Expression of the progestin-induced fatty acid synthetase in benign mastopathies and breast cancer as measured by RNA in situ hybridization.

We have shown previously that fatty acid synthetase (FAS) is specifically induced by progestins in human breast cancer cell lines. To test the potential value of FAS as a clinical marker in breast diseases, we measured FAS expression in frozen sections of 22 benign and 27 malignant mammary tumors using in situ hybridization with the [35S]UTP alpha S-labeled FAS anti-sense mRNA. The hybridized RNA was quantified with an IMSTAR computerized image analyzer. We found FAS RNA in epithelial cells, but no labeling was detected in the connective tissue. In breast cancer, we found no correlation between FAS expression and estrogen receptor and progesterone receptor concentrations or status. However, the level of FAS was significantly (P less than .02) higher in premenopausal than in post-menopausal patients and increased with the grade of tumor differentiation (P less than .005 between the poorly and well-differentiated tumors). In benign mastopathies, high levels of FAS RNA were found in some cysts (mostly with apocrine metaplasia). In lobules, the FAS RNA level increased proportionally to the degree of proliferation determined by histological examination (P less than .015) and correlated with the H4 histone level measured in an adjacent section using in situ hybridization (r = 0.85, P less than .001). In ductal structures, a lower correlation (r = 0.64, P less than .01) was found between FAS and H4 RNA levels. We conclude that FAS RNA is overexpressed in some mammary tumors and may be useful in predicting high-risk mastopathies and less aggressive breast cancers.

Identification and androgen regulation of two proteins released by T47D human breast cancer cells.

Three [35S]methionine-labeled polypeptides released by T47D human breast cancer cells have been identified as corresponding to two proteins previously described in breast gross cystic disease fluid. A Mr 43,000 protein was immunoprecipitated by polyclonal antibodies to the Zn-alpha 2-glycoprotein. A Mr 18,000 and a Mr 13,000 polypeptide were both immunoprecipitated by four monoclonal antibodies directed against four separate epitopes of Mr 15,000 gross cystic disease fluid protein, a major protein that is characteristic of apocrine gland secretions. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, purified Mr 15,000 gross cystic disease fluid protein migrated at the level of the Mr 18,000 protein of T47D cells. The proteins were regulated by androgens and progestins. In addition to a general stimulation of protein secretion, 5 alpha-dihydrotestosterone (DHT) specifically increased, by 2- to 20-fold, the release of the Mr 43,000 and Mr 18,000 proteins into the medium. DHT also increased the cellular level of the Mr 18,000 protein, as shown by immunoprecipitation with a Mr 15,000 gross cystic disease fluid protein antibody, which suggests a stimulation of protein synthesis. The progestin 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione also increased the release in the medium of the Mr 43,000 and Mr 18,000 proteins, but higher molar concentrations were required than in the case of DHT. The induction of these proteins by DHT and 17,21-dimethyl-19nor,4,9-pregnadiene-3,20-dione was specifically inhibited by the antiandrogen flutamide which has no effect on other progestin-regulated proteins. This suggests an effect mediated by the androgen receptor. This is the first report on the identification of two proteins induced by androgens in a human breast cancer cell line. These proteins should be useful in studying the role of androgens in human mammary tumors and their mechanism of action in cell culture.

Anti-growth factor activities of benzothiophenes in human breast cancer cells.

