epidermal-growth-factor and Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is a disease with a high incidence and a dreary prognosis. Its lack of symptomatology and late diagnosis contribute to the dearth and inefficiency of therapeutic schemes. Studies show that overexpressed epidermal growth factor receptor (EGFR) is a common occurrence, linking this to the progression of pancreatic cancer, although the association between its expression and the survival rate is rather controversial. EGFR-targeted therapy has not shown the results expected, leaving at hand more questions than answers; clearly, there is a need for a better understanding of the molecular pathways involved. Nanoparticles have been used in trying to improve the efficacy of antitumor treatment; thus, using EGFR's ligand, EGF, for nanoconjugation, showed promising results in increasing the cellular uptake mechanisms and apoptosis of the targeted cells.

Topics: Adenocarcinoma; Animals; Apoptosis; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Targeted Therapy; Nanoparticles; Pancreatic Neoplasms

To summarize the available knowledge about nonsmall cell lung cancer (NSCLC) in never smokers in terms of biological and clinical-pathological findings.. Overall in newly diagnosed NSCLC, 10% of men and 20% of women, with a much higher proportion among Asiatic women, are never smokers and among them an overwhelming proportion have adenocarcinoma. Several environmental, genetic, hormonal and viral factors have been associated with an increased risk of NSCLC in never smokers, but for none of them there is definitive evidence. The incidence of epidermal growth factor receptor mutations is higher in never smokers, whereas K-ras mutations are rarely detected in this group of never smoking patients. The role of never smoking status in NSCLC as a positive prognostic factor or predictive of a better chemosensitivity to systemic treatments is still undefined.. Epidemiological, molecular and clinical-pathological features indicate NSCLC in never smokers as a distinct entity. Future preclinical studies should address more deeply the biological differences between NSCLC in smokers and never smokers and, to avoid biased results due to differences in survival outcomes, smoking status should be considered among stratification factors in future clinical studies.

Topics: Adenocarcinoma; Asian People; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Risk Factors; Smoking

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cytokines; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Mutation; NF-kappa B; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins c-akt; Signal Transduction; Vascular Endothelial Growth Factor A

Pancreas cancer is a highly aggressive and rapidly fatal disease. The current standard of care for advanced disease improves survival modestly at best and provides palliation for a minority of patients. The need for new therapies is undisputed. This article describes new therapeutic strategies currently under investigation and discusses possible reasons that others have failed. New potential targets in the treatment of this formidable disease are suggested based on recent findings.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Genes, Tumor Suppressor; Humans; Immunotherapy; Macrophages; Matrix Metalloproteinase Inhibitors; Pancreatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Serine Endopeptidases; Signal Transduction

Barrett's esophagus is an acquired metaplastic change that occurs in the distal esophagus secondary to chronic gastroesophageal reflux. This premalignant condition forms the most important risk factor for developing esophageal adenocarcinoma, which is an extremely aggressive tumor with a 5-year survival rate of less than 25%. Carcinomas that arise in the setting of Barrett's esophagus are thought to develop as part of the metaplasia-dysplasia-carcinoma sequence.. To review the current knowledge on the genomic alterations involved in the development of Barrett's esophagus and its progression to dysplasia and/or cancer.. Several changes in gene structure, gene expression, and protein structure are associated with the progression of Barrett's esophagus to adenocarcinoma. Accumulation of these changes seems to be essential, rather than the exact sequence of these changes. Multiple molecular pathways are involved and interact with each other. Alterations in tumor suppressor genes, amongst which p53 and p16, are early events in the metaplasia-dysplasia-adenocarcinoma sequence, followed by loss of cell cycle checkpoints. Ongoing genomic instability leads to cumulative genetic errors and thereby the generation of multiple clones of transformed cells.. Within the multistep process of esophageal adenocarcinogenesis, to date no single molecular marker came forward able to predict who will and who will not develop cancer in the setting of Barrett's esophagus. Instead, panels of markers need to be developed in the future allowing to indicate disease progression. Identification of crucial molecular pathways involved in esophageal adenocarcinogenesis would ultimately improve therapy and facilitate development of new treatment strategies.

Topics: Adenocarcinoma; Apoptosis; Barrett Esophagus; Chromosome Aberrations; Cyclin D1; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Gastroesophageal Reflux; Gene Expression Regulation, Neoplastic; Humans; Metaplasia; Microsatellite Repeats; Precancerous Conditions; Receptor, ErbB-2; Tumor Suppressor Protein p53

Despite advances in our understanding of the molecular and genetic basis of pancreatic cancer, the disease remains a clinical challenge. Gemcitabine, the standard chemotherapy for pancreatic cancer, offers modest improvement of tumor-related symptoms and marginal advantage of survival. New approaches, alone and in combination with gemcitabine, are being developed to combat this cancer. In this article we review the current status of investigations into several classes of agents: matrix metalloproteinase inhibitors; farnesyl transferase inhibitors; epidermal growth factor receptor inhibitors, including monoclonal antibodies and tyrosine kinase inhibitors; cyclooxygenase-2 inhibitors, and others. The scientific rationale, mechanism of action, and clinical trial data for these novel agents are discussed.

Topics: Adenocarcinoma; Angiogenesis Inhibitors; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Cysteine Endopeptidases; Deoxycytidine; Epidermal Growth Factor; Gemcitabine; Humans; Hydroxamic Acids; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Multienzyme Complexes; Pancreatic Neoplasms; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; RNA, Antisense

Topics: Adenocarcinoma; Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, erbB-1; Humans; Lung Neoplasms; Mutation; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-raf; Quinazolines

Topics: Adenocarcinoma; Amides; Aniline Compounds; Animals; Antineoplastic Agents; Breast Neoplasms; Drug Design; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Genistein; Humans; Leukemia; Macaca fascicularis; Mice; Mice, Inbred BALB C; Mice, SCID; Models, Molecular; Molecular Structure; Neoplasm Proteins; Neoplasms; Nitriles; Protein Conformation; Protein Structure, Tertiary; Quinazolines; Recombinant Fusion Proteins; Sequence Alignment; Species Specificity; Structure-Activity Relationship; Tumor Cells, Cultured; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Prostate growth and development are primarily under the control of androgens; however, other factors can also influence prostatic growth through alternative pathways. This article discusses some of the major nonandrogenic mediators of prostate growth. Information on the pathways by which these factors exert their effects is also reviewed.

Topics: Adenocarcinoma; Androgens; Animals; APUD Cells; Bombesin; Carcinoma, Small Cell; Chromosomes, Human; Cytokines; Endothelin-1; Epidermal Growth Factor; Fibroblast Growth Factors; Growth Substances; Hormones; Humans; Insulin-Like Growth Factor Binding Proteins; Male; Neoplasms, Hormone-Dependent; Organ Specificity; Prostate; Prostatic Neoplasms; Receptors, Fibroblast Growth Factor; Receptors, Growth Factor; Receptors, Transforming Growth Factor beta; Somatomedins; Transforming Growth Factor beta; Tumor Cells, Cultured

A case of pancreatic carcinoma associated with the Leser-Trélat sign is reported. A 53-yr-old male had complained of mild epigastric discomfort and back pain accompanied by seborrheic keratoses, which had increased in size and number over the previous 6 mo. A tumor was detected in the head of the pancreas and macroscopically curatively resected. His skin lesions diminished after surgery, but progressed again when the tumor recurred. Immunohistology for EGF showed a low level in the pancreatic carcinoma cells but a higher EGF content was recognized in the hyperkeratinized portions of the seborrheic keratoses. Of 130 underlying malignancies described in the 125 reported patients with the Leser-Trélat sign, neoplasms of the gastrointestinal tract were most common, comprising 47.7% of the total. The present case is the third case showing an association between a pancreatic carcinoma and the Leser-Trélat sign, but the first case for which the tumor of the pancreas was diagnosed in an early stage and resected surgically, as a result of the suggestive nature of this sign.

Topics: Adenocarcinoma; Epidermal Growth Factor; Humans; Immunohistochemistry; Keratosis, Seborrheic; Male; Middle Aged; Pancreatic Neoplasms; Tomography, X-Ray Computed

Topics: Adenocarcinoma; Barrett Esophagus; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Growth Substances; Humans; Transforming Growth Factor alpha; Transforming Growth Factor beta

Growth and development of the prostate depend on androgen action and other factors. Androgens act directly by specific nuclear receptors and induce or control many effects mediated by peptide growth factors on both epithelial and stromal cells. The major important peptide growth factors detected in the prostate are Fibroblast Growth Factors, Transforming Growth Factors, and Epidermal Growth Factor. These factors are not prostate specific. They are produced by epithelial or stromal cells and regulate the growth of these two cell compartments through autocrine or paracrine pathways. Overproduction of these factors or modification in affinity or number of cell receptors may participate in the two main prostatic pathologies: benign hyperplasia or adenocarcinoma. Recently, other factors have been evidenced in the prostate: Interleukine 6, Nerve Growth Factor or Insulin-like Growth factors. The study of the localization and the effects of these factors may be crucial to understand the prostatic development and diseases and may suggest new targets for therapy.

Topics: Adenocarcinoma; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Interleukin-6; Male; Nerve Growth Factors; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Suramin; Transforming Growth Factor alpha; Transforming Growth Factor beta

Topics: Adenocarcinoma; Chromosome Deletion; Cytokines; Epidermal Growth Factor; Genes, Tumor Suppressor; Humans; Neoplasm Staging; Oncogenes; Stomach Neoplasms; Transforming Growth Factor alpha; Transforming Growth Factor beta

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

To investigate the anti-tumor activity and toxicity of the epidermal growth factor receptor (EGFR) inhibitor gefitinib (ZD1839 or Iressa; AstraZeneca Pharmaceuticals, Wilmington, DE), in patients with advanced non-small cell lung cancer (NSCLC).. This was an open label, expanded access program (EAP) of oral gefitinib administered at 250 mg per day continuously until evidence of undue toxicity or disease progression.. Two hundred consecutive patients with advanced NSCLC were enrolled in this study. The median number of prior chemotherapy regimens was 2 (range 0-6). One hundred seventy-two patients were treated with gefitinib; 23 expired from disease progression prior to treatment and 5 withdrew their consent. One hundred fifty-four patients are evaluable for toxicity; 8 (5.2%) experienced grade 3/4 toxicity; 2 patients discontinued therapy for grade 3 rash and one for grade 3 nausea. Of 172 patients evaluable for efficacy, 7 (4.1%; 95% CI; 1.7-8.2%) experienced a partial response (PR); 60 patients (34.9%) had stable disease (SD) as their best response. Median survival for all patients was 4.5 months (95% CI; 4.1-4.9 months). One-year survival was 29%. Significant independent prognostic factors associated with improved survival were female gender, good performance status (0/1), and adenocarcinoma histology.. Gefitinib has anti-tumor activity, in a heterogeneous population of NSCLC patients treated on the EAP study. Treatment with gefitinib in this population is associated with a longer survival in women, those with good performance status and in patients with adenocarcinomas. These findings need to be further validated in additional clinical studies.

Topics: Adenocarcinoma; Administration, Oral; Adult; Aged; Aged, 80 and over; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Disease Progression; Epidermal Growth Factor; Female; Gefitinib; Health Status; Humans; Lung Neoplasms; Male; Middle Aged; Prognosis; Protein-Tyrosine Kinases; Quinazolines; Sex Factors; Survival Analysis

Serum Epidermal Growth Factor (EGF) concentrations were estimated in 40 women with endometrial carcinoma and in 10 women with proliferative endometrium. Medium EGF level in study group was 0.97 +/- 0.41 ng/ml whereas 0.78 +/- 0.19 ng/ml in control group. The highest values of EGF (1.39 +/- 0.44 ng/ml) were found in moderately differentiated adenocarcinomas (G2) whereas the low values (0.65 +/- 0.18 ng/ml) in poorly differentiated adenocarcinomas were noted. Statistically significant differences were observed among control group and moderately differentiated adenocarcinomas and between all groups of adenocarcinomas. In two patients with sarcoma uteri the lowest EGF (0.49 and 0.58 ng/ml) serum concentrations were found. Our results confirm that low EGF serum concentrations as well as EGF receptor on the cell surface probably may increase the risk of cancerogenesis in human endometrium.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Endometrial Neoplasms; Epidermal Growth Factor; Female; Humans; Middle Aged; Sarcoma

The value of tumor angiogenesis, EGFR and c-erbB-2 oncoprotein, a long with p 53 protein expression for predicting relapse-free survival was investigated in 110 node-negative breast cancer patients. The grade of neovascularization was assessed by the microvessel density which was obtained by an immunocytochemical staining by factor VIII-related antigen. EGFR, c-erbB-2 oncoprotein and p 53 oncoprotein were also determined by immunocytochemical assay. Univariate analysis showed no statistical significance of EGFR, c-erbB-2 and p53 status as a prognostic indicator. However, the microvessel density was a significant predictor of relapse-free survival. Patients with over 100 counts of factor VIII-RA positive cells per mm2 field in the most active areas of neovascularization showed significantly poorer prognosis compared to those with less than 100 counts (p < 0.005). Multivariate analysis demonstrated that microvessel density was an independent prognostic indicator in node-negative breast cancer patients (p < 0.0005). It was suggested that microvessel density might be of use in selecting the high-risk group in node-negative breast cancer patients needing adjuvant therapies.

Topics: Adenocarcinoma; Adenocarcinoma, Scirrhous; Axilla; Bone Neoplasms; Breast Neoplasms; Carcinoma, Papillary; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Lymph Nodes; Neovascularization, Pathologic; Prognosis; Receptor, ErbB-2; Regression Analysis; Tumor Suppressor Protein p53

The aim of this study was to investigate the correlation between Silva pattern system and clinicopathological features of endocervical adenocarcinoma. Moreover, it was to find molecular markers helpful for Silva classification, and thus we also explored the expression levels of invasion, adhesion and proliferation biomarkers in cases of Silva non-invasive and invasive types. The survival based on Silva pattern system was analysed by Kaplan-Meier survival analysis, Log-rank test and  a COX risk proportionality model. Sixty samples were chosen to detect the MMP-2, MMP-9, u-PA, E-cadherin, β-catenin, EGF, TGF-α, HDGF, c-Met and RGN expression by immunohistochemistry. Multivariate analysis showed that pattern A/pattern B/pattern C Silva pattern system provided independent risk factors for prognosis. Our results found the levels of MMP-2, MMP-9 and u-PA were significantly higher in endocervical adenocarcinoma with destructive growth than in the  nondestructive group. The levels of E-cadherin and β-catenin were significantly lower in endocervical adenocarcinoma with destructive growth than in the nondestructive group. The levels of EGF, TGF-α and HDGF were significantly higher in endocervical adenocarcinoma with destructive growth than in the nondestructive group. Compared with 'non-invasive/invasive Silva pattern', this study suggests 'pattern A/pattern B/pattern C Silva pattern' could be a better criteria for predicting the prognosis. Furthermore, the dual-marker combination of 'MMP-2 and u-PA' and 'E-cadherin and β-catenin' is very important in the diagnosis of Silva pattern classification.

Topics: Adenocarcinoma; beta Catenin; Cadherins; Epidermal Growth Factor; Female; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Prognosis; Transforming Growth Factor alpha; Urokinase-Type Plasminogen Activator; Uterine Cervical Neoplasms

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) resistance may be acquired via genotypic and/or phenotypic transformations. Herein, we report an extremely uncommon case with sequential small cell transformation and EGFR T790M mutation, in an elderly female with EGFR exon 21 L858R-mutant lung adenocarcinoma, following treatment with a first-generation EGFR-TKI.. A 67-year-old female never-smoker presented with a cough and dyspnoea of 2 months' duration. Computerised tomography revealed a 39 mm lesion in the upper lobe of the right lung with pleural effusion. Pleural fluid cytology revealed metastatic lung adenocarcinoma, and EGFR testing revealed exon 21 L858R mutation. She was started on gefitinib. After a progression-free survival of 31 months, she presented with disease progression and multiple extra-thoracic metastases. Fine needle aspiration cytology of a chest wall lesion revealed metastatic small cell carcinoma. EGFR testing on this aspirate revealed persistent L858R mutation only. In view of small cell transformation, chemotherapy (etoposide and carboplatin) was administered. After 4 months, ascitic fluid cytology revealed metastatic adenocarcinoma with persistent L858R mutation and an acquired T790M mutation (both detected on liquid biopsy as well) indicating amplification of the adenocarcinoma clone and regression of the small cell carcinoma clone. She was then initiated on osimertinib.. The index case highlights the significance of serial EGFR genotyping along with repeated tissue and/or blood sampling in the prompt detection of genetic and phenotypic resistance mechanisms to EGFR-TKIs. Furthermore, it lends evidence in support of the upfront treatment approaches targeting the heterogeneity of acquired EGFR-TKI resistance mechanisms.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Aged; Carboplatin; Carcinoma, Small Cell; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Etoposide; Female; Gefitinib; Humans; Lung Neoplasms; Mutation; Protein Kinase Inhibitors

The aim of this study was to examine the antitumor activity of hinokitiol for its clinical application in the treatment of human cervical carcinoma.. Cervical carcinoma HeLa cells were treated by different concentrations of hinokitiol. Flow cytometry was used to analyze cell cycle. Senescence-associated β-galactosidase (SA-β-gal) assay was used to identify senescent cells. The effects of hinokitiol on EGF-induced cell migration were determined by wound healing and transwell migration assays. Western blot was used to detect proteins involved in cell cycle progression, apoptosis, autophagy, and EGF-induced signaling pathways.. Hinokitiol suppressed cell viability in a dose-dependent manner. Flow cytometric analysis indicated that hinokitiol treatment resulted in cell cycle arrest at G1 phase, with reduced number of cells in the G2/M phase. Western blot analysis further demonstrated that hinokitiol treatment increased the levels of p53 and p21, and concomitantly reduced the expression of cell cycle regulatory proteins, including cyclin D and cyclin E. SA-β-gal assay showed that hinokitiol treatment significantly induced β-galactosidase activity. In addition, treatment with hinokitiol increased the accumulation of the autophagy regulators, beclin 1 and microtubule-associated protein 1 light chain 3 (LC3-II), in a dose-dependent manner; however, it did not induce caspase-3 activation and poly ADP ribose polymerase (PARP) cleavage. In addition, epidermal growth factor-induced cell migration and c-Jun N-terminal kinase (JNK) and focal adhesion kinase (FAK) phosphorylation were significantly inhibited by hinokitiol.. Our findings revealed that hinokitiol might serve as a potential therapeutic agent for cervical carcinoma therapy.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Movement; Epidermal Growth Factor; Female; HeLa Cells; Humans; Monoterpenes; Tropolone; Uterine Cervical Neoplasms

Interaction of cancer cells with cancer-associated fibroblasts (CAFs) plays critical roles in tumor progression. Recently we proposed a new tumor invasion mechanism in which invasive cancer cells individually migrate on elongate protrusions of CAFs (CAF fibers) in 3-D collagen matrix. In this mechanism, cancer cells interact with fibronectin fibrils assembled on CAFs mainly through integrin-α5β1. Here we tested whether this mechanism is applicable to the collective invasion of cancer cells, using two E-cadherin-expressing adenocarcinoma cell lines, DLD-1 (colon) and MCF-7 (breast). When hybrid spheroids of DLD-1 cells with CAFs were embedded into collagen gel, DLD-1 cells collectively but very slowly migrated through the collagen matrix in contact with CAFs. Epidermal growth factor and tumor necrosis factor-α promoted the collective invasion, possibly by reducing the E-cadherin junction, as did the transforming growth factor-β inhibitor SB431542 by stimulating the outgrowth of CAFs. Transforming growth factor-β itself inhibited the cancer cell invasion. Efficient collective invasion of DLD-1 cells required large CAF fibers or their assembly as stable adhesion substrates. Experiments with function-blocking Abs and siRNAs confirmed that DLD-1 cells adhered to fibronectin fibrils on CAFs mainly through integrin-α5β1. Anti-E-cadherin Ab promoted the single cell invasion of DLD-1 cells by dissociating the E-cadherin junction. Although the binding affinity of MCF-7 cells to CAFs was lower than DLD-1, they also collectively invaded the collagen matrix in a similar fashion to DLD-1 cells. Our results suggest that the direct interaction with CAFs, as well as environmental cytokines, contributes to the collective invasion of cancers.

Topics: A549 Cells; Adenocarcinoma; Amides; Benzamides; Cancer-Associated Fibroblasts; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chromones; Collagen; Colonic Neoplasms; Connective Tissue; Dioxoles; Epidermal Growth Factor; Fibroblasts; Fibronectins; Humans; Immunohistochemistry; Integrin alpha5beta1; MCF-7 Cells; Morpholines; Neoplasm Invasiveness; Pyridines; Spheroids, Cellular; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Pancreatic cancer is one of the most malignant cancers with a high mortality rate. Some types of pancreatic cancer cells overexpress epidermal growth factor receptor (EGFR), which is a potential target for anticancer agents. In this study, we examined the effect of epidermal growth factor (EGF)-conjugated liposomes containing curcumin (EGF-LP-Cur) on three different EGFR-expressed human pancreatic cancer cell lines, BxPC-3, Panc-1 and Mia Paca-2. We have demonstrated that it is feasible to prepare liposomal vesicles of EGF-LP-Cur and that it is stable in the liquid vehicle at ambient conditions for three weeks. In addition, the formulation of curcumin had higher cytotoxicity on BxPC-3 than on any other cells. It is also shown that the cellular uptake of curcumin on BxPC-3, which is essential for the cytotoxicity, is associated with EGFR-mediated mechanism of action. In summary, our results have showed that targeting EGFR with EGF-conjugated curcumin liposomes enhanced the antitumor activity of curcumin against human pancreatic cancer cells.

Topics: Adenocarcinoma; Antineoplastic Agents; Curcumin; Epidermal Growth Factor; Humans; Liposomes; Pancreatic Neoplasms

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane receptor that plays a key role in cell-cell adhesion. CAR is found in normal epithelial cells and is increased in abundance in various human tumors, including lung carcinomas. We investigated the potential mechanisms by which CAR contributes to cancer cell growth and found that depletion of CAR in human lung cancer cells reduced anchorage-independent growth, epidermal growth factor (EGF)-dependent proliferation, and tumor growth in vivo. EGF induced the phosphorylation of CAR and its subsequent relocalization to cell junctions through the activation of the kinase PKCδ. EGF promoted the binding of CAR to the chromokinesin KIF22. KIF22-dependent regulation of microtubule dynamics led to delayed EGFR internalization, enhanced EGFR signaling, and coordination of CAR dynamics at cell-cell junctions. These data suggest a role for KIF22 in the coordination of membrane receptors and provide potential new therapeutic strategies to combat lung tumor growth.

Topics: Adenocarcinoma; Animals; Apoptosis; Cell Proliferation; Coxsackie and Adenovirus Receptor-Like Membrane Protein; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinesins; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant cancer that lacks effective targets for therapy. Alteration of epidermal growth factor (EGF) expression has been recognized as an essential molecular event in pancreatic carcinogenesis. Accumulating studies have demonstrated that miRNAs play critical roles in EGF signaling regulation, tumor initiation, cell proliferation and apoptosis. Here, we demonstrated that miR-21 expression was induced by EGF in pancreatic cancer cells. miR-21 promoted EGF-induced proliferation, inhibited cell apoptosis and accelerated cell cycle progression. In vivo experiments confirmed the influence of miR-21 on tumor growth. Mechanistic studies revealed that miR-21 targeted MAPK/ERK and PI3K/AKT signaling pathways to modulate cell proliferation. In addition, Spry2 was proven to be a target of miR-21. Furthermore, miR-21 and Spry2 were significantly related to clinical features and may be valuable predictors of PDAC patient prognosis.

Topics: Adenocarcinoma; Aged; Animals; Apoptosis; Carcinogenesis; Carcinoma, Pancreatic Ductal; Cell Cycle; Cell Movement; Cell Proliferation; Disease-Free Survival; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Heterografts; Humans; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Male; Membrane Proteins; Mice; MicroRNAs; Middle Aged; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

The intracellular environment is composed of a filamentous network that exhibits dynamic turnover of cytoskeletal components and internal force generation from molecular motors. Particle tracking microrheology enables a means to probe the internal mechanics and dynamics. Here, we develop an analytical model to capture the basic features of the active intracellular mechanical environment, including both thermal and motor-driven effects, and show consistency with a diverse range of experimental microrheology data. We further perform microrheology experiments, integrated with Brownian dynamics simulations of the active cytoskeleton, on metastatic breast cancer cells embedded in a three-dimensional collagen matrix with and without the presence of epidermal growth factor to probe the intracellular mechanical response in a physiologically mimicking scenario. Our results demonstrate that EGF stimulation can alter intracellular stiffness and power output from molecular motor-driven fluctuations in cells overexpressing an invasive isoform of the actin-associated protein Mena.

Topics: Adenocarcinoma; Algorithms; Breast Neoplasms; Cell Line, Tumor; Collagen; Computer Simulation; Cytoskeleton; Epidermal Growth Factor; Humans; Intracellular Space; Microfilament Proteins; Mitochondria; Models, Biological; Motion; Rheology; Tissue Scaffolds

Long non-coding RNAs are being increasingly recognised as important molecules involved in regulating a diverse array of biological functions. For example, many long non-coding RNAs have been associated with tumourigenesis and in this context their molecular functions often involves impacting on chromatin and transcriptional control processes. One important cellular control system that is often deregulated in cancer cells is the ERK MAP kinase pathway. Here we have investigated whether ERK pathway signaling in response to EGF stimulation, leads to changes in the production of long non-coding RNAs. We identify several different classes of EGF pathway-regulated lncRNAs. We focus on one of the inducible lincRNAs, EGF inducible long intergenic non-coding RNA 1 (EINCR1). EINCR1 is predominantly nuclear and shows delayed activation kinetics compared to other immediate-early EGF-inducible genes. In humans it is expressed in a tissue-specific manner and is mainly confined to the heart but it exhibits little evolutionary conservation. Importantly, in several cancers EINCR1 shows elevated expression levels which correlate with poor survival in lung adenocarcinoma patients. In the context of lung adenocarcinomas, EINCR1 expression is anti-correlated with the expression of several protein coding EGF-regulated genes. A potential functional connection is demonstrated as EINCR1 overexpression is shown to reduce the expression of EGF-regulated protein coding genes including FOS and FOSB.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Cell Line; Cell Line, Tumor; Chromatin; Epidermal Growth Factor; Gene Expression Regulation; HEK293 Cells; Humans; Lung Neoplasms; MAP Kinase Signaling System; RNA, Long Noncoding

In a patient with previous radically treated lung adenocarcinoma, the detection of a new lung cancer raises the question whether recurrence or a second primary lung cancer is involved. Current criteria for differentiating multiple lung tumors lack a biologic and molecular basis and may lead to misclassification with impact on survival.. We report the case of a female patient with a recent diagnosis of lung adenocarcinoma and a previous lung adenocarcinoma submitted to curative surgical therapy 4 years before. As both lesions were resected, were of the same histologic subtype and presented the same immunohistochemistry profile; we decided to perform mutational analysis of the epidermal growth factor (EGFR) gene to differentiate between recurrence and second primary lung cancer.. The EGFR gene was screened for mutations in exons 18, 19, 20 and 21 using direct sequencing of polymerase chain reaction products in DNA obtained from paraffin preserved cells from both tumors.. Mutational analysis of the EGFR gene, revealed different mutations in each tumor (both on exon 19) allowing the confirmation of the diagnosis of two metachronous primary lung cancers.. In this patient, mutational analysis of the EGFR gene was superior to histologic and immunohistochemistry characterization in differentiating between recurrent lung cancer and second primary lung cancer; allowing confirmation of the diagnosis of two metachronous primary lung cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; DNA Mutational Analysis; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Lung Neoplasms; Middle Aged; Mutation; Neoplasm Recurrence, Local; Neoplasms, Second Primary; Tomography, X-Ray Computed

Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes.. The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined.. CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors.. EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.

Topics: Adenocarcinoma; Cell Line, Tumor; Cetuximab; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; fms-Like Tyrosine Kinase 3; Gefitinib; Gene Rearrangement; Hepatocyte Growth Factor; Humans; Indoles; Lung Neoplasms; MAP Kinase Signaling System; Mutation; Niacinamide; Phenylurea Compounds; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Mas; Proto-Oncogene Proteins c-ret; Pyrroles; Quinazolines; RNA, Small Interfering; Signal Transduction; Sorafenib; Sunitinib

The molecular events in the malignant progression of colon adenoma after loss of adenomatous polyposis coli (APC) are not fully understood. KITENIN (KAI1 C-terminal interacting tetraspanin) increases the invasiveness of colorectal cancer cells, and we identified a novel EGFR-independent oncogenic signal of EGF that works under coexpressed KITENIN and ErbB4. Here we tested whether elevated KITENIN and ErbB4 contribute to further progression of intestinal adenoma following APC loss.. The intestinal tissues of villin-KITENIN transgenic mice in which villin-driven KITENIN expression induces increased c-Jun expression exhibit mild epithelial cell proliferation but no epithelial lineage changes compared with those of nontransgenic mice. Among the four ErbB4 isoforms, JM-a/CYT-2 and JM-b/CYT-2 exhibited the highest AP-1 activity when cells coexpressing KITENIN and each isoform were stimulated by EGF. Interestingly, predominant overexpression of the ErB4-CYT-2 mRNA as well as increased EGFR expression were observed in intestinal adenoma of APC(min/+) mice, which makes the microenvironment of activated EGF signaling. When we crossed villin-KITENIN mice with APC(min/+) mice, intestinal tumor tissues in the crossed mice showed the characteristics of early-stage invading adenocarcinoma. In patients with colorectal cancer, ErbB4-CYT-2 mRNA expression was significantly greater in tumor tissues than in normal adjacent tissues, but no significant differences in tumor tissue expression were found between different colorectal cancer stages. Furthermore, the mRNA expression of KITENIN and that of ErbB4-CYT-2 were positively correlated in human colorectal cancer tissue.. Elevated coexpression of KITENIN and ErbB4-CYT-2 promotes the transition of colon adenoma to adenocarcinoma within an APC loss-associated tumor microenvironment.

Topics: Adenocarcinoma; Adenoma; Adenomatous Polyposis Coli Protein; Animals; Biomarkers, Tumor; Carrier Proteins; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; JNK Mitogen-Activated Protein Kinases; Male; Membrane Proteins; Mice; Mice, Transgenic; Microfilament Proteins; Protein Isoforms; Receptor, ErbB-4; Tumor Microenvironment

Tetrandrine has been shown to reduce cancer cell proliferation and to inhibit metastatic effects in multiple cancer models in vitro and in vivo. However, the effects of tetrandrine on the underlying mechanism of HT29 human colorectal adenocarcinoma cell metastasis remain to be fully elucidated. The aim of the present study was focused on tetrandrine‑treated HT29 cells following epidermal growth factor (EGF) treatment, and Transwell, gelatin zymography, gene expression and immunoblotting assays were performed to investigate metastatic effects in vitro. Tetrandrine was observed to dose‑dependently inhibit EGF‑induced HT29 cell invasion and migration, however, no effect on cell viability occurred following exposure to tetradrine between 0.5 and 2 µM. Tetrandrine treatment inhibited the enzymatic activity of matrix metalloprotease (MMP)‑2 and MMP‑9 in a concentration‑dependent manner. The present study also found a reduction in the mRNA expression levels of MMP‑2 and MMP‑9 in the tetrandrine‑treated HT29 cells. Tetrandrine also suppressed the phosphorylation of EGF receptor (EGFR) and its downstream pathway, including phosphoinositide‑dependent kinase 1, phosphatidylinositol 3‑kinase and phosphorylated AKT, suppressing the gene expression of MMP‑2 and MMP‑9. Furthermore, tetrandrine triggered mitogen‑activated protein kinase signaling through the suppressing the activation of phosphorylated extracellular signal‑regulated protein kinase. These data suggested that targeting EGFR signaling and its downstream molecules contributed to the inhibition of EGF‑induced HT29 cell metastasis caused by tetrandrine, eventually leading to a reduction in the mRNA and gelatinase activities of MMP-2 and MMP-9, respectively.

Topics: Adenocarcinoma; Benzylisoquinolines; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; HT29 Cells; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Signal Transduction

Pancreatic ductal adenocarcinoma (PDAC) is one of the most potent and perilous diseases known, with a median survival rate of 3-5 months due to the combination of only advanced stage diagnosis and ineffective therapeutic options. Metformin (1,1-Dimethylbiguanide hydrochloride), the leading drug used for type 2 diabetes mellitus, emerges as a potential therapy for PDAC and other human cancers. Metformin exerts its anticancer action via a variety of adenosine monophosphate (AMP)-activated protein kinase (AMPK)- dependent and/or AMPK-independent mechanisms. We present data here showing that metformin downregulated pancreatic transcription factor pancreatic duodenal homeobox-1 (PDX-1), suggesting a potential novel mechanism by which metformin exerts its anticancer action. Metformin inhibited PDX-1 expression at both protein and mRNA levels and PDX-1 transactivity as well in PDAC cells. Extracellular signal-regulated kinase (ERK) was identified as a PDX-1-interacting protein by antibody array screening in GFP-PDX-1 stable HEK293 cells. Co-transfection of ERK1 with PDX-1 resulted in an enhanced PDX-1 expression in HEK293 cells in a dose-dependent manner. Immunoprecipitation/Western blotting analysis confirmed the ERK-PDX-1 interaction in PANC-1 cells stimulated by epidermal growth factor (EGF). EGF induced an enhanced PDX-1 expression in PANC-1 cells and this stimulation was inhibited by MEK inhibitor PD0325901. Metformin inhibited EGF-stimulated PDX-1 expression with an accompanied inhibition of ERK kinase activation in PANC- 1 cells. Taken together, our studies show that PDX-1 is a potential novel target for metformin in PDAC cells and that metformin may exert its anticancer action in PDAC by down-regulating PDX-1 via a mechanism involving inhibition of ERK signaling.

Topics: Adenocarcinoma; Carcinoma, Pancreatic Ductal; Cell Line; Cell Line, Tumor; Diabetes Mellitus, Type 2; Down-Regulation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Genes, Homeobox; HEK293 Cells; Homeodomain Proteins; Humans; MAP Kinase Signaling System; Metformin; Pancreas; Pancreatic Neoplasms; Protein Kinase Inhibitors; Signal Transduction; Trans-Activators

The transcription factor c-Fos controls many important cellular processes, including cell growth and apoptosis. c-Fos expression is rapidly elevated in the prostate upon castration-mediated androgen withdrawal through an undefined mechanism. Here we show that androgens (5α-dihydrotestosterone and R1881) suppress c-Fos protein and mRNA expression induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) or EGF in human prostate cancer (PCa) cell lines. Such suppression transpires through a transcriptional mechanism, predominantly at the proximal serum response element of the c-fos promoter. We show that androgen signaling suppresses TPA-induced c-Fos expression through repressing a PKC/MEK/ERK/ELK-1 signaling pathway. Moreover, our results support the hypothesis that p38(MAPK), PI3K, and PKCδ are involved in the androgenic regulation of c-Fos through controlling MEK/ERK. Stable silencing of c-Fos and PKCδ with shRNAs suggests that R1881 promotes cell death induced by low-dose TPA through a mechanism that is dependent on both PKCδ and loss of c-Fos expression. Reciprocally, loss of either PKCδ or c-Fos activates p38(MAPK) while suppressing the activation of ERK1/2. We also provide the first demonstration that R1881 permits cell death induced by low-dose TPA in the LNCaP androgen-dependent PCa cell line and that TPA-induced cell death is independent of exogenous androgen in the castration-resistant variants of LNCaP, C4-2 and C4-2B. Acquisition of androgen-independent killing by TPA correlates with activation of p38(MAPK), suppression of ERK1/2, and loss of c-Fos. These results provide new insights into androgenic control of c-Fos and use of PKC inhibitors in PCa therapy.

Topics: Adenocarcinoma; Androgens; Cell Death; Cell Line, Tumor; Dihydrotestosterone; Down-Regulation; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Metribolone; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Prostate; Prostatic Neoplasms; Protein Kinase C; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate

The efficacy of EGFR-tyrosine kinase inhibitors (TKIs) is significantly limited by various resistance mechanisms to those drugs. The resistance to EGFR-TKI is largely divided by two classes; acquired resistance after EGFR-TKI treatment, and primary resistance marked by cancer cell's dependence on other oncogene, such as KRAS. YAP has emerged as critical oncogene in conferring drug resistance against targeted therapy. In this study, we evaluated the role of YAP in primary and acquired EGFR-TKI resistance using gefitinib-resistant A549 and PC9 cells and their parental cell lines. Our study revealed that EGFR-TKI resistance is associated with enhanced YAP activity. Notably, YAP activation was independent of the Hippo pathway. We confirmed that AXL is a downstream target of YAP that confers EGFR-TKI resistance. And our results showed that YAP can induce ERK activation in lung adenocarcinoma. The combination of YAP inhibition with EGFR-TKI overcomes primary and acquired EGFR-TKI resistance. We also found increased YAP expression in human lung cancer after acquiring EGFR-TKI resistance. Collectively, we suggest a novel EGFR-TKI resistance mechanism involving YAP activation and suggest targeting YAP and EGFR simultaneously may be a breakthrough treatment of primary and acquired EGFR-TKI resistant lung cancer.

Topics: A549 Cells; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Epidermal Growth Factor; Hippo Signaling Pathway; Humans; Lung Neoplasms; Phosphoproteins; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Signal Transduction; Transcription Factors; Treatment Outcome; YAP-Signaling Proteins

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a very poor prognosis, due to the influence of the tumor stroma, which promotes tumor growth, early invasion and chemoradiation resistance. Efforts to develop models for identifying novel anticancer therapeutic compounds have been hampered by the limited ability of in vitro models to mimic these in vivo tumor-stroma interactions. This has led to the development of various three-dimensional (3D) culture platforms recapitulating the in vivo tumor-stroma crosstalk and designed to better understand basic cancer processes and screen drug action. However, a consensus for different experimental 3D platforms is still missing in PDAC. We compared four PDAC cell lines of different malignancy grown in 2D monolayers to three of the more commonly used 3D techniques (ultralow adhesion concave microwells, Matrigel inclusion and organotypic systems) and to tumors derived from their orthotopic implantation in mice. In these 3D platforms, we observed that cells grow with very different tumor morphologies and the organotypic setting most closely resembles the tumor cytoarchitecture obtained by orthotopically implanting the four cell lines in mice. We then analyzed the molecular and cellular responses of one of these cell lines to epidermal growth factor receptor (EGFR) stimulation with EGF and inhibition with erlotinib and found that only in the 3D platforms, and especially the organotypic, cells: i) responded to EGF by changing the expression of signalling components underlying cell-stroma crosstalk and tissue architecture, growth, invasion and drug resistance (E-cadherin, EGFR, ezrin, β1 integrin, NHERF1 and HIF-1α) similar to those reported in vivo; ii) had stimulated growth and increased erlotinib sensitivity in response to EGF, more faithfully mimicking their known in vivo behaviour. Altogether, these results, indicate the organotypic as the most relevant physiological 3D system to study the complex tumor stroma interactions driving progression and determining chemio-resistance.

Topics: Adenocarcinoma; Animals; Carcinoma, Pancreatic Ductal; Cell Culture Techniques; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Mice; Stromal Cells

Epidermal Growth Factor (EGF) receptor overexpression, KRAS, TP53, CDKN2A and SMAD4 mutations characterize pancreatic ductal adenocarcinoma. This mutational landscape might influence cancer cells response to EGF, Transforming Growth Factor β1 (TGFβ1) and stromal inflammatory calcium binding proteins S100A8/A9. We investigated whether chronic exposure to EGF modifies in a SMAD4-dependent manner pancreatic cancer cell signalling, proliferation and invasion in response to EGF, TGFβ1 and S100A8/A9. BxPC3, homozigously deleted (HD) for SMAD4, and BxPC3-SMAD4+ cells were or not stimulated with EGF (100 ng/mL) for three days. EGF pre-treated and non pretreated cells were stimulated with a single dose of EGF (100 ng/mL), TGFβ1 (0,02 ng/mL), S100A8/A9 (10 nM). Signalling pathways (Reverse Phase Protein Array and western blot), cell migration (Matrigel) and cell proliferation (XTT) were evaluated. SMAD4 HD constitutively activated ERK and Wnt/β-catenin, while inhibiting PI3K/AKT pathways. These effects were antagonized by chronic EGF, which increased p-BAD (anti-apoptotic) in response to combined TGFβ1 and S100A8/A9 stimulation. SMAD4 HD underlied the inhibition of NF-κB and PI3K/AKT in response to TGFβ1 and S100A8/A9, which also induced cell migration. Chronic EGF exposure enhanced cell migration of both BxPC3 and BxPC3-SMAD4+, rendering the cells less sensitive to the other inflammatory stimuli. In conclusion, SMAD4 HD is associated with the constitutive activation of the ERK and Wnt/β-catenin signalling pathways, and favors the EGF-induced activation of multiple signalling pathways critical to cancer proliferation and invasion. TGFβ1 and S100A8/A9 mainly inhibit NF-κB and PI3K/AKT pathways and, when combined, sinergize with EGF in enhancing anti-apoptotic p-BAD in a SMAD4-dependent manner.

Topics: Adenocarcinoma; Apoptosis; Calgranulin A; Calgranulin B; Cell Line, Tumor; Cell Movement; Critical Pathways; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; NF-kappa B; Pancreatic Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Smad4 Protein; Transforming Growth Factor beta1

Topics: Adenocarcinoma; Aged; Anaplastic Lymphoma Kinase; Brain Neoplasms; Endometrial Neoplasms; Epidermal Growth Factor; Female; Genotype; Humans; Lung Neoplasms; Precision Medicine; Radiography; Receptor Protein-Tyrosine Kinases

The epidermal growth factor (EGF) plays important roles in non-small cell lung cancer (NSCLC) susceptibility and functional polymorphism in the EGF (+61A/G) gene has been linked to increased risk of NSCLC. This study aimed to evaluate the role of the EGF +61A/G polymorphism in risk of NSCLC adenocarcinoma (ADC) occurrence and survival in an Indian population.. This case- control study included 100 histopathologically confirmed NSCLC (ADC) patients and 100 healthy controls. EGF (A61G) was genotyped by AS-PCR to elucidate putative associations with clinical outcomes. The association of the polymorphism with the survival of NSCLC patients was estimated by Kaplan-Meier curves.. It was found that EGF 61AG heterozygous and GG homozygous genotype is significantly associated with increased risk of NSCLC (ADC) occurrence compared to AA genotype, [OR 2.61 (1.31-5.18) and 3.25 (1.31-8.06), RR 1.51(1.15-2.0) and 1.72 (1.08-2.73) and RD 23.2 (6.90-39.5) and 28.53(7.0-50.1) for heterozygous AG (p=0.005) and homozygous GG (p=0.009)]. Patients homozygous for the G allele exhibited a significantly poor overall survival. The median survival time for patients with EGF 61 AA, AG, and GG genotypes was 10.5, 7.4, and 7.1 months (p=0.02), respectively. NSCLC (ADC) patients with GG + AG exhibited 7.3 months median survival compared to the AA genotype (p=0.009).. The present study revealed that the EGF A61G genotype may be a novel independent prognostic marker to identify patients at higher risk of occurrence and an unfavourable clinical outcome.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Epidermal Growth Factor; Female; Genetic Predisposition to Disease; Genotype; Humans; India; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Risk

Allyl isothiocyanate (AITC) has been found to present sources from consumed cruciferous vegetables. AITC is known to possess pharmacological and anticancer activities. The present study was designed to test the hypothesis that AITC suppressed the invasion and migration of epidermal growth factor (EGF)-stimulated HT29 cells and to elucidate the mechanisms for the antimetastatic abilities in vitro. The invasion and migration of EGF-stimulated HT29 cells were determined individually by Transwell cell invasion and wound-healing assays. Our results showed that AITC effectively inhibited both the invasive and migratory ability of HT29 cells. Furthermore, AITC downregulated the protein levels of matrix metalloproteinase-2 (MMP-2), MMP-9 and mitogen-activated protein kinases (MAPKs) (p-JNK, p-ERK and p-p38) by western blot analysis in HT29 cells following EGF induction. Thus, the metastatic responses in AITC-treated HT29 cells after EGF stimulation were mediated by the MMP-2/-9 and MAPK signaling pathways. We also used gene expression microarrays to investigate the gene levels related to cell growth, G-protein coupled receptor, angiogenesis, cell adhesion, cell cycle and mitosis, cell migration, cytoskeleton organization, DNA damage and repair, transcription and translation, EGFR and PKB/mTOR signals. In summary, it is possible that AITC suppresses the invasion and migration of EGF-induced HT29 cells, resulting from MMP-2/-9 and MAPKs. Hence, AITC may be beneficial in the treatment of human colorectal adenocarcinoma in the future.

Topics: Adenocarcinoma; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Down-Regulation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Food Preservatives; Humans; Isothiocyanates; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; p38 Mitogen-Activated Protein Kinases; Wound Healing

Circulating factors in patients with gastric/gastroesophageal junction (GEJ) cancers may promote tumor progression and metastasis and may be targeted for therapy.. Serum levels of ligands-vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), hepatocyte growth factor (HGF)-from four targetable pathways were measured before surgery, and levels were correlated to clinicopathologic characteristics and overall survival (OS).. In 147 patients who underwent potentially curative resection for gastric/GEJ adenocarcinoma, VEGF-A levels were higher in patients with R1 versus R0 resection (p = 0.037). High EGF levels were associated with poorly differentiated tumors (p = 0.02). Elevated FGF2 levels were found in Lauren diffuse-type tumors (p = 0.017) and tumors with seven or more metastatic nodes (N3) (p < 0.042). Patients with advanced-staged tumors had higher HGF levels (p = 0.012). At a median follow-up of 35 months, 46 patients (31 %) had died. Increased VEGF and HGF levels were correlated with decreased OS (p = 0.009 and 0.005). An adjusted total value (ATV) of all factors was better than any single factor in stratifying patients into good and poor prognosis groups (5-year OS 84.1 vs. 53.9 %, p = 0.005). By multivariate analysis, serum VEGF-A and ATV were significant independent prognostic factors (along with T and N category) for OS (p = 0.028 and 0.013, respectively).. In patients undergoing resection for gastric and GEJ cancer, high levels of angiogenic and growth factors are associated with unfavorable tumor characteristics and poorer overall survival. Thus levels of these factors can help delineate tumor biology and stratify prognosis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Esophageal Neoplasms; Esophagogastric Junction; Female; Fibroblast Growth Factor 2; Follow-Up Studies; Hepatocyte Growth Factor; Humans; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Retrospective Studies; Stomach Neoplasms; Survival Rate; Vascular Endothelial Growth Factor A

A new method based on Atomic Force Microscopy (AFM) was developed to real-time and in-situ detect epidermal growth factor receptor (EGFR) expression levels on living MCF-7 cells for evaluating the anticancer activity of resveratrol. Here, the inhibition effect of resveratrol on EGFR expression levels on MCF-7 cells was probed by epidermal growth factor (EGF)-functionalized tips for the first time. Changes in morphology and stiffness of single cell stimulated by resveratrol at different concentrations were detected by AFM. The consequences showed that resveratrol influenced the cellular state and reduced expression of EGFR on the cell surface, which were also interpreted by MTT assay and confocal microscopy assay. AFM, which was used to investigate potential targets for anti-tumor drug on living cells and realize a better understanding of drug action mechanism, was expected to be developed into a promising tool for screening of drugs.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immobilized Proteins; MCF-7 Cells; Microscopy, Atomic Force; Optical Imaging; Resveratrol; Stilbenes

Ras guanine nucleotide exchange factors (RasGEFs) mediate the activation of the Ras signaling pathway that is over activated in many human cancers. The RasGRP3, an activator of H-Ras and R-Ras protein exerts oncogenic effects and the overexpression of the protein is observed in numerous malignant cancer types. Here, we investigated the putative alteration of expression and potential function of RasGRP3 in the formation and progression of human breast cancer.. The RasGRP3 and phosphoRasGRP3 expressions were examined in human invasive ductal adenocarcinoma derived samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) both in mRNA (Q-PCR) and protein (Western blot; immunohistochemistry) levels. To explore the biological function of the protein, RasGRP3 knockdown cultures were established. To assess the role of RasGRP3 in the viability of cells, annexin-V/PI staining and MitoProbe™ DilC1 (5) assay were performed. To clarify the function of the protein in cell proliferation and in the development of chemotherapeutic resistance, CyQuant assay was performed. To observe the RasGRP3 function in tumor formation, the Severe combined immunodeficiency (SCID) mouse model was used. To investigate the role of the protein in Ras-related signaling Q-PCR and Western blot experiments were performed.. RasGRP3 expression was elevated in human breast tumor tissue samples as well as in multiple human breast cancer cell lines. Down-regulation of RasGRP3 expression in breast cancer cells decreased cell proliferation, induced apoptosis in MCF7 cells, and sensitized T-47D cells to the action of drugs Tamoxifen and trastuzumab (Herceptin). Gene silencing of RasGRP3 reduced tumor formation in mouse xenografts as well. Inhibition of RasGRP3 expression also reduced Akt, ERK1/2 and estrogen receptor alpha phosphorylation downstream from IGF-I insulin like growth factor-I (IGF-I) or epidermal growth factor (EGF) stimulation confirming the functional role of RasGRP3 in the altered behavior of these cells.. Taken together, our results suggest that the Ras activator RasGRP3 may have a role in the pathological behavior of breast cancer cells and may constitute a therapeutic target for human breast cancer.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Guanine Nucleotide Exchange Factors; Humans; Insulin-Like Growth Factor I; Mice; Mice, SCID; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Proto-Oncogene Proteins c-akt; ras Guanine Nucleotide Exchange Factors; RNA, Small Interfering; Signal Transduction; Tamoxifen; Trastuzumab; Xenograft Model Antitumor Assays

Growth and inflammatory factors are associated with poor prognosis in gastric adenocarcinoma (GA); however, the additive effects of growth and inflammatory factors in GA remain unclear. In this study, we investigated the ability of epidermal growth factor (EGF) and interleukin (IL-1β) to activate extracellular signal-regulated kinase (ERK)1/2 in GA cells, and correlated the relationships between their roles with the metastatic potential both in GA cells and GA tissues. The effects of EGF, IL-1β and EGF plus IL-1β in AGS and MKN-45 GA cells were examined using western blotting, Transwell migration and invasion assays, immunocytochemical staining and an activator protein (AP)-1 luciferase reporter gene assay, and was further characterized in GA tissues by immunohistochemistry. The results exhibited that EGF and IL-1β additively activated ERK1/2, increased migration and invasion than either EGF or IL-1β alone in AGS and MKN-45 cells. The mechanisms were involved in upregulating MMP-9 expression through increasing AP-1 transcriptional activity via ERK1/2 pathway; these effects were dose-dependently inhibited by silencing ERK1/2 or using U0126. In vivo data also confirmed that the overexpression of p-ERK1/2 in GA tissues correlated well with the EGF, IL-1β, EGF plus IL-1β, and was associated with metastasis, which was well correlation with the expression of MMP-9 and c-fos (AP-1). The results demonstrate that growth and inflammatory factors play an important role in metastasis of GA by additively activating ERK-1/2 and AP-1, and upregulating MMP-9. As both cytokines contribute to the migration and invasion of GA cells, EGF/IL-1β/ERK1/2 pathways may be key pathways closely associated with GA progression.

Topics: Adenocarcinoma; Butadienes; Cell Line, Tumor; Cell Movement; Enzyme Inhibitors; Epidermal Growth Factor; Female; Humans; Interleukin-1beta; Male; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Nitriles; Phosphorylation; Stomach Neoplasms; Transcription Factor AP-1

Ligands of transmembrane receptor tyrosine kinases have important roles in cell proliferation, survival, migration and differentiation in solid tumours. We conducted this study to evaluate the relationship between concentration of serum ligands and prognosis of patients with metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (EGFR) antibodies.. Between August 2008 and August 2011, serum samples were obtained from KRAS wild-type patients who met the inclusion criteria and received an anti-EGFR antibody treatment. Serum concentration of ligands was measured by an enzyme-linked immunosorbent assay, and somatic mutations of KRAS, BRAF, PIK3CA and BRAF were analysed by direct sequencing.. A total of 103 patients were enrolled in the present study. At the pretreatment serum levels, patients with high levels of hepatocyte growth factor (HGF) had shorter progression-free survival (PFS) and overall survival (OS) compared with those with low levels of HGF (median PFS: 6.4 months vs 4.4 months; P<0.001, median OS: 15.3 months vs 8.0 months; P<0.001, respectively). Patients with high levels of epiregulin (EREG) also had shorter PFS and OS compared with those with low levels of EREG (median PFS: 6.6 months vs 4.9 months; P=0.016, median OS: 13.8 months vs 7.4 months; P=0.048, respectively). In addition, patients whose serum levels of ligands were elevated at progressive disease had shorter PFS and OS compared with other patients.. Our study indicated that high levels of HGF and EREG were associated with resistance to treatment with anti-EGFR antibodies in KRAS wild-type patients with mCRC. Our findings will contribute to the newly combination therapy on the treatment of anti-EGFR antibodies.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cetuximab; Colorectal Neoplasms; Disease-Free Survival; DNA Mutational Analysis; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Hepatocyte Growth Factor; Humans; Male; Middle Aged; Prognosis; Proportional Hazards Models; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); ras Proteins; Retrospective Studies; ROC Curve; Treatment Outcome

The endocannabinoid anandamide (AEA), a neurotransmitter was shown to have anti-cancer effects. Fatty acid amide hydrolase (FAAH) metabolizes AEA and decreases its anti-tumorigenic activity. In this study, we have analyzed the role of FAAH inhibition in non-small cell lung cancer (NSCLC). We have shown that FAAH and CB1 receptor which is activated by AEA are expressed in lung adenocarcinoma patient samples and NSCLC cell lines A549 and H460. Since the synthetic analogue of anandamide (Met-F-AEA) did not possess significant anti-tumorigenic effects, we used Met-F-AEA in combination with FAAH inhibitor URB597 which significantly reduced EGF (epidermal growth factor)-induced proliferative and chemotactic activities in vitro when compared to anti-tumorigenic activity of Met-F-AEA alone. Further analysis of signaling mechanisms revealed that Met-F-AEA in combination with URB597 inhibits activation of EGFR and its downstream signaling ERK, AKT and NF-kB. In addition, it inhibited MMP2 secretion and stress fiber formation. We have also shown that the Met-F-AEA in combination with URB597 induces G0/G1 cell cycle arrest by downregulating cyclin D1 and CDK4 expressions, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, the combination treatment inhibited tumor growth in a xenograft nude mouse model system. Tumors derived from Met-F-AEA and URB597 combination treated mice showed reduced EGFR, AKT and ERK activation and MMP2/MMP9 expressions when compared to Met-F-AEA or URB597 alone. Taken together, these data suggest in EGFR overexpressing NSCLC that the combination of Met-F-AEA with FAAH inhibitor resulted in superior therapeutic response compared to individual compound activity alone.

Topics: Adenocarcinoma; Amidohydrolases; Animals; Apoptosis; Arachidonic Acids; Blotting, Western; Calcium Channel Blockers; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Chemotaxis; Drug Resistance, Neoplasm; Endocannabinoids; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Immunoenzyme Techniques; Lung Neoplasms; Male; Mice; Mice, Nude; NF-kappa B; Polyunsaturated Alkamides; Real-Time Polymerase Chain Reaction; Receptor, Cannabinoid, CB1; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Tissue Array Analysis; Tumor Cells, Cultured; Wound Healing; Xenograft Model Antitumor Assays

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFRTKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.

Topics: Adenocarcinoma; Aspartate Carbamoyltransferase; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Cell Line, Tumor; Dihydroorotase; Epidermal Growth Factor; ErbB Receptors; Glucose; Glucose Transporter Type 3; Glycolysis; Humans; Lactic Acid; Lung Neoplasms; Neoplasm Proteins; Pentose Phosphate Pathway; Signal Transduction

Aberrant epidermal growth factor (EGF)-dependent signaling plays a key role in the progression of human carcinomas. We found that TIP30, a tumor suppressor protein, translocated into the nucleus of human lung adenocarcinoma cells following EGF treatment, and the selective inhibitors of EGFR signaling pathways blocked this effect. Chromatin immunoprecipitation assays revealed that TIP30 negatively regulated EGF-dependent transcriptional activation of CCND1 through a HDAC1-dependent mechanism. In lung adenocarcinoma patients, the level of nuclear TIP30 was inversely correlated with that of EGFR and cyclin D1. These findings suggest that nuclear TIP30-induced downregulation of cyclin D1 transcription antagonizes EGFR signaling and suppresses tumorigenesis.

Topics: Acetyltransferases; Active Transport, Cell Nucleus; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Lung Neoplasms; Mice; Protein Binding; Signal Transduction; Transcription Factors; Transcription, Genetic

Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-β (IFNβ) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFNβ induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.

Topics: Adenocarcinoma; Animals; Base Sequence; Binding Sites; Cell Line, Tumor; Cell Nucleus; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; HEK293 Cells; Histones; Humans; Interferon-beta; Lung Neoplasms; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Metastasis; Nuclear Proteins; Promoter Regions, Genetic; Promyelocytic Leukemia Protein; RNA, Small Interfering; STAT3 Transcription Factor; Transcription Factors; Transcriptional Activation; Tumor Suppressor Proteins; Up-Regulation

Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein with putative functions in cell proliferation, angiogenesis and differentiation. Expression of ECM1 in several types of carcinoma suggests that it may promote tumor development. In this study, we investigated the role of ECM1 in oncogenic cell signaling in breast cancer, and potential mechanisms for its effects.. In order to find out the functional role of ECM1, we used the recombinant human ECM1 and viral transduction systems which stably regulated the expression level of ECM1. We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo. Moreover, tissues and sera of patients with breast cancer were used to confirm the effect of ECM1.. ECM1 protein was increased in trastuzumab-resistant (TR) cells, in association with trastuzumab resistance and cell proliferation. Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment. Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling.. ECM1 has important roles in both cancer development and trastuzumab resistance in breast cancer through activation of EGFR signaling.

