological findings, including degree of differentiation, diameter of tumor, and lymph node metastasis. However, unlike previous reports on breast and bladder cancers, there was no obvious correlation between these pathological characteristics and the EGF receptor levels of lung cancer.

Topics: Adenocarcinoma; Blotting, Southern; Carcinoma; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Lung Neoplasms

In a previously published report (Exp. Cell. Res. 161:421 (1985] we have demonstrated that cultured normal and transformed keratinocytes exhibit two classes of EGF binding sites after growth under normal Ca2+ conditions but only low-affinity binding sites after growth under low Ca2+ conditions. Here we demonstrate the presence of high-affinity binding sites in transformed keratinocytes grown under low Ca2+ conditions, using a specific monoclonal anti EGF-receptor antibody.

Topics: Antibodies, Monoclonal; Calcium; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Viral; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Humans; Keratinocytes; Simian virus 40; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A novel class of low molecular weight protein tyrosine kinase inhibitors is described. These compounds constitute a systematic series of molecules with a progressive increase in affinity toward the substrate site of the EGF receptor kinase domain. These competitive inhibitors also effectively block the EGF-dependent autophosphorylation of the receptor. The potent EGF receptor kinase blockers examined were found to competitively inhibit the homologous insulin receptor kinase at 10(2)-10(3) higher inhibitor concentrations in spite of the significant homology between these protein tyrosine kinases. These results demonstrate the ability to synthesize selective tyrosine kinase inhibitors. The most potent EGF receptor kinase inhibitors also inhibit the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on EGF independent cell growth. These results demonstrate the potential use of protein tyrosine kinase inhibitors as selective antiproliferative agents for proliferative diseases caused by the hyperactivity of protein tyrosine kinases. We have suggested the name "tyrphostins" for this class of antiproliferative compounds which act as protein tyrosine kinase blockers.

Topics: Benzylidene Compounds; Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Structure; Nitriles; Phosphorylation; Protein-Tyrosine Kinases; Structure-Activity Relationship

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Lung Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms; Uterine Cervical Neoplasms

The cell-attached version of patch-clamp technique was used to search for calcium permeable channels in human carcinoma A-431 cells. With 100 mM CaCl2 in pipette, the inward currents were recorded having the mean unitary conductance of 2.8 pS and the reversal potential (obtained by linear extrapolation) equal to +25.5 mV. Application of the epidermal growth factor (EGF) into the bath extracellular solution produced a transient increase in probability for these channels to be open. The effect developed with a delay of about 20 seconds to last thereafter for 36 seconds (mean values). We propose that these channels mediate EGF-induced increase in concentration of cytosolic free calcium.

Topics: Calcium Channels; Calcium Chloride; Carcinoma, Squamous Cell; Cell Line; Cytological Techniques; Epidermal Growth Factor; Humans; Membrane Potentials; Tumor Cells, Cultured

The KU-8 cell line was established from a lymph node metastasis of human squamous cell carcinoma (SCC) of the penis. The cells grew rapidly (doubling time 20 hours) as adherent monolayers, and were tumorigenic in nude mice. The carcinoma cells showed epithelial characteristics by observation with a phase contrast microscope. The cells retained the ultrastructural characteristics of squamous epithelium including tonofilaments and desmosomes. In addition to the morphological characteristics of SCC, this cell line preserved specific molecular markers of epithelium, such as desmoplakin, cytokeratin, and involucrin, all of which were demonstrated by immunofluorescent studies. Furthermore, SCC-related antigen (SCC-RA), a tumor marker for SCC, was produced in KU-8. Moreover, immunofluorescent study showed that KU-8 cell line expressed the specific receptor of epidermal growth factor (EGF), which turned out to increase the cell growth of KU-8. These results indicate that this new cell line could provide an excellent model for the basic research and development of new therapeutic modalities of penile carcinoma.

Topics: Animals; Biomarkers; Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; Humans; Male; Mice; Mice, Nude; Neoplasm Transplantation; Penile Neoplasms; Tumor Cells, Cultured

We have recently reported that extracellular ATP induces a transient rise in cytosolic free Ca2+ [( Ca2+]i) in individual human epidermoid carcinoma A431 cells (Gonzalez et al: Journal of Cellular Physiology 135:269-276, 1988). We have now studied nucleotide specificity and desensitization for several early responses. Extracellular ATP (5-100 microM) caused the rapid formation of inositol trisphosphate and later its metabolites, inositol bisphosphate and inositol monophosphate. ATP also induced the efflux of 45Ca2+ from pre-loaded cells. In addition, an increase in the rate of influx of 45Ca2+ stimulated by extracellular ATP was detected. Based on measurements of 45Ca2+ efflux and influx, desensitization studies, and chlortetracycline fluorimetry, we conclude that ATP mobilizes Ca2+ from internal stores and also stimulates entry across the plasma membrane. These effects were also displayed by UTP and to a lesser extent by ITP, while other nucleoside triphosphates as well as ADP, AMP, and adenosine, were inactive. Furthermore, desensitization of the response to ATP and UTP was seen after prolonged exposure to either nucleotide. This was specific for the nucleotide receptor since a response to bradykinin was not affected by the ATP pretreatment, although pretreatment with phorbol ester inhibited responses to both the nucleotides and bradykinin. Quantitative data on rate of recovery from the desensitized state and the response of desensitized cells to greatly elevated levels of ATP are presented. Extracellular ATP stimulated another early change previously reported for epidermal growth factor, namely, the phosphorylation of an 81-kDa cytoskeletal protein. The stimulation of these events involves an ATP receptor whose properties differ from other ATP receptors that have been described.

Topics: Adenosine Triphosphate; Calcium; Carcinoma, Squamous Cell; Chlortetracycline; Cytoskeletal Proteins; Down-Regulation; Epidermal Growth Factor; Fluorescence; Humans; Inositol Phosphates; Nucleotides; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Purinergic; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uridine Triphosphate

Epidermal growth factor (EGF) receptor capping results from the interaction between the receptors and polyvalent ligands in A-431 cells examined in suspension at 22 degrees C. Colocalization of actin and spectrin with the ligand-receptor complexes during the redistribution was shown using double immunofluorescence. The obtained data show that the cortical microfilaments are involved in capping. EGF receptors become associated with the Triton-insoluble cytoskeleton as a consequence of ligand binding. EGF-receptor capping is not sensitive to the action of cytochalasin B. Capping in A-431 cells is discussed as a new model for studying the redistribution of the ligand-receptor complex.

Topics: Actins; Antibodies; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line; Cytochalasin B; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Sera; Ligands; Receptor Aggregation; Spectrin; Tumor Cells, Cultured

Two cell lines established from tumors of the head and neck area at different clinical stages were found to differ in the expression and in the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Cell line 183A was derived from an early-stage tumor and cell line 1483 was derived from a tumor that had metastasized to lymph nodes. The 1483 cells displayed a higher plating efficiency and clonogenicity in soft agar, suggesting a more tumorigenic phenotype over the 183A cells. Analyses of EGF receptor levels by using R1 anti-EGF receptor serum indicated that the 1483 cells expressed 5-fold more receptor than did the 183A cells. EGF receptors isolated from each cell line were active for kinase activity in an immune complex kinase assay, using monoclonal R1 anti-EGF receptor antibody. The autophosphorylation activity of both receptors was stimulated by addition of EGF to isolated membrane preparations and intact cells, although the EGF receptor of the 1483 cells was much less responsive to EGF than the receptor from 183A cells. In addition, the 1483 receptor consistently incorporated about twice as much phosphate as did the 183A receptor in an immune complex kinase assay. These data suggest that the basal tyrosine kinase activity of the EGF receptor from 1483 cells may be more active than the EGF receptor kinase from 183 cells.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Head and Neck Neoplasms; Humans; Kinetics; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Treatment of A431 human epidermoid carcinoma cells with 4-phorbol 12-myristate 13-acetate (PMA) causes an inhibition of the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an inhibition of the EGF receptor tyrosine protein kinase activity. The hypothesis that PMA controls EGF receptor function by regulating the oligomeric state of the receptor was tested. Dimeric EGF receptors bound to 125I-EGF were identified by covalent cross-linking analysis using disuccinimidyl suberimidate. Treatment of cells with PMA in the presence of 20 nM 125I-EGF caused no significant change in the level of labeled cross-linked monomeric and dimeric receptor species. Investigation of the in vitro autophosphorylation of receptor monomers and dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide demonstrated that the treatment of cells with PMA caused an inhibition of the tyrosine phosphorylation of both monomeric and dimeric EGF receptors. We conclude that the inhibition of the EGF receptor tyrosine protein kinase activity caused by PMA is not associated with the regulation of the oligomeric state of the EGF receptor.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Macromolecular Substances; Molecular Weight; Phosphorylation; Protein Kinase C; Protein-Tyrosine Kinases; Tetradecanoylphorbol Acetate

The clinical implications of the epidermal growth factor (EGF) and its receptor (EGF-R) were studied in 52 squamous cell carcinomas of the uterine cervix. In comparison to 40 biopsies of the normal cervix EGF-R capacity was significantly increased in the carcinomas, while the affinity was unchanged. The amount of EGF-like substances extracted from the tumors was increased in patients with lymph node metastases, in whom 5-year survival is reduced. Irrespective of tumor stage patients with a very high level of EGF-R (greater than 100 fmole/mg protein) were more likely to have recurrences later or to die from disease: recurrence or death occurred in 5 of 7 patients with high capacity and in 2 of 45 patients with low capacity. Our data suggest that the level of EGF-R is indicative of the biological aggressiveness of cervical carcinomas.

Topics: Adult; Carcinoma, Squamous Cell; Cervix Uteri; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lymphatic Metastasis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Uterine Cervical Neoplasms

Experiments were done to determine the effect of murine epidermal growth factor (EGF) on the radiation responses of the human squamous carcinoma cell line CaSki grown as an exponential monolayer culture. The radiation responses studied included recovery from potentially lethal damage (PLDR) and recovery from sublethal damage (SLDR). The presence of EGF in the cell culture either after irradiation (during the clonogenic assay period) or continuously before, during, and after irradiation enhanced the radiosensitivity of the cells and reduced their plating efficiency (PE). However, these effects were not as great when EGF was present in the cell culture only before and during irradiation. This enhancement of radiosensitivity was associated with a reduction in the shoulder region of the cell survival curves. The PE reduction and radiosensitivity enhancement were maximum with 10 ng of EGF/mL. However, EGF present continuously in the cell culture, including during the 6-hour repair period, had no effect on cellular PLDR or SLDR. The single-stranded DNA breaks present in large numbers in cells immediately after irradiation returned to control levels by 6 hours. Moreover, EGF present in cell cultures for the 48 hours before irradiation and during the 6-hour repair period had no effect on the DNA alkaline elution profiles of either control or irradiated cells. EGF did not affect the growth of unirradiated cells; however, it extended the lag-phase period for growth of irradiated cells. In summary, this EGF-induced radiosensitivity enhancement was not correlated with the effects of EGF on PE, cell growth, PLDR, or SLDR.

Topics: Carcinoma, Squamous Cell; Cell Division; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Humans; In Vitro Techniques; Radiation-Sensitizing Agents; Time Factors; Tumor Cells, Cultured

The binding of epidermal growth factor (EGF) by A-431 human epidermoid carcinoma cells was reduced after exposure of the cells to low concentrations (0.01-1 mM) of ATP and other nucleoside 5'-triphosphates at 37 degrees C, but not at 0 degree C. This was due to loss of high-affinity EGF binding sites. The modulation was associated with transient increases in inositol phosphate synthesis and intracellular Ca2+ and with phosphorylation of the EGF receptor on serine and threonine. There was no evidence for entry of labeled ATP into the cells. ATP appeared to bind to specific cell surface receptors. Such binding was demonstrated directly with the nonmetabolizable ATP analogue adenosine 5'-[beta,gamma-imido]triphosphate.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Phosphates; Phosphorylation; Ribonucleotides

Epidermal growth factor inhibits proliferation of A431 cells when added to the cultural medium. Strains of A431 cells, resistant to EGF (800 ng/ml), were obtained by one-step selection after the treatment of these cells by 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). Two of the obtained strains differ from the initial line in the EGF reception.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Depression, Chemical; Dose-Response Relationship, Drug; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Methylnitronitrosoguanidine; Tumor Cells, Cultured

Topics: Animals; Antineoplastic Agents; Carcinoma, Squamous Cell; Epidermal Growth Factor; Esophageal Neoplasms; Humans; Male; Mice; Mice, Inbred ICR; Mice, Nude; Middle Aged; Neoplasm Transplantation

Extracellular ATP in concentrations of 5-50 microM displayed very little mitogenic activity by itself but it caused synergistic stimulation of [3H]thymidine incorporation in the presence of phorbol 12-tetradecanoate 13-acetate, epidermal growth factor, platelet-derived growth factor, insulin, adenosine, or 5'-(N-ethyl)carboxamidoadenosine. Cultures of Swiss 3T3, Swiss 3T6, A431, DDT1-MF2, and HFF cells were used. The percent of cell nuclei labeled with [3H]thymidine and cell number were also increased. ADP was equally mitogenic, while UTP and ITP were much less active. The effect of ATP was not due to hydrolysis by ectoenzymes to form adenosine, a known growth factor. Thus, the nonhydrolyzable analogue adenosine 5'-[beta, gamma-imido]triphosphate was mitogenic. In addition, it was found that ATP showed synergism in 3T6 and 3T3 cells when present for only the first hour of an incorporation assay, during which time no significant hydrolysis occurred. Furthermore, prolonged preincubation of cells with ATP reduced the mitogenic response to ATP but not to adenosine; preincubation with adenosine or N6-(R-phenylisopropyl)adenosine had the reverse effect. Finally, the effect of adenosine, but not of ATP, was inhibited by aminophylline. We conclude that extracellular ATP is a mitogen that interacts with P2 purinoceptors on the plasma membrane.

Topics: Adenosine; Adenosine Triphosphate; Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cells, Cultured; DNA Replication; Drug Synergism; Epidermal Growth Factor; Growth Substances; Humans; Kinetics; Mice; Mitogens

The cheek pouch of the Syrian hamster is an excellent tissue for the experimental induction of oral cancer by carcinogenic chemicals. Lysate prepared from a cell line (HCPC-1) derived from one of these hamster oral tumors greatly increased the growth of these oral tumor cells in vitro. We now show that the mitogenic substance, transforming growth factor alpha (TGF-alpha), is present in all of the chemically transformed hamster oral tumors examined (in vitro and in vivo). In no adult normal tissue of the Syrian hamster can we detect expression of TGF-alpha. TGF-alpha could be partly or wholly responsible for the mitogenic activity detected in the lysate of the chemically transformed hamster oral keratinocytes. Both normal and chemically transformed hamster oral keratinocytes express the receptor to epidermal growth factor. The consistent detection of TGF-alpha and epidermal growth factor receptor mRNAs in these hamster oral tumor cells suggests that an autocrine growth mechanism might be operative. This hamster cheek pouch oral cancer model can be used for the molecular analysis of how TGF-alpha and epidermal growth factor receptor might be involved in the malignant transformation of epithelial tissues.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; Cricetinae; DNA Replication; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Growth Substances; Keratins; Mouth Neoplasms; Peptides; Transforming Growth Factors

A tenfold increase in phospholipase C activity specific for phosphatidylinositol 4,5-bisphosphate (PIP2) was immunopurified from extracts of A-431 epidermoid carcinoma cells stimulated with epidermal growth factor. This finding suggests a biochemical link between growth factor-stimulated tyrosine kinase activity and PIP2 hydrolysis.

Topics: Antibodies; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Cytosol; Epidermal Growth Factor; Hydrolysis; Inositol Phosphates; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Substrate Specificity; Tumor Cells, Cultured; Type C Phospholipases; Tyrosine

Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca2+ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Epidermal Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrocortisone; Keratins; Retinoids; Tumor Cells, Cultured

Human squamous cell carcinoma cell lines often possess increased levels of epidermal growth factor (EGF) receptor. The growth of these EGF receptor-hyperproducing cells is usually inhibited by EGF. To investigate the mechanism of EGF-mediated inhibition of cell growth, variants displaying alternate responses to EGF were isolated from two squamous cell carcinoma lines, NA and Ca9-22; these cell lines possess high numbers of the EGF receptor and an amplified EGF receptor (EGFR) gene. The variants were isolated from NA cells after several cycles of EGF treatment and they have acquired EGF-dependent growth. Scatchard plot analysis revealed a decreased level of EGF receptor in these ER variants as compared with parental NA cells. Southern blot analysis and RNA dot blot analysis demonstrated that the ER variants had lost the amplified EGFR gene. One variant isolated from Ca9-22 cells, CER-1, grew without being affected by EGF. CER-1 cells had higher numbers of EGF receptor than parental Ca9-22 but similar EGFR gene copy number. Flow cytometric analysis indicated an increase in ploidy and cell volume which may give rise to the increase in receptor number per cell. The EGF receptors on both Ca9-22 and CER-1 cells were autophosphorylated upon EGF exposure in a similar manner suggesting no obvious alteration in receptor tyrosine kinase. However, very efficient down-regulation of the EGF receptor occurred in CER-1 cells. These data suggest two independent mechanisms by which EGF receptor-hyperproducing cells escape EGF-mediated growth inhibition: one mechanism is common and involves the loss of the amplified EGFR genes, and another is novel and involves the efficient down-regulation of the cell-surface receptor.

Topics: Carcinoma, Squamous Cell; Cell Division; DNA; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Gene Amplification; Gene Expression Regulation; Humans; Karyotyping; Nucleic Acid Hybridization; Phosphorylation; Ploidies; RNA; Tumor Cells, Cultured

Epidermal growth factor- (EGF) dependent tyrosine phosphorylation of the EGF receptor was inhibited by the exogenous addition of GM3 to a membrane preparation and to purified EGF receptor adsorbed to antireceptor-antibody-Sepharose (Bremer, E. G., Schlessinger, J., and Hakomori, S. (1986) J. Biol. Chem. 261, 2434-2440). A specific functional correlation between GM3 and EGF receptor function has been further assessed in this study, employing two variant clones of A431 cells showing completely different growth responses to EGF. The A1S clone showed EGF cell growth stimulation and contained GM3 whereas the A5I clone, whose growth was completely inhibited by EGF addition, lacked detectable GM3. Both the endogenous and EGF-dependent receptor tyrosine-kinase activities were low in the A1S clone and were only minimally inhibited by the exogenous addition of GM3. In contrast the EGF receptor kinase activity in A5I cells was much higher and was more strongly inhibited by GM3 than it was in A1S cells. The EGF receptor fraction prepared from A1S cells, eluted from an anti-EGF receptor antibody-Sepharose column, contained GM3, in contrast to the fraction prepared from A5I cells, which lacked detectable GM3. The receptor kinase activity in vitro was greatly influenced by detergent and ATP concentration. GM3 affected the receptor kinase in a biphasic manner, i.e. GM3 was inhibitory at a low concentration of detergent under a physiological concentration of ATP and stimulatory at a high concentration of detergent. In contrast lyso-GM3 displayed a monophasic inhibitory effect under a wide range of detergent concentrations. Lyso-CDH (lactosylsphingosine) had no detectable effect on the receptor kinase activity. The presence of a small quantity of lyso-GM3 in A431 cells was detected after DEAE-Sepharose chromatography followed by high performance liquid chromatography in a n-propanolyl alcohol-ammonia system. It is possible that de-N-fatty acylation of gangliosides could be an effective means to modulate EGF receptor function in membranes.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Membrane; Dogs; Epidermal Growth Factor; ErbB Receptors; Erythrocytes; G(M3) Ganglioside; Gangliosides; Humans; Kinetics; Mutation; Phosphorylation; Protein-Tyrosine Kinases

Topics: Adult; Aminoisobutyric Acids; Biological Transport, Active; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Infant, Newborn; Insulin; Kinetics; Simian virus 40; Sodium; Tumor Cells, Cultured

A new cell line designated 584A2 has been recently established from a patient with squamous cell carcinoma of the larynx. Cytogenetic analysis of the cell line revealed multiple copies of chromosome 7, as well as a homogeneous staining region (HSR) on one chromosome 7. Since overexpression of epidermal growth factor (EGF) cell surface receptors (EGFr) often occurs in other squamous carcinoma cell lines, it was predicted that 584A2 might overexpress EFGr. This was confirmed by: (1) metabolic labeling, with subsequent immunoprecipitation of EGFr and comparing autoradiographs to a cell line without an HSR and fewer copies of chromosome 7, and (2) performing EGF binding assays with Scatchard analysis. Since overexpression of EGFr correlates with an inhibitory effect of EGF on cell culture, the biological effects of EGF on 584A2 were examined in this study. At 5 ng/ml (serum-free medium), EFG stimulated incorporation of [3H] thymidine into trichloroacetic acid-precipitable material compared with controls. Incorporation increased between days 0 to 1 and 1 to 2 days with a 6- to 7-fold maximum. Dose-response studies (0 to 100 ng/ml) indicated maximum incorporation (6- to 7-fold) occurred between 0.1 ng/ml and 1.0 ng/ml. Cell growth was monitored over 7 days and, during this time, 5 ng/ml EGF produced a 10- to 12-fold increase in absolute cell numbers when compared with controls. We concluded that, unlike other squamous carcinoma lines with elevated EGFr, EGF stimulates rather than inhibits 584A2 cell proliferation.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Chromosomes, Human, Pair 7; Epidermal Growth Factor; Humans; Laryngeal Neoplasms

Epidermal growth factor (EGF)-receptor is a transmembrane glycoprotein whose intracellular degradation is known to be enhanced by EGF. We tested whether the receptor is replenished during this process by an enhanced rate of synthesis. Human A431 epidermoid carcinoma cells and primary cultures of human placental cytotrophoblasts were used in these studies. Cells were labeled with [35S]methionine, and EGF-receptor biosynthesis was quantitated by immunoprecipitation using a monoclonal anti-EGF-receptor antibody. EGF stimulated receptor biosynthesis at concentrations of 0.1 to 1 nM. The effect was seen within 2 h of EGF addition. At high EGF concentrations the stimulatory effect was diminished. In contrast, the effect of EGF on receptor degradation in these cells was negligible at low nanomolar concentrations and was pronounced only at saturating concentrations (greater than or equal to 10 nM). These results show that occupation of the cell surface EGF-receptor by its ligand can lead to the production of more receptor protein, thus counterbalancing the negative effect on receptor degradation. At low nanomolar concentrations of EGF the stimulatory effect on receptor synthesis predominates over degradation, indicating a positive regulatory role of EGF in receptor action.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Stimulation, Chemical; Trophoblasts; Tumor Cells, Cultured

We have used the soluble covalent cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDAC) to examine the capacity of epidermal growth factor (EGF) to stimulate the dimerization of purified EGF receptor, of EGF receptor in membrane preparations and in intact A431 cells. The addition of EGF either to membranes from A431 cells or to EGF receptor which was purified from A431 cells by immunoaffinity chromatography caused the appearance of a cross-linked product of Mr 340,000 which was identified using EGF receptor-specific antibodies as an EGF receptor dimer. Three independent approaches including biosynthetic labeling, surface iodination, and immunoblotting experiments were utilized to follow EGF receptor dimerization in living A431 cells. These approaches provided consistent results indicating that EGF induced rapid dimerization of EGF receptor in living cells, suggesting that this process may play a role in transmembrane signalling mediated by EGF.

Topics: Carbodiimides; Carcinoma, Squamous Cell; Chromatography, Affinity; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Ethyldimethylaminopropyl Carbodiimide; Humans; Immunoassay; Kinetics; Macromolecular Substances; Molecular Weight; Phosphorylation; Tumor Cells, Cultured

SqCC/Y1 cells grow as a monolayer in culture and differentiate when maintained in the plateau phase; in the absence of serum these cells differentiate more rapidly. The differentiation is characterized by the stratification of the culture to form a structure consisting of several cellular layers, synthesis of specific keratins, and the attainment of the capacity to form a cornified cell membrane. The stratification process is indicative of the importance of cell-cell interactions during maturation. To study the relationship between membrane glycosphingolipids (GSLs) and the state of differentiation of SqCC/Y1 cells, GSLs were measured in cultures grown in the presence or absence of fetal calf serum. Glycolipids were isolated by diethylaminoethyl-Sephadex and Iatrobeads column chromatographies, and their distributions were determined by high-performance thin-layer chromatography. GM3 was the major ganglioside present in these cells. Other ganglioside components were tentatively identified as GM2, GM1, and GD3. Differences in ganglioside patterns were observed in differentiated cultures; the major changes were accumulation of GD3 and depletion of GM1. The predominant neutral GSLs in SqCC/Y1 cells were identified as Glc beta 1-1Cer, Gal beta 1-4Glc beta 1-1Cer, Gal beta 1-4Gal alpha 1-4Glc beta 1-1Cer, Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, and three unknown complex GSLs. Differentiated cultures, however, showed variations in banding patterns, which include an increase in Glc beta 1-1Cer and Gal beta 1-4Glc beta 1-1Cer and a decrease in Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer and Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta-1Cer. These changes, however, were not observed when the cells were grown in the presence of epidermal growth factor or retinoic acid, factors which inhibit the differentiation process. The findings demonstrate significant changes in glycolipid composition of differentiated SqCC/Y1 cells grown in the absence of serum, suggesting that these lipids may be important to the differentiated state.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Epidermal Growth Factor; Fatty Acids; Glycosphingolipids; Humans; Tretinoin; Tumor Cells, Cultured

Several observations have indicated that clustering of growth factor receptors plays an important role in the action of growth factors. In this investigation, we have used the label fracture method to study the effects of epidermal growth factor (EGF) on the lateral distribution of its receptors in A431 epidermoid carcinoma cells. This method allows a direct visualization of immunogold-labeled plasma membrane receptors on ultrastructural level and in addition permits an quantitative analysis of their lateral distribution. EGF receptors were immunogold-labeled according to standard procedures with the monoclonal anti-EGF receptor antibody 2E9 (IgG1), which binds to the EGF receptor in a 1:1 ratio. In the absence of EGF, EGF receptors located on the surface of A431 cells were found to be clustered, as deduced from Poisson variance analysis (p less than 0.001). Following treatment of A431 cells with EGF, receptor clustering increased rapidly, reaching the maximum within 10 min. Maximal clustering was maintained for 1 h, after which the lateral distribution of receptors returned to the control situation within another hour.

Topics: Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Freeze Fracturing; Humans; Immunohistochemistry; Membrane Proteins; Microscopy, Electron; Receptor Aggregation; Tumor Cells, Cultured

Plasma membranes have been isolated from the human epidermoid carcinoma cell line A431 by a rapid fractionation of lysate on Percoll density gradient at pH 9.6. Endoplasmic reticulum, lysosomes and mitochondria sedimented at the bottom of gradient whereas plasma membranes focused at low density, as shown with specific markers. Plasma membranes displayed a 4.5- and 4.4-fold enrichment in [3H]concanavalin A and 5'-nucleotidase, respectively. This proteic fraction was further characterized by its lipid composition and phospholipid analysis. The cholesterol/phospholipid molar ratio was 0.45 in plasma membranes against 0.19 in lysate. Sphingomyelin increased from 7.5% of total phospholipids in lysate to 16.2% in plasma membranes, as well as phosphatidylserine which displayed a 1.5-fold enrichment in the plasma membrane fraction. This was at the expense of phosphatidylcholine (45.2% in lysate, against 35% in plasma membranes). Electron microscopy of the isolated material showed vesicles essentially free from endoplasmic reticulum and organelles. These plasma membranes retained the ability to bind 125I-labelled epidermal growth factor (125I-EGF) with a Kd = 4.7 nM and Bmax = 63 pmol/mg protein. EGF binding resulted in a stimulation of the phosphorylation protein reaction in the presence of [gamma-32P]ATP and sodium dodecyl sulfate polyacrylamide gels of phosphorylated proteins indicated that the radioactivity of the major band of molecular weight 170,000 was clearly enhanced by EGF binding. These results indicate that the EGF receptor and its intrinsic protein kinase activity were preserved during our plasma membrane isolation procedure.