We have tested the effects of two Eli-Lilly compounds, LY 117, 018 and raloxifene, on E2-regulated and IGF-I-induced proliferation or AP-1 activity in human breast cancer cells. We now demonstrate that both molecules have strong antiestrogenic and anti-growth factor inhibitory effects in MCF7 cells. They were as potent as ICI 182, 780 and more efficient than OH-Tam to prevent estradiol action whereas their inhibition on IGF-I stimulation was less than with ICI 182, 780 and equivalent to that of OH-Tam. Moreover, raloxifene was the most efficient molecule to prevent IGF-I-induced AP-1 activity, with a significant effect observed with a concentration as low as 5 x 10(-11)M in the presence of IGF-I alone. Similar dose-response curves were obtained with a combined treatment of IGF-I and E2 with a 2log shift. Their action on IGF-I-induced proliferation was completely abrogated in MCF7 transfectants in which the expression of an antiestrogen-regulated protein tyrosine phosphatase, PTPL1, was abolished by antisense RNA transfection. Accordingly, they were both able to dose-dependently regulate the expression of PTPL1 and to interfere with the PI3-K/Akt pathway by drastically decreasing Akt phosphorylation exclusively in wild-type PTPL1 expressing cells. Our data altogether demonstrate that raloxifene has a potent inhibitory effect on IGF-I action, with a drastic effect on AP-1 triggered responses as well as on Akt phosphorylation, suggesting that it might be a useful therapeutic agent in tumors in which these signalling pathways become constitutively active.

Progestin increases gene transcription and messenger ribonucleic acid stability of fatty acid synthetase in breast cancer cells.

The cDNA clone (Pg8) corresponding to the 3'-end of fatty acid synthetase (FAS) mRNA is one of the few probes to study in cell lines the mechanism by which an endogenous gene is regulated by progestins. The steady-state level of FAS mRNA is known to be increased by the synthetic progestin R5020 in the human breast cancer cell line MCF7. We show here that it is also increased in another progesterone-receptor-positive cell line T47D but not in progesterone-receptor-negative cell lines BT20, MDA-MB231, and HBL100. In R5020-treated MCF7 cells, the FAS mRNA concentration increased with cell density. Protein synthesis inhibitors did not abolish progestin induction of FAS suggesting a primary effect of the hormone. In vitro nuclear run-on transcription assays showed that the FAS gene transcription rate was increased about 4-fold by progestin. This stimulation of transcription was detectable 30 min after addition of R5020 and was maximal after 4 h, but could not totally account for the total increase in the mRNA steady state level. Chase experiments in the presence of Actinomycin D or Cordycepin showed that progestin also increased the half-life of FAS mRNA from 6-9 h to 24-33 h. We conclude that progestin stimulates the FAS concentration both by increasing the transcription rate of the gene and by stabilizing the mRNA.

Unliganded and liganded estrogen receptors protect against cancer invasion via different mechanisms.

While estrogens are mitogenic in breast cancer cells, the presence of estrogen receptor a (ERalpha) clinically indicates a favorable prognosis in breast carcinoma. To improve our understanding of ERalpha action in breast cancer, we used an original in vitro method, which combines transient transfection and Matrigel invasion assays to examine its effects on cell invasiveness. ERalpha expression in MDA-MB-231 breast cancer cells reduced their invasiveness by 3-fold in the absence of hormone and by 7-fold in its presence. Integrity of hormone and DNA-binding domains and activating function 2 were required for estradiol-induced inhibition, suggesting that transcriptional activation of estrogen target genes was involved. In contrast, these domains were dispensable for hormone-independent inhibition. Analysis of deletion mutants of ERalpha indicated that amino acids 179-215, containing the N-terminal zinc finger of the DNA-binding domain, were required for ligand-independent receptor action. Among different members of the nuclear receptor family, only unliganded ERalpha and ERbeta reduced invasion. Calreticulin, a Ca2+-binding protein that could interact with amino acids 206-211 of ERalpha, reversed hormone-independent ERalpha inhibition of invasion. However, since calreticulin alone also inhibited invasion, we propose that this protein probably prevents ERalpha interaction with another unidentified invasion-regulating factor. The inhibitor role of the unliganded ER was also suggested in three ERalpha-positive cell lines, where ERalpha content was inversely correlated with cell migration. We conclude that ERalpha protects against cancer invasion in its unliganded form, probably by protein-protein interactions with the N-terminal zinc finger region, and after hormone binding by activation of specific gene transcription.

FRA-1 expression level modulates regulation of activator protein-1 activity by estradiol in breast cancer cells.

