epidermal-growth-factor and afimoxifene

In the present study, we evaluated the ability of 4-hydroxytamoxifen (OHT) and epidermal growth factor (EGF) to regulate homotypic adhesion in MCF7 breast cancer cells. Our results demonstrate that OHT and EGF activate the E-cadherin promoter, increase E-cadherin mRNA and protein expression and enhance homotypic aggregation of MCF7 cells. Interestingly, an ERalpha and EGFR cross-talk is involved in the E-cadherin expression by OHT and EGF, as demonstrated by knocking down either receptor. On the basis of our findings, the well-established cross-talk between ERalpha and EGFR could be extended to the modulation of E-cadherin expression by OHT and EGF. Thus, the potential ability of tamoxifen to induce cell-cell aggregation may contribute to the biologic response of pharmacologic intervention in patients with breast cancer.

Topics: Antineoplastic Agents, Hormonal; Blotting, Western; Breast Neoplasms; Cadherins; Cell Adhesion; Cell Aggregation; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Estrogen Receptor alpha; Female; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Oligonucleotide Array Sequence Analysis; Receptor Cross-Talk; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tamoxifen; Transcriptional Activation; Transfection

Tamoxifen (Tam), and its active metabolite, 4-hydroxytamoxifen (OHT), compete with estrogens for binding to the estrogen receptor (ER). Tam and OHT can also induce ER-dependent apoptosis of cancer cells. 10-100nM OHT induces ER-dependent apoptosis in approximately 3 days. Using HeLaER6 cells, we examined the role of OHT activation of signal transduction pathways in OHT-ER-mediated apoptosis. OHT-ER activated the p38, JNK and ERK1/2 pathways. Inhibition of p38 activation with SB203580, or RNAi-knockdown of p38alpha, moderately reduced OHT-ER mediated cell death. A JNK inhibitor partly reduced cell death. Surprisingly, the MEK1/2 inhibitor, PD98059, completely blocked OHT-ER induced apoptosis. EGF, an ERK1/2 activator, enhanced OHT-induced apoptosis. OHT induced a delayed and persistent phosphorylation of ERK1/2 that persisted for >80h. Addition of PD98059 as late as 24h after OHT largely blocked OHT-ER mediated apoptosis. The antagonist, ICI 182,780, blocked both the long-term OHT-mediated phosphorylation of ERK1/2 and OHT-induced apoptosis. Our data suggests that the p38 and JNK pathways, which often play a central role in apoptosis, have only a limited role in OHT-ER-mediated cell death. Although rapid activation of the ERK1/2 pathway is often associated with cell growth, persistent activation of the ERK1/2 pathway is essential for OHT-ER induced cell death.

Topics: Caspase 3; Caspase 9; Cell Death; Enzyme Activation; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Flavonoids; Fulvestrant; HeLa Cells; Humans; Imidazoles; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Receptors, Estrogen; Tamoxifen; Time Factors

Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim(EL) is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim(EL) turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim(EL) is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim(EL) does not involve its tyrosine phosphorylation; indeed, Bim(EL) is not a tyrosine phosphoprotein. Furthermore, Bim(EL) fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim(EL) turnover proceeds normally in Cbl-containing or Cbl-/- fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim(EL) turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.

Topics: Animals; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Cell Line; Enzyme Inhibitors; Epidermal Growth Factor; Epithelial Cells; Fibroblasts; Humans; Membrane Proteins; Mice; Mice, Knockout; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mutation; Phosphorylation; Proteasome Endopeptidase Complex; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-cbl; Serine; Tamoxifen; Tyrosine; Ubiquitin; Vanadates

Vascular endothelial cells (EC) are an important target of estrogen action through both the classical genomic (i.e. nuclear-initiated) activities of estrogen receptors alpha and beta (ERalpha and ERbeta) and the rapid "non-genomic" (i.e. membrane-initiated) activation of ER that stimulates intracellular phosphorylation pathways. We tested the hypothesis that the red wine polyphenol trans-resveratrol activates MAPK signaling via rapid ER activation in bovine aortic EC, human umbilical vein EC, and human microvascular EC. We report that bovine aortic EC, human umbilical vein EC, and human microvascular EC express ERalpha and ERbeta. We demonstrate that resveratrol and estradiol (E(2)) rapidly activated MAPK in a MEK-1, Src, matrix metalloproteinase, and epidermal growth factor receptor-dependent manner. Importantly, resveratrol activated MAPK and endothelial nitric-oxide synthase (eNOS) at nm concentrations (i.e. an order of magnitude less than that required for ER genomic activity) and concentrations possibly achieved transiently in serum following oral red wine consumption. Co-treatment with ER antagonists ICI 182,780 or 4-hydroxytamoxifen blocked resveratrol- or E(2)-induced MAPK and eNOS activation, indicating ER dependence. We demonstrate for the first time that ERalpha-and ERbeta-selective agonists propylpyrazole triol and diarylpropionitrile, respectively, stimulate MAPK and eNOS activity. A red but not a white wine extract also activated MAPK, and activity was directly correlated with the resveratrol concentration. These data suggest that ER may play a role in the rapid effects of resveratrol in EC and that some of the atheroprotective effects of resveratrol may be mediated through rapid activation of ER signaling in EC.