Topics: Carcinoma, Squamous Cell; Cell Fractionation; Cell Membrane; Centrifugation, Density Gradient; Cholesterol; Epidermal Growth Factor; ErbB Receptors; Humans; Membrane Lipids; Membrane Proteins; Microscopy, Electron; Phosphatidylcholines; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Phosphorylation; Protein Kinases; Sphingomyelins; Tumor Cells, Cultured

The plasma membrane ultrastructural changes after the action of epidermal growth factor were studied in A-431 cells using freeze-fracture methods. The incubation with EGF (100 ng/ml, 0 degree C, 60 min) led to a decrease in density of intramembrane particles on the P surface of ventral cell membrane, while the number of coated pits increased there. The revealed effects of EGF may be related to direct consequences of EGF-receptor complex formation, because all the temperature dependent steps of its processing were blocked. The data obtained testify to an active involvement of the membrane ventral surface in the formation of cell response towards growth factors.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Dose-Response Relationship, Drug; Epidermal Growth Factor; Freeze Fracturing; Humans; Microscopy, Electron; Surface Properties; Tumor Cells, Cultured

In a previous study [(1984) J. Cell Biol. 98, 725-731] we showed that the level of human MHC, HLA antigens on A431 carcinoma cells is reduced after these cells bind epidermal growth factor (EGF). Here we use flow cytometry to determine the effects of various doses and times of EGF treatment on HLA expression. We then show that the reduction in HLA expression is associated with a reduction in the level of phosphorylation of immunoprecipitable surface HLA antigens, although longer exposure of cells with EGF increased both surface HLA expression and their phosphorylation levels. Lateral diffusion of HLA antigens is lower in EGF-treated than in control cells. The lower diffusion coefficients measured may be causally related to the decreased phosphorylation of HLA antigens.

Topics: Aged; Aged, 80 and over; Antigens, Neoplasm; Carcinoma, Squamous Cell; Diffusion; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; HLA Antigens; Humans; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Expression of the epidermal growth factor (EGF) receptor gene is stimulated by EGF and the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). PMA elevates EGF receptor mRNA levels in human KB epidermoid carcinoma cells, but does not significantly affect the half-life of this mRNA when its decay is examined after the addition of actinomycin D. In contrast, EGF greatly prolongs the half-life of EGF receptor mRNA suggesting a possible mechanism for the stimulatory effect of EGF on EGF receptor mRNA levels. EGF also stabilizes beta-tubulin and beta-actin mRNAs but has very little effect on the degradation of total mRNA.

Topics: Actins; Carcinoma, Squamous Cell; Dactinomycin; Epidermal Growth Factor; ErbB Receptors; Half-Life; Humans; RNA Processing, Post-Transcriptional; RNA, Messenger; Tubulin; Tumor Cells, Cultured

The compartmentalization of the epidermal growth factor (EGF) receptors in A-431 cells was studied using centrifugation of the microsomal fraction of these cells in continuous Percoll gradient. The existence of an intact (non-degraded) EGF receptor in plasma membrane and endosome fraction was demonstrated by electrophoretic analysis of in vitro phosphorylated Percoll fractions. No phosphorylated receptor was revealed in lysosomal fraction by this method. The existence of non dissociated EGF-receptor complexes in intracellular compartments 30 minutes after the start of internalization was proven using a synthesized photoreactive labeled EGF derivative (125I-EGF-SANAH). The removing of pH gradient in organellar membranes by 10 mkM of monensin did not affect dissociation from its receptor. The data obtained proved the existence of non-dissociated and non-degraded EGF-receptor complexes in the endosomal compartment of A-431 cells.

Topics: Carcinoma, Squamous Cell; Cell Compartmentation; Cell Fractionation; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Microscopy, Electron; Organoids; Phosphorylation; Tumor Cells, Cultured

Effects of EGF on proliferation and tumor marker secretion of cervical cancer cells are reported together with the characteristics of EGF receptors on the cells. TA-4 producing cell line (OMC-1) originating from cervical squamous cell carcinoma and CA-125 producing cell line (OMC-4) originating from cervical adenocarcinoma, were used. Scatchard plot of EGF binding to OMC-1 indicated a single class of binding sites with a dissociation constant (Kd) of 360pM, whereas that of OMC-4 was curvilinear suggesting two classes of binding sites with a Kd of 170pM and 510pM. The theoretical maximum number of binding sites of OMC-1 and OMC-4 was 2.4 X 10(4) and 1.6 X 10(5), respectively. Effects of EGF on growth were studied by monitoring cell number and the incorporation of 3H-thymidine into the DNA of the cells. OMC-1 was stimulated by EGF at low concentrations (0.01-0.1nM) and inhibited at higher concentrations. OMC-4 was not stimulated by EGF. The TA-4 secretion of OMC-1 was slightly stimulated by EGF at low concentrations (0.01-1nM) and significantly stimulated at high concentration (10nM). The CA-125 secretion of OMC-4 was not stimulated by EGF. These results suggest that there are some differences between cervical squamous cell carcinoma and adenocarcinoma in the mechanisms of regulation of proliferation and tumor marker secretion by EGF.

Topics: Adenocarcinoma; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Binding Sites; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Division; Cell Line; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Uterine Cervical Neoplasms

Epidermal growth factor (EGF) is a cell-regulating polypeptide that appears important to the maintenance and function of some benign tissues and to the transformation and proliferation of certain malignancies. In humans the highest concentrations of EGF are found in the urine. We investigated possible interactions between EGF and normal and neoplastic tissues of the urinary system with indirect immunohistochemical staining of paraffin-embedded tissue sections. A polyclonal antibody directed against mouse EGF but shown to react with human EGF was used in the assays. Positive staining was granular in nature and confined to the cytoplasm. Staining of the renal parenchyma (N = 5) was observed in the epithelium of the proximal and distal tubules and the collecting ducts. There was staining of clear cell (N = 6) and papillary (N = 3) carcinomas of the kidney. Staining of the normal urothelium (N = 5) was limited to superficial cells. All transitional cell (N = 21) and squamous (N = 2) carcinomas of the bladder stained. Subjectively, the staining intensity of the transitional cell carcinomas correlated inversely with tumor differentiation. In light of evidence that internalized, receptor-bound EGF is rapidly degraded, the striking immunohistochemical demonstration of cytoplasmic EGF suggests active synthesis. EGF synthesized by urothelial and renal carcinomas may be involved in an autocrine mechanism of malignant proliferation.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Papillary; Carcinoma, Squamous Cell; Carcinoma, Transitional Cell; Epidermal Growth Factor; Humans; Immunoenzyme Techniques; Kidney; Kidney Neoplasms; Urinary Bladder; Urinary Bladder Neoplasms

We have developed the computer-aided image processing analysis to visualize and quantitate the cell motility and morphological changes in living cultured cells. The effect of growth factors on cell motility and morphology has been analyzed by this method. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) elicited membrane rufflings in rat embryo fibroblast 3Y1 cells as well as in human epidermoid carcinoma KB cells. Insulin and IGF-I also induced ruffling membranes in Balb/c-3T3 cells. The quantitative analysis by the programmed trace mode of the AVEC system has shown that the motion of the membrane rufflings was observed within 2 min, reached the maximum level within 4-8 min, and rapidly decreased within 10-15 min after the addition of these growth factors. The analysis also revealed the temperature- and growth factor concentration-dependent changes in the motion of membrane rufflings elicited by these growth factors. 12-O-Tetradecanoylphorbol-13-acetate, one of the well-known tumor promoters, rapidly induced cell rounding in Balb/c-3T3 cells. This change of cell morphology could be also quantified by the trace mode analysis. The fluorescent phalloidin staining experiment indicated that these growth factor- or phorbol ester-induced morphological changes were accompanied by the reorganization of filamentous actin. Furthermore, we were able to visualize actin stress fibers in living EBTr cells by enhancing the video image and to follow the reduction of stress fibers induced by cytochalasin B without any fixation or fluorescent probes.

Topics: Actins; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Movement; Epidermal Growth Factor; Fibroblasts; Growth Substances; Humans; Image Processing, Computer-Assisted; Insulin; Somatomedins; Temperature; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Manoalide is a marine natural product that has anti-inflammatory and anti-proliferative activities and is an irreversible inhibitor of phospholipase A2 and phospholipase C. It is now shown that the compound is a potent inhibitor of Ca2+ mobilization in several cell types. In A431 cells the increase in epidermal growth factor receptor-mediated Ca2+ entry and release from intracellular Ca2+ stores were blocked by manoalide in a time-dependent manner with an IC50 of 0.4 microM. The effect of manoalide on phosphoinositide metabolism, namely the production of inositol monophosphate, did not coincide with its effect on the epidermal growth factor response. In GH# cells, manoalide blocked the thyrotropin-releasing hormone-dependent release of Ca2+ from intracellular stores without inhibition of the formation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate. Manoalide also blocked the K+ depolarization-activated Ca2+ channel in these cells as well as the activation of the channel by Bay K8644 with an IC50 of 1 microM. In addition, manoalide also inhibited the Ca2+ influx induced by concanavalin A in mouse spleen cells in a time- and temperature-sensitive manner with an IC50 of 0.07 microM. However, neither forskolin-activated adenylate cyclase in A431 cells nor the distribution of the potential sensitive dye, 3,3'-dipropylthiodicarbocyanide iodide in GH3 cells was affected by manoalide. Thus, manoalide acts as a Ca2+ channel inhibitor in all cells examined. This action may account for its effects on inflammation and proliferation and may be independent of its effect on phospholipases.

Topics: Animals; Calcium; Calcium Channel Blockers; Carcinoma, Squamous Cell; Cell Line; Colforsin; Concanavalin A; Cyclic AMP; Epidermal Growth Factor; Humans; Inositol Phosphates; Ion Channels; Kinetics; Lymphocytes; Mice; Terpenes

KB cells respond to insulin and insulin-like growth factor I (IGF-I) in a closely similar way (induction of membrane ruffling, stimulation of pinocytosis, and amino acid transport) but respond to epidermal growth factors (EGF) in a similar but distinct way. In the KB cells, using phosphotyrosine-specific antibody we have found that: the receptors for insulin (beta subunit), IGF-I (beta subunit), and EGF undergo tyrosine phosphorylation as early as 10 s after addition of their respective ligands; a 185-kDa protein is rapidly (less than 10 s) tyrosine phosphorylated by insulin and IGF-I through their respective receptor kinases but not EGF; tyrosine phosphorylation of a 190-kDa glycoprotein is rapidly (less than 10 s) induced by EGF through EGF receptor kinase; and tyrosine phosphorylation of a 240-kDa protein is stimulated within 30 s by all three growth factors. These patterns of tyrosine phosphorylation could be causally related to biological responses induced by the three growth factors.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Immunosorbent Techniques; Insulin; Insulin-Like Growth Factor I; Molecular Weight; Phosphorylation; Phosphotyrosine; Proteins; Receptor, Insulin; Receptors, Somatomedin; Somatomedins; Tyrosine

A number of peptide growth factors have been shown to induce the secretion of type I collagenase into the medium of human fibroblast cultures (Chua et al., 1985). In this study the ability of eye-derived growth factor, lectin and tumor-promoting agents on collagenase induction in human fibroblast cells were examined. These agents were found to be able to induce collagenase production to a similar extent as epidermal growth factor. Dexamethasone at 10-100 nM was found to suppress collagenase induction in human fibroblast cells. The cell-type specificity of this enzyme induction by growth factors was studied by using a human epidermoid carcinoma cell line, A-431. An Mr 55,000 band appeared in the medium of A-431 cells upon 22 h exposure to EGF. Two-dimensional peptide pattern of the Mr 55,000 band in A-431 cells was identical to the counterpart in HF cells. Our results indicated that the induction of collagenase was not unique to human fibroblast cultures.

Topics: Carcinoma, Squamous Cell; Cell Line; Concanavalin A; Dexamethasone; Epidermal Growth Factor; Fibroblast Growth Factors; Fibroblasts; Growth Substances; Humans; Microbial Collagenase; Molecular Weight; Ocular Physiological Phenomena; Tetradecanoylphorbol Acetate

EGF receptor-hyperproducing cells of squamous carcinoma origin were inoculated s.c. into the bilatero-abdominal regions of athymic mice and a mini-osmotic pump containing EGF was implanted on teh back. After 2 weeks the tumors formed from 5 different cell lines in the presence of EGF weighed 3 to 6 times more than those formed in the absence of EGF. The cells recovered from these tumors maintained their original characteristics such as the amplified EGF receptor gene, high numbers of EGF receptors and a sensitivity to EGF-mediated inhibition of growth in vitro. These results suggest that EGF plays an important role in promoting growth of squamous-cell carcinoma in vivo.

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; Gene Amplification; Humans; Mice; Mice, Inbred Strains; Mice, Nude; Receptors, Cell Surface; Tumor Cells, Cultured

The effects of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) and epidermal growth factor (EGF) on the synthesis and secretion of human chorionic gonadotropin (hCG) and its subunits by normal and malignant trophoblasts as well as by non-trophoblastic cells were investigated in vitro. The explants of normal early placental tissues, choriocarcinoma cell line BeWo and non-trophoblastic tumor cell line CaSki from epidermoid carcinoma of the cervix, respectively, were cultured in the presence or absence of dibutyryl cAMP or EGF. The addition of either dibutyryl cAMP (1 mM) or EGF (100 ng/ml) caused significant increases in the synthesis and secretion of hCG and its subunits in cultures of normal and malignant trophoblasts, but had no stimulatory effect on hCG beta synthesis and secretion in culture of non-trophoblastic cell line CaSki that secretes predominantly hCG beta-like material. The magnitude of the stimulatory effects of dibutyryl cAMP and EGF on hCG (alpha,beta) synthesis and secretion by BeWo cells was much greater than that observed in normal trophoblasts. The time course of these stimulatory effects indicated that EGF-stimulated increase in hCG synthesis and secretion required a lag period longer than that for the dibutyryl cAMP-stimulated increase. These results suggest that there were no differences in normal and malignant trophoblasts in the mechanism for the stimulatory regulation of hCG (alpha, beta) synthesis and secretion, but immunoreactive hCG beta synthesis and secretion in non-trophoblastic tumor cells are regulated by a mechanism different from that in trophoblastic cells.

Topics: Bucladesine; Carcinoma, Squamous Cell; Cell Line; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Pregnancy; Trophoblastic Neoplasms; Uterine Cervical Neoplasms; Uterine Neoplasms

Previous studies have indicated that an autostimulatory transforming growth factor was required for the optimal growth of SW-13 adrenal carcinoma cells in soft agar. The production of SW-13 colony-stimulating activity by other human malignant cell lines of both epithelial and mesenchymal origin has been demonstrated. Evidence was presented indicating that the stimulating activity detected in crude acid-ethanol extracts was an acid- and heat-stable polypeptide requiring disulfide bonds for full activity. This activity was detected more frequently in tumors and human cancer cells in culture of epithelial origin than of mesenchymal origin and in a variety of nonneoplastic tissues. In the present study, this activity, termed epithelial transforming growth factor (TGFe) because of its ability to stimulate soft agar growth of certain epithelial cells, was partially purified from bovine kidney. Fourfold purification of the kidney acid-ethanol extract with 50% maximal growth-stimulatory activity of 10 micrograms was achieved using molecular sieve chromatography where TGFe eluted with an apparent molecular weight of 20,000-25,000. The next purification step, molecular sieve high performance liquid chromatography, yielded a 50% maximal growth-stimulatory activity of 50 ng and an 800-fold purification from the initial acid-ethanol extract. TGFe eluted in the Mr 11,000 range. Reversed phase high performance liquid chromatography with a C18 column was then used, yielding a single or double peak of SW-13 colony-stimulating activity at 30-35% acetonitrile. The degree of purification was 11,000-fold with a 50% maximal growth-stimulatory activity of 3.5 ng. Analysis of the peak on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major and sometimes single band with a molecular weight of 23,000-25,000. Extraction of protein from the polyacrylamide gel demonstrated that only the Mr 23,000-25,000 band stimulated soft agar growth of SW-13 cells. The biological activity of the partially purified TGFe was found to differ from other known growth factors with regard to its ability to stimulate soft agar growth of SW-13 cells with the exception of basic fibroblast growth factor (FGF). The acid lability of FGF, the different molecular weights of these two growth factors, the lack of stimulation of soft agar growth of A431 cells, and the lack of binding of TGFe to FGF receptors indicated that TGFe was not related to basic FGF. Partially purified TGFe was al

Topics: Adrenal Gland Neoplasms; Carcinoma, Squamous Cell; Cell Division; Cell Line; Chromatography, Gel; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Molecular Weight; Peptides; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factors

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin, and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3). Maximal concentrations of EGF (316 ng/ml; approximately 50 nM), bradykinin (1 microM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K0.5 values for stimulation were approximately 10 nM, 3 nM, and 10 microM for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid accumulation of the two isomers of InsP3, Ins(1,3,4)P3, and Ins(1,4,5)P3 as determined by high performance liquid chromatography analysis; maximal accumulation of Ins(1,4,5)P3 occurred within 15 s. EGF and bradykinin also stimulated a rapid (maximal levels attained within 30 s after addition of hormone) and a sustained 4- and 6-fold rise, respectively, in cytosolic free Ca2+ levels as measured by Fura-2 fluorescence. EGF and bradykinin also produced a rapid, although transient, 3- and 5-fold increase, respectively, in cytosolic free Ca2+ after chelation of extracellular Ca2+ with 3 mM EGTA. These data are consistent with the idea that EGF elevates intracellular Ca2+ levels in A431 cells, at least in part, as a result of the rapid formation of Ins(1,4,5)P3 and the consequential release of Ca2+ from intracellular stores.

Topics: Bradykinin; Calcium; Carcinoma, Squamous Cell; Cell Line; Chromatography, High Pressure Liquid; Cytosol; Egtazic Acid; Epidermal Growth Factor; Histamine; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Sugar Phosphates

A monoclonal antibody of IgG2a isotype (425) is described that reacts with the epidermal growth factor receptor on human cells of different tissue origins. Monoclonal antibody 425 mediates tumor cytotoxicity in vitro using mouse and human effector cells and suppresses in vivo tumor cell growth of epidermoid (A 431) and colorectal (SW 948) carcinoma-derived cell lines. The tumoricidal effects in vitro are proportional to the antigen density on target cells. At concentrations higher than 1 nM, monoclonal antibody 425 inhibits growth of epidermal growth factor receptor-bearing A 431 cells, showing an epidermal growth factor-like agonist activity on the growth properties of these cells. A 431 cultures grown in the presence of growth-inhibiting doses of antibody or epidermal growth factor reveal a clear decrease of the relative number of cells in S phase. Additionally, cells treated with the antibody show a decrease of G2-M-phase cells in some, but not all, cultures tested.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line; Colonic Neoplasms; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Humans; Immunoenzyme Techniques; Interphase; Macrophages; Mice; Monocytes; Recombinant Proteins; Rectal Neoplasms

The epidermal growth factor receptor (EGFR) and insulin receptor undergo slow post-translational modification by which they acquire hormone binding and tyrosine kinase (EGFR) function. The half-time for acquisition of EGF or insulin binding activity is 30-40 min and of tyrosine kinase activity (EGFR), is 10-15 min. Tunicamycin, an inhibitor of N-linked oligosaccharide addition, blocks acquisition of both EGF and insulin binding activity. With EGFR, activation precedes acquisition of resistance to endoglucosaminidase H (t1/2 approximately equal to 75 min), a medial Golgi event. Treatment of active high mannose receptor with endo H generates fully active aglyco-receptor; thus, core oligosaccharide addition is a prerequisite for activation, but not for EGF binding per se. EGFR is activated in and translocated from the endoplasmic reticulum (ER) slowly (t1/2 approximately equal to 75 min). Since translocation rate equals the rate for acquisition of endo H resistance, transit from the ER is rate limiting for EGFR maturation. Tunicamycin inhibits exit from the ER parallel to its effect on acquisition of binding activity. Insulin proreceptor, a 210 kDa high-mannose glycopolypeptide, acquires insulin binding function (t1/2 approximately equal to 45 min) then is proteolytically cleaved (t1/2 approximately equal to 3 hr) into subunits of the mature alpha 2 beta 2 receptor. Modification giving rise to insulin binding activity is due to a conformational change in the binding domain, since human autoimmune antibody recognizes only the active species, while rabbit polyclonal antibody recognizes all forms. Newly-translated EGF proreceptor lacks a functional tyrosine domain capable of autophosphorylation; 30-40 min after translation, while still in the ER, tyrosine kinase activity is acquired. Since the kinase domain is cytoplasmic, the receptor may become phosphorylated on tyrosine before reaching the plasma membrane.

Topics: Adipose Tissue; Binding Sites; Carcinoma, Squamous Cell; Cell Line; Endoplasmic Reticulum; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Humans; Insulin; Kinetics; Models, Biological; Phosphorylation; Protein Binding; Protein Conformation; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Receptor, Insulin; Tunicamycin

Dynamics of compartmentalization of epidermal growth factor (EGF) in human carcinoma A431 cells during the first hour after initiation of endocytosis was examined by methods of the organelle fractionation on a 20% Percoll gradient and of the microfluorimetric visualization of endocytosis of rhodamine-labeled EGF (EGF-R). EGF was revealed in small vesicles localized in the peripheral region of cytoplasm in a few minutes after endocytosis initiation. During centrifugation in Percoll these vesicles (endosomes), with an average density of 1.038 g/ml, were seen co-sedimented with Golgi membranes. By one hour after initiation of endocytosis, EGF-R was accumulated in perinuclear zone, in a trans-Golgi region, as numerous big luminous centres that were apparently MB-endosomes and had the same density in Percoll as did small peripheral endosomes. Such centres appeared in several cells already within 5-10 minutes. In A431 cells EGF did not reach lysosomes within 60 minutes, because no accumulation of 125I-EGF was shown in lysosome corresponding regions of Percoll gradient (average density 1.070 g/ml).

Topics: Animals; Carcinoma, Squamous Cell; Cell Compartmentation; Cell Line; Cytological Techniques; Endocytosis; Epidermal Growth Factor; Humans; Mice; Microscopy, Fluorescence; Organoids; Time Factors; Tumor Cells, Cultured

EGF receptor levels were investigated in esophageal squamous-cell carcinoma tissues from 31 patients. Twenty-two (71%) of these cancer tissues exhibited significantly higher 125I-EGF binding activity than normal mucosa in the adjacent non-cancerous tissues. These EGF receptor levels were then compared on the basis of pathological findings including lymph-node metastasis, depth of invasion, differentiation type, vascular invasion, infiltration and location of the lesion. Unlike previous reports on breast and bladder cancers, our study showed no obvious correlation between these pathological characteristics and the EGF receptor levels in esophageal carcinomas. Immunohistochemical staining with the anti-EGF receptor monoclonal antibody detected EGF receptors in squamous cells of the cancer tissues as well as in the basal cells of nearby normal epithelium. Since the basal cells have proliferative potential in the esophagus, the increase in EGF receptor levels in these cells may possibly be associated with the development of human esophageal squamous-cell cancer.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Epidermal Growth Factor; Esophageal Neoplasms; Humans; Lymphatic Metastasis; Neoplasm Invasiveness

The CaSki cell line derived from an epidermoid carcinoma of the uterine cervix produces and releases two types of tumor-associated antigen. One is a eutopic antigen, TA-4 and the other is an ectopic antigen, hCG beta-like material. The aim of the present investigation was to elucidate a possible difference in the induction mechanism of production of TA-4 and hCG beta-like material in the CaSki cells in relation to cellular differentiation and gene modulation. The exposure to epidermal growth factor (EGF, 100ng/ml) for two days greatly increased TA-4 production by the cultured CaSki cells without affecting cell growth and immunoreactive hCG beta production. The EGF-stimulated increase in TA-4 production required a lag period of approximately 24 hours. The exposure to sodium butyrate (2.5mM) stimulated immunoreactive hCG beta production by the cells, but decreased TA-4 production. The stimulatory effect of sodium butyrate on immunoreactive hCG beta production occurred only during the exposure to the agent. These discrepant effects of EGF and sodium butyrate on the production of TA-4 and hCG beta-like material by the CaSki cells suggest that a fundamental difference exists in the induction mechanism for the expression of these two types of tumor-associated antigen in cervical squamous carcinoma cells.

Topics: Antigens, Neoplasm; Butyrates; Butyric Acid; Carcinoma, Squamous Cell; Cells, Cultured; Chorionic Gonadotropin; Culture Media; Epidermal Growth Factor; Female; Humans; Uterine Cervical Neoplasms

A newly synthesized isoprenoid, N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine, has a verapamil-like structure but no calcium channel blocking activity. The isoprenoid enhanced the cytotoxic effect of a conjugate of epidermal growth factor coupled with Pseudomonas exotoxin in human KB cells. By using iodinated epidermal growth factor ([125I]EGF), the effect of the isoprenoid on intracellular transport of EGF was examined. The isoprenoid did not affect the binding and uptake of [125I]EGF by KB cells. The release of radioactivity associated with [125I]EGF into medium was slow in the presence of the isoprenoid. Density gradient fractionation studies using cell homogenates suggest that [125I]EGF accumulates in an undegraded form in lysosomes when cells are treated with the isoprenoid. The pH value in lysosomes of KB cells was 5.5, and SDB did not affect significantly the pH value at the concentrations used to potentiate the cytotoxicity of chimeric toxins. Electron microscopy showed an increased number of electron-dense bodies in KB cells grown for 24 hr with 17-51 micrograms/ml isoprenoid. The potentiating action of chimeric toxins by the isoprenoid is discussed in relation to the altered lysosomal function in treated cells.

Topics: ADP Ribose Transferases; Bacterial Toxins; Carcinoma, Squamous Cell; Cell Line; Drug Resistance; Drug Synergism; Epidermal Growth Factor; Ethylenediamines; Exotoxins; Humans; Kinetics; Lysosomes; Microscopy, Electron; Pseudomonas aeruginosa Exotoxin A; Terpenes; Virulence Factors

Vanadate can activate the uptake of Ca in A431 epidermal carcinoma cells by two- to fivefold with no detectable lag period. Preincubation with epidermal growth factor (EGF) to down-regulate the EGF receptor prevents subsequent stimulation by EGF but not that by vanadate. Ca uptake is sodium-independent and is not activated by depolarization in high KCl. On the contrary, vanadate-stimulated uptake is completely inhibited by decreasing the plasma membrane potential from about -65 to -30 mV. These results demonstrate that the EGF receptor is not itself functioning as a Ca channel, that vanadate is not acting at the level of EGF receptor, and that the Ca transport system exhibits an unusual potential sensitivity in that it is inhibited by depolarization of the plasma membrane.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Membrane Potentials; Vanadates; Vanadium

The c-erbB-2 gene was first identified by virtue of its cross-hybridization with v-erbB. Nucleotide sequence analysis of complementary DNA clones suggested that the c-erbB-2 gene encodes a growth factor receptor similar to that for EGF. Antibodies against the carboxyl terminal sequence of the c-erbB-2 protein immunoprecipitated a 185-kDa glycoprotein which showed protein-tyrosine kinase activity in vitro. Despite the extensive similarity between the c-erbB-2 protein and EGF receptor, neither EGF nor TGF-alpha bound to the c-erbB-2 protein. Phosphorylation of the c-erbB-2 protein was stimulated by TPA via protein kinase C in vivo. EGF also induced phosphorylation of the c-erbB-2 protein. This phosphorylation occurred not only on serine and threonine residues but also on tyrosine residues. Preliminary data suggested that the latter was mediated by the kinase activity of the EGF receptor. Southern blot analysis of DNAs from primary tumors revealed that the c-erbB-2 gene tends to be amplified in adenocarcinomas, mostly of the stomach and the breast. By screening both human genomic and cDNA libraries using v-yes DNA as a probe, we obtained DNA clones of the c-yes gene, the pseudogene of c-yes, c-fgr gene and c-src gene and two novel yes-related genes, fyn and lyn. Complete nucleotide sequence analysis of the cDNA clones of c-yes, fyn and lyn revealed that these genes encode proteins similar to p60src both in size and sequence.

