epidermal-growth-factor and Carcinoma

Oncogenic viruses may have a significant impact on the therapeutic management of several malignancies besides their well-known role in tumor pathogenesis. Epstein-Barr virus (EBV) induces neoplastic transformation of epithelial cells of the nasopharynx by various molecular mechanisms mostly involving activation of oncogenes and inactivation of tumor-suppressor genes. EBV infection can also induce the expression of several immunogenic peptides on the plasma membrane of the infected cells. Importantly, these virus-related antigens may be used as targets for antitumor immunotherapy-based treatment strategies. Two different immunotherapy strategies, namely adoptive and active immunotherapy, have been developed and strongly improved in the recent years. Furthermore, EBV infection may influence the use of targeted therapies for nasopharyngeal carcinoma (NPC) considering that the presence of EBV can induce important modifications in cell signaling. As an example, latent membrane protein type 1 is a viral transmembrane protein mainly involved in the cancerogenesis process, which can also mediate overexpression of the epidermal growth factor receptor (EGFR) in NPC cells, rendering them more sensitive to anti-EGFR therapy. Finally, EBV may induce epigenetic changes in the infected cells, such as DNA hypermethylation and histone deacetylation, that can sustain tumor growth and can thus be considered potential targets for novel therapies. In conclusion, EBV infection can modify important biological features of NPC cells, rendering them more vulnerable to both immunotherapy and targeted therapy.

Topics: Antigens, Viral; Carcinoma; Epidermal Growth Factor; Epigenesis, Genetic; Epstein-Barr Virus Infections; Herpesvirus 4, Human; Humans; Immunotherapy, Adoptive; Molecular Targeted Therapy; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Viral Matrix Proteins

Topics: Carcinoma; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Drug Resistance, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Genes, erbB-1; Humans; Ligands; Neoplasm Metastasis; Neoplasm Proteins; Ovarian Neoplasms; Prognosis

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.

Topics: Animals; Carcinoma; Cell Transformation, Neoplastic; Dimerization; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; Mutation; Protein Structure, Tertiary; Signal Transduction

The ability of neoplastic cells to dissemination from a primary tumor to lymphatic nodes and to adjacent and distant tissues and organs is an inseparable feature of malignant tumors and the main cause of failure in their treatment. Metastasis formation is a multistage process which includes proteolysis, the motility and migration of cells, proliferation, and neoangiogenesis. In the first step, the cells released from the primary tumor have to penetrate to the blood or lymphatic vessels (intravasation), the road which dissemination follows. Circulating cells can then migrate through the walls of vessels to surrounding tissues (extravasation) where they settle, proliferate, and induce angiogenesis, creating metastases. Indispensable in the process of intra- and extravasation is the activation of proteolytic enzymes capable of degrading the extracellular matrix (ECM) surrounding the endothelium or creating the basement membrane of epithelial tissue in different organs. In this stage, the activation of proteolytic enzymes, such as proteinases of the plasmin system, serine proteinases, and matrix metalloproteinases (MMPs), is necessary. Simultaneously, changes occur in the expression of many superficial glycoproteins and factors responsible for cell adhesion (integrins) and intercellular communication (cadherins). Neoangiogenesis is connected with the expression of many markers of this process, among them vascular endothelial growth factor (VEGF), endoglin (CD105), a transmembranous glycoprotein which is a component of the receptor for transforming growth factor beta (TGFbeta), as well as neuropilin (NRP), the co-receptor for VEGF. Conventionally, the prognosis of neoplastic disease and its treatment are based mainly on exact clinical and histopathological staging. This prognosis could, however, be improved by measuring the molecular and cellular markers which play key roles in tumor progression. Understanding the cellular processes responsible for tumor dissemination can be useful not only in the diagnosis and prognosis of treatment results, but also in developing targeted drugs, selectively directed towards those factors responsible for tumor invasiveness, as well as in creating new therapeutic strategies permitting the use of such drugs. In the present review the authors concentrate mainly on one tumor type, colorectal carcinoma, in which distant metastases, predominantly to the liver, are the main cause of failure, in spite of surgical curing of the primar

Topics: Biomarkers, Tumor; Carcinoma; Cell Movement; Cell Transformation, Neoplastic; Colorectal Neoplasms; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Humans; Lymphatic Metastasis; Neoplasm Invasiveness; Neovascularization, Pathologic

Targeting the estrogen receptor (ER) is the oldest form of molecular targeted therapy, and the widespread use of the selective estrogen receptor modulator tamoxifen in breast cancer is responsible for major improvements in cure rates, quality of life, and disease prevention in the last 25 years. Newer forms of endocrine therapy now available for the management of endocrine responsive breast cancer include a new generation of aromatase inhibitors, which lower the estrogen ligand for ER, and pure ER antagonists which destroy the receptor. Despite these recent clinical advances, intrinsic and acquired resistance to these endocrine therapies is still a common feature that limits the success of this therapeutic strategy. Recent research into the molecular biology of ER signaling has revealed a remarkably complex interactive signaling with other growth factor signaling pathways in breast cancer cells, potentially explaining some of the reasons behind endocrine therapy action as well as resistance. This view of a more complex ER signaling system has uncovered new molecular targets which, if present in a cancer cell, might be additionally targeted using various signal transduction inhibitors to overcome or prevent resistance to endocrine therapy. In addition, the dynamic inverse relationship between the expression of ER and growth factor receptors brings more excitement to the potential of restoring ER expression in apparently ER-negative cells by inhibition of growth factor signaling. Ongoing clinical trials of endocrine therapy combined with growth factor pathway inhibitors or their downstream signaling elements promise to further improve the present care for breast cancer patients.

Topics: Animals; Antineoplastic Agents, Hormonal; Breast Neoplasms; Carcinoma; Drug Resistance, Neoplasm; Epidermal Growth Factor; Humans; Models, Biological; Receptor Cross-Talk; Receptors, Estrogen; Signal Transduction

Endocrine therapy is the treatment of choice in hormone receptor-positive breast cancer. However, the effectiveness of anti-hormone drugs, such as tamoxifen, is limited because of the development of resistance, ultimately leading to disease progression and patient mortality. Using in vitro cell models of anti-hormone resistance, we have previously demonstrated that altered growth factor signalling contributes to an endocrine insensitive phenotype. Significantly, our recent studies have revealed that the acquisition of endocrine resistance in breast cancer is accompanied by a greatly enhanced migratory and invasive phenotype. Furthermore, therapeutic intervention using anti-growth factor monotherapies, despite an initial growth suppressive phase, again results in the development of a resistant state and a further augmentation of their invasive phenotype. Using the dual specific Src/Abl kinase inhibitor, AZD0530, we have highlighted a central role for Src kinase in promoting the invasive phenotype that accompanies both anti-hormone and anti-growth factor resistance. Importantly, the use of Src inhibitors in combination with anti-growth factor therapies appears to be additive, producing a marked inhibitory effect on cell growth, migration and invasion and ultimately prevents the emergence of a resistant phenotype. These observations suggest that the inhibition of Src activity may present a novel therapeutic intervention strategy, particularly when used as an adjuvant in endocrine-resistant breast disease, with the potential to delay or prevent the acquisition of subsequent resistance to anti-growth factor therapies.

Topics: Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cadherins; Carcinoma; Catenins; Cell Adhesion; Cell-Matrix Junctions; Drug Resistance, Neoplasm; Epidermal Growth Factor; Humans; Neoplasm Invasiveness; Proto-Oncogene Proteins pp60(c-src)

Activation of the epidermal growth receptor (ErbB1) occurs within minutes of a radiation exposure. Immediate downstream consequences of this activation are currently indistinguishable from those obtained with growth factors (GF), e.g. stimulation of the pro-proliferative mitogen-activated protein kinase (MAPK). To identify potential differences, the effects of GFs and radiation on other members of the ErbB family have been compared in mammary carcinoma cell lines differing in their ErbB expression profiles. Treatment of cells with EGF (ErbB1-specific) or heregulin (ErbB4-specific) resulted in a hierarchic transactivations of ErbB2 and ErbB3 dependent on GF binding specificity. In contrast, radiation indiscriminately activated all ErbB species with the activation profile reflecting that cell's ErbB expression profile. Downstream consequences of these ErbB interactions were examined with MAPK after specifically inhibiting ErbB1 (or 4) with tyrphostin AG1478 or ErbB2 with tyrphostin AG825. MAPK activation by GFs or radiation was completely inhibited by AG1478 indicating total dependance on ErbB1 (or 4) depending on which ErbB is expressed. Inhibiting ErbB2 caused an enhanced MAPK response simulating an amplified ErbB1 (or 4) response. Thus ErbB2 is a modulator of ErbB1 (or 4) function leading to different MAPK response profiles to GF or radiation exposure.

Topics: Autocrine Communication; Benzothiazoles; Breast Neoplasms; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Genes, erbB; Growth Substances; Humans; Neuregulin-1; Quinazolines; Radiation, Ionizing; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Receptor, ErbB-3; Receptor, ErbB-4; Signal Transduction; Tumor Cells, Cultured; Tyrphostins

Previous work carried out in the authors' laboratory has shown that LHRH agonists directly inhibit the proliferation of hormone-responsive and hormone-independent human prostatic cancer cell lines (respectively LNCaP and DU145). In addition, the hormone-dependent LNCaP cells respond to a challenge with testosterone with an increase in growth rate. The following experiments have been performed to investigate whether the LHRH agonists might act by interfering with the stimulatory actions of either the EGF/TGF alpha system or androgens. The results obtained in LNCaP and DU145 cells show that LHRH agonists counteract the mitogenic action of the EGF/TGF alpha system. This effect is mediated by a decrease in the concentration of EGF receptors. In addition, in the hormone-dependent LNCaP cells, the treatment with LHRH agonists antagonizes the proliferation promoting effect of testosterone, which in turn appears to be mediated by the activation of the locally expressed EGF/TGF alpha system. Finally, the results suggest the presence in LNCaP cells of a soluble peptidase able to degrade LHRH. In conclusion, the present data suggest an intimate interplay among the actions of LHRH agonists, of androgens and of growth factors, thus, supporting the hypothesis that LHRH agonists may interfere with the EGF/TGF alpha stimulatory loop and with androgens in the control of the proliferation of human prostatic tumors.

Topics: Androgen Antagonists; Androgens; Antineoplastic Agents, Hormonal; Brain Neoplasms; Carcinoma; Cell Division; Endopeptidases; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Lymphatic Metastasis; Male; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Prostatic Neoplasms; Somatostatin; Testosterone; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Transforming Growth Factors

Topics: 5-alpha Reductase Inhibitors; Androgens; Androstenes; Azasteroids; Carcinoma; Cell Division; Cells, Cultured; Dihydrotestosterone; Epidermal Growth Factor; Finasteride; Gonadotropin-Releasing Hormone; Growth Substances; Humans; Insulin-Like Growth Factor I; Male; Peptides; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Tumor Cells, Cultured

To study the effect of Qingre Liyan Decoction (QRLYD) in the prevention and treatment of acute radiative oral mucositis (AROM), and to explore the mechanism of QRLYD by detecting epidermal growth factor (EGF) and T lymphocytes (CD3, CD4, and CD8).. Sixty patients conforming with the standard were randomly assigned to two groups, 30 patients in each group. Patients in the trial group were treated with QRLYD, and those in the control group were treated with Dobell's solution, both groups receiving conventional radiation treatment. The treatment course for both groups was 6 weeks on average. Blood routine test, CD3, CD4, and CD8 in the peripheral blood and EGF in the saliva were detected one day before and on the 14th and 28th day of radio-therapy.. Patients in the trial group were in good condition with normal spirits and intake of food and drinks. The incidence of AROM is lower and the effect in preventing AROM is higher in the trial group than those in the control group (P<0.05). The EGF in saliva, and CD4 and CD8 in the blood of patients in the trial group were higher than those in the control group (P<0.05).. QRLYD can cure and prevent AROM. The mechanism may be related with its effects in enhancing body immunity and promoting salivary EGF.

Topics: Acute Disease; Adult; Carcinoma; Drugs, Chinese Herbal; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Incidence; Leukocyte Count; Male; Middle Aged; Phytotherapy; Platelet Count; Radiation Injuries; Stomatitis; T-Lymphocyte Subsets; Treatment Outcome

There is evidence of a relationship between epidermal growth factor (EGF) and tumor cell proliferation, such as the overexpression of EGF receptor (EGF-R) in different human tumors, which makes this system an interesting target for cancer treatment. Up to now, passive immunotherapy with monoclonal antibodies against the EGF-R has been assayed in clinics. Our approach consists of active immunotherapy with human EGF (hu-EGF). We conducted a pilot clinical trial to define the safety, toxicity and immunogenicity of vaccination with hu-EGF coupled to a carrier protein.. Ten patients with histologically-proven malignant carcinomas (colon, lung, stomach and prostate) in advanced clinical stages were enrolled. Patients were immunized twice (on days 0 and 15) with hu-EGF linked to either tetanic toxoid (TT, five patients) or P64K Neisseria Meningitidis recombinant protein (P64k, five patients), intradermically, using aluminium hydroxyde as adjuvant.. In both groups 60% of patients developed anti-EGF antibody titers without evidence of toxicity. Secondary reactions were very mild, limited to erythema and itching at the site of injection, which disappeared without medication.. We conclude that the proposed vaccination with hu-EGF was well tolerated and that antibody titers against self EGF were developed. The results of this trial may be useful in the design of new clinical trials with higher dose immunization protocols and using more effective adjuvants.

Topics: Aged; Cancer Vaccines; Carcinoma; Carrier Proteins; Colonic Neoplasms; Epidermal Growth Factor; Female; Humans; Immunotherapy; Lung Neoplasms; Male; Middle Aged; Pilot Projects; Prostatic Neoplasms; Stomach Neoplasms; Treatment Outcome

Somatostatin analogues (SMS-A) have been found to inhibit the growth of experimental tumors, as of prostate cancer, via several mechanisms as antihormonal and direct antimitogenic actions. It was demonstrated also that several SMS-A induce greater prostatic tumor regression with more pronounced histological changes if combined with LHRH analogues or in association with complete androgen blockade (CAB). In a phase II clinical trial we administered, in addition to CAB, SMS-A octreotide in 14 patients with stage D2 (group B) prostate cancer-8 previously hormonally treated (PHT) and 6 without any previous hormone treatment (NPHT); 4 other patients, 3 NPHT and one PHT, were treated with CAB only (group A). Antiandrogen and antitumoral activity followed assaying a) plasma testosterone b) prostatic specific antigen (PSA) c) prostatic acid phosphatase (PAP) levels and d) objective (o) and subjective (s) clinical improvement according to WHO criteria. Somatostatin activity was evaluated assaying Insulin like Growth Factor-1 (IGF-1) and Epidermal Growth Factor (EGF). In group B we observed 3 responses, with the best quality of response (oPR/sCR) among the 6 NPHT-patients (50%) and 3 responses among the PHT-patients (37,5%), two of them with an incomplete PHT. In group A, 2 out of 3 NPHT-patients had a response (oPR/sPR). Among group B patients we observed long symptom-free survival, when they responded (17 months), in comparison to group A patients (12 months), but almost the same total duration of survival in the two groups, 18.5 and 18 months, respectively. EGF and IGF-1 serum levels showed a distinct drop parallel to the decrease of PSA serum levels, among the patients with response vs. nonrespondent patients of group B during the treatment. Although our results showed that octreotide in small doses, in addition to CAB, having mild toxicity, enhance number, quality and perhaps the duration of symptom-free responses in patients with stage 2 prostate cancer, the therapeutic efficacy of this combined treatment remains to be ascertained in wider and better randomized clinical trials.

Topics: Acid Phosphatase; Aged; Aged, 80 and over; Androgen Antagonists; Antineoplastic Agents, Hormonal; Carcinoma; Epidermal Growth Factor; Humans; Insulin-Like Growth Factor I; Male; Middle Aged; Octreotide; Prospective Studies; Prostate-Specific Antigen; Prostatic Neoplasms; Somatostatin; Survival Analysis

We have shown that decursin, a coumarin compound, induces cell cycle arrest and apoptosis in human prostate cancer cells (PCa); however, its molecular mechanisms are largely unexplored. We studied the mechanisms associated with its anticancer activity in advanced human prostate carcinoma cells. We found that decursin inhibited epidermal growth factor receptor (EGFR) signaling by inhibiting its activating phosphorylation at tyrosine 1068 residue in DU145 and 22Rv1 cells. This inhibition of EGFR was associated with the downregulation of ERK1/2 phosphorylation. Both EGFR and ERK1/2 are known to be deregulated/activated in many human malignancies. Consistent with our earlier study, decursin (25-100 µM) treatment for 24-72 h inhibited DU145 cell proliferation by 49%-87% (p < 0.001) which was associated with strong G1 phase arrest and cell death. It also decreased (p < 0.001) the number of surviving colonies. Decursin moderately increased the expression of Rb-related proteins p107 and p130 but decreased the levels of E2F family transcription factors including E2F-3, E2F-4 and E2F-5. Further, decursin strongly inhibited the growth of androgen-dependent prostate carcinoma 22Rv1 cells from 61% to 79% (p < 0.001) and arrested these cells at G1 phase via induction of cyclin-dependent kinase inhibitor p27/Kip1 and downregulation of CDK2 and CDK4 protein expression. Additionally, EGFR inhibitor erlotinib- and EGF ligand-modulated EGFR activation validated EGFR signaling as a target of decursin-mediated cell growth inhibition and cytotoxicity. Decursin decreased EGF ligand-induced phosphorylation of EGFR (Y-1068) as well as activation of its downstream mediator, ERK1/2. Furthermore, inhibitory targeting of EGFR-ERK1/2 axis by combinatorial treatment of decursin and erlotinib further sensitized DU145 cells for the decursin-induced growth inhibition and cell death. Overall, these findings strongly suggest that anticancer efficacy of decursin against human PCa involves inhibitory targeting of EGFR-ERK1/2 signaling axis, a pathway constitutively active in advanced PCa.

Topics: Carcinoma; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Humans; Ligands; Male; MAP Kinase Signaling System; Phosphorylation; Prostate; Prostatic Neoplasms

The present research work describes development of dual drug-loaded lipid-polymer hybrid nanoparticles (LPHNPs) of anticancer therapeutics for the management of colon cancer. The epidermal growth factor (EGF)-functionalized LPHNPs coloaded with 5-fluorouracil (FU) and sulforaphane (SFN) were prepared by one-step nanoprecipitation method. Box-Behnken design was applied for optimizing the material attributes and process parameters. The optimized LPHNPs revealed particle size 198 nm, polydispersity index 0.3, zeta potential -25.3 mV, and drug loading efficiency 19-20.3% for 5-FU and SFN, respectively. EGF functionalization on LPHNPs was confirmed from positive magnitude of zeta potential to 21.3 mV as compared with the plain LPHNPs. In vitro drug release performance indicated sustained and non-Fickian mechanism release nature of the drugs from LPHNPs. Anticancer activity evaluation in HCT-15 colon cancer cells showed significant reduction (p < 0.001) in the cell growth and cytotoxicity of the investigated drugs from various treatments in the order: EGF-functionalized LPHNPs > plain LPHNPs > free drug suspensions. Overall, the research work corroborated improved treatment efficacy of EGF-functionalized LPHNPs for delivering chemotherapeutic agents for the management of colon carcinoma.

Topics: Biological Availability; Carcinoma; Cell Survival; Colonic Neoplasms; Drug Carriers; Drug Delivery Systems; Epidermal Growth Factor; Fluorouracil; Humans; Lipids; Nanoparticles; Particle Size; Polymers

Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR : EGF stoichiometry of approximately 4 : 1 for plasma membrane-colocalized EGFR-EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.

Topics: Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphorylation; Receptor, ErbB-2; Signal Transduction

Duodenal cancer in familial adenomatous polyposis (FAP) arises from adenomas. Differentially expressed genes (DEGs) in the duodenal adenoma-carcinoma pathway have been identified in murine FAP models, but similar data in patients with FAP are limited. Identifying such changes may have significance in understanding duodenal polyposis therapies and identifying cancer biomarkers. We performed a genome-wide transcriptional analysis to describe the duodenal adenoma-carcinoma sequence and determine changes distinguishing patients with FAP with and without duodenal cancer.. Transcriptional profiling was performed with the Affymetrix Human Transcriptome Array 2.0 on duodenal biopsies from 12 FAP patients with duodenal cancer (FAP cases) and 12 FAP patients without cancer (FAP controls). DEGs were compared between cancer-normal, adenoma-normal, and cancer-adenoma in FAP cases and between adenomas from FAP cases and FAP controls. Significant results at P < 0.05 were filtered using fold change > 2.. Two hundred twenty-four DEGs were identified at an absolute fold change > 2. In adenoma-normal, downregulation of DEGs involved in metabolism of brush border proteins (LCT), lipids (APOB/A4), reactive oxygen species (GSTA2), and retinol (RBP2) was observed. In the cancer-adenoma comparison, upregulation of DEGs involved in cell invasion/migration (POSTN, SPP1) and downregulation of DEGs involved in Paneth differentiation (DEFA5/6) were observed. In the adenoma-adenoma comparison, downregulation of several DEGs (CLCA1, ADH1C, ANXA10) in FAP case adenomas was observed. DEGs with therapeutic potential include SPP1, which is involved in both cyclooxygenase and epidermal growth factor receptor pathways targeted by the sulindac/erlotinib combination for duodenal polyposis.. We describe DEGs in the human duodenal adenoma-carcinoma sequence in FAP, which may have prognostic and therapeutic significance. Validation studies are needed to confirm these findings.

Topics: Adenoma; Adenomatous Polyposis Coli; Adult; Animals; Carcinoma; Cell Adhesion Molecules; Duodenal Neoplasms; Epidermal Growth Factor; Female; Gene Expression Profiling; Humans; Male; Mice; Middle Aged; Osteopontin

Adverse events (AEs) of epidermal growth factor inhibitors (EGFRi) influence well-being with a risk to dose modifications (DMs). Hereby, clinical benefit of treatment might be affected. This retrospective cohort study was set up to gain insight into the suitability and added value of a patient-reported outcome measurement tool (PROM), together with a stepwise intervention management plan for EGFRi-related AEs in daily practice. The primary objective was to gain insight into total treatment duration and DMs, and the secondary objective to gain insight into patient-reported symptoms and well-being as well as healthcare professional-reported AEs. Sixty-eight patients on cetuximab and 19 on panitumumab treatment were included for analysis; 69% had squamous cell carcinoma of head and neck (SCCHN) and 26% metastatic colorectal carcinoma. DMs due to AEs occurred in 39% of the patients and dose discontinuations in 22%. Especially anorexia, dysphagia, oral pain and skin changes led to a decreased well-being. In patients on EGFRi, application of PROMs together with a stepwise symptom management plan enhances early recognition of symptom burden, pro-active symptom management and effect evaluation of interventions performed whereby well-being recovers. Since only SCCHN patients discontinued treatment due to AEs, patient-centred care focused on radiotherapy-related AEs, creates opportunities for amelioration.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antineoplastic Agents, Immunological; Carcinoma; Cetuximab; Colorectal Neoplasms; Epidermal Growth Factor; Female; Head and Neck Neoplasms; Humans; Male; Middle Aged; Panitumumab; Retrospective Studies

Most sporadic carcinomas with high metastatic activity show an increased rate of changes in chromosome structure and number, known as chromosomal instability (CIN). However, the role of CIN in driving invasiveness remains unclear. Using an epithelial model in Drosophila, we present evidence that CIN promotes a rapid and general invasive behavior. Cells with an abnormal number of chromosomes delaminate from the epithelium, extend actin-based cellular protrusions, form membrane blebs, and invade neighboring tissues. This behavior is governed by the activation of non-muscle Myosin II by Rho kinase and by the expression of the secreted EGF/Spitz ligand. We unravel fundamental roles of the mitogen-activated protein kinase pathways mediated by the Fos proto-oncogene and the Capicua tumor suppressor gene in the invasive behavior of CIN-induced aneuploid cells. Our results support the proposal that the simple production of unbalanced karyotypes contributes to CIN-induced metastatic progression.

Topics: Aneuploidy; Animals; Apoptosis; Blister; Carcinoma; Chromosomal Instability; Drosophila melanogaster; Drosophila Proteins; Epidermal Growth Factor; Epithelium; ErbB Receptors; HMGB Proteins; Membrane Proteins; Mitogen-Activated Protein Kinases; Myosin Type II; Neoplasm Invasiveness; Proto-Oncogene Proteins c-fos; Repressor Proteins; rho-Associated Kinases

Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling.

Topics: Animals; Apoptosis; Carcinoma; Cell Line, Tumor; Ectopic Gene Expression; Epidermal Growth Factor; ErbB Receptors; Gene Expression; Gene Expression Regulation, Neoplastic; Humans; Mice; NIH 3T3 Cells; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Binding; Protein Transport; Proteins; Proteolysis; Signal Transduction

Sprouty (SPRY) proteins are well-characterized factors that inhibit receptor tyrosine kinase signaling. Our Human Exonic Evidence-Based Oligonucleotide (HEEBO) microarray results showed that the mRNA levels of SPRY2, but not of SPRY1 or SPRY4, are down-regulated in high-grade serous ovarian carcinoma (HGSC) tissues and epithelial ovarian cancer (EOC) cell lines. Molecular inversion probe (MIP) copy number analysis showed the deletion of the SPRY2 locus in HGSC. Overexpression of SPRY2 reduced EGF-induced cell invasion by attenuating EGF-induced E-cadherin down-regulation. Moreover, a positive correlation between SPRY2 and E-cadherin protein levels was observed in HGSC tissues. This study reveals the loss of SPRY2 in HGSC and indicates an important tumor-suppressive role for SPRY2 in mediating the stimulatory effect of EGF on human EOC progression.

Topics: Cadherins; Carcinoma; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Signal Transduction

Hypercortisolism is a common endocrine disorder in dogs, caused by a cortisol-secreting adrenocortical tumor (AT) in approximately 15% of cases. In adrenocortical carcinomas of humans, activation of the phosphatidylinositol 3 kinase (PI3K) signaling pathway by insulin-like growth factor (IGF) signaling represents a promising therapeutic target.. To investigate the involvement of PI3K signaling in the pathogenesis of ATs in dogs and to identify pathway components that may hold promise as future therapeutic targets or as prognostic markers.. Analyses were performed on 36 canine cortisol-secreting ATs (11 adenomas and 25 carcinomas) and 15 normal adrenal glands of dogs.. mRNA expression analysis was performed for PI3K target genes, PI3K inhibitor phosphatase and tensin homolog (PTEN), IGFs, IGF receptors, IGF binding proteins and epidermal growth factor receptors. Mutation analysis was performed on genes encoding PTEN and PI3K catalytic subunit (PIK3CA).. Target gene expression indicated PI3K activation in carcinomas, but not in adenomas. No amino acid-changing mutations were detected in PTEN or PIK3CA and no significant alterations in IGF-II or IGFR1 expression were detected. In carcinomas, ERBB2 expression tended to be higher than in normal adrenal glands, and higher expression of inhibitor of differentiation 1 and 2 (ID1 and ID2) was detected in carcinomas with recurrence within 2.5 years after adrenalectomy.. Based on these results, ERBB2 might be a promising therapeutic target in ATs in dogs, whereas ID1 and 2 might be valuable as prognostic markers and therapeutic targets.

Topics: Adenoma; Adrenal Cortex Neoplasms; Animals; Carcinoma; Dog Diseases; Dogs; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Hydrocortisone; Male; Phosphatidylinositol 3-Kinase; PTEN Phosphohydrolase; RNA, Messenger; Signal Transduction; Somatomedins

The role of EGF and TGF-β1 in thyroid cancer is still not clearly defined. TGF-β1 inhibited the cellular growth and migration of follicular (FTC-133) and papillary (B-CPAP) thyroid carcinoma cell lines. Co-treatments of TGF-β1 and EGF inhibited proliferation in both cell lines, but displayed opposite effect on their migratory capability, leading to inhibition in B-CPAP and promotion in FTC-133 cells, by a MAPK-dependent mechanism. TGF-β1, TβRII and EGFR expressions were evaluated in benign and malignant thyroid tumors. Both positivity (51.7% and 60.0% and 80.0% in FA and PTC and FTC) and overexpression (60.0%, 77.7% and 75.0% in FA, PTC and FTC) of EGFR mRNA correlates with the aggressive tumor behavior. The moderate overexpression of TGF-β1 and TβRII mRNA in PTC tissues (61.5% and 62.5%, respectively), counteracted their high overexpression in FTC tissues (100% and 100%, respectively), while EGFR overexpression was similar in both carcinomas. Papillary carcinomas were positive to E-cadherin expression, while the follicular carcinomas lose E-cadherin staining. Our findings of TGF-β1/TβRII and EGFR overexpressions together with a loss of E-cadherin observed in human follicular thyroid carcinomas, and of increased migration ability MAPK-dependent after EGF/TGF-β1 treatments in the follicular thyroid carcinoma cell line, reinforced the hypothesis of a cross-talk between EGF and TGF-β1 systems in follicular thyroid carcinomas phenotype.

Topics: Adult; Aged; Cadherins; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression; Humans; Male; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA, Messenger; Thyroid Neoplasms; Transforming Growth Factor beta1; Young Adult

The molecular mechanism underlying cancer invasiveness and metastasis of larynx carcinoma remains elusive. Here we reported a strong correlation between phosphorylated epidermal growth factor receptor (EGFR) and matrix metalloproteinase-7 (MMP7) levels in larynx carcinoma patients. To examine whether a causal link exists, we used a human larynx carcinoma line, Hep-2, to study the molecular basis of EGFR signaling and MMP7 activation. We found that EGF-induced EGFR phosphorylation in Hep-2 cells resulted in activation of MMP7 and, consequently, an increase in cancer invasiveness. An EGFR inhibitor efficiently blocked this EGF-induced activation of MMP7. Moreover, an inhibitor for PI3 kinase (PI3K)/Akt, but not an inhibitor for mitogen-activated protein kinase (MAPK) or an inhibitor for c-Jun N-terminal kinase (JNK), significantly inhibited the EGF-induced activation of MMP7, suggesting that PI3K/Akt signaling cascades may be responsible for EGF-activated MMP7. Further dissection of the pathway revealed that nuclear exclusion of Akt downstream target, FoxO1, was induced by EGF-induced Akt activation and could be inhibited by either the EGFR inhibitor or by the PI3K/Akt inhibitor. Expression of a constitutive nuclear form of FoxO1 significantly inhibited MMP7 activation induced by EGF. Taken together, these findings suggest that EGF/EGFR signaling activates downstream PI3K/Akt to induce FoxO1 nuclear exclusion, which activates MMP7 to promote larynx carcinoma metastasis. Thus, Akt and FoxO1 appear to be promising therapeutic targets for preventing the metastasis of larynx carcinoma.

Topics: Blotting, Western; Carcinoma; Cell Line, Tumor; Cell Nucleus; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Forkhead Box Protein O1; Forkhead Transcription Factors; Humans; Laryngeal Neoplasms; Matrix Metalloproteinase 7; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Protein Transport; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; Transfection

The intention of this work was to lift saponin supported tumor targeted therapies onto the next level by using targeted toxins in nude mice xenotransplant models.. Combined application of dianthin coupled to EGF and saponin SO-1861 was tested in a xenograft model of colon carcinoma. In vitro cytotoxicity was tested in real-time in NIH3T3 cells (no human EGF receptor expression), HER14 and human colon carcinoma HCT116 (both EGF receptor overexpressing) cells. A xenograft model was established using HCT116 cells and tumor-bearing animals were treated with SO-1861 (30 µg/treatment) and dianthin coupled to EGF (0.35 µg/treatment). Tumor progression was monitored, using (18)F-2-fluor-2-desoxy-d-glucose, by small animal PET and by x-ray computed tomography.. In vitro results demonstrated a high-receptor specificity and the in vivo experiment showed a progressive reduction of the tumor volume and glycolytic activity in the treated group (>95% reduction; p < 0.05).. This therapy has great advantage because of high specificity, low side effects and great effectiveness for future development in the treatment of colon cancer.

Topics: Animals; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Dianthus; Disease Models, Animal; Drug Therapy, Combination; Epidermal Growth Factor; Hemolysis; Humans; Immunotoxins; Male; Mice; Positron-Emission Tomography; Ribosome Inactivating Proteins, Type 1; Saponins; Tumor Burden; Xenograft Model Antitumor Assays

Invadopodia or invasive feet, which are actin-rich membrane protrusions with matrix degradation activity formed by invasive cancer cells, are a key determinant in the malignant invasive progression of tumors and represent an important target for cancer therapies. In this work, we presented a microfluidic 3D culture device with continuous supplement of fresh media via a syringe pump. The device mimicked tumor microenvironment in vivo and could be used to assay invadopodia formation and to study the mechanism of human lung cancer invasion. With this device, we investigated the effects of epidermal growth factor (EGF) and matrix metalloproteinase (MMP) inhibitor, GM6001 on invadopodia formation by human non-small cell lung cancer cell line A549 in 3D matrix model. This device was composed of three units that were capable of achieving the assays on one control group and two experimental groups' cells, which were simultaneously pretreated with EGF or GM6001 in parallel. Immunofluorescence analysis of invadopodia formation and extracellular matrix degradation was conducted using confocal imaging system. We observed that EGF promoted invadopodia formation by A549 cells in 3D matrix and that GM6001 inhibited the process. These results demonstrated that epidermal growth factor receptor (EGFR) signaling played a significant role in invadopodia formation and related ECM degradation activity. Meanwhile, it was suggested that MMP inhibitor (GM6001) might be a powerful therapeutic agent targeting invadopodia formation in tumor invasion. This work clearly demonstrated that the microfluidic-based 3D culture device provided an applicable platform for elucidating the mechanism of cancer invasion and could be used in testing other anti-invasion agents.

Topics: Apoptosis; Carcinoma; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Dipeptides; Epidermal Growth Factor; Extracellular Matrix; Humans; Lung Neoplasms; Microfluidic Analytical Techniques; Tumor Cells, Cultured

We sought to explore the mechanisms of cervical carcinoma response to epidermal growth factor (EGF), and then identify biologically active small molecules capable of targeting the sub-pathways that were dysregulated in cervical cancer cells in the response to EGF.. Differentially expressed genes and pathways were analyzed based on the transcription profile of GSE6783, and then the differentially expressed molecules were further analyzed by several bioinformatics methods.. Our results suggested that EGF could promote cervical cancer cell proliferation through triggering the dysregulation of certain sub-pathways in the mitogen-activated protein kinase signaling pathway, p53 signaling pathway and pathways in cancer. Furthermore, our bioinformatics analysis revealed a total of 49 small molecules which may play a role in perturbing the response to EGF of cervical cancer cells.. Candidate drugs identified by our approach may provide the groundwork for a combination therapy approach for cervical cancer; however, further studies are still needed to make sure that the use of parthenolide or other anti-cancer agents is effective without inhibiting important host defense mechanisms in cervical cancer.

Topics: Antineoplastic Agents; Carcinoma; Cell Proliferation; Computational Biology; Epidermal Growth Factor; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Molecular Targeted Therapy; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Tumor-associated macrophages (TAM) promote malignant progression, yet the repertoire of oncogenic factors secreted by TAM has not been clearly defined. We sought to analyze which EGFR- and STAT3-activating factors are secreted by monocytes/macrophages exposed to tumor cell-secreted factors.. Following exposure of primary human monocytes and macrophages to supernatants of a variety of tumor cell lines, we have analyzed transcript and secreted protein levels of EGFR family ligands and of STAT3 activators. To validate our findings, we have analyzed TAM infiltration levels, systemic and local protein levels as well as clinical data of primary breast cancer patients.. Primary human monocytes and macrophages respond to tumor cell-derived factors by secreting EGFR- and STAT3-activating ligands, thus inducing two important oncogenic pathways in carcinoma cells. Tumor cell-secreted factors trigger two stereotype secretory profiles in peripheral blood monocytes and differentiated macrophages: monocytes secrete epiregulin (EREG) and oncostatin-M (OSM), while macrophages secrete heparin-binding EGF-like growth factor (HB-EGF) and OSM. HB-EGF and OSM cooperatively induce tumor cell chemotaxis. HB-EGF and OSM are co-expressed by TAM in breast carcinoma patients, and plasma levels of both ligands correlate strongly. Elevated HB-EGF levels accompany TAM infiltration, tumor growth and dissemination in patients with invasive disease.. Our work identifies systemic markers for TAM involvement in cancer progression, with the potential to be developed into molecular targets in cancer therapy.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cells, Cultured; Chemotaxis; Epidermal Growth Factor; Epiregulin; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Lymphatic Metastasis; Macrophages; Monocytes; Oncostatin M; Paracrine Communication; STAT3 Transcription Factor

Store-operated Ca(2+) entry (SOCE) mediates Ca(2+) responses evoked by extracellular signaling molecules to promote increases in cytosolic Ca(2+), thereby triggering downstream signal transduction. Here we demonstrated that either the pharmacological blockage of Ca(2+) influx through SOCE or the knockdown of Orai1, a key molecule of SOCE, suppressed the epidermal growth factor-induced migration by disturbing Ca(2+) signaling in nasopharyngeal carcinoma (NPC) cell. Furthermore, Orai1 depletion led to a delayed cell attachment to the extracellular matrix surface in vitro and eliminated the extravasation of microinjected cells from vasculature in a zebrafish hematogenous metastasis model. Our findings thus indicate that SOCE acts as a predominant Ca(2+) signaling involved in NPC cell metastasis, and may serve as a candidate target for anti-metastasis therapy in NPC.

Topics: Animals; Animals, Genetically Modified; Calcium; Calcium Channel Blockers; Calcium Signaling; Carcinoma; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Humans; Intracellular Calcium-Sensing Proteins; Ion Transport; Membrane Proteins; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Signal Transduction; Zebrafish

Increasing the efficiency of gene transfer using non-viral vectors, which have the potential to be safe and economical, would improve upon available options for gene therapy. We previously reported that the third EGF motif of the extracellular matrix protein Del1 (E3) increases the transfection efficiency of non-viral vector methods. Here, we asked if E3 could increase the in vivo transfection efficiency of a polyplex-based approach. To test this, cDNA encoding a heat-stable alkaline phosphatase (AP) was first injected intravenously into mice along with recombinant E3. After 24 h, exogenous AP activity in serum was measured. We found that the introduction of E3 resulted in 50 % more AP activity as compared to the control. We next tested transfection into a tumour explant of SCCKN cells, an oral carcinoma-derived cell line. To do this, a cDNA encoding yellow fluorescent protein was locally injected into a tumour explant, followed by local injection of recombinant E3. Use of E3 increased the number of transfected cells to 2.5 times that of the control. Histochemical staining revealed that E3-induced apoptosis in a tumour explant. The data suggest that E3 might be a useful tool for cancer gene therapy using non-viral vectors.

Topics: Alkaline Phosphatase; Animals; Apoptosis; Bacterial Proteins; Carcinoma; Carrier Proteins; Cell Line, Tumor; Epidermal Growth Factor; Extracellular Matrix Proteins; Gene Transfer Techniques; Genetic Vectors; Humans; Luminescent Proteins; Mice; Mice, Inbred BALB C; Mouth Neoplasms; Recombinant Proteins; Transfection

Aquaporins (AQPs) are a family of small, integral membrane proteins that have been shown to play an important role in tumor development and metastasis. Several studies have demonstrated that expression of AQP3 contributes to the enhanced migration of epithelial cells and is related to differentiation, metastasis and vascular invasion in lung and gastric cancer. Therefore, we investigated whether AQP3 could enhance human colorectal carcinoma cell migration and we examined the role of AQP3 in the prognosis of colorectal carcinoma. Our results showed that human epidermal growth factor (hEGF) increased the expression of AQP3 and, subsequently, the migration ability of human colorectal carcinoma cells HCT116 in a dose- and time-dependent manner. The enhanced migration ability of HCT116 cells was blocked by the AQP3 inhibitor, CuSO(4). Overexpression of AQP3 induced by hEGF was inhibited by a PI3K/AKT inhibitor, LY294002, but the ERK inhibitor U0126 had a minor effect on the hEGF-induced AQP3 upregulation. Immunohistochemical staining of the cancer tissues and corresponding normal tissues showed that AQP3 expression in cancer tissue was higher compared to that in normal tissue. The expression intensity of AQP3 was associated with the differentiation, lymph node and distant metastasis of colorectal carcinoma patients. Our results suggest that AQP3 overexpression could facilitate colorectal carcinoma cell migration and AQP3 may be considered a potential indicator and therapeutic target for colon tumor metastasis and prognosis.

Topics: Adult; Aged; Aged, 80 and over; Analysis of Variance; Aquaporin 3; Butadienes; Carcinoma; Cell Movement; Chi-Square Distribution; Chromones; Colorectal Neoplasms; Copper Sulfate; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; HCT116 Cells; Humans; Lymphatic Metastasis; Male; MAP Kinase Signaling System; Middle Aged; Morpholines; Nitriles; Phosphatidylinositol 3-Kinases; Prognosis; Proto-Oncogene Proteins c-akt; Up-Regulation

Cervical carcinoma is the second most prevalent and the fifth most deadly malignancy seen in women worldwide. Dysregulated activation of EGF ErbB system has been implicated in diverse types of human cancer; however, it is elusive how it is regulated in human cervical cancer cells. We herein aimed to explore the mechanisms of cervical carcinoma response to epidermal growth factor (EGF), with a view of the pathways activated by EGF.. Using the GSE6783 affymetrix microarray data accessible from gene expression omnibus database, we first identified the differentially expressed genes between EGF-stimulated and -unstimulated samples. Then we constructed a regulation network and identified the network motifs. We also performed biological process and pathway enrichment analyses to functionally classify the genes in the regulation network.. A total of 11 network motifs were identified in the regulation network. EGF treatment could increase the risk of cancer via dysregulation of cancer-related pathways and immune response pathways.. Network motif analysis is useful in mining the useful information underlying the network. We hope our work could serve as a basis for further experimentation.

Topics: Carcinoma; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; HeLa Cells; Humans; Uterine Cervical Neoplasms

Regulatory T cells (Treg) cells play a crucial role in tumor progression by suppressing anti-tumor immunity, but are not well-documented in veterinary oncology. To identify the characteristics of Treg cells in tumor microenvironments, the numbers of Treg cells were analyzed and compared with histological prognostic factors and molecular biomarkers in canine mammary carcinoma (MC) tissues (n=37). Abundant Treg cells were associated with high histological grade and lymphatic invasion. The numbers of Treg cells infiltrating intratumoral areas markedly increased in tumors with poor prognostic factors, such as high histological grade, lymphatic invasion, and necrosis. These findings suggest that Treg cells play a role in canine MC progression. Furthermore, Treg cell numbers in intratumoral compartments may provide a potential prognostic factor when assessing canine MCs, which may in turn lead to the development of new immunologic therapeutics.

Topics: Animals; Biomarkers, Tumor; Carcinoma; Disease Progression; Dog Diseases; Dogs; Epidermal Growth Factor; Female; Forkhead Transcription Factors; Mammary Neoplasms, Animal; Prognosis; Receptors, Estrogen; T-Lymphocytes, Regulatory

Topics: Angiogenesis Inhibitors; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Bevacizumab; Carcinoma; Colorectal Neoplasms; Epidermal Growth Factor; Gastroenterology; Humans; Medical Oncology; Molecular Targeted Therapy; Neoplasm Metastasis

Obesity confers risks to cancer development and progression but the mechanisms underlying these risks remain unclear. In this study, we identify a role for the obesity cytokine leptin, which has been implicated previously in breast cancer development, as a determinant for the tumor-promoting activity of cancer-associated fibroblasts (CAF) in both wild-type (WT) and K303R mutant estrogen receptor-α (ERα)-expressing breast cancer cells. Human CAFs stimulated a greater increase in the proliferation and migration of breast cancer cells expressing the K303R-ERα hyperactive receptor than WT-ERα-expressing cells. A concomitant increase was seen in leptin receptor isoform expression and activation of the leptin signaling pathway in cells expressing K303R-ERα compared with WT-ERα, correlating with leptin effects on cell growth, motility, and invasiveness in mutant cells. Epidermal growth factor and other factors secreted by K303R-ERα cells stimulated CAF proliferation, migration, and subsequent leptin secretion. Moreover, K303R-ERα expression generated a leptin hypersensitive phenotype in vivo. Together, our results reveal a bidirectional cross-talk between breast cancer cells and "educated" CAFs that drives tumor progression via leptin signaling. In elucidating a mechanism that connects obesity and cancer, these findings reinforce the concept that blocking cancer-stromal cell communication may represent an effective strategy for targeted therapy of breast cancer.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; Estrogen Receptor alpha; Female; Fibroblasts; Gene Expression Profiling; Humans; Leptin; Neoplasm Invasiveness; Receptors, Leptin; Signal Transduction; Stromal Cells

Epithelial Mesenchymal Transition (EMT) is a crucial mechanism for carcinoma progression, as it provides routes for in situ carcinoma cells to dissociate and become motile, leading to localized invasion and metastatic spread. Targeting EMT therefore represents an important therapeutic strategy for cancer treatment. The discovery of oncogene addiction in sustaining tumor growth has led to the rapid development of targeted therapeutics. Whilst initially optimized as anti-proliferative agents, it is likely that some of these compounds may inhibit EMT initiation or sustenance, since EMT is also modulated by similar signaling pathways that these compounds were designed to target. We have developed a novel screening assay that can lead to the identification of compounds that can inhibit EMT initiated by growth factor signaling. This assay is designed as a high-content screening assay where both cell growth and cell migration can be analyzed simultaneously via time-course imaging in multi-well plates. Using this assay, we have validated several compounds as viable EMT inhibitors. In particular, we have identified compounds targeting ALK5, MEK, and SRC as potent inhibitors that can interfere with EGF, HGF, and IGF-1 induced EMT signaling. Overall, this EMT screening method provides a foundation for improving the therapeutic value of recently developed compounds in advanced stage carcinoma.

Topics: Antineoplastic Agents; Biological Assay; Carcinoma; Cell Count; Cell Line, Tumor; Drug Evaluation, Preclinical; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Hepatocyte Growth Factor; Humans; Image Processing, Computer-Assisted; Inhibitory Concentration 50; Insulin-Like Growth Factor I; Reproducibility of Results; Small Molecule Libraries

Tumor growth and angiogenic factors are usually overexpressed in colorectal carcinomas. We aimed to assess the prognostic role of VEGF, bFGF, PDGF-AA, EGF, HGF, and E-selectin in patients with metastatic colorectal cancer treated with capecitabine and oxaliplatin (XELOX) chemotherapy protocol. Thirty-eight colorectal cancer patients who had evidence of distant metastasis were enrolled in the study. Angiogenic factors were measured before and after third cycle of chemotherapy. Patients were randomized into three groups, partial response (PR), stable disease (SD), and progressive disease (PD) groups, according to their clinical and radiologic evaluation after three cycles of XELOX chemotherapy. Eighteen patients (47.3 %) achieved partial response, 10 (26.3 %) stable disease, and 10 (26.3 %) progressive disease. VEGF (63.20 Pg/ml vs. 19,79 Pg/ml; p < 0.001), EGF (7.29 ± 3.08 Pg/ml vs. 4.79 ± 2.05 Pg/ml; p < 0.011), HGF (618.16 ± 340.39 Pg/ml vs. 452.02 ± 217.18 Pg/ml; p < 0.049), and PDGF-AA (691.68 ± 187.10 Pg/ml vs. 404.89 ± 168.47 Pg/ml; p < 0.001) were significantly decreased in PR group. PDGF-AA levels were also decreased in SD group (706.66 ± 206.66 Pg/ml vs. 389.79 ± 143.16 Pg/ml; p < 0,001). HGF levels were significantly increased in PD disease group (449.99 Pg/ml vs. 682.22 Pg/ml; p < 0.046). The baseline E-selectin levels were inversely proportional with overall survival that could be an important prognostic factor at the time of diagnosis. This study demonstrated that tumor growth factors can be helpful to determine colorectal cancer prognosis and overall survival in patients with metastatic disease. VEGF, HGF, EGF, and PDGF-AA levels were decreased in PR group. However, meaningful increment was seen HGF levels in PD group. Angiogenic factors and E-selectin provided unique prognostic information in advanced colorectal carcinoma patients.

Topics: Angiogenesis Inducing Agents; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Capecitabine; Carcinoma; Colorectal Neoplasms; Deoxycytidine; E-Selectin; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Fluorouracil; Hepatocyte Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Oxaloacetates; Platelet-Derived Growth Factor; Prognosis; Vascular Endothelial Growth Factor A

Receptor tyrosine kinases and integrins play an essential role in tumor cell invasion and metastasis. We previously showed that EGF and other growth factors induce human carcinoma cell invasion and metastasis mediated by integrin αvβ5 that is prevented by Src blockade. MUC1, a transmembrane glycoprotein, is expressed in most epithelial tumors as a heterodimer consisting of an extracellular and a transmembrane subunit. The MUC1 cytoplasmic domain of the transmembrane subunit (MUC1.CD) translocates to the nucleus where it promotes the transcription of a metastatic gene signature associated with epithelial to mesenchymal transition. Here, we demonstrate a requirement for MUC1 in carcinoma cell metastasis dependent on EGFR and Src without affecting primary tumor growth. EGF stimulates Src-dependent MUC1 cleavage and nuclear localization leading to the expression of genes linked to metastasis. Moreover, expression of MUC1.CD results in its nuclear localization and is sufficient for transcription of the metastatic gene signature and tumor cell metastasis. These results demonstrate that EGFR and Src activity contribute to carcinoma cell invasion and metastasis mediated by integrin αvβ5 in part by promoting proteolytic cleavage of MUC1 and highlight the ability of MUC1.CD to promote metastasis in a context-dependent manner. Our findings may have implications for the use and future design of targeted therapies in cancers known to express EGFR, Src, or MUC1.

Topics: Animals; Carcinoma; Cell Line, Tumor; Chick Embryo; CSK Tyrosine-Protein Kinase; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Mucin-1; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Protein-Tyrosine Kinases; Receptors, Vitronectin; Signal Transduction; src-Family Kinases

Carcinoma-associated fibroblasts (CAFs) play a pivotal role in promoting the growth, invasion and metastasis of tumor cells. However, to date little is known about the oncogenic mechanisms of CAFs. This study aimed to identify the microenvironmental factors involved in tumor development and progression directed by CAFs in liver metastases. Tissue samples collected from 20 patients with colorectal liver metastases were used in this study. Histological and morphological characterization of the samples was performed using hybridization and immunohistological assays. The mRNA expression of α-smooth muscle actin (α-SMA) was measured by northern blotting. The expression of plasminogen activator inhibitor type 1 (PAI-1) was measured by enzyme-linked immunosorbent assay (ELISA). As a result, co-expression of Thy-1 (CD90) and α-SMA was identified in CAFs, while normal liver samples were negative for α-SMA and Thy-1. Compared with epidermal growth factor (EGF) and tumor necrosis factor (TNF) incubation, the expression of α-SMA increased significantly following transforming growth factor-1 (TGF-1) incubation (P<0.05), while platelet-derived growth factor (PDGF) caused a significant suppression of α-SMA expression (P<0.05). PAI-1 expression was significantly lower in unstimulated fibroblasts compared to TGF-1-treated fibroblasts (P<0.01). The levels of PAI-1 transcription were significantly higher in CAFs from the patient samples compared with the healthy controls. Taken together, our findings suggest that CAFs may be important in migration, matrix degradation, invasion and angiogenesis of tumors, and TGF-1 may promote the activation of PAI-1 transcription in CAFs.

Topics: Actins; Carcinoma; Cells, Cultured; Colorectal Neoplasms; Epidermal Growth Factor; Fibroblasts; Humans; Liver Neoplasms; Plasminogen Activator Inhibitor 1; Platelet-Derived Growth Factor; RNA, Messenger; Thy-1 Antigens; Transcriptional Activation; Transforming Growth Factor beta1; Up-Regulation

Altered cell motility is considered to be a key factor in determining tumor invasion and metastasis. Epidermal growth factor (EGF) signaling has been implicated in this process by affecting cytoskeletal organization and dynamics in multiple ways. To sort the temporal and spatial regulation of EGF-dependent cytoskeletal re-organization in relation to a cell's motile behavior time-lapse microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells co-expressing different fluorescently labeled cytoskeletal filaments and focal adhesion components in various combinations. The experiments showed that EGF almost instantaneously induces a considerable increase in membrane ruffling and lamellipodial activity that can be inhibited by Cetuximab EGF receptor antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells. The transient cell extensions are rich in actin but lack microtubules and keratin intermediate filaments. We show that this EGF-induced increase in membrane motility can be measured by a simple image processing routine. Microtubule plus-ends subsequently invade growing cell extensions, which start to accumulate focal complexes at the lamellipodium-lamellum junction. Such paxillin-positive complexes mature into focal adhesions by tyrosine phosphorylation and recruitment of zyxin. These adhesions then serve as nucleation sites for keratin filaments which are used to enlarge the neighboring peripheral keratin network. Focal adhesions are either disassembled or give rise to stable zyxin-rich fibrillar adhesions which disassemble in the presence of EGF to support formation of new focal adhesion sites in the cell periphery. Taken together the results serve as a basis for modeling the early cytoskeletal EGF response as a tightly coordinated and step-wise process which is relevant for the prediction of the effectiveness of anti-EGF receptor-based tumor therapy.

Topics: Actins; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma; Cell Movement; Cetuximab; Epidermal Growth Factor; ErbB Receptors; Focal Adhesions; Gene Expression; Humans; Keratins; Microtubules; Paxillin; Phosphorylation; Pseudopodia; Signal Transduction; Stomach Neoplasms; Time Factors; Time-Lapse Imaging; Tumor Cells, Cultured; Zyxin

Human epithelial cancers account for approximately 50% of all cancer deaths. This type of cancer is characterized by excessive activation and expression of the epidermal growth factor receptor (EGFR). The EGFR pathway is critical for cancer cell proliferation, survival, metastasis and angiogenesis. The EGF-EGFR signaling pathway has been validated as an important anticancer drug target. Increasing numbers of targeted therapies against this pathway have been either approved or are currently under development. Here, we adopted a prodrug system that uses 5-fluorocytosine (5-FC) and human EGF (hEGF) fused with yeast cytosine deaminase (Fcy) to target EGFR-overexpressing cancer cells and to convert 5-FC to a significantly more toxic chemotherapeutic, 5-fluorouracil (5-FU). We cloned and purified the Fcy-hEGF fusion protein from Pichia pastoris yeast. This fusion protein specifically binds to EGFR with a similar affinity as hEGF, approximately 10 nM. Fcy-hEGF binds tightly to A431 and MDA-MB-468 cells, which overexpress EGFR, but it binds with a lower affinity to MDA-MB-231 and MCF-7, which express lower levels of EGFR. Similarly, the viability of EGFR-expressing cells was suppressed by Fcy-hEGF in the presence of increasing concentrations of 5-FC, and the IC(50) values for A431 and MDA-MB-468 were approximately 10-fold lower than those of MDA-MB-231 and MCF-7. This novel prodrug system, Fcy-hEGF/5-FC, might represent a promising addition to the available class of inhibitors that specifically target EGFR-expressing cancers.

Topics: Carcinoma; Cell Line, Tumor; Cloning, Molecular; Cytosine Deaminase; Epidermal Growth Factor; ErbB Receptors; Flucytosine; Humans; Inhibitory Concentration 50; Mitogens; Prodrugs; Recombinant Fusion Proteins

The hedgehog (Hh) signaling pathway is essential for the development of tissues and organs. Hyperactive Hh signaling has been implicated in many gastric cancers, including esophageal cancer. However, the interaction between the Hh pathway and other potential signaling pathways in primary esophageal tumorigenesis has not been well investigated. In our study, we found that esophageal cancer cells expressed Hh signaling molecules and that the hyperexpression of Hh target genes was related to protein kinase B (AKT) activation but not extracellular signal-regulated kinase activation. We analyzed the relationship between Gli1 or p-AKT expression and clinicopathological features in esophageal carcinoma samples and found that Gli1 expression was associated with lymph vessel invasion (p = 0.016), blood vessel invasion (p = 0.006) and a poor prognosis (p = 0.003), and p-AKT expression was associated with blood vessel invasion (p = 0.031) and a poor prognosis (p = 0.031). We also studied the relationship between Hh and phosphinositide-3 kinase (PI3K)/AKT or mitogen-activated protein kinase (MAPK) signaling pathways in both TE-1 and TE-10 cell lines. We found that the PI3K/AKT pathway played a critical role in Hh signaling after stimulation with epidermal growth factor, Gβγ and N-Shh. Conversely, PI3K/AKT and MAPK signaling cooperated with the Shh pathway to promote esophageal cancer cell survival and proliferation. The results from esophageal cancer cells shed light on the significance of Hh signaling in esophageal tumor formation and the crosstalk of the Hh pathway with other basic signaling pathways, which is consistent with that observed in human tumor samples.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Cell Proliferation; Cell Survival; Epidermal Growth Factor; Esophageal Neoplasms; Extracellular Signal-Regulated MAP Kinases; Female; Hedgehog Proteins; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors; Zinc Finger Protein GLI1

An epithelial cell line, referred to as A163, was established from breast carcinoma derived from a patient with a strong family history of breast cancer but no known breast cancer susceptibility mutation. A163 was propagated in a serum-free culture medium including the epidermal growth factor. Immunophenotypic characterization demonstrated a mixed luminal and basal-like phenotype. When epidermal growth factor was excluded from the culture medium, A163 entered a quiescent period followed by a period of increased cell proliferation in a subpopulation of the cells. The epidermal growth factor-independent subpopulation retained the basal-like phenotype of the parental cell line. Karyotype and fluorescent in situ hybridization analysis showed an amplification of epidermal growth factor receptor on 7q in A163-S1 only, resulting in high expression of total and phosphorylated epidermal growth factor receptor. The A163-S1 sub-line piles up in culture, indicating a loss of contact inhibition. When grown on transwell filters, A163 shows basal expression of P63 and cytokeratin 14, whereas A163-S1 expresses P63 ubiquitously, and has lost the basal specific expression of cytokeratin 14, indicating a loss of polarity. Furthermore, when cultured in reconstituted basement membrane matrix, A163 form polarized normal like acini. In contrast, A163-S1 form large disorganized structures with lack of polarity. These cell lines may prove useful to understand molecular changes in breast cancer progression, in particular basal-like breast cancer subtype with bad prognosis and no current treatment options.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Proliferation; Culture Media, Serum-Free; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Amplification; Humans; Middle Aged; Neoplasms, Basal Cell; Selection, Genetic

Immunohistochemistry is used in both research and clinical settings to identify proteins in tissue samples. Despite the power and versatility of immunohistochemistry, limitations are imposed by the slow diffusion of antibodies through tissue and the need for secondary staining or signal amplification. Aptamers can circumvent these limitations, but their application has been hindered by nonspecific binding to cellular components, particularly in the nucleus. Here we describe unique slow off-rate modified aptamers that facilitate rapid and selective binding to target proteins in tissue. Specifically, we have developed a fluorescent aptamer that binds to the human epidermal growth factor receptor 2 (HER2) in breast carcinomas quickly and specifically, and we have shown that the slow off-rate of the aptamer from the HER2 protein contributes to its selectivity. These findings open the door to aptamer histochemistry applications in both research and clinical settings, including intraoperative diagnostics in which speed and accuracy are paramount.

Topics: Aptamers, Peptide; Breast Neoplasms; Carcinoma; Epidermal Growth Factor; Female; Fluorescent Dyes; Humans; Molecular Diagnostic Techniques; Protein Binding; Sensitivity and Specificity

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Female; Humans; Neoplasm Invasiveness; Proto-Oncogene Proteins p21(ras); rac1 GTP-Binding Protein

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

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

The functional interaction between integrin α6β4 and growth factor receptors has been implicated in key signaling pathways important for cancer cell function. However, few attempts have been made to selectively target this interaction for therapeutic intervention. Previous studies showed that curcumin, a yellow pigment isolated from turmeric, inhibits integrin α6β4 signaling important for breast carcinoma cell motility and invasion, but the mechanism is not currently known. To address this issue, we tested the hypothesis that curcumin inhibits the functional interaction between α6β4 and the epidermal growth factor receptor (EGFR). In this study, we found that curcumin disrupts functional and physical interactions between α6β4 and EGFR, and blocks α6β4/EGFR-dependent functions of carcinoma cells expressing the signaling competent form of α6β4. We further showed that curcumin inhibits EGF-dependent mobilization of α6β4 from hemidesmosomes to the leading edges of migrating cells such as lammelipodia and filopodia, and thereby prevents α6β4 distribution to lipid rafts where functional interactions between α6β4 and EGFR occur. These data suggest a novel paradigm in which curcumin inhibits α6β4 signaling and functions by altering intracellular localization of α6β4, thus preventing its association with signaling receptors such as EGFR.

Topics: Antineoplastic Agents; Carcinoma; Cell Line, Tumor; Curcumin; Epidermal Growth Factor; ErbB Receptors; Hemidesmosomes; Humans; Integrin alpha6beta4; Membrane Microdomains; Protein Binding; Protein Transport; Pseudopodia; Signal Transduction

Rho-associated coiled-coil containing protein kinase (Rho-kinase/ROCK) is involved in various cellular functions including cell proliferation, and is generally considered to be oncogenic, while some studies show that ROCK functions as a negative regulator of cancer progression. As a result, the precise role of ROCK remains controversial. We have previously reported that Rho-kinase/ROCK negatively regulates epidermal growth factor (EGF)-induced cell proliferation in SW480 colon cancer cells. In the present study, we investigated the role of ROCK in EGF receptor (EGFR) signaling in the pancreatic cancer cell lines, Panc1, KP3 and AsPc1.. In these cells, Y27632, a specific ROCK inhibitor, enhanced EGF-induced BrdU incorporation. The blockade of EGF stimulation utilizing anti-EGFR-neutralizing antibodies suppressed Panc1 cell proliferation. EGF induced RhoA activity, as well as the phosphorylation of cofilin and myosin light chain (MLC), both targets of ROCK signaling, and Y27632 suppressed both of these processes, indicating that the phosphorylation of cofilin and MLC by EGF occurs through ROCK in Panc1 cells. EGF-induced phosphorylation of EGFR at tyrosine residues was augmented when the cells were pretreated with Y27632 or were subjected to gene silencing using ROCK-siRNA. We also obtained similar results using transforming growth factor-α. In addition, EGF-induced phosphorylation of p44/p42 mitogen-activated protein kinase and Akt were also enhanced by Y27632 or ROCK-siRNA. Moreover, an immunofluorescence microscope study revealed that pretreatment with Y27632 delayed EGF-induced internalization of EGFR. Taken together, these data indicate that ROCK functions to switch off EGFR signaling by promoting the internalization of the EGFR.. While EGF first stimulates the activation of the EGFR and subsequently increases cancer cell proliferation, EGF concurrently induces the activation of ROCK, which then turns off the activated EGFR pathway via a negative feedback system.

Topics: Amides; Antibodies, Neutralizing; Carcinoma; Cell Line, Tumor; Cell Proliferation; Drug Evaluation, Preclinical; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Pancreatic Neoplasms; Protein Kinase Inhibitors; Pyridines; rho-Associated Kinases; Up-Regulation

Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogen-activated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer.

Topics: Adaptor Proteins, Signal Transducing; Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Estrogens; Female; Fibronectins; Humans; Lymphatic Metastasis; Matrix Metalloproteinases; MicroRNAs; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Neoplasm Metastasis

FK506 binding proteins (FKBPs) represent a subfamily of peptidyl prolyl cis/trans isomerases that can control receptor-mediated intracellular signaling. The prototypic PPIase FKBP12 functionally interacts with EGFR. FKBP12 was shown to inhibit EGF-induced EGFR autophosphorylation with all internal phosphorylation sites equally affected. The inhibition of EGFR catalytic activity is conducted by targeting the EGFR kinase domain. The change of intracellular FKBP12 levels resulted in a change of EGFR autophosphorylation level. Collectively, our results demonstrate that FKBP12 forms an endogenous inhibitor of EGFR phosphorylation directly involved in the control of cellular EGFR activity.

Topics: Antibodies, Phospho-Specific; Carcinoma; Cell Line, Tumor; Cross-Linking Reagents; Dimerization; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gene Silencing; HeLa Cells; Humans; Kinetics; Neoplasm Proteins; Phosphorylation; Protein Kinase Inhibitors; Protein Transport; Recombinant Fusion Proteins; Recombinant Proteins; RNA, Small Interfering; Signal Transduction; Tacrolimus Binding Protein 1A

We analyzed the influence of 8 germinal polymorphisms of candidate genes potentially related to EGFR signalling (EGFR, EGF, CCND1) or antibody-directed cell cytotoxicity (FCGR2A and FCGR3A) on outcome of colorectal cancer (CRC) patients receiving cetuximab-based therapy.. Fifty-eight advanced CRC patients treated with cetuximab-irinotecan salvage therapy between 2001 and 2007 were analyzed (mean age 60; 50 PS 0-1). The following polymorphisms were analyzed on blood DNA: EGFR (CA repeats in intron 1, -216 G > T, -191C > A, R497K), EGF (A61G), CCND1 (A870G), FCGR2A (R131H), FCGR3A (F158V). Statistical analyses were conducted on the total population and on patients with wt KRas tumors. All SNPs were considered as ternary variables (wt/wt vs wt/mut vs mut/mut), with the exception of -191C > A EGFR polymorphism (AA patient merged with CA patients).. Analysis of skin toxicity as a function of EGFR intron 1 polymorphism showed a tendency for higher toxicity in patients with a low number of CA-repeats (p = 0.058). CCND1 A870G polymorphism was significantly related to clinical response, both in the entire population and in KRas wt patients, with the G allele being associated with a lack of response. In wt KRas patients, time to progression (TTP) was significantly related to EGFR -191C > A polymorphism with a longer TTP in CC patients as compared to others, and to CCND1 A870G polymorphism with the G allele being associated with a shorter TTP; a multivariate analysis including these two polymorphisms only retained CCND1 polymorphism. Overall survival was significantly related to CCND1 polymorphism with a shorter survival in patients bearing the G allele, and to FCGR3A F158V polymorphism with a shorter survival in VV patients (in the entire population and in KRas wt patients). FCGR3A F158V and CCND1 A870G polymorphisms were significant independent predictors of overall survival.. Present original data obtained in wt KRas patients corresponding to the current cetuximab-treated population clearly suggest that CCND1 A870G polymorphism may be used as an additional marker for predicting cetuximab efficacy, TTP and overall survival. In addition, FCGR3A F158V polymorphism was a significant independent predictor of overall survival.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Carcinoma; Cetuximab; Colorectal Neoplasms; Cyclin D1; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Introns; Male; Middle Aged; Polymorphism, Genetic; Receptors, IgG; Skin Diseases; Survival Analysis

Mitogen-inducible gene 6 (Mig-6) is a putative tumor suppressor gene and prognostic biomarker in papillary thyroid cancer. We hypothesized that Mig-6 knockout would activate pro-oncogenic signaling in mouse thyrocytes.. We performed a thyroid-specific knockout using the Cre/loxP recombinase system.. Four knockout and 4 control mouse thyroids were harvested at 2 months of age. Immunoblotting confirmed Mig-6 ablation in knockout mice thyrocytes. Epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) phosphorylation levels were increased in Mig-6 knockout compared to wild-type mice. Total EGFR levels were similar in knockout and wild-type mice. However, EGFR was absent in the caveolae-containing membrane fraction of knockout mice, indicating that Mig-6 depletion is associated with a change in the membrane distribution of EGFR. Although p65 localized to the nucleus in wild-type mice, it was distributed in both cytoplasm and nucleus in knockouts, suggesting that Mig-6 loss decreases p65 activity.. Our results confirm the feasibility of targeted, thyroid-specific gene knockout as a strategy for studying the relevance of specific genes in thyroid oncogenesis. We suggest that the loss of Mig-6 alters the membrane distribution of EGFR, which may limit receptor degradation and activate this oncogenic signaling pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; Biomarkers; Carcinoma; Carcinoma, Papillary; Epidermal Growth Factor; Gene Knockout Techniques; Genes, Tumor Suppressor; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; NF-kappa B; Signal Transduction; Thyroid Cancer, Papillary; Thyroid Gland; Thyroid Neoplasms; Transcription Factor RelA

Twelve human cancer cell lines and one non-malignant cell line were investigated with respect to a potential antiproliferative/cytotoxic activity of molecular iodine and iodolactones. Except CCL221 colon carcinoma cells, the growth of all cancer cell lines decreased if the cells were cultured in the presence of 10 microM molecular iodine (I(2)) for at least two days. delta-iodolactone (IL, 5 microM) was found to have a similar effect. SH-SY5Y neuroblastoma cells turned out to be most susceptible to both iodine compounds (total inhibition), followed by MCF-7 mammary carcinoma cells (60% and 77.7% inhibition in the presence of I(2) respect. IL) and HS24 lung carcinoma cells (36.3% respect. 40.3% inhibition). In contrast, MCF-10 normal mammary epithelial cells were much less affected by the iodine treatment. In both, SH-SY5Y and MCF-7 cells, I(2) and IL also abolished EGF-induced promotion of cell growth completely. This effect was, however, not due to an interfering with EGF-signaling, because I(2) and IL did not affect the phosphorylation of EGF-receptors, EGF-induced activation of MAP-kinase (Erk(1/2)), or EGF-induced lamellar actin protrusion. A disruption by molecular iodine of mitochondrial transmembrane electrical potential, which was prevented by a pre-treatment of the cells with N-acetyl-cysteine, supports a mitochondria-mediated apoptotic mechanism.

Topics: Antineoplastic Agents; Apoptosis; Arachidonic Acids; Carcinoma; Cell Line, Tumor; Cell Proliferation; Cytotoxins; Epidermal Growth Factor; Humans; Iodine; Lactones; Membrane Potential, Mitochondrial; Mitogen-Activated Protein Kinases

Identification of molecular mechanisms responsible for brain metastatic breast cancer (BMBC) is imperative to develop novel therapies. However, current understanding of the molecular circuitry that governs BMBC dissemination remains fragmentary. Heparanase (HPSE) is the only functional mammalian endoglycosidase whose activity correlates with cancer metastasis, angiogenesis, and the reduced postoperative survival of cancer patients, making it an active target for anticancer therapeutics. We hypothesized that human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) activation promotes HPSE function in human BMBC. To address this, we examined HPSE content, activity, and intracellular trafficking in a HER2/EGFR-expressing BMBC model system and show that HPSE is present, functional, and correlates with HER2 status. Further, we showed that EGF induced nucleolar translocation of HPSE in these cells in a dose- and time-dependent manner upon activation of HER2/EGFR. Knockdowns of HER2/EGFR by small interference RNA abolished EGF-induced HPSE nucleolar translocalization. It was also noted that nucleolar HPSE modulates DNA topoisomerase I (Topo I), an enzyme that is highly present in nucleoli, essential for DNA replication and transcription in a variety of tumors, and inhibited by heparan sulfate. Evidence is provided that HPSE can regulate Topo I activity, which subsequently affects BMBC cell proliferation. Finally, we showed that the nucleolar presence of HPSE with Topo I colocalization is detected only in HER2-overexpressing BMBC patient specimens. Altogether, these findings support the notion that HPSE is a critical downstream target of HER2 mechanisms driving BMBC and is potentially relevant for BMBC therapeutic interventions.

Topics: Active Transport, Cell Nucleus; Animals; Brain Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Nucleolus; DNA Topoisomerases, Type I; Down-Regulation; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Glucuronidase; Humans; Mice; Mice, Nude; Models, Biological; Neoplasm Metastasis; Protein Transport; Receptor, ErbB-2; RNA Interference; Signal Transduction

Previously, we showed that basal activity of nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway protects against spontaneous apoptosis and confers resistance to cisplatin-induced apoptosis in human ovarian cancer cells. The present study determines whether basal PKG kinase activity regulates Src family kinase (SFK) activity and proliferation in these cells. PKG-Ialpha was identified as predominant isoform in both OV2008 (cisplatin-sensitive, wild-type p53) and A2780cp (cisplatin-resistant, mutated p53) ovarian cancer cells. In both cell lines, ODQ (inhibitor of endogenous NO-induced cGMP biosynthesis), DT-2 (highly specific inhibitor of PKG-Ialpha kinase activity), and PKG-Ialpha knockdown (using small interfering RNA) caused concentration-dependent inhibition of DNA synthesis (assessed by bromodeoxyuridine incorporation), indicating an important role of basal cGMP/PKG-Ialpha kinase activity in promoting cell proliferation. DNA synthesis in OV2008 cells was dependent on SFK activity, determined using highly selective SFK inhibitor, 4-(4'-phenoxyanilino)-6,7-dimethoxyquinazoline (SKI-1). Studies using DT-2 and PKG-Ialpha small interfering RNA revealed that SFK activity was dependent on PKG-Ialpha kinase activity. Furthermore, SFK activity contributed to endogenous tyrosine phosphorylation of PKG-Ialpha in OV2008 and A2780cp cells. In vitro coincubation of recombinant human c-Src and PKG-Ialpha resulted in c-Src-mediated tyrosine phosphorylation of PKG-Ialpha and enhanced c-Src autophosphorylation/activation, suggesting that human c-Src directly tyrosine phosphorylates PKG-Ialpha and the c-Src/PKG-Ialpha interaction enhances Src kinase activity. Epidermal growth factor-induced stimulation of SFK activity in OV2008 cells increased PKG-Ialpha kinase activity (indicated by Ser(239) phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein), which was blocked by both SKI-1 and SU6656. The data suggest an important role of Src/PKG-Ialpha interaction in promoting DNA synthesis/cell proliferation in human ovarian cancer cells. The NO/cGMP/PKG-Ialpha signaling pathway may provide a novel therapeutic target for disrupting ovarian cancer cell proliferation.

Topics: Carcinoma; Cell Line, Tumor; Cell Proliferation; CSK Tyrosine-Protein Kinase; Cyclic GMP; Cyclic GMP-Dependent Protein Kinase Type I; Cyclic GMP-Dependent Protein Kinases; DNA Replication; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Nitric Oxide; Ovarian Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; RNA Interference; src-Family Kinases

Transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) have critical roles in regulating the metastasis of aggressive breast cancers, yet the impact of epithelial-mesenchymal transition (EMT) induced by TGF-β in altering the response of breast cancer cells to EGF remains unknown. We show in this study that murine metastatic 4T1 breast cancer cells formed compact and dense spheroids when cultured under three-dimensional (3D) conditions, which was in sharp contrast to the branching phenotypes exhibited by their nonmetastatic counterparts. Using the human MCF10A series, we show that epithelial-type and nonmetastatic breast cancer cells were unable to invade to EGF, whereas their mesenchymal-type and metastatic counterparts readily invaded to EGF. Furthermore, EMT induced by TGF-β was sufficient to manifest dense spheroid morphologies, a phenotype that increased primary tumor exit and invasion to EGF. Post-EMT invasion to EGF was dependent on increased activation of EGF receptor (EGFR) and p38 mitogen-activated protein kinase, all of which could be abrogated either by pharmacologic (PF-562271) or by genetic (shRNA) targeting of focal adhesion kinase (FAK). Mechanistically, EMT induced by TGF-β increased cell-surface levels of EGFR and prevented its physical interaction with E-cadherin, leading instead to the formation of oncogenic signaling complexes with TβR-II. Elevated EGFR expression was sufficient to transform normal mammary epithelial cells, and to progress their 3D morphology from that of hollow acini to branched structures characteristic of nonmetastatic breast cancer cells. Importantly, we show that TGF-β stimulation of EMT enabled this EGFR-driven breast cancer model to abandon their inherent branching architecture and form large, undifferentiated masses that were hyperinvasive to EGF and showed increased pulmonary tumor growth upon tail vein injection. Finally, chemotherapeutic targeting of FAK was sufficient to revert the aggressive behaviors of these structures. Collectively, this investigation has identified a novel EMT-based approach to neutralize the oncogenic activities of EGF and TGF-β in aggressive and invasive forms of breast cancer.

Topics: Animals; Breast Neoplasms; Cadherins; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Focal Adhesion Protein-Tyrosine Kinases; Humans; Indoles; Mammary Neoplasms, Experimental; Mice; p38 Mitogen-Activated Protein Kinases; Receptors, Transforming Growth Factor beta; RNA, Small Interfering; Sulfonamides; Transforming Growth Factor beta

Cell migration is fundamental for invasion and metastasis and is modulated by the reversible phosphorylation of tyrosine residues on target proteins. Here we report that the tyrosine phosphatase HD-PTP has a role in modulating the motility of T24 bladder carcinoma cells. Indeed, HD-PTP silencing by RNA interference (RNAi) markedly induced cell migration in a Src dependent fashion. We therefore investigated the interaction and the regulation of Src and HD-PTP. We found that, in Epidermal Growth Factor (EGF) stimulated cells, Src binds to and phosphorylates HD-PTP on tyrosine residues. On the contrary, HD-PTP does not modulate the levels of Src phosphorylation. Interestingly, HD-PTP also binds to FAK, another regulator of cell migration, and this interaction is inhibited after exposure to EGF. FAK phosphorylates HD-PTP and this event reduced the interactions between the two proteins. Interestingly, in cells silencing HD-PTP the phosphorylation of FAK is enhanced and this correlates with its localization in focal complexes both in the presence and in the absence of EGF. We hypothesize that in unstimulated T24 cells HD-PTP does not interact with Src, while it binds to FAK. Following stimulation with EGF, HD-PTP is tyrosine-phosphorylated and releases FAK which will ultimately contribute to the turn-over of focal adhesion and, therefore, to cell motility.

Topics: Blotting, Western; Carcinoma; Cell Line, Tumor; Cell Movement; Cells, Cultured; Epidermal Growth Factor; Focal Adhesion Kinase 1; Humans; Protein Tyrosine Phosphatases, Non-Receptor; RNA Interference; RNA, Small Interfering; src-Family Kinases; Urinary Bladder Neoplasms

Tyrosine kinase receptors and integrins play essential roles in tumor cell invasion and metastasis. Previously, we showed that epidermal growth factor (EGF) stimulation of pancreatic carcinoma cells led to invasion and metastasis that was blocked by antagonists of integrin alpha(v)beta(5). Here, we show that EGF stimulates metastasis of carcinoma cells via a Src-dependent phosphorylation of p130 CAS leading to activation of Rap1, a small GTPase involved in integrin activation. Specifically, EGF receptor (EGFR)-induced Src activity leads to phosphorylation of a region within the CAS substrate domain, which is essential for Rap1 and alpha(v)beta(5) activation. This pathway induces alpha(v)beta(5)-mediated invasion and metastasis in vivo yet does not influence primary tumor growth or activation of other integrins on these cells. These findings show cross-talk between a tyrosine kinase receptor and an integrin involved in carcinoma cell invasion and metastasis and may explain in part how inhibitors of EGFR affect malignant disease.