Topics: Angiogenesis Inhibitors; Animals; Aorta; Blotting, Western; Cattle; Cell Nucleus; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Epidermal Growth Factor; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Flavonoids; Fulvestrant; Humans; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mice; Microcirculation; Models, Biological; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phenols; Phosphorylation; Polyphenols; Resveratrol; Serine; Signal Transduction; Stilbenes; Tamoxifen; Time Factors; Transfection; Umbilical Veins; Wine

Protein kinase B (PKB/Akt) has been shown to play a role in protection from apoptosis, cell proliferation and cell growth. It is also involved in mediating the effects of insulin, such as lipogenesis, glucose uptake and conversion of glucose into fatty acids and cholesterol. Sterol-regulatory element binding proteins (SREBPs) are the major transcription factors that regulate genes involved in fatty acid and cholesterol synthesis. It has been postulated that constitutive activation of the phosphatidylinositol 3 kinase/Akt pathway may be involved in fatty acid and cholesterol accumulation that has been described in several tumour types. In this study, we have analysed changes in gene expression in response to Akt activation using DNA microarrays. We identified several enzymes involved in fatty acid and cholesterol synthesis as targets for Akt-regulated transcription. Expression of these enzymes has previously been shown to be regulated by the SREBP family of transcription factors. Activation of Akt induces synthesis of full-length SREBP-1 and SREBP-2 proteins as well as expression of fatty acid synthase (FAS), the key regulatory enzyme in lipid biosynthesis. We also show that Akt leads to the accumulation of nuclear SREBP-1 but not SREBP-2, and that activation of SREBP is required for Akt-induced activation of the FAS promoter. Finally, activation of Akt induces an increase in the concentration of cellular fatty acids as well as phosphoglycerides, the components of cellular membranes. Our data indicate that activation of SREBP by Akt leads to the induction of key enzymes of the cholesterol and fatty acid biosynthesis pathways, and thus membrane lipid biosynthesis.

Topics: CCAAT-Enhancer-Binding Proteins; Cell Membrane; Cell Nucleus; Cells, Cultured; Cholesterol; DNA-Binding Proteins; Enzymes; Epidermal Growth Factor; Fatty Acid Synthases; Fatty Acids; Gene Expression Profiling; Humans; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Synthase; Insulin; Pigment Epithelium of Eye; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Sterol Regulatory Element Binding Protein 1; Sterol Regulatory Element Binding Protein 2; Tamoxifen; Transcription Factors; Transcription, Genetic

About 50-64% of human breast cancers express receptors for GnRH-I. Direct antiproliferative effects of analogs of GnRH-I on human breast cancer cell lines have been shown. They are at least in part mediated by antagonizing growth promoting effects of estradiol, epidermal growth factor (EGF) or insulin-like growth factor. Recently, expression of a putative receptor for GnRH-II in human tissues was demonstrated. Antiproliferative effects of GnRH-II in human endometrial and ovarian cancer cells were shown not to be mediated through the GnRH-I receptor. Now we demonstrate direct anti-proliferative effects of the GnRH-I analog Triptorelin and the GnRH-II analog [d-Lys(6)]GnRH-II in MCF-7 and T47D human breast cancer cells expressing GnRH-I receptors and putative GnRH-II receptors. Pretreatment with Triptorelin or [d-Lys(6)]GnRH-II blocked EGF-induced autophosphoryla-tion of EGF receptor and activation of mitogen-activated protein kinase (extracellular-signal-regulated kinase 1/2 (ERK1/2)) in these cells. In sublines of MCF-7 and T47D cells, which were developed to be resistant to 4OH-tamoxifen, HER-2/p185 was overexpressed. Pretreatment of these cell lines with Triptorelin or [d-Lys(6)]GnRH-II completely abolished resistance to 4OH-tamoxifen, assessed by 4OH-tamoxifen-induced apoptosis. Analogs of GnRH-I and GnRH-II counteract EGF-dependent signal transduction in human breast cancer cells with expression of receptors for GnRH-I and GnRH-II. Through this mechanism, they probably reverse acquired resistance to 4OH-tamoxifen mediated through overexpression or activation of receptors of the c-erbB family.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Estrogen Antagonists; Extracellular Signal-Regulated MAP Kinases; Female; Gefitinib; Gonadotropin-Releasing Hormone; Humans; Phosphorylation; Quinazolines; Signal Transduction; Tamoxifen; Triptorelin Pamoate