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix Proteins; Female; Galectin 3; Humans; Matrix Metalloproteinase 9; MCF-7 Cells; Mice, Nude; Mucin-1; Neoplasm Transplantation; Receptor, ErbB-3; RNA, Messenger; Signal Transduction; Trastuzumab

It has been proved that cyclooxygenase-2 (COX-2) is a key factor in lung cancer oncogenesis. COX-2 can be induced by a number of cytokines and growth factors and can be regulated by the JAK/STAT signaling pathway. Inhibiting the expression of COX-2 can prevent the development of lung cancer. The aim fo this study is to investigate whether the epidermal growth factor (EGF) can stimulate the signal transducers and activators of transcription 5 (STAT5) as well as to discover the effects of the STAT5 signaling pathway on the COX-2 in human lung adenocarcinoma A549 cells.. The phenomenon of STAT5 activation stimulated by the EGF was assayed through immunofluorescence and Western blot. The adenovirus containing the wild-type (WT)-STAT5 (AdWT-STAT5) plasmid, dominant-negative (DN)-STAT5 (Ad-CMV5Stat5aΔ740) plasmid, and STAT5 siRNA were transfected into A549 cells. The latter two groups were stimulated using EGF. Reverse transcriptase polymerase chain reaction was used to detect the mRNA expression of COX-2.. STAT5 was not activated in A549 cells in vitro. EGF stimulation significantly increased the level of the p-STAT5 protein and induces the shuttling of p-STAT5 from the cytoplasm into the nucleus. STAT5 activation was crucial for the COX-2 expression induced by the EGF. STAT5 was required for COX-2 expression, but can mediated the effects of the COX-2 expression through pathways that were independent of transcriptional activation.. COX-2 expression is dependent on STAT5 phosphorylation. A second pathway does not require STAT5 phosphorylation.. 背景与目的 已有的研究表明COX-2在肺癌发生发展过程中起关键作用，它被一些细胞因子和生长因子所诱导产生，并受到JAK/STAT等信号通路的调控，抑制COX-2的表达能阻止肺癌的发展。本研究旨在探讨表皮生长因子（epidermal growth factor, EGF）在人肺腺癌A549细胞中对STAT5激活效应，以及STAT5信号通路对COX-2调控机制。方法 应用免疫荧光法及Western印迹法检测人肺腺癌A549细胞中EGF对STAT5的激活现象。分别用野生型STAT5（AdWT STAT5），STAT5显性负突变体（AdCMV5 Stat5a△740）以及STAT5 siRNA转染A549细胞，并用EGF对后两组转染细胞加以刺激，使STAT5及p-STAT5的表达发生变化，再用RT-PCR检测A549细胞中的COX-2 mRNA表达。结果 在体外A549细胞中STAT5无激活；EGF可以诱导STAT5的激活，促使磷酸化的STAT5穿梭入核；STAT5的激活是EGF诱导COX-2上调表达的必要条件；非磷酸化的STAT5可能通过非转录激活的途径参与了COX-2表达的调控。结论 在A549细胞中STAT5可以通过磷酸化和非磷酸化两种途径来实现对COX-2的调控。

Topics: Adenocarcinoma; Blotting, Western; Cell Line, Tumor; Cyclooxygenase 2; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Microscopy, Fluorescence; Models, Genetic; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; STAT5 Transcription Factor

Tid1 (DNAJA3), a DnaJ cochaperone, may promote degradation of oncogenic kinases. Tid1 has 2 isoforms, Tid1-L and Tid1-S, that may function differently. In this study, we investigated the role of the Tid1 isoforms in regulating EGF receptor (EGFR) signaling and lung cancer progression. We found that both Tid1-L and Tid1-S expressions were reduced in patients with non-small cell lung cancer compared with normal counterparts. Tid1-L expression correlated inversely with EGFR expression. Low Tid1-L/high EGFR expression predicted poor overall survival in patients with lung adenocarcinoma. Tid1-L overexpression in lung cancer cells attenuated EGFR signaling and inhibited cell proliferation, colony formation, and tumor growth in subcutaneous and orthotropic xenograft models. Conversely, depletion of Tid1 restored EGFR signaling and increased cell proliferation and colony formation. Tid1-L, but not Tid1-S, interacted with EGFR/HSP70/HSP90 through the DnaJ domain, counteracting the EGFR regulatory function of HSP90 by causing EGFR ubiquitinylation and proteasomal degradation. Tid1-L inhibited EGFR signaling even more than the HSP90 inhibitor 17-allylamino-demethoxy geldanamycin. We concluded that Tid1-L acted as a tumor suppressor by inhibiting EGFR signaling through interaction with EGFR/HSP70/HSP90 and enhancing EGFR ubiquitinylation and degradation.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Kaplan-Meier Estimate; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Multivariate Analysis; Neoplasm Transplantation; Polyubiquitin; Protein Isoforms; Protein Structure, Tertiary; Proteolysis; Signal Transduction; Ubiquitination

We have recently identified tumor necrosis factor (TNF)-α-induced phosphorylation of epidermal growth factor receptor (EGFR) at Thr-669 and Ser-1046/1047 via ERK and p38 pathways, respectively. In the present study, we investigated the roles of ligand-induced phosphorylation of serine and threonine residues in EGFR-overexpressing MDA-MB-468 breast cancer cells. Epidermal growth factor and heregulin, an ErbB3 ligand, induced the phosphorylation of Thr-669 and Ser-1046/1047. Inversely, constitutive tyrosine phosphorylation of the C-terminal domain, including Tyr-1068, was significantly downregulated on ligand stimulation. Inhibition of the ERK pathway by U0126 blocked ligand-induced Thr-669 phosphorylation as well as Tyr-1068 dephosphorylation. Downregulation of constitutive tyrosine phosphorylation of EGFR in HEK293 cells stably expressing the wild type was abolished by substitution of Thr-669 for Ala. In an asymmetric EGFR homodimer structure, one Thr-669 in the receiver kinase of the dimer was involved in downregulation. Similarly, Thr-669 in an EGFR-ErbB3 heterodimer also participated in tyrosine dephosphorylation. These results indicate that ERK-mediated Thr-669 phosphorylation suppresses constitutive tyrosine phosphosphorylation in the homo- and heterodimer asymmetric conformations of the EGFR.

Topics: Adenocarcinoma; Breast Neoplasms; Butadienes; Cell Line, Tumor; Dimerization; Epidermal Growth Factor; ErbB Receptors; Feedback, Physiological; Female; Humans; Ligands; MAP Kinase Signaling System; Neoplasm Proteins; Neuregulin-1; Nitriles; Phosphorylation; Phosphothreonine; Protein Conformation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Recombinant Proteins; Tumor Necrosis Factor-alpha

Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair, and endocytosis. While some reports have implicated myoferlin in cancer, the extent of its expression in and contributions to cancer are not well established. In this study, we show that myoferlin is overexpressed in human breast cancers and that it has a critical role in controlling degradation of the epidermal growth factor (EGF) receptor (EGFR) after its activation and internalization in breast cancer cells. Myoferlin depletion blocked EGF-induced cell migration and epithelial-to-mesenchymal transition. Both effects were induced as a result of impaired degradation of phosphorylated EGFR via dysfunctional plasma membrane caveolae and alteration of caveolin homo-oligomerization. In parallel, myoferlin depletion reduced tumor development in a chicken chorioallantoic membrane xenograft model of human breast cancer. Considering the therapeutic significance of EGFR targeting, our findings identify myoferlin as a novel candidate function to target for future drug development.

Topics: Adenocarcinoma; Animals; Apoptosis; Breast Neoplasms; Calcium-Binding Proteins; Carcinoma, Ductal, Breast; Carcinoma, Lobular; Caveolae; Cell Movement; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Immunoenzyme Techniques; Membrane Proteins; Muscle Proteins; Neoplasm Invasiveness; Proteomics; RNA, Small Interfering; Tumor Cells, Cultured

Physiological electric field (EF) plays a pivotal role in tissue development and regeneration. In vitro, cells under direct-current electric field (dcEF) stimulation may demonstrate directional migration (electrotaxis) and long axis reorientation (electro-alignment). Although the biophysical models and biochemical signaling pathways behind cell electrotaxis have been investigated in numerous normal cells and cancer cells, the molecular signaling mechanisms in CL1 lung adenocarcinoma cells have not been identified. Two subclones of CL1 cells, the low invasive CL1-0 cells and the highly invasive CL 1-5 cells, were investigated in the present study. CL1-0 cells are non-electrotactic while the CL 1-5 cells are anodally electrotactic and have high expression level of epidermal growth factor receptor (EGFR), in this study, we investigated the generally accepted hypothesis of receptor tyrosine kinase (RTK) activation in the two cell lines under dcEF stimulation. Erbitux, a therapeutic drug containing an anti-EGFR monoclonal antibody, cetuximab, was used to investigate the EGFR signaling in the electrotaxis of CL 1-5 cells. To investigate RTK phosphorylation and intracellular signaling in the CL1 cells, large amount of cellular proteins were collected in an airtight dcEF stimulation device, which has advantages of large culture area, uniform EF distribution, easy operation, easy cell collection, no contamination, and no medium evaporation. Commercial antibody arrays and Western blotting were used to study the phosphorylation profiles of major proteins in CL1 cells under dcEF stimulation. We found that electrotaxis of CL 1-5 cells is serum independent and EGFR independent. Moreover, the phosphorylation of Akt and S6 ribosomal protein (rpS6) in dcEF-stimulated CL1 cells are different from that in EGF-stimulated cells. This result suggests that CL1 cells' response to dcEF stimulation is not through EGFR-triggered pathways. The new large-scale dcEF stimulation device developed in the present work will aid the sample preparation for protein-based experiments.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cetuximab; Electric Stimulation; Electromagnetic Fields; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Lung Neoplasms; Phosphorylation; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6; Signal Transduction

In this study, we aimed to determine EGF, c-erbB-2 and EGFR expression in 25 specimens of intestinal gastric adenocarcinomas by standardized immunohistochemistry and to establish correlations with the major clinico-morphological parameters of these patients. We observed EGF reactivity in 22 (88%) cases, a c-erbB-2 protein expression in eight (32%) cases and an EGFR reactivity in 13 (46.42%) cases. The EGF expression was significantly correlated with the tumor degree of differentiation, but not with other investigated clinico-morphological parameters and nor with c-erbB-2 and EGFR1 expression. However, we noticed the existence of a dependence between c-erbB-2 and EGFR1 expression in the main tumor mass. Such immunoprofile suggests the possible intervention of autocrine and paracrine loops in the developing of intestinal variant of gastric adenocarcinomas.

Topics: Adenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Receptor, ErbB-2; Stomach Neoplasms

ADF/cofilin proteins are key modulators of actin dynamics in metastasis and invasion of cancer cells. Here we focused on the roles of ADF and cofilin-1 individually in the development of polarized migration of rat mammary adenocarcinoma (MTLn3) cells, which express nearly equal amounts of each protein. Small interference RNA (siRNA) technology was used to knockdown (KD) the expression of ADF and cofilin-1 independently.. Either ADF KD or cofilin KD caused cell elongation, a reduction in cell area, a decreased ability to form invadopodia, and a decreased percentage of polarized cells after 180 s of epidermal growth factor stimulation. Moreover, ADF KD or cofilin KD increased the rate of cell migration and the time of lamellipodia protrusion but through different mechanisms: lamellipodia protrude more frequently in ADF KD cells and are more persistent in cofilin KD cells. ADF KD cells showed a significant increase in F-actin aggregates, whereas cofilin KD cells showed a significant increase in prominent F-actin bundles and increased cell adhesion. Focal adhesion area and cell adhesion in cofilin KD cells were returned to control levels by expressing exogenous cofilin but not ADF. Return to control rates of cell migration in ADF KD cells was achieved by expression of exogenous ADF but not cofilin, whereas in cofilin KD cells, expression of cofilin efficiently rescued control migration rates.. Although ADF and cofilin have many redundant functions, each of these isoforms has functional differences that affect F-actin structures, cell adhesion and lamellipodial dynamics, all of which are important determinants of cell migration.

Topics: Actin Cytoskeleton; Actins; Adenocarcinoma; Animals; Cell Adhesion; Cell Movement; Cofilin 1; Destrin; Epidermal Growth Factor; Female; Focal Adhesions; Gene Expression Regulation, Neoplastic; Mammary Neoplasms, Animal; Neoplasm Metastasis; Rats; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinogenesis; Cell Line; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Gene Knockout Techniques; Genomics; Humans; Lung Neoplasms; Male; Mice; Mice, Transgenic; Mutation; Phosphoproteins; Proto-Oncogene Mas; ras Proteins; Tumor Suppressor Proteins

Metastatic breast adenocarcinomas display activation signatures for signaling pathways that trigger cell motility and tissue invasion. Here, we report that the adaptor protein transducer of Cdc42-dependent actin assembly-1 (Toca-1) is expressed in highly invasive breast cancers and regulates their metastatic phenotypes. We show that Toca-1 localizes to the filamentous actin-rich core of invadopodial protrusions actively degrading the extracellular matrix (ECM). Toca-1 colocalizes with Cortactin, and we show that this interaction is mediated by the SH3 domain of Toca-1. Stable knockdown (KD) of Toca-1 expression in MDA-MB-231 cells led to a significant defect in epidermal growth factor (EGF)-induced cell migration and invasion. Toca-1 KD cells also showed significant defects in EGF- and Src-induced ECM digestion and formation of invadopodial membrane protrusions. To test the role of Toca-1 in metastasis, we achieved stable Toca-1 KD in both human and rat metastatic breast adenocarcinoma cell lines. Orthotopic tumor xenografting of control and Toca-1 KD cells in natural-killer /B-/T-cell-deficient mice revealed a significant defect in spontaneous lung metastases with Toca-1 silencing in vivo. In contrast, no defects in primary tumor growth or lung seeding following tail vein injection of Toca-1 KD cells was observed, suggesting that Toca-1 functions at an early step in the dissemination of metastatic breast tumor cells. Taken together, our results identify Toca-1 as a proinvasive protein in breast adenocarcinoma and a potential therapeutic target to limit tumor metastasis.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Cortactin; Epidermal Growth Factor; Extracellular Matrix; Female; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Rats

Gastroesophageal reflux disease (GERD) is a pathology with a wide range of clinical and endoscopic manifestations. Epidermal growth factor receptor (EGFR), found in the epithelium of the digestive tract, plays an important role in epithelial repair and shows increased expression in different neoplasms, including esophageal tumors.. The purpose of this study was to evaluate EGFR expression using immunohistochemistry in esophageal biopsies obtained from patients with GERD, Barrett's esophagus, and adenocarcinoma of the esophagus.. EGFR expression was immunohistochemically determined in biopsies from 194 patients with symptoms suggestive of GERD or adenocarcinoma of the esophagus, seen at two Brazilian university hospitals between January 2003 and December 2008. Based on histopathological analysis, patients were divided into three groups: GERD, Barrett's esophagus and adenocarcinoma of the esophagus. EGFR expression was considered positive when staining was detected in the membrane.. Mean age was 55.25 years (range 30-90). Patients with GERD (n = 127) accounted for 65.5% of the sample, compared with 12.4% (n = 24) of patients with Barrett's esophagus and 22.2% (n = 43) of patients with esophageal adenocarcinoma. Immunohistochemical analysis was positive for EGFR in 19.1% of the patients (37/194), divided as follows: 8.7% (11/127) in the GERD group, 25% (6/24) in the Barrett's esophagus group, and 46.5% (20/43) in the esophageal adenocarcinoma group. Statistical analysis revealed significant differences between the three groups (p = 0.0001).. GERD patients showed lower levels of EGFR expression than patients with Barrett's esophagus or patients with adenocarcinoma of the esophagus, suggesting a direct relationship between EGFR expression and disease progression.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Barrett Esophagus; Epidermal Growth Factor; Esophageal Neoplasms; Female; Gastroesophageal Reflux; Gene Expression Regulation; Humans; Male; Middle Aged

This study was to determine preoperative serum levels of epidermal growth factor (EGF), interleukin-6 (IL-6), and C-reactive protein (CRP) in stage III colon cancer and correlate them with disease status and prognosis. The circulating EGF in correlation with primary site epidermal growth factor receptor (EGFR) was also evaluated.. Seventy-seven patients with curatively resected stage III colon cancer were selected for analysis. Enzyme-linked immunosorbent assay was used to determine EGF and IL-6 serum levels, and serum CRP levels were measured via immunoturbidimetry. EGFR expression was observed with immunohistochemical studies.. The median levels of EGFR, IL-6, and CRP were 189.4 pg/mL, 9.09 pg/mL, and 1.4 mg/mL, respectively. The factors related to recurrence with statistical significance included positive node status (P = 0.041), lymphovascular invasion (P = 0.001), and preoperative IL-6 level ≥9 pg/mL (P = 0.020). CRP and EGF levels were not significantly associated with disease-free survival rates (P = 0.438 and P = 0.309, respectively). Multivariate analysis using Cox's proportion model revealed that lymph node status was the single independent prognostic factor for predicting time until recurrence (odds ratio, 4.99; 95% confidence interval, 1.09-22.91; P = 0.038).. IL-6 expression in stage III colon cancer patients appears to be a prognostic marker of tumor behavior. No correlations between serum EGF concentrations and tumor EGFR positivity were found in this study.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; C-Reactive Protein; Colonic Neoplasms; Cytokines; Disease-Free Survival; Epidermal Growth Factor; Female; Follow-Up Studies; Humans; Interleukin-6; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Prognosis; Retrospective Studies

Targeted toxin-based therapeutics are hindered by poor intracellular uptake, limited stability and non-specific immune stimulation. To address these problems, ligand-targeted toxins in combination with low dose saponin mixtures have been adapted and tested in vivo in the past, however, undefined saponin raw mixtures are not suitable for use in clinical development. In the present work we therefore used a targeted toxin (Sap3-EGF, i.e. saporin fused to epidermal growth factor) in combination with a structurally defined isolated saponin m/z 1861 (SO-1861). In vitro evaluation confirmed a 6900-fold enhancement in the cytotoxic efficacy of Sap3-EGF against TSA-EGFR target cells. The required dose of the targeted toxin was appreciably reduced and there was a highly synergistic effect observed. An ex vivo hemolysis assay showed no or very less hemolysis up to 10 μg/mL of SO-1861. In the acute toxicity studies SO-1861 was found to be non-toxic up to a dose of 100 μg/treatment. The enzymes aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase did not show any statistically significant liver damage, which was further confirmed by histological examination. Additionally, creatinine was also similar to the control group thus ruling out damage to kidney. In vivo studies in a syngeneic BALB/c tumor model characterized by EGFR overexpression were done by applying 30 μg SO-1861 and 0.1 μg Sap3-EGF per treatment. A more than 90% reduction (p < 0.05) in the average tumor volume was observed by this combined therapy.

Topics: Adenocarcinoma; Animals; Breast; Breast Neoplasms; Cell Line, Tumor; Drug Delivery Systems; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunotoxins; Mice; Mice, Inbred BALB C; NIH 3T3 Cells; Pharmaceutical Vehicles; Saponaria; Saponins

More than half of patients with KRAS-wild type advanced colorectal cancer (CRC) fail anti-EGFR monoclonal antibodies. We studied EGFR-axis messenger RNA (mRNA) expression and RAS, RAF, PIK3CA mutations in order to identify additional biomarkers of cetuximab efficacy.. Previously genotyped (KRAS, NRAS, BRAF, PIK3CA mutations) formalin-fixed paraffin-embedded tumour biopsies of 226 cetuximab-treated CRC patients (1st to 3rd line therapy) were assessed for mRNA expression of epidermal growth factor receptor (EGFR) and its ligands EGF, Transofrming Growth Factor-a (TGFA), Amphiregulin (AREG) and Epiregulin (EREG) with real time quantitative PCR. Mutations were detected in 72 (31.9%) tumours for KRAS, in 6 (2.65%) for BRAF, in 7 (3.1%) for NRAS and in 37 (16.4%) for PIK3CA.. Only PIK3CA mutations occasionally coexisted with other gene mutations. In univariate analysis, prognostic significance for survival ( from metastases until death) was seen for BRAF mutations (Hazard Ratio HR 8.1, 95% CI 3.4-19), codon 12-only KRAS mutations (HR 1.62, 95% CI 1.1-2.4), high AREG mRNA expression only in KRAS wild type CRC (HR 0.47, 95% CI 0.3-0.7) and high EREG mRNA expression irrespective of KRAS mutation status (HR 0.45, 95% CI 0.28-0.7). EREG tumoural mRNA expression was significantly associated with a 2.26-fold increased likelihood of objective response to cetuximab therapy (RECIST 1.1). In multivariate analysis, favourable predictive factors were high AREG mRNA in KRAS wild type tumours, high EREG mRNA, low Ephrin A2 receptor mRNA. Cetuximab-treated patients with AREG-low KRAS wild type CRC fared very poorly, their survival being similar to KRAS mutant CRC. Patients with KRAS codon 13 or other non-codon 12 mutations had a median survival (30 months, 95% CI 20-35) similar to that of patients with KRAS wild-type (median survival 29 months, 95% CI 25-35), in contrast to patients with KRAS codon 12 mutations who fared worse (median survival 19 months, 95% CI 15-26).. BRAF and codon 12 KRAS mutations predict for adverse outcome of CRC patients receiving cetuximab. AREG mRNA reflects EGFR signalling in KRAS wild type tumours, predicting for cetuximab efficacy when high and failure when low. EREG may have a prognostic role independent of KRAS mutation.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Amphiregulin; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Biomarkers, Tumor; Cetuximab; Colorectal Neoplasms; DNA Mutational Analysis; EGF Family of Proteins; Epidermal Growth Factor; Epiregulin; ErbB Receptors; Female; Genes, ras; Genetic Predisposition to Disease; Genotype; Glycoproteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Phosphatidylinositol 3-Kinase; Proto-Oncogene Proteins B-raf; Retrospective Studies; RNA, Messenger

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

We have recently shown that the adaptor protein p140Cap regulates tumor properties in terms of cell motility and growth. Here, by using the highly metastatic rat adenocarcinoma cell line MTLn3-epidermal growth factor receptor (EGFR), we assess the role of p140Cap in metastasis formation. Orthotopic transplantation of MTLn3-EGFR cells over-expressing p140Cap in Rag2(-/-)γ(c)(-/-) mice resulted in normal primary tumor growth compared with the controls. Strikingly, p140Cap over-expression causes an 80% inhibition in the number of lung metastases. p140Cap over-expressing cells display a 50% reduction in directional cell migration, an increased number and size of focal adhesions, and a strong impairment in the ability to invade in a 3D matrix. p140Cap over-expression affects EGFR signaling and tyrosine phosphorylation of cortactin in response to EGF stimulation. Intriguingly, p140Cap associates with cortactin via interaction with its second proline-rich domain to the cortactin SH3 domain. The phosphomimetic cortactin tyrosine 421 mutant rescues migration and invasive properties in p140Cap over-expressing cells. Taken together, these data demonstrate that p140Cap suppresses the invasive properties of highly metastatic breast carcinoma cells by inhibiting cortactin-dependent cell motility.

Topics: Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Animals; Cell Line, Tumor; Cell Movement; Cortactin; Epidermal Growth Factor; ErbB Receptors; Green Fluorescent Proteins; HEK293 Cells; Humans; Immunohistochemistry; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Knockout; Microscopy, Fluorescence, Multiphoton; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Protein Binding; Rats; RNA Interference; Transplantation, Heterologous

There has been recent widespread enthusiasm in epidermal growth factor (EGFR) as a molecularly active target in esophageal adenocarcinoma (EAC). However, there is limited data on the extent of EGFR expression in EAC. Thus, the aim of this study was to evaluated EGFR, pErk1/2, and total Erk1/2 expression in malignant and benign specimens.. Baseline expression of EGFR in the human normal squamous, Barrett's, and EAC cell lines were determined as well as after bile acid treatment and curcumin pretreatment. In addition, EGFR expression was also evaluated in 60 matched normal and malignant EAC resected specimens.. The in vitro studies in the Het-1a, BarT, and OE19 cell lines failed to show any measurable expression of EGFR via Western blot technique. The marker serving as the positive control for the study, MnSOD, showed expression in each cell line for all three treatment regimens at approximately 24 kDa EGFR, showing moderate staining in the malignant tumor specimens and low staining in the benign tissue specimens. pErk1/2 showed low staining in the malignant tumor specimens and no staining in the benign tissue specimens. Total Erk1/2 showed high staining in both the malignant tumor specimens and benign tissue specimens. The differences in the mean staining scores for the malignant versus benign tissue specimens for pErk1/2 and total Erk1/2 are not statistically significant (p = 0.0726 and p = 0.7054, respectively).. Thus, in conclusion, EGFR expression has been confirmed to be limited to non-existent in EAC and thus its use as a clinically active target is limited at best. Prior to the use of these expensive anti-EGFR therapies, confirmation of overexpression should be verified.

Topics: Adenocarcinoma; Blotting, Western; Epidermal Growth Factor; Esophageal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Vitro Techniques; Reference Values; Sampling Studies; Sensitivity and Specificity; Statistics, Nonparametric; Tumor Cells, Cultured

Reflux esophagitis (RO) and Barrett's esophagus (BO) can cause esophageal adenocarcinoma (OAC). The esophageal mucosa in the RO-BO-OAC cascade is chronically exposed to gastro-esophageal reflux. Epidermal growth factor (EGF) has an important role in the protection and repair of mucosal damage, and non-physiologic levels are associated with gastrointestinal tumors. The aim is to determine the functional effect of EGF gene polymorphisms on RO, BO and OAC development. A cohort of 871 unrelated Dutch Caucasians consisted of 198 healthy controls, 298 RO patients, 246 BO patients and 129 OAC patients. The frequency of the EGF-production-associated 5'UTR A+61G polymorphism was determined in these four groups. EGF immunohistochemistry was performed on BO biopsies. EGF expression was significantly lower in the G/G genotype compared with the A/G (P=0.008) and A/A (P=0.002) group. The G/G genotype was significantly more prevalent in RO (odds ratios (OR)=2.6; 95% confidence intervals (95% CI): 1.3-5.2), BO (OR=3.0; 95% CI: 1.5-6.2) and OAC (OR=4.1; 95% CI: 1.8-9.7) than in controls. The G allele is associated with reduced EGF expression and increased risk for RO, BO and OAC development. This indicates that reduced mucosal protection resulting from genetically decreased EGF expression enhances esophageal tumor development.

Topics: Adenocarcinoma; Barrett Esophagus; Cohort Studies; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Esophagitis, Peptic; Female; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Immunohistochemistry; Logistic Models; Male; Middle Aged; Polymorphism, Single Nucleotide; Sex Characteristics

Metastasis is a multistep process and the main cause of mortality in lung cancer patients. We previously showed that EGFR mutations were associated with a copy number gain at a locus encompassing the TWIST1 gene on chromosome 7. TWIST1 is a highly conserved developmental gene involved in embryogenesis that may be reactivated in cancers promoting both malignant conversion and cancer progression through an epithelial to mesenchymal transition (EMT). The aim of this study was to investigate the possible implication of TWIST1 reactivation on the acquisition of a mesenchymal phenotype in EGFR mutated lung cancer. We studied a series of consecutive lung adenocarcinoma from Caucasian non-smokers for which surgical frozen samples were available (n = 33) and showed that TWIST1 expression was linked to EGFR mutations (P<0.001), to low CDH1 expression (P<0.05) and low disease free survival (P = 0.044). To validate that TWIST1 is a driver of EMT in EGFR mutated lung cancer, we used five human lung cancer cell lines and demonstrated that EMT and the associated cell mobility were dependent upon TWIST1 expression in cells with EGFR mutation. Moreover a decrease of EGFR pathway stimulation through EGF retrieval or an inhibition of TWIST1 expression by small RNA technology reversed the phenomenon. Collectively, our in vivo and in vitro findings support that TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR mutated lung adenocarcinoma and that large series of EGFR mutated lung cancer patients are needed to further define the prognostic role of TWIST1 reactivation in this subgroup.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Antigens, CD; Blotting, Western; Cadherins; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Lung Neoplasms; Male; Middle Aged; Mutation; Nuclear Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Twist-Related Protein 1

Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions. However, the role of S1P signaling in tumorigenesis remains to be elucidated. In this study, we show that S1P receptor subtype 3 (S1P₃) is markedly up-regulated in a subset of lung adenocarcinoma cells compared to normal lung epithelial cells. Specific knockdown of S1P₃ receptors inhibits proliferation and anchorage-independent growth of lung adenocarcinoma cells. Mechanistically, we demonstrate that S1P₃ signaling increases epidermal growth factor receptor (EGFR) expression via the Rho kinase (ROCK) pathway in lung adenocarcinoma cells. Nuclear run-off analysis indicates that S1P/S1P₃ signaling transcriptionally increases EGFR expression. Knockdown of S1P₃ receptors diminishes the S1P-stimulated EGFR expression in lung adenocarcinoma cells. Moreover, S1P treatment greatly enhances EGF-stimulated colony formation, proliferation and invasion of lung adenocarcinoma cells. Together, these results suggest that the enhanced S1P₃-EGFR signaling axis may contribute to the tumorigenesis or progression of lung adenocarcinomas.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lysophospholipids; Mice; Neoplasm Invasiveness; Receptors, Lysosphingolipid; rho-Associated Kinases; RNA Interference; RNA, Messenger; Signal Transduction; Sphingosine; Sphingosine-1-Phosphate Receptors; Time Factors; Transcriptional Activation; Transfection; Up-Regulation

In human adenocarcinoma, claudin-2 expression is higher than that in normal lung tissue, but the regulatory mechanism of its expression has not been clarified. In human adenocarcinoma A549 cells, claudin-2 level time-dependently increased under the control conditions. In contrast, claudin-1 expression remained constant for 24h. The concentration of epidermal growth factor (EGF) in medium time-dependently increased, which was inhibited by matrix metalloproteinase (MMP) inhibitor II, an inhibitor of MMP-1, 3, 7, and 9. MMP inhibitor II decreased claudin-2 and phosphorylated ERK1/2 (p-ERK1/2) levels, which were recovered by EGF. Both claudin-2 and p-ERK1/2 levels were decreased by EGF neutralizing antibody, EGF receptor (EGFR) siRNA, AG1478, an inhibitor of EGFR, U0126, an inhibitor of MEK, and the exogenous expression of dominant negative-MEK. These results suggest that EGF is secreted from A549 cells by MMP and increases claudin-2 expression mediated via the activation of an EGFR/MEK/ERK pathway. The inhibition of the signaling pathway decreased phosphorylated c-Fos and nuclear c-Fos levels. The introduction of c-Fos siRNA decreased claudin-2 level without affecting claudin-1. The promoter activity of human claudin-2 was decreased by AG1478 and U0126. Furthermore, the activity was decreased by the deletion or mutation of the AP-1 binding site of claudin-2 promoter. Chromatin immunoprecipitation and avidin-biotin conjugated DNA assays showed that c-Fos binds to the AP-1 binding site. We suggest that a secreted EGF up-regulates the transcriptional activity of claudin-2 mediated by the activation of an EGFR/MEK/ERK/c-Fos pathway in A549 cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Binding Sites; Butadienes; Cell Line, Tumor; Claudins; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; MAP Kinase Signaling System; Nitriles; Promoter Regions, Genetic; Protease Inhibitors; Protein Binding; Proto-Oncogene Proteins c-fos; Quinazolines; RNA, Messenger; RNA, Small Interfering; Transcription Factor AP-1; Tyrphostins

To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy.. Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF) in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK)-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM). "Gefitinib-sensitive" genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer.. The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS) with a hazard ratio (HR) of 7.16 (P = 0.029) and 3.26 (P = 0.0072), respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC) histology in a Japanese cohort for OS and recurrence-free survival (RFS) with HRs of 8.79 (P = 0.001) and 3.72 (P = 0.0049), respectively.. The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis.. The Gene Expression Omnibus (GEO) GSE31210.

Topics: Adenocarcinoma; Cell Line, Tumor; Computational Biology; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Neoplasm Staging; Prognosis; Quinazolines; Reproducibility of Results

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

The assessment of cancer risk in patients with Barrett's esophagus (BE) is currently fraught with difficulty. The current gold standard method of assessing cancer risk is histological assessment, with the appearance of high-grade dysplasia (HGD) as the key event monitored. Sampling error during endoscopy limits the usefulness of this approach, and there has been much recent interest in supplementing histological assessment with molecular markers, which may aid in patient stratification.. No molecular marker has been yet validated to accurately correlate with esophageal histological progression. Here, we assessed the suitability of several membranous proteins as biomarkers by correlating their abundance with histological progression. In all, 107 patient samples, from 100 patients, were arranged on a tissue microarray (TMA) and represented the various stages of histological progression in BE. This TMA was probed with antibodies for eight receptor proteins (mostly membranous).. Epidermal growth factor receptor (EGFR) staining was found to be the most promising biomarker identified with clear increases in staining accompanying histological progression. Further, immunohistochemistry was performed using the full-tissue sections from BE, HGD, and adenocarcinoma tissues, which confirmed the stepwise increase in EGFR abundance. Using a robust H-score analysis, EGFR abundance was shown to increase 13-fold in the adenocarcinoma tissues compared to the BE tissues. EGFR was "overexpressed" in 35% of HGD specimens and 80% of adenocarcinoma specimens when using the H-score of the BE patients (plus 3 s.d.) as the threshold to define overexpression. EGFR staining was also noted to be higher in BE tissues adjacent to HGD/adenocarcinoma. Western blotting, although showing more EGFR protein in the adenocarcinomas compared to the BE tissue, was highly variable. EGFR overexpression was accompanied by aneuploidy (gain) of chromosome 7, plus amplification of the EGFR locus. Finally, the bile acid deoxycholic acid (DCA) (at neutral and acidic pH) and acid alone was capable of upregulating EGFR mRNA in vitro, and in the case of neutral pH DCA, this was NF-κB dependent.. EGFR is overexpressed during the histological progression in BE tissues and hence may be useful as a biomarker of histological progression. Furthermore, as EGFR is a membranous protein expressed on the luminal surface of the esophageal mucosa, it may also be a useful target for biopsy guidance during endoscopy.

Topics: Adenocarcinoma; Aged; Barrett Esophagus; Biomarkers, Tumor; Biopsy, Needle; Blotting, Western; Cell Transformation, Neoplastic; Cohort Studies; Disease Progression; Epidermal Growth Factor; Esophageal Neoplasms; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Middle Aged; Neoplasm Staging; Polymerase Chain Reaction; Precancerous Conditions; Predictive Value of Tests; Prognosis; RNA, Messenger; Up-Regulation

MTLn3 cells are highly invasive breast adenoacarcinoma cells. The relative level of the epidermal-growth-factor-stimulated invasion of this cell line is greater than two other breast cancer cell lines (MDA-MB-231 and MCF-7) and one non-small cell lung cancer cell line (H1299). We have determined that the mechanism of cancer cell invasion involves the presence of an enzymatically active phospholipase D (PLD), with the PLD2 isoform being more relevant than PLD1. PLD2 silencing abrogated invasion, whereas ectopic expression of PLD2 augmented cell invasion in all four cell lines, with an efficacy (MTLn3±MDA-MB-231>H1299±MCF-7) that correlated well with their abilities to invade Matrigel in vitro. We also report that PLD2 is under the control of Janus kinase 3 (JAK3), with the kinase phosphorylating PLD2 at the Y415 residue, thus enabling its activation. Y415 is located downstream of a PH domain and upstream of the catalytic HKD-1 domain of PLD2. JAK3 knockdown abrogated lipase activity and epidermal-growth-factor-stimulated cell invasion directly. For the purposes of activating PLD2 for cell invasion, JAK3 operates via an alternative pathway that is independent of STAT, at least in MTLn3 cells. We also consistently found that JAK3 and PLD2 pathways are utilized at the maximum efficiency (phosphorylation and activity) in highly invasive MTLn3 cells versus a relatively low utilization in the less invasive MCF-7 cell line. In summary, a high level of cell invasiveness of cancer cells can be explained for the first time by combined high JAK3/PLD2 phosphorylation and activity involving PLD2's Y415 residue, which might constitute a novel target to inhibit cancer cell invasion.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Line, Tumor; Epidermal Growth Factor; Female; Gene Silencing; Humans; Janus Kinase 3; Neoplasm Invasiveness; Phospholipase D; Phosphorylation; Rats

Estrogens contribute to the pathogenesis of female lung cancer and function mainly through estrogen receptor-β (ERβ). However, the way in which ERβ expression is regulated in lung cancer cells remains to be explored. We have found that signal transducer and activator of transcription 3 (Stat3) activation up-regulates ERβ expression in PC14PE6/AS2 lung cancer cells in a preliminary Affymetrix oligonucleotide array study, and we sought to confirm the findings. In this study, we show that IL-6 induced ERβ mRNA and protein expression in lung cancer cells. The induction of ERβ in response to IL-6 was abolished by Janus kinase 2 inhibitor-AG490, dominant-negative mutant of Stat3, and Stat3-targeting short interfering RNA. The luciferase reporter assay and chromatin immunoprecipitation assay confirmed that IL-6-activated Stat3 binds to the ERβ promoter. Besides the Janus kinase 2/Stat3 pathway, the MEK/Erk pathway contributes to ERβ up-regulation induced by IL-6; however, the phosphoinositide 3'-kinase/Akt pathway does not. We also found that epidermal growth factor (EGF) stimulation or L858R mutation in EGF receptor (EGFR) induced Stat3 activation as well as ERβ expression in lung cancer cells. Inhibiting Stat3 activity by pharmacological or genetic approaches reduced EGF- and L858R mutant EGFR-induced ERβ expression, indicating that Stat3 activation is required for EGFR signaling-mediated ERβ up-regulation. Silencing ERβ decreased cell proliferation in lung cancer cells that overexpress L858R mutant EGFR. In conclusion, we have identified that Stat3 activation is essential for ERβ induction by IL-6, EGF, and the presence of EGFR mutation. The findings shed light on new therapeutic targets for female lung cancer, especially for those with EGFR mutations.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor beta; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Lung Neoplasms; MAP Kinase Signaling System; Mutation, Missense; Neoplasms, Hormone-Dependent; Phosphorylation; Protein Binding; RNA Stability; STAT3 Transcription Factor; Transcription, Genetic; Up-Regulation

The epidermal growth factor receptor (EGFR) can be activated by several growth factors within the tumor microenvironment, and it can activate several signaling pathways. For tumor development, these EGFR-related signaling pathways may converge on several common nuclear transcription factors, one such transcription factor being STAT5. STAT5 plays an important role in the oncogenic signal transduction pathway in non-small cell lung cancer. In this study, we examined whether the epidermal growth factor (EGF) can stimulate cyclooxygenase-2 (COX-2) expression in human lung adeno-carcinoma A549 cells transfected with or without STAT5 siRNA or dominant-negative (DN)-STAT5, and identified the pathways involved in this response. We found that STAT5 siRNA significantly reduced EGF-induced COX-2 expression, and STAT5 phosphorylation. STAT5 phosphorylation predominantly mediates EGF-induced COX-2 promoter activity. STAT5 siRNA was found to inhibit COX-2 expression in resting A549 cells despite the absence of detectable activated phosphorylated STAT5. Using an adenoviral system, we expressed DN-STAT5 in human lung adenocarcinoma A549 cells in order to broaden the investigation and to determine the role of STAT5 in EGF-mediated COX-2 gene expression. The overexpression of DN-STAT5 significantly inhibited EGF-induced COX-2 expression, and we found that EGF induced the tyrosine phosphorylation of STAT5 and up- regulated COX-2 expression. DN-STAT5 also blocked COX-2 promoter activity. Our results demonstrate that EGF stimulates COX-2 expression in human lung adenocarcinoma A549 cells via the activation of the STAT5 pathway and that COX-2 expression may be independent of phosphorylated STAT5 in A549 cells in vitro.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cell Line, Tumor; Cyclooxygenase 2; DNA-Binding Proteins; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Phosphorylation; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction; STAT5 Transcription Factor

Lung cancer is one of the leading causes of death in the world. Some tumor events are attributed to an important group of molecules (cadherins and integrins). We evaluated the interactions of cell adhesion molecules in cell lines from lung cancer. Two lung cancer cell lines were nonmetastatic (H358 and H441) and two were metastatic (H1299 and H292). All cell lines were treated with epidermal growth factor (EGF), and Western blot analysis was performed to assess the interactions between these proteins. The bronchoalveolar cells H358 showed the three analyzed proteins: E-cadherin, β-catenin, and p120 catenin. The adenocarcinoma cells H441 did not present p120 catenin, and carcinoma cells did not show E-cadherin (H1299) or p120 catenin (H292). FAK (pTyr925) was dephosphorylated in adenocarcinoma cells H441, absent in carcinoma cells H1299, and upregulated in the other carcinoma cells H292. p130Cas showed no difference when the cell lines were treated with EGF for 30 min; it was absent in the metastatic carcinoma cells H1299. Paxillin was dephosphorylated in adenocarcinoma cells H441 and also absent in other metastatic carcinoma cells H292. Vinculin showed the same results, and talin was downregulated in adenocarcinoma cells H441 when the cells were treated with EGF. Rap1 was downregulated and PYK2 was upregulated in the same cell line. Our data help to comprehend the mechanism involved in cell migration to the blood and metastasis generation. In conclusion, the expression patterns of cell-cell adhesion were not affected by EGF treatment but it affected cell-extracellular matrix adhesion.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; beta Catenin; Cadherins; Carcinoma, Non-Small-Cell Lung; Catenins; Cell Adhesion Molecules; Cell Line, Tumor; Crk-Associated Substrate Protein; Delta Catenin; Epidermal Growth Factor; Focal Adhesion Kinase 1; Humans; Lung Neoplasms; Paxillin; rap1 GTP-Binding Proteins; Talin; Vinculin

Topics: Adenocarcinoma; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Camptothecin; Cetuximab; Colorectal Neoplasms; Epidermal Growth Factor; Female; Humans; Irinotecan; Keratitis; Lung Neoplasms; Ophthalmic Solutions

Gastric secretion can provide valuable information especially when Helicobacter pylori (Hp) infection results in chronic atrophic gastritis (CAG) and intestinal metaplasia (IM) preceding adenocarcinoma (AdCa).. Looking for a potential biomarker of malignant transformation in the setting of chronic inflammation we studied the levels of prostaglandin E2 (PGE(2)), as well as peptide growth factors [epidermal growth factor (EGF) and transforming growth factor α (TGFα)], harbingers of injury and repair, in gastric juice aspirated at endoscopy from patients with CAG, CAG/IM, AdCa, and controls.. The PGE(2), EGF and TGFα concentrations in the gastric juice were measured using radioimmunoassays (RIAs).. In patients with AdCa gastric juice PGE(2) increased fivefold versus controls (P < 0.01) and almost threefold versus patients with CAG (P < 0.05). The EGF levels in patients with AdCa were fourfold higher versus controls (P < 0.001) and almost threefold higher versus CAG (P < 0.05). In patients with CAG/IM the EGF levels were also almost 3 times higher versus controls. The TGFα levels in patients with AdCa were half the value of controls and CAG (P < 0.05). In patients with CAG/IM the levels were as low as 1/5 of controls or CAG (P < 0.05).. Testing the gastric juice for PGE(2), EGF, and TGFα in patients with endoscopy and biopsy proven CAG, may be helpful in follow up of patients who may potentially progress to IM and ultimately AdCa. This could be considered as an adjunct to histologic assessment especially that even the best surveillance biopsy specimen regimens are inherited with sampling errors.

Topics: Adenocarcinoma; Adult; Aged; Biomarkers, Tumor; Digestive System Neoplasms; Dinoprostone; Epidermal Growth Factor; Female; Gastric Juice; Gastritis, Atrophic; Helicobacter Infections; Humans; Intestines; Male; Metaplasia; Middle Aged; Pilot Projects; Transforming Growth Factor alpha

Radiotherapy combined with androgen depletion is generally successful for treating locally advanced prostate cancer. However, radioresistance that contributes to recurrence remains a major therapeutic problem in many patients. In this study, we define the high-affinity neurotensin receptor 1 (NTR1) as a tractable new molecular target to radiosensitize prostate cancers. The selective NTR1 antagonist SR48692 sensitized prostate cancer cells in a dose- and time-dependent manner, increasing apoptotic cell death and decreasing clonogenic survival. The observed cancer selectivity for combinations of SR48692 and radiation reflected differential expression of NTR1, which is highly expressed in prostate cancer cells but not in normal prostate epithelial cells. Radiosensitization was not affected by androgen dependence or androgen receptor expression status. NTR1 inhibition in cancer cell-attenuated epidermal growth factor receptor activation and downstream signaling, whether induced by neurotensin or ionizing radiation, establish a molecular mechanism for sensitization. Most notably, SR48692 efficiently radiosensitized PC-3M orthotopic human tumor xenografts in mice, and significantly reduced tumor burden. Taken together, our findings offer preclinical proof of concept for targeting the NTR1 receptor as a strategy to improve efficacy and outcomes of prostate cancer treatments using radiotherapy.

Topics: Adenocarcinoma; Androgens; Animals; Apoptosis; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Phosphorylation; Prostatic Neoplasms; Protein Processing, Post-Translational; Pyrazoles; Quinolines; Radiation Tolerance; Radiation-Sensitizing Agents; Receptors, Androgen; Receptors, Neurotensin; Tumor Stem Cell Assay; Xenograft Model Antitumor Assays

Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

Topics: Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Cell Proliferation; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; HCT116 Cells; HT29 Cells; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurotensin; Phosphorylation; Receptors, Neurotensin

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca(2+) from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Blotting, Western; Calcium; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Humans; Lung Neoplasms; Membrane Potentials; Patch-Clamp Techniques; Protein Serine-Threonine Kinases; RNA, Small Interfering; TRPM Cation Channels; Up-Regulation

The purpose of the present study was to investigate polymorphisms related to the metabolism of fluoropyrimidine and oxaliplatin, thymidylate synthase (TS) and excision repair cross-complementing gene 1 (ERCC1) 118, in metastatic colorectal cancer patients treated with capecitabine and oxaliplatin (XELOX). We also investigated the importance of the EGF61A>G polymorphism, which holds a functional influence on the tyrosine kinase receptor regulation.. We included 68 patients treated with first-line XELOX. Polymorphism analyses were carried out on pretreatment blood samples. Response was evaluated according to the RECIST. Survival analysis was described by the Kaplan-Meier method and log-rank testing.. The overall response rate was 38% and the median overall survival 19.4 months. A favorable outcome was seen in patients with the EGF61A/G genotype compared with the combined group of A/A and G/G, with response rates of 57% and 18%, respectively (P = 0.001). There was a significantly different progression-free survival (P = 0.018) in favor of the A/G group. The TS and ERCC1 genotypes failed to provide any significant impact on the outcome.. Polymorphism analysis of a simple blood sample is a feasible approach to biomarker analysis and the EGF61A>G polymorphism may influence the effect of first-line XELOX. Consequently, this marker deserves further investigation.

Topics: Adenocarcinoma; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Capecitabine; Colorectal Neoplasms; Deoxycytidine; DNA-Binding Proteins; Endonucleases; Epidermal Growth Factor; Feasibility Studies; Female; Fluorouracil; Humans; Male; Middle Aged; Neoplasm Staging; Organoplatinum Compounds; Oxaliplatin; Polymorphism, Genetic; Survival Rate; Thymidylate Synthase; Treatment Outcome

The aim of this study was to determine whether the risk of systemic disease after esophagectomy could be predicted by angiogenesis-related gene polymorphisms.. Systemic tumor recurrence after curative resection continues to impose a significant problem in the management of patients with localized esophageal adenocarcinoma (EA). The identification of molecular markers of prognosis will help to better define tumor stage, indicate disease progression, identify novel therapeutic targets, and monitor response to therapy. Proteinase-activated-receptor 1 (PAR-1) and epidermal growth factor (EGF) have been shown to mediate the regulation of local and early-onset angiogenesis, and in turn may impact the process of tumor growth and disease progression.. We investigated tissue samples from 239 patients with localized EA treated with surgery alone. DNA was isolated from formalin-fixed paraffin-embedded normal esophageal tissue samples and polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism and 5'-end [gamma-P] ATP-labeled polymerase chain reaction methods.. PAR-1 -506 ins/del (adjusted P value=0.011) and EGF +61 A>G (adjusted P value=0.035) showed to be adverse prognostic markers, in both univariate and multivariable analyses. In combined analysis, grouping alleles into favorable versus nonfavorable alleles, high expression variants of PAR-1 -506 ins/del (any insertion allele) and EGF +61 A>G (A/A) were associated with a higher likelihood of developing tumor recurrence (adjusted P value<0.001).. This study supports the role of functional PAR-1 and EGF polymorphisms as independent prognostic markers in localized EA and may therefore help to identify patient subgroups at high risk for tumor recurrence.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Endostatins; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Esophagectomy; Female; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Male; Middle Aged; Multivariate Analysis; Neoplasm Recurrence, Local; Neovascularization, Pathologic; Prognosis; Receptor, PAR-1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

The diagnosis of multiple primary lung cancer is sometimes difficult when multiple lung tumors with the same histologic type are identified. We now present a case of synchronous double primary lung adenocarcinomas (one in the right upper lobe and another in the right middle lobe) diagnosed based on mutational analysis of the epidermal growth factor receptor (EGFR) gene, although clinico-pathological findings suggested the diagnosis of intrapulmonary metastasis. After complete resection, pathological sections revealed the similar pathological features of two adenocarcinomas and unexpected subcarinal nodal metastasis. As the L858R mutation within exon 21 of the EGFR gene was identified in the middle-lobe tumor and the subcarinal node but not in the upper-lobe tumor, we diagnosed as double primary cancers. Local mediastinal recurrence after operation has been well-controlled with administration of gefitinib, a EGFR-tyrosine kinase inhibitor, and mutational analysis of the EGFR gene provided important information not only in the diagnosis of double primary cancers but also in decision-making of selection of chemotherapeutic agent.

Topics: Adenocarcinoma; Aged; Diagnosis, Differential; DNA Mutational Analysis; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Neoplasm Metastasis; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasms, Multiple Primary; Pneumonectomy; Quinazolines; Radiography, Thoracic

Four cardiac hormones synthesized by the same gene, i.e. atrial natriuretic peptide, vessel dilator, kaliuretic peptide and long-acting natriuretic peptide, have anticancer effects in vitro and in vivo. Epidermal growth factor's mechanism of cancer formation involves the activation of Ras.. These four cardiac hormones were evaluated for their ability to inhibit mitogen (epidermal growth factor) activation of Ras.. Epidermal growth factor increased the activation of Ras by 68%, 85% and 90% at its 1, 2 and 5 ng mL(-1) concentrations. Vessel dilator, long-acting natriuretic peptide, atrial natriuretic peptide and kaliuretic peptide inhibited 5 ng mL(-1) epidermal growth factor's stimulation of Ras by 73%, 79%, 33% and 45%, respectively, at their 1 microM concentrations. Their effects on epidermal growth factor's activation of Ras were specific with addition of the cardiac hormones' respective antibodies (5 microM) blocking 95%, 93%, 100% and 100% (P < 0.001 for each) of their ability to inhibit epidermal growth factor's stimulation of Ras.. Four cardiac hormones specifically inhibit epidermal growth factor's activation of Ras. This investigation would suggest that these cardiac hormones' anticancer effects involve the inhibition of mitogens such as epidermal growth factor's ability to activate Ras as well as inhibiting unstimulated basal activity of Ras.

Topics: Adenocarcinoma; Antineoplastic Agents; Atrial Natriuretic Factor; Dose-Response Relationship, Drug; Epidermal Growth Factor; Genes, ras; Humans; Male; Prostatic Neoplasms

Aquaporins (AQPs) are expressed in many different tumor cell types in human. New evidence for the involvement of AQPs in cell migration and proliferation adds AQPs to an expanding list of effectors in tumor biology.. The aim of this study was to investigate whether AQP3 expression in the human gastric carcinoma cell lines, AGS and SGC7901, enhances cell migration and proliferation.. Here, we showed that AQP3 is expressed in the human gastric cancer cell lines, AGS and SGC7901. The hEGF induced AQP3 expression in a time- and dose-dependent manner and increased gastric cancer cell migration and proliferation. AQP3 knockdown by siRNA inhibited hEGF-induced AQP3 expression and thus cell migration and proliferation. Furthermore, a mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 inhibited hEGF-induced AQP3 expression and cell migration or proliferation.. Cultured AGS or SGC7901 cells were treated with human epidermal growth factor (hEGF) and subjected to cell migration assay and cell proliferation assay. The expression or activation level of proteins was analyzed by western blot. AQP3 knockdown was obtained by small interfering (si)RNA.. Collectively, our findings provide for the first time that AQP3 plays a critical role in hEGF-induced cancer cell migration and proliferation and that hEGF induces AQP3 expression via ERK signal transduction pathways. These finds provide evidence for a novel role of AQP3 in human gastric carcinoma as a potentially important determinant of tumor growth and spread.

Topics: Adenocarcinoma; Aquaporin 3; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Humans; MAP Kinase Signaling System; Stomach Neoplasms

Amphiregulin (AREG) and epiregulin (EREG) have been found to play pivotal roles in several malignancies. However, the correlation between their expression and clinicopathological factors in colorectal carcinoma (CRC) is yet to be further investigated. To clarify the clinical significance of AREG and EREG expression in CRC, we detected serum and tissue levels of AREG and EREG.. We detected serum AREG and EREG levels by ELISA, and tissue levels by immunohistochemical test in 73 patients with CRC. The correlation between each independent clinicopathological characteristic and AREG and EREG levels was examined.. There was significant correlation between serum AREG level and vascular invasion. There was no correlation between EREG serum level and any clinicopathological characteristics. Among the 73 primary lesions, 51 were AREG-positive, and 48 were EREG-positive. AREG-positive status was significantly correlated with depth of tumor invasion, distant metastases, and nerve invasion. EREG-positive status was significantly correlated with depth of tumor invasion and distant metastases. Coexpression analysis showed that 46 patients were both AREG-positive and EREG-positive.. High serum and tissue levels of AREG and high tissue level of EREG are predictors of a poor prognosis in patients with CRC.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Amphiregulin; Biomarkers, Tumor; China; Colorectal Neoplasms; Disease Progression; EGF Family of Proteins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epiregulin; Female; Glycoproteins; Humans; Hyperplasia; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Neoplasm Invasiveness; Neoplastic Cells, Circulating; Precancerous Conditions; Prognosis

Invasion and metastasis are the primary causes of death in patients with pulmonary carcinoma. The epidermal growth factor (EGF) stimulates A549 cells invasion greatly through activating ERK and PI3K-Akt signaling pathway. The aim of this study is to elucidate the inhibitory effect of Resveratrol on EGF-induced invasive ability of A549 cells in vitro and explore the molecular mechanism.. The cytotoxicity of Resveratrol was evaluated by methyl thiazolyltetrazolium (MTT) assay. Then, the A549 cells were treated with EGF and non-cytotoxic concentration of Resveratrol. The cells' invasion were detected by Boyden chamber assay; MMP-2 activity was determined by gelatine zymography assay; the changes of the related proteins were detected by Western blot.. Resveratrol was not toxic to A549 cells at the concentration between 0 to 30 microM. The invasion ability of EGF-induced A549 cells was decreased after treatment with 20 microM resveratrol for 24 h, accompanied by the inhibition of MMP-2 secretion. And the levels of p-ERK1/2, PI3K (within 6 h) were suppressed too.. 20 microM Resveratrol inhibits A549 cells' invasion possibly through the suppression of the activation of ERK and PI3K-Akt signaling pathways, subsequently exerting inhibitory effect on MMP-2.

Topics: Adenocarcinoma; Anticarcinogenic Agents; Blotting, Western; Cell Line, Tumor; Epidermal Growth Factor; Humans; Lung Neoplasms; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phosphatidylinositol 3-Kinases; Resveratrol; Signal Transduction; Stilbenes

PAI-1 (plasminogen activator inhibitor-1) in breast cancer cells is involved in tumour development and metastasis of breast cancer cells. PAI-1 function and the regulation of its expression have been precisely investigated. Here we report that EGF, which promotes breast cancer tumour growth and survival, rapidly induces PAI-1 expression in the breast adenocarcinoma cell line MCF-7 through the activation of the transcription factor Elk-1. We have found that the PAI-1 promoter fragment (-140 to +173) containing the Ets consensus binding site is activated by Elk-1. Chromatin immunoprecipitation analysis confirms in vivo binding of Elk-1 to the PAI-1 promoter and demonstrates that Elk-1 phosphorylation on the Ets binding site is EGF-dependent.

Topics: Adenocarcinoma; Binding Sites; Breast Neoplasms; Epidermal Growth Factor; ets-Domain Protein Elk-1; Female; Gene Expression Regulation, Neoplastic; Humans; Phosphorylation; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Protein Binding; Protein Kinases; Proto-Oncogene Protein c-ets-1; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Bone metastasis occurs frequently in advanced prostate cancer (PCa) patients; however, it is not known why this happens. The epidermal growth factor receptor (EGFR) ligand EGF is available to early stage PCa; whereas, TGF-alpha is available when PCa metastasizes. Since the microenvironment of metastases has been shown to play a role in the survival of the tumor, we examined whether the ligands had effects on cell survival and proliferation in early and late PCa.. We used LNCaP cells as a model of early stage, non-metastatic PCa and the isogenic C4-2B cells as a model of late stage, metastatic PCa.. We found that the proliferation factor MAPK and the survival factor AKT were differentially activated in the presence of different ligands. TGF-alpha induced growth of C4-2B cells and not of the parental LNCaP cells; however, LNCaP cells expressing a constitutively active AKT did proliferate with TGF-alpha. Therefore, AKT appeared to be the TGF-alpha-responsive factor for survival of the late stage PCa cells. LNCaP cells exposed to EGF produced more osteoprotegerin (OPG), an inhibitor of bone remodeling, than C4-2B cells with TGF-alpha, which had increased expression of RANKL, an activator of bone remodeling. In concordance, TGF-alpha-treated C4-2B conditioned medium was able to differentiate an osteoclast precursor line to a greater extent than EGF-treated C4-2B or TGF-alpha-treated LNCaP conditioned media.. The switch in EGFR ligand availability as PCa progresses affects cell survival and contributes to bone remodeling.

Topics: Adenocarcinoma; Bone Neoplasms; Bone Remodeling; Cell Line, Tumor; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Male; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Neoplasm Staging; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; RANK Ligand; Signal Transduction; Transforming Growth Factor alpha

ErbB receptors and their cognate ligands are implicated in cancer progression. Their expression in gastrointestinal cancer, however, has not been systemically studied.. The expression of four ErbB receptors and a panel of ErbB ligands were determined by reverse transcription-PCR in two gastric (TMK1, MKN-45) and two colon (SW1116, HT-29) cancer cell lines. Cell proliferation was measured by MTT assay while gene knockdown was achieved by RNA interference.. ErbB1, ErbB2 and ErbB3 receptors and five known or putative ErbB ligands, namely, epiregulin, epidermal growth factor (EGF), heparin-binding EGF, transforming growth factor alpha (TGFalpha) and neuroglycan-C were expressed in all four cell lines. Knockdown of neuroglycan-C, however, did not affect cell proliferation.. This study profiles the expression of ErbB receptors and their cognate ligands in gastric and colon cancer cells. These findings might lay the basis for the development of ErbB pathway-directed therapeutics for gastrointestinal cancer.

Topics: Adenocarcinoma; Cell Growth Processes; Cell Line, Tumor; Chondroitin Sulfate Proteoglycans; Colonic Neoplasms; Epidermal Growth Factor; Epiregulin; Heparin-binding EGF-like Growth Factor; HT29 Cells; Humans; Intercellular Signaling Peptides and Proteins; Ligands; Neuregulins; Oncogene Proteins v-erbB; Stomach Neoplasms

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

Topics: Adenocarcinoma; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Estradiol; Female; Humans; Ovarian Neoplasms; Receptors, Estrogen; Receptors, G-Protein-Coupled; Receptors, Urokinase Plasminogen Activator; Up-Regulation

Adenovirus early region 4 open reading frame 4 (E4orf4) protein is a novel cell death factor that selectively induces apoptosis in cancer cells. This study evaluated tumor inhibitory effects of a protein made by fusion E4orf4 and human epidermal growth factor (EGF). EGF was used to ensure the selective targeting of EGF receptor (EGFR)-overexpressing tumor cells. Results showed that EGF-E4orf4 stimulated EGFR phosphorylation in a time- and dose-dependent manner. Confocal microscopy analysis showed both EGF-E4orf4 and EGF could be internalized via EGFR but they had different intercellular trafficking pathways. In vitro study showed that EGF-E4orf4 significantly inhibited the proliferation of BGC823 and in vivo study showed EGF-E4orf4 suppressed tumor growth in a dose-dependent fashion with an inhibition rate of 79% for MDA-MB-231 and 49% for BGC 823 (p < 0.05). No toxic effects were observed in the nude mice with a dose as high as 10 mg/kg of EGF-E4orf4. These results indicated that EGF-E4orf4 could be a potential drug for cancer therapy.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Proliferation; Colony-Forming Units Assay; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Humans; Immunoblotting; Immunoprecipitation; Mice; Mice, Inbred BALB C; Mice, Nude; Phosphorylation; Recombinant Fusion Proteins; Stomach Neoplasms; Viral Proteins; Xenograft Model Antitumor Assays

Single-nucleotide polymorphisms of key cancer genes, such as EGF A61G, are associated with an elevated risk of esophageal adenocarcinoma (EAC). As gastroesophageal reflux disease (GERD) is an established risk factor for EAC, we evaluated whether the association between epidermal growth factor (EGF) polymorphism and EAC development is altered by the presence of GERD.. EGF genotyping of DNA samples was performed and GERD history was collected for 309 EAC patients and 275 matched healthy controls. Associations between genotypes and EAC risk were evaluated using adjusted logistic regression. Genotype-GERD relationships were explored using analyses stratified by GERD history and joint effects models that considered severity and duration of GERD symptoms.. EGF variants (A/G or G/G) were more common (P = 0.02) and GERD was more prevalent (P < 0.001) in cases than in controls. When compared with the EGF wild-type A/A genotype, the G/G variant was associated with a substantial increase in EAC risk among individuals with GERD [Odds ratio 9.7; 95% confidence interval (CI), 3.8-25.0; P < 0.001] and a slight decrease in risk for GERD-free individuals (odds ratio 0.4; 95% CI = 0.22-0.90; P = 0.02). In the joint effects models, the odds of EAC was also highest for G/G patients (when compared with A/A) who either experienced frequent GERD of more than once per week (odds ratio 21.8; 95% CI = 5.1-94.0; P < 0.001) or suffered GERD for longer than 15 years (odds ratio 22.4; 95% CI = 6.5-77.6; P < 0.001). There was a highly significant interaction between the G/G genotype and the presence of GERD (P < 0.001).. EGF A61G polymorphism may alter EAC susceptibility through an interaction with GERD.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Case-Control Studies; Epidermal Growth Factor; Esophageal Neoplasms; Female; Gastroesophageal Reflux; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; Risk Factors; Survival Rate; Young Adult

Vasculogenesis, or recruitment of progenitors able to differentiate into endothelial-like cells, may provide an important contribution to neovessel formation in tumors. However, the factors involved in the vasculogenic process and in particular the role of the epithelial-mesenchymal transition of tumor cells have not yet been investigated. We found a CD14(+)/KDR(+) angiogenic monocyte population in undifferentiated ovarian tumors, significantly increased in the corresponding tumor metastasis. In vitro, monocyte differentiation into CD14(+)/KDR(+) cells was induced by conditioned media from the primary ovarian tumor cells expressing a mesenchymal phenotype. In contrast, the ovarian tumor cell line SKOV3 expressing an epithelial phenotype was unable to stimulate the differentiation of monocytes into CD14(+)/KDR(+) cells. When an epithelial-mesenchymal transition was induced in SKOV3, they acquired this differentiative ability. Moreover, after mesenchymal transition pleiotrophin expression by SKOV3 was increased and conversely its blockade significantly reduced monocyte differentiation. The obtained CD14(+)/KDR(+) cell population showed the expression of endothelial markers, increased the formation of capillary-like structures by endothelial cells and promoted the migration of ovarian tumor cells in vitro. In conclusion, we showed that the epithelial-mesenchymal transition of ovarian tumor cells induced differentiation of monocytes into the pro-angiogenic CD14(+)/KDR(+) population and thus it may provide a tumor microenvironment that favours vasculogenesis and metastatization of the ovarian cancer.

Topics: Adenocarcinoma; Adult; Aged; Antigens, CD; Cell Differentiation; Cell Division; Cell Line, Tumor; Epidermal Growth Factor; Epithelial Cells; Female; Flow Cytometry; Humans; Hydrocortisone; Lipopolysaccharide Receptors; Mesoderm; Middle Aged; Monocytes; Neoplasm Metastasis; Neovascularization, Pathologic; Ovarian Diseases; Ovarian Neoplasms

During breast cancer development, increased presence of leukocytes in neoplastic stroma parallels disease progression; however, the functional significance of leukocytes in regulating protumor versus antitumor immunity in the breast remains poorly understood. Utilizing the MMTV-PyMT model of mammary carcinogenesis, we demonstrate that IL-4-expressing CD4(+) T lymphocytes indirectly promote invasion and subsequent metastasis of mammary adenocarcinomas by directly regulating the phenotype and effector function of tumor-associated CD11b(+)Gr1(-)F4/80(+) macrophages that in turn enhance metastasis through activation of epidermal growth factor receptor signaling in malignant mammary epithelial cells. Together, these data indicate that antitumor acquired immune programs can be usurped in protumor microenvironments and instead promote malignancy by engaging cellular components of the innate immune system functionally involved in regulating epithelial cell behavior.

Topics: Adenocarcinoma; Animals; CD4-Positive T-Lymphocytes; Epidermal Growth Factor; Female; Interleukin-4; Lung Neoplasms; Macrophages; Mammary Neoplasms, Experimental; Mice; Mice, Transgenic; Myeloid Cells; Phenotype; Signal Transduction; Th2 Cells

The chemokine receptor CXCR4 and its ligand CXCL12 play an important role in breast cancer invasion and metastasis, and induce the chemotaxis of various types of cancer cells. Previous studies of CXCL12-induced chemotaxis have, for the most part, relied on endpoint assays (e.g., transwell assays) that provide poor control over the cell microenvironment. Specifically, these assays lacked the ability to dissect the role that autocrine and paracrine growth factors play in chemokine-induced cancer cell chemotaxis. Here, we employ a microfluidic chemotaxis chamber that allows the effects of specific exogenous factors on cell migration to be directly characterized, without the interference of autocrine/paracrine signaling. Using this approach, we investigated the migration of MDA-MB-231 breast cancer cells in well-defined CXCL12 gradients. We found that CXCL12 alone failed to stimulate chemotaxis of these cells; however, when the CXCL12 gradient was supplemented with a uniform stimulus of either EGF or conditioned media, a directional response was induced. This dependence on growth factor signaling points to the importance of autocrine and paracrine factors in determining the migratory response of the cells, and may play an important role in cancer metastasis.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Chemokine CXCL12; Chemotaxis; Culture Media, Conditioned; Epidermal Growth Factor; Female; Humans; Microfluidic Analytical Techniques; Receptors, CXCR4; Research Design; Signal Transduction

The androgen receptor (AR) is a ligand-activated transcription factor that interacts with coregulatory proteins during androgen-dependent gene regulation. Melanoma antigen gene protein 11 (MAGE-11) is an AR coregulator that specifically binds the AR NH(2)-terminal FXXLF motif and modulates the AR NH(2)- and carboxyl-terminal N/C interaction to increase AR transcriptional activity. Here we demonstrate that epidermal growth factor (EGF) signaling increases androgen-dependent AR transcriptional activity through the posttranslational modification of MAGE-11. EGF in the presence of dihydrotestosterone stabilizes the AR-MAGE complex through the site-specific phosphorylation of MAGE-11 at Thr-360 and ubiquitinylation at Lys-240 and Lys-245. The time-dependent EGF-induced increase in AR transcriptional activity by MAGE-11 is mediated through AR activation functions 1 and 2 in association with the increased turnover of AR and MAGE-11. The results reveal a dynamic mechanism whereby growth factor signaling increases AR transcriptional activity through the covalent modification of an AR-specific coregulatory protein. Sequence conservation of the MAGE-11 phosphorylation and ubiquitinylation sites throughout the MAGE gene family suggests common regulatory mechanisms for this group of cancer-testis antigens.

Topics: Adenocarcinoma; Amino Acid Motifs; Animals; Antigens, Neoplasm; Cell Line; Chlorocebus aethiops; COS Cells; Dihydrotestosterone; Endometrial Neoplasms; Epidermal Growth Factor; Female; HeLa Cells; Humans; Multiprotein Complexes; Neoplasm Proteins; Phosphorylation; Protein Interaction Mapping; Protein Processing, Post-Translational; Receptors, Androgen; Recombinant Fusion Proteins; Transcription, Genetic; Transfection; Ubiquitination

Cripto-1 may be capable of up-regulating signalling molecules associated with epithelial-to-mesenchymal transition (EMT), an important event characterized by loss of E-cadherin during malignant tumour progression and metastasis. The aim was to investigate the expression of Cripto-1 and E-cadherin in relation to clinicopathological features and patient prognosis of gastric cancer.. The expression of Cripto-1 and E-cadherin was studied by immunohistochemistry in 118 gastric cancer cases. Up-regulated Cripto-1 (CR+) was found in 54% (64/118) of cases, whereas down-regulated E-cadherin (E-cad-) was found in 70% (83/118) of cases. Either CR+ or E-cad- was associated with lymph node metastasis, liver metastasis and late TNM stage (P < 0.05). Patients with either CR- or E-cad+ showed higher 5-year survival rates than those with CR+ or E-cad- (P = 0.0012 and P = 0.0017, respectively). When combined, evaluation of these two proteins, simultaneous CR+ and E-cad- (CR+/E-cad-) in cancer was strongly associated with the above three aggressive clinicopathological features (P < 0.001) and indicated the worst patient survival (P = 0.0001). Multivariate analysis revealed that CR+/E-cad- was an independent prognostic factor in gastric cancer.. Combined analysis of Cripto-1 and E-cadherin has significant value in evaluating the metastatic potential of gastric cancer and predicting patient prognosis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cadherins; Disease Progression; Down-Regulation; Epidermal Growth Factor; Female; Gastrectomy; GPI-Linked Proteins; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Lymph Nodes; Male; Membrane Glycoproteins; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Prognosis; Stomach Neoplasms; Survival Rate; Up-Regulation

Cetuximab (Erbitux), a monoclonal epidermal growth factor receptor (EGFR) antibody, has been used for the treatment of advanced colorectal carcinoma over the last two years. Inhibition of EGFR also influences corneal wound healing as EGF stimulates the proliferation of epithelial cells.. Extensive corneal erosion was seen in both eyes of a 62-year-old patient under treatment with cetuximab for a metastasized colorectal carcinoma. Progression was fast despite vigorous conservative treatment. The application of autologous serum could not be considered because of the antibody treatment. Human EGF was applied topically several times daily in order to utilize the proliferative effect on corneal epithelial cells and to antagonize the inhibition of EGF receptors.. Improvement was seen shortly after the onset of therapy with EGF eye drops.. The epithelial defect was closed 7 days (left eye) and 19 days (right eye), respectively, after the onset of therapy. During this time treatment with cetuximab was continued.. Cetuximab (Erbitux) can cause persisting epithelial defects. Patients with an impairment of corneal wound healing under cetuximab treatment can benefit from the topical application of human EGF. Consequently, surgical measures or complications such as infections can be avoided.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Cell Differentiation; Cetuximab; Colorectal Neoplasms; Corneal Diseases; Epidermal Growth Factor; Epithelium, Corneal; ErbB Receptors; Female; Humans; Middle Aged; Wound Healing

The epidermal growth factor (EGF) pathway is important in esophageal adenocarcinoma (EAC) tumorigenesis. We hypothesized that the EGF A61G homozygous variant genotype (GG) is (a) both a risk and poor prognostic factor for EAC and (b) associated with higher EGF serum levels in individuals with gastroesophageal reflux disease (GERD).. Using unconditional logistic regression, we compared EGF A61G in 312 EAC cases and 447 GERD-free controls, adjusting for age, gender, smoking history, and healthy adult body mass index. Using the method of Kaplan and Meier, log-rank tests, and Cox proportional hazard models, we correlated EGF A61G with overall and failure-free survival in the EAC cases. Serum EGF levels and EGF genotype (G/G versus others) were correlated in 144 GERD patients using Wilcoxon rank sum tests.. The EGF A61G G/G genotype conferred increased EAC risk, with an adjusted odds ratio of 1.81 (95% confidence interval, 1.2-2.7), and was even higher in the subgroup of EAC patients with concurrent Barrett's esophagus (adjusted odds ratio, 2.18; 95% confidence interval, 1.3-3.7). However, EGF A61G was not associated with a more aggressive phenotype or prognosis in EAC patients. Higher serum EGF levels were found in GERD patients carrying G/G compared with A/A or A/G (P = 0.03, Wilcoxon rank sum test).. The EGF A61G G/G genotype is associated with a near 2-fold greater risk of EAC. The G/G allele was also associated with higher EGF levels in tumor-free patients with GERD. EGF genotyping can potentially identify high-risk patients with GERD and Barrett's metaplasia who might benefit from increased surveillance.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Barrett Esophagus; Case-Control Studies; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Esophageal Neoplasms; Female; Gastroesophageal Reflux; Genetic Predisposition to Disease; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Polymorphism, Single Nucleotide; Precancerous Conditions; Risk Factors; Treatment Outcome

In this study, we evaluated, for the first time, the antiproliferative and cytotoxic effects induced by a combination of a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib, with other chemotherapeutic drugs including estrogen receptor-beta (ER-beta) antagonist (tamoxifen) and topoisomerase II inhibitor (etoposide) on some metastatic prostate cancer (PC) cell lines. Immunohistochemial analyses revealed that EGFR expression was enhanced in 38% of primary prostatic adenocarcinomas (Gleason scores 4-10) as compared to the corresponding normal tissues of the same prostate gland from 32 PC patients. The RT-PCR and Western blot data have also indicated the higher expression levels of EGFR and ER-beta transcripts and proteins in metastatic LNCaP, DU145 and PC3 cells relative to nonmalignant normal prostate cells. Moreover, the results from MTT and FACS analyses revealed that the drugs, alone or in combination at lower concentrations, inhibited the growth of 17beta-estradiol (E2) plus EGF and serum-stimulated androgen-responsive LNCaP-C33 and androgen-independent LNCaP-C81, DU145 and PC3 cells. Importantly, the combined gefitinib, tamoxifen and etoposide also caused a higher rate of apoptotic death of PC cells as compared to single agents. The cytotoxic effects induced by these drugs in PC3 cells appear to be mediated through the accumulation of cellular ceramide and activation of caspase cascades via a mitochondrial pathway. These findings indicate that the combined use of inhibitors of EGF-EGFR and E2-ER-beta signaling with etoposide, which act by increasing the cellular ceramide levels and caspase activity, represents a promising strategy for a more effective treatment of metastatic PC forms.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Caspases; Cell Proliferation; Cells, Cultured; Ceramides; Cytochromes c; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Etoposide; Flow Cytometry; Gefitinib; Humans; Immunoenzyme Techniques; Male; Membrane Potential, Mitochondrial; Neoplasms, Hormone-Dependent; Prostate; Prostatic Neoplasms; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tamoxifen

Ceramide-1-phosphate (C1P) is emerging as a new addition to the family of bioactive sphingolipid metabolites. At low concentrations, C1P enhanced survival of NIH 3T3 fibroblasts and A549 lung cancer cells, while at high concentrations, it reduced survival and induced apoptosis. Apoptosis correlated with degradation of C1P to pro-apoptotic ceramide. To examine the role of endogenous C1P, expression of ceramide kinase, the enzyme that produces C1P, was downregulated, which reduced cellular proliferation, progression into S phase and enhanced apoptosis induced by serum starvation. Our results suggest that ceramide kinase determines the balance between pro-apoptotic ceramide and anti-apoptotic C1P to regulate cell fate, reminiscent of its function in plants.

Topics: Adenocarcinoma; Animals; Apoptosis; Cattle; Cell Cycle; Cell Proliferation; Cell Survival; Ceramides; Down-Regulation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Mice; NIH 3T3 Cells; Phosphotransferases (Alcohol Group Acceptor); RNA, Messenger; RNA, Small Interfering

Estrogen and structurally related molecules play critical roles in breast cancer. We reported that resveratrol (50 microM), an estrogen-like phytosterol from grapes, acts in an antiestrogenic manner in breast cancer cells to reduce cell migration and to induce a global and sustained extension of actin structures called filopodia. Herein, we report that resveratrol-induced filopodia formation is time-dependent and concentration-dependent. In contrast to resveratrol at 50 microM, resveratrol at 5 microM acts in a manner similar to estrogen by increasing lamellipodia, as well as cell migration and invasion. Because Rho GTPases regulate the extension of actin structures, we investigated a role for Rac and Cdc42 in estrogen and resveratrol signaling. Our results demonstrate that 50 microM resveratrol decreases Rac and Cdc42 activity, whereas estrogen and 5 microM resveratrol increase Rac activity in breast cancer cells. MDA-MB-231 cells expressing dominant-negative Cdc42 or dominant-negative Rac retain filopodia response to 50 microM resveratrol. Lamellipodia response to 5 microM resveratrol, estrogen, or epidermal growth factor is inhibited in cells expressing dominant-negative Rac, indicating that Rac regulates estrogen and resveratrol (5 microM) signaling to the actin cytoskeleton. These results indicate that signaling to the actin cytoskeleton by low and high concentrations of resveratrol may be differentially regulated by Rac and Cdc42.

Topics: Actins; Adenocarcinoma; Breast Neoplasms; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Movement; Cytoskeleton; Dose-Response Relationship, Drug; Epidermal Growth Factor; Estradiol; Estrogens; Female; Genes, Dominant; Humans; Neoplasm Invasiveness; Neoplasms, Hormone-Dependent; Pseudopodia; rac GTP-Binding Proteins; Recombinant Fusion Proteins; Resveratrol; Stilbenes; Transfection

Overexpression of epidermal growth factor (EGF) receptor and constitutive activation of nuclear factor-kappaB (NF-kappaB) are frequently encountered in tumor cells. Although EGF has been shown to induce NF-kappaB activation, the mechanism is poorly understood. EGF activated NF-kappaB DNA binding, induced NF-kappaB reporter activity and the expression of antiapoptotic and cell-proliferative gene products. Interestingly, non-small cell lung adenocarcinoma cell lines (HCC827 and H3255), which exhibit EGFR amplification, showed ligand-independent activation of NF-kappaB. Unlike tumor-necrosis factor (TNF), however, EGF failed to induce IkappaBalpha phosphorylation and ubiquitination and the activation of IkappaBalpha kinase (IKK). Although DN-IKKbeta inhibited TNF-induced NF-kappaB activity, DN-IKKbeta had no effect on EGF-induced NF-kappaB activation, suggesting that EGF-induced NF-kappaB activation is IKK independent. Using dominant-negative plasmids, we also demonstrated the role of TRADD, TRAF2, NIK and Ras in EGF-induced NF-kappaB activation. By using specific antibodies and IkappaBalpha plasmid, which is mutated at tyrosine 42 to phenylalanine, we show that EGF induced the tyrosine phosphorylation of IkappaBalpha at residue 42. Furthermore, EGF receptor kinase inhibitor blocked IkappaBalpha phosphorylation and consequent NF-kappaB activation. Overall, our results indicate that tyrosine phosphorylation of IkappaBalpha at residue 42 is critical for EGF-induced NF-kappaB activation pathway.

Topics: Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Cell Nucleus; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; I-kappa B Kinase; NF-kappa B; Phosphorylation; Tumor Necrosis Factor-alpha; Tyrosine; Ubiquitination

Obesity and gastro-oesophageal reflux are the main predisposing factors for oesophageal adenocarcinoma. We have examined the effects of transient acid exposure and leptin on OE33 oesophageal adenocarcinoma cells. Leptin and acid individually stimulated proliferation and inhibited apoptosis and the combination was synergistic. Leptin receptor protein levels were unchanged by acid exposure. The COX-2 inhibitor NS 398 blocked the effects of acid and leptin but while both acid and leptin individually significantly increased PGE2 production and COX-2 mRNA levels, the combination was not more effective than either stimulant alone. Leptin synergistically enhanced acid-stimulated EGFR and ERK phosphorylation but did not further increase JNK or p38 MAP kinase phosphorylation. Specific EGFR and ERK inhibitors reduced the effects of leptin and acid alone and in combination. The combination of increased circulating leptin levels in obesity and transient reflux of gastric acid may promote oesophageal carcinogenesis by increasing proliferation and inhibiting apoptosis.

Topics: Acids; Adenocarcinoma; Apoptosis; Cell Proliferation; Cyclooxygenase 2; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Humans; Leptin; Receptors, Cell Surface; Receptors, Leptin; Tumor Cells, Cultured

Sphingosine kinases (SK) catalyze the formation of sphingosine-1-phosphate (S1P) which plays a crucial role in cell growth and survival. Here, we show that prolactin (PRL) biphasically activates the SK-1, but not the SK-2 subtype, in the breast adenocarcinoma cell-line MCF7. A first peak occurs after minutes of stimulation and is followed by a second delayed activation after hours of stimulation. A similar biphasic effect on SK-1 activity is seen for 17beta-estradiol (E(2)). The delayed activation of SK-1 derives from an upregulated mRNA and protein expression and is due to increased SK-1 promoter activity and mechanistically involves STAT5 activation as well as protein kinase C and the classical mitogen-activated protein kinases. Furthermore, glucocorticoids also block both hormone-induced SK-1 expression and activity. Functionally, long-term stimulation of MCF7 cells with PRL or E(2) is well known to trigger increased cell proliferation and migration. Both hormone-induced cell responses critically involve SK-1 activation since the depletion of SK-1, but not SK-2, by siRNA transfection abolishes the hormone-induced cell proliferation and migration. In summary, our data show that PRL and E(2) cause a pronounced delayed SK-1 activation which is due to increased gene transcription, and critically determines the capability of cells to grow and move. Thus, the SK-1 may represent a novel attractive target for anti-tumor therapy.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Estradiol; Humans; Phosphotransferases (Alcohol Group Acceptor); Prolactin; Promoter Regions, Genetic; RNA, Small Interfering; Up-Regulation

Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P(3) production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P(3) production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P(3) production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Enzyme Activation; Epidermal Growth Factor; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; rac GTP-Binding Proteins; ras Proteins; Rats; Recombinant Fusion Proteins

Cripto-1 (CR-1) is a glycosylphosphatidylinositol (GPI)-anchored membrane glycoprotein that has been shown to play an important role in embryogenesis and cellular transformation. CR-1 is reported to function as a membrane-bound co-receptor and as a soluble ligand. Although a number of studies implicate the role of CR-1 as a soluble ligand in tumor progression, it is unclear how transition from the membrane-bound to the soluble form is physiologically regulated and whether differences in biological activity exist between these forms. Here, we demonstrate that CR-1 protein is secreted from tumor cells into the conditioned medium after treatment with serum, epidermal growth factor, or lysophosphatidic acid, and this soluble form of CR-1 exhibits the ability to promote endothelial cell migration as a paracrine chemoattractant. On the other hand, membrane-bound CR-1 can stimulate endothelial cell sprouting through direct cell-cell interaction. Shedding of CR-1 occurs at the GPI-anchorage site by the activity of GPI-phospholipase D (GPI-PLD), because CR-1 shedding was suppressed by siRNA knockdown of GPI-PLD and enhanced by overexpression of GPI-PLD. These findings describe a novel molecular mechanism of CR-1 shedding, which may contribute to endothelial cell migration and possibly tumor angiogenesis.

Topics: Adenocarcinoma; Animals; Cell Line; Cell Line, Tumor; Cell Movement; Chlorocebus aethiops; Coculture Techniques; Colonic Neoplasms; COS Cells; Dogs; Endothelial Cells; Endothelium, Vascular; Epidermal Growth Factor; Fluorescent Dyes; Glycosylphosphatidylinositols; GPI-Linked Proteins; Growth Substances; Humans; Indoles; Intercellular Signaling Peptides and Proteins; Kidney; Mass Spectrometry; Membrane Glycoproteins; Neoplasm Proteins; Phalloidine; Phospholipase D; Rhodamines; RNA, Small Interfering; Umbilical Veins

EGF/EGFR interactions are important mechanisms behind colorectal tumour development and growth. Recently a single nucleotide polymorphism in the EGF gene has been identified (EGF A61G). It may be a potential predictor for survival of patients receiving EGFR-inhibitor cetuximab treatment, but the clinical importance and the functional influence on EGF gene expression levels in colorectal cancer (CRC) patients have not yet been further assessed. The aim of the present study was to investigate the relationship between EGF A61G genotype and EGF gene expression levels in colorectal adenocarcinomas and normal colon tissue.. Eighty-one CRC patients were included in the study. Tissue samples from normal colon, adenocacinomas and corresponding blood samples were analysed by real-time PCR for EGF gene expression and EGF A61G genotype, respectively.. Thirty-three percent were AA, 48% and 19% A/G and G/G respectively. We found a significantly lower median age in the A/A group compared to the G/G group, suggesting a later time of diagnosis in the G/G patients. There was a significant difference between the median EGF gene expression among the three genotypes in normal colon (p < 0.001) but not in adenocarcinomas. Furthermore, the median EGF gene expression was lower in CRC tissue than in normal colon samples, (0.13 (range 0.01-6.4) vs. 0.76, (range 0.013-5.55)).. We suggest that EGF A61G genotype has a functional influence on EGF gene expression in normal colon in CRC patients. The clinical implications warrant further investigations in prospective trials.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Colon; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Gene Frequency; Genotype; Humans; Male; Middle Aged; Polymorphism, Single Nucleotide

The primary focus of this investigation was to study the relationship between neuroendocrine (NE) differentiation and epidermal growth factor (EGF) because both have been implicated in the progression of prostate cancer. For this purpose, we used gefitinib and trastuzumab, which are inhibitors of EGF receptor (EGFR) and ErbB2, respectively. EGF prevents NE differentiation induced by androgen depletion. This effect is prevented by gefitinib, which blocks the activation of EGFR and ErbB2, stimulation of mitogen-activated protein kinase (MAPK), and cell proliferation induced by EGF. Conversely, trastuzumab does not inhibit the effect of EGF on EGFR phosphorylation, MAPK activity, cell proliferation, and NE differentiation, although it reduces ErbB2 levels specifically, suggesting that ErbB2 is not necessary to inhibit NE differentiation. Prevention of NE differentiation by EGF is mediated by a MAPK-dependent mechanism and requires constitutive Akt activation. The abrogation of the PI3K/Akt pathway changes the role of EGF from inhibitor to inductor of NE differentiation. We show that EGFR tyrosine kinase, MAPK, and PI3K inhibitors inhibit the cell proliferation stimulated by EGF but induce the acquisition of NE phenotype. Altogether, the present data should be borne in mind when designing new clinical schedules for the treatment of prostate cancer, including the use of ErbB receptors and associated signaling pathway inhibitors.

Topics: Adenocarcinoma; Androgens; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cell Differentiation; Cell Line, Tumor; Culture Media, Serum-Free; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Phosphatidylinositol 3-Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor, ErbB-2; Trastuzumab

Epidermal growth factor receptor (EGFR) has been extensively targeted in the treatment of non-small cell lung cancer, producing responses in a small number of patients. To study the role of ligand expression in mediating response to EGFR antagonism, we injected NCI-H441 [EGFR and EGF/transforming growth factor-alpha (TGF-alpha) positive] or PC14-PE6 (EGFR positive and EGF/TGF-alpha negative) human lung adenocarcinoma cells into the lungs of nude mice. We randomized the mice to receive treatment with the EGFR tyrosine kinase inhibitors gefitinib or AEE788 or vehicle. Treatment of mice bearing NCI-H441 but not PC14-PE6 lung tumors resulted in a significant reduction in primary tumor growth, pleural effusion, and lymph node metastasis. Immunohistochemical analyses revealed that NCI-H441 and PC14-PE6 cells expressed EGFR but that the expression of EGF/TGF-alpha was high in NCI-H441 cells and very low in PC14-PE6 cells. Consequently, EGFR was activated in both tumor and tumor-associated endothelial cells in the NCI-H441 tumors but not in the PC14-PE6 tumors. Antagonism of EGFR signaling by treatment of mice with AEE788 decreased proliferation and increased apoptosis of both tumor cells and tumor-associated endothelial cells in NCI-H441 tumors but not in PC14-PE6 tumors. However, after transfection of PC14-PE6 cells with TGF-alpha, lung tumors derived from the transfected cells expressed and activated EGFR in both tumor and tumor-associated endothelial cells and tumors responded to treatment with AEE788. Collectively, these results strongly suggest that the response of human lung cancers growing orthotopically in mice to the inhibition of EGFR signaling is determined by ligand (EGF/TGF-alpha) expression by tumor cells. Our findings provide an additional explanation for the susceptibility of lung cancers to treatment with EGFR tyrosine kinase inhibitors.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Blotting, Western; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Gene Dosage; Humans; Lung Neoplasms; Male; Mice; Mice, Nude; Phosphorylation; Purines; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha; Xenograft Model Antitumor Assays

Topics: Adenocarcinoma; Back; Carcinoma, Non-Small-Cell Lung; Chemotherapy, Adjuvant; Drug Eruptions; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Lymphatic Metastasis; Middle Aged; Protein Kinase Inhibitors; Quinazolines; Radiotherapy, Adjuvant; Skin

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

The EGFR is expressed in malignant ovarian tumor tissue, and tissue content of EGFR has been directly associated with poor prognosis in patients with ovarian cancer. The uPA system plays a role in pericellular proteolysis, cell migration, invasion, and is over-expressed in ovarian cancer. This study explored the effects of EGF on uPAR expression in the ovarian cancer cell line OVCAR-3.. We used OVCAR-3 cells and the following methods: cell migration assay, time-lapse video microscopy, real-time PCR, assays for cellular binding of 125I-uPA and cellular degradation of 125I-uPA:PAI-1 complex, biosynthetic labeling using 35S-methionin, Western blot, Northern blot, and ELISAs for uPA, PAI-1, and uPAR.. EGF up-regulates both protein and mRNA not only for uPAR, but also for the ligand uPA and its inhibitor PAI-1. Cell surface uPAR, in control as well as EGF-stimulated cells, is present only in the intact, not the cleaved, form. Ligand binding experiments showed an increase of endogenously occupied uPAR, whereas non-occupied receptor sites were not increased. In addition, EGF treatment resulted in decreased degradation of radiolabeled uPA:PAI-1 complex. This suggests decreased internalization of uPAR, since the complex is internalized together with uPAR. Like EGF, colchicine, which inhibits endocytosis, increased cell surface expression of uPAR. In addition, we found an immediate increase of uPAR after exposing the cells to EGF and this was accompanied by a transient increase of cell migration. The increase of cell surface uPAR in response to EGF is accompanied by increased release of the soluble form of uPAR (suPAR) to the medium as well as by increased cell migration. Both uPAR and suPAR increased in cells treated with the endocytosis inhibitor colchicine even though cell migration was inhibited, suggesting that the mechanism of uPAR shedding is not related to cell migration.. Increased cell surface uPAR in response to EGF stimulation results from mobilization of uPAR from detergent-resistant domains, increased expression of uPAR mRNA, and decreased internalization and degradation of uPAR. Both the anti-uPAR antibody R3, which inhibits binding of uPA, and the EGFR phosphorylation inhibitor Iressa inhibited cell migration in response to uPA as well as to EGF, suggesting that EGFR and uPAR are engaged in the same multiprotein assembly on the cell surface.

Topics: Adenocarcinoma; Blotting, Western; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; RNA, Messenger

Glioblastoma multiforme (GBM) is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC]), a strong activator of apoptosis, selectively to cancer cells.. Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF). EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and "bystander killing" of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors.. The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

Topics: Adenocarcinoma; Animals; Apoptosis; Bystander Effect; Cell Line, Tumor; eIF-2 Kinase; Epidermal Growth Factor; ErbB Receptors; Glioblastoma; Humans; Imines; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Poly I-C; Polyethylene Glycols; Polyethylenes; RNA, Double-Stranded; Time Factors; Transfection; Transplantation, Heterologous

The epidermal growth factor has long been known to be strictly correlated with the highly proliferating activities of cancer cells and primary tumors. Moreover, in the nucleus, the epidermal growth factor/epidermal growth factor receptor complex (EGF/EGFR) functions as a transcriptional regulator that activates the cyclin D1 gene. 9-hydroxystearic acid (9-HSA) induces cell proliferation arrest and differentiation in HT29 colon cancer cells by inhibiting histone deacetylase 1 (HDAC1). 9-HSA-treated HT29, when stimulated with EGF, are not responsive and surprisingly undergo a further arrest. In order to understand the mechanisms of this effect, we analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. It appears that HDAC1, as modified by 9-HSA, is unable to associate with cyclin D1, interfering with the cell proliferation program, and sequesters the EGF/EGFR complex interrupting the transduction of the mitogenic signal.

Topics: Adenocarcinoma; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Histone Deacetylase 1; Histone Deacetylases; HT29 Cells; Humans; Signal Transduction; Stearic Acids

This study was conducted to gain insight into the relationship between expression profiles and underlying genetic changes, which are known to be important for the pathogenesis of lung cancers.. Expression profiles of 18,175 unique genes and three major targets for genetic changes, p53, epidermal growth factor receptor (EGFR), and K-ras, were investigated in 149 patients with non-small-cell lung cancer, including 90 patients with adenocarcinoma to determine their relationships with various clinicopathologic features and Gene Ontology (GO) terms.. This study successfully established a basis for expression profile-defined classification, which can classify adenocarcinomas into two major types, terminal respiratory unit (TRU) type and non-TRU type. Our GO term-based identifier of particular biologic processes, molecular functions, and cellular compartments clearly showed characteristic retention of normal peripheral lung features in TRU type, in sharp contrast to the significant association of non-TRU type with cell cycling and proliferation-related features. While significantly higher frequency of EGFR mutation was observed in TRU type, we found that the presence of EGFR mutations was a significant predictor of shorter postoperative survival for TRU type, independent of disease stage. We were also able to identify a set of genes in vivo with significant upregulation in the presence of EGFR mutations.. This study has shed light on heterogeneity in lung cancers, especially in adenocarcinomas, by establishing a molecularly, genetically, and clinically relevant, expression profile-defined classification. Future studies using independent patient cohorts are warranted to confirm the prognostic significance of EGFR mutations in TRU-type adenocarcinoma.

Topics: Adenocarcinoma; Epidermal Growth Factor; Genes, ras; Humans; Lung Neoplasms; Molecular Biology; Mutation; Oligonucleotide Array Sequence Analysis

Both gastrin and Helicobacter pylori have been shown capable of up-regulating gene expression and protein shedding of heparin-binding epidermal growth factor (HB-EGF). Furthermore, the bacteria have previously been shown to induce serum hypergastrinemia in infected individuals. The aim of this work was to assess the extent to which the ability of H. pylori to up-regulate expression of HB-EGF can be attributed to its effect on gastrin. Gastric cells, transfected with either gastrin small interfering RNA or antisense plasmid or the gastrin/cholecystokinin-2 receptor (CCK-2R), were cultured for 24 hours with H. pylori(+/-), a CCK-2R antagonist. Gene expression levels were measured using reverse transcription-PCR, whereas protein changes were measured using ELISA, Western blotting, and immunofluorescence. H. pylori induced significantly higher levels of HB-EGF gene expression and ectodomain shedding in the CCK-2R-transfected cells than the vector control (P < 0.01). Addition of the CCK-2R inhibitor significantly decreased gene and shedding up-regulation. Gastrin down-regulation reduced the effect of the bacteria on HB-EGF gene and protein expression levels. Endogenous gastrin and CCK-2R expression were also found to be significantly up-regulated in all cell lines as a result of exposure to H. pylori (P < 0.02). Gastric mucosal tissue from H. pylori-infected individuals had significantly higher CCK-2R expression levels than noninfected (P < 0.003), and in hypergastrinemic mice, there was an increase in HB-EGF-expressing cells in the gastric mucosa and colocalization of HB-EGF with CCK-2R-positive enterochromaffin-like cells. In conclusion, gastrin and the CCK-2R play significant roles in the induction of HB-EGF gene and protein expression and ectodomain shedding by H. pylori.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Disease Models, Animal; DNA, Antisense; Enterochromaffin Cells; Epidermal Growth Factor; Gastrins; Helicobacter Infections; Helicobacter pylori; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Mice; Plasmids; Receptor, Cholecystokinin B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Stomach Neoplasms; Transfection; Up-Regulation

We investigated the survival signals of epidermal growth factor (EGF) in human gastric adenocarcinoma cell line TMK-1. Treatment of TMK-1 cells with adriamycin (ADR) caused apoptosis and apoptosis-related reactions such as the release of cytochrome c from mitochondria and the activation of caspase 9. However, EGF treatment greatly reduced the ADR-induced apoptosis as well as these reactions. We previously reported that hepatocyte growth factor transmitted protective signals against ADR-induced apoptosis by causing activation of the phosphatidylinositol-3'-OH kinase (PtdIns3-K)/Akt signaling pathway in human epithelial cell line MKN74 [Takeuchi K & Ito F (2004) J Biol Chem279, 892-900]. However, PtdIns3-K/Akt signaling did not mediate the antiapoptotic action of EGF in TMK-1 cells. EGF increased the expression of the Bcl-X(L) protein, an antiapoptotic member of the Bcl-2 family, but not that of other anti (Bcl-2) or proapoptotic (Bad and Bax) protein members. Expression of the c-Fos and c-Jun, components of activator protein 1 (AP1), which are known to regulate bcl-X(L) gene transcription, were increased in response to EGF. Pretreatment of the cells with PD98059, an inhibitor of MAP kinase kinase, inhibited the EGF-induced c-Fos and c-Jun expression, AP1 DNA binding, Bcl-X(L) expression, and the resistance against ADR-induced apoptosis, suggesting that EGF transmitted the antiapoptotic signal in such a way that it activated AP1 via a MAP kinase signaling pathway. TMK-1 cells stably transfected with TAM67, c-Jun dominant-negative mutant, did not display EGF-induced Bcl-X(L) expression or resistance against ADR-induced apoptosis. These results indicate that AP1-mediated upregulation of Bcl-X(L) expression is critical for protection of TMK-1 cells against ADR-induced apoptosis.

Topics: Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Line, Tumor; Cell Survival; Cells, Cultured; DNA Damage; Doxorubicin; Epidermal Growth Factor; Humans; Mice; Stomach Neoplasms; Transcription Factor AP-1

Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.

Topics: Adenocarcinoma; Amphiregulin; Animals; Antibodies, Blocking; Cell Line, Tumor; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Cyclin D1; Cyclin E; EGF Family of Proteins; Enzyme Activation; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Glycoproteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Thiazoles; Transforming Growth Factor alpha

Herein, we show that the hematopoietic-specific GEF VAV1 is ectopically expressed in primary pancreatic adenocarcinomas due to demethylation of the gene promoter. Interestingly, VAV1-positive tumors had a worse survival rate compared to VAV1-negative tumors. Surprisingly, even in the presence of oncogenic KRAS, VAV1 RNAi abrogates neoplastic cellular proliferation in vitro and in vivo, thus identifying Vav1 as a growth-stimulatory protein in this disease. Vav1 acts synergistically with the EGF receptor to stimulate pancreatic tumor cell proliferation. Mechanistically, the effects of Vav1 require its GEF activity and the activation of Rac1, PAK1, and NF-kappaB and involve cyclin D1 upregulation. Thus, the discovery of prooncogenic pathways regulated by Vav1 makes it an attractive target for therapeutic intervention.

Topics: Adenocarcinoma; Animals; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Cyclin D1; DNA Methylation; Epidermal Growth Factor; Humans; Male; Mice; Mice, Nude; p21-Activated Kinases; Pancreatic Neoplasms; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-vav; rac1 GTP-Binding Protein; Recombinant Fusion Proteins; Signal Transduction; Survival Rate

Topics: Adenocarcinoma; Antineoplastic Agents; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Ligands; Lung Neoplasms; Mutation, Missense; Quinazolines; Sequence Deletion; Stromal Cells; Transforming Growth Factor alpha

Human Cripto-1 (CR-1) is overexpressed in approximately 80% of human breast, colon and lung carcinomas. Mouse Cr-1 upregulation is also observed in a number of transgenic (Tg) mouse mammary tumors. To determine whether CR-1 can alter mammary gland development and/or may contribute to tumorigenesis in vivo, we have generated Tg mouse lines that express human CR-1 under the transcriptional control of the mouse mammary tumor virus (MMTV). Stable Tg MMTV/CR-1 FVB/N lines expressing different levels of CR-1 were analysed. Virgin female MMTV/CR-1 Tg mice exhibited enhanced ductal branching, dilated ducts, intraductal hyperplasia, hyperplastic alveolar nodules and condensation of the mammary stroma. Virgin aged MMTV/CR-1 Tg mice also possessed persistent end buds. In aged multiparous MMTV/CR-1 mice, the hyperplastic phenotype was most pronounced with multifocal hyperplasias. In the highest CR-1-expressing subline, G4, 38% (12/31) of the multiparous animals aged 12-20 months developed hyperplasias and approximately 33% (11/31) developed papillary adenocarcinomas. The long latency period suggests that additional genetic alterations are required to facilitate mammary tumor formation in conjunction with CR-1. This is the first in vivo study that shows hyperplasia and tumor growth in CR-1-overexpressing animals.

Topics: Adenocarcinoma; Animals; Cell Division; DNA Primers; DNA, Complementary; Epidermal Growth Factor; Female; GPI-Linked Proteins; Growth Substances; Hyperplasia; Intercellular Signaling Peptides and Proteins; Mammary Glands, Animal; Mammary Neoplasms, Animal; Membrane Glycoproteins; Mice; Mice, Transgenic; Neoplasm Proteins

Tissue Factor (TF), the initiator of the extrinsic coagulation cascade, is overexpressed in a variety of cancers. TF is also expressed in normal human endometrium but little is known about its expression or regulation in endometrial cancer. We demonstrate herein that TF is expressed in the endometrial adenocarcinoma cell line Ishikawa. Furthermore, epidermal growth factor (EGF) induces a rapid and sustained increase in TF expression. Estradiol and progesterone had no effect on basal or EGF-induced TF expression in Ishikawa cells. In contrast to the pronounced and sustained upregulation at the protein level, EGF treatment elicited only a modest and transient increase in TF mRNA levels. This activity corresponded to the response observed from an exogenous TF promoter construct. However, the induction of TF was abrogated by cycloheximide as well as actinomycin-D, inhibitors or protein- and mRNA-synthesis, respectively, demonstrating that EGF mediates its effect through activation of the TF gene. Fractionation experiments showed that EGF increases TF presence in caveolin-I containing membrane fractions. Coagulation and invasion assays were used to explore the physiological implications of TF regulation. The results demonstrate that EGF-mediated induction of TF increases the procoagulant activity and invasive potential of Ishikawa cells. Furthermore, immunocytochemistry confirmed that TF is regulated by EGF in primary cultures of normal endometrial epithelial cells and malignant tumor cells. In conclusion, EGF-mediated upregulation of TF results in accumulation of this glycoprotein in caveolae-like membrane fractions and increased coagulative and invasive potential. Our results suggest that TF may play an integral role in endometrial carcinogenesis.

Topics: Adenocarcinoma; Anticoagulants; Blotting, Western; Cell Line, Tumor; Cells, Cultured; Coagulants; Cycloheximide; Dactinomycin; Detergents; Endometrial Neoplasms; Endometrium; Epidermal Growth Factor; Estradiol; Female; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Immunohistochemistry; Nucleic Acid Synthesis Inhibitors; Octoxynol; Progesterone; Promoter Regions, Genetic; RNA, Messenger; Sucrose; Thromboplastin; Time Factors; Transfection; Ultracentrifugation; Up-Regulation

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

Urokinase-type plasminogen activator receptor (uPAR) and epidermal growth factor receptor (EGFR) are ubiquitous receptors involved in the control of a variety of cellular processes frequently found altered in cancer cells. The EGFR has been recently described to play a transduction role of uPAR stimuli, mediating uPA-induced proliferation in highly malignant cells that overexpress uPAR. In the present work, we found for the first time that uPAR stimulation with the amino-terminal fragment (ATF) of urokinase devoid of proteolytic activity transactivates the EGFR in mammary MCF-7 cells through a mechanism involving Src and a metalloproteinase, as indicated by its sensitivity to selected inhibitors. In these cells, which express low levels of uPAR and malignancy, both ATF and EGF stimuli induced an interaction of the EGFR with uPAR and ERK activation. However, EGFR activation by uPAR stimuli mediated cellular invasion rather than proliferation, while EGFR activation by EGF led to a proliferative response. These results revealed a complex modulation of EGFR function toward different cellular responses according to the status of uPAR activity. On the other hand, we also found that MMP-mediated activation of EGFR can occur in an autocrine manner in cells which secrete uPA. All this reveals novel regulatory systems operating through autocrine loops involving uPAR stimuli, Src, MMP and EGFR activation which could mediate fine control of physiological processes as well as contribute to the expression of proliferative and invasive phenotypes of cancerous cells.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Female; Genes, src; Humans; Kinetics; Matrix Metalloproteinases; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Peptide Fragments; Phosphorylation; Quinazolines; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Transcriptional Activation; Tyrphostins; Urokinase-Type Plasminogen Activator

The ability to determine the spatial localization of signals can assist in defining the regulation of specific signaling pathways within the cell. Localized application of a stimulus, followed by imaging of various proteins or their phosphorylated products, allows visualization of the propagation scales of responses to the stimulus. To obtain a localized stimulus of epidermal growth factor (EGF), we bound the ligand to magnetic beads, which could be rapidly pulled onto cell surfaces by a magnet. This method allows the spatial analysis of EGF-induced signaling pathways.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Chemotaxis; Epidermal Growth Factor; Image Interpretation, Computer-Assisted; Magnetics; Mammary Neoplasms, Animal; Microspheres; Rats; Research Design; Signal Transduction

The study investigated if EGF signaling inhibitors, EGF antibody and tyrphostin 51 (a tyrosine kinase inhibitor), mediated the action of EGF on apoptosis and the expression of EGF receptors and p21 (a cyclin-dependent kinase inhibitor) of human colorectal cancer cells.. Human colorectal adenocarcinoma cells (SW480) were incubated with 0.6 mL/L dimethyl sulfoxide (DMSO, the control group), 225 ng/mL (37.5 nmol/L) EGF in 0.6 mL/L DMSO, 225 ng/mL EGF+2.5 microg/mL (17 nmol/L) EGF antibody in 0.6 mL/L DMSO, or 225 ng/mL EGF+215 ng/mL (0.8 micromol/L) tyrphostin 51 in 0.6 mL/L DMSO.. After 48 h incubation, the levels of EGF in medium significantly increased (P<0.05) in the EGF-treated groups. The numbers of apoptotic cells were significantly fewer (P<0.05) in the EGF + EGF antibody and EGF + tyrphostin 51 groups than those in the control and EGF groups after 12 h treatments. The expression of phosphorylated EGF receptors in the EGF, EGF + EGF antibody, and EGF + tyrphostin 51 groups was 176.8%, 62.4%, and 138.1% of the control group, respectively. The expression of p21 protein in the EGF, EGF + EGF antibody, and EGF + tyrphostin 51 groups was 115.7%, 4.8%, and 61.5% of the control group, respectively.. The data suggest that EGF antibody and tyrphostin 51 can inhibit the action of EGF on apoptosis in human colorectal cancer cells through down-regulation of EGF receptor and p21 expression.

Topics: Adenocarcinoma; Antibodies; Apoptosis; Cell Line, Tumor; Colorectal Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Humans; Signal Transduction; Tyrphostins

Although the effects of progesterone on cell cycle progression are well known, its role in spreading and adhesion of breast cancer cells has not attracted much attention until recently. Indeed, by controlling cell adhesion proteins, progesterone may play a direct role in breast cancer invasion and metastasis. Progesterone has also been shown to modulate epidermal growth factor (EGF) effects in neoplasia, although EGF effects on progesterone pathways and targets are less well understood. In the present study we identify an effect of EGF on a progesterone target, namely desmoplakin.. Initially flow cytometry was used to establish the growing conditions and demonstrate that the T47D breast cancer cell line was responding to progesterone and EGF in a classical manner. Differential display RT-PCR was employed to identify differentially expressed genes affected by progesterone and EGF. Western and Northern blotting were used to verify interactions between EGF and progesterone in three breast cancer cell lines: T47D, MCF-7, and ZR-75.. We found the cell adhesion protein desmoplakin to be upregulated by progesterone - a process that was suppressed by EGF. This appears to be a general but not universal effect in breast cancer cell lines.. Our findings suggest that progesterone and EGF may play opposing roles in metastasis. They also suggest that desmoplakin may be a useful biomarker for mechanistic studies designed to analyze the crosstalk between EGF and progesterone dependent events. Our work may help to bridge the fields of metastasis and differentiation, and the mechanisms of steroid action.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Adhesion; Cell Differentiation; Cell Line, Tumor; Cell Size; Cytoskeletal Proteins; Desmoplakins; Epidermal Growth Factor; Female; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Progesterone; Promegestone; S Phase

Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown.. We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells.. Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib.. A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Amino Acid Sequence; Antineoplastic Agents; Base Sequence; Carcinoma, Non-Small-Cell Lung; DNA Mutational Analysis; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Gefitinib; Genes, erbB-1; Heterozygote; Humans; Lung Neoplasms; Male; Middle Aged; Molecular Sequence Data; Mutation; Protein-Tyrosine Kinases; Quinazolines; Sequence Deletion

Neuropilin-1 (NRP-1), a recently identified co-receptor for vascular endothelial growth factor, is expressed by several nongastrointestinal tumor types and enhances prostate cancer angiogenesis and growth in preclinical models. We investigated the expression and regulation of NRP-1 and the effect of NRP-1 overexpression on angiogenesis and growth of human colon adenocarcinoma by immunohistochemistry and in situ hybridization. NRP-1 was expressed in 20 of 20 human colon adenocarcinoma specimens but not in the adjacent nonmalignant colonic mucosa. By reverse transcriptase-polymerase chain reaction analysis, NRP-1 mRNA was expressed in seven of seven colon adenocarcinoma cell lines. Subcutaneous xenografts of stably transfected KM12SM/LM2 human colon cancer cells overexpressing NRP-1 led to increased tumor growth and angiogenesis in nude mice. In in vitro assays, conditioned medium from NRP-1-transfected cell lines led to an increase in endothelial cell migration, but did not affect endothelial cell growth. Epidermal growth factor (EGF) led to induction of NRP-1 in human colon adenocarcinoma cells and selective blockade of the epidermal growth factor receptor (EGFR) decreased constitutive and EGF-induced NRP-1 expression. Blockade of the Erk 1/2 and P38 mitogen-activated protein kinase signaling pathways also led to a decrease in constitutive and EGF-induced NRP-1 expression. These findings demonstrate the ubiquitous expression of NRP-1 in human colon cancer and suggest that NRP-1 may contribute to colon cancer angiogenesis and growth. This study also suggests that EGF and mitogen-activated protein kinase signaling pathways play an important role in NRP-1 regulation in colon cancer cells.

Topics: Adenocarcinoma; Animals; Cell Division; Cell Line, Tumor; Cloning, Molecular; Colonic Neoplasms; DNA, Complementary; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Intestinal Mucosa; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neovascularization, Pathologic; Neuropilin-1; Phosphorylation; Recombinant Proteins; Transfection; Transplantation, Heterologous

A431 cells escape EGF-induced apoptosis by forming cell aggregates. We show that these clusters migrate and merge with neighboring ones, resulting in larger structures composed of a multilayer central (3D) population surrounded by a cell monolayer (2D). We found that after 48 hr of 10 nM EGF treatment, 3D structure formation correlates with alpha2beta1 integrin upregulation. Blockade of alpha2 integrin impairs 3D structure formation. We studied the involvement of reactive oxygen species (ROS) in this process. We show that A431 cells express the NADPH oxidase catalytic subunits Nox1. EGF-induced dose-dependent ROS production was inhibited by the NADPH oxidase inhibitor, diphenylene iodonium (DPI), in these cells while rotenone was ineffective. Inhibition of ROS level in A431 cells with DPI or ebselen (glutathione peroxydase mimic) as well as P38 MAP kinase inhibition by SB203580 decreases alpha2 integrin subunit expression and induces a shift to 3D versus 2D populations. Cell cycle analysis of 2D cells shows that DPI, ebselen and SB203580 decrease the number of cells in S/G2 phase without affecting the cell number in mitosis phase. On the contrary, for 3D cells, these treatments increased the proportion of cells in mitosis without modification of the cell number in S/G2 phase. For both populations, apoptosis was increased by DPI and ebselen. Resistance of cell aggregates by paclitaxel to cell death is usually described. We show that DPI abolishes paclitaxel resistance of 3D cell aggregates. We observed a greater than additive effect between paclitaxel and DPI resulting in an increased proportion of cells in S/G2 phase for 3D populations. These results suggested that the ROS-P38 MAP kinase-alpha2beta1 integrin pathway was implicated in the A431 survival process by modulating the balance between 2D/3D cells.

Topics: Adenocarcinoma; Apoptosis; Cell Division; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; Humans; Integrin alpha2beta1; Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction

Topics: Adenocarcinoma; Amino Acid Substitution; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Controlled Clinical Trials as Topic; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Genes, erbB-1; Humans; Japan; Ligands; Lung Neoplasms; Mutation; Phosphorylation; Protein Structure, Tertiary; Quinazolines; Sequence Deletion; Smoking; Treatment Outcome; United States

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

S100A6 (Calcyclin) is a calcium-binding protein that has been implicated in a variety of biological functions as well as tumorigenesis. The aim of our study was to investigate the involvement of S100A6 during prostate cancer development and progression. Using immunohistochemistry, the expression of S100A6 was examined in benign (n=66), premalignant (n=10), malignant (n=66) and metastatic prostate (n=5) tissues arranged in a tissue-microarray or whole sections as well as in prostate cancer cell lines. The S100A6 immunostaining pattern in tissues was compared with that of cytokeratin 5 (a basal cell marker) and 18 (a benign luminal cell marker). In all cases of benign epithelium, intense S100A6 expression was seen in the basal cell layer with absent staining in luminal cells. In all cases of prostatic adenocarcinoma (matched), metastatic lesions and 3/10 high-grade prostatic intraepithelial neoplasia lesions, an absence of S100A6 was seen. Western blotting and RT-PCR analysis of cell lines showed S100A6 expression to be absent in LNCaP, LNCaP-LN3 and LNCaP-Pro5 but present in Du145, PC3, PC-3M and PC-3M-LN4. LNCaP cells treated with 5-Azacytidine, caused re-expression of S100A6 mRNA. Sequencing of bisulphite modified DNA showed CpG methylation within the S100A6 promoter region and exon 1 of LNCaP, LNCaP-LN3 and LNCaP-Pro5 cell lines but not in Du145 cells. Our data suggest that loss of S100A6 protein expression is common in prostate cancer development and may occur at an early stage. The mechanism of loss of expression may involve hypermethylation of CpG sites. The finding of intense S100A6 expression in the basal cells of benign glands but loss of expression in cancer could be useful as a novel diagnostic marker for prostate cancer.

Topics: Adenocarcinoma; Blotting, Western; Cell Cycle Proteins; Diagnosis, Differential; DNA Methylation; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Male; Oligonucleotide Array Sequence Analysis; Precancerous Conditions; Prostate; Prostatic Intraepithelial Neoplasia; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; S100 Calcium Binding Protein A6; S100 Proteins; Tumor Cells, Cultured

Enzymes which exhibit core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity play important roles in physiologic processes including the inflammatory response and immune system function, and C2GnT activity is regulated during processes, such as T cell activation and cellular differentiation. In this study, we have examined the regulation of C2GnT activity in the H292 airway epithelial cell line by epidermal growth factor (EGF), which has been previously shown to upregulate expression of the airway mucin MUC5AC in this cell line. We found that EGF suppressed C2GnT activity in a time- and dose-dependent fashion, and also suppressed core 4 beta1,6 N-acetylglucosaminyltransferase (C4GnT) activity. Consistent with the suppression of C4GnT activity, Northern blotting results showed that EGF preferentially inhibited the M isoform of C2GnT, which forms core 2, core 4, and blood group I beta1,6 branched carbohydrate structures, while the L isoform, which forms only the core 2 structure, was only modestly affected. Furthermore, EGF treatment resulted in a shift in the carbohydrate structure of FLAG-tagged MUC1 expressed in the cells from core 2-based toward core 1-based structures, consistent with the inhibitory effects of EGF on C2GnT. Transforming growth factor alpha mimicked the effect of EGF on C2GnT, implicating the EGF receptor (EGF-R) in C2GnT suppression, and the EGF-R tyrosine kinase inhibitor AG1478 blocked C2GnT suppression, confirming the role of EGF-R in the inhibition of C2GnT expression. Also, PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1/2 in the Ras-mitogen-activated protein kinase pathway, completely blocked the EGF suppressive effect, suggesting possible involvement of the Ras-mitogen-activated protein kinase pathway in EGF-mediated downregulation of C2GnT. The results of this study suggest that exposure of airway cells to EGF may result in remodeling of mucin carbohydrate structure, potentially altering the biological properties of the cells.

Topics: Adenocarcinoma; Carbohydrate Sequence; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Flavonoids; Humans; Imidazoles; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Mucin-1; Mucins; N-Acetylglucosaminyltransferases; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Pyridines; Respiratory Tract Neoplasms; RNA, Messenger; Tumor Cells, Cultured

Osteopontin (OPN) is a secreted, integrin-binding glycophosphoprotein that has been implicated in breast cancer. We previously showed that OPN-induced cell migration of mammary epithelial cells (MEC) depends on binding to cell surface integrins and involves activation of the hepatocyte growth factor (HGF) receptor, Met. Here, we show that OPN-induced migration of MEC also requires activation of the epidermal growth factor (EGF) pathway. Synergism was seen between EGF and OPN in inducing cell migration. Furthermore, incubation of cells with exogenous OPN increased ligand (TGFalpha> EGF) and EGF receptor (EGFR) mRNA expression, as well as EGFR kinase activity. Treatment of cells with anti-TGFalpha or anti-EGFR antibody, or with tyrphostin-25 (EGFR inhibitor), significantly impaired the cell migration response to OPN. Other more broad-spectrum tyrosine kinase inhibitors and the growth factor/ receptor interaction inhibitor, suramin, also inhibited OPN-induced migration. Using specific signal transduction pathway inhibitors, we have screened for involvement of MEK (MAP kinase kinase), phosphatidylinositol 3-kinase, phospholipase C (PLC), and protein kinase C (PKC). Results implicated all of these pathways in OPN-induced cell migration, the most pronounced effect being seen with PLC and PKC inhibitors. These results suggest that induction of MEC migration by OPN involves a cascade of events including at least two growth factor/receptor pathways and multiple downstream signal transduction pathways. A number of potential targets are thus provided for strategies aimed at blocking the malignancy-promoting effects of OPN.