We compared the effect of estradiol on activator protein-1 (AP-1) activity in estrogen receptor positive (ER alpha+) and estrogen receptor negative (ER alpha-) human breast cancer cell lines transiently transfected with the AP-1-responsive reporter plasmid AP-1-TK-CAT and an ER alpha expression vector. While estradiol increased AP-1 activity in the ER alpha+ cell lines MCF7, ZR75.1, and T47D, it decreased (MDA-MB231 and BT20 cells) or had no significant effect (MDA-MB435 cells) on AP-1-mediated transcription in ER alpha- cells. Estradiol also inhibited AP-1 activity in ER alpha-MDA-MB231 cells stably transfected with ER alpha and in which ER alpha levels are close to those found in MCF7. Use of ER alpha mutant expression vectors demonstrated that the DNA-binding domain of ER alpha was needed for stimulation or inhibition of AP-1 activity by estradiol but suggested that ER alpha binding to estrogen-responsive elements was not required for these effects. Changes in regulation paralleled quantitative and qualitative changes in protein binding to AP-1 sites, as demonstrated by gel shift assay: protein binding was greater and DNA/protein complexes migrated faster for ER alpha--than for ER alpha+ cells. In fact, by Northern blot, a high level of Fra-1 mRNA was found in BT20 and MDA-MB231 cells as compared with ER alpha+ cells, and MDA-MB435 cells showed an intermediary level of expression. The differential expression of Fra-1 in MCF7 and MDA-MB231 cells was confirmed at the protein level by supershift experiments. In addition, overexpression of Fra-1 in MCF7 cells decreased the positive effect of estradiol while inhibition of Fra-1 expression in MDA-MB231 cells, by transient transfection of the Fra-1 antisense expression vector, abolished the negative effect of the hormone. In conclusion, we demonstrated that ER alpha- breast cancer cell lines differ from ER+ cells by a high level of AP-1 DNA-binding activity due, at least in part, to high Fra-1 constitutive expression. High Fra-1 concentration is crucial for the negative regulation of AP-1 activity by estradiol and thus may take part in estradiol-induced inhibition of cell proliferation in ER alpha- breast cancer cells transfected with ER alpha expression construct.

Synthetic antiestrogens modulate induction of pS2 and cathepsin-D messenger ribonucleic acid by growth factors and adenosine 3',5'-monophosphate in MCF7 cells.

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.

Effects of progestins and menstrual cycle on fatty acid synthetase and progesterone receptor in human mammary glands.

Fatty acid synthetase (FAS) is induced by progestins in human breast cancer cell lines. To study its regulation in normal mammary glands, the FAS level was estimated by immunohistochemistry, using the biotin-streptavidin method, in ducts and lobules of normal tissues adjacent to nonproliferative benign breast lesions collected by biopsy. Rabbit polyclonal antibodies to human FAS specifically recognized the 250-kDa FAS from MCF7 cells, as shown by Western immunoblotting. An excess of purified FAS totally switched off FAS immunostaining of R5020-treated MCF7 cells, demonstrating the validity of FAS immunocytochemical detection. FAS labeling was quantified using a computer-aided image analyzer (SAMBA 2005) in 18 patients receiving progestin therapy from the 15th to the 25th day of the menstrual cycle and 26 untreated patients. In the 2 groups, FAS staining, absent of fibroblasts, was observed in the cytoplasm of epithelial cells. It was higher in lobules than in ducts and increased significantly from the follicular to the luteal phase in both structures. Progestin treatment increased FAS expression in both structures. Using monoclonal antibodies, progesterone receptor expression was measured in frozen serial sections. In patients receiving progestin treatment, the progesterone receptor level increased from the beginning of the cycle to day 14 and then decreased during the second part of the menstrual cycle, probably down-regulated by progestin, indicating a regulation similar to that in the endometrium. We conclude that FAS is induced by progestins in the ducts and lobules of human normal mammary glands as it is in human breast cancer cells. FAS may, therefore, be useful for studying the effect of progesterone in normal human mammary glands.

Antiestrogenic effect of R5020, a synthetic progestin in human breast cancer cells in culture.

