epidermal-growth-factor and Uterine-Cervical-Neoplasms

The sub-committee constituted by the Indian Council of Medical Research (ICMR) for the management of cervical cancer (CC) detailed in the consensus document (2016) reported CC as a significant cause of morbidity and mortality in women. The incidence of an increase in CC and associated mortality in women is a major cause of cancer. To date, human papilloma viral (HPV) infection accounts for more than 99% of CC. However, there are individuals infected with HPV do not develop CC. There is a greater correlation between HPV infection and upregulation of the epidermal growth factor receptor (EGFR) signaling cascade during the initiation, sustenance, and progression of CC. Therefore, EGFR is often targeted to treat CC using tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAB). The current review analyzed the existing clinical/pre-clinical studies and the significance of EGFR abundance using the Kaplan-Meier (KM) survival plot analysis for disease-free survival (DFS) and overall survival (OS). We performed a series of bioinformatics analyses to screen the crucial role of the EGFR gene in CC. Further, different transcription factors that are dysregulated due to EGFR abundance and their relevance were determined using computational tools in this review. Endogenous microRNAs (miRNA) that undergo changes due to alterations in EGFR during CC were identified using computational database and consolidated the information obtained with the published in the area of miRNA and EGFR with special reference to the initiation, sustenance and progression of CC. The current review aims to consolidate contemporary approaches for targeting CC using EGFR and highlight the current role of miRNA and genes that are differently regulated during CC involving EGFR mutations. Potential resistance to the available EGFR therapies such as TKIs and mABs and the need for better therapies are also extensively reviewed for the development of newer therapeutic molecules with better efficacy.

Topics: Biomarkers; Biomarkers, Tumor; Disease Management; Disease Progression; Disease Susceptibility; Drug Development; Epidermal Growth Factor; ErbB Receptors; Female; Humans; MicroRNAs; Molecular Targeted Therapy; Papillomavirus Infections; Signal Transduction; Treatment Outcome; Uterine Cervical Neoplasms

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

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

Our studies highlight the importance of dietary vitamin A (retinol) and other retinoids in maintaining normal cervical cell function and in inhibiting the growth of cervical tumors. Based on our results we conclude that 1) HPV 16-immortalization enhances cervical cell sensitivity to retinoids, 2) cytokeratin expression may be useful as a marker for evaluating the success of retinoid therapy in vivo, 3) retinoids do not necessarily act to inhibit proliferation of HPV-immortalized cervical cells via effects on HPV E6 and E7 RNA levels and 4) retinoids may act to inhibit cervical proliferation by "suppressing" the activity of the EGF and IGF signalling pathways. Based on these and other results, it is worth considering the possibility that vitamin A or related retinoids could be administered therapeutically, early in the neoplastic process (either systemically or locally), to inhibit the progress of the disease. These results also suggest that combined interferon/retinoid therapy may provide an enhanced beneficial effect to reduce cervical tumor size due to the fact that each agent is inhibiting cervical cell proliferation via distinct, but reinforcing, pathways (i.e., IFN gamma reduces E6/E7 expression, RA inhibits the function of the EGF and IGF1 signalling pathways).

Topics: Epidermal Growth Factor; Female; Humans; Insulin-Like Growth Factor I; Interferons; Papillomaviridae; Papillomavirus Infections; Retinoids; Signal Transduction; Tumor Virus Infections; Uterine Cervical Neoplasms

Evidence has indicated that lysine methyltransferase 2B (KMT2B), a major H3K4 tri-methyltransferase (H3K4me3), contributes to the development of various cancers; however, its role in cervical cancer (CC) is unclear. In this study, increased KMT2B expression was observed in human CC specimens and significantly associated with poor prognosis. The condition medium of KMT2B-overexpressing cells facilitated angiogenesis

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; HeLa Cells; Histone Methyltransferases; Histone-Lysine N-Methyltransferase; Humans; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Uterine Cervical Neoplasms

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

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

Lymphocyte antigen 6 complex locus K (LY6K), a glycosylphosphatidylinositol-anchored protein, plays a dynamic role in cancer metastasis. In the current study, we deciphered the effects of LY6K on transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) signaling through clathrin- and caveolin-1 (CAV-1)-mediated endocytosis.. Analysis of the TCGA and GTEx dataset were performed to explore the expression and survival of LY6K in cancer patients. Short interfering RNA (siRNA) was used to knockdown the expression of LY6K in human cervical cancer patients. The effect of lack of LY6K on cell proliferation, migration, and invasion was performed, and RT-qPCR and immunoblotting were performed to identify LY6K-affected TGF-β and EGF signaling pathways. Additionally, Immunofluorescence (IF) and transmission electron microscope (TEM) were performed to identify the role of LY6K in CAV-1- and Clathrin-mediated endocytosis.. Lymphocyte antigen 6 complex locus K expression level is elevated in higher grade cervical cancer patients correlating with poor overall survival, progression-free survival, and disease-free survival. LY6K-depletion in HeLa and SiHa cancer cells suppressed EGF-induced proliferation and TGF-β-enhanced migration and invasion. Both TGF-β receptor-I (TβRI) and EGF receptor (EGFR) localized at the plasma membrane regardless of LY6K expression, and LY6K bound TβRI irrespective of the presence of TGF-β; however, LY6K did not bind EGFR. LY6K-depleted cells showed impaired Smad2 phosphorylation upon TGF-β treatment and lower proliferation rates following long-term treatment with EGF. We revealed the atypical movement of TβRI and EGFR from plasma membrane upon ligand stimulation in LY6K-depleted cells and an impaired movement of the endocytic proteins clathrin and CAV-1.. Our study demonstrates the key role of LY6K in both clathrin- and CAV-1-mediated endocytic pathways regulated by TGF-β and EGF, and it suggests a correlation between LY6K overexpression in cervical cancer cells and poor overall survival.

Topics: Antigens, Ly; Clathrin; Epidermal Growth Factor; ErbB Receptors; Female; GPI-Linked Proteins; Humans; Transforming Growth Factor beta; Uterine Cervical Neoplasms

Our previous laboratory findings suggested the beneficial effects of epigallocatechin gallate (EGCG) against cervical cancer (CC) cells survival. The present study is aimed at identifying the effects of EGCG in preventing the actions of epidermal growth factor (EGF) in human papilloma virus (HPV) 68 positive ME180 and HPV negative C33A CC cells. An elevated level of EGF in tumor micro-environment (TME) is linked to the metastasis of several cancers including CC. We hypothesized that EGCG has the ability to block the actions of EGF. To test this, survival assay was performed in cells treated with or without EGF and EGCG. The mitochondrial activity of cells was ascertained using MTT assay and mitored staining. Protein and non-protein components in the extracellular matrix such as collagen and sulphated glycosaminoglycans (GAGs) were evaluated using sirius red and alcian blue staining, respectively. Matrix metalloproteinase-2 (MMP-2) gene expression and enzymatic activity were assessed using real time-reverse transcriptase-polymerase chain reaction (RT-PCR) and gelatin zymography. Wound healing assay was performed to assess the EGF induced migratory ability and its inhibition by EGCG pre-treatment. Clonogenic assay showed that EGCG pre-treatment blocked the EGF driven colony formation. In silico analysis performed identified the efficacy of EGCG in binding with different domains of EGF receptor (EGFR). EGCG pre-treatment prevented the epithelial-mesenchymal transition (EMT) and metabolic activity induced by EGF, this is associated with concomitant reduction in the gene expression and enzyme activity of MMP-2. Further, reduced migration and ability to form colonies were observed in EGCG pre-treated cells when stimulated with EGF. HPV positive ME180 cells showed increased migratory and clonogenic ability upon EGF stimulation, whose effects were not much significant in HPV negative C33A cells. EGCG effectively blocked the actions of EGF in both HPV positive and HPV negative conditions and can be advocated as supplementary therapy for the management of EGF driven CC. However, further studies using cell line-derived xenograft (CDX)/patient-derived xenograft (PDX) model system is warranted to validate the therapeutic utility of EGCG.

Topics: Catechin; Epidermal Growth Factor; Female; Humans; Matrix Metalloproteinase 2; Papillomavirus Infections; Tumor Microenvironment; Uterine Cervical Neoplasms

This study aimed to assess the effectiveness and safety of intravesical instillation treatment of Kangfuxin liquid (KFL) combined with thrombin and epidermal growth factor (EGF) for radiation-induced hemorrhagic cystitis (HC) in patients with cervical cancer. A total of 34 patients with radiation-induced HC of grade 2-4 were treated with intravesical instillation of KFL combined with thrombin and EGF until the complete disappearance of hematuria and lower urinary tract symptoms (LUTS). Gentamicin was added if white blood cells were detected and bacterial culture was positive in the urine. All patients were followed up for 2 years to evaluate the clinical efficacy and safety of the treatment regimen. Patients with and without recurrent hematuria (n = 3, 9% and n = 31, 91%, respectively) were completely recovered from hematuria and LUTS by intravesical instillation treatment for 6-22 days. No adverse event was reported during the treatment and the 2-year follow-up for all patients. Thus, intravesical instillation of KFL combined with thrombin and EGF is an effective and safe therapeutic regimen for radiation-induced HC of grade 2-4 in patients with cervical cancer.

Topics: Administration, Intravesical; Adult; Aged; Cystitis; Epidermal Growth Factor; Female; Hemorrhage; Hemostatics; Humans; Materia Medica; Middle Aged; Radiation Injuries; Radiotherapy; Thrombin; Uterine Cervical Neoplasms

Synaptonemal complex protein 3 (SCP3), a member of the Cor1 family, has been implicated in cancer progression, and therapeutic resistance, as well as cancer stem cell (CSC)-like properties. Previously, we demonstrated that SCP3 promotes these aggressive phenotypes via hyperactivation of the AKT signaling pathway; however, the underlying mechanisms responsible for SCP3-induced AKT activation remain to be elucidated. In this study, we demonstrated that the EGF-EGFR axis is the primary route through which SCP3 acts to activate AKT signaling. SCP3 triggers the EGFR-AKT pathway through transcriptional activation of EGF. Notably, neutralization of secreted EGF by its specific monoclonal antibody reversed SCP3-mediated aggressive phenotypes with a concomitant reversal of EGFR-AKT activation. In an effort to elucidate the molecular mechanisms underlying SCP3-induced transcriptional activation of EGF, we identified Jun activation domain-binding protein 1 (JAB1) as a binding partner of SCP3 using a yeast two-hybrid (Y2H) assay system, and we demonstrated that SCP3 induces EGF transcription through physical interaction with JAB1. Thus, our findings establish a firm molecular link among SCP3, EGFR, and AKT by identifying the novel roles of SCP3 in transcriptional regulation. We believe that these findings hold important implications for controlling SCP3

Topics: Cell Cycle Proteins; COP9 Signalosome Complex; DNA-Binding Proteins; Drug Resistance, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Intracellular Signaling Peptides and Proteins; Mutation; Neoplastic Stem Cells; Peptide Hydrolases; Phosphorylation; Protein Interaction Domains and Motifs; Signal Transduction; Tumor Cells, Cultured; Uterine Cervical Neoplasms

As a transcription factor, SNAIL plays a crucial role in embryonic development and cancer progression by mediating epithelial‑mesenchymal transition (EMT); however, post‑translational modifications, such as ubiquitination, which control the degradation of SNAIL have been observed to affect its functional role in EMT. In a previous study by the authors, it was demonstrated that the HECT domain E3 ubiquitin ligase 1 (HECTD1) regulated the dynamic nature of adhesive structures. In the present study, HECTD1 was observed to interact with SNAIL and regulate its stability through ubiquitination, and the knockdown of HECTD1 increased the expression levels of SNAIL. HECTD1 was discovered to contain putative nuclear localization and export signals that facilitated its translocation between the cytoplasm and nucleus, a process regulated by epidermal growth factor (EGF). Treatment with leptomycin B resulted in the nuclear retention of HECTD1, which was associated with the loss of SNAIL expression. The knockdown of HECTD1 in HeLa cells increased cell migration and induced a mesenchymal phenotype, in addition to demonstrating sustained EGF signaling, which was observed through increased phosphorylated ERK expression levels. Under hypoxic conditions, HECTD1 expression levels were decreased by microRNA (miRNA or miR)‑210. Upon the observation of genetic abnormalities in the HECTD1 gene in cervical cancer specimens, it was observed that the decreased expression levels of HECTD1 were significantly associated with a poor patient survival. Thus, it was hypothesized that HECTD1 may regulate EMT through the hypoxia/hypoxia inducible factor 1α/miR‑210/HECTD1/SNAIL signaling pathway and the EGF/EGF receptor/HECTD1/ERK/SNAIL signaling pathway in cervical cancer. On the whole, the data of the present study indicated that HECTD1 serves as an E3 ubiquitin ligase to mediate the stability of SNAIL proteins.

Topics: Cell Movement; Down-Regulation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Fatty Acids, Unsaturated; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HeLa Cells; Humans; Nuclear Export Signals; Nuclear Localization Signals; Protein Stability; Protein Transport; Snail Family Transcription Factors; Ubiquitin-Protein Ligases; Ubiquitination; Uterine Cervical Neoplasms

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

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

Topics: Carcinogenesis; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Female; HeLa Cells; Humans; Interleukin-6; Phosphorylation; Phytotherapy; Plant Extracts; Signal Transduction; STAT3 Transcription Factor; Uterine Cervical Neoplasms; Zingiberaceae

The characteristics of cancer stem cells (CSCs) and the genes responsible for their maintenance are highly variable in different cancers. Here, we identify the coordination among miRNAs and EGF pathway genes which is critical for the maintenance of CSCs in cervical cancer. The transcript analysis of CSCs enriched from cervical cancer cell lines (CaSki and HeLa) revealed a significant upregulation of SOX2. Since EGF receptor is frequently over expressed in cervical cancer, we hypothesized that EGF pathway may be responsible for the upregulation of SOX2. Also, the media used for CSC enrichment was supplemented with EGF. The hypothesis was validated as inhibiting the EGF/PI3K pathway suppressed the expression of SOX2 and reduced the CSC population. In addition, miRNA profiling identified miR-181a-2-3p and let-7i-5p as markedly reduced in CSCs. The exogenous expression of either of these miRNAs in CaSki cells inhibited the expression of SOX2 and subsequently reduced CSC population. In conclusion, this study highlights for the first time the contrasting role of let-7i-5p/ miR-181a-2-3p and EGF/PI3K/SOX2 axis in maintaining cervical CSCs. While the EGF pathway promotes CSC formation in cervical cancer by inducing SOX2, miR-181a-2-3p/let-7i-5p counteracts the EGF pathway by inhibiting SOX2, thereby reducing the CSC population.

Topics: Cell Line, Tumor; Chromones; Epidermal Growth Factor; Erlotinib Hydrochloride; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; MicroRNAs; Morpholines; Neoplastic Stem Cells; Phosphatidylinositol 3-Kinases; Signal Transduction; SOXB1 Transcription Factors; Up-Regulation; Uterine Cervical Neoplasms

Cyclin G2 (CCNG2) is an atypical cyclin that functions to inhibit cell cycle progression and is often dysregulated in human cancers. We have previously shown that cyclin G2 is highly unstable and can be degraded through the ubiquitin/proteasome pathway. Furthermore, cyclin G2 contains a PEST domain, which has been suggested to act as a signal for degradation by multiple proteases. In this study, we determined if calpains, a family of calcium-dependent proteases, are also involved in cyclin G2 degradation. The addition of calpain inhibitors or silencing of calpain expression by siRNAs strongly enhanced cyclin G2 levels. On the other hand, incubation of cell lysates with purified calpains or increasing the intracellular calcium concentration resulted in a decrease in cyclin G2 levels. Interestingly, the effect of calpain was found to be dependent on the phosphorylation of cyclin G2. Using a kinase inhibitor library, we found that Epidermal Growth Factor (EGF) Receptor is involved in cyclin G2 degradation and treatment with its ligand, EGF, induced cyclin G2 degradation. In addition, the presence of the PEST domain is necessary for calpain and EGF action. When the PEST domain was completely removed, calpain or EGF treatment failed to trigger degradation of cyclin G2. Taken together, these novel findings demonstrate that EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2 and that the PEST domain is critical for EGF/calpain actions.

Topics: Amino Acid Sequence; Animals; Calpain; Cell Line, Tumor; Cyclin G2; Drug Screening Assays, Antitumor; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Phosphorylation; Protein Domains; Proteolysis; Uterine Cervical Neoplasms

The role of Fused Toes Homolog (FTS) in epidermal growth factor (EGF) induced epithelial-mesenchymal transition (EMT) in cervical cancer cells was studied. EGF treatment induced the change of EMT markers and increased cell migration. EGF treatment also increased phosphorylated EGFR and ERK and nuclear level of ATF-2. The binding of ATF-2 to the promoter region of FTS was evidenced after EGF treatment. Pretreatment with PD98059 and gefitinib prevented EGF-induced FTS expression. FTS silencing reduced EMT and cell migration by EGF treatment. These results demonstrate a novel function for FTS in EGF-mediated EMT process.

Topics: Adaptor Proteins, Signal Transducing; Apoptosis Regulatory Proteins; Cell Culture Techniques; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Humans; RNA, Messenger; RNA, Small Interfering; Transfection; Uterine Cervical Neoplasms

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

Melittin (MEL), a major component of bee venom, has been associated with various diseases including arthritis, rheumatism and various cancers. In this study, the anti-angiogenic effects of MEL in CaSki cells that were responsive to the epidermal growth factor (EGF) were examined.. MEL decreased the EGF-induced hypoxia-inducible factor-1α (HIF-1α) protein and significantly regulated angiogenesis and tumor progression. We found that inhibition of the HIF-1α protein level is due to the shortened half-life by MEL. Mechanistically, MEL specifically inhibited the EGF-induced HIF-1α expression by suppressing the phosphorylation of ERK, mTOR and p70S6K. It also blocked the EGF-induced DNA binding activity of HIF-1α and the secretion of the vascular endothelial growth factor (VEGF). Furthermore, the chromatin immunoprecipitation (ChIP) assay revealed that MEL reduced the binding of HIF-1α to the VEGF promoter HRE region. The anti-angiogenesis effects of MEL were confirmed through a matrigel plus assay.. MEL specifically suppressed EGF-induced VEGF secretion and new blood vessel formation by inhibiting HIF-1α. These results suggest that MEL may inhibit human cervical cancer progression and angiogenesis by inhibiting HIF-1α and VEGF expression.

Topics: Animals; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Melitten; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Protein Biosynthesis; Protein Stability; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; TOR Serine-Threonine Kinases; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A

The third member of transforming acidic coiled-coil protein (TACC) family, TACC3, has been shown to be an important player in the regulation of centrosome/microtubule dynamics during mitosis and found to be deregulated in a variety of human malignancies. Our previous studies have suggested that TACC3 may be involved in cervical cancer progression and chemoresistance, and its overexpression can induce epithelial-mesenchymal transition (EMT) by activating the phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated protein kinases (ERKs) signal transduction pathways. However, the upstream mechanisms of TACC3-mediated EMT and its functional/clinical importance in human cervical cancer remain elusive. Epidermal growth factor (EGF) has been shown to be a potent inducer of EMT in cervical cancer and associated with tumor invasion and metastasis. In this study, we found that TACC3 is overexpressed in cervical cancer and can be induced upon EGF stimulation. The induction of TACC3 by EGF is dependent on the tyrosine kinase activity of the EGF receptor (EGFR). Intriguingly, depletion of TACC3 abolishes EGF-mediated EMT, suggesting that TACC3 is required for EGF/EGFR-driven EMT process. Moreover, Snail, a key player in EGF-mediated EMT, is found to be correlated with the expression of TACC3 in cervical cancer. Collectively, our study highlights a novel function for TACC3 in EGF-mediated EMT process and suggests that targeting of TACC3 may be an attractive strategy to treat cervical cancers driven by EGF/EGFR signaling pathways.

Topics: Cell Line, Tumor; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Microtubule-Associated Proteins; Middle Aged; Snail Family Transcription Factors; Transcription Factors; Uterine Cervical Neoplasms

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

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

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

Cervical cancer constitutes the second most common cancer in women. It is evident from earlier studies that epidermal growth factor (EGF) is a mitogen, in that it mimics the function of estrogen by mediating cross-talk with other oncoproteins. Although epidermal growth factor receptor (EGFR) is highly expressed in breast and ovarian tumor tissues, its regulation by the exogenous source of its ligand EGF in human papillomavirus (HPV)-associated cervical cancer remains unclear. In this study, we addressed the question of whether EGF is required for the proliferation of HPV-positive cervical cancer cells and what mechanisms are involved. To determine this, we conducted a series of studies using HPV-positive human cervical cancer cells, CaSki and HeLa, and stimulated the cells with EGF. Our findings suggest that 6 h of stimulation with 10 ng/ml of EGF is sufficient to induce cell cycle progression associated with a significant increase in DNA synthesis, EGFR, COX-2 and cyclin D1 levels. Consistently, cellular localization and Western blot analysis for p-EGFR (Try-1045) protein showed an increase after EGF stimulation. Using siRNA gene knockdown assays we have shown that cyclin D1 siRNA has a significant negative effect on EGFR and inhibit cell growth independent of COX-2 levels. In summary, our findings reveal that an exogenous EGF stimulation may enhance HPV-related cervical cancer cell proliferation by activating EGFR and cyclin D1 that is independent of COX-2 levels, suggesting that the inhibitors of EGFR and cyclin D1 may be effective against cervical cancer cell proliferation.

Topics: Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin D1; Cyclooxygenase 2; Epidermal Growth Factor; ErbB Receptors; Female; HeLa Cells; Human papillomavirus 16; Human papillomavirus 18; Humans; Molecular Targeted Therapy; Papillomavirus Infections; Transcription, Genetic; Uterine Cervical Neoplasms

Malignant cell transformation requires changes in the ability of cells to migrate. The disruption of actin cytoskeleton and intercellular adhesions is an important component of the acquisition of invasive properties in epithelial malignancies. The invasive ability of carcinoma cells is associated with reduced expression of adhesion junction molecules and increased expression of mesenchymal markers, frequently referred to as epithelial-to-mesenchymal transition (EMT). Standard features of the EMT program in cancer cells include fibroblastic phenotype, downregulation of the epithelial marker E-cadherin, induction of Snail-family transcription factors, as well as expression of mesenchymal proteins. We compared the epithelial and mesenchymal marker profiles of nonmalignant HaCaT keratinocytes to the corresponding profiles of cervical carcinoma cell lines C-33A, SiHa, and CaSki. The characteristics of the EMT appeared to be more developed in SiHa and CaSki cervical cancer cells. Further activation of the EMT program in cancer cells was induced by epidermal growth factor. Decreased epithelial marker E-cadherin in CaSki cells was accompanied by increased mesenchymal markers N-cadherin and vimentin. Downregulated expression of E-cadherin in SiHa and CaSki cells was associated with increased expression of Snail transcription factor. Our goal was to study actin reorganization in the EMT process in cell cultures and in tissue. We found that β-cytoplasmic actin structures are disorganized in the cervical cancer cells. The expression of β-cytoplasmic actin was downregulated.

Topics: Actin Cytoskeleton; Actins; Adherens Junctions; Cadherins; Cell Line, Tumor; Down-Regulation; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Humans; Protein Isoforms; Snail Family Transcription Factors; Transcription Factors; Uterine Cervical Neoplasms; Vimentin

The epithelial-mesenchymal transition (EMT) induced by EGF promotes cervical cancer progression; however, the mechanisms underlying the EGF-induced EMT remain unclear. In this study, we reported that miR155 overexpression suppressed EGF-induced EMT, decreased migration/invasion capacities, inhibited cell proliferation and increased the chemo-sensitivity to DDP in human Caski cervical cancer cells. Further, the overexpression of miR155 increased TP53 expression but reduced SMAD2, and CCND1 expression levels. These data suggest that miR155 negatively regulates EGF-induced EMT. We conclude that miR155 does not act as an oncogene but as a tumour suppressor in Caski cells.

Topics: Blotting, Western; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cisplatin; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; Female; Fluorescent Antibody Technique; Humans; MicroRNAs; Uterine Cervical Neoplasms

Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.

Topics: Animals; Calcium; Calcium Channels; Cell Cycle; Cell Movement; Cell Proliferation; Disease Models, Animal; Epidermal Growth Factor; Female; Focal Adhesions; Humans; Membrane Proteins; Mice; Neoplasm Metastasis; Neoplasm Proteins; Neovascularization, Pathologic; ORAI1 Protein; Signal Transduction; Stromal Interaction Molecule 1; Treatment Outcome; Uterine Cervical Neoplasms

The epidermal growth factor receptor (EGFR) overexpressed in many epithelial tumors is an attractive target for tumor therapy since numerous blocking agents of EGFR signaling have proven their anti-tumor activity. Here we report a novel monoclonal antibody (mAb), A13, which was generated from mice immunized with human cervical carcinoma A431 cells. In addition to binding to soluble EGFR with affinity of K(D) approximately 5.8nM, mAb A13 specifically bound to a variety of tumor cells and human placenta tissues expressing EGFR. A13 efficiently inhibited both EGF-dependant EGFR tyrosine phosphorylation in cervical and breast tumor cells and also in vitro colony formation of EGFR-overexpressing lung tumors. Competition and sandwich ELISAs, competitive surface plasmon resonance, and domain-level epitope mapping analyses demonstrated that mAb A13 competitively bound to the domain III (amino acids 302-503) of EGFR with EGF, but recognized distinct epitopes from those of cetuximab (Erbitux). Our results demonstrated that anti-EGFR mAb A13 interfered with EGFR proliferation signaling by blocking EGF binding to EGFR with different epitopes from those of cetuximab, suggesting that combination therapies of mAb A13 with cetuximab may prove beneficial for anti-tumor therapy.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Base Sequence; Binding Sites, Antibody; Cell Line, Tumor; Cell Proliferation; Cetuximab; Epidermal Growth Factor; Epitope Mapping; Epitopes; ErbB Receptors; Female; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Neoplasms; Phosphorylation; Uterine Cervical Neoplasms

Certain types of human papillomaviruses (HPVs) are etiologically linked to cervical cancer. Their transforming capacity is encoded by a polycistronic premRNA, where alternative splicing leads to the translation of functional distinct proteins such as E6, E6*, and E7. Here we show that splicing of HPV16 E6/E7 ORF cassette is regulated by the epidermal growth factor (EGF) pathway. The presence of EGF was coupled to preferential E6 expression, whereas depletion of EGF, or treatment with EGF receptor (EGFR) neutralizing antibodies or the EGFR inhibitor tyrphostin AG1478, resulted in E6 exon exclusion in favor of E6*. As a consequence, increased p53 levels and enhanced translation of E7 with a subsequent reduction of the retinoblastoma protein pRb could be discerned. E6 exon exclusion upon EGF depletion was independent from promoter usage, mRNA stability, or selective mRNA transport. Time-course experiments and incubation with cycloheximide demonstrated that E6 alternative splicing is a direct and reversible effect of EGF signal transduction, not depending on de novo protein synthesis. Within this process, Erk1/2-kinase activation was the critical event for E6 exon inclusion, mediated by the upstream MAP kinase MEK1/2. Moreover, siRNA knockdown experiments revealed an involvement of splicing factors hnRNPA1 and hnRNPA2 in E6 exon exclusion, whereas the splicing factors Brm and Sam68 were found to promote E6 exon inclusion. Because there is a natural gradient of EGF and EGF receptor expression in the stratified epithelium, it is reasonable to assume that EGF modulates E6/E7 splicing during the viral life cycle and transformation.

Topics: Alternative Splicing; Cell Differentiation; Epidermal Growth Factor; Exons; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Oncogene Proteins, Viral; Repressor Proteins; Retinoblastoma Protein; RNA, Messenger; Signal Transduction; Time Factors; Uterine Cervical Neoplasms

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

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

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 KCl cotransporter (KCC) is a major determinant of osmotic homeostasis and plays an emerging role in tumor biology. This study stresses the important role of KCC4 in tumor malignant behavior. Real-time reverse transcription-PCR on samples collected by laser microdissection and immunofluorescent stainings with different KCC isoform antibodies indicate that KCC4 is abundant in metastatic cervical and ovarian cancer tissues. Insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulate KCC4 recruitment from a presumably inactive cytoplasmic pool of endoplasmic reticulum and Golgi to plasma membrane along actin cytoskeleton that is significantly inhibited by LY294002 and wortmannin. Throughout the trafficking process, KCC4 is incorporated into lipid rafts that function as a platform for the association between KCC4 and myosin Va, an actin-dependent motor protein. KCC4 and ezrin, a membrane cytoskeleton linker, colocalize at lamellipodia of migratory cancer cells. Interference with KCC activity by either an inhibitor or a dominant-negative loss-of-function mutant profoundly suppressed the IGF-I-induced membrane trafficking of KCC4 and the structural interaction between KCC4 and ezrin near the cell surface. Endogenous cancer cell invasiveness was significantly attenuated by small interfering RNA targeting KCC4, and the residual invasiveness was much less sensitive to IGF-I or EGF stimulation. In the metastatic cancer tissues, KCC4 colocalizes with IGF-I or EGF, indicating a likely in vivo stimulation of KCC4 function by growth factors. Thus, blockade of KCC4 trafficking and surface expression may provide a potential target for the prevention of IGF-I- or EGF-dependent cancer spread.

Topics: Adult; Aged; Cell Line, Tumor; Cell Membrane; Epidermal Growth Factor; Female; Fluorescent Antibody Technique; Humans; Insulin-Like Growth Factor I; Lasers; Microdissection; Middle Aged; Molecular Motor Proteins; Neoplasm Invasiveness; Ovarian Neoplasms; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Symporters; Uterine Cervical Neoplasms

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

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

Na+/H+ exchanger 1 (NHE1) is involved in cell migration but little is known about the signal pathways that regulate NHE1 activity and that are associated with tumor cell invasiveness. This study is to investigate the mechanisms by which epidermal growth factor (EGF) regulates NHE1 expression to promote cervical cancer cell invasiveness and the clinical significance in early-stage cervical cancer. NHE1 protein was scanty in normal or noncancerous cervical tissues of all surgical specimens examined (n = 92). Tumor tissues clearly expressed NHE1 protein with different amounts. The differential expression level of NHE1 is associated with the clinical outcome. NHE1 protein was also differentially expressed between normal cervical epithelial cells and two cervical cancer cell lines. Cervical cancer cells benefit some enhanced cellular functions from NHE1 abundance, such as cell volume regulation, migration, and invasion. Interestingly, NHE1 colocalized with EGF in cervical cancer tissues. Studies in cell culture systems indicated that EGF-stimulated NHE1 abundance in a time-dependent manner by post-translational regulation. This implies a likely autocrine or paracrine EGF stimulation of NHE1 production in vivo. In addition, the phosphoinositide 3-kinase pathway is the dominant signal controlling EGF-stimulated NHE1 abundance. Pharmacological inhibition of NHE1 activity markedly inhibited the basal and EGF-stimulated cervical cancer cell migration. Image studies and immunoprecipitaion experiments suggest that EGF-induced NHE1 translocation to the leading-edge lamellipodia, where NHE1 interacted with actin-associated protein Ezrin, thereby remodeling cytoskeleton and stimulating cervical cancer cell migration. In conclusion, EGF upregulates NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness.

Topics: Adult; Aged; Cation Transport Proteins; Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial Cells; Female; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neoplasm Invasiveness; Protein Processing, Post-Translational; Protein Transport; Pseudopodia; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Treatment Outcome; Up-Regulation; Uterine Cervical Neoplasms

Multicellular tumor spheroids (MCTS) are three dimensional cell culture systems induced by suspension culture. MCTS are widely used in cancer research because of their similarity to solid tumors. CaSki cells are derived from a metastatic cervical cancer containing human papillomavirus 16 (HPV16). Cell death of CaSki cells in MCTS has been previously reported, and our model is used to better characterize the mechanisms of cell death of HPV16-positive keratinocytes. In this study, we found that apoptosis of CaSki cells was induced by suspension culture along with the formation of MCTS after 24 h of incubation. In suspended CaSki cells, monoclonal antibodies blocking E-cadherin function inhibited MCTS formation and suppressed suspension-induced apoptosis in a dose-dependent manner. Western blot for E-cadherin detected upregulation of the authentic 120 kDa band from MCTS of CaSki cells as well as a shorter 100 kDa band. Addition of EGF, whose receptor is known to form a complex with E-cadherin, abrogated apoptosis of suspended CaSki cells in a dose-dependent manner. These findings suggest that E-cadherin-dependent cell-cell contact, directly or indirectly, mediates the signal to undergo apoptosis of CaSki cells during MCTS formation, and thus provides new information on the role of E-cadherin in cervical cancer cell apoptosis.

Topics: Apoptosis; Cadherins; Calcium; Cell Line, Tumor; Epidermal Growth Factor; Female; Human papillomavirus 16; Humans; Microscopy, Electron, Transmission; Oncogene Proteins, Viral; Spheroids, Cellular; Uterine Cervical Neoplasms

Curcumin (diferuloyl methane), the major yellow pigment from the rhizomes of turmeric (Curcuma longa Linn), has anticancer properties. Infection with high-risk human papillomaviruses (HPV) leads to development of cervical carcinoma, predominantly through the action of viral oncoproteins E6 and E7. The present study aims at analyzing the antitumor and antiviral properties of curcumin, on HPV associated cervical cancer cells. Our findings indicate curcumin to be cytotoxic to cervical cancer cells in a concentration-dependent and time-dependent manner. The cytotoxic activity was selectively more in HPV16 and HPV18 infected cells compared to non-HPV infected cells. Balance between tumor cell proliferation and spontaneous cell death via apoptosis had an important role in regulation of tumor cell growth. Curcumin-induced apoptosis in cervical cancer cells. Morphological hallmarks of apoptosis such as nuclear fragmentation and internucleosomal fragmentation of DNA were observed. Curcumin also selectively inhibited expression of viral oncogenes E6 and E7, evident from RT-PCR and Western blotting data. Electrophoretic mobility shift assay revealed that activation of NFkappaB-induced by TNFalpha is down regulated by curcumin. Curcumin blocked IkBalpha phosphorylation and degradation, leading to abrogation of NFkappaB activation. Curcumin also down regulated the expression of COX-2, a gene regulated by NFkappaB. Binding of AP-1, an indispensable component for efficient epithelial tissue-specific gene expression of HPV was also selectively down regulated by curcumin. These results provide attractive data for the possible use of curcumin in the management of HPV associated tumors.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Proliferation; Curcumin; Cyclooxygenase 2; DNA-Binding Proteins; Down-Regulation; Electrophoretic Mobility Shift Assay; Epidermal Growth Factor; Female; Humans; NF-kappa B; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus Infections; Protein Transport; Protein-Tyrosine Kinases; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms

Clinical trials using rapamycin analogues or HER1/epidermal growth factor receptor (EGFR) inhibitors show that each class of agent has activity against a range of human solid tumors. Because blockade of mitogen-activated protein kinase signaling occurs following HER1/EGFR inhibition in some cell types, we tested the combination of rapamycin and erlotinib in SiHa, Me180, and CaSki human cervical carcinomas xenografts in severe combined immunodeficient mice. In tissue culture, all three cell lines showed decreased phosphorylated S6 ribosomal protein and decreased phosphorylated extracellular signal-regulated kinase (ERK) following treatment with rapamycin and erlotinib, respectively. In SiHa tumors, suppression of phosphorylated S6 was induced by either drug alone, whereas phosphorylated ERK decreased with erlotinib, and enhancement of these effects was obtained with the combination. Continuous treatment of xenografts for 3 weeks led to significant tumor growth delay compared with vehicle control for rapamycin as single agent (P = 0.003) and greater for the combination (P = 0.04 versus rapamycin). Significant antiangiogenic effect was obtained in SiHa xenografts using the drugs together (measured by microvascular density and vascular endothelial growth factor plasma levels) but not for the single agents. Me180 and CaSki xenografts showed significant growth delay with rapamycin but not with erlotinib. Erlotinib treatment resulted in decreased phosphorylated ERK, associated with enhanced suppression of phosphorylated S6 and improved growth delay in Me180 but not in CaSki tumors. These results support the further clinical investigation of rapamycin and EGFR inhibitor combinations in anticancer therapy but highlight the problem of intertumoral heterogeneity in the prediction of in vivo response.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Proliferation; Drug Interactions; Epidermal Growth Factor; Erlotinib Hydrochloride; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Mice; Mice, SCID; Neoplasm Transplantation; Neoplasms, Experimental; Phosphorylation; Quinazolines; Ribosomal Protein S6; Signal Transduction; Sirolimus; Transplantation, Heterologous; Uterine Cervical Neoplasms

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

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

The tumor suppressor protein p53 is the most frequently mutated gene in human cancer. The function of p53 is not restricted to "guarding" against oncogenic stress, but also p53 can guard against the presence of DNA damage. One of the principal mechanisms by which cells achieve this is by regulating the p53 protein level although its phosphorylation and cellular localization also contribute to the regulation of its function. Since many tumors secrete growth factor(s) that inhibit apoptosis and support the growth of cancer cells, we investigated the effects of human epidermal growth factor (EGF) on human TNF-alpha-mediated induction of p53 and its transcriptional target, p21 in TNF-alpha sensitive human cervical carcinoma cell line, ME180S. We found that TNF-alpha can increase the cellular levels of p53, p21 and induce apoptosis in ME180S cells. However, pretreatment of cells with EGF can suppress all these effects of TNF-alpha. To determine which kinase(s) pathway was utilized by EGF to show these suppressive effects, cells were pretreated with inhibitors of MAPK, PI3K and PKC pathways. Among these only PKC inhibitor reversed all the suppressive effects of EGF. We also found that ME180S cells express only zeta, lambda, epsilon, iota, delta, theta, beta PKC subtypes and among these EGF treatment activate only PKC-delta redistribution to the membrane from the cytosol. An inhibitor of PKC, GF 109203X inhibited EGF-mediated suppression of TNF-alpha-induced accumulation of p53, p21 and induction of apoptosis. In summary, we concluded that EGF can protect ME180S cells from TNF-alpha-induced apoptosis through activation of PKC-delta.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Cytosol; DNA Damage; Enzyme Inhibitors; Epidermal Growth Factor; Female; Genes, p53; Humans; Immunoblotting; Indoles; Maleimides; Phosphorylation; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Time Factors; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms

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

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

The peroxisome proliferator-activated receptor-gamma (PPARgamma), a ligand-dependent transcription factor belonging to the family of nuclear receptors, has been implicated in the control of cyclooxygenase (COX) 2 expression in some tissue, although the exact mechanism(s) of this activity has not been elucidated. In this study we explored the possible mechanism(s) of control of COX-2 gene expression through PPARgamma signaling in human cervical cancer.. Using primary human cervical tissues and the CaSki human cervical cancer cell line, we assayed for PPARgamma and COX-2 mRNA expression by reverse transcription-PCR. Nuclear protein binding activities to three response elements located in the COX-2 promoter [nuclear factor kappaB (NFkappaB), cyclic AMP response element, and activator protein (AP)-2] were measured by gel mobility shift assays. We used transient transfection assays with COX-2 promoter reporter gene constructs to determine the regulatory sites in this promoter, which mediates PPARgamma regulation of COX-2 activity.. We showed, for the first time, that primary human cervical cancer tissues express PPARgamma. Using CaSki cells, we demonstrated that COX-2 and PPARgamma mRNA levels were inversely regulated by PPARgamma ligands in that these compounds up-regulated PPARgamma but down-regulated COX-2. In contrast, epidermal growth factor (EGF), a potent activator of COX-2, decreased PPARgamma mRNA levels. This down-regulation of PPARgamma mRNA by EGF was blocked in the presence of NS-398, a selective COX-2 inhibitor. PPARgamma ligands suppressed the binding activities of AP-1 (binding to CRE) and NFkappaB but not AP-2. Transient transfection results indicated that EGF stimulated whereas PPARgamma ligands inhibited COX-2 promoter (-327/+59) activity. This effect by PPARgamma ligands on the COX-2 promoter was blocked when the CRE, but not the NFkappaB, binding site was mutagenized.. Cervical cancer cells express readily detectable levels of PPARgamma. There is reciprocal negative regulation between COX-2 and PPARgamma signaling in human cervical cancer cells. The ability of PPARgamma ligands to inhibit COX-2 appears to be mediated predominantly through inhibition of AP-1 protein binding to the CRE site in the COX-2 promoter.

Topics: Cyclic AMP Response Element-Binding Protein; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Epidermal Growth Factor; Female; Gene Expression Regulation, Enzymologic; Humans; Isoenzymes; Membrane Proteins; NF-kappa B; Nitrobenzenes; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; Response Elements; RNA, Messenger; Sulfonamides; Transcription Factor AP-1; Transcription Factor AP-2; Transcription Factors; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Uterine Cervical Neoplasms

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

The cadherin/catenin adhesion complex is fundamentally involved in epithelial cancer invasion and metastasis. Much evidence suggesting that epidermal growth factor (EGF) induced the scattering and invasion of cancer cells, probably by affecting E-cadherin function, has been reported. The present study aimed to confirm the hypothesis that EGF/epidermal growth factor receptor (EGFR) was related with the E-cadherin adhesion system in cervical cancer cells and that EGF might induce tyrosine phosphorylation of beta- and gamma-catenin.. Cervical cancer cells were treated for different time durations with 30 ng/ml of EGF. Alteration of the cell morphology was examined by light microscopy and the expression of E-cadherin, beta-catenin, gamma-catenin, EGFR, and activated EGFR was assayed using Western blotting. Tyrosine phosphorylation of beta- and gamma-catenin was also examined using immunoprecipitation.. E-cadherin and EGFR were expressed in CaSki, HT-3, and ME-180 cell lines, which showed epithelial contact growth. The expression of E-cadherin and beta- and gamma-catenin did not change after treatment with EGF. The expression of EGFR decreased and activated EGFR expression increased in 30 min and then decreased subsequently. The simultaneous expression of activated EGFR and tyrosine phosphorylation of beta- and gamma-catenin was found.. EGF-induced scattering of the E-cadherin-positive cervical cancer cells might be the result of tyrosine phosphorylation of the beta- and gamma-catenin. Phosphorylation of the beta- and gamma-catenin may hamper the adhesive function of the E-cadherin-catenin complex.

Topics: beta Catenin; Blotting, Western; Cadherins; Cell Adhesion; Cytoskeletal Proteins; Desmoplakins; Epidermal Growth Factor; ErbB Receptors; Female; gamma Catenin; Humans; Phosphorylation; Precipitin Tests; Trans-Activators; Tumor Cells, Cultured; Tyrosine; Uterine Cervical Neoplasms

We investigated the signalling pathways by which epidermal growth factor (EGF) modulates paclitaxel-induced apoptosis in SiHa human cervical cancer cells. SiHa cells exposed to paclitaxel underwent apoptosis, which was strongly inhibited by EGF. This inhibition of apoptosis by EGF was not altered by pharmacological blockade of phosphatidylinositol 3'-OH kinase (PI-3K) with the PI-3K specific inhibitor LY294002 or blockade of the mitogen-activated protein kinase (MAPK) kinase (MEK) with the MEK specific inhibitor PD98059, or by transfection of the cells with PI-3K or MEK dominant-negative expression vectors. EGF did not stimulate PI-3K/Akt, MEK/MAPK, or p38 MAPK activity in SiHa cells but did transiently activate the c-Jun NH2-terminal kinase (JNK). Co-exposure of SiHa cells to SB202190 at concentrations that inhibit JNK abolished the protective effect of EGF on SiHa cells against paclitaxel-induced apoptosis. Our findings indicate that the JNK signaling pathway plays an important role in EGF-mediated protection from paclitaxel-induced apoptosis in SiHa cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Enzyme Activation; Epidermal Growth Factor; Female; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinases; Paclitaxel; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Cell cycle regulators and signal transduction pathways can influence apoptotic sensitivity of tumor cells, and we previously described an association between EGFr overexpression, reduced DNA repair activity, and increased apoptotic sensitivity of ME-180 cervical carcinoma cells toward cis-diammedichloroplatinum (cDDP; K. Nishikawa, et al., Cancer Res., 52: 4758-4765, 1992). In the present study, the characteristics of ME-180 cells selected for high or low apoptotic sensitivity to cDDP (or camptothecin) were examined and compared to determine whether signal transduction components and cell cycle regulation were distinct in these isogenic drug response variant populations. As ME-180 cells progressed from high to low cDDP sensitivity [IC50 approximately 80 ng/ml in cDDP sensitive (PT-S) to approximately 2000 ng/ml in cDDP-resistant (Pt-R) cells], there was a significant decrease in EGFr expression that paralleled the relative reduction in cDDP apoptotic responsiveness (approximately 30-fold). cDDP-resistant cells had the slowest rate of growth and more effectively reduced DNA adduct levels following cDDP exposure than parental cells. Cellular levels of the cell cycle inhibitor p21WAF1 inversely correlated with cDDP responsiveness with high levels of p21WAF1 expressed in drug-resistant Pt-R cells in the absence of elevated p53. cDDP stimulated a 2-fold increase in p53 levels in both drug-sensitive and drug-resistant cells but caused a delayed reduction in p21WAF1 levels, suggesting p53-independent regulation of p21WAF1 in ME-180 cells. Activation of EGFr in Pt-R cells stimulated cell cycle progression (2-fold), reduced p21WAF1 levels (>2-fold), and increased sensitivity to cDDP (3-fold), suggesting that receptor signaling enhanced the efficacy of cDDP to induce cell death by relieving cell cycle restriction. These results demonstrate that the transition of ME-180 cells from a drug-sensitive to drug-resistant phenotype correlates with reciprocal changes in EGFr and p21WAF1 expression and provides additional evidence that the pathways controlled by these proteins may contribute to some forms of drug resistance.

Topics: Apoptosis; Cell Cycle; Cell Division; Cell Survival; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Adducts; Drug Resistance, Neoplasm; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Cripto-1 (CR-1), a member of the EGF-CFC peptide family, plays an essential role during mesoderm formation in vertebrates as well as in cancer development. Using cDNA gene expression array, Western blot, and indirect immunofluorescence, an increase in vimentin expression was demonstrated in CR-1-transfected human Caski cervical carcinoma cells compared to control vector-transfected cells. In parental Caski cells, recombinant CR-1 induced a dose-dependent increase of vimentin protein expression within 24 h. Since vimentin expression has been demonstrated to correlate with a more aggressive phenotype in human cervical cancer, the migration capacity of CR-1-transfected or CR-1-treated Caski cells was studied in the Boyden chamber assay. Compared to the vector-transfected or untreated Caski cells, CR-1-transfected cells or cells treated with recombinant CR-1 exhibit enhanced migration, both through collagen- and through gelatin-coated membranes. Additionally, CR-1 can function as a chemoattractant for Caski cells. These findings are of biological significance since CR-1 is overexpressed in several types of human carcinomas. The present data demonstrate that CR-1 can increase vimentin expression and modulate migration in human cervical carcinoma cells.

Topics: Cell Movement; Epidermal Growth Factor; Female; GPI-Linked Proteins; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Neoplasm Proteins; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vimentin

This study evaluates the expression of cripto (CR-1) protein in matched sets of non-neoplastic cervical epithelium, primary cervical carcinoma and metastatic tumours in the lymph nodes to investigate its role in uterine cervical cancer development and progression. Ninety-four primary cervical carcinomas in an early clinical stage and having the same surgical treatment modality were analysed. Immunoreactivity in the primary tumour was compared with that of non-neoplastic cervical epithelium and metastatic lymph nodes. The conventional clinicopathological prognostic variables for cervical carcinomas such as grade, tumour size, depth of invasion, parametrial and endometrial extension, lymphovascular space involvement and lymph node metastasis status were also compared with CR-1 expression of the primary tumour. Strong CR-1 immunopositivity was significantly correlated with tumour size and lymphovascular space involvement (P < 0.05). Furthermore, a significant relationship was found between CR-1 immunoreactivity and endometrial extension as well as parametrial involvement (P < 0.05). Interestingly, the CR-1 expression level was increased in metastatic lymph nodes compared with their primary tumours. These results suggest that CR-1 may contribute to disease progression in cervical carcinomas.

Topics: Epidermal Growth Factor; Female; Follow-Up Studies; GPI-Linked Proteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lymphatic Metastasis; Membrane Glycoproteins; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Recurrence, Local; Neoplasm Staging; Prognosis; Survival Analysis; Uterine Cervical Neoplasms

Over-expression of epidermal growth factor-receptors (EGF-R) has been described in a variety of cancers, including cervical cancer. Nicotine may increase cellular proliferation rates through a mechanism involving EGF or EGF-R. In this study, we ascertain the effect of EGF antibodies on nicotine-enhanced proliferation rates in two cervical cancer cell lines.. We studied (a) nicotine-induced increase in the cellular expression of EGF-R in human papillomavirus (HPV)-positive ME-180 and HPV-negative HT-3 cervical cancer cell line cultures, using a semi-quantitative immunofluorescent antibody assay; (b) alterations in cellular proliferation in association with changes in EGF-R levels; and (c) the EGF-R mediation by EGF.. Nicotine exposure at physiologically attainable plasma concentrations caused increased expression of EGF-R in both cervical cancer cell lines. Up-regulation of EGF-R was associated with increased cellular proliferation. Decreased expression of EGF-R was associated with decreased cellular proliferation. These data were consistent with EGF-R expression as a mechanism for the control of proliferation of the cervical cancer cells. The action of nicotine was abrogated when antibodies to EGF were added, implying that nicotine up-regulation of EGF-R may be mediated by EGF.. Our data show that nicotine-induced proliferation of cervical cancer cells is mediated through EGF-R over-expression and that this action of nicotine utilizes EGF.

Topics: Cell Division; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Nicotine; Tumor Cells, Cultured; Up-Regulation; Uterine Cervical Neoplasms

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

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

Cripto-1 (CR-1), a member of the epidermal growth factor-CFC peptide family, activates the ras/raf/mitogen-activated protein/extracellular signal-regulated kinase/mitogen-activated protein kinase pathway. In the present study, the role of CR-1 in the phosphatidylinositol 3'-kinase (PI3K)/AKT (protein kinase B)/glycogen synthase kinase 3beta (GSK-3beta)-dependent signaling pathway was evaluated in human SiHa cervical carcinoma cells. Our data demonstrate that CR-1 can enhance the tyrosine phosphorylation of the p85 regulatory subunit of PI3K and transiently induce the phosphorylation of AKT in a time- and dose-dependent manner. In addition, CR-1 was found to induce the phosphorylation of GSK-3beta. Phosphorylation of AKT and GSK-3beta by CR-1 can be blocked by LY294002, a specific inhibitor of PI3K, thus leading to apoptosis. Finally, the apoptotic effect of LY294002 can be partially rescued by exogenous CR-1. In summary, our data suggest that human CR-1 may function as a survival factor through a PI3K-dependent signaling pathway involving AKT and GSK-3beta.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Chromones; Culture Media, Serum-Free; Enzyme Inhibitors; Epidermal Growth Factor; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Morpholines; Neoplasm Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Transfection; Tumor Cells, Cultured; Uterine Cervical Neoplasms

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

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

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

Epidermal growth factor (EGF) is a mitogen for most epithelial cells. Paradoxically, the growth of some cultured cell lines, containing high numbers of EGF receptors, are inhibited by EGF. Here we demonstrate that growth inhibition by EGF in several cell lines correlates with the activation of the signal transducer and activator of transcription (Stat) 1. In contrast, in normal fibroblasts and several cell lines that are growth stimulated by EGF, we observed no or very transient activation of Stat1. A causal association between Stat1 activation by EGF and growth inhibition was suggested by the expression of a dominant-negative Stat1 in A431 cells, resulting in the loss of Stat1 DNA binding and concomitant resistance to growth inhibition by EGF. We conclude that, in the cells examined, EGF-induced arrest of growth requires activated Stat1.

Topics: Cell Cycle; Cell Division; Cell Line; DNA-Binding Proteins; Epidermal Growth Factor; Female; Fibroblasts; Growth Inhibitors; Humans; Interferon-gamma; Phosphorylation; STAT1 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Uterine Cervical Neoplasms

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

Interleukin 1alpha (IL-1alpha), Interleukin 6 (IL-6) and epidermal growth factor (EGF) were tested for their ability to regulate epithelial cervical cell cytokine production and secretion and to induce proliferation of human normal and neoplastic epithelial cervical cells. IL-1alpha, and IL-6 enhanced tumour and normal cell growth by 20-120%. The interleukins efficacy was similar to that of EGF for some cell lines but not for normal esocervical cells. The stimulatory effects of the interleukins were observed in both human papilloma virus (HPV)-infected and HPV-non-infected cervical cells. Normal cells constitutively expressed IL-1alpha, IL-6 and EGF mRNA. All cell lines except C33A expressed IL-1alpha mRNA. CaSki, C-4II and HT-3 expressed mRNA for IL-6. IL-1alpha induced or increased IL-6 mRNA levels in the Me-180 and HT-3 lines and in normal cervical cells. IL-6 induced: (1) the expression of its own mRNA only in Me-180 cells that constitutively lacked IL-6 mRNA; (2) the expression of IL-1alpha mRNA in C-33A and increased IL-1alpha mRNA level in the case of Me180 cells. Increased amounts of IL-6 mRNA were found in normal cells when treated with IL-1alpha. In spite of the pattern of mRNA expression, only HT-3 and normal cervical cells constitutively secreted IL-6, and only normal cells were able to produce IL-1alpha protein. A significant IL-1alpha-dependent increase of IL-6 secretion was found in Me-1 80, HT-3 and normal cells. IL-1alpha- and IL-6-driven cell proliferations were almost completely inhibited by the addition of neutralizing anti-IL-6 antibodies. Taken together, these data suggest that interleukins play a role in cervical carcinogenesis as autocrine and/or paracrine stimuli.

Topics: Cell Division; Cervix Uteri; Epidermal Growth Factor; Female; Humans; Interleukin-1; Interleukin-6; Neoplasm Proteins; RNA, Messenger; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Expression of epidermal growth factor receptor (EGFR) was quantified by 2-color flow cytometry in cervical cancer (n = 73) and normal cervical epithelium (n = 11). EGFR was determined using a murine monoclonal EGFR antibody, and number of bound antibodies was quantified adding calibration beads with defined antigenic binding sites. Tumor cells were identified by simultaneous DNA staining. Epithelium of normal cervical tissue was detected by labeling for cytokeratin. Results were compared with EGFR quantification by autoradiography on cryostat sections using a radioligand binding assay. A high degree of correlation was found between the 2 methods. In cervical carcinomas 14,600 binding sites/cell (median; range, 160-283,000 binding sites/cell) were detected, considerably less compared with normal cervical squamous epithelium, which was 30,700 binding sites/cell (median; range, 19,900-44,000 binding sites/cell). This finding clearly contrasts with other reports of enhanced EGFR expression in cervical cancer. The discrepancy may be explained by contamination of tissue homogenates used for radioligand or enzyme immunosorbent assays by non-epithelial tissue elements with low or absent EGFR expression. Interference with quantitative EGFR determination in epithelial cells may result in false low estimates of EGFR expression predominantly in normal cervical tissue. This should be avoided by identifying tumor and normal epithelial cells prior to analysis. In our study, 63% of cervical cancers expressed low levels of EGFR compared with normal cervical epithelium, and only 10% showed overexpression. There is evidence that cervical carcinomas overexpressing EGFR represent a small, but biologically distinct group of cervical cancers exhibiting enhanced aggressiveness associated with poor survival of the patients.

Topics: Antibodies, Monoclonal; Autoradiography; Binding Sites, Antibody; Biomarkers; Cervix Uteri; Down-Regulation; Epidermal Growth Factor; Epithelial Cells; Epithelium; ErbB Receptors; Female; Flow Cytometry; Humans; Iodine Radioisotopes; Keratins; Prospective Studies; Regression Analysis; Uterine Cervical Neoplasms

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

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

We characterized mechanisms of growth control involving insulin-like growth factor-1 (IGF-1), IGF-2, and IGF-1 receptor (IGF-1R) by investigating their expression in human cervical cancer cell lines, primary cervical tumor cell cultures, and normal ectocervical epithelial cells maintained in short-term culture. By reverse transcription followed by PCR, IGF-1 mRNA was not detected in any of the cell lines, whereas IGF-2 mRNA transcripts were detected in all of them. Using the RNase protection assay, low levels of IGF-2 mRNA were also detected in all of the cervical cancer cell lines, primary cervical tumor cell cultures, and normal ectocervical cultures tested, but no IGF-1 transcripts were detected. Scatchard analysis revealed 3- and 5-fold increases in IGF-1R expression by the primary cervical cancer cell cultures and cervical cancer cell lines, respectively, compared with the normal ectocervical cells. In proliferation assays, epidermal growth factor (EGF) consistently enhanced cervical cancer cell growth, but an antisense oligonucleotide to IGF-2 uniformly inhibited the EGF-induced mitogenic effect. These studies suggest that autocrine production of IGF-2 and overexpression of the IGF-1R are important components controlling the proliferation of cervical carcinoma cells, and that autocrine IGF-2 production in cervical cancer cells may participate in the mitogenic signaling of EGF.

Topics: Base Sequence; Cell Division; Clone Cells; DNA Primers; Epidermal Growth Factor; Female; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Kinetics; Molecular Sequence Data; Oligonucleotides, Antisense; Polymerase Chain Reaction; Receptor, IGF Type 1; RNA, Messenger; Thionucleotides; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Crotoxin (CT), a phospholipase A2 (PLA2) derived from the venom of Crotalus durissus terrificus, is a heterodimeric protein composed of subunit B with enzymatic activity and a binding regulatory subunit (A) without enzyme activity. Although the PLA2 activity of CT may be important in its anti-proliferative activity, its cytostatic mechanism is unknown. In this study, we examined the cytostatic effect of PLA2-associated CT activity on squamous carcinoma cells expressing distinct levels of epidermal growth factor receptor (EGFr). CT was most effective in suppressing growth on cells expressing high intrinsic levels of EGFr. Cardiotoxin, another membrane active toxin with no intrinsic PLA2 activity, had no differential anti-proliferative activity on cells expressing high EGFr levels, suggesting a correlation between EGFr expression and CT-directed anti-proliferative activity. Both chemically modified CT (MCT) devoid of PLA2 activity and covalently cross-linked CT (CCT), which is functionally unable to utilize cellular membranes as PLA2 substrate, were also without growth inhibitory activity. No evidence for direct binding of CT to EGFr was found, although pretreatment with EGF was able to partially suppress the anti-proliferative activity of CT. Tyrosine phosphorylation of EGFr, however, was stimulated by CT in intact A431 cells. Tyrosine phosphorylation of EGFr was concentration-dependently stimulated (3- to 8-fold) in cellular membranes of A431 cells treated in vitro with CT but not with anti-proliferatively inactive MCT or CCT. The data provide evidence for transmembrane receptors involved in growth signaling (namely EGFr) as cellular targets and potential effectors of PLA2-mediated anti-proliferative activity of snake venom.

Topics: Animals; Cell Division; Crotoxin; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Phospholipases A; Phospholipases A2; Tumor Cells, Cultured; Uterine Cervical Neoplasms

We used newly established cervical dysplasia-derived cell lines to elucidate a molecular mechanism of the preventive action of beta-carotene in cervical multi-step carcinogenesis. Liposomal beta-carotene was added to the culture medium for human cervical dysplasia cell lines, CICCN-2 from cervical intraepithelial neoplasia grade I (CIN I), CICCN-3 from CIN II, and CICCN-4 from CIN III, and human cervical carcinoma-derived cell lines such as CICCN-6, CICCN-18, and HeLa cells. beta-Carotene (10 mumol/L) induced significant growth retardation in three cervical dysplasia cell lines but not in three cervical carcinoma-derived cell lines. Binding activities of epidermal growth factor (EGF) and cellular amounts of either messenger RNA for EGF receptor gene or EGF receptor protein were all highest in CICCN-4 cells. Cell surface binding, as well as internalization, of 125I-labeled EGF was rapidly reduced after beta-carotene treatment in dysplasia cell lines and 170-kD protein bands of EGF receptor disappeared from protein immunoblots at day 3 of the treatment. Cellular amounts of EGF receptor messenger RNA remained constant until day 3 of the treatment and were substantially reduced after day 7. Chromatin condensations, morphologic evidence for apoptotic cell death, were observed at day 1 by staining. From these results, we contend that prevention of cervical carcinogenesis by beta-carotene is due to induction of apoptosis in cervical dysplastic cells, which are premalignant cells in cervical multi-step carcinogenesis, via down-regulation of EGF receptor protein.

Topics: Anticarcinogenic Agents; Antioxidants; Apoptosis; beta Carotene; Carotenoids; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; RNA, Messenger; Tumor Cells, Cultured; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

The early genes E6 and E7 from human papillomaviruses (HPVs) play a key role in the development of cervical cancer. Modulation of E6 and E7 gene expression may alter tumour progression; therefore, modifiers of viral transcription such as hormones or growth factors are potential risk factors in cancer development. We have analysed the effects of epidermal growth factor (EGF) on E6/E7 mRNA from human papillomavirus type 16 (HPV-16) by Northern blot in two cell lines, SiHa cervical carcinoma cells, and HPK IA, an HPV-16-immortalized keratinocyte cell line. E6/E7 mRNA is EGF-inducible in SiHa cells, with the earliest response after 2 h. In contrast, in HPK IA cells no increase in E6/E7 RNA is observed, suggesting a differential EGF response of viral transcription in tumour cells compared with keratinocytes. We demonstrate that the cell type-specific HPV-16 enhancer is a target of EGF-induced signals, as its activity is amplified by EGF in SiHa cell transfections. However, when transfected into HPK IA keratinocytes, the viral enhancer shows no EGF response. The enhancer contains two binding sites for the transcription factor AP-1, a potential mediator of the EGF signalling cascade. Enhancer subfragments with single AP-1 binding sites are also EGF-responsive in SiHa cells. Mutating either AP-1 site in the complete enhancer decreases the EGF response, whereas a double mutation causes a complete loss of EGF regulation, suggesting that the EGF induction of HPV-16 early transcription requires AP-1 activation. We conclude that alterations of EGF responsiveness that increase viral oncogene expression may contribute to cervical cancer progression.

Topics: Base Sequence; Cell Line, Transformed; Disease Progression; DNA, Neoplasm; Enhancer Elements, Genetic; Epidermal Growth Factor; Female; Gene Expression Regulation, Viral; Humans; Keratinocytes; Molecular Sequence Data; Mutation; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Repressor Proteins; RNA, Messenger; RNA, Viral; Signal Transduction; Transcription Factor AP-1; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Uterine Cervical Neoplasms

There is increasing evidence that activation of the insulin-like growth factor I (IGF-I) receptor plays a major role in the control of cellular proliferation of many cell types. We studied the mitogenic effects of IGF-I, IGF-II, and epidermal growth factor (EGF) on growth-arrested HT-3 cells, a human cervical cancer cell line. All three growth factors promoted dose-dependent increases in cell proliferation. In untransformed cells, EGF usually requires stimulation by a "progression" factor such as IGF-I, IGF-II, or insulin (in supraphysiologic concentrations) in order to exert a mitogenic effect. Accordingly, we investigated whether an autocrine pathway involving IGF-I or IGF-II participated in the EGF-induced mitogenesis of HT-3 cells. With the RNase protection assay, IGF-I mRNA was not detected. However, IGF-II mRNA increased in a time-dependent manner following EGF stimulation. The EGF-induced mitogenesis was abrogated in a dose-dependent manner by IGF-binding protein 5 (IGFBP-5), which binds to IGF-II and neutralizes it. An antisense oligonucleotide to IGF-II also inhibited the proliferative response to EGF. In addition, prolonged, but not short-term, stimulation with EGF resulted in autophosphorylation of the IGF-I receptor, and coincubations with both EGF and IGFBP-5 attenuated this effect. These data demonstrate that autocrine secretion of IGF-II in HT-3 cervical cancer cells can participate in EGF-induced mitogenesis and suggest that autocrine signals involving the IGF-I receptor occur "downstream" of competence growth factor receptors such as the EGF receptor.

Topics: Base Sequence; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Gene Expression; Humans; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Kinetics; Mitosis; Molecular Sequence Data; Oligodeoxyribonucleotides; Oligonucleotides, Antisense; Receptor, IGF Type 1; RNA, Messenger; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Human ectocervical epithelial cells are a primary target for infection by oncogenic papillomaviruses, which are strongly implicated as causative agents in the genesis of cervical cancer. Growth factors have been implicated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human papillomavirus (HPV) type 16-immortalized ectocervical epithelial cell lines to investigate the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cell proliferation and the production of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immortalized/nontumorigenic cell line, maintained in defined medium, produce and release high levels of IGFBP-3 (38/42 kDa) as well as smaller amounts of a 24-kDa IGFBP. Supplementation of defined medium with EGF causes a dose-dependent increase in cell growth and a concomitant decrease in the levels of IGFBP-3 released into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is suppressed 67% due to the simultaneous presence of 3 ng/ml of TGF beta 1, indicating that EGF suppression of IGFBP-3 levels is independent of EGF effects on cell growth. EGF suppression of IGFBP-3 production is correlated with a reduction in IGFBP-3 mRNA level. In the presence of EGF, the growth response of the cells to ng amounts of IGF-I is significantly enhanced. Moreover, the simultaneous presence of both EGF and IGF-I reduces the level of IGFBP-3 more efficiently than EGF alone. We also observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level is increased in HPV16-positive tumorigenic versus nontumorigenic cell lines. This is the first report of EGF acting as a positive regulator of IGF-I action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) stimulating growth directly via the EGF mitogenic pathway and (b) stimulating growth indirectly by reducing the levels of inhibitory IGFBPs and thereby potentiating the effects of IGF-I. In addition, the observation that more highly transformed cell types produce lower levels of IGFBP-3 and higher levels of 24-kDa IGFBP suggests that tumor cells in more advanced cervical cancers may have an altered response to IGF-I.

Topics: Antibodies; Carrier Proteins; Cell Division; Cells, Cultured; Cervix Uteri; Drug Synergism; Epidermal Growth Factor; Epithelial Cells; Female; Growth Inhibitors; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Models, Biological; Papillomaviridae; Signal Transduction; Somatomedins; Transfection; Uterine Cervical Neoplasms

Reverse transcription polymerase chain reaction (RT-PCR) revealed that the Fallopian tubes express epidermal growth factor (EGF), transforming growth factor (TGF alpha), and EGF receptor (EGF-R) mRNA. The RT-PCR product was verified by restriction enzyme digestion analysis. Immunohistochemically, EGF, TGF alpha, and EGF-R were localized in Fallopian tubes by use of specific antibodies to human EGF, mature fragments of human TGF alpha, and monoclonal antibodies to the extracellular binding domain of EGF-R. The tubal epithelial cells were the primary site of immunoreactive EGF, TGF alpha, and EGF-R, which were present to a lesser extent in the stromal cells, smooth muscle cell layers, fibroblasts of serosal tissue, and arterial endothelial and smooth muscle cells. Using antibodies generated against the amino and carboxy termini of TGF alpha precursor produced a similar cellular distribution to that observed for mature TGF alpha. The intensity of immunoreactive TGF alpha with these antibodies was similar to that seen with EGF. The ciliated and nonciliated epithelial cells in the ampullary and isthmus regions immunostained with similar intensity for EGF, TGF alpha, and EGF-R. The immunostaining for EGF, TGF alpha, and EGF-R was cycle-dependent, was considerably higher during late proliferative and early-to-mid-secretory phases than during early proliferative and late secretory phases of the menstrual cycle, and was reduced during the postmenopausal period. Specimens obtained 5-12 yr after tubal ligation immunostained for EGF, TGF alpha, and EGF-R similarly to sections from unligated tubes taken during the same phase of the cycle. Quantitative autoradiography of 125I-EGF binding generated a pattern similar to that of immunostaining for EGF-R binding. Net grain density/100 microns 2 calculated for different cell types indicated that the epithelial cells had a significantly higher grain density than did other tubal cell types (p < 0.05) without the cycle dependency seen in the immunohistochemical study. In summary, the results demonstrate that the human Fallopian tube expresses mRNA and contains immunoreactive proteins for EGF, TGF alpha, and EGF-R as well as binding sites for 125I-EGF. The cycle dependency and lower immunostaining in postmenopausal tubes suggest a potential regulation of their expression by ovarian steroids. The results imply the importance of EGF/TGF alpha in a variety of tubal biochemical and physiological functions and possibly early embryon

Topics: Autoradiography; DNA Restriction Enzymes; Endothelium, Vascular; Epidermal Growth Factor; ErbB Receptors; Fallopian Tubes; Female; Gene Expression; Humans; Immunohistochemistry; Male; Muscle, Smooth; Ovarian Neoplasms; Polymerase Chain Reaction; Pregnancy; RNA, Messenger; Transforming Growth Factor alpha; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

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

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

In the present study, we examine the effects of all-trans-retinoic acid (RA) and interferons-alpha and -gamma (IFN-alpha and IFN-gamma) on the growth of HPV16-immortalized cell lines, ECE16-1 and CaSki. Treating proliferating ECE16-1 cells with RA causes a concentration-dependent decrease in cell number. At 1 microM RA, cell growth is suppressed by 65% and the level of mRNA encoding cytokeratin K5, a biochemical marker of retinoid action, is also suppressed. In contrast, the level of transcript encoding the HPV16 oncogenes, E6 and E7, is reduced by only 5 to 10%. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml suppresses growth by 70%. This growth suppression by IFN-gamma is correlated with a > 90% reduction in E6/E7 mRNA levels. Additional growth suppression is observed upon simultaneous treatment with retinoid and interferon. Optimal suppression is observed in the presence of 200 IU/ml IFN-gamma and 1 microM RA. The rank order of effectiveness is IFN-gamma/RA > IFN-alpha/RA = IFN-gamma > RA > IFN-alpha. In contrast to the suppression of ECE16-1 cell growth, RA causes a concentration-dependent increase in CaSki cell number (50-60%) which is optimal at 1 microM RA. Cytokeratin K5 mRNA levels are markedly suppressed, and E6/E7 mRNA levels increased by 5% under these conditions. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml decreases CaSki cell growth by 20 and 45%, respectively, and 200 IU/ml of IFN-gamma reduce E6/E7 expression to undetectable levels. Addition of RA (1 microM) partially counters the IFN-dependent suppression of growth and E6/E7 mRNA levels. Our results suggest that retinoid-dependent changes in human papillomavirus-immortalized cervical cell proliferation are not always correlated with changes in E6/E7 transcript levels.

Topics: Animals; Cell Division; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; Cervix Uteri; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epithelial Cells; Epithelium; Female; Gene Expression; Humans; Interferon-alpha; Interferon-gamma; Kinetics; Mice; Mice, Nude; Oncogenes; Papillomaviridae; RNA, Messenger; Transcription, Genetic; Transplantation, Heterologous; Tretinoin; Uterine Cervical Neoplasms

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

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

Immunohistochemical studies were performed using specific antibodies to epidermal growth factor (EGF) and EGF receptor to determine their presence and cellular localization in the human ovary during follicular growth and regression. There was no immunostaining for EGF or EGF receptor in primordial follicles. In the preantral follicle stage, immunostaining for EGF and EGF receptor was observed only in the oocyte. The staining intensity of the oocyte increased as the oocyte reached the preovulatory stage. In the antral follicle stage, immunostaining for EGF and EGF receptor became apparent in the granulosa and theca interna cell layers, without appreciable staining in the surrounding stromal cells. The immunostaining for EGF and EGF receptor in the granulosa cells and theca interna cells persisted in preovulatory follicles and corpus luteum, and intensified in the midluteal phase. The stromal cells surrounding the corpus luteum were negative for EGF and EGF receptor staining. In the regressing corpus luteum, immunostaining for EGF and EGF receptor was present in the peripheral lutein cells adjacent to the central core of scar tissue, but absent in the scar tissue of the central core. Corpus albicans showed no staining for EGF and EGF receptor. By contrast, the stromal cells surrounding the corpus albicans in the cortex region demonstrated intense staining for EGF and EGF receptor, while the stromal cells surrounding the corpus albicans in the medullary region were negative for immunostaining. In the case of atretic follicles, the theca interna cells showed intense staining for EGF and EGF receptor, but immunostaining in the scattered granulosa cells was negligible. This is the first study to demonstrate a remarkable change in the expression of EGF and EGF receptor in the oocyte, granulosa cells, thecal cells, and surrounding stromal cells over the course of follicular growth and regression. The results obtained support EGF participation in oocyte maturation and in follicular growth and atresia. The intense immunostaining for EGF and EGF receptor observed in the theca interna cells in atretic follicles and the stromal cells surrounding corpus albicans in the cortex region raises the possibility of EGF involvement in transformation of thecal cells into stromal cells. Furthermore, the cell type-specific simultaneous expression of EGF and EGF receptor in follicular and stromal compartments in the various stages of follicular development suggests that an autocri

Topics: Adult; Endometriosis; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Hysterectomy; Immunohistochemistry; Leiomyoma; Menstrual Cycle; Ovarian Follicle; Ovariectomy; Ovary; Uterine Cervical Neoplasms

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

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

A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent Km approximately 0.45 mM) for phosphorylation by purified p44mpk, a MAP kinase from sea star oocytes. The peptide was also phosphorylated by a related human MAP kinase, which was identified by immunological criteria as p42mapk. Within 5 min of treatment of human cervical carcinoma A431 cells with EGF or phorbol myristate acetate (PMA), a greater than 3-fold activation of p42mapk was measured. However, Mono Q chromatography of A431 cells extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mono Q, phosphorylated myelin basic protein (MBP) and other MAP kinase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a approximately 45 kDa protein upon Superose 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mono Q than p42mapk (peak II), exhibited a nearly identical substrate specificity profile to that of p42mapk, but it immunoreacted as a 40 kDa protein only with anti-p44mpk antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mono Q peak III was tentatively identified as an active form of p34cdc2 associated with cyclin A. The Mono Q peaks III and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42mapk. These findings indicated that multiple proline-directed kinases may mediate phosphorylation of the EGF receptor.

Topics: Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Calcium-Calmodulin-Dependent Protein Kinases; CDC2 Protein Kinase; Cyclins; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Molecular Sequence Data; Oocytes; Peptide Fragments; Phosphorylation; Phosphothreonine; Protein Kinases; Starfish; Substrate Specificity; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Several malignant neoplasms express high levels of epidermal growth factor receptors. The aim of this study was to assess whether 123I-labeled epidermal growth factor can concentrate in lymph node metastases of squamous cell carcinomas of the cervix. 14 patients with advanced cervical cancer were selected because of their high probability of lymph node metastases. Planar scintigrams were recorded from the lower and upper abdomen following subcutaneous injection of 123I-labeled epidermal growth factor into the web space of each foot. Scintigraphic images were interpreted without knowledge of computerized tomography scan (n = 13) and ultrasound (n = 9) results from the pelvic lymph nodes. In 2 patients, histological verification was performed by diagnostic biopsy of pelvic lymph nodes. Nodal involvement was confirmed by computerized tomography for 4 of the 11 positive scans and by ultrasound for 2. In 11 out of 14 patients an increased uptake of 123I-labeled epidermal growth factor could also be seen in the primary tumour. Our findings suggest that targeting of cervical cancer lymph node metastases can be achieved by in vivo binding of 123I-labeled epidermal growth factor with receptors on tumour cell surfaces.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Iodine Radioisotopes; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Radionuclide Imaging; Uterine Cervical Neoplasms

The effect of epidermal growth factor on the radiation response of two human squamous carcinoma cell lines, A431 (from vulva) and SiHa (from cervix), was examined. In both lines, cells in S phase were more radioresistant than cells in other cell cycle phases. Epidermal growth factor present after irradiation enhanced the radiation response of A431 cells in different cell cycle phases, whereas no effect was seen for SiHa cells. The enhancement was maximum with 10 ng/ml epidermal growth factor and was associated mainly with a reduction in the shoulder region of the cell survival curve. The ratio between the n values of the control and epidermal growth factor treated total cell population, G1, S, and G2M cells is 2.2, 4.1, 1.7, and 2.2, respectively. Epidermal growth factor reduced plating efficiency by about 50% for A431 cells in different cell cycle phases whereas a slight increase in plating efficiency was seen for SiHa cells. The present results indicate that epidermal growth factor related radiosensitization is dependent on both cell line and cell cycle.

Topics: Carcinoma, Squamous Cell; Cell Cycle; Cell Survival; Dose-Response Relationship, Radiation; Epidermal Growth Factor; Female; Humans; In Vitro Techniques; Radiation Tolerance; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Vulvar Neoplasms

We have used 123I-labelled epidermal growth factor (EGF) scans to study 14 patients with advanced cervical cancer. Abnormal lymph node imaging was seen most clearly 6-8 h after the injection and revealed abnormal uptake by pelvic lymph nodes in 11 patients. 4 of these 11 had abnormal computerised tomographic and ultrasound scans; in the other 7 conventional radiology did not confirm the presence of disease.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Evaluation Studies as Topic; Female; Humans; Iodine Radioisotopes; Lymphatic Metastasis; Pelvis; Tomography, Emission-Computed; Uterine Cervical Neoplasms

Fourteen patients suffering from advanced inoperable cervical cancer were investigated by planar scintigraphy after subcutaneous administration of a radiolabelled (I-123) epidermal growth factor (EGF). The objective of the study was to test whether labelled EGF concentrates in lymph node metastases of squamous cell cancer of the cervix uteri. Scintigraphic results were correlated with the gynecological findings, computed tomography (CT), ultrasound (US) and in two patients with histology. A series of scintigrams were performed up to 24 hours post injection. Slight activity in liver and kidneys was found already 30 min after s.c. injection of EGF. Optimal imaging quality for the lymphatics was obtained at 6-8 hours post injection. Accumulation in the pelvic lymph nodes was documented by the region of interest technique (ROI). Lymph nodes of the inguinal and iliac communis region were marked in all cases. Due to this, accumulation of EGF could not be called selective. In 11/14 patients hot spots were correlated to other pelvic lymph nodes. In 4/11 patients with positive EGF-scanning metastatic disease was confirmed by CT scan and/or US examination. 2/11 patients underwent a Probatoria operation. The respective histological reports confirmed our scintigraphic results. In conclusion, labelled EGF did not fulfil our theoretical expectations of excellent accumulation in lymph node metastases and cannot at present be recommended for routine clinical use.

Topics: Carcinoma, Squamous Cell; Epidermal Growth Factor; Female; Humans; Lymph Nodes; Lymphatic Metastasis; Neoplasm Staging; Radionuclide Imaging; Uterine Cervical Neoplasms

The effect of epidermal growth factor on radiosensitivity of cells in spheroids and its relationship to radioresistance associated with cell-cell interactions was examined. A human squamous carcinoma cell lines, CaSki, was used. Epidermal growth factor present for 48 hr before irradiation reduced the plating efficiency but did not affect the radiosensitivity of cells. However, epidermal growth factor present after irradiation, that is, during the period of colony formation, reduced the plating efficiency and increased the radiosensitivity of cells from spheroids. Both effects were maximum at 10 ng/ml epidermal growth factor. The enhancement in radiation response was not related to epidermal growth factor effects on potentially lethal and sublethal damage repair. In the absence of cell-cell interactions, such as monolayer cultures and spheroids disaggregated for 15 hr before irradiation, radiosensitivity enhancement by epidermal growth factor was associated with reduced shoulder of the cell survival curve. However, in the presence of cell-cell interactions, such as intact spheroids and spheroids disaggregated immediately before irradiation, in addition to reduced shoulder, epidermal growth factor treatment increased the slope compared to that of the monolayer cultures. The results indicate that epidermal growth factor enhances cellular radiosensitivity and modifies effects of cell-cell interactions.

Topics: Carcinoma, Squamous Cell; Cell Communication; Clone Cells; Epidermal Growth Factor; Female; Humans; Radiation Tolerance; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Retinoic acid (RA) increases epidermal growth factor (EGF) receptors in many cells; in ME180 cells, a human epidermoid carcinoma, RA resulted in a dose- and time-dependent reduction of EGF binding. In RA-treated ME180 cells, binding was 41% of the control. The reduction of EGF binding was due to a decrease in the number of receptors, from 8.7 x 10(4) to 3.6 x 10(4) per cell. The difference was present 8 h after the addition of RA and was reversible 3 days after its removal. Scatchard analysis indicated that RA did not change the binding affinity of EGF (Kd = 1 nM). Also, RA did not alter the rate of EGF internalization or the down-regulation induced by exogenous EGF. Flow-cytometric analysis revealed that RA did not alter the cell cycle. Soluble cell membrane extracts were prepared in a Tris buffer with protease inhibitors, immunoprecipitated, electrophoresed, and immunoblotted with an antiserum to EGF receptors. The EGF receptor band of Mr 170,000 was decreased in RA-treated cells. These results suggest that RA reduces the synthesis of EGF receptors in ME180 cells.

Topics: Carcinoma, Squamous Cell; Cell Division; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Time Factors; Tretinoin; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Stearic acid and iodo-stearic and inhibited cell growth in a cervical cancer cell line (HOG-1) in a dose-related manner, with a half maximal effect at 50 microM stearic acid. Addition of oleic acid abrogated the effect of stearic acid. EGF-stimulated DNA synthesis and growth of HOG-1 cells was inhibited in the presence of stearic acid without any apparent effect on EGF receptor number or affinity.

Topics: Cell Division; Epidermal Growth Factor; Female; Humans; Stearic Acids; Tumor Cells, Cultured; Uterine Cervical Neoplasms; Uterine Neoplasms

Effects of epidermal growth factor (EGF) on growth and tumor antigen-4 (TA-4) production of newly established uterine cervical cancer cell line (OMC-1) are reported. OMC -1 was established from a metastatic lesion of Virchow's lymph node of a large cell non-keratinizing squamous cell carcinoma of the uterine cervix and successively subcultured for about 4 years. Scatchard analysis of EGF binding to OMC-1 cells indicated a single class of binding sites with a dissociation constant (Kd) of 360 pM. The theoretical maximum number of binding sites was 2.4 x 10(4) sites/cell. The growth of OMC-1 cells was stimulated by EGF at 0.01-0.1 nM and inhibited at higher concentrations. The TA-4 production of OMC-1 cells was slightly stimulated by EGF at 0.01-1 nM and significantly stimulated at 10 nM. OMC-1 may serve as one of the available model systems for studies of regulation of proliferation and tumor marker production by EGF, particularly in cervical squamous cell carcinomas.

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

Tumor necrosis factor (TNF) is a cytokine which induces cytotoxicity in some but not all tumor cells. Initial studies of five tumor cell lines demonstrated that TNF was able to rapidly (within 30 min) modulate tyrosine protein kinase activity of epidermal growth factor (EGF) receptors on tumor cell lines which were sensitive to the cytotoxic effects of TNF but not alter EGF receptor kinase activity in TNF-resistant tumor cells. Two tumor cell lines (ME-180 cervical carcinoma and T24 bladder carcinoma) which have been shown to express similar TNF-binding characteristics but differ in their sensitivity to the cytotoxic actions of TNF were chosen for further characterization. Treatment of TNF-sensitive ME-180 cells with 1 nM TNF resulted in a 3-fold stimulation of EGF receptor tyrosine protein kinase activity within 10 min which correlated with increased phosphorylation of EGF receptor protein itself. In addition, dose-response studies indicate that similar concentrations of TNF modulate both ME-180 cell growth and EGF receptor kinase activity. Treatment of TNF-resistant T24 cells showed that TNF had no significant effect on their growth, EGF receptor tyrosine protein kinase activity, or phosphorylation of EGF receptor protein although EGF receptor kinase activity was stimulated by EGF. Quantitation of receptors expressed on the surface of ME-180 and T24 cells demonstrated a 3-fold difference between the number of EGF-binding sites on T24 (100,000) versus ME-180 cells (300,000), suggesting the relative abundance of EGF receptor does not solely account for differential effects of TNF on EGF receptor activation in these two cell lines. Phosphoamino acid analysis of EGF receptor from 32P-equilibrated ME-180 cells demonstrated that TNF-induced phosphorylation of amino acids which was quantitatively similar to that of EGF but distinct from the effects of phorbol ester. However, unlike EGF, TNF was unable to stimulate EGF receptor kinase activity in ME-180 cell lysates. The kinetics of EGF receptor activation and the metabolic consequence of activation of EGF receptor activity by TNF appear to be distinct from those induced by EGF. These results suggest that TNF-induced modulation of EGF receptor occurs through a unique mechanism and may play a role in the cytotoxic actions of TNF.

Topics: Carcinoma, Squamous Cell; Cell Line; Cell Survival; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Kinetics; Lung Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urinary Bladder Neoplasms; Uterine Cervical Neoplasms

The clinical implications of the epidermal growth factor (EGF) and its receptor (EGF-R) were studied in 52 squamous cell carcinomas of the uterine cervix. In comparison to 40 biopsies of the normal cervix EGF-R capacity was significantly increased in the carcinomas, while the affinity was unchanged. The amount of EGF-like substances extracted from the tumors was increased in patients with lymph node metastases, in whom 5-year survival is reduced. Irrespective of tumor stage patients with a very high level of EGF-R (greater than 100 fmole/mg protein) were more likely to have recurrences later or to die from disease: recurrence or death occurred in 5 of 7 patients with high capacity and in 2 of 45 patients with low capacity. Our data suggest that the level of EGF-R is indicative of the biological aggressiveness of cervical carcinomas.

Topics: Adult; Carcinoma, Squamous Cell; Cervix Uteri; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Lymphatic Metastasis; Neoplasm Metastasis; Neoplasm Recurrence, Local; Uterine Cervical Neoplasms

In this study we investigated the presence of epidermal growth factor receptors (EGF-R) and the tissue levels of EGF-like factors (EGF-F) in ovarian, endometrial, cervical and breast carcinomas. EGF-R were found in 33/40 (83%) cervical, 15/26 (58%) endometrial, 64/141 (45%) ovarian, and 19/59 (33%) breast carcinomas. The highest number of EGF-R binding sites was detected in cervical carcinomas followed by endometrial, breast and ovarian carcinomas. The tissue concentrations of EGF-like factors, were investigated in extracts of 63 ovarian, 25 breast, 12 cervical, 14 endometrial carcinomas and in 21 biopsies of nonmalignant tissue such as myometrium and ovaries. The extracts of nonmalignant tissues had a mean EGF-F level of 1.5 +/- 0.7 ng/mg with a concentration range from 0 to 4 ng/mg. The mean EGF-F levels of malignant tissues were: ovarian carcinomas 4.2 +/- 1.5 ng/mg (range 0-15 ng), endometrial carcinomas 4.5 +/- 1.7 ng/mg (range 0-12 ng), cervical carcinomas 4.15 +/- 1.1 ng/mg (range 0-8) and breast carcinomas 3.16 +/- 1.1 ng/mg (range 0-10 ng). About 30% ovarian, endometrial and cervical carcinomas and 16% breast carcinomas, respectively, had enhanced EGF levels from 5 ng/mg to 15 ng/mg compared to nonmalignant tissues. The EGF-F of tissue extracts consists of EGF and transforming growth factor TGF alpha) as shown by the results of EGF and TGF alpha radioimmunoassays. It is assumed that in some tumors the EGF-F tissue levels influence the number of biochemically detectable EGF binding sites.

Topics: Binding Sites; Breast Neoplasms; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Ovarian Neoplasms; Prognosis; Radioimmunoassay; Transforming Growth Factors; Uterine Cervical Neoplasms; Uterine Neoplasms

Effects of EGF on proliferation and tumor marker secretion of cervical cancer cells are reported together with the characteristics of EGF receptors on the cells. TA-4 producing cell line (OMC-1) originating from cervical squamous cell carcinoma and CA-125 producing cell line (OMC-4) originating from cervical adenocarcinoma, were used. Scatchard plot of EGF binding to OMC-1 indicated a single class of binding sites with a dissociation constant (Kd) of 360pM, whereas that of OMC-4 was curvilinear suggesting two classes of binding sites with a Kd of 170pM and 510pM. The theoretical maximum number of binding sites of OMC-1 and OMC-4 was 2.4 X 10(4) and 1.6 X 10(5), respectively. Effects of EGF on growth were studied by monitoring cell number and the incorporation of 3H-thymidine into the DNA of the cells. OMC-1 was stimulated by EGF at low concentrations (0.01-0.1nM) and inhibited at higher concentrations. OMC-4 was not stimulated by EGF. The TA-4 secretion of OMC-1 was slightly stimulated by EGF at low concentrations (0.01-1nM) and significantly stimulated at high concentration (10nM). The CA-125 secretion of OMC-4 was not stimulated by EGF. These results suggest that there are some differences between cervical squamous cell carcinoma and adenocarcinoma in the mechanisms of regulation of proliferation and tumor marker secretion by EGF.

Topics: Adenocarcinoma; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Binding Sites; Biomarkers, Tumor; Carcinoma, Squamous Cell; Cell Division; Cell Line; DNA, Neoplasm; Epidermal Growth Factor; ErbB Receptors; Female; Humans; Uterine Cervical Neoplasms

The effects of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) and epidermal growth factor (EGF) on the synthesis and secretion of human chorionic gonadotropin (hCG) and its subunits by normal and malignant trophoblasts as well as by non-trophoblastic cells were investigated in vitro. The explants of normal early placental tissues, choriocarcinoma cell line BeWo and non-trophoblastic tumor cell line CaSki from epidermoid carcinoma of the cervix, respectively, were cultured in the presence or absence of dibutyryl cAMP or EGF. The addition of either dibutyryl cAMP (1 mM) or EGF (100 ng/ml) caused significant increases in the synthesis and secretion of hCG and its subunits in cultures of normal and malignant trophoblasts, but had no stimulatory effect on hCG beta synthesis and secretion in culture of non-trophoblastic cell line CaSki that secretes predominantly hCG beta-like material. The magnitude of the stimulatory effects of dibutyryl cAMP and EGF on hCG (alpha,beta) synthesis and secretion by BeWo cells was much greater than that observed in normal trophoblasts. The time course of these stimulatory effects indicated that EGF-stimulated increase in hCG synthesis and secretion required a lag period longer than that for the dibutyryl cAMP-stimulated increase. These results suggest that there were no differences in normal and malignant trophoblasts in the mechanism for the stimulatory regulation of hCG (alpha, beta) synthesis and secretion, but immunoreactive hCG beta synthesis and secretion in non-trophoblastic tumor cells are regulated by a mechanism different from that in trophoblastic cells.

Topics: Bucladesine; Carcinoma, Squamous Cell; Cell Line; Chorionic Gonadotropin; Epidermal Growth Factor; Female; Humans; Pregnancy; Trophoblastic Neoplasms; Uterine Cervical Neoplasms; Uterine Neoplasms

The CaSki cell line derived from an epidermoid carcinoma of the uterine cervix produces and releases two types of tumor-associated antigen. One is a eutopic antigen, TA-4 and the other is an ectopic antigen, hCG beta-like material. The aim of the present investigation was to elucidate a possible difference in the induction mechanism of production of TA-4 and hCG beta-like material in the CaSki cells in relation to cellular differentiation and gene modulation. The exposure to epidermal growth factor (EGF, 100ng/ml) for two days greatly increased TA-4 production by the cultured CaSki cells without affecting cell growth and immunoreactive hCG beta production. The EGF-stimulated increase in TA-4 production required a lag period of approximately 24 hours. The exposure to sodium butyrate (2.5mM) stimulated immunoreactive hCG beta production by the cells, but decreased TA-4 production. The stimulatory effect of sodium butyrate on immunoreactive hCG beta production occurred only during the exposure to the agent. These discrepant effects of EGF and sodium butyrate on the production of TA-4 and hCG beta-like material by the CaSki cells suggest that a fundamental difference exists in the induction mechanism for the expression of these two types of tumor-associated antigen in cervical squamous carcinoma cells.

Topics: Antigens, Neoplasm; Butyrates; Butyric Acid; Carcinoma, Squamous Cell; Cells, Cultured; Chorionic Gonadotropin; Culture Media; Epidermal Growth Factor; Female; Humans; Uterine Cervical Neoplasms

We describe the properties of two monoclonal antibodies produced to a synthetic peptide consisting of residues 985 to 996 from the cytoplasmic domain of the epidermal growth factor (EGF) receptor. We have examined a group of ten human tumors including cervical, ovarian, and vulval carcinomas for expression of EGF receptors by immunohistological staining using one of these antibodies and another monoclonal antibody to the extracellular domain of the molecule. The tumors were examined using a sensitive amplified enzyme system and a less sensitive indirect staining method. There was generally a good correlation in staining intensity with the two monoclonal antibody reagents. Both antibodies showed strong staining of squamous cell carcinomas and usually weak or heterogeneous patterns with the adenocarcinomas. Samples of each tumor were solubilized in detergent and analyzed for the presence of functional EGF receptors by immunoprecipitation and autophosphorylation. Three of the squamous cell tumors gave labeled bands, Mr 170,000, on sodium dodecyl sulfate:polyacrylamide gels. DNA was extracted from seven of the tumors and digested with two restriction endonucleases, and the fragments were analyzed on Southern blots using probes representing the extracellular and cytoplasmic domains of the molecule. The tumor DNA showed no apparent rearrangements or amplifications when compared to the EGF receptor gene in human placental DNA. These results suggest that there is a high level of EGF receptors on some squamous cell tumors.

Topics: Antibodies, Monoclonal; Epidermal Growth Factor; ErbB Receptors; Female; Gene Expression Regulation; Humans; Ovarian Neoplasms; Phosphoproteins; Phosphorylation; Receptors, Cell Surface; Uterine Cervical Neoplasms; Vulvar Neoplasms