Topics: Adenocarcinoma; Breast; Breast Neoplasms; Cell Movement; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Neoplasm Proteins; Oligonucleotides, Antisense; Osteopontin; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Proto-Oncogene Proteins c-met; Recombinant Fusion Proteins; Recombinant Proteins; RNA, Messenger; Sialoglycoproteins; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured; Type C Phospholipases

A loss of functional androgen receptor and an enhanced expression of growth factor receptors and associated ligands are causal genetic events in prostate cancer (PCA) progression. These genetic alterations lead to an epigenetic mechanism where a feedback autocrine loop between membrane receptor and ligand (e.g. EGFR-TGFalpha) results in a constitutive activation of MAPK-Elk1-AP1-mediated mitogenic signaling in human PCA at an advanced and androgen-independent stage. We rationalized that inhibiting these epigenetic events could be useful in controlling advanced PCA growth. Recently, we found that grape seed extract (GSE), a dietary supplement rich in flavonoid procyanidins, inhibits advanced and androgen-independent human PCA DU145 cell growth in culture and nude mice. Here, we performed detailed mechanistic studies to define the effect of GSE on EGFR-Shc-MAPK-Elk1-AP1-mediated mitogenic signaling in DU145 cells. Pretreatment of serum-starved cells with GSE resulted in 70% to almost complete inhibition of EGF-induced EGFR activation and 50% to complete inhibition of Shc activation, which corroborated with a comparable decrease in EGF-induced Shc binding to EGFR. Conversely, EGF-induced ERK1/2 phosphorylation was inhibited only by lower doses of GSE; in fact, higher doses showed an increase. Additional studies showed that GSE alone causes a dose- and time-dependent increase in ERK1/2 phosphorylation in starved DU145 cells that is inhibited by an MEK1 inhibitor PD98059. Independent of this increase in ERK1/2 phosphorylation, GSE showed a strong inhibition of ERK1/2 kinase activity to Elk1 in both cellular and cell-free systems. GSE treatment of cells also inhibited both EGF-induced and constitutively active Elk1 phosphorylation and AP1 activation. GSE treatment also showed DNA synthesis inhibition in starved and EGF-stimulated cells as well as loss of cell viability and apoptotic death that was further increased by adding MEK1 inhibitor. Since GSE strongly induced apoptosis independent of its affect on an increase in phospho-ERK1/2, we hypothesized that apoptotic effect of GSE could be by other mechanism(s) including its effect on stress-associated MAPK, the JNK. Indeed, GSE-treated cells showed a strong and sustained increase in phospho-JNK1/JNK2 levels, JNK activity and phospho-cJun levels. An inhibition of GSE-induced JNK activation by a novel JNK inhibitor SP600125 resulted in a significant reversal of GSE-induced apoptotic death suggesting the involveme

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Anthracenes; Apoptosis; Cell Division; Cell-Free System; Culture Media, Serum-Free; DNA-Binding Proteins; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; ets-Domain Protein Elk-1; Flavonoids; Humans; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Phosphorylation; Plant Extracts; Prostatic Neoplasms; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins; Seeds; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Transcription Factor AP-1; Transcription Factors; Tumor Cells, Cultured; Vitis

This work examined the importance of radiation-induced and ligand-induced EGFR-ERK signaling for the regulation of DNA repair proteins XRCC1 and ERCC1 in prostate carcinoma cells, DU145 (TP53(mut)), displaying EGFR-TGFA-dependent autocrine growth and high MAPK (ERK1/2) activity, and LNCaP (TP53(wt)) cells expressing low constitutive levels of ERK1/2 activity. Using quantitative RT-PCR and Western analyses, we determined that ionizing radiation activated the DNA repair genes XRCC1 and ERCC1 in an ERK1/2-dependent fashion for each cell line. After irradiation, a rapid increase followed by a decrease in ERK1/2 activity preceded the increase in XRCC1/ERCC1 expression in DU145 cells, while only the rapid decrease in ERK1/2 preceded the increase in XRCC1/ERCC1 expression in LNCaP cells. Administration of EGF, however, markedly increased the up-regulation of phospho-ERK, ERCC1 and XRCC1 in both cell lines. Although the EGFR inhibitor tyrphostin (AG-1478) and the MEK inhibitor PD90859 both attenuated EGF-induced levels of the ERCC1 and XRCC1 protein, PD98059 blocked the induction of ERCC1 and XRCC1 by radiation more effectively in both cell lines. Inhibition of ERK at a level that reduced the up-regulation of DNA repair led to the persistence of apurinic/apyrimidinic (AP) sites of DNA damage and increased cell killing. Taken together, these data imply a complex control of DNA repair activation that may be more generally dependent on MAPK (ERK1/2) signaling than was previously noted. These data provide novel insights into the capacity of the EGFR-ERK signaling to modulate DNA repair in cancer cells and into the functional significance of this signaling.

Topics: Adenocarcinoma; Autocrine Communication; Cobalt Radioisotopes; DNA Damage; DNA Repair; DNA-Binding Proteins; Endonucleases; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Gamma Rays; Gene Expression Regulation, Neoplastic; Humans; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Prostatic Neoplasms; Protein Biosynthesis; Proteins; Quinazolines; Tumor Cells, Cultured; Tyrphostins; X-ray Repair Cross Complementing Protein 1

We describe the case of a 52-year-old Japanese woman with advanced adenocarcinoma of the lung, in whom once-daily treatment with 250 mg ZD1839 ('Iressa') demonstrated a marked antitumour effect. She had initially achieved a partial response with cisplatin-based combination chemotherapy, but had subsequently progressed and had failed to respond to salvage chemotherapy. She had also received whole-brain irradiation for brain metastases. On admission, the patient was confined to bed due to dyspnoea and had rapidly progressing hypoxia secondary to lymphangitis carcinomatosa and a massive right pleural effusion. She was treated with oxygen supplementation and oral ZD1839, which, within a week, led to marked tumour regression and gradually improving dyspnoea. The main adverse event observed was a grade 2 rash. A month after starting ZD1839 treatment, the patient was discharged without the need for oxygen supplementation and had since returned to full-time work. This is a demonstration of ZD1839 producing a dramatic clinical response when administered to a patient with poor performance status who had received extensive prior treatment with cytotoxic agents.'Iressa' is a trademark of the AstraZeneca group of companies.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Carcinoma, Non-Small-Cell Lung; Cisplatin; Docetaxel; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gefitinib; Humans; Irinotecan; Lung Neoplasms; Middle Aged; Neoplasm Staging; Paclitaxel; Protein-Tyrosine Kinases; Quinazolines; Salvage Therapy; Taxoids; Treatment Outcome

Epidermal growth factor (EGF) receptor (EGFR) is involved in various basic biochemical pathways and is thus thought to play an important role in cell migration. We examined the effect of EGF on motility, migration, and morphology of a human adenocarcinoma cell line CAC-1. EGF treatment increased the motility of cervical adenocarcinoma cells and promoted migration of the cells on fibronectin and type IV collagen. EGF induced morphological changes with lamellipodia during EGFR-mediated motility. The results of an immunoprecipitation study showed that EGF up-regulated the expression of alpha2beta1-integrin in a dose-dependent manner. EGF-induced cell migration was blocked by alpha2beta1-integrin antibody. Our results also showed that EGF treatment stimulated the level of tyrosine dephosphorylation of FAK, which is required for EGF-induced changes in motility, migration, and cell morphology. A tyrosine kinase inhibitor (ZD1839) blocked EGF-induced changes in cervical adenocarcinoma cells. The results suggest that EGF promotes cell motility and migration and increases the expression of alpha2beta1-integrin, possibly by decreasing FAK phosphorylation.

Topics: Adenocarcinoma; Antibodies; Cell Movement; Cell Size; Collagen Type IV; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Fibronectins; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha2beta1; Phosphorylation; Protein-Tyrosine Kinases; Pseudopodia; Signal Transduction; Tumor Cells, Cultured; Tyrosine; Uterine Cervical Neoplasms

EMR2 and CD97, members of the epidermal growth factor (EGF)-TM7 family, show a very high homology. CD97, whose expression is closely related to clinical tumor stage in colorectal carcinomas, potentially functions as an adhesion molecule. Nothing is known about the presence of EMR2 in these tumors. We systematically examined the expression of EMR2 in colorectal carcinoma cell lines and adenocarcinomas. Of 18 cell lines, 10 were only slightly positive for EMR2 according to flow cytometry. Various EMR2 splice variants, including a new isoform, have been detected at the mRNA level. EMR2 expression did not correlate with in vitro migration or invasion capacity of the cell lines. Normal colorectal epithelial cells were EMR2 negative. In contrast to CD97, which is found in most colorectal adenocarcinomas, only 8 of 81 of these tumors expressed EMR2. No correlation was found between EMR2 expression and clinicopathological parameters of the tumors. In summary, a significant but low number of colorectal carcinomas are positive for EMR2, indicating different roles for this molecule and CD97 in these tumors.

Topics: Adenocarcinoma; Alternative Splicing; Antigens, CD; Cell Movement; Colorectal Neoplasms; Epidermal Growth Factor; Flow Cytometry; Gene Expression; Humans; Immunoenzyme Techniques; Membrane Glycoproteins; Neoplasm Invasiveness; Neoplasm Staging; Protein Isoforms; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

MST4, a member of the Sterile 20 serine/threonine kinase family, was found to be expressed in prostate carcinoma tumor samples and cell lines. In addition, expression levels appeared to correlate with tumorigenicity and androgen receptor status of the cells. Ectopic expression of wild-type and kinase-inactive MST4 was used to alter cellular MST4 activity levels in three widely studied human prostate tumor cell lines: LNCaP, DU 145, and PC-3. Overexpression of wild-type MST4 induced anchorage-independent growth of the LNCaP cell line, and increased both in vitro proliferation and in vivo tumorigenesis of the DU 145 cell line. On the other hand, expression of a kinase-inactive form reverted the anchorage-independent growth phenotype and highly tumorigenic behavior of the PC-3 cell line. MST4 kinase activity was stimulated significantly by epidermal growth factor receptor ligands, which are known to promote growth of prostate cancer cells. Together, our studies suggest a potential role for MST4 in the signal transduction pathways involved in prostate cancer progression.

Topics: Adenocarcinoma; Adult; Cell Adhesion; Cell Division; Disease Progression; Enzyme Induction; Epidermal Growth Factor; Extracellular Matrix; Humans; Lymphatic Metastasis; Male; Neoplasm Proteins; Organ Specificity; Prostate; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured

Pancreatic cancer remains a devastating disease, with 95% of all patients diagnosed with the disease dying within 2 years. The combined therapy using Erbitux, gemcitabine, and radiation caused complete tumor regression using a nude mouse model inoculated with pancreatic MiaPaCa-2 cells but only a delay in tumor growth with BxPC-3. We investigated the effect of prolonged Erbitux treatment to the sensitivity to gemcitabine and/or radiation and the epidermal growth factor receptor (EGFR) signal transduction pathway.. MiaPaCa-2 and BxPC-3 cells were cultured with or without Erbitux for 6 weeks. Cells were then treated with gemcitabine and/or radiation, harvested 48 h after treatment, and counted. Differences in EGFR expression after exposure to Erbitux were analyzed by FACS. Internalization rates of EGFR induced by Erbitux on these cell lines were determined using 125I-EGF binding assay after removal of Erbitux by acidic wash. Cell lysates were harvested after cells were stimulated with EGF, FGF, or IGF-1 respectively, and EGFR was immunoprecipitated using Erbitux. Samples were separated using SDS-PAGE and transferred to PVDF membrane. The membranes were probed with antibody against human growth factor receptor binding protein (Grb2) to detect the association of this Ras-MAPK upstream adaptor protein to EGFR. Cell lysates were also separated with SDS-PAGE and probed with rabbit anti-human PARP after samples were transferred to PVDF membrane. Expression of BAX and Bcl-(XL) were probed in the cells treated with or without Erbitux.. Proliferation assays indicated that prolonged exposure to Erbitux increased the sensitivities of MiaPaCa-2 to gemcitabine and radiation therapy (41 +/- 16% vs 52 +/- 9% for gemcitation, 28 +/- 9 vs 39 +/- 9% for combination; P = 0.015) but not for BxPC-3. FACS analysis showed that the expressed EGFR level decreased by about 42% on MiaPaCa 2 whereas no loss was seen on BxPC-3. Expression of BAX was upregulated on MiaPaCa-2. Poly (ADP-ribose) polymerase cleavage indicated the killing was mediated by apoptosis. Immunoblots showed that Grb2 was co-immunoprecipitated with EGFR after EGF stimulation. Incubation with Erbitux blocked Grb2 binding in MiaPaCa-2 but not BxPC 3. FGF transactivated EGFR down stream Ras-MAPK in the presence or absence of Erbitux. Internalization of EGFR induced by Erbitux did not differ between MiaPaCa-2 and BxPC-3.. 1) Association of Grb2 to EGFR in BxPC-3 induced by EGF in the presence of Erbitux indicates an alternate pathway of Ras-MAPK activation, which may be related with the tumor resistance to treatment; 2) transactivation of EGFR downstream Ras-MAPK pathway by FGF contributes the resistance to treatment; and 3) downregulation of EGFR may increase the response to therapy.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Cell Division; Cetuximab; Cobalt Radioisotopes; Deoxycytidine; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Flow Cytometry; Gemcitabine; Gene Expression Regulation, Neoplastic; GRB2 Adaptor Protein; Humans; Immunosorbent Techniques; Iodine Radioisotopes; Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

It was recently shown that neuropilin-1 (NRP-1), which was described originally as a receptor for the semaphorins/collapsins (ligands involved in neuronal guidance), is a coreceptor for vascular endothelial growth factor (VEGF) and increases the affinity of specific isoforms of VEGF to its receptor, VEGF-R2.. The authors investigated the expression and regulation of NRP-1 in human pancreatic adenocarcinoma specimens and cell lines.. Immunohistochemical analysis revealed that NRP-1 was expressed in 12 of 12 human pancreatic adenocarcinoma specimens but was absent in nonmalignant pancreatic tissue. Northern blot analysis revealed NRP-1 mRNA expression in 8 of 11 human pancreatic adenocarcinoma cell lines. NRP-1 mRNA expression was increased by epidermal growth factor (EGF) but not by tumor necrosis factor alpha in several of the human pancreatic adenocarcinoma cell lines studied. Treating human Panc-48 adenocarcinoma cells with EGF activated Akt and Erk but not P-38. Blockade of the phosphatidylinositol-3 kinase (PI-3K)/Akt, mitogen-activated protein kinase (MAPK)/Erk, or P-38 pathways abrogated EGF-induced NRP-1 expression. Finally, EGF receptor blockade in vivo led to a decrease in NRP-1 expression in an orthotopic model of human pancreatic carcinoma.. NRP-1 is expressed in most human pancreatic adenocarcinomas and cell lines but not in nonmalignant pancreatic tissue. EGF regulates NRP-1 expression through the PI-3K/Akt and MAPK/Erk signaling pathways, and blockade of the EGF receptor is associated with decreased expression of NRP-1 in vivo. NRP-1 may act as a coreceptor for VEGF in pancreatic carcinoma, as it does in other tumor systems, thereby enhancing angiogenesis and the effect of VEGF on the growth of pancreatic adenocarcinoma.

Topics: Adenocarcinoma; Blotting, Northern; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Immunohistochemistry; Keratins; Mitogen-Activated Protein Kinases; Neuropilin-1; Pancreas; Pancreatic Neoplasms; Phosphatidylinositol 3-Kinases; Receptors, Vascular Endothelial Growth Factor; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Angiotensin II (A-II) receptor (AT(1) receptor) blockers (ARB) are a class of antihypertensive agent. It is known that they suppress signal transduction pathways mediated by growth factors [e.g., epidermal growth factor (EGF)] through the AT(1) receptor in vascular endothelial cells. In the present study, we demonstrated that A-II activates the cell proliferation of prostate cancer as well as EGF. In addition, we showed that A-II induces the phosphorylations of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) in prostate cancer cells. In contrast, ARB was shown to inhibit the proliferation of prostate cancer cells stimulated with EGF or A-II, the mechanism of which is through the suppression of MAPK or STAT3 phosphorylation by ARB. Oral administration of ARB to nude mice inhibited the growth of prostate cancer xenografts in both androgen-dependent and androgen-independent cells in a dose-dependent manner. Microvessel density was reduced in xenografts treated with ARB, which means ARB inhibits the vascularization of xenografts. Expression of AT(1) receptor mRNA was examined by reverse transcription-PCR using 10 pairs of human prostate cancer and normal prostate tissues. AT(1) receptor expression in human prostate cancer tissue was higher (9 of 10 cases) than that in normal prostate tissue. These results suggest the possibility that ARB is a novel therapeutic class of agents for prostate cancer, especially hormone-independent tumors.

Topics: Adenocarcinoma; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Antineoplastic Agents; Cell Division; DNA-Binding Proteins; Epidermal Growth Factor; Humans; Male; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neoplasm Transplantation; Phosphorylation; Prostatic Neoplasms; Protein-Tyrosine Kinases; Receptors, Angiotensin; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

The specificity of a novel epidermal growth factor (EGF)-Cy5.5 fluorescent optical probe in the detection of EGF receptor (EGFr) was assessed using continuous-wave fluorescence imaging accomplished via an intensified charge-coupled device (CCD) camera. Human mammary MDA-MB-468 (EGFr+) and MDA-MB-435 (EGFr-) cancer cells were incubated with Cy5.5, EGF-Cy5.5, or the anti-EGFr monoclonal antibody C225 or EGF followed by EGF-Cy5.5 and examined under a fluorescence microscope. In vivo imaging was performed on mice with s.c. MDA-MB-468 and MDA-MB-435 tumors. Images were obtained every 6 s for 20 min after i.v. injection of each agent and every 24 h after injection for up to 192 h. Additionally, mice with MDA-MB-468 tumors were injected i.v. with C225 24 h before injection of EGF-Cy5.5. EGF-Cy5.5, but not Cy5.5 or indocyanine green dye (ICG), bound to MDA-MB-468 cells. Binding of EGF-Cy5.5 was blocked by C225 and by EGF. In contrast, binding of EGF-Cy5.5 to MDA-MB-435 cells was not observed. Monitoring of the time-fluorescence intensity in mice confirmed that ICG and Cy5.5 had no favorable binding to tumor regardless of EGFr expression level. In contrast, EGF-Cy5.5 accumulated only in MDA-MB-468 tumors. Moreover, tumor uptake of EGF-Cy5.5 was blocked by C225. ICG and Cy5.5 fluorescence was completely absent from the tumor site, regardless of EGFr expression level, 24 h after injection. Little EGF-Cy5.5 fluorescence was detected in MDA-MB-435 tumors 24 h after injection. In MDA-MB-468 tumors, our data suggest that EGF-Cy5.5 may be used as a specific NIR contrast agent for noninvasive imaging of EGFr expression and monitoring of responses to molecularly targeted therapy.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Fluorescent Dyes; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Spectroscopy, Near-Infrared; Substrate Specificity; Transplantation, Heterologous

We described a 70 years old patient with pericardial effusion due to adenocarcinoma of the lung, in whom gefitinib, which is an oral selective inhibitor of the epidermal growth factor receptor of tyrosine kinase, demonstrated a marked antitumor effect. We recommend possible consideration of a treatment with gefitinib for female patients with pericarditis carcinomatosa due to lung adenocarcinoma, even if they have a poor performance status and are not indicated for other intensive therapy.

Topics: Adenocarcinoma; Aged; Antineoplastic Agents; Epidermal Growth Factor; Female; Gefitinib; Humans; Lung Neoplasms; Pericardial Effusion; Protein-Tyrosine Kinases; Quinazolines

S100A6 and S100A4, two of S100 protein family, have been suggested to be associated with cancer tumorigenesis and metastasis. The aim of this study was to evaluate the expression levels of S100A6 and S100A4 in matched samples of primary human colorectal adenocarcinomas (T), adjacent normal colorectal mucosa (N) and liver metastases (M). This gave us the advantage of directly comparing levels of S100A6 and S100A4 expression within the same genetic background.. In matched samples of N, T and M from 10 colorectal adenocarcinoma patients, expressions of S100A6 and S100A4 were studied by Western blot and immunohistochemical analyses using specific antibodies against each protein.. The expression levels of S100A6 were significantly higher in T than in N (p < 0.05), while those of S100A4 showed no difference between T and N. There were no significant differences in the expression levels of S100A6 or S100A4 between M and T. Similar results were obtained by immunohistochemical analysis. Moreover, S100A6 was stained more intensely in invading fronts than in central portions of both T and M.. The observed differential expression of S100A6 and S100A4 suggests that S100A6, rather than S100A4, is associated with human colorectal adenocarcinoma tumorigenesis and invasion/metastasis.

Topics: Adenocarcinoma; Aged; Cell Cycle Proteins; Chromosome Mapping; Chromosomes, Human, Pair 1; Colonic Neoplasms; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Liver Neoplasms; Male; Middle Aged; Neoplasm Staging; Rectal Neoplasms; S100 Calcium Binding Protein A6; S100 Calcium-Binding Protein A4; S100 Proteins

Cyr61 is a secreted pro-angiogenic factor that belongs to an emerging family of growth regulators classified as CCN (CTGF/Cyr61/NOV). Work in our laboratory has focused on sex steroid regulation of Cyr61 and its role in hormonal carcinogenesis. In this study, both Cyr61 mRNA and protein were induced by the progestin, R5020, in T47D mammary adenocarcinoma cells in a dose- and time-dependent fashion. Cyr61 gene induction by R5020 was transcriptionally regulated by progesterone receptor (PR) as the antiprogestin, RU486, and actinomycin D blocked induction completely. Moreover, Cyr61 was upregulated by epidermal growth factor (EGF) but not by R5020 in the PR-MDA-MB-431 mammary adenocarcinoma cell line, underscoring the necessity of PR. The functional significance of progestin induction of Cyr61 in breast cancer cell growth was demonstrated by anti-Cyr61 neutralizing antibodies, which diminished R5020 and EGF-dependent DNA synthesis by 30%. Moreover, anti-Cyr61 neutralizing antibodies reduced the synergistic effects of R5020 and EGF on T47D cell growth by 30%. Accordingly, protein lysates generated from stage II invasive ductal carcinomas (n = 20) were analyzed in order to determine the relevance of Cyr61 expression in the context of breast tumorigenesis. Remarkably, increased Cyr61 protein expression was observed in greater than 50% of primary breast tumor lysates that were progesterone receptor (PR)+ but estrogen receptor negative. Taken together, our data suggest that in addition to its proangiogenic activity, Cyr61 may be a novel mediator of progesterone activity in enhancing growth-factor-driven tumor growth in breast cancer.

Topics: Adenocarcinoma; Antibodies; Breast Neoplasms; Cell Division; Cysteine-Rich Protein 61; Drug Synergism; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Neovascularization, Pathologic; Progesterone Congeners; Promegestone; S Phase; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured; Up-Regulation

PRL is essential for normal lobulo-alveolar growth of the mammary gland and may contribute to mammary cancer development or progression. However, analysis of the mechanism of action of PRL in these processes is complicated by the production of PRL within mammary epithelia. To examine PRL actions in a mammary cell-specific context, we selected MCF-7 cells that lacked endogenous PRL synthesis, using PRL stimulation of interferon-gamma-activated sequence-related PRL response elements. Derived clones exhibited a greater proliferative response to PRL than control cells. To understand the mechanism, we examined, by Western analysis, levels of proteins essential for cell cycle progression as well as phosphorylation of retinoblastoma protein. The expression of cyclin D1, a critical regulator of the G1/S transition, was significantly increased by PRL and was associated with hyperphosphorylation of retinoblastoma protein at Ser(780). Cyclin B1 was also increased by PRL. In contrast, PRL decreased the Cip/Kip family inhibitor, p21, but not p16 or p27. These studies demonstrate that PRL can stimulate the cell cycle in mammary epithelia and identify specific targets in this process. This model system will enable further molecular dissection of the pathways involved in PRL-induced proliferation, increasing our understanding of this hormone and its interactions with other factors in normal and pathogenic processes.

Topics: Adenocarcinoma; Base Sequence; Breast Neoplasms; Cell Cycle Proteins; Cyclin B; Cyclin B1; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Female; Humans; Interferon-gamma; MAP Kinase Signaling System; Molecular Sequence Data; Oligonucleotides, Antisense; Phosphorylation; Prolactin; Retinoblastoma Protein; Signal Transduction

Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase blocks the mevalonate metabolic pathway, which is necessary for the isoprenylation of a number of small guanosine triphosphatases. We examined the effects of HMG-CoA reductase inhibitors, fluvastatin and lovastatin, on human pancreatic cancer cell invasion in vitro and experimental liver metastasis in vivo.. Cell invasion was studied in a modified Boyden chamber assay. The translocation of RhoA was assessed by immunoblotting. Experimental liver metastases were induced in nude mice by intrasplenic inoculation of ASPC-1 human pancreatic cancer cells.. Fluvastatin and lovastatin inhibited the in vitro cancer cell invasion induced by epidermal growth factor (EGF) in a manner sensitive to C3 transferase, a specific inhibitor of Rho. Treatment of ASPC-1 cells with fluvastatin markedly attenuated the EGF-induced translocation of RhoA from the cytosol to the membrane fraction and caused cell rounding. The effects of fluvastatin could be reversed by the addition of all-trans-geranylgeraniol. Administration of fluvastatin to nude mice reduced both metastatic tumor formation in the liver and the growth of established liver metastases at doses recommended for the treatment of hypercholesterolemia in humans.. HMG-CoA reductase inhibitors can be antimetastatic agents with the potential for useful clinical applications.

Topics: Adenocarcinoma; Animals; Cell Division; Cell Membrane; Cytosol; Epidermal Growth Factor; Fatty Acids, Monounsaturated; Fluvastatin; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Indoles; Liver Neoplasms, Experimental; Lovastatin; Mice; Mice, Nude; Neoplasm Invasiveness; Pancreatic Neoplasms; rhoA GTP-Binding Protein; Tumor Cells, Cultured

Epidermal growth factor receptor (EGF-R) autophosphorylation is essential for its intracellular mitogenic signaling via the MAPK pathway and for interaction in other cellular processes. Inhibition of this activity in tumor cells that predominantly utilise EGF-R therefore offers an alternative approach to therapy.. The ability of a specific inhibitor of EGF-R tyrosine kinase, ZM 252868, (TKI) to alter various parameters related to growth in DU145 and PC3 cell lines was investigated, by immunocytochemistry, Northern blotting, Western blotting and invasion assays.. In DU145 cultures, the total cell population and number of cells in cell cycle decreased in the presence of TKI whilst the apoptotic rate was significantly increased. Reduction in autophosphorylation of the EGF-R, membrane expression of EGF-R, activation of the MAPK, p38, and JNK enzymes and the invasive capacity of DU145 cells was observed in the TKI treated cells. Under the same conditions, PC3 cell growth and EGF-R expression and MAPK activation were not affected. The use of inhibitors of intracellular signaling indicated that the DU145 cells, in contrast to PC3 cells, predominantly utilize EGF-R activation of the MAPK signaling pathway for growth.. In prostatic cancer patients, in whom androgen resistance has developed and whose tumors have upregulated EGF-R for growth, specific TKI's may offer an important therapy option.

Topics: Adenocarcinoma; Apoptosis; Blotting, Northern; Blotting, Western; Cell Count; Cell Cycle; Cell Division; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Male; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Phosphorylation; Prostatic Neoplasms; Quinazolines; RNA, Messenger; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Growth factors can enhance the malignant potential of tumor cells. To examine the relationship between growth factors and tumor progression, we previously established a weakly malignant cell line, ER-1. We found that a 24-hour exposure of ER-1 cells to epidermal growth factor (EGF) induced malignant properties (tumor progression) that were reversible but that, after a 1-month exposure, these changes were irreversible. In this study, we investigated the irreversible changes induced in ER-1 cells by a 1-month exposure to EGF and the possible involvement of oxidative stress.. ER-1 cells were treated with EGF (100 ng/mL) for 1 month in the presence or absence of an antioxidant, N-acetylcysteine or selenium, and compared with untreated control ER-1 cells. We assessed tumor progression by measuring intracellular peroxide levels, 8-hydroxydeoxyguanosine (a marker for oxidative DNA damage) levels, in vitro invasiveness, and in vivo tumorigenicity and metastatic ability. All statistical tests are two-sided.. After ER-1 cells were treated for 1 month with EGF, levels of intracellular peroxide and 8-hydroxyguanosine in the DNA of treated cells were higher than those in the DNA of control cells, and treated ER-1 cells were more tumorigenic and metastatic in vivo and more invasive in vitro than untreated control cells (all P<.001). Levels of 8-hydroxyguanosine in DNA increased as the length of the EGF treatment increased (P<.001). However, when N-acetylcysteine or selenium was added with EGF for 1 month, levels of intracellular peroxide and 8-hydroxyguanosine in DNA were comparable to those in control cells (r =.795). Both tumorigenicity (P =.008) and metastatic ability (P<.001) decreased after addition of N-acetylcysteine or selenium.. The irreversible changes caused by continuous EGF stimulation of ER-1 cells result from increased oxidative damage in the DNA, which generates tumor cells with more malignant characteristics.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Acetylcysteine; Adenocarcinoma; Animals; Antioxidants; Deoxyguanosine; Disease Progression; DNA Damage; Epidermal Growth Factor; Female; Free Radical Scavengers; Glutathione Peroxidase; Mammary Neoplasms, Experimental; Microscopy, Confocal; Oxidative Stress; Rats; Rats, Inbred SHR; Selenium; Tumor Cells, Cultured

The extension of lamellipodia has been triggered by the application of epidermal growth factor (EGF). We have used an atomic force microscope (AFM) to investigate this lamellipodial extension. During extension we could detect an increase in height from about 500 nm for the stable lamellipodium to typical values of 600-800 nm for the extending lamellipodium. The AFM was also used to determine the mechanical properties of the lamellipodium where we found a decrease of the elastic modulus by a factor of 1.4 at the same location within the same cell. Both findings are consistent with the cortical expansion hypothesis, suggesting that severing of actin filaments, leading to a swelling of the cytoskeleton, generates the protrusive force during lamellipodial extension.

Topics: Adenocarcinoma; Animals; Chemotaxis; Elasticity; Epidermal Growth Factor; Lung Neoplasms; Microscopy, Atomic Force; Pseudopodia; Rats; Tumor Cells, Cultured

Transgenic mouse models were established to study tumorigenesis of bronchiolo-alveolar adenocarcinomas derived from alveolar type II pneumocytes (AT-II cells). Transgenic lines expressing the murine oncogene c- myc under the control of the lung-specific surfactant protein C promoter developed multifocal bronchiolo-alveolar hyperplasias, adenomas and carcinomas respectively, whereas transgenic lines expressing a secretable form of the epidermal growth factor (IgEGF), a structural and functional homologue of transforming growth factor alpha (TGF alpha), developed hyperplasias of the alveolar epithelium. Since the oncogenes c- myc and TGF alpha are frequently overexpressed in human lung bronchiolo-alveolar adenocarcinomas, these mouse lines are useful as models for human lung bronchiolo-alveolar adenocarcinomas. The average life expectancies of hemizygous and homozygous c- myc transgenics were 14.25 months and 9.2 months, respectively, suggesting that a dosage effect of c- myc caused an accelerated bronchiolo-alveolar adenocarcinoma formation. First analyses of double transgenics, hemizygous for both c- myc and IgEGF, show that these mice develop bronchiolo-alveolar adenocarcinomas at the average age of 9 months, indicating that these oncogenes cooperate during the lung cancer formation. Our results demonstrate that c- myc and EGF are directly involved and cooperate with one another during formation of bronchiolo-alveolar adenocarcinomas in the lung.

Topics: Adenocarcinoma; Animals; Bronchial Neoplasms; Cloning, Molecular; Epidermal Growth Factor; Gene Expression; Genes, myc; Lung Neoplasms; Mice; Mice, Transgenic; Phenotype; Pulmonary Alveoli

Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading edge of the cell. We have now examined the role of the p85alpha regulatory subunit in greater detail. Microinjection of recombinant p85alpha into MTLn3 cells blocked both EGF-stimulated mitogenic signaling and lamellipod extension. In contrast, a truncated p85(1-333), which lacks the SH2 and iSH2 domains and does not bind p110, had no effect on EGF-stimulated mitogenesis but still blocked EGF-stimulated lamellipod extension. Additional deletional analysis showed that the SH3 domain was not required for inhibition of lamellipod extension, as a construct containing only the proline-rich and breakpoint cluster region (BCR) homology domains was sufficient for inhibition. Although the BCR domain of p85 binds Rac, the effects of the p85 constructs were not because of a general inhibition of Rac signaling, because sorbitol-induced JNK activation in MTLn3 cells was not inhibited. These data show that the proline-rich and BCR homology domains of p85 are involved in the coupling of p85/p110 PI 3-kinases to regulation of the actin cytoskeleton. These data provide evidence of a distinct cellular function for the N-terminal domains of p85.

Topics: Adenocarcinoma; Animals; Cytoskeleton; Epidermal Growth Factor; JNK Mitogen-Activated Protein Kinases; Kinetics; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Protein Subunits; Pseudopodia; Rats; Recombinant Fusion Proteins; Recombinant Proteins; Sequence Deletion; Signal Transduction; Tumor Cells, Cultured

Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10(-8)M EGF stimulated AR expression in HMECs to 140-300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5-10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system.

Topics: Adenocarcinoma; Adolescent; Adult; Amphiregulin; Breast; Breast Neoplasms; Cell Division; Cells, Cultured; EGF Family of Proteins; Epidermal Growth Factor; Epithelial Cells; Female; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycoproteins; Growth Substances; Humans; Immunophenotyping; Intercellular Signaling Peptides and Proteins; Neoplasm Invasiveness; Plasminogen Activator Inhibitor 1; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Cyr61, a member of the CCN (CTGF/Cyr61/NOV) family of growth regulators, is a secreted cysteine-rich proangiogenic factor that has been implicated in tumorigenesis. Previous studies have also demonstrated that Cyr61 is regulated by 17beta-estradiol (E(2)) in the uterus. Therefore, we hypothesized that hormonal regulation of Cyr61 may be important in estrogen-dependent pathogenic processes such as breast tumorigenesis. Our study demonstrates that both Cyr61 messenger RNA and protein are induced by E(2) in MCF-7 mammary adenocarcinoma cells that primarily overexpress estrogen receptor alpha (ERalpha) in a dose-dependent and immediate early fashion. Cyr61 gene induction by E(2) is transcriptionally regulated by ERalpha as the antiestrogen, ICI 182,780, and actinomycin D blocked induction completely. In addition, Cyr61 is up-regulated in MCF-7 cells by epidermal growth factor (EGF) in an immediate early fashion as well. The functional relevance of steroid induction of Cyr61 in breast cancer cell growth is demonstrated by anti-Cyr61 neutralizing antibodies, which diminished E(2) and EGF-dependent DNA synthesis and dramatically reduced E(2)-driven cell proliferation by more than 70%. Most importantly, Cyr61 is overexpressed in 70% (28 of 40) of breast cancer patients with infiltrating ductal carcinoma and is localized exclusively to hyperplastic ductal epithelial cells. Moreover, the levels of Cyr61 protein are higher in breast tumors that are ER(+)/EGF receptor(+) than those that are ER(-)/EGF receptor(+), suggesting that estrogens may mediate Cyr61 expression in vivo. Collectively, our data suggest that Cyr61 may play a critical role in estrogen- as well as growth factor-dependent breast tumor growth.

Topics: Adenocarcinoma; Antibodies; Breast Neoplasms; Cell Division; Cysteine-Rich Protein 61; Dactinomycin; DNA, Neoplasm; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Fulvestrant; Gene Expression Regulation, Neoplastic; Growth Substances; Humans; Immediate-Early Proteins; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Receptors, Estrogen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcriptional Activation; Tumor Cells, Cultured

To evaluate the role of the NF-kappaB signaling pathway in oncogenic transformation, we expressed IkappaBbeta, a specific inhibitor of NF-kappaB, in two human lung adenocarcinoma cell lines, A549 and H441. Expression of IkappaBbeta significantly reduced NF-kappaB activation induced by cotransfection with p65/RelA or TNF-alpha and abrogated the basal NF-kappaB activity in A549 cells. Transfection of IkappaBbeta into A549, H441 and K-ras-transformed NIH3T3 cells suppressed anchorage-independent growth as measured by colony formation in soft agar. Anchorage-independent growth of vector-transfected A549 cells in reduced serum could be enhanced by both EGF and IGF-I. In contrast, only EGF but not IGF-I could induce anchorage-independent growth of IkappaBbeta-expressing A549 cells, suggesting that the IGF-I signaling pathway regulating growth and survival may be blocked by IkappaBbeta. Interestingly, expression of IkappaBbeta suppressed growth of A549 cells in low serum in vitro without affecting in vivo growth subcutaneously in nude mice. However, metastatic growth of IkappaBbeta-expressing A549 cells in the lungs of nude mice was significantly inhibited. These results provide evidence that NFkappaB plays an important role in anchorage-independent growth and metastatic growth of lung carcinoma cells.

Topics: 3T3 Cells; Adenocarcinoma; Animals; Cell Adhesion; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; DNA-Binding Proteins; Epidermal Growth Factor; Humans; I-kappa B Proteins; Lung Neoplasms; Mice; Mice, Nude; NF-kappa B; Signal Transduction; Transcription Factor RelA; Transfection; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Previous studies demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) contributes to carcinogenesis and carcinoma progression. In this study, we investigated its expression in human pancreatic adenocarcinoma.. We immunohistochemically investigated the expression of HB-EGF in 40 cases of pancreatic adenocarcinoma.. HB-EGF was only occasionally and faintly expressed in normal and hyperplastic pancreas duct epithelia. In pancreatic adenocarcinoma, 22 (55.0%) of the 40 cases were classified as positive for HB-EGF. Its expression was more frequently observed in cases with a low Ki-67 labeling index, well differentiated. early stage, small size, without lymph node metastasis and low EGF-R expression.. These results suggest that HB-EGF mainly plays a role in early phase of the progression of pancreatic adenocarcinoma.

Topics: Adenocarcinoma; Aged; Epidermal Growth Factor; ErbB Receptors; Female; Heparin-binding EGF-like Growth Factor; Humans; Hyperplasia; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Ki-67 Antigen; Male; Middle Aged; Pancreas; Pancreatic Neoplasms; Reference Values

Elevated focal adhesion kinase (FAK) expression in human tumor cells has been correlated with an increased cell invasion potential. In cell culture, studies with FAK-null fibroblasts have shown that FAK function is required for cell migration. To determine the role of elevated FAK expression in facilitating epidermal growth factor (EGF)-stimulated human adenocarcinoma (A549) cell motility, antisense oligonucleotides were used to reduce FAK protein expression >75%. Treatment of A549 cells with FAK antisense (ISIS 15421) but not a mismatched control (ISIS 17636) oligonucleotide resulted in reduced EGF-stimulated p130(Cas)-Src complex formation, c-Jun NH(2)-terminal kinase (JNK) activation, directed cell motility, and serum-stimulated cell invasion through Matrigel. Because residual FAK protein in ISIS 15421-treated A549 cells was highly phosphorylated at the Tyr-397/Src homology (SH)2 binding site, expression of the FAK COOH-terminal domain (FRNK) was also used as an inhibitor of FAK function. Adenoviral-mediated infection and expression of FRNK promoted FAK dephosphorylation at Tyr-397, resulted in reduced EGF-stimulated JNK as well as extracellular-regulated kinase 2 (ERK2) kinase activation, inhibited matrix metalloproteinase-9 (MMP-9) secretion, and potently blocked both random and EGF-stimulated A549 cell motility. Equivalent expression of a FRNK (S-1034) point-mutant that did not promote FAK dephosphorylation also did not affect EGF-stimulated signaling or cell motility. Dose-dependent reduction in EGF-stimulated A549 motility was observed with the PD98059 MEK1 inhibitor and the batimastat (BB-94) inhibitor of MMP activity, but not with the SB203580 inhibitor of p38 kinase. Finally, comparisons between normal, FAK-null, and FAK-reconstituted fibroblasts revealed that FAK enhanced EGF-stimulated JNK and ERK2 kinase activation that was required for cell motility. These data indicate that FAK functions as an important signaling platform to coordinate EGF-stimulated cell migration in human tumor cells and support a role for inhibitors of FAK expression or activity in the control of neoplastic cell invasion.

Topics: Adenocarcinoma; Cell Movement; Enzyme Activation; Epidermal Growth Factor; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Oligonucleotides, Antisense; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Recent data demonstrate that endothelin-1 (ET-1) concentration increases in plasma of men with advanced, hormone-refractory prostate adenocarcinoma. In addition, ET-1 is involved in osteblastic remodelling and new bone formation, suggesting a role for this vasoactive peptide in the metastatic progression of prostate cancer to the bone.. We investigated the regulation of ET-1 expression in androgen-sensitive and insensitive prostate cancer cell lines by androgens and several factors involved in progression of prostate cancer (EGF) and bone remodelling (TGFbeta-1, IL1-alpha and IGF-1).. Northern analysis and radio immunoassay demonstrated that all the ET-1 pathways are tuned off in the androgen-sensitive LNCaP cell line when compared to the androgen-insensitive PC-3 and DU145. In PC-3 cells transfected with a full-length androgen receptor expression vector (PC-3-AR), treatment with androgens reduced gene expression and secretion of ET-1 without affecting the gene expression of ET-3. Collectively, these data support a role for androgens in the regulation of ET-1 production by prostate adenocarcinoma cells. In PC-3 and DU145 cells, ET-1 gene expression and secretion were up-regulated by TGFbeta-1, EGF and IL1-alpha, whereas IGF-1 was ineffective. Conversely, none of the treatments affected ECE-1 or ET-3 gene expression.. In conclusion, ET-1 production by prostate adenocarcinoma cells is down-regulated by androgens and up-regulated by factors involved in tumour progression indicating a role for this peptide in the biology of prostate cancer. In view of the role exerted by ET-1 in the process of bone metastasis, our data suggest the use of ET-1 receptor antagonists in the treatment of advanced prostate cancer.

Topics: Adenocarcinoma; Androgens; Blotting, Northern; Bone Neoplasms; Cytokines; Endothelin-1; Endothelin-3; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Male; Metalloendopeptidases; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Relatively few genes have been shown to directly affect the metastatic phenotype of breast cancer epithelial cells in vivo. The Rho family of proteins, incluing the Rho, Rac and Cdc42 subfamilies, are related to the small GTP binding protein Ras and regulated diverse biological processes including gene transcription, cytoskeletal organization, cell proliferation and transformation. The effects of Cdc42, Rac and Rho on the actin cytoskeleton suggested a possible role for Rho proteins in cellular motility and metastasis; however, a formal analysis of the role of Rho proteins in breast cancer cellular growth and metastasis in vivo had not previously been performed.. We generated a panel of MTLn3 rat mammary adenocarcinoma cells that expressed similar levels of dominant inhibitory mutants of Cdc42-, Rac- and Rho-dependent signaling, to examine the contribution of these GTPases to cell spreading, guided chemotaxis, and metastasis in vivo. The ability of Rho proteins to regulate intravasation into the peripheral blood was determined by implanting MTLn3 cell stable dominant negative lines in nude mice and measuring the formation of breast cancer cell colonies grown from the peripheral blood. Serial sectioning of the lungs was performed to determine the presence of metastasis in mice in which mammary tumors expressing the dominant negative Rho family proteins had grown to a similar size.. Cell spreading of MTLn3 cells was selectively abrogated by N17Rac1. N19RhoA and N17Cdc42 reduced the number of focal contacts (FCs) and disrupted the co-localization of vinculin with phosphotyrosine at FCs. While N17Rac1 and N17Cdc42 preferentially inhibited colony formation in soft agar, all three GTPases affected cell growth in vivo. To distinguish effects on tumorigenicity from intravasation into the bloodstream, implanted tumors were grown to the same size in nude mice. Each dominant inhibitory Rho protein reduced intravasation into the peripheral blood. Lung metastasis of MTLn3 cells was also abrogated by the dominant inhibitory Rho proteins, despite the presence of residual CFU.. These studies demonstrate for the first time a critical role for the Rho GTPases involving independent signaling pathways to limit mammary tumor cellular growth and metastasis in vivo.

Topics: Adenocarcinoma; Animals; cdc42 GTP-Binding Protein; Cell Division; Chemotaxis; Epidermal Growth Factor; Epithelium; Lung Neoplasms; Mammary Neoplasms, Experimental; Multigene Family; Rats; rho GTP-Binding Proteins; Signal Transduction; Stress Fibers; Transfection; Tumor Cells, Cultured; Tyrosine

The epidermal growth factor (EGF) family of tyrosine kinase receptors (ErbB1, -2, -3, and -4) and their ligands are involved in cell differentiation, proliferation, migration, and carcinogenesis. However, it has proven difficult to link a given ErbB receptor to a specific biological process since most cells express multiple ErbB members that heterodimerize, leading to receptor cross-activation. In this study, we utilize carcinoma cells depleted of ErbB2, but not other ErbB receptor members, to specifically examine the role of ErbB2 in carcinoma cell migration and invasion. Cells stimulated with EGF-related peptides show increased invasion of the extracellular matrix, whereas cells devoid of functional ErbB2 receptors do not. ErbB2 facilitates cell invasion through extracellular regulated kinase (ERK) activation and coupling of the adaptor proteins, p130CAS and c-CrkII, which regulate the actin-myosin cytoskeleton of migratory cells. Overexpression of ErbB2 in cells devoid of other ErbB receptor members is sufficient to promote ERK activation and CAS/Crk coupling, leading to cell migration. Thus, ErbB2 serves as a critical component that couples ErbB receptor tyrosine kinases to the migration/invasion machinery of carcinoma cells.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Movement; Crk-Associated Substrate Protein; Enzyme Activation; Epidermal Growth Factor; Female; Humans; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Peptide Fragments; Phosphoproteins; Protein Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-crk; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Receptors, Growth Factor; Recombinant Proteins; Retinoblastoma-Like Protein p130; Signal Transduction

The expression of S100A6 (also known as Calcyclin/2A9/ 5B10/PRA) in surgically resected human colorectal adenocarcinomas was examined to investigate whether S100A6 plays a role in the malignancy of human tumor cells. Western blot analysis using the lysates from colorectal adenocarcinomas and adjacent normal mucosa from 10 patients revealed that the average S100A6 level of adenocarcinomas was significantly higher (about 2.4-fold) than that of normal mucosa. Immunohistochemical analysis using formalin-fixed paraffin-embedded surgical specimens and monoclonal anti-S100A6 antibody (mAbA6) demonstrated that 2(5%) of 42 normal mucosa and 6 (46%) of 13 adenoma specimens were mAbA6-positive and showed granular staining localized at the supranuclear regions of epithelial cells, whereas 23 (55%) of 42 adenocarcinomas and 13 (100%) of 13 carcinoma cells that metastasized to the liver were mAbA6-positive and showed diffuse cytoplasmic staining. A significant correlation between S100A6 expression and Dukes' tumor stage or lymphatic permeation but not with other clinicopathological factors was shown. S100A6 was stained more intensely in peripheral portions than in central portions of adenocarcinomas, whereas Ki-67 (a growth marker) was stained equally in these two portions. These results suggest that S100A6 may be involved in the progression and invasive process of human colorectal adenocarcinomas.

Topics: Adenocarcinoma; Adenoma; Blotting, Western; Cell Cycle Proteins; Colonic Neoplasms; Colorectal Neoplasms; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Intestinal Mucosa; Male; Middle Aged; Neoplasm Staging; Rectal Neoplasms; S100 Calcium Binding Protein A6; S100 Proteins

We have studied the role of phosphatidylinositol 3-kinases (PI 3-kinases) in the regulation of the actin cytoskeleton in MTLn3 rat adenocarcinoma cells. Stimulation of MTLn3 cells with epidermal growth factor (EGF) induced a rapid increase in actin polymerization, with production of lamellipodia within 3 min. EGF-stimulated lamellipodia were blocked by 100 nM wortmannin, suggesting the involvement of a class Ia PI 3-kinase. MTLn3 cells contain equal amounts of p110alpha and p110beta, and do not contain p110delta. Injection of specific inhibitory antibodies to p110alpha induced cell rounding and blocked EGF-stimulated lamellipod extension, whereas control or anti-p110beta antibodies had no effect. In contrast, both antibodies inhibited EGF-stimulated DNA synthesis. An in situ assay for actin nucleation showed that EGF-stimulated formation of new barbed ends was blocked by injection of anti-p110alpha antibodies. In summary, the p110alpha isoform of PI 3-kinase is specifically required for EGF-stimulated actin nucleation during lamellipod extension in breast cancer cells.

Topics: Actins; Adenocarcinoma; Amino Acid Sequence; Androstadienes; Animals; Antibodies; Breast Neoplasms; Cytoskeleton; DNA Replication; Epidermal Growth Factor; Isoenzymes; Microinjections; Microscopy, Fluorescence; Molecular Sequence Data; Phosphatidylinositol 3-Kinases; Rats; Tumor Cells, Cultured; Wortmannin

Caveolin-1 is a principal component of caveolae membranes that may function as a transformation suppressor. For example, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (D7S522; 7q31.1) that is deleted in human cancers, including mammary carcinomas. However, little is known about the role of caveolins in regulating cell movement, a critical parameter in determining metastatic potential. Here, we examine the role of caveolin-1 in cell movement. For this purpose, we employed an established cellular model, MTLn3, a metastatic rat mammary adenocarcinoma cell line. In this system, epidermal growth factor (EGF) stimulation induces rapid lamellipod extension and cell migration. Interestingly, we find that MTLn3 cells fail to express detectable levels of endogenous caveolin-1. To restore caveolin-1 expression in MTLn3 cells efficiently, we employed an inducible adenoviral gene delivery system to achieve tightly controlled expression of caveolin-1. We show here that caveolin-1 expression in MTLn3 cells inhibits EGF-stimulated lamellipod extension and cell migration and blocks their anchorage-independent growth. Under these conditions, EGF-induced activation of the p42/44 mitogen-activated protein kinase cascade is also blunted. Our results suggest that caveolin-1 expression in motile MTLn3 cells induces a non-motile phenotype.

Topics: Adenocarcinoma; Adenoviridae; Animals; Caveolin 1; Caveolin 2; Caveolins; Cell Adhesion; Cell Movement; Cell Size; Epidermal Growth Factor; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Mammary Neoplasms, Animal; Membrane Proteins; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Rats; Time Factors; Transfection; Tumor Cells, Cultured

Certain tumor cells, such as squamous carcinoma cells, express an increased number of epidermal growth factor (EGF) receptors. Therefore, we studied the targeted delivery of the photocytotoxic compound Sn-(IV)chlorin e6 monoethylenediamine [SnCe6(ED)] to tumors that overexpress the EGF receptor. EGF was conjugated to SnCe6(ED) through a carrier, such as dextran (Dex) and human serum albumin (HSA), and the photocytotoxicity on the EGF receptor-overexpressing MDA-MB-468 breast adenocarcinoma cell line was evaluated. The photobiological activities of these EGF conjugates, of the conjugates of the photosensitizer to HSA or Dex, or of the photosensitizer alone were compared. The affinity of EGF for its receptor was substantially impaired when conjugated in EGF-Dex-SnCe6(ED), in contrast to EGF-HSA-SnCe6(ED). In corresponding results, EGF-HSA-SnCe6(ED) displayed a high photocytotoxicity (IC50, 63 nM) on MDA-MB-468 cells at a light dose of 27 kJ/m2, whereas EGF-Dex-SnCe6(ED) showed very limited photocytotoxicity. EGF-HSA-SnCe6(ED) was no longer photocytotoxic in the presence of a competing EGF concentration. The high photocytotoxicity of EGF-HSA-SnCe6(ED) was shown to be the result of a high intracellular concentration in MDA-MB-468 cells, which could be lowered dramatically by incubating the conjugate with a competing EGF concentration. In contrast, EGF-Dex-SnCe6(ED) accumulated poorly in MDA-MB-468 cells, in agreement with its low EGF receptor affinity and photocytotoxicity. EGF-HSA-SnCe6(ED) produced much more intracellular reactive oxygen species on light irradiation than EGF-Dex-SnCe6(ED). It is concluded that the photodynamic activity of the EGF-HSA conjugate of SnCe6(ED) on MDA-MB-468 breast adenocarcinoma cells is EGF specific and is much more potent than EGF-Dex-SnCe6(ED) or free SnCe6.

Topics: Adenocarcinoma; Binding, Competitive; Biological Transport; Breast Neoplasms; Cell Division; Chlorophyllides; Dextrans; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Humans; Inhibitory Concentration 50; Photochemotherapy; Porphyrins; Radiation-Sensitizing Agents; Reactive Oxygen Species; Recombinant Fusion Proteins; Serum Albumin; Substrate Specificity; Transfection; Tumor Cells, Cultured

We have shown that moderately differentiated endometrial adenocarcinoma (RL95-2) cells differentiate in response to retinoic acid treatment, illustrated by their reorganization of actin filaments and cell enlargement (Carter et al., Anticancer Res. 16, 17-24, 1996). Tyrphostin, an inhibitor of epidermal growth factor receptor (EGF-R)-associated protein tyrosine kinases, caused a dramatic reorganization of actin filaments in RL95-2 cells, similar to retinoic-acid-treated cells (Carter and Bellido, J. Cell. Physiol. 178, 320-332, 1999). We evaluated the possibility that the differentiating effects of retinoids are due to retinoic-acid-induced decreases in phosphorylation of EGF-R and changes in downstream effector proteins. Retinoic acid caused a decrease in tyrosine phosphorylation of EGF-R. Retinoic acid treatment induced a dramatic actin filament reorganization and cell enlargement. Treatment with EGF reversed this effect, because cells treated with retinoic acid followed by EGF only possessed disrupted actin aggregates and appeared small, thus resembling medium controls. Retinoic acid induced a relocalization and decrease in the amount of Shc protein, another actin-binding protein which is an adaptor protein for EGF-R signaling. In addition, retinoic acid induced a relocalization of gelsolin from the plasma membrane to the cytoplasm. Retinoic acid decreased cell detachment in detachment assays; one-half as many retinoic-acid-treated cells detached as in controls. These results are consistent with the idea that retinoic acid induces differentiation of RL95-2 cells by interfering with the EGF-R signaling pathway.

Topics: Actin Cytoskeleton; Actins; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Cell Adhesion; Cell Differentiation; Cell Membrane; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Cytoskeleton; Endometrial Neoplasms; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gelsolin; GRB2 Adaptor Protein; Humans; Isotretinoin; Membrane Proteins; Neoplasm Proteins; Phosphorylation; Protein Processing, Post-Translational; Proteins; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; Tetradecanoylphorbol Acetate; Tretinoin; 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

Epiregulin belongs to the epidermal growth factor (EGF) family of polypeptides. Previous studies have underscored the important role of the EGF family of ligands and receptors in the pathology of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). It is not known, however, whether epiregulin may also have a role in these diseases. Therefore, in the present study we investigated the expression and function of epiregulin in five pancreatic cancer cell lines and in PDAC and CP tissue samples. Epiregulin mRNA was present at high (MIA-PaCa-2 cells) or moderate levels (ASPC-1, CAPAN-1, and T3M4) in most cells, but was below detection levels in PANC-1 cells. All the cell lines exhibited a dose-dependent increase in growth in response to recombinant human epiregulin. Epiregulin mRNA levels were increased 2.1-fold in PDAC samples (P < 0.01) and 1.7-fold in CP samples (P < 0.01), when compared with the normal controls. There was no correlation between epiregulin mRNA levels and tumor stage or grade. By in situ hybridization, a moderate to intense epiregulin mRNA signal was present in most pancreatic cancer cells in PDAC. In contrast, only a weak (normal pancreas) to moderate (CP) signals were present in the ductal and acinar cells in CP. These findings suggest that epiregulin may contribute to the pathobiology of PDAC, and may also have a role in CP.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; Epidermal Growth Factor; Epiregulin; Humans; In Situ Hybridization; Pancreatic Neoplasms; RNA, Messenger; Up-Regulation

Recent evidence suggests that both estrogens and growth factors play an important role in the growth of gastrointestinal tumors. The expression of estrogen receptors (ER) and epidermal growth factor receptors (EGFR) in the gastrointestinal tract might therefore result in functional cross-talk between estrogens and EGF. The aim of the present study was to evaluate in vitro the effects of 17beta-estradiol and EGF administration on cell proliferation of a human gastric adenocarcinoma cell line (AGS) and investigate whether any interaction of these compounds may play a role in regulating gastric cancer cell proliferation.. Estrogen and EGFRs were detected by enzyme immunoassay. Cell proliferation was assessed with the MTT test.. Exposure of AGS cells to increasing concentrations of 17beta-estradiol showed an anti-proliferative action at concentrations of 2 microM or higher. The addition of increasing concentrations of EGF stimulated cell growth, with a maximal response at 50 ng/ml EGF. The effect of increasing 17beta-estradiol concentrations combined with 50 ng/ml EGF was to increase cell growth at the lower estradiol concentrations. At the highest estradiol concentration the EGF proliferative effect was suppressed, and a decrease in proliferation rates occurred. Moreover, a significant negative correlation was found between 17beta-estradiol concentrations and EGFR expression.. These findings suggest that growth of cultured gastric cancer cells (AGS) might be modulated by sex steroid hormones through interaction with EGF.

Topics: Adenocarcinoma; Cell Division; Epidermal Growth Factor; ErbB Receptors; Estradiol; Humans; Receptors, Estrogen; Stomach Neoplasms; Tumor Cells, Cultured

The EWS/FLI-1 fusion gene is characteristic of most cases of Ewing's sarcoma and has been shown to be crucial for tumor transformation and cell growth. In this study we demonstrate a drastic down-regulation of the EWS/FLI-1 protein, and a growth arrest, following serum depletion of Ewing's sarcoma cells. This indicates that growth factor circuits may be involved in regulation of the fusion gene product. Of four different growth factors tested, basic fibroblast growth factor (bFGF) was found to be of particular significance. In fact, upon treatment of serum-depleted cells with bFGF, expression of the EWS/FLI-1 protein and growth of the Ewing's sarcoma cells were restored. In addition, a bFGF-neutralizing antibody, which was confirmed to inhibit FGF receptor (FGFR) phosphorylation, caused down-regulation of EWS/FLI-1. Experiments using specific cell cycle blockers (thymidine and colcemide) suggest that EWS/FLI-1 is directly linked to bFGF stimulation, and not indirectly to cell proliferation. We also demonstrated expression of FGFRs in several tumor samples of Ewing's sarcoma. Taken together, our data suggest that expression of FGFR is a common feature of Ewing's sarcoma and, in particular, that the bFGF pathway may be important for the maintenance of a malignant phenotype of Ewing's sarcoma cells through up-regulating the EWS/FLI-1 protein. Oncogene (2000) 19, 4298 - 4301

Topics: Adenocarcinoma; Antibodies, Monoclonal; Bone Neoplasms; Cell Cycle; Cell Division; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 22; Culture Media, Serum-Free; Demecolcine; Drug Synergism; Epidermal Growth Factor; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Male; Neoplasm Proteins; Oncogene Proteins, Fusion; Platelet-Derived Growth Factor; Prostatic Neoplasms; Proto-Oncogene Protein c-fli-1; Receptors, Fibroblast Growth Factor; RNA-Binding Protein EWS; Sarcoma, Ewing; Thymidine; Transcription Factors; Translocation, Genetic; Tumor Cells, Cultured

Certain tumor cells, such as squamous carcinoma cells, express an increased number of epidermal growth factor (EGF) receptors. The goal of this study was the targeted delivery of the photocytotoxic compound Sn(IV)chlorine e6 monoethylenediamine++ (SnCe6(ED)) to tumors that overexpress the EGF receptor. Therefore EGF was conjugated to SnCe6(ED) through a carrier, such as dextran (Dex) and human serum albumin (HSA), followed by the evaluation of the photocytotoxicity on the EGF receptor overexpressing MDA-MB-468 cell line. The photobiologic activity of these conjugates was then compared to a conjugate of the photosensitizer to HSA or dextran, or to the photosensitizer alone. In contrast to EGF-HSA-SnCe6(ED), the affinity of EGF for its receptor was substantially impaired upon conjugation in EGF-Dex-SnCe6(ED). In correlation with these results, EGF-HSA-SnCe6(ED) displayed a high cytotoxicity (IC50, 63 nM) on MDA-MB-468 cells at a light dose of 27 kJ/m2, whereas EGF-Dex-SnCe6(ED) showed very limited photocytotoxicity. In the presence of a competing EGF concentration (10 microM), EGF-HSA-SnCe6(ED) was not cytotoxic anymore. The high photocytoxicity of EGF-HSA-SnCe6(ED) was shown to be a result of a high intracellular concentration in MDA-MB-468 cells, which could be lowered dramatically by incubating the conjugate with a competing EGF concentration. In contrast, EGF-Dex-SnCe6(ED) displayed very poor accumulation in MDA-MB-468 cells, in agreement with its low EGF receptor affinity and photocytoxicity. Besides, it could be demonstrated that EGF-HSA-SnCe6(ED) produced intracellularly ROS (reactive oxygen species) upon light irradiation, more than EGF-Dex-SnCe6(ED) did. It was concluded that, in contrast to EGF-Dex-SnCe6(ED) the photodynamic activity of the EGF-HSA conjugate of SnCe6(ED) on MDA-MB-468 breast adenocarcinoma cells is EGF-specific and more potent than free SnCe6(ED).

Topics: Adenocarcinoma; Animals; Binding, Competitive; Breast Neoplasms; Chlorophyllides; Dextrans; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Mice; Photochemotherapy; Porphyrins; Radiation-Sensitizing Agents; Serum Albumin; Tumor Cells, Cultured

We have previously documented that the vast majority of high-grade gliomas over-express binding sites for interleukin 13 (IL13) in situ. We now extend this analysis to evaluate the distribution of the binding of IL13 among other brain tumors. Tumor specimens from patients with low-grade gliomas, oligodendrogliomas, ependymomas, pilocytic astrocytomas, gliosarcomas, medulloblastomas, meningiomas, and metastases to the brain were analyzed and compared to a new series of glioblastoma multiforme (GBM) samples. Serial tumor tissue sections were incubated with 125I-labeled (i) IL13, (ii) antibody against transferrin (Tf) receptor, and (iii) epidermal growth factor (EGF). Most (17/18) GBMs stained specifically for IL13 binding sites while sections from 3/11 low-grade gliomas, 5/5 high-grade gliomas (grade III), 3/5 oligodendrogliomas (all three were anaplastic), and 1/2 gliosarcomas also showed specific binding for IL13. We did not detect IL13 binding sites in medulloblastomas (0/4) and found them only in 2/20 meningiomas. Metastases to the brain (4/12, i.e., lung adenocarcinomas and renal cell carcinoma) showed some binding of 125I-IL13. The presence of receptors for Tf was ubiquitous among all studied tumors while EGF receptor expression was much more variable. Since it appears that primarily the least differentiated forms of gliomas possess IL13 binding sites in abundance, it is plausible that IL 13 receptor expressed in low-grade gliomas might be a prognostically significant marker associated with their progression to high-grade gliomas. Finally, we demonstrate that the glioma-associated IL13 receptor is truly more restrictive in nature also due to its selective representation among brain tumors of glial origin.

Topics: Adenocarcinoma; Biomarkers, Tumor; Brain Neoplasms; Carcinoma; Cell Transformation, Neoplastic; Disease Progression; Ependymoma; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioma; Gliosarcoma; Humans; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Interleukin-4; Medulloblastoma; Meningeal Neoplasms; Meningioma; Neoplasm Proteins; Oligodendroglioma; Receptors, Interleukin; Receptors, Interleukin-13; Receptors, Transferrin; Recombinant Proteins; Substrate Specificity

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.

Topics: Actin Depolymerizing Factors; Actins; Adenocarcinoma; Animals; Cell Movement; Epidermal Growth Factor; Female; Gene Expression; Genes, Reporter; Green Fluorescent Proteins; Indicators and Reagents; Lim Kinases; Luminescent Proteins; Mammary Neoplasms, Experimental; Microfilament Proteins; Mutagenesis; Phosphorylation; Protein Kinases; Protein Structure, Tertiary; Pseudopodia; Rats; Tumor Cells, Cultured

The four main cell functions, proliferation, apoptosis, differentiation and migration, are tightly regulated by external signals that initiate intracellular signal transduction pathways and determine the cellular behaviour. The concentration and composition of such external signals are at least important for the decision of cells as to which function has to be executed. Interleukin-8 is a well known inducing signal for neutrophil granulocyte migration, while the epidermal growth factor is an inducing signal for breast carcinoma cell migration. Depending on the concentrations of interleukin-8, the neutrophil granulocytes are capable of migration. However, at high concentration of interleukin-8 the migratory activity of each single cell is reduced, indicating that high concentrations of the chemokine inhibit migration and promote the performance of other cell functions. Concerning breast carcinoma cells, the epidermal growth factor is not only an inducer of migration but also an inhibitor of proliferation. These two examples provide evidence for a dose dependent action of external signals for several cell functions in parallel. This versatility of the effects of one ligand might be based on several intracellular signal transduction pathways that are turned on. For the dose-dependent differences of the effect of interleukin-8 we propose a two wheel model of an inositolphosphate-mediated, ATP-independent release of calcium from intracellular stores and a cyclic AMP-mediated, ATP-dependent uptake of calcium into the endoplasmatic reticulum.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Cell Differentiation; Cell Division; Cell Movement; Cell Physiological Phenomena; Chemotaxis, Leukocyte; Epidermal Growth Factor; Flow Cytometry; Humans; Interleukin-8; Microscopy, Video; Neutrophils; Tumor Cells, Cultured

EGF stimulates gene expression through a variety of signal transduction pathways that include the ras-Erk pathway. We have shown previously that EGF receptor activation stimulates gastrin gene expression through a GC-rich element called gERE. This element binds Sp1 family members and raises the possibility that the ras-Erk signal transduction cascade may target this novel EGF responsive element. Moreover, it is known that Erk 2 is capable of phosphorylating other mitogen-inducible transcription factors, e.g., Elk, Sap suggesting that Erk may also inducibly phosphorylate Sp1. To test this hypothesis directly using cotransfection experiments, we show that ras and Erk 2 activation indeed target the gERE element. The Mek 1 kinase inhibitor, PD98059, blocks 50% of EGF-inducible gastrin promoter activity. Pretreatment of the extracts with recombinant Erk2 stimulated Sp1 binding; whereas dephosphorylation reduced but did not eliminate Sp1 binding. Together, these studies demonstrate the novel finding that inducible binding of Sp1 is regulated by its state of phosphorylation. Further, gastrin promoter activation is mediated in part by the ras-Erk signaling cascade that targets Sp1.

Topics: Adenocarcinoma; Base Sequence; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Inhibitors; Epidermal Growth Factor; Flavonoids; Gastrins; Gene Expression Regulation, Neoplastic; Genes, ras; Humans; Mitogen-Activated Protein Kinase 1; Oligodeoxyribonucleotides; Phosphorylation; Promoter Regions, Genetic; Signal Transduction; Sp1 Transcription Factor; Stomach Neoplasms; Transcription Factors; Tumor Cells, Cultured

The effects of chronic EGF exposure on expression of the alpha2beta1 collagen and alpha5beta1 fibronectin receptor in a pair of human carcinoma cell lines (A431 and A549) with differential responses to EGF in a short-term ECM-cell adhesion assay were investigated. Treatment with EGF at 10 ng/ml for 24 hr increased on both cell lines the expression of the alpha2- but not the beta1- or alpha5-integrin sub-units, and concomitantly cellular adhesion was increased on collagen IV but not on fibronectin. Increased collagen adhesion of A549 cells could be blocked by alpha2- and beta1-integrin-sub-unit antibodies down to control levels, while it was blocked by alpha2-integrin-sub-unit antibody only by 60% and completely by the beta1-integrin-sub-unit antibody on A431 cells. EGF induced disparate shifts in cell morphologies (dome-like structures, A431, vs. spindle-like fibroblastoid, A549) with concomitant opposite changes in the expression/localization of E-cadherin in cell-cell contacts. This could be taken as an indication for cell-type-specific differential changes in the ratio of cell-ECM vs. cell-cell contacts. The EGF-induced up-regulation of the alpha2beta1 integrin was instrumental in increasing collagen adhesion of A549 but only partly in the case of A431 cells, in which cells the alpha2beta1 integrin may have additional functions besides serving as cell-ECM receptor.

Topics: Adenocarcinoma; Antigens, CD; Carcinoma; Cell Adhesion; Cell Communication; Epidermal Growth Factor; ErbB Receptors; Humans; Integrin alpha2; Integrin alpha5; Neoplasm Proteins; Neoplasms; Tumor Cells, Cultured

Protein kinase CK2, a messenger-independent serine/threonine kinase, has been implicated in cell growth. Androgenic stimulus in rat prostate modulates its association with nuclear matrix (NM) and chromatin. Because the growth of human prostate carcinoma cells is influenced by androgens and/or growth factors, we determined the nature of CK2 signaling in the NM in response to androgen and growth factor stimuli. Androgen-sensitive LNCaP and androgen-insensitive PC-3 cells were cultured in media to regulate their growth in the presence of 5alpha-dihydrotestosterone (5alpha-DHT) or growth factors (epidermal growth factor, keratinocyte growth factor, and transforming growth factor alpha). The activity of CK2 was measured in the cytosolic and NM fractions isolated from these cells after treatment with growth stimuli. The changes in CK2 in various fractions were also confirmed by immunoblotting with a specific antibody. LNCaP cells responded to both 5alpha-DHT and growth factors for growth. The presence of these agents in the culture medium evoked a translocation of CK2 to the NM from the cytosol. The PC-3 cells did not respond to 5alpha-DHT for growth but did respond to growth factors. Under these conditions, there was also a translocation of CK2 to the NM concomitant with a decrease in the cytosolic fraction. These results suggest that CK2 translocation to the NM occurs in response to various growth stimuli in cells in culture. Thus, CK2 is a common downstream signal transducer in response to diverse growth stimuli that may relate to the pathobiology of prostate cancer cells.

Topics: Adenocarcinoma; Animals; Casein Kinase II; Cell Division; Chromatin; Cytosol; Dihydrotestosterone; DNA-Binding Proteins; Epidermal Growth Factor; Fibroblast Growth Factor 10; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Growth Substances; Humans; Kinetics; Male; Nuclear Matrix; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Rats; Signal Transduction; Transforming Growth Factor alpha; Tumor Cells, Cultured

Salicylates inhibit signaling by tumor necrosis factor (TNF), including TNF-induced activation of mitogen-activated protein kinases (MAPKs). On the other hand, we recently showed that in normal human diploid fibroblasts sodium salicylate (NaSal) elicits activation of p38 MAPK but not activation of c-Jun N-terminal kinase (JNK). Here we show that NaSal treatment of COS-1 or HT-29 cells produced a sustained c-Jun N-terminal kinase (JNK) activation. Activation of JNK or p38 MAPK by NaSal (or aspirin) was not due to a nonspecific hyperosmotic effect because much higher molar concentrations of sorbitol or NaCl were required to produce a similar activation. Three structurally unrelated nonsteroidal antiinflammatory drugs (ibuprofen, acetaminophen, and indomethacin) failed to induce significant activation of JNK or p38 MAPK, suggesting that cyclooxygenase inhibition is not the underlying mechanism whereby salicylates induce p38 MAPK and JNK activation. Activation of JNK and p38 MAPKs may be relevant for some antiinflammatory actions of salicylates.

Topics: Acetaminophen; Adenocarcinoma; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Calcium-Calmodulin-Dependent Protein Kinases; Chlorocebus aethiops; Colonic Neoplasms; COS Cells; Cyclooxygenase Inhibitors; Enzyme Activation; Epidermal Growth Factor; Fibroblasts; Humans; Hypertonic Solutions; Ibuprofen; Indomethacin; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; NF-kappa B; Organ Specificity; Osmotic Pressure; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-jun; Recombinant Fusion Proteins; Saline Solution, Hypertonic; Signal Transduction; Sodium Salicylate; Sorbitol; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The importance of epidermal growth factor (EGF) in both normal and malignant mammary gland development are presented in these studies. Initial findings demonstrated that in the absence of ovarian hormones, EGF had a significant proliferative effect on mammary epithelial cells. To determine whether mammary epithelial cells grown with EGF, in the absence of ovarian hormones, could be transformed by N-methyl-N-nitrosourea (MNU), female ovariectomized Lewis rats were implanted with pellets containing EGF for 1 week and then treated with MNU for initiation. Two days after MNU treatment, ovaries were implanted and EGF pellets were removed from all ovariectomized groups in order to promote carcinogenesis. The mammary carcinoma incidence of the EGF-stimulated group (90%) was not significantly different from the intact group (100%). The mammary cancer morphology of EGF-treated carcinomas was either ductal carcinoma or cribriform adenocarcinoma, whereas intact animals developed mainly papillary and occasional cribriform carcinomas. Fifty-eight percent of the carcinomas from the EGF group were ovarian hormone-independent compared with 10% of carcinomas from the intact group. These results demonstrate that EGF-induced proliferation during initiation with MNU was sufficient to induce the transformation of mammary carcinomas in the absence of ovarian hormones. The hormonal dependency of these EGF-induced carcinomas were different compared with MNU-initiated mammary carcinomas in intact rats.

Topics: Adenocarcinoma; Animals; Carcinoma, Ductal, Breast; Carcinoma, Papillary; Cell Division; DNA Mutational Analysis; Epidermal Growth Factor; Estradiol; Estrogens; Female; Genes, ras; Mammary Neoplasms, Experimental; Methylnitrosourea; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Ovariectomy; Ovary; Polymerase Chain Reaction; Progesterone; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Receptors, Estrogen; Receptors, Progesterone

Matrix metalloproteinases (MMPs) are regulated both positively and negatively at the transcriptional level by a variety of growth factors, oncogenes, and tumor promoters. Induction of the MMP, matrilysin, by epidermal growth factor (EGF) was investigated in a human prostate cancer cell line.. Secreted protein and messenger RNA were detected using Western and Northern methods, respectively. EGF receptor antibodies were used for neutralization of the EGF receptor to determine the role of the EGF growth factor family (EGF, transforming growth factor alpha (TGFalpha), or amphiregulin) in the basal induction of matrilysin.. EGF increased mRNA and secreted protein levels for the MMP matrilysin in LNCaP cells, in a concentration- and time-dependent manner. Transforming growth factor beta1 (TGFbeta1) had no inhibitory effect on the levels of mRNA or secreted protein induced by EGF in LNCaP cells. Decay of matrilysin mRNA after the addition of actinomycin D indicated that the half-life of matrilysin mRNA was not altered by EGF. Blocking with a neutralizing antibody to the EGF receptor did not alter the basal level of secreted matrilysin.. Exogenously added EGF increased matrilysin mRNA, perhaps at a transcriptional level. Growth factors, other than the members of the EGF family which act through the EGF receptor, may be involved in the regulation of the basal level of secreted matrilysin in LNCaP cells. Our data with LNCaP cells suggest that paracrine regulation of matrilysin expression in human prostate carcinoma cells could be via the EGF receptor.

Topics: Adenocarcinoma; Dactinomycin; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 7; Metalloendopeptidases; Prostatic Neoplasms; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

HMG-CoA reductase is the key enzyme for the biosynthesis of isoprenoid compounds essential for cell growth and differentiation. Its tyrosine kinase-dependent modulation has recently been suggested and described in the ErbB-2 overexpressing cell line SKBR-3 [Asslan et al. (1998) Biochem. J. 330, 241-246]. Epidermal growth factor (EGF) increased the HMG-CoA reductase activity, protein, and mRNA levels only in ErbB-2-expressing cells (SKBR-3 and MCF-7) but not in MDA-MB-468 cells that do not express ErbB-2 even though their EGF receptor was efficiently phosphorylated. Tyrphostin AG 879, a specific inhibitor of ErbB-2 tyrosine kinase activity, decreased HMG-CoA reductase activity only in cells that expressed ErbB-2. A functional EGF receptor appeared to be necessary since its inhibition by the specific tyrphostin AG 1478 abolished the EGF effects. Phosphatidylinositol 3-kinase (PI 3-kinase) might be a crucial enzyme in the signaling pathway since the specific inhibitor, LY 294002, was shown to inhibit HMG-CoA reductase activity and to completely abolish the stimulation by EGF in SKBR-3 cells.

Topics: Adenocarcinoma; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Chromones; Epidermal Growth Factor; ErbB Receptors; Female; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA-Reductases, NADP-dependent; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein-Tyrosine Kinases; Quinazolines; Receptor, ErbB-2; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

The invasive and metastatic transformation of cancers often results in death. However, the mechanisms that promote this transformation remain unclear. Two closely related receptors, the epidermal growth factor receptor (EGFR) and ErbB2, are overexpressed in a significant percentage of breast and prostate carcinomas, among others, with this up-regulated signaling correlating with tumor progression. Previous studies in our laboratory have demonstrated that an EGFR-phospholipase C (PLC)gamma-mediated motility-associated signaling pathway is rate-limiting for tumor cell invasion in vitro and in vivo in one model of prostate carcinoma. Therefore, we investigated whether this PLCgamma signaling pathway also was rate-limiting for invasion in other tumor cell lines and types and whether this EGFR activity is subsumed by the closely related ErbB2. We determined the effects of PLCgamma signal abrogation by pharmacological (U73122) and molecular (expression of the dominant-negative PLCz) means on the in vitro invasiveness of tumor cells. Inhibition of PLCgamma signaling concomitantly decreased invasiveness of de novo-occurring transgenic adenocarcinoma mouse prostate (TRAMP) lines and the human breast cancer cell lines MDA-468 and MDA-231; these lines present up-regulated EGFR signaling. Because the prostate and breast cancer lines usually present autocrine stimulatory loops involving EGFR, we also examined transgenic adenocarcinoma mouse prostate C1 and MDA-468 treated with the EGFR-specific kinase inhibitor PD153035 to determine whether invasiveness is dependent on EGFR signaling. PD153035 reduced invasiveness to levels similar to those seen with U73122, suggesting that the autocrine EGFR stimulatory loop is functioning to promote invasiveness. To determine whether this signaling pathway also promotes invasiveness of ErbB2-overexpressing tumors, we examined the human breast carcinoma line MDA-361; again, U73122 inhibition of PLCgamma decreased invasiveness. In all situations, the inhibition of PLCgamma signaling did not decrease mitogenic signaling. Thus, the motility-associated PLCgamma signaling pathway is a generalizable rate-limiting step for tumor cell progression.

Topics: Adenocarcinoma; Animals; Autocrine Communication; Breast Neoplasms; Cell Line; Diffusion Chambers, Culture; Epidermal Growth Factor; ErbB Receptors; Estrenes; Female; Humans; Immunoblotting; Isoenzymes; Male; Mice; Neoplasm Invasiveness; Phosphodiesterase Inhibitors; Phospholipase C gamma; Phosphorylation; Prostatic Neoplasms; Pyrrolidinones; Quinazolines; Receptor, ErbB-2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Type C Phospholipases

Immunoreaction to TGF-alpha was limited to the basal epithelial cells of focal areas in the normal prostates. In benign prostatic hyperplasia (BPH) the immunostained areas were more widespread and immunolabelling was observed in both basal and columnar (secretory) cells of the epithelium. Some cells in the connective tissue stroma were also stained. In prostatic adenocarcinoma, epithelial immunostaining was even more extensive and intense than in BPH, and some stromal cells were also stained. Epidermal growth factor (EGF) immunostaining was only present in some basal cells in normal prostates. In BPH, this immunoreaction was strong in the basal cells and even stronger in the secretory cells. In prostatic cancer, the intensity of epithelial cell immunoreactivity was intermediate between that of normal prostates and that of BPH specimens. EGF-receptor immunostaining was focal and located in the basal cells in normal prostates. In BPH, labelling was also localized in basal cells but extended to wider areas. Some stromal cells appeared weakly labelled. In the prostatic carcinoma, both basal and columnar cells appeared stained and the number of immunolabelled stromal cells was higher than in BPH. The results presented suggest that, in normal conditions, EGF and TGF-alpha act as autocrine growth factors for the basal cells of the prostatic epithelium. In BPH this action is maintained and, in addition, the columnar cells start to secrete both factors which are bound by the basal cell receptors, giving rise to a paracrine regulation which probably overstimulates basal cell proliferation. In prostatic carcinoma, besides these regulatory mechanisms, the acquisition of EGF-receptors by the secretory cells develops an autocrine regulation which might induce their proliferation.

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Animals; Antibody Specificity; Connective Tissue; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Humans; Immunoenzyme Techniques; Male; Mice; Middle Aged; Neoplasm Proteins; Organ Specificity; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Rabbits; Stromal Cells; Transforming Growth Factor alpha

Cyclooxygenase (COX)-2 mRNA and protein expression were found to be frequently elevated in human pancreatic adenocarcinomas and cell lines derived from such tumors. Immunohistochemistry demonstrated cytoplasmic COX-2 expression in 14 of 21 (67%) pancreatic carcinomas. The level of COX-2 mRNA was found to be elevated in carcinomas, relative to histologically normal pancreas from a healthy individual, as assessed by reverse transcription-PCR. COX-2 protein expression was detected by the Western blot assay in three of five pancreatic carcinoma cell lines (BxPC-3, Capan-1, and MDAPanc-3), whereas COX-1 protein was detected in two of the five cell lines (BxPC-3 and Capan-1). Increased levels of COX-2 mRNA were found in four of five cell lines, and only in PANC-1 cells was the low level of transcript comparable to that in the normal pancreas. The level of COX-2 mRNA was positively correlated with the differentiation status of the tumor of origin for each cell line, COX-2 protein expression was up-regulated by epidermal growth factor when the cells were grown in absence of serum. Finally, two nonsteroidal anti-inflammatory drugs, sulindac sulfide and NS398, produced a dose-dependent inhibition of cell proliferation in all pancreatic cell lines tested. No correlation was found between the level of COX-2 or COX-1 expression and the extent of growth inhibition. Treatment of BxPC-3 cells with sulindac sulfide and NS398 resulted in an induction of COX-2 expression. Our findings indicate that COX-2 up-regulation is a frequent event in pancreatic cancers and suggest that nonsteroidal anti-inflammatory drugs may be useful in the chemoprevention and therapy of pancreatic carcinoma.

Topics: Adenocarcinoma; Anti-Inflammatory Agents, Non-Steroidal; Cell Division; Cyclooxygenase 1; Cyclooxygenase 2; Epidermal Growth Factor; Humans; Isoenzymes; Membrane Proteins; Nitrobenzenes; Pancreatic Neoplasms; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Sulfonamides; Sulindac; Tumor Cells, Cultured

Tumorigenesis is related to the dysregulation of cell growth or cell death pathways. Hence, elucidation of the mechanisms involved in the modulation of pro- or anti-apoptotic proteins is important in furthering understanding of breast cancer aetiology and may aid in designing prevention and treatment strategies. In the present study, we examined the role of 17beta-oestradiol on the regulation of apoptosis in the breast cancer cell line MCF-7. Using multi-probe RNAase protection assays, we found changes in the mRNA levels of several Bcl-2 family proteins upon treatment of MCF-7 cells with 17beta-oestradiol. Unexpectedly, we found a paradoxical effects of 17beta-oestradiol on two anti-apoptotic proteins Bcl-2 and Bcl-x. Treatment with 17beta-oestradiol resulted in up-regulation of Bcl-2 mRNA and protein, but down-regulated Bcl-x(L) mRNA and protein. The effect of 17beta-oestradiol on Bcl-x(L) occurred at concentration-dependent fashion. The effect was specific to 17beta-oestradiol since other steroid hormones exert no effect on Bcl-x(L). Tamoxifen, an anti-oestrogen, blocked the down-regulation of Bcl-x(L) by 17beta-oestradiol demonstrating this effect is oestrogen receptor-dependent. We speculate that different members of the Bcl-2 family proteins may be regulated through different pathway and these pathways may be modulated by 17beta-oestradiol.

Topics: Adenocarcinoma; Apoptosis; bcl-X Protein; Breast Neoplasms; Cell Count; Dihydrotestosterone; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Estrogens; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Insulin-Like Growth Factor I; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; RNA, Messenger; RNA, Neoplasm; Tamoxifen

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

Cigarette smoking has been shown to aggravate ulceration and delay ulcer healing. Smokers had a lower level of mucus in their stomachs. In the present study, we examined whether cigarette smoke or its extract reduced mucus production through the suppression of epidermal growth factor (EGF) associated with the reduction of polyamine biosynthesis both in vivo and in vitro. Ornithine decarboxylase (ODC) activities and mucus synthesis were determined in rat gastric mucosa and in human MKN-28 cells. Incubation of MKN-28 cells with EGF (0.01-1.00 ng/mL) significantly increased mucus synthesis in vitro, which was accompanied by an increase of ODC activity. Removal of salivary glands decreased the circulated EGF level and induced a significant reduction of mucus-secreting layer thickness in the gastric mucosa. Cigarette smoke or its extract markedly decreased mucus synthesis in vivo and in vitro, both of which could be completely reversed by intravenous administration of EGF (20 microg/kg) in rats or co-incubation with EGF (1 and 2 ng/mL) in MKN-28 cells. However, ODC activities, which were suppressed by cigarette smoke or its extract, were unaffected by intravenous administration of EGF in rats, or only partially reversed by co-incubation with EGF in MKN-28 cells. These findings indicate that both EGF and ODC activity represent two different entities in the modulation of cigarette smoking on gastric mucus synthesis. The action of EGF on mucus synthesis may only be partially if not dependent on ODC activity in the stomach.

Topics: Adenocarcinoma; Animals; Epidermal Growth Factor; Gastric Mucosa; Humans; Male; Mucus; Nicotiana; Ornithine Decarboxylase; Plants, Toxic; Rats; Rats, Sprague-Dawley; Salivary Glands; Smoke; Stomach Neoplasms; Tumor Cells, Cultured

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/-0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.

Topics: Actins; Adenocarcinoma; Animals; Cell Membrane Permeability; Epidermal Growth Factor; Fluorescent Dyes; Rats; Rhodamines; Time Factors; Tumor Cells, Cultured

We examined the effects of epidermal growth factor (EGF) on tumor progression of a weakly malignant rat mammary carcinoma cell line, ER-1. In vitro treatment with EGF enhanced tumorigenicity, metastatic capacity and in vitro invasive capacity of ER-1 cells. The increased malignancy of ER-1 cells was reversible, when the cells were pretreated with EGF for 24 h, whereas it was irreversible when pretreated with EGF for 1 month. EGF treatment elevated the intracellular peroxide level in ER-1 cells. When ER-1 cells were treated with EGF in the presence of N-acetylcysteine, a chemical antioxidant, the reversible or irreversible EGF-induced progression was inhibited. These results suggest that the reversible or irreversible tumor progression in ER-1 cells occur in accordance with the duration of exposure to EGF, and that reactive oxygen species may be involved in the progression.

Topics: Acetylcysteine; Adenocarcinoma; Animals; Disease Progression; DNA Damage; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Female; Flow Cytometry; Free Radical Scavengers; In Vitro Techniques; Mammary Neoplasms, Experimental; Microscopy, Confocal; Neoplasm Invasiveness; Oxidative Stress; Peroxides; Rats; Rats, Inbred SHR; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is the major rate-limiting enzyme in sterol and non-sterol isoprenoid synthesis. Isoprenoids are involved in the mechanisms of cell proliferation and transformation leading notably to crucial post-translational maturation of small G-proteins of the Ras superfamily. HMG-CoA reductase is among the most highly regulated enzymes. It is controlled by several feedback regulation mechanisms induced by sterol and non-sterol metabolites. The present results show that tyrosine kinase activity is also involved in the regulation of HMG-CoA reductase activity in the human breast cancer cell line SKBR-3. Incubation of SKBR-3 cells with the tyrosine kinase inhibitor, herbimycin A, induces a concentration-dependent reduction of HMG-CoA reductase activity with an IC50 of 80nM. The inhibition of HMG-CoA reductase activity by herbimycin A is also time-dependent. A similar effect of herbimycin A was obtained on the steady-state level of the HMG-CoA reductase protein. The effect of herbimycin A is probably specific as it abolished the stimulation of reductase activity by epidermal growth factor. To elucidate the molecular basis of the inhibition of HMG-CoA reductase activity and protein level by herbimycin A, we performed experiments to study the metabolic turnover of this enzyme using [35S]methionine and [35]cysteine. Herbimycin A (1 microM) did not have any significant effect on the rate of HMG-CoA reductase protein degradation but did affect its rate of synthesis and mRNA levels. The decrease in protein synthesis rate correlates with the lower reductase protein level but is more pronounced than the decrease in mRNA levels. Taken together, the results reveal a novel pathway of regulation of HMG-CoA reductase expression and activity by cellular tyrosine kinase activities.

Topics: Adenocarcinoma; Benzoquinones; Breast Neoplasms; Down-Regulation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl CoA Reductases; Lactams, Macrocyclic; Protein-Tyrosine Kinases; Quinones; Rifabutin; RNA, Messenger

Epidermal growth factor (EGF) has a wide variety of biological activities in the protection of gastric mucosa against acute injury and the healing of gastric ulcer. However, the molecular basis of the EGF action remains unknown. EGF-induced activation of ornithine decarboxylase (ODC) was examined, because ODC is a key enzyme for the cell proliferation.. The effects of epidermal growth factor (EGF) and indomethacin on ornithine decarboxylase (ODC) mRNA and enzyme activity were examined in gastric cancer derived KATO-III cells.. EGF induced both ODC mRNA expression and enzyme activation in a biphasic manner with the peaks at 0.5 and 3 h for mRNA and at 5 and 9 h for enzyme activity, respectively. Indomethacin pretreatment significantly reduced EGF-induced ODC activation and was completely abolished for the first 5 h.. Since it has been reported that both EGF and ODC are involved in cell migration and proliferation, two actions of EGF for gastric mucosal healing may be through, at least in part, this biphasic activation of ODC. Indomethacin suppresses and changes the response of ODC induced by EGF.

Topics: Adenocarcinoma; Anti-Inflammatory Agents, Non-Steroidal; Cell Division; Cell Movement; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gastric Mucosa; Gene Expression Regulation, Neoplastic; Humans; Indomethacin; Ornithine Decarboxylase; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms; Time Factors; Tumor Cells, Cultured

The cellular responses of a newly established and early-passage human prostate adenocarcinoma cell line, MDA PCa2a, to transforming growth factor (TGF) beta1, epidermal growth factor (EGF), and TGFalpha were characterized in terms of proliferation, production of prostate-specific antigen (PSA), fibronectin (FN) and laminin (LM). The responses of the MDA PCa2a cells were compared with those of the well-established human prostate carcinoma cell lines LNCap, PC3, and DU145. The MDA PCa2a cells were more responsive to the growth-inhibitory effect of TGFbeta1 than the established cell lines. The androgen-responsive cell lines (MDA PCa2a and LNCap) were relatively responsive to the growth-stimulatory effect of EGF and TGFalpha whereas the androgen-independent lines (PC3 and DU145) were not. Only the androgen-responsive cells produced PSA, which was further upregulated by treatment with growth factors. The androgen-independent cells did not produce PSA, and growth factors had no effect on PSA production. However, all cell lines produced abundant amounts of FN and LM, and the levels of production of these molecules were subject to modulation by growth factors. It is concluded that each growth factor elicits diverse and distinct responses in prostate carcinoma cells, which may reflect the involvement of diverse post-receptor signal pathways.

Topics: Adenocarcinoma; Cell Division; Epidermal Growth Factor; Fibronectins; Growth Substances; Humans; Laminin; Male; Prostate-Specific Antigen; Prostatic Neoplasms; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.

Topics: Adenocarcinoma; Amphiregulin; Cell Division; Cell Line; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Gastric Mucosa; Gastritis; Glycoproteins; Growth Substances; Helicobacter Infections; Helicobacter pylori; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Peptic Ulcer; Recombinant Proteins; RNA, Messenger; Stomach Neoplasms; Transforming Growth Factor alpha; Up-Regulation; Virulence

In previous papers we have demonstrated that sialoadenectomy inhibited MPA-induced mammary tumorigenesis in BALB/c mice. To further explore the role of EGF in this experimental model, we evaluated its effects on mammary glands of sialoadenectomized (sialox) MPA-treated female mice and on tumor growth. MPA-treated sialox mice were injected s.c. (n = 3) or not (n = 6) with 5 microg EGF every 36 h for 45 days; MPA-treated sham-operated mice were used as controls (n = 6). Mammary glands from sialox MPA-treated mice are considerably less developed as compared with sham-operated animals. The exogenous administration of EGF restores the usual MPA-induced growth pattern of the glands, thus confirming a role for EGF either in mediating or cooperating with MPA in inducing the mammary architectural changes observed in MPA-treated mice. On the other hand, primary cultures of progestin-dependent (PD) ductal mammary adenocarcinoma in vivo tumor lines and of lobular progestin-independent (PI) tumor lines were used to evaluate the effect of EGF on tumor growth. In vitro EGF was found to stimulate cell proliferation of lobular PI tumor cells and of fibroblastic cells from both types of tumors at concentrations higher than 0.1-0.5 ng/ml and in the presence of 1-5% of charcoal-stripped fetal calf serum. Conversely, no proliferative effects were observed in ductal PD cells under the same experimental conditions, regardless of the presence of 10 nM MPA. It can be concluded that although EGF plays an important role in MPA-induced mammary carcinogenesis, it is not necessary in PD tumor growth.

Topics: Adenocarcinoma; Animals; Carcinogens; Cell Division; Epidermal Growth Factor; Female; Hyperplasia; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Salivary Glands; Tumor Cells, Cultured

We investigated the ability of a variety of growth factors to regulate the differentiation of prostatic fibroblasts into smooth muscle cells.. Smooth muscle actin levels were monitored by immunoblot analysis and immunocytochemistry. Proliferation was measured in clonal growth assays and by cell counts.. We determined that TGFbeta inhibited proliferation and induced smooth muscle differentiation of stromal cells derived from prostatic adenocarcinomas, as we previously reported for cells derived from the normal peripheral zone. Basic FGF, EGF, TGFalpha, and PDGF, but not IGF, retinoic acid, 1,25-dihydroxyvitamin D3, or androgen, attenuated induction of differentiation by TGFbeta, by a mechanism apparently unrelated to proliferation.. Regulation of growth and differentiation occurs equivalently in prostatic stromal cells derived from adenocarcinomas and normal peripheral zone. TGFbeta is a potent inducer of the smooth muscle phenotype. Basic FGF, EGF and/or TGFalpha, and PDGF attenuate TGFbeta's activity, and promote a fibroblastic phenotype. Our studies provide an in vitro model system in which fibroblastic or smooth muscle cells can be promoted, maintained, and investigated in a defined manner. The results suggest that the ratio of fibroblasts to smooth muscle cells in the stroma reflects the relative levels of growth factors, which may be altered in diseased states.

Topics: Actins; Adenocarcinoma; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Immunohistochemistry; Male; Muscle, Smooth; Phenotype; Platelet-Derived Growth Factor; Prostatic Neoplasms; Transforming Growth Factor alpha

We investigated the expression of interleukin 1beta-converting enzyme (ICE; caspase-1) in human adenocarcinomas of the pancreas. Immunohistochemistry and Western blot analyses revealed an overexpression of ICE in 71 and 80% of tumor cells, respectively. Also, on a mRNA level, ICE mRNA was overexpressed in 45% of the cases, as compared to normal pancreatic tissue. Interestingly, the overexpression of ICE in tumor cells correlated significantly with the overexpression of cyclin D1, epidermal growth factor, and epidermal growth factor receptor (P < 0.0005, P < 0.05, and P < 0.002, respectively), which are involved in cell cycle progression and proliferation in human pancreatic carcinoma. This is the first report concerning ICE expression in human carcinomas; however, the exact mechanism underlying these close correlations warrant further research.

Topics: Adenocarcinoma; Adult; Aged; Blotting, Western; Caspase 1; Cyclin D1; Cysteine Endopeptidases; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Pancreas; Pancreatic Neoplasms; RNA, Messenger

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a secreted protein which may play a pivotal role in tumor-associated microvascular angiogenesis and hyperpermeability. The expression of mRNA for VEGF was examined in eight gastric carcinoma cell lines and 30 gastric carcinoma tissues as well as corresponding normal mucosa. All the cell lines expressed VEGF mRNA at various levels that correlated well with the amounts of VEGF secreted into the condition medium. The expression of VEGF mRNA by TMK-1 cells was increased by the treatment of epidermal growth factor (EGF) or interleukin-1alpha (IL-1alpha), whereas it was decreased by the treatment of interferon-beta (IFN-beta). In gastric carcinoma tissues, the level of VEGF mRNA in primary tumors was higher than that in the corresponding normal mucosas in six (46%) of 13 well-differentiated adenocarcinomas and in two (12%) of 17 poorly differentiated adenocarcinomas, respectively. Vessel counts in well-differentiated adenocarcinomas had a tendency to be higher than those in poorly differentiated adenocarcinomas. In well-differentiated adenocarcinomas, the levels of VEGF mRNA expression tended to be higher in carcinomas of advanced stage than in early stage carcinomas. Both in situ mRNA hybridization and immunohistochemistry demonstrated the presence of VEGF expression within the tumor cells. These results suggest that VEGF may confer angiogenesis and progression of human gastric carcinomas, especially of the well-differentiated type.

Topics: Adenocarcinoma; Adult; Aged; Blood Vessels; Blotting, Northern; DNA Probes; DNA, Neoplasm; Endothelial Growth Factors; Epidermal Growth Factor; Female; Humans; In Situ Hybridization; Interferon-beta; Interleukin-1; Lymphokines; Male; Middle Aged; RNA; Stomach Neoplasms; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The role of estrogen and estrogen-related growth factors in the mechanism of hormone dependency of endometrial adenocarcinoma cells was investigated. The proliferation of hormone-responsive human endometrial adenocarcinoma cells (Ishikawa cells), which possess both estrogen and progesterone receptors, was optimally stimulated by 10 nM estradiol. Both transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), added to the culture media, stimulated the proliferation of Ishikawa cells in a dose-dependent manner. Anti-TGF-alpha antibody completely eliminated the stimulatory effects of TGF-alpha. Anti-EGF receptor antibody inhibited the proliferation of these cells. The production of TGF-alpha into culture media was 5-40 pg/10 cells/24 h in 9 human endometrial adenocarcinoma cells. Ten nanomoles of estradiol increased the TGF-alpha production of Ishikawa cells by approximately 2.5-fold of the control level. In contrast, the production of TGF-alpha in hormone-unresponsive HEC-50 cels was not influenced by estradiol. C-erbB-2 oncoprotein expression of human endometrial adenocarcinoma cells, detected by both immunocytochemical staining and Western blot analysis, was associated with the tumor grade of the original tumor tissues. Ten nanomoles of estradiol clearly increased the c-erbB-2 oncoprotein levels at an optimal incubation period of 72 h, whereas estradiol did not affect the expression in HEC-50 cells.

Topics: Adenocarcinoma; Blotting, Western; Dose-Response Relationship, Drug; Endometrial Neoplasms; Epidermal Growth Factor; Estrogens; Female; Humans; Immunohistochemistry; Neoplasms, Hormone-Dependent; Receptor, ErbB-2; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

The biologic significance of epidermal growth factor (EGF)-related proteins and EGF receptor (EGFR) in the development and progression of human ovarian carcinoma was studied in 7 ovarian cystadenomas, 6 mucinous tumors of low malignant potential (LMP), and 25 invasive adenocarcinomas by immunohistochemistry. Results were correlated with clinicopathologic features. We also examined immunoreactivity in five serous adenocarcinomas both before and after cisplatin chemotherapy. Amphiregulin (AR) expression was observed only in mucinous tumors (4 of 8 cystadenomas, 2 of 6 tumors of LMP, and 6 of 10 cystadenocarcinomas), but was not detected in the serous tumors or clear cell adenocarcinomas. EGF, cripto, and EGFR expression was significantly higher in mucinous cystadenocarcinomas than in mucinous cystadenomas or mucinous tumors of LMP. Three of five specimens obtained at a second operation after chemotherapy had more intense or diffuse immunostaining for transforming growth factor alpha (TGF-alpha) than the initial specimens did. Coexpression of more than two of the EGF-related proteins or EGFR significantly correlated with increased surgical stage in serous and clear cell carcinoma. AR expression seems to correlate with mucinous differentiation rather than with advanced stages of ovarian tumors. Our results indicate that expression of some EGF-related proteins is greater in certain subtypes of ovarian carcinomas than in their benign counterparts and that coexpression of these proteins is associated with advanced stage in serous and clear cell carcinoma. Increased TGF-alpha expression may also be related to ovarian tumor resistance to cisplatin chemotherapy.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Biomarkers, Tumor; Cystadenoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Middle Aged; Ovarian Neoplasms; Transforming Growth Factor alpha

Growth of the EGF receptor-expressing non-small-cell lung carcinoma cell line H125 seems to be at least partially driven by autocrine activation of the resident EGF receptors. Thus, the possibility of an EGF receptor-directed antiproliferative treatment was investigated in vitro using a monoclonal antibody (alpha EGFR ior egf/r3) against the human EGF receptor and gangliosides which are known to possess antiproliferative and anti-tyrosine kinase activity. The moderate growth-inhibitory effect of alpha EGFR ior egf/r3 was strongly potentiated by the addition of monosialoganglioside GM3. Likewise, the combination of alpha EGFR ior egf/r3 and GM3 inhibited EGF receptor autophosphorylation activity in H125 cells more strongly than either agent alone. A synergistic inhibition of EGF receptor autophosphorylation by alpha EGFR ior egf/r3 and GM3 was also observed in the human epidermoid carcinoma cell line A431. In both cell lines, the inhibition of EGF receptor autophosphorylation by GM3 was prevented by pretreatment of the cells with pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases). Also, GM3 accelerated EGF receptor dephosphorylation in isolated A431 cell membranes. These findings indicate that GM3 has the capacity to activate EGF receptor-directed PTPase activity and suggest a novel possible mechanism for the regulation of cellular PTPases.

Topics: Adenocarcinoma; Benzylidene Compounds; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Membrane; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; G(M3) Ganglioside; Humans; Lung Neoplasms; Nitriles; Phosphorylation; Protein Tyrosine Phosphatases; Quinazolines; Radioligand Assay; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

This report describes the isolation and characterization of a new human breast cancer cell line, SUM-102PT, obtained from a minimally invasive human breast carcinoma. SUM-102PT cells have a near diploid karyotype, and early-passage cells had minor chromosomal abnormalities including a 5, 12 and a 6, 16 reciprocal translocation. The cells were isolated and have been continually cultured in three defined media, one of which contains exogenous epidermal growth factor (EGF). SUM-102PT cells have also been carried in an EGF-free medium supplemented with progesterone. All SUM-102PT cells require EGF receptor (EGFR) activation for continuous growth, because incubation of the cells with EGFR-neutralizing antibodies or with EGFR kinase inhibitors blocks growth of these cells. Southern analysis indicates that the EGFR gene is not amplified in these cells; however, these cells express high levels of EGFR mRNA. Thus, SUM-102PT is representative of a class of human breast cancers characterized by high level EGFR expression in the absence of gene amplification. SUM-102PT cells cultured in EGF-free, progesterone-containing medium express high levels of constitutively active EGFR. Conditioned medium from SUM-102PT cells contains an EGF-like mitogen that binds to a heparin-agarose affinity matrix with high affinity. Northern analysis for various EGF family members indicates that SUM-102PT cells synthesize heparin binding (HB)-EGF mRNA. HB-EGF protein is detectable on the surface of these cells by immunohistochemistry, and SUM-102PT cells are killed by diphtheria toxin, which acts by binding to HB-EGF. Furthermore, HB-EGF antibodies partially neutralize the mitogenic activity of the conditioned medium. Thus, EGFR activation in SUM-102PT cells is mediated, at least in part, by autocrine/juxtacrine stimulation by HB-EGF. SUM-102PT cells also express constitutively active STAT-3 homodimers. Constitutively tyrosine-phosphorylated STAT-3 homodimers were also detected in another breast cancer cell line, MDA468, which has an EGFR amplification and also has constitutive EGFR activity. Thus, SUM-102PT is a new human breast cancer cell line that expresses activated EGFR as a result of an autocrine/juxtacrine interaction with HB-EGF which, in turn, results in activation of STAT-3.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Division; DNA-Binding Proteins; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Middle Aged; Phosphotyrosine; Receptor Protein-Tyrosine Kinases; Receptors, Estrogen; RNA, Neoplasm; Signal Transduction; STAT3 Transcription Factor; Trans-Activators; Tumor Cells, Cultured

Amplification of the c-erbB2 gene and overexpression of p185erbB2 is found in approximately one-third of primary breast and ovarian cancers and also in some colon carcinomas. Moreover, a single point mutation in erbB2(V 664 E) confers transforming potential to erbB2 in NIH3T3 cells, even when expressed at low levels. To examine the transformation potential of erbB2 or erbB2(V-E) in colon epithelial cells, we have transfected a nontumorigenic clone of SW 613-S cells with either wild-type p185erbB2 or mutated p185erbB2(V-E). In contrast to p185erbB2, p185erbB2(V-E) associated constitutively with members of the Shc protein family, leading to phosphorylation of Shc and to stimulation of mitogen-activated protein kinase (MAP kinase). However, constitutive activation of MAP kinase activation in p185erbB2(V-E) expressing cells did not result in a tumorigenic phenotype. In addition, p185erbB2(V-E) expressing cells displayed a reduced ability to grow in soft agar compared to the parental cell line. In contrast these transfected cells were able to grow in three-dimensional collagen gels, whereas parental cells were not. Thus, expression of erbB2(V-E) in SW 613-S cells induced multiple changes in intracellular signaling and in growth requirement phenotype, particularly in response to the extracellular environment.

Topics: Adaptor Proteins, Signal Transducing; Adaptor Proteins, Vesicular Transport; Adenocarcinoma; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Collagen; Colonic Neoplasms; Enzyme Activation; Enzyme Induction; Epidermal Growth Factor; Epithelial Cells; Epithelium; Female; Gels; Humans; Intestinal Mucosa; Mice; Neoplasm Proteins; Neoplasm Transplantation; Oncogenes; Phosphorylation; Point Mutation; Protein Processing, Post-Translational; Proteins; Receptor, ErbB-2; Recombinant Fusion Proteins; Shc Signaling Adaptor Proteins; Src Homology 2 Domain-Containing, Transforming Protein 1; Transfection; Tumor Cells, Cultured

Growth factor regulation of normal and cancerous cell proliferation has been well-documented and may be mediated by proto-oncogene activity. The purpose of this study was to assess changes in proliferation and mitogen-induced c-fos mRNA expression of an endometrial carcinoma cell line, HEC-1-A, in response to TGF-beta, a potent growth-inhibitory peptide. HEC-1-A cells were incubated in the presence or absence of TGF-beta. Mitogen-stimulated cells were additionally treated with epidermal growth factor (EGF). Changes in proliferation were measured by [3H]thymidine uptake assays. Alterations in EGF-induced c-fos expression following TGF-beta pretreatment were assessed by Northern blot using a 32P-labeled human c-fos probe. Finally, chloramphenicol acetyltransferase assays were performed to evaluate c-fos promoter activity in response to treatment conditions. Basal and EGF-stimulated proliferation was inhibited by TGF-beta in a dose- and time-dependent manner. TGF-beta also reversibly decreased EGF-induced c-fos mRNA expression in a dose- and time-dependent manner. Sequences in the c-fos promoter that were stimulated by EGF showed suppressed activity when preincubated with TGF-beta. These results show that TGF-beta negatively modulates EGF-induced c-fos expression, which may be related to the observed inhibition of carcinoma cell proliferation.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; DNA, Neoplasm; Dose-Response Relationship, Drug; Drug Interactions; Endometrial Neoplasms; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genes, fos; Humans; Promoter Regions, Genetic; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; RNA, Messenger; Time Factors; Transfection; Transforming Growth Factor beta; Tritium; Tumor Cells, Cultured

Urokinase-type plasminogen activator (uPA) initiates a proteolytic cascade with which invasive cells eliminate barriers to movement. The signaling pathways regulating uPA production in tumor cells remain unclear. We first studied the effects of n-butanol, a phospholipase D (PLD) and protein kinase C (PKC) inhibitor, on the production of uPA in murine mammary adenocarcinoma cells. Tumor cell monolayers treated during 24 h with 0.3% v/v n-butanol, secreted 45-50% less uPA to the culture medium than control monolayers (P < 0.001) as determined by radial caseinolysis, zymography and western blot. This inhibition occurred also with 5-h treatments and remained up to 5 h after the removal of the alcohol. Treatment with the phorbol ester PMA or with EGF, strongly increased uPA production (P < 0.001). Interestingly, a mild inhibition of uPA production was observed when PMA stimulation was assayed in cotreatments with n-butanol. In contrast EGF was unable to reverse the inhibition induced by n-butanol. H7 significantly inhibited uPA activity (P < 0.001) secreted to the culture media. Furthermore, phosphatidic acid significantly stimulated uPA production meanwhile propranolol, which blocks phosphatidic acid availability, reduced it, suggesting a main regulatory role for this intermediary metabolite. These results suggest for the first time that uPA production is regulated by PLD and PKC signal transduction pathways in murine mammary adenocarcinoma cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 1-Butanol; Adenocarcinoma; Animals; Butanols; Epidermal Growth Factor; Female; Mammary Neoplasms, Experimental; Mice; Phosphatidic Acids; Phospholipase D; Protein Kinase C; Signal Transduction; Temperature; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured; Up-Regulation; Urokinase-Type Plasminogen Activator

The factors regulating human endometrial receptivity remain poorly understood. The alpha v beta 3 integrin cell adhesion molecule appears to be regulated in the human endometrium, appearing on postovulatory days 5-6, corresponding to the time of initial embryo attachment. This integrin has been extensively studied as a potential marker of endometrial receptivity and is aberrantly expressed in the endometrial epithelium of some infertile women. Ishikawa cells are a well differentiated endometrial adenocarcinoma cell line that maintain functional estrogen and progesterone receptors and are a useful model to study steroid-mediated events in human endometrial epithelium. This cell line expresses most of the normal endometrial epithelial integrins, including the alpha v beta 3 vitronectin receptor. The regulation of this integrin was studied with fluorescence immunocytochemistry, flow cytometry, and Northern blot analysis. Estrogen with or without progesterone treatment down-regulates alpha v beta 3 in this cell line. Several growth factors, including epidermal growth factor and the closely related transforming growth factor-alpha significantly increase the expression of this integrin. We conclude that endometrial differentiation is influenced by both steroid hormones and growth factors. The alpha v beta 3 integrin appears to be an excellent marker to study the molecular events leading to the establishment of uterine receptivity and successful implantation.

Topics: Adenocarcinoma; Biomarkers; Blotting, Northern; Cytokines; Dose-Response Relationship, Drug; Endometrial Neoplasms; Epidermal Growth Factor; Estrogens; Female; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Progesterone; Receptors, Vitronectin; Tumor Cells, Cultured

Epidermal growth factor (EGF) is a potent morphogen affecting cell shape and motility through regulation of adhesive interactions. We have characterized the morphological effects of EGF on GP2d and GP5d colon carcinoma cell lines and have compared the ability of the heparin-binding EGF receptor ligand amphiregulin (AR) to elicit the same effects. EGF induced a marked epithelial-mesenchymal transition in both cell lines. This effect was evident at 7 pM EGF and was associated with a reduction in cellular adherens junctions and diminished cell-cell contact; it was also associated with an increase in expression of alpha2-integrin as well as enhanced adhesion to the substratum and cell spreading. These changes in adhesion molecule expression were accompanied by enhanced migration on collagen. Blockade of cell growth with mitomycin C did not prevent the EGF-induced morphological change, showing that the mitogenic and morphogenic responses of the GP cells were separable. The phosphatidyl inositol (PI) 3-kinase inhibitor wortmannin inhibited basal proliferation but had no effect on the EGF-induced morphological change, further suggesting that the PI 3-kinase pathway was not involved in the morphogenic response of these cells. Amphiregulin stimulated proliferation of both cell lines, but could only elicit a modest morphological change if used at considerably higher doses or if growth was blocked with mitomycin C. In cells treated with 55 nM AR, alpha2-integrin expression was slightly increased; however, unlike the EGF case, adherens junctions remained intact. These differences in the ability of EGF and amphiregulin to affect cellular adhesion and migration may be significant factors influencing normal and tumor cell behavior.

Topics: Adenocarcinoma; Amphiregulin; Androstadienes; Antigens, CD; Cadherins; Cell Adhesion; Cell Adhesion Molecules; Cell Division; Cell Movement; Colonic Neoplasms; Enzyme Inhibitors; Epidermal Growth Factor; Epithelium; ErbB Receptors; Glycoproteins; Growth Substances; Integrin alpha2; Intercellular Junctions; Intercellular Signaling Peptides and Proteins; Ligands; Mesoderm; Mitomycin; Nucleic Acid Synthesis Inhibitors; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Wortmannin

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

Topics: Adenocarcinoma; Animals; Cell Division; Colonic Neoplasms; Cytokines; DNA Primers; Ephrin-A1; Epidermal Growth Factor; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Growth Substances; Humans; Interferon-gamma; Interleukin-1; Interleukins; Intestinal Mucosa; Membrane Proteins; Polymerase Chain Reaction; Protein Biosynthesis; Protein-Tyrosine Kinases; Receptor, EphA2; Recombinant Proteins; RNA, Messenger; Swine; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

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

A software was designed to simulate the calcium signal following hormone or growth factor stimulation in epithelial cells. The software written in C runs on a PC under Windows environment. It is based on a Markov process where the dynamic of the system is characterised by phenomenological transition probabilities. Moreover a minimal model is proposed to analyse the role of plasma channels and IP3 receptors, together with the opposite action of the CaATPase pumps, in the cytosolic and endoplasmic reticulum (ER) calcium signal control. The simulation is applied on the calcium response following stimulation by carbacol (protein G coupled receptors) or epidermal growth factor (tyrosine kinase type receptors) in A431 epithelial cells. The experimental calcium signals can be grouped in three classes; a spike and a return to the basal level (signal A), a spike and a decrease to a plateau level (signal B) or a slow increase to a plateau (signal C). Epidermal growth factor induces signal A and B while carbacol gives signal B and C. When a 'pseudo' steady state is reached oscillations occur. Computer simulations show that signal A can result from the activation of IP3 receptors while signal C would result from the activation of the plasma channels; signal B appears as the additive contribution of both channels, while oscillations are compatible with a calcium induced calcium release mechanism. Simulations suggest that the calcium dynamic in the ER is a mirror of cytosolic calcium but that a simple way to produce similar calcium elevation in these two compartments is to activate plasma channels. Implications of such a mechanism is discussed.

Topics: Adenocarcinoma; Algorithms; Calcium; Calcium Channels; Calcium-Transporting ATPases; Carbachol; Cell Membrane; Computer Simulation; Cytosol; Endoplasmic Reticulum; Epidermal Growth Factor; Epithelial Cells; GTP-Binding Proteins; Humans; Image Processing, Computer-Assisted; Inositol Phosphates; Ion Pumps; Markov Chains; Models, Biological; Models, Chemical; Protein-Tyrosine Kinases; Receptor Protein-Tyrosine Kinases; Receptors, Cell Surface; Signal Transduction; Software; Stochastic Processes; Tumor Cells, Cultured

We have studied the involvement of growth factors (GF), their receptors (GF-R) and oncogenes in modulating tumor growth in the medroxyprogesterone acetate (MPA)-induced mammary tumor model in BALB/c mice. We demonstrated the presence of both ligands of the insulin-like growth factor family (IGF-I, IGF-II) and the two types of receptors (IGF-RI, IGF-RII). MPA upregulated IGF-II mRNA and protein levels in hormone-dependent lines (MPA-D). The progression to a hormone-independent phenotype was accompanied by a high constitutive expression of IGF-II and by a significant decrease in IGF-IIR number. An antisense strategy used to evaluate the role of IGF in the MPA-induced growth of epithelial MPA-D cells showed that IGF mediate progestin-induced mammary tumor growth by autocrine/intracrine pathways. We also studied the role of heregulins (HRG), the recently identified ligands for the c-erbB3 and c-erbB4 oncogenes. HRG mRNA expression was restricted to tumors of ductal origin. MPA induced an in vivo up-regulation of HRG expression. Finally, we also found that MPA may be exerting its proliferative effect on MPA-D lines by inhibiting the expression of transforming growth factor beta 1, (TGF-beta 1) and the lack of expression of TGF-beta 1 in hormone-independent tumors may be related to the acquisition of autonomous growth.

Topics: Adenocarcinoma; Animals; Cell Transformation, Neoplastic; Epidermal Growth Factor; Female; Growth Substances; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Mammary Neoplasms, Experimental; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Oncogenes; Receptors, Growth Factor; Transforming Growth Factor beta

Changes in lamellipod extension and chemotaxis in response to EGF were analysed for MTLn3 cells (a metastatic cell line derived from the 13762NF rat mammary adenocarcinoma). Addition of EGF produced a cessation of ruffling followed by extension of hyaline lamellipods containing increased amounts of F-actin at the growing edge. A non-metastatic cell line (MTC) derived from the same tumor did not show such responses. Lamellipod extension was maximal within 5 min, followed by retraction and resumption of ruffling. Maximal area increases due to lamellipod extension occurred at about 5 nM EGF. Chemotactic and chemokinetic responses, measured using a microchemotaxis chamber, were also greatest at 5 nM. Cytochalasin D inhibited EGF-stimulated responses including lamellipod extension, increases in F-actin in lamellipods, and chemotaxis. Nocodazole affected chemotaxis at higher concentrations but not EGF-induced lamellipod extension. We conclude that polymerization of F-actin at the leading edges of lamellipods is necessary for extension of lamellipods and chemotaxis of MTLn3 cells in response to EGF. The motility and chemotaxis responses of this metastatic cell line have strong similarities to those seen in well-characterized chemotactic cells such as Dictyostelium and neutrophils.

Topics: Actins; Adenocarcinoma; Animals; Cell Movement; Chemotaxis; Cytochalasin D; Dose-Response Relationship, Drug; Epidermal Growth Factor; Mammary Neoplasms, Experimental; Microtubules; Nocodazole; Pseudopodia; Rats; Tumor Cells, Cultured

Overexpression of the erbB-2 gene contributes to aggressive behavior of various human adenocarcinomas, including breast cancer, through an unknown molecular mechanism. The erbB-2-encoded protein is a member of the ErbB family of growth factor receptors, but no direct ligand of ErbB-2 has been reported. We show that in various cells ErbB-2 can form heterodimers with both EGF receptor (ErbB-1) and NDF receptors (ErbB-3 and ErbB-4), suggesting that it may affect the action of heterologous ligands without the involvement of a direct ErbB-2 ligand. This possibility was addressed in breast cancer cells through either overexpression of ErbB-2 or by blocking its delivery to the cell surface by means of an endoplasmic reticulum-trapped antibody. We report that ErbB-2 overexpression enhanced binding affinities to both EGF and NDF, through deceleration of ligand dissociation rates. Likewise, removal of ErbB-2 from the cell surface almost completely abolished ligand binding by accelerating dissociation of both growth factors. The kinetic effects resulted in enhancement and prolongation of the stimulation of two major cytoplasmic signaling pathways, namely: MAP kinase (ERK) and c-Jun kinase (SAPK), by either ligand. Our results imply that ErbB-2 is a pan-ErbB subunit of the high affinity heterodimeric receptors for NDF and EGF. Therefore, the oncogenic action of ErbB-2 in human cancers may be due to its ability to potentiate in trans growth factor signaling.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; CHO Cells; Cricetinae; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Genes, erbB-2; Glycoproteins; Humans; Kinetics; Macromolecular Substances; Neuregulins; Proto-Oncogene Proteins; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Recombinant Proteins; Signal Transduction; Transfection

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of polypeptide growth factors, which includes EGF, transforming growth factor alpha(TGF-alpha), amphiregulin (AR) and betacellulin (BTC). To assess the potential role of HB-EGF in human gastric carcinomas, the expression of HB-EGF and EGF receptor (EGF-R) was examined in normal and cancerous gastric tissues and cultured gastric cancer cell lines. By Northern blot analysis, there was a 4.7-fold increase in HB-EGF mRNA levels in human gastric cancers compared with normal gastric tissues. There was a concomitant 3.9-fold increase in EGF-R mRNA levels in these cancers. Immunostaining revealed co-localization in 72% of the cancer cells of HB-EGF and EGF-R. AR and BTC moieties were not evident by Northern blot analysis. However, using PCR, both AR and BTC mRNA species were demonstrated in normal and cancerous gastric tissues. By Northern blot analysis, HB-EGF, TGF-alpha, AR, BTC and EGF-R mRNA moieties were co-expressed in KATO III and NCI-N87 gastric cancer cell lines. Furthermore, HB-EGF, EGF and TGF-alpha enhanced the growth of both cell lines in a dose-dependent manner. Our findings suggest that HB-EGF is relatively abundant in human gastric cancers and that co-expression of the EGF ligand family may lead to excessive activation of EGF-R in this disorder.

Topics: Adenocarcinoma; Adult; Aged; Base Sequence; Blotting, Northern; Blotting, Southern; Cell Line; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Heparin; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms; Transcription, Genetic; Tumor Cells, Cultured

Using immunohistochemistry, Northern blotting and a semi-quantitative PCR technique, epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) expression were studied in the pancreas of N-nitrosobis(2-oxopropyl)-amine (BOP)-treated hamsters. After initiation pancreatic carcinogenesis was modulated by a high fat diet or by injections with the cholecystokinin analogue caerulein. Autopsies were performed 6 and 12 months after the last injection with BOP. Immunohistochemistry revealed a weak expression of TGF-alpha in nomal acinar cells and a stronger expression in ductular and centro-acinar cells. Over-expression of TGF-alpha was observed in advanced putative preneoplastic lesions (classified as borderline lesions) and in ductular adenocarcinomas. EGFR immunoreactivity was present only in ductular adenocarcinomas. EGF peptide expression was observed both in acinar and ductular normal and tumorous cells and the level of expression did not change significantly during carcinogenesis. Moreover, the post-initiation treatments did not cause differences in EGF, TGF-alpha or EGFR peptide or mRNA levels, except for a significantly lower expression of TGF-alpha mRNA in hamsters fed a high fat diet when compared with those fed a low fat diet. TGF-alpha mRNA levels increased, whereas EGF mRNA levels decreased significantly in total pancreatic homogenates of BOP-treated hamsters in comparison with untreated controls. Also, in ductular adenocarcinomas TGF-alpha and EGFR (but not EGF) mRNA levels were significantly higher than in normal pancreatic homogenates. In pancreatic homogenates obtained 6 months after the last BOP injection, these differences were less pronounced in comparison with those obtained after 12 months. The present results indicate that TGF-alpha (but not EGF) might act in a paracrine or autocrine manner in pancreatic tumours in BOP-treated hamsters via simultaneously expressed EGFR. However, TGF-alpha, EGF and EGFR do not seem to be involved in the modulating effects of a high fat diet or caerulein treatment on pancreatic carcinogenesis in BOP-treated hamsters.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Body Weight; Carcinogens; Cricetinae; Epidermal Growth Factor; ErbB Receptors; Immunohistochemistry; Mesocricetus; Nitrosamines; Organ Size; Pancreas; Pancreatic Neoplasms; Polymerase Chain Reaction; Transforming Growth Factor alpha

After acute lung injury, altered bronchioloalveolar epithelia must be repaired quickly in order to restore lung function. During reepithelialization, type II cells initially appear to migrate and spread over a remodeled matrix; then a secondary proliferative phase occurs. It was hypothesized that 1) type II cells can develop locomotion in vitro that is modulated by growth factors, proinflammatory cytokines, and substrate adhesion molecules and 2) migration and proliferation of type II cells can occur as distinctive processes. Chemotaxis assays were elaborated using short term cultures of rat type II pneumocytes. Epidermal growth factor (EGF), transforming growth factor-alpha, laminin, fibronectin were found to be the main attractants for type II cells with respective increases of approximately 8.5-, 10.5-, 8-, and 7-fold in cell migration (P<0.05 vs. control). Laminin induced gradient-dependent and random cell migration. Addition of laminin with EGF had a synergistic effect in promoting cell migration (approximately 30-fold increase over control, P<0.05). Interferon-gamma and interleukin-6 inhibited EGF-induced type II cell migration, whereas tumor necrosis factor-alpha and interleukin-1beta acted as primers for type II cell migration (approximately 1.5-fold increase over control, P<0.05. Type II cells did not need to be in a proliferative phase in order to exhibit motility. New insights regarding the regulatory processes for type II cell migration are especially relevant in our understanding of early events occurring during epithelial repair after acute lung injury.

Topics: Adenocarcinoma; Analysis of Variance; Animals; Cells, Cultured; Chemotaxis; Collagen; Cytokines; Epidermal Growth Factor; Epithelial Cells; Epithelium; Extracellular Matrix Proteins; Fibronectins; Growth Substances; Humans; Interferon-gamma; Interleukin-6; Kinetics; Laminin; Lung Neoplasms; Macrophages, Alveolar; Platelet-Derived Growth Factor; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Tumor Cells, Cultured

Biological effects of sex steroids (estradiol-17beta, E2; progesterone, P; medroxyprogesterone acetate, MPA; Danazol, DZ) and growth factors (epidermal growth factor, EGF; transforming growth factor, TGF-alpha,beta) on migration and invasion of endometrial adenocarcinoma SNG-M cells were investigated by haptotactic migration and haptoinvasion assay. The enzymatic degradation of the extracellular matrix by tumor cells was also examined. Tumor cell migration along a gradient of substratum-bound fibronectin was inhibited by 0.1-10 microM MPA and DZ, but promoted by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. E2, P and TGF-beta did not have any effect on the motility of tumor cells. These effects were also confirmed by wound assay. The invasive activity of SNG-M cells into reconstituted basement membrane (Matrigel) was inhibited by the presence of 0.1-10 microM MPA and DZ, but promoted by 0.1-10 nM EGF and TGF-alpha in a concentration-dependent manner. E2, P and TGF-beta did not have any effect on tumor cell invasion. The zymography of tumor-conditioned medium showed that the treatment of SNG-M cells with EGF and TGF-alpha resulted in the increase of the 68, 72 and 92 kDa type IV collagenases (matrix metalloproteinase, MMP-2 and 9). Sex steroids and TGF-beta did not have significant effects on MMP-2 and 9. Stromelysin (MMP-3), also secreted by SNG-M cells, was not affected by sex steroids and growth factors. These results suggest that EGF and TGF-alpha act as positive regulators on the invasion process of endometrial adenocarcinoma cells, which may partly be associated with the induction of type IV collagenase secretion by tumor cells. The inhibitory effects of MPA and DZ on tumor cell invasion may depend at least partly on their inhibitory action on the motility of tumor cells.

Topics: Adenocarcinoma; Caseins; Cell Division; Cell Movement; Culture Media, Conditioned; Danazol; Endometrial Neoplasms; Epidermal Growth Factor; Estrogen Antagonists; Female; Gelatin; Humans; Neoplasm Invasiveness; Progestins; Transforming Growth Factor alpha; Tumor Cells, Cultured

Parathyroid hormone-related protein (PTHrP) has previously been shown to be expressed in human prostatic tissue and in prostatic cancer cell lines. In the present study, PTHrP immunoreactivity was detected in the glandular epithelium of normal prostate and benign prostatic hyperplasia (BPH), as well as in prostatic adenocarcinoma (CaP). Epithelial cell cultures derived from normal, BPH, and CaP tissues were also stained by antibodies against PTHrP, and northern analysis revealed multiple transcripts of PTHrP in the cellular RNA. PTHrP (1-34) was measurable by radioimmunoassay (RIA) in media conditioned by the prostatic epithelial cell cultures, and PTHrP accumulated in conditioned media during a 72 hr time course. Addition of complete growth medium to starved cells resulted in increased PTHrP mRNA levels by 1 hr, with maximal stimulation at 8-24 hr. Several individual factors contained in the complete growth medium were tested for their ability to regulate PTHrP expression. Epidermal growth factor (EGF) was the major inducer of PTHrP expression, while cholera toxin, bovine pituitary extract, hydrocortisone, and insulin had minimal or no effect on PTHrP transcript levels. Since each of these factors is growth stimulatory, the unique ability of EGF to induce PTHrP is apparently unrelated to mitogenicity. 1,25-Dihydroxyvitamin D3[1,25(OH)2D3], an inhibitor of PTHrP expression in several other cell types, had no effect on steady-state levels of PTHrP mRNA expressed by epithelial cells in complete growth medium, although prostate cells have vitamin D receptors and are responsive to 1,25(OH)2D3 in other ways. Our results indicate that PTHrP expression is not confined to the neuroendocrine cells of the human prostate and that our culture system can be used as a model to investigate the role of PTHrP in the prostate.

Topics: Adenocarcinoma; Calcitriol; Cells, Cultured; Culture Media; Epidermal Growth Factor; Epithelium; Gene Expression Regulation; Humans; Immunohistochemistry; Kinetics; Male; Parathyroid Hormone-Related Protein; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Proteins; RNA, Messenger

Epidermal growth factor (EGF) rapidly stimulates the release of arachidonic acid in A549 cells by a mechanism that is sensitive to pertussis toxin [1]. We show that EGF treatment of A549 cells stimulates phosphorylation of cytosolic phospholipase A2 (cPLA2) through a mechanism that is similarly inhibited by pertussis toxin. The level of cPLA2 expression is, apparently, not changed during this period. Pretreatment of cells with dexamethasone (10-100 nM) for 3 hr prevents this activation of cPLA2 by EFG, without changing the level of cPLA21 expression. The effect of dexamethasone is reversed in the presence of the neutralizing antilipocortin Mab 1A but not by the nonneutralizing antilipocortin 1 control Mab 1B. This strongly suggests that lipocortin 1 mediates the effect of dexamethasone by inhibiting activation of cPLA2. This concept is supported by the fact that a peptide Lc13-25 (10-200 micrograms/mL), derived from the N-terminus of lipocortin 1, also inhibits activation of cPLA2 by EGF in these cells.

Topics: Adenocarcinoma; Amino Acid Sequence; Annexin A1; Cell Line; Dexamethasone; Enzyme Activation; Epidermal Growth Factor; Glucocorticoids; GTP-Binding Proteins; Humans; Molecular Sequence Data; Peptide Fragments; Phospholipases A; Phospholipases A2

Somatostatin inhibits proliferation of many solid tumors. The current study examines whether inhibition of the growth of pancreatic cancer by the somatostatin analog, octreotide, requires tumor expression of somatostatin receptors.. We studied five human pancreatic cancer cell lines, Capan-1, Capan-2, CAV, MIA PaCa-2, and Panc-1. Solid tumors were established in nude mice (n = 20/cell line) by flank injection of tumor cells. Subcutaneous octreotide (500 micrograms/kg/day) was administered by osmotic pumps to 10 of the animals in each group, and the other 10 received control infusions of saline solution. On day 36, the tumors were excised and weighed. Plasma levels of the putative trophic peptides cholecystokinin, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin were assessed by radioimmunoassay. Each of the five cell lines was assayed for the presence of cell surface somatostatin receptors by using whole cell competitive binding assays with 125I-somatostatin. Expression of the somatostatin receptor subtype-2 (SSR2) gene was determined with reverse transcriptase-polymerase chain reactions. Southern blot hybridization was used to assess the presence of the SSR2 gene.. Octreotide inhibited tumor growth in the MIA PaCa-2 group (512 +/- 75 mg control versus 285 +/- 71 mg treated; p < 0.05) but had no significant effect on tumor weight in the other four cell lines. Plasma levels of cholecystokinin, epidermal growth factor, insulin-like growth factor-1, and insulin were not altered by chronic octreotide infusion. Cell surface somatostatin receptors and SSR2 gene expression were detected only in the MIA PaCa-2 tumors. The gene for the SSR2 receptor was found in all five tumor lines.. Octreotide-mediated inhibition of pancreatic cancer growth is dependent on expression of somatostatin receptors. The expression of somatostatin receptors should be considered in the design and interpretation of clinical trials with somatostatin analogs for treatment of pancreatic cancer.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Hormonal; Base Sequence; Binding, Competitive; Blotting, Southern; Cell Division; Cholecystokinin; DNA, Neoplasm; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Insulin; Insulin-Like Growth Factor I; Mice; Mice, Nude; Molecular Sequence Data; Octreotide; Pancreatic Neoplasms; Peptides; Polymerase Chain Reaction; Receptors, Somatostatin; Tumor Cells, Cultured

The epidermal growth factor (EGF) signal transduction pathway, frequently activated in pancreatic cancer, is an important regulator of cellular growth and transformation. This study examined whether activation of the cyclic adenosine monophosphate protein kinase A pathway may inhibit the EGF signal transduction pathway in pancreatic cancer cell lines.. Human pancreatic cancer lines BxPC-3 and AsPC-1 were stimulated with EGF, forskolin, or both. Forskolin is a compound that increases cyclic adenosine monophosphate levels. Assays of cell lines were then obtained for cellular growth (MTT assay), anchorage-independent growth (soft agar), and EGF-induced mitogen-activated protein kinase activation as measured by an in-gel kinase assay.. Treatment with forskolin resulted in inhibition of EGF-induced activation of mitogen-activated protein kinase activity (BxPC-3 78% inhibition and AsPC-1 70% inhibition, p < 0.005), diminished cellular proliferation (BxPC-3 92% inhibition and AsPC-1 86% inhibition, p < 0.001), and formation of colonies in soft agar (BxPC-3 98% inhibition and AsPC-1 76% inhibition, p < 0.001). Forskolin did not inhibit EGF receptor autophosphorylation or tyrosine kinase signaling in response to EGF.. Forskolin-induced inhibition of mitogen-activated protein kinase is associated with diminished pancreatic cancer cell proliferation in vitro. Use of strategies to increase cyclic adenosine monophosphate levels may have therapeutic application in pancreatic cancer.

Topics: Adenocarcinoma; Calcium-Calmodulin-Dependent Protein Kinases; Cell Culture Techniques; Cell Division; Cyclic AMP; Epidermal Growth Factor; ErbB Receptors; Humans; Pancreatic Neoplasms; Phosphorylation; Phosphotyrosine; Signal Transduction; Tumor Cells, Cultured

We examined the proliferative signal transduction by EGF in HSG-AZA 3, a subclone of HSG cell line. The treatment of cells with EGF resulted in an increase in [3H]thymidine incorporation into DNA depending upon EGF concentrations. In addition, the nuclear proto-oncogene c-fos was rapidly induced by EGF. Moreover, EGF induced transient expression of EGF receptor mRNA followed by the de novo synthesis of EGF receptor protein. On the other hand, treatment of the cells with EGF occurred phosphorylation by tyrosine kinase comprised in the EGF receptor, autophosphorylation, followed by activation of MAP kinase. These results indicate that the proliferative response to EGF is modulated by the phosphorylation cascade mediating EGF receptor-associated tyrosine kinase and MAP kinase, and transient activation of c-fos protein is implicated in the cell proliferation.

Topics: Adenocarcinoma; Base Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Sequence Data; Phosphorylation; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Receptor Protein-Tyrosine Kinases; RNA, Messenger; Salivary Gland Neoplasms; Signal Transduction; Tumor Cells, Cultured

Corticotropin-releasing hormone (CRH) is expressed in several peripheral tissues, including normal epithelial cells of the human and rodent uterus. However, the biological role of endometrial CRH is known in neither species. As a first step to clarify this role, we studied the regulation of CRH promoter in endometrial cells. We performed homologous transfection experiments in Ishikawa cells, a human endometrial cell line, using a 0.9-kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase. Transfected cells were exposed for 18 h to 8-bromo cyclic adenosine monophosphate, forskolin, epidermal growth factor, steroids (estradiol, progesterone, and the synthetic glucocorticoid dexamethasone and their antagonists), and prostaglandin E2; then the activity of the luciferase reporter was determined in the cell lysates. We found that the activity of the 5'-flanking region of the CRH gene was stimulated by cyclic adenosine monophosphate and epidermal growth factor and inhibited in a receptor-mediated, dose-dependent fashion by estradiol and dexamethasone. The antiglucocorticoid RU 486 acted as a glucocorticoid agonist, suppressing the CRH gene activation, while progesterone was devoid of any activity. Prostaglandin E2 stimulated the CRH activation, and the prostanoid inhibitor indomethacin suppressed it, most probably by inhibiting endogenous prostaglandins. These findings suggest that endometrial CRH gene expression may be under the negative control of estrogens and glucocorticoids and under the positive control of prostaglandin E2.

Topics: Adenocarcinoma; Corticotropin-Releasing Hormone; Cyclic AMP; Dexamethasone; Dinoprostone; Endometrial Neoplasms; Endometrium; Epidermal Growth Factor; Estradiol; Female; Gene Expression Regulation; Humans; Indomethacin; Luciferases; Mifepristone; Promoter Regions, Genetic; Recombinant Fusion Proteins; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Human colon tumor cell lines that were adapted to grow in chemically defined medium were tested for their growth sensitivity to exogenous transferrin, insulin, and epidermal growth factor (EGF). Less differentiated cell lines, C and HCT116, were able to grow in the presence of a single peptide supplement, to respond synergistically to transferrin and insulin in combination, and to be insensitive to supplementation with exogenous EGF. The more differentiated cell lines, CBS and GEO, were found to respond to exogenous EGF in a concentration-dependent manner, to require at least two peptide factor supplements to support growth, and to respond synergistically when EGF was added to chemically defined medium that already contained transferrin and insulin. To investigate whether changes in the number or the affinity-classes of the EGF-receptor might be involved in any of these growth responses, changes in EGF-receptor number and dissociation constant were determined as a function of cell growth condition. The poorly differentiated HCT cell line HCT116 was found to undergo 28 to 64% increases in [125I]EGF-binding when its medium was supplemented with transferrin, insulin, or transferrin plus insulin. The more differentiated CBS cell line responded to all peptide supplements with decreases in [125I]EGF-binding ranging from 12 to 73%. These findings indicate that the actions of transferrin and insulin are fundamental to the growth regulatory mechanisms of these two differentiation classes of human colon tumor cell lines, but that their mechanisms are different.

Topics: Adenocarcinoma; Cell Differentiation; Colonic Neoplasms; Culture Media, Serum-Free; Drug Synergism; Epidermal Growth Factor; Humans; Insulin; Transferrin; Tumor Cells, Cultured

Metastasis is a multistep process that involves alterations in a tumour cell's invasion, motility and adhesive capabilities. This study examined the effect of EGF on the in vitro invasion, motility and adhesion of the primary renal adenocarcinoma cell line, A704. Stimulation of the tumour cells by EGF (40 ng/ml) for a period of 24 h increased the in vitro invasion (P = 0.040) and motility (P = 0.039). Cell adhesion was examined on fibronectin, laminin, collagen IV and a 1:1:1 mix of the three extracellular matrix components. After EGF (40 ng/ml) stimulation, adhesion was significantly decreased on fibronectin (P = 0.022) and collagen type IV (P = 0.026), but increased on the 1:1:1 mix of extracellular matrix components (P = 0.022). The 92 kDa matrix metalloproteinase (MMP-9) present in the cell-conditioned medium was also increased after a 24 h stimulation with EGF (40 ng/ml) when measured. Hence, EGF can modulate the in vitro invasion, motility, adhesiveness and matrix metalloproteinase production in the A704 cell line, and subsequently may have a role in the metastatic potential of some renal carcinomas.

Topics: Adenocarcinoma; Cell Adhesion; Cell Movement; Collagenases; Epidermal Growth Factor; Humans; Kidney Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Tumor Cells, Cultured

The expression of cripto, a member of the epidermal growth factor (EGF) family, was examined by immunohistochemistry in benign lesions and carcinomas of the gall bladder. Cripto expression was detected in 6 (67 percent) of 9 hyperplasias, 4 (58 percent) of 7 adenomas, and 89 (68 percent) of 132 adenocarcinomas of the gall bladder. The degree of cripto expression was not correlated with depth of tumour invasion, tumour stage or patient prognosis. The incidence of cases with cripto expression was significantly higher in papillary and well-differentiated adenocarcinomas (positive 73 percent; strongly positive 38 percent) than in moderately and poorly differentiated adenocarcinomas (positive 54 percent; strongly positive 17 percent) (P < 0.05). These results suggest that cripto expression may not relate to progression in gall bladder carcinomas, but may be associated with tumour differentiation.

Topics: Adenocarcinoma; Adenoma; Epidermal Growth Factor; Gallbladder; Gallbladder Neoplasms; Gene Amplification; Gene Rearrangement; GPI-Linked Proteins; Humans; Hyperplasia; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins

The adenocarcinoma cell line derived from an intercalated ductal epithelium of a human salivary gland (HSG) proliferates autonomously mediated by an epidermal growth factor-(EGF)-like molecule with a molecular weight of 46 kDa and an EGF receptor (EGFR). The c-erbB2 protein, a member of EGFR family was also expressed in HSG cells and was involved in the growth signal pathway of HSG cells as well as EGFR. The autocrine growth is regulated by glucocorticoid and retinoic acid (RA) via their receptors. Retinoic acid receptor (RAR) of HSG cells revealed a transcriptional activity in vivo, and the heterodimerization between RAR and 9-cis retinoic acid receptor (RXR) is requisite for the binding with a specific DNA element termed RA response element in vitro. RXR alpha and RXR beta were cloned from HSG cells, and these RXRs, together with RAR, seemed to play a physiological role in RA signaling in vivo.

Topics: Adenocarcinoma; Bucladesine; Cell Division; Cell Transformation, Neoplastic; Epidermal Growth Factor; Humans; Receptors, Retinoic Acid; Salivary Gland Neoplasms; Signal Transduction; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

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

Overproduction of uPA by prostate cancer cells in vivo results in tumor invasiveness and osteoblastic skeletal metastasis due to its mitogenic actions in osteoblasts. In the present study we have examined the effect of several growth factors and steroid hormones on regulating uPA gene expression in the human prostate cancer cell line (PC-3). Treatment of these cells with dexamethasone (Dex) caused a decrease, whereas epidermal growth factor (EGF) and fetal bovine serum (FBS) increased uPA expression in a dose-dependent manner. Trans retinoic acid (RA) also induced uPA mRNA and protein production in a dose-dependent manner (10(-6) to 10(-9) M). This increase was seen as early as 2 hr of treatment until 48 hr. Dex treatment resulted in decreased tumor cells invasiveness, whereas exposure to EGF and RA caused an increase in the invasive capacity of PC-3 cells. These studies should help to better understand the control mechanism of uPA expression in prostate cancer, where uPA has been implicated as a major pathogenetic factor.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents, Hormonal; Cattle; Dexamethasone; Dose-Response Relationship, Drug; Enzyme Induction; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Invasiveness; Prostatic Neoplasms; RNA, Messenger; Serum Albumin, Bovine; Time Factors; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Human colon cancer cell lines express epidermal growth factor (EGF) mRNA, secrete EGF and may respond to it via the cell-surface EGF receptor (EGFR). Expression of these molecules in human colon and colon tumor, however, is not clear. Reverse transcription-polymerase chain reaction (RT-PCR) analyses of RNA prepared from paired normal human colon and colon tumor samples from 12 individuals followed by Southern blotting analyses of the RT-PCR products revealed a major fragment of 527 bp and a minor fragment of 404 bp that hybridized to a human EGF cDNA probe under stringent conditions. Identical results were obtained from 8 human colon cancer cell lines. Cloning and sequencing of PCR products confirmed that both fragments were from the human EGF gene; the 527-bp fragment corresponded exactly to nucleotides 2,891 to 3,417 of the human EGF mRNA reported by others. A deletion of 123 nucleotides (nucleotides 3,172 to 3,294) was found in the 404-bp fragment. Immunohistochemical studies using cyostat sections of human colon specimens showed that EGF was expressed in the human colon and that expression was restricted to the epithelial colonic crypt cells and epithelium-derived cancer cells. Since EGF and EGF-related molecules are potent mitogens that mediated their effect through the EGFR, we also determined the efficacy of anti-sense EGFR RNA in circumventing the EGFR-related pathway of proliferation. Expression of anti-sense EGFR RNA, by transfection with an inducible anti-sense EGFR expression vector, down-regulated cell-surface EGFR expression and proliferation of these cells and their ability to grow in soft agar. Anti-sense EGFR RNA was found to be an anti-proliferative agent in both relatively non-aggressive and highly aggressive human colon cancer cells.

Topics: Adenocarcinoma; Base Sequence; Blotting, Southern; Cell Division; Cloning, Molecular; Colon; Colonic Neoplasms; Down-Regulation; Epidermal Growth Factor; Epithelium; ErbB Receptors; Humans; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Antisense; RNA, Messenger; Sepharose; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The effects of hormones and synthetic analogues have been examined on the growth of 2 human pancreatic cancer cell lines, MiaPaCa2 a well-established cell line and PANI which was derived in our own laboratories from a tumour specimen. The hormones/growth factors included gastrin (G-17), epidermal growth factor (EGF) and bombesin, while the synthetic analogues used were a gastrin receptor antagonist (CR 1718), a somatostatin analogue (RC-160) and a bombesin receptor antagonist (ICI 216,140). Cell proliferation was assessed by the [75Se]selenomethionine uptake method which has been shown to correlate with cell counts. The effect of each hormone or growth factor on growth was expressed as a percentage of the untreated control. There were 5 replicates in each experiment, and each one was repeated at least 3 times. In vitro growth of both cell lines was unaffected by gastrin, bombesin or the respective antagonists (CR1718 and ICI 216140). The somatostatin analogue RC-160 also had no effect on basal growth. Significant growth stimulation of both MiaPaCa2 and PANI was seen with epidermal growth factor. We tested the hypothesis that somatostatin analogues may inhibit EGF-stimulated growth on both MiaPaCa2, a somatostatin receptor positive cell line, and on PANI which is negative for somatostatin receptors. RC-160 did not inhibit EGF-stimulated growth of either MiaPaCA2 or PANI. Both cell lines were established in vivo as xenografts in nude mice. The effect of RC-160 on tumour growth was measured. RC-160 inhibited the growth of MiaPaCa2, the somatostatin receptor-positive cell line, but not of PANI.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cell Division; Epidermal Growth Factor; Gastrointestinal Hormones; Humans; In Vitro Techniques; Mice; Mice, Nude; Neoplasm Transplantation; Pancreatic Neoplasms; Receptors, Cholecystokinin; Somatostatin; Transplantation, Heterologous; Tumor Cells, Cultured

Local application of growth factors promote wound healing and may find clinical application for use in high-risk intestinal anastomoses such as that following anterior resection. Since viable tumour cells are present in the bowel lumen and circulation after curative colorectal cancer surgery, it is unclear what effect such factors may have on tumour recurrence. The aim of this study was to examine the effect of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in a collagen suspension on perianastomotic tumour growth in an animal model. Significantly (P < 0.05) more animals in the collagen treated groups developed anastomotic tumours. The area of tumour growth at the anastomosis was also significantly greater for the collagen (median 14.7 mm2) and collagen + EGF (median 10.8 mm2) groups compared with controls (median 0.78 mm2). We were unable to demonstrate any promotion of tumour by growth factors alone. Collagen promotes perianastomotic tumour growth in this model and is not a suitable vehicle for growth factor application in colorectal cancer surgery.

Topics: Adenocarcinoma; Anastomosis, Surgical; Animals; Collagen; Colon; Epidermal Growth Factor; Fibroblast Growth Factor 2; Neoplasm Seeding; Rats; Rats, Inbred F344; Tumor Cells, Cultured

Endometrial fibroblasts derived from uterine endometrium as controls and endometrial cancer cells (Ishikawa and HHUA cells) were used to analyze the manner of induction of c-Ha-ras transcripts in endometrial cancers, some of which are estrogen-dependent in growth. Estrogen increased c-Ha-ras expression and tyrosine kinase (TK) activity in fibroblast and Ishikawa cells, but not in HHUA cells. Progesterone diminished c-Ha-ras expression and tyrosine kinase (TK) activity induced by estradiol in the fibroblasts, but not in Ishikawa cells, which persistently overexpressed c-Ha-ras. In these cells, epidermal growth factor (EGF) increased c-Ha-ras expression as did estradiol. Pretreatment with tyrphostin, an inhibitor of TK, abolished estrogen-inducible overexpression of c-Ha-ras. The combination of both estradiol and EGF at maximum effective concentration exerted no additive or synergistic effect on induction of c-Ha-ras expression. In conclusion, persistent activation of TK might lead to overexpression of c-Ha-ras in some endometrial cancer cells under estrogen predominant milieu, which might be associated with the transformation or growth potential.

Topics: Adenocarcinoma; Adult; Base Sequence; Carcinoma; Endometrial Neoplasms; Endometrium; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Estradiol; Female; Fibroblasts; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Progesterone; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); RNA, Messenger; Tumor Cells, Cultured

ErbB-2 and EGFR (epidermal growth factor receptor) are expressed in lung adenocarcinomas and associated with a poor prognosis. Immunocytochemical analysis revealed erbB-2 and EGFR coexperession as a characteristic feature of most lung adenocarcinomas, and at levels of receptor expression present in bronchial epithelial cells. In primary lung tumours and cell lines, erbB-2 detected using Western blot analysis demonstrated low-level phosphotyrosine staining of the 185 kDa band, as compared with breast cancer cell lines. A549 and A427 lung adenocarcinoma cells treated with neu differentiation factor (NDF) showed increased erbB-2 phosphotyrosine staining, but to a much lesser extent than breast cancer cells. The lung cells were examined for expression of the potential autocrine growth factors NDF and transforming growth factor alpha (TGF-alpha) by Northern blot analysis. Both NDF and TFG-alpha mRNA were abundantly expressed in the A549 cells. NDF mRNA was highest during active cell proliferation and decreased in confluent cells or after treatment with the growth-inhibitory steroid dexamethasone. Primary tumours and cell lines expressed EGFR, showing higher basal level phosphotyrosine staining than erbB-2. Treatment with NDF and EGF (epidermal growth factor) stimulated cell growth, and in A549 cells the presence of both factors provided an additive increase in cell growth. The growth stimulus that ligand-activated erbB-2 and EGFR provides to lung adenocarcinoma cells may establish a background of continued cell proliferation over which other critical transforming events may occur.

Topics: Adenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Humans; Lung Neoplasms; Neuregulins; Receptor, ErbB-2; Tumor Cells, Cultured

Two morphologically distinct cell lines, GP2d and GP5d, derived from the same adenocarcinoma of the colon, have been established and characterised. Both clones have the same genetic changes, consistent with the usual pattern of tumour progression in colon cancer. The cells also have an inverted duplication of bands 10q11 to 10q21, but Southern blot analysis failed to identify any translocations involving the ret protooncogene, which maps to this region. GP2d grew by spreading from the edges of microcolonies to form a confluent layer of cells. GP5d grew in discrete islands of cells forming multi-layered colonies. These differing patterns of growth correlated with variation in expression or cellular distribution of alpha 2-integrin, desmoplakin and e-cadherin. Only GP2d responded to exogenously added epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha) or insulin with an increase in cell numbers, even though both cell lines possessed similar numbers of EGF receptors. Analysis of EGF receptor ligand expression showed that GP5d cells expressed relatively more TGF alpha mRNA than did GP2d; in contrast, amphiregulin mRNA, which was abundant in GP2d, was virtually undetectable in GP5d. Even though GP5d failed to exhibit a growth response to EGF, it underwent a marked epithelial-mesenchymal transition when treated with EGF, indicating separation of growth and morphological responses to EGF.

Topics: Adenocarcinoma; Base Sequence; Cell Adhesion Molecules; Cell Division; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; Molecular Sequence Data; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

The Caco-2 cell line was utilized to analyze the role of nutrient factors such as calcium, vitamin D and epidermal growth factor (EGF) in epigenetic control of human colon carcinoma cell growth. Proliferative signals from either low extracellular calcium or EGF, respectively, are transduced in Caco-2 cells via an increase in c-myc proto-oncogene mRNA and nuclear protein expression levels. Activation of the EGF receptor is associated also with down-regulation of the cytoplasmic high-affinity vitamin D receptor (VDR). This would allow colon carcinoma cells to escape from the VDR-mediated anti-mitogenic action of 1 alpha, 25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). However, Caco-2 cells have the unique property to synthesize the vitamin D hormone from 25-hydroxyvitamin D3. 1 alpha,25(OH)2D3, in turn, counteracts the negative effect of EGF on VDR abundancy and slow down tumor cell proliferation through a c-myc-independent pathway.

Topics: Adenocarcinoma; Calcifediol; Calcitriol; Calcium; Cell Division; Colonic Neoplasms; Epidermal Growth Factor; Humans; Proto-Oncogene Mas; Proto-Oncogene Proteins c-myc; Receptors, Calcitriol; Tumor Cells, Cultured; Vitamin D

Androgens are required for the optimal growth and development of both the normal prostate and steroid-sensitive prostate cancer. PC3 prostate cancer cell lines stably expressing the human androgen receptor (AR) and possessing an androgen-sensitive phenotype (PC3-hAR) were used to examine the role of the epidermal growth factor receptor (EGFR) in androgen-stimulated prostate cancer cell growth. Epidermal growth factor (EGF) and dihydrotestosterone (DHT) independently induced the growth of PC3-hAR cells. Moreover, EGF and DHT in combination exerted a synergistic effect on PC3-hAR cell growth. DHT-exposed PC3-hAR cells expressed a greater than 2-fold increase in EGFR mRNA and 50% more EGFR protein than controls. Time course radioligand-binding assays confirmed these findings by showing an elevation in EGF binding in the DHT-exposed PC3-hAR cells. In addition, radioligand competition-binding studies revealed a 2-fold increase in EGFR-EGF binding affinity in the PC3-hAR cells after DHT treatment. However, no enhancement of transforming growth factor alpha or EGF expression was detected because DHT did not affect the levels of these cytokines in the PC3-hAR cell lysate or conditioned media. Our observations suggest that DHT increases both EGFR number and receptor-ligand affinity in androgen-sensitive prostate cancer cells and that these effects correlate with increased EGF binding and an enhanced mitogenic response to EGF.

Topics: Adenocarcinoma; Androgens; Animals; Bone Neoplasms; Cell Division; Dihydrotestosterone; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Lymphatic Metastasis; Male; Mice; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Receptors, Androgen; Stimulation, Chemical; Transforming Growth Factor alpha; Tumor Cells, Cultured; Up-Regulation

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are potent mitogens that contribute to abnormal growth regulation in colon cancer. Growth factors have been shown to regulate transmembrane nutrient uptake as an adaptive response to support cellular proliferation.. The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized by assaying the uptake of [3H]L-arginine in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transport affinity (Km) and maximal transport velocity (Vmax). To further characterize the specific transporters, [3H]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To investigate the effects of EGF and TGF alpha, cells were incubated with increasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine transport was measured at various time intervals (8, 12, 24 h). Proliferation was assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 3 days after growth factor stimulation.. The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65-70%), whereas the remainder was sodium-dependent (28-30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport revealed a single high-affinity Na(+)-independent transporter with a Km = 55.8 +/- 5.8 microM and a Vmax = 710.6 +/- 87.3 pM/mg protein/30 s. Na(+)-independent arginine uptake was pH-insensitive and markedly inhibited by system y+ substrates L-homoarginine, L-lysine, and L-ornithine. A single Na(+)-dependent transporter with a Km = 19.8 +/- 2.3 microM and a Vmax = 159.1 +/- 8.9 pM/mg protein/30 s was identified. Na(+)-dependent arginine uptake was inhibited by system BO,+ substrates L-lysine, L-ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. In addition, Na(+)-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGF alpha had no effect on Na(+)-independent L-arginine uptake; however, Na(+)-dependent uptake was enhanced 60% by EGF (10 ng/ml, p < 0.05) and 100% by TGF alpha (10 ng/ml, p < 0.05), whereas cellular proliferation was increased 27% by EGF (10 ng/ml, p < 0.05) and 37% by TGF alpha (10 ng/ml, p < 0.01).. L-arginine transport in the SW480 colon cancer cell line is principally mediated by the Na(+)-independent system y+ and to a lesser extent by the Na(+)-dependent system BO,+. Furthermore, EGF and TGF alpha preferentially stimulate L-arginine uptake via the Na(+)-dependent transporter, ostensibly to accommodate for the mitogenic stimulus.

Topics: Adenocarcinoma; Amino Acids; Arginine; Carrier Proteins; Cell Division; Cell Membrane Permeability; Colonic Neoplasms; Epidermal Growth Factor; Humans; Hydrogen-Ion Concentration; Ion Channel Gating; Nitric Oxide; Radioisotopes; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

Membrane extracts of surgically resected colorectal cancer and nearby non-cancerous tissues were prepared and the expression of epidermal growth factor receptor (EGF-R) was examined by 125I-EGF binding assay. Scatchard analysis indicated that a single class of EGF-R was present in the cancer as well as the non-cancerous tissues. EGF-R was found over-expressed in cancer (38.07 +/- 4.75 fmol/mg protein) as compared to that in the non-cancerous tissue (29.31 +/- 1.64 fmol/mg protein) (P < 0.05). However, the difference in binding affinities of EGF-R in the cancer and non-cancerous tissues was not statistically significant. The results suggest an important role of an autocrine/paracrine loop of growth regulation in colorectal cancer.

Topics: Adenocarcinoma; Adult; Aged; Cell Membrane; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Male; Middle Aged; Rectal Neoplasms

We examined immunohistochemically 111 cases of primary adenocarcinoma of the lung, for transforming growth factor alpha (TGF alpha) or epidermal growth factor (EGF), and argyrophilic nucleolar organizer regions (AgNORs). The presence of more than 75% positive cells for both growth factors was designated as a high-GF, while all others were considered to be a low-GF. If AgNORs counts were more than 5.00, it was considered to be a high-AgNORs group, while less than 5.00 was designated as a low-AgNORs group. In our 111 examined specimens, there were 51 (46%) cases of high-GF, and 64 (58%) with high AgNORs. The 5-year survival rates of the patients with a high-GF and low-GF were 34% and 57% (P < 0.05) respectively, while those with high-AgNORs and low-AgNORs were 21% and 81% (P < 0.001), respectively. In the cases of high-AgNORs, the 5-year survival rates of the patients with high-GF and low-GF were 0% and 36% (P < 0.05), respectively. However, in the cases of low-AgNORs, the 5-year survival rates of the patients with high-GF and low-GF were 83% and 79%, respectively. These data suggest that growth factors might be related to the biological malignancy of tumours with a high cell proliferation.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cell Division; Epidermal Growth Factor; Female; Humans; Lung Neoplasms; Male; Middle Aged; Nucleolus Organizer Region; Silver Staining; Survival Analysis; Transforming Growth Factor alpha

Attachment of highly metastatic rat mammary adenocarcinoma MTLn3 cells to matrix proteins and its modulation by EGF was examined. Plastic plates were coated with varying amounts of collagen or fibronectin. MTLn3 cells exhibited a dose-dependent adhesion to both matrix proteins, however, they attached more efficiently to collagen than to fibronectin. When EGF or TGF alpha were added at 0.3 to 10 ng/ml for 30 min, a dose-dependent increase in adhesion to both matrix proteins was observed. Maximal stimulation (2-fold) was seen with 10 ng/ml of either growth factor. However, EGF was more potent at lower concentrations (0.3-3 ng/ml) than TGF alpha. The ability of growth factors to stimulate adhesion was also dependent on the amount of matrix the cells were exposed to. While EGF increased rapid attachment of MTLn3 cells to both matrix proteins similarly, subsequent cell spreading and formation of lamellar extensions was faster in cells plated on collagen. These results are suggestive of a functional link between EGF receptor and specific integrin activities.

Topics: Adenocarcinoma; Animals; Cattle; Cell Adhesion; Collagen; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix Proteins; Fibronectins; Integrins; Mammary Neoplasms, Animal; Microscopy; Rats; Time Factors; Transforming Growth Factor alpha; Tumor Cells, Cultured

We compared morphological, biological and molecular biological patterns of a newly established, spontaneously immortalized pancreatic ductal cell line, TAKA-1, with a hamster pancreatic ductal adenocarcinoma cell line, PC-1. PC-1 cells grew in a monolayer on plastic tissue culture flasks, whereas TAKA-1 cells required type I collagen gel matrix to propagate. The growth rate and argyrophilic nuclear organizer region (Ag-NOR) counts were greater in PC-1 cells than in TAKA-1 cells. More TAKA-1 cells were in G0/G1 and less were in the S cell cycle phase than PC-1 cells. Karyotypically, the consistent change in TAKA-1 cells was an abnormal no. 3 chromosome, whereas additional chromosomal abnormalities were found in PC-1 cells. Ultrastructurally, TAKA-1 cells formed ductal structures and were composed of two types of cells, as in the normal hamster pancreatic ducts, whereas PC-1 cells were pleomorphic, showed evidence for loss of differentiation and contained intracytoplasmic lumens. Unlike the PC-1, TAKA-1 cells did not show a point mutation at codon 12 in the c-Ki-ras oncogene and did not grow in soft agar. Receptor binding assay showed specific epidermal growth factor binding to both cell lines, but secretin binding only to TAKA-1 cells. Both cells produced and released transforming growth factor-alpha in serum-free medium. Both cell lines expressed blood group A antigen, carbonic anhydrase, coexpressed cytokeratin and vimentin, and reacted with tomato and Phaseolus vulgaris leucoagglutinin (L-PHA) lectins. The results demonstrate that chromosomal abnormalities, cell cycle patterns, expression of cytokeratin 18, lectin bindings and the c-Ki-ras mutation are the features that distinguish the benign from the malignant pancreatic ductal cells in Syrian hamster.

Topics: Adenocarcinoma; Animals; Base Sequence; Cell Adhesion; Cell Cycle; Cell Division; Cell Line; Cholecystokinin; Cricetinae; Epidermal Growth Factor; Flow Cytometry; Gastrin-Releasing Peptide; Genes, ras; Immunohistochemistry; Karyotyping; Kinetics; Light; Mesocricetus; Microscopy; Microscopy, Electron; Molecular Sequence Data; Pancreas; Pancreatic Ducts; Pancreatic Neoplasms; Peptides; Point Mutation; Secretin; Tumor Cells, Cultured

Increased expression of EGF receptor (EGFR) in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive causative link, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of functional receptors. A full length cDNA of the human EGFR (HER) was introduced by infection with a retroviral vector into MTC cells. Expression of HER was stable and receptors were functional with respect to surface expression, ligand binding and EGF-stimulated phosphorylation. Independent clones of the transfectants were isolated and characterized. Ligand stimulation of MTC HER cells and derived clones led to enhanced adhesion of cells to extracellular matrix proteins. Implantation of cells intravenously into female nu/nu mice revealed ligand-dependent enhancement of lung colonizing potential of EGFR-expressing cells.

Topics: Adenocarcinoma; Animals; Cell Adhesion; Epidermal Growth Factor; ErbB Receptors; Extracellular Matrix; Female; Ligands; Lung Neoplasms; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Phosphorylation; Rats; Transfection

The metastatic variants of human lung adenocarcinoma cell line DMS4C were established by selection in vivo. Lung, brain, spleen and liver metastatic tumors derived from intracarotid inoculation of athymic BALB/c mice were collected, and their corresponding variant cell lines established. The epidermal growth factor (EGF) receptor expression of the parental cell line as well as the metastatic variant cell lines were investigated. 125I-labeled EGF binding assays showed that there were two types of EGF receptors in both parental and metastatic variants. Compared to DMS4C, the EGF binding capacities were found to be down-regulated by 70, 79, 85 and 89% for lung, spleen, liver and brain variant, respectively. The dissociation constants of spleen, liver and brain EGF receptors were distinct from that of the parental cell line. The EGF receptor autophosphorylation activity of lung variant was shown to be down-regulated as shown by immune complex kinase assay that corresponded to EGF receptor numbers whereas kinase activities of the liver, spleen and brain variants EGF receptors were completely abolished. However, the 170 kilodalton EGF receptor was shown to be unaltered during metastasis. The results indicated that, during metastasis progression, the proliferation of adenocarcinoma cells may have adopted a different growth regulation that is independent of EGF receptor kinase-modulated autocrine pathway. The result also implies that other oncogene may emerge as the major growth regulator for distant metastasis of adenocarcinoma cancer cells. This work provides a model for understanding tumor metastasis progression of human lung cancer.

Topics: Adenocarcinoma; Animals; Brain; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Liver; Lung; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Spleen; Tumor Cells, Cultured

To study the regulation of c-erbB, EFG and TGF-alpha in endometrium, we examined the expression of their mRNA in 5 normal endometrial and 5 endometrial carcinoma tissues by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR). By Northern blot with 10 micrograms of total RNA, c-erbB mRNA was not detected in the normal endometrium, but it was detected in 3 samples of endometrial carcinoma tissues. On the other hand RT-PCR identified c-erbB mRNA in all the normal endometrium and endometrial carcinoma tissues by amplifying cDNA derived from c-erbB mRNA. EGF mRNA was detected by RT-PCR in normal endometrium except in the early follicular phase. It was detected in all cases of endometrial carcinoma tissues. TGF-alpha mRNA was also detected in all the normal and endometrial carcinoma tissues by RT-PCR. Our study suggests that an autocrine/paracrine mechanism of EGF may regulate the endometrial cycle. Because some endometrial carcinoma tissues express the c-erbB mRNA much more than normal endometrium, disruption of the autocrine/paracrine mechanism may trigger subsequent endometrial carcinogenesis.

Topics: Adenocarcinoma; Adult; Base Sequence; Blotting, Northern; Endometrial Neoplasms; Endometrium; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Middle Aged; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

Poorly differentiated MATLyLu rat prostate cancer cells are resistant to the growth inhibitory effect of transforming growth factor (TGF) beta 1 in vivo, but are inhibited by TGF-beta 1 in vitro. However, TGF-beta 1 inhibited proliferation only when the cells were plated at low density in serum-free medium (concentration for 50% of maximum inhibition, 0.1 ng/ml). TGF-beta 1 was not growth inhibitory when cells were plated at high density, or at low density in 0.5% serum. At low cell density in serum-free medium, 0.5 ng/ml TGF-beta 1 caused maximum inhibition. In the presence of basic fibroblast growth factor (10 ng/ml), TGF-beta 1 did not inhibit proliferation. In the presence of epidermal growth factor (50 ng/ml), TGF-beta 1 inhibited proliferation by only 18%. Growth inhibition by TGF-beta 1 was less effective on extracellular matrix than on plastic. The ability of high cell density, serum, growth factors, or extracellular matrix to prevent or blunt the growth inhibitory effect of TGF-beta 1 in vitro probably explains why TGF-beta 1 does not inhibit tumor growth in vivo. Thus, prostate cancer cells express high levels of TGF-beta and retain exquisite sensitivity to the growth inhibitory effect of TGF-beta, but have devised a way to protect themselves from growth inhibition by TGF-beta in vivo. TGF-beta 1 stimulated MATLyLu cell motility even at high cell density, suggesting that TGF-beta 1 might affect motility even in vivo and contribute to the aggressiveness of the tumor, without affecting proliferation.

Topics: Adenocarcinoma; Animals; Cell Adhesion; Cell Division; Cell Movement; Clone Cells; Culture Media; Culture Media, Serum-Free; Drug Interactions; Epidermal Growth Factor; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblast Growth Factor 2; Male; Prostatic Neoplasms; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

Gene expression of growth factors including epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGFR), oncogenes such as c-myc, N-ras, c-erbB2 and tumor suppressor gene P53 were studied in 4 human lung cancer cell lines using Northern blot technique. Among these cell lines were 2 adenocarcinoma cell lines, one large cell carcinoma cell line and one small cell carcinoma cell line. Expression of EGF and TGF alpha mRNAs were found in all 4 cell lines and EFGR mRNA was seen in 3 out of 4 cell lines. Among these cell lines, 2 cell lines with weaker expression of EGF and TGF alpha, expressed c-myc mRNA. Another 2 cell lines had no c-myc but expressed large amounts of EGF and TGF alpha mRNA. No expression of N-ras, c-erbB2 and p53 were found in these cell lines. The results indicate the presence of autocrine loop of growth factors in these cancer cells. The autostimulation of growth factors may be the main cause for the uncontrolled growth of cancer cells. After treating the cancer cells with EGF, anti-EGF and anti-EGFR antibodies, EGF was found to exert a mild stimulating effect on the growth of one cell line, but no effect on the other cell lines. Anti-EGF and anti-EGFR antibodies inhibited the cell growth on all cell lines. These results provided further evidence for the presence of autocrine loop of growth factors in these lung cancer cell lines.

Topics: Adenocarcinoma; Antibodies, Neoplasm; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Genes, Tumor Suppressor; Humans; Lung Neoplasms; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

We investigated the effects of epidermal growth factor (EGF) on polyamine uptake in Caco-2 cell monolayers. Cells were grown until confluence (day 7) or until differentiation (day 14). Polyamine uptake into Caco-2 cells was stimulated by EGF in a dose-dependent manner. Both basal and EGF-stimulated uptake rates were higher in 7- than in 14-day-old Caco-2 cells. Stimulation with EGF resulted in a significant increase in Vmax and an increased affinity for putrescine and spermine. Polyamine uptake was not inhibited when protein synthesis was blocked by cycloheximide, implying that no additional protein synthesis occured for stimulatory effect of EGF on polyamine uptake. The tyrosine kinase inhibitor, genistein, completely inhibited EGF-stimulated polyamine uptake, indicating that tyrosine phosphorylation plays a role in EGF-stimulated polyamine uptake in Caco-2 cells. The effect of EGF on polyamine uptake into Caco-2 cells, therefore, could be due to translocation of intracellular proteins which were not previously incorporated into the membrane, or direct alteration of polyamine transporter.

Topics: Adenocarcinoma; Biological Transport; Cell Differentiation; Colonic Neoplasms; Cycloheximide; Epidermal Growth Factor; Genistein; Humans; Isoflavones; Kinetics; Polyamines; Putrescine; Spermine; Tumor Cells, Cultured

The frequency of expression and localization of cripto-1 (CR-1), amphiregulin (AR), transforming growth factor alpha (TGF alpha), epidermal growth factor receptor (EGFR) and erbB-2 were examined by immunohistochemistry in 45 carcinomas and adjacent non-involved normal colon mucosa. Thirty (66.7%), 24 (53.3%), 23 (51.1%), 23 (51.1%) and 13 (28.9%) of the 45 carcinomas showed positive staining for CR-1, AR, TGF alpha, EGFR and erbB-2, respectively, whereas 7 (15.5%), 17 (37.7%), 15 (33.3%), 20 (44.4%) and 0 (0%) of the corresponding non-involved normal mucosa specimens were reactive. Among 13 carcinomas with lymph node involvement, 10 (76.9%), 8 (61.5%), 10 (76.9%), 8 (61.5%) and 7 (53.8%) exhibited positive staining for CR-1, AR, TGF-alpha, EGFR and erbB-2, respectively. There was a statistically significant association between the frequency of either TGF alpha (P < 0.05) or erbB-2 (P < 0.05) expression and lymph node metastasis. In addition, a significantly higher frequency of positive staining for TGF alpha was observed in Dukes' grade C carcinomas (P < 0.05). Finally, significant trends for coexpression of EGFR and either TGF alpha (P < 0.01) or AR (P < 0.05) were detected in carcinomas. These data suggest that AR and TGF alpha may play an important role in the development of colorectal carcinomas through an autocrine mechanism involving EGFR, and demonstrate that TGF alpha and erbB-2 may be more reliable indicators of metastasis or prognosis than CR-1, AR or EGFR in human colon cancers.

Topics: Adenocarcinoma; Amphiregulin; Colorectal Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lymphatic Metastasis; Membrane Glycoproteins; Neoplasm Proteins; Peptides; Receptor, ErbB-2; Transforming Growth Factor alpha

A 150 bp epidermal growth factor (EGF) cDNA fragment and a 1024 bp epidermal growth factor receptor (EGFR) cDNA fragment were inserted into 5.05 kb pBabe-puro retroviral vectors between BamH I and EcoR I sites in 3'-5' and/or 5'-3' orientation. The vectors were ligated with EGF and EGFR fragments by T-4 Ligase. The recombinant retroviral vectors were then packaged with packaging cell line PA317 through calcium phosphate mediated transfection. The viral supernatant of transfected PA317 cell lines were used to infect the human pancreatic carcinoma cell line PC-7. The resultant transformant cell lines: PC-7/AS-EGF, PC-7/S-EGFR, PC-7/AS-EGFR and PC-7/pBabe were tested for their endogenous EGF and EGFR mRNA expressions, cell growth rate, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice. The results showed that there were noticeable inhibitions of cell growth, 3H-TdR incorporation rate, soft agar colony formation and tumorigenicity in nude mice in PC-7/AS-EGF and PC-7/AS-EGFR transformant cell lines. The endogenous EGF mRNA expression was blocked in PC-7/AS-EGF cell line and the endogenous EGFR mRNA was significantly down-regulated in PC-7/AS-EGFR cell line.

Topics: Adenocarcinoma; Animals; Cell Division; DNA, Antisense; DNA, Recombinant; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Genetic Vectors; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Pancreatic Neoplasms; Retroviridae; Tumor Cells, Cultured

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses the Int-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.

Topics: Adenocarcinoma; Animals; Antigens, Viral, Tumor; Cattle; Choroid Plexus; Cricetinae; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Kidney; Kidney Tubules, Distal; Male; Mice; Mice, Transgenic; Proto-Oncogene Proteins; Simian virus 40; Submandibular Gland; Submandibular Gland Neoplasms; Wnt Proteins; Wnt1 Protein; Zebrafish Proteins

Gliomas, squamous carcinomas and different adenocarcinomas from breast, colon and prostate might have an increased number of epidermal growth factor (EGF) receptors. The receptors are, in these cases, candidates for binding of receptor specific toxic conjugates that might inactivate cellular proliferation. The purpose of this study was to evaluate whether it is reasonable to try ligand-dextran based conjugates for therapy.. EGF or TGF alpha were conjugated to dextran and binding, internalization, retention and degradation of eight types of such conjugates were analyzed in EGF-receptor amplified glioma cells. The conjugates were labelled with radioactive nuclides to allow detection and two of the conjugates were carrying boron in the form of carboranyl amino acids or aminoalkyl-carboranes. Comparative binding tests, applying 125I-EGF, were made with cultured breast, colon and prostate adenocarcinoma, glioma and squamous carcinoma cells. Some introductory tests to label with 76Br for positron emission tomography and with 131I for radionuclide therapy were also made.. The dextran part of the conjugates did not prevent receptor specific binding. The amount of receptor specific binding varied between the different types of conjugates and between the tested cell types. The dextran part improved intracellular retention and radioactive nuclides were retained for at least 20-24 h. The therapeutical effect improved when 131I was attached to EGF-dextran instead of native EGF.. The improved cellular retention of the ligand-dextran conjugates is an important property since it gives extended exposure time when radionuclides are applied and flexibility in the choice of time for application of neutrons in boron neutron capture therapy (BNCT). It is possible that ligand-dextran mediated BNCT might allow, if the applied neutron fields covers rather wide areas around the primary tumor, locally spread cells that otherwise would escape treatment to be inactivated.

Topics: Adenocarcinoma; Animals; Boron Compounds; Boron Neutron Capture Therapy; Carcinoma; Colonic Neoplasms; Dextrans; Drug Carriers; Epidermal Growth Factor; ErbB Receptors; Glioma; Humans; Iodine Radioisotopes; Male; Mammary Neoplasms, Experimental; Models, Biological; Neoplasms; Neoplasms, Experimental; Prostatic Neoplasms; Rats; Rats, Sprague-Dawley; Transforming Growth Factor alpha; Tumor Cells, Cultured

Histochemical activity and immunoreactivity of nitric oxide synthase (NOS, EC 1.14.13.39) have been recently demonstrated in human lung epithelium. However, the molecular nature of NOS and the regulation and function of the enzyme(s) in the airway is not known. A549 cells (human alveolar type II epithelium-like), BEAS 2B cells (transformed human bronchial epithelial cells), and primary cultures of human bronchial epithelial cells all exhibited constitutive NOS activity that was calcium dependent and inhibitable by the NOS inhibitor NG-monomethyl-L-arginine. Nitric oxide production by epithelial cells was enhanced by culture in the presence of interferon gamma, interleukin 1 beta, tumor necrosis factor alpha, and lipopolysaccharide; the NOS activity expressed under these conditions showed less dependence on calcium, reminiscent of other inducible forms of NOS. Two distinct NOS mRNA species, homologous to previously identified constitutive brain (type I) and inducible hepatic (type II) NOS, were demonstrated by reverse transcription-polymerase chain reaction in all cell lines. Northern analysis confirmed the expression of inducible NOS mRNA. Cell culture with epidermal growth factor, a principal regulator of epithelial cell function, decreased inducible NOS activity by posttranscriptional action but did not affect constitutive NOS activity. The coexistence of constitutive and inducible NOS in human alveolar and bronchial epithelial cells is consistent with a complex mechanism evolved by epithelial cells to protect the host from microbial assault at the air/surface interface while shielding the host from the induction of airway hyperreactivity.

Topics: Adenocarcinoma; Amino Acid Oxidoreductases; Base Sequence; Cell Line; Cytokines; DNA Primers; Enzyme Induction; Epidermal Growth Factor; Epithelium; Gene Expression Regulation, Enzymologic; Humans; Interferon-gamma; Interleukin-1; Lung; Lung Neoplasms; Molecular Sequence Data; Nitric Oxide Synthase; Polymerase Chain Reaction; Pulmonary Alveoli; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Soybean trypsin inhibitor was added to the primary cell culture for establishing a novel moderately differentiated human pancreatic adenocarcinoma cell line, PC-7. The PC-7 cell line was confirmed to be a malignant human pancreatic adenocarcinoma by morphological and biological studies. Chromosomal analysis and DNA content of PC-7 determined by flow cytometry showed a hypodiploid karyotypic pattern. Cultured PC-7 cells were inoculated into the back of athymic nude mice to create a transplanted tumor model. The effects of epidermal growth factor (EGF) and anti-EGF antiserum on PC-7 transplanted tumor were observed by injecting EGF or anti-EGF at the periphery of the solid tumors. In comparing to the control group, the tumor weights of the EGF group and the anti-EGF group were 138% and 67% respectively. Histological and electron microscopic studies higher mitotic rate in the EGF group and a lower rate in the anti-EGF group compared to the control. The results indicate that EGF stimulated and anti-EGF partially inhibited the growth of transplanted PC-7 solid tumors in athymic nude mice.

Topics: Adenocarcinoma; Animals; Epidermal Growth Factor; Humans; Immune Sera; Immunization, Passive; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Pancreatic Neoplasms; Tumor Cells, Cultured

Nude mice bearing xenografts of the gastrin-responsive human gastric carcinoma MKN45 cell line were treated for 4 to 5 weeks with bombesin/gastrin-releasing-peptide(GRP) antagonist (RC-3095), somatostatin analogues RC-160 and SMS 201-995, or the combination of RC-3095 and RC-160. Tumor volumes and weights were reduced significantly to a similar extent by RC-160 and SMS 201-995, administered by daily s.c. injections at a dose of 100 micrograms/day. Bombesin/GRP antagonist RC-3095, given s.c. at a dose of 20 micrograms/day, had the greatest inhibitory effect on tumor growth. The combination of RC-3095 with RC-160 did not further potentiate the suppression of tumor growth. Histologically, the number of mitotic cels decreased significantly in the groups treated with RC-160 or the combination of RC-3095 with RC-160. Serum gastrin levels were significantly diminished in all treated groups. Therapy with RC-160 or the combination also significantly decreased levels of serum growth hormone. Receptor assays on tumor membranes showed that bombesin was bound to 2 classes of receptor sites, one with high affinity and low capacity, the other with low affinity and high capacity. Binding sites for epidermal growth factor (EGF) were down-regulated in tumor cells after treatment with RC-3095, RC-160 or the combination of RC-3095 with RC-160. In studies in vitro, both RC-160 and RC-3095 significantly inhibited the proliferation of MKN45 cells in culture as measured by cell number. These data demonstrate, for the first time, that the growth of human gastric cancer in nude mice can be inhibited not only by somatostatin analogues, but also by administration of modern bombesin/GRP antagonists, such as RC-3095.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Antineoplastic Agents; Binding Sites; Body Weight; Bombesin; Cell Division; Epidermal Growth Factor; Female; Humans; Insulin-Like Growth Factor I; Male; Mice; Mice, Nude; Middle Aged; Molecular Sequence Data; Neoplasm Transplantation; Octreotide; Peptide Fragments; Somatostatin; Stomach Neoplasms; Transplantation, Heterologous; Tumor Cells, Cultured

Immunohistochemical studies were performed to clarify the significance of the expression or overexpression of epidermal growth factor (EGF), EGF-receptor (EGFR), p53, v-erb B, ras p21 in 23 cases each of tubular adenoma and adenocarcinoma. The expression of EGF, EGFR, p53, v-erb B, and ras p21 in paraffin-embedded tissues, from 46 patients with colorectal tumors (adenoma: 23 cases; 14 mild dysplasia, six moderate dysplasia, three severe dysplasia, adenocarcinoma: 23 cases; 17 well differentiated, two moderately differentiated, three poorly differentiated, one mucinous carcinoma was analyzed immunohistochemically using anti-EGF, EGFR, p53, v-erb B and ras p21 antibodies. The EGF and ras p21 tended to express more strongly in carcinoma cases than in the adenoma cases, and in severe and moderate dysplasia than in mild dysplasia (EGF: stained positive in five adenomas [21.74%] and 17 adenocarcinomas [73.91%]; ras p21: stained positive in six adenomas [26.09%] and 14 adenocarcinomas [60.87%]. The EGFR stained positive in two adenomas (8.70%) and two adenocarcinomas (8.70%). The p53 and v-erb B showed positive staining only in the carcinoma cases (p53: stained positive in four cases [17.39%]; v-erb B: stained positive in eight cases [34.78%]). This study suggests that these factors seem to have some role in the progression of colon neoplasms. It suggests that genetic alteration is not always equal to the overexpression of protein products, but that it reflects them well, and that the staining makes some contribution to differential diagnosis in colorectal neoplasms.

Topics: Adenocarcinoma; Adenoma; Adult; Aged; Aged, 80 and over; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Neoplasms, Glandular and Epithelial; Oncogene Proteins v-erbB; Precancerous Conditions; Proto-Oncogene Proteins p21(ras); Retroviridae Proteins, Oncogenic; Tumor Suppressor Protein p53

The binding characteristics and steroidal regulation of the EGF receptor were investigated in the human endometrial adenocarcinoma cell line HEC-1-B. The cell line was shown to possess a single, high affinity binding site for epidermal growth factor receptor (EGF) with a Kd of 3.09 +/- 1.39 nM (mean +/- SD, n = 6) and binding of 845 +/- 311 fmol/mg protein (mean +/- SD, n = 6). The protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA) increased the Kd of the EGF receptor in a dose dependent manner (PMA: 0, 1, 10, 100 nM; Kd: 4.1, 5, 10, 50 nM, respectively). The effect of PMA (10 nM) was overcome by preincubating the cells with the protein kinase C inhibitor staurosporine (1 microM) prior to the addition of PMA. The effect of the ovarian steroids oestradiol and progresterone on EGF receptor accumulation was studied by pretreating the cells for 6 days with oestradiol or progesterone in phenol red free DMEM:F12, 1:1 supplemented with 5% charcoal stripped fetal calf serum. Both steroids were shown to increase EGF receptor number with a maximum 5- and 7-fold increase in the presence of 1 nM oestradiol or 1 microM progresterone, respectively. The study demonstrates the presence of a high affinity binding site for EGF in HEC-1-B cells which is regulated by oestradiol and progresterone.

Topics: Adenocarcinoma; Alkaloids; Binding Sites; Endometrial Neoplasms; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Humans; Kinetics; Progesterone; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

We recently established epidermal growth factor (EGF) receptor-hyperproducing human gastric cancer xenografts in nude mice. The present study was designed to examine whether the growth of a xenograft having 1,098 +/- 276 fmol/mg protein of EGF receptor would either be stimulated by the administration of EGF or inhibited by the removal of the submandibular glands (sialoadenectomy) which contain a large amount of EGF. A miniosmotic pump containing 2 micrograms or 20 micrograms of EGF was implanted on the back of the animals in the EGF stimulation experiments. The tumor growth was stimulated by the administration of EGF (P < 0.01), and the doubling time of the tumor was reduced relative to the controls (P < 0.01). Both the mitotic indices and the bromodeoxyuridine (BrdU)-labeling indices of the tumor were higher than those of the controls (P < 0.01). Tumor growth inhibited by the sialoadenectomy (P < 0.05) while the tumor doubling time was prolonged compared with the sham-operated mice (P < 0.05). These results suggest that the growth of a human gastric cancer xenograft may be modulated by EGF.

Topics: Adenocarcinoma; Animals; Cell Cycle; Epidermal Growth Factor; ErbB Receptors; Growth; Humans; Immunohistochemistry; Mice; Mice, Nude; Neoplasm Transplantation; Stimulation, Chemical; Stomach Neoplasms; Submandibular Gland; Transplantation, Heterologous; Tumor Cells, Cultured

We detect the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in 104 specimens of gastric cancer by avidin biotin-peroxides complex technique (ABC). The positive rates of EGF, EGFR and synchronous EGF and EGFR were 35.6%, 42.3%, 30.8% respectively. The positive expression of EGF and EGFR to gastric cancer tissue was inclined to occur in patients who were in advanced stages, of poorly-differentiated types, Borrmann III, IV types, scirrhous type, seroinvasive type or lymph node metastasic types. The survival rates at 1, 3, 5 years after gastrectomy in the patients with expression of EGF were significantly lower than the survival rates of the patients with negative expression (P < 0.05). The patients with synchronous expression of EGF and EGFR had the worst prognosis. After gastrectomy, all of them died within 4 years. It is indicated that EGF and EGFR could serve as biological indicators of gastric cancer malignancy and an index for evaluating, the prognosis of the patients with gastric cancer.

Topics: Adenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Prognosis; Stomach Neoplasms; Survival Rate

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Topics: Adenocarcinoma; Animals; Aorta; Cattle; Cell Division; Collagenases; Culture Media, Conditioned; Endothelium, Vascular; Epidermal Growth Factor; Glycoproteins; Humans; Matrix Metalloproteinase Inhibitors; Neoplasm Metastasis; Salivary Gland Neoplasms; Tissue Inhibitor of Metalloproteinases; 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

Retinoic acid (RA) is a natural derivative of vitamin A which regulates the growth and differentiation of epithelia. We have previously proposed that RA participates in compensatory kidney growth and reported that RA inhibits rat mesangial cell growth. This paper describes the effects of RA on a human renal adenocarcinoma cell line (PAD) under different growth conditions, and its interactions with epidermal growth factor (EGF). PAD cells were shown to express RA receptors alpha and beta by Northern blot analysis. In serum free cultures, addition of RA (10(-7) M) markedly increased thymidine incorporation by PAD cells (155 +/- 7% mean +/- SE vs. control in 6 separate experiments; P < 0.0001). RA also caused a significant increase in thymidine incorporation by PAD cells under conditions of rapid growth in serum supplemented medium (115 +/- 2% vs. control; P < 0.001). RA by itself was unable to reverse contact inhibition of PAD cell growth (NS vs. control), but it synergistically enhanced the mitogenic effect of EGF on confluent monolayers (110 +/- 0.6% vs. EGF alone; P < 0.05). Northern blot analysis demonstrated that PAD cells express EGF receptor mRNA, and this was not significantly modified by the addition of RA. Growth arrested (serum starved) PAD cells expressed RAR-alpha mRNA which was upregulated eightfold at three hours following the addition of 10% FCS. Thus, our data show that RA is directly mitogenic for serum starved human renal adenocarcinoma cells and that it exerts complex modulation of cell growth in the presence of EGF and serum components.

Topics: Adenocarcinoma; Base Sequence; Cell Division; Culture Media; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Interactions; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney Neoplasms; Molecular Sequence Data; Oligonucleotide Probes; RNA, Messenger; Tretinoin; Tumor Cells, Cultured; Vitamin A

The role of EGF in the proliferation of the poorly differentiated endometrial adenocarcinoma cell line (KLE) was examined. EGF (10 ng/ml) and the tumor promoter, protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA) were potent stimulators (P < 0.01) of DNA synthesis in this cell line as determined by [3H]thymidine incorporation into DNA. Staurosporine, a protein kinase C inhibitor, partially blocked the EGF effects on [3H]thymidine incorporation. Similarly, downregulation of protein kinase C also failed to completely abolish EGF effect on DNA synthesis and cell division. These results suggest that in the KLE cell line EGF stimulation of cell growth is exerted through both protein kinase C-dependent and -independent pathways.

Topics: Adenocarcinoma; Alkaloids; Animals; Cell Division; Dose-Response Relationship, Drug; Endometrial Neoplasms; Epidermal Growth Factor; Female; Humans; Mice; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate; Thymidine; 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

The present immunohistochemical study examines the expression of TGF alpha, EGF, and the EGF receptor in prostatic tissues obtained from patients with either benign prostatic hyperplasia (BPH) or adenocarcinoma of the prostate. Frozen sections were cut at 5 microns and were incubated with monoclonal antibodies directed against TGF alpha, EGF, or the EGF receptor. The remainder of the staining procedure was performed with an avidin-biotin-complex peroxidase procedure. Strong TGF alpha expression was not detected in any of the 15 BPH specimens examined or in the normal/hyperplastic tissue intermixed within the adenocarcinomas. In contrast, malignant cells in 9 of 11 adenocarcinomas strongly expressed TGF alpha. EGF reactivity was observed in both hyperplastic and malignant epithelial tissues. EGF receptor expression was strongest along the basal aspects of cells of normal or hyperplastic glands. Although most malignant cells also were positive for the EGF receptor the level of staining was less than that observed in cells forming normal or hyperplastic glands. These results suggest that EGF and the EGF receptor may be components of an autocrine/paracrine regulatory pathway involved in hyperplastic and/or malignant disease of the prostate. The finding that TGF alpha is expressed only in malignant cells suggests that TGF alpha may represent a locally active autocrine regulator of malignant prostate cells.

Topics: Adenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Male; Prostatic Hyperplasia; Prostatic Neoplasms; Staining and Labeling; Transforming Growth Factor alpha

During the host response to inflammation/tissue injury there are many changes in intermediary metabolism including a dramatic change in the concentrations of many "acute phase" plasma proteins. Although many of these acute phase proteins are predominantly derived from the liver and the response can be elicited from liver cells incubated in tissue culture with cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), tumor necrosis factor-alpha, interferon-gamma, leukemia inhibitory factor, interleukin-11 (IL-11), and oncostatin M, there is now evidence that the response can also be elicited in extrahepatic tissues and cell types. In this study, we show that many of the acute phase plasma proteins are expressed in human intestinal epithelial cell lines Caco2 and T84 and that their expression is induced or regulated by cytokines IL-6, IL-1, interferon, and tumor necrosis factor in a manner characteristic of the acute phase response. In fact, effects of IL-1 and IL-6 which are additive, synergistic, and antagonistic in liver cell lines are also observed in these intestinal epithelial cell lines. Responses to IL-6 and IL-1 are seen at all stages of differentiation of Caco2 cells from crypt-like enterocytes to villus-like enterocytes. Caco2 cells express binding sites for IL-6 at both poles, for IL-1 at the basolateral pole and, to a lesser extent, at the apical pole. T84 cells have IL-1 and IL-6 receptor binding sites only at the basolateral pole. IL-6 and IL-1 also regulate the expression of enterocyte-specific integral membrane proteins as exemplified by down-regulation of sucrase-isomaltase gene expression in response to IL-6. These data raise the possibility that enterocytes are involved in a local response to injury/inflammation at the epithelial surface and establish a model system for examining coordination of the acute phase response in a bipolar cell.

Topics: Acute-Phase Proteins; Adenocarcinoma; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Cell Differentiation; Cell Line; Cell Membrane; Colonic Neoplasms; Complement Factor B; Cytokines; Epidermal Growth Factor; Epithelium; Humans; Interferon-gamma; Interleukin-1; Interleukin-6; Intestinal Mucosa; Kinetics; Methionine; Recombinant Proteins; Sulfur Radioisotopes; Transferrin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The cycle of phospholipid turnover has been found to be under the negative control of hormonal cytostatics (progesterone) and under the positive control of proliferation stimulants (17 beta-estradiol, epidermal growth factor). Specific changes in the synthesis of phospholipids are shown when tamoxiphen, an antiestrogen and an inhibitor of protein kinase C, was used. The findings suggest that changes in the turnover rate of phospholipids are one of the key stages of steroid action on target cells and may be regarded as an additional criterion of tumor genetic sensibility.

Topics: Adenocarcinoma; Breast Neoplasms; Drug Screening Assays, Antitumor; Epidermal Growth Factor; Estradiol; Female; Humans; Phospholipids; Phosphorus Radioisotopes; Progesterone; Protein Kinase C; Tamoxifen; Tumor Cells, Cultured; Uterine Neoplasms

The actions of insulin-like growth factors (IGFs) are modulated by interaction with a family of secreted binding proteins (IGFBPs). We have now demonstrated in intestinal epithelial cells (Caco-2) that the secretion of different members of this family depends on the cell surface secreting them. Polarized monolayers of cells secreted IGFBP-3 mainly into the medium adjacent to the apical surface, while IGFBP-2 was secreted predominantly through the basolateral surface. The secretion of IGFBP-1 and -4 was equivalent from both surfaces. However, administration of epidermal growth factor (EGF) induced polarized secretion of IGFBP-4 by increasing secretion from the apical surface more than from the basolateral aspect. It did not affect the polarity of the other IGFBPs. We believe that this is the first evidence that epithelial cells can interact with extrinsic agents in a polarized fashion at sites other than the membrane surface.

Topics: Adenocarcinoma; Carrier Proteins; Cell Line; Cell Membrane; Colonic Neoplasms; Epidermal Growth Factor; Epithelium; Humans; Insulin-Like Growth Factor Binding Protein 1; Insulin-Like Growth Factor Binding Protein 2; Insulin-Like Growth Factor Binding Protein 4; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Intestinal Mucosa; Molecular Weight; Recombinant Proteins; Tumor Cells, Cultured

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

Immunohistochemical staining for EGF, EGFR, c-erbB-2, p53, K-ras and PCNA was performed on the formalin-fixed, paraffin embedded sections of resected gastric carcinomas. A relatively high positive rate was observed for EGFR and c-erbB-2 in the well-differentiated adenocarcinomas and p53 in the poorly-differentiated adenocarcinomas. The positive rate of these factor was higher in the advanced cases than in the early cases, and also in the deep invasive area than the superficial area. According to the PCNA staining, a relatively high positive rate was observed in the well-differentiated adenocarcinomas compared with the early cases of poorly-differentiated adenocarcinomas, but the positive rate was markedly higher in the advanced cases of the latter. Typical signet-ring cell carcinomas showed the lowest positivity rate compared with the other histological types of gastric carcinomas.

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Epidermal Growth Factor; ErbB Receptors; Genes, p53; Genes, ras; Humans; Immunohistochemistry; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Stomach Neoplasms

Epidermal growth factor (EGF) elicits estrogen receptor (ER)-dependent physiological sequelae and estrogen-like biochemical effects on the ER in the mouse uterus. These in vivo observations indicate that EGF may elicit some of its actions by activation of the ER. The effect of peptide growth factors on activation of a consensus estrogen-responsive element was assessed in a strain of Ishikawa human endometrial adenocarcinoma cells with negligible levels of ERs, as determined by Western blot and [3H]estradiol binding, and in BG-1 human ovarian adenocarcinoma cells, which contain abundant ERs. EGF and transforming growth factor-alpha induced transcriptional activation of a consensus ERE in an ER-dependent manner in both cell types. Transcriptional activation by the growth factors was inhibited by ICI 164,384, an ER receptor antagonist, and neutralizing antibodies to the EGF receptor. Immunodetection of the ER in BG-1 cells demonstrated that receptor levels were not induced by transforming growth factor-alpha vs. untreated cells. ER deletion mutants containing amino acids 1-339 and 121-599 were transfected into Ishikawa cells. The 1-339 mutant was more active in inducing transcription after EGF treatment than the 121-599 mutant. Estrogen only stimulated transcription in the presence of the 121-599 mutant, while 1-339 was inactive. Interestingly, synergism between a physiological dose of estrogen and peptide growth factors was observed. The presence of cross-talk between EGF receptor and ER signaling pathways suggests that interactions between growth factors and steroid receptors may modulate hormonal activity influencing normal and aberrant function in mammalian cells.

Topics: Adenocarcinoma; Animals; Base Sequence; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Female; Gene Expression Regulation; Humans; Mice; Molecular Sequence Data; Ovarian Neoplasms; Polyunsaturated Alkamides; Promoter Regions, Genetic; Receptors, Estrogen; Recombinant Fusion Proteins; Recombinant Proteins; Regulatory Sequences, Nucleic Acid; Signal Transduction; Simian virus 40; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured; Uterine Neoplasms; Vitellogenins

The biological role of 1,25(OH)2D3 in controlling Ca++ homeostasis in the body has been identified and widely investigated for a long time. More recently its effect in regulating cell proliferation or differentiated activity was described in a variety of normal and malignant cells. The present study was carried out to investigate the different aspects and biological mechanisms of this activity and to determine if the use of 1,25(OH)2D3 in the treatment of breast cancer patients could be considered. It is found that 1,25(OH)2D3 reduces the proliferation of MCF-7 and BT-20 cells lines regardless of their sex steroid receptor status. This effect is related to the concentration, from 10(-12) M to 10(-8) M. Its amplitude is less in other cell lines, but it opposes the EGF-induced increase of proliferation. It is observed that the proliferation rate of MCF-7 and BT-20 cells is increased when these tumor cells are cocultured with fibroblasts derived from breast tumor biopsies and that 1,25(OH)2D3 reverses this process. Moreover, experiments on DMBA induced mammary tumors in Sprague Dawley rats found that 1,25(OH)2D3 given at non toxic doses reduces significantly the tumor proliferation. These data showed that 1,25(OH)2D3 at low doses is effective on the proliferation of BT-20 and MCF-7 cells and on the paracrine growth stimulatory effect observed in the presence of fibroblasts. They suggest that 1,25(OH)2D3 or related synthetic molecules which are less active on Ca++ metabolism could be useful in the treatment of breast cancer patients.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Adenocarcinoma; Animals; Breast Neoplasms; Calcitriol; Cell Division; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Mammary Neoplasms, Experimental; Rats; Rats, Sprague-Dawley; Tumor Cells, Cultured

We have studied the effects of growth factors and cytokines on the tumorigenicity and invasion capacity of tumor cells by using regressor and progressor tumor cell lines (ER-1 and ERpP, respectively) derived from an SHR rat mammary adenocarcinoma. ER-1 cells regress spontaneously whereas ERpP cells show invasive growth and high metastasis to lung and other organs in syngeneic SHR rats. When ER-1 cells were pretreated with either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) for 24 h in vitro, and intraperitoneally transplanted into SHR rats, they grew and killed the host, whereas ER-1 cells pretreated with tumor necrosis factor-alpha did not. Tumorigenicity and invasion capacity of ERpP cells were also enhanced by treatment with EGF and TGF-beta. The ER-1 cells pretreated with EGF, once grown in vivo, had acquired irreversible tumorigenicity and invasion capacity without requiring further EGF treatment, and the enhanced malignancy was irreversible. These findings suggest that growth factors play an important role in acquisition of malignancy of tumor cells.

Topics: Adenocarcinoma; Animals; Epidermal Growth Factor; Female; Mammary Neoplasms, Experimental; Neoplasm Invasiveness; Rats; Rats, Inbred SHR; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In this paper, we investigated how epidermal growth factor (EGF) acts on growth and regulation of extracellular matrix components and their degenerative enzymes in uterine cervical adenocarcinoma cells in vitro. Effects of EGF on cell growth, DNA synthesis, and laminin, collagen IV, and tissue plasminogen activator (t-PA) production of human uterine cervical adenocarcinoma cell line OMC-4 were examined, together with the characteristics of its EGF receptors. Scatchard plot of EGF binding to OMC-4 indicated two classes of binding sites with a dissociation constant of 170 pM and 510 pM. The total binding sites were 1.6 x 10(5)sites/cell. The number of OMC-4 cells did not increase in the presence of EGF, whereas 3H-thymidine incorporation was inhibited by EGF at concentrations of 1 and 10 nM. The production of laminin and collagen IV by OMC-4 cells was inhibited by EGF, whereas that of t-PA was significantly promoted at the physiological concentration of 0.1 nM. These results suggest that EGF is closely associated with regulation of proliferation and extracellular matrix degradation of uterine cervical adenocarcinoma cells.

Topics: Adenocarcinoma; Cell Division; Collagen; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Laminin; Tissue Plasminogen Activator; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Endometrial adenocarcinoma is the most common gynecologic malignancy occurring in the United States. Evidence is accumulating that links peptide growth factors with malignant proliferation. Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) are known mitogens for endometrial adenocarcinoma in vitro. However, the biological activity of other growth factors in this malignancy is unclear. This study was undertaken to determine the influence of growth factors on the mitogenic activity of the human endometrial adenocarcinoma cell lines HEC-1-A and KLE. Incubation with EGF, IGF-I, insulin-like growth factor II (IGF-II), or insulin stimulated a time-dependent mitogenic response in both cell lines, with the peak response occurring at 24 hr for HEC-1-A and 48 hr for KLE. After two doubling intervals, the number of HEC-1-A cells was increased 3.5-fold by EGF (100 ng/ml), 2.7-fold by IGF-I (100 ng/ml), 2.3-fold by IGF-II (100 ng/ml), and 2.2-fold by insulin (1000 ng/ml) when compared to untreated controls (P < 0.05). The number of KLE cells was increased 2.6-fold by EGF (100 ng/ml), 2.3-fold by IGF-I (100 ng/ml), 2.1-fold by IGF-II (100 ng/ml), and 2.0-fold by insulin (1000 ng/ml) when compared to untreated controls (P < 0.05). Similar results were obtained when DNA content was measured. PDGF failed to stimulate any mitogenic response in either cell line at all concentrations tested (0.1-100 ng/ml). These findings suggest that EGF, IGF-I, IGF-II, and insulin may play a regulatory role in the proliferation of endometrial adenocarcinoma.

Topics: Adenocarcinoma; Analysis of Variance; DNA, Neoplasm; Endometrial Neoplasms; Epidermal Growth Factor; Female; Growth Substances; Humans; Mitosis; Platelet-Derived Growth Factor; Somatomedins; Thymidine; Time Factors; Tumor Cells, Cultured

Immunohistochemical study for epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) was performed on paraffin-embedded tissue specimens from 39 colorectal and 24 gastric carcinomas. The carcinomas were placed in one of the following 3 groups: group 1, neither EGF nor EGFR was stained (11 gastric and 21 colorectal carcinomas); group 2, either EGF or EGFR was stained (4 gastric and 4 colorectal carcinomas); and group 3, both EGF and EGFR were stained (9 gastric and 14 colorectal carcinomas). Compared with the carcinomas in groups 1 and 2, those in group 3 had significantly higher rates of lymph node spread and serosal invasion of the gastrointestinal wall. In contrast, no significant differences were found between the EGF and/or EGFR expression and histological differentiation of carcinomas. These results suggest that gastrointestinal carcinomas expressing both EGF and EGFR display pathological features of more aggressive disease. Furthermore, the synchronous expression of EGF and EGFR indicates that these carcinomas may regulate their growth by an autocrine and/or paracrine mechanism.

Topics: Adenocarcinoma; Cell Differentiation; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Neoplasm Invasiveness; Retrospective Studies; Stomach Neoplasms

The in vitro effects of the epidermal growth factor (EGF) and progesterone on phospholipid turnover in cells of 19 human adenocarcinomas (postsurgical material) have been studied. In 58% of tumours EGF increased the 32P incorporation into two basic cell phospholipids--phosphatidylcholine and phosphoinositides. In EGF-insensitive cells progesterone induced no noticeable changes in the basal level of phospholipid metabolism. However, in 10 out of 11 positively responding to EGF adenocarcinomas progesterone inhibited the EGF-dependent activation of 32P incorporation into the phospholipids already on the 15th min after its addition to the cells. Analysis of effects of EGF and the anti-estrogen drug tamoxifen on phospholipid turnover in 22 human mammary tumours did not reveal any significant differences in tamoxifen effect on tumour cells differing in their sensitivity to EGF. Independently of cell sensitivity to EGF, tamoxifen caused some decrease in the 32P incorporation into phosphatidylcholine but increased the label incorporation into phosphoinositides. Tamoxifen added to tumour cells prestimulated with EGF or 17 beta-estradiol failed to abrogate the effect of these compounds on phospholipid turnover. At the same time, treatment of cells with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate fully inhibited the effect of tamoxifen on phospholipid metabolism. The results obtained suggest that the EGF-dependent activation of intracellular phospholipid turnover is under the negative control of progesterone. As for tamoxifen, its effect on cells is independent of EGF and consists, apparently, in the inhibition of protein kinase C activity.

Topics: Adenocarcinoma; Breast Neoplasms; Chromatography, Thin Layer; Epidermal Growth Factor; Estradiol; Female; Humans; Phospholipids; Phosphorus Radioisotopes; Progesterone; Tamoxifen; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Neoplasms

We report that cytosine arabinoside (Ara-C), a cytosine analogue that at low doses causes phenotypical changes on human leukemia cells in vitro and in vivo, induces growth inhibition of oropharyngeal cancer KB and lung adenocarcinoma A549 cell lines. An increase in the number of epidermal growth factor and transferrin receptors (EGFR, TrfR) is induced by Ara-C on these cells. Maximal EGFR up-regulation occurs 96 h after the beginning of Ara-C exposure while maximal TrfR up-regulation is detected 24 h later. These effects occur without changes in the affinity of EGFR and TrfR for their ligands. Two classes of EGF-binding sites with a Kd of 0.055 nM and 2.3 nM respectively, and one class of transferrin-binding sites with a Kd of about 4 nM are detected on both untreated and Ara-C-treated KB cells. [3H]Thymidine uptake is clearly stimulated on KB cells by nanomolar concentrations of EGF and transferrin, whereas in Ara-C-treated cells [3H]thymidine uptake is not increased by EGF and transferrin under conditions where maximal EGFR and TrfR up-regulation occurs. The enhanced EGF and transferrin binding is paralleled by a twofold increase of in vitro targeting of Ara-C-treated KB and A549 cells with anti-EGFR 108.1 mAb and anti-TrfR OKT9 mAb. We propose that Ara-C could provide a new approach for the improvement of the therapeutic index of anti-EGFR and anti-TrfR immunoconjugates.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Cell Division; Cytarabine; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy; Iodine Radioisotopes; KB Cells; Lung Neoplasms; Neoplasms; Radioimmunoassay; Receptors, Cell Surface; Receptors, Transferrin; Time Factors; Transferrin; Tumor Cells, Cultured; Up-Regulation

To study the early effects of steroid hormones on cells we investigated the influence of the sex steroids and tamoxifen on phospholipid turnover in endometrial carcinoma and breast cancer cells. Studies were performed on 19 human uterine adenocarcinomas and 29 breast cancer tumors. Progesterone in a final concentration of 10(-7) mol/l caused a twofold decrease of 32P incorporation into phospholipids (phosphatidylcholine and phosphoinositides) in 85% of the uterine adenocarcinomas where the progesterone receptor (PR) content was more than 100 nmol/kg and only in 30% of the tumors where the PR content was less than 100 nmol/kg. Treatment of the cells with 10(-8) mol/l 17 beta-estradiol or 10(-8) mol/l epidermal growth factor led to an increase in 32P incorporation into phospholipids. Analysis of the hormonal responsiveness of 29 human breast cancers showed that 17 beta-estradiol increased 32P incorporation into phospholipids in 47% of the tumors where the estradiol receptor (ER) content was more than 10 nmol/kg and in 21% of the receptor-negative tumors (ER < 10 nmol/kg) The results show that phospholipid turnover in uterine and breast cells can be regulated by sex steroids. Treatment of the breast cancer cells with the antiestrogen tamoxifen (10(-6) mol/l) led to an increase of 32P incorporation into phosphoinositides and a decrease of 32P incorporation into phosphatidylcholine. Addition of an activator of protein kinase C, i.e. 2 x 10(-7) mol/l 12-0-tetradecanoylphorbol-13-acetate, weakened the inhibitory effect of tamoxifen on phosphatidylcholine turnover. These findings suggest that tamoxifen action can be mediated via an alteration of the growth signal transducing system.

Topics: Adenocarcinoma; Breast Neoplasms; Endometrial Neoplasms; Epidermal Growth Factor; Estradiol; Female; Gonadal Steroid Hormones; Humans; Neoplasms, Hormone-Dependent; Phosphatidylcholines; Phosphatidylinositols; Phospholipids; Progesterone; Tamoxifen; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Galactosyl beta-1,3-N-acetyl galactosamine (Gal beta-1,3-GalNAc) (Thomsen Friedenreich antigen), the Class I core sequence in O-linked oligosaccharide chains, behaves as an oncofetal antigen showing increased expression in many epithelial malignancies. Previous work has shown that peanut agglutinin (PNA), a lectin that binds Gal beta-1,3-GalNAc, stimulates proliferation in HT-29 (human colon cancer) cells and normal human colonic epithelium and this implies that cell surface glycoproteins which express Gal beta-1,3-GalNAc may play an important role in the regulation of epithelial cell proliferation. We have now studied the effect on epithelial cells of another dietary Gal beta-1,3-GalNAc-binding lectin, the edible mushroom Agaricus bisporus lectin (ABL). This differs from PNA in its ability to bind also to sialylated Gal beta-1,3-GalNAc. In contrast to PNA, ABL (25 micrograms/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87% (95% confidence limit, 85-89%), Caco-2 colon cancer cells by 16% (95% confidence limit, 12-20%), MCF-7 breast cancer cells by 50% (95% confidence limit, 47-52%), and Rama-27 rat mammary fibroblasts by 55% (95% confidence limit, 51-60%) when these cells were grown for 24 h in serum-free medium. When assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. In the presence of 2% fetal calf serum (which contains the ABL-binding glycoprotein fetuin), the inhibitory effect of ABL on cell proliferation was still demonstrable but at increased ABL concentration (60 micrograms/ml for 49% inhibition). Ten micrograms/ml ABL completely abolished the stimulatory effect on [3H]thymidine incorporation of epidermal growth factor (100 pg/ml) and PNA (25 micrograms/ml) and markedly inhibited the stimulatory effect of insulin (50 ng/ml). ABL (0.2 mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50 micrograms/ml ABL was reversible after removal of the lectin. Binding studies with 125I-labeled ABL suggested a single class of binding site with an apparent Kd value of (4.12 +/- 0.29) x 10(-7) M with (3.6 +/- 0.3) x 10(7) binding sites/cell. A. bisporus lectin is a reversible noncytotoxic inhibitor of epithelial cell proliferation which deserves study as a potential agent for cancer therapy.

Topics: Adenocarcinoma; Adult; Agaricus; Aged; Animals; Arachis; Binding Sites; Breast Neoplasms; Carbohydrate Sequence; Cell Division; Cell Membrane; Cell Survival; Colonic Neoplasms; Disaccharides; DNA, Neoplasm; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial Cells; Epithelium; Female; Humans; Lectins; Male; Mammary Glands, Animal; Molecular Sequence Data; Peanut Agglutinin; Plant Lectins; Rats; Thymidine; Tumor Cells, Cultured

In the highly metastatic rat mammary adenocarcinoma cell clone MTLn3, EGF induced increased adhesion to fibronectin while in the human epidermoid carcinoma cell line A431 EGF induced diminished adhesive properties. Flattening of cells with extensive formation of filopodia was observed in MTLn3 cells within 5 min of EGF addition, while in A431 cells EGF induced rounding up and only occasional formation of filopodia. Immunofluorescent analysis revealed extension of microtubules (MT) into the filopodia and Western blot analysis demonstrated an EGF-induced 2- to 3-fold increase in the amount of assembled tubulin in MTLn3 but not in A431 cells. In MTLn3, but only marginally in A431 cells, EGF treatment resulted in phosphorylation of a 280 kD cytoskeleton-associated protein, which was rapid and dose-dependent. These results suggest differential signal transduction pathway of cytoskeleton-associated EGFRs in highly metastatic MTLn3 as compared with A431 cells.

Topics: Adenocarcinoma; Animals; Cell Adhesion; Cell Division; Cytoskeleton; Epidermal Growth Factor; ErbB Receptors; Humans; Mammary Neoplasms, Experimental; Neoplasm Proteins; Phosphorylation; Rats; 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

Lipocortin-1 mediates growth inhibition of glucocorticoids in A549 cells by suppressing the release of PGE2 necessary for their proliferation. We now show that 2 peptide fragments derived from the N-terminal portion of lipocortin-1 corresponding to amino-acids 13-25 and 21-33 also inhibited A549 cell growth and suppressed release of PGE2, whereas peptides 1-12 and 13-25 (Phe21; in which the tyrosine at position 21 was replaced by a phenylalanine residue) were inactive. Similarly, peptide 21-33 (Phe21) and a scrambled sequence of 13-25 failed to inhibit cell growth. Moreover, the EGF-induced stimulation of cell proliferation and PGE2 release in these cells was blocked by peptides 13-25 and 21-33, and also by peptides 1-12, 13-25 (Phe21) and 21-33 (Phe21), but not by a scrambled sequence of peptide 13-25.

Topics: Adenocarcinoma; Amino Acid Sequence; Annexin A1; Cell Division; Dinoprostone; Epidermal Growth Factor; Humans; Lung Neoplasms; Molecular Sequence Data; Peptide Fragments; 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

Basic fibroblast growth factor (bFGF) has been shown to be mitogenic to many different eukaryotic cell lines of mesodermal and neuroectodermal origin. Addition of exogenous bFGF to the chemically defined media of five characterized human colon tumor cell lines, cultured in the absence of epidermal growth factor (EGF), resulted in stimulation of growth from 24% to 146% in four of five cell lines, as measured by a colorimetric MTT assay. A positive dose-response relationship was observed when colon cells were treated with bFGF concentrations from 1 pM to 1 nM. bFGF showed a cumulative effect with EGF in stimulating the proliferation of colon tumor cells. The growth-inhibitory effect of exogenous transforming growth factor-beta (TGF-beta) on these cells was abolished by bFGF. When colon tumor cells were examined on immunoblots with a fibroblast growth factor (FGF) receptor-specific antibody, bands were detected at apparent molecular weights of 131 and 145 kDa. Conditioned media and cell lysates from the same human colon tumor cell lines were immunoprecipitated with a bFGF-specific antibody. An immunoreactive band was detected that comigrated with authentic human recombinant bFGF (16 kDa). Furthermore, preabsorption of anti-bFGF antibody with authentic ligand blocked immunodetection of the 16 kDa band on immunoblots. Documentation of a bFGF response, receptor, and ligand expression in human colon tumor cell lines is novel, and may represent a more widespread role for FGF that extends to epithelial cells and tumors of endodermal germ layer origin. The expression of both ligand and receptors by these cells indicates that bFGF could be involved in their growth regulation at the autocrine level.

Topics: Adenocarcinoma; Blotting, Western; Colonic Neoplasms; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Ligands; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

The conventional assessment of the premalignant potential of Barrett's oesophagus is unsatisfactory. However, it has recently been shown that abnormalities of growth-regulatory peptides and their receptors may be important in the pathogenesis of this condition. In an attempt to improve the diagnostic and prognostic criteria we have studied 21 consecutive patients with Barrett's oesophagus and 7 others with adenocarcinoma of the oesophagus. In each patient biopsy specimens were taken from the columnarlined oesophagus or the adenocarcinoma and from the gastric cardiac mucosa for routine histologic evaluation. Immediately adjacent specimens were taken from both the Barrett's mucosa or adenocarcinoma and from the gastric mucosa for flow-cytometric study. The latter samples were disaggregated and labelled with antibodies to epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and epidermal growth factor receptor (EGF-R). The flow cytometer selected cells labelled with each antibody and expressed them as a percentage of the total number of disaggregated cells (average, 5500 cells). Epidermal growth factor receptors were expressed in a greater number of cells from Barrett's mucosa, with the intestinal type or those with dysplasia, than in gastric cardiac mucosa (p less than 0.05). All seven adenocarcinoma had many more cells expressing EGF, TGF-alpha, and EGF-R than normal gastric mucosa (p less than 0.01). We conclude that flow-cytometric evaluation of EGF-R can help in the understanding of the pathogenesis of Barrett's oesophagus.

Topics: Adenocarcinoma; Adult; Aged; Autoantigens; Barrett Esophagus; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Flow Cytometry; Humans; Middle Aged; Nuclear Proteins; Proliferating Cell Nuclear Antigen; Transforming Growth Factor alpha

Two new human cholangiocarcinoma (CC) cell lines (CC-SW-I and CC-LP-I) were established and maintained in culture for 2 years. Histologically, both original liver tumors were adenocarcinomas, and the cell lines exhibited morphologic features of moderately differentiated adenocarcinoma. Immunohistochemistry showed that both cell lines were strongly positive for cytokeratin AEI but negative for carbohydrate tumor-associated antigen, CA19-9. Ultrastructural analysis of both cell lines showed the presence of tight junctional complexes and focally formed microvilli. Both CC cell lines were tumorigenic in nude mice. Cytogenetic analysis showed that both cell lines expressed highly aneuploid karyotypes with numerous structural and numerical deviations. CC-SW-I was hypodiploid with numerous chromosome losses and structural rearrangements, while CC-LP-I was hyperdiploid and displayed multiple additional chromosomes. Doubling times for the CC-SW-I and CC-LP-I cell lines in the presence of 15% fetal bovine serum were 72 hr and 180 hr, respectively. Growth of the CC-SW-I cell line was significantly stimulated in the presence of insulin, while that of the CC-LP-I cell line was significantly augmented by epidermal growth factor (EGF). In contrast, dexamethasone strongly inhibited proliferation of both cell lines in a dose-dependent manner. Among various recombinant cytokines examined for effects on growth or surface antigen expression on CC cell lines, only interleukin I-beta (ILI-beta) strongly inhibited growth of the CC-LP-I cell line, while interferons (IFNs) or tumor necrosis factor-alpha (TNF-alpha) were mildly inhibitory. Both tumor cell lines were resistant to natural killer (NK) cells but sensitive to lymphokine-activated killer (LAK) cells. Preincubation of tumor cells with IFN-gamma, IFN-alpha or TNF-alpha significantly decreased the susceptibility of each tumor cell line to lysis by LAK cells, and the change in sensitivity did not correlate with the expression of HLA antigens or intercellular adhesion molecule-I (ICAM-I) on the surface of tumor cells. These 2 CC cell lines are expected to provide valuable information about cell biology of human CC.

Topics: Adenocarcinoma; Adenoma, Bile Duct; Animals; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Division; Dexamethasone; Epidermal Growth Factor; Female; Glucagon; Histocompatibility Antigens Class I; Humans; Insulin; Insulin-Like Growth Factor I; Intercellular Adhesion Molecule-1; Karyotyping; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Transplantation, Heterologous; Tumor Cells, Cultured

Ascites sublines of the 13762 rat mammary adenocarcinoma have a cell surface sialomucin complex composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the membrane-associated glycoprotein ASGP-2. The sialomucin complex is synthesized as a high M(r) precursor, pre-sialomucin complex (pSMC-1). To characterize the structure of the membrane-associated component of this complex, a lambda gt11 cDNA expression library was constructed using mRNA from 13762 rat mammary adenocarcinoma cells and screened with polyclonal antibody against ASGP-2. The strongest antibody-binding clone, designated lambda ASGP2.9-1, had a 1.3-kilobase (kb) insert, and hybridized to a 9-kb transcript in 13762 cell mRNA. The large size of this transcript was expected, since the estimated molecular mass of pSMC-1 is greater than 250 kDa. To obtain the full sequence of ASGP-2, a longer cDNA (5.4 kb), designated pASGP1/2.1, was subsequently cloned by screening a plasmid library with an oligonucleotide complementary to the 5' end of the phage insert. The amino acid sequence derived from nucleotide sequence of pASGP1/2.1 showed a 12-amino acid identity with amino acid sequence obtained from the NH2 terminus of ASGP-2, indicating the entire ASGP-2 coding region was included in the cDNA. Furthermore, an 18-amino acid identity with the NH2 terminus of a 6-kDa CNBr fragment of ASGP-2 was also observed in the cDNA sequence. The polypeptide contains several distinct domains, including a hydrophobic transmembrane domain, a short (20 residue) COOH-terminal cytoplasmic tail, and a large extracellular domain with 24 potential N-glycosylation sites. These properties correspond to features of ASGP-2 and pSMC-1 predicted by previous biochemical studies. Most interestingly, the extracellular domain contains two cysteine-rich sequences, each of which has a segment with strong similarities to proteins with epidermal growth factor activity. Since our recent studies show that ASGP-2 can modulate epidermal growth factor receptor phosphorylation activity, these results provide structural evidence to support the role of the heterodimeric sialomucin complex as a bifunctional modulator of cellular interactions and cell proliferation.

Topics: Adenocarcinoma; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA, Neoplasm; Epidermal Growth Factor; Gene Library; Humans; Mammary Neoplasms, Experimental; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Mucins; Multigene Family; Poly A; Protein Conformation; Rats; Recombinant Fusion Proteins; RNA; RNA, Messenger; RNA, Neoplasm; Sequence Homology, Nucleic Acid; Sialomucins; Tumor Cells, Cultured

A human adenocarcinoma cell line designated as GAC-1, was established from ascites of the 56-year old male patient with rapidly progressive gastric cancer. The doubling time was about 18.5 hours in vitro, and cell cycle analysis using flow cytometry showed marked increase of S phase (46.1%). Immunohistochemical demonstration of GAC-1 cells revealed positive staining of TGF-alpha, EGF, EGF-R, FN-R, laminin and negative staining of fibronectin. Histogram of them indicated aneuploidy with modal number 57 and they formed tumors in nude mice.

Topics: Adenocarcinoma; Animals; Cell Cycle; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Karyotyping; Laminin; Male; Mice; Middle Aged; Receptors, Fibronectin; Stomach Neoplasms; Tumor Cells, Cultured

In this study, 3 human pancreatic adenocarcinoma cell lines (PC-1, PC-2 and PC-3) and 3 other human cancer cell lines (adenocarcinoma of lung, LETP; gastric carcinoma, SGL7901; and breast carcinoma, BCP37) were investigated by adding EGF, anti-EGF antiserum and anti-EGFR monoclonal antibody into culture medium. EGF was found to exert a mild stimulating effect on the growth of PC-1 and LETP cells, but had no effect on the other 4 cell lines. Anti-EGF and anti-EGFR antibodies inhibited the proliferation of PC-1, LETP and SGL7901 cells. No significant effect on the other 3 cell lines was seen. By using the Northern blot technique, expression of EGFR mRNA was identified in all 6 cell lines. There were 3 bands (10.5 kb, 5.8 kb and 2.8 kb) of EGFR mRNA in all cell lines except for LETP, in which the 10.5 kb band was absent. The results indicate that the effect of EGF on the growth of cancer cells is very complicated and may involve an unknown regulatory mechanism of cancer cell growth. EGF may exert stimulating or inhibiting effects on cancer cell proliferation, or it may have no effect at all, even though the EGFR gene was expressed in these cell lines.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Immune Sera; Lung Neoplasms; Pancreatic Neoplasms; RNA, Messenger; Stomach Neoplasms; Tumor Cells, Cultured

Epidermal growth factor (EGF), at 10(-11) M and 10(-10) M, stimulated [methyl-3H]thymidine incorporation into DNA and cell growth of R3230AC mammary adenocarcinomas in primary cultures, whereas at higher concentrations (10(-9) M and 10(-8) M) EGF inhibited DNA synthesis and cell growth in vitro. To determine whether these responses were receptor-mediated, 125I-EGF binding to freshly dissociated cells and primary cultures of R3230AC cells was measured and found to be time- and temperature-dependent. Specificity of EGF binding was demonstrated by 50% displacement occurring at an EGF concentration of 0.46 nM. Using 125I-EGF concentrations from 0.05 nM to 10 nM, saturable binding sites were documented; Scatchard analysis of these data produced curvilinear plots, suggesting the presence of high affinity (0.44-0.93 nM) and low affinity (1.5-4.8 nM) sites. 125I-EGF was rapidly internalized by cultured R3230AC tumor cells. By 30 min, 73% (25 degrees C) and 77% (37 degrees C) of total 125I-EGF was internalized (resistance to acid/salt extraction), whereas at 4 degrees C, cells internalized only 20% of EGF over a 3 hr incubation period. Following incubation with 125I-EGF for 2 hr at 4 degrees C, 25 degrees C, or 37 degrees C, the majority of cell-associated radioactivity eluted with intact 125I-EGF. However, when the material that dissociated from R3230AC cells after the 2 hr incubation was analyzed, 38% (25 degrees C) and 46% (37 degrees C) of the radioactivity migrated as lower molecular weight products, indicating that 125I-EGF was partially degraded intracellularly by R3230AC cells in primary culture. Pre-incubation of cells in primary culture with EGF (1-100 nM) for 30 min at 37 degrees C, led to "down-regulation" of EGF receptors; 1 nM EGF reduced the specific binding of 125I-EGF by 54% with higher concentrations (10 nM and 100 nM) reducing it further. Scatchard analysis of down-regulated cells showed a reduced number of high affinity binding sites with no change in the Kd of binding. Sialoadenectomy of rats had no effect on R3230AC tumor growth or EGF receptor levels in the tumor, liver, or uterus. Experiments to determine whether perturbations of the insulin milieu or ovariectomy would alter EGF receptors were performed. 125I-EGF binding was significantly elevated in tumors from diabetic rats (152% increase vs controls) and binding was returned to control (77% of intact rats) levels after administration of insulin to diabetic rats.(ABSTRACT TRUNCATED AT 400

Topics: Adenocarcinoma; Animals; Cell Division; Cell Line; Cell Membrane; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Female; Insulin; Insulin-Like Growth Factor I; Kinetics; Liver; Mammary Neoplasms, Experimental; Radioligand Assay; Rats; Rats, Inbred F344; Salivary Glands; Thymidine; Tumor Cells, Cultured; Uterus

The role of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) in the growth modulation of three human ovarian adenocarcinoma cell lines, PEO1, PEO4 and PEO14, has been examined by measuring responses of the cells growing in monolayer culture to exogenous addition of the growth factors. The presence of EGF receptors in the cell lines has been confirmed by ligand binding and immunocytochemical staining using a monoclonal antibody directed against the EGF receptor. The growth of all three cell lines was stimulated by both EGF and TGF-alpha. Dose-response effects were noted with the greatest growth stimulation occurring at concentrations between 0.1 and 10 nmol/l. The stimulatory effects of EGF and TGF-alpha were accompanied by changes in the cell cycle distribution as detected by flow cytometric analysis. It is concluded that EGF and TGF-alpha are important growth regulators in these EGF-receptor positive ovarian cancer cells.

Topics: Adenocarcinoma; Cell Cycle; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Neoplasm Proteins; Ovarian Neoplasms; Transforming Growth Factor alpha

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; DNA; Endometrial Neoplasms; Epidermal Growth Factor; Estrogen Antagonists; Female; Humans; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The spontaneous expression of HLA class I and class II molecules in two human breast carcinoma cell lines (MCF7, T47D) and their modulation during epidermal growth factor treatment are reported. Transcription was analysed by Northern blot and hybridisation with HLA class II and class I cDNA specific probes. The expression of cell surface determinants was examined by internal protein labelling with 35s-methionine, immunoprecipitation with monoclonal antibodies specific for HLA class I or class II, followed by isolation of the immune complex on protein A-Sepharose; at least a quantification of glycoprotein was performed by chromatofocusing. Glycoprotein quantification showed a significant increase of HLA class I and class II (DR) antigen expression after stimulation by epidermal growth factor (0.02 microgram ml-1) in the two cell lines, when compared with untreated cell controls. However, with epidermal growth factor treatment of MCF7 and T47D cells, low increases in the amounts of HLA class I and class II RNA were obtained. These differences between expressed antigens and correspondent RNA amounts would be explained by the fact that EGF in these two cell lines acts more in post-transcription for HLA class I and class II antigens.

Topics: Adenocarcinoma; Blotting, Northern; Breast Neoplasms; Cell Line; DNA Probes; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Genes, MHC Class I; Genes, MHC Class II; Histocompatibility Antigens Class I; HLA-D Antigens; Humans; RNA, Neoplasm

Transforming growth factor alpha production by renal tumors, acting through the epidermal growth factor receptor, has been implicated in malignant transformation by studies which compared gene expression in neoplastic and normal human tissue. We sought confirmation of this hypothesis by measuring the growth responses of a human renal tumor cell line to the addition of epidermal growth factor and transforming growth factor alpha. Surprisingly, it was found that both growth factors could induce either mitogenic or inhibitory signals depending on the growth status of the cultures. Confluent cultures were stimulated by both growth factors, and nonconfluent cultures were inhibited, as determined by thymidine incorporation, cell cycle analysis, and direct cell counting. These signals appear to use different transduction pathways, as growth factor induced inhibition was reversed by Bordetella pertussis toxin (which affects G protein signaling), whereas the stimulatory effects were not reversed. Two clones isolated from these cells responded in the same manner as the main cell isolate. These data show that the same cell may display opposite responses to equivalent concentrations of the same growth factor, depending on the transduction pathway used after triggering by receptor occupancy of either ligand (epidermal growth factor or transforming growth factor alpha).

Topics: Adenocarcinoma; Cell Count; Cell Division; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Kidney Neoplasms; Pertussis Toxin; Transforming Growth Factor alpha; Tumor Cells, Cultured; Virulence Factors, Bordetella

In this study, evidence was obtained that endothelin-1 (ET-1) is produced by an established endometrial cancer (HEC-1A) cell line. PreproET-1 mRNA is present in HEC-1A cells, and immunoreactive endothelin is secreted into the medium of these cells maintained in culture. Cycloheximide treatment of these cells caused superinduction of preproET-1 mRNA. Transforming growth factor-beta acts in these cells to increase the levels of preproET-1 mRNA. This effect of transforming growth factor-beta on preproET-1 mRNA accumulation was accompanied by an increase in the amount of immunoreactive endothelin secreted into the culture medium. ET-1, added to the culture medium, did not act as a mitogen in HEC-1A cells. We speculate that ET-1 (which is known to stimulate fibroblast proliferation) produced by endometrial adenocarcinoma cells may participate in the angiogenic process that occurs during the establishment of this carcinoma in vivo.

Topics: Adenocarcinoma; DNA Replication; DNA, Neoplasm; Endometrial Neoplasms; Endothelin-1; Endothelins; Epidermal Growth Factor; Female; Fibroblast Growth Factors; Gene Expression; Humans; Insulin; Interleukin-1; Kinetics; Platelet-Derived Growth Factor; Protein Precursors; RNA, Messenger; Transforming Growth Factor beta

Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Base Sequence; Cell Division; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Sequence Data; Radioimmunoassay; Radioligand Assay; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

A panel of rat colon adenocarcinoma cell lines (the Per series) were used to investigate the phenotype and karyotype changes induced by in vivo passage in the subcutis of athymic nude mice. One poorly and one well-differentiated tumor cell line were serially passaged through the athymic nude mouse and then back to the syngeneic rat host. Each of the primary and xenograft cell lines expressed fetal crypt cell ("CaCo") antigens. The well differentiated primary and xenograft lines (Per305, Per305N1, and Per305N2a) were different in each of their growth factor responsiveness in vitro [i.e. epidermal growth factor (EGF), bombesin, vasoactive intestinal peptide], their EGF receptor expression, their secretion of transforming growth factor-alpha, and their exhibition of anchorage independent (A-I) growth capabilities. The poorly differentiated primary and xenograft cell lines were also different but were all capable of A-I growth; their responsiveness to exogenous growth factor stimulation decreased with progressive in vivo passage, as did their basal unstimulated proliferation rate. Cytogenetic alterations detected were those associated with clinical specimens from various stages of malignancy, i. e. aneuploidy, structural aberrations, and marker chromosomes. Genetic and mitogenic individuality of each line demonstrated the diversity of the growth control mechanisms in neoplasms at different stages of progression.

Topics: Adenocarcinoma; Animals; Biomarkers, Tumor; Bombesin; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Karyotyping; Male; Mice; Mice, Nude; Neoplasm Transplantation; Phenotype; Receptors, Bombesin; Receptors, Neurotransmitter; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Retinoic acid (RA) binding has been detected in the nuclei of a subclone (CL-1) of human submandibular adenocarcinoma cell line HSG conditioned to grow in a serum-free defined medium. Competition assay confirmed the specificity of the RA binding. Scatchard analysis showed the binding molecule to have a high affinity and low capacity. From the analyses by gel-filtration and glycerol density gradient centrifugation, the nuclear binding molecule appears to be distinct from cellular RA binding protein (CRABP) in terms of molecular weight. Furthermore, immunoblotting analysis revealed a band (Mr 47,000) reactive with specific antibody to RA receptor (RAR) alpha in the gel containing the nuclear fraction of CL-1 cells. Northern blotting analysis with specific cDNA probes revealed the expression of RAR alpha and RAR gamma in CL-1 cells. These results indicate that CL-1 cells express two types of RAR subtype, suggesting that these receptor molecules may mediate biological effects of RA. Treatment of CL-1 cells with RA resulted in an increase in the incorporation of [3H]thymidine into TCA-insoluble materials. The maximal increase was observed at 10(-6) M around 48 h. Previously, we demonstrated the autocrine growth of HSG cells mediated by epidermal growth factor (EGF) receptors and EGF-like molecules (Kurokawa et al. (1989) Cancer Res. 49, 5136-5142) and showed that RA had no significant effect on the secretion of the EGF-like molecule. RA induced an increase in [125I]EGF binding to CL-1 cells. The increase in the EGF binding was maximal at 24 h at 10(-6) M RA. RA also increased the amount of [3H]leucine-labeled EGF receptor dose-dependently. No significant change was observed in total protein synthesis of CL-1 cells by treatment with RA. These results suggest that RA stimulates the growth of CL-1 cells by increasing EGF receptor levels.

Topics: Adenocarcinoma; Carrier Proteins; Cell Division; Cell Nucleus; Clone Cells; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Receptors, Retinoic Acid; RNA, Messenger; Salivary Gland Neoplasms; Tretinoin; Tumor Cells, Cultured

1. In order to assess potential abnormalities in the control of mucosal proliferation, 30 patients with Barrett's oesophagus were studied in order to evaluate the presence and distribution of epidermal growth factor, transforming growth factor-alpha and epidermal growth factor receptor to determine the Ki-67 labelling index in the affected oesophageal mucosa. Serial sections were analysed immunohistochemically. Ten of the patients had adenocarcinoma in the Barrett's mucosa and the other 20 had differing histological types of Barrett's mucosa (10, intestinal-type; 10, fundic- or cardiac-type). 2. The expression of transforming growth factor-alpha, epidermal growth factor and epidermal growth factor receptor was increased and the Ki-67 labelling index was higher in Barrett's mucosa compared with normal gastric mucosa. The 'intestinal-type' of Barrett's mucosa had the greatest expression of transforming growth factor-alpha, epidermal growth factor receptor and the highest Ki-67 labelling index compared with the other types of Barrett's metaplasia. Five cases of 'intestinal-type' Barrett's metaplasia had especially high Ki-67 labelling indices and these patients over-expressed both transforming growth factor-alpha and epidermal growth factor receptor. The patients with adenocarcinomas in the Barrett's mucosa also over-expressed transforming growth factor-alpha and epidermal growth factor receptor, but not epidermal growth factor, compared with normal gastric mucosa. 3. In conclusion, both normal gastric mucosa and Barrett's mucosa have potential autocrine growth regulatory mechanisms, but Barrett's mucosa has increased expression of both of the measured ligands and of the epidermal growth factor receptor.

Topics: Adenocarcinoma; Adult; Aged; Barrett Esophagus; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Female; Gastric Mucosa; Humans; Male; Middle Aged; Mucous Membrane; Transforming Growth Factor alpha

Overexpression of the epidermal growth factor receptor (EGFR) has been reported as an important molecular abnormality in human pancreatic cancer. There is in vitro evidence that simultaneous overproduction of one of its ligands, transforming growth factor alpha (TGF-alpha), might result in an autocrine loop with an increased proliferation signal. We analysed by immunocytochemical staining a retrospective series of human pancreatic cancers, chronic pancreatitis, and normal fetal and adult pancreatic tissues for the presence of TGF-alpha and epidermal growth factor (EGF). Ductal epithelial cells showed TGF-alpha immunoreactivity in both normal tissue and chronic pancreatitis, and 95 per cent of tumours showed strong immunoreactivity. In contrast, EGF immunoreactivity was not found in normal pancreas, but was expressed in 12 per cent of pancreatic carcinomas. Well-defined areas of EGF immunoreactivity in exocrine ducts showing reactive changes in pancreatitis might represent a benign response to tissue damage similar to that previously described in the gastric mucosa.

Topics: Adenocarcinoma; Chronic Disease; Epidermal Growth Factor; ErbB Receptors; Fetus; Humans; Immunoenzyme Techniques; Pancreas; Pancreatic Neoplasms; Pancreatitis; Staining and Labeling; Transforming Growth Factor alpha

Amphiregulin (AR) and cripto are proteins that are structurally related to epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). AR is also functionally related to this family of growth regulatory molecules and is able to bind and activate the 170-kDa EGF receptor (EGFR). Human EGFR-3 (HER3)/ERBB3 is a recently identified protein related to the EGFR that is widely expressed in breast carcinomas and is a candidate receptor for EGF-like growth factors. Differential expression of these putative ligands and receptors in transformed cells suggests that they may function in an autocrine manner to regulate tumor cell growth. Specific mRNA transcripts for TGF-alpha [4.8 kilobases (kb)], AR (1.4 kb), cripto (2.2 kb), and HER3 (6.2 kb) were expressed in a majority of human colon cancer cell lines. HER3 mRNA was detected in 55% of primary or metastatic human colorectal carcinomas but in only 22% of normal colon mucosa and 32% of normal liver samples. In contrast, cripto and AR mRNA were expressed in 60-70% of primary or metastatic human colorectal cancers but in only 2-7% of normal human colonic mucosa. Immunostaining also detected AR protein in primary and metastatic colorectal tumors but not in normal colon or uninvolved liver. These findings suggest that cripto and AR may be useful markers to discriminate between normal and malignant colonic epithelium and may provide a selective growth advantage for colorectal carcinomas.

Topics: Actins; Adenocarcinoma; Amphiregulin; Blotting, Northern; Cell Line; Colon; Colonic Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Liver; Membrane Glycoproteins; Neoplasm Proteins; Restriction Mapping; RNA; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha

We examined 35 primary human ovarian adenocarcinomas for the presence of epidermal growth factor (EGF) receptor (EGFR) in plasma membranes from cancer tissues by using 125I-EGF as a ligand. Specific 125I-EGF bindings were observed in 20 (57%) of these 35 cases. Scatchard analysis showed a class of high affinity EGF receptor: Kd 5.0 +/- 1.0 x 10(-10) M; Bmax, 83.3 +/- 12.1 fmol/mg protein (mean +/- SE, n = 20). Northern analysis in polyadenylated RNA from 15 EGFR(+) cancers using pretransforming growth factor alpha (pre-TGF alpha), prepro-EGF complementary DNA, and pE7, a complementary DNA clone of human EGFR, as probes revealed that pre-TGF alpha and EGFR mRNAs but not prepro-EGF mRNA were expressed in all cases examined. Immunocytochemical studies using monoclonal antibodies (mAbs) against TGF alpha, EGF, and EGFR showed that TGF alpha and EGFR but not EGF proteins were present on ovarian cancer cells in all cases. These data suggested a possible TGF alpha/EGFR autocrine mechanism in EGFR(+) ovarian cancers. We, therefore, examined the biological significance of this autocrine mechanism by using primary monolayer cell cultures. In primary cultures from EGFR (+) cancers, TGF alpha added to the culture medium stimulated the [3H]thymidine incorporation dose dependently. Moreover, the addition of mAbs against TGF alpha and EGFR but not EGF inhibited [3H]thymidine incorporation dose dependently in EGFR(+) cancer cells. On the other hand, in primary cultures from EGFR(-) cancers, TGF alpha and anti-TGF alpha, -EGFR, and -EGF mAbs did not show any effects on [3H]thymidine incorporation. All these results suggested the possible crucial role of a TGF alpha/EGFR autocrine growth mechanism in primary human ovarian cancers which express EGFR.

Topics: Adenocarcinoma; Blotting, Northern; DNA; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Humans; Ovarian Neoplasms; RNA; Transcription, Genetic; Transforming Growth Factor alpha; Tumor Cells, Cultured

The growth-promoting effect of several hormones and growth factors on eight human colon tumor cell lines (SW 48, SW 403, SW 480, SW 620, SW 948, HT29, LS174T and Caco-2) was studied using seven different chemically defined serum-free media [GF3: Chee's essential medium plus insulin, transferrin and selenium; GF3F: GF3 plus fetuin; GF4: GF3 plus linoleic acid/bovine-serum albumin (BSA); GF5: GF4 plus fetuin, GF5E, GF5 plus EGF; GF5T: GF5 plus triiodothyronine; GF7: GF3 plus EGF, transferrin, insulin, linoleic acid/BSA, oleic acid/BSA and fetuin]. GF5 appears to be the best serum-free medium as it supported continuous growth of all of the colon tumor cell lines. GF5 also supported growth of five of the seven human colon and stomach tumor xenografts as primary tissue cultures. However, the stomach xenograft cells had a very slow growth rate as compared to the colon xenograft cells in the medium. Cells grown in GF5 retained their tumorigenicity in athymic (nude) mice and characteristic cellular morphology. GF7 was the poorest of all of the serum-free media studied as none of the cell lines or xenografts grew in this medium.

Topics: Adenocarcinoma; alpha-Fetoproteins; Cell Division; Cell Line; Colonic Neoplasms; Culture Media, Serum-Free; Culture Techniques; Epidermal Growth Factor; Humans; Insulin; Linoleic Acid; Linoleic Acids; Neoplasm Transplantation; Oleic Acid; Oleic Acids; Transferrin; Triiodothyronine; Tumor Cells, Cultured

Production of immunoreactive (ir-) endothelin-2 (ET-2) in renal adenocarcinoma cells, ACHN, was reduced by transforming growth factor-beta, basic fibroblast growth factor, transforming growth factor-alpha and, most strikingly, by epidermal growth factor (EGF). These growth factors did not show such inhibitory effects on the secretion of ir-ET-1 in ET-1-producing cells, indicating that the production of ET-2 and ET-1 is regulated differently by the growth factors. EGF specifically reduced the secretion of not only ir-ET-2 but also ir-big ET-2 with only a small decrease in total protein synthesis. Northern blot analysis indicated that EGF controls the ET-2-production at the transcription levels of ET-2 gene.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Line; Endothelins; Epidermal Growth Factor; Growth Substances; Humans; Immunoenzyme Techniques; Kidney Neoplasms; Kinetics; Leucine; Poly A; RNA; RNA, Messenger

To investigate the biosynthetic basis for the mosaic expression of brush border enzymes in confluent Caco-2 cells, a human colon carcinoma cell line exhibiting characteristics of adult small intestinal enterocytes, we have obtained a series of clones differing markedly in their growth rates, amounts of transforming growth factor-alpha/epidermal growth factor-like activity released into the culture medium, and sucrase-isomaltase (SI) activity. Other intestinal markers (aminopeptidase N, dipeptidylpeptidase IV, lactase, alkaline phosphatase and 'crypt cell antigen') displayed a much more limited variability in expression, suggesting that the Caco-2 cell clones we have obtained did not differ in their overall ability to differentiate. Immunofluorescence staining, metabolic labelling with radioactive methionine and hybridization analysis of SI mRNA abundance were used to investigate SI synthesis and its regulation in clones endowed with low, intermediate or high sucrase activity. The results obtained have demonstrated heterogeneous SI expression, even in clonal cell lines, and a negative correlation between SI expression and growth factor concentrations in the culture medium, suggesting an autocrine regulation of cell proliferation and differentiation in confluent Caco-2 cells. Pulse-chase experiments using the two clones endowed with the lowest and highest levels of SI activity, followed by immunoprecipitation of labelled SI with epitope-specific antibodies and SDS/PAGE analysis, suggested that both transcriptional and post-translational mechanisms play a role in the regulation of SI expression in intestinal cells.

Topics: Adenocarcinoma; Aminopeptidases; Antibodies, Monoclonal; Clone Cells; Epidermal Growth Factor; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Humans; RNA, Messenger; Sucrase-Isomaltase Complex; Transforming Growth Factors; Tumor Cells, Cultured

To investigate the role and mechanism of action of epidermal growth factor (EGF) in the intestinal epithelium, we have studied its influence on proliferation and differentiation of Caco-2 cells, a human colon adenocarcinoma cell line exhibiting several characteristics of adult small intestinal enterocytes. A clone of Caco-2 cells synthesizing minimal amounts of transforming growth factor-alpha (TGF-alpha)/epidermal growth factor (EGF)-like activity was used in these studies. Cells grown in the presence of 20-200 ng EGF/ml exhibited increased DNA synthesis and proliferation; formation of morphologically poorly differentiated multilayers was observed at 200 ng EGF/ml. At all concentrations tested EGF produced a significant and marked reduction in sucrase activity, whereas other brush-border enzymes (aminopeptidase N, alkaline phosphatase, dipeptidylpeptidase IV) were only marginally affected. EGF influenced sucrase expression at two different levels. At 20 ng/ml, it affected primarily sucrase-isomaltase processing in the endoplasmic reticulum and/or increased its degradation. At 200 ng EGF/ml, a significant and marked reduction in sucrase-isomaltase mRNA levels and biosynthesis was observed. These results demonstrated that EGF has important and selective effects on Caco-2 cell proliferation and differentiation and may affect different cellular activities depending on its concentration.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Cell Differentiation; Cell Division; Colonic Neoplasms; Epidermal Growth Factor; Humans; Microscopy, Electron; Microvilli; Precipitin Tests; Sucrase-Isomaltase Complex; 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

The epidermal-growth-factor receptor (EGF-r) and its ligands are involved in the control of proliferation of both normal and neoplastic thyroid epithelium. Autocrine stimulation of growth involving this receptor system has been identified in several types of human neoplasia and we were interested to determine whether it might occur in human thyroid tumours. We have therefore examined an archival series of thyroid tumours and non-neoplastic pathologies for expression of the EGF-r and 2 of its ligands, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF), using immunohistochemistry. We found evidence of expression of both the EGF-r and the TGF-alpha in the majority of thyroid tumours, with a trend to higher expression in more malignant neoplasms. We also found variable levels of expression of EGF-r and TGF-alpha in all cases of thyroiditis examined. We conclude that there is a potential autocrine loop involving the EGF-r system in both neoplastic and non-neoplastic conditions of the human thyroid.

Topics: Adenocarcinoma; Carcinoma; Carcinoma, Papillary; Epidermal Growth Factor; ErbB Receptors; Humans; Retrospective Studies; Thyroid Gland; Thyroid Neoplasms; Thyroiditis; Transforming Growth Factor alpha

Tumorigenesis and invasive capacity of tumor cells are affected by the interactions between the tumor cells and the host cells; in particular, they are regulated by growth factors released from host cells. We have studied the effect of growth factors and cytokine on tumorigenesis and invasive capacity of tumor cells by examining a regressor tumor cell line (ER-1) derived from SHR rat mammary adenocarcinoma. The regressor tumor cells grew progressively in immunosuppressed rats, but spontaneously regressed in normal rats. We studied in vitro effect of growth factors on invasive capacity of ER-1 tumor cells into mesothelial cells obtained from SHR rats. As the results, pretreatment with EGF or TGF-beta significantly enhanced invasive capacity of ER-1 cells, whereas TNF-alpha did not show any effect. We pretreated ER-1 cells with EGF or TGF-beta for 24 hours in vitro, and then intraperitoneally transplanted them into SHR rats. The treated regressor tumor cells grew and killed the host. These results suggest that tumorigenesis and invasive capacity of regressor tumor cells are mediated by growth factors which promote growth and invasive abilities of tumor cells.

Topics: Adenocarcinoma; Animals; Cell Division; Epidermal Growth Factor; Female; Growth Substances; Mammary Neoplasms, Animal; Neoplasm Invasiveness; Rats; Rats, Inbred SHR; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Dexamethasone (1 microM) decreased the distribution of cells in S phase (about 75%) and increased that of G1 cells (1.1-fold) in the DNA histogram of human submandibular salivary gland adenocarcinoma cells (HSG) reversibly. In synchronized cells at G1 phase, glucocorticoid delayed the initiation of DNA synthesis by about 3-4 h. The conditioned medium (50%) or exogenous human epidermal growth factor (EGF, 10 ng/ml) significantly nullified these effects by glucocorticoids. These results suggested that glucocorticoids arrested the cells at G1 phase, which implied the inhibition of production of some progressive factor, probably EGF, in the cell cycle of HSG.

Topics: Adenocarcinoma; Cell Division; Culture Media; Dexamethasone; DNA; Epidermal Growth Factor; G1 Phase; Humans; S Phase; Salivary Gland Neoplasms; Triamcinolone Acetonide; Tumor Cells, Cultured

Steroid hormones and peptide growth factors promote growth and development of normal mammary tissues and some types of breast cancer. Ovarian steroids may influence mammary growth directly or indirectly. The epidermal growth factor (EGF) family of proteins may also regulate mammary growth. These two pathways may function independently of each other or they may act in concert, with steroids inducing transcription of genes that encode growth factors or growth factor receptors. We used a feline mammary adenocarcinoma cell line (K12) to address whether there was an interrelation between progesterone (PGN) and EGF-associated growth pathways. K12 cells responded to EGF by a dose-dependent increase in proliferation. PGN or promegestone (R5020, a synthetic progestagen) alone did not stimulate K12 growth, but when EGF and PGN, or EGF and R5020 were combined, they were synergistic. This synergistic response was abrogated by the PGN receptor antagonist RU486 or by antibodies that blocked binding of EGF to its receptor. K12 cells expressed characteristic double-affinity EGF receptors, as well as p185 (a functionally and structurally related protein, product of the neu gene) on their surface. PGN receptors were also found on intact cells and in cleared cytosols. Stimulation of K12 cells by PGN or by R5020 induced a two- to threefold increase in the number of high-affinity surface EGF receptors after 24 h. Stimulation of these cells by PGN also affected the relative levels of phosphorylation of the EGF receptor and p185 within minutes, but not of other cellular phosphoproteins. Our results show that PGN enhances the EGF-induced growth of K12 cells and suggest that this effect may be mediated at least partly via an increase in the number or function of high-affinity EGF receptors.

Topics: Adenocarcinoma; Animals; Cat Diseases; Cats; Cell Division; DNA Replication; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Female; Mammary Neoplasms, Animal; Mifepristone; Neoplasms, Hormone-Dependent; Phosphorylation; Progesterone; Progestins; Promegestone; Receptors, Progesterone; Tumor Cells, Cultured

Thirty-two surgical specimens and three cell lines of human gastric cancers were used for subcutaneous transplantation into nude mice, resulting in the establishment of eight (25%) xenografts from the surgical specimens and two (67%) from the cell lines. The localization of epidermal growth factor (EGF) in the surgical specimens and cell lines of the gastric cancers and their xenografts in nude mice was then investigated immunohistochemically. Epidermal growth factor was stained in the cytoplasm of the cancer cells, being detected in 16 (50%) of the 32 surgical specimens and in all of the cell lines. Seven (44%) of the sixteen EGF-positive surgical specimens and one (6%) of the 16 EGF-negative ones were tumorigenic in nude mice. All of the xenografts in nude mice were positive for EGF. The tumorigenicity of human gastric cancer xenografts in nude mice may, therefore, be correlated with the presence of EGF in cancer cells.

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Animals; Carcinoma, Renal Cell; Epidermal Growth Factor; Female; Humans; Kidney Neoplasms; Male; Mice; Mice, Nude; Middle Aged; Neoplasm Metastasis; Neoplasm Transplantation; Staining and Labeling; Stomach Neoplasms; Tumor Cells, Cultured; Wilms Tumor

To evaluate the distribution and density of epidermal growth factor (EGF) receptors (EGF-Rs) on urothelium, immunohistological studies using a monoclonal antibody to the binding portion of the human EGF-R were performed on frozen specimens of normal urothelium (N = 20), urothelium from patients with nonurothelial urological malignancies (N = 15) and inflammatory diseases (N = 8), low grade superficial transitional cell carcinomas (TCC) (N = 13), high grade superficial or invasive TCC (N = 28), and endoscopically normal appearing urothelium from patients with low grade superficial (N = 5) or high grade (N = 21) TCC elsewhere in the bladder (or ipsilateral renal pelvis/ureter). EGF-Rs are found only on the basal layer of epithelial cells (with scattered representation on intermediate cells) in 95% of normal urothelial specimens and 100% of pathological specimens without urothelial malignancy. Alternatively, 92.3% of specimens of low grade superficial TCC and 100% of high grade TCCs had EGF-Rs richly expressed on the superficial as well as the deeper layers of urothelium. This "malignant" distribution of EGF-Rs was also found on all specimens of endoscopically normal appearing urothelium in patients with TCC elsewhere. The density of EGF-Rs correlated closely with tumor grade on both "premalignant" and frankly neoplastic urothelium. We conclude that the expression of EGF-Rs on urothelium favors the interaction of premalignant and malignant tissue with urinary EGF. To determine if altering the physiochemical environment of urine could interfere with this interaction, the effects of pH on the binding of and growth responses to EGF were assessed on four human TCC cell lines. Scatchard plots demonstrated that varying pH from 5.0 to 7.5 did not significantly change the total number of receptors, but EGF-R affinity was reduced approximately 20-fold as pH decreased from 7.5 to 5 in each TCC target. Similarly, significant growth stimulation by EGF at pH 7.5 was abrogated at pH less than or equal to 7.0 while growth rates in the absence of EGF remained unchanged at lower pHs. It thus appears that urinary acidification may hold promise in the management and prevention of recurrent bladder cancer.

Topics: Adenocarcinoma; Carcinoma, Transitional Cell; Cell Division; Epidermal Growth Factor; Epithelium; ErbB Receptors; Female; Humans; Kinetics; Male; Neoplasm Invasiveness; Prostatic Hyperplasia; Prostatic Neoplasms; Tumor Cells, Cultured; Ureter; Urinary Bladder; Urinary Bladder Neoplasms

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

We have determined the presence of transforming growth factor-beta (TGF-beta)-like polypeptides in mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in BALB/c mice. In hormone-dependent tumors (HD) from nontreated and MPA-treated mice a high molecular weight (43 kDa) transforming activity was purified by Bio-Gel P-60 chromatography. This TGF was able to confer the neoplastic phenotype on NRK-49F cells without the addition of epidermal growth factor (EGF), though its activity was potentiated by EGF. It did not compete for binding to the EGF receptor, had no mitogenic activity on monolayer cultures of NRK fibroblasts, and was a potent inhibitor of DNA synthesis induced in these cells by EGF and insulin. In HD and hormone-independent tumors (HI) another TGF with a Mr of 13 kDa was isolated. This transforming activity showed the same biological properties as 43 kDa TGF, with the exception that in the absence of EGF it did not stimulate soft agar growth of NRK-49F cells. The synthesis of both factors in 'in vivo' HD tumors seems to be under MPA control, since it is much lower in HD tumors from MPA-treated mice. Further purification of the 13 and 43 kDa TGFs by hydrophobic interaction HPLC demonstrated that each one eluted in a different position, and that their elution profile differed from the TGF-beta from human platelets. The biological activity of the 13 and 43 kDa TGFs was not neutralized by a specific anti-TGF-beta antibody.

Topics: Adenocarcinoma; Animals; Cell Division; Cell Transformation, Neoplastic; Chromatography, High Pressure Liquid; DNA Replication; Drug Interactions; Epidermal Growth Factor; Fibroblasts; Gene Expression Regulation, Neoplastic; Insulin; Mammary Neoplasms, Experimental; Medroxyprogesterone; Medroxyprogesterone Acetate; Mice; Mice, Inbred BALB C; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Rats; Transforming Growth Factor beta

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.

Topics: Adenocarcinoma; Carcinoembryonic Antigen; Cell Count; Cell Differentiation; Cell Division; Clone Cells; Colonic Neoplasms; Culture Media; Cyclic AMP; Epidermal Growth Factor; HLA Antigens; Humans; Suramin; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

We immunohistochemically examined 131 primary human lung adenocarcinomas for the possible presence of autocrine factors. Transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) were considered growth factors with epidermal growth factor receptor (EGFR) as the receptor. Of these tumors, 87 (66%) showed a high expression of TGF alpha, 66 (50%) showed a high expression of EGF, and 55 (42%) were positive for EGFR reactivity. In the EGFR-positive cases, the 5-year survival rates of patients with high TGF alpha and low TGF alpha were 36% and 85%, respectively (P less than 0.05). The 5-year survival rates of patients with high EGF and low EGF were 25% and 77%, respectively (P less than 0.05). In contrast, in the EGFR-negative cases, there was no statistical difference between the 5-year survival rates of patients with either high TGF alpha or EGF and low TGF alpha or EGF. Because autocrine growth mechanisms are present in adenocarcinoma of the human lung, these events may contribute to clarification of tumor development, and perhaps even to a better prognosis.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Cell Membrane; Cytoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Lung Neoplasms; Male; Middle Aged; Transforming Growth Factor alpha

Growth factors, including epidermal growth factor (EGF), have been implicated in the growth of several types of cancer. This study compares EGF receptors in normal and neoplastic endometrium. Membrane fractions were isolated from surgical specimens. Radioreceptor assays demonstrated the presence of receptors with a dissociation constant of 0.64 nmol/l in normal endometrium. Affinity cross-linking revealed receptor molecular weight of 150 to 170 kiloDaltons (KD). A survey of samples (n = 37) revealed progressive decrease of EGF receptors in cancers of increasing grade: Grade 1-2 adenocarcinoma decreased 34% from control (n = 6, P less than 0.01), whereas Grade 3 adenocarcinoma decreased 90% (n = 7, P less than 0.01) and sarcoma decreased by 72% (n = 3, P less than 0.01). The dissociation constant and molecular weight of the receptor in neoplastic endometrium did not differ significantly from normal. The inverse relationship with grade suggests receptor alteration or down regulation by hormones and/or growth factors.

Topics: Adenocarcinoma; Animals; Endometrium; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Mice; Molecular Weight; Uterine Neoplasms

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

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

The expression of epidermal growth factor (EGF) was studied immunohistochemically in a total of 92 human colorectal cancers to examine whether EGF was expressed in relation to its degrees of malignancy. Significantly high incidence of EGF was observed in advanced cancers than in early cancers. In the tissues of advanced cancers, tumor cells having EGF-immunoreactivity tended to be more frequently and intensely observed in deeply invasive tumor cells than in superficial tumor cells. Regarding macroscopic and histologic types, incidence of EGF-immunoreactivity was significantly higher in infiltrating types and in differentiated types than in localized types and in poorly differentiated types, respectively. Cases with poor prognosis and in advanced stages showed high positive rate of EGF-immunoreactivity. These results suggest that the expression of EGF may serve as a biologic marker for the degree of malignancy in human colorectal cancers.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colorectal Neoplasms; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Staging; Prognosis

Dedifferentiation of human thyroid tumors is frequently found in humans. The effect of retinoids (13 cis-RA) was studied on the proliferation and differentiation of a human follicular cell line in vitro (UCLA R0 82 W-1). A significant and dose-dependent reduction (P less than 0.001) in cell number and [3H] thymidine uptake was found in cells exposed to 13 cis-RA up to 10 microM. Higher concentrations of 13 cis-RA, however, led to a dose-dependent restoration of cell proliferation. Various parameters of differentiation increased under the influence of 13 cis-RA (10 microM) over nonexposed cells. The 125I uptake increased 4-fold over that in control nonexposed cells (P less than 0.05). [125I] Epidermal growth factor binding increased 5-fold, and [125I] human TSH binding increased significantly after exposure to 13 cis-RA (P less than 0.02). Deiodinase activity, however, was significantly lower in 13 cis-RA exposed cells than in control cells. The present study shows that 13 cis-RA (10 microM) drives the tumor cells toward a more normal state of proliferation and differentiation.

Topics: Adenocarcinoma; Binding Sites; Cell Cycle; Cell Differentiation; Cell Survival; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Iodide Peroxidase; Iodine; Thymidine; Thyroid Neoplasms; Thyrotropin; Tretinoin; Tumor Cells, Cultured

The responsiveness and action mechanisms of steroid hormones and epidermal growth factor on human endometrial carcinoma cells are analyzed by using in vitro culture system. 1) The Ishikawa cells, derived from a well differentiated endometrial adenocarcinoma and possess ER and PR, are shown to respond to estrogens by increasing a variety of parameters, viz cell proliferation, PR levels, ALP and DNA polymerase activities. 2) ER and PR of those cells are localized in the nuclei by immunocytochemical staining using the monoclonal antibodies against to ER and PR, confirming the correctness of Gorski and Greene's one step theory involving the action mechanisms of steroid hormones. 3) Progestins reduced the ER level and stimulate E2DH activities and glycogen content, which are completely abolished by anti-progestin (RU486), suggesting that PR of those cells should be functional. 4) These responses to steroid hormones of Ishikawa cells are synergistically enhanced or appeared earlier by addition of EGF. 5) The main metabolite of E2 incubated with Ishikawa cells is E2-3-sulfate instead of E1, indicate that the higher estrogenic status may be persisted in endometrial cancer tissues.

Topics: Adenocarcinoma; Alkaline Phosphatase; Cell Division; DNA Polymerase II; Endometrial Neoplasms; Epidermal Growth Factor; ErbB Receptors; Estradiol Dehydrogenases; Estrogens; Female; Glycogen; Humans; Progesterone; Receptors, Estrogen; Receptors, Progesterone; Tumor Cells, Cultured

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.

Topics: Adenocarcinoma; Antibodies; Breast Neoplasms; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Estradiol; Estrogens; Female; Humans; Laminin; Peptides; Receptors, Immunologic; Receptors, Laminin; Transforming Growth Factors; Tumor Cells, Cultured

Topics: Adenocarcinoma; Adenocarcinoma, Mucinous; Cell Division; Cell Line; Cell Survival; Drug Screening Assays, Antitumor; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Fluorouracil; Humans; Stomach Neoplasms; Tumor Cells, Cultured

The binding of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) to cell membranes was determined in 14 renal cancers and in 13 normal kidney tissues adjacent to the tumors. The soluble 34K IGF binding protein (34K IGF-BP) content and the phosphotyrosyl-protein phosphatase activity in renal cancer tissue and adjacent normal tissue were also determined. The specific EGF receptor binding in renal cancers was 12.7 +/- 2.5% (mean +/- SEM) as compared to 2.6 +/- 0.2% (mean +/- SEM) in normal tissues (p less than 0.01). Phosphotyrosyl-protein phosphatase activity in renal cancer tissue was less than half of that observed in normal renal tissue (p less than 0.01). The highest IGF-I binding was observed in 5 renal cancers although no consistent differences between IGF-I binding to tumor and normal tissues were observed. Both EGF and IGF binding to kidney tissue were higher than binding to gastro-intestinal tissue irrespective of whether normal or malignant tissues were compared. All normal kidney tissues and 7 of 8 kidney tumors contained measurable amounts of 34K IGF-BP as determined by RIA and the cross-linking technique. In 2 tumor tissue samples the 34K IGF-BP content was increased 8- and 15-fold over that seen in adjacent normal kidney tissue, whereas in the 6 other renal cancers the 34K IGF-BP was similar to that observed in normal kidney tissue.

Topics: Adenocarcinoma; Aged; Colorectal Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Insulin-Like Growth Factor I; Kidney; Kidney Neoplasms; Male; Middle Aged; Phosphoprotein Phosphatases; Protein Binding; Protein Tyrosine Phosphatases; Receptors, Cell Surface; Receptors, Somatomedin; Solubility; Somatomedins; Stomach Neoplasms

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

Immunohistochemical study for epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) was performed on 222 specimens of gastric carcinoma. The authors placed each carcinoma into one of the following three groups: group 1, neither EGF nor EGFR was stained (123 cases); group 2, either EGF or EGFR was stained (64 cases); and group 3, both EGF and EGFR were stained (35 cases). Compared with the carcinomas in groups 1 and 2, those in group 3 had significantly higher rates of infiltrative gross type, microscopically infiltrative type, poorly differentiated type, scirrhous type, and deep invading type. These results suggest that carcinomas in group 3 may have more proliferative and invasive activity and thus may have an autocrine mechanism, that is, the ability of cancer cells to produce and respond to their own growth factor.

Topics: Adenocarcinoma; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Neoplasm Invasiveness; Stomach Neoplasms

We previously reported that two human lung adenocarcinoma cell lines (A-549 and PC-9) produce human transforming growth factor-alpha (hTGF-alpha) and express its receptors. In the present study an exogenously added monoclonal antibody against recombinant hTGF-alpha inhibited growth of these cell lines in vitro. This result indicated that endogenous hTGF-alpha produced by the cancer cells can function as an autocrine growth factor.

Topics: Adenocarcinoma; Antibodies, Monoclonal; Antibody Specificity; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Immunotherapy; In Vitro Techniques; Lung Neoplasms; Radioligand Assay; Recombinant Proteins; Transforming Growth Factors; Tumor Cells, Cultured

Malignant prostatic carcinoma, a major cause of cancer mortality in males, most often metastasizes to secondary sites in bone. Frequently, the growth rate of the secondary tumor in bone marrow is considerably greater than that of the slowly growing primary prostatic tumor. We now report that two lines of human prostatic carcinoma cells proliferate in response to conditioned media from unstimulated human, rat, or bovine bone marrow. Nonprostatic tumor cell lines showed little or no growth response to the same medium. The proliferative activity found in bone marrow was not duplicated by any of a variety of purified growth factors including epidermal growth factor (EGF), acidic or basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF) alpha or beta, interleukins 1, 2, 3, 4 or 6, granulocyte (G), macrophage (M) or granulocyte-macrophage (GM) colony stimulating factor (CSF). Whereas a mixture of G-CSF, M-CSF, and IL 3 produced a mitogenic response in the prostatic carcinoma cells, these three factors were not present in our bone marrow samples in sufficient quantities to promote the observed proliferative response. To further identify the cellular source of the proliferative activity present in bone marrow-conditioned medium, we tested conditioned media made from human bone marrow stromal cells. The stromal cell conditioned medium stimulated increased growth of the prostatic carcinoma cells to levels equivalent to those observed with the bone marrow conditioned medium. These results suggest that novel mitogenic factors that are produced by bone marrow stromal cells and remain in the bone marrow cavity may account, in part, for the preferential growth of prostatic metastases in bone.

Topics: Adenocarcinoma; Animals; Bone Marrow; Bone Marrow Cells; Bone Neoplasms; Cattle; Cell Line; Colony-Stimulating Factors; Culture Media; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Male; Platelet-Derived Growth Factor; Prostatic Neoplasms; Rats; Transforming Growth Factors

Human salivary gland adenocarcinoma cell line HSG secretes an epidermal growth factor (EGF)-like molecule and contains EGF receptors. Growth of HSG cells is inhibited by glucocorticoid. We have identified that the growth inhibition by glucocorticoid is induced by the reduced secretion of the EGF-like molecule and that addition of anti-human EGF antibody to the culture specifically inhibits the growth of HSG cells, suggesting that autocrine secretion is involved in the growth of HSG cells. To prove that autocrine secretion functions in glucocorticoid-regulated growth of the HSG cell line, we purified the EGF-like molecule from serum-free, defined medium conditioned by the HSG cells and examined the growth-stimulatory effect of the purified molecule. The cultivation of HSG cells in serum-free defined medium, which contains insulin (10 micrograms/ml) and transferrin (10 micrograms/ml) only as proteinaceous components, resulted in establishment of a new cell line (HSG-SF) which had different morphological features from the parental HSG cell line. HSG-SF cells were found to have basically the same responsiveness to glucocorticoid as parental HSG cells. Parental HSG cells secreted high molecular weight EGF-like molecules (Mr 46,000 and 57,000), which were recognized by specific antibody to low molecular weight human EGF (Mr 6,201). From conditioned, serum-free medium of HSG-SF cells, an EGF-like molecule (Mr 46,000) was purified by using an anti-human EGF antibody-coupled Sepharose CL-4B column. This EGF-like molecule induced a maximal increase (36%) in incorporation of [3H]thymidine into DNA of parental HSG cells as well as low molecular weight human EGF. These observations demonstrate that growth of the HSG cell line is regulated by autocrine secretion.

Topics: Adenocarcinoma; Cell Division; Cell Line; Culture Media; DNA Replication; Epidermal Growth Factor; Fibronectins; Humans; Salivary Gland Neoplasms; Triamcinolone Acetonide; Tumor Cells, Cultured

Associations between epidermal growth factor (EGF) and carcinoma of the prostate (CAP) have not been systematically investigated. We used indirect immunohistochemical techniques to demonstrate cytoplasmic EGF in paraffin-embedded sections of the following primary prostatic tissues: benign prostatic hyperplasia (BPH) (N = 10), BPH adjacent to CAP (N = 42), clinically localized CAP (N = 45), untreated metastatic CAP (N = 10), and metastatic CAP after varying periods of androgen deprivation (N = 10). In six of the latter 10 cases biopsies of the primary tumor obtained before androgen deprivation therapy were also available for study. Three of the BPH specimens (6%) and 44 of the CAP specimens (68%) stained. Forty per cent of the localized tumors stained but all untreated and treated metastatic tumors stained (p less than 0.01). There were direct but statistically insignificant correlations between the demonstration of EGF and both the Gleason score of localized and untreated metastatic tumors and the pathologic stage of localized tumors. The proportion of malignant cells stained in EGF positive tumors was similar regardless of Gleason score, pathologic stage or the presence or absence of metastases. However, the proportion of cells stained was greater in five of six specimens obtained during hormonal deprivation compared to specimens of the same tumor obtained before treatment. These data suggest that some prostatic cancers interact with EGF and that the interaction may be influenced by the androgenic milieu.

Topics: Adenocarcinoma; Epidermal Growth Factor; Humans; Immunoenzyme Techniques; Male; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms

The expressions of epidermal growth factor (EGF) and its receptor were studied immunohistochemically in a total of 156 gastric carcinomas; 26 early and 130 advanced. No EGF immunoreactivity was found in early carcinomas, while EGF-positive tumor cells were detected in 38 (29.2%) of the 130 advanced carcinomas. EGF receptor immunoreactivity was detected in one (3.8%) of the 26 early carcinomas and in 44 (33.8%) of the 130 advanced carcinomas, the incidence being significantly different (p less than 0.01). Out of the 130 advanced carcinomas, 17 (13.1%) had synchronous expression of EGF and its receptor and most of the tumors with strong expression of EGF were positive to EGF receptor. A significant correlation was observed between the depth of tumor invasion and EGF or its receptor immunoreactivity in tumor cells (p less than 0.05). Furthermore, a good correlation was demonstrated between the synchronous expression of EGF and its receptor and the depth of tumor invasion or the tumor staging. The incidence of cases with EGF in metastatic tumors was significantly higher than that in primary tumors (p less than 0.05). Patients with synchronous expression of EGF and its receptor had a far poorer prognosis than those without EGF and receptor.

Topics: Adenocarcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Staging; Protein-Tyrosine Kinases; Stomach Neoplasms; Time Factors

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.

Topics: Adenocarcinoma; Autoradiography; Chromatography, Thin Layer; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Humans; Immunoassay; Peptide Mapping; Phosphorylation; Proto-Oncogene Proteins; Receptor, ErbB-2; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

RA 233, a pyrimido-pyrimidine analogue developed originally as an antiplatelet agent, has reduced the incidence of tumor metastases in clinical trials. However, in animal tumor models antimetastatic therapy using RA 233 has been inconsistent. We therefore tested RA 233 for additional effects, such as its direct action on tumor cells. Using the rat 13726NF mammary adenocarcinoma tumor system, low, nontoxic concentrations of RA 233 had pleiotropic and differential effects on two 13762NF tumor cell clones. The growth of MTC cells (low spontaneous metastatic potential) was not affected by low concentrations of RA 233 (50 microM) or epidermal growth factor (EGF) (up to 10 ng/ml) for 3 days in 0.5-10% fetal bovine serum. In contrast, MTLn3 (high spontaneous metastatic potential) cell cultures maintained for 3 days in low (0.5-1%) serum in the presence of 1.25-10 ng/ml EGF doubled in cell numbers compared with control cultures, and addition of 50 microM RA 233 abrogated the growth-stimulatory effect of EGF. The inhibitory effect of RA 233 on MTLn3 cells was dose dependent and not due to cell toxicity as determined by cell viability, cell growth, and colony formation properties after drug removal. In addition, incubation of MTLn3 cells with 50 microM RA 233 resulted in an increase of p21ras protein expression, whereas there was no effect on the level of p21ras in identically treated MTC cells or when either clone was treated with 10 ng/ml EGF. The results suggest that among the heterogeneous effects of RA 233 on tumor cells, modulation of growth factor responses and regulatory molecules may be important.

Topics: Adenocarcinoma; Animals; Cell Division; Epidermal Growth Factor; Mammary Neoplasms, Experimental; Mopidamol; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Pyrimidines; Rats

The treatment of a human submandibular gland adenocarcinoma cell line (HSG cell line) for 48 h with triamcinolone acetonide (TA; 1-100 nmol/l) reduced the secretion of epidermal growth factor (EGF) in a closely related manner to a maximum of 66%. The reduction in the level of EGF secreted resulted in the suppression of DNA synthesis in the HSG cells to a similar extent. When the cells were incubated with TA and exogenous human EGF (hEGF), DNA synthesis was 1.7-fold higher than that without added hEGF. The removal of EGF by the addition of hEGF antibody reduced DNA synthesis in HSG cell cultures to the same extent as did TA. These results suggest that the growth inhibition of HSG cells by TA is due to the reduction in the amount of EGF secreted.

Topics: Adenocarcinoma; Cell Line; DNA, Neoplasm; Epidermal Growth Factor; Humans; Salivary Gland Neoplasms; Submandibular Gland Neoplasms; Triamcinolone Acetonide; Tumor Cells, Cultured

The effect of human epidermal growth factor (hEGF) on the growth of various histological types of six human gastric carcinoma cell lines was examined. The cell lines had relatively high affinity EGF receptors (dissociation constant Kd = 10(-9) to 10(-10) M). One gastric cancer cell line, MKN-74 (well differentiated adenocarcinoma) showed no response to hEGF, in cell growth, DNA synthesis or 125I-hEGF cell binding. There were no apparent correlations between histological type and cell growth, DNA synthesis or number of EGF receptors in these cells. The number of EGF receptors and the Kd value of the gastric carcinoma cell lines varied with their internal and external environments. hEGF concentrations corresponding to maximum stimulation in DNA synthesis varied between cell lines. The results suggest some gastric carcinoma cells to have EGF receptors and their growth seemingly to be stimulated by EGF in vitro. There are, however, no obvious correlations between the effect of hEGF on the growth of human gastric carcinoma cell lines or their histological type.

Topics: Adenocarcinoma; Cell Division; Cell Line; DNA Replication; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Stomach Neoplasms; Temperature; Time Factors

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Alkaline Phosphatase; Animals; Cell Division; Epidermal Growth Factor; Female; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Stomach Neoplasms

Two hundred and twenty one gastric carcinomas were immunohistochemically stained for h-EGF and we examined the correlation between h-EGF immunoreactivities and histologic findings. Regarding macroscopic and histologic types, incidence of h-EGF immunoreactivities in infiltrating type and in poorly differentiated type was significantly higher than those in localized type and in differentiated type, respectively. In addition, h-EGF producing carcinomas showed high positive rate in prognostic serosal involvement and scirrhous type in stroma. Prognosis in patients with h-EGF producing tumors was poorer than that in those with h-EGF non-producing tumors, especially in stages II and III. These results suggest that h-EGF immunoreactivities serves as a biological marker of high malignancy.

Topics: Adenocarcinoma; Biomarkers, Tumor; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Staging; Prognosis; Stomach Neoplasms

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

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.

Topics: Adenocarcinoma; Animals; Blotting, Northern; Cell Differentiation; Collagen; Epidermal Growth Factor; Estradiol; Mammary Neoplasms, Experimental; Neoplasms, Hormone-Dependent; Poly A; Radioimmunoassay; Rats; RNA; RNA, Messenger; Transforming Growth Factor alpha; Tumor Cells, Cultured

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 radioimmunological and radioreceptor methods have been used to show that sialadenectomy leads to the stable decrease of the epidermal growth factor (EGF) concentration in saliva and blood serum. The mean number of colon tumours per rat was significantly lower among the rats which had been sialadenectomized before injections of the carcinogen, than in the control. But a sharp stimulation of carcinogenesis in the duodenal mucosa was observed after sialadenectomy. The production of the alpha-transforming growth factor with the EGF-competing activity for the EGF-receptors was found in the chemically-induced rat colon tumours.

Topics: 1,2-Dimethylhydrazine; Adenocarcinoma; Animals; Dimethylhydrazines; Epidermal Growth Factor; Intestinal Mucosa; Intestinal Neoplasms; Male; Peptides; Rats; Submandibular Gland; Transforming Growth Factors

Human peripheral blood monocytes and tumor-associated macrophages release a factor that enhances the clonal growth of a human epithelial tumor cell line (SW-13) in soft agar. We now demonstrate that purified interleukin 1 (IL-1) may account for part of this colony-stimulating activity. Purified IL-1 (0.5 to 8 units/ml) was added to SW-13 cells cultured in soft agar. IL-1 increased colony growth in a dose-dependent manner and did not inhibit colony formation at the highest doses tested. Other purified human monocyte products (alpha-interferon, tumor necrosis factor, transforming growth factor beta, fibronectin) did not stimulate colony growth. Antibody to IL-1 only partially inhibited the ability of monocyte-conditioned medium to stimulate SW-13 colony growth. This antibody did, however, completely inhibit the ability of purified IL-1 to support the growth of SW-13 colonies in soft agar. IL-1 increased growth of quiescent SW-13 cells cultured in monolayers as assessed by tritiated thymidine incorporation assays. The results of this study indicate that IL-1 can enhance clonogenic growth of an epithelial cell line in soft agar. However, other uncharacterized activities in monocyte conditioned medium also promote colony growth. These studies add to an increasing body of evidence indicating that inflammatory products play a role in maintaining the transformed phenotype.

Topics: Adenocarcinoma; Culture Media; Dose-Response Relationship, Drug; Epidermal Growth Factor; Humans; Interleukin-1; Monokines; Peptides; Proteins; Thymidine; Transforming Growth Factors; Tumor Cells, Cultured

The conditioned medium from the HT-29 human colonic adenocarcinoma cell line contains a potent mitogenic activity that can markedly stimulate the proliferation of both rat and human fibroblasts in the absence of serum. Fractionation of conditioned medium on Bio-Gel P-100 shows that HT-29 cells simultaneously produce 2 different types of endogenous growth factors. The first one (molecular mass of 35, 8 and 5.5 kDa) exhibits an IGF-I competing activity which is positively correlated to mitogenic activity. This mitogen is recognized by anti-IGF-I antibodies but is resistant to reducing agents. It is distinct from IGF-II, insulin and PDGF. The second one (molecular mass of 40- and 20-kDa) is able to displace EGF binding to its receptor. This factor is immunologically recognized by anti-EGF antibodies but with a lower affinity as compared to EGF. This suggests that this endogenous HT-29-growth factor is related to but distinct from native EGF. Although more active in radioreceptor assay than in radioimmunoassay, the EGF-competing factor is distinct from TGF alpha or beta since it is unable to induce anchorage-independent growth of NRK or FR3T3 target cells in the presence or absence of exogenous EGF. Moreover, free functional EGF receptors are available at the HT-29 cell surface.

Topics: Adenocarcinoma; Antibodies; Binding, Competitive; Cell Division; Cell Line; Colonic Neoplasms; Culture Media; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor I; Radioimmunoassay; Somatomedins

Expression of epidermal growth factor (EGF) receptor by human breast cancer tissues has an inverse relationship with expression of the estrogen receptor and may be associated with a poor clinical response. We have studied the regulation of EGF receptor expression in a series of human breast cancer cell lines with varying degrees of estrogen responsiveness. Three estrogen receptor-positive lines, MCF-7, ZR-75-1, and T47D, were found to have less than 70,000 EGF binding sites per cell by radioreceptor assay and were growth stimulated in vitro by EGF. Four estrogen receptor-negative lines, MDA-MB-231, Hs578T, EVSA-T, and BT-20, contained greater than 70,000 EGF binding sites per cell and showed no in vitro growth stimulation by EGF. In all cell lines EGF receptor number was correlated with the amount of EGF receptor protein and RNA. Differences in EGF receptor expression between the cell types was not due to amplification of the EGF receptor gene. Rather, variations in EGF receptor expression between lines were due, at least in part, to differences in the rate of EGF gene transcription as determined by nuclear run-off studies. Our data confirm the previously described inverse relationship between expression of EGF and estrogen receptors. We show here that the absence of estrogen receptor expression in human breast cancer cell lines is associated with higher levels of functional EGF receptor protein and mRNA.

Topics: Adenocarcinoma; Breast Neoplasms; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Fractional Precipitation; Humans; Nucleic Acid Hybridization; Receptors, Estrogen; RNA, Messenger; Tumor Cells, Cultured

The presence of human epidermal growth factor (hEGF) was studied in a total of 210 gastric carcinomas comprising 52 early carcinomas, 113 advanced carcinomas and 45 scirrhous carcinomas. An immunohistochemical study revealed no hEGF-immunoreactivity in early gastric carcinomas, while hEGF-positive tumor cells were detected in 24 (21.2%) of the 113 advanced carcinomas and in 15 (33.3%) of the 45 scirrhous carcinomas. The incidence of hEGF-immunoreactivity in well-differentiated adenocarcinomas was significantly higher than that in poorly differentiated adenocarcinomas (P less than 0.05). Moreover, hEGF-immunoreactive tumor cells were observed in 13 (30.4%) of the 42 scirrhous poorly differentiated adenocarcinomas, the incidence being significantly higher than that in non-scirrhous poorly differentiated adenocarcinomas (P less than 0.05). The average hEGF content in the tumor tissue estimated by radioimmunoassay was 3.77 +/- 0.61 (mean +/- SE) ng/g wet weight in immunohistochemical hEGF-positive tumors and 2.19 +/- 0.18 ng/g wet weight in hEGF-negative tumors, the difference being significant (P less than 0.05). Patients with hEGF-positive carcinomas (excluding scirrhous carcinomas) had much worse prognosis than those with hEGF-negative carcinomas. These results suggest that EGF produced by tumor cells plays an important role in the invasive growth and productive fibrosis of gastric carcinoma and also serves as a biologic marker of high malignancy in patients with gastric cancers.

Topics: Adenocarcinoma; Adenocarcinoma, Scirrhous; Aged; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Histocytochemistry; Humans; Male; Middle Aged; Prognosis; Receptors, Cell Surface; Staining and Labeling; Stomach Neoplasms

The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor.

Topics: Adenocarcinoma; Animals; Cell Line; Clone Cells; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Hormones; Hydrocortisone; Mammary Neoplasms, Experimental; Neoplasm Transplantation; Neoplasms, Hormone-Dependent; Progesterone; Prolactin; Rats; Rats, Inbred F344; Receptors, Cell Surface; Receptors, Glucocorticoid; Receptors, Progesterone

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

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

Because epidermal growth factor (EGF) is rapidly bound and internalized into rat pancreas, stimulates uptake of tritiated thymidine, and increases pancreatic weight, a cocarcinogenic effect on pancreatic cancer seemed likely. Pancreatic adenocarcinomas were induced in 70 female Syrian hamsters by 19 weekly s.c. injections of N-nitrosobis(2-oxopropyl)amine (BOP) (10 mg/kg). From Wk 5 through Wk 8 of BOP injections, additional s.c. injections of EGF (5 micrograms every 3 days for 10 injections) were given to 45 animals, while 25 received saline solution. An additional group of 10 received EGF alone, and another 10 animals received saline solution alone (controls). Eleven wk later, the mean body weight of EGF-treated animals increased by 29% as compared with that of controls, and their mean pancreatic weight relative to body weight increased by 44% as compared with controls. The mean body weight of EGF + BOP-treated animals increased by 10%, and their pancreatic weight relative to body weight increased by 22% as compared with that of animals treated with BOP alone. The incidence of pancreatic cancer in the EGF + BOP-treated animals was 75% versus 44% in those treated with BOP alone (P = 0.016). No tumors developed in either animals treated with EGF alone or control animals. EGF augments pancreatic carcinogenesis induced by BOP. The incidence of bronchial carcinomas doubles.

Topics: Adenocarcinoma; Animals; Body Weight; Bronchial Neoplasms; Carcinogens; Cocarcinogenesis; Cricetinae; Epidermal Growth Factor; ErbB Receptors; Female; Mesocricetus; Nitrosamines; Oncogenes; Pancreatic Neoplasms; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor

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 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

Neoplastic intercalated duct cells that originated from a human submandibular salivary gland release transforming growth factor and human epidermal growth factor into serum-free medium. The transforming growth factor with the soft agar colony-forming activity when assayed in the presence of mouse epidermal growth factor on normal rat kidney clone 49F indicator cells, but without mitogenic action to quiescent 3T3 cells, does not compete with epidermal growth factor for receptor binding, is heat and acid resistant but trypsin and dithiothreitol sensitive, and therefore is of the transforming growth factor-beta class. The immunoreactive human epidermal growth factor is detected by radioimmunoassay on certain fractions obtained by the Bio-Gel P-60 chromatography of neoplastic epithelial duct cells originated from a human submandibular salivary gland-conditioned medium and is biologically very similar to mouse epidermal growth factor. Moreover, the presence of human epidermal growth factor in cultured neoplastic epithelial duct cells originated from a human submandibular salivary gland is demonstrated by the peroxidase:antiperoxidase method.

Topics: Adenocarcinoma; Cell Division; Cell Line; Culture Media; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Peptide Biosynthesis; Peptides; Receptors, Cell Surface; Salivary Gland Neoplasms; Transforming Growth Factors

The submandibular gland is a rich source of epidermal growth factor (EGF) in mice. The concentration of EGF in the gland of virgin female mice of C3H/HeN strain increased as much as 9-fold from the age of 30 to 52 weeks. During this period, the incidence of mammary tumor in virgin females increased markedly to a maximal level of 62.5% (n = 48) at 52 weeks of age. Removal of the submandibular gland (sialoadenectomy) of virgin mice 14-22 weeks old reduced the tumor incidence to 12.8% (n = 39) at the age of 52 weeks and also increased the latency period of mammary tumor development as much as 14 weeks when compared to that of normal mice. Long-term treatment of sialoadenectomized virgin mice with EGF (5 micrograms per mouse every other day) increased the tumor incidence to 33.3%. Moreover, sialoadenectomy of mammary tumor-bearing animals caused a rapid and sustained cessation of tumor growth, but EGF administration (5 micrograms per mouse per day) quickly restored the rate of tumor growth to a normal level. These results indicate that submandibular gland EGF plays a crucial role in mouse mammary tumorigenesis.

Topics: Adenocarcinoma; Age Factors; Animals; Epidermal Growth Factor; Female; Mammary Neoplasms, Experimental; Mice; Submandibular Gland

The R3327H-G8-A1 cell line derived from the Dunning rat prostate adenocarcinoma contains both androgen and glucocorticoid receptors. Following steroid deprivation, androgens specifically increase the concentration of their receptors in these cells by approximately 2-fold within 6 h and 3-4-fold in 24 h. In the presence of potent glucocorticoids, androgen receptor augmentation is reduced by 40-50% in the first 6 h and completely inhibited during the subsequent 24 h. This event, which is specific for glucocorticoids, appears to be due to an inhibition of androgen receptor synthesis. Furthermore, glucocorticoids inhibit proliferation of these cells by inhibiting the release of growth factors and arresting them in the G0 or A state of the cell cycle. This inhibition can be overcome by addition of low concentrations of either epidermal growth factor or platelet-derived growth factor; however, the inhibitory effect of the glucocorticoid on androgen receptor augmentation is not released. These results suggest that glucocorticoids arrest cellular proliferation by altering the autoregulation of growth and that this event is not dependent upon inhibition of androgen receptor augmentation.

Topics: Adenocarcinoma; Androgens; Animals; Blood; Cell Division; Cell Line; Cell Nucleus; Cycloheximide; Dihydrotestosterone; Epidermal Growth Factor; Estrenes; Glucocorticoids; Interphase; Kinetics; Male; Metribolone; Platelet-Derived Growth Factor; Prostatic Neoplasms; Rats; Receptors, Androgen; Receptors, Glucocorticoid; Triamcinolone Acetonide

A completely defined medium has been designed to promote cell proliferation of 2 colonic adenocarcinoma cell lines of epithelial origin (HT 29 and HRT 18). The spreading of both cell types, especially of HT 29 cells, was not possible in a serum-free medium supplemented with growth factors. Spreading was obtained in a defined medium (a 1/1 mixture of DMEM and F12 media supplemented with 5 micrograms/ml transferrin, 5 ng/ml EGF, 10 ng/ml selenite and 15 mM HEPES pH 7.3) with an extracellular matrix-like material (ECM) secreted by the cells themselves. The properties of the ECM have been studied: ECM secreted by the 2 cell lines induced very quick spreading of HT 29 and HRT 18 cells (1 to 2 hr vs. 12 to 24 hr in serum-supplemented medium). ECM induced morphological differentiation of a rat pheochromocytoma cell line (PC 12). PC-12 cells grown under these conditions began to develop neurite extensions as early as 2 hr after seeding.

Topics: Adenocarcinoma; Adrenal Gland Neoplasms; Animals; Cell Differentiation; Cell Line; Colonic Neoplasms; Culture Media; Epidermal Growth Factor; Extracellular Matrix; Humans; Pheochromocytoma; Rats

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

Transforming growth factor-alpha (TGF-alpha) is secreted by many human tumors and can induce the reversible transformation of nontransformed cell lines. Using long synthetic deoxyoligonucleotides as hybridization probes we isolated an exon coding for a portion of TGF-alpha from a human genomic DNA library. Utilizing this exon as a probe, a cell line derived from a human renal cell carcinoma was identified as a source of TGF-alpha mRNA. A cloned TGF-alpha cDNA was isolated from a cDNA library prepared using RNA from this cell line, and was found to encode a precursor polypeptide of 160 amino acids. The 50 amino acid mature TGF-alpha produced by expression of the appropriate coding sequence in E. coli binds to the epidermal growth factor receptor and induces the anchorage independence of normal mammalian cells in culture.

Topics: Adenocarcinoma; Amino Acid Sequence; Base Sequence; Cell Line; Cell Transformation, Neoplastic; Cloning, Molecular; DNA; Epidermal Growth Factor; ErbB Receptors; Escherichia coli; Genes; Humans; Kidney Neoplasms; Neoplasm Proteins; Nucleic Acid Hybridization; Peptides; Plasmids; Receptors, Cell Surface; RNA, Messenger; Transforming Growth Factors

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

Rat mammary epithelium and adenocarcinomas derived from it synthesize type IV collagen, a structural protein of basement membranes. In cultures of cells, net production of collagen is stimulated 2-fold more than total cell protein by epidermal growth factor. Mammary adenocarcinoma cells also respond to epidermal growth factor but to a much reduced extent. This difference in growth factor responsiveness appears to be due to the production of collagen synthesis stimulating factors by the mammary tumor cells. Such factors have been partially purified and shown to differentially stimulate the incorporation of proline into collagenase-sensitive protein by 2.5-10-fold in normal rat mammary epithelium, normal rat kidney, and mouse 3T3 cells. The tumor factors do not stimulate net collagen production in cultures of tumor cells from which the factors are derived, suggesting that tumor cells produce sufficient stimulatory factors for optimal synthesis of collagen. Pulse-chase studies indicate that the tumor factors stimulate collagen synthesis rather than block collagen turnover. The activities in the extract have been partially purified by gel filtration, ion exchange column chromatography, and isoelectric focusing. The major species has a molecular weight of about 68,000 and a pI of 5.9. A smaller peak of activity with a molecular weight of 6,000 is also present. Since collagen synthesis appears to be necessary for the growth of mammary adenocarcinomas in vivo, production of these collagen synthesis stimulating factors may be important for tumor growth.

Topics: Adenocarcinoma; Amino Acids; Animals; Cells, Cultured; Chromatography, Gel; Chromatography, Ion Exchange; Collagen; Epidermal Growth Factor; Isoelectric Focusing; Mammary Neoplasms, Experimental; Molecular Weight; Rats

Incubation of membranes prepared from the human colon adenocarcinoma cell line LoVo in vitro with [gamma-32P]ATP demonstrated numerous components whose phosphorylation was stimulated several fold by epidermal growth factor (EGF). One major component of Mn 170 K, which was either undetectable or very weakly phosphorylated in the absence of EGF, was primarily affected by exposure to EGF. The phosphorylation of the 170 K Mr membrane component required the presence of Mn2+; Mg2+ was ineffective. Although the phosphorylation of many LoVo membrane components was significantly modified by cAMP or dibutyryl-cAMP, none of these cyclic nucleotides appeared to be involved in the phosphorylation of the 170 K membrane component in the presence or absence of EGF. The phosphorylation system of the LoVo membranes efficiently utilized [gamma-32P]GTP in both the basal and EGF-stimulated reactions. All the membrane components phosphorylated in the absence or presence of EGF, except a band comigrating with the tracking dye front, were digested by trypsin. The possible glycoprotein nature of the 170 K dalton phosphoprotein was indicated by the fact that the 170 K dalton band comigrated with a periodic acid-Schiff base-stained band. These findings suggest that one of the biochemical steps in the mechanism of action of EGF in LoVo cells is enhanced phosphorylation of several membrane proteins, especially that of a glycoprotein of Mr 170 K, by a membrane-bound cyclic nucleotide independent protein kinase.

Topics: Adenocarcinoma; Cell Line; Cell Membrane; Colonic Neoplasms; Epidermal Growth Factor; Glycoproteins; Humans; Membrane Proteins; Phosphorylation

Topics: Adenocarcinoma; Cell Aggregation; Cell Line; Cell Survival; Culture Media; Epidermal Growth Factor; Epithelial Cells; Humans; Karyotyping; Male; Microscopy, Electron, Scanning; Microvilli; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms

Topics: Adenocarcinoma; Calcium; Cell Division; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; Growth Substances; Humans; Hydrocortisone; Insulin; Male; Prostate; Prostatic Neoplasms; Tetradecanoylphorbol Acetate