Topics: Adenocarcinoma; Amino Acid Sequence; Breast Neoplasms; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Protein-Tyrosine Kinases; Proto-Oncogenes; Stomach Neoplasms; Transcription, Genetic

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Membrane; Epidermal Growth Factor; Humans; Hydrocortisone; Lipids; Lipoproteins, LDL; Retinoids; Tretinoin

The fate of epidermal growth factor (EGF) after internalization by A431 cells was studied. First, cells containing 125I-EGF-receptor complexes in endosomes were obtained. Subsequent incubation of the cells at 37 degrees C resulted in the recycling of 125I-EGF from endosomes to the cell surface in the receptor-bound state and the gradual release of recycled ligand into the medium. The excess of unlabeled EGF blocked both rebinding and re-internalization of recycled 125I-EGF to produce enhanced accumulation of ligand in the medium. The rate of recycling was shown to be much higher than that of EGF degradation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Fractionation; Cell Line; Cell Membrane; Centrifugation, Density Gradient; Cytoplasm; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Mice

The transferrin receptor cycles rapidly between cell surface and endosomal membrane compartments. Treatment of cultured cells with epidermal growth factor (EGF) or insulin-like growth factor I (IGF-I) at 37 degrees C causes a rapid redistribution of transferrin receptors from an intracellular compartment to the cell surface. The effects of EGF and IGF-I on the kinetics of the cycling of the transferrin receptor in A431 human epidermoid carcinoma cells were compared. The primary site of EGF action was found to be an increase in the rate of transferrin receptor exocytosis. The exocytotic rate constant was measured to be 0.11 min-1 in control cells and 0.33 min-1 in EGF-treated cells. In contrast, IGF-I was found to increase the cell surface expression of transferrin receptors by causing a small increase in the rate of exocytosis (from 0.11 to 0.17 min-1) and a decrease in the rate of endocytosis (from 0.33 to 0.24 min-1). It is concluded that the mechanisms for EGF and IGF-I action to increase the cell surface expression of the transferrin receptor are distinct. A kinetic model of the cycling of the transferrin receptor based on experimentally determined rate constants is presented. The model predicts that a consequence of IGF-I action on transferrin receptor cycling is to decrease the apparent Km for the uptake of diferric transferrin by cells. This prediction is confirmed by direct measurement of the accumulation of 59Fe-labeled diferric transferrin by A431 cells. These data demonstrate that the accumulation of iron by cultured cells is a complex function of the rate of cycling of the transferrin receptor and that this process is under acute regulation by growth factors.

Topics: Carcinoma, Squamous Cell; Cell Membrane; Epidermal Growth Factor; Exocytosis; Humans; Insulin-Like Growth Factor I; Iron; Kinetics; Models, Biological; Receptors, Transferrin; Somatomedins

The transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF-induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth-inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti-EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down-regulation, effects on transferrin receptor expression were independent of these events. EGF-induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P-labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6-fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGE induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in

Topics: Animals; Antibodies; Binding Sites; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; Epithelium; ErbB Receptors; Methionine; Phosphorylation; Receptors, Transferrin; Tetradecanoylphorbol Acetate; Vanadates

Human and mouse epidermal growth factor (hEGF; mEGF) bind to the same two classes of receptor sites on cell membranes prepared from the human epidermoid carcinoma cell line A431. However, the affinities of mEGF for the low affinity receptors (K(d) 3.5 X 10-9 M) and high affinity receptors (K(d) 2.7 X 10-10 M) are lower than those of hEGF (K(d) 1.2 X 10-9 M and 1.9 X 10-10 M respectively). In consequence, artefactually high results are obtained when mEGF is used for radioreceptor assays of EGF-like proteins in human biological fluids. Both species of EGF stimulated A431 cell growth at low concentrations and inhibited at high concentrations; however, hEGF enhanced cell growth over a wider concentration range (up to 1.6 nM), and had a higher maximal stimulating effect (38% vs. 25%).

Topics: Animals; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Radioligand Assay; Species Specificity; Tumor Cells, Cultured

The effects of epidermal growth factor (EGF) and transforming growth factor beta (TGF beta) on the growth of A431 epidermoid carcinoma cells were studied. Whereas the monolayer growth of A431 cells was inhibited by EGF, it was stimulated by TGF beta. Contrary to the effects on the monolayer growth, EGF stimulated the soft agar growth of A431 cells. The stimulatory effects of TGF beta on the anchorage-dependent growth were antagonized by EGF and those of EGF on anchorage-independent growth were antagonized by TGF beta. These results suggest that both factors not only convey the proliferative signals to A431 cells but also induce phenotypic changes, resulting in a preference for either anchorage-dependent or anchorage-independent growth. Moreover, as the stimulatory effects of EGF on the soft agar growth of A431 cells paralleled its reported stimulatory effects on their in vivo growth, it is also suggested that in vivo responses of cells to certain growth factors may correlate better with their responses in soft agar culture than with those in monolayer culture.

Topics: Agar; Carcinoma, Squamous Cell; Cell Adhesion; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Peptides; Transforming Growth Factors; Tumor Cells, Cultured

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Culture Media; Culture Techniques; Epidermal Growth Factor; Humans

Activation of the EGF receptor in A431 cells induces the hydrolysis of phosphoinositides and a transient rise of the cytosolic Ca2+ concentration, [Ca2+]i, which are completely inhibited by acute pretreatment with activators of protein kinase C, such as phorbol esters. Down regulation of the enzyme (by long-term pretreatment of the cells with phorbol esters) causes the [Ca2+]i response to EGF to increase in magnitude and, especially, to become much more persistent (average t1/2 of [Ca2+]i decline 9 min with respect to 2.3 min in controls). These results demonstrate that the activation of protein kinase C induced by EGF in intact A431 cells is sufficient to trigger a feed back, autolimitative regulation of the EGF receptor that might play a prominent physiological role in the definition of the mitogenic activity of the growth factor.

Topics: Calcium; Carcinoma, Squamous Cell; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Feedback; Inositol Phosphates; Kinetics; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Monoclonal antibodies which bind to the EGF receptor have the capacity to inhibit EGF-induced effects upon proliferation and biochemical functions in cultured human cells. Some EGF receptor-bearing tumor cells are prevented from growth by treatment with antireceptor antibody. Evidence is presented which suggests that antibody-mediated antiproliferative activity may result from effects upon growth factor-dependent processes in the receptor-bearing cells.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans

Transforming growth factors (TGFs) have been implicated in the pathogenesis of the hypercalcemia in malignancy (HM). In order to evaluate the role of these growth factors (epidermal growth factor (EGF) and TGF-alpha acting via the EGF receptor) in the development of HM, we studied the effect of 2 doses of EGF (0.1 and 0.3 microgram/g/day) given for 7 days as a continuous infusion on serum and urine calcium in athymic mice. These infusions had no effect on serum and urine Ca values in this study. In order to assess the biological activity of the infused EGF, other known effects on gastric and pancreatic weights were evaluated. EGF-infused animals had significantly greater gastric and pancreatic weights than controls. Thus, EGF infusion into mice in doses which elicited known biological effects failed to have an effect on serum and urine Ca. An infusion of bovine parathyroid hormone 1-34 at the dose of 0.1 microgram/g/day resulted in significant hypercalcemia.

Topics: Animals; Calcium; Carcinoma, Squamous Cell; Carnitine; Dose-Response Relationship, Drug; Epidermal Growth Factor; Hypercalcemia; Male; Mice; Mice, Nude; Thymectomy

Epidermal growth factor (EGF) induced the formation of thin sheetlike extensions (lamellipodia) and filamentous extensions at the edges of colonies of A431 cells. To determine the necessary processes for the induction of the morphological changes mediated by EGF, the effects of a variety of ions on these changes were examined. In a NaCl solution supplemented with CaCl2, MgCl2 and glucose, no EGF-induced morphological changes were observed. However, when the NaCl was replaced with LiCl, fingerlike extensions were formed, but sheetlike extensions were not. Addition of vanadate to the NaCl solution also induced fingerlike extensions in cells treated with EGF. In contrast, sheetlike lamellipodia were formed in EGF-treated cells by the addition of K+ or PO4(3-) to the NaCl solution or by the addition of PO4(3-) to the LiCl solution. These findings indicate that Li+, K+, PO4(3-) and vanadate are involved in the processes of EGF-induced morphological changes. Since vanadate and Li+ have been shown to inhibit phosphatases, an EGF-dependent phosphorylation step may play an important role in the induction of the morphological changes observed.

Topics: Carcinoma, Squamous Cell; Cell Line; Cytoplasm; Epidermal Growth Factor; Humans; Inositol; Lithium; Microscopy, Phase-Contrast; Phosphates; Potassium; Skin Neoplasms; Sodium; Vanadium

Glycosphingolipids added exogenously to 3T3 cells in culture were shown to inhibit cell growth, alter the membrane affinity to platelet-derived growth factor binding, and reduce platelet-derived growth factor-stimulated membrane phosphorylation (Bremer, E., Hakomori, S., Bowen-Pope, D. F., Raines, E., and Ross, R. (1984) J. Biol. Chem. 259, 6818-6825). This approach has been extended to the epidermal growth factor (EGF) receptor of human epidermoid carcinoma cell lines KB and A431. GM3 and GM1 gangliosides inhibited both KB cell and A431 cell growth, although GM3 was a much stronger inhibitor of both KB and A431 cell growth. Neither GM3 nor GM1 had any affect on the binding of 125I-EGF to its cell surface receptor. However, GM3 and, to a much lower extent, GM1 were capable of inhibiting EGF-stimulated phosphorylation of the EGF receptor in membrane preparations of both KB and A431 cells. Further characterization of GM3-sensitive receptor phosphorylation was performed in A431 cells, which had a higher content of the EGF receptor. The following results were of particular interest. (i) EGF-dependent tyrosine phosphorylation of the EGF receptor and its inhibition by GM3 were also demonstrated on isolated EGF receptor after adsorption on the anti-receptor antibody-Sepharose complex, and the receptor phosphorylation was enhanced on addition of phosphatidylethanolamine. (ii) Phosphoamino acid analysis of the EGF receptor indicated that the reduction of phosphorylation induced by GM3 was entirely in the phosphotyrosine and not in the phosphoserine nor phosphothreonine content. (iii) The inhibitory effect of GM3 on EGF-dependent receptor phosphorylation could be reproduced in membranes isolated from A431 cells that had been cultured in medium containing 50 nmol/ml GM3 to effect cell growth inhibition. The membrane fraction isolated from such growth-arrested cells was found to be less responsive to EGF-stimulated receptor phosphorylation. These results suggest that membrane lipids, especially GM3, can modulate EGF receptor phosphorylation in vitro as well as in situ.

Topics: Amino Acids; Animals; Carcinoma, Squamous Cell; Cattle; Cell Division; Cell Line; Depression, Chemical; Dogs; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; G(M1) Ganglioside; G(M3) Ganglioside; Gangliosides; Humans; Immunosorbent Techniques; Mice; Mouth Neoplasms; Ovarian Neoplasms; Phosphorylation; Phosphotyrosine; Receptors, Cell Surface; Tyrosine

Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Chloroquine; Chromatography, High Pressure Liquid; Epidermal Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Keratins; Receptors, Cell Surface; Time Factors

Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal growth factor (EGF) receptors. Since EGF has an important role in epithelial growth and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (2- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal keratinocytes, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the growth of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Cells; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Esophagus; Female; Humans; Japan; Keratins; Kinetics; Receptors, Cell Surface; South Africa; Vulvar Neoplasms

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Deoxyribonucleotides; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation; Humans; Keratins; Receptors, Cell Surface; RNA, Messenger

An association has been observed between squamous cell carcinomas and elevated EGF receptor levels. The approach taken here to analyze this problem was to construct vectors which express antisense RNA for the EGF receptor. The plasmid vector used was derived from pSV2-neo, which is readily selectable. Three EGF receptor cDNA fragments, a 5'-, a middle and a 3'-region were tested for differences in efficiency as antisense RNA. The results suggest that the 5'-fragment was the most efficient.

Topics: Carcinoma, Squamous Cell; Cell Line; DNA; DNA Restriction Enzymes; DNA, Recombinant; Epidermal Growth Factor; ErbB Receptors; RNA; RNA, Messenger; Transfection

A431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10(-3) M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells when compared to normal human keratinocytes. In other respects, EGF, which inhibits the proliferation of A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Membrane; Cells, Cultured; Culture Media; Epidermal Growth Factor; Epidermis; Female; Humans; Protein Precursors; Transglutaminases; Tretinoin

Southern blot-hybridization analysis of DNAs from human tumors demonstrated amplification of the epidermal growth factor (EGF) receptor gene in 10 of 12 squamous cell carcinoma cell lines tested and in none of 18 tumor cell lines of nonsquamous cell carcinomas. The degree of amplification in the squamous cells varied from 2- to 50-fold relative to the epidermal keratinocyte. Hybridization analysis of the RNA showed that the amplification of the EGF receptor gene is accompanied with an increase of the 5.6 kilobases of EGF receptor mRNA. Scatchard plot analysis and sodium dodecyl sulfate-polyacrylamide gel analysis of the EGF receptor revealed that the synthesis of the EGF receptor is also greater in the cells with amplified EGF receptor gene. In contrast, Southern blot analysis of DNAs of primary tumors showed that incidence of amplification of the EGF receptor gene in squamous cells (1 of 6) was almost as frequent as in nonsquamous cells (1 of 4). These results show that amplification of the EGF receptor gene is commonly found in various tumors. In addition, our data suggest that primary squamous cell carcinomas with amplified EGF receptor gene may readily adapt to growth in tissue culture.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation; Humans; Receptors, Cell Surface

The role of epidermal growth factor (EGF) and its receptors in human cancers was studied using 24 human cell cultures including 15 of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the growth and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal fibroblasts. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the growth of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs.

Topics: Carcinoma, Squamous Cell; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gene Amplification; Humans; Mouth Neoplasms; Proto-Oncogenes; Receptors, Cell Surface; Skin Neoplasms

We have initiated an in-depth, comprehensive study of non-SCLC in the expectation that improvements in clinical management and diagnosis of lung cancer patients will follow. We have established and characterized over 30 such tumors in long-term culture and as xenografts in athymic nude mice. These models usually retain the morphologic and other properties of the tumors from which they were derived. With the use of fully defined medium, most SCLC and adenocarcinomas can be established as long-term cultures. The growth conditions for squamous cell carcinomas have not been fully defined, and the success rate is low. In contrast to SCLC, there is considerable heterogeneity of non-SCLC tumors, with more than 12 phenotypes identified among the first 30 lines established. In addition, there is considerable overlap of properties, both within the non-SCLC types as well as between non-SCLC and SCLC. These findings indicate a common origin for all lung cancers. Radiation and drug sensitivity studies suggest that cell line data may correlate with clinical responses. Clinical protocols utilizing our ability to culture and drug test individual tumors are currently under way. They represent the first steps in a combined clinico-laboratory approach to the management of lung cancer.

Topics: Adenocarcinoma; Animals; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Division; Cell Line; DNA, Neoplasm; Epidermal Growth Factor; Humans; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Ploidies; Transplantation, Heterologous

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Survival; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Sera; Interphase; Kinetics; Mitosis; Neoplasm Proteins; Phosphorylation; Receptors, Cell Surface; Vinblastine

Specific epidermal growth factor (EGF) receptors were measured in RL95-2 human endometrial carcinoma cells. At 37 C, binding of 125I-labeled EGF was associated with marked ligand internalization. Maximal cell surface binding occurred within 10 min. Bound [125I]EGF was partially degraded to low mol wt products, and this degradation was blocked by the lysosomotropic compound ammonium chloride. At 4 C, maximal cell surface binding occurred at 3 h. Scatchard analysis of data obtained after 3 h at 4 C revealed a single order of binding sites with a Kd of 1.8 nM and approximately 150,000 surface receptors/cell. EGF, at a concentration of 0.83 nM, inhibited the proliferation of RL95-2 cells. Inhibition was reversible and was associated with morphological changes leading to a fusiform appearance of the growth-arrested cells. These findings indicate that RL95-2 human endometrial carcinoma cells bind, internalize, and degrade EGF in a manner similar to that described for other target cells for EGF action. The ability of EGF to inhibit the growth of RL95-2 cells supports the hypothesis that this type of inhibition is dependent on postreceptor events, rather than on the presence of an overabundance of EGF receptors.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Receptors, Cell Surface; Temperature; Uterine Neoplasms

The rotational diffusion of the complexes of epidermal growth factor (EGF) with its specific receptor on plasma membrane vesicles prepared from human epidermoid carcinoma A431 cells was studied using the time-resolved polarization of phosphorescence of erythrosin-labeled hormone. The measured rotational correlation times of 16-20 microseconds at 4 degrees C are consistent with monomeric freely diffusing EGF receptor. Upon increasing the temperature to 37 degrees C, the rate of rotational diffusion slows down as evidenced by an increase in the correlation time to 75 microseconds. This finding suggests that small clusters of the occupied EGF receptor (microaggregation) form at the higher temperature, a property we have reported previously for occupied receptors on living A431 cells. Subsequent cooling of the membranes leads to a partial reversal of the microaggregation. We conclude that clustering of occupied EGF receptors can proceed at 37 degrees C in the absence of metabolic energy and external interactions, e.g. with components of the cytoskeleton, and thus reflects inherent properties of the receptor protein in its natural environment. A lag phase in the time course of microaggregation observed with the isolated membrane preparations may reflect cooperativity in the process of receptor association.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Diffusion; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Luminescent Measurements; Male; Mice; Receptors, Cell Surface; Rotation; Submandibular Gland; Temperature

Both epidermal growth factor (EGF) and vanadate can activate 45Ca2+ influx into A431 epidermal carcinoma cells, without a detectable lag period possibly via a voltage-independent calcium channel. 22Na+/H+ exchange and 45Ca2+ uptake are mutually independent. Neither EGF nor vanadate induce any significant change in the steady-state levels of [1,3-3H]glycerol-labeled diacylglycerol, myo-[2-3H]inositol-labeled inositol trisphosphate or in 32P-labeled polyphosphoinositides or phosphatidic acid over the first 10 min of treatment, suggesting that the EGF receptor is not directly coupled to phosphatidylinositol turnover and that the two ion fluxes are not induced via a kinase C-dependent pathway. An increase in turnover of polyphosphoinositides can be detected in EGF-stimulated cells by nonequilibrium labeling with [32P]phosphate, but the increase shows a lag of about 1 min under the conditions used to detect 45Ca2+ influx. Chelation of free Ca2+ decreases but does not abolish the EGF-stimulated turnover. Preincubation with tetradecanoylphorbol acetate or 1-oleoyl-2-acetylglycerol inhibits the increase in 45Ca2+ uptake by both EGF and vanadate. Tetradecanoylphorbol acetate alone does not alter the basal rate of influx when added together with 45Ca2+. Surprisingly, the activation by vanadate and its inhibition by phorbol 12-myristate 13-acetate are unaffected by down-regulation of the EGF receptors through prior incubation with growth factor. Therefore, in A431 cells the activation of Na+/H+ exchange and Ca2+ influx appear to be independent of phosphatidylinositol turnover, and the EGF receptor does not itself function as a Ca2+ channel. Vanadate apparently activates influx through a mechanism distinct from or distal to the EGF receptor.

Topics: Calcium; Calcium Radioisotopes; Carcinoma, Squamous Cell; Cell Line; Diglycerides; Epidermal Growth Factor; Humans; Kinetics; Phosphatidylinositols; Protons; Sodium; Sodium Radioisotopes; Tetradecanoylphorbol Acetate; Vanadates; Vanadium

Treatment of [32P]phosphate prelabeled intact human A431 epidermoid carcinoma cells with epidermal growth factor (EGF, 100 ng/ml) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7)M) resulted in a selective enhancement in the phosphorylation of the following soluble acidic proteins: a phosphoprotein with a molecular weight of 17,000 (pp17; similar notation used throughout) pI approximately 5.5); pp27 (pI approximately 5.5); pp34 (pI approximately 6.2); and pp80 (pI approximately 4.5) as detected by two-dimensional gel electrophoresis. EGF or TPA induced a 4- to 6-fold increase in the phosphorylation of pp17 and a 2- to 4-fold increase in the phosphorylation of pp27, pp34, and pp80 within 15 min after treatment of subconfluent A431 cells. Alkali treatment of the gels removed most of the incorporated [32P] phosphate from the phosphoproteins, including pp27, pp34, and pp80; however, the phosphoester bond in pp17 was stable to alkaline hydrolysis since there was no removal of [32P]phosphate from this protein. Treatment of A431 cells with dibutyryl cyclic adenosine 3':5'-monophosphate (1 mM) also increased the phosphorylation of pp17, pp27, and pp34 but not of pp80. Activation of endogenous calcium- and phospholipid-dependent protein kinase C in the cytosol of A431 cells in a cell-free system resulted in the enhanced phosphorylation of pp27, pp34, and pp80 but not of pp17 while exogenous addition of the catalytic subunit of cyclic adenosine 3':5'-monophosphate-dependent protein kinase to cytosol preparations resulted in the phosphorylation of pp17, pp27, and pp34, but not of pp80. These results demonstrate that at least four soluble acidic proteins are phosphorylated in A431 cells in response to either EGF or TPA in vivo suggesting that these two agents may exert part of their biological effects on A431 cells through a biochemical pathway involving the phosphorylation of several common proteins; moreover, the studies suggest that these four acidic proteins may be substrates in vitro for protein kinase C and/or a cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

Topics: Bucladesine; Carcinoma, Squamous Cell; Cells, Cultured; Cytosol; Epidermal Growth Factor; Humans; Molecular Weight; Phorbols; Phosphoproteins; Phosphorylation; Protein Kinase C; Protein Kinases; Proteins; Tetradecanoylphorbol Acetate

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Fluorescein-5-isothiocyanate; Fluoresceins; Humans; Kinetics; Receptors, Cell Surface; Spectrometry, Fluorescence; Thiocyanates

Three biologically active monoclonal antibodies against the human epidermal growth factor (EGF) receptor (2E9, 2D11 and 2G5) have been used to analyse the interrelationship between various cellular responses to EGF. Antibody 2E9 (IgG1) is directed against the protein core of the receptor, close to or at the EGF binding site, while 2D11 (IgG3) and 2G5 (IgG2a) recognize blood-group A-related carbohydrate determinants of the receptor. These antibodies have EGF-like effects in that they can activate the receptor tyrosine kinase both in vitro and in vivo. Cross-linking of the receptor-bound antibodies by a second antibody mimics EGF in inducing a rapid aggregation of receptors on the cell surface. However, all three antibodies fail to mimic EGF in raising cytoplasmic pH and free Ca2+ and do not stimulate DNA synthesis in quiescent fibroblasts, even after external cross-linking of the occupied receptors. It is concluded that EGF-R tyrosine kinase activity as well as substrate specificity can be modulated by ligands other than EGF, even if they bind to sites distinct from the EGF binding domain; activation of the receptor tyrosine kinase, receptor clustering and induction of the ionic signals are causally unrelated events; and tyrosine kinase activation and receptor cross-linking are not sufficient for stimulation of DNA synthesis.

Topics: Amino Acids; Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Phosphorus Radioisotopes; Phosphorylation; Receptors, Cell Surface

Epidermal growth factor receptor (EGF-R) expression was assessed in 63 lung tumour samples with a monoclonal antibody (EGF-R1) by indirect immunoperoxidase staining on cryostat sections. All 15 small cell lung cancer samples were negative whereas over 80% of the 48 non small lung cancer stained positively. In 30 bronchial biopsies two monoclonal antibodies against the cytoplasmic part of the EGF-R were evaluated. These antibodies showed weaker staining than EGF-R1. No additional or enhanced staining as compared with EGF-R1 was observed, suggesting a lack of enhanced expression of a truncated EGF-R analogous to the v-erb-B oncogene product. Monoclonal antibodies against the EGF-R may be helpful diagnostically in differentiating small cell from non small cell lung cancer and may also be important in elucidating biological differences in primary lung cancer.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Middle Aged; Receptors, Cell Surface

Using specific antibodies directed against the external and internal domains of the epidermal growth factor (EGF) receptor, we have directly localized by the protein A gold technique at the electron microscopic level these receptor regions in A-431 epidermoid carcinoma cells. With all antibodies tested, 80-85% of the EGF receptors are found inside the cells, where they preferentially associate with lysosome-like structures, a tubulovesicular system, the rough endoplasmic reticulum and the nuclear envelope. The same distribution pattern is observed for antibodies directed against the external carbohydrate region of the receptor, an antibody against the protein core of the external segment of the receptor, and an antibody reacting with the internal kinase domain of the receptor, suggesting that both receptor segments are similarly distributed intracellularly.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Nucleus; Endoplasmic Reticulum; Epidermal Growth Factor; ErbB Receptors; Humans; Immunologic Techniques; Lysosomes; Microscopy, Electron; Mitochondria; Nuclear Envelope; Organoids; Receptors, Cell Surface

Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. We measured the activity of protein kinase C in A431 cells by determining the incorporation of [32P]phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in [32P] incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phosphorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the [32P] incorporation into threonine-654 reached 50% of the [32P] in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na+/H+ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, we measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na+/H+ exchange by hypertonic medium is independent of protein kinase C activity. Since stimulation of protein kinase C by phorbol diesters results in a decrease in EGF receptor activity, the stimulation of protein kinase C activity by addition of EGF to A431 cells contributes to a feedback mechanism which results in the attenuation of EGF receptor function.

Topics: Carcinoma, Squamous Cell; Cell Line; Culture Media; Diglycerides; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Feedback; Humans; Hypertonic Solutions; Phosphatidylinositols; Phosphorylation; Protein Kinase C; Threonine

We have identified a factor in nuclear extracts that binds to the c-fos enhancer. Treatment of A431 cells with epidermal growth factor results in a rapid increase in the level of transcription and a concomitant increase in binding of the factor to the enhancer. Surprisingly, as transcription decreases rapidly, the enhancer-binding activity remains elevated. In addition, although HeLa cells exhibit no detectable transcription of the c-fos gene, they contain significant amounts of binding activity, comparable to those in induced A431 cells. These results suggest that regulation of c-fos transcription involves more than simply an increased level of a factor capable of binding the enhancer. Finally, transcription of c-fos in A431 cells is markedly induced by the tumor promoter TPA and the calcium ionophore A23187, yet neither induced an increased level of the enhancer-binding activity. These agents thus appear to activate c-fos transcription via a mechanism distinct from that used by epidermal growth factors.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Line; DNA-Binding Proteins; DNA, Neoplasm; Enhancer Elements, Genetic; Epidermal Growth Factor; Genes, Regulator; HeLa Cells; Humans; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Tetradecanoylphorbol Acetate; Transcription, Genetic

Addition of epidermal growth factor (EGF) to human A431 cells causes a 2-4-fold increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured by quin-2 fluorescence. The EGF effect is rapid but transient: [Ca2+]i reaches a maximum within 30-60 s and then returns to its resting value (182 +/- 3 nM) over a 5-8-min period. The EGF-induced [Ca2+]i rise is completely dependent on extracellular Ca2+, is abolished by La3+ and Mn2+, and is not accompanied by changes in membrane potential (mean values of -64 mV). Serum also elicits a transient [Ca2+]i rise in A431 cells, but this response is not dependent on the presence of extracellular Ca2+. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate completely inhibits the EGF- and serum-induced increases in [Ca2+]i without affecting basal [Ca2+]i levels. Our results, together with previous 45Ca2+ uptake data (Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20, 6280-6286), suggest that while serum factors trigger the release of Ca2+ from internal stores, EGF acts by opening a voltage-independent Ca2+ channel in the plasma membrane. The data further suggest a role for protein kinase C in attenuating the Ca2+-mobilizing mechanisms of EGF and serum.

Topics: Aminoquinolines; Blood; Calcium; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Humans; Lanthanum; Manganese; Mathematics; Membrane Potentials; Potassium; Protein Kinase C; Tetradecanoylphorbol Acetate

Human epidermoid carcinoma A431 cells attach more rapidly to collagen type-I and -IV substrates than to surfaces coated with laminin or fibronectin. The diminished intercellular interaction and rounding up manifested when these cells are exposed to epidermal growth factor (EGF) in tissue culture plastic or collagen films is not shown when the assay is performed on 3-dimensional collagen. In the latter substrate, cells exposed to EGF reveal greater cell cohesion with interdigitations and desmosomal junctions, compared to the limited intercellular interaction detected in similarly treated cells assayed in tissue culture substrate. Although the protein synthesis inhibitor, cycloheximide, did not prevent these EGF-mediated changes, metabolic labelling indicated a substrate-dependent effect of EGF on the synthesis of proteins associated with the Triton-insoluble cytoskeletal matrix. The involvement of cytoskeleton components in the EGF effects on intercellular adhesion was shown by its susceptibility to cytochalasin B, known to disorganize actin-containing microfilaments. Some of the mechanisms of epithelial morphogenesis and the influence of extracellular matrix components on cell-receptor/growth-factor interactions may now be suitably analyzed by examining the EGF effects on A431 cells grown on different substrata.

