Progestin effects on long-term growth, death, and Bcl-xL in breast cancer cells

The effect of progestins on proliferation of breast cancer has been a controversial area. We have consistently reported progestin stimulation of proliferation in the progesterone receptor-rich human breast cancer cell line T47D. Other authors, under other conditions, have agreed that progestins stimulate, but for just one turn of the cell cycle. To our knowledge, this is the first in vitro report to show progestin stimulation of human breast cancer cell growth for multiple, probably unlimited, cell cycles, while control cells are dying. This is accompanied by progestin up-regulation of the antiapoptotic protein bcl-xL, no effect on the proapoptotic protein bax, and, surprisingly, diminution of the antiapoptotic protein bcl-2. Thus, progestins both serve as survival factors and stimulate proliferation, implying a possible similar role in breast cancer patients. The data support the notion that many patients may benefit more from combined antiprogestin-antiestrogen therapy than from antiestrogen alone, and suggest bcl-x(L) as a possible target for breast cancer therapy.     

Ligand-modulated regulation of progesterone receptor messenger ribonucleic acid and protein in human breast cancer cell lines

We have examined the effects of estrogen and progestin agonist and antagonist ligands on regulation of progesterone receptor (PR) protein and mRNA levels in a variety of human breast cancer cell lines. By Northern blot analysis, using human PR cDNA probes, PR mRNA in T47D and MCF-7 cells appears as five species of approximately 11.4, 5.8, 5.3, 3.5, and 2.8 kilobases. PR mRNA species are not detected in the PR protein-negative breast cancer cell lines MDA-MB-231 and LY2. T47D cells contain high levels of PR mRNA and protein (detected by hormone binding assay or Western blot analysis), and the PR protein and mRNA content of T47D cells are reduced to about 10% of the control level within 48 h of treatment with 10 nM promegestone; 17, 21-dimethyl-19-nor-pregna-4,9-diene-3, 20-dione (R5020) or 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione (ORG2058), both potent progestins. In contrast, treatment of T47D cells with the antiprogestin 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]-17 alpha-(1-propynyl)-estra- 4, 9-dien-3-one) (RU38486) reduces PR protein and mRNA levels only transiently. PR protein and mRNA are virtually undetectable in control MCF-7 cells grown in the absence of estrogens. When estradiol is administered to MCF-7 cells, the PR mRNA and protein levels increase gradually and proportionately (10- or 40-fold, respectively, in 3 days).(ABSTRACT TRUNCATED AT 250 WORDS)     

Progestin stimulation of thymidine kinase in the human breast cancer cell line T47D

Our laboratory has previously reported that progestins stimulate growth of the human breast cancer cell line T47D. In an attempt to probe further into this stimulation, we are investigating progestin effects on thymidine kinase (EC 2.7.1.21), an enzyme known to be involved in growth regulation. This report relates our finding that progestins stimulate thymidine kinase activity, at physiological progestin concentrations, in a dose-responsive manner. Estradiol-17 beta also stimulates, but testosterone, hydrocortisone and aldosterone do not. The antiprogestin RU486 inhibits progestin stimulation, but also stimulates on its own. Maximal by 24 h, the progestin stimulation then falls off with time. Experiments with actinomycin D and cycloheximide suggest that the thymidine kinase stimulation depends on new RNA and protein synthesis. These data shed further light on progestin stimulation of the growth of human breast cancer. To our knowledge, this is the first report of progestin stimulation of thymidine kinase in human breast cancer cells.     

Progestin stimulation of thymidine kinase in the human breast cancer cell line T74D

Our laboratory has previously reported that progestins stimulate growth of the human breast cancer cell line T47D. In an attempt to probe further into this stimulation, we are investigating progestin effects of thymidine kinase (EC 2.7.1.21), an enzyme known to be involved in growth regulation. This report relates our finding that progestins stimulate thymidine kinase activity, at physiological progestin concentrations, in a dose-responsive manner. Estradiol-17β also stimulates, but testosterone, hydrocortisone and aldosterone do not. The antiprogestin RU486 inhibits progestin stimulation, but also stimulates on its own. Maximal by 24 h, the progestin stimulation then falls off with time. Experiments with actinomycin D and cycloheximide suggest that the thymidine kinase stimulation depends on new RNA and protein synthesis. These data shed further light on progestin stimulation of the growth of human breast cancer. To our knowledge, this is the first report of progestin stimulation of thymidine kinase in human breast cancer cells.     

