Exogenous TGF-alpha Research Articles

Expression of transforming growth factor alpha and its receptor in human neuroendocrine tumours

Transforming growth-factor-alpha (TGF-alpha) is a 50-amino-acid polypeptide that binds to the epidermal growth factor (EGF) receptor and stimulates cell growth. It has been suggested that enhanced production of TGF-alpha and EGF receptors by tumour cells promote tumour-cell growth by autocrine mechanisms. In the present study we have investigated the expression of TGF-alpha and EGF receptors in human neuroendocrine tumours, including midgut carcinoid tumours, phaeochromocytomas and medullary thyroid carcinomas. TGF-alpha expression was demonstrated in biopsies of all tumours examined (n = 30) and EGF receptors in a majority of tumours by Northern analysis and/or immunocytochemistry. Expression of TGF-alpha and EGF receptors was also demonstrated in primary cultures of tumour cells. Carcinoid tumours and phaeochromocytomas in culture secreted detectable amounts of TGF-alpha into the culture medium (400-700 pM). The amount of secreted TGF-alpha could be suppressed by octreotide treatment in individual tumours. Administration of exogenous TGF-alpha stimulated carcinoid tumour growth in vitro as determined by the DNA contents of cell cultures. The growth-stimulatory effect of TGF-alpha could be partially blocked by the use of neutralizing anti-EGF receptor monoclonal antibodies (MAbs). In conclusion, several human neuroendocrine tumours express both TGF-alpha and EGF receptors in in vivo and in vitro, suggesting that TGF-alpha may regulate tumour-cell growth by autocrine mechanisms.

Regulation of plasminogen activator activity in transitional carcinoma cell lines by wound site growth factors.

The effect of two growth factors ellicited in response to surgical woundings (transforming growth factor alpha [TGF alpha] and beta 1) on the regulation of cellular plasminogen activator activity (PAA) in human transitional carcinoma cell lines, with differing baseline PAA (253J--high; 647V--low), was studied. mRNA transcript levels of PA regulatory proteins in both cell lines were responsive to TGF alpha and TGF beta 1. However, both the magnitude and nature of the response differed markedly between the two lines. TGF alpha increased uPA transcript levels in both cell lines in a dose-dependent fashion. In the case of uPA receptor, exogenous TGF alpha concentrations which increased receptor levels over fivefold in the 253J line had no effect on this transcript in the 647V line. This differential responsiveness was even more pronounced for TGF beta 1. TGF beta 1 appeared to increase uPA transcript in the 253J line in a dose dependent manner while decreasing transcript levels in the 647V line. uPAr mRNA in 253J cells increased over a 19-fold range in response to TGF beta 1 while this same transcript was decreased 14-fold in 647V cells. The most pronounced effect of TGF beta 1 was seen on PAI1 transcript levels in the 253J line. This transcript increased in a concentration dependent fashion from non-detectable levels. These findings demonstrate that growth factors ellicited by surgical wounding may alter the biology of neoplastic cells. Both the growth factor milieu, and intrinsic cellular regulatory mechanism, appear important in determing net PAA in transitional carcinoma cell lines.

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.

Localization of transforming growth factor alpha in the human placenta and decidua: role in trophoblast growth.

Transforming growth factor alpha (TGF alpha) is an important growth regulatory molecule, the location and function of which at the human fetomaternal interface remain to be determined. The present study examined the presence of TGF alpha in the human placenta, decidua, and fetal membranes throughout gestation (from a total of 29 subjects) as well as its functional role in the proliferation of first trimester trophoblasts. The peptide was localized immunocytochemically with a monoclonal anti-TGF alpha antibody (Ab) (MF9) on paraffin-embedded tissues via the avidin-biotin complex-peroxidase technique with diaminobenzidine (DAB) as the chromogen. Omission or TGF alpha absorption of the primary Ab served as negative controls. Specific (cytoplasmic) staining was noted in typical stromal-type decidual cells, including cells of the decidua basalis and parietalis and chorionic decidua, throughout gestation. Villous trophoblast cells (syncytiotrophoblast and to a minor extent cytotrophoblast) at all gestational ages as well as extravillous cytotrophoblast cells (intermediate and cytotrophoblastic shell) also showed specific cytoplasmic staining. Chorionic trophoblasts showed variable staining, and little or no immunoreactivity was seen in the amniocytes. Second-passage first trimester human trophoblast cells (characterized by their expression of cytokeratin as well as other markers) were cultured in the presence of TGF alpha or neutralizing anti-TGF alpha Ab (TAb-1) or no additive for 18 h prior to exposure to 3H-TdR for 6 h to measure 3H-TdR uptake. TGF alpha (0-100 ng/ml) caused a dose-dependent stimulation of proliferation, reaching a near plateau at 6-100 ng/ml to slightly more than double the basal level. The presence of anti-TGF alpha Ab alone (25 micrograms/ml) did not significantly influence the proliferation of the cells, indicating the absence of significant endogenous TGF alpha in these cultures; however, the Ab was able to abolish the stimulatory function of exogenous TGF alpha. Exogenous TGF alpha also increased the number of trophoblast nuclei immunoreactive for proliferating cell nuclear antigen and reduced the incidence of multinucleate cells in culture. These results indicate that TGF alpha is present in the cells of the fetomaternal interface throughout human gestation and may function as a stimulator of trophoblastic growth in situ.

Autocrine/paracrine regulation of keratinocyte urokinase plasminogen activator through the TGF‐α/EGF receptor

Transforming growth factor-alpha (TGF-alpha) appears to be an important autocrine/paracrine regulator of keratinocyte function. Not only does TGF-alpha induce keratinocyte proliferation and migration in vitro, but it also has been detected in normal human epidermis and at elevated levels in hyperproliferative epidermis. In the present study we report that exogenous TGF-alpha increases urokinase-type plasminogen activator (uPA) in cultured human keratinocytes. Furthermore, in the absence of exogenous growth factors, the "basal" levels of uPA are decreased by an antagonist monoclonal antibody to the receptor shared by TGF-alpha and epidermal growth factor (EGF). These results suggest that an endogenous factor serves as an autocrine/paracrine regulator of keratinocyte uPA. We hypothesize that activation of the TGF-alpha/EGF receptor may coordinately regulate the keratinocyte response to cutaneous wounding, which includes enhanced uPA expression, migration, and proliferation.

Transforming growth factor-alpha and epidermal growth factor expression in human fetal gastrointestinal tract.

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) are mitogenic to the intestinal epithelium. To further clarify their role in the developing human fetal gut, their expression was studied in fetuses at 15 to 20 wk of gestation. TGF-alpha mRNA was present throughout the gastrointestinal tract, most abundantly in the duodenum. EGF mRNA could be detected only with ribonuclease protection assay and reverse transcription-polymerase chain reaction analysis. The effect of EGF and TGF-alpha on TGF-alpha mRNA expression was studied by culturing explants of fetal jejunum, ileum, and colon for 7 d in Leibowitz L-15 medium supplemented with 100 micrograms/L of either EGF or TGF-alpha. EGF receptor-like immunoreactivity was detected in both the villi and the crypts. In the jejunum, exogenous EGF up-regulated TGF-alpha mRNA 3-fold. However, exogenous TGF-alpha reduced its own mRNA by 40%. No mature 6-kD TGF-alpha was detected in the culture medium by Western blotting, but precursor forms of approximately 30 and 68 kD were present. The ileum and colon did not respond to either growth factor. Besides the gut, TGF-alpha was expressed in the gallbladder, salivary gland, adrenals, brain, kidney, liver, and placenta. The data imply an important role for TGF-alpha and EGF in the developing intestine.

Interferon-induced enhancement of transforming growth factor-alpha expression in a human breast cancer cell line.

Previous work from our laboratory has demonstrated that gamma-interferon (IFN) inhibits growth of the human breast carcinoma cell line MDA 468, while enhancing expression of epidermal growth factor receptor (EGFR). Epidermal growth factor at high levels is known to inhibit growth of this cell line. Because MDA 468 cells produce low levels of transforming growth factor (TGF)-alpha (a ligand for epidermal growth factor receptor), we reasoned that IFN-induced cytotoxicity could be partially mediated by enhanced secretion of TGF-alpha. Therefore, we determined the ability of IFN to modulate the endogenous expression of TGF-alpha by MDA 468 cells. IFN-gamma, at 500 units/ml, increased the levels of TGF-alpha in serum-free conditioned media of MDA 468 cells 3-fold as measured by radioimmunoassay. TGF-alpha mRNA was similarly increased approximately 3-fold after 5 days of IFN treatment as determined by dot blot and Northern analysis. IFN increased expression of TGF-alpha in conditioned media in a dose-dependent fashion. Increased secretion of TGF-alpha into conditioned media was not observed at Days 1 and 3. Similarly, increases in TGF-alpha mRNA were not observed at those time points. These results demonstrate that IFN-gamma enhanced secretion of TGF-alpha by MDA 468 cells. Although exogenous TGF-alpha inhibited MDA 468 cell growth, the role that the enhanced endogenous production of TGF-alpha plays in the cessation of cell growth induced by IFN remains to be determined.

Analysis of phospholipid metabolism in murine keratinocytes transformed by the v-ras oncogene: relationship of phosphatidylinositol turnover and cytokine stimulation to the transformed phenotype.

Introduction of a v-rasHa oncogene into cultured mouse keratinocytes by transduction with a defective retrovirus is sufficient to transform keratinocytes to the benign phenotype. Transduced keratinocytes overexpress TGF alpha and hyperproliferate in culture medium with 0.05 mM Ca2+. Whereas normal keratinocytes respond to elevated medium Ca2+ by cessation of proliferation and induction of terminal differentiation, v-rasHa keratinocytes are not induced to differentiate by Ca2+. We now demonstrate that v-rasHa keratinocytes have elevated basal levels of phosphatidylinositol, inositol phosphates and diacylglycerols in 0.05 mM Ca2+ medium. Basal turnover of phosphatidylcholine is not altered by the rasHa oncogene. The generation of inositol phosphates is even further stimulated in v-rasHa cells by an increase in extracellular Ca2+ or by exposure to aluminum fluoride. Thus, the v-rasHa gene product does not stimulate the inositol phospholipid pathway maximally and additional phosphatidylinositol is available for turnover in response to inducers of phospholipase C activity. TGF alpha and medium conditioned by v-rasHa keratinocytes, both of which stimulate proliferation of normal cells in 0.05 mM Ca2+, transiently increased phosphatidylinositol turnover in normal keratinocytes but did not inhibit Ca(2+)-induced terminal differentiation. In contrast, sustained elevation in basal phosphatidylinositol metabolism was produced by aluminum fluoride. Combined exposure to aluminum fluoride and exogenous TGF alpha caused hyperproliferation, resistance to Ca(2+)-induced differentiation and morphological changes identical to those of v-rasHa keratinocytes. These results provide a link between the biological consequences of v-rasHa gene expression and biochemical changes which are known to alter the keratinocyte phenotype.

Polyamine involvement in the secretion and action of TGF-α in hormone sensitive human breast cancer cells in culture

These experiments were designed to test polyamine (PA) involvement in the secretion and action of transforming growth factor alpha (TGF-alpha) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E2) and TGF-alpha stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E2-stimulated growth, administration of the PA synthesis inhibitor alpha-difluoromethylornithine (DFMO) did not influence either basal or E2-induced TGF-alpha secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-alpha. However, when the culture conditions were changed to serum-free medium, TGF-alpha and E2-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E2-stimulated TGF-alpha secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF-alpha and E2-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.

Expression of transfected transforming growth factor alpha induces a motile fibroblast-like phenotype with extracellular matrix-degrading potential in a rat bladder carcinoma cell line.

Acquisition of cell motility is often correlated with the malignant progression of a transformed cell. To investigate some of the mechanisms involved in the development of a migratory state, we transfected the NBTII rat carcinoma cell line, which forms stationary epithelial clusters in culture, with the gene encoding human transforming growth factor alpha (TGF alpha). Expression of TGF alpha in NBTII cells resulted in cells of motile and vimentin-positive phenotype with internalized desmosomal components, analogous to the treatment of cells with exogenous TGF alpha. The clones expressed a 5.2-kb TGF alpha message and synthesized an 18-kDa form of TGF alpha. Supernatants of TGF alpha-producing clones induced the internalization of desmosomal components, the production of vimentin, and increased motility in untransfected epithelial NBTII cells, indicating that the factor produced by the clones was in a biologically active form. TGF alpha-producing clones secreted significant levels of a 95-kDa gelatinolytic metal-loproteinase, virtually absent in untransfected cell supernatants. In contrast, levels of inhibitors of metalloproteinases and of a plasminogen activator were similar in untransfected and TGF alpha-transfected NBTII cells. These results suggest that expression of TGF alpha in an epithelial tumor cell results in the development of a motile, fibroblast-like phenotype with matrix-degrading potential, which could result in a more aggressive tumor in vivo.

Production and response of a human prostatic cancer line to transforming growth factor-like molecules

Serum-free media conditioned by the androgen insensitive human prostate cancer cell line DU145 showed immunological transforming growth factor-alpha (TGF alpha) activity, as well as competing activity in epidermal growth factor (EGF) radioreceptor assays (RRA). Furthermore, there were factors in the conditioned media which inhibited and stimulated DNA synthesis by DU145 cells in a dose-dependent fashion. Fractionation of the concentrated conditioned media by reverse-phase high performance liquid chromatography revealed several peaks containing EGF-like competitive activity only one of which demonstrated TGF alpha activity. However, none of the peaks corresponded to immunoreactive EGF. Measurement of EGF receptors on DU145 cells by competition and saturation analysis revealed high levels of receptors (mean +/- s.d. = 2.5 +/- 1 x 10(5) surface receptors per cell) which were of high affinity (Kd +/- s.d. = 1.0 +/- 0.5 nmol l-1). Although DU145 cells express high levels of EGF receptors, DNA synthesis was only minimally affected by exogenous EGF and TGF alpha.

Potential role of the epidermal growth factor receptor in human pancreatic cancer

The epidermal growth factor (EGF) receptor is a transmembrane protein that has tyrosine kinase activity. It is activated by both EGF and transforming growth factor-alpha (TGF-alpha). Human pancreatic cancer cells overexpress the EGF receptor and exhibit a parallel increase in EGF receptor mRNA without a detectable increase in the number of gene copies coding for the receptor. These cells also produce TGF-alpha and are capable of binding exogenous TGF-alpha. They often recycle EGF, but markedly and rapidly degrade TGF-alpha. However, TGF-alpha is 10-100-fold more potent than EGF in enhancing their anchorage-independent growth. Both growth factors induce EGF receptor down-regulation, but EGF is more efficient than TGF-alpha in this regard. The concomitant overexpression of the EGF receptor and production of TGF-alpha, the recycling of EGF, and the attenuated ability of TGF-alpha to down-regulate the EGF receptor may combine to provide a distinct growth advantage to human pancreatic cancer cells.

Growth stimulation by coexpression of transforming growth factor-alpha and epidermal growth factor-receptor in normal and adenomatous human colon epithelium.

Autocrine stimulation of the epidermal growth factor receptor (EGF-R), by coexpression of transforming growth factor-alpha (TGF-alpha), causes malignant transformation of some fibroblast cell lines. TGF-alpha and EGF-R are both known to be expressed in colon carcinoma tissue and have been shown coexpressed in colon carcinoma cell lines. TGF-alpha autocrine activation of EGF-R has been suggested as a potential mechanism contributing to abnormal growth control in colon cancer. We now report coexpression of TGF-alpha and EGF-R transcripts in morphologically normal colonic epithelium from five individuals, in colonic adenomas from three individuals, and in a nontumorigenic colon adenoma cell line, VACO-330. Functional studies demonstrate VACO-330 growth is stimulated by exogenous TGF-alpha and is completely abolished by a blocking anti-EGF-R antibody. Autocrine stimulation of EGF-R by TGF-alpha is therefore required for growth of the adenoma cell line. Autocrine stimulation of EGF-R by TGF-alpha does not cause malignant transformation of the colonic epithelial cell. In normal and adenomatous human colon TGF-alpha, via either an autocrine or paracrine mechanism, is likely an important physiologic stimulant of epithelial proliferation.

Induction of transforming growth factor alpha expression in mouse mammary epithelial cells after transformation with a point-mutated c-Ha-ras protooncogene.

NOG-8 ras cells are a normal mouse mammary epithelial cell line transfected with a plasmid containing a glucocorticoid-inducible mouse mammary tumor virus long terminal repeat linked to the activated c-Ha-ras protooncogene. After addition of dexamethasone, there is a rapid induction (within 1-3 h) of p21ras protein that is concomitant with a parallel induction of the c-Ha-ras specific mRNA. After 4-6 days of dexamethasone treatment, NOG-8 ras cells are able to grow as colonies in semisolid medium. Between 9 and 12 days of dexamethasone treatment, there is a 5- to 6-fold increase of transforming growth factor alpha (TGF alpha) activity in the conditioned medium from NOG-8 ras cells. A 60-65% reduction in epidermal growth factor cell surface receptors on NOG-8 ras cells also occurs during this time interval. A 3- to 4-fold increase of the expression of a specific TGF alpha mRNA can be detected within 2 days of dexamethasone treatment, preceding the increase in TGF alpha protein found in the conditioned medium. Exogenous TGF alpha is able to stimulate in a dose-dependent fashion the anchorage-dependent and anchorage-independent growth of NOG-8 ras cells to a level comparable to that observed in dexamethasone treated ras-transformed NOG-8 ras cells. These results suggest that the enhanced expression of TGF alpha after induction of an activated ras protooncogene may be necessary for the anchorage-independent growth and subsequent morphological changes and the enhanced growth rate observed in ras-transformed mammary epithelial cells.

Blockade of the epidermal growth factor receptor inhibits transforming growth factor alpha-induced but not estrogen-induced growth of hormone-dependent human breast cancer.

Transforming growth factor alpha (TGF alpha), a polypeptide that binds to the epidermal growth factor (EGF) receptor, is expressed and secreted by human breast cancer cells and has been proposed as an autocrine growth factor and as a mediator of the mitogenic effect of estrogen. We investigated the potential importance of secreted TGF alpha in estrogen-responsive MCF-7 human breast cancer cells using monoclonal (528ab and 225ab) and polyclonal antibodies that block the EGF/TGF alpha receptor. Confirming other studies, these MCF-7 cells expressed TGF alpha with mRNA transcripts of 4.8 kilobases identified by Northern analysis, and they secreted TGF alpha activity measured by normal rat kidney colony-forming assay and an EGF RRA of conditioned medium. This activity was increased 3-fold by 1 nM 17 beta-estradiol and decreased by 1 microM tamoxifen. 528ab and 225ab bound to EGF receptors in MCF-7 cells with high affinity [dissociation constant (Kd) 0.1-0.5 nM] and blocked the binding of EGF/TGF alpha. These antibodies failed to inhibit baseline DNA synthesis or growth of MCF-7 cells although they were potent inhibitors of EGF/TGF alpha-induced growth of these cells. We hypothesized that if secreted TGF alpha mediates estrogen-induced growth, then EGF/TGF alpha receptor blockade should inhibit estrogen stimulation. MCF-7 cells were first treated with tamoxifen to inhibit growth and to reduce TGF alpha expression. Under these conditions, estrogen replenishment induced a marked dose-dependent rescue of TGF alpha secretion, DNA synthesis, and cell proliferation. Exogenous TGF alpha also partially restored growth of tamoxifen-inhibited cells. Although the simultaneous addition of 528ab or 225ab blocked TGF alpha-induced rescue of MCF-7 cells, it had no effect on rescue by estradiol.(ABSTRACT TRUNCATED AT 250 WORDS)