Topics: Carcinoma, Squamous Cell; Cell Adhesion; Cell Communication; Cells, Cultured; Collagen; Cytochalasin B; Epidermal Growth Factor; Extracellular Matrix; Gels; Humans; Neoplasm Proteins; Polyethylene Glycols; Tritium; Trypsin

A squamous cell carcinoma of 33-yr-old patient who developed marked leukocytosis and hypercalcemia was transplanted into nude mice in which more marked leukocytosis and hypercalcemia also developed. This tumor (LJC-1-JCK) produced a colony-stimulating factor (CSF) and formed a cyst in the tumor from which a CSF-producing cell line (T3M-1) was established. The CSF causes predominantly formation of granulocytic colonies in addition to macrophage colonies. Bone-resorbing activity (BRA) was detected in the cystic fluid and was eluted as two separate peaks with proteins of an apparent molecular weight of 30,000-50,000 and 10,000-20,000. Colony-stimulating activity (CSA) was eluted at an apparent 30,000 mol wt. The conditioned medium of the T3M-1 cells also contained a BRA with an apparent 14,000 mol wt, whereas CSA eluted at an apparent 30,000 mol wt. PTH, epidermal growth factor, transforming growth factor-alpha, prostaglandin Es, and vitamin D could not account for the powerful BRA. In contrast to CSA, BRA was not inactivated by trypsin and more stable at 70 degrees C. When T3M-1 cells were transplanted into nude mice, marked hypercalcemia developed in addition to granulocytosis. Our findings suggest that the tumor produces and secretes a powerful BRA in vivo and in vitro, which is different from CSA in terms of molecular weight, heat stability, and trypsin treatment. We speculate that the synergistic action of CSF that stimulates macrophage colony formation and recruits osteoclast precursors, and BRA, which stimulates mononuclear phagocytes and/or osteoclasts were responsible for a marked increase in osteoclastic bone resorption and humoral hypercalcemia in the patient.

Topics: Animals; Bone Resorption; Carcinoma, Squamous Cell; Chromatography, Gel; Colony-Stimulating Factors; Culture Media; Epidermal Growth Factor; Exudates and Transudates; Hot Temperature; Humans; Hydrocortisone; Hypercalcemia; Indomethacin; Interleukin-1; Leukocytosis; Mice; Molecular Weight; Parathyroid Hormone; Prostaglandins; Prostaglandins E; Trypsin; Vitamin D

Using the adjacent histologically normal tissues obtained from the same patients as controls, six human lung tumors were studied for the activities of epidermal growth factor (EGF) receptor binding, and receptor autophosphorylation. There was a 1.2- to 2.8-fold increase in EGF receptor activities in lung tumors due to an increase in the number of receptors without changes in their affinity. The increase had no direct correlation with the degree of differentiation or the type of lung tumors. The elevated expression of EGF receptor may be one of the characteristics in lung tumors. Epidermal growth factor and its receptor also may play a role in the regulatory mechanisms during tumorigenesis.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lung Neoplasms; Phosphorylation

Terminal differentiation of normal and malignant keratinocytes is routinely determined by the ability of these cells to form cornified envelopes after incubation with a calcium ionophore. We have used the human squamous cell carcinoma, SqCC/Y1, to quantify cellular differentiation by the formation of detergent-insoluble protein. The methodology developed employs the metabolic labeling of detergent-insoluble cellular protein with [35S]methionine in the presence of a calcium ionophore. The ratio of filter-retainable radioactivity to that of total cellular protein was shown to be closely correlated to the results obtained by measuring the number of envelope-competent cells when cells were induced to enter a pathway of terminal differentiation in culture by serum deprivation or by treatment with hydrocortisone, and during the inhibition of maturation by either retinoic acid (RA) or epidermal growth factor (EGF). This way of measuring the degree of terminal differentiation of epidermal cells is a relatively simple one that readily allows the simultaneous measurement of multiple samples.

Topics: Blood; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Culture Media; Epidermal Growth Factor; Epidermis; Humans; Hydrocortisone; Membrane Proteins; Skin Neoplasms; Solubility; Tretinoin

The replication of human epidermoid carcinoma A431 cells is inhibited by epidermal growth factor (EGF), with 5 ng/ml of EGF causing 50% inhibition of cellular proliferation. EGF resistant clones isolated from A431 cells were able to replicate in the presence of 100 ng/ml of EGF. That this insensitivity to EGF was probably due to a decrease in the expression of EGF receptors (EGFR) on the cell surface was shown using an EGFR cDNA probe to detect a 68% to 85% decrease relative to parental cells in the amount of EGFR mRNA in the EGF resistant clones. A corresponding decrease in surface EGFR levels was also detected in EGF resistant clones as measured by 125I-EGF binding. Eighteen percent of A431 cells cultured in serum-free medium for 6 days entered a pathway of terminal differentiation, as expressed by the formation of envelope-competent cells, whereas EGF resistant clones exhibited a considerably greater capacity to mature, even when cultured in serum-containing medium. The findings suggest that the concentration of EGFRs is important for the capacity of epidermal cells to undergo terminal differentiation in vitro.

Topics: Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; Clone Cells; Drug Resistance; Epidermal Growth Factor; ErbB Receptors; Humans; Phenotype; RNA, Messenger

Synthesis of the epidermal growth factor (EGF) receptor has been analyzed in a series of variant A431 human epidermoid carcinoma cell clones reported to contain different amounts of EGF binding sites. The amount of EGF receptor protein, quantitated by immunoaffinity chromatography, and EGF receptor mRNA, quantitated by cDNA hybridization, were closely correlated to the extent of EGF receptor gene amplification. This correlation existed in variants selected for reduced EGF receptors and in revertants from those variants with increased EGF receptors. There was also a correlation between the frequency of translocation of chromosome 7, containing the EGF receptor gene, and EGF receptor protein. These results support gene amplification as the mechanism enhancing A431 cell EGF receptor protein and determining growth responses.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Chromosomes, Human, 6-12 and X; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Humans; Neoplasm Proteins; Phosphoproteins; Phosphotyrosine; Receptors, Cell Surface; RNA, Messenger; RNA, Neoplasm; Translocation, Genetic; Tyrosine

The epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells, A431, was found to be predominantly associated with the detergent-insoluble cytoskeleton, where it retained both a functional ligand-binding domain and an intrinsic tyrosine kinase activity. The EGF-R was constitutively associated with the A431 cytoskeleton; this association was not a consequence of adventitious binding. The EGF-R was associated with cytoskeletal elements both at the cell surface, within intracellular vesicles mediating the internalization of the hormone-receptor complex, and within lysosomes. The EGF-R became more stably associated with cytoskeletal elements after its internalization. The cytoskeletal association of the EGF-R was partially disrupted on suspension of adherent cells, indicating that alteration of cellular morphology influences the structural association of the EGF-R, and that the EGF-R is not intrinsically insoluble. Cytoskeletons prepared from EGF-treated A431 cells, when incubated with gamma-32P-ATP, demonstrated enhanced autophosphorylation of the EGF-R in situ as well as the phosphorylation of several high molecular weight proteins. In this system, phosphorylation occurs between immobilized kinase and substrate. The EGF-R and several high molecular weight cytoskeletal proteins were phosphorylated on tyrosine residues; two of the latter proteins were phosphorylated transiently as a consequence of EGF action, suggesting that EGF caused the active redistribution of the protein substrates relative to protein kinases. The ability of EGF to stimulate protein phosphorylation in situ required treatment of intact cells at physiological temperatures; addition of EGF directly to cytoskeletons had no effect. These data suggest that the structural association of the EGF-R may play a role in cellular processing of the hormone, as well as in regulation of the EGF-R kinase activity and in specifying its cellular substrates.

Topics: Carcinoma, Squamous Cell; Cell Line; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cell Surface; Tyrosine

Topics: ABO Blood-Group System; Animals; Antibodies, Monoclonal; Carbohydrate Conformation; Carbohydrate Sequence; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; Epitopes; ErbB Receptors; Glycoproteins; H-Y Antigen; Humans; Hybridomas; Mice; Receptors, Cell Surface

Antisera were prepared against three synthetic peptides with amino acid sequences identical to those surrounding the three major autophosphorylation sites of the epidermal growth factor (EGF) receptor. The affinity-purified antibodies reacted strongly in an enzyme-linked immunosorbent assay against the immunizing peptide but showed little cross-reaction with the other two phosphorylation site peptides. EGF receptors labelled by autophosphorylation could be specifically precipitated by each of the phosphorylation site antibodies. The antibodies recognised EGF receptors labelled at each of the autophosphorylation sites, indicating that they could bind to the immunizing sequences irrespective of their states of phosphorylation. The antibodies were able to inhibit EGF receptor autophosphorylation without affecting EGF-stimulated tyrosine kinase activity towards exogenous peptide substrates, suggesting that the kinase and autophosphorylation sites were in distinct domains. Immunofluorescent staining of A431 cells showed that the autophosphorylation site sequences resided inside the cell. The autophosphorylation sites were shown to be within a domain of 20 000 mol. wt. which could be cleaved from the receptor through limited proteolysis by the calcium-dependent protease, calpain. The position of cleavage of the EGF receptor by the protease was mapped to lie between residues 996 and 1059. These results are discussed in the context of a model for the structure and function of the human EGF receptor.

Topics: Amino Acid Sequence; Antibodies; Calpain; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Epitopes; ErbB Receptors; Fluorescent Antibody Technique; Humans; Methionine; Peptide Fragments; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Cell Surface; Sulfur Radioisotopes

A method is described for a rapid two-step purification of the membrane receptor for epidermal growth factor (EGF) from cultured human A-431 cells. After solubilization of the cells with Triton X-100, the receptor is immobilized on an immunoaffinity column containing a monoclonal antibody directed against the receptor. In the second step of purification, the receptor, eluted from the antibody column, is adsorbed and specifically eluted from a lectin-agarose column. The molecular species obtained is mainly the 170,000-dalton EGF receptor polypeptide. The activity of the pure receptor depends on the conditions used for the desorption from the immunoaffinity beads. High-yield elution is obtained with acidic buffer and the receptor so purified specifically binds EGF, but is devoid of the kinase activity. When the elution is done with alkaline buffers or with buffer containing urea, a fully active receptor kinase is purified (yield of 10%). The pure receptor binds 125I-EGF with a Kd of 4 X 10(-8) M and retains EGF-sensitive protein kinase activity which phosphorylates tyrosine residues on the receptor itself. An additional protocol is described for large-scale purification (yield of 55%) of EGF receptor for the analysis of its primary structure. In this procedure, the EGF receptor is first purified by immunoaffinity chromatography which is followed by preparative gel electrophoresis of the 32P internally labeled receptor to remove minor protein contaminants.

Topics: Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Epitopes; ErbB Receptors; Humans; Kinetics; Molecular Weight; Protein Kinases; Receptors, Cell Surface

4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment of KB cells at 37 degrees C rapidly induces a 50% reduction in epidermal growth factor (EGF) binding that is maximal by 30 min. EGF binding activity returns to the original value by 1 hr and remains constant for 2 hr thereafter. Using a polyclonal antibody directed against the cytoplasmic domain of the EGF receptor (EGF-R), we examined the fate of the receptor after PMA treatment. Immunofluorescent and electron microscopic localization of the EGF-R after PMA treatment demonstrated that about 50% of the receptor became internalized into endocytic vesicles (receptosomes) and Golgi-associated structures. Unlike EGF-induced internalization, PMA-induced internalization did not cause delivery of EGF-R to lysosomes or receptor degradation. Rather, receptor reappeared on the cell surface. No stimulation of EGF-R synthesis was observed after 1 hr of PMA treatment. Loss of cell surface binding correlated with the internalization of the EGF-R observed morphologically. A possible explanation for these observations is that PMA, an activator of protein kinase C, confers a signal sufficient for EGF-R clustering and internalization but not for transport to lysosomes.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Endosomes; Epidermal Growth Factor; ErbB Receptors; Golgi Apparatus; Histocytochemistry; Humans; Lysosomes; Phorbols; Protein Kinase C; Protein Kinases; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

The human epidermal growth factor receptor has been purified and partial amino acid sequence obtained. A synthetic oligonucleotide was used to select complementary DNA clones from placental and A431 clone banks. The nucleotide sequence of a 5.8 kilobase transcript was determined and used to predict the total amino acid sequence of the receptor. We have predicted a model for the receptor which has an external ligand binding domain of 621 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 542 amino acids having protein tyrosine kinase activity. The kinase autophosphorylation sites have been mapped onto the primary amino acid sequence. Analysis of protein sequence databases have shown that the erb-B oncogene of avian erythroblastosis virus has acquired part of the avian EGF receptor gene. The hypothesis has been proposed that transformation by this virus is the result of expression of a truncated EGF receptor which lacks the majority of the EGF binding domain and delivers a continuous proliferation signal to transformed cells. We describe here the production of polyclonal and monoclonal antibodies to selected synthetic peptides from the EGF receptor and v-erb B sequences. Antisera to sequences encompassing the three major sites of autophosphorylation and the putative ATP binding site all recognize the native EGF receptor molecule. We have used these reagents to test our model of EGF receptor structure and v-erb B function.

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cloning, Molecular; DNA; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; ErbB Receptors; Humans; Placenta; Receptors, Cell Surface

The post-translational processing of the epidermal growth factor receptor in human A431 epidermoid carcinoma cells has been investigated. By employing the affinity matrix epidermal growth factor Affi-Gel in conjunction with immunoprecipitation, it has been demonstrated that core oligosaccharide addition is essential for the acquisition of epidermal growth factor-binding activity. Furthermore, the initial 160-kDa translation product was observed to undergo a processing step by which ligand-binding activity was acquired with a half-time of approximately 30 min while exhibiting no apparent change in mobility on sodium dodecyl sulfate-polyacrylamide gels. This was shown not to involve the conversion of high-mannose chains to complex chains which have been capped with fucose and sialic acid. Possible explanations for this activation in terms of translocation of intermediates and/or formation of disulfide bonds are discussed.

Topics: Carbohydrate Metabolism; Carcinoma, Squamous Cell; Cell Line; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Weight; Protein Processing, Post-Translational; Receptors, Cell Surface; Tunicamycin

The cell-permeable diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), is shown to mimic the effect of tumor promoting phorbol diesters on epidermal growth factor (EGF) binding and action in intact cells. DiC8 inhibited the binding of [3H]phorbol dibutyrate to A431 cell monolayers indicating that the diacylglycerol interacts with the phorbol diester receptor. At 0.3 microM, DiC8 half-maximally inhibited the high affinity binding of 125I-EGF to A431 human epidermoid carcinoma cells. Scatchard analysis indicated that the inhibition of 125I-EGF binding was very similar to that observed in the presence of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). DiC8 also mimicked the action of PMA to increase the phosphorylation state of the EGF receptor in 32P-labeled cells. Phosphoamino acid analysis demonstrated that DiC8 and PMA caused an increase in the level of EGF-receptor phosphoserine and phosphothreonine, whereas EGF caused an increase in the level of phosphoserine, phosphothreonine, and phosphotyrosine. Phosphopeptide mapping of the EGF receptor showed that DiC8 and PMA enhanced the phosphorylation of the same tryptic peptides. DiC8 inhibited the EGF-dependent tyrosine phosphorylation of the EGF receptor in A431 cells in a similar manner to that observed with PMA. In further experiments with quiescent Swiss 3T3 fibroblasts, DiC8 mimicked the ability of PMA to stimulate the incorporation of [methyl-3H]thymidine synergistically with low concentrations of EGF. This result indicates that DiC8 will mimic the long-term effects of PMA to regulate mitogenesis and raises the possibility that it may be active in two stage carcinogenesis. As both DiC8 and PMA stimulate the Ca2+- and phospholipid-dependent protein kinase (C-kinase) in vitro, the results support the hypothesis that the activation of C-kinase is a critical component of phorbol diester action on EGF receptor modulation and cell proliferation.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Diglycerides; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Glycerides; Humans; Mice; Mitosis; Phorbol Esters; Phorbols; Phospholipids; Phosphoproteins; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

Addition of amiloride to A431 human epidermoid carcinoma cell membranes inhibited autophosphorylation of the epidermal growth factor (EGF) receptor. The tyrosine phosphorylation of histone H2B catalyzed by an affinity-purified preparation of EGF receptor was also inhibited by amiloride. The inhibition was noncompetitive with respect to histone but competitive with ATP, suggesting that amiloride may act as an ATP analogue which causes the formation of nonproductive enzyme-substrate complexes. The tyrosine phosphorylation of histone H2B catalyzed by the purified EGF receptor was inhibited by amiloride at concentrations identical to those previously reported to block EGF action on cell proliferation (Ki = 350 microM). Amiloride similarly inhibited the tyrosine phosphorylation of the human placental insulin receptor and the platelet-derived growth factor receptor of Swiss 3T3 cells. Immunoprecipitation of the EGF receptor from A431 cells labeled for 24 h with [32P]phosphate demonstrated that amiloride decreased the phosphorylation of the EGF receptor on serine and threonine residues and blocked the effect of EGF to cause phosphorylation of the receptor on tyrosine residues. Phosphoamino acid analysis of total cell proteins indicated that amiloride inhibited the increase in phosphotyrosine levels caused by EGF. We conclude that amiloride directly inhibits the tyrosine kinase activity of the receptors for EGF, insulin, and platelet-derived growth factor in in vitro and can mediate such actions in vivo. This effect of amiloride demonstrates that it is unsuitable as a drug to test the hypothesis that the stimulation of the Na+/H+ antiporter is essential for mitogenic signaling by growth factor receptors.

Topics: Adenosine Triphosphate; Amiloride; Animals; Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Histones; Humans; Kinetics; Mice; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazines; Receptor, Insulin; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; Sodium

The human epidermal growth factor (EGF) receptor is known to be homologous to the v-erb B oncogene protein of the avian erythroblastosis virus. Overexpression of the EGF receptor gene in A431 epidermoid carcinoma cells is due to gene amplification. In this study, a variety of squamous cell carcinomas were examined and one, SCC-15, contained high levels of the EGF receptor as determined by immunoprecipitation via an EGF receptor-specific polyclonal antibody. Using a cloned EGF receptor complementary DNA as a probe, the level of EGF receptor RNA was found to be elevated four-fold in SCC-15 relative to normal cultured keratinocytes. When the same probe was used to identify EGF receptor gene fragments on a genomic DNA blot, the SCC-15 cell line was shown to possess an EGF receptor gene copy number amplified four to five times. Gene amplification results in the enhancement in the level of the EGF receptor in several carcinomas and could be responsible for the appearance of the transformed phenotype in these cells.

Topics: Carcinoma, Squamous Cell; Cell Line; Cloning, Molecular; Epidermal Growth Factor; ErbB Receptors; Genes; Humans; Receptors, Cell Surface

Phorbol esters specifically reduce the binding of epidermal growth factor to surface receptors in intact cells, but not when added directly to isolated membranes. We show that after treatment of intact cells with phorbol myristate acetate, 125I-EGF binding is reduced in membranes prepared subsequently. High-affinity binding of 125I-EGF is modulated by an intracellular calcium-dependent regulatory process. Preventing calcium entry with EGTA or enhancing intracellular calcium with A23187 in intact cells modulates EGF receptor affinity in membranes isolated subsequently. Also, EGTA attenuates the usual inhibition of EGF binding caused by phorbol esters. Membrane preparations do not respond to phorbol ester treatment because the calcium- and phospholipid-dependent protein kinase C is removed or inactivated during membrane isolation. Reconstitution of unresponsive membranes with purified C kinase alters phosphorylation of the EGF receptor and restores the inhibitory effect of phorbol esters on 125I-EGF binding previously observed only in intact cells. Thus, activation of the Ca++-dependent enzyme, C kinase, modulates EGF receptor affinity, possibly via altered receptor phosphorylation.

Topics: Animals; Calcimycin; Calcium; Carcinoma, Squamous Cell; Cell Line; Egtazic Acid; Epidermal Growth Factor; ErbB Receptors; Phosphorylation; Protein Kinase C; Protein Kinases; Rats; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

Culture medium which was conditioned by tissue of a CE mouse breast tumor in vitro contained dose-dependent osteolytic activity. The osteolytic activity was not soluble in dichloromethane and ethylacetate, indicating that it was not attributable to vitamin D metabolites or prostaglandins. However, breast tumor-conditioned medium stimulated production and release of prostaglandin E2 from mouse calvaria in vitro, and the stimulation of bone resorption in vitro by breast tumor-conditioned medium was blocked by a dose of indomethacin that prevented stimulation of mouse calvarial prostaglandin E2 production and release. The resorptive activity of parathyroid hormone (PTH) was not affected by the same dose of indomethacin, suggesting that the osteolytic factor was not PTH. This was further supported by observation that mouse kidney cell cAMP production was stimulated by PTH, but not by the aqueous phase of ethylacetate-extracted breast tumor-conditioned medium. In addition to osteolytic activity, breast tumor-conditioned medium contained a dose-dependent bone cell mitogenic activity, demonstrated by the stimulation of [3H]thymidine incorporation into trichloroacetic acid-insoluble macromolecules and a corresponding increase in bone cell number in monolayer cultures of bone cells. Breast tumor-conditioned medium also contained a dose-dependent transforming growth factor-(TGF-) like activity as defined by its ability to transform anchorage-dependent growth of nontransformed cells to anchorage-independent growth. The TGF in breast tumor-conditioned medium did not compete with epidermal growth factor (EGF) for EGF receptor binding, but its transforming activity was greatly enhanced by EGF, indicating that it was a beta-type TGF. Both the osteolytic and mitogenic activities were nondialyzable, sensitive to reducing agent, and not removable by dichloromethane and ethylacetate extractions. Furthermore, the TGF activity was not removed by ethylacetate extraction. Thus, the possibility that these activities in breast tumor-conditioned medium might be mediated by the same molecule must be considered. In summary, our data suggest that the CE mouse mammary carcinoma cells produce and secrete into the culture medium an osteolytic factor which is neither PTH nor prostaglandin and which stimulates local synthesis in bone of prostaglandin E2 which in turn increases bone resorption in vitro.

Topics: Animals; Animals, Newborn; Bone and Bones; Bone Resorption; Calcium; Carcinoma, Squamous Cell; Cell Line; Culture Media; Cyclic AMP; Dinoprostone; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Kidney; Mammary Neoplasms, Experimental; Mice; Organ Culture Techniques; Osteolysis; Peptides; Prostaglandins E; Rats; Receptors, Cell Surface; Transforming Growth Factors

Cryo-ultramicrotomy in combination with immuno-gold labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. We have applied this method to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. However, both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative, whereas under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, gold particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multi-vesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the electron microscopic level, independent of the internalization process of labeled ligands.

Topics: Carcinoma, Squamous Cell; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Gold; Histocytochemistry; Humans; Immunochemistry; Microscopy, Electron; Receptors, Cell Surface

After a chance observation that multiple cutaneous papillomas and squamous cell carcinomas occurred in 2 adult mice heterozygous for the repeated epilation gene Er, we surveyed a panel of 10 +/+ (wild type) and 30 Er/+ (heterozygous) mice from birth to over 2 years of age. Homozygous Er/Er mice could not be included since their defect is lethal at birth. Whereas no cutaneous tumors developed in the +/+ mice, 20 of the Er/+ mice, males and females, had developed 1-5 cutaneous papillomas and at least 1 cutaneous invasive squamous cell carcinoma by 2 years of age. No lesions were seen in mice younger than 6 months old. Although almost all Er/+ mice died with their tumor burden, no metastases have yet been proven histologically. The Er/+ mouse should serve as a useful model for the exploration of genetic factors in cutaneous squamous cell carcinomas in humans.

Topics: Alopecia; Animals; Antigens, Viral; Carcinoma, Squamous Cell; DNA, Neoplasm; DNA, Viral; Epidermal Growth Factor; Female; Genes, Lethal; Heterozygote; Male; Mice; Mice, Mutant Strains; Neoplasms, Multiple Primary; Papillomaviridae; Rodent Diseases; Skin Neoplasms

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.

Topics: Amino Acids; Animals; Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney; Kinetics; Melanoma; Molecular Weight; Neoplasm Proteins; Peptides; Rats; Receptors, Cell Surface; Transforming Growth Factors

Specific binding of 125I-labeled epidermal growth factor (EGF) was measured in 62 skin tumors of different severity. Within a group of 28 benign tumors, 11 of 15 condylomata acuminata were receptor positive, whereas the investigated mesenchymal tumors and normal skin as a control were receptor negative. 6 of 18 basal cell epitheliomas bound EGF specifically. In the group of precancerous and malignant skin tumors, 7 of 8 squamous cell carcinomas had the highest number of EGF binding sites and a high affinity state, whereas 5 malignant melanomas were receptor negative. The clinical relevance of these findings is not yet clear due to the short follow-up of the patients.

Topics: Binding Sites; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Condylomata Acuminata; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Melanoma; Receptors, Cell Surface; Skin; Skin Diseases; Skin Neoplasms

This study investigated a possible role for the epidermal growth factor (EGF) receptor protein tyrosine kinase in phosphoinositide metabolism with plasma membrane vesicles from human epidermoid carcinoma (A431) cells. We found a novel chloroform-soluble product radiolabeled with [gamma-32P]ATP that did not migrate from the origin in the thin layer system designed to separate the phosphoinositides, appeared as a single band of Mr = 3500 on polyacrylamide gels in the presence of dodecyl sulfate, had an ultraviolet absorbance spectrum with a maximum at 275 nm and stained with Coomassie dye. Based on these properties this phosphorylation product is referred to as a proteolipid. The 32P label was not detected in phosphotyrosine [Tyr(P)], phosphoserine [Ser(P)] or phosphothreonine [Thr(P)] and was lost during acid or base hydrolysis. Phosphorylation of proteolipid was increased significantly by EGF, whereas phosphorylation of phosphatidic acid was decreased and labeling of phosphoinositides was unaffected. Thus, it appears that in A431 membranes the EGF receptor/kinase does not utilize phosphatidylinositol as a substrate, but does phosphorylate a membrane proteolipid.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chloroform; Chromatography, Thin Layer; Epidermal Growth Factor; Humans; Kinetics; Membrane Lipids; Phosphatidylinositols; Phosphorus Radioisotopes; Phosphorylation; Solubility

The epidermal growth factor (EGF) receptor is a transmembrane polypeptide of 170 000 daltons (Da) with a cytoplasmically facing protein kinase domain. The regulation of the tyrosine kinase activity of the EGF receptor by added EGF and by receptor association state was studied in an in vitro system. The rate of autophosphorylation of the solubilized and purified EGF receptor was found to be independent of receptor concentration. To determine whether the zero-order kinetics observed point to intrapeptide phosphorylation, we measured the sedimentation characteristics of the undenatured solubilized receptor. The receptor was found to exist in two association-dissociation states-a monomeric 7.7S form and a dimeric 12S form. The 7.7S form is an active tyrosine kinase; it has high basal activity, and the activity is not further stimulated by EGF; it appears to be an EGF-independent form of the receptor kinase. The 12S form is devoid of catalytic activity, but in the presence of EGF it dissociates into the active monomeric form. Freshly purified receptor preparations contain mainly the monomeric receptor, have high basal kinase activity, and show low EGF stimulatability (less than 1.3-fold). Aging of the receptor results in progressive dimerization and decay of EGF-independent kinase activity (and increase in EGF stimulatability). All of these processes are reversed in the presence of EGF or dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Macromolecular Substances; Mice; Models, Biological; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface; Submandibular Gland

Human squamous cell carcinoma tissue was screened to determine the epidermal growth factor (EGF) receptor level. In 9 out of 15 cases, increased EGF binding was observed relative to normal adjacent tissue. In two tissue samples (SCE1 and SCL1), amplification of the EGF receptor gene and increased mRNA level were also observed. In two other cases (SCE2 and SCL3), EGF receptor gene amplification did not occur. We propose that there is an alternative mechanism for EGF receptor hyperproduction, independent of gene content, active in these tissues. A possible role of EGF receptor hyperproduction is envisioned in human neoplasia.

Topics: Carcinoma, Squamous Cell; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gene Amplification; Genes; Humans; Lung Neoplasms; Receptors, Cell Surface; RNA, Messenger; RNA, Neoplasm

The receptor for epidermal growth factor (EGF) in the human epidermoid carcinoma cell line A-431 is a glycoprotein of apparent molecular weight = 170,000. During biosynthesis, the receptor is first detected as a precursor of apparent Mr = 160,000. In this report we describe our studies on the structures of the oligosaccharide moieties of the mature receptor and its precursor. A-431 cells were grown in medium containing radioactive sugars and the radiolabeled receptors were purified by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiolabeled glycopeptides were prepared from the purified receptor by proteolysis, and their structures were examined by a variety of techniques. The mature EGF receptor contains both complex-type and high mannose-type Asn-linked oligosaccharides in the approximate ratio of 2 to 1, while the precursor contains only high mannose-type chains. A number of experimental results demonstrate that the mature receptor does not contain oligosaccharides in O-linkage through N-acetylgalactosamine to either serine or threonine. The high mannose-type oligosaccharides in both precursor and mature receptor can be cleaved by endo-beta-N-acetylglucosaminidase H and occur in the mature receptor as Man9GlcNAc2 (6%), Man8GlcNAc2 (49%), Man7GlcNAc2 (25%), and Man6GlcNAc2 (20%), whereas, in the receptor precursor the high mannose chains occur primarily as Man8GlcNAc2 (70%). The complex-type oligosaccharides in the mature receptor are predominantly tri- or tetraantennary species and are unusual in several respects. (i) Many of the chains do not contain sialic acid, while the remaining chains contain 1-2 sialic acid residues. (ii) Half of the [3H] mannose-derived radioactivity was recovered as [3H] fucose and the remaining half as [3H] mannose, indicating that there may be an average of 3 fucose residues/chain. (iii) About one-third of the [3H] glucosamine-derived radioactivity in these glycopeptides was recovered as N-acetylgalactosamine and these residues are all alpha-linked and occur at the nonreducing termini. These data demonstrate that the complex-type Asn-linked oligosaccharides in the EGF receptor from A-431 cells contain sugar residues related to human blood type A. In light of other recent studies, these results suggest that in A-431 cells blood group determinants in surface glycoproteins are contained in Asn-linked but not O-linked oligosaccharides.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Glucosamine; Glycopeptides; Glycoside Hydrolases; Humans; Mannose; Molecular Weight; Oligosaccharides; Receptors, Cell Surface; Tritium

We have isolated and partially purified an intracellular vesicle fraction from A-431 cells that contains both epidermal growth factor (EGF) and enzymatically active EGF:receptor/kinase. Exposure of intact A-431 cells to EGF leads to an accumulation of both EGF and kinase activity in this vesicle fraction. The accumulation is time- and temperature-dependent and is blocked by inhibitors of energy production. The EGF receptor in internalized vesicles is capable of autophosphorylation and, in the presence of Ca2+, of phosphorylation of the previously isolated 35-kDa protein (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). The demonstration of an EGF-induced increase in kinase activity of an internalized vesicle fraction lends credence to the hypothesis that EGF-induced endocytosis of the receptor is of physiological significance in the response of cells to this ligand. In addition, these results are consistent with the suggestion that the phosphorylation of the 35-kDa protein is associated with internalization of the EGF:receptor/kinase complex.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Weight; Peptide Fragments; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Trypsin

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Mouth Neoplasms; Oncogenes; Receptors, Cell Surface

The effect of autophosphorylation and protein kinase C-catalyzed phosphorylation on the tyrosine-protein kinase activity and ligand binding affinity of the epidermal growth factor (EGF) receptor has been studied. Kinetic parameters for the phosphorylation by the receptor kinase of synthetic peptide substrates having sequences related to the 3 in vitro receptor autophosphorylation sites (tyrosine residues 1173 (P1), 1148 (P2), and 1068 (P3)) were measured. The Km of peptide P1 (residues 1164-1176) was significantly lower than that for peptides P2 (residues 1141-1151) or P3 (residues 1059-1072). The tyrosine residue 1173 was also the most rapidly autophosphorylated in purified receptor preparations, consistent with previous observations for the receptor in intact cells (Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485). Variation in the extent of receptor autophosphorylation from 0.1 to 2.8 mol of phosphate/mol of receptor did not influence kinase activity or EGF binding affinity either for purified receptor or receptor in membrane preparations. Phosphorylation of the EGF receptor by protein kinase C was shown to cause a 3-fold decrease in the affinity of purified EGF receptor for EGF and to reduce the receptor kinase activity. In membrane preparations, phosphorylation of the EGF receptor by protein kinase C resulted in conversion of high affinity EGF binding sites to a low affinity state. This suggests that activation of protein kinase C by certain growth promoting agents and tumor promoters is directly responsible for modulation of the affinity of the EGF receptor for its ligand EGF. The regulation of the EGF receptor function by protein kinase C is discussed.

Topics: Amino Acids; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Phosphorylation; Protein Binding; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface

The effect of self-phosphorylation on the protein-tyrosine kinase activity of the epidermal growth factor receptor has been investigated using immunoaffinity-purified protein. Enzyme was first incubated for various times with excess ATP to phosphorylate it to differing extents; the ability of the enzyme to phosphorylate exogenous peptide substrates was then measured as a function of its self-phosphorylation state. Increasing self-phosphorylation to 1.3-1.8 mol of phosphate mol-1 of epidermal growth factor receptor enhanced protein-tyrosine kinase activity 2-3-fold. Comparison of the kinetics of protein-tyrosine kinase activity at different ATP concentrations revealed significant differences between unphosphorylated and phosphorylated enzyme. At low levels of ATP, a double reciprocal plot of the protein-tyrosine kinase activity of the unphosphorylated enzyme was hyperbolic, suggesting that ATP may act as an activator of the enzyme. At higher ATP concentrations, where greater levels of self-phosphorylation occurred during the reaction, the kinetics appeared linear and similar to those of the phosphorylated enzyme. Dose-response studies using three different peptide substrates (angiotensin II, gastrin, and a synthetic peptide corresponding to the self-phosphorylation site in p60v-src) showed that exogenous substrates inhibit receptor self-phosphorylation. In each case, half-maximal inhibition was observed at a peptide concentration approximately equal to the substrate's Km. A kinetic analysis comparing peptide phosphorylation using unphosphorylated and prephosphorylated enzyme indicated that the self-phosphorylation site can act as a competitive inhibitor (alternate substrate) versus peptide substrates. These results suggest that self-phosphorylation of the epidermal growth factor receptor removes a competitive constraint so that exogenous substrates can be more readily phosphorylated.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Cell Surface; Substrate Specificity

Two established human tumor cell lines, epidermoid carcinoma line A431 and glioblastoma line SF268, were studied to compare the interaction of each with epidermal growth factor (EGF). SF268 cells bound [125I] EGF with 35-40 fold higher affinity than did the A431 cells. The EGF binding sites of both lines were photoaffinity labeled using 2,4-NAPS-[125I] EGF, a photoreactive derivative of EGF. Extracts of photolysed cells analyzed by SDS-PAGE showed a difference between the two cell lines in the high molecular weight component corresponding to the EGF receptor. EGF in a dose range from 0.3-200 nM had no effect on thymidine incorporation by SF268 cells, whereas thymidine incorporation by A431 cells was markedly inhibited by EGF.

Topics: Affinity Labels; Binding Sites; Carcinoma, Squamous Cell; Cell Line; Chromatography, Gel; Chromatography, High Pressure Liquid; DNA Replication; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Photochemistry; Receptors, Cell Surface

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Differentiation; Cells, Cultured; Child; Epidermal Cells; Epidermal Growth Factor; Epidermis; ErbB Receptors; Humans; Receptors, Cell Surface; Skin Neoplasms

A monoclonal antibody specific for human EGF receptors was cross-linked to subunit A of toxic ricin. Using this conjugate, we isolated a variant of A431 cells, designated C1-B7, with approximately 40 times less EGF binding capacity. Unlike the parental cells, the C1-B7 variant was resistant to EGF-induced suppression of cell growth. The EGF receptors retained in this variant were of high-affinity type and susceptible to EGF-induced autophosphorylation. Membrane prepared from C1-B7 cells was highly phosphorylated in the presence of 2 microM [gamma-32P]-ATP, primarily on the lipid components shown as phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. This same level of lipid phosphorylation was observed on A431 membrane only in the presence of higher ATP concentrations. After addition of EGF to A431 membrane, phosphatidylinositol phosphorylation was significantly decreased with a concomitant increase in EGF-dependent protein phosphorylation. Thus, the EGF-dependent receptor-mediated protein phosphorylation precedes phosphatidylinositol phosphorylation. These observations support the idea that the growth inhibitory effect of EGF on A431 cells is caused by high ATP consumption due to the EGF-induced protein phosphorylation and reduction of phosphatidylinositol turnover.

Topics: Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Separation; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Genetic Variation; Humans; Kinetics; Phosphatidylinositols; Phosphorylation; Receptors, Cell Surface; Ricin

Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor.

Topics: Animals; Calpain; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Kinetics; Mice; Molecular Weight; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Cell Surface

In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell-surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo-ultramicrotomy in combination with immuno-gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell-surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label-efficiency. The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno-gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface-replication method involves, however, dehydration and critical-point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO4 fixation and dehydration caused a significant loss of cell-associated antibodies. This disadvantage is overcome by freeze etching and the label-fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell-surface located EGF-receptors.

Topics: Antigen-Antibody Reactions; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cytoplasm; Epidermal Growth Factor; ErbB Receptors; Freeze Etching; Frozen Sections; Gold; Histocytochemistry; Humans; Microscopy, Electron; Receptors, Cell Surface; Staphylococcal Protein A

Because epidermal growth factor (EGF) can modify cell proliferation in the gastrointestinal tract, effects of EGF were studied on the development of colonic, rectal, and anal neoplasms in male mice treated with 1,2-dimethylhydrazine (DMH) (20 mg/kg/wk for 20 weeks). DMH treatment caused a 13% increase in colonic RNA content, a 16% increase in DNA content, and 28% greater crypt depth. EGF (5 micrograms on alternate days during weeks 20 to 22) administered to DMH-treated mice produced no additional changes in colonic mucosa. At 30 weeks colorectal tumors were present in 13 of 20 mice treated with DMH (mean number of tumors per mouse 2.3 +/- 0.5) and 18 of 24 mice (mean 2.6 +/- 0.7) treated with DMH and EGF. Anal tumors were present in two of 20 DMH-treated mice (mean 0.1 +/- 0.07) but in eight of 24 DMH-EGF-treated mice (mean 0.33 +/- 0.1) (X2 = 4.84; p less than 0.05 for prevalence). Although EGF in this dose has no effect on the frequency of colorectal adenocarcinomas, the frequency of anal squamous cell carcinomas is increased more than three fold.

Topics: 1,2-Dimethylhydrazine; Adenoma; Animals; Anus Neoplasms; Body Weight; Carcinoma, Squamous Cell; Dimethylhydrazines; DNA, Neoplasm; Epidermal Growth Factor; Male; Methylhydrazines; Mice; RNA, Neoplasm

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) on type-IV-procollagen (basement-membrane collagen) and laminin synthesis, turnover, and secretion was studied in human A431 squamous epidermoid carcinoma cells. Type-IV procollagen and laminin were biochemically and immunologically identified in the medium and cell extracts using immuno-precipitation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. EGF or TPA produced a sixfold increase in type-IV-procollagen and laminin secretion within 2 h; this was accompanied by a three- to fourfold increase in the levels of cell-associated type-IV procollagen and laminin, respectively. The level of type-IV-procollagen and laminin synthesis and secretion remained elevated for at least 16 h after the administration of either EGF or TPA. A combination of EGF and TPA was more effective than either agent alone in promoting the secretion of laminin but not of type-IV procollagen. EGF and/or TPA did not, however, produce a selective increase in the synthesis of collagen and laminin, since total protein synthesis was also increased to the same degree by these agents. As determined by labeling and chase studies, neither EGF nor TPA had any appreciable effect upon type-IV-procollagen or laminin degradation. These results indicate that the synthesis of components associated with the basement membrane in A431 cells (i.e., type-IV procollagen and laminin) can be rapidly modulated by EGF and a tumor promoter, i.e., TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Basement Membrane; Carcinogens; Carcinoma, Squamous Cell; Cell Division; Cell Line; Drug Synergism; Epidermal Growth Factor; Epithelium; Extracellular Matrix; Humans; Laminin; Neoplasm Proteins; Phorbols; Procollagen; Tetradecanoylphorbol Acetate; Time Factors

Growth of the human epidermoid carcinoma cell line A431 in vitro is stimulated by low concentrations of epidermal growth factor (EGF; 0.1-10 pM) and inhibited by high concentrations (0.1-10 nM). This cell also grows as a solid tumor in female athymic mice. Sustained high levels of EGF in vivo can be achieved by the administration of testosterone to female mice via a cholesterol-based pellet inserted subcutaneously. This chronic elevation of EGF levels (serum concentration = 90 ng/ml), however, does not affect growth of the tumor. In contrast, low levels of the growth factor (0.5 micrograms/g body wt by injection 5 times/week; serum concentration = 8.25 ng/ml) stimulate growth of the tumor. These data suggest that the mechanism(s) involved in the inhibition of A431 cell growth by EGF in vitro does not function in vivo and the physiologically significant effect of EGF in vivo is growth promotion.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Female; Mice; Mice, Nude; Testosterone

Immunohistochemical identification of epidermal growth factor (EGF) is described in experimental carcinoma of the submandibular gland of mice given testosterone before sacrifice. EGF in the submandibular gland was confined to the granular convoluted tubule (GCT) cells, and its level was enhanced following testosterone injection. In the initial phase of carcinogenesis of the gland, degranulation of the GCT cells occurred as well as decreased EGF staining in the degranulated cells. In the testosterone-treated animals, changed GCT cells showed intense EGF deposition. Histological aspects during carcinogenesis in submandibular glands indicated duct-like structures, squamous metaplasia, and squamous cell types of carcinoma with different keratinization. Immunohistochemically detectable EGF was characterized by positive staining in pre-neoplastic or early neoplastic epithelial structures in testosterone-treated mice. However, tumour epithelia did not show any EGF reaction.

Topics: Animals; Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Histocytochemistry; Male; Mice; Mice, Inbred Strains; Salivary Gland Neoplasms; Submandibular Gland Neoplasms; Testosterone

Addition of epidermal growth factor (EGF) to cultures of A431 human epidermoid carcinoma cells produces an increase in the rate of intracellular protein breakdown that cannot be accounted for by increased proteolysis in lysates from EGF-treated cells. In support of this observation, inhibition of protein synthesis with cycloheximide does not reduce the EGF response in cell monolayers. On the other hand, inhibitors of lysosomal proteolytic function such as leupeptin, vinblastine and especially the weak base, ammonia, are able to block the ability of EGF to increase protein breakdown. Additional results suggest that the EGF effect is mediated via a stimulation of autophagy. First, the autophagocytosis inhibitor, 3-methyladenine, reduces the EGF response, and second, the ability of insulin to inhibit protein breakdown by preventing the formation of autophagic vacuoles is overcome by EGF. Moreover, the actions of inhibitors and competing hormones are similar to those reported for glucagon, a hormone known to increase autophagy. The EGF response on protein breakdown persists for at least 6 h after thorough washing of the A431 monolayers. This result contrasts with the rapid reversal of EGF effects in other cell lines. Examination of the fate of bound EGF in cells washed and incubated for 2 h at 37 degrees C shows that some 500-fold more EGF per mg protein is retained on the surface of A431 cells compared to AG2804-transformed fibroblasts, a difference which probably explains the unusual persistence of the EGF effect on protein breakdown.

Topics: Adenine; Ammonium Chloride; Autolysis; Carcinoma, Squamous Cell; Cell Line; Cycloheximide; Epidermal Growth Factor; Fibroblasts; Humans; Hydrogen-Ion Concentration; Insulin; Leupeptins; Lung; Proteins; Time Factors; Vinblastine

Previous studies have documented the activation of Na+/H+ exchange in A431 cells by the addition of epidermal growth factor or serum (Rothenberg et al., 1983b). Here we show that exposure of A4 31 cells to medium of increased osmolarity also leads to activation of Na+/H+ exchange and to an increase in intracellular pH (pHi), which under a variety of conditions displays similar kinetics to that observed upon addition of mitogens to the cells. Measurements of cell volume using the 3-0-methylglucose equilibration technique clearly show that mitogens do not activate Na+/H+ exchange by an osmotic mechanism (i.e., a decrease in cell volume). In fact, mitogens can induce further intracellular alkalinization if added to cells which have been shrunken in hypertonic medium. Activation of the Na+/H+ antiport does not lead to an obligatory change in pHi. Addition of epidermal growth factor of hypertonic solution to A431 cells in bicarbonate buffer activates Na+/H+ exchange without a concomitant increase in pHi. Under these conditions the increased proton efflux via Na+/H+ exchange must therefore be compensated by other mechanisms that control cytoplasmic pH.

Topics: Bicarbonates; Buffers; Carbon Dioxide; Carcinoma, Squamous Cell; Cell Count; Cell Line; Epidermal Growth Factor; Humans; Hydrogen; Hydrogen-Ion Concentration; Hypertonic Solutions; Ion Exchange; Mitogens; Osmolar Concentration; Sodium

Addition of epidermal growth factor (EGF) to quiescent cultured cells was found to stimulate the activity of glycogen synthase (UDPglucose:glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11), an enzyme subjected to regulation by covalent modification. In Swiss mouse 3T3 cells, the activation by EGF paralleled the effect seen with insulin; the time course and dose-response curves of the two polypeptide factors were similar. Stimulation of enzyme activity ratio [(activity in the absence of glucose 6-phosphate)/(activity in the presence of glucose 6-phosphate)] was maximal after 20-30 min of incubation. Both factors caused a maximal stimulation of 2.5-fold in synthase activity ratio at approximately equal to 10 nM, and the half-maximal effect was observed at 0.1-1 nM. Insulin and EGF exhibited partial additivity in effecting this enzyme activation. In contrast, human A431 cells showed no response to insulin. Although quantitatively different, the EGF effect in the latter cells was time dependent, reaching a maximum at 90 min, and dose dependent, with a maximal stimulation of 4-fold in synthase activity ratio at 10 nM. Half-maximal effect was observed at 0.3 nM EGF. Direct quantitation of allosteric effectors (glucose 6-phosphate, adenine nucleotides, and Pi) present in the enzyme assay mixtures indicated that the observed activation was not simply a consequence of changes in metabolite concentrations. These results suggest that EGF may be important in regulating glycogen synthesis through phosphorylation/dephosphorylation mechanisms.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cells, Cultured; Epidermal Growth Factor; Glycogen Synthase; Humans; Insulin; Kinetics; Mice

We have used a variety of methods, including lactoperoxidase-catalyzed iodination, proteolysis, and photolabel incorporation, to determine whether exposure to the acidic pH encountered during receptor-mediated endocytosis causes observable conformational changes in receptor proteins. Two receptor systems were chosen for this study: the asialoglycoprotein receptor and the epidermal growth factor (EGF) receptor. The purified asialoglycoprotein receptor protein was reconstituted into lipid membranes by spontaneous incorporation into phosphatidylcholine liposomes with the binding site facing outward. The EGF receptor was studied in living A-431 cells and was identified by immunoprecipitation using monoclonal antibodies. Lactoperoxidase-catalyzed iodination of both receptor systems, carried out with the external pH equal to 7.4 or 5.6, showed that the extent of receptor protein iodination was less at the lower pH even though lactoperoxidase has an acidic pH optimum. Using the nonspecific hydrophilic photolabeling agent [35S]N-(4-azido-3-nitrophenyl)-2-aminoethylsulfonic acid-taurine, we observed less incorporation into both the asialoglycoprotein receptor in liposomes and the EGF-receptor in A-431 cells when the external pH was reduced to 5.6. Also, using the enzyme papain, we have found that both receptors become resistant to proteolysis when the external pH is lowered from 7.0 to 5.6. These results suggest a conformational change in both of these receptors in which they become less exposed to the external aqueous environment at low pH. Such a conformational change may be responsible for the pH dependence of binding for both of these ligands. Also, this conformational change may serve to protect receptors from enzymatic degradation within endocytic or lysosomal compartments.

Topics: Asialoglycoprotein Receptor; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrogen-Ion Concentration; Kinetics; Liposomes; Membrane Proteins; Molecular Weight; Orosomucoid; Protein Conformation; Receptors, Cell Surface

The metabolism of the receptor for epidermal growth factor (EGF) in A-431 cells has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. The rate of EGF receptor degradation (t1/2 = 20 hr) was faster than the rate of degradation of total cell protein (t1/2 = 52 hr). When EGF was added at the beginning of the chase, the half-life of prelabeled receptor decreased to 8.9 hr. This decrease was specific, as the level of total cellular protein and another plasma membrane protein, the transferrin receptor, were relatively unaffected by EGF. The carbohydrate portion of the receptor is degraded, in the presence or absence of EGF, at approximately the same rate as the protein moiety. The amount of EGF receptor protein in A-431 cells has been quantitated by radiolabeling total cellular protein and quantitating the immunoprecipitable receptor. The EGF receptor constitutes approximately 0.15% of the total cell protein in A-431 cells. These cells, therefore, have approximately 30 times more EGF receptor protein than fibroblasts. The EGF receptor constitutes an even higher proportion of 3H-glucosamine- or 3H-mannose-labeled macromolecules in A-431 cells, 1.5% or 5.2%, respectively. The EGF receptor from A-431 cells can easily be identified by submitting carbohydrate-labeled, solubilized cells to electrophoresis as described by Laemmli (1970).

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Glucosamine; Kinetics; Mannose; Receptors, Cell Surface; Receptors, Transferrin; Time Factors

Epidermal growth factor (EGF) promotes the growth of cultured benign and malignant cells. Recent studies have demonstrated that the amount of EGF receptor is elevated in squamous cell carcinoma cells in tissue culture when compared with normal epidermal cells. This study demonstrates that elevated levels of EGF receptor are detected in biopsy specimens of human squamous cell carcinomas of the lung with a murine monoclonal antibody, EGF-R1, which binds specifically to the receptor. The increased receptor ranged from 2.5- to 5-fold that of normal skin. These findings have been observed in 11 of 11 squamous carcinomas and two of two epidermoid head and neck cancers. Seven of eight adenocarcinomas, two of two small cell, and four of eight undifferentiated lung cancers had negligible amounts of EGF receptor. The EGF receptor antibody did not bind significantly to normal lung tissues and 35 nonepidermoid tumors. Therefore, EGF receptor may be an excellent marker for epidermoid malignancies.

Topics: Adenocarcinoma; Autoradiography; Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Lung Neoplasms; Radioimmunoassay; Receptors, Cell Surface; Skin

Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H+ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H+ exchange system of A431 cells. Stimulation of Na+/H+ exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promoters also do not modulate the activation of Na+/H+ exchange. By contrast, the stimulation of Na+/H+ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H+ antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H+ exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca2+. The inhibition BY PMA of EGF-promoted Na+/H+ exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H+ exchange by polypeptide growth factors.

Topics: Carcinoma, Squamous Cell; Carrier Proteins; Cell Line; Culture Media; Epidermal Growth Factor; Humans; Hydrogen-Ion Concentration; Mannitol; Mitogens; Phorbols; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Vanadates; Vanadium

Rhodamine-labelled epidermal growth factor (Rh-EGF) was shown to bind to A431 cells grown at low density both to a small number of high affinity receptors (KD = 2.8 X 10(-10) M; fraction of total binding sites approximately 0.12) and also to a large number of low affinity receptors (KD = 4 X 10(-9) M; fraction of total binding sites approximately 0.88). Measurements of the lateral diffusion of EGF receptors on the cell surface were made using Rh-EGF and the technique of fluorescence photobleaching recovery. The high affinity receptors (labelled with 1.6 X 10(-10) M Rh-EGF, 5% of EGF binding sites occupied) did not show lateral mobility over the temperature range 3 degrees-37 degrees C. The low affinity receptors (labelled with 2.4 X 10(-7) M Rh-EGF, 90% of EGF sites occupied) showed at least 75% fluorescence recovery after photobleaching, and lateral diffusion coefficients of approximately 2 X 10(-10) cm2/s. These results show that the two populations of EGF receptors defined by binding studies differ in their freedom to diffuse laterally. The observation that the high affinity receptors are immobile indicates that lateral diffusion of receptors, at least over a distance of a few hundred nanometres or more, may not be required for the action of low concentrations of EGF.

Topics: Carcinoma, Squamous Cell; Cell Line; Diffusion; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Membrane Fluidity; Photolysis; Receptors, Cell Surface

EGF binding capacity was examined in 9 different human cell lines which were derived from colon, rectum and pancreas tumors. Among these cell lines, a pancreatic carcinoma cell line, UCVA-1, was found to possess a high number (0.9 X 10(6)/cell) of EGF receptors. This number is comparable to that of EGF receptors in human vulva epidermoid carcinoma A431 cells (2 X 10(6)/cell). However, it was found that, unlike A431 cells, the growth of UCVA-1 cells, in serum-containing and serum-free conditions, was not inhibited by EGF. The UCVA-1 cells have EGF receptor of Mr = 170 K and of two affinity types: Kd1 = 72 X 10(-9) M and Kd2 = 2 X 10(-8) M. The EGF receptors in UCVA-1 cells are less susceptible to proteolytic cleavage than those in A431 cells. In UCVA-1 cells, EGF is apparently processed via a receptor-mediated endocytosis. The UCVA-1 cell membrane contained EGF-stimulated protein kinase as was found in A431 cells. The stimulation of phosphorylation by EGF was only approximately 20% in UCVA-1 while it was over 100% in A431. When angiotensin II was used as a substrate, the relative activity of EGF-dependent tyrosine-specific protein phosphorylation was approximately 8 times less in UCVA-1 cell membrane. The EGF-stimulated phosphorylation was mostly on EGF receptors for both cell lines. However, several other components (Mr = 100 K, 80 K, 72 K and 65 K) were readily detected in A431 cells. These observations indicate that the EGF receptor/protein kinase relation differs in these two cell lines and suggests that it may be related to the growth-inhibitory effect of EGF seen in A431.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Pancreatic Neoplasms; Phosphoproteins; Phosphorylation; Protein Kinases; Receptors, Cell Surface; Vulvar Neoplasms

Tumor promoters cause a variety of effects in cultured cells, at least some of which are thought to result from activation of the Ca2+-phospholipid-stimulated protein kinase C. One action of tumor promoters is the modulation of the binding and phosphorylation of the epidermal growth factor (EGF) receptor in A431 cells. To determine if these compounds act on the EGF receptor by substituting for the endogenous activator of C kinase, diacylglycerol, we compared the effects of the potent tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) with those of the synthetic diacylglycerol analog 1-oleyl 2-acetyl diglycerol (OADG). When A431 cells were treated with TPA, the subcellular distribution of C kinase activity shifted from a predominantly cytosolic location to a membrane-associated state; OADG also caused the disappearance of cytosolic C kinase activity. The shift in the subcellular distribution of C kinase, caused by TPA or OADG, correlated with changes in binding and phosphorylation of the EGF receptor. OADG, like TPA, caused loss of binding to an apparent high affinity class of receptors, blocked EGF-induced tyrosine phosphorylation of the EGF receptor, and stimulated phosphorylation of the EGF receptor at both serine and threonine residues. No difference between the phosphopeptide maps of receptors from cells treated with OADG or TPA was observed. Thus, it appears that tumor promoters can exert their effects on the EGF receptors by substituting for diacylglycerol, presumably by activating protein kinase C. Further, these results suggest that endogenously produced diacylglycerol may have a role in normal growth regulatory pathways.

Topics: Amino Acids; Carcinoma, Squamous Cell; Cell Line; Diglycerides; Epidermal Growth Factor; ErbB Receptors; Glycerides; Humans; Peptide Fragments; Phosphopeptides; Phosphorylation; Protein Binding; Protein Kinase C; Protein Kinases; Receptors, Cell Surface

A-431 cells were treated with inhibitors of either N-linked glycosylation (tunicamycin or glucosamine) or of N-linked oligosaccharide processing (swainsonine or monensin) to examine the glycosylation of epidermal growth factor (EGF) receptors and to determine the effect of glycosylation modification on receptor function. The receptor was found to be an Mr = 130,000 polypeptide to which a relatively large amount of carbohydrate is added co-translationally in the form of N-linked oligosaccharides. Processing of these oligosaccharides accounts for the 10,000-dalton difference in electrophoretic migration between the Mr = 160,000 precursor and Mr = 170,000 mature forms of the receptor. No evidence was found for O-linked oligosaccharides on the receptor. Mr = 160,000 receptors resulting from swainsonine or monensin treatment were present on the cell surface and retained full function, as judged by 125I-EGF binding to intact cells and detergent-solubilized extracts and by in vitro phosphorylation in the absence or presence of EGF. On the other hand, when cells were treated with tunicamycin or glucosamine, ligand binding was reduced by more than 50% in either intact cells or solubilized cell extracts. The Mr = 130,000 receptors synthesized in the presence of these inhibitors were not found on the cell surface. In addition, no Mr = 130,000 phosphoprotein was detected in the in vitro phosphorylation of tunicamycin or glucosamine-treated cells. It appears, therefore, that although terminal processing of N-linked oligosaccharides is not necessary for proper translocation or function of the EGF receptor, the addition of N-linked oligosaccharides is required.

Topics: Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Glycoside Hydrolases; Glycosides; Humans; Kinetics; Molecular Weight; Protein Processing, Post-Translational; Receptors, Cell Surface

Complementary DNA corresponding to total poly(A)+-RNA from the human A431 epidermoid carcinoma cell line was cloned in the phage expression vector lambda gt 11. An epidermal growth factor (EGF) receptor cDNA clone was obtained by screening of the expression library with a rabbit polyclonal antibody (IgG), raised to the purified A431 EGF receptor, in combination with [125I]protein A of S. aureus. The cloned cDNA was able to select, by hybridization, messenger RNA which was translated in Xenopus oocytes and yielded an immunoprecipitable EGF receptor protein of Mr = 160,000. The insert of this cDNA (phEGFR-1), is approximately 880 base pairs in length and encodes the carboxyterminal portion of the EGF receptor protein. Its sequence is evolutionarily conserved among vertebrates as shown by hybridization to unique chromosomal DNA sequences from human, baboon, dog, rat, mouse and frog.

Topics: Amino Acid Sequence; Animals; Base Sequence; Biological Evolution; Carcinoma, Squamous Cell; Cell Line; Cloning, Molecular; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Nucleic Acid Hybridization; Oocytes; Plasmids; Protein Biosynthesis; Receptors, Cell Surface; RNA, Messenger; Xenopus

Epidermal growth factor (EGF) receptor protein has been purified in a single high-yield step by immunoaffinity chromatography of extracts of A431 cells. A monoclonal antibody directed against the EGF binding site of the receptor was immobilized to Sepharose 4B as a specific immune absorbent and competitive elution with EGF was used to obtain purified EGF receptor protein with tyrosine kinase activity. The stoichiometry of EGF binding was determined by comparing 125I-EGF binding to A431 cells with the mass of EGF receptor protein in those cells as measured by immunoaffinity chromatography, radioimmunoassay, and immune precipitation. Each measurement indicated one EGF binding site/EGF receptor protein molecule. Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups. The autophosphorylation reaction has a Km for ATP (0.2 microM) which is about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation is an intramolecular reaction.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Male; Mice; Molecular Weight; Phosphorylation; Receptors, Cell Surface

Immunohistochemical demonstration of epidermal growth factor (EGF) and nerve growth factor (NGF) was made during chemical carcinogenesis in the mouse submandibular gland. The granular convoluted tubule cells in the normal male submandibular gland contained larger amounts of EGF and NGF than in the female. The initial phase and early stages in chemical carcinogenesis showed degranulation of the granular convoluted tubule cells with a marked decrease in EGF and NGF. Premalignant lesions such as duct-like structures and multicystic lesions showed variable staining for EGF and were usually negative for NGF. Material secreted into the luminal spaces revealed increased staining for EGF and NGF. Scattered tumor cells of the poorly differentiated squamous-cell carcinoma type and desquamated tumor cells contained abundant EGF, but not NGF. No positive reaction for EGF or NGF was found in the induced squamous-cell carcinoma cells.

Topics: Animals; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cytoplasmic Granules; Epidermal Growth Factor; Female; Male; Mice; Nerve Growth Factors; Salivary Gland Neoplasms; Submandibular Gland Neoplasms

The sequence of the human epidermal growth factor (EGF) receptor shows great homology with the avian erythroblastosis virus v-erb B oncogene, raising the possibility that the receptor gene is identical to the c-erb B protooncogene. Human A431 epidermoid carcinoma cells, which have an unusually high number of EGF receptors, were examined to determine whether elevated EGF receptor levels correlate with gene amplification. Southern blots of genomic DNA's from A431 and other human cell lines were probed with either a v-erb B gene fragment or a human EGF receptor complementary DNA clone (pE7), previously isolated from an A431 complementary DNA library. When either probe was used to analyze Eco RI- or Hind III-generated DNA fragments, EGF receptor DNA sequences were amplified about 30-fold in A431. Differences in the banding pattern of A431 DNA fragments relative to normal fibroblast DNA indicate the occurrence of a rearrangement in the region of the receptor gene. Furthermore, A431 cells contain a characteristic, prominent 2.9-kilobase RNA. These results are consistent with the hypothesis that, in A431 cells, gene amplification, possibly associated with a translocation event, may result in the overproduction of EGF receptor protein or the appearance of the transformed phenotype (or both).

Topics: Alpharetrovirus; Base Sequence; Carcinoma, Squamous Cell; Cell Line; DNA; DNA Restriction Enzymes; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Genes, Viral; Humans; Nucleic Acid Hybridization; Oncogenes; Poly A; Receptors, Cell Surface; RNA; RNA, Messenger; Translocation, Genetic

Antibodies generated against two synthetic peptides corresponding to two defined regions on the transforming protein of Rous sarcoma virus, pp60src, interact specifically with the epidermal growth factor (EGF)-receptor kinase. An antibody directed against a synthetic peptide corresponding to the major phosphorylation site of pp60src interacts specifically with EGF receptor and immunoprecipitates a functional EGF-receptor kinase. The second antibody, which binds close to a region on the src molecule that is required for its kinase activity, also binds to EGF-receptor kinase and prevents the autophosphorylation of the receptor molecules. Neither antibody binds to intact cells, but they do recognize various forms of the solubilized receptor. It is concluded that at least two cytoplasmic domains of the EGF receptor are antigenically and presumably also structurally related to specific domains on pp60src.

Topics: Amino Acid Sequence; Antibodies; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epitopes; ErbB Receptors; Humans; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinases; Receptors, Cell Surface; Viral Proteins

Monoclonal antibodies (MoAbs) were raised against epidermal growth factor (EGF) receptors on a human epidermoid carcinoma cell line, A431. Administration of anti-EGF receptor MoAbs inhibited tumor formation in athymic mice by A431 cells and by another epidermal carcinoma cell line, T222. When one of the same MoAbs was used in therapy against Li-7 (a human hepatoma) and HeLa cells (a cervical carcinoma), tumor growth was not affected. The number of EGF receptors on A431 cells was about 100-fold higher than on T222, Li-7, and HeLa cells, suggesting that the number of EGF receptors may not be an important determinant in suppressing tumor growth. Three anti-EGF receptor MoAbs were used in the present studies. MoAbs 528 (immunoglobulin G2a) and 225 (immunoglobulin G1) are capable of competing with EGF for receptor binding and inhibit proliferation of A431 cells in culture. The other MoAb, 455 (immunoglobulin G1), is incapable of blocking the binding of EGF to its receptors and has no effect on the proliferation of cultured A431 cells. All three MoAbs inhibited A431 tumor growth in athymic mice, indicating that the antibody isotype and the site of binding on the EGF receptor are not the determinants of antiproliferative activity in vivo. The observation that MoAb against the receptor for EGF is cytostatic rather than cytocidal in vitro against A431 cells, yet completely prevents tumor growth in vivo, suggests that some host animal responses also may be involved in the antitumor effect. MoAbs against growth factor receptors could provide useful immunotherapeutic agents.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; HeLa Cells; Humans; Immunotherapy; Kinetics; Mice; Mice, Nude; Neoplasm Transplantation; Receptors, Cell Surface; Transplantation, Heterologous

It has been suggested that products of the major histocompatibility complex, the MHC, of vertebrates function in many processes of recognition and ligand binding at the cell surface. Here we show that binding of polyclonal and monoclonal antibodies against human MHC antigens, HLA, reduced the binding of epidermal growth factor (EGF) to its membrane receptors on A-431 tumor cells and on normal human fibroblasts. Binding of EGF at 37 degrees C similarly inhibited the binding of Fab fragments and intact Ig anti-HLA to human cells. The inhibitory effect of anti-HLA antibodies was rapid and dependent upon temperature and antibody concentration and valence. Fluorescence microscopy qualitatively confirmed the binding data and showed that MHC antigens and EGF-receptors do not co-cluster in the membrane.

Topics: Animals; Antibodies; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; HLA Antigens; Humans; Major Histocompatibility Complex; Male; Mice; Microscopy, Fluorescence; Receptors, Cell Surface

Addition of epidermal growth factor (EGF) to serum-free or serum-supplemented cultures of A431 cells stimulates protein breakdown without affecting rates of protein synthesis. These effects are atypical because in other cell lines, including AG2804-transformed human fibroblasts examined for comparison, EGF inhibits protein breakdown and stimulates protein synthesis. The response to EGF in A431 cells does not reflect a general post-receptor modification in growth factor action, since addition of insulin to the cells leads to the normal inhibition of protein breakdown. These findings indicate that the unusual growth inhibition produced by EGF in A431 cells can be explained by an increased rate of intracellular protein breakdown.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Insulin; Proteins; Receptors, Cell Surface

When human epidermoid carcinoma A431 cells labeled with 32Pi to steady state specific activity were treated either with epidermal growth factor (EGF) or with active phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or phorbol 12,13-dibutyrate, labeling of 160 kDa EGF receptors isolated by immunoprecipitation with monoclonal anti-EGF receptor IgG was increased 2- to 3-fold. These treatments produced no significant increase in 32Pi labeling of acid-precipitable material present in detergent extracts of the cells. Phosphoamino acid analysis of radiolabeled EGF receptors isolated from these cells revealed several differences: the relative abundance of phosphotyrosine in EGF receptors was increased in cells treated with EGF, but decreased in cells treated with TPA; the overall relative abundance of phosphothreonine in EGF receptors was decreased in cells treated with EGF, but remained constant within the limits of experimental detection in cells treated with TPA. Two-dimensional mapping of the radiolabeled phosphopeptides produced from EGF receptors isolated by immunoprecipitation and treated with trypsin revealed 9 independent labeled regions, 2 of which contained phosphothreonine and were present only in EGF- or TPA-treated cells. These two phosphopeptide regions were more highly labeled in cells treated with TPA than with EGF.

Topics: Carcinogens; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Peptide Fragments; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Phosphorylation; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

The Ca2+- and phospholipid-dependent protein kinase (C-kinase) binds tightly in the presence of Ca2+ to purified membranes of A431 human epidermoid carcinoma cells. The major membrane substrate for C-kinase is the epidermal growth factor (EGF) receptor. Phosphorylation of the EGF receptor is Ca2+-dependent and occurs at threonine and serine residues. After tryptic digestion of the receptor, three major phosphothreonine-containing peptides were identified. These are identical with three new phosphopeptides present in the EGF receptor isolated from A431 cells treated with either of the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. C-kinase catalyzes phosphorylation at these same sites in purified EGF receptor protein. These results indicate that, in A431 cells exposed to tumor promoters, C-kinase catalyzes phosphorylation of a significant population of EGF receptor molecules. This phosphorylation of EGF receptors results in decreased self-phosphorylation of the EGF receptor at tyrosine residues both in vivo and in vitro and in decreased EGF-stimulated tyrosine kinase activity in vivo.

Topics: Animals; Brain; Calcium; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Weight; Peptide Fragments; Phosphorylation; Protein Kinase C; Protein Kinases; Protein-Tyrosine Kinases; Rats; Receptors, Cell Surface

Human epidermoid carcinoma A431 cells, possessing an extraordinarily high number of epidermal growth factor (EGF) receptors (1), were found to be hypotetraploid in their chromosome constitution and to contain two copies of intact chromosome 7 and two types of the translocation chromosomes involving chromosome 7 (M4 and M14) as well as several other rearranged chromosomes. The A431 cells were fused with mouse A9 cells, which lack EGF receptors (2) and are deficient in hypoxanthine phosphoribosyltransferase (3), and the human-mouse cell hybrid (AA series) were selected in HAT/ouabain medium (3, 4). The expression of high EGF binding ability was correlated with the presence of human translocation chromosome M4. AA hybrid clones that contained intact human chromosome 7 but not the marker chromosome M4 expressed only ordinary levels of EGF receptors. The EGF receptors expressed in the AA hybrids were proven to be of human nature by immunoprecipitation of the receptors cross-linked with [125I]EGF. These observations and our previous gene assignment of the EGF receptor to human chromosome 7 (2, 5) suggest that the marker chromosome M4 may carry an alteration(s) in the gene(s) involved in EGF receptor biosynthesis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Chromosomes, Human, 13-15; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Hybrid Cells; Hypoxanthine Phosphoribosyltransferase; Karyotyping; Mice; Polyploidy; Receptors, Cell Surface

Recent studies have established that polypeptide growth factors cause an elevation of the cytoplasmic pH (pHi) in cultured mammalian cells by stimulating Na+/H+ exchange. We show that vanadate, previously found to act as a mitogen for a number of cells, reversibly activates Na+/H+ exchange at micromolar concentrations in A431 cells, leading to a large increase of pHi. The stimulation of Na+/H+ exchange by vanadate is not due to inhibition of the Na+/K+ ATPase and is unrelated to possible effects of vanadate on cAMP levels. Elevation of pHi by vanadate and by epidermal growth factor (EGF) both display similar kinetics, and both EGF and vanadate stimulate the rate of pHi recovery following an acute acid load, suggesting that vanadate stimulates Na+/H+ exchange by a mechanism similar to that of polypeptide growth factor stimulation. Thus, stimulation of Na+/H+ exchange may be a common property not only of polypeptide growth factors but also of other, chemically unrelated mitogens.

Topics: Carcinoma, Squamous Cell; Carrier Proteins; Cell Line; Epidermal Growth Factor; Humans; Hydrogen-Ion Concentration; Kinetics; Sodium; Sodium-Hydrogen Exchangers; Vanadates; Vanadium

The binding of radiolabeled epidermal growth factor (EGF) to immobilized A-431 target cell membranes coupled to polyvinyl chloride microtiter wells is described. Saturation curves and Scatchard analysis of the data indicate that the observed binding parameters are consistent with those previously reported. Binding capacity of the membranes are approx. 6.6 pmol EGF per mg membrane protein. Kinetics of 125I-EGF binding were slower, however, than reported for binding to membranes in suspension, although binding constants were not greatly different. The high- and low-affinity binding constants for 125I-EGF were calculated to be approximately 1 X 10(12) M-1 and 2.5 X 10(9) M-1, respectively. Application of this technique in a competitive binding assay requires no more than 2.5 micrograms of membrane protein per assay, is essentially complete after 60 min, and facilitates screening of a large number of samples in a short time. Therefore, this will assist in the evaluation and quantitation of EGF and EGF-related transforming growth factor activity in physiological fluids. This technique may also be applied to analyses of other hormone-receptor systems.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chromatography, High Pressure Liquid; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Peptides; Receptors, Cell Surface; Transforming Growth Factors

Epidermal growth factor (EGF) receptors can be spontaneously and selectively transferred from donor plasma membranes to recipient receptorless fibroblasts in the absence of any added fusogenic agent. Studies on the time and temperature dependence of this transfer indicate that it is due to preferential insertion of the EGF receptor over the other plasma membrane proteins. The inserted receptor is exceptionally stable to dissociation or damage. The number of receptors inserted increased with increasing amounts of donor membranes and then reached a plateau, which also suggests the existence of saturable receptor 'docking' sites in recipient cells. It is interesting that both human and murine receptors are selectively inserted into the mutant mouse cell membrane. This suggests that the parts of the receptor molecule responsible for insertion are similar in murine and human receptors, and that a 'docking' factor present in the mouse recipient cells may accept both human and murine receptors.

Topics: Animals; Biological Transport; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Colchicine; Cycloheximide; Cytochalasin B; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Liver; Membrane Proteins; Mice; Receptors, Cell Surface; Temperature; Tunicamycin

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Weight; Phosphopeptides; Phosphorylation; Protein Kinases; Receptors, Cell Surface

A primary receptor-binding region of mouse epidermal growth factor (EGF) was identified by comparing the relative affinities of selected synthetic fragments with overlapping sequences in the EGF receptor-binding assay, using human foreskin fibroblasts. Only synthetic peptides containing the amino acid residues 20-31 in the mouse EGF sequence showed the ability to compete with 125I-labeled EGF in binding to EGF receptors. The affinities of the cyclic EGF fragment [Ala20]EGF-(14-31) and the linear [(S-acetamidomethyl)-Cys20,31]-EGF-(20-31) were approximately 1/10(4) of the affinity of EGF. Despite their reduced receptor affinities, these two peptides exhibited the in vitro biological activities of native EGF, while fragments from other regions of the EGF molecule were devoid of these biological properties. The peptides induced DNA synthesis in human foreskin fibroblasts as measured by [3H]thymidine incorporation into DNA. They also induced EGF receptor clustering and activated the EGF-sensitive kinase, enhancing the autophosphorylation of EGF receptors in a dose-related manner. Moreover, a major antigenic determinant of EGF for rabbit anti-EGF antibodies was identified within this same localized region of the EGF molecule by competition experiments utilizing the synthetic EGF fragments. The predominant EGF antigenic determinant(s) was also found within the fragment [(S-acetamidomethyl)Cys20,31]-EGF-(20-31). The accessibility of the residues in positions 20-31 for antibody recognition is consistent with the conclusion that these residues constitute or contain a major receptor-binding region for EGF.

Topics: Binding, Competitive; Carcinoma, Squamous Cell; Cell Line; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Male; Peptide Fragments; Receptors, Cell Surface; Skin

Human epidermoid carcinoma A431 cells in culture produce a soluble 105-kilodalton protein which, by the criteria of epidermal growth factor (EGF) binding, recognition by monoclonal and polyclonal antibodies to the EGF receptor, amino-terminal sequence analysis and carbohydrate content, is related to the cell surface domain of the EGF receptor. The high rate of production and the finding that with biosynthetic labeling the specific activity of this 105-kilodalton protein exceeds that of the intact receptor indicate that it is not derived from membrane-bound mature receptor but is separately produced by the cell. These cells thus separately synthesize an EGF receptor that is inserted into the membrane and an EGF receptor-related protein that is secreted.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Carbohydrates; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Humans; Kinetics; Molecular Weight; Receptors, Cell Surface

Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [gamma-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 4 degrees C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumor-promoting phorbol ester can mimic a critical early response usually elicited by EGF.

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Molecular Weight; Peptide Fragments; Phorbols; Phosphorus Radioisotopes; Phosphorylation; Receptors, Cell Surface; Tetradecanoylphorbol Acetate; Tyrosine

A fusion gene containing 5' rat prolactin genomic sequences ligated to the structural portion of the rat growth hormone gene ( grl ) was introduced by DNA-mediated gene transfer into mammalian cells by using a chimeric plasmid vector. Clonal transfected cell lines produced a mRNA that used the authentic 5' initiation site and that was processed to the predicted size. The intracellular levels of this RNA product were increased 2.5- to 5-fold by exposure of the cells to epidermal growth factor (EGF) and 2- to 3-fold by exposure of the cells to a potent phorbol ester, phorbol 12-myristate 13-acetate, apparently due to regulation at the level of gene transcription. Substitution of the 5' prolactin DNA sequences by 5' growth hormone DNA sequences resulted in the loss of EGF inducibility. A genomic sequence in or near the 5' flanking portion of the prolactin gene therefore appears to confer polypeptide hormone transcriptional regulation upon the gene.

Topics: Base Sequence; Carcinoma, Squamous Cell; Cell Line; Clone Cells; Cloning, Molecular; DNA Restriction Enzymes; Epidermal Growth Factor; Genes; Genetic Vectors; Growth Hormone; Humans; Phorbols; Prolactin; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection

Tyrosine-specific phosphorylation of the epidermal growth factor (EGF) receptor in hormonally stimulated A431 cells is blocked by three chemically distinct classes of tumor promoters. Tumor-promoting esters of the diterpene phorbol (phorbol 12-myristate 13-acetate, beta-phorbol 12,13-dibutyrate, and beta-phorbol 12,13-didecanoate), indole alkaloids (teleocidin and lyngbyatoxin A), and polyacetates ( aplysiatoxin and debromoaplysiatoxin ) all inhibited EGF-stimulated phosphorylation of the receptor. Non-tumor-promoting analogs (beta-phorbol, alpha-phorbol 12,13-didecanoate, and hydrolyzed teleocidin) had no effect on the levels of receptor phosphorylation. The ED50 values of the inhibitory effect (0.1-3 ng/ml) reflected the relative tumor-promoting abilities of these compounds in vivo. None of the tumor promoters tested significantly decreased the overall specific binding of 125I-labeled EGF to A431 cells. Scatchard analysis, however, revealed two apparent EGF receptors in this cell type. The dose-responses for tumor-promoter inhibition of EGF receptor tyrosine phosphorylation and high-affinity EGF binding were similar, suggesting that the same initial event is responsible for both effects. This demonstrates a correlation between modulation of EGF receptor binding and phosphorylation of tyrosine by tumor promoters. The data suggest a possible role for protein kinase C, the putative cellular receptor for these tumor promoters, in the mechanism of action.

Topics: Amino Acids; Carcinogens; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Phorbol Esters; Phorbols; Phosphorylation; Protein Kinase C; Protein Kinases; Receptors, Cell Surface; Tyrosine

Topics: Animals; Carcinoma, Squamous Cell; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Nerve Growth Factors; Pheochromocytoma; Rats; Receptors, Cell Surface

A monoclonal antibody, EGR/ G49 , raised against the receptor for epidermal growth factor (EGF) present in A431 cells inhibits EGF binding by decreasing the affinity of the major population of low affinity receptors while leaving the minor high affinity population relatively unperturbed. The antibody, which binds to a carbohydrate determinant at a site distinct from the EGF binding site, induces clustering and internalisation of the receptor without stimulating the EGF receptor-kinase or affecting its ability to undergo stimulation by EGF. It is toxic to A431 cells and induces morphological changes similar to those seen when these cells are challenged with EGF in the concentration range 1-10 nM. These results suggest that high and low affinity EGF receptors can be distinguished and that they may serve different functions.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Hybridomas; Kinetics; Mice; Receptors, Cell Surface

Four mouse monoclonal antibodies specific for human epidermal growth factor (EGF) receptors have been prepared using EGF receptor protein from human A431 epidermoid carcinoma cells as immunogen. We have determined the effect of these antibodies on two known functions of the EGF receptor: EGF binding and tyrosine kinase. Three of these antibodies (225, 528, and 579) are inhibitors of EGF binding, whereas the fourth (455) does not compete for binding but immunoprecipitates the EGF receptor. Inhibition is of the mixed competitive and noncompetitive type. The three competing monoclonal antibodies are antagonists of EGF-stimulated tyrosine protein kinase activity assayed both in intact cells and using an exogenous peptide substrate in solubilized membranes. These immunoglobulins are partial agonists in self-phosphorylation of the EGF receptor in solubilized membranes but exhibit only antagonist activity for this reaction in intact cells. The three competing monoclonal immunoglobulins recognize receptors in variant A431 cells with the same efficiency as in parental A431 cells. Such antagonist monoclonal antibodies can be used to control the concentration of receptors which can be activated by EGF.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoglobulin G; Mice; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Receptors, Cell Surface

4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Phorbols; Phosphorylation; Receptors, Cell Surface; Succinimides; Tetradecanoylphorbol Acetate

This paper demonstrates spontaneous transfer of epidermal growth factor (EGF) receptor mitogenicity from human A431 carcinoma cells to receptor negative mouse cells. The fact of transfer was documented by the demonstration in the recipient cell of normal EGF-binding affinity, covalent labeling of transferred receptor by 125I-EGF, and reactivity with specific anti-human receptor antibody. Phosphorylation experiments suggest that in the transferred receptor the protein kinase site tends to face the inside of the cell. The transferred receptor imposed upon the normally unresponsive cells a mitogenic responsiveness to EGF in the nM concentration range, as measured by stimulation of DNA replication. EGF-dependent mitogenesis in the recipient cells was abolished by both anti-EGF antibody and anti-human receptor antibody, indicating that the transferred mitogenicity is both EGF and receptor specific. For NR-6 cells without transplanted receptor, the same antibody had no effect on non-EGF-dependent mitogenesis. Interestingly, although the transferred receptor displayed normal behavior in several respects, it did not mediate rapid internalization and degradation of bound EGF.

Topics: Animals; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoglobulin G; Kinetics; Mice; Mitogens; Molecular Weight; Phosphorylation; Protein Kinases; Receptors, Cell Surface

Reverse-phase high-performance liquid chromatography (HPLC) performed on urine from cancer patients and normal controls revealed the presence of seven chromatographically distinct peaks of transforming growth factor (TGF) activity, as measured by colony formation of normal rat kidney cells in soft agar. Comparison of urines from normal donors and cancer patients showed no differences in EGF (epidermal growth factor)-dependent beta-TGF-like activity but did reveal distinct patterns of EGF-related, EGF-independent alpha-TGF-like activity. All urine samples contained at least two chromatographically distinguishable forms of EGF-dependent TGF activity, eluting from HPLC as broad peaks with 30 and 43% acetonitrile. The remaining five TGFs eluted as sharp peaks with 32, 34, 35, 37, and 38% acetonitrile, demonstrated EGF-competing activity, and thus were functionally related to EGF. Two of the five EGF-related TGFs were consistently elevated only in the urine of cancer patients and eluted with 32% (TGFA) and 37% (TGFD) acetonitrile Two of the other EGF-related TGFs, eluting with 34% (TGFB) and 35% (TGFC) acetonitrile, were commonly found in both normals and cancer patients. The fifth EGF-related TGF, TGFE, eluting with 38% acetonitrile, was found only in normal donor specimens. TGFA corresponded to the unique Mr 30,000 TGF activity previously identified only in the urine of cancer patients. These observations demonstrate that cancer patients produce high levels of EGF-related TGF activities which can be readily distinguished, using reverse-phase HPLC, from EGF-related TGFs produced by normal individuals. Using a solid-phase competitive radioreceptor binding assay for EGF, we demonstrated that quantitation of EGF-competing activity is as sensitive and effective as the soft-agar colony formation assay for distinguishing HPLC profiles of urinary TGF from cancer patients versus that from normal individuals.

Topics: Adolescent; Adult; Carcinoma, Squamous Cell; Cell Line; Chromatography, High Pressure Liquid; Epidermal Growth Factor; ErbB Receptors; Female; Growth Substances; Humans; Kinetics; Male; Middle Aged; Neoplasm Proteins; Neoplasms; Peptides; Receptors, Cell Surface; Reference Values; Transforming Growth Factors

To analyze the influence of ricin B-chain on the toxicity of hybrid-protein conjugates, the rate of cellular uptake of conjugates, and the rate at which ricin A-chain (RTA) is delivered to the cytoplasm, we have constructed toxic hybrid proteins consisting of epidermal growth factor (EGF) coupled in disulfide linkage either to ricin or to RTA. EGF-ricin is no more toxic on A431 cells than EGF-RTA. The two conjugates demonstrate similar kinetics of cellular uptake (defined as antibody irreversible toxicity). EGF-RTA and EGF-ricin, like ricin, required a 2-2 1/2 hour period at 37 degrees before the onset of protein synthesis inhibition occurred. Our results suggest that RTA determines the processes which carry it, either in conjugate or toxin, from the plasma membrane binding site to the cytoplasm following endocytosis, and the ricin B chain is not required for these processes.

Topics: Biological Transport; Carcinoma, Squamous Cell; Cell Line; Cell Survival; Epidermal Growth Factor; Humans; Kinetics; Plant Proteins; Protein Biosynthesis; Protein Multimerization; Receptors, Mitogen; Ricin; Structure-Activity Relationship

A431 human epidermoid carcinoma cells in culture may be grown in the absence of serum in a one to one mixture of Ham's F12 and Dulbecco-modified Eagle's medium supplemented with 10 micrograms/ml bovine insulin, 10 micrograms/ml human transferrin, 5 micrograms/ml human cold-insoluble globulin and 0.5 mM ethanolamine. Growth rate of the cells in this serum-free medium is essentially identical to that of cells in medium containing 10% fetal calf serum. Addition of epidermal growth factor at concentrations which are stimulatory for the growth of other cell types in culture causes and inhibition of A431 cell growth in both serum-free and serum-containing medium. The inhibitory effect of epidermal growth factor is reversible upon replacement of epidermal growth factor-containing medium with medium containing no epidermal growth factor. Simultaneous addition to the medium of antibody to epidermal growth factor along with epidermal growth factor prevents the inhibitory effect.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Culture Media; Culture Techniques; Epidermal Growth Factor; Humans; Kinetics

We have studied the action of phorbol, 12-myristate, 13-acetate (PMA) on human keratinocytes grown with lethally-irradiated 3T3 cells using medium supplemented with hydrocortisone, cholera toxin and epidermal growth factor. Normal keratinocyte cultures show a heterogeneous response to PMA; 90-93% of the colony-forming cells lose their colony-forming ability and form cornified envelopes when treated for 24 h with doses of 100 nM or less, but the remainder are resistant to doses of 1000 nM. The resistant cells are the precursors of the sensitive ones and heterogeneity is restored to those cells and their progeny after 8 days culture in the absence of PMA. Cultures of 3 squamous cell carcinoma lines, a SV40-transformed human keratinocyte line, and three clones of these lines were found to contain 3-17 times more PMA-resistant keratinocytes than the normal strains, and the size of the PMA-resistant fraction in each line was inversely related to the competence of that line to lose colony-forming efficiency when placed in suspension culture (which is the first detectable change in an ordered programme of events resembling terminal differentiation of the keratinocyte). The number of cells with cornified envelopes in surface cultures of normal human keratinocytes increased from approximately 3% in control cultures to approximately 70% in those treated for 6 days with 100 nM PMA. The transformed human keratinocyte cultures showed a 3-25-fold smaller increase in cornified envelope formation when treated with 100 nM PMA, and the increase in envelope formation by each line when exposed to this dose of PMA was related to the competence of that line to lose cloning efficiency in suspension culture. No relationship was found between the ability of any human keratinocyte strain or line we studied to metabolically inactivate PMA and their resulting response to the compound. The results are discussed in relation to the mechanism of action of PMA as a promoter of epidermal carcinogenesis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Child; Cholera Toxin; Epidermal Growth Factor; Female; Fetus; Humans; Hydrocortisone; Keratins; Male; Mice; Phorbols; Pregnancy; Simian virus 40; Skin; Skin Physiological Phenomena; Tetradecanoylphorbol Acetate

Epidermal growth factor (EGF) increases Na+ uptake in several cell types through an electroneutral, amiloride-sensitive pathway putatively identified as Na+/H+ countertransport. We have previously shown (Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983) J. Biol. Chem. 257, 4883-4889) that EGF rapidly activates amiloride-sensitive net Na+ influx in the A431 human epidermoid carcinoma cell line. We also described the presence of transmembrane, amiloride-sensitive Na+/H+ exchange in A431 cells using a new fluorescence technique for the measurement of intracellular pH (pHi) based on the incorporation of fluorescein-dextran into the cell cytoplasm. The low pKa of fluorescein (approximately 6.4) prevented the direct assessment of the inferred, EGF-induced cytoplasmic alkalinization, mediated by stimulated Na+/H+ exchange. In this paper, 4',5'-dimethylfluorescein (pKa 6.75) was coupled to dextran, allowing increased pH sensitivity of the fluorescence assay in the physiological range. Using this improved assay, basic features of pHi regulation in A431 cells are documented, including the role of Na+/H+ exchange and Na+-linked C1-/HCO3-exchange in acid extrusion. We directly demonstrate a rapid elevation of pHi by addition of EGF as well as by serum in A431 cells. The pHi increase is half-maximal at 5-10 ng/ml of EGF, is dependent on external Na+, independent of external Ca2+, and inhibited by millimolar amiloride. EGF and serum also enhance Na+/H+ exchange-mediated cytosolic acidification when the transmembrane Na+ concentration gradient favors Na+ efflux from the cells. An alkaline pHi shift, caused by activation of Na+/H+ exchange, may be an important primary event in the mechanism of EGF action.

Topics: Amiloride; Biological Transport, Active; Carcinoma, Squamous Cell; Carrier Proteins; Cell Line; Dextrans; Epidermal Growth Factor; Fluoresceins; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Isothiocyanates; Kinetics; Microscopy, Fluorescence; Ouabain; Sodium-Hydrogen Exchangers; Spectrometry, Fluorescence; Thiocyanates

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Mice; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Phosphotyrosine; Protein Kinases; Protein-Tyrosine Kinases; Radioisotope Dilution Technique; Receptors, Cell Surface; Tyrosine

A Mr = 34,000 protein present in the 100,000 X g supernatant fraction from A431 human epidermoid carcinoma cells is the major radiolabeled phosphate acceptor from [gamma-32P]ATP in a cell-free system requiring epidermal growth factor (EGF) and EGF receptor kinase. This protein is immunoprecipitated by IgG directed against avian Mr = 34,000 cellular substrate for pp60src. Phosphoamino acid analysis of the Mr = 34,000 protein labeled with 32Pi from [gamma-32P]ATP in a cell-free system requiring EGF and EGF receptor kinase yielded radiolabeled phosphotyrosine with no detectable radioactivity in phosphoserine or phosphothreonine.

Topics: Carcinoma, Squamous Cell; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Weight; Neoplasm Proteins; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinases; Receptors, Cell Surface; Viral Proteins

Topics: Avian Sarcoma Viruses; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Viral; Epidermal Growth Factor; Glycopeptides; Humans; Membrane Proteins; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinases; Substrate Specificity; Viral Proteins

Epidermal growth factor (EGF) at 3 nM maximally inhibits the proliferation of A431 epidermoid carcinoma cells. We show that at lower concentrations, in the range of 3-100 pM, EGF has a mitogenic effect on A431 cells. In the presence of 100 nM anti-EGF-receptor monoclonal IgG (designated 528), which inhibits A431 cell proliferation and blocks greater than 95% of EGF binding, EGF becomes mitogenic for A431 cells at concentrations up to 3 nM. These results suggest that a minor population of high-affinity EGF receptors may be involved in stimulation of A431 cell proliferation. Saturation binding assays with 125I-labeled EGF indicate that approximately equal to 0.1-0.2% of receptors for EGF are high-affinity receptors that bind EGF with an estimated Kd of 7 X 10(-11) M. This affinity is nearly 2 orders of magnitude higher than that of the remaining EGF receptors. Although A431 cell proliferation is maximally inhibited by nonsaturating amounts of EGF (3 nM), maximal inhibition by 528 IgG (approximately equal to 70% of maximal inhibition by EGF) requires saturating concentrations of antibody (approximately equal to 15 nM). Unlike EGF, rapid down-regulation is not observed with 528 IgG. These results indicate different mechanisms of growth inhibition of A431 cells by EGF and 528 IgG.

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Division; Epidermal Growth Factor; ErbB Receptors; Mice; Mice, Inbred BALB C; Receptors, Cell Surface

Epidermal growth factor (EGF) increases Na+ uptake in several cell types through an electroneutral, amiloride-sensitive pathway putatively identified as Na+/H+ countertransport. The inferred cytosolic alkalinization resulting from this process has been proposed to be an important component of mitogenic stimulation. We studied the effect of EGF on the Na+/H+ exchange system of A431 cells, a cell line having a very high EGF receptor density but which is not mitogenically stimulated by EGF. We demonstrate that EGF rapidly activates net Na+ influx in A431 cells. Amiloride inhibits the EGF-dependent Na+ uptake (65% inhibition at 3 mM, ID50 approximately 0.3 mM) and inhibits much less the EGF-independent uptake. EGF is known to enhance 45Ca+ accumulation in A431 cells (Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20, 6280-6286). The following findings indicate that EGF-dependent 22Na+ and 45Ca2+ uptake are two independent processes. 1) EGF effectively stimulates an amiloride-sensitive 22Na+ uptake in the absence of external Ca2+. 2) EGF-dependent 45Ca2+ uptake is not inhibited by amiloride. A new fluorescence technique is described for intracellular pH determination based on the introduction of fluorescein-labeled dextran into the cell cytoplasm. Using this method, the presence of amiloride-sensitive Na+/H+ exchange in A431 cells is documented. Although the lack of pH sensitivity of fluorescein fluorescence above pH 7.3-7.4 prevents a direct assessment of an EGF-induced increase of intracellular pH, the combined results of 22Na+ flux and intracellular pH measurements suggest that EGF activates Na+/H+ exchange in A431 cells. We conclude that enhanced Na+/H+ exchange may not necessarily be coupled to mitogenic triggering in different cell types, although the stimulation of Na+/H+ exchange may constitute a primary event in the mechanism of EGF action.

Topics: Amiloride; Calcium; Carcinoma, Squamous Cell; Carrier Proteins; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Ouabain; Pyrazines; Sodium; Sodium-Hydrogen Exchangers

Human A431 epidermoid carcinoma cells in culture exhibit epidermal growth factor (EGF)-induced "down-regulation" of cell-surface and total cellular (Triton X-100 extractable) EGF receptors caused entirely by an enhanced rate (4-fold) of receptor inactivation [Krupp, M. N., Connolly, D. T. & Lane, M. D. (1982) J. Biol. Chem. 257, 11489-11496]. The following observations show that this enhanced rate of EGF receptor inactivation is closely correlated with an increased cellular activity of plasminogen activator (PA), a serine protease. First, EGF-induced down-regulation of cell-surface and total cellular EGF receptors and the concomitant increase in cellular PA activity occur with identical kinetics, the t 1/2 for both processes being 3-3.5 hr. Second, the EGF dose-response curves for down-regulation of total cellular EGF receptor and increased PA activity are similar. The EGF concentrations for half-maximal responses of both processes are 10-15 nM and 20 nM, respectively. Third, the removal of EGF from previously down-regulated cells results in the recovery of total cellular EGF binding activity with a concurrent loss of cellular PA activity. Fourth, blocking PA synthesis or activity with cycloheximide or dexamethasone prevents down-regulation of the EGF receptor. Fifth, the addition of leupeptin, an inhibitor of PA and plasmin action, blocks EGF-induced receptor down-regulation as well as the increase of PA activity. That EGF receptor down-regulation is independent of plasminogen per se in the culture medium suggests that PA-mediated events may initiate the rapid inactivation of the EGF receptor that occurs during down-regulation.

Topics: Carcinoma, Squamous Cell; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Fibrinolysin; Humans; Hydrolysis; Kinetics; Plasminogen Activators; Protein Binding; Receptors, Cell Surface

Human epidermoid carcinoma A431 cell clones have been obtained whose growth is inhibited, stimulated, or unaffected by epidermal growth factor (EGF). In clones exhibiting each type of growth response, EGF induced similar morphologic changes consisting of aggregation of cells into dense clusters with baring of large areas of the culture dish. The similarity of the clones' morphologic responses, despite their differing growth responses, indicates that the effects of EGF on morphology are distinct from effects on growth. Cells whose growth was inhibited by EGF contained high numbers of EGF receptors, whereas the concentration of EGF receptors was reduced in cells whose growth was stimulated or unaffected by EGF. There were, however, no consistent differences in EGF receptor concentrations between stimulated or null clones. Cells that exhibited each type of growth response displayed similar rates of EGF binding to receptors, rates of internalization of EGF, and rates and extent of EGF-induced receptor down-regulation. Changes in EGF-stimulated tyrosine-specific protein kinase activity paralleled changes in EGF receptors, both between clones and upon down-regulation. These studies indicate that a reduction in the concentration of EGF receptors in A431 cells allows escape from the growth inhibitory effects of EGF, but suggest that the pattern of growth response depends on biochemical events subsequent to EGF-receptor metabolism and activation of tyrosine-specific protein kinase.

Topics: Carcinoma, Squamous Cell; Cell Aggregation; Cell Division; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Genetic Variation; Humans; Protein Kinases; Receptors, Cell Surface

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; Fibroblasts; Humans; Kinetics; Magnetic Resonance Spectroscopy; Mice; NAD; Simian virus 40

Treatment of membrane vesicles from A431 cells, a human epidermoid carcinoma line, with the affinity label 5'-p-fluorosulfonylbenzoyl [8-14C]adenosine (5'-p-FSO2Bz[14C]Ado) results in an inhibition of the epidermal growth factor (EGF)-stimulable protein kinase and in the modification of proteins having the same molecular weight (Mr = 170,000 and 150,000) as the receptor for EGF (Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. 257, 4019-4022). Modification of the vesicles with 5'-p-FSO2BzAdo inhibits not only the EGF-stimulated phosphorylation of endogenous membrane proteins but also the EGF-stimulated phosphorylation of an exogenous synthetic tyrosine-containing peptide substrate. This indicates that the EGF-stimulable protein kinase is modified by 5'-p-FSO2BzAdo at a site affecting catalytic activity. Membrane vesicles were treated with 5'-p-FSO2Bz-[14C]Ado to affinity label the kinase, then the EGF receptor was purified by affinity chromatography on immobilized EGF. The EGF receptor thus purified contains the 5'-p-SO2Bz[14C]Ado moiety. These data strongly support our hypothesis that the EGF receptor and EGF-stimulable kinase are two parts of the same polypeptide chain.

Topics: Adenosine; Affinity Labels; Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Molecular Weight; Protein Kinases; Rats; Receptors, Cell Surface

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoma; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Mice; Mice, Inbred BALB C; Molecular Weight; Mouth Neoplasms; Receptors, Cell Surface

The effects of cholecystokinin-octapeptide (CCK8), the biologically active C-terminal moiety of cholecystokinin (CCK), on the binding of epidermal growth factor (EGF) were studied in isolated rat pancreatic acini. CCK8 inhibited 125I-EGF binding in a dose-dependent manner. One-half maximal inhibition occurred at 5 X 10(-10)M, and maximal inhibition at 10(-8)M CCK8. This inhibitory effect was detectable within 5 minutes of addition of CCK8, and was not associated with enhanced degradation of 125I-EGF in incubation media. Unlabeled EGF exerted only a slightly greater inhibitory effect than CCK8 on 125I-EGF binding at equivalent molar concentrations. In contrast to CCK8, the gastrointestinal hormone vasoactive intestinal polypeptide (VIP) did not significantly alter EGF binding. CCK8 also inhibited EGF binding in mouse pancreatic acini, but did not alter binding in A-431 human carcinoma cells. These findings suggest that physiological levels of CCK may regulate EGF binding in the pancreas and other tissues with receptors for both hormones. They thus point to a previously unrecognized mechanism for hormonal interaction.

Topics: Animals; Appetite Depressants; Carcinoma, Squamous Cell; Cell Line; Cholecystokinin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Male; Pancreas; Peptide Fragments; Rats; Rats, Inbred Strains; Receptors, Cell Surface; Sincalide; Vulvar Neoplasms

The transforming proteins (p21) of Harvey and Kirsten sarcoma viruses threonine kinase activity, which phosphorylates threonine 59 of the p21 proteins themselves. A tridecapeptide: Arg-Arg-Leu56-Asp-Thr-Thr59-Gly-Gln-Glu-Tyr-Ser-Ala66 containing residues 56-66 of p21 is phosphorylated solely on tyrosine by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Km-Values of 240 and 80 microM and Vmax values of 1.7 and 0.1 nmol.min-1.mg-1 were obtained in the presence and absence of EGF, respectively.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Kinetics; Kirsten murine sarcoma virus; Peptides; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Sarcoma Viruses, Murine; Transforming Growth Factors

Epidermal growth factor (EGF) inhibited the growth of A431 human epidermoid carcinoma cells. The tumor promoting, phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also retarded A431 cell growth. Addition of both TPA and EGF inhibited cell growth in an additive or synergistic manner depending upon the initial plating density of the cultures. EGF increased the production of diacylglycerol (60-70%) and stimulated the synthesis of phosphatidylinositol (PI) from 3H-inositol (three- to fourfold increase). Both of these responses were attenuated in the presence of TPA. TPA alone stimulated the production of diacylglycerol (DG) but had little effect on PI synthesis. The biological effect of TPA appeared to be mediated by the presence of a high-affinity receptor for phorbol esters on A431 cells. Moreover, the binding of 125I-EGF to A431 cells was unaffected by TPA, suggesting that the antagonistic effects of TPA were occurring distal to the EGF receptor. These findings also indicated that although TPA and EGF both inhibited A431 cell growth, this effect could be dissociated from changes in PI synthesis but may be dependent upon transient changes in DG production.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Diglycerides; Epidermal Growth Factor; ErbB Receptors; Humans; Phorbol Esters; Phorbols; Phosphatidylinositols; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

Human platelet ionophore release-products (IRP) inhibit the binding of 125I-labelled epidermal growth factor (125I-EGF) to its receptors on Swiss 3T3 cells. The inhibition appears to be caused by platelet-derived growth factor (PDGF) in the IRP and results from a decrease in the apparent affinity of cellular receptors for 125I-EGF. However, our results indicate that PDGF does not bind directly to EGF receptors, since (1) PDGF does not down-regulate EGF receptors; (2) the PDGF-mediated inhibition of 125I-EGF binding is temperature-dependent; (3) cells which possess EGF receptors but lack PDGF receptors do not exhibit a PDGF-mediated inhibition of 125I-EGF binding.

Topics: Animals; Blood Platelets; Carcinoma, Squamous Cell; Cells, Cultured; Epidermal Growth Factor; Epithelium; ErbB Receptors; Fibroblasts; Humans; Male; Mice; Platelet-Derived Growth Factor; Rats; Receptors, Cell Surface

Epidermal growth factor (EGF) stimulates the phosphorylation by A431 membranes of tyrosine residues in the myosin regulatory light chain (LC20) from chicken gizzard smooth muscle. In the presence of EGF, the Km of the EGF receptor kinase for LC20 is 73 microM and the V max is 17 nmol/min/mg. Two moles of phosphate are incorporated into tyrosine per mol of LC20. Trypsin digestion of the phosphorylated LC20 produces two phosphopeptides which are phosphorylated to approximately equal extents. Sequential Edman degradation of the separated phosphopeptides shows that tyrosines 142 and 155 of the protein are phosphorylated. Tyrosine 142 is located within a sequence similar to that of autophosphorylated tyrosine kinases in that five of the seven amino acids on the NH2-terminal side are acidic. Tyrosine 155 has no acidic amino acids near its NH2-terminal side. A comparison of the initial rates of phosphorylation of the two tyrosines shows that tyrosine 142 is phosphorylated at three times the rate of tyrosine 155.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Chickens; Epidermal Growth Factor; ErbB Receptors; Gizzard, Avian; Humans; Kinetics; Muscle, Smooth; Myosins; Phosphorylation; Protein Kinases; Receptors, Cell Surface; Tyrosine

A 125I-labeled derivative of rat epidermal growth factor-like transforming growth factor (125I-eTGF) that retains the chemical and biological properties of native eTGF has been prepared to characterize the interaction of eTGF with epidermal growth factor (EGF) membrane receptors. Binding of 125I-eTGF and 125I-labeled mouse EGF (125I-EGF) to membrane preparations from human placenta and the A431 human epidermoid carcinoma cell line was competed for to the same extent and with the same potency by either unlabeled eTGF or EGF. Components of placenta membranes and A431 membranes (145-170 kDa) were the major species affinity-labeled by cross-linking with membrane-bound 125I-eTGF or 125I-EGF. Affinity labeling of these receptor species was fully inhibited by both native eTGF and native EGF when present in excess during incubation of membranes with 125I-eTGF or 125I-EGF. The binding affinity and capacity of receptors in A431 membranes and placenta membranes were strikingly similar for eTGF and EGF. However, binding of 125I-eTGF to membrane receptors was highly sensitive to pH, being optimal only within the pH 8.0-8.5 range whereas EGF binding was optimal at pH 7.0-9.0. eTGF binding and EGF binding were modified in parallel by exposure of placental membranes to phytohemagglutinin, concanavalin A, or wheat germ agglutinin. Prolonged exposure of A431 cells to eTGF or EGF induced the same extent of down-regulation of both eTGF binding and EGF binding. eTGF-induced down-regulation of receptors in A431 cells followed the same kinetics as EGF-induced receptor down-regulation. The data indicate that despite the limited sequence homology between eTGF and EGF, these two ligands exhibit a remarkably similar mode of interaction with a common eTGF/EGF membrane receptor type.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Peptides; Placenta; Pregnancy; Receptors, Cell Surface; Transforming Growth Factors

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Liver; Mice; Mutation; Radioligand Assay; Rats; Receptors, Cell Surface

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Diffusion; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Humans; Membrane Fluidity; Protein Conformation; Receptors, Cell Surface

Topics: Antibodies; Antigen-Antibody Complex; Carcinoma, Squamous Cell; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface; Research

The factors controlling the growth of human tumor cells in soft agar are poorly understood. However, it has been demonstrated that serum provides factors which promote anchorage-independent growth. We tested 58 tumor specimens, which were obtained from patients with adenocarcinoma of the lung, colon, ovary or squamous cell carcinoma, for their ability to form colonies in soft agar in serum-free or serum-supplemented media. The cells were unable to replicate, and none of the hormones or growth factors tested: insulin (I), transferrin (T), selenium (S), estradiol (E), hydrocortisone (H) or epidermal growth factor (EGF) could substitute for serum. Examination of the serum dose-response curves indicated that growth factors reduced the serum concentrations needed to support anchorage-independent growth. The addition of the supplements and the lowering of serum concentrations increased cloning efficiencies (C.E.) in 38/51 trials, when cells were able to grow initially. The addition of ITS increased C.E. in 18/21 cases, HITES in 15/17 cases and EGF in 12/18 cases as compared to controls. ITS and HITES increased the number of colonies only when serum was the limiting factor. EGF, however, increased the number of colonies even when serum was not the limiting factor. The ability of the supplements to enhance growth could not be correlated to tumor type or initial cloning efficiencies. However, in only 1/25 cases were cells that were unable to form colonies under standard conditions induced to form colonies in the presence of the growth factors. Normal and tumor-derived human fibroblasts did not form colonies in soft agar in the presence of these growth factors. The results suggest that human tumor cells may require the presence of serum-derived factors for growth in soft agar.

Topics: Adenocarcinoma; Agar; Blood; Carcinoma, Squamous Cell; Cell Division; Clone Cells; Colonic Neoplasms; Epidermal Growth Factor; Estradiol; Female; Humans; Hydrocortisone; Insulin; Lung Neoplasms; Ovarian Neoplasms; Selenium; Transferrin

Tyrosine phosphorylation seems to be a key event in the control of cellular growth. Several viral transforming proteins, including the src protein of Rous sarcoma virus, the p120 protein of Abelson leukaemia virus and the middle T antigen of polyoma virus, are phosphorylated by associated tyrosine kinases. The levels of kinase activity correlate with the transforming efficiency of the virus. The receptors for epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin are also phosphorylated by associated tyrosine kinase activities, which are stimulated by EGF, PDGF and insulin, respectively. The EGF-stimulated kinase and the src protein share similar substrate specificity for tyrosines immediately C-terminal to a sequence of acidic amino acids. Such a sequence is also found adjacent to the phosphotyrosine of middle T antigen, and in the homologous region of the hormone gastrin, adjacent to a tyrosine which is sulphated in approximately half the gastrin isolated from gastric mucosa. Reports that gastrin acts as a growth factor for cells of the gastrointestinal tract suggested that phosphorylation of this tyrosine might be physiologically more relevant than sulphation. We report here that synthetic human gastrin 17 is phosphorylated by the EGF-stimulated tyrosine kinase of A431 cell membranes. The Km values of 53-87 and 223-547 microM obtained in the presence and absence of EGF, respectively, are the lowest reported so far for this enzyme.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chromatography, High Pressure Liquid; Epidermal Growth Factor; Gastrins; Humans; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases

In the present study, we have demonstrated that human growth hormone (hGH) can be phosphorylated by the epidermal growth factor (EGF)-stimulated tyrosine kinase of A431 cell membranes. Phosphotyrosine was the predominant phosphoamino acid released from phosphorylated hGH on partial acid hydrolysis. All five tyrosine-containing tryptic peptides of hGH are also phosphorylated by the EGF-stimulated tyrosine kinase. The highest phosphate incorporation was found for peptide T4 (residues 20-38), which is distinguished by a high frequency of acidic amino acids. The phosphorylated peptides have been characterized by HPLC and two-dimensional mapping on paper. Comparison with the labeled peptides obtained on tryptic digestion of phosphorylated hGH suggests that tyrosine phosphorylation is restricted to two tryptic peptides, T4 (tyrosine-28 or -35) and T6 (tyrosine-42). It is suggested that the absence of early insulin-like activity in the naturally occurring Mr 20,000 variant of hGH, which has an internal deletion spanning residues 32-46, may be a consequence of the loss of the tyrosine phosphorylation sites at residues 35 and 42.

Topics: Amino Acid Sequence; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Growth Hormone; Humans; Kinetics; Peptide Fragments; Phosphorylation; Protein Kinases; Protein-Tyrosine Kinases; Trypsin

We have used monolayers of bromodeoxyuridine (BrdU)-grown and control human epidermoid carcinoma (A431) cells to investigate the polypeptide changes resulting when cells are rounded by epidermal growth factor (EGF). Whereas no significant change was detected in Triton-soluble components, the urea-solubilized matrix fraction revealed greater levels of a 20 kd species in cells exposed to EGF, compared with the same cells not exposed to the growth factor. The corresponding urea fraction from BrdU-grown cells showed decreased levels of the 20 kd species as a result of exposure to EGF. Further evidence for a differential effect of EGF resulting from growth of the cells with the pyrimidine analog was observed in the matrix fraction soluble in SDS, which revealed a decrease in a 20 kd species resulting from exposure of control cells to EGF, with no comparable effect in BrdU-grown cells. Our results suggest that EGF induces a change in the properties of matrix-associated components of low molecular weight in an effect which appears to be modified by prior growth of cells with BrdU.

Topics: Bromodeoxyuridine; Carcinoma, Squamous Cell; Cell Line; Cytoplasm; Epidermal Growth Factor; Humans; Mercaptoethanol; Octoxynol; Polyethylene Glycols; Proteins; Sodium Dodecyl Sulfate; Solubility; Urea

A431 cell membranes phosphorylate a synthetic peptide (Arg-Arg-Leu-Ile-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-Arg-Gly) in which residues 2--12 correspond to the sequence of the reported site of tyrosine phosphorylation in pp60src. Epidermal growth factor stimulates the phosphorylation of this peptide 2-fold over basal levels in a dose-dependent fashion. Phosphorylation is linear for approximately 3 min at 30 degrees C and occurs on the tyrosine residue. Kinetic analysis of the phosphorylation reaction indicates that epidermal growth factor increases the average Vmax from 3.8 to 7.5 nmol/min per mg and slightly decreases the average Km from 0.53 mM to 0.28 mM. A number of other peptides analogous to this tridecapeptide are also phosphorylated by A431 membranes. The data suggest that peptides with sequences similar to the site of tyrosine phosphorylation in pp60src are preferred substrates for the kinase in these membranes. Thus, the epidermal growth factor-stimulated protein kinase has the potential to interact with and phosphorylate pp60src. However, the A431 membranes also phosphorylate a tyrosine-containing peptide of totally unrelated sequence, suggesting that the kinase possesses a broad specificity for peptide phosphorylation that may not reflect its specificity with protein substrates.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; Oncogene Protein pp60(v-src); Peptide Fragments; Phosphoproteins; Phosphotyrosine; Protein Kinases; Tyrosine; Viral Proteins

Topics: Carcinoma, Squamous Cell; Cell Line; Diglycerides; Drug Antagonism; Epidermal Growth Factor; Humans; Kinetics; Phorbols; Phospholipids; Protein Kinase C; Protein Kinases; Structure-Activity Relationship; Tetradecanoylphorbol Acetate

The patching and endocytosis of EGF (epidermal growth factor) bound to A-431 cells (a human epidermoid carcinoma line) are temperature-sensitive processes which are completely inhibited at 4 degrees C. Receptor-mediated endocytosis generally occurs through coated regions, and EGF bound to its membrane receptor must diffuse laterally to these points of internalization. In this work we investigated the thermal sensitivity of the lateral diffusion of EGF receptor complexes and the thermal sensitivity of the patching and endocytosis of the hormone receptor complexes. Using the fluorescence photobleach recovery technique, we measured the lateral diffusion coefficients of a fluorescent derivative of EGF as a function of temperature. The lateral diffusion coefficient (D) increased gradually from 2.8 X 10(-10) cm2/s at 5 degrees C to 8.5 X 10(-10) cm2/s at 37 degrees C, and no phase transition was detected. Neither was a phase transition detected when we measured the diffusion coefficient of fluorescent lipid probes over this temperature range. From a calculation of the collision frequency of the occupied EGF receptors with coated regions using our measured values of D at 5 and 37 degrees C, we conclude that diffusion is not the rate-limiting step for either endocytosis or patching.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Diffusion; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Histocytochemistry; Humans; Kinetics; Male; Mice; Microscopy, Fluorescence; Receptors, Cell Surface; Temperature; Thermodynamics

To determine the role of epidermal growth factor (EGF)-stimulated protein kinase in the biological effects caused by EGF, tyrosine-specific kinase activity has been quantitated in A431 human epidermoid carcinoma cells and six variant cell lines. Because EGF inhibited proliferation of A431 cells, variants resistant to this inhibition were selected by treatment with mutagen and maintenance for 1 month in 0.1 muM EGF. After cloning and growth for 6-20 generations without EGF, the resistance of the variants to the growth-inhibitory effect of EGF was confirmed. Whereas EGF increased cellular phosphotyrosine content approximately 10-fold in parental A431 cells, EGF caused smaller or undetectable increases in the six variant cell lines. Solubilized membranes from the six variants displayed diminished EGF-stimulated phosphorylation of the EGF receptor and of antibodies to p60(src) (the product of the Rous sarcoma virus transforming gene), which act as an exogenous substrate. The decrease in EGF-stimulated tyrosine-specific protein kinase activity varied from approximately 40% (clone 16) to approximately 8% (clone 18) of parental A431 activity. Phosphorylated EGF receptors from parental and variant cells migrated identically on sodium dodecyl sulfate/polyacrylamide gels. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity, so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in the six variant cell lines with reductions in both activities to as low as 10%. These results suggest that this tyrosine-specific protein kinase activity mediates the growth-inhibitory effect of EGF on A431 cells and that both EGF binding and kinase activities reside in the same or tightly associated molecules.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Genetic Variation; Humans; Kinetics; Protein Kinases; Receptors, Cell Surface

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.