Prolonged progestin treatment induces the promoter of CDK inhibitor p21 Cip1,Waf1 through activation of p53 in human breast and endometrial tumor cells

Progestins are frequently used in the treatment of advanced breast and endometrial cancer. The human breast carcinoma cell line T47D shows a biphasic response to progestins. Short-term progestin treatment leads to enhanced DNA synthesis, while this line is growth inhibited upon prolonged exposure. An important protein involved in growth regulation by progestins in this cell is the CDK inhibitor p21(Cip1,Waf1). We show that after 1 day of progestin treatment in T47D cells, the p21 promoter-proximal region containing Sp1 binding sites is crucial in the induction by progestins. However, after 3 days the activity of the promoter-distal region becomes predominant in T47D cells or the endometrial carcinoma cell line ECC1. This is dependent upon two domains within this region that contain p53 response elements. In ECC1 and T47D cells 3-day progestin treatment induces a reporter containing a p53 response element, but not a mutated version. This induction is due to activation of p53 by progestin, which may be caused by nuclear translocation of p53. These data indicate that upon prolonged exposure, progestins activate p53, in human breast and endometrial tumor cells, which up-regulates the p21(Cip1,Waf1) promoter. This may be an important mechanism involved in progestin-inhibited cellular proliferation in these cells.     

Progestins induce down-regulation of insulin-like growth factor-I (IGF-I) receptors in human breast cancer cells: potential autocrine role of IGF-II.

Insulin-like growth factor-I (IGF-I) receptors are present in breast cancer cells and may play a role in breast cancer cell growth. We have studied the effect of progestins on IGF-I receptors in T47D human breast cancer cells. T47D cells constitutively express high levels of progesterone receptors and are a model for studying the regulation of cellular functions by progestins. Treatment of T47D cells with either progesterone or the synthetic progestin promegestone (R5020) decreased IGF-I receptor content by approximately 50%, as measured by Scatchard analysis and receptor biosynthesis studies. In contrast to progestins, estradiol, dexamethasone, and dihydrotestosterone did not influence IGF-I receptor content. No effect of R5020 was seen after 12 h of incubation, a near-maximal effect was seen after 24 h, and greatest effects were seen after 72 h. R5020 decreased IGF-I receptor mRNA abundance, indicating that progestins acted at the level of gene expression. However, progestins also increased the secretion of IGF-II, a ligand for the IGF-I receptor. In contrast to IGF-II, T47D cells did not express IGF-I. The addition of exogenous IGF-II to T47D cells down-regulated both IGF-I receptor binding and IGF-I receptor mRNA abundance. This study indicates, therefore, that progestins regulate IGF-I receptors in breast cancer cells and suggests that this regulation occurs via an autocrine pathway involving enhanced IGF-II secretion.     

Progestins and androgens stimulate lipid accumulation in T47D breast cancer cells via their own receptors

Using electron microscopy, in the human breast cancer cell line T47D, the synthetic progestin R5020, and 5 alpha-dihydrotestosterone were shown to increase significantly the number of lipid droplets per cell section compared to control cells or estradiol- and dexamethasone-treated cells. Lipid accumulation, as measured by Oil Red O dying and by [2-14C]acetate incorporation, was observed at concentrations as low as 10 pM R5020 and 1 nM 5 alpha-dihydrotestosterone, and was always more abundant after progestin treatment. The progestin antagonist RU486 inhibited, in a dose-dependent manner, lipid accumulation initiated by the two hormones, whereas the androgen antagonist flutamide inhibited only the effect initiated by 5 alpha-dihydrotestosterone. Cytoplasmic lipid droplets accumulation was not observed in the BT20 breast cancer cell line, which contains neither progesterone nor androgen receptors. These results indicate that progestins and androgens increase lipid accumulation by interacting with their own receptor. Chromatographic analysis of [2-14C]acetate labeled lipids showed that R5020 and 5 alpha-dihydrotestosterone enhanced the accumulation of cellular triglycerides at least in part by increasing their synthesis and decreased the quantity of lipids released into the medium. To conclude, we have shown that progestins and androgens, via their own receptor, can induce the same triglyceride accumulation in T47D cells. This effect follows fatty acid synthetase induction and precedes cell growth inhibition, two responses also triggered by progestin and androgen in these cells.     

Progestin stimulation of manganese superoxide dismutase and invasive properties in T47D human breast cancer cells

Superoxide dismutase (SOD) occurs in two intracellular forms in mammals, copper–zinc SOD (CuZnSOD), found in the cytoplasm, mitochondria and nucleus, and manganese superoxide dismutase (MnSOD), in mitochondria. Changes in MnSOD expression (as compared to normal cells) have been reported in several forms of cancer, and these changes have been associated with regulation of cell proliferation, cell death, and metastasis. We have found that progestins stimulate MnSOD in T47D human breast cancer cells in a time and physiological concentration-dependent manner, exhibiting specificity for progestins and inhibition by the antiprogestin RU486. Progestin stimulation occurs at the level of mRNA, protein, and enzyme activity. Cycloheximide inhibits stimulation at the mRNA level, suggesting that progestin induction of MnSOD mRNA depends on synthesis of protein. Experiments with the MEK inhibitor UO126 suggest involvement of the MAP kinase signal transduction pathway. Finally, MnSOD-directed siRNA lowers progestin-stimulated MnSOD and inhibits progestin stimulation of migration and invasion, suggesting that up-regulation of MnSOD may be involved in the mechanism of progestin stimulation of invasive properties. To our knowledge, this is the first characterization of progestin stimulation of MnSOD in human breast cancer cells.     

Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor alpha, epidermal growth factor receptor, c-fos, and c-myc genes.

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.     

Heregulin induces transcriptional activation of the progesterone receptor by a mechanism that requires functional ErbB-2 and mitogen-activated protein kinase activation in breast cancer cells

The present study addresses the capacity of heregulin (HRG), a ligand of type I receptor tyrosine kinases, to transactivate the progesterone receptor (PR). For this purpose, we studied, on the one hand, an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female BALB/c mice and, on the other hand, the human breast cancer cell line T47D. HRG was able to exquisitely regulate biochemical attributes of PR in a way that mimicked PR activation by progestins. Thus, HRG treatment of primary cultures of epithelial cells of the progestin-dependent C4HD murine mammary tumor line and of T47D cells induced a decrease of protein levels of PRA and -B isoforms and the downregulation of progesterone-binding sites. HRG also promoted a significant increase in the percentage of PR localized in the nucleus in both cell types. DNA mobility shift assay revealed that HRG was able to induce PR binding to a progesterone response element (PRE) in C4HD and T47D cells. Transient transfections of C4HD and T47D cells with a plasmid containing a PRE upstream of a chloramphenicol acetyltransferase (CAT) gene demonstrated that HRG promoted a significant increase in CAT activity. In order to assess the molecular mechanisms underlying PR transactivation by HRG, we blocked ErbB-2 expression in C4HD and T47D cells by using antisense oligodeoxynucleotides to ErbB-2 mRNA, which resulted in the abolishment of HRG's capacity to induce PR binding to a PRE, as well as CAT activity in the transient-transfection assays. Although the inhibition of HRG binding to ErbB-3 by an anti-ErbB-3 monoclonal antibody suppressed HRG-induced PR activation, the abolishment of HRG binding to ErbB-4 had no effect on HRG activation of PR. To investigate the role of mitogen-activated protein kinases (MAPKs), we used the selective MEK1/MAPK inhibitor PD98059. Blockage of MAPK activation resulted in complete abrogation of HRG's capacity to induce PR binding to a PRE, as well as CAT activity. Finally, we demonstrate here for the first time that HRG-activated MAPK can phosphorylate both human and mouse PR in vitro.     

Progesterone receptor regulation by retinoic acid in the human breast cancer cell line T-47D

Data are presented which document the first known effect of retinoic acid on progesterone receptor (PR) gene expression. Treatment of T-47D human breast cancer cells with retinoic acid for 48 h resulted in a marked concentration-dependent decrease in the level of PR mRNA and immunoreactive protein which was similar to the known effect of progestins on these parameters. Retinoic acid, however, did not bind to PR, nor did it cause the previously demonstrated increase in PR molecular weight observed after progestin exposure. When T-47D cells were treated with retinoic acid for 6 h rather than 48 h, no reduction in the level of PR protein was noted at any retinoic acid concentration whereas the effects of retinoic acid on PR mRNA at 6 and 48 h were the same. Examination of the time course of the effects of retinoic acid revealed a rapid decrease in PR mRNA levels detectable 1 h after and maximal 6 h after treatment of T-47D cells with retinoic acid. These effects of retinoic acid contrasted with previously demonstrated progestin effects on PR mRNA which were not apparent until 3 h after and were not maximal until 12 h after treatment. As expected, the PR protein concentration was unaffected for at least 6 h but was maximally decreased 24-48 h after retinoic acid treatment. In summary, retinoic acid treatment of T-47D cells caused a decrease in the cellular PR concentration by decreasing levels of receptor mRNA and protein, suggesting that retinoic acid is capable of modulating sensitivity to progestins in human breast cancer cells.     

Progestin stimulation of lactate dehydrogenase in the human breast cancer cell line T-47D

We have previously reported that physiological levels of progestins alone stimulate lactate dehydrogenase in a dose-responsive manner in the progesterone-receptor-rich human breast cancer cell line T-47D. Using isozyme electrophoresis, we have not found that lactate dehydrogenase isozyme 5 is the only isozyme detectable in these cells, as has been reported for other human breast cancer cells in long-term tissue culture. Upon treatment with progestins, isozyme 5 remains the only isozyme detectable. T-47D cells were plated in charcoal-stripped serum-containing medium and grown for 2 days before treatment with progestin. Lactate dehydrogenase stimulation then plateaued after around 2-3 days of treatment with progestin and was maintained until around day 5, following which a decline in enzyme activity occurred. The effect is specific for progestins, and inhibited by the anti-progestin RU-38486 (17 beta-hydroxy-11 beta-(4-dimethyl-aminophenyl-1)-17 alpha-(prop-1-ynil)-estra-4,9-dien-3-one). Experiments using actinomycin D and cycloheximide suggests that the effect is dependent on RNA and protein synthesis, respectively. Lactate dehydrogenase stimulation occurs regardless of the presence of the estrogenic pH indicator Phenol red, and of whether it was analyzed per mg DNA or per mg protein.     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)