Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Inability of anti-epidermal growth factor receptor monoclonal antibody to block "autocrine" growth stimulation in transforming growth factor-secreting melanoma cells

Mouse monoclonal antibodies to the human epidermal growth factor (EGF) receptor were raised by immunizing with plasma membrane vesicles prepared from A431 cells. This paper describes the characterization of one of the IgG anti-receptor monoclonal antibodies generated and its use to probe the role of transforming growth factor (TGF) in the autonomous growth of a melanoma cell line in culture. This antibody blocks: 1) the binding of 125I-EGF to the A431 EGF receptor; 2) the EGF stimulation of the EGF-dependent protein kinase in vitro; and 3) human fibroblast DNA synthesis and proliferation in culture. It can precipitate the EGF receptor from metabolically labeled A431 cells and human fibroblasts and these receptors have indistinguishable peptide maps. No EGF receptor could be detected by immunoprecipitation after fibroblasts were treated with EGF or conditioned medium from the melanoma cells which secrete EGF-like TGF (alpha TGF). The antibody itself did not down-regulate the receptor but could block down-regulation caused by EGF and alpha TGF. Despite its ability to block EGF-stimulated growth and down-regulation in fibroblasts, the antibody was unable to block the growth and soft agar colony formation of alpha TGF-secreting melanoma cells, nor could the antibody detect EGF receptor in these cells under the conditions developed to prevent down-regulation and lysosomal degradation of the EGF receptor. These studies suggest that these melanoma cells do not have the intact EGF receptor and that the secretion of alpha TGF by these cells plays no role in their growth in culture. The absence of receptor cannot be explained by down-regulation by secreted alpha TGF.     

Modulation of transforming growth factor alpha-dependent expression of epidermal growth factor receptor gene by transforming growth factor beta, triiodothyronine, and retinoic acid

We have investigated the actions of transforming growth factor (TGF) type alpha on epidermal growth factor (EGF) receptor mRNA expression in MDA-468 human mammary carcinoma cells in serum-free media. We found that exposure of MDA-468 cells to TGF alpha results in elevated levels of EGF receptor mRNA. This increase in mRNA accumulation showed time and dose dependence. Addition of TGF beta 1 enhanced the accumulation of EGF receptor mRNA induced by TGF alpha in a time- and dose-dependent manner. We also found that triiodothyronine at physiological concentrations exerts synergistic control on the action of TGF alpha alone, or in association with TGF beta 1, on EGF receptor mRNA expression. Similarly, retinoic acid treatment also enhanced in a time- and dose-dependent manner the TGF alpha-dependent response of EGF receptor mRNA and acted synergistically with TGF beta 1. The results described here suggest that optimum regulation of EGF receptor gene expression by TGF alpha is a complex process involving synergistic interactions with heterologous growth factors and hormones.     

Purification and characterization of a novel growth factor from human breast cancer cells.

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.     

Anti-sense transforming growth factor alpha oligonucleotides inhibit autocrine stimulated proliferation of a colon carcinoma cell line.

Many carcinoma cells secrete transforming growth factor alpha (TGF alpha). A 23 base anti-sense oligonucleotide that recognizes the TGF alpha mRNA inhibits both DNA synthesis and the proliferation of the colon carcinoma cell line LIM 1215. The effects of the anti-sense TGF alpha oligonucleotide are reversed by epidermal growth factor (EGF) at 20 ng/ml. When the LIM 1215 cells are grown under serum free conditions, the anti-sense TGF alpha oligonucleotides have their greatest effects at high cell density (2 x 10(5) cells/cm2), indicating that the secreted TGF alpha is acting as an exogenous growth stimulus. In addition, at higher cell densities, the kinase activity of the EGF receptor is activated and the receptor is down-modulated. The cell density dependent activation of the EGF receptor is inhibited by the application of the antisense TGF alpha oligonucleotides.     

Epidermal growth factor and transforming growth factor alpha bind differently to the epidermal growth factor receptor

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) compete with each other for binding to the EGF receptor. These two growth factors have similar actions, but there are distinguishable differences in their biological activities. It has never been clear how this one receptor can mediate different responses. A monoclonal antibody to the EGF receptor (13A9) has been identified which has only small effects on the binding of EGF to the EGF receptor, but which has very large effects on the binding of TGF alpha to the EGF receptor; 5 micrograms/mL antibody has been shown to totally block 0.87 microM TGF alpha from binding to purified EGF receptor and to lower both the high- and low-affinity binding constants of TGF alpha binding to EGF receptor on A431 cells by about 10-fold. The 13A9 antibody causes a 2.5-fold stimulation of the tyrosine kinase activity of partially purified EGF receptor, compared to a 4.0-fold stimulation of the tyrosine kinase activity by EGF under the same conditions. The data suggest either that the antibody stabilizes a conformation of the EGF receptor which is not favorable for TGF alpha binding or that it blocks a part of the surface of the receptor which is necessary for TGF alpha binding but not EGF binding.     