To see whether progestins prevent estrogen action in breast cancer cells, we have studied in vitro the effect of R5020 on the cell growth and the synthesis of secreted proteins in T47D and R27 human breast cancer cells. While R5020 had no effect on cell growth when tested alone, it significantly inhibited the growth of both cell lines in the presence of estradiol (1 nM). The effect was most clear-cut after 10-12 days of treatment and was dose dependent, a half-maximal inhibition occurred with 1 nM R5020. R27, a cloned MCF7 variant resistant to Tamoxifen, remained responsive to R5020, which prevented the effect of 17 beta-estradiol (E2) and inhibited cell growth in the presence of Tamoxifen. This suggests that the two antiestrogens are acting through different mechanisms. Dihydrotestosterone and dexamethasone did not reproduce or inhibit the effect of R5020 on cell growth. R5020 was ineffective in a rat tumor cell line containing androgen and glucocorticoid receptors but lacking progesterone receptors and estrogen receptors. These results suggest that R5020 is probably acting via progesterone receptors rather than via the androgen or glucocorticoid receptors. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have shown that R5020 specifically decreases the production of the 52K protein, a major protein released by R27 cells after E2 stimulation. We conclude that R5020 has an antiestrogenic activity on breast cancer cells in culture, since it prevents the stimulation of cell growth and protein synthesis by E2.

RU486, a progestin and glucocorticoid antagonist, inhibits the growth of breast cancer cells via the progesterone receptor.

The progestin and glucocorticoid antagonist RU486 was tested on the growth of several cell lines in culture. RU486 inhibited the growth of two progesterone receptor (RP) positive human breast cancer cell lines (MCF7 and T47D). The antiproliferative effect was dose dependent and its magnitude correlated with the RP content of the tested cells (T47D greater than estradiol-primed MCF7 greater than withdrawn MCF7). Cell growth inhibition was not prevented by the addition of dexamethasone, dihydrotestosterone, or estradiol, but the cells were rescued by low concentrations of the progestin R5020. RU486 had no effect on the growth of two RP negative human breast cancer cell lines and a rat fibroblast cell line. Moreover, RU486 had no progestin agonist activity in T47D cells when evaluated by measuring the 35S-labeling of two progestin-regulated proteins with mol wts of 48,000 and 250,000, but it totally prevented the induction of these two proteins by R5020. In conclusion, RU486 selectively inhibited the growth of human breast cancer cell lines with unoccupied RP sites and its effect was correlated with the RP concentration of these cells. We propose that RU486 is a RP-targeted drug of potential utility in breast cancer treatment.

Estrogens stimulate cell proliferation and induce secretory proteins in a human breast cancer cell line (T47D).

The effects of estradiol (E2) on two cloned sublines derived from the T47D human breast cancer cell line have been studied in vitro. Cell proliferation was evaluated by DNA assay, cell counts, and thymidine incorporation. The rate of synthesis of proteins released into the cell culture medium was assayed by [35S]methionine incorporation and polyacrylamide gel electrophoresis, followed by fluorography. In clone 11, which contains estrogen and progesterone receptors, estradiol (1 pM-1 nM) stimulated cell proliferation 2- to 5-fold after a lag period of 6 days. Maximal stimulation was observed with 1% or 3% fetal calf serum and without added insulin. The effect of E2 was biphasic, since the growth rate was stimulated for E2 concentrations less than 10 nM and then progressively inhibited for higher concentrations. Dexamethasone, dihydrotestosterone, progesterone, and R5020 (at 1 nM or 1 microM) did not modify cell growth. The antiestrogen Tamoxifen (1 microM) inhibited the E2-induced stimulation and decreased the growth of control cells. Estrogen also stimulated 2- to 3-fold the synthesis of approximately 60K molecular weight dalton proteins which were released into the medium. This effect was seen as early as 1 day of E2 treatment, and a plateau of stimulation was reached with 0.1 nM E2. By contrast, in clone 8, which contains low concentrations of estrogen and progesterone receptors, E2 had no effect on cell growth and stimulated slightly the synthesis of a 55K protein. These results demonstrate that E2 is able to directly stimulate the proliferation of human epithelