Expression and prognostic value of epidermal growth factor receptor, transforming growth factor-alpha, and c-erb B-2 in nephroblastoma.

BACKGROUND: Wilms tumor is one of the most common solid tumors in children. A transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) autocrine loop plays an important role in tumor growth. Abnormal expression of TGF-alpha, EGF-R and c-erb B-2 has been demonstrated in several human malignancies. METHODS: The immunohistochemical expression of TGF-alpha, EGF-R, and c-erb B-2 was studied in paraffin material of 62 clinical Wilms tumors. Patients had a mean follow-up of 5.7 years. RESULTS: Generally, TGF-alpha, EGF-R, and c-erb B-2 were expressed in tissue of the normal kidney and at variable levels in the three cell types of Wilms tumor, i.e., blastemal, epithelial, and stromal cells. Immunoreactive blastema cells were found in 48%, 44%, and 34% of tumors for TGF-alpha, EGF-R, and c-erb B-2, respectively. It was found that TGF-alpha, EGF-R, and c-erb B-2 blastemal and epithelial expression gradually increased from T1 to T3. The blastemal expression of TGF-alpha was statistically significantly correlated with clinicopathologic stages. Both univariate and multivariate analysis showed that blastemal TGF-alpha expression was indicative for clinical progression, but neither blastemal TGF-alpha, nor EGF-R or c-erb B-2 expression correlated with patients survival. Epithelial staining was of no prognostic value. The simultaneous expression of TGF-alpha/EGF-R was indicative for clinical progression at univariate level. CONCLUSIONS: Increased expression of TGF-alpha in the blastemal part of Wilms tumor correlated with tumor classification and clinical progression. These findings suggest that significant expression of TGF-alpha and EGF-R may play a role in promoting transformation and/or proliferation of Wilms tumor, perhaps by an autocrine mechanism. Therefore, their expression may be of value in identifying patients at high risk of tumor recurrence.     

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.     

Prevention of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the MCF-10A cell line: correlation with increased transforming growth factor alpha production

We have recently reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits epidermal growth factor (EGF) withdrawal-induced apoptosis in the human mammary epithelial cell line MCF-10A. We hypothesized that TCDD-mediated inhibition of apoptosis was due to its ability to stimulate the EGF receptor (EGFR) pathway. Indeed, in the present studies, the EGFR inhibitor AG1478 was able to prevent TCDD-, EGF-, and transforming growth factor alpha (TGF-alpha)-dependent cell recovery and inhibition of apoptosis. These effects were specific for an EGFR-mediated pathway because cotreatment with AG825, an erbB2 inhibitor, had little effect on apoptosis. In addition, TCDD was able to mimic the EGF and TGF-alpha signaling as demonstrated by increasing Akt and extracellular signal-regulated kinase 1,2 phosphorylation. These effects were dependent on EGFR activity because AG1478, but not AG825, was able to prevent EGF-, TGF-alpha, or TCDD-mediated Akt and extracellular signal-regulated kinase 1,2 phosphorylation. The ability of TCDD to stimulate the EGFR pathway and inhibit apoptosis may be due to the ability of TCDD to increase expression of TGF-alpha, a ligand for EGFR. Treatment with 10 nM TCDD increased TGF-alpha mRNA at 2 h and TGF-alpha protein at 6 h. These data suggest a mechanism whereby TCDD is able to inhibit apoptosis in human mammary epithelial cells by stimulating TGF-alpha production, resulting in an autocrine effect.     

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.     

Inhibition of ligand-induced activation of epidermal growth factor receptor tyrosine phosphorylation by curcumin

We explored the regulation of epidermal growth factor (EGF)-mediatedactivation of EGF receptor (EGF-R) phosphorylation by curcumin(diferuloyl-methane), a recently identified kinase inhibitor,in cultured NIH 3T3 cells expressing human EGF-R. Treatmentof cells with a saturating concentration of EGF for 5–15min induced increased EGF-R tyrosine phosphorylation by 4- to11-fold and this was inhibited in a dose- and time-dependentmanner by up to 90% by curcumin, which also inhibited the growthof EGF-stimulated cells. There was no effect of curcumin treatmenton the amount of surface expression of labeled EGF-R and inhibitionof EGF-mediated tyrosine phosphorylation of EGF-R by curcuminwas mediated by a reversible mechanism. In addition, curcuminalso inhibited EGF-induced, but not bradykinin-induced, calciumrelease. These findings demonstrate that curcumin is a potentinhibitor of a growth stimulatory pathway, the ligand-inducedactivation of EGF-R, and may potentially be useful in developinganti-proliferative strategies to control tumor cell growth.     

Expression of transforming growth factor alpha (TGF alpha) in breast cancer

Transforming growth factor alpha (TGF alpha) is one growth factor that has been circumstantially implicated in regulating the autocrine growth of breast cancer cells. Expression of TGF alpha can be modulated by activated cellular protooncogenes such as ras and by estrogens. For example, the epidermal growth factor (EGF)-responsive normal NOG-8 mouse and human MCF-10A mammary epithelial cell lines can be transformed with either a point-mutated c-Ha-ras protooncogene or with a normal or point-mutated c-neu (erbB-2) protooncogene. In ras transformed NOG-8 and MCF-10A cells but not in neu transformed cells there is a loss in or an attenuated response to the mitogenic effects of EGF. This response may be due in part to an enhanced production of endogenous TGF alpha that is coordinately and temporally linked to the expression of the activated ras gene and to the acquisition of transformation-associated properties in these cells. TGF alpha mRNA and TGF alpha protein can also be detected in approximately 50-70% of primary human breast tumors. In addition, approximately 2- to 3-fold higher levels of biologically active and immunoreactive TGF alpha can also be detected in the pleural effusions from breast cancer patients as compared with the TGF alpha levels in the serous effusions of noncancer patients. Over-expression of a full-length TGF alpha cDNA in NOG-8 and MCF-10A cells is capable of transforming these cells. Finally, expression of TGF alpha mRNA and production of biologically active TGF alpha protein is also found in normal rodent and human mammary epithelial cells.     

Epidermal growth factor stimulates colony formation and non-neuronal marker protein expression by human neuroblastoma in methylcellulose culture

Growth of the human neuroblastoma IMR-32 in methylcellulose culture was studied. The number of colonies was proportional to the number of seeded cells in all conditions tested: control cultures (CT) and test cultures with epidermal growth factor (EGF), hydrocortisone (HC), combined EGF/HC, fibroblast growth factor (FGF) or nerve growth factor (NGF). A portion of IMR-32 cells formed colonies and all factors were without effect when tested individually. In contrast, the combination of EGF/HC at low cell densities enhanced the number of colonies two-fold as compared to controls. Differentiation in IMR-32 colonies was examined by immunocytochemical detection of cell specific marker proteins. As determined by staining with different markers, at least two cells subpopulations could be established within the same colony. One of them expressed NSE (neuron specific enolase) and was designated as neuronal. The other subpopulation was called non-neuronal since it consisted of vimentin and S-100 protein positive cells which were considerably enhanced in the presence of EGF or EGF/HC. In vitro, the IMR-32 neuroblastoma cell line contains pluripotent stem cells from which are derived distinct phenotypes sensitive to different extrinsic factors. Increasing time in culture enhanced neuronal differentiation. EGF, on the other hand, targeted preferentially the non-neuronal phenotype, and stimulated colony formation and its differentiation.     

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.     

Expression of transforming growth factor alpha, epidermal growth factor receptor and epidermal growth factor in precursor lesions to gastric carcinoma.

Epidermal growth factor (EGF), its related peptide transforming growth factor (TGF-alpha) and their common receptor (EGFR) have been implicated in the control of cell proliferation and differentiation in the gastrointestinal epithelium and may play an important role in gastric carcinogenesis. We compared the immunohistochemical expression and topographic distribution of these peptides using Western blot analysis in gastric carcinoma precursor lesions and in non-cancer tissue. We observed: (i) increased and extended expression of TGF-alpha in normal mucosa and hyperplasia in carcinoma fields compared with non-cancer controls; (ii) increased expression of EGFR in intestinal metaplasia (IM) from carcinoma fields compared with controls; (iii) EGF expression was not detected in normal mucosa and only weakly in IM; (iv) coexpression of TGF-alpha/EGFR and EGF/EGFR was higher in intestinal metaplasia in carcinoma fields than in non-cancer controls. We conclude that altered expression of TGF-alpha/EGFR is associated with morphological changes during gastric carcinogenesis. In this regard increased expression of TGF-alpha is a very early event which is subsequently followed by up-regulation of EGFR and this has important biological and clinical implications.     

Additive effects of c-erbB-2, c-Ha-ras, and transforming growth factor-alpha genes on in vitro transformation of human mammary epithelial cells.

MCF-10A cells are a spontaneously immortalized untransformed human mammary epithelial cell line. We have previously shown that overexpression of a human point-mutated c-Ha-ras proto-oncogene, the rat c-neu (c-erbB-2) proto-oncogene, or the human transforming growth factor-alpha (TGF-alpha) gene in MCF-10A cells leads to in vitro transformation of such cells. To ascertain whether the introduction of two of these genes into MCF-10A human mammary epithelial cells induces a completely tumorigenic phenotype, we infected MCF-10A Ha-ras and MCF-10A TGF-alpha cells with a recombinant retroviral vector containing the human c-erbB-2 proto-oncogene and the hygromycin-resistance gene. Ten MCF-10A TGF-alpha/c-erbB-2 (MCF-10A TE) and 10 MCF-10A Ha-ras/c-erbB-2 (MCF-10A HE) hygromycin-resistant clones were randomly selected and expanded into cell lines. MCF-10A TE and MCF-10A HE clones expressed a 10-fold to 40-fold increase in p185 erbB-2 protein levels compared with parental uninfected cells. These cells exhibited a fourfold increase in their growth rate in serum-free medium and showed a strongly reduced mitogenic response to exogenous epidermal growth factor or TGF-alpha compared with MCF-10A cells. Moreover, both MCF-10A TE and MCF-10A HE clones exhibited a fivefold to 20-fold higher cloning efficiency in soft agar than MCF-10A Ha-ras, MCF-10A c-erbB-2, or MCF-10A TGF-alpha clones. However, neither MCF-10A TE nor MCF-10A HE cells were able to grow as tumors in vivo when they were injected into nude mice. These results suggest that c-Ha-ras, c-erbB-2, and TGF-alpha genes have an additive effect on the in vitro transformation of an immortalized human mammary epithelial cell line, but that additional genetic changes such as activation of other proto-oncogenes or inactivation of a tumor suppressor gene may be necessary to elicit a fully tumorigenic phenotype.     

Co-expression of epidermal growth factor receptor and transforming growth factor-alpha predicts worse prognosis in breast-cancer patients

Epidermal growth factor receptor (EGF-R) and its ligand, transforming growth factor-alpha (TGF-alpha), play an important role through the autocrine growth-regulation system in several human cancers, including breast cancer. However, the clinical significance of co-expression of EGF-R and TGF-alpha has not been elucidated. One hundred seventy-three female patients diagnosed as invasive ductal carcinoma who had undergone a mastectomy (159 patients) or breast-conserving surgery (14 patients) were followed up for 81 to 119 months (median 94 months) post-operatively. Immunoreactivity for EGF-R, TGF-alpha, p53 and c-erbB-2 with paraffin-embedded carcinoma tissue was investigated using labeled streptavidin-biotin methods. Positive rates of carcinoma cells were 27%, 33%, 32% and 26% for EGF-R, TGF-alpha, p53 and c-erbB-2, respectively. Expression of EGF-R only was observed in 16% (28/173), of TGF-alpha only in 22% (38/173), of both EGF-R and TGF-alpha in 11% (19/173) and of neither in 51% (88/173). By univariate analysis, significant differences in overall survival and disease-free survival were noted according to the co-expression of EGF-R and TGF-alpha (p< 0.0001, p<0.0001), co-expression of EGF-R and c-erbB-2 (p = 0.0029, p = 0.0028), nodal status (p = 0.0028, p = 0.0001), tumor size (p = 0.0001, p<0.0001) and c-erbB-2 expression (p = 0.0034, p = 0.018), respectively. The status of p53 expression (p = 0.01), estrogen receptor (p = 0.042) and progesterone receptor (p = 0.046) showed significant differences in overall survival. According to Cox's multivariate analysis, co-expression of EGF-R and TGF-alpha had the most significant effect on disease-free survival (p<0.0001) and overall survival (p<0.0001), followed by nodal status. Co-expression of EGF-R and TGF-alpha by immunohistochemical detection is an independent prognostic indicator, and it may be helpful for determining the group of breast-cancer patients with an aggressive phenotype.     

Overexpression of epidermal growth factor and insulin-like growth factor-I receptors and autocrine stimulation in human esophageal carcinoma cells

The growth-stimulatory effects of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and insulin-like growth factor-I (IGF-I) on the human esophageal carcinoma cell line CE48T/VGH were evaluated. Under serum-free conditions, EGF, TGF-alpha, and IGF-I promoted 3.6- to 4.1-fold increased cell proliferation. Scatchard analyses and Northern blot hybridization revealed that both the EGF/TGF-alpha receptor and the IGF-I receptor were overexpressed in CE48T/VGH cells. Furthermore, ligand-dependent autophosphorylation of the EGF receptor and the IGF-I receptor was clearly detected using antireceptor and antiphosphotyrosine antibodies. Autocrine regulation was strongly indicated by the following evidence: (a) CE48T/VGH cells were found to express TGF-alpha and IGF-I genes, (b) serum-free conditioned medium promoted the growth of CE48T/VGH cells and stimulated the autophosphorylation of the EGF/TGF-alpha receptor and the IGF-I receptor, and (c) the addition of IGF-I receptor antibodies significantly suppressed CE48T/VGH cell growth under serum-free conditions. Our studies suggest that the overexpression of EGF and IGF-I receptors and autocrine growth regulation may concertedly control the proliferation of esophageal carcinoma cells.     

RAS transformation causes sustained activation of epidermal growth factor receptor and elevation of mitogen-activated protein kinase in human mammary epithelial cells

Activation of the ras oncogene is an important step in carcinogenesis. Human MCF-10A mammary epithelial cells were transformed with a point-mutated form of the Ha-ras oncogene. Epidermal growth factor receptor (EGFR) phosphorylation levels were chronically elevated after EGF induction and the EGFR ligand-driven internalization rate was slower in Ha-ras transformed MCF-10A cells. Additionally, basal levels of p42/44 mitogen-activated protein kinase (MAPK) expression and enzyme activity were significantly higher in Ha-ras transformed cells, localized predominantly in the nucleus. The anti-EGFR monoclonal antibody (MAb) 225 and the EGFR tyrosine kinase inhibitor PD153035 blocked anchorage-independent growth of Ha-ras transformed cells in soft agar and were more effective when used in combination. The MEK inhibitor PD98059 and anti-erbB-2 MAb L26 also suppressed colony formation of Ha-ras transformed cells in soft agar. Therefore, Ha-ras transformation leads to an augmentation in signaling through the EGFR as a result of an increase in ligand-dependent phosphorylation, a decrease in its internalization and an up-regulation in basal p44/42 MAPK levels. These effects may contribute to uncontrolled growth of Ha-ras-transformed human mammary epithelial cells.     

Induction of neuropilin-1 and vascular endothelial growth factor by epidermal growth factor in human gastric cancer cells

The epidermal growth factor receptor (EGF-R) pathway plays a pivotal role in the progression of human gastric cancer. The angiogenic factor vascular endothelial growth factor (VEGF) has been shown to be induced by EGF in various cancer cell lines. Neuropilin-1 (NRP-1) acts as a coreceptor for VEGF-165 and increases its affinity for VEGF receptor 2 (VEGFR-2) in endothelial cells. Furthermore, NRP-1 has been found to be expressed by tumour cells and has been shown to enhance tumour angiogenesis and growth in preclinical models. We examined the expression of NRP-1 mRNA and EGF-R protein in seven human gastric cancer cell lines. NRP-1 expression was expressed in five of seven cell lines, and EGF-R expression closely mirrored NRP-1 expression. Moreover, in EGF-R-positive NCI-N87 and ST-2 cells, EGF induced both NRP-1 and VEGF mRNA expression. C225, a monoclonal antibody to EGF-R, blocked EGF-induced NRP-1 and VEGF expression in NCI-N87 cells in a dose-dependent manner. The treatment of NCI-N87 cells with EGF resulted in increases in phosphorylation of Erk1/2, Akt, and P38. Blockade of the Erk, phosphatidylinositol-3 kinase/Akt, or P38 pathways in this cell line prevented EGF induction of NRP-1 and VEGF. These results suggest that regulation of NRP-1 expression in human gastric cancer is intimately associated with the EGF/EGF-R system. Activation of EGF-R might contribute to gastric cancer angiogenesis by a mechanism that involves upregulation of VEGF and NRP-1 expression via multiple signalling pathways.     

Induction of growth factor-receptor and metalloproteinase genes by epidermal growth factor and/or transforming growth factor-alpha in human gastric carcinoma cell line MKN-28

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

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: 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.     

Epidermal growth factor and its receptor in cervical cancer

Epidermal growth factor (EGF) is an important growth factor regulating both normal and malignant cells. The present study analyzes the expression pattern of EGF and its receptor (EGF-R) in 424 cases of various stages of tumor progression in the uterine cervix. In all the samples studied, EGF and EGF-R expression was predominant in the basal or basaloid cells. Increased expression of the two proteins correlated with increasing histological abnormality (dysplasia). In invasive cancer, alteration of EGF expression was more evident than EGF-R. The expression pattern showed significant correlation in the basal and spinal cell layers. This increased expression of EGF observed in dysplastic and malignant cells may be due to overexpression of EGF-R and suggests its role in cellular proliferation.     

8-Cl-cAMP antagonizes mitogen-activated protein kinase activation and cell growth stimulation induced by epidermal growth factor

The growth factor-activated mitogenic pathways are often disregulated in tumour cells and, therefore, they can provide specific molecular targets for novel anti-tumour approaches. 8-Chloro-cAMP (8-Cl-cAMP), a synthetic cAMP analogue, is a novel anti-tumour agent that has recently undergone clinical evaluation. We investigated the effects of 8-Cl-cAMP on the epidermal growth factor (EGF)/EGF receptor (EGF-R) signalling in human epidermoid cancer KB cells, which are responsive to the mitogenic stimulus of EGF. We found that the growth-promoting activity of EGF was completely abolished when EGF treatment was performed in combination with 8-Cl-cAMP. The inhibition of the EGF-induced proliferation by 8-Cl-cAMP was paralleled by the blockade of the EGF-stimulated activation of mitogen-activated protein kinases (MAPK), ERK-1 and ERK-2. Conversely, we found an increase of EGF-R expression and EGF-R tyrosine phosphorylation when KB cells were growth inhibited by 8-Cl-cAMP. Moreover, the activity of Raf-1 and MEK-1 protein kinases, the activators upstream MAPK in the phosphorylation cascade induced by EGF, was not modified in 8-Cl-cAMP-treated cells. We concluded that the impairment of KB cell response to EGF, induced by 8-Cl-cAMP, resides in the specific inhibition of MAPK/ERKs activity while the function of the upstream elements in the EGF-R signalling is preserved.     

Divergent effects of epidermal growth factor and transforming growth factors on a human endometrial carcinoma cell line

Epidermal growth factor (EGF), at concentrations ranging from 0.83 to 4.98 nM, markedly inhibited the proliferation of RL95-2 cells that were seeded at low plating densities (4.7 X 10(3) cells/cm2). Under the same incubation conditions, 16.6 pM EGF enhanced cell proliferation. At high plating densities (2.5 X 10(4) cells/cm2) 0.83 nM EGF also stimulated cell proliferation. Both the inhibitory and stimulatory effects of EGF were mimicked by transforming growth factor-alpha (TGF-alpha). However, the inhibitory action of TGF-alpha was always greater that of EGF. Binding studies with 125I-labeled TGF-alpha indicated that maximal cell surface binding of TGF-alpha occurred at 15 min, whereas maximal internalization occurred at 45 min. Both cell surface and internalized radioactivity declined sharply thereafter. Analysis of radioactivity released into the incubation medium during pulse-chase experiments indicated that RL95-2 cells extensively degraded both TGF-alpha and EGF. The lysosomotropic compound methylamine arrested the generation of low-molecular-weight degradation products of EGF, but not of TGF-alpha. In contrast to EGF and TGF-alpha, transforming growth factor-beta (TGF-beta) inhibited the proliferation of RL95-2 cells that were seeded at either low or high plating densities. Further, transforming growth factor-beta induced the appearance of large cuboidal cells that were readily distinguished from cells treated with either EGF or TGF-alpha. These findings point to complex regulatory actions of growth factors on the proliferation of RL95-2 cells and suggest that the processing of TGF-alpha following EGF receptor activation is distinct from the processing of EGF.