Although many estrogen receptor-positive breast cancers initially respond to antihormones, responses are commonly incomplete with resistance ultimately emerging. Delineation of signaling mechanisms underlying these phenomena would allow development of therapies to improve antihormone response and compromise resistance. This in vitro investigation in MCF-7 breast cancer cells examines whether epidermal growth factor receptor (EGFR) signaling limits antiproliferative and proapoptotic activity of antihormones and ultimately supports development of resistance. It addresses whether the anti-EGFR agent gefitinib (ZD1839/Iressa; TKI: 1 mum) combined with the antihormones 4-hydroxytamoxifen (TAM: 0.1 mum) or fulvestrant (Faslodex; 0.1 mum) enhances growth inhibition and prevents resistance. TAM significantly suppressed MCF-7 growth over wk 2-5, reducing proliferation detected by immunocytochemistry and fluorescence-activated cell sorter cell cycle analysis. A modest apoptotic increase was observed by fluorescence-activated cell sorter and fluorescence microscopy, with incomplete bcl-2 suppression. EGFR induction occurred during TAM response, as measured by immunocytochemistry and Western blotting, with EGFR-positive, highly proliferative resistant growth subsequently emerging. Although TKI alone was ineffective on growth, TAM plus TKI cotreatment exhibited superior antigrowth activity vs. TAM, with no viable cells by wk 12. Cotreatment was more effective in inhibiting proliferation, promoting apoptosis, and eliminating bcl-2. Cotreatment blocked EGFR induction, markedly depleted ERK1/2 MAPK and protein kinase B phosphorylation, and prevented emergence of EGFR-positive resistance. Faslodex plus TKI cotreatment was also a superior antitumor strategy. Thus, increased EGFR evolves during treatment with antihormones, limiting their efficacy and promoting resistance. Gefitinib addition to antihormonal therapy could prove more effective in treating estrogen receptor-positive breast cancer and may combat development of resistance.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Breast Neoplasms; Cell Division; Cell Line, Tumor; Drug Combinations; Drug Resistance; Drug Synergism; Epidermal Growth Factor; ErbB Receptors; Estradiol; Estrogen Antagonists; Female; Fulvestrant; Gefitinib; Humans; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Tamoxifen

Inhibitors of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, such as Simvastatin and Lovastatin, reduce the rate of DNA synthesis and proliferation of a wide variety of cell types in vitro, by inducing a cell cycle arrest in G1. In estrogen-free medium, DNA synthesis is reduced by more that 90% following exposure of normal and transformed human breast epithelia] cells to 20 microM Simvastatin or Lovastatin for 24 to 42 hrs. We show here that stimulation of estrogen responsive MCF-7 cells with nanomolar concentrations of 17beta-estradiol (E2) prevents inhibition of DNA synthesis by these compounds. The effect of the hormone is antagonized by both steroidal and non steroidal antiestrogens, and it is not detectable in estrogen receptor-negative MCF-10a cells. Cell cycle analysis demonstrates that HMG-CoA reductase inhibitors are unable to induce G1 arrest of MCF-7 cells in the presence of E2.

Topics: Breast Neoplasms; Cell Cycle; Cell Line; DNA, Neoplasm; Enzyme Inhibitors; Epidermal Growth Factor; Estradiol; Estradiol Congeners; Estrogen Antagonists; Female; Fulvestrant; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Kinetics; Lovastatin; RNA, Messenger; Simvastatin; Tamoxifen; Tumor Cells, Cultured

The phosphorylation of the human estrogen receptor (ER) serine residue at position 118 is required for full activity of the ER activation function 1 (AF-1). This Ser118 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro and in cells treated with epidermal growth factor (EGF) and insulin-like growth factor (IGF) in vivo. Overexpression of MAPK kinase (MAPKK) or of the guanine nucleotide binding protein Ras, both of which activate MAPK, enhanced estrogen-induced and antiestrogen (tamoxifen)-induced transcriptional activity of wild-type ER, but not that of a mutant ER with an alanine in place of Ser118. Thus, the activity of the amino-terminal AF-1 of the ER is modulated by the phosphorylation of Ser118 through the Ras-MAPK cascade of the growth factor signaling pathways.