Topics: Calcium; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Iodoacetates; Iodoacetic Acid; Kinetics; Peptide Hydrolases; Phosphorylation; Receptors, Cell Surface

Topics: Autoradiography; Azo Compounds; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chromatography, Affinity; Epidermal Growth Factor; ErbB Receptors; Humans; Membrane Proteins; Paper; Phosphorylation; Receptors, Cell Surface

Epidermal growth factor (EGF) receptors extracted with Triton X-100 from human skin fibroblasts and A431 epidermoid carcinoma cells rapidly lose EGF-binding activity precipitable with polyethylene glycol. The presence of concanavalin A which can cross-link and, thereby, aggregate the receptors, allowed quantitative recovery of the lost EGF-binding activity. Scatchard analysis of EGF binding of Triton X-100-solubilized receptors showed that A431 cells and skin fibroblasts possess approximately 1.5 X 10(6) and 7 X 10(4) EGF-binding sites/cell, respectively, which exhibit similar affinities for the ligand. The heavy isotope density-shift method was employed to determine whether differences in rates of receptor synthesis or decay account for the large difference in number of receptors/cell between the two cell types. After shifting cells to medium containing heavy (15N, 13C, and 2H) amino acids, light and heavy receptors, solubilized from total cellular membranes, were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that A431 cells synthesize EGF receptors at a rate 12 times faster than skin fibroblasts and that the half-life for receptor decay of A431 cells is somewhat longer (t1/2 = 16 h) than that (t1/2 = 9 h) of fibroblasts. Down-regulation of cell surface and total cellular EGF-binding capacity in A431 cells occurs with a t1/2 of 2-3 h and results in a 70-83% decrease in receptor level in 12 h. Scatchard analysis revealed that these changes in EGF binding were due to an alteration of receptor number and not EGF-binding affinity. Rates of EGF receptor synthesis and inactivation/decay were determined by the heavy isotope density-shift method. No change in the rate of receptor synthesis occurred as a consequence of EGF receptor down-regulation. Down-regulation, however, caused a decrease in receptor half-life from 16 to 4.5 h. These results indicate that EGF-dependent regulation of EGF receptor level in A431 cells involves an alteration of the rate of receptor inactivation.

Topics: Carcinoma, Squamous Cell; Cell Line; Cells, Cultured; Concanavalin A; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Kinetics; Molecular Weight; Receptors, Cell Surface; Skin

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; Humans; Kinetics; Phosphoprotein Phosphatases; Protein Kinases; Vanadates; Vanadium

Topics: Animals; Antibodies, Monoclonal; Carcinoma, Squamous Cell; Cell Line; Cyanogen Bromide; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Male; Mice; Mitogens; Peptide Fragments; Receptors, Cell Surface; Skin Physiological Phenomena

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.

Topics: Avian Sarcoma Viruses; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Ketones; Kinetics; Phosphorylation; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Retroviridae; Structure-Activity Relationship

Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A431 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains approximately 50% and clone 4 contains approximately 20% of the EGF-stimulated protein kinase activity of the parental A431 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:1 mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.

Topics: Carcinoma, Squamous Cell; Cell Division; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Humans; Mutation; Protein Kinases; Protein-Tyrosine Kinases; Receptors, Cell Surface

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Cell Membrane; Chromatography, Affinity; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphorylation; Protein Binding; Protein Kinases; Receptors, Cell Surface

Thirteen primary squamous cell carcinomas of the epidermis and of the oral and pharyngeal epithelium were cultured with a 3T3 fibroblast feeder layer, a system originally developed for clonal growth and long-term serial cultivation of normal human keratinocytes. Six of these tumors could be propagated indefinitely as established cell lines. They formed rapidly growing well-differentiated squamous cell carcinomas when injected sc into athymic (nude) mice. The squamous cell carcinoma lines possessed different aneuploid karyotypes. They displayed subtle differences in colony morphology such that the were visually distinguishable from one another as well as from normal keratinocytes. The lines also varied greatly in their dependence on the fibroblast feeder layer for clonal growth in surface culture. Only 1 line could form large colonies with high efficiency in semisolid medium; the others grew only abortively under this condition and eventually differentiated terminally to form cornified envelopes. Progressive growth in semisolid medium, therefore, was not a useful in vitro marker of malignant transformation for these cancer cells of keratinocyte origin. However, a property shared by all the squamous cell carcinoma lines was a subnormal rate of commitment to terminal differentiation during incubation in suspension culture. Deprivation of anchorage triggers commitment so rapidly in normal keratinocyte populations that no cell remain viable after 2 days in semisolid medium. In contrast, after this same-period, all 6 lines retained more than 20% of their original colony-forming ability when replated in surface culture. This phenotype of increased survival capacity in semisolid medium promises to be a useful selective marker for the detection of rare malignant keratinocytes within large normal keratinocyte populations.

Topics: Aneuploidy; Carcinoma, Squamous Cell; Cell Survival; Cells, Cultured; Culture Media; Epidermal Growth Factor; Fibroblasts; Humans; In Vitro Techniques; Karyotyping; Neoplasm Transplantation; Phenotype; Skin

Serum-free culture conditions that permit the continuous growth of A-431 human epidermoid carcinoma cells were developed. In Dulbecco's modified Eagle's synthetic nutritional medium (DME) supplemented with fetuin, insulin, transferrin, biotin, and oleic acid-fatty acid-free bovine serum albumin complex A-431 cells grew at a rate comparable to that observed in the presence of calf or fetal calf serum. Of the factors tested, oleic acid had the most pronounced stimulatory effect on the growth and [3H]-thymidine incorporation of A-431 cells in serum-free medium. A-431 cells have a high number of receptors for epidermal growth factor (EGF); they bind and rapidly internalize EGF. Nevertheless, EGF did not stimulate either the growth or the [3H]-thymidine incorporation of these cells. Analyses of [125I]-EGF binding data indicated that A-431 cells grown in the presence of calf serum had about 3.2-3.9 X 10(6) specific, saturable EGF receptor sites on their surface. Linear Scatchard plots indicated a single class of noninteracting receptors with an apparent equilibrium dissociation constant of about 2.8 X 10(-9) M. The average number of receptors of A-431 cells maintained in DME supplemented with only fetuin, insulin, and transferrin for several months was significantly less, 1.54 X 10(6), than that of A-431 stock cells cultured in the same medium for 2 days only (2.68 X 10(6)). The apparent dissociation constants for the same cell populations were, however, similar, 4.5 X 10(-9) M and 4.1 X 10(-9) M, respectively. Stimulation of growth by oleic acid resulted in about 20% decrease in the average number of receptor sites, with an increase in the apparent equilibrium dissociation constant.

Topics: Biotin; Carcinoma, Squamous Cell; Cell Division; Cells, Cultured; Culture Media; Epidermal Growth Factor; Humans; Iodine Radioisotopes; Linoleic Acids; Oleic Acids; Thymidine; Tritium

A partially purified preparation of a transforming growth factor (TGF) obtained from serum-free growth medium conditioned by a human melanoma tumor line was found to stimulate the phosphorylation of a synthetic tyrosine-containing peptide. The sequence of the peptide is related to that of the known site of tyrosine phosphorylation in the Rous sarcoma virus-encoded transforming protein, pp60src. In A431 membranes, the characteristics of TGF- and epidermal growth factor (EGF)-stimulated peptide phosphorylation are nearly identical. The effects of the two growth factors are not additive, suggesting that TGF and EGF stimulate peptide phosphorylation through the same EGF receptor system. This conclusion is supported by the finding that both TGF and EGF stimulate peptide phosphorylation in wild type Swiss 3T3 cell membranes, but neither factor is effective in stimulating peptide phosphorylation in membranes prepared from EGF receptor-deficient NR6 3T3 cells.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Epidermal Growth Factor; Humans; Kinetics; Melanoma; Membrane Proteins; Mice; Peptides; Phosphorylation; Transforming Growth Factors; Tyrosine

A medium consisting of a rich basal nutrient mixture supplemented with bovine insulin (10 micrograms/ml), human transferrin (10 micrograms/ml), human cold-insoluble globulin (5 micrograms/ml), and ethanolamine (0.5 mM) supported the growth of the A431 human epidermoid cell line in the absence of serum with a generation time equal to that of cells in serum-containing medium. Addition of epidermal growth factor (EGF) to this culture medium at concentration mitogenic for other cell types resulted in a marked inhibition of A431 cell growth. Inhibitory effects of EGF were observed at 1 ng/ml and near-maximal effects were observed at 10 ng/ml. The inhibitory effect of EGF could be reversed by the omission of EGF in subsequent medium changes and could be prevented by the addition of anti-EGF antibody to the culture medium. Inhibition of A431 cell growth by EGF also could be demonstrated in serum-containing medium.

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; Humans; Insulin; Kinetics; Transferrin

Epidermal growth factor (EGF) induces rapid rounding of A-431 human epidermoid carcinoma cells in Ca(++)-free medium. Cell rounding is not induced by a variety of other polypeptide hormones, antiserum to cell membranes, local anesthetics, colchicine, cytochalasin B, or cyclic nucleotides. However, trypsin, like EGF, induces rounding of A- 431 cells in the absence of Ca(++). Both trypsin- and EGF-induced rounding are temperature dependent, appear to be energy dependent, and are inhibited by cytochalasins, suggesting that the active participation of microfilaments in cell rounding. However, a medium transfer experiment suggests that EGF-induced rounding is not attributable to secretion of a protease, and a number of serine protease inhibitors have no effect on the EGF-induced rounding process. Cell rounding is not attributable to the slight stimulation by EGF of the release of Ca(++) that is observed in the Ca(++)-free medium, as stimulation of such release by the ionophore A23187 neither induces cell rounding nor blocks EGF-induced rounding. Cells that have rounded up after treatment with EGF or trypsin spread out upon addition of Ca(++) to the medium, even in the continuing presence of EGF or typsin. Like the cell-rounding process, the cell-spreading process is temperature dependent, appears to be energy dependent, and is inhibited by cytochalasin B. Thus, EGF does not destroy the ability of the cell to spread; rather, in the presence of the EGF (or trypsin), cell spreading and the maintenance of the flattened state become dependent on external Ca(++). Because untreated cells remain flattened in the absence of Ca(++), the data suggest that EGF may disrupt Ca(++)-independent mechanisms of adhesion normally present in A-431 cells.

Topics: Adenosine Triphosphate; Calcium; Carcinoma, Squamous Cell; Cell Line; Cells, Cultured; Cyclic AMP; Cyclic GMP; Cytoskeleton; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Microtubules; Peptides; Trypsin

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Line; Contact Inhibition; Epidermal Growth Factor; ErbB Receptors; Humans; Protein Kinases; Receptors, Cell Surface

Treatment of A-431 human epidermoid carcinoma cells with epidermal growth factor (EGF) was shown to enhance the phosphorylation of a Mr = 34,000 protein. Because the phosphorylation of an analogous protein is enhanced in various cell lines transformed by Rous sarcoma virus (RSV) (Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836), we characterized the phosphorylation of the A-431 Mr = 34,000 protein under these two conditions in order to determine whether there are common pathways between viral transformation and EGF stimulation. The results of tryptic phosphopeptide mapping and phosphoamino acid analysis showed that the Mr = 34,000 protein was phosphorylated in an identical manner by the EGF-stimulated protein kinase activity and by the protein kinase activity of the RSV transformation-specific protein or of its normal cell homolog. Although the specific protein kinase that phosphorylates the Mr = 34,000 protein under conditions of EGF-stimulation is not yet identified, these studies demonstrate that at least one consequence of EGF stimulation is identical with one of the consequences of viral transformation.

Topics: Avian Sarcoma Viruses; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; Humans; Molecular Weight; Neoplasm Proteins; Oncogene Protein pp60(v-src); Peptide Fragments; Phosphopeptides; Phosphorylation; Trypsin; Viral Proteins

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cyanogen Bromide; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Male; Membrane Proteins; Mice; Molecular Weight; Peptide Fragments; Phosphorylation; Receptors, Cell Surface; Submandibular Gland

Epidermal growth factor (EGF) stimulates the incorporation of 32Pi and [3H]inositol into phosphatidylinositol (5-10-fold) in A-431 cells. EGF also stimulates the incorporation of 32Pi into phosphatidic acid (up to 10-fold). These effects are attributed to an acceleration of the turnover of phosphatidylinositol as a consequence of the binding of EGF to its membrane receptor. The extent of the phosphatidylinositol response to EGF parallels the extent of hormone binding. The phosphatidylinositol response to EGF appears to be dependent on an influx of calcium since (a) external calcium is required for the enhancement of phosphatidylinositol turnover, (2) the accumulation of 45Ca by A-431 cells is stimulated by EGF, (3) blockage of calcium influx with LaCl3 inhibits stimulation of phosphatidylinositol turnover, and (4) calcium influx via ionophore A23187 is sufficient to stimulate phosphatidylinositol turnover. Since the binding, internalization, and degradation of 125I-labeled EGF in A-431 cells are unaffected by the omission of calcium from the medium, external calcium and phosphatidylinositol turnover are not necessary for the internalization and degradation of the EGF-receptor complex.

Topics: Biological Transport, Active; Calcimycin; Calcium; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lanthanum; Magnesium; Phosphates; Phosphatidylinositols; Receptors, Cell Surface

Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating specific endogenous membrane proteins, as well as exogenously added histone. The binding of EGF to membranes in vitro resulted in a several-fold stimulation of the phosphorylation reaction. The phosphorylation reaction was not dependent on cyclic AMP or cyclic GMP. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system. The activation of the membrane-associated protein kinase by EGF appears to be a reversible phenomenon. The membrane preparation could be solubilized by a number of non-ionic detergents with the retention of both 125I-labeled EGF binding activity and EGF-enhanced phosphorylation of specific membrane proteins. The solubilized membrane preparation was purified by affinity chromatography. The purified preparation retained both EGF-binding activity and EGF-enhanced phosphorylation activity. Analysis of the affinity-purified preparation by SDS gel electrophoresis indicated the presence of one major protein band of molecular weight 150,000 and several trace bands. The evidence suggests that the major 150,000 protein band is the receptor for EGF and is a substrate of the phosphorylation reaction. The co-purification of EGF-binding activity and EGF-stimulated phosphorylation activity suggests an inherent close relationship. The EGF-stimulated phosphorylation reaction appears to be specific for tyrosine residues and in this regard resembles the src protein kinase.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Membrane Proteins; Molecular Weight; Phosphorylation; Protein Kinases; Receptors, Cell Surface

Topics: Actinin; Actins; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cytoplasm; Cytoskeleton; Epidermal Growth Factor; Humans; Muscle Proteins; Time Factors

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; Epithelium; Kinetics; Mice; Microscopy, Electron, Scanning; Microvilli; Pinocytosis; Pseudopodia

Epidermal growth factor (EGF)-enhanced protein kinase activity in plasma membrane preparations of A-431 human epidermoid carcinoma cells was shown to involve the phosphorylation of tyrosine residues. Phosphotyrosine was detected in both endogenous membrane proteins and in histone when added as an exogenous protein substrate. The major phosphorylated amino acid in partial acid hydrolysates of 32P-labeled A-431 membranes was identified as phosphotyrosine on the basis of its identical behavior to authentic phosphotyrosine on paper electrophoresis and thin layer chromatography; its 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) derivative was indistinguishable from that of the authentic compound. Only traces, if any, of phosphoserine or phosphothreonine were detected. [32P]Phosphotyrosine was also detected in pronase digests of 32P-labeled membrane proteins. The EGF receptor . protein kinase complex, which was solubilized with Triton X-100 and purified by EGF affinity chromatography, was shown to phosphorylate tyrosine residues of the isolated membrane protein.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chromatography, Thin Layer; Electrophoresis, Paper; Enzyme Activation; Epidermal Growth Factor; Humans; Membrane Proteins; Peptides; Phosphotyrosine; Protein Kinases; Tyrosine

Membranes may be prepared from A-431 human epidermoid carcinoma cells which have the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner and which, in the presence of EGF, catalyze the phosphorylation of a number of endogenous membrane proteins. The activation of the membrane associated protein kinase by EGF appears to be a reversible phenomenon. The membrane preparation may be solubilized by a number of nonionic detergents with the retention of both 125I-labeled EGF-binding activity and EGF-enhanced phosphorylation of specific membrane proteins. The solubilized membrane preparation may be purified by affinity chromatography using EGF covalently linked to Affi-Gel. The purified preparation retains both EGF-binding activity and EGF-enhanced phosphorylation activity. Analysis of the affinity-purified preparation by sodium dodecyl sulfate-gel electrophoresis indicates the presence of one major protein band of molecular weight 150,000 and several trace bands. The evidence suggests that the major 150,000 protein band is the receptor for EGF and is a substrate of the phosphorylation reaction. The co-purification of EGF-binding activity and EGF-stimulated phosphorylation activity suggests an inherent close relationship.

Topics: Animals; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Chromatography, Affinity; Epidermal Growth Factor; Humans; Immune Sera; Immunoassay; Kinetics; Mice; Molecular Weight; Peptides; Phosphorylation; Protein Kinases; Receptors, Cell Surface

Topics: Adenosine Triphosphate; Carcinoma, Squamous Cell; Cell Membrane; Cyclic AMP; Cyclic GMP; Electrophoresis; Epidermal Growth Factor; Humans; In Vitro Techniques; Membrane Proteins; Neoplasms, Experimental; Peptides; Phosphorylation

Three different human tumor lines in culture, a rhabdomyosarcoma, a bronchogenic carcinoma and a metastatic melanoma, release proteins (transforming growth factors, TGFs) into the medium that confer the transformed phenotype on untransformed fibroblasts. These proteins are acid and heat-stable; produce profound morphologic changes in rat and human fibroblasts; and enable normal anchorage-dependent cells to grow in agar. Removal of the transforming protein results in a reversion of cell phenotype. The major activity interacts with epidermal growth factor (EGF) cell membrane receptors. The peptides from these tumor cells are similar in their action to the sarcoma growth factor (SGF) released by murine sarcoma virus-transformed rodent cells. The most anchorage-independent tumor cells released the most TGFs. EGF-related TGFs were not detectable in fluids from cultures of cells with high numbers of free EGF membrane receptors (normal human fibroblasts and human carcinomas).

Topics: Adult; Aged; Animals; Binding, Competitive; Carcinoma, Bronchogenic; Carcinoma, Squamous Cell; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; DNA; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Growth Substances; Humans; Lung Neoplasms; Male; Melanoma; Peptides; Rats; Receptors, Cell Surface; Rhabdomyosarcoma

A small portion of the 125I-EGF that binds specifically to intact cells or isolated membrane from a variety of sources becomes directly and irreversibly linked to EGF receptors. This provides a simple technique for affinity labeling the EGF receptor. Membranes isolated from the human epidermoid carcinoma cell line A431, which possesses extraordinarily high numbers of EGF receptors, gave rise to three major direct linkage complexes of MW = 160,000, 145,000, and 115,000. The time course for information of each is similar, showing that 125I-EGF can form direct linkage complexes with several preexisting forms of the EGF receptor. The direct linkage of EGF to receptor is slow in comparison to 125I-EGF binding, but both processes have similar susceptibilities to competition by unlabeled EGF. EGF was modified chemically with the amino site-specific reagent, N-hydroxysuccinimidyl biotin. The biotinyl-EGF had a reduced capacity to engage in direct linkage complex formation with no concomitant reduction in its ability to bind to EGF receptors. Since native and biotinyl EGF have identical abilities to stimulate the uptake of 3H-thymidine into DNA when incubated with cultured murine 3T3 cells, the direct linkage of EGF to its receptor does not appear to play an important role in EGF-stimulated mitogenesis.

Topics: Animals; Carcinoma, Squamous Cell; Cell Membrane; Chemical Phenomena; Chemistry; DNA; Drug Stability; Epidermal Growth Factor; ErbB Receptors; Humans; Hydrogen-Ion Concentration; Male; Mice; Peptides; Receptors, Cell Surface

A small quantity of the 125I-EGF (epidermal growth factor) bound specifically to EGF receptors on the human epidermoid carcinoma cell line A431 associates covalently. The direct linkage complex formed migrates during gel electrophoresis as a single diffuse band of MW = 160,000-170,000. In contrast, direct linkage complexes of 160,000, 145,000, and 115,000 daltons are formed when EGF is incubated with membranes isolated from these cells; these arise from EGF receptor modification during membrane isolation. None of these modifications affected the affinity of the EGF binding site for 125I-EGF. The electrophoretic mobilities of the MW = 160,000 and 145,000 direct linkage complexes were similar to those of the major 32Pi-labeled products of the EGF-stimulated phosphorylation reaction described by Carpenter et al [Nature 276:409-410, 1978], indicating that proteolytic fragments of EGF receptors are the major phosphate acceptors in this reaction. EGF receptors on intact A431 cells accepted phosphate effectively from gamma-32Pi-ATP only when the cells were permeabilized with lysolecithin. This shows that the EGF binding and phosphate acceptor sites lie on opposing faces of the membrane. When the 145,000 dalton form of receptor is labeled with EGF or 32Pi and the labeled peptides subjects to tryptic hydrolysis under identical conditions, all phosphate is lost from high molecular weight products under conditions where the EGF-receptor covalent complex is converted largely to a 115,000 dalton form. This suggests that the phosphate acceptor site lies on the cytoplasmic side of the membrane on a region of receptor extending 30,000 daltons from the 115,000 dalton fragment containing the EGF binding site.

Topics: Binding Sites; Carcinoma, Squamous Cell; Cell Membrane; Electrophoresis, Agar Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphates; Phosphorylation; Receptors, Cell Surface; Trypsin

Between 60% and 100% of epidermal growth factor (EGF) binding activity was recovered from membranes of the A431 human epidermoid carcinoma cell line treated with solutions containing the nonionic detergent Triton X-100. Approximately half of the recovered binding activity was sedimented at low centrifugal force and hence was operationally insoluble in nonionic detergent solution. Receptors in both the detergent-soluble and -insoluble fractions displayed similar affinities for 125I-EGF, and the values were in good agreement with those obtained for receptors in untreated membranes. The receptors in both fractions also formed identical direct linkage complexes with 125I-EGF in similar yield, providing no evidence for partitioning of different molecular species of EGF receptors in the detergent-soluble membrane fraction of Sepharose 6-B revealed heterogeneity of 125I-EGF binding activity; the smallest and most monodisperse peak of activity resolved by this technique was eluted at a Strokes radius of 95 A. Operationally soluble 125I-EGF binding activity also behaved heterogeneously during velocity sedimentation; more than half the activity sedimented more rapidly than the apparently monidisperse, 7S form. An average of less than half the nonionic detergent-solubilized activity recovered from 10 independent membrane preparations behaved as an apparently monodisperse entity. Since a maximum of 60% of 125I-EGF binding activity was operationally soluble, less than 25% of the total EGF binding activity was recovered in an apparently monodisperse form. The remaining 75% of the EGF receptors displayed a marked tendency to exist as aggregates in nonionic detergent solutions.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Centrifugation, Density Gradient; Chromatography, Gel; Detergents; Epidermal Growth Factor; ErbB Receptors; Humans; Macromolecular Substances; Octoxynol; Polyethylene Glycols; Receptors, Cell Surface; Solubility; Surface-Active Agents

Membranes prepared from A-431 human epidermoid carcinoma cells retained the ability to bind 125I-labeled epidermal growth factor (EGF) in a specific manner. In the presence of [gamma-32P]ATP and Mn2+ or Mg2+, this membrane preparation was capable of phosphorylating endogenous membrane components, including membrane-associated proteins; the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. The binding of EGF to these membranes in vitro resulted in a severalfold stimulation of the phosphorylation reaction; again, the major phosphorylated amino acid residue detected in partial acid hydrolysates was phosphothreonine. Membrane-associated dephosphorylation reactions did not appear to be affected by EGF. The phosphorylation reaction was not stimulated by cyclic AMP or cyclic GMP in the absence or presence of EGF. The phosphorylation system of the membrane was able to utilize [gamma-32P]GTP in both the basal and EGF-stimulated reactions. The enhanced membrane phosphorylation was specific for EGF and its derivatives; a wide variety of other peptide hormones were ineffective. The A-431 membrane preparation also was capable of phosphorylating exogenous proteins, such as histone, phosvitin, and ribonuclease, by a process which was stimulated by EGF. These findings suggest that one of the biochemical consequences of the binding of EGF to membranes is a rapid activation of a cyclic AMP-independent phosphorylating system.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; Hormones; Humans; Kinetics; Membrane Proteins; Peptides; Phosphorylation

We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; Ferritins; Humans; Peptides; Pinocytosis; Receptors, Drug; Temperature

The morphological effects of epidermal growth factor (EGF) on human carcinoma cells A-431 have been examined by scanning electron microscopy. These flat polygonal cells normally exhibit only small membrane folds, but show extensive ruffling and extension of filopodia within 5 min of exposure to EGF at 37 degrees C. This ruffling activity is transient, subsiding within another 5--15 min, but several other changes in surface morphology follow. Within the first hour of exposure to the hormone, the cell surface becomes exceedingly smooth and the nuclei seem to protrude above the plane of the otherwise thin monolayer, giving the cells a "fried egg" appearance. Cells at the edges of colonies gradually retract from the substrate, leading to reorganization, by 12 h, of the monolayer into multilayered colonies. EGF thus induces both rapid and long-term alterations in the morphology of these epidermoid cells.

Topics: Carcinoma, Squamous Cell; Cell Membrane; Cell Nucleus; Epidermal Growth Factor; Humans; Microscopy, Electron, Scanning; Neoplasms, Experimental; Peptides; Time Factors

Horseradish peroxidase (HRP) uptake was used to measure fluid-phase pinocytosis in monolayers of human epithelioid carcinoma cells (A-431). Histochemistry confirmed that cell-associated HRP was restricted to intracellular vesicles. Biochemical methods showed that HRP uptake in control cultures was directly proportional to the duration of exposure. The addition of low concentrations of epidermal growth factor (EGF) to the incubation media produced a 10-fold increase in the initial rate of pinocytosis. The EGF effect was rapid (within 30 s) but transient; the rate of pinocytosis returned to control levels within 15 min. Metabolic inhibitors reduced the EGF-stimulated rate of pinocytosis by greater than 90%. A conjugate of EGF and ferritin (F:EGF) was used to simultaneously compare the intracellular locations of EGF and HRP. Much of F:EGF was internalized in approximately 100-nm vesicles, while most of the HRP was located in much larger vesicles (range 0.1--1.2 micrometer) which also contained F:EGF. The tumor-promoter 12-0-tetradecanoyl-phorbol-13-acetate, which shares several biological activities with EGF, was also effective in stimulating an increase in the rate of pinocytosis.

Topics: Animals; Carcinoma, Squamous Cell; Dose-Response Relationship, Drug; Epidermal Growth Factor; Histocytochemistry; Horseradish Peroxidase; Humans; Mice; Neoplasms, Experimental; Peptides; Pinocytosis

The binding and internalization of epidermal growth factor (EGF) in human epithelioid carcinoma cells (A-431), which have approximately 2.6 X 10(6) receptors per cell, has been followed with 125I-labeled EGF and by fluorescence microscopy. We have prepared a fluorescent derivative of EGF that is biologically active and retains substantial binding affinity for cell receptors. After binding of this derivative to cells at 6 degrees, the cellular borders were prominently stained and the fluorescence on the remainder of the membrane was uniform. Upon warming of these cells to 37 degrees for 10 min, the surface fluorescence diminished and randomly distributed endocytotic vesicles appeared in the cytoplasm. After 20 min at 37 degrees these fluorescent vesicles formed a perinuclear ring. The binding of EGF to the surface of these cells was also visualized by immunofluorescence using rabbit antibodies to EGF and rhodamine-labeled goat anti-rabbit antibodies. We did not detect large fluorescent clusters or cap formation in these experiments. These data provide direct confirmation of the previous biochemical data that suggested that cell membrane-bound EGF is rapidly internalized.

Topics: Biological Transport; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Fluorescent Antibody Technique; Humans; Neoplasms, Experimental; Peptides

Topics: Animals; Autoradiography; Bone Marrow Cells; Carcinoma, Squamous Cell; Cell Count; Cell Division; Cells, Cultured; Diffusion; DNA; Epidermal Cells; Epidermal Growth Factor; Epidermis; Growth Inhibitors; Humans; Interferons; Mice; Peptides; Psoriasis; Skin Neoplasms