A synthetic fragment of rat transforming growth factor alpha with receptor binding and antigenic properties

A fragment of rat transforming growth factor alpha (TGF alpha) comprising the third disulfide loop (residues 34-43) was selected as a potential antigenic and receptor binding region. Immunization of rabbits with a peptide conjugate resulted in antibodies which were specific for both the peptide and rat TGF alpha, but not for the homologous epidermal growth factor (EGF). The synthetic decapeptide exhibited low affinity for EGF receptors on human cells. Affinity was increased 100x to 0.2% of EGF or TGF alpha binding by blocking the peptide ends. The blocked decapeptide had no mitogenic activity but prevented the mitogenic effect of EGF and TGF alpha on fibroblasts. This decapeptide is an antagonist and contains an important receptor binding region of TGF alpha.     

Inhibition of epidermal growth factor/transforming growth factor-alpha-stimulated cell growth by a synthetic peptide

Estrogen-stimulated growth of the human mammary adenocarcinoma cell line MCF-7 is significantly inhibited by monoclonal antibodies to the epidermal growth factor (EGF) receptor that act as antagonists of EGF's mitogenic events by competing for high-affinity EGF receptor binding sites. These antibodies likewise inhibit the EGF or transforming growth factor-alpha (TGF-alpha)-stimulated growth of these MCF-7 cells. An analogous pattern of specific EGF or TGF-alpha growth inhibitory activity was obtained using a synthetic peptide analog encompassing the third disulfide loop region of TGF-alpha, but containing additional modifications designed for increased membrane affinity [( Ac-D-hArg(Et)2(31),Gly32,33]HuTGF-alpha(31-43)NH2). The growth factor antagonism by this synthetic peptide was specific in that it inhibited EGF, TGF-alpha, or estrogen-stimulated growth of MCF-7 cells but did not inhibit insulin-like growth factor-1 (IGF-1)-stimulated cell growth. Altogether, these results suggest that a significant portion of the estrogen-stimulated growth of these MCF-7 cells is mediated in an autocrine/paracrine manner by release of EGF or TGF-alpha-like growth factors. The TGF-alpha peptide likewise inhibited EGF- but not fibroblast growth factor (FGF)- or platelet-derived growth factor (PDGF)-stimulated growth of NIH-3T3 cells in completely defined media; but had no effect on growth or DNA synthesis of G0-arrested cells, nor did it effect growth of NR-6 cells, which are nonresponsive to EGF. Although this synthetic peptide did not directly compete with EGF for cell surface receptor binding, it exhibited binding to a cell surface component (followed by internalization), which likewise was not competed by EGF. The peptide did not directly inhibit EGF-stimulated phosphorylation of the EGF receptor, nor did it inhibit phosphorylation of an exogenous substrate, angiotensin II, by activated EGF receptor. The TGF-alpha peptide did, however, affect the structure of laminin as manifested by laminin self-aggregation; this affect on laminin may, in turn, have a modulatory effect on EGF-mediated cell growth.     

Regulation of heparin-binding EGF-like growth factor expression in Ha-ras transformed human mammary epithelial cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA and protein expression is induced by EGF in MCF-10A nontransformed and Ha-ras transfected human mammary epithelial cells. The anti-EGF receptor (EGFR) blocking monoclonal antibody (MAb) 225 and the EGFR tyrosine kinase inhibitor PD153035 were able to inhibit the induction of HB-EGF mRNA levels in MCF-10A cells. However, the Ha-ras transformed MCF-10A cells were more refractory to inhibition by these agents and only a combination of the 225 MAb and PD153035 was able to significantly abrogate HB-EGF induction by EGF. The anti-erbB2 MAb L26 which interferes with heterodimer formation was able to block HB-EGF induction in response to EGF in MCF-10A cells and in the Ha-ras transformed cells only when used in combination with either the 225 MAb or PD153035. The MEK inhibitor PD90859 completely blocked EGF induction of HB-EGF mRNA levels in the nontransformed and Ha-ras transformed MCF-10A cells, which indicates that MAPK is involved in the signaling pathway of HB-EGF induction by EGF. An increase in the levels of HB-EGF may, therefore, be an important contributor to oncogenic transformation that is caused by Ha-ras overexpression in mammary epithelial cells. J. Cell. Physiol. 186:233-242, 2001. Published 2001 Wiley-Liss, Inc.     

Epidermal growth factor dependence and TGF alpha autocrine growth regulation in primary rat tracheal epithelial cells.

We have examined dependence of primary rat tracheal epithelial (RTE) on exogenous epidermal growth factor (EGF) and determined whether a TGF alpha autocrine pathway is operating in these cells. Primary RTE cells plated in serum free media (SFM) without EGF and bovine pituitary factor (BPE) show little proliferation compared to cultures propagated in media containing EGF/BPE (CSFM). Removal of EGF/BPE shortly after plating, however, results in significant proliferation, although plateau cell densities are reduced and cell morphology is significantly altered compared to cells propagated in CSFM. Addition of EGF and/or BPE to cultures propagated in SFM minus EGF/BPE restores maximum cell density. The concentration of TGF alpha peptide in media conditioned by cells propagated without EGF/BPE is lower than the concentration in the media of CSFM cultures. TGF alpha mRNA and protein levels are also significantly lower in cells late in culture compared to logarithmically growing cells regardless of the presence or absence of EGF/BPE. The proliferation of primary RTE cells propagated without EGF/BPE is inhibited by neutralizing TGF alpha antiserum and by a tyrphostin compound that blocks TGF alpha/EGF receptor tyrosine kinase activity. These results indicate that primary RTE cells utilize TGF alpha as an autocrine growth factor and that the autocrine pathway is regulated as a function of growth state of the cells. However, this pathway does not provide growth autonomy to primary RTE cells, since cultures remain dependent on exogenous EGF/BPE for sustained proliferation.     

Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation

MCF-10A cells are a spontaneously immortalized normal human mammary epithelial cell line. MCF-10A cells were transfected with two expression vector plasmids containing either a human point-mutated c-Ha-ras protooncogene or the rat c-neu protooncogene. c-Ha-ras-transfected MCF-10A cells grow as colonies in soft agar, exhibit a 3- to 4-fold increase in their growth rate in serum-free medium, and show a reduced mitogenic response to exogenous epidermal growth factor (EGF) or transforming growth factor-alpha (TGF alpha) as compared to MCF-10A cells. c-Ha-ras-transfected MCF-10A cells express a 4- to 8-fold increase in TGF alpha mRNA levels and secrete 4- to 6-fold more TGF alpha protein as compared to MCF-10A cells. Addition of either an anti-TGF alpha neutralizing monoclonal antibody or an anti-EGF receptor blocking monoclonal antibody to the Ha-ras-transformed MCF-10A cells produces a 50 to 80% inhibition of colony formation of these cells in soft agar. c-neu-transfected MCF-10A cells grown in soft agar and exhibit an increase in their growth rate in serum-free medium at a level comparable to that observed in Ha-ras-transformed MCF-10A cells. Addition of an anti-c-erbB-2 monoclonal antibody inhibits the anchorage-independent growth of these cells in soft agar. However, c-neu-transformed MCF-10A cells show no increase in TGF alpha secretion and no change in their responsiveness to exogenous EGF or TGF alpha. A recombinant retroviral vector containing the human TGF alpha gene was also introduced into MCF-10A cells. TGF alpha-infected MCF-10A cells secrete 15- to 20-fold more TGF alpha protein than MCF-10A cells, form colonies in soft agar, exhibit an enhanced growth rate in serum-free medium, and show a decreased mitogenic response to exogenous EGF or TGF alpha at a level equivalent to Ha-ras-transformed MCF-10A cells. Growth of TGF alpha-infected MCF-10A cells in soft agar is completely inhibited by anti-TGF alpha neutralizing or anti-EGF receptor blocking monoclonal antibodies. These results suggest that TGF alpha is an intermediary in the transformation of human mammary epithelial cells by an activated c-Ha-ras gene, but not by the c-neu gene, and demonstrate that overexpression of this growth factor is able to transform immortalized human mammary epithelial cells which also express a sufficient complement of functional EGF receptors.     

Reduced growth rate accompanied by aberrant epidermal growth factor signaling in drug resistant human breast cancer cells

We examined transforming growth factor (TGF) alpha, epidermal growth factor (EGF) and EGF receptor (EGFR) expression and signaling in three drug resistant MCF-7 human breast cancer sublines and asked whether these pathways contribute to the drug resistance phenotype. In the resistant sublines, upregulation of both TGFalpha and EGFR mRNA was observed. In an apparent contrast with upregulated growth factor and receptor gene expression, the drug resistant sublines displayed a reduced growth rate. Defects in the EGFR signaling pathway cascade were found in all examined drug resistant sublines, including altered EGF-induced Shc, Raf-1, or mitogen-activated protein kinase phosphorylation. Induction of c-fos mRNA expression by EGF was impaired in the sublines compared to parental MCF-7 cells. In contrast, the induction of the stress-activated protein kinase activity was similar in both parental and drug resistant cells. Evaluating the link between the reduced growth rate and drug resistance, serum starvation experiments were performed. These studies demonstrated that a reduced proliferative activity resulted in a marked reduction in sensitivity to cytotoxic agents in the parental MCF-7 cells. We propose that the altered EGFR levels frequently observed in drug resistant breast cancer cells are associated with perturbations in the signaling pathway that mediate a reduced proliferative rate and thereby contribute to drug resistance.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

Purification and characterization of vaccinia virus growth factor

A growth factor detected in the medium of vaccinia-virus-infected cells was purified to homogeneity. Sequence analysis shows it to be a processed form of a polypeptide encoded in the vaccinia virus genome which is related to epidermal growth factor (EGF) and alpha-transforming growth factor (alpha TGF). The amino terminus of the processed vaccinia virus growth factor (VVGF) begins at residue 20 of the primary translation product, indicating a signal sequence has been removed. The amino acid composition of purified VVGF predicts the carboxyl terminus is at, or near, residue 96, suggesting a transmembrane sequence has also been removed from secreted VVGF, which is, therefore, approximately 77 amino acids. VVGF, unlike EGF or alpha TGF, is glycosylated. VVGF binds to the EGF receptor and stimulates its autophosphorylation, suggesting that vaccinia virus has acquired sequences encoding a growth factor that may allow it to subvert EGF receptor-dependent functions.     

Estrogen stimulates growth of mammary tumor cells ZR-75 without activation of S6 kinase and S6 phosphorylation. Difference from epidermal growth factor and alpha-transforming growth-factor-induced proliferation

Growth of the human mammary tumor cell line ZR-75-1 is stimulated by epidermal growth factor (EGF) and alpha-type transforming growth factor (alpha TGF), as well as by estradiol (E2). The role of activation of S6 kinase and S6 phosphorylation in the EGF(alpha TGF)-induced and E2-induced growth was investigated. Maximal effects on growth are observed at 10 nM EGF or alpha TGF. EGF as well as alpha TGF treatment of serum-starved cells leads to rapid activation of S6 kinase; the activity is increased about tenfold after 30 min of EGF treatment and declines with the time reaching about 25% of the maximal activity after 2 h of EGF treatment. Similar to the growth response, S6 kinase is activated at lower doses of EGF than alpha TGF and shows a maximal response at 10 nM for both growth factors. In contrast to this finding the incubation of serum-starved cells with E2 over a concentration range between 1 pM and 10 nM and times from 30 min to 4 h does not lead to increased S6 kinase activity. On investigating whether this lack of response to E2 is due to desensitization of the system by induction of alpha TGF it was found that preincubation of cells with alpha TGF for 2-6 h desensitizes them to reactivation of S6 kinase by alpha TGF, whereas preincubation with E2 does not. When S6 phosphorylation is monitored over times from 1 h to 6 h, it is observed that EGF leads to increased S6 phosphorylation, whereas E2 does not. The rate of onset of protein synthesis in the first 2 h of stimulation, when EGF-induced S6 phosphorylation is maximal, is more rapid with EGF than with E2. The results suggest that different pathway are involved in E2-induced and EGF(alpha TGF)-induced proliferation.     

Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta

Activation of mitogen-activated protein kinase (Erk/MAPK) is a critical signal transduction event for estrogen (E(2))-mediated cell proliferation. Recent studies from our group and others have shown that persistent activation of Erk plays a major role in cell migration and tumor progression. The signaling mechanism(s) responsible for persistent Erk activation are not fully characterized, however. In this study, we have shown that E(2) induces a slow but persistent activation of Erk in MCF-7 breast carcinoma cells. The E(2)-induced Erk activation is dependent on new protein synthesis, suggesting that E(2)-induced growth factors play a major role in Erk activation. When MCF-7 cells were treated with E(2) in the presence of an anti-HER-2 monoclonal antibody (herceptin), 60-70% of E(2)-induced Erk activation is blocked. In addition, when untreated MCF-7 cells were exposed to conditioned medium from E(2)-treated cells, Erk activity was significantly enhanced. Furthermore Erk activity was blocked by an antibody against HER-2 or by heregulin (HRG) depletion from the conditioned medium through immunoprecipitation. In contrast, epidermal growth factor receptor (Ab528) antibody only blocked 10-20% of E(2)-induced Erk activation, suggesting that E(2)-induced Erk activation is predominantly mediated through the secretion of HRG and activation of HER-2 by an autoctine/paracrine mechanism. Inhibition of PKC-delta-mediated signaling by a dominant negative mutant or the relatively specific PKC-delta inhibitor rottlerin blocked most of the E(2)-induced Erk activation but had no effect on TGF alpha-induced Erk activation. By contrast inhibition of Ras, by inhibition of farnesyl transferase (Ftase-1) or dominant negative (N17)-Ras, significantly inhibited both E(2)- and TGF alpha-induced Erk activation. This evaluation of downstream signaling revealed that E(2)-induced Erk activation is mediated by a HRG/HER-2/PKC-delta/Ras pathway that could be crucial for E(2)-dependent growth-promoting effects in early stages of tumor progression.     

Transforming growth factor alpha and a PC12-derived growth factor induce neurites in PC12 cells and enhance the survival of embryonic brain neurons.

We have identified and characterized a 5000-Da protein that induces neurite outgrowth from PC12 pheochromocytoma cells, enhances the survival of embryonic rat brain neurons in primary culture, and induces the multiplication of embryonic rat brain astrocytes in primary culture. The factor is produced by a flat cell PC12 variant that expresses the activated ras oncogene after transfection of the gene. The factor resembles transforming growth factor alpha (TGF alpha) and epidermal growth factor (EGF) in that it induces anchorage-independent colony formation of normal rat kidney cells in soft agar and competes with EGF for binding to the EGF receptor. Rat TGF alpha and human TGF alpha also induce neurite outgrowth from PC12 and enhance the survival of embryonic brain neurons. The PC12 variant-derived factor can be distinguished from TGF alpha and EGF immunologically and by migration rates on reversed-phase high-performance liquid chromatography.     

Alterations in phosphoinositide metabolism associated with 17 beta-estradiol and growth factor treatment of MCF-7 breast cancer cells

Steady-state levels of phosphatidyl inositol (PtdIns) turnover are examined in MCF-7 human breast cancer cells in response to estradiol treatment. Elevated levels of PtdIns are observed 12-24 h after estradiol treatment, occur at estradiol concentrations as low as 10(-12) M, and are competitively blocked by the antiestrogen LY117018. MCF-7 cells secrete a transforming growth factor (TGF) alpha-like material which can partly replace estradiol in conferring tumorgenicity in nude mice. We show that acute or chronic treatment of MCF-7 cells with TGF alpha results in elevated PtdIns turnover and that chronic treatment increases growth rate. In contrast TGF beta is growth inhibitory and blocks estradiol-induced increases in PtdIns turnover. A phosphatidyl inositol 4,5-bisphosphate specific phospholipase-C activity has been identified and is elevated in association with estradiol treatment. These data are consistent with estradiol-induced autocrine growth factors, including TGF alpha, acting through the PtdIns turnover pathway as part of their mechanism of action.     

Transforming growth factor alpha stimulates prostacyclin production by cultured human vascular endothelial cells more potently than epidermal growth factor

Transforming growth factor alpha (TGF alpha) induces dose- and time-dependent stimulation of prostacyclin (PGI2) production by cultured human umbilical vein endothelial cells. The lowest stimulatory concentration of TGF alpha was 0.1 ng/ml and the maximal response, a 2.7-fold rise, was obtained with 10 ng/ml. The stimulation, which lasted at least 24 h, was blocked by cycloheximide and by indomethacin. TGF alpha induced PGI2 production at 10-100 times lower concentrations than did epidermal growth factor (EGF), although in stimulating endothelial cell growth the two factors were equipotent. This is the first demonstration that TGF alpha enhances PGI2 production by human cells. Moreover, this is the first evidence that it acts as both an agonist (growth) and a superagonist (PGI2 production) of EGF in the same cell type. I suggest that this phenomenon may be involved with the angiogenic activity of TGF alpha.