Epidermal growth factor and transforming growth factor alpha characteristics of human oral carcinoma cell lines.

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

Expression of EGF, TGF-alpha and EGFR in squamous cell lung carcinomas.

Immunohistochemical study of epidermal growth factor (EGF), epidermal growth factor receptor (EGFR) and transforming growth factor-alpha (TGF-alpha) expression was performed on paraffin-embedded tissue specimens of 70 squamous cell lung carcinomas. The carcinomas were placed to one of the following eight groups, according to the results of EGF, TGF-alpha and EGFR expression: group 1: none, group 2: only EGFR, group 3: EGFR and TGF-alpha, group 4: EGFR and EGF, group 5: TGF-alpha and EGF, group 6: all three, group 7: only TGF-alpha and finally group 8: only EGF. Statistical analysis of the results revealed that the ratio of squamous cell lung carcinomas with lymph node metastasis was significantly higher in groups 4, 5 and 6 (P less than 0.01). We also examined whether EGF receptors were truncated with the use of two monoclonal antibodies directed against different portions of the receptor (EGFR1 and F4). No truncated EGF receptors were detected. These results suggest that lung carcinomas expressing the molecules EGF/EGFR, TFG-alpha/EGFR or TGF/alpha/EGF/EGFR display pathologic features of more aggressive disease.     

Low mitogenic response to EGF and TGF-alpha: a characteristic feature of cultured Kaposi's sarcoma derived cells

We have investigated the mitogenicity of Epidermal Growth Factor (EGF) and Transforming Growth Factor alpha (TGF-alpha) for cultured Kaposi's sarcoma derived cells (KS cells). In contrast to control fibroblasts from the same patients, KS cells revealed only a weak mitogenic response to these growth factors. Neither EGF nor TGF-alpha were able to substitute for Platelet-derived growth factor (PDGF) when KS cells were grown in PDGF-depleted Platelet-Poor-Plasma serum (PPPS)-supplemented medium. The low mitogenicity of EGF and TGF-alpha for KS cells is not based upon a reduced expression of EGF receptor mRNA and protein in KS cells. However, the binding of EGF to KS cells is about 50% lower than that to fibroblasts. This reduced binding is not due to an occupation of the receptors by TGF-alpha since the expression level of this mitogen in different KS cell lines does not correlate with their capacity to bind EGF. In contrast, the low EGF binding seems to be an intrinsic feature of the EGF receptors of KS cells. In spite of the low mitogenicity of EGF for the tumor cells, the expression of c-myc, PDGF-A and Fibroblast growth factor 5 (FGF-5) mRNA is equally induced by purified EGF in KS cells and fibroblasts. This shows that at least the signal transduction pathways which lead to the expression of these genes are functional in KS cells.     

EGF and TGF-alpha, the ligands of hyperproduced EGFR in human esophageal carcinoma cells, act as autocrine growth factors

In order to ascertain autocrine growth factors in esophageal carcinomas, we analysed expression of mRNAs and proteins for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR) in 6 esophageal carcinoma cell lines. Gene alterations were also examined. All of the esophageal carcinoma cell lines expressed mRNA for EGFR and TGF-alpha genes. Interestingly, EGF mRNA of about 5.0 kb was also detected in TE-1, TE-2, and TE-8 cells. Production of protein was also confirmed by binding assay and ELISA on 3 of the 6 cell lines. The cells had a relatively high number of EGFRs and produced TGF-alpha and EGF protein at the same time. Furthermore, anti-EGF (KEM-10) and anti-TGF-alpha (WA-3) monoclonal antibodies (MAbs) inhibited spontaneous uptake of tritiated thymidine (3H-TdR) by TE-1 cells which expressed EGF, TGF-alpha and EGFR mRNA and protein. These results strongly suggest that EGF and/or TGF-alpha produced by carcinoma cells function as autocrine growth factors for human esophageal carcinomas.     

Growth control by epidermal growth factor and transforming growth factor-alpha in human lung squamous carcinoma cells.

Although EGF receptor expression is generally elevated in human lung squamous carcinoma, the biological significance of this phenomenon and the role of EGF and TGF-alpha in this disease are poorly understood. We have investigated three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) and have shown, using an antibody (EGFR1) directed against the EGF receptor, that the majority of cells in all three lines express the EGF receptor. Using a ligand binding assay, Scatchard analysis indicated high concentrations (1,300-2,700 fmol mg-1 protein) of a single low affinity binding site (Kd = 3-5 nM) within these lines. Addition of EGF or TGF-alpha at concentrations greater than 0.1 nM resulted in growth inhibition of all three lines and this was associated with an accumulation of cells in the G2/M phase of the cell cycle. Growth inhibitory effects were not explained by an enhancement of cellular differentiation as monitored by involucrin expression and the ability to form cornified envelopes. While the presence of EGF could not be detected in medium conditioned by the NX002 cell line, mRNA for TGF-alpha was detected in all three lines suggesting the possibility of an autocrine loop. These results together with reports of growth inhibition by EGF and TGF-alpha in other systems suggest that EGF and similar molecules might have a growth regulatory role in lung cancer cells and modulation of such may have therapeutic potential.     

Transforming growth factor gene expression in human endometrial adenocarcinoma cells: regulation by progestins.

In an attempt to understand the antiproliferative effects of progestins in endometrial cancer, we have examined the effects of the potent progestin, medroxyprogesterone acetate (MPA), on the cell proliferation and the expression of transforming growth factor (TGF) alpha and beta genes in human endometrial adenocarcinoma cell lines. The two cell lines used were Ishikawa, var 1, and HEC-50. In addition, the effects of exogenous TGF-alpha and anti-epidermal growth factor (EGF) receptor monoclonal antibody on cell proliferation were determined. Incubation of both cell lines with MPA resulted in a time- and dose-dependent inhibition of cell proliferation. Half-maximal growth inhibition was observed at 0.6 nM. In Ishikawa cells, the relative abundance of TGF-alpha was significantly reduced by MPA. A significant decrease in TGF-alpha mRNA was apparent 6 h after exposure to MPA and a further decrease was seen 12-24 h after addition of the progestin. The concentration of TGF-alpha immunoreactivity in conditioned medium of MPA-treated cells was also significantly reduced compared to control cultures. MPA had no effect on TGF-alpha expression by HEC-50 cells. EGF mRNA was not detected by Northern blot analysis in either cell type. MPA had no significant effect on EGF receptor mRNA abundance but resulted in a small increase in EGF receptor number in Ishikawa cells. Anti-EGF receptor monoclonal antibody (0.6-6 nM) inhibited Ishikawa cell growth but had no effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines, but Ishikawa cells were significantly more sensitive to exogenous TGF-alpha than HEC-50 cells. Furthermore, TGF-alpha could reverse the growth inhibitory effects of MPA on Ishikawa cells. A decrease in TGF-beta mRNA abundance was also observed in MPA-treated Ishikawa and HEC-50 cells. This effect was of small magnitude, variable, and only observed after prolonged exposure to MPA. These observations are consistent with the hypothesis that the antiproliferative effects of progestins on Ishikawa cells are mediated by decreased expression and autocrine action of TGF-alpha. Since similar growth inhibition is also seen in the HEC-50 cells in which progestins have no effect on TGF-alpha expression, additional mechanisms are likely to be involved in the antiproliferative effects of progestins in human endometrial cancer.     

Role of interleukin-8 secreted from human oral squamous cell carcinoma cell lines

Interleukin-8 (IL-8) is an important cytokine involved in tumor growth and angiogenesis in a variety of malignancies. Furthermore, matrix metalloptoteinases (MMPs) also play important roles in the invasion and metastasis of carcinomas including oral squamous cell carcinoma (OSCC). We studied whether IL-8 and MMPs participate in tumorigenesis and metastasis of OSCC. First, we investigated the gene and protein expressions of IL-8 and IL-8 receptor (IL-8R), and the effect of IL-8 on proliferation, migration and invasion of OSCC. Second, we thus also investigated the effect of IL-8 on MMP release in OSCC cells. OSCC cell lines NA and HSC-4 constitutively expressed IL-8 mRNA and secreted its protein in vitro. The production of IL-8 was significantly enhanced by the addition of tumor necrosis factor (TNF)-alpha and IL-beta, but not interferon (IFN)-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-2. Flow cytometric analysis revealed the constitutive expression of both receptors of IL-8, IL-8RA and IL-8RB, in OSCC cell lines. The expression of IL-8 receptors in HSC-4 cells was stronger than that in NA cells. The intensity of IL-8RA expression was stronger than that of IL-8RB expression in each cell line. The expression of IL-8 receptors was not altered by the addition of cytokines such as TNF-alpha and IL-1beta. The conditioned medium containing IL-8 from OSCC cell lines induced migration and invasion of OSCC cells, but did not change cell proliferation. The differences in migrational and invasive ability between NA cells and HSC-4 cells were correlated with the expression intensity of IL-8 receptors in each cell line. Neutralizing antibodies to IL-8, IL-8RA and IL-8RB partially inhibited the chemotactic activity induced by conditioned medium. The expression of MMP-2, -7 and -9 was detected in culture supernatants from these OSCC cell lines. The expressions of MMP-7 protein and mRNA were enhanced by the addition of rIL-8, but that of other MMPs was not observed in a similar manner. These results suggest that IL-8 secreted from OSCC may contribute to the invasion of OSCC through the regulation of MMP-7 expression.     

Growth regulation of human renal carcinoma cells: role of transforming growth factor alpha.

Findings of increased numbers of epidermal growth factor receptors (EGF-R) and increased expression of transforming growth factor alpha (TGF-alpha) in surgical specimens of human renal cell carcinoma have led to the proposal that growth of these tumors may be regulated by TGF-alpha in an autocrine manner. In the studies presented here, we have examined this hypothesis using two human renal carcinoma cell lines, SKRC-4 and SKRC-29. We demonstrated that both SKRC-4 and SKRC-29 cells were growth stimulated by greater than 35% when cultured in the presence of TGF-alpha or EGF and were inhibited by 29% to 46% if cultured in the presence of anti-EGF-R monoclonal antibody 225. Treatment of cells with TGF-alpha enhanced the levels of expression of EGF-R mRNA and TGF-alpha mRNA. In addition, incubation of cells with monoclonal antibody 225 significantly elevated the levels of excreted TGF-alpha species in the culture medium. Our findings suggest that proliferation of human renal carcinoma cells may be regulated by endogenously produced TGF-alpha and that this regulatory pathway can be interrupted using antibody to its receptor, EGF-R.     

Type I collagen degradation by invasive oral squamous cell carcinoma

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

Expression of epidermal growth factor, transforming growth factor-alpha and their receptor genes in human gastric carcinomas; implication for autocrine growth

The expressions of mRNA for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and EGF receptor (EGFR) genes were examined in 7 human gastric carcinoma cell lines and 15 gastric carcinoma tissues and the corresponding normal mucosas. All of the gastric carcinoma cell lines expressed mRNA for EGFR and TGF-alpha genes. TMK-1 and MKN-28 cells also expressed EGF mRNA. Production of EGF, TGF-alpha and EGFR protein by gastric carcinoma cell lines was also confirmed by EGF and TGF-alpha specific monoclonal antibody binding. As for surgical specimens, EGFR and TGF-alpha mRNA were detected at high levels in all the tumor tissues. Interestingly, EGF mRNA was detected in 5 (33.3%) of the 15 gastric carcinomas but it was not detected in normal tissues. Moreover, anti-EGF and anti-TGF-alpha monoclonal antibodies inhibited the spontaneous 3H-TdR uptake by gastric carcinoma cells. These results suggest that EGF and/or TGF-alpha produced by tumor cells act as autocrine growth factors for gastric carcinomas.     

Epidermal growth factor: receptor and ligand expression in human breast cancer

The epidermal growth factor (EGF) gene is expressed by most human breast cancer cell lines as well as 83% of human breast cancers in vivo. Furthermore, EGF mRNA is detectable in normal human breast tissue. These data suggest that EGF may have a functional role in both normal and neoplastic human breast tissue. Expression of EGF was generally highest in steroid receptor positive human breast tumor biopsies and cell lines. EGF expression was increased by progestins in T-47D and ZR 75 human breast cancer cells. Furthermore, progestins specifically increased the level of TGF-alpha and EGF-receptor mRNA in T-47D cells. Under these same conditions progestins inhibit growth of the cells. Regulation of expression of EGF, TGF-alpha and the EGF-receptor is unlikely to be directly related to the mechanism of progestin induced growth inhibition in T47-D cells. T-47D-5 cells are more sensitive than T-47D cells to progestin and antiestrogen induced growth inhibition. T-47D-5 cells do not express EGF and contain very low levels of TGF-alpha mRNA. The higher level of EGF and TGF-alpha expression in T-47D cells may be one mechanism by which these cells decrease their sensitivity to growth inhibition by progestins and antiestrogens.     

Autonomous growth of androgen-independent human prostatic carcinoma cells: role of transforming growth factor alpha

The androgen-independent prostatic carcinoma cell line PC3 is known to exhibit autonomous growth in vitro and in vivo. The purpose of the present study was to investigate the role of transforming growth factor alpha (TGF-alpha) and its receptor, the epidermal growth factor (EGF) receptor, in the regulation of PC3 cell proliferation. Results showed that PC3 cells secrete factors into conditioned medium that are mitogenic for the less aggressive prostatic carcinoma lines DU145 and LNCaP. Gel filtration chromatography of PC3-conditioned medium revealed a major peak of mitogenic activity at a molecular weight of 5,000 to 10,000 which was inhibited by the addition of antibody to TGF-alpha. The synthesis and secretion of TGF-alpha by PC3 cells were further demonstrated by immunoblotting and radioimmunoassay. Radioreceptor analysis showed a single class (Kd 5.3 nM) of EGF receptors on PC3 cells. The presence of Mr 170,000 EGF receptors on PC3 cells was further demonstrated by immunoprecipitation of metabolically labeled proteins. TGF-alpha was effective in stimulating the growth of low-density, but not high-density, PC3 cultures. In addition, the proliferation of PC3 cells under serum-free defined conditions was inhibited by antibodies to TGF-alpha and/or the EGF receptor. These data indicate that TGF-alpha/EGF receptor interactions are partially responsible for autonomous growth of the PC3 cell line and may explain one mechanism of escape from androgen-dependent growth in human prostatic carcinoma.     

Expression of epidermal growth factor receptor gene in cultured human lung cancer cells

The expression and organization of epidermal growth factor (EGF) receptor gene in cultured human lung cancer cell lines (5 adenocarcinomas, 3 squamous cell carcinomas, 2 small cell carcinomas and 1 large cell carcinoma) have been studied. Two (PC-8 and PC-9) of the adenocarcinomas overproduced EGF receptor mRNA and protein, and exhibited gene amplification, the magnitude of which was comparable to that of A431 cells. Six cell lines (3 adenocarcinomas, 2 squamous cell carcinomas and 1 small cell carcinoma) expressed EGF receptor gene and its product to a significant level without gene amplification, and the other three cell lines were found to be negative as regards expression.     

Interleukin 1alpha and interleukin 6 promote the in vitro growth of both normal and neoplastic human cervical epithelial cells.

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.     

The effect of hypoxic microenvironment on matrix metalloproteinase expression in xenografts of human oral squamous cell carcinoma

Hypoxia is a common environmental stress factor and is associated with physiological and pathological conditions related to cancer invasion and metastasis. The process of cancer cell invasion involves degradation of the extracellular matrix. Here, we examine the effect of hypoxic microenvironment on matrix metalloproteinase expression in human oral squamous cell carcinoma under in vitro and in vivo conditions. At first, the expression levels of HIF-1alpha and matrix metalloproteinase (MMP) proteins in human oral squamous cell carcinoma cell lines, SAS and HSC-2 cultured under hypoxic or normoxic condition, were assessed by Western blotting. Enzyme activity and mRNA of MMP under hypoxic or normoxic condition were also investigated. Then the SAS and HSC-2 cells were transplanted subcutaneously into immunodeficient mice and the correlation between hypoxia and protein expression for MMPs, HIF-1alpha and Ki-67 were assessed. Hypoxic region was detected by in situ hypoxic probe, pimonidazole. MMP proteins and mRNA in both SAS and HSC-2 cells were increased under hypoxic condition. In xenograft, MMP-2 was expressed in tumor tissue, especially in hypoxic region. In contrast, MMP-9 expression was recognized in tumor tissue, especially neighboring stromal tissues containing blood vessels. Our study suggests that the hypoxic microenvironment in human oral squamous cell carcinoma plays important roles in expression for HIF-1alpha and MMPs, and proliferative activity of tumor cells.     

Restoration of BRAK / CXCL14 gene expression by gefitinib is associated with antitumor efficacy of the drug in head and neck squamous cell carcinoma

Clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for epidermal growth factor (EGF) receptor tyrosine kinase, has been shown in non-small-cell lung carcinoma patients with EGF receptor mutations, so these mutations are useful marker(s) to find a responder for the drug. Recent studies have shown that the EGF receptor gene mutation is rare in squamous cell carcinoma in the esophageal and head and neck regions. We previously reported that the expression of the chemokine BRAK/CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells was down-regulated by EGF treatment, and that forced expression of BRAK in tumor cells decreased the tumorigenicity of the cells in xenografts. Thus, we investigated the relationship between restoration of BRAK expression by gefitinib and the efficacy of the drug for tumor suppression. We found that EGF down-regulated BRAK expression through the MEK–extracellular signal regulated kinase pathway and that this down-regulated expression was restored by gefitinib in vitro . Oral administration of gefitinib significantly ( P  <   0.001) reduced tumor growth of xenografts of three HNSCC cell lines (HSC-2, HSC-3, and HSC-4), in female athymic nude mice, accompanied by an increase in BRAK expression specifically in tumor tissue. This tumor-suppressing effect of the drug was not observed in the case of BRAK non-expressing cells. Furthermore introduction of BRAK shRNA vector reduced both the expression levels of BRAK in HSC-3 cells and the antitumor efficacy of gefitinib in vivo . Our data showing an inverse relationship between BRAK expression levels in tumor cells and the tumor growth rate indicate that the gefitinib-induced increase in BRAK expression is beneficial for tumor suppression in vivo. ( Cancer Sci 2009)     

Inhibition of transforming growth factor alpha (TGF-alpha)-mediated growth effects in ovarian cancer cell lines by a tyrosine kinase inhibitor ZM 252868

The modulating effects of the epidermal growth factor (EGF) receptor-specific tyrosine kinase inhibitor ZM 252868 on cell growth and signalling have been evaluated in four ovarian carcinoma cell lines PE01, PE04, SKOV-3 and PE01CDDP. Transforming growth factor alpha (TGF-alpha)-stimulated growth was completely inhibited by concentrations > or =0.3 microM in the PE01 and PE04 cell lines and by > or =0.1 microM in SKOV-3 cells. TGF-alpha inhibition of PE01CDDP growth was reversed by concentrations > or =0.1 microM ZM 252868. TGF-alpha-stimulated tyrosine phosphorylation of both the EGF receptor and c-erbB2 receptor in all four cell lines. The inhibitor ZM 252868, at concentrations > or =0.3 microM, completely inhibited TGF-alpha-stimulated tyrosine phosphorylation of the EGF receptor and reduced phosphorylation of the c-erbB2 protein. EGF-activated EGF receptor tyrosine kinase activity was completely inhibited by 3 microM ZM 252868 in PE01, SKOV-3 and PE01CDDP cells. These data indicate that the EGF receptor-targeted TK inhibitor ZM 252868 can inhibit growth of ovarian carcinoma cells in vitro consistent with inhibition of tyrosine phosphorylation at the EGF receptor.