Transforming growth-factor-Beta (tgf-Beta) messenger-RNA expression and growth-control in human lung squamous carcinoma cell-lines

The pattern of TGF-beta mRNA expression and response to TGF-beta isoforms has been examined in three lung squamous carcinoma cell lines (NX002, CX140 and CX143). Expression for TGF-beta1 and TGF-beta2 but not TGF-beta3 was found in all 3 lines. TGF-beta1 and TGF-beta3 (but not TGF-beta2) inhibited the growth of the NX002 cell line in culture. The effect of TGF-beta1 on growth was accompanied with changes in the cell cycle. These data indicate that the potential for autocrine regulation by TGF-beta is present in lung squamous carcinoma cells.     

12-O-tetradecanoylphorbol 13-acetate induced differentiation in human lung squamous carcinoma cells.

Three human lung squamous carcinoma cell lines (NX002, CX140 and CX143) demonstrate features of squamous differentiation including involucrin synthesis and competence to form cornified envelopes. 12-O-Tetradecanoylphorbol 13-acetate inhibits growth of these cell lines and this growth inhibition is associated with enhanced differentiation.     

Autocrine growth stimulation by transforming growth factor-alpha in human non-small cell lung cancer.

We studied the biological response to and production of transforming growth factor-alpha (TGF-alpha) by the non-small cell lung carcinoma (NSCLC) clonal cell lines H226b, H322a, H460a, H596b. Each of these cell lines expressed epidermal growth factor receptor (EGFR) as determined by [125I]EGF competitive binding and Scatchard analysis and by phosphorylation. The receptors were functionally active as determined in immune complex kinase assays. H322a, H226b, H460a, and H596b cells showed stimulated [3H]thymidine (Thd) uptake in response to TGF-alpha. Exogenously added TGF-alpha increased colony formation in soft agar for three of the cell lines in media containing serum. All cell lines expressed TGF-alpha detected by immunohistochemistry and TGF-alpha mRNA, although to differing degrees. Cell lysates and spent media competed for EGFR binding with EGF, thus demonstrating production of TGF-alpha-like activity. The anti-TGF-alpha monoclonal antibody AB-3 inhibited the uptake of [3H]Thd by proliferating H322a and H226b cells but not H460a and H596b cells. No inhibition occurred with MOPC21 antibody and inhibition was completely reversed by addition of TGF-alpha to the culture. Suramin inhibited cell proliferation and [3H]Thd uptake by all cell lines. Inhibition of H460a and H596b cells was reversed with exogenous TGF-alpha but not PDGF. Our data suggests that TGF-alpha is a mediator of autocrine growth stimulation for NSCLC cells, and that for some NSCLC cells cytoplasmic binding of receptor and ligand is the primary mechanism for autocrine growth stimulation.     

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.     

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.     

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.     

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.     

Autocrine stimulation of a human lung mesothelioma cell line is mediated through the transforming growth factor alpha/epidermal growth factor receptor mitogenic pathway.

Malignant cells frequently acquire a certain independency of exogenous growth factors via the coexpression of epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF)-related molecules. In the present study we investigate a possible involvement of EGF-related molecules in the growth of human lung mesothelioma. Four well-characterised cell lines are analysed for their responsiveness to exogenous EGF and transforming growth factor alpha (TGF-alpha) as well as for coexpression of EGFR and EGF/TGF-alpha. Both growth factors are able to stimulate DNA synthesis in three cell lines, although the degree of responsiveness is very variable, but neither EGF nor TGF-alpha has an effect on the cell line ZL34. In contrast, no heterogeneity is observed in the expression of EGFR, which is similarly high in all cell lines. Analysis of cell supernatants reveals that, whereas no EGF is detected, TGF-alpha is released by two cell lines. Furthermore, these two cell lines, ZL5 and ZL34, are shown to express the membrane anchored precursor pro-TGF-alpha. Thus, coexpression of EGFR and TGF-alpha is observed on two mesothelioma cell lines. The potential autocrine mitogenic role of TGF-alpha in these two cell lines was tested using neutralising antibodies against TGF-alpha and EGFR. In ZL5 cells DNA synthesis was not affected by the presence of neutralising antibodies, indicating that an external autocrine mitogenic pathway is not active in these cells. In ZL34 cells, however, the potential autocrine loop could be disrupted, as DNA synthesis was significantly reduced in the presence of neutralising antibodies. This result gives strong evidence for an autocrine role of TGF-alpha in the growth of the mesothelioma cell line ZL34.     

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 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.     

Expression of TGF-alpha/EGF and TGF-beta receptors in human colon carcinoma cell lines

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

Epidermal growth factor receptor as a target for therapy with antireceptor monoclonal antibodies.

The epidermal growth factor (EGF) receptor is a potential target for antitumor therapy. Recent studies from many laboratories have found that this receptor is expressed in high levels on a variety of human tumor cells. Furthermore, the EGF receptor has been implicated in autocrine stimulation of cell growth in a number of experimental studies. We have produced anti-EGF receptor monoclonal antibodies (MAbs), which block the binding of EGF and transforming growth factor alpha (TGF-alpha), and can prevent ligand-stimulated activation of EGF receptor tyrosine kinase. These MAbs have been useful in studies of EGF receptor function. Experiments utilizing the MAbs to block ligand binding have demonstrated that autocrine stimulation of EGF receptor phosphorylation can occur via an extracellular pathway, involving TGF-alpha-mediated activation of EGF receptor on the surface of the cell. The capacity of anti-EGF receptor MAbs to inhibit cell proliferation has provided evidence of an autocrine stimulatory pathway in cultures of malignant human skin, breast, colon, and lung cells. Growth of a variety of human tumor xenografts can be inhibited in situations where autocrine dependency is demonstrable in cell culture. Imaging studies with anti-EGF receptor MAb labeled with indium 111 (111In) demonstrated selective uptake in xenografts expressing high receptor levels. Based on these observations, a phase I trial was carried out with 111In-labeled anti-EGF receptor MAb 225 IgG1 in patients with advanced squamous cell lung carcinoma, a tumor that invariably expresses large numbers of EGF receptors. In the case of squamous lung carcinoma, there is evidence that overexpression of EGF receptors correlates with worse clinical stage and worse prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth stimulatory effect of interleukin (IL)-1 alpha produced by oral squamous carcinoma cell lines

The expression of IL-1 alpha and its effect on the cell growth were examined in six human oral squamous carcinoma cell lines. All the cell lines expressed IL-1 alpha mRNA and protein at various levels. Particularly, HSC-2 and HSC-3 cells showed high level of the mRNA expression and secreted large amounts of IL-1 alpha into the culture fluid. Scatchard plot analysis of IL-1 alpha binding revealed that HSC-2 cells had high-and low-affinity receptors, whereas IL-1 alpha receptors on HSC-3 cells were of undetectable level. The cell growth of HSC-2 and HSC-3 cells was stimulated by IL-1 alpha and inhibited by anti-IL-1 alpha antibody or IL-1 receptor antagonist. The expression of IL-1 alpha mRNA by these cell lines was induced by either IL-1 alpha, epidermal growth factor (EGF) or transforming growth factor alpha (TGF-alpha). On the other hand, IL-1 alpha promoted the mRNA expression of TGF-alpha and EGF receptor. These findings indicate that IL-1 alpha acts as an autocrine growth stimulator for oral squamous carcinoma cells in vitro and its interaction with EGF/TGF-alpha/receptor system may play a role in this enhanced growth by IL-1 alpha.     

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.     

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.     

Differential role of transforming growth factor-alpha in two human colon-carcinoma cell lines

Antibodies to epidermal-growth-factor receptor (EGF) and transforming growth factor-alpha (TGF-alpha) were used to determine the role of endogenous TGF-alpha in the growth of 2 human colon-carcinoma cell lines. Both the GEO and HCT 116 colon-carcinoma cell lines secrete similar levels of TGF-alpha and have similar numbers of low-affinity binding sites for EGF. However, the HCT 116 cells lack the high-affinity EGF binding site present on the GEO cells. The anti-EGF receptor antibodies effectively blocked the binding of 125I-EGF to the GEO and HCT 116 cell lines. Growth of the GEO cell line was inhibited 50-80% by the anti-EGF receptor and anti-TGF-alpha antibodies. When the same antibodies, in sufficient amounts to block binding of TGF-alpha to the cells, were added to the HCT 116 cell line, no effect on growth was seen. These results suggest that while the GEO cell line utilizes TGF-alpha in an autocrine manner, the TGF-alpha secreted by the HCT 116 cells apparently does not play a role in the growth of these cells.     

Epidermal growth factor reduces HER-2 protein level in human ovarian carcinoma cells.

Over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial, and mammary carcinoma is an indicator of poor prognosis. Interactions between the epidermal growth factor (EGF) receptor and the HER-2 protein have been described. The aim of this study was to elucidate the effects of EGF on HER-2 expression. In the human ovarian carcinoma cell lines HTB-77, OVCAR-3, 2780, SKOV-6, SKOV-8 and 2774, and the human mammary tumor cell line SKBR-3, total cellular p185HER-2 was determined by an ELISA, whereas the surface p185HER-2 was measured with a living-cell RIA. Stimulation of these cell lines with either EGF (0.1-30 nM) or TGF-alpha (0.1-30 nM) led to a significant reduction in p185HER-2 expression. The effect was more pronounced in cells with normal HER-2 expression. A reduction of mRNA levels for p185HER-2 by EGF was observed in OVCAR-3 cells but not in the over-expressing lines HTB-77 and SKBR-3. Interestingly, the EGF-induced effect was not always associated with growth stimulation and was not correlated with the number of EGF binding sites detected by a radioligand assay. Our data indicate that EGF treatment results in a down-regulation of p185HER-2.     

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.     

The epidermal growth factor receptor as a target for therapy with antireceptor monoclonal antibodies

The epidermal growth factor (EGF) receptor is a potential target for antitumor therapy, because it is expressed at high levels on many human tumor cells and appears to be involved in autocrine stimulation of cell growth in a number of experimental studies. Anti-EGF receptor monoclonal antibodies (MAbs) which block ligand binding can prevent the growth in culture of cells that are stimulated by EGF or TGF-alpha. Growth of human tumor xenografts bearing high levels of EGF receptors is also inhibited. A Phase I trial in patients with squamous cell carcinoma of the lung has demonstrated the capacity of a single dose of 120 mg anti-EGF receptor MAb to localize in such tumors and to achieve saturating concentrations in the blood for more than 3 days, without causing toxicity.     

Transforming growth factor release by human tumor cell lines and their interactions with other growth factors

We have studied the production of transforming growth factor (TGF) by several human tumor cell lines and their interactions with exogenously added epidermal growth factor (EGF) and insulin. TGF-like activities were present in all the conditioned media tested. The clonogenic capacity of the tumor cell lines had no correlation with the TGF-like activity production. EGF and insulin had a promoting colony-forming activity on tumor cells but this effect was not additive. Moreover, an inverse statistically significant correlation (-0.817, p less than 0.05) was found between the response to exogenous EGF and the EGF or TGF-alpha production by tumor cell lines. The EGF receptor (EGF-R) was not detected in any of the melanomas studied, nor in breast adenocarcinoma cell lines which were producers of EGF or TGF-alpha.