Topics: Amino Acid Sequence; Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Enzyme Activation; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Humans; Mitogen-Activated Protein Kinase Kinases; Molecular Sequence Data; Mutation; Phosphorylation; Polyunsaturated Alkamides; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras); Receptors, Estrogen; Recombinant Fusion Proteins; Serine; Somatomedins; Tamoxifen; Transcriptional Activation; Transfection

In uterine tissue, estrogen regulates various components of the insulin-like growth factor system; however, there are few suitable in vitro systems to examine these effects. Here we have examined the effects of 17-beta estradiol (E2) on expression and synthesis of insulin-like growth factors (IGF-I and IGF-II) and the insulin-like growth factor binding proteins (IGFBPs) by Ishikawa human endometrial cancer cells. Using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay, we demonstrated that both E2 and 4-hydroxy-tamoxifen (OHT) enhanced IGF-I expression but had no effect on IGF-II expression. The pure antiestrogen ICI 182,780 had no effect on IGF-I expression and partially blocked the E2 and OHT effect on IGF-I expression. The effect of epidermal growth factor (EGF), which is able to mimic some of the effects of E2 in Ishikawa cells and uterine tissue, was also examined. EGF, unlike E2, did not increase IGF-I expression but rather resulted in a significant decrease in IGF-I messenger RNA (mRNA) levels. EGF also resulted in a small, nonsignificant increase in IGF-II mRNA levels. IGFBP-3, -5, and -6 mRNAs were detected by Northern blot analyses of Ishikawa cells RNA. However, only IGFBP-3 was consistently detected by ligand blotting of conditioned medium. E2 had no significant effect on expression of any of the binding proteins, whereas EGF increased IGFBP-5 mRNA levels. These data provide the first in vitro demonstration of regulation of IGF-I expression by E2. The Ishikawa cell line may provide a useful model to further investigate the molecular mechanisms underlying E2 regulation of IGF-I expression. Furthermore, we have demonstrated a clear dissociation of the effects of E2 and EGF on IGF-I expression in this cell line.

Topics: Base Sequence; Carrier Proteins; Endometrial Neoplasms; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Female; Gene Expression; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Molecular Probes; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Somatomedins; Tamoxifen; Transcription, Genetic; Tumor Cells, Cultured

Growth of human breast cancer cells is controlled by multiple interacting factors that trigger different intracellular signaling pathways. The nonsteroidal antagonist 4-hydroxytamoxifen (OH-Tam), which acts as an antiestrogen, is also able to inhibit the mitogenic activity of epidermal growth factor (EGF) on hormone-responsive MCF7 cells. To further characterize the mechanism of this antigrowth factor activity, which is accompanied by an increase of high-affinity EGF binding and a drastic decrease in EGF receptor autophosphorylation, we studied the effect of OH-Tam on protein tyrosine phosphatase (PTPase) activity with specific in vitro assays using two different substrates. OH-Tam increased membrane PTPase activity in a time- and dose-dependent fashion whereas cytoplasmic enzyme activity remained unchanged. The increase in PTPase activity was mediated by the estrogen receptor (ER) since it was restricted to ER-positive cells, and the optimal OH-Tam concentration (ED50 = 1 nM) was correlated with the ligand affinity for ER. The increase in enzyme activity was selectively obtained with nuclear receptor ligands (OH-Tam, ICI 164,384) that inhibited growth factor-induced proliferation, whereas other inhibitors of estrogenic responses such as synthetic progestins and antiprogestins had no effect. The time course of stimulation (maximal stimulation at day 4) was concomitant to the loss of EGF mitogenic response. Moreover, addition of a specific PTPase inhibitor (5 microM sodium orthovanadate) to intact cells in culture prevented OH-Tam inhibition of cell proliferation, suggesting that these two events are closely associated.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Binding Sites; Breast Neoplasms; Cell Division; Chromatography, High Pressure Liquid; Epidermal Growth Factor; ErbB Receptors; Estrogen Antagonists; Homeostasis; Humans; Kinetics; Molecular Sequence Data; Phosphorylation; Protein Tyrosine Phosphatases; Receptors, Estrogen; Tamoxifen; Tumor Cells, Cultured; Vanadates

11 beta-Amidoalkoxyphenyl estradiols, a series of new antiestrogens, have been prepared and compared with tamoxifen (TAM) and 4-hydroxytamoxifen (OH-TAM). In vitro, these compounds were up to 20 times as active as OH-TAM on estradiol (E2)-stimulated MCF-7 cells. Unlike TAM or OH-TAM which were inactive, they displayed potent growth inhibitory effects on MCF-7 cells stimulated by a cocktail of epidermal growth factor and platelet derived growth factor. One of the most active compounds, 5e, was tested in vivo for its antiuterotrophic and antitumoral activities: it proved to be fully antiuterotrophic at 3 mg/kg subcutaneously in mice while being devoid of any uterotrophic activity. It inhibited the E2-induced growth of MCF-7 tumors implanted in nude mice and prevented the partial agonistic activity of TAM on MCF-7 tumor growth in ovariectomized mice. Moreover, on MCF-7 variant tumors, 5e, unlike TAM, did not display any proliferative activity, but inhibited the TAM-induced growth. Overall, these results show that this new series of compounds displays an improved activity profile compared with that of TAM, on tests relevant to human breast cancer treatment.

Topics: Animals; Antineoplastic Agents; Cell Line; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Structure; Platelet-Derived Growth Factor; Rats; Rats, Sprague-Dawley; Tamoxifen

In estrogen receptor positive human breast cancer cells, anti-estrogens inhibit the mitogenic effect of growth factors in the absence of estrogens. As activator protein-1 (AP-1) activity is one of the first nuclear events following growth factor receptor activation, we studied the effects of estrogens and anti-estrogens on growth factor-induced AP-1 activity using transient transfection of the AP-1-responsive gene (AP-1)4-TK-CAT into MCF7 cells. The growth factor-induced AP-1 response was increased by estradiol and inhibited by anti-estrogens in conditions where growth factor-induced c-fos and c-jun mRNA levels were unchanged by hormone and anti-hormone treatments. The same regulations were obtained when the AP-1 response was directly induced by co-transfection of c-fos and c-jun expression vectors. Co-transfection of the wild-type estrogen receptor HEGO amplified both effects. Inhibition of AP-1 activity by anti-estrogens was unlikely to be explained by the presence of residual estrogens in MCF7 cells. (i) anti-estrogens inhibited AP-1 activity in conditions where they had no effect on basal ERE-mediated activity levels, whereas estradiol was as efficient in stimulating both activities. (ii) The relative efficacy of the two anti-estrogens, OH-tamoxifen and ICI 164,384 in inhibiting these two activities was different; OH-tamoxifen was more efficient in inhibiting ERE-mediated activity, whereas ICI 164,384 was more efficient in trans-repressing AP-1-mediated activity. We conclude that in conditions where c-fos and c-jun syntheses were not affected, the estrogen receptor cooperated with growth factors to stimulate the AP-1 response when activated by estrogens but inhibited AP-1-mediated transcription when occupied by anti-estrogens.

Topics: Breast Neoplasms; Chloramphenicol O-Acetyltransferase; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Female; Genes, fos; Genes, jun; Humans; Insulin-Like Growth Factor I; Kinetics; Polyunsaturated Alkamides; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Receptors, Estrogen; Recombinant Fusion Proteins; RNA, Messenger; Tamoxifen; Thymidine Kinase; Time Factors; Transfection; Tumor Cells, Cultured

In MCF7 human breast cancer cells, the antiestrogens 4-hydroxy-tamoxifen and ICI 164,384 inhibit the mitogenic activity of epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). These growth factors also stimulate the expression of cathepsin-D and pS2 genes. Therefore, we studied the effects of antiestrogens on growth factor induction of pS2 and cathepsin-D mRNA. The two antiestrogens strongly inhibited the transcriptional induction of pS2 by growth factors. On the contrary, estradiol and IGF-I or EGF had an additive effect on pS2 mRNA accumulation. Growth factor induction of cathepsin-D was also inhibited by ICI 164,384. By contrast, 4-hydroxytamoxifen had an agonist effect on cathepsin-D and an additive effect on IGF-I-induced mRNA. When 12-O-tetradecanoylphorbol-13-acetate or 8-bromo-cAMP (8-Br-cAMP) was used instead of growth factors, similar effects of 4-hydroxytamoxifen and ICI 164,384 were obtained on pS2 (12-O-tetradecanoylphorbol-13-acetate and 8-Br-cAMP) and cathepsin-D (8-Br-cAMP) induction. A mechanism based on the classical competitive inhibition by antiestrogens of estrogen binding and action on the estrogen receptor was very unlikely, as 1) no antigrowth factor activity was obtained with R5020, which was a potent inhibitor of estrogen induction of pS2 and cathepsin-D mRNA; 2) in the Ishikawa endometrial cancer cell line, the cathepsin-D gene is unresponsive to estrogen, but was inhibited by antiestrogen after its induction by EGF or 8-Br-cAMP; and 3) the residual estrogen concentration in cells was too low to induce the expression of estrogen-specific genes. However, antiestrogens did not inhibit the expression of all genes induced by growth factors, as they were without effect on IGF-I induction of glyceraldehyde-3-phosphate dehydrogenase mRNA. These results demonstrate that antiestrogens can modulate the transcription of some growth factor-induced genes and strongly suggest that this effect is not due to interference with residual estrogens.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Breast Neoplasms; Cathepsin D; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Growth Substances; Humans; Insulin-Like Growth Factor I; Neoplasm Proteins; Polyunsaturated Alkamides; Proteins; RNA, Messenger; Tamoxifen; Tetradecanoylphorbol Acetate; Transcription, Genetic; Trefoil Factor-1; Tumor Cells, Cultured; Tumor Suppressor Proteins

The effects of the transforming growth factor-beta 1 (TGF-beta 1) and epidermal growth factor (EGF) on the growth of cells from 2 endometrial cancer lines, Ishikawa and HEC-50 were evaluated by measuring rates of DNA synthesis and changes in cell numbers during culture. EGF at 17 and 1.7 nM concentrations consistently enhanced HEC-50 cell proliferation. TGF-beta 1 inhibited Ishikawa cell proliferation but, unexpectedly for epithelium-derived cells, stimulated HEC-50 cell growth. This effect is of interest as it indicates that endometrial cells can acquire an altered responsiveness to a growth inhibitor during the process of malignant transformation. Northern blot analyses showed expression of TGF-alpha, TGF-beta 1 and EGF receptors mRNA in both cell lines. Neither estradiol (E2) nor 4-hydroxytamoxifen (OHTam) affected mRNA levels for either TGF-alpha or TGF-beta in HEC-50 cells, a line unresponsive to E2 for proliferation. In Ishikawa cells, previously shown to respond to both E2 and OHTam by increasing proliferation rates, E2 increased TGF-alpha mRNA and reduced TGF-beta mRNA levels. OHTam lowered the levels of both mRNA species, although the effect was greater on TGF-beta than TGF-alpha mRNA. These data are consistent with, but do not prove, the existence of a possible autocrine regulation by TGF-alpha and TGF-beta of human cancer cell proliferation, which might be under E2 influence in Ishikawa cells.

Topics: Adenocarcinoma; Blotting, Northern; Cell Division; DNA; Endometrial Neoplasms; Epidermal Growth Factor; Estrogen Antagonists; Female; Humans; RNA, Messenger; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured

The hormone dependency of the MCF-7 breast cancer cell line, while extensively tested in liquid culture, has not been previously evaluated under conditions of anchorage-independent growth in serum-free media. Using the soft agar clonogenic assay, we demonstrate that physiologically relevant concentrations of estradiol (E2), progesterone (Pg), and prolactin (PRL) similarly stimulated MCF-7 cell colony formation in the absence of serum. Addition of an anti-insulin-like growth factor-I (IGF-I) antibody inhibited E2- and Pg-stimulated growth, while PRL action was not affected. Similar results were obtained with an anti-IGF-I receptor antibody, except that its inhibitory effect on Pg-induced colony formation was modest and not statistically significant. Administration of either an anti-transforming growth factor-alpha (TGF-alpha) antibody or an anti-epidermal growth factor (EGF) receptor antibody similarly inhibited E2-stimulated MCF-7 cell growth in soft agar, while neither antibody influenced Pg or PRL effects. Addition of TGF-beta 1, -beta 2, -beta 3 similarly suppressed MCF-7 cell colony formation in a dose dependent manner to a degree comparable to that observed with 4-OH-tamoxifen (4-OH-T). Furthermore, the growth inhibitory effect of 4-OH-T was completely reversed by an anti-TGF-beta antibody. We conclude that IGFs and TGF-alpha are important mediators of E2-stimulated MCF-7 cell growth in soft agar. IGFs may also be playing a role in Pg action, while neither growth factor is involved in PRL-stimulated colony formation. Finally, TGF-beta appears to be an important mediator of antiestrogen-induced inhibition of tumor growth.

Topics: Agar; Antibodies; Breast Neoplasms; Culture Media; Drug Interactions; Epidermal Growth Factor; Estradiol; Female; Growth Substances; Hormones; Humans; Insulin-Like Growth Factor I; Neoplasms, Hormone-Dependent; Progesterone; Prolactin; Tamoxifen; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay

The role of growth factor signal transducers in the induction of the progesterone receptor by epidermal growth factor (EGF) and the potential sites of EGF antagonism by an antiestrogen were studied in fetal uterine cells in culture. The effects of EGF and estradiol were not additive, suggesting that EGF and estradiol are acting through common mechanisms where antiestrogens could possibly intervene. Fetal uterine cells in culture were found to contain specific, high affinity binding sites for [125I]EGF. Estradiol treatment of the cells led to a higher number of binding sites, but the site of action of 4-hydroxytamoxifen is not the EGF receptor because this antiestrogen had no effect on EGF binding. Activation of protein kinase C by a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) increased progesterone receptor levels to a similar extent as EGF or estradiol. Increasing the intracellular cAMP concentrations by either adding dibutyryl cyclic AMP or activating adenylate cyclase with forskolin also raised progesterone receptor concentrations. Neither the phorbol ester nor dibutyryl cAMP had any effect on cell proliferation. 4-Hydroxytamoxifen completely abolished the effects of the phorbol ester and cAMP. In conclusion, the levels of an estrogen-induced steroid hormone receptor can be regulated by molecules involved in the signal transduction pathway of peptide factors. Moreover, in fetal uterine cells, a potent antiestrogen appears to act as a multiple antagonist but only on an estrogen-inducible response.

Topics: Animals; Bucladesine; Cell Division; Cells, Cultured; Colforsin; Cyclic AMP; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Estradiol; Female; Guinea Pigs; Kinetics; Protein Kinase C; Receptors, Progesterone; Tamoxifen; Tetradecanoylphorbol Acetate; Uterus

Cells isolated from the uterus of the guinea pig foetus can be maintained in culture even through several sub-cultures. In these cells, estradiol increases progesterone receptor concentrations 2 o 3 times, although estradiol has no effect on cell proliferation. Epidermal growth factor (EGF) stimulates both cell proliferation and the progesterone receptor 4-Hydroxytamoxifen, a tamoxifen metabolite and potent anti-oestrogen, completely inhibits the stimulatory effect of EGF on the progesterone receptor but has no effect on the EGF-induced cell growth. These cells have specific binding sites with high affinity for 125I-EGF. Estradiol increases the number of binding sites but does not affect the affinity for EGF. 4-Hydroxytamoxifen has no significant effect on either the number of binding sites or the binding affinity. In conclusion, EGF could be an autocrine or paracrine factor in estrogen-sensitive cells not only as a potent mitogen but also as a factor capable of increasing an estrogen-induced protein like the progesterone receptor. The observation that an anti-estrogen can also act as an "anti-growth factor" suggests a close relationship between estrogens and growth factors.

Topics: Animals; Cell Division; Cells, Cultured; Epidermal Growth Factor; ErbB Receptors; Estradiol; Estrogen Antagonists; Female; Guinea Pigs; Receptors, Progesterone; Tamoxifen; Uterus

To determine the functional significance of the endogenous expression of the epidermal growth factor (EGF) and transforming growth factor-alpha genes in T-47D, human breast cancer cells, we have examined the effects of two types of antiproliferative agents, progestins and antiestrogens, on the expression of these growth factors and the effects of exogenous EGF on the antiproliferative action of these agents. Using Northern blot analysis, the regulation of expression of these two genes by the antiproliferative agents, tamoxifen and monohydroxytamoxifen, was examined. In T-47D cells the two antiestrogens did not affect the accumulation of EGF mRNA and decreased the accumulation of TGF alpha mRNA. As we have shown before, the progestin, medroxyprogesterone acetate, increased the level of both EGF mRNA and TGF alpha mRNA in this cell line. The regulation of expression of these endogenous growth factor genes was unrelated to the proliferative behavior of T-47D cells since both antiestrogens and progestins were antiproliferative under the conditions of the experiments. The variant cell line, T-47D-5, had no detectable EGF mRNA and contained about 1/10th the level of TGF alpha mRNA expressed by "wild type" T-47D cells. T-47D-5 cells were 2.5 times more sensitive to the antiproliferative effects of both progestins and antiestrogens when compared to the growth factor expressing T-47D cells. Exogenously added murine EGF was able to decrease slightly the sensitivity of both cell lines to the antiproliferative effects of both progestins and antiestrogens as well as increase the proliferation of T-47D but not T-47D-5 cells. These data suggest that endogenous expression of growth factors may be associated with decreased sensitivity of the cells to growth inhibitory agents.

Topics: Breast Neoplasms; Cell Line; Dose-Response Relationship, Drug; Epidermal Growth Factor; Estrogen Antagonists; Gene Expression Regulation; Humans; Progestins; RNA, Messenger; Tamoxifen; Transforming Growth Factors

In fetal uterine cells in culture, epidermal growth factor (EGF) increased progesterone receptor concentrations more than 2-fold. Two other growth factors, transforming growth factor-alpha and fibroblast growth factor, were not able to cause the same increase. This response to EGF was dose dependent; a half-maximal effect was obtained at 10(-10) M. The antiestrogens tamoxifen and 4-hydroxytamoxifen were able to antagonize the stimulatory effect of EGF on progesterone receptor concentrations, but they did not affect its mitogenic effect. The inhibitory effect of 4-hydroxytamoxifen depended on concentration; half-maximal inhibition was observed between 0.5-1 X 10(-9) M. 4-Hydroxytamoxifen could completely inhibit the progesterone receptor increase due to EGF even when added to cells already exposed to the growth factor for 6 days. EGF seems to be acting as an estrogen in increasing progesterone receptors in fetal uterine cells, and antiestrogens are potent antagonists of this response, indicating that growth factors may also be involved in some protein-inducing effects of estrogens. Since estrogen receptor levels were at the limits of detectability under all of the experimental conditions studied, nonestrogen receptor-mediated pathways may be involved. These observations show the potential importance of other factors acting in combination with estrogens in the modulation of progesterone receptor levels.

Topics: Animals; Cells, Cultured; Epidermal Growth Factor; Estrogen Antagonists; Female; Fetus; Fibroblast Growth Factors; Guinea Pigs; Kinetics; Receptors, Progesterone; Tamoxifen; Transforming Growth Factors; Uterus

Regulation of breast tumor proliferation depends in a large part on a variety of hormones and growth factors. In this report we show that estrogen and antiestrogen modulate epidermal growth factor-receptor (EGF-R) level in the human breast cancer MCF-7 cells with opposite mechanisms. Although a short-term treatment (24h to 48h) with estradiol leads to a decrease in EGF-R number, the addition of hormone in cell culture for 5 days increases EGF-R level with a maximal effect observed at 10(-10) M estradiol. In contrast, when cells are treated with the antiestrogen hydroxytamoxifen, a dose-dependent decrease in EGF-R level occurs. We also report that EGF is able to induce estrogen receptors and, to a lesser extent, progesterone receptors when added to MCF-7 cell cultures. These results demonstrate an interaction between both estrogen receptor and EGF receptor growth promoting systems in target cells. The implications of such an interaction in the understanding of human breast cancer hormone responsiveness and, in the development of therapies, are discussed.

Topics: Breast Neoplasms; Dose-Response Relationship, Drug; Epidermal Growth Factor; ErbB Receptors; Estradiol; Humans; Kinetics; Receptors, Estrogen; Receptors, Progesterone; Tamoxifen; Tumor Cells, Cultured

We have previously reported that the cloned cell line (B-1-A-2) derived from an estrogen-responsive mouse Leydig cell tumor shows an estrogen-dependent enhancement of cell proliferation in medium supplemented with charcoal-dextran-stripped fetal bovine serum. To avoid the involvement of unknown factors present in the serum in the pathway for estrogen-dependent cell growth, the present study was designed to establish a serum-free culture system to which growth factors could be added. To this end, we subcloned B-1 cells from the parental tumor cell line. The proliferation of B-1 cells was markedly stimulated by the addition of 10(-11)-10(-8) M estradiol into the serum-free medium [Eagle's Minimum Essential Medium-Ham's F-12 (1:1, vol/vol) containing 0.2% (wt/vol) BSA]. Epidermal growth factor (0.1-50 ng/ml) or insulin (0.1-50 micrograms/ml) alone or in combination with 10(-8) M estradiol did not affect the proliferation rate of B-1 cells. In contrast, a greater than 10-fold molar excess of 4-hydroxytamoxifen blocked estradiol-induced cell proliferation, while 4-hydroxytamoxifen alone failed to show a stimulatory effect on cell multiplication. Additionally, the conditioned medium collected from estradiol-stimulated cells was found to contain a growth-promoting factor(s) whose activity was not antagonized by 4-hydroxytamoxifen. Nonstimulated cells secreted a significant but low level of the growth-promoting factor. Finally, B-1 cells were found to be estrogen dependent for cell proliferation in BALB/c mice. Their growth was markedly inhibited by the administration of tamoxifen to the host mice. These results indicate that the serum-free culture system presented here is suitable for studying the autocrine mechanisms of cell growth regulated by estrogens as well as triphenylethylene compounds.

Topics: Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Culture Media; Epidermal Growth Factor; Estradiol; Estrogen Antagonists; Insulin; Kinetics; Leydig Cell Tumor; Male; Mice; Mice, Inbred BALB C; Receptors, Estrogen; Tamoxifen; Testicular Neoplasms

When T 47D human breast cancer cells were treated with 10 nM of the potent antiestrogen, 4-hydroxyclomiphene, growth rate was reduced to about 50% of control. Simultaneous treatment with epidermal growth factor (EGF) and 4-hydroxyclomiphene led to a partial reversal of the growth inhibitory effect of the antiestrogen. The effect of EGF was concentration-dependent being half-maximal at 0.10 ng/ml (0.02 nM) and maximal at concentrations greater than 0.5 ng/ml (greater than 0.08 nM). Furthermore, EGF partially reversed the growth inhibitory effects of several other antiestrogens including tamoxifen, 4-hydroxytamoxifen, and LY 117018. These results are compatible with the hypothesis that part of the growth inhibitory effects of antiestrogens on breast cancer cell proliferation are mediated by inhibition of autocrine secretion of growth stimulatory peptides acting through the EGF receptor.

Topics: Breast Neoplasms; Cell Division; Cell Line; Clomiphene; Epidermal Growth Factor; Estrogen Antagonists; Humans; Pyrrolidines; Tamoxifen; Thiophenes