Topics: Animals; Carcinoma; Cell Movement; Chick Embryo; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Gene Knockdown Techniques; Humans; Inverted Repeat Sequences; Lung; Lung Neoplasms; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Pancreatic Neoplasms; Polymerase Chain Reaction; Receptor Cross-Talk; Receptors, Vitronectin; RNA, Neoplasm; Tumor Cells, Cultured

Human Cripto-1, a member of the EGF-CFC family, is indispensable for early embryonic development. Cripto-1 plays an important oncogenic role during tumorigenesis and is overexpressed in a wide range of epithelial carcinomas, yet little is known about Cripto-1 in nasopharyngeal carcinoma (NPC). The aim of this study was to analyze the roles of Cripto-1 in the progression and clinical characteristics in NPC clinical samples and cell lines.. The expression of Cripto-1 at mRNA level was detected by the reverse transcription-polymerase chain reaction (RT-PCR) and real time RT-PCR, and western blot was used to examine the protein expression. Cripto-1 expression and its clinical characteristics were investigated by performing immunohistochemical analysis on a total of 37 NPC clinical tissue samples. Lentiviral vectors were constructed to get an efficient expression of anti-Cripto-1 siRNA in CNE-2 and C666-1 cells, with invalid RNAi sequence as control. After the inhibition of the endogenous Cripto-1, the growth, cell cycle and invasion of cells were detected by MTT, FACS and Boyden chamber assay respectively. Moreover, in vivo, the proliferation of the tumor cells was evaluated in xenotransplant nude mice model with whole-body visualizing instrument.. The results of real-time RT-PCR and western blot showed that the expression level of Cripto-1 was markedly higher in NPC cell lines than that in the immortalized nasopharyngeal epithelial cell at both mRNA and protein levels. RT-PCR of 17 NPC tissues showed a high expression rate in 76.5% (13/17) cases. In an immunohistochemical study, Cripto-1 was found to express in 54.1% (20/37) cases of NPC. In addition, Cripto-1 overexpression was significantly associated with N classification (p = 0.034), distant metastasis (p = 0.036), and clinical stage (p = 0.007). Inhibition of endogenous Cripto-1 by lentivirus-mediated RNAi silencing technique suppressed NPC cell growth and invasion in vitro. In vivo, the average weight (p = 0.026) and volume (p = 0.044) of tumor in CNE-2/GFP+/Cripto-1- xenotransplant mice group were significantly lower than those in the control group. The Ki67 index was obviously lower in Cripto-1 RNAi treated tumors (p < 0.01).. Data of this study suggest that Cripto-1 overexpression is connected with the tumorigenesis and progression of NPC, lentivector-mediated RNAi might be feasible for the inhibition of the growth and invasion of NPC.

Topics: Adult; Aged; Aged, 80 and over; Animals; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Membrane Glycoproteins; Mice; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Proteins; Neoplasm Transplantation; Neoplastic Processes

To develop a targeted biological drug that when systemically injected can penetrate to metastatic breast cancer tumors, one needs a drug of high potency and reduced immunogenicity. Thus, we bioengineered a novel bispecific ligand-directed toxin (BLT) targeted by dual high-affinity cytokines with a PE(38)KDEL COOH terminus. Our purpose was to reduce toxin immunogenicity using mutagenesis, measure the ability of mutated drug to elicit B-cell antitoxin antibody responses, and show that mutated drug was effective against systemic breast cancer in vivo.. A new BLT was created in which both human epidermal growth factor (EGF) and interleukin 4 cytokines were cloned onto the same single-chain molecule with truncated Pseudomonas exotoxin (PE(38)). Site-specific mutagenesis was used to mutate amino acids in seven key epitopic toxin regions that dictate B-cell generation of neutralizing antitoxin antibodies. Bioassays were used to determine whether mutation reduced potency, and ELISA studies were done to determine whether antitoxin antibodies were reduced. Finally, a genetically altered luciferase xenograft model was used; this model could be imaged in real time to determine the effect on the systemic malignant human breast cancer MDA-MB-231.. EGF4KDEL 7mut was significantly effective against established systemic human breast cancer and prevented metastatic spread. Mutagenesis reduced immunogenicity by approximately 90% with no apparent loss in in vitro or in vivo activity.. Because EGF4KDEL 7mut was highly effective even when we waited 26 days to begin therapy and because immunogenicity was significantly reduced, we can now give multiple drug treatments for chemotherapy-refractory breast cancer in clinical trials.

Topics: Animals; Breast Neoplasms; Carcinoma; Drug Resistance, Neoplasm; Epidermal Growth Factor; Female; Humans; Immunotoxins; Lymphocyte Activation; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Mutagenesis, Site-Directed; Neoplasm Metastasis; Receptors, Interleukin-4; Recombinant Fusion Proteins; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Matrix metalloproteinase 2 (MMP-2) and membrane type 1 matrix metalloproteinase (MT1-MMP) have been identified as important participants in tumor invasion, metastasis, and angiogenesis. Membrane type 1 matrix metalloproteinase has also been recognized as a major activator of MMP-2. The purpose of this study was to investigate epidermal growth factor (EGF) mediating signal pathways in the regulation of MMP-2 and MT1-MMP in SiHa cells, a cervical cancer cell line. We showed here that EGF induced the expression of MT1-MMP and inhibited the expression of MMP-2 at both the mRNA and protein levels. Membrane type 1 matrix metalloproteinase induction was blocked by mitogen-activated protein kinase or extracellular signal-regulated kinase inhibitors PD98059 and U0126 but not by phosphatidylinositol-3 kinase (PI3-K) inhibitors LY294002 and wortmannin. Interestingly, the mitogen-activated protein kinase or extracellular signal-regulated kinase inhibitors PD98059 and U0126 actually increased MMP-2 mRNA and protein synthesis, whereas the PI3-K inhibitors LY294002 and wortmannin further suppressed the expression of MMP-2. Our results suggest that EGF receptor up-regulated the expression of MT1-MMP and down-regulated the synthesis of MMP-2 through the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway while concomitantly transmitting a mild positive regulatory signal to the expression of MMP-2 via the PI3-K/AKT pathway in SiHa cells. Furthermore, we found that EGF elevated the activity of MMP-2 in culture media.

Topics: Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphorylation; Signal Transduction; Uterine Cervical Neoplasms

The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development.. In this study, the expression of Nodal, Cripto (co-receptor) and Lefty A (antagonist) was examined by RT-PCR and immunohistochemistry across the menstrual cycle and in endometrial carcinomas.. Nodal and Cripto were found to be expressed at high levels in both stromal and epithelial cells during the proliferative phase of the menstrual cycle. Although immunoreactivity for both proteins in surface and glandular epithelium was maintained at relatively steady-state levels across the cycle, their expression was significantly decreased within the stromal compartment by the mid-secretory phase. Lefty expression, as has previously been reported, was primarily restricted to glandular epithelium and surrounding stroma during the late secretory and menstrual phases. In line with recent studies that have shown that Nodal pathway activity is upregulated in many human cancers, we found that Nodal and Cripto immunoreactivity increased dramatically in the transition from histologic Grade 1 to histologic Grades 2 and 3 endometrial carcinomas. Strikingly, Lefty expression was low or absent in all cancer tissues.. The expression of Nodal in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the tissue remodelling events that occur across the menstrual cycle and in tumourogenesis.

Topics: Adult; Body Fluids; Carcinoma; Endometrial Neoplasms; Endometrium; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Left-Right Determination Factors; Membrane Glycoproteins; Menstrual Cycle; Neoplasm Proteins; Nodal Protein; Signal Transduction; Uterus

The family of epidermal growth factor (EGF, EGFR, c-erbB-2) plays a pivotal role in gastric cancer progression, invasion and metastasizing. Helicobacter pylori infection is known to contribute significantly to the formation and progression of gastric cancer. However, the mechanisms responsible for this process have not been yet elucidated. We analysed the relationship between H. pylori infection and expression of proteins belonging to the family of epidermal growth factor (EGF, EGFR, c-erbB-2). Fifty-five patients with gastric cancer were analysed for Helicobacter pylori infection. The expressions of EGF, EGFR, c-erbB-2 proteins were determined using an immunohistochemical method. No statistically significant correlation was found between the degree of H. pylori infection and the expressions of EGF, EGFR and c-erbB-2 in gastric cancer. However, c-erbB-2 expression in the main mass of tumour correlated with tumour expression of EGF and EGFR and with c-erbB-2 expression in local lymph nodes. The expression of c-erbB-2 in lymph nodes was statistically significantly related to the expressions of EGF and EGFR both in the main mass of tumour and in lymph nodes. The expression of EGF was found to correlate with EGFR in the main mass of tumour and the expression of EGF in lymph nodes was related to lymph node EGFR level. Our study did not confirm the relationship between H. pylori infection and the expression of epidermal growth factor in gastric cancer.

Topics: Carcinoma; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Helicobacter Infections; Helicobacter pylori; Humans; Immunohistochemistry; Receptor, ErbB-2; Stomach Neoplasms

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor beta1 (TGFbeta1) and transcription factors including Snail and Slug. We utilized the ARCaP(E)/ARCaP(M) prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaP(E) cells, the highly tumorigenic mesenchymal ARCaP(M) and ARCaP(M1) variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaP(M) and ARCaP(M1) expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-kappaB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFbeta1 treatment and Snail overexpression induced EMT in ARCaP(E) and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Topics: Animals; Biomarkers, Tumor; Bone Remodeling; Cadherins; Carcinoma; Cell Dedifferentiation; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Epidermal Growth Factor; Epithelial Cells; Humans; Male; Mesoderm; Mice; Mice, SCID; Neoplasm Metastasis; Neoplasm Transplantation; Osteoclasts; Prostatic Neoplasms; RANK Ligand; Snail Family Transcription Factors; Transcription Factors; Transforming Growth Factor beta1; Vimentin

The intracellular domains of the membrane-anchoring regions of some precursors of epidermal growth factor (EGF) family members have intrinsic biologic activities. We have determined the role of the human proEGF cytoplasmic domain (proEGFcyt) as part of the proEGF transmembrane-anchored region (proEGFctF) in the regulation of motility and elastinolytic invasion in human thyroid cancer cells. We found proEGFctF to act as a negative regulator of motility and elastin matrix penetration and the presence of proEGFcyt or proEGF22.23 resulted in a similar reduction in motility and elastinolytic migration. This activity was counteracted by EGF-induced activation of EGF receptor signaling. Decreased elastinolytic migratory activity in the presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with decreased secretion of elastinolytic procathepsin L. The presence of proEGFctF and proEGFcyt/proEGF22.23 coincided with the specific transcriptional up-regulation of t-SNARE member SNAP25. Treatment with siRNA-SNAP25 resulted in motility and elastin migration being restored to normal levels. Epidermal growth factor treatment down-regulated SNAP25 protein by activating EGF receptor-mediated proteasomal degradation of SNAP25. These data provide first evidence for an important function of the cytoplasmic domain of the human proEGF transmembrane region as a novel suppressor of motility and cathepsin L-mediated elastinolytic invasion in human thyroid carcinoma cells and suggest important clinical implications for EGF-expressing tumors.

Topics: Animals; Carcinoma; Cathepsin L; Cathepsins; Cell Movement; Cysteine Endopeptidases; Cytoplasm; Down-Regulation; Elastin; Epidermal Growth Factor; Female; Humans; Hydrolysis; Male; Models, Biological; Neoplasm Invasiveness; Protein Precursors; Protein Structure, Tertiary; Thyroid Neoplasms; Transfection; Tumor Cells, Cultured

In thyroid cancer (TC) endostatin was identified as a powerful negative regulator of tumor angiogenesis in vitro. It is currently being evaluated in phase I trials for antiangiogenic therapy in various solid tumors. The aim of this study was to evaluate endostatin expression in archival TC specimens and its secretion following stimulation with thyrotropin (TSH) and epidermal growth factor (EGF) in TC cell lines.. Tissue microarrays of 44 differentiated and 7 anaplastic TC and their metastasis were immunostained for endostatin protein expression and compared with corresponding non-neoplastic thyroid tissue (NT). In vitro, six differentiated (FTC133, FTC236, HTC, HTC-TSHr, XTC, and TPC1) and three anaplastic (C643, Hth74, Kat4.0) TC cell lines were evaluated for basal as well as TSH (1-100 mU/ml) and EGF stimulated (1-100 ng/ml) endostatin.. Endostatin was detected in all TC and more than half of the NT. Endostatin expression was more frequent and intense in differentiated as compared to anaplastic TC. In vitro, basal endostatin secretion varied between 33 +/- 5 pg/ml (FTC236) and 549 +/- 65 pg/ml (TPC1) and was doubled in FTC, when the "primary" (FTC133) was compared with the metastasis (FTC236). Some cell lines showed TSH-induced (e.g., 60% in XTC) or EGF-induced (e.g., 120% in TPC1) upregulation of endostatin secretion, while others did not, despite documented receptor expression.. This study demonstrates endostatin expression in TC, metastasis and--less frequently and intensely--in NT, suggesting a possible association to tumor progression. In vitro, endostatin secretion of some cell lines is regulated by TSH and EGF, however the individual differences deserve further functional studies. These results support rather tumor-specific than histotype-specific expression and regulation of endostatin in TC.

Topics: Adenocarcinoma, Follicular; Angiogenesis Inhibitors; Carcinoma; Carcinoma, Papillary; Cell Differentiation; Endostatins; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Humans; Immunoenzyme Techniques; Lymphatic Metastasis; Paraffin Embedding; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

Electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) has been introduced recently for phosphopeptide enrichment. Here we compared ERLIC with the well-established SCX-IMAC for identifying phosphopeptides in EGF-treated A431 cells. The ERLIC approach detected a higher number of phosphopeptides (17 311) than SCX-IMAC (4850), but it only detected 926 unique phosphopeptides compared to 1315 in SCX-IMAC. Only 12% unique phosphopeptides were common to both approaches, suggesting that more comprehensive phosphoproteomes could be generated by complementing SCX-IMAC with ERLIC.

Topics: Carcinoma; Cell Line, Tumor; Chromatography; Chromatography, Affinity; Epidermal Growth Factor; Humans; Ion Exchange; Models, Biological; Phosphopeptides; Static Electricity; Substrate Specificity

Breast tumor kinase (Brk), an Src-like nonreceptor tyrosine kinase, is overexpressed in breast cancer and several other cancer types. Our previous study indicates that Brk promotes cell migration and tumor invasion by phosphorylating the focal adhesion protein paxillin. Here, we report the identification of p190RhoGAP-A (p190) as a Brk substrate. Brk phosphorylates p190 at the Y(1105) residue both in vitro and in vivo, thereby promoting the association of p190 with p120RasGAP (p120). As a consequence, Brk stimulates p190 and attenuates p120 functions, leading to RhoA inactivation and Ras activation, respectively. In carcinoma cells expressing high levels of Brk, endogenous Brk functions as a key contributor to epidermal growth factor-induced p190 tyrosine phosphorylation. We present evidence showing that p190 phosphorylation plays essential roles in both migratory and proliferative effects of Brk. Furthermore, disruption of p190 phosphorylation-induced p190/p120 complex in breast cancer cells abolishes not only the abilities of Brk to regulate RhoA and Ras but also the stimulatory effects of Brk on proliferation, migration, invasion, transformation, and tumorigenicity. Together, our findings reveal a previously unknown function of Brk in regulating both RhoA and Ras by phosphorylating p190 and provide evidence for the crucial roles of this Brk-elicited signaling pathway in promoting breast malignancy.

Topics: Animals; Breast Neoplasms; Carcinoma; Cell Movement; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Guanine Nucleotide Exchange Factors; HeLa Cells; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Proteins; Phosphorylation; Protein-Tyrosine Kinases; ras Proteins; Repressor Proteins; rho GTP-Binding Proteins; RNA, Small Interfering; Transplantation, Heterologous

Overexpression of epidermal growth factor (EGF) and urokinase plasminogen activator receptor (uPAR) have been observed in human gastric cancers. However, the interaction between EGF and uPAR in gastric cancer has not been well elucidated. In this study, we investigated the effect of EGF on uPAR expression and the underlying signal pathways in human gastric cancer AGS cells. EGF induced uPAR mRNA expression in a time- and concentration-dependent manner. EGF also induced uPAR promoter activity. In addition, EGF induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2) and P38 mitogen-activated protein kinase (MAPK) but not the activation of c-Jun amino terminal kinase. A specific inhibitor of MEK-1 (an upstream effector of ERK-1/2) and a dominant negative MEK-1 were able to suppress the EGF-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift assays demonstrated that the binding sites of transcription factors, activator protein-1 (AP-1) and nuclear factor (NF)-kappaB, are involved in the EGF-induced uPAR transcription. Suppression of the EGF-induced uPAR promoter activity by the AP-1 decoy oligonuclotide, as well as expression vectors encoding mutated-type NF-kappaB-inducting kinase and I-kappaB, confirmed that the activation of AP-1 and NF-kappaB are essential for the EGF-induced uPAR upregulation. The AGS cells pretreated with EGF showed a remarkably enhanced invasiveness and this effect was partially abrogated by uPAR neutralizing antibodies and by the inhibitors of ERK-1/2, AP-1, and NF-kappaB. The above results suggest that EGF induces uPAR expression via ERK-1/2, AP-1, and NF-kappaB signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

Topics: Carcinoma; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; NF-kappa B; Oligonucleotides; Promoter Regions, Genetic; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Stomach Neoplasms; Transcription Factor AP-1

The spread of cancer during metastatic disease requires that tumor cells subvert normal regulatory networks governing cell motility to invade surrounding tissues and migrate toward blood and lymphatic vessels. Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) proteins regulate cell motility by controlling the geometry of assembling actin networks. Mena, an Ena/VASP protein, is upregulated in the invasive subpopulation of breast cancer cells. In addition, Mena is alternately spliced to produce an invasion isoform, Mena(INV). Here we show that Mena and Mena(INV) promote carcinoma cell motility and invasiveness in vivo and in vitro, and increase lung metastasis. Mena and Mena(INV) potentiate epidermal growth factor (EGF)-induced membrane protrusion and increase the matrix degradation activity of tumor cells. Interestingly, Mena(INV) is significantly more effective than Mena in driving metastases and sensitizing cells to EGF-dependent invasion and protrusion. Upregulation of Mena(INV) could therefore enable tumor cells to invade in response to otherwise benign EGF stimulus levels.

Topics: Alternative Splicing; Animals; Carcinoma; Cell Movement; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Macrophages; Mice; Microfilament Proteins; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Protein Isoforms

The expression of epidermal growth factor receptors in normal and tumor cells of the pancreas, the type and incidence of EGFR gene polymorphism were studied. EGFR gene expression in pancreatic adenocarcinoma cells significantly surpassed that in normal pancreatic cells. On the other hand, AA genome and A allele polymorphism in the EGF gene nucleotide pair G-A 61 is a significant risk factor for pancreatic cancer. The effect of AG-1478 preparation (a new-generation inhibitor of EGFR) on apoptosis and cell proliferation in pancreatic cancer was evaluated. This preparation is not inferior to 5FU by its apoptotic effect and significantly reduces cell proliferation, its antiproliferative effect being 1.5 times higher than that of 5FU.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma; Cell Proliferation; Drug Evaluation, Preclinical; Epidermal Growth Factor; ErbB Receptors; Fluorouracil; Gene Frequency; Genetic Predisposition to Disease; Humans; Pancreatic Neoplasms; Polymorphism, Restriction Fragment Length; Polymorphism, Single Nucleotide; Quinazolines; Time Factors; Tumor Cells, Cultured; Tyrphostins

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.

Topics: Amides; Animals; Carcinoma; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Membrane; Cell Movement; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Focal Adhesions; Humans; Protein Transport; Pseudopodia; Pyridines; rac1 GTP-Binding Protein; Rats; rho-Associated Kinases; rhoA GTP-Binding Protein

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous-related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.

Topics: Actin Cytoskeleton; Animals; Carcinoma; Carrier Proteins; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Cytochrome-B(5) Reductase; Down-Regulation; Epidermal Growth Factor; Formins; Neoplasm Invasiveness; Neoplasms; Pseudopodia; Rats; rhoA GTP-Binding Protein; Wiskott-Aldrich Syndrome Protein Family; Wiskott-Aldrich Syndrome Protein, Neuronal

The human gene Teratocarcinoma-derived growth factor 1 (TDGF1)/Cripto-1/CR-1 which is expressed in a wide variety of human carcinomas is a member of the EGF-cripto FRL1 cryptic (EGF-CFC) gene family. A majority of human colorectal tumors and hepatomas are known to possess a constitutively active canonical Wnt/beta-catenin/TCF signaling pathway, also express CR-1. Expression of a short form of CR-1 mRNA in colon carcinoma and hepatoma cell lines suggests that there may be differential regulation of CR-1 expression by the canonical Wnt signaling pathway in colon cancer as well as hepatoma cell lines. The present study demonstrates a direct transcriptional regulation of the short form CR-1 expression by the canonical Wnt signaling pathway through an intronic-exonic enhancer element, containing three tandem TCF/LEF binding sites within the CR-1 gene.

Topics: beta Catenin; Binding Sites; Carcinoma; Carcinoma, Hepatocellular; Cell Line, Tumor; Colonic Neoplasms; Enhancer Elements, Genetic; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Genetic Vectors; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Liver Neoplasms; Lymphoid Enhancer-Binding Factor 1; Membrane Glycoproteins; Neoplasm Proteins; Protein Isoforms; Recombinant Proteins; RNA, Messenger; TCF Transcription Factors; Transcription, Genetic

The effects and mechanisms of exogenous phosphatase and tensin homolog deleted from chromosome ten (PTEN) gene on phosphatase activity-dependent apoptosis of breast cancer cell line MDA468 were investigated. PTEN gene packaged with lipofectin was transferred into breast cancer cell line MDA468 and parental MDA468 cells served as controls. RT-PCR and Western blot were done to detect the expression of target genes. The expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by epidermal growth factor (EGF) was also detected. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. MDA468 cells transfected with PTEN gene could express PTEN mRNA and protein. PTEN decreased the phosphorylation level of AKT protein and down-regulated FAK protein expression in MDA468 stimulated by EGF. The apoptosis rate was 21.68%. PTEN induced breast cancer apoptosis phosphatase activity-dependently. The mechanism is possibly related with phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/AKT signaling pathway. Those results may provide new clues on the gene therapy in breast cancer.

Topics: Apoptosis; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genetic Therapy; Humans; Phosphorylation; Plasmids; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Recombinant Proteins; Reference Standards; RNA, Messenger; Transfection

Serum levels of the soluble epidermal growth factor receptor (sEGFR) and its ligands epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and amphiregulin (AR) were measured in healthy donors and patients with non-small cell lung cancer (NSCLC) and head and neck carcinoma (HNC). In NSCLC, we found sEGFR and EGF levels significantly lowered in patients with respect to healthy donors. In HNC patients, significantly diminished levels were found in the case of sEGFR, EGF and also AR. In both malignancies, no significant association was found between the serum levels of the molecules and the patients' gender, age or smoking habit. Only a significant association was found between the decrease of sEGFR and the absence of distant metastasis in NSCLC and the tumour stage in HNC. The most interesting result was that combining sEGFR and EGF, sensitivities of 88% in NSCLC and 100% in HNC were reached without losing specificity (97.8% in both cases). The use of discriminant analysis and logistic regression improved the sensitivity for NSCLC and the specificity for HNC. These data demonstrate a potentially interesting value of the serum levels of sEGFR and EGF, especially when combined, as markers for NSCLC and HNC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma; Carcinoma, Non-Small-Cell Lung; Epidermal Growth Factor; ErbB Receptors; Female; Head and Neck Neoplasms; Humans; Ligands; Lung Neoplasms; Male; Middle Aged; Receptors, Androgen; Transforming Growth Factor alpha

Epidermal growth factor (EGF) is involved in cancer development and proliferation. We measured preoperative serum EGF, and serologically investigated the clinical significance of EGF expression in gastric cancer. We also performed immunohistological staining at the same time, and investigated its relationship with serum EGF.. There were 79 patients who underwent surgery for gastric cancer. For the measurement, one-step sandwich EIA was performed. Of 79 cases of gastric cancer in which the serum EGF level was measured, EGF immunostaining was performed in 50 cases.. In Serology, the EGF level was 345.6 +/- 260.6 pg/mL in m-sm cases, and 212.2 +/- 170.4 pg/mL in mp-si cases (p < 0.05). The EGF level was 294.4 +/- 236.0 pg/mL in ly0 cases, and 194.2 +/- 142.5 pg/mL in ly1-3 cases (p < 0.05). The EGF level was 323.5 +/- 233.4 pg/mL in cases staged IA-IB, and 202.8 +/- 176.8 pg/mL in cases staged II-IV (p < 0.05). In immunohistology the EGF positivity rate was 36.4% in the differentiated types, and 67.9% in the poorly differentiated types (p < 0.05). The EGF positivity rate was 25.0% in m-sm cases, and 63.1% in mp-si cases (p < 0.05).. The above findings suggest that EGF uptake by cancer cells increases when cancer cells are poorly differentiated, and that invasion in the surrounding tissue is severe.

Topics: Carcinoma; Epidermal Growth Factor; Female; Humans; Immunohistochemistry; Male; Stomach Neoplasms

Ovarian cancer is the primary cause of death from gynecological malignancies with a poor prognosis characterized by widespread peritoneal dissemination. However, mechanisms of invasion and metastasis in ovarian cancer remain poorly understood. Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) are often both overexpressed and contribute to the growth of ovarian cancer by activating autocrine pathways. In the present study, we investigated the mechanisms of invasive activity of EGF, HGF, and their synergistic effects in human ovarian cancer cells. Here our data suggest that EGF and HGF may use unique and overlapping signaling cascades leading to the invasive phenotype. We revealed that HGF-mediated cell migration and invasion required the coordinate activation of the phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2. Although EGF-dependent invasive phenotype appeared to have similar requirements for phosphatidylinositol 3-kinase, this growth factor used the alternative p38 MAPK pathway for cell invasion. A significant role of p38 MAPK was further supported by the observation that expression of dominant negative p38 MAPK likewise inhibited EGF-dependent invasiveness and cell motility. We also showed that EGF cooperated with HGF to promote a highly invasive phenotype via the increased secretion of matrix metalloproteinase (MMP)-9. The coincident induction of MMP-9 was functionally significant because inclusion of MMP-9 inhibitor or an anti-MMP-9 neutralizing antibody abolished EGF- and HGF-induced cellular invasion. These findings provide insights into the mechanism of the malignant progression of ovarian cancer.

Topics: Carcinoma; Cell Movement; Collagen; Drug Combinations; Drug Evaluation, Preclinical; Drug Synergism; Epidermal Growth Factor; Female; Hepatocyte Growth Factor; Humans; Laminin; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasm Invasiveness; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Proteoglycans; Tumor Cells, Cultured

Cdc42 plays a central role in regulating the actin cytoskeleton and maintaining cell polarity. Here, we show that Cdc42 is crucial for epidermal growth factor (EGF)-stimulated protrusion in MTLn3 carcinoma cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the protruding edge of the cells. siRNA-mediated knockdown of Cdc42 expression caused a decrease in EGF-stimulated protrusion and reduced cell motility in time-lapse studies. These changes were correlated with a decrease in barbed-end formation and Arp2/3 localization at the cell edge, and a marked defect in actin filament branching, as revealed by rotary-shadowing scanning electron microscopy. Upstream of Arp2/3, Cdc42 knockdown inhibited EGF-stimulated activation of PI 3-kinase at early (within 1 minute) but not late (within 3 minutes) time points. Membrane targeting of N-WASP, WAVE2 and IRSp53 were also inhibited. Effects on WAVE2 were not owing to Rac1 inhibition, because WAVE2 recruitment is unaffected by Rac1 knockdown. Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac.

Topics: Actin-Related Protein 2-3 Complex; Actins; Adaptor Proteins, Signal Transducing; Animals; Carcinoma; cdc42 GTP-Binding Protein; Cell Line, Tumor; Cell Movement; Cell Surface Extensions; Enzyme Activation; Epidermal Growth Factor; Nerve Tissue Proteins; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; rac GTP-Binding Proteins; ras Proteins; Rats; Recombinant Fusion Proteins; RNA, Small Interfering; Signal Transduction; Tropomyosin

Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium.

Topics: Biopsy; Carcinoma; Cell Nucleus; Contrast Media; Cytoplasm; Epidermal Growth Factor; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Mouth Neoplasms; Sensitivity and Specificity

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP(2); however, this is mainly based on in vitro-binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells. We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Topics: Actin Depolymerizing Factors; Actins; Animals; Breast Neoplasms; Carcinoma; Cell Line; Cell Membrane; Epidermal Growth Factor; Female; Hydrolysis; Phosphatidylinositol 4,5-Diphosphate; Protein Transport; Rats

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cdelta (PKCdelta)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCdelta is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCdelta using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCdelta and phosphorylated PKCdelta protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCdelta inhibition can limit migration and invasion of prostate cancer cells.

Topics: Carcinoma; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; ErbB Receptors; Humans; Male; Myosin Light Chains; Neoplasm Invasiveness; Prostatic Neoplasms; Protein Kinase C-delta; Signal Transduction

Ras family GTPases (RFGs) are known to share many regulatory and effector proteins. How signaling and biological specificity is achieved is poorly understood. Using a proteomics approach, we have identified a complex comprised of Shoc2/Sur-8 and the catalytic subunit of protein phosphatase 1 (PP1c) as a highly specific M-Ras effector. M-Ras targets Shoc2-PP1c to stimulate Raf activity by dephosphorylating the S259 inhibitory site of Raf proteins bound to other molecules of M-Ras or Ras. Therefore, distinct RFGs, through independent effectors, can regulate different steps in the activation of Raf kinases. Shoc2 function is essential for activation of the MAPK pathway by growth factors. Furthermore, in tumor cells with Ras gene mutations, inhibition of Shoc2 expression inhibits MAPK, but not PI3K activity. We propose that the Shoc2-PP1c holoenzyme provides an attractive therapeutic target for inhibition of the MAPK pathway in cancer.

Topics: Blotting, Western; Carcinoma; Catalytic Domain; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epidermal Growth Factor; Fibroblast Growth Factors; Green Fluorescent Proteins; HCT116 Cells; HeLa Cells; Holoenzymes; Humans; Intracellular Signaling Peptides and Proteins; Mass Spectrometry; Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Phosphoric Monoester Hydrolases; Precipitin Tests; Protein Phosphatase 1; Protein Structure, Tertiary; Proteomics; Proto-Oncogene Proteins c-raf; ras Proteins; Repressor Proteins; Retroviridae; RNA Interference; RNA, Small Interfering

A better understanding of pathways involved in survival of prostate cancer cells is the key to develop effective and target-selective therapies. Presence of serum or epidermal growth factor in the culture medium of LNCaP cells decreases apoptosis induced by the inhibition of phosphatidylinositol 3-kinase with LY294002. However, intracellular pathway(s) involved in this survival signaling are not well defined. Here, we investigated the mechanism(s) involved in serum or epidermal growth factor-mediated inhibition of LY294002-induced death in LNCaP cells. Cell death was assessed by the percentage of cells in sub-G1 phase and caspase 3 activity. Phosphorylation status of BAD, ERK1/2 and RSKs were assessed by Western blot. Specific gene expression knock down of BAD, BAX, RSK1 and RSK2 were performed using siRNA transfections. Our results demonstrate that cell death induced by LY294002 is mediated by translocation of BAD and BAX proteins from the cytosol to the mitochondria. Whereas, epidermal growth factor activates a MAPK/ERK/RSK1 module leading to inactivation of BAD via Ser(75) phosphorylation, the presence of serum, on the other hand, induces a nonconducive intracellular environment for mitochondrial translocation of dephosphorylated BAD. Taken together, these results indicate that phosphorylation of BAD or inhibition of its translocation to the mitochondria are critical phosphatidylinositol 3-kinase-independent survival pathways in LNCaP cells.

Topics: bcl-Associated Death Protein; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Humans; Male; Mitochondria; Phosphorylation; Prostatic Neoplasms; Protein Transport; Serum; Signal Transduction

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

Topics: Amino Acid Sequence; Annexin A5; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Melanoma; Molecular Sequence Data; Up-Regulation

The epidermal growth factor (EGF)-like domain is involved in receptor-ligand interactions, extracellular matrix formation, cell adhesion and chemotaxis. Nasopharyngeal carcinoma associated gene 6 (NGX6) is a novel EGF-like domain-containing gene located at the high frequent loss of heterozygosity (LOH) region 9p21-22 associated with nasopharyngeal carcinoma (NPC). It is down-regulated in NPC and its over-expression can delay the cell cycle G(0)-G(1) progression in NPC cells. In the present study, in situ hybridization analysis, using NPC tissue microarrays, showed that loss of NGX6 expression was associated with NPC lymph node metastasis. The Tet-on gene expression system and cDNA array techniques were used to profile the potential targets of NGX6. We found that NGX6 can influence the expression of some cell adhesion molecules in NPC cells. NGX6 can associate with ezrin, a linkage between the cell membrane and cytoskeleton. The NGX6 protein was expressed on the cell surface as a glycoprotein. Ectopic induction of NGX6 can impair NPC cell migration and invasive ability as well as improve cell adhesion and gap junctional intercellular communication, and can suppress tumor formation in vivo. The data revealed that NGX6 plays a role in cell adhesion modulation in NPC cells.

Topics: Animals; Carcinoma; Cell Adhesion; Cell Adhesion Molecules; Cell Cycle; Cell Membrane; Cell Movement; Chlorocebus aethiops; COS Cells; Cytoskeletal Proteins; Cytoskeleton; Epidermal Growth Factor; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Nasopharyngeal Neoplasms; Phosphoproteins; Tumor Cells, Cultured; Tumor Suppressor Proteins

The over-expression of epidermal growth factor receptor (EGFR) and its ligands, epidermal growth factor (EGF) and transforming growth factor-alpha, is a common feature of epithelial carcinomas and correlates with neoplastic progression. Secretory leukocyte protease inhibitor (SLPI), a member of the Kazal superfamily of serine anti-proteases, induces proliferation and promotes malignancy of epithelial cells and is expressed at high levels in multiple tumor types. In the present study, we have demonstrated that EGF increases SLPI expression in the human endometrial epithelial cell line Ishikawa in a dose- and time-dependent manner. We have shown that this effect of EGF occurs, in part, at the level of the SLPI promoter and involves the MAP kinase signaling pathway. We have further shown that EGF promotion of cell proliferation, but not induction of cyclin D1 gene expression, involves SLPI. Our results suggest that the regulation of SLPI expression by EGFR ligand(s) may represent a 'feed-forward' mechanism by which the enhanced proliferative and migratory properties of EGFR over-expressing cancer cells are sustained. Increased SLPI expression is likely an important component of altered EGFR signaling in human tumors and may have significant therapeutic implications in cancer progression.

Topics: Carcinoma; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Endometrial Neoplasms; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Gene Expression Regulation; Humans; Proteinase Inhibitory Proteins, Secretory; Proteins; Secretory Leukocyte Peptidase Inhibitor; Time Factors; Transfection; Transforming Growth Factor alpha

Thrombospondin-1 (TSP-1) is a multidomain extracellular macromolecule that was first identified as natural modulator of angiogenesis and tumor growth. In the present study, we found that epidermal growth factor (EGF) up-regulated TSP-1 expression in FTC-133 (primary tumor) but not in FTC-238 (lung metastasis) thyroid cancer cells. Both EGF and TSP-1 induced expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner. In FTC-133 cells, EGF induced proliferation in a TSP-1- and TIMP-1-dependent manner. In addition, we determined that re-expression of the tumor suppressor protein PTEN induced cell death, an effect that correlated with a block of Akt kinase phosphorylation. EGF-induced TSP-1 and TIMP-1 promoter activity and protein expression were inhibited in FTC-133 cells stably expressing wtPTEN but not in cells expressing mutant PTEN. Furthermore, we found that wtPTEN inhibited EGF--but not TSP-1--stimulated FTC-133 cell migration and also inhibited invasion induced by EGF and by TSP-1. Finally, an antibody against TSP-1 reversed EGF-stimulated FTC-133 cell invasion as well as the constitutive invasive potential of FTC-238 cells. Overall, our results suggest that PTEN can function as an important modulator of extracellular matrix proteins in thyroid cancer. Therefore, analyzing differential regulation of TSP-1 by growth factors such as EGF can be helpful in understanding thyroid cancer development.

Topics: Apoptosis; Carcinoma; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; Humans; MAP Kinase Kinase 1; Phosphatidylinositol 3-Kinases; Phosphoric Monoester Hydrolases; Promoter Regions, Genetic; PTEN Phosphohydrolase; Thrombospondin 1; Thyroid Neoplasms; Tissue Inhibitor of Metalloproteinase-1; Tumor Suppressor Proteins; Up-Regulation

Growth factors and Herceptin specifically and differentially modulate cell proliferation of tumor cells. However, the mechanism of action on erbB-receptor level is incompletely understood. We evaluated Herceptin's capacity to modulate erbB-receptor activation and interaction on the cell surface level and thereby potentially impair cell proliferation of HER2/neu (c-erbB2) overexpressing breast cancer cells, both in the presence and absence of relevant growth factors.. BT474 and SK-BR-3 breast cancer cell lines were treated with Epidermal Growth Factor (EGF), Heregulin, and with Herceptin in different combinations. Kinetics of cell proliferation were evaluated flow cytometrically based on BrdU-labeling. Fluorescence Resonance Energy Transfer, ELISAs and phosphorylation site specific Western Blotting was performed to investigate erbB-receptor interaction and activation.. EGF induced EGFR/EGFR and EGFR/c-erbB2 interactions correlate with stimulation of cell proliferation in BT474 cells. Both homo- and heterodimerization are considerably less pronounced in SK-BR-3 cells and heterointeraction is additionally reduced by EGF treatment, causing inhibition of cell proliferation. Heregulin stimulates cell proliferation extensively in both cell lines. Herceptin drives BT474 cells more efficiently into quiescence than it does with SK-BR-3 cells and thereby blocks cell cycle progress. In SK-BR-3 Herceptin treatment causes c-erbB2 phosphorylation of Y877 and Y1248, EGF induces Y877 and Y1112 phosphorylation. The Y1112 phosphorylation site, activated by EGF in SK-BR-3 cell, is bypassed in BT474. In addition the inhibitory capacity of Herceptin on BT474 and SK-BR-3 cell proliferation depends on the presence and absence of growth factors to a various extent.. The growth inhibitory effect of Herceptin on c-erbB2 overexpressing breast cancer cells is considerably modulated by EGFR coexpression and consequently EGFR/c-erbB2 homo- and heterointeractions, as well as the presence or absence of growth factors. C-erbB2 overexpression alone is insufficient to predict the impact of growth factors and antibodies on cell proliferation. The optimization and specification of therapeutic approaches based on erbB-receptor targeting requires to account for EGFR coexpression as well as the potential presence of erbB-receptor relevant growth factors.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Female; Growth Inhibitors; Humans; Neuregulin-1; Phosphorylation; Receptor, ErbB-2; Receptors, Cell Surface; Trastuzumab

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.

Topics: Caco-2 Cells; Carcinoma; Cell Movement; Cell Proliferation; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-8; Metalloendopeptidases; Mitogen-Activated Protein Kinase Kinases; Protein Kinase Inhibitors; Receptors, Cell Surface; Signal Transduction

An emerging paradigm holds that loss of negative signalling to receptor tyrosine kinases (RTKs) is permissive for their oncogenic activity. Herein, we have addressed tumor suppression by RALT/MIG-6, a transcriptionally controlled feedback inhibitor of ErbB RTKs, in breast cancer cells. Knockdown of RALT expression by RNAi enhanced the EGF-dependent proliferation of normal breast epithelial cells, indicating that loss of RALT signalling in breast epithelium may represent an advantageous condition during ErbB-driven tumorigenesis. Although mutational inactivation of the RALT gene was not detected in human breast carcinomas, RALT mRNA and protein expression was strongly and selectively reduced in ERBB2-amplified breast cancer cell lines. Reconstitution of RALT expression in ERBB2-amplified SKBr-3 and BT474 cells inhibited ErbB-2-dependent mitogenic signalling and counteracted the ability of ErbB ligands to promote resistance to the ErbB-2-targeting drug Herceptin. Thus, loss of RALT expression cooperates with ERBB2 gene amplification to drive full oncogenic signalling by the ErbB-2 receptor. Moreover, loss of RALT signalling may adversely affect tumor responses to ErbB-2-targeting agents.

Topics: Adaptor Proteins, Signal Transducing; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Line, Tumor; DNA Mutational Analysis; Drug Resistance, Neoplasm; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Loss of Heterozygosity; Receptor, ErbB-2; Signal Transduction; Trastuzumab; Tumor Suppressor Proteins

Sphingosine 1-phosphate (S1P) the product of sphingosine kinase (SK) action plays an important role in various pathological conditions like inflammation and cancer. In this study, we show that in the human breast cancer cell line MCF7, epidermal growth factor (EGF) stimulates SK-1 activity in a biphasic manner with a first peak after 15 min and a second delayed activation occurring after 1 h up to 18 h and thereafter declining again. This delayed activation is accompanied by increased mRNA and protein expression of SK-1, but not SK-2. Mechanistically, the transcriptional upregulation is dependent on the classical mitogen-activated protein kinase, protein kinase C (PKC) and the phosphoinositide 3-kinase, since specific inhibitors of these enzymes all abolish the EGF-induced mRNA upregulation and activity of SK-1. Moreover, dexamethasone also suppressed EGF-induced SK-1 mRNA expression and activity which is reversed by the glucocorticoid receptor antagonist RU486. To see whether EGF-induced upregulation of SK-1 is of relevance for tumor progression, we investigated two hallmarks of carcinogenesis, i.e., cell proliferation and migration. Stimulation of cells with EGF leads to enhanced [(3)H]thymidine incorporation into DNA and also to stimulated migration in a modified Boyden chamber assay. When cells are depleted of SK-1, but not SK-2, by siRNA transfection or by dexamethasone treatment, EGF-induced proliferation and migration are drastically reduced. In summary, these data show that EGF causes an acute stimulation of SK-1 activity and, moreover, triggers a delayed SK activation which is due to increased gene transcription and de novo synthesis of SK-1, which in turn directs cells towards growth and increased motility. Thus, the sphingosine kinase-1 may represent a novel attractive target for cancer therapy.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Enzyme Inhibitors; Epidermal Growth Factor; Female; Humans; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; ras Proteins

Oral epithelial tumour tissue, and cultured cervical epithelial carcinoma cells have been studied using synchrotron infrared microspectroscopy. Mid infrared absorption spectra collected at cellular spatial resolution from within oral tumours were found to be sufficiently distinct, when analysed by principal component analysis, to distinguish between three different cell types within the tumour. The resulting data were sufficiently robust to allow correct classification of spectra from cells within subsequent tissue samples. These results go some way to demonstrate the potential of infrared spectroscopy as a tool in the post-operative screening of oral cancer patients by the examination of exfoliated epithelial cells. To gain a better understanding of the inherent variability in the infrared spectra of such epithelial cells, we have studied A431 carcinoma cells under the stimulus of the growth-stimulating hormone EGF. We have detected key changes in the infrared spectrum that relate to the activation of the growth factor signalling mechanism.

Topics: Carcinoma; Cells, Cultured; Culture Techniques; Epidermal Growth Factor; Humans; Infrared Rays; Mouth Mucosa; Mouth Neoplasms; Spectrophotometry, Infrared; Synchrotrons

The MUC1 tumor antigen is overexpressed on most breast tumors and metastases. It interacts with signaling proteins such as the ErbB kinases and beta-catenin, and is involved in mammary gland oncogenesis and tumor progression. Herein, we report a novel interaction between MUC1 and adenomatous polyposis coli (APC), a tumor suppressor involved in downregulating beta-catenin signaling. Initially identified in colorectal cancer, APC is also downregulated in breast tumors and presumably involved in mammary carcinogenesis. MUC1 and APC co-immunoprecipitate from the ZR-75-1 human breast carcinoma cell line and co-localize in mouse mammary glands and tumors. These studies also indicate that the association of MUC1 and APC may be increased by epidermal growth factor stimulation. Intriguingly, the co-immunoprecipitation of MUC1 and APC increases in human breast tumors and metastases as compared to adjacent normal tissues, indicating that this association may play a role in the formation and progression of breast tumors.

Topics: Adenomatous Polyposis Coli Protein; Animals; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Epidermal Growth Factor; Female; Humans; Mammary Glands, Human; Mammary Neoplasms, Animal; Mice; Mucin-1; Neoplasm Metastasis; Precipitin Tests

Autotaxin (ATX/NPP2) is a tumor cell motility-stimulating factor that displays both a nucleotide pyrophosphatase/phosphodiesterase activity and a recently described lysophospholipase D (lysoPLD) activity. The precise function of ATX in tumor cells and the role of ATX in thyroid carcinoma remains unclear. We have quantified ATX mRNA expression in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. ATX gene activity was significantly higher in undifferentiated anaplastic thyroid carcinoma cell lines (UTC) and tumor tissues as compared to follicular thyroid carcinoma (FTC) cell lines, FTC tissues or goiter tissues that were used as a control. In the thyroid carcinoma cell line 1736, EGF and bFGF stimulated ATX mRNA expression, whereas the cytokines IL-4, IL-1beta and TGF-beta reduced ATX transcriptional levels. FTC-133 cells, stably transfected with an expression vector for ATX, showed a higher lysoPLD activity, a higher proliferation rate and an increased migratory behavior. In addition, ATX also displayed a paracrine stimulatory effect on the motility of different thyroid carcinoma cell lines. Overexpression of ATX in the stably transfected FTC-133 resulted in down-regulation of CD54/ intercellular adhesion molecule-1 (ICAM-1) gene expression and augmented gene activity of the pro-angiogenic chemokine IL-8. We conclude that ATX may be regarded as a new tissue marker for undifferentiated human thyroid carcinoma cells. ATX increases the proliferation and migration of thyroid carcinoma cell lines and may also affect the angiogenic potential of thyroid carcinoma cells. Further studies are needed to provide insight into the role of ATX in the normal and neoplastic thyroid gland.

Topics: Adenocarcinoma, Follicular; Adult; Aged; Aged, 80 and over; Carcinoma; Carcinoma, Papillary; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Glucose-6-Phosphate Isomerase; Glycoproteins; Goiter; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-4; Interleukin-8; Male; Middle Aged; Multienzyme Complexes; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thyroid Neoplasms; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Cross-communication between the Met receptor tyrosine kinase and the epidermal growth factor receptor (EGFR) has been proposed to involve direct association of both receptors and EGFR kinase-dependent phosphorylation. Here, we demonstrate that in human hepatocellular and pancreatic carcinoma cells the Met receptor becomes tyrosine phosphorylated not only upon EGF stimulation but also in response to G protein-coupled receptor (GPCR) agonists. Whereas specific inhibition of the EGFR kinase activity blocked EGF- but not GPCR agonist-induced Met receptor transactivation, it was abrogated in the presence of a reducing agent or treatment of cells with a NADPH oxidase inhibitor. Both GPCR ligands and EGF are further shown to increase the level of reactive oxygen species within the cell. Interestingly, stimulation of the Met receptor by either GPCR agonists, EGF or its cognate ligand HGF, resulted in release of Met-associated beta-catenin and in its Met-dependent translocation into the nucleus, as analyzed by small interfering RNA-mediated knockdown of the Met receptor. Our results provide a new molecular explanation for cell surface receptor cross-talk involving the Met receptor and thereby link the wide diversity of GPCRs and the EGFR to the oncogenic potential of Met signaling in human carcinoma cells.

Topics: Animals; beta Catenin; Carcinoma; Cell Fractionation; Cell Line, Tumor; Cytoskeletal Proteins; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Ligands; MAP Kinase Signaling System; NADPH Oxidases; Phosphorylation; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-met; Reactive Oxygen Species; Receptor Protein-Tyrosine Kinases; Receptors, G-Protein-Coupled; Receptors, Growth Factor; RNA, Small Interfering; Trans-Activators; Transcriptional Activation

Focal adhesion kinase (FAK), a member of a growing family of structurally distinct protein tyrosine kinases (PTK), has been linked to specific phosphorylation events, and the elevation of FAK activity in human carcinoma cells correlated with increased invasive potential. Transactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase activity is proposed to stimulate cell migration and the subsequent activation of downstream signaling pathways. Quercetin (Qu) and luteolin (Lu), are potent PTK inhibitors as well as putative chemopreventive agents. The present work, we demonstrate that Qu and Lu at concentration of 20 microM transinactivated EGFR tyrosine kinase activity with marked reduction in phosphotyrosyl level of 170, 125, 65, 60 and 42 kDa cellular proteins, and induced apoptosis in MiaPaCa-2 cells. The 125 kDa protein was further identified as a FAK by immunoprecipitation and immunoblotting analyses. Tumor cells treated with Lu or Qu dampened the phosphorylation of FAK. In addition, our data clearly demonstrated that tumor cells responded to Qu and Lu by parallel reductions in the levels of phosphorylated FAK and the secreted matrix metalloproteinase (MMP) that may lead to the suppression of invasive potential and cell migration in vitro. While the molecular mechanism of FAK regulation of MMP secretion in tumor cells remains unclear, our results suggested that blockade of the EGFR-signaling pathway may contributed to the net effect. As suggested in the current study, targeting EGFR and FAK with the objective of modulating their regulatory pathways could offer prospects for the treatment of EGFR-responsive cancers in the future.

Topics: Carcinoma; Cell Movement; Diet; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression; Humans; Luteolin; Matrix Metalloproteinases; Neoplasm Invasiveness; Phosphorylation; Protein-Tyrosine Kinases; Quercetin; Subcellular Fractions; Transcriptional Activation; Tumor Cells, Cultured; Tyrosine

Selection of phage libraries against complex living targets such as whole cells or organs can yield valuable targeting ligands without prior knowledge of the targeted receptor. Our previous studies have shown that noninfective multivalent ligand display phagemids internalize into mammalian cells more efficiently than their monovalent counterparts suggesting that cell-based selection of internalizing ligands might be improved using multivalently displayed peptides, antibodies or cDNAs. However, alternative methods of phage recovery are needed to select phage from noninfective libraries. To this end, we reasoned that rolling circle amplification (RCA) of phage DNA could be used to recover noninfective phage. In feasibility studies, we obtained up to 1.5 million-fold enrichment of internalizing EGF-targeted phage using RCA. When RCA was applied to a large random peptide library, eight distinct human prostate carcinoma cell-internalizing peptides were isolated within three selection rounds. These data establish RCA as an alternative to infection for phage recovery that can be used to identify peptides from noninfective phage display libraries or infective libraries under conditions where there is the potential for loss of phage infectivity.

Topics: Bacteriophages; Carcinoma; Epidermal Growth Factor; Gene Library; Humans; Ligands; Male; Molecular Biology; Peptide Library; Prostatic Neoplasms; Tumor Cells, Cultured

Tumor cell motility and invasion have been linked to upregulated signaling from both the epidermal growth factor receptor (EGFR) and that for urokinase-type plasminogen activator (uPAR). However, we do not know whether these events are interdependent or unrelated, despite the obvious diagnostic and therapeutic implications. Gene microarray analyses have suggested that EGFR signaling via phospholipase C-gamma (PLCgamma) induces uPAR transcription. We utilized two sublines of the DU145 human prostate carcinoma cell line that are genetically engineered to differentially activate the EGFR/PLCgamma cascade and are variously invasive in vitro and in vivo. uPAR protein levels in these cells were found to be dependent on PLC signaling, pharmacologic inhibition of PLC signaling reduced uPAR expression. To determine whether uPAR was a required element in EGFR-mediated invasion, we stably expressed uPAR cDNA in either sense or antisense orientation in the two DU145 sublines. Interestingly, uPA production was modulated in parallel, although to a lesser degree, with uPAR in these sublines. Antisense to uPAR significantly restricted invasion of the highly invasive DU145 WT cells through Matrigel and reduced aggressiveness of tumors in nude mice. Up-regulation of uPAR significantly increased the invasiveness of the moderately invasive DU145 parental (DU145 P) cells through Matrigel, but this increased invasiveness was not seen in mice. uPA activity appears to contribute to invasiveness at least through Matrigel, as antibody to uPA or amiloride limited the transmigration. These results support a model of tumor invasion promoted by autocrine EGFR signaling involving reinforcing altered gene expression, of uPAR at least, that further induces cell motility. Herein, a number of key molecules whose expression levels are interrelated, including both EGFR and uPAR, are required but none are sufficient in the absence of other keys molecules in promoting tumor progression.

Topics: Amiloride; Animals; Antisense Elements (Genetics); Autocrine Communication; Carcinoma; Cell Movement; DNA, Complementary; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Phospholipase C gamma; Prostatic Neoplasms; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Signal Transduction; Type C Phospholipases; Up-Regulation; Urokinase-Type Plasminogen Activator; Xenograft Model Antitumor Assays

The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

Topics: Actin Cytoskeleton; Actin Depolymerizing Factors; Actins; Animals; Antibodies; Carcinoma; Cell Line, Tumor; Chemotaxis; Enzyme Inhibitors; Epidermal Growth Factor; Microfilament Proteins; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Pseudopodia; Rats; RNA Interference; Type C Phospholipases

Chemotaxis, directed cell migration in a gradient of chemoattractant, is an important biological phenomenon that plays pivotal roles in cancer metastasis. Newly developed microfluidic chemotaxis chambers (MCC) were used to study chemotaxis of metastatic breast cancer cells, MDA-MB-231, in EGF gradients of well-defined profiles. Migration behaviors of MDA-MB-231 cells in uniform concentrations of EGF (0, 25, 50, and 100 ng/ml) and EGF (0-25, 0-50, and 0-100 ng/ml) with linear and nonlinear polynomial profiles were investigated. MDA-MB-231 cells exhibited increased speed and directionality upon stimulation with uniform concentrations of EGF. The cells were viable and motile for over 24 h, confirming the compatibility of MCC with cancer cells. Linear concentration gradients of different ranges were not effective in inducing chemotactic movement as compared to nonlinear gradients. MDA-MB-231 cells migrating in EGF gradient of 0-50 ng/ml nonlinear polynomial profile exhibited marked directional movement toward higher EGF concentration. This result suggests that MDA-MB-231 cancer cell chemotaxis depends on the shape of gradient profile as well as on the range of EGF concentrations.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Chemotaxis; Collagen; Diffusion Chambers, Culture; Dose-Response Relationship, Drug; Drug Combinations; Epidermal Growth Factor; Extracellular Matrix; Female; Humans; Laminin; Neoplasm Metastasis; Nonlinear Dynamics; Proteoglycans; Vitronectin

No effective treatment options currently are available to patients with anaplastic thyroid cancer (ATC), resulting in high mortality rates. Epidermal growth factor (EGF) has been shown to play a role in the pathogenesis of many types of cancer, and its receptor (EGFR) provides an attractive target for molecular therapy.. The expression of EGFR was determined in ATC in vitro and in vivo and in human tissue arrays of ATC. We assessed the potential of the EGFR inhibitor gefitinib ("Iressa," ZD1839) to inhibit EGFR activation in vitro and in vivo, inhibit ATC cellular proliferation, induce apoptosis, and reduce the growth of ATC cells in vivo when administered alone and in combination with paclitaxel.. EGFR was overexpressed in ATC cell lines in vitro and in vivo and in human ATC specimens. Activation of EGFR by EGF was blocked by the addition of gefitinib. In vitro studies showed that gefitinib greatly inhibited cellular proliferation and induced apoptosis in ATC cell lines and slowed tumor growth in a nude mouse model of thyroid carcinoma cells injected subcutaneously.. ATC cells consistently overexpress EGFR, rendering this receptor a potential target for molecular therapy. Gefitinib effectively blocks activation of EGFR by EGF, inhibits ATC cellular proliferation, and induces apoptosis in vitro. Our in vivo results show that gefitinib has significant antitumor activity against ATC in a subcutaneous nude mouse tumor model and therefore is a potential candidate for human clinical trials.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma; Carcinoma, Papillary; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Gefitinib; Humans; Mice; Mice, Nude; Paclitaxel; Phosphorylation; Quinazolines; Thyroid Neoplasms; Transforming Growth Factor alpha; Tumor Cells, Cultured

Topics: Biomarkers, Tumor; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoenzyme Techniques; Salivary Gland Neoplasms; Salivary Glands

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and beta-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, beta-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Topics: Actins; Breast Neoplasms; Cadherins; Carcinoma; Cell Adhesion; Cell Transformation, Neoplastic; Chemotaxis; Dose-Response Relationship, Drug; Epidermal Growth Factor; Extracellular Matrix Proteins; Keratins; Neoplasm Proteins; Tumor Cells, Cultured; Vimentin; Vinculin

Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.

Topics: Animals; Basement Membrane; Breast; Breast Neoplasms; Carcinoma; Cell Adhesion Molecules; Cell Movement; Cells, Cultured; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Extracellular Matrix Proteins; Female; Kalinin; Matrix Metalloproteinase 2; Matrix Metalloproteinases; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Peptide Fragments; Protein Binding; Protein Structure, Tertiary; Receptor Protein-Tyrosine Kinases; Tissue Inhibitor of Metalloproteinase-3

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

Topics: Adrenal Cortex Neoplasms; Carcinoma; DNA-Binding Proteins; Dose-Response Relationship, Drug; Epidermal Growth Factor; HeLa Cells; Humans; Hydrocortisone; Isoenzymes; Milk Proteins; Multienzyme Complexes; Progesterone Reductase; Response Elements; RNA, Messenger; STAT5 Transcription Factor; Steroid Isomerases; Trans-Activators; Transcription, Genetic; Transduction, Genetic; Tumor Cells, Cultured; Tumor Suppressor Proteins

Imbalances in the epithelial-stromal interactions are important in the pathogenesis of prostate cancer. However, we know little about androgenic regulation in the stroma of prostate cancer. We examined the cancer-stromal interaction paying attention to androgen responsiveness of stromal side. In co-culture, PC3 and LNCaP cells did not affect dihydrotestosterone (DHT)-dependent growth of prostate stromal cells (PrSCs), but DU145 cells significantly reduced it. Conditioned medium from DU145 cells (DU145-CM) also inhibited DHT-dependent PrSCs growth, androgen receptor (AR) expression, and prostate specific antigen transcription. Although the inhibitory effect of DU145-CM was not affected by neutralizing antibody against EGF, FGF-2, or TNF-alpha, pretreatment with testosterone-Sepharose partially reduced the inhibitory ability of DU145-CM. These results suggest that DU145 cells produce inhibitory factors for androgen responsiveness, including steroid-binding protein(s), and these may participate in crosstalk between DU145 cells and PrSCs as modulators of androgen.

Topics: Carcinoma; Coculture Techniques; Culture Media, Conditioned; Dihydrotestosterone; Epidermal Growth Factor; Fibroblast Growth Factor 2; Humans; Male; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Sepharose; Stromal Cells; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Pancreatic carcinoma still has the highest mortality rate in comparison to any other malignancy. Major reasons are late detection of disease, highly aggressive tumor growth and the early formation of metastases. Thus, novel effective therapies are urgently needed to improve the outcome of the patients. Overexpression of the epidermal growth factor receptor (EGFR) and its ligands has been implicated in the oncogenesis of pancreatic carcinoma and associated with an unfavorable prognosis. Consequently, the EGFR represents a specific target antigen suitable for immunotherapy. We generated a recombinant immunotoxin by fusing the anti-EGFR single chain fragment 425(scFv) to a truncated mutant of Pseudomonas Exotoxin A (ETA'). Using the expression vector pBM1.1, functional 425(scFv)-ETA' was periplasmically expressed under osmotic stress conditions in the presence of compatible solutes. The 72 kDa His10-tagged fusion protein was purified by a combination of metal-ion affinity and molecular size chromatography. Binding activity and specificity of the immunotoxin to the EGFR-positive pancreatic carcinoma cell line L3.6pl was confirmed by flow cytometry and ELISA. Finally, 425(scFv)-ETA' showed significant toxicity toward this cell line reaching 50% inhibition of cell proliferation at a concentration (IC50) of 7.5 ng/ml. This is the first report documenting the specific cytotoxicity of a recombinant immunotoxin towards metastatic pancreatic carcinoma cells, suggesting that EGFR-specific antibody toxins may become valuable therapeutic reagents for the treatment of pancreatic carcinoma.

Topics: Carcinoma; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Escherichia coli; Flow Cytometry; Humans; Immunoglobulin Variable Region; Immunotherapy; Immunotoxins; Inhibitory Concentration 50; Ligands; Models, Genetic; Mutation; Neoplasm Metastasis; Pancreatic Neoplasms; Plasmids; Prognosis; Recombinant Fusion Proteins; Recombinant Proteins; Single-Chain Antibodies

Aminopeptidase N (APN)/CD13 is a transmembrane ectopeptidase expressed on a wide variety of cells. However, the precise function of APN/CD13 in tumor cells and the relationship of APN/CD13 to thyroid cancer remain unclear. In our study, we quantified the expression of APN/CD13 and additionally dipeptidyl peptidase IV (DPIV)/CD26 in thyroid carcinoma cell lines and in tissues of patients with thyroid carcinomas. Undifferentiated anaplastic thyroid carcinomas expressed more APN/CD13 than differentiated thyroid carcinomas. DPIV/CD26 showed an opposite expression pattern. We detected higher levels of DPIV/CD26 in follicular thyroid carcinomas (FTCs) and papillary thyroid carcinomas than in undifferentiated anaplastic thyroid carcinomas. In the undifferentiated thyroid carcinoma cell line 1736, APN/CD13 mRNA expression could be increased by epidermal growth factor, basic fibroblast growth factor, interleukin-6, and tumor necrosis factor alpha. FTC-133 cells stably transfected with an expression vector for APN-enhanced green fluorescent protein showed a higher migration rate than FTC-133 cells transfected with the enhanced green fluorescent protein-control plasmid. Overexpression of APN/CD13 in stably transfected cells is associated with down-regulation of N-myc down-regulated gene (NDRG)-1, melanoma-associated antigen ME491/CD63, and DPIV/CD26 gene expression. Inhibition of APN/CD13 mRNA expression by small interfering RNA induced NDRG-1, ME491/CD63, and DPIV/CD26 mRNA expression in cells of the undifferentiated thyroid carcinoma cell line C643. We conclude that APN/CD13-associated down-regulation of NDRG-1, ME491/CD63, and DPIV/CD26 in thyroid carcinoma cells is an important step of tumor progression to more malignant phenotypes, and we underline the important role of APN/CD13 as mediator in a multimolecular process regulating cell migration.

Topics: Adult; Aged; Carcinoma; CD13 Antigens; Cell Line, Tumor; Cell Movement; Dipeptidyl Peptidase 4; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Male; Middle Aged; Thyroid Neoplasms; Transfection; Tumor Cells, Cultured

Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express alpha6beta4 integrin. Here, we show that autocrine LM-5 mediates anchorage-independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail-truncated alpha6beta4 integrin. alpha6beta4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of beta4 integrin is necessary for basal and epidermal growth factor-induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional beta1 and beta4 integrin through activation of NFkappaB, and overexpression of NFkappaB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5-alpha6beta4 integrin-RAC-NFkappaB signaling.

Topics: Autocrine Communication; Basement Membrane; Breast Neoplasms; Carcinoma; Cell Adhesion; Cell Adhesion Molecules; Cell Line, Tumor; Cell Survival; Epidermal Growth Factor; Humans; Integrin alpha6beta4; Kalinin; MAP Kinase Signaling System; Neoplasm Invasiveness; NF-kappa B; Protein Structure, Tertiary; rac GTP-Binding Proteins

We analyzed lateral propagation of epidermal growth factor (EGF) signaling in single live COS cells following local stimulation, achieved by the use of laminar flows containing rhodamine-labeled EGF. The spatiotemporal pattern of EGF signaling was visualized by fluorescent indicators for Ras activation and tyrosine phosphorylation. Contrary to the findings in previous reports, both signals were localized to the stimulated regions in control COS cells expressing EGF receptor at the basal level. However, the signals spread over the entire cell when EGF receptors were overexpressed or when receptor/ligand endocytosis was blocked. We thus present evidence that ligand-independent propagation of EGF signaling occurs only when the receptor density on the plasma membrane is high, such as in carcinoma cells.

Topics: Animals; Bacterial Proteins; Carcinoma; Cell Membrane; COS Cells; Diffusion Chambers, Culture; Dose-Response Relationship, Drug; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Eukaryotic Cells; Gene Expression Regulation; Green Fluorescent Proteins; GTP Phosphohydrolases; Humans; Indicators and Reagents; Luminescent Proteins; Macromolecular Substances; Phosphorylation; Protozoan Proteins; ras Proteins; Recombinant Proteins; Signal Transduction; Tumor Cells, Cultured; Tyrosine

The stimulation of human tumor cells overexpressing epidermal growth factor receptor (EGFR) with EGF enhances tumor development and malignancy. Therefore, compounds that modulate the EGF-mediated signal inducing apoptosis in EGFR-overexpressing cells would represent a new class of antitumor drug and might be useful in the treatment of a subset of human tumors. In the course of screening for compounds that induce apoptosis in EGFR-overexpressing human epidermal carcinoma A431 cells from secondary metabolites of microorganisms, we found that vacuolar-type H(+)-ATPase (V-ATPase) inhibitors, such as concanamycin B and destruxin E, induced apoptosis only when the cells were stimulated with EGF. The EGF-dependent apoptosis by V-ATPase inhibitors was not observed in other types of human tumor cells which do not overexpress EGFR. The apoptosis in A431 cells was inhibited by anti-FasL antibody which neutralized the cytotoxic effect of FasL, indicating that the Fas/FasL system was involved. The expression of cell surface FasL was upregulated by stimulation with EGF and increased further by V-ATPase inhibitors. Moreover, EGF inhibited cytotoxic Fas antibody-induced apoptosis, whereas V-ATPase inhibitors disrupted the protective effect of EGF on apoptosis in A431 cells. Taken together, these results suggested that V-ATPase inhibitors induced EGF-dependent apoptosis in A431 cells, possibly through both the enhancement of EGF-induced cell surface expression of FasL and the disruption of an EGF-induced survival signal.

Topics: Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Nucleus; Cell Survival; Colonic Neoplasms; Depsipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Fas Ligand Protein; fas Receptor; Fungal Proteins; Humans; Macrolides; Membrane Glycoproteins; Peptides, Cyclic; Tumor Cells, Cultured; Vacuolar Proton-Translocating ATPases

Buccal mucosa carcinoma-derived cell line, HO-1-N-1, epithelial-like cells, was obtained in order to investigate the characteristics of oral cancer cells and examine the [Ca2+]i responses to stimulants, such as bradykinin (BK), histamine (HIST), thapsigargin (TG), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha ). Intracellular Ca2+ influx was observed by all stimulants that enhanced the [Ca2+]i response. However, intracellular Ca2+ release was not observed in response to growth factors. The [Ca2+]i response of BK (100 nM) was inhibited by 10 micro M of the BKB2 antagonist, D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK, and HIST (1 mM) was completely inhibited by 100 nM of the H1 antagonist, (+)-chlorpheniramine, in the presence and absence of extracellular Ca2+ (1.5 mM).

Topics: Bradykinin; Calcium; Calcium Signaling; Carcinoma; Epidermal Growth Factor; Epithelial Cells; Histamine; Histamine H1 Antagonists; Humans; Mouth Mucosa; Mouth Neoplasms; Receptors, Cell Surface; Stimulation, Chemical; Thapsigargin; Transforming Growth Factor alpha; Tumor Cells, Cultured

Cell migration requires precise control, which is altered or lost when tumor cells become invasive and metastatic. beta-catenin plays a dual role in this process: as a member of adherens junctions it is essential to link cadherins to the cytoskeleton thereby allowing tight intercellular adhesion, and as a member of the Wnt-signaling pathway, beta-catenin is translocated into the nucleus and serves together with the LEF1/TCF-transcription factors to drive gene expression necessary for the epithelial-to-mesenchymal transition (EMT). Activated beta-catenin signaling has been implicated in the genesis of a variety of tumors. Here we demonstrate a pivotal function for beta-catenin signaling in epithelial cell migration and tumorigenesis. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) induce beta-catenin signaling under conditions where they stimulate cell motility. Ectopic expression of either stabilized beta-catenin or a regulatable form of activated beta-catenin induces cell migration in different cell types and cooperates with EGF and HGF in this process. Activation of beta-catenin signaling induces expression of the new target gene osteopontin during migration. Cells expressing stabilized beta-catenin also exhibit significantly increased capability to form tumors in a nude mouse xenograft model. The data suggest that a critical threshold of beta-catenin signaling, activated by cooperative mechanisms, may be important during the EMT and tumorigenesis.

Topics: Animals; Aphidicolin; beta Catenin; Carcinoma; Cell Line; Cell Movement; Cycloheximide; Cytoskeletal Proteins; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Gene Targeting; Mice; Mice, Nude; Neoplasms; Osteopontin; Protein Synthesis Inhibitors; Rats; Sialoglycoproteins; Signal Transduction; Trans-Activators; Transcriptional Activation; Tumor Cells, Cultured; Urinary Bladder Neoplasms

CD97 expression is related closely to the dedifferentiation and tumor stage in thyroid carcinomas. We systematically examined the role of CD97 and its closest relative, EMR2, in normal and malignant gastric, esophageal, and pancreatic tissue. The normal tissues were EMR2-, whereas CD97 was expressed slightly in the parietal cells of gastric mucosa and in exocrine pancreatic cells. Interestingly, intralobular and interlobular pancreatic ducts were CD97+. All tumors were EMR2-. CD97 was expressed by 44 of 50 gastric, 14 of 18 pancreatic, and 10 of 13 esophageal carcinomas. Of the 44 gastric cancers, 27 showed disseminated or scattered tumor cells at the invasion front with stronger CD97 expression than tumor cells located in solid tumor formations. There was no correlation between CD97 levels in the tumors or soluble CD97 in the serum samples and the clinicopathologic features of the patients. Taken together, significant numbers of gastric, esophageal, and pancreatic carcinomas are CD97+, whereas its homolog, EMR2, does not have any role in such tumors.

Topics: Aged; Antigens, CD; Biomarkers, Tumor; Carcinoma; Epidermal Growth Factor; Esophageal Neoplasms; Female; Humans; Immunohistochemistry; Male; Membrane Glycoproteins; Middle Aged; Pancreatic Neoplasms; Receptors, G-Protein-Coupled; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Stomach Neoplasms; Tumor Cells, Cultured

Engineered phage-based vectors are an attractive alternative strategy for gene delivery because they possess no natural mammalian cell tropism and can be genetically modified for specific applications. Genotoxic treatments that increase the transduction efficiency of single-stranded adeno-associated virus were tested on cells transfected by single-stranded phage. Indeed, green fluorescent protein transgene expression by epidermal growth factor-targeted phagemid particles increased with heat shock, UV irradiation, and camptothecin (CPT) treatment. CPT resulted in transduction efficiencies of 30-45% in certain human carcinoma cell lines and reduced the minimal dose needed to detect green fluorescent protein-expressing cells to as low as 1-10 particles/cell. Targeted phage transduction was effective in many tumor cell lines and in prostate tumor xenografts with CPT treatment. Taken together, these data suggest the feasibility of using phage-based vectors for therapeutic gene delivery to cancer cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Bacteriophages; Camptothecin; Carcinoma; Combined Modality Therapy; Enzyme Inhibitors; Epidermal Growth Factor; Genetic Therapy; Genetic Vectors; Humans; Male; Mice; Mice, Nude; Neoplasms; Plasmids; Transduction, Genetic; Transfection; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR) and epidermal growth factor (EGF)-EGF receptor (EGFR) expression is highly correlated with breast cancer metastasis. Phosphoinositide 3-kinase (PI3K), small Rho GTPases, such as Cdc42 and Rac1, and neuronal Wiskott Aldrich syndrome protein (N-WASP) are key effectors that regulate dynamic changes in the actin cytoskeleton and cell migration. uPA- and EGF-stimulated chemotaxis, cytoskeletal rearrangements and activation of Cdc42, Rac1 and N-WASP were studied in the highly metastatic human breast cancer cell line MDA MB 231. These studies reveal that divergent signalling occurs downstream of PI3K. The activity of PI3K was not necessary for uPA-induced chemotactic responses, but those induced by EGF were entirely dependent upon PI3K. Furthermore, PI3K-independent chemotactic signalling by uPA was shown to involve disruption of an interaction between beta(1)-integrins and N-WASP and translocation of N-WASP to the actin cytoskeleton.

Topics: Actins; Breast Neoplasms; Carcinoma; Chemotaxis; Cytoskeleton; Epidermal Growth Factor; Female; Humans; Integrin beta1; Nerve Tissue Proteins; Phosphatidylinositol 3-Kinases; Protein Transport; rho GTP-Binding Proteins; Signal Transduction; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Wiskott-Aldrich Syndrome Protein, Neuronal

Human colon carcinoma cells express 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP27B1) and thus produce the vitamin D receptor (VDR) ligand 1alpha,25-dihydroxyvitamin D(3) (1,25-D3), which can be metabolized by 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24). Expression of VDR, CYP27B1, and CYP24 determines the efficacy of the antimitotic action of 1,25-D3 and is distinctly related to the degree of differentiation of cancerous lesions. In the present study we addressed the question of whether the effects of epidermal growth factor (EGF) and of 1,25-D3 on VDR, CYP27B1, and CYP24 gene expression in human colon carcinoma cell lines also depend on the degree of cellular differentiation. We were able to show that slowly dividing, highly differentiated Caco-2/15 cells responded in a dose-dependent manner to both EGF and 1,25-D3 by up-regulation of VDR and CYP27B1 expression, whereas in highly proliferative, less differentiated cell lines, such as Caco-2/AQ and COGA-1A and -1E, negative regulation was observed. CYP24 mRNA was inducible in all clones by 1,25-D3 but not by EGF. From the observed clonal differences in the regulatory effects of EGF and 1,25-D3 on VDR and CYP27B1 gene expression we suggest that VDR-mediated growth inhibition by 1,25-D3 would be efficient only in highly differentiated carcinomas even when under mitogenic stimulation by EGF.

Topics: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Calcitriol; Carcinoma; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Colonic Neoplasms; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Receptors, Calcitriol; RNA, Messenger; Steroid Hydroxylases; Tumor Cells, Cultured; Vitamin D3 24-Hydroxylase

We analyzed 149 women (81 with cervical intraepithelial neoplasia and with invasive carcinoma of the cervix and 68--as a control group). The influence of Chlamydia trachomatis (Cht) infection into expression of EGFR, TGF-alpha, Ki 67, HPV 16 and 18 was examined. IS-PCR was used to measure the level of antibodies in the serum. We detected that chlamydial infection may cause cervical hypertrophy in women with and without cervical intraepithelial neoplasia and invasive carcinoma. Infections of both Cht and HPV correlate with high expession of Ki 67 in epithelium. Cht infection also increased the expression of HPV16 in CIN I. These results suggest that Cht infection modifies the activity of viruses. In our research we have confimed that Cht infection increases the expression of EGFR and TGF-alpha. These facts may explain variants other than the HPV-mechanism of cervical carcinogenesis.

Topics: Adult; Antibodies, Bacterial; Carcinoma; Case-Control Studies; Chlamydia Infections; Chlamydia trachomatis; Epidermal Growth Factor; Female; Humans; Ki-67 Antigen; Middle Aged; Neoplasm Invasiveness; Papillomaviridae; Polymerase Chain Reaction; Transforming Growth Factor alpha; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Epidermal growth factor (EGF) has widespread growth effects, and in some tissues proliferation is associated with the nuclear localization of EGF and epidermal growth factor receptor (EGFR). In the thyroid, EGF promotes growth but differs from thyrotropin (TSH) in inhibiting rather than stimulating functional parameters. We have therefore studied the occurrence and cellular distribution of EGF and EGFR in normal thyroid, in Graves' disease, where growth is mediated through the thyrotropin receptor (TSHR), and in a variety of human thyroid tumors. In the normal gland the staining was variable, but largely cytoplasmic, for both EGF and EGFR. In Graves' disease there was strong cytoplasmic staining for both EGF and EGFR, with frequent positive nuclei. Nuclear positivity for EGF and particularly for EGFR was also a feature of both follicular adenomas and follicular carcinomas. Interestingly, nuclear staining was almost absent in papillary carcinomas. These findings document for the first time the presence of nuclear EGF and EGFR in thyroid. Their predominant occurrence in tissues with increased growth (Graves' disease, follicular adenoma, and carcinoma) may indicate that nuclear EGF and EGFR play a role in growth regulation in these conditions. The absence of nuclear EGF and EGFR in papillary carcinomas would suggest that the role played by EGF in growth control differs between papillary carcinoma and follicular adenomas/carcinomas of the thyroid.

Topics: Adenoma; Carcinoma; Carcinoma, Papillary; Cell Nucleus; Epidermal Growth Factor; ErbB Receptors; Goiter, Nodular; Graves Disease; Humans; Immunohistochemistry; Reference Values; Thyroid Gland; Thyroid Neoplasms; Tissue Distribution

Signals from the epidermal growth factor (EGF) receptor and integrin-dependent adhesion to laminin contribute to the progression and metastasis of colonic tumors. However, little is know about the mechanisms by which these signals cooperate. Recently, we have reported that the colon cancer cell line LIM1215 secretes and adhere to autocrine laminin-10 via multiple integrin receptors and that EGF stimulates spreading of these cells on the same substrate. In this report, we investigate the effect of EGF and laminin-10 on colon cancer cell migration in vitro. EGF stimulates migration of LIM1215 cells in a wound healing assay. The response to EGF is inhibited by anti-EGF receptor antibody 528, the EGF receptor kinase inhibitor AG-1478, or the MAP kinase kinase inhibitor PD98059 but not the PI3-K inhibitor wortmannin. Using Transwell migration chambers, we demonstrate that laminin-10 but not collagen-I, collagen-IV, or a commercial preparation of human placental laminin is a potent motility factor for LIM1215 cells. The migration response to laminin-10 is increased upon stimulation of the cells with EGF and correlates with the up-regulation of alpha(6)beta(4) integrin expression as measured by analysis of Triton X-100-soluble cellular extracts. The results from integrin inhibition experiments indicate that basal migration on laminin-10 is mediated by alpha(3)beta(1) but not alpha(2)beta(1) nor alpha(6)beta(4) integrins. Alpha(3) blocking antibodies also inhibited EGF-stimulated chemokinetic migration of LIM1215 cells on laminin-10. However, in contrast to unstimulated cells, alpha(6) or beta(4) integrin-blocking antibodies inhibited the migration of EGF-stimulated cells by up to 50%. Taken together, these results support the cooperative role of EGF receptor and laminin-10 on colon cancer cell motility and suggest a critical role for both the alpha(3)beta(1) and the alpha(6)beta(4) integrins in this process.

Topics: Actins; Antigens, Surface; Biological Assay; Carcinoma; Cell Movement; Colonic Neoplasms; Cytoskeleton; Epidermal Growth Factor; Humans; Integrin alpha3beta1; Integrin alpha6beta4; Integrins; Laminin; Neoplasm Metastasis; Time Factors; Tumor Cells, Cultured

CD97 is a founding member of the EGF-TM7 family of class II seven-span transmembrane (7-TM) receptors. CD97 has an extended extracellular region with several N-terminal epidermal growth factor (EGF)-like domains, which mediate binding to CD55. Previous studies demonstrated the expression of CD97 on activated lymphocytes, monocytes, macrophages, granulocytes, and numerous haematopoietic and nonhaematopoietic cell lines. Here, we determined the cellular distribution of human CD97 in situ by immunohistochemistry (IH) and immunofluorescence (IF). Abundant expression of CD97 was detected on all types of macrophages and dendritic cells, except for microglia. Within the lymphoid lineage, most T cells but only a few B cells express CD97. Germinal centre B cells do not express the molecule. Except for smooth muscle cells, no staining was found on other cells outside the immune system. However, analysis of a restricted set of epithelial tumors revealed CD97 expression on the malignant cells in thyroid and gastrointestinal tract cancer.

Topics: Antibodies, Monoclonal; Antigens, CD; Binding Sites, Antibody; Carcinoma; CD55 Antigens; Epidermal Growth Factor; Humans; Lymphoid Tissue; Membrane Glycoproteins; Muscle, Smooth; Myeloid Cells; Organ Specificity; Receptors, G-Protein-Coupled

The expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated for 76 cases of breast carcinoma. HB-EGF was expressed in 71.8% of the carcinoma cases but only slightly in normal mammary glands. Interestingly, its expression was inversely related to biological aggressiveness of the breast carcinoma. These results suggest that HB-EGF may play a crucial role in the early stage of this carcinoma.

Topics: Adult; Aged; Breast Neoplasms; Carcinoma; Epidermal Growth Factor; Female; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Middle Aged; Neoplasm Proteins; Prognosis

Ezrin is a member of the ezrin, radixin, and moesin family. These proteins are membrane-actin cross-linking proteins. Furthermore, ezrin is an important signal transduction protein that undergoes phosphorylation and translocation on stimulation by growth factors. Ezrin phosphorylation and translocation are thought to be correlated with cell motility, invasion, and carcinoma metastasis. Recently, the authors reported that an ezrin antisense phosphorothionate could significantly inhibit endometrial carcinoma cells' penetration in the Matrigel membrane cancer invasion assay. In the current study, the authors measured ezrin content in clinical ovarian epithelial carcinoma (OVCA) specimens and cell lines and investigated whether interleukin (IL)-1alpha and epidermal growth factor (EGF) induce an invasive phenotype in OVCA cells via ezrin phosphorylation and translocation.. Twenty-five normal ovary, 25 primary OVCA, 21 metastatic OVCA tissue (7 in omentum, 16 in ascites), and 3 OVCA cell lines were collected for Western blot detection of ezrin content. The OVCA cell line SKOV3 was treated with IL-1alpha or EGF. Indirect immunofluorescence staining followed by confocal laser scanning and double-staining electron microscopic immunohistochemistry were used to investigate changes in the intracellular distribution of ezrin and cell morphology after IL-1alpha or EGF treatment. The content of ezrin was measured by Western blotting and analyzed by the National Institutes of Health Image computer program. Immunoprecipitation and Western blot techniques were used for ezrin phosphorylation studies. Genistein was used to block tyrosine phosphorylation.. (1) Ezrin was overexpressed in OVCA, with the highest values in metastases. (2) Interleukin-1alpha and EGF significantly increased OVCA tyrosine phosphorylation, ezrin translocation, and cell growth. (3) These effects were abolished by treatment with the tyrosine kinase inhibitor, genistein. (4) Treatment with IL-1alpha or EGF induced an invasive phenotype, i.e., membrane ruffling, and process formation.. High expression and activation of ezrin appear to be related to OVCA metastatic behavior. Interleukin-1alpha and EGF may regulate OVCA invasive behavior by activating ezrin tyrosine phosphorylation, translocation, and cancer cell proliferation. The authors' results may partially explain why OVCA patients with positive macrophage colony stimulating factor (a chemoattractant of IL-1alpha secreting monocytes) or EGF receptors (c-erb B-2) have a poor prognosis.

Topics: Blotting, Western; Carcinoma; Cell Division; Cytoskeletal Proteins; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Interleukin-1; Neoplasm Invasiveness; Neoplasm Metastasis; Ovarian Neoplasms; Phenotype; Phosphoproteins; Phosphorylation; Translocation, Genetic; Tumor Cells, Cultured; Tyrosine

Intestinal trefoil factor (ITF) has a role in gastrointestinal mucosal integrity and the repair of damaged mucosa. However, little is known about its role in tumors. To analyze the role of ITF in colon carcinomas, overexpression of the ITF gene in colon carcinoma cells was used.. Human colon carcinoma cell lines LoVo and SW837, expressing no endogenous ITF, and WiDr expressing a low level of ITF were stably transfected with an expression vector harboring human ITF complementary DNA. The effects of ITF overexpression on in vitro growth, morphology in collagen gel, response to epidermal growth factor (EGF), mitogen-activated protein kinase (MAPK) activity, and growth in nude mice were assessed.. Overexpression of ITF in LoVo and SW837 resulted in significantly reduced growth in vitro and in vivo. In collagen gels, the ITF-expressing LoVo clones formed smaller, more dispersed colonies. EGF-induced phosphorylation of MAPKs was modestly reduced in the ITF-expressing clones. The growth of WiDr was modestly suppressed only in vivo by ITF overexpression.. Overexpression of ITF suppressed the growth of colon carcinoma cells. ITF may function as an inhibitory factor for the growth of colonic neoplasm.

Topics: Animals; Carcinoma; Clone Cells; Colonic Neoplasms; Epidermal Growth Factor; Growth Substances; Humans; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Mucins; Muscle Proteins; Neuropeptides; Peptides; Phosphorylation; RNA, Messenger; Trefoil Factor-2; Trefoil Factor-3; Tumor Cells, Cultured

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Topics: Antibodies, Monoclonal; Calcium; Carbocyanines; Carcinoma; Cell Membrane; Dimerization; Energy Transfer; Epidermal Growth Factor; ErbB Receptors; Fluorescent Dyes; Humans; Intracellular Fluid; Microscopy, Fluorescence; Phosphorylation; Rhodamines; Signal Transduction; Tumor Cells, Cultured

Overexpression of epidermal growth factor receptor (EGFR) and establishment of transforming growth factor alpha (TGF alpha)/EGF autocrine system are frequently detected in tumor cells. In addition to mitogenic ability, we demonstrate in this report that EGF protects a human esophageal carcinoma (CE) cell line, CE81T/VGH, from staurosporine-induced apoptosis. The anti-apoptotic signal of EGF is alleviated by a MEK inhibitor PD98059 or an ERK2 dominant negative mutant but not by a phosphatidylinositol-3'-kinase (PI-3K) inhibitor wortmannin. Furthermore, v-raf blocks apoptosis induced by staurosporine. This evidence implies that the survival signal of EGF is mediated via the Raf-MEK-ERK pathway but not the PI3-K pathway. The survival effect of EGF is coincident with the induction of mcl-1, an antiapoptotic gene in the bcl-2 family. PD98059 also suppresses the induction of Mcl-1 by EGF, implying that EGF may up-regulate Mcl-1 via the MAP kinase pathway. Overexpression of mcl-1 is sufficient to protect against apoptosis, while transfection of a mcl-1 antisense plasmid causes cell death. The expression of mcl-1 antisense plasmid also suppresses the anti-apoptotic effect of EGF. Taken together, these results indicate that EGF may up-regulate Mcl-1 through the MAP kinase pathway to suppress apoptosis.

Topics: Androstadienes; Apoptosis; Carcinoma; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA, Antisense; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Phosphoinositide-3 Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-raf; Recombinant Fusion Proteins; Staurosporine; Transfection; Tumor Cells, Cultured; Wortmannin

The epidermal growth factor (EGF)-like peptides CRIPTO (CR), amphiregulin (AR) and transforming growth factor alpha (TGFalpha) are expressed in human ovarian carcinomas.. The expression of AR, CR and TGFalpha in ovarian carcinoma cell lines was assessed by immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR). The antiproliferative effects of antisense phosphorothioate oligodeoxynucleotides (AS S-Oligos) directed against either AR, CR or TGFalpha was evaluated by using a clonogenic assay.. A majority of the ovarian carcinoma cell lines was found to express TGFalpha, AR and CR mRNAs and proteins. AS S-Oligos directed against either AR, CR or TGFalpha were able to inhibit the anchorage-independent growth of NIH:OVCAR3 and NIH:OVCAR8 cells in a dose dependent manner. A 30%-50% growth inhibition was observed at a 2 microM concentration of the AS S-Oligos. Treatment of these cells with combinations of EGF-related AS S-Oligos resulted in a more significant growth inhibition when compared to treatment with a single AS S-oligo. A 60%-75% growth inhibition was observed using combinations of AR, CR and TGFalpha AS S-oligos at a total concentration of 2 microM. An additive growth-inhibitory effect occurred when ovarian carcinoma cells were exposed to the AS S-Oligos after treatment with either paclitaxel or cis-platinum.. These data suggest that EGF-related peptides function as autocrine growth factors in ovarian carcinoma cells, and that they might represent targets for experimental therapy of ovarian carcinoma.

Topics: Amphiregulin; Carcinoma; EGF Family of Proteins; Epidermal Growth Factor; Female; Glycoproteins; Growth Substances; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Oligonucleotides, Antisense; Ovarian Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thionucleotides; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Adenocarcinoma, Follicular; Carcinoma; Cathepsin L; Cathepsins; Cell Differentiation; Cell Division; Colforsin; Cysteine Endopeptidases; Endopeptidases; Enzyme Induction; Enzyme Precursors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Insulin; Lysosomes; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Signal Transduction; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

Increased expression of fatty acid synthase (FAS) is observed in a clinically aggressive subset of various common cancers and interference with FAS offers promising opportunities for selective chemotherapeutic intervention. The mechanisms by which FAS expression is (up)-regulated in these tumors remain, however, largely unknown. Recently we demonstrated that in LNCaP prostate cancer cells FAS expression is markedly elevated by androgens via an indirect pathway involving sterol regulatory element-binding proteins (SREBPs). Here, we also show that growth factors such as EGF are able to stimulate FAS mRNA, protein and activity. Several observations also indicate that the effects of EGF on FAS expression are ultimately mediated by SREBPs. EGF stimulates SREBP-1c mRNA expression and induces an increase in mature nuclear SREBP-1. Moreover, in transient transfection studies EGF stimulates the transcriptional activity of a 178 bp FAS promoter fragment harboring a complex SREBP-binding site. Deletion or mutation of this binding site abolishes these effects and ectopic expression of dominant negative SREBP-1 inhibits FAS expression and induction in intact LNCaP cells. Given the frequent dysregulation of growth factor signaling in cancer and the key role of SREBP-1 in lipid homeostasis, growth factor-induced activation of the SREBP pathway is proposed as one of the mechanisms responsible for up-regulation of lipogenic gene expression in a subset of cancer cells.

Topics: Binding Sites; Carcinoma; CCAAT-Enhancer-Binding Proteins; DNA-Binding Proteins; Epidermal Growth Factor; Fatty Acid Synthases; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Regulatory Sequences, Nucleic Acid; RNA, Messenger; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Transcription Factors; Tumor Cells, Cultured; Up-Regulation

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

There is evidence that vitamin D receptor (VDR)-mediated action of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) could limit colon cancer cell growth particularly when induced by activation of the epidermal growth factor receptor (EGFR). We therefore wanted to ascertain the relevance of this observation for human colon cancerogenesis. Utilizing in situ mRNA hybridization and immunocytochemical techniques, we analyzed cell-specific expression of VDR and EGFR in normal and malignant human colonic mucosa. In normal mucosa, VDR positivity is weak and observed only in a small number of luminal surface colonocytes. In contrast, EGFR expression at a relatively high level is also found in cells at the crypt base. The number of VDR-positive colonocytes increases remarkably during tumor progression. It reaches its maximum in low grade adenocarcinomas and returns to lower levels in highly malignant cancers. In both low- and high grade carcinomas, the great majority of tumor cells contain the EGFR message. The relative abundance of EGFR over VDR in normal mucosa and in high grade carcinomas would create a situation in which mitogenic effects from EGFR activation are only ineffectively counteracted by signaling from 1 alpha,25-(OH)2D3/VDR. In contrast, in well to moderately differentiated tumors, upregulation of VDR could retard further tumor progression.

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; Humans; Immunohistochemistry; In Situ Hybridization; Intestinal Mucosa; Receptors, Calcitriol; Reference Values; RNA, Messenger

This study investigated whether epidermal growth factor (EGF) administration was capable of modifying salivary gland carcinogenesis. Two groups of mice were given 1 mg of 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland, and then Group 1 mice received 2 microg of EGF and Group 2 mice received vehicle subcutaneously for 8 weeks. Mice in two other groups, 3 and 4, received either EGF or vehicle alone. Twelve weeks after the start of the experiment, the incidences of submandibular gland carcinomas in Groups 1 and 2 were 39% and 58%, respectively, although this difference was not statistically significant. Duct- and cyst-like structures and carcinomas in the left submandibular glands were weakly stained by anti-EGF receptor (EGFR) antibody. Immunoblot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed the expression of EGFR in the submandibular glands and carcinomas. However, EGFR was undetectable in YT cells that were derived from a submandibular gland undifferentiated carcinoma of a Group 2 mouse. These findings indicate that EGF does not promote tumor induction in mouse salivary gland carcinogenesis. This may be ascribed in part to the low expression level of EGFR in tumor cells.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Immunoblotting; Incidence; Injections; Injections, Subcutaneous; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Mice, Nude; Neoplasm Transplantation; Pharmaceutical Vehicles; Polymerase Chain Reaction; Salivary Ducts; Submandibular Gland; Submandibular Gland Neoplasms; 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

Epithelial ovarian cancer (EOC) has a high mortality rate, which is due primarily to the fact that early clinical symptoms are vague and nonspecific; hence, this disease often goes undetected and untreated until in its advanced stages. Sensitive and reliable methods for detecting earlier stages of EOC are, therefore, urgently needed. Epidermal growth factor (EGF) is a ligand for EGF receptor (ErbB1); this receptor is the product of the c-erbB1 proto-oncogene. ErbB1 overexpression is common in human ovarian carcinoma-derived cell lines and tumors, in which overexpression is thought to play a critical role in tumor etiology and progression. Furthermore, ErbB1 overexpression is associated with disease recurrence and decreased patient survival. Recently, we have developed an acridinium-linked immunosorbent assay that detects a approximately 110-kDa soluble analogue of ErbB1, ie., sErbB1, in serum samples from healthy men and women (A. T. Baron, et al., J. Immunol. Methods, 219: 23-43, 1998). Here, we demonstrate that serum p110 sErbB1 levels are significantly lower in EOC patients with stage III or IV disease prior to (P < 0.0001) and shortly after (P < 0.0001) cytoreductive staging laparotomy than in healthy women of similar ages, whereas EGF levels are significantly higher than those of age-matched healthy women only in serum samples collected shortly after tumor debulking surgery (P < 0.0001). We observe that the preoperative serum sErbB1 concentration range of advanced stage EOC patients barely overlaps with the serum sErbB1 concentration range of healthy women. In addition, we show that serum sErbB1 and EGF levels changed temporally for some EOC patients who were surgically debulked of tumor and who provided a second serum sample during the course of combination chemotherapy. Finally, we observe a significant positive association between sErbB1 and EGF levels only in serum samples of EOC patients collected prior to cytoreductive surgery (correlation coefficient = 0.61968; P = 0.0027). These data suggest that epithelial ovarian tumors concomitantly affect serum sErbB1 and EGF levels. In conclusion, these data indicate that serum sErbB1 and EGF (postoperative only) levels are significantly different between EOC patients and healthy women and that altered and/or changing serum sErbB1 and EGF levels may provide important diagnostic and/or prognostic information useful for the management of patients with EOC.

Topics: Acridines; Adult; Aged; Biomarkers, Tumor; Carcinoma; Case-Control Studies; Chemotherapy, Adjuvant; Disease Progression; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Immunosorbent Techniques; Laparotomy; Male; Middle Aged; Neoplasm Recurrence, Local; Neoplasm Staging; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Prognosis; Proto-Oncogene Mas; Reproducibility of Results; Sensitivity and Specificity; Survival Rate; Tumor Cells, Cultured

Several members of the epidermal growth factor (EGF) family of growth factors that contain EGF-like units at their carboxy portion have been isolated and characterized. Schwannoma-derived growth factor (SDGF) and amphiregulin (AR) are members of this family. SDGF has high sequence homology to AR, and is known to be not only a potent mitogen for astrocytes and fibroblasts but also a neurotrophic factor. We previously confirmed that the synthetic EGF-like peptides SDGF(38-80) and AR(44-84), corresponding to the EGF-like domain of mouse SDGF and human AR, respectively, formed similar disulfide bond patterns to that of EGF. In the present study, we further investigated the biological actions of these two EGF-like peptides on several cultured cell lines. We found that SDGF(38-80) and AR(44-84) have weak mitogenic activity in NIH/3T3 cells and weak binding affinity to the EGF receptor on the surface of A431 cells compared with EGF. However, SDGF(38-80) and EGF induced short neurite outgrowth in PC12 h cells, a subclone of PC12 cells, at 100 nM. In addition, a significant increase in acetylcholinesterase (AChE) activity induced by SDGF(38-80) was observed at a concentration similar range to that of EGF, which is known as a differentiation marker of these cells. The effect of AR(44-84) in PC12 h cells was weaker than those of SDGF(38-80) and EGF, but the AChE activity was significantly increased by the addition of 100 nM AR(44-84), which did not stimulate NIH/3T3 cell growth. These results also suggest that SDGF(38-80) and AR(44-84) may be effective for neuronal differentiation rather than proliferation.

Topics: 3T3 Cells; Acetylcholinesterase; Amphiregulin; Animals; Antineoplastic Agents; Carcinoma; Cell Differentiation; Cell Division; EGF Family of Proteins; Epidermal Growth Factor; ErbB Receptors; Glycoproteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Mice; PC12 Cells; Peptide Fragments; Peptides; Rats; Tumor Cells, Cultured

A model using chemically permeabilized cells was developed to examine mechanisms that regulate protein tyrosine phosphorylation in osteoblastic cells. Using either permeabilized UMR106 osteoblastic or A431 (reference) cells, epidermal growth factor (EGF)-induced cellular tyrosine phosphorylation, and whether there are previously unrecognized interactions between this transduction pathway and Ca2+- or G protein-dependent signalling pathways, were investigated. Both permeabilized cell types, when maintained in non-supplemented cytoplasmic substitution solution (basic CSS), responded to EGF (1-100 ng/ml) with dose-dependent increases in tyrosine phosphorylation. A complex and time-dependent pattern of phosphotyrosine-containing proteins resulted, but the profile of tyrosine phosphorylated proteins was appreciably less complex than in intact cells. Supplementation of basic CSS with MgATP restored the normal complexity of the profiles for EGF-induced tyrosine phosphorylation proteins in both permeabilized cell lines and produced a more sustained accumulation of phosphoprotein products in A431 cells. Adding Ca2+ (< or = 10(-6) M), with or without exogenous MgATP, dose-dependently attenuated EGF-induced tyrosine phosphorylation of EGF receptors (EGFR) and other substrates in UMR106 cells, but was less effective in A431 cells. In both cell types, genistein, an inhibitor of tyrosine kinases, was more effective in attenuating EGF-induced receptor tyrosine phosphorylation in permeabilized cells. Similarly, orthovanadate, an inhibitor of protein tyrosine phosphatases, stimulated the accumulation of phosphoprotein products more effectively in permeabilized cells. Thus, the permeabilization preserves many features of intact cells while facilitating manipulation of intracellular conditions. NaF reproducibly produced a significant vanadate-like action in permeabilized cells that was somewhat stronger than its effect on intact cells. In contrast, the well-known inhibition of tyrosine phosphorylation by phorbol 12-myristate 13-acetate (PMA) was less effective in permeabilized cells than in intact cells; these actions of PMA were Ca2+-dependent. In addition, guanylyl-imidodiphosphate (Gpp(NH)p) attenuated tyrosine phosphorylation in UMR106 cells, and this effect was specifically blocked by guanosine 5'-O-(2-thiodiphosphate) (GDPbetas). These results strongly suggest that there is crosstalk between EGFR-activated tyrosine phosphorylation/dephosphorylation pathways and both C

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Signaling; Carcinoma; Cell Membrane Permeability; Culture Media; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Genistein; GTP-Binding Proteins; Humans; Osteoblasts; Osteosarcoma; Phosphoproteins; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Signal Transduction; Time Factors; Tumor Cells, Cultured; Tyrosine; Vanadates

The erbB-2/HER2 oncogene is overexpressed in a significant fraction of human carcinomas of the breast, ovary, and lung in a manner that correlates with poor prognosis. Although the encoded protein resembles several receptors for growth factors, no high affinity ligand of ErbB-2 has so far been fully characterized. However, several lines of evidence have raised the possibility that ErbB-2 can augment signal transduction initiated by binding of certain growth factors to their direct receptors. Here, we contrasted these two models of ErbB-2 function: First, examination of a large series of epidermal growth factor (EGF)-like ligands and neuregulins, including virus-encoded ligands as well as related motifs derived from the precursor of EGF, failed to detect interactions with ErbB-2 when this protein was singly expressed. Second, by using antibodies that block inter-ErbB interactions and cells devoid of surface ErbB-2, we learned that signaling by all ligands examined, except those derived from the precursor of EGF, was enhanced by the oncoprotein. These results imply that ErbB-2 evolved as a shared receptor subunit of all ErbB-specific growth factors. Thus, oncogenicity of ErbB-2 in human epithelia may not rely on the existence of a specific ligand but rather on its ability to act as a coreceptor for multiple stroma-derived growth factors.

Topics: Breast Neoplasms; Carcinoma; Epidermal Growth Factor; Female; Genes, erbB-2; Glycoproteins; Humans; Ligands; Lung Neoplasms; Nerve Growth Factors; Neuregulins; Ovarian Neoplasms; Receptor, ErbB-2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Stromal Cells; Tumor Cells, Cultured

The mechanism(s) whereby nonsteroidal anti-inflammatory drugs (NSAIDs) attenuate colorectal tumor growth remains poorly understood. This study determined if NSAIDs decreased epidermal growth factor (EGF)-induced proliferation in human colonic tumor (Caco-2) cells and whether this process involved the inhibition of prostaglandin (PG) synthesis.. Caco-2 cells were serum-starved (48 h) and subsequently treated (48 h) with either serum-free media or EGF (10 ng/ml) +/- physiologic and noninjurious (as determined by LDH release) concentrations of aspirin, indomethacin, and ibuprofen. PG synthesis was measured by EIA. Proliferation was quantitated with two assays: cellular protein and nucleic acid content.. NSAID treatment did not inhibit growth in cells treated with only serum-free media. Cells exposed to EGF demonstrated a significant increase in PGE2, protein, and nucleic acid. Levels of other eicosanoids (PGI2, TXA2) were minimal both before and after EGF treatment. Despite varying degrees of PGE2 inhibition, each NSAID group equally attenuated EGF-induced protein and nucleic acid synthesis. The correlation between PGE2 levels and protein (R2 = 0.56) or nucleic acid (R2 = 0.54) was poor. Finally, the addition of a physiologically appropriate concentration of exogenous PGE2 failed to reverse NSAID-induced growth inhibition.. These data suggest that NSAIDs, independent of PG synthesis inhibition, attenuate EGF-induced proliferation in Caco-2 cells. This may provide one explanation for how NSAIDs limit colonic neoplasia.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Biological Transport; Caco-2 Cells; Calcium; Carcinoma; Cell Division; Colonic Neoplasms; Eicosanoids; Epidermal Growth Factor; Humans; Prostaglandin Antagonists

An imbalance between the activity of matrix metalloproteinases (MMPs) (proteolytic enzymes that degrade protein components of the extracellular matrix) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), may be one of the mechanisms responsible for tumor cell invasion. We have investigated the regulation of MMP-1 and TIMP-1 gene expression in benign and malignant (follicular, anaplastic, and papillary) human thyroid cells. As expected of cells with invasive potential, detectable MMP-1 messenger RNA (mRNA) levels were observed in malignant cells under basal conditions, in contrast to undetectable levels in benign cells. Exposure of these cells, for 1 h, to the active phorbol ester, phorbol 12-myristate 13-acetate (TPA, 100 nmol/L), acting via protein kinase C (PKC), elicited an increase in MMP-1 mRNA, with a peak stimulation after a 3- to 4-h culture period. Epidermal growth factor (EGF, 25 ng/mL), however, acting via protein tyrosine kinase (PTK), stimulated such gene expression in malignant cells but failed to do so in benign cells. TIMP-1 mRNA was not significantly altered by the TPA-PKC, EGF-PTK, or TSH-protein kinase A (PKA) pathways in malignant cells. In benign cells, however, TPA induced a small, though significant, increase in TIMP-1. The MMP-1 stimulation by EGF and lack of TPA-induced rise in TIMP-1 in malignant cells, in sharp contrast to the effects obtained in benign thyrocytes, seems to indicate that the MMP: TIMP balance favors a more extensive extracellular matrix protein breakdown by malignant thyrocytes, as expected of cells exhibiting invasive capacity. TSH (10-500 microU/mL) failed to significantly influence basal MMP-1 or TIMP-1 mRNA levels, but it caused a dose-dependent inhibition in TPA- and EGF-induced MMP-1 mRNA in malignant cells, and TPA-stimulated MMP-1 and TIMP-1 in benign cells. The repressive action of TSH on MMP-1 mRNA was mimicked by forskolin and 8-bromo-cAMP and was abrogated by the PKA inhibitor, H-89, suggesting that the TSH inhibitory action is PKA-mediated. In conclusion, the present study provides novel data on MMP-1 and TIMP-1 gene expression and their modulation by the major signal transduction pathways operating in human thyroid cells. Similar and divergent patterns have emerged in the regulation of such gene expression in benign and malignant human thyrocytes, in many instances in accord with the concept of MMP playing the role of stimulating, and TIMP inhibiting, cell invasion. Although

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenocarcinoma, Follicular; Carcinoma; Carcinoma, Papillary; Cells, Cultured; Colforsin; Collagenases; Cyclic AMP-Dependent Protein Kinases; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Humans; Matrix Metalloproteinase 1; Protein Kinase C; Protein-Tyrosine Kinases; RNA, Messenger; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Thyrotropin; Tissue Inhibitor of Metalloproteinase-1

Interleukin (IL)-8 is a multifunctional cytokine that can stimulate the division of endothelial cells. We examined the expression of IL-8 mRNA using Northern blot analysis and in situ mRNA hybridization (ISH) and protein production using enzyme-linked immunosorbent assay and immunohistochemistry in 8 human gastric carcinoma cell lines and 39 gastric carcinomas and corresponding normal mucosa (34 surgical specimens and 5 biopsy specimens). Of the 8 human gastric carcinoma cell lines, 6 expressed 1.8-kb IL-8 mRNA and secreted various levels of IL-8 protein. The expression of IL-8 by TMK-1 cells was induced by exposure to IL-1 alpha, epidermal growth factor, and transforming growth factor-alpha, shown previously to be autocrine growth stimulators for human gastric carcinoma cells. In tumor tissues, most of the tumors (28 of 34 surgical specimens and 4 of 5 biopsy specimens) expressed IL-8 at higher levels than the corresponding normal mucosa. ISH and immunohistochemical analyses revealed that IL-8 mRNA and protein were localized in the cytoplasm of tumor cells. The number of blood vessels in the gastric carcinomas was determined by using antibodies against CD34. The level of IL-8 mRNA in the neoplasms strongly correlated with vascularization (Spearman correlation, r = 0.812; P = 0.001). The data suggest that IL-8 produced by tumor cells may regulate neovascularization and, hence, the growth and spread of human gastric carcinoma.

Topics: Adult; Aged; Blood Vessels; Carcinoma; Cell Division; Epidermal Growth Factor; Female; Humans; Interleukin-1; Interleukin-8; Male; Middle Aged; RNA, Messenger; Stomach Neoplasms; Tissue Distribution; Transforming Growth Factor alpha; Tumor Cells, Cultured

The role of the epidermal-growth-factor receptor (EGFR) in cervical cancer is controversial, due to technical difficulties in localizing or in quantifying EGFR by homogenate assays or immunohistochemistry. Our autoradiographic approach, in combination with morphometry, allowed cell-type-specific quantification of EGFR, leading to the following observations: (i) In normal cervical epithelium, EGFR levels per cell were high in non-dividing squamous cells of the upper layers of normal epithelium, where a mitogenic function of these EGFRs can be excluded. (ii) In contrast to earlier findings in tissue homogenates, but consistent with our observation in normal cervical epithelium that cells of the proliferating strata (basal and parabasal cells) express intermediate and comparatively reduced levels of EGFR per cell, cervical cancers displayed a significant reduction both of specific EGF binding and of EGFR levels per cell as compared with normal epithelium. (iii) A significant negative correlation of cell density and EGFR number per cell was obtained. In normal cervical epithelium, cervical intra-epithelial neoplasia and invasive cervical cancer (p = 0.002). This negative correlation was most evident in normal epithelium, where large changes of cell density occur within one slide (p < 0.001). (iv) Specific EGF-binding was also significantly reduced in endometrial cancers when compared with normal endometrium. It is proposed that in uterine tissues low or intermediate levels of EGFR do not exclude their function as mediators of cell proliferation.

Topics: Autoradiography; Carcinoma; Cervix Uteri; Down-Regulation; Endometrium; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Humans; Radioligand Assay; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms; Uterine Neoplasms

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is an activating ligand for the EGF receptor (HER1/ErbB1) and the high-affinity receptor for diphtheria toxin (DT) in its transmembrane form (proHB-EGF). HB-EGF was immunolocalized within human benign and malignant prostatic tissues, using monospecific antibodies directed against the mature protein and against the cytoplasmic domain of proHB-EGF. Prostate carcinoma cells, normal glandular epithelial cells, undifferentiated fibroblasts, and inflammatory cells were not decorated by the anti-HB-EGF antibodies; however, interstitial and vascular smooth muscle cells were highly reactive, indicating that the smooth muscle compartments are the major sites of synthesis and localization of HB-EGF within the prostate. In marked contrast to prostatic epithelium, proHB-EGF was immunolocalized to seminal vesicle epithelium, indicating differential regulation of HB-EGF synthesis within various epithelia of the reproductive tract. HB-EGF was not overexpressed in this series of cancer tissues, in comparison to the benign tissues. In experiments with LNCaP human prostate carcinoma cells, HB-EGF was similar in potency to epidermal growth factor (EGF) in stimulating cell growth. Exogenous HB-EGF and EGF each activated HER1 and HER3 receptor tyrosine kinases and induced tyrosine phosphorylation of cellular proteins to a similar extent. LNCaP cells expressed detectable but low levels of HB-EGF mRNA; however, proHB-EGF was detected at the cell surface indirectly by demonstration of specific sensitivity to DT. HB-EGF is the first HER1 ligand to be identified predominantly as a smooth muscle cell product in the human prostate. Further, the observation that HB-EGF is similar to EGF in mitogenic potency for human prostate carcinoma cells suggests that it may be one of the hypothesized stromal mediators of prostate cancer growth.

Topics: Carcinoma; Cell Division; Epidermal Growth Factor; Gene Expression; Heparin-binding EGF-like Growth Factor; Humans; Intercellular Signaling Peptides and Proteins; Male; Mitogens; Muscle, Smooth, Vascular; Prostate; Protein Precursors; Tumor Cells, Cultured

The molecular mechanisms by which ovarian hormones stimulate growth of breast tumors are unclear. It has been reported previously that estrogens activate the signal-transducing Src/p21(ras)/Erk pathway in human breast cancer cells via an interaction of estrogen receptor (ER) with c-Src. We now show that progestins stimulate human breast cancer T47D cell proliferation and induce a similar rapid and transient activation of the pathway which, surprisingly, is blocked not only by anti-progestins but also by anti-estrogens. In Cos-7 cells transfected with the B isoform of progesterone receptor (PRB), progestin activation of the MAP kinase pathway depends on co-transfection of ER. A transcriptionally inactive PRB mutant also activates the signaling pathway, demonstrating that this activity is independent of transcriptional effects. PRB does not interact with c-Src but associates via the N-terminal 168 amino acids with ER. This association is required for the signaling pathway activation by progestins. We propose that ER transmits to the Src/p21(ras)/Erk pathway signals received from the agonist-activated PRB. These findings reveal a hitherto unrecognized cross-talk between ovarian hormones which could be crucial for their growth-promoting effects on cancer cells.

Topics: Animals; Breast Neoplasms; Carcinoma; COS Cells; CSK Tyrosine-Protein Kinase; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; Estradiol; Gene Expression Regulation; Guanosine Triphosphate; Hormone Antagonists; Humans; Mitogen-Activated Protein Kinase 1; Point Mutation; Promegestone; Protein-Tyrosine Kinases; Proto-Oncogene Proteins p21(ras); Receptors, Estrogen; Receptors, Progesterone; Signal Transduction; src-Family Kinases; Tamoxifen; Transcriptional Activation; Tumor Cells, Cultured

We report here the first direct observation of chemotaxis to EGF by rat mammary carcinoma cells. When exposed to a gradient of EGF diffusing from a micropipette, MTLn3 cells displayed typical ameboid chemotaxis, extending a lamellipod-like protrusion and moving toward the pipette. Using a homogeneous upshift in EGF to model stimulated lamellipod extension (J. E. Segall et al., 1996, Clin. Exp. Metastasis 14, 61-72), we analyzed the relationship between adhesion and chemoattractant-stimulated protrusion. Exposure to EGF led to a rapid remodeling of the adhesive contacts on adherent cells, in synchrony with extension of a flat lamellipod over the substratum. EGF-stimulated lamellipods still extended in the presence of adhesion-blocking peptides or over nonadhesive surfaces. They were, however, slightly shorter and retracted rapidly under those conditions. The major protrusive structure observed on well-spread, adherent cells, after EGF stimulation was a flat broad lamellipod, whether or not in contact with the substratum, while cells in suspension showed transient protrusive activity over the entire cell surface. We conclude that the initial adhesive status of the cell conditions the shape of the outcoming protrusion. Altogether our results suggest that, although adhesive contacts are not necessary for lamellipod extension, they play a role in stabilizing the protrusion as well as in the control of its final shape and amplitude.

Topics: Animals; Carcinoma; Cell Adhesion; Cell Size; Chemotactic Factors; Chemotaxis; Epidermal Growth Factor; Mammary Neoplasms, Animal; Neoplasm Metastasis; Rats; Tumor Cells, Cultured

Many oncogenes and tumor suppressor genes are involved in multistep carcinogenesis. Cripto is an epidermal growth factor (EGF) related gene which shares homology with EGF and TGFalpha. The aim of this study was to evaluate the role of abnormal p53 and cripto oncogene expression in endometrial carcinogenesis and progression using a hyperplasia carcinoma sequence model. Ninety-six primary endometrial adenocarcinomas and 30 hyperplastic tissues of which 7 were atypical (AH), were immunohistochemically examined for the presence of cripto and abnormal p53 protein. Immunopositivity was compared in hyperplastic and carcinoma tissues and analysed for conventional clinicopathological prognostic variables such as grade, depth of myometrial invasion, lymphovascular invasion, lymph node metastases and clinical stage. Cripto immunoreactivity was strong in most cases of AH, and endometrial carcinomas revealed 71% overall and 41% strong positivity, while hyperplasias without atypia were weakly stained. There was no correlation between cripto expression and clinicopathological prognosticators. Abnormal p53 was not observed in hyperplasias but AH and carcinomas expressed 14% and 25% overall positivity, respectively. There was a statistically significant correlation between the stage of the disease and abnormal p53 accumulation. Our results suggest that both cripto and p53 may play a role in endometrial carcinogenesis while abnormal p53 expression is an important parameter for disease progression.

Topics: Carcinoma; Disease Progression; Endometrial Hyperplasia; Endometrial Neoplasms; Epidermal Growth Factor; Female; GPI-Linked Proteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Tumor Suppressor Protein p53

This study seeks to define the role of pretreatment of evaluation of tumour growth fraction in cervical cancer and its relationship to the clinical course of the disease. In addition, it also seeks to explain whether cell kinetics and growth factor expression have an association with tumour response to radiotherapy and hence could be of value in the management of patients. All pre-treatment biopsies were analysed for the tumour-proliferative compartment by evaluation of Ki67 antigen expression and argyrophilic nucleolar organiser region (AgNOR) counts. Growth factor analysis was done by analysing for expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGF-R) and transforming growth factors alpha and beta (TGFalpha, TGFbeta). A total of 152 patients were evaluated and a correlation obtained between pre-treatment status of the tumour-growth-fraction-associated markers and clinical outcome following radiotherapy. Such patients were either disease-free (group 1, n=106) or with residual/recurrent disease (group 2, n=46) at a 16-month follow-up. Pre-treatment analysis of AgNOR significantly correlated to disease status after treatment (r=-0.517, P=0.0000). This may be due to an effect of cell proliferation. Lower AgNOR counts were significantly associated with recurrent/residual tumours, suggesting that increased proliferative activity may be a positive prognostic indicator. Similar results were also obtained for the other proliferation-associated marker Ki67 (r=-0.443, P=0.0000). Expression of EGF and EGF-R also showed significant pre-treatment correlations with the final disease outcome (r=0.248, P=0.031 and r=0.503, P=0.0000 respectively). Both these markers were expressed more by patients belonging to group 2. The opposite was the case for TGFalpha, where patients belonging to group 1 showed higher values (r=0.417, P=0.0001). The other growth factor investigated, TGFbeta, also showed a conspicuous differential expression in the two groups of patients (r=-0.604, P=0.0000). Group 1 patients showed mostly mild to moderate expression while most group 2 patients were negative for the growth factor. It therefore appears that tumours with high AgNOR counts and Ki67 index, along with expression of the two types of transforming growth factor (alpha and beta), responded better to radiotherapy.

Topics: Biomarkers, Tumor; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Prognosis; Transforming Growth Factor alpha; Transforming Growth Factor beta; Uterine Cervical Neoplasms

We generally choose transhiatal esophagectomy (THE) for patients with high risk for postoperative complications and for carcinoma of the lower thoracic esophagus, even if the tumor is in the advanced stage. In order to define indications for THE in esophageal cancer patients, we investigated 40 THE cancer patients according to the expressions of EGF/EGFR, p53 and p21. In patients with stage I, II, III and IV tumors, 5-year survival rates were 66.7%, 28.6%, 30.0% and 11.4%, respectively. The sites of first recurrence were the lymph nodes (n = 10) and single organs (n = 10). Dissemination (n = 3) and local recurrence (n = 2) were also seen as a first recurrence. According to EGF/EGFR, 5-year survival rate was 69% and 14% in the low and high EGF/EGFR groups, respectively. According to p53 expression, 5-year survival was 60% and 30% in the negative and positive groups, respectively; according to p21 expression, 5-year survival was 71% and 0% in the negative and positive groups, respectively. Significant difference was seen in EGF/EGFR and p21 groups. These data support less invasive surgery for some patients even for esophageal cancer patients. THE is a less invasive surgery, that also implies fewer curative procedure. Our results also showed that THE alone will be the only curative procedure necessary for some patients. We can determine therapeutic procedures using these new factors, and thus avoid unnecessary excess surgical stress in esophageal cancer patients.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Esophagectomy; Female; Humans; Lymphatic Metastasis; Male; Middle Aged; Oncogene Protein p21(ras); Prognosis; Survival Rate; Tumor Suppressor Protein p53

Previously, we have shown that IGF-1, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and aurintricarboxylic acid (ATA) protected MCF-7 cells against death induced by the protein synthesis inhibitor cycloheximide (CHX). We proposed that phosphorylation of a putative cellular proteins(s) may be involved in this survival mechanism. In the present study we investigated the ability of several agents to induce phosphorylation of cellular proteins and correlated this ability to their survival effect. We found that TPA, ATA, and IGF-1 increased the degree of phosphorylation of a 27-kDa protein in a dose- and time-dependent manner in CHX-treated MCF-7 cells. The ED50 values observed were 25 ng/ml, 40 micrograms/ml and 15 ng/ml for TPA, ATA, and IGF-1, respectively. The effect was measured upon 10 min of cell treatment with each agent; it reached maximum at 60 min and thereafter decreased continuously to control levels. The 27-kDa protein was found in the cytosolic fraction as a phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphorylated serine residue. Further characterization with two-dimensional electrophoresis indicated that the 27-kDa phosphoprotein was resolved into two isoforms with pI 5.7 and 5.9. Such characteristics were observed for the small molecular weight heat shock protein HSP27. Indeed, a single band of 27 kDa was detected immunologically with rabbit polyclonal anti-human HSP27. The inactive phorbol ester alpha TPA, epidermal growth factor (EGF), and 8-bromoadenosine 3'5'-cyclic monophosphate (Br-cAMP) did not increase phosphorylation of the 27-kDa protein. Cell survival was measured by exposure of the CHX-pretreated cells to increasing concentrations of the various agents for 60 min, followed by a further incubation for 48 h in the presence of CHX only. TPA, ATA, and IGF-1 were found to enhance cell survival, whereas alpha-TPA, EGF and Br-cAMP did not. Our results indicate a correlation between phosphorylation of a 27-kDa protein, probably HSP27, and enhanced cell survival, suggesting a role for this phosphoprotein in the survival mechanism.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Aurintricarboxylic Acid; Breast Neoplasms; Carcinoma; Cell Survival; Cycloheximide; Cytosol; Epidermal Growth Factor; Heat-Shock Proteins; Humans; Insulin-Like Growth Factor I; Isoelectric Point; Mitogens; Molecular Weight; Phosphoproteins; Phosphorylation; Phosphoserine; Protein Synthesis Inhibitors; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured

The expression of c-myc, c-erbB-1 and c-erbB-2 in 24 cases of urothelial carcinoma by Southern and northern blot analysis, and immunohistochemistry was examined. The results were compared with the pathological grade and stage. We found elevated mRNA expressions of c-myc and c-erbB-1 in 19 and 11 of 21 cases, respectively, but there was no apparent amplification or rearrangement of these oncogenes in any of the cases examined. By immunohistochemistry using anti-epidermal growth factor receptor antibody, most of the cases showed positive immunoreactivity on the cancer cell membranes, and cancers of higher pathological grade and stage showed more intense staining. By contrast, amplification of c-erbB-2 was detected in four of 24 cases, all of which were assigned to a high pathological grade (G3). Elevated c-erbB-2 mRNA levels appeared to correlate with the pathological grade of the cancers. Positive immunohistochemical reactions to c-erbB-2 were found in the cancer cell membranes in three of 24 cases, which were accompanied by amplification and elevated mRNA levels of c-erbB-2. In conclusion, expressions of c-myc, c-erbB-1 and c-erbB-2 were all elevated in the majority of urothelial carcinomas, but the amplification was not universal.

Topics: Blotting, Northern; Blotting, Southern; Carcinoma; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Mucous Membrane; Proto-Oncogene Proteins c-myc; Receptor, ErbB-2; RNA, Messenger; Urologic Neoplasms

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

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

A major problem in the management of bladder cancer is the high risk for recurrence of bladder tumors after transurethral resection. This has generally been attributed to the attachment and subsequent expansion of exfoliated tumor cells to the traumatized bladder wall. An in vitro cocultivation model was used to study the implantation and growth of human tumor cells in traumatized murine urothelium. Furthermore, we investigated in a time-course experiment whether stimulation of the regenerative activity of the normal urothelium by a growth factor could affect implantation and subsequent growth of bladder tumor cells. After inoculation on injured confluent cultures of murine urothelium, human T24 and SD bladder carcinoma cells preferentially attached to the denuded areas. SD cells expanded into the normal urothelium as a sharply demarcated tumor, while T24 cells infiltrated as single cells. Treatment of the primary urothelium with epidermal growth factor (EGF) stimulated the proliferation of the primary urothelium and reduced the implantation and growth of T24 considerably. EGF reduced the implantation of the SD tumor cells but could not prevent the further expansion at the expense of surrounding normal urothelium. Since EGF had no effect on migration or proliferation of SD or T24 cells, its modulation of expansive growth is most probably due to an increase in the regeneration of normal urothelium. This study suggests that recurrence of transitional cell carcinomas might in some instances be inhibited by stimulation of the regeneration of traumatized urothelium. The reported in vitro cocultivation model may be useful for studying additional factors involved in intraepithelial expansion of carcinoma cells.

Topics: Animals; Carcinoma; Cell Division; Cell Movement; Coculture Techniques; Epidermal Growth Factor; Female; Humans; Mice; Mice, Inbred C3H; Neoplasm Invasiveness; Regeneration; Tumor Cells, Cultured; Urinary Bladder; Urinary Bladder Neoplasms; Urothelium

Epidemiological studies have consistently shown an association between infection of Helicobacter pylori (Hp) and duodenal ulcer (DU) and gastric cancer. The mechanism of the ulcerogenic effect of Hp has been related to excessive gastrin release, gastric acid hypersecretion and gastric metaplasia in duodenum. The implication of Hp in gastric carcinogenesis has not been explained. In this study, mucosal expression of EGF and TGF alpha and luminal release of EGF as well as basal and pentagastrin-stimulated acid secretion and plasma gastrin levels have been determined in healthy subjects, gastric carcinoma and DU patients. It was found that Hp positive DU patients show excessive gastrin release and gastric acid secretion combined with increased expression and luminal release of EGF and TGF alpha. These changes returned to normal values two years after the eradication of Hp. Gastric cancer patients also showed increased expression of EGF and TGF alpha and highly increased plasma gastrin but their gastric acid secretion was markedly reduced possibly due to atrophy of oxyntic mucosa. This study indicates that overexpression of growth factors in gastric mucosa may be implicated in the pathogenesis of both duodenal ulcer and gastric cancer and that Hp positive hypochlorhydric and hypergastrinemic patients may be predisposed to development of gastric cancer.

Topics: Adult; Aged; Carcinoma; Duodenal Ulcer; Epidermal Growth Factor; Gastric Acid; Gastric Juice; Gastric Mucosa; Helicobacter Infections; Helicobacter pylori; Humans; Immunochemistry; Middle Aged; Pentagastrin; Stomach Neoplasms; Transforming Growth Factor alpha

Receptors for luteinizing hormone-releasing hormone (LH-RH) are found in nearly 80% of human ovarian cancers. The chemotherapeutic agent doxorubicin can be linked to [D-lysine6]LH-RH to form a cytotoxic analogue (AN-152) that may have greater specificity for tumor cells. This study was conducted to investigate the effects of AN-152 on the growth of LH-RH receptor-positive OV-1063 human epithelial ovarian cancers.. Nude mice bearing human ovarian tumors, OV-1063 or UCI-107 (LH-RH receptor negative), were injected intraperitoneally with saline (control) or with equimolar doses of AN-152 or doxorubicin; experiments involving mice with OV-1063 tumors also included groups that were administered [D-lysine6]LH-RH either alone or in combination with doxorubicin. Tumor volume, weight, doubling time, and burden (i.e., tumor weight/body weight) as well as tumor apoptotic and mitotic indices were determined. The levels of receptors for LH-RH and epidermal growth factor (EGF) and their messenger RNAs were measured by use of radioreceptor and reverse transcription-polymerase chain reaction assays, respectively.. The growth of OV-1063 ovarian tumors in nude mice, as based on reduction in tumor volume, was inhibited significantly (all P<.05, two-sided) 4 weeks after treatment with AN-152, even at the lowest dose tested (413 nmol/20 g weight); the toxic effects of an equivalent dose of doxorubicin caused substantial mortality. High-affinity receptors for LH-RH and EGF were found on cell membranes of OV-1063 cancers; however, after in vivo treatment with AN-152, LH-RH receptor-binding sites were not detectable and EGF receptors were reduced in number. The growth of UCI-107 ovarian cancers was not inhibited by AN-152.. In nude mice bearing LH-RH receptor positive OV-1063 epithelial ovarian cancers, systemic administration of AN-152 is less toxic and inhibits tumor growth better than equimolar doses of doxorubicin.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Southern; Carcinoma; Doxorubicin; Epidermal Growth Factor; ErbB Receptors; Female; Gonadotropin-Releasing Hormone; Humans; Luteinizing Hormone; Mice; Mice, Nude; Mitotic Index; Ovarian Neoplasms; Polymerase Chain Reaction; Receptors, LHRH; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured

The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer.

Topics: Base Sequence; Carcinoma; DNA Primers; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Molecular Sequence Data; Multivariate Analysis; Ovarian Neoplasms; Prognosis; RNA, Messenger; RNA, Neoplasm; Survival Analysis; Teratoma; Tumor Necrosis Factor-alpha

Hepatocyte growth factor (HGF) stimulated cell migration of human gastric carcinoma cell lines MKN1, MKN7, and MKN28. Epidermal growth factor (EGF) also stimulated the cell migration of these three cell lines. In MKN7 cells, HGF-stimulated cell migration was rather reduced in the presence of EGF, whereas such an observation was not made with MKN1 and MKN28 cells. Therefore, we compared the effect of EGF on HGF-stimulated HGF receptor phosphorylation in these cell lines. HGF induced a rapid tyrosine phosphorylation of the HGF receptor in all these cell lines. In MKN7 cells, the increased phosphorylation was further enhanced by EGF, although EGF alone did not affect tyrosine phosphorylation of the HGF receptor. In MKN1 and MKN28 cells, EGF did not influence tyrosine phosphorylation of the HGF receptor, whether HGF was present or not. The data presented here suggest that EGF negatively modulates the cellular response to HGF by increasing tyrosine phosphorylation of the HGF receptor in certain types of epithelial cells, e.g., MKN7 cells.

Topics: Carcinoma; Cell Movement; Epidermal Growth Factor; Hepatocyte Growth Factor; Humans; Phosphorylation; Proto-Oncogene Proteins c-met; Receptor Protein-Tyrosine Kinases; Stomach Neoplasms; Tumor Cells, Cultured; Tyrosine

Chemoattractants expressed at bony sites and pelvic lymph nodes are thought to promote the preferential metastasis of human prostate tumor cells to these organs. Epidermal growth factor (EGF) is a potent chemoattractant for several human metastatic prostate tumor cell lines, including the TSU-pr1 cell line, and EGF has been localized to the stroma of both bony sites and pelvic lymph nodes in humans. Hence, we investigated whether the TSU-pr1 cell line expresses a functional EGF receptor (EGFR), which when antagonized reduces EGF-mediated chemomigration of this cell line. In this context, the EGFR immunoprecipitated from cell lysates of TSU-pr1 cells comigrated with the EGFR from A431 cells at a molecular weight of 170 kD. Addition of human EGF (hEGF) to the TSU-pr1 cells for 5 min stimulated the dose-dependent biphasic phosphorylation of the EGFR, with maximal stimulation of EGFR phosphorylation occurring at 2 ng/ml hEGF. In addition, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with 0.5 microgram/ml anti-hEGF monoclonal antibody or 100 nM staurosporine inhibited EGFR phosphorylation. Conversely, as negative controls, treatment of hEGF-stimulated (2 ng/ml) TSU-pr1 cells with K252a or dimethyl sulfoxide (DMSO) vehicle did not inhibit EGFR phosphorylation. TSU-pr1 cells were stimulated to migration in 4 hr across Boyden chambers in response to 10 ng/ml hEGF. Treatment of the TSU-pr1 cells with anti-hEGFR monoclonal antibody inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. Similarly, treatment of the TSU-pr1 cells with staurosporine inhibited in a dose-dependent manner the chemomigration of the TSU-pr1 cells across Boyden chambers. These results demonstrate that antagonists of hEGF-mediated hEGFR phosphorylation also antagonize chemomigration of the TSU-pr1 cells across Boyden chambers, suggesting that antagonists of the EGFR in prostate cancer may be useful in the treatment of metastatic disease.

Topics: Alkaloids; Antibodies, Monoclonal; Carbazoles; Carcinogens; Carcinoma; Cell Movement; Dimethyl Sulfoxide; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Immunoblotting; Indole Alkaloids; Male; Neoplasm Metastasis; Phosphorylation; Prostatic Neoplasms; Protein Kinase C; Staurosporine; Tumor Cells, Cultured

In order to study the potential of non-invasive scintigraphic evaluation of the epidermal growth factor (EGF) receptor status in vivo, the biokinetics and tumor binding of 125I-EGF and anti-(EGF receptor) mAb 425 were investigated in nude mice bearing human tumor xenografts with different EGF-receptor densities as determined by a radioreceptor assay. The results demonstrated a tumor uptake for both substances depending on the receptor level. The EGF receptor status, however, was reflected slightly better by the binding of EGF to tumor tissue compared to the mAb. The rapid blood clearance of EGF with a plasma half-life of less than 1 min led to a tumor-to-blood ratio of approximately 3 within 6 h after injection in tumors with a high receptor expression. A similar ratio for the mAb was not obtained before day 6 after injection. The absolute concentration of EGF, however, was low compared to the mAb. Therefore, it can be concluded that the EGF receptor status as a target for (radio)immunotherapy can be evaluated in vivo with EGF labeled with a short-life positron-emitting radionuclide or with monoclonal antibodies to the EGF receptor or their fragments.

Topics: Animals; Antibodies, Monoclonal; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Tissue Distribution; Transplantation, Heterologous

Plasminogen activators (PAs) play a key role in malignant transformation. PA secretion by tumoral cells is strongly correlated with their aggressive phenotype. Regulation of invasive potential by growth factors has been also demonstrated. This study was designed to investigate the effects of 5alpha-dihydrotestosterone (DHT), epidermal growth factor (EGF), transforming growth factor beta1 (TGFbeta1), retinoic acid and basic fibroblastic growth factor (bFGF) on cell growth and PA expression and secretion in DU145 and PC3 cells, 2 human prostatic-cancer cell lines. The proliferation of 2 cell lines was significantly increased only by EGF (about 30%), but decreased by TGFbeta1 (40% inhibition). However, EGF-treated cells showed significant enhancement (about 400%) of u-PA secretion. A similar effect was observed when cells were cultured with DHT (200%) and with TGFbeta1 (300%). Nevertheless, u-PA mRNA level in EGF-, TGFbeta1 - or DHT-treated cells was amplified only between 110 and 180% of control, suggesting that growth factors differently controlled the steps of PA expression. Furthermore, our results clearly showed the divergent effect of TGFbeta1, i.e., an inhibition of prostatic-cell-line growth accompanied by an increase in proteolytic activity.

Topics: Bone Marrow; Brain Neoplasms; Carcinoma; Cell Division; Culture Media; Dihydrotestosterone; Enzyme Activation; Epidermal Growth Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Male; Neoplasm Metastasis; Neoplasm Proteins; Plasminogen Activator Inhibitor 1; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

The kidney is one of the major sites of EGF production and there it seems to play several biological functions, such as modulation of cell growth, renal repair following injury, regulation of cellular metabolism and glomerular haemodinamics. The present study was first aimed at localizing EGF and its receptor (R) in normal human kidney by immunohistochemical and in situ hybridization techniques. Then, the distribution of the growth factor and its R was explored in biopsy specimens from eight patients with acute tubulointerstitial damage. In the normal human kidney, both EGF immunoreactivity and EGF mRNA were localized in tubular profiles corresponding to Henle's loop and, although to a lesser intensity, to distal convoluted tubule. EGF immunostaining was remarkable mainly at the apical surface of tubular cells. EGF-R protein expression was detected in glomerular endothelial cells, in peritubular capillaries and arteriolar walls, as well as along the thick ascending limb of Henle's lop and distal convoluted tubule, where it colocalized with Tamm-Horsfall protein. Immunohistochemical analysis of tubular profiles revealed that EGF-R was located especially along the basolateral membrane of tubular cells and within the basal part of cytoplasm. Endogenous alkaline phosphatase and CHIP28 positive tubules did not show any signal for EGF and its receptor. Kidneys with acute tubulointerstitial injury exhibited a dramatic decrease of EGF expression, whereas EGF-R showed only minor modifications. Interestingly, EGF-R was localized to both apical and antiluminal membranes of positive tubular cells. It is concluded that EGF-EGF receptor loop may be relevant in the pathogenesis of acute tubulointerstitial damage and recovery from tubular injury, while its role in the physiological renewal of the urothelium remains speculative.

Topics: Biopsy; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; In Situ Hybridization; Kidney; Kidney Neoplasms; RNA, Messenger

Androgens stimulate the growth of prostatic carcinoma, possibly by modulating the activity of locally expressed growth factors. Recently, we have shown that an LHRH (or LHRH-like) system exerting an inhibitory action on cell proliferation is present in the human androgen-dependent prostatic tumor cell lines LNCaP. The following experiments have been performed in LNCaP cells to clarify whether LHRH might inhibit cell proliferation by interfering with the two major mitogenic factors for these cells: (a) testosterone (T), the major exogenous stimulating factor, and (b) epidermal growth factor (EGF), one of the locally produced growth factors. (a) It has been shown that an LHRH agonist (LHRH-A, Zoladex) counteracts the proliferative action of T in a dose-dependent way. To clarify whether LHRH might interfere with the activity of T in prostate tumors, LNCaP cells were treated with LHRH agonist over different time intervals, and the effects of treatment evaluated in terms of expression of androgen receptor mRNA. The data obtained indicate that LHRH-A does not affect androgen receptor expression at any time interval examined. (b) LHRH-A inhibits the mitogenic action of EGF on LNCaP cells and significantly reduces the concentration of EGF receptors in these cells. Experiments have been performed to explore whether LHRH-A might alter intracellular signaling mechanisms mediating the activity of EGF. In LNCaP cells LHRH-A blocks EGF-induced expression of the c-fos proto-oncogene but does not modify EGF-induced tyrosine phosphorylation of the EGF receptor. These data suggest that, in androgen-dependent prostate tumors, LHRH might inhibit cell proliferation by interfering with some but not all of the mechanisms mediating the mitogenic action of EGF. Possible interactions between LHRH and T-activated events still remain to be elucidated.

Topics: Binding Sites; Carcinoma; Cell Division; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Humans; Male; Mitogens; Prostatic Neoplasms; Proto-Oncogene Mas; Testosterone; Tumor Cells, Cultured

The mouse lactoferrin gene responded to forskolin, 12-O-tetradecanoyl phorbol-13-acetate, and epidermal growth factor (EGF) stimulation via two adjacent enhancer elements, the cAMP response element (CRE) and EGF response element (EGFRE), collectively referred to as the mitogen response unit. In this report, we examined the minimal promoter and enhancer elements of the mouse lactoferrin gene that are required for EGF-induced transcriptional activation. We found that the CRE and noncanonical TATA box (ATAAA) are the minimal promoter elements for basal activity of the chloramphenicol acetyltransferase (CAT) reporter construct whereas the EGFRE is needed for an additional activity induced by EGF in transiently transfected human endometrial carcinoma RL95-2 cells (RL95-2). The EGFRE, however, did not function in heterologous promoters [SV 40 and thymidine kinase (TK)]. Therefore, EGF-stimulated lactoferrin gene activity is promoter specific in RL95-2 cells. In transiently transfected cells, EGF and forskolin showed synergistic effects on the CAT reporter that contained both response elements. Mutation made at either element or insertion of extra nucleotides between the two elements severely affected EGF-stimulated activity. Nuclear protein prepared from RL95-2 cells formed three complexes (A, B, and C) with the oligonucleotides containing both EGFRE and CRE in electrophoretic mobility shift assay. A new complex (E) was detected with the nuclear protein of EGF-treated cells. By oligonucleotide competition experiments, we demonstrated that the complex E was generated by protein bound to CRE. EGF-induced binding activity could be abolished by calf intestinal alkaline phosphatase but not by the protein synthesis inhibitor, cycloheximide. Therefore, binding of a preexisting phosphoprotein to the CRE region could be one of the requirements for EGF-induced mouse lactoferrin gene promoter activity.

Topics: Animals; Binding Sites; Carcinoma; Electrophoresis; Endometrial Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation; Humans; Lactoferrin; Mice; Nuclear Proteins; Promoter Regions, Genetic; Receptors, Cyclic AMP; Recombinant Proteins; TATA Box; Transfection; Tumor Cells, Cultured

Previous studies have shown increasing evidence that TGF-alpha, a ligand for the often overexpressed EGF-receptor may be important for the oncogenesis and autocrine stimulation of the proliferation in head and neck cancers. The occurrence of TGF-alpha and its relation to the EGF-receptor still remain unclear. Twenty six specimens (primaries and metastasis) of oropharyngeal squamous cell carcinomas were investigated for the presence of transforming growth factor alpha (TGF-alpha), epidermal growth factor (EGF) and the EGF-receptor using a tissue extraction method and a "sandwich" immuno absorbent assay. In 77% of the specimens we found TGF-alpha, all had a significant amount of EGF-receptors, no EGF was found. No significant difference was noted for metastasis and primaries. No correlation was seen to the TNM stage and to the histological grading. There was an inverse statistical correlation between the TGF-alpha and the EGF-receptor concentration. High TGF-alpha concentrations were associated with low EGF-receptor concentration. Interestingly, even high TGF-alpha concentrations showed a lower limit of EGF-receptor concentrations which could not be passed. The present investigation gives a quantitative determination of the EGF-receptors and TGF-alpha in oropharyngeal carcinomas. The results indicate that a EGF-receptor/TGF-alpha complex could be functionally important for autocrine/paracrine stimulation.

Topics: Carcinoma; Epidermal Growth Factor; Humans; Oropharyngeal Neoplasms; Oropharynx; Transforming Growth Factor alpha

In this study, we analyze effects of IFN-gamma on the proliferation of normal human mammary epithelial cells (MECs) and several mammary carcinoma cell lines. We demonstrate that IFN-gamma blocks the proliferation of MECs in a time- and concentration-dependent manner. This growth arrest is irreversible and occurs at a specific stage in the G1 phase of the cell cycle. IFN-gamma caused a rapid (within 12-24 h) down-regulation of cyclin A, c-myc, and cdc2 proteins, as well as a disappearance of hyperphosphorylated forms of the retinoblastoma family proteins, Rb and p130. The synthesis of several other growth control proteins, p53, p21/Waf1, and proliferating cell nuclear antigen, was down-regulated between 24 and 48 h. In MECs synchronized by epidermal growth factor deprivation and released for cell cycle traverse by re-addition of epidermal growth factor to the medium, IFN-gamma was able to block DNA synthesis only if added in the first 6 to 7 h after epidermal growth factor. The block in Rb phosphorylation and cyclin A expression was coordinately regulated during the same narrow window of G1. Several mammary carcinoma cell lines demonstrated resistance to the growth-inhibitory effects of IFN-gamma and did not exhibit down-regulation of cdc2 and cyclin A expression or a change in hyperphosphorylation of Rb when treated with IFN-gamma. Initial studies suggest, in some carcinoma cell lines, that resistance to IFN-gamma may be caused by defects in the IFN-gamma signal transduction pathway (measured by expression of the IFN-gamma-responsive gene GBP), while resistance in others may be due to defects in cell cycle regulatory proteins that are the targets of IFN-gamma action.

Topics: Breast; Breast Neoplasms; Carcinoma; Cell Cycle Proteins; Cell Division; Cell Line; DNA; Down-Regulation; Epidermal Growth Factor; Epithelial Cells; Epithelium; G1 Phase; Humans; Interferon-gamma; Phosphoproteins; Phosphorylation; Proliferating Cell Nuclear Antigen; Proteins; Proto-Oncogene Proteins c-myc; Retinoblastoma Protein; Retinoblastoma-Like Protein p130; Signal Transduction; Tumor Cells, Cultured

The proliferation of mammary carcinoma cells can be stimulated by estrogens and various growth factors such as EGF and IGF-I. Steroid hormones and growth factors are understood to exert their effects via different receptors and signal transduction pathways. Recently, it has been shown that growth factors can utilize the unliganded estrogen receptor (ER) as a transcription factor. This study was aimed at identifying the growth factors that can act via the estrogen receptor, and finding new estrogen antagonists that block this activity. Originally, a transcription assay was used in which HeLa cells had been transiently co-transfected with the expression vector for the human ER and a reporter plasmid EREwtc luc. EGF and, to a lesser extent, insulin stimulated the expression of the reporter gene in the absence of estradiol (E2), whereas IGF-I was inactive. The stimulatory effect of E2 and insulin was suppressed when the ER was blocked by the pure antiestrogen ICI 182,780. In ER-positive MCF-7 cells, transfected transiently with the reporter plasmid, EGF had no stimulatory effect on luciferase expression. IGF-I stimulated the transcription to about 50% of the E2 value. Similar activity was found for insulin. The effect of both growth factors was only partly reversed by the addition of a pure antiestrogen. The combination of E2 and IGF-I or insulin led to a synergistic activation of transcription. Because transiently transfected cells do not allow one to study the influence of chromatin structure on gene expression, an MCF-7 subline (MCF-7/2a) was established, in which the reporter construct had been integrated in the genome. IGF-I stimulated luciferase expression in these cells, but showed no overadditive effect with E2. The effects of both agents were completely suppressed by the pure antiestrogen ICI 182,780. These data suggest the existence of an ER-independent mechanism for the activation of the reporter gene in transiently transfected cells, but not in stable transfectants.

Topics: Breast Neoplasms; Carcinoma; Cell Division; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Fulvestrant; Gene Expression Regulation, Neoplastic; Genes, Reporter; Growth Substances; HeLa Cells; Humans; Insulin; Insulin-Like Growth Factor I; Luciferases; Receptors, Estrogen; Transfection; Tumor Cells, Cultured; Vitellogenins

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

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

Transforming growth factor beta (TGF-beta) is released by a variety of cells and known to be involved in many different processes including the immune response, wound healing and carcinogenesis. As most experimental investigations have been based on quantitative analysis of TGF-beta production using a bioassay, it seemed important to test the validity and limitations of this method. This paper analyses several parameters that may impair TGF-beta quantification by bioassay. Recommendations are made concerning the influence of technical parameters and the presence of other cytokines (EGF and bFGF) commonly released by cultured cells to which the Mv1Lu mink lung epithelial cell line (CCL64) is sensitive.

Topics: Animals; Biological Assay; Blood Physiological Phenomena; Carcinoma; Cattle; Cell Line; Colonic Neoplasms; Culture Media, Conditioned; Culture Media, Serum-Free; Cytokines; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelium; Fibroblast Growth Factor 2; Growth Inhibitors; Humans; Lung; Mink; Preservation, Biological; Recombinant Proteins; Sensitivity and Specificity; Swine; Transforming Growth Factor beta

Using differential display polymerase chain reaction, early growth response gene alpha (EGR alpha) was first isolated as a 291-base pair 3'-cDNA clone, which was highly expressed in the androgen-independent prostate carcinoma cell lines PC3 and DU145, as compared with the androgen-responsive prostate carcinoma cell line LNCaP. Full length cloning of the EGR alpha coding region revealed that EGR alpha was a new member of an important subfamily of nuclear zinc finger transcription factors (others members e.g. Sp1, EGR-2, and Wilms' tumor gene). Moreover, it was observed that EGR alpha, as with most Sp1 subfamily members, was conserved between mammalian species ranging from human to rabbit. Two hormones important for prostate development and differentiation were found to be potent regulators of EGR alpha mRNA expression. Androgens were observed to induce a down-regulation of EGR alpha mRNA expression (70% in 72 h), while epidermal growth factor induced a rapid transient up-regulation (6-fold in 100 min). The up-regulation was controlled at the transcriptional level and effectively blocked by staurosporine (which suggests the involvement of the protein kinase C pathway). Functional analysis demonstrated that EGR alpha could bind to, and stimulate transcription from, a basic transcription element (BTE) consensus sequence on DNA (BTE is a transcription-modulating sequence in the promoter region of some cytochrome P450 family members). Furthermore, in stage-synchronized prostate cells, EGR alpha mRNA was highly expressed in the early G1 phase of the cell cycle, similar to c-fos mRNA expression. These results indicated that the zinc finger transcription factor EGR alpha seems to play a role in cell cycle regulation.

Topics: Alkaloids; Amino Acid Sequence; Androgens; Animals; Base Sequence; Binding Sites; Carcinoma; Cell Cycle; Consensus Sequence; DNA; Early Growth Response Transcription Factors; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Humans; Kruppel-Like Transcription Factors; Male; Molecular Sequence Data; Neoplasms, Hormone-Dependent; Polymerase Chain Reaction; Prostatic Neoplasms; Protein Kinase C; Rabbits; Rats; Regulatory Sequences, Nucleic Acid; Sequence Alignment; Sequence Homology, Amino Acid; Species Specificity; Staurosporine; Transcription Factors; Zinc Fingers

In the search for a substance which would specifically block a particular step in the signal transduction cascade, we identified glucopiericidin A produced by Streptomyces sp. as an inhibitor of phosphoinositide (PI)-turnover in phospholipase-Cgamma1 (PLC-gamma1) overexpressing NIH 3T3 fibroblasts (NIH 3T3gamma1). Glucopiericidin A inhibited the formation of inositol phosphate (IPt) in platelet-derived growth factor (PDGF)-stimulated NIH 3T3gamma1 cells with an IC50 of 5.0 microM. In vitro enzyme assay showed the compound had no inhibitory effect on PLC-gamma1 even at 100 microM concentration. Glucopiericidin A reduced PDGF-induced tyrosine phosphorylations of proteins, including PDGF receptor and PLC-gamma1, in the cells. In contrast, glucopiericidin A showed only a slight inhibitory effect on epidermal growth factor (EGF)-induced IPt production and protein tyrosine phosphorylations in A431 cells. These results suggest that glucopiericidin A inhibits PDGF-induced activation of PLC-gamma1 by reducing the tyrosine kinase activity of the PDGF receptor and it more potently inhibits PI-turnover induced by PDGF than by EGF.

Topics: 3T3 Cells; Aminoglycosides; Animals; Anti-Bacterial Agents; Carcinoma; Epidermal Growth Factor; Humans; Immunoblotting; Isoenzymes; Mice; Phosphatidylinositols; Phospholipase C gamma; Phosphorylation; Platelet-Derived Growth Factor; Precipitin Tests; Receptors, Platelet-Derived Growth Factor; Tumor Cells, Cultured; Type C Phospholipases; Tyrosine

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

Overexpression of the EGF receptor in breast cancer correlates with poor prognosis and failure on endocrine therapy for both ER-/EGFR+ and ER+/EGFR+ tumors, suggesting a role for EGFR in the progression to hormone independence. The identification of specific DNAse I hypersensitive site patterns for the EGFR gene in ER+ vs. ER- cells implicates regions of the EGFR first intron in up-regulation of EGFR, while estrogen regulation studies indicate the involvement of a repressor(s) in the maintenance of low levels of EGFR. Based on these findings, a multi-step model is proposed for the progression of breast cancer from a hormone-dependent, ER+/EGFR-phenotype to an aggressive, hormone-independent, ER-/EGFR+ stage.

Topics: Breast Neoplasms; Carcinoma; Disease Progression; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Prognosis; Receptors, Estrogen; Tumor Cells, Cultured; Vulvar Neoplasms

Antiproliferative effects of somatostatin (SRIH) analogs were investigated in human thyroid carcinoma cell lines. Membrane preparations from six human thyroid cell lines, including follicular (RO 87-M-1 and RO 82-W-1), papillary (NPA87), and anaplastic (RO 90-D-1) carcinomas, a follicular adenoma, as well as benign and malignant human thyroid tissues, contained high affinity SRIH-binding sites. Ligand-binding studies ([125I]Tyr11-SRIH) demonstrated mean dissociation constants ranging from 114-224 pmol/L, and 20-154 fmol/mg membrane protein mean receptor sites, in cell lines. Four cell lines were grown for 3 days in monolayers with SRIH analogs (octreotide or MK0678) at concentrations from 0.05-100 nmol/L in serum-free medium to assess changes in cell numbers. At the highest dose, MK0678 produced dose-dependent inhibition of growth in RO 87-M-1 and NPA87, each to about 70% of that in control cells. Octreotide produced dose-dependent stimulation of growth in RO 87-M-1 cells, but caused growth inhibition in NPA87 cells, with loss of effect at the highest dose. RO 82-W-1 did not respond to MK0678, yet caused biphasic inhibition with octreotide (75% and 45% of control cell numbers at 0.05 and 100 nmol/L doses, respectively). Anaplastic cells, RO 90-D-1, did not respond to either analog despite similar ligand binding. Addition of epidermal growth factor (100 micrograms/L) or TSH (200 mU/L) increased the sensitivity of RO 87-M-1 cells to growth inhibition by the lowest dose of MK0678, producing biphasic dose-response curves. In conclusion, the present data demonstrate specific SRIH binding to membranes of thyroid carcinoma cells and tissues as well as discordant growth effects of different SRIH analogs on the same cell lines. This may be a result of differential stimulation and regulation of distinct SRIH receptor subtypes.

Topics: Carcinoma; Cell Division; Epidermal Growth Factor; Humans; Receptors, Somatotropin; Somatostatin; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

The prognostic value of epidermal growth factor receptor (EGF-R) was prospectively assessed in a series of 229 clinical T1-T2, N0-N1 breast carcinomas diagnosed between May 1987 and October 1989. EGF-R expression was determined by measuring the specific Bmax of 125I EGF to tumor plasma membrane preparations. Tumor with a Bmax > or = 3 fmol/mg of protein were considered positive with regard to EGF-R expression. With a median follow-up of 34 months, the 3-year overall and disease-free survivals are respectively 92% and 88% for EGF-R < or = 3, and 91% and 86% EGF-R > 3 fmol, showing no significant difference, even when comparing axillary lymph node status. We did not succeed in finding an EGF-R cut-off value which might be significant in univariate analysis. Multivariate analysis of our data indicates that pT (p = 0.001), pN (p = 0.04), and Scarff-Bloom grade (p = 0.04) are the only significant predictors of disease-free survival among the parameters investigated in this study.

Topics: Analysis of Variance; Breast Neoplasms; Carcinoma; Combined Modality Therapy; Epidermal Growth Factor; ErbB Receptors; Female; Follow-Up Studies; Humans; Middle Aged; Prognosis; Prospective Studies; Survival Rate

CG-1 human nasopharyngeal carcinoma cells in monolayer culture formed both cohesive, epithelial-like colonies and scattered, fibroblastic-like colonies in mixed proportions. In the presence of exogenously added bFGF (4 ng/ml), about 85% of the colonies formed were fibroblastic-like. CG-1 cells were capable of synthesizing and releasing bFGF, and, when compared by the immunological method, cells in fibroblastic-lke colonies were found to contain higher levels of endogenous bFGF than cells in the epithelial-like colonies. Furthermore, cells in the peripheral region of the epithelial-like colonies, which were fibroblastic-like in morphology, also appeared to contain higher levels of endogenous bFGF. In addition, in the presence of suramin, neutralizing antibody to bFGF, or neutralizing antibodies to bFGF and EGF, the number of cohesive colonies formed was greatly increased. Moreover, addition of the 2 M NaCl-eluted heparin-Sepharose fraction of the CG-1 cell-coditioned medium promoted the formation of dispersed colony in a dose-dependent manner. The results suggest that bFGF can regulate CG-1 cell phenotype in an autocrine manner.

Topics: Antibodies; Carcinoma; Epidermal Growth Factor; Epithelial Cells; Fibroblast Growth Factor 2; Fibroblasts; Humans; Mesoderm; Nasopharyngeal Neoplasms; Neoplastic Stem Cells; Suramin; Tumor Cells, Cultured

The expression of cripto gene product was examined immunohistochemically in 45 surgically resected pancreatic tumors, including 32 invasive ductal carcinomas, 4 intraductal papillary adenocarcinomas, 4 intraductal papillary adenomas, 2 mucinous cystadenomas, 2 islet cell tumors, and one solid and cystic tumor, and compared with that in 32 areas of accompanying chronic pancreatitis present in the cases of invasive ductal carcinomas and 5 non-tumorous areas of pancreas without pancreatitis. All pancreatic ductal tumors including adenomas and carcinomas showed positive staining with no difference in terms of staining intensity among intraductal tumors and invasive carcinomas with or without mucin hypersecretion. Islet cell tumors were positively stained but the solid and cystic tumor was negative. Duct epithelial cells and acinar cells were negative but islet cells were positive in the pancreas tissues without pancreatitis. Cells arranged in duct-like structures in areas of accompanying chronic pancreatitis were positively stained. The results suggest that cripto expression might be associated with a growth advantage of tumor cells and also with differentiation to form duct-like structures.

Topics: Biomarkers, Tumor; Carcinoma; Carcinoma, Islet Cell; Cell Transformation, Neoplastic; Cystadenoma, Mucinous; Epidermal Growth Factor; Gene Expression; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Membrane Glycoproteins; Neoplasm Proteins; Pancreatic Ducts; Pancreatic Neoplasms; Pancreatitis

A series of rat monoclonal antibodies (MAbs) has been generated against the extracellular domain of the receptor for EGF which block the binding of EGF and TGF alpha to the receptor and inhibit the growth in vitro of a range of carcinoma cell lines that over-express the receptor for EGF. Some of these antibodies were able also to induce the complete regression of xenografts of EGFR-over-expressing tumours when treatment was started, either at the time of tumour inoculation or later when the tumours were established. The most effective of these antibodies was ICR62, which was also able to activate host immune effector functions. We conclude that antibodies which block growth-factor-ligand interaction can have a profound influence on the proliferative capacity of tumour cells in vivo and may have useful clinical application.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Epidermal Growth Factor; ErbB Receptors; Female; Fibroblasts; Head and Neck Neoplasms; Humans; Immunotherapy; Lung Neoplasms; Mice; Mice, Nude; Ovarian Neoplasms; Rats; Rats, Inbred Strains; Recombinant Proteins; Transforming Growth Factor alpha; Transplantation, Heterologous; Tumor Cells, Cultured; Urinary Bladder Neoplasms; Vulvar Neoplasms

In order to characterize the effects of growth factors on the regulation of expression of the genes coding for prostatic differentiation markers, prostatic acid phosphatase and prostate-specific antigen, we studied changes occurring in the biosynthesis of these enzymes in LNCaP prostatic cancer cells treated with growth factors. Epidermal growth factor was found to reduce the secretion of prostatic acid phosphatase and prostate-specific antigen by the cells, as the result of lowered steady-state levels of the corresponding messenger RNAs (mRNAs). In addition, epidermal growth factor (EGF) interfered with the androgen regulation of these genes. EGF evoked these changes in a concentration- and time-dependent fashion, in both the presence and absence of serum and most likely through interactions with the epidermal growth factor receptor, inasmuch as similar effects were achieved by treating the cells with transforming growth factor alpha. The regulation of the human glandular kallikrein 1 gene was quite similar to the regulation of the prostate-specific antigen gene. In addition to the expression of the genes coding for prostatic secretory proteins, the amount of the human androgen receptor mRNA was down-regulated by EGF. This reduction was more pronounced than the autologous down-regulation of human androgen receptor (hAR) mRNA by androgen and could be maintained for at least 5 days. In the presence of androgen, some of the effects of EGF and transforming growth factor alpha on the levels of androgen-regulated mRNAs may be due to down-regulation of the expression of the hAR gene. Transforming growth factor beta 1, which blocked the growth induction of LNCaP cells by EGF, increased the level of prostatic acid phosphatase and hAR mRNAs, but when given to the cells together with EGF its up-regulatory effect could not be discerned. In summary, regulation of the prostatic acid phosphatase and prostate-specific antigen genes is a complex matter, inasmuch as androgens and growth factors regulate the levels of the mRNAs originating from them. Furthermore, the interactions between the androgen-regulatory system and the growth factor-regulatory systems are likely to be at multiple levels in prostatic cells, as suggested by the modulation of the hAR gene expression by these growth factors.

Topics: Acid Phosphatase; Base Sequence; Carcinoma; Epidermal Growth Factor; Gene Expression Regulation; Humans; Kallikreins; Male; Molecular Sequence Data; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The growth of a recently established human nasopharyngeal carcinoma cell line, CG-1, and 5 randomly selected, single-cell-derived sub-lines in serum-free medium and in fetal-bovine-serum(FBS)-containing medium was investigated. In basal medium supplemented with insulin, transferrin, fibronectin and high-density lipoprotein, cell growth was moderately stimulated by aFGF and EGF in a dose-dependent manner. In contrast, in medium containing as little as 0.5% FBS, most of the stimulatory effect of the aforementioned growth factors observed was masked. Western blotting analysis of the cell lysates and conditioned media showed that CG-1 and sub-line cells were all capable of synthesizing and releasing aFGF- and EGF-immunoreactive proteins. The amounts of these 2 growth factors synthesized and released appeared to vary among the parental cell line and sub-line cells. Moreover, the rate of basal proliferation of these cells appeared to be positively correlated with the amounts of aFGF- and EGF-immunoreactive proteins produced. Addition of the neutralizing antibodies to aFGF and EGF exerted a dose-dependent suppression on cell growth in medium containing 0.5% FBS. The results suggest a role of aFGF and EGF in autocrine growth stimulation of CG-1 and sub-line cells, and may explain the moderate response of these cells to exogenously added aFGF and EGF.

Topics: Carcinoma; Cell Division; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factor 1; Humans; Nasopharyngeal Neoplasms; Tumor Cells, Cultured

Intratumoral regional differences in DNA ploidy patterns, and expression of epidermal growth factor (EGF) and EGF receptor (EGF-R) were studied to evaluate the biological and clinical significance of intratumoral DNA heterogeneity in 23 cases of esophageal carcinoma. Multiple specimens were subjected to histologic grading of carcinoma, DNA analysis and immunohistochemistry. DNA heterogeneity was found in 34.8% of the cases. Expression of EGF and EGF-R within a single tumor was observed in 69.6% and 73.9% of the lesions examined, respectively. A positive correlation was noted between the expression of EGF and that of the EGF-R. EGF expression showed positive correlation with the DNA index, but not with the proliferative index. There was no relationship between DNA heterogeneity and the degree of histopathological differentiation of carcinoma. Cases with DNA heterogeneity showed better prognosis than those with DNA homogeneity. The present study suggests that esophageal carcinoma consists of carcinoma cells with intratumoral regional polymorphism and variable types of clones. Furthermore, the autocrine mechanism of EGF and EGF-R could be one of the contributory factors on the development of DNA abnormalities during tumor proliferation.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Female; Humans; Male; Middle Aged; Ploidies; Prognosis

There are conflicting data about the effect of the epidermal growth factor (EGF) on protein kinase C (PKC) enzyme activity. The aim of our study was to find out which type of phospholipids [phosphatidylinositol 4,5-bisphosphate PI4,5P2 or the other phospholipids-phosphatidylcholine (PC) or phosphatidic acid (PA)] could be the source of 1,2-diacylglycerol (1,2-DAG) in PKC activation. In colon carcinoma cells (HT29) we observed a more than 2-fold increase in the PC pool and at the same time decreased tyrosine kinase activity (50%). With increasing incubation time EGF affects the pools of both phosphatidylinositols and other phospholipids parallel with the activation of the tyrosine kinase activity. EGF increases the activity of PKC in the HT29 cell line and PC could be the source of 1,2-DAG which may stimulate PKC activity.

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; Humans; Phospholipids; Phosphorus Radioisotopes; Protein Kinase C; Protein-Tyrosine Kinases; Signal Transduction; Time Factors; Tumor Cells, Cultured

Female Syrian golden hamsters with N-nitroso-bis (2-oxopropyl) amine (BOP)-induced pancreatic cancers were treated for 2 months with bombesin/gastrin-releasing peptide (GRP) antagonist D-Tpi6,Leu13 psi(CH2NH)Leu14 bombesin(6-14) (RC-3095). Bombesin and GRP(14-27) were also administered alone and in combination with the antagonist RC-3095. RC-3095 exerted a dose-dependent inhibitory effect on growth of pancreatic cancers. The number of animals with pancreatic cancers was significantly lower in the group treated with 60 micrograms/day of RC-3095 and the weight of tumorous pancreata was reduced. Administration of bombesin or GRP alone did not stimulate the growth of pancreatic tumors and, in fact, had a slightly suppressive effect on cancers which was significant only in Experiment I. Bombesin and GRP (14-27) given together with RC-3095 did not nullify the inhibitory effect of the antagonist on pancreatic cancer growth. Actually, a greater inhibition of pancreatic tumors was observed after administration of RC-3095 together with bombesin or GRP, than with RC-3095 alone. The mechanism of action of bombesin, GRP, and bombesin antagonists on pancreatic cancers appears to be complex. The inhibitory effect of bombesin antagonists on pancreatic cancer growth was accompanied by a decrease in the binding capacity of EGF receptors in tumor membranes. Administration of bombesin also caused a down-regulation of EGF receptors and the greatest decrease in binding capacity of EGF receptors was observed after treatment with RC-3095 in combination with GRP. Inhibition of pancreatic cancer can thus be tentatively explained by some common pathways in the action of bombesin, GRP and their antagonists, that could be mediated by interference with EGF-receptor mechanisms.

Topics: Animals; Body Weight; Bombesin; Carcinoma; Cricetinae; Dose-Response Relationship, Drug; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Gastrin-Releasing Peptide; Gastrins; Growth Hormone; Insulin-Like Growth Factor I; Mesocricetus; Nitrosamines; Pancreatic Neoplasms; Peptide Fragments; Peptides; Receptors, Bombesin; Receptors, Neurotransmitter

In 19 patients with advanced gastric cancer the expression of epidermal growth factor receptor and epidermal growth factor (EGF) were studied using immunohistochemical techniques. There were 12 males and the mean age of the group was 54.8 years. Most cases belonged in Borrmann's types III or IV. Tumoral cells were positive for EGF receptor in 9 patients (47%), with a strong reaction in 6. Thirteen of 18 subjects were positive for EGF. The reaction for EGF receptor was positive in 20% of 12 patients with intestinal type tumors and in 67% of 7 patients with diffuse tumors. Reaction for EGF was positive in 80% of intestinal type tumors and 64% of diffuse tumors. Simultaneous positive reactions for both antigens was observed in 5 of 18 patients, all with diffuse type tumors. Signet ring cell tumors showed less positivity than less differentiated ones. Thus, the expression of EGF and EGF receptor was higher in our patients with advanced gastric cancer than reported elsewhere.

Topics: Adult; Aged; Aged, 80 and over; Antibodies, Monoclonal; Biomarkers, Tumor; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Middle Aged; Stomach Neoplasms

A panel of eight cell lines has been derived from colon carcinomas. These cell lines have both been characterized according to standard criteria of growth rate, response to mitogens (epidermal growth factor and basic fibroblast growth factor), xenograft growth and growth in soft agar, and according to the ability of the cells to express epitopes known to be expressed by cells in the normal intestinal mucosa. The expression of epitopes present in columnar (absorptive) cells has been assessed using a panel of monoclonal antibodies to brush border peptidases and disaccharidases, villin and brush border-specific peptides. Goblet cell epitopes have been determined by monoclonal antibodies to mucin and carcinoembryonic antigen. An antibody to chromogranin was used to identify endocrine cells. Using these antibodies we found that all the cell lines reacted with at least one of the antibodies to columnar cells. Similarly, varying proportions of cells in six of the eight cell lines stained with antibodies to mucin. None of the cells expressed chromogranin. Expression of a differentiated colonic phenotype, as measured from antibody staining, did not correlate with measurements of malignancy, such as the ability of the cells to grow in soft agar or as xenografts. Similarly, there was no correlation between retention of a colonic phenotype and the initial pathological stage of the tumour from which the cell lines were derived.

Topics: Animals; Antigens, Neoplasm; Carcinoma; Carrier Proteins; Cell Differentiation; Cell Division; Cell Polarity; Colonic Neoplasms; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Immunohistochemistry; Intestinal Mucosa; Mice; Mice, Nude; Microfilament Proteins; Mucins; Neoplasm Transplantation; Transplantation, Heterologous; Tumor Cells, Cultured

A total of 117 colorectal tissue specimens were examined immunohistochemically for the production of immunoreactive (IR-) transforming growth factor (TGF)-alpha and IR-epidermal growth factor (EGF). IR-TGF-alpha was detected in 26/32 (81.3%) invasive cancers, 14/27 (51.9%) carcinomas in situ, and 14/58 (24.1%) adenomas. IR-EGF was detected in 14/32 (43.8%) invasive cancers, 12/27 (44.4%) carcinomas in situ, and 12/58 (20.7%) adenomas. The staining intensity of IR-TGF-alpha was related to the histologic grade of malignancy, but that of IR-EGF was not. These suggest that IR-TGF-alpha plays a more important role than IR-EGF in the growth of colorectal neoplasms, and that further study of these growth factors would be helpful in understanding the biology of colorectal carcinoma.

Topics: Adenoma; Carcinoma; Carcinoma in Situ; Colorectal Neoplasms; Epidermal Growth Factor; Humans; Immunohistochemistry; Transforming Growth Factor alpha

The effects of sodium butyrate (NaB) on the response of the RCA human colon carcinoma cell line to transforming growth factor-beta 1 (TGF-beta 1) were examined. NaB induced differentiation, as judged by an increase in cellular alkaline phosphatase, in the RCA cells and this differentiation was accompanied by a decreased growth rate. TGF-beta 1 did not significantly alter the growth or state of differentiation of the RCA cells. The growth rate of cells treated simultaneously with NaB and TGF-beta 1 was similar to that of control untreated cells while the alkaline phosphatase levels remained comparable to cells treated with NaB. Addition of TGF-beta 1 to cells grown in the presence of NaB resulted in a stimulation of growth. Cells pretreated with TGF-beta 1 remained sensitive to the growth inhibitory and differentiation inducing effects of NaB. These results suggest that NaB may alter the expression of proteins responsible for a stimulatory signal response to TGF-beta 1 in RCA cells.

Topics: Alkaline Phosphatase; Butyrates; Butyric Acid; Carcinoma; Cell Differentiation; Cell Division; Colonic Neoplasms; Drug Screening Assays, Antitumor; Enzyme Induction; Epidermal Growth Factor; Humans; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Thirty-six primary human colorectal tumors, 43 noninvolved colon samples that were adjacent to either carcinomas of adenomas, 22 adenomas, and nine normal colon specimens were immunohistochemically examined for the presence and localization of two epidermal growth factor-related peptides, amphiregulin (AR) and cripto. Within the primary tumors, 18 (50%) showed moderate levels of AR expression. Approximately 60% of the tubular and tubulovillous adenomas were positive for AR expression, whereas only 15% of the adjacent, noninvolved colon mucosa expressed AR. A greater proportion of well-differentiated tumors (71%) were positive for AR expression than were poorly differentiated tumors (18%). All of the nine normal colon specimens were positive. Consequently, AR expression appeared to be associated with both normal and malignant epithelial cells that were more differentiated. The distribution of cripto expression was different. Seventy-nine % of the colon tumors expressed cripto with a frequency of expression that was approximately equivalent between well-differentiated and poorly differentiated tumors. Approximately 86% of the tubulovillous adenomas, but only 43% of the tubular adenomas, were positive for cripto expression. In contrast, whereas AR was expressed in normal colon specimens, none of these tissues expressed cripto, and only 12% of the noninvolved normal colon samples adjacent to tumors or adenomas were positive for cripto. Cripto expression therefore appeared related to neoplasia. These data suggest that AR and cripto may be functioning as potential autocrine and/or paracrine growth factors in the colon and that the differential expression of cripto may serve as a potential tumor marker for colonic carcinogenesis.

Topics: Adenoma; Amphiregulin; Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Colon; Colonic Polyps; Colorectal Neoplasms; EGF Family of Proteins; Epidermal Growth Factor; Glycoproteins; GPI-Linked Proteins; Growth Substances; Humans; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Membrane Glycoproteins; Neoplasm Proteins; Phenotype; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured

Expression of epidermal growth factor (EGF) receptor is often increased in various human carcinomas. Therefore, inhibition of the EGF/EGF receptor-induced signaling pathway may help to suppress these carcinomas. In the presence of Ca2+, EGF induces elongation of A431 cells in approximately 30 min. The cell elongation was shown to be accompanied by a reorganization of actin filaments. These phenotypical changes were specifically inhibited by a tyrosine kinase inhibitor, erbstatin, and inhibitors of phosphatidylinositol (PI) turnover such as psi-tectorigenin and inostamycin. The amount of filamentous actin was increased by EGF, which was also inhibited by these compounds. Long-term treatment of A431 cells with EGF induced the disappearance of cytoskeleton and aggregation of the cells, which was again inhibited by the PI turnover inhibitors. Thus tyrosine kinase and phosphatidylinositol turnover inhibitors were shown to inhibit the signaling pathways of EGF-induced cytoskeletal organization of A431 cells.

Topics: Actins; Calcium; Carcinoma; Cell Aggregation; Cytoskeleton; Epidermal Growth Factor; Furans; Humans; Hydroquinones; Isoflavones; Microscopy, Phase-Contrast; Phosphatidylinositols; Protein-Tyrosine Kinases; Tumor Cells, Cultured

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Kinetics; Neoplasm Proteins; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Signal Transduction; Tumor Cells, Cultured; Tyrosine

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.

Topics: Animals; Carcinoma; Culture Media; Epidermal Growth Factor; ErbB Receptors; Female; Growth Substances; Mammary Neoplasms, Experimental; Neoplasm Proteins; Phenotype; Phosphoproteins; Phosphotyrosine; Rats; Tyrosine

The antiparasitic drug, suramin, has antiproliferative effects in human carcinoma cells. It has been suggested that this occurs through blockade of growth factor-receptor interactions. Three types of evidence that suramin rapidly inhibits cellular respiration or disrupts cellular energy balance in intact cells of the human prostate carcinoma cell line, DU145, are presented. Beginning at approximately 10(-4) M, suramin rapidly causes dose-dependent inhibition of tetrazolium conversion by mitochondrial dehydrogenases in intact cells, demonstrating an inhibition of respiration. This effect is reversed by exchange with suramin-free media but not by pretreatment with serum, epidermal growth factor, insulin-like growth factor I, acidic and basic fibroblast growth factors, or calcium. Rhodamine 123 (10 micrograms/ml) uptake by mitochondria in intact DU145 cells is inhibited in the presence of 10(-3) M suramin. Treatment with 10(-4)-10(-3) M suramin causes the loss of rhodamine 123 from cells with mitochondria prestained with rhodamine 123, indicating that suramin is acting as an ionophore or respiratory poison. Also shown by electron microscopy are progressive toxic changes in mitochondria of DU145 cells within 1 h after treatment with 10(-4) M suramin. These data indicate that in intact DU145 cells 10(-4) M suramin rapidly disrupts cellular energy balance or respiration as seen by three studies of mitochondrial state. Disruption of energy balance or respiration represents a likely antiproliferative mechanism, as is thought to be a primary mechanism for the action of suramin in parasitic diseases. This proposed mechanism of action for suramin can explain the most prominent observed clinical toxicities of nephrotoxicity, adrenal toxicity, coagulopathy, and demyelinating neuropathy.

Topics: Biological Transport; Calcium; Carcinoma; Cell Division; Energy Metabolism; Epidermal Growth Factor; Humans; In Vitro Techniques; Male; Microscopy, Electron; Mitochondria; Oxidoreductases; Prostatic Neoplasms; Rhodamine 123; Rhodamines; Suramin; Tetrazolium Salts; Thiazoles; Tumor Cells, Cultured

Epidermal growth factor and transforming growth factor alpha are two peptides which bind to the epidermal growth factor receptor. One hundred and seventy-four samples from 133 patients with ovarian cancer were examined for EGF and TGF alpha. EGF was detected in only 27.6% of samples while TGF alpha was present in 88.5%. The median values for TGF alpha presence were at least 10-fold greater than those of EGF. There was no statistical difference between either TGF alpha or EGF levels and degree of differentiation of the tumours. There was no statistical difference between stage three and four in relation to concentration of either peptide. Median concentration did not differ significantly among the histological sub-groups.

Topics: Carcinoma; Cell Differentiation; Epidermal Growth Factor; Female; Humans; Middle Aged; Ovarian Neoplasms; Radioimmunoassay; Transforming Growth Factor alpha

The receptor binding and cellular growth responses to exogenous epidermal growth factor (EGF) were studied using the DiFi cell line established from a familial adenomatous polyposis patient. The number of cell membrane EGF receptors on DiFi cells, as measured by competitive radioligand binding assays and Scatchard analysis of 125I-EGF binding isotherms, was calculated to be 4.8 x 10(6) receptors/cell. An acid prewash step performed prior to ligand binding assays did not reveal additional receptor numbers. A single, low-affinity receptor population was identified by Scatchard analysis, with an apparent Kd of 4.6 nM. This result was confirmed by radioligand binding studies performed in the presence and absence of the receptor-antagonist monoclonal antibody 528 IgG that binds predominantly to the low-affinity form of the EGF receptor. DiFi cells at 50-60% confluence, when exposed to 50 nM exogenous EGF, exhibited a rapid but partial (30%) reduction in their cell membrane-associated receptor, characteristic of sequestration. Exposure of DiFi cells to 50 nM EGF for longer periods of time (4 h) did not result in any further reduction in EGF-receptor number. The cellular growth response of DiFi cells to exogenous EGF was studied in monolayer cultures as well as in a soft agarose assay. Inhibition of soft agar colony formation was observed at exogenous EGF concentrations greater than 1.7 nM, and inhibition of monolayer growth occurred at EGF concentrations greater than 1 nM. In immune complex kinase assays, the DiFi receptor showed similar specific activity to that from the well-characterized A431 cell line. Additionally, phosphorylation of the receptor on tyrosine was qualitatively similar to that of A431 cells, further suggesting that the DiFi receptors identified by EGF-binding studies were biologically functional.

Topics: Adenomatous Polyposis Coli; Antibodies, Monoclonal; Carcinoma; Colonic Neoplasms; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Humans; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured

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

The epidermal growth factor receptor (EGFR) level in 56 esophageal cancer tissues was measured by 125I-EGF binding assay to elucidate its role in tumor progression. The survival rate of patients with high EGFR level (more than 50 fmol/mg protein) was significantly lower than that of patients with low EGFR level (less than 50 fmol/mg protein, P less than 0.01), although a correlation between EGFR level and the pathologic findings was not observed. The expression of EGF was examined immunohistochemically using anti-EGF monoclonal antibody in 100 esophageal cancer tissues; EGF-positive tumor cells were detected in 92.0%. The immunoreactivity of EGF was classified arbitrarily into four grades according to the number of stained tumor cells. The expression of EGF significantly correlated with the differentiation of esophageal squamous cell carcinoma (P less than 0.01, by chi-square test). The survival rate of patients with high EGF immunoreactivity (Grade 2 or 3) was much lower than in those with lower grade (0 or 1) tumors, (P less than 0.01). Patients with both high EGFR level and EGF immunoreactivity had a much worse prognosis than if both were low. Furthermore, the mitotic index was higher in groups with both high EGFR and EGF than if both were low (16.39 +/- 5.35 versus 6.90 +/- 3.31). These results suggest that EGF and EGFR in the autocrine system may play an important role in tumor progression in esophageal cancer and their expression could be of prognostic significance.

Topics: Antibodies, Monoclonal; Carcinoma; Carcinoma, Squamous Cell; Cell Differentiation; Epidermal Growth Factor; ErbB Receptors; Esophageal Neoplasms; Follow-Up Studies; Humans; Immunohistochemistry; Lymphatic Metastasis; Melanoma; Mitotic Index; Neoplasm Staging; Survival Rate

We examined the effects of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) on EGF receptor (EGFR) phosphorylation and the expression of mRNAs for oncogenes, growth factors, their receptors and metalloproteinase genes by MKN-28 gastric carcinoma cells which express EGF, TGF-alpha and EGFR genes. Both EGF and TGF-alpha stimulated EGFR phosphorylation, EGF and TGF-alpha induced FOS, MYC and ERBB-2 oncogene expression. Interestingly, EGF increased the expression of mRNAs for TGF-alpha and EGFR. On the other hand, TGF-alpha increased TGF-alpha mRNA but decreased the expression of mRNAs for EGFR and TGF-beta. Furthermore, mRNAs for interstitial collagenase, stromelysin and procollagen type I genes were also enhanced after treatment with EGF and TGF-alpha. These results indicate that EGF and TGF-alpha successively evoke cascade phenomena which favor tumor progression, invasion and extracellular matrix formation, acting as autocrine growth regulators for gastric carcinomas.

Topics: Carcinogens; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 3; Metalloendopeptidases; Microbial Collagenase; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; RNA, Messenger; Stomach Neoplasms; Transforming Growth Factors; Tumor Cells, Cultured

Patch clamp experiments on human carcinoma A431 cells have revealed two types of Ca2(+)-permeable channels, the activity of which can be increased by the application of non-hydrolyzable analogues of GTP to the intracellular side of the membrane. With 105 mM Ca2+ in recording pipette at 30-33 degrees C their unitary conductances (in pS) are 1.3 (SG-channels) and 2.4 (G-channels). G- and, possibly, SG-channels are activated from the extracellular side of the membrane with epidermal growth factor (EGF). The data are consistent with the hypothesis that both channels are activated via guanine nucleotide binding (G) proteins.

Topics: Calcium Channels; Carcinoma; Electrophysiology; Epidermal Growth Factor; GTP-Binding Proteins; Guanine Nucleotides; Guanosine 5'-O-(3-Thiotriphosphate); Humans; In Vitro Techniques; Tumor Cells, Cultured

Several studies have reported effects of gastrointestinal regulatory peptides on growth of experimentally induced pancreatic neoplasms and human cancer cell lines. The growth of human pancreatic cancer lines PANC-1 and MIA PaCa-2 was characterized in vitro, and the effects of cholecystokinin, bombesin, insulin, epidermal growth factor, secretin, vasoactive intestinal peptide, and somatostatin were determined. Fetal bovine serum was required for initiation of growth in both cell lines. Growth effects of peptides were determined by incubating cells with peptides in serum-free medium after a 72-h preincubation in 10% serum-supplemented medium alone. Epidermal growth factor (3.4 x 10(-9) M) and insulin (10(-6) M) significantly (p less than 0.001) increased growth of both cell lines as determined by increases in deoxyribonucleic acid and protein. Bombesin, secretin, vasoactive intestinal peptide, and somatostatin (all 10(-8) M) did not affect growth of either cell line. Neither cholecystokinin-8 nor [Thr4, Nle7] cholecystokinin-9 altered growth in concentrations from 10(-12)-10(-6) M. Anchorage-dependent clonogenic growth of both cell lines was also not altered by cholecystokinin-8. Cholecystokinin added to cultures was degraded by separate effects of serum and cells. Addition of cholecystokinin-8 to cultures every 8 h maintained cholecystokinin levels but did not alter cell growth. These data support roles for epidermal growth factor and insulin as growth factors for human pancreatic cancer cell lines.

Topics: Blood; Carcinoma; Cell Division; Cholecystokinin; Clone Cells; Culture Media; DNA, Neoplasm; Epidermal Growth Factor; Gastrointestinal Hormones; Humans; Insulin; Neoplasm Proteins; Pancreatic Neoplasms; Tumor Cells, Cultured

Carcinoma cells frequently coexpress transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor (EGF) receptor, implicating an autocrine function of carcinoma-derived TGF-alpha. Using a monoclonal antibody (425) to the EGF-receptor, we investigated the role of exogenous and tumor cell-derived EGF/TGF-alpha mitogenic activities in proliferation of cell lines derived from solid tumors. Monoclonal antibody 425 was chosen for these studies because it inhibits binding of EGF/TGF-alpha to the EGF-receptor and effectively blocks activation of the EGF-receptor by EGF/TGF-alpha. Seven malignant cell lines originating from carcinomas of colon, pancreas, breast, squamous epithelia, and bladder expressed surface EGF-receptor and secreted EGF/TGF-alpha-like mitogenic activities into their tissue culture media. All cell lines were maintained in a defined medium free of exogenous EGF/TGF-alpha. EGF and TGF-alpha added to the culture medium stimulated proliferation of five cell lines to comparable levels. EGF/TGF-alpha-dependent proliferation was significantly reduced by addition of MAb 425 to culture media. In addition, monoclonal antibody 425 reduced proliferation of the five EGF/TGF-alpha responsive cell lines in the absence of exogenous EGF/TGF-alpha. Antiproliferative effects induced by monoclonal antibody 425 were reversible and could be overcome by addition of EGF to culture media. Our results indicate that tumor-derived EGF-receptor-reactive mitogens can promote proliferation of carcinoma cells in an autocrine fashion.

Topics: Antibodies, Monoclonal; Carcinoma; Cell Division; Epidermal Growth Factor; ErbB Receptors; Humans; Neoplasm Proteins; Organ Specificity; Transforming Growth Factor alpha; Tumor Cells, Cultured

The epidermal growth factor and the homologous alpha-tumor growth factor are mitogenic polypeptides that act by binding to the epidermal growth factor receptor. The present study investigated whether increased production of epidermal growth factor/alpha-tumor growth factor or increased density of epidermal growth factor receptors may occur in gastric carcinomas as compared with normal mucosa from the same individuals. Epidermal growth factor receptors were measurable by (125I)EGF-binding assays in 13 of 15 normal mucosas and in 15 of 15 carcinomas. The epidermal growth factor-binding capacity was significantly higher in carcinomas than in mucosa. A comparison of pairs of mucosa and carcinomas showed an increase of epidermal growth factor receptors in 9 of 15 carcinomas, no change in 3, and a decrease in 2 carcinomas. One mucinous adenocarcinoma contained extreme numbers of epidermal growth factor receptors (2445 fmol/mg protein) corresponding to a 320-fold increase over normal mucosa. Epidermal growth factor-like activity was increased in 2 of 22 carcinomas compared with mucosa. We conclude that relative overexpression of epidermal growth factor receptors occurs in a fraction of gastric carcinomas. Whether increased expression of epidermal growth factor receptors is associated with particular patterns of tumor progression needs to be investigated.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Gastric Mucosa; Humans; Male; Middle Aged; Radioligand Assay; Stomach Neoplasms; Transforming Growth Factors

In order to document a possible involvement of structural alterations of FGF (Fibroblast Growth Factor)-like genes in human oncogenesis, we have screened a large series of human tumors for amplification of five FGF-related genes (Basic-FGF, INT2, HST, FGF5 and FGF6). None of 37 hematopoietic neoplasms, one out of 13 melanomas (8%), three out of 43 bladder tumors (7%) and 41 out of 238 breast carcinomas (17%) contained amplified FGF-related sequences, namely HST and INT2. Only these two genes, both located on band q13 of chromosome 11 have been found amplified. In all cases they were co-amplified and in only one instance did amplification extend to the ETS1 locus at position 11q23. INT2 and HST RNA could be evidenced by RNA/RNA in situ hybridization in breast carcinomas. Our results indicate a correlation between RNA expression and gene amplification in the case of HST but not of INT2. Although evaluation of the clinical significance of HST amplification and expression must await long-term follow-up of the patients, we suggest that HST gene product could play a role in development and/or progression of human breast cancer.

Topics: Breast Neoplasms; Carcinoma; DNA, Neoplasm; Epidermal Growth Factor; Female; Gene Amplification; Humans; RNA

The regulation of urokinase secretion and receptor display in a well-differentiated colon carcinoma cell line, GEO, adapted to serum-free conditions was examined. In protein-free medium, the cell line secreted 0.8 +/- 0.1 ng/ml/10(6) cells of urokinase in a 3-day period as determined by an enzyme-linked immunosorbent assay. This value was elevated 4-fold when the cells were cultivated in the presence of epidermal growth factor (EGF) but not insulin or transferrin. Propagation of the cell line with any combination of these growth factors was not superior to EGF alone in inducing urokinase secretion. The presence of EGF raised the radioactive laminin-solubilizing activity of the conditioned medium. In the absence of the growth factor, spent medium supplemented with plasminogen solubilized 23,000 +/- 7,000 dpm/10(6) cells of the immobilized laminin. This value was increased to 95,000 +/- 10,000 dpm/10(6) cells when the cultures were grown with EGF. Northern analysis indicated that the elevated level of the plasminogen activator protein by EGF was a consequence of a more abundant urokinase transcript. The stimulation of urokinase secretion by EGF was accompanied by a reduction of radioactive urokinase binding to the cell line. The reduction in plasminogen activator binding was not further enhanced by insulin or transferrin. In addition, these latter growth factors, by themselves, were ineffective in altering the amount of plasminogen activator bound. The attenuation in 125I-labeled urokinase binding did not reflect occupation of the receptors with endogenous ligand as acid pretreatment was without effect on the binding profile. Scatchard analysis revealed that the altered urokinase binding by EGF reflected a decrease in receptor number from 14,000 +/- 1,500 to 8,000 +/- 1,500 sites per cell. The temporal relationship of urokinase secretion and receptor display was examined. Changes in either parameter required an EGF exposure period of 10 h or more. Further amplification of the EGF effects was seen with longer incubation times with the growth peptide. These opposite effects of EGF on urokinase secretion and receptor display may suggest a homeostatic control mechanism for keeping the plasminogen activator system in check. The ability of the cell line to express biological characteristics associated with a well-differentiated colon cell type may reflect its capacity to suppress a system which is usually associated with the transformed state.

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; Extracellular Matrix; Humans; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Previous studies have established that colon carcinoma cells secrete several polypeptide growth factors, including TGF-alpha/EGF and TGF-beta, suggesting that these and related molecules function in an autocrine/paracrine fashion to modulate tumor-cell growth. To investigate this possibility, we have studied the expression of transforming growth factor receptors in a panel of human colon carcinoma cell lines and in several untransformed epithelial cell populations. The results have revealed that neoplastic colon cells express receptors for both TGF-alpha/EGF and TGF-beta. Immunoprecipitation identified the TGF-alpha/EGF receptor as a structurally intact 170-kDa protein. No evidence for over-expression was found. TGF-alpha (and EGF) enhanced receptor autophosphorylation, indicating that these receptors were biochemically functional. TGF-beta blocked DNA synthesis in non-neoplastic epithelial cells but not in tumorigenic colon populations. There was no correlation with TGF-beta receptor number or dissociation constant. However, chemical cross-linking studies revealed a TGF-beta receptor subtype of 75 kDa in 3 of the 4 colon carcinoma cells which was undetectable in normal IEC epithelial cultures, suggesting a possible association between 75-kDa receptor expression and refractoriness to growth inhibition of TGF-beta. Together, these data support the concept that locally-produced growth regulators can function in an autocrine or paracrine manner to influence the proliferation of colon carcinoma cells.

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Transforming Growth Factors; Tumor Cells, Cultured

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

Southern blot analysis of 6 human bladder carcinoma cell lines revealed amplification of the epidermal growth factor receptor (EGFR) gene in the KU1 cell line. The amplification of the gene was about 4-fold as compared with that of human placental DNA. Several restriction endonuclease digestions revealed that there was no gross rearrangement of the EGFR gene in KU1. Northern blot analysis showed normal 10 and 5.6 kb of EGFR gene-related mRNA species. 125I-EGF binding revealed 2 distinct EGF binding sites on KU1 cells: high-affinity sites 5.7 X 10(5) receptors per cell with 1.1 nM Kd and low-affinity sites 2.3 X 10(6) receptors per cell with 7.4 nM Kd. The number of the EGFR was compatible with that of the A431 squamous carcinoma cell ine which has an amplified, rearranged and over-expressed EGFR gene. Solid-phase immuno-isolation analysis showed a single 170 kDa EGFR protein in KU1 as well as in A431. Unlike other cell lines with amplified and over-expressed EGFR gene, anchorage-dependent growth of KU1 was stimulated but not inhibited by EGF. Moreover, anchorage-independent growth of KU1 was stimulated by EGF.

Topics: Antibodies, Monoclonal; Blotting, Northern; Blotting, Southern; Carcinoma; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Gene Amplification; Gene Expression Regulation, Neoplastic; Humans; RNA, Neoplasm; Tumor Cells, Cultured; Urinary Bladder 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 demonstrate the differential sensitivity of poorly differentiated and well differentiated human colon carcinoma cells to nutrients alone or to nutrients and polypeptide growth factors under completely serum-free conditions. 3H-Thymidine incorporation into trichloroacetic acid precipitable material and autoradiographic analysis indicated that nutrient replenishment alone was sufficient to initiate DNA synthesis in quiescent poorly differentiated cells, whereas defined polypeptide growth factors produced no additional effect. In contrast, well differentiated cells were mitogenically stimulated to a much greater extent by growth factors (epidermal growth factor + insulin + transferrin), than by nutrient replenishment alone. Expression of the c-myc protooncogene was increased approximately 5-fold after growth factor addition to the well differentiated cells. Maximal expression of c-myc occurred at 4 h post stimulation. In contrast, nutrients resulted in only a slight up-regulation of c-myc (1.8-fold) at approximately 90 min after addition. Addition of nutrients and/or growth factors to the poorly differentiated colon carcinoma cells resulted in an initial decline in c-myc expression (90 min), presumably due to removal of endogenous growth stimulators. Expression of c-myc returned to baseline levels by 24 h after additions. The results indicate that differential sensitivity to polypeptide growth factors is related to differentiation status in this model system and suggest that the insensitivity of poorly differentiated cells to exogenous growth factors may be due to a greater production of autocrine growth stimulators.

Topics: Carcinoma; Cell Differentiation; Cell Division; Colonic Neoplasms; Culture Media; DNA Replication; Epidermal Growth Factor; Gene Expression Regulation; Humans; Insulin; Kinetics; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; RNA, Neoplasm; Transferrin; Tumor Cells, Cultured

A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.

Topics: Amino Acids; Animals; Antigens, Neoplasm; Carcinoma; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Female; Insulin; Integrin alpha6beta4; Integrins; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Phosphorylation

Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controlling factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type beta transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF exhibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma.

Topics: Carcinoma; DNA; Epidermal Growth Factor; Fibroblast Growth Factors; Humans; Neoplasms; Transforming Growth Factors; Tumor Cells, Cultured

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.

Topics: Adenosine Triphosphate; Antifungal Agents; Azides; Benzimidazoles; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Microscopy; Microscopy, Electron; Microtubules; Nocodazole; Sodium Azide; Tumor Cells, Cultured

The effects of epidermal growth factor (EGF) on membrane potential were investigated in suspensions of the following three cell types endowed with a large complement of specific receptors: EGFR-T17 (a clone of mouse NIH-3T3 fibroblasts overexpressing EGF receptors); A431 and KB (two human carcinoma lines). In all these lines EGF induced a rapid and marked hyperpolarization constituted by an initial peak (in all three cell lines) and a subsequent sustained plateau phase, concomitant with the well-known increase of [Ca2+]i. The time course and phorbol ester inhibitability of the membrane potential effects were the same as for the [Ca2+]i response. Experiments with Na+-free and chloride-free media excluded a major role of the latter ions in the EGF-induced hyperpolarization. In contrast, experiments with high K+ media, with the monovalent cation ionophore gramicidin and with Ca2+-free media together with either a Ca2+ ionophore (ionomycin, in A431 and EGFR-T17), or an agonist (bradykinin, in A431) addressed to a receptor coupled to phosphoinositide hydrolysis, were consistent with the involvement of Ca2+-activated K+ channels. The EGF-induced hyperpolarization was completely blocked by the K+ channel blocker, quinidine, and unaffected by a variety of other drugs. Patch clamping of individual EGFR-T17 cells confirmed the initial hyperpolarization (from approximately -30 mV, the resting potential, to -60, -80 mV) was due to activation of an outward current. This initial hyperpolarization was followed by fluctuations (period approximately 1 min) persisting as long as the cells could be analyzed. Thus, the changes of membrane potential appear to be not only novel members of the group of early events triggered by EGF in target cells but also long-lasting effects of the growth factor, which continue for unexpectedly long periods of time after EGF application.

Topics: Animals; Calcium; Carcinoma; Cell Line; Cytosol; Electric Conductivity; Epidermal Growth Factor; Fibroblasts; Humans; Membrane Potentials; Mice; Potassium Channels; Tumor Cells, Cultured

Abnormally high expression of epidermal growth factor receptors (EGF-receptors) may contribute to the unregulated growth of some tumors. We here report the EGF-receptor numbers and the effects of epidermal growth factor (EGF) on two human cell lines. The glioblastoma cell line T-MG1 had 135,000 EGF-receptors per cell, was slightly growth stimulated by EGF and showed no obvious change in morphology after exposure to EGF. The carcinoma cell line T-CAR1, derived from a brain metastasis of a carcinoma of the adrenal cortex, had approximately 7 million EGF-receptors per cell. EGF had a significant antiproliferative effect on these cells and caused rounding and detachment of cells in adherent cultures. The cell lines may become useful in future studies concerning the role of the EGF-receptors in malignant growth.

Topics: Brain Neoplasms; Carcinoma; Cell Division; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation; Humans; Neoplasm Metastasis; Time Factors; Tumor Cells, Cultured

A high false negative rate from endoscopic forceps biopsy is well-known in gastric carcinoma. The initial aim of the present study was to determine whether possible thickening of adjacent nontumorous mucosa by nonspecific or specific trophic factors could contribute to this observation; 167 gastrectomy specimens (77 carcinomas, 14 lymphomas, 76 gastric ulcers) were examined and mucosal thickness measured. Mean thickness of uninvolved mucosa near carcinoma (1.4 +/- 0.08 mm, mean +/- SEM) and near lymphoma (1.5 +/- 0.1 mm) was in each case significantly greater than mucosal thickness near ulcer (1.14 +/- 0.05 mm) or at a distance in the same specimen (P less than 0.01 for each comparison). A subset of specimens representing 20% of carcinomas, showed marked mucosal thickening (2.01 +/- 0.05 mm) above the control mean. Immunohistochemical evaluation for intratumoral epidermal growth factor content (EGF) correlated with mucosal thickness in all groups examined (R = 0.67). Immunostaining for EGF receptor showed similar patterns of expression to those of EGF. EGF and EGF receptor contents were also correlated with depth of invasion when possible. In conclusion, the mucosal thickening adjacent to gastric malignancy may well contribute to the insensitivity of endoscopic forceps biopsy. More importantly, the higher tumor EGF and EGF-receptor contents seen in these lesions may prove to be a useful marker of biologic behavior and predictor of prognosis in these tumors.

Topics: Biomarkers, Tumor; Carcinoma; Epidermal Growth Factor; ErbB Receptors; False Negative Reactions; Female; Gastrectomy; Gastric Mucosa; Humans; Lymphoma; Male; Stomach Neoplasms

T3M4 human pancreatic carcinoma cells avidly bound and internalized 125I-labeled epidermal growth factor (EGF) but did not readily degrade the ligand. Pulse-chase experiments in which the cell-bound radioactivity was allowed to dissociate into the incubation medium in the presence of unlabeled EGF indicated that the majority of the released 125I-EGF consisted of intact EGF and a slightly processed species that readily bound to the cell. Omission of unlabeled EGF during the chase period markedly decreased the amount of radioactivity in the incubation medium, mainly as a result of the rebinding of EGF to the cells. In contrast, T3M4 cells readily degraded 125I-labeled transforming growth factor-alpha (TGF-alpha), and the released radiolabeled products did not rebind to the cells. Both ligands were released from T3M4 cells under acidic conditions, complete dissociation occurring at a pH of 4.5 for EGF, and a pH of 6.5 for TGF-alpha. A 431 human epidermoid carcinoma cells and ASPC-1 human pancreatic carcinoma cells also failed to extensively degrade 125I-EGF, whereas Rat-1 fibroblasts markedly degraded the growth factor. As in the case of T3M4 cells, ASPC-1 cells extensively degraded 125I-TGF-alpha. Degradation of either ligand was blocked by the lysosomotropic compound methylamine in all the tested cell lines. Immunoprecipitation of the EGF receptor with specific polyclonal antibodies and Western blot analysis revealed the anticipated 170-kDa protein in T3M4 cells. Both EGF and TGF-alpha enhanced EGF receptor degradation, but TGF-alpha was less effective than EGF. These findings indicate that in certain cell types EGF and TGF-alpha may be differentially processed.

Topics: Animals; Binding, Competitive; Carcinoma; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Iodine Radioisotopes; Kinetics; Mice; Pancreatic Neoplasms; Transforming Growth Factors; Tumor Cells, Cultured

The biological effects of 1.25 (OH)2D3 on epidermal growth receptor (EGF-R) and on EGF internalization were examined in human mammary carcinoma BT-20 cells. In this cell line, with known amplification of the epidermal growth factor receptor gene. EGF was not stimulatory for growth. Biological assay and quantitative EM autoradiography combined with iodinated ligand binding to specific receptors demonstrated that the number of binding sites unit of length of plasma membrane was 2.48-fold higher in treated than in control cells. I-EGF was progressively internalized in a time-and temperature-dependent manner after selective association with the membrane-coated pits. No modification of the time course of I-EGF internalization was noted in the control and in the treated cells, but a different distribution of the labeling in the subcellular compartment was observed in treated cells. In 1.25(OH)2D3-treated batches, the grain density remained low in the receptosomes throughout the experiment, whereas it was high and occurred early in the lysosomes. On the other hand, in control cells, the grain density of the receptosomes was high, whereas it occurred late and was relatively low in the lysosomes. These data suggest that 1.25(OH)2D3 is a regulator of EGF-R level in BT-20 cell line, but it cannot affirmed whether this effect is direct or mediated by other parameters.

Topics: Autoradiography; Breast; Breast Neoplasms; Calcitriol; Carcinoma; Cell Division; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Iodine Radioisotopes; Microscopy, Electron; Organelles; Tumor Cells, Cultured

The expression of epidermal growth factor (EGF) receptor was examined immunohistochemically in a total of 122 gastric and 61 colonic carcinomas, out of which 16 gastric and 8 colonic carcinomas were also examined by 125I-labeled EGF binding analysis and Western blotting. The values of EGF binding were 12.68 +/- 1.98 (SE; n = 16) fmol/mg protein in gastric carcinomas and 5.72 +/- 2.15 (n = 8) fmol/mg protein in nonneoplastic gastric mucosa, the difference being significant (P less than 0.01). In the colonic tissue, the binding capacities in carcinomas and nonneoplastic mucosa were 13.29 +/- 4.17 (n = 8) and 10.68 +/- 0.41 (n = 3) fmol/mg protein, respectively. Scatchard analysis of 125I-labeled EGF binding indicated a single class of receptors in gastric and colonic carcinomas with an apparent Kd value of from 111 to 277 (n = 4) and from 87.4 to 341 fM (n = 5), respectively, except for one gastric carcinoma having two classes of receptors (Kd = 15.9 and 896 fM). In Western blotting using monoclonal anti-EGF receptor antibody, various levels of EGF receptor expression were detected in 12 (85.7%) of the 14 gastric carcinomas and in 7 (87.5%) of the 8 colonic carcinomas. Immunohistochemically, EGF receptor immunoreactivity was detected in one (3.8%) of the 26 early gastric carcinomas, while it was observed in 33 (34.4%) of the 96 advanced gastric carcinomas, the incidence between the two being significantly different (P less than 0.01). In the colonic carcinomas, 47 (77.1%) of the 61 cases showed positive immunoreactivity to EGF receptor, which did not differ by histological type.

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Humans; Immunohistochemistry; Iodine Radioisotopes; Staining and Labeling; Stomach Neoplasms

Epidermal growth factor (EGF) receptors on primary-cultured human thyroid cells from 27 neoplasias (nine adenomas and 18 differentiated carcinomas) were analyzed and compared with those on the cultured nonneoplastic part of human thyroid cells. Total binding of 125I-EGF to the nonneoplastic part, adenoma, and carcinoma cells did not differ significantly. Scatchard analysis showed that the neoplastic human thyroid cells, like their adjacent nonneoplastic counterparts, consistently possessed EGF receptors with two components. In a paired study of five patients, the association constant of the carcinoma cells' high-affinity component (Ka1) was found to be significantly lower than that of adjacent nonneoplastic thyroid cells (P less than 0.05). Furthermore, a study of the cells from 18 carcinomas revealed that overall their Ka1s (4.15 +/- 0.82 x 10(9) M-1, mean +/- SEM) were significantly lower than those of adenoma cells (10.34 +/- 1.51 x 10(9) M-1, n = 9) and of nonneoplastic cells adjacent to them (8.32 +/- 0.84 x 10(9) M-1, n = 23). The difference in Ka1s for adenoma and nonneoplastic thyroid cells was not statistically significant. The number of receptor sites (Cmax) per cell was not significantly different in any of the three. Incorporation of [3H]thymidine (dThd) increased significantly in all kinds of thyroid cells examined following the addition of 10 nM EGF, and the paired study showed that the size of this increase was not significantly different in neoplastic and adjacent nonneoplastic cells. The addition of 300 microunits/ml of thyroid-stimulating hormone caused a significant increase in dThd incorporation by adenoma cells but not by carcinoma or nonneoplastic cells. Furthermore, combined treatment with EGF and thyroid-stimulating hormone additively promoted adenoma cell growth only. A close inverse relationship was observed between Ka1 and the stimulatory effect of EGF on the dThd uptake in both nonneoplastic thyroid cells and adenoma cells. Carcinoma cells also showed similar profiles, but Ka1 relative to dThd increases were much smaller than the other two.

Topics: Adenoma; Carcinoma; Cell Division; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Humans; Thyroid Neoplasms; Thyrotropin; Tumor Cells, Cultured

Tissue extracts prepared from human renal tissue, renal cell carcinoma and serum-free conditioned media of ACHN cells and A498 cells, cell line originated from human renal cell carcinoma, stimulated DNA synthesis of BALB/c 3T3 cells. The activity (growth factor activity) was significantly higher in renal cell carcinoma than in normal tissues. Radioreceptor assay revealed that the contents of epidermal growth factor and type alpha transforming growth factor in the tissue extracts from renal cell carcinoma and conditioned media from renal cell carcinoma cell lines were below detectable level. Most of growth factor activity of the tissue extracts and conditioned media showed high affinity for heparin-Ultrogel, indicating that the major growth factor activity was due to heparin-binding growth factor(s). In addition, renal cell carcinoma contained growth factor activity for ACHN cells, which did not show specific affinity for heparin-Ultrogel.

Topics: Binding, Competitive; Carcinoma; Carcinoma, Renal Cell; Chromatography, Affinity; DNA; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Heparin; Humans; Kidney Neoplasms; Tumor Cells, Cultured

The human colon cancer cell line HT-29 produces a growth factor (CRDGF; Mr = 25,000) which inhibits EGF binding to a wide variety of different normal and tumoral cell types in culture. Scatchard analysis of EGF binding shows that CRDGF induces a decrease in EGF receptor affinity. In contrast, EGF binding to any of the human colorectal cancer cell lines tested, i.e., HT-29, HT-29 (clone D4), HRT-18 or CAL-14, remains unaltered in the presence of exogenous CRDGF. However, the inhibitory effect of CRDGF becomes apparent on HT-29 cells after overnight exposure of these to suramin (at 37 degrees C). A short exposure to suramin (1 hr at 4 degrees C) or a mild acid washing of HT-29 cells can partially restore the inhibitory activity of CRDGF. These observations suggest that the action of suramin results in an unmasking of substantial levels of CRDGF receptors on HT-29 cells. Scatchard analysis of EGF binding on suramin-treated HT-29 cells shows that CRDGF inhibits EGF binding by decreasing EGF receptor affinity, as previously observed with the non-colonic cell types. A similar unmasking of CRDGF receptors is observed when the other colorectal cell lines are exposed to suramin. These results provide evidence for a model in which the colorectal cell lines have the property of secreting a unique growth factor that binds to its receptor by an autocrine mechanism.

Topics: Carcinoma; Colonic Neoplasms; Epidermal Growth Factor; ErbB Receptors; Growth Substances; Humans; Suramin; Tumor Cells, Cultured

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

Addition of epidermal growth factor (EGF) to cultures of the urinary bladder carcinoma cell line 5637 regulated proliferation and production of colony stimulating activity (CSA). The optimal concentration range of EGF for stimulation of cell proliferation was 5-20 ng/ml EGF and for production of CSA 2-20 ng/ml EGF. High EGF concentrations (100-200 ng/ml) showed inhibitory effects on proliferation and to a greater extent on CSA production. Also, EGF binding sites of high affinity (kd:3.25 nM) were demonstrated on the cell surface. In the optimal concentration range for stimulation (5-20 ng/ml EGF) EGF binding sites were occupied half-maximally. The loss in EGF binding after long incubation at 37 degrees C was prevented by the lysosomal inhibitory agent, chloroquine. Nonspecific binding of EGF was very low, the amount of maximally bound EGF was 1430 fmol/mg protein (130,000 bound EGF molecules/cell). A strong band of approximately 170,000 daltons could be detected by means of an anti-erbB serum which recognizes the EGF receptor protein. The protein became phosphorylated upon addition of gamma-32P ATP. The data suggest that EGF initiates its action by binding to specific high affinity receptors and plays a role in growth regulation and differentiation of the urinary bladder carcinoma cell line 5637.

Topics: Carcinoma; Cell Division; Chemical Precipitation; Colony-Stimulating Factors; Epidermal Growth Factor; ErbB Receptors; Humans; Immunologic Techniques; Kinetics; Phosphoproteins; Temperature; Tumor Cells, Cultured; Urinary Bladder Neoplasms

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.

Topics: Antineoplastic Agents; Breast Neoplasms; Carcinoma; Cell Division; Cell Line; Cell Survival; Cells, Cultured; Culture Media; Epidermal Growth Factor; Extracellular Matrix; Gastrointestinal Neoplasms; Humans; Lung Neoplasms; Melanoma; Neoplasms; Sarcoma; Urogenital Neoplasms

We have developed a defined serum-free medium for the in vitro growth of normal and neoplastic urothelium. Growth of epithelial cells in this medium was stimulated while proliferation of fibroblasts was inhibited. The medium allowed for the isolation of a new line from a high grade human bladder carcinoma. Several strains of normal human urothelium that could be passaged several times in culture were also obtained. Although growth of normal and neoplastic urothelial cells in the defined medium was not stimulated by epidermal growth factor, the presence of 50 ng/ml epidermal growth factor induced morphological changes suggestive of terminal maturation of normal urothelium, implying that epidermal growth factor either acted as an epithelial maturation factor or induced the secretion of such a factor.

Topics: Adult; Blood Physiological Phenomena; Calcium; Carcinoma; Cell Line; Culture Media; Epidermal Growth Factor; Humans; Urinary Bladder; Urinary Bladder Neoplasms

Modulation of epidermal growth factor (EGF) receptor expression determines cellular responsiveness to EGF and might play an important role in growth inhibition. We have investigated the actions of EGF and/or transforming growth factor type beta (TGF beta) on EGF receptor gene expression in MDA-468 human breast carcinoma cell line, which responds to EGF and/or TGF beta with growth inhibition. Using the cDNA clone pE7, which encodes 2.4 kilobases of the human EGF receptor mRNA, as a hybridization probe, we have found that exposure of MDA-468 cells to EGF results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta enhances the accumulation of EGF receptor mRNA induced by EGF. Under this condition, stimulation could be detected after 1 h exposure to TGF beta with a maximum at 6-8 h. A concentration of 10 pM TGF beta gave detectable stimulation with maximal stimulation occurring at 300 pM in the presence of EGF (50 ng/ml). In contrast, TGF beta alone had no significant effect on EGF receptor mRNA accumulation. In the presence of cycloheximide, the EGF receptor mRNA was super-induced in response to EGF. Treatment of the cells with TGF beta enhances the EGF-dependent superinduction of EGF receptor mRNA produced by cycloheximide, suggesting that the stimulatory action of TGF beta does not depend on continuous protein synthesis. The results described here are consistent with the hypothesis that the growth inhibitory action of TGF beta in MDA-468 cells may be mediated, at least in part, by modulation of EGF receptor gene expression.

Topics: Breast Neoplasms; Carcinoma; Cell Line; Cycloheximide; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation; Humans; Kinetics; Peptides; RNA, Messenger; Transforming Growth Factors

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

Epidermal growth factor (EGF)-like factors with EGF competing and cell growth stimulating activity were investigated in malignant and nonmalignant tissues. About 37% of ovarian carcinomas present an increased factor activity between 9.0 and 19.3 ng EGF units/mg protein. In one tumor 175.0 ng EGF units/mg protein were found. In extracts of nonmalignant tissues, the factor concentration was about 1.0-6.4 ng EGF units/mg protein. Isoelectric focusing was performed to characterize these factors. In normal ovaries and ovarian carcinomas, factors with EGF competing activity focus at pH 8.0-9.0, pH 5.7-6.3, and pH 3.6-4.9. In ovarian carcinomas, an additional peak with EGF competing and cell growth stimulating activity was found between pH 6.5 and 7.2. Similar results could be achieved in other malignant tissues investigated. These data indicate the presence of EGF-like factors. EGF itself focuses at pH 4.6 (G. Carpenter and S. Cohen, Annu. Rev. Biochem., 48: 193-216, 1979). Specific EGF binding was determined in 12 ovarian carcinomas. In five of these cases EGF receptors could be detected. In the EGF receptor positive carcinomas, the content of EGF-like growth factors varied between 0 and 9 ng EGF units/mg protein. In EGF receptor negative cases, the content of EGF-like growth factors varied between 0 and 19.3 ng EGF units/mg protein. The clinical data of 19 patients are also demonstrated.

Topics: Animals; Binding, Competitive; Biological Assay; Breast Neoplasms; Carcinoma; Epidermal Growth Factor; ErbB Receptors; Female; Growth Substances; Humans; Isoelectric Point; Melanoma; Mice; Ovarian Neoplasms; Radioligand Assay; Receptors, Cell Surface; Sarcoma

c-Ha-ras oncogene product in human gastric carcinomas was examined by Western blotting and immunohistochemistry using anti-Ha-ras p21 antibody. In Western blotting, high levels of c-Ha-ras p21s were found in gastric carcinomas. Immunohistochemically, c-Ha-ras p21 was detected in 3 (11.1%) of 27 early carcinomas and in 63 (43.8%) of 144 advanced carcinomas. In advanced carcinomas, c-Ha-ras p21-immunoreactivity was correlated with the depth of tumor invasion and was stronger in metastatic tumors than in primary tumors. Patients with c-Ha-ras p21-positive carcinomas had a significantly worse prognosis than those with p21-negative carcinomas.

Topics: Amino Acid Sequence; Carcinoma; Epidermal Growth Factor; Histocytochemistry; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Oncogenes; Prognosis; Proto-Oncogene Proteins; Stomach Neoplasms

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

Two distinctly different clonal cell lines were isolated from a mammary tumor induced by ingestion of 7,12-dimethylbenz[a]anthracene (CAS: 57-97-6) in a SD rat. Cells of one of the clones, RMT-1 clone E4, showed typical epithelial characters; it was concluded that they were derived from neoplastic mammary epithelial cells. The other clone, M2, exhibited characters consistent with its derivation from the normal mammary myoepithelial component. The 2 cell lines had different proliferative responses to growth factors (GF) such as cholera toxin, dexamethasone, and epidermal GF. The epithelioid E4 cells were found to produce potent growth-promoting activity in culture medium that stimulated proliferation of myoepithelial M2 cells as well as that of stromal fibroblasts. The present work provides supporting evidence that the mechanism of "paracrine stimulation" is operative in the mammary tumorigenesis of the rat.

Topics: Animals; Carcinoma; Cell Line; Culture Media; Cytoskeleton; Epidermal Growth Factor; Female; Glycoproteins; Growth Substances; Keratins; Mammary Neoplasms, Experimental; Rats; Rats, Inbred Strains

Several ligands, including epidermal growth factor (EGF), have been found to negatively modulate or down-regulate their specific plasma membrane receptors. Using both 125I-EGF and a monoclonal antibody against the EGF-receptor (EGF-R1), we studied the down-regulation of the EGF-receptor in the human adenocarcinoma cell line KB. The results presented here demonstrate that incubating KB cells at 37 degrees C with EGF rapidly decreases the number of plasma membrane EGF-receptors. In addition, there is a concomitant rise of equal magnitude in the number of EGF molecules taken up. The latter result argues strongly that there is negligible recycling of the EGF-receptor in KB cells and that the major portion of internalized EGF-receptor complexes are transported to lysosomes and subsequently degraded. The fate of the EGF-receptor is markedly different from that of receptors not subject to down-regulation. The biochemical signals that operate to regulate such diverse receptor traffic in cells remains to be elucidated.

Topics: Carcinoma; Epidermal Growth Factor; ErbB Receptors; Humans; KB Cells; Kinetics; Mouth Neoplasms; Receptors, Cell Surface

A 160,000 mol wt precursor of the epidermal growth factor (EGF) receptor has been identified in human A-431 carcinoma cells and skin fibroblasts. The presence of one discrete precursor band indicates the presence of a slow processing step. We have determined that this slow processing step involves the conversion of high mannose N-linked oligosaccharides on the receptor precursor to primarily complex oligosaccharides on the mature form of the receptor. This is shown by 1) the presence of fucose, a characteristic terminal sugar of complex oligosaccharides, in only the mature receptor and by 2) the susceptibility of the precursor to digestion with endoglycosidase H, which cleaves high mannose N-linked oligosaccharides, but not complex oligosaccharides from glycoproteins. The precursor to mature receptor transition half-time is 1.7 h in A-431 cells. This long transition half-time causes an accumulation of approximately 7.2 X 10(5) precursor molecules per cell (approximately 12% of the total population of EGF receptors). The net quantity of mature EGF receptors, but not of receptor precursors, is reduced when EGF is added to the culture medium of A-431 cells. The presence of EGF in the growth medium also decreases electrophoretic migration (as a result of increased phosphate incorporation) of the mature receptor, but not that of the precursor. The EGF-insensitive state of the precursor is most likely due to its intracellular location.

Topics: Carcinoma; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; In Vitro Techniques; Kinetics; Molecular Weight; Protein Precursors; Receptors, Cell Surface; Skin; Trypsin

PANC-1 human pancreatic carcinoma cells readily bound and internalized 125I-labeled epidermal growth factor (EGF). Bound 125I-labeled EGF was then partially processed to a number of high molecular weight acidic species. Percoll gradient centrifugation of cell homogenates indicated that the majority of 125I activity localized to several intracellular vesicular compartments. Both intact EGF and its processed species were subsequently released into the incubation medium. A major portion of the released radioactivity was capable of rebinding to the cell. Only a small amount of bound 125I-labeled EGF was degraded to low molecular weight products, and this degradation was completely blocked by methylamine. This lysosomotropic compound did not arrest either the generation or the extrusion of the major high molecular weight species of processed EGF (pI 4.2). These findings suggest that in PANC-1 cells, bound EGF undergoes only limited processing. Both intact EGF and its major processed species bypass the cellular degradative pathways, are slowly released from the cell, and then rebind to the cell.

Topics: Biological Transport; Carcinoma; Cell Line; Culture Media; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Exocytosis; Humans; Isoelectric Point; Lysosomes; Molecular Weight; Pancreatic Neoplasms; Peptide Fragments; Receptors, Cell Surface

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.

Topics: Carbohydrates; Carcinoma; Cell Line; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; ErbB Receptors; Humans; Molecular Weight; Peptide Fragments; Phosphorylation; Receptors, Cell Surface; Trypsin; Tyrosine

The effect of epidermal growth factor (EGF) on the synthesis of the nuclear protein cyclin and its relationship with cell proliferation has been studied in human carcinoma A431 cells. Quantitative two-dimensional gel electrophoretic analysis of [35S]methionine-labeled polypeptides from EGF-treated and untreated A431 cells showed that the synthesis of cyclin (0.07% of total labeled protein) could be decreased to 20% (less than 0.01% of total labeled protein) by EGF at a concentration of 100 ng/ml. A good correlation between the effect of different concentrations of EGF on cyclin synthesis and A431 cell proliferation was found. A431-derived cells resistant to the growth-inhibitory effect of EGF showed no significant changes in cyclin synthesis after EGF treatment. Taken together, these results support the idea that cyclin may be an important protein that is involved in the control of cell proliferation.

Topics: Carcinoma; Cell Cycle; Cell Nucleus; Cells, Cultured; Drug Resistance; Epidermal Growth Factor; Humans; Isoelectric Point; Molecular Weight; Neoplasm Proteins; Nucleoproteins; Proliferating Cell Nuclear Antigen

The kinetics of association of epidermal growth factor (EGF) and diferric-transferrin (TF) with clathrin-coated membranes of KB cells were examined using anti-clathrin antibody bound to Staphylococcus aureus. The ligands were bound to cells at 4 degrees C and after warming to 30 degrees C for 5 min, both ligands were found concentrated in a membrane fraction immunoadsorbed with anti-clathrin antibody. Fifteen minutes after entry both ligands moved to a non-clathrin-associated compartment. Twenty to 25 min after entry, EGF, but not TF, appeared in a second clathrin-associated compartment. In parallel morphologic experiments, EGF-horseradish peroxidase (EGF-HRP) and TF-HRP were both found at 6 min in coated pits associated with the plasma membrane. At 22 min EGF-HRP, but not TF-HRP, was localized in Golgi-associated coated pits. These studies provide biochemical and morphological evidence suggesting that coated membranes in the Golgi region are involved at some stage in the transfer of EGF to lysosomes. The method should be of general utility in the study of receptor-mediated endocytosis.

Topics: Biological Transport; Carcinoma; Cell Line; Clathrin; Coated Pits, Cell-Membrane; Endosomes; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Microscopy, Electron; Mouth Neoplasms; Receptors, Cell Surface; Receptors, Transferrin; Transferrin

Using a monoclonal antibody to the human epidermal growth factor (EGF) receptor (EGF-R1), we have followed the metabolism of the receptor and the pathway of its internalization in KB cells after the addition of EGF. Measurement of surface binding of 125I-labeled EGF showed that about 80% of EGF binding activity disappeared from the plasma membrane after a 10-min exposure to EGF at 37 degrees C. Immunoprecipitation of the receptor from [35S]methionine-labeled cell extracts with EGF-R1 showed that EGF caused the receptor to be degraded with a half-life of 40 min. Immunofluorescence using EGF-R1 showed an EGF-dependent redistribution of the EGF receptor. In cells not exposed to EGF, almost all of the receptor was diffusely distributed on the cell surface. After EGF addition, the receptor was rapidly internalized, first appearing in small punctate organelles characteristic of receptosomes and then in larger perinuclear lysosome-like structures. By 120 min almost all of the immunoreactive EGF receptor had disappeared from the cells. Immunocytochemistry at the electron microscopic level confirmed these light microscopic findings. The diffusely distributed receptor on the cell surface first clustered into clathrin-coated pits in the presence of EGF, next was internalized into receptosomes, appeared transiently in transreticular Golgi elements, and finally was seen in lysosomes. This EGF-dependent down-regulation and degradation of the EGF receptor in KB cells provides a striking example of ligand-dependent clustering and internalization of a receptor, followed by degradation in lysosomes of both ligand and receptor.

Topics: Antibodies, Monoclonal; Biological Transport; Carcinoma; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; KB Cells; Kinetics; Lysosomes; Microscopy, Electron; Mouth Neoplasms; Receptors, Cell Surface

A conjugate of Pseudomonas exotoxin and epidermal growth factor (PE-EGF) inhibits proteins synthesis in KB cells, and this inhibition is increased by adenovirus. Protein synthesis inhibition is dependent on the amount of adenovirus and PE-EGF used and the time of incubation of cells with these agents. With 1 microgram of adenovirus and 0.5 micrograms of PE-EGF per ml, protein synthesis is inhibited about 80% in a 60-min experiment. Under these conditions neither adenovirus nor PE-EGF alone has any effect. In the presence of several weak bases or monensin, the enhancement of toxicity was substantially inhibited; half-maximal inhibition was achieved with 40 microM chloroquine, 10 mM ammonium chloride, 5 mM methylamine, 0.1 mM N-hexylamine and 1 microM monensin. At the concentrations employed, none of the inhibitors affected the amount of virus taken up or bound to the cell surface, and chloroquine had no effect on the amount of EGF taken up in 60 min. Chloroquine did not prevent the toxicity of the PE-EGF (5 micrograms/ml) alone. Because these compounds are known to elevate the pH in receptosomes, it seems likely that the acidification of the receptosome either enhances the lysis of the membrane by adenovirus or enhances some other step in the release of PE-EGF.

Topics: Adenoviruses, Human; Amines; Ammonium Chloride; Carcinoma; Cell Line; Chloroquine; Drug Synergism; Epidermal Growth Factor; Exotoxins; Humans; Hydrogen-Ion Concentration; Kinetics; Methylamines; Monensin; Mouth Neoplasms; Protein Biosynthesis; Pseudomonas; Receptors, Virus

When KB cells labeled with 51Cr (1 muCi/ml) were treated with adenovirus type 2 (Ad2) at pH 6, 51Cr was released in a concentration-dependent manner with half-maximal release at 1 microgram/ml of virus. The 51Cr release was maximum at 10 micrograms/ml and represented nearly 20% of cell-associated 51Cr. 51Cr release depended on the length of incubation with Ad2 and the pH of the medium; maximum release was at pH 6 with very little release at pH 7.5. An antiserum against the penton base of Ad2 specifically neutralized the ability of Ad2 to release 51Cr. Chloroquine up to 200 microM did not block Ad2-dependent 51Cr release. DEAE-dextran stimulated Ad2-dependent 51Cr release; 4-5-fold stimulation was observed at 50 micrograms/ml of DEAE-dextran. We have compared the ability of Ad2 to release 51Cr with its ability to disrupt endocytic vesicles. Vesicle disruption was independent of pH of the medium in the range of pH 6 to 7.5 and was blocked by chloroquine, whereas, 51Cr release was much greater at pH 6 than at pH 7.5 and was not affected by chloroquine. These results suggest that Ad2 possess a lytic activity which ordinarily lyses acidic endocytic vesicles but, if the cells are maintained at acidic pH, can also directly disrupt the plasma membrane.

Topics: Adenoviruses, Human; ADP Ribose Transferases; Bacterial Toxins; Carcinoma; Cell Transformation, Viral; Chloroquine; Chromium Radioisotopes; Epidermal Growth Factor; Exotoxins; Humans; Hydrogen-Ion Concentration; KB Cells; Kinetics; Mouth Neoplasms; Pseudomonas aeruginosa Exotoxin A; Virulence Factors

A monoclonal antibody to the epidermal growth factor (EGF) receptor of A431 cells was obtained after fusion of immunized BALB/c mouse spleen cells with NS-1 myeloma cells. Specific binding of the antibody to the plasma membrane of A431 cells was demonstrated by indirect immunofluorescence and electron microscopy. The antibody did not react with human KB cells, normal rat kidney cells, or Swiss 3T3 cells. The antibody is an IgG3K; it specifically immunoprecipitated a Mr approximately 170,000 protein from radiolabeled A431 cell extracts. This protein is phosphorylated in a EGF-dependent manner in intact A431 cells and in Triton X-100-solubilized plasma membranes. The specificity of the interaction of the antibody with the Mr = 170,000 protein was confirmed by electrophoretic transfer of A431 cell proteins to nitrocellulose followed by incubation with the antibody and 125I-protein A. When 125I-EGF was covalently cross-linked to its receptor, the 125I-EGF-receptor complex was specifically precipitated by the antibody. The monoclonal antibody did not inhibit the binding of 125I-EGF to its receptor in intact A431 cells and also failed to stimulate the phosphorylation of the Triton X-100-solubilized EGF receptor. The results indicate that the antibody and EGF bind to different sites on the EGF receptor. The antibody will be useful for isolating the EGF receptor in an unactivated form.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Carcinoma; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Mice; Mice, Inbred BALB C; Molecular Weight; Mouth Neoplasms; Receptors, Cell Surface

Urine from 22 patients with a variety of disseminated cancers and from an equivalent number of nonmalignant controls of similar age and sex was tested for the presence of transforming growth factor (TGF) activity as measured by the ability to promote the growth in soft agar of nontransformed indicator cells. Cancer patients included those with carcinomas of the lung, breast, colon, and ovary, as well as melanomas and sarcomas. The nonmalignant controls included both normals and individuals with a variety of inflammatory and infectious disorders. Aliquots of unfrozen urine were acid extracted, chromatographed on a Bio-Gel P-30 column, and then tested for TGF activity using normal rat kidney fibroblasts and epidermal growth factor (EGF)-competing activity with human carcinoma A431 cells. These assays revealed that a high-molecular-weight TGF activity (Mr 30,000 to 35,000) which coelutes with EGF-competing activity was present in 18 of 22 cancer patients but present in only five of 22 nonmalignant controls (p less than 0.01). In contrast, a low-molecular-weight TGF activity (Mr 6000 to 8000) which does not coelute with EGF-competing activity was found in all urines tested. These results indicate that an EGF-related, high-molecular-weight TGF activity is found in the urine of cancer patients and may be a useful tumor marker. Unlike other tumor markers described previously, high-molecular-weight TGF activity has a biological activity which is related to the expression of the transformed phenotype.

Topics: Binding, Competitive; Carcinoma; Cell Transformation, Neoplastic; Chromatography, Gel; Epidermal Growth Factor; Humans; Molecular Weight; Neoplasms; Peptides; Transforming Growth Factors

A new human breast carcinoma cell line (PMC42) has been further characterized. The cells can grow either as monolayers or as floating cords of cells. The cords grow in suspension for long periods but may spontaneously attach and grow out to form a typical PMC42 monolayer. Ultrastructurally, the cells resemble breast ductal cells in many respects. Both epidermal growth factor (EGF) and prolactin induce ultrastructural changes, and lipid production is stimulated markedly by both factors. EGF also promoted the attachment of the floating cords and the growth of cells from these cords as monolayer cultures. The karyotype of the cord cells is different from that previously described for the monolayer cultures. Cord cells are hypodiploid (mode 39), whereas the monolayer cultures are subtriploid (mode 66). Although the ploidy is different, the karyotypes are related with 9 marker chromosomes being common to both populations. In addition, cultures in which cords have attached and in which cells are growing out as monolayers are bimodal with 10-20% of the cells becoming pseudotetraploid with a mode of 77.

Topics: Breast Neoplasms; Carcinoma; Cell Line; Cells, Cultured; Epidermal Growth Factor; Female; Humans; Karyotyping; Microscopy, Electron; Neoplastic Stem Cells; Organoids; Ploidies; Prolactin; Stem Cells

Topics: Carcinoma; Cell Line; Cycloheximide; Epidermal Growth Factor; ErbB Receptors; Freezing; Humans; Kinetics; Macromolecular Substances; Mouth Neoplasms; Phorbols; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

Using the direct conjugate of epidermal growth factor (EGF) and horseradish peroxidase, we have followed the entry of EGF into KB (human carcinoma) cells. EGF initially was found bound diffusely to the entire cell surface at 4 degrees C; on warming to 37 degrees C, EGF was found clustered in clathrin-coated pits on the plasma membrane in 1 min or less. Within 1-2 min at 37 degrees C, EGF began to accumulate in receptosomes within the cell and remained there for up to 10 min. At 10-13 min after warming to 37 degrees C, EGF was found in thin reticular membranous elements of the Golgi system, as well as concentrated in the clathrin-coated pits present on these membranes. By 15 min after warming, EGF began to be delivered to lysosomes located near the Golgi system. These findings suggest that clathrin-coated pits in the Golgi reticular system accumulate EGF before delivery to lysosomes.

Topics: Carcinoma; Cell Membrane; Cells, Cultured; Cytoplasmic Granules; Epidermal Growth Factor; ErbB Receptors; Golgi Apparatus; Humans; Intracellular Membranes; Kinetics; Lysosomes; Neoplasms, Experimental; Receptors, Cell Surface; Temperature

Membrane components that interact with epidermal growth factor (EGF) and transforming growth factors (TGFs) have been identified by covalent crosslinking to their respective 125I-labeled ligands. Under appropriate conditions, disuccinimidyl suberate or hydroxysuccinimidyl p-azidobenzoate cross-link receptor-bound 125I-labeled EGF to a 140- to 170-kilodalton (kDal) receptor species in membranes from both A431 human carcinoma cells and normal rat kidney cells. 125I-Labeled sarcoma growth factor (SGF), a TGF from virally transformed mouse 3T3 cells, also can be affinity-crosslinked to the 140- to 170-kDal EGF receptor species in membranes from A431 and rat kidney cells. The labeling of this receptor is inhibited when either excess unlabeled EGF or SGF is present during incubation of membranes with either 125I-labeled EGF or 125I-labeled SGF. In contrast, a second receptor species of 60 kDal is affinity-labeled with 125I-labeled SGF but not with 125I-labeled EGF in membranes from both A431 and rat kidney cells. SGF and a TGF from virally transformed rat embryo cells inhibit the labeling of the 60-kDal species when present in excess during incubation of membranes with 125I-labeled SGF, whereas EGF is completely ineffective in inhibiting the labeling of this receptor. The data suggest that a specific 60-kDal receptor that displays high affinity for TGFs but not for EGF may mediate induction of the transformed phenotype. In addition, SGF and other TGFs interact with the 140- to 170-kDal EGF receptor that appears to mediate normal cell growth effects.

Topics: Affinity Labels; Animals; Carcinoma; Cell Line; Cell Transformation, Neoplastic; Epidermal Growth Factor; ErbB Receptors; Humans; Kidney; Peptides; Rats; Receptors, Cell Surface; Transforming Growth Factors

Topics: Carcinoma; Cells, Cultured; Epidermal Growth Factor; Fibronectins; Humans; Membrane Proteins; Phosphorylation; Protein Kinases; Tetradecanoylphorbol Acetate; Urea

Epidermal growth factor (EGF) stimulates phosphorylation of its own receptor at a tyrosine residue. Similarly, the viral gene product pp60src, which is responsible for cellular transformation by avian sarcoma virus (ASV), phosphorylates itself and immunoglobulin directed against pp60src at tyrosine residues. This unusual site of phosphorylation catalysed by two membrane-associated protein kinases involved in growth control prompted us to study the immunological relatedness of the EGF-stimulated protein kinase and the pp60src. Using anti-pp60src antisera, we attempted to immunoprecipitate the EGF-stimulated protein kinase solubilized from plasma membranes. We report here that neither the EGF-stimulated kinase nor the EGF receptor were immunoprecipitable by anti-pp60src sera. However, anti-pp60src IgG served as a specific substrate for the EGF-stimulated kinase, suggesting a close similarity between the EGF-stimulated kinase and pp60src.

Topics: Antibodies, Neoplasm; Carcinoma; Cross Reactions; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Humans; Oncogene Protein pp60(v-src); Peptides; Phosphoproteins; Phosphorylation; Protein Kinases; Receptors, Cell Surface; Tyrosine; Viral Proteins

Topics: Animals; Carcinoma; Cell Line; Cell Transformation, Viral; Epidermal Growth Factor; Growth Substances; Humans; Kinetics; Mice; Peptides; Receptors, Cell Surface; Sarcoma Viruses, Murine; Submandibular Gland; Transforming Growth Factors

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes. The immunoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5 degrees C). Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.

Topics: Animals; Antigen-Antibody Reactions; Carcinoma; Cell Line; Cell Membrane; DNA Replication; Epidermal Growth Factor; Humans; Immunoassay; Immunoglobulin Fab Fragments; Immunoglobulin G; Mice; Peptides; Rabbits; Receptors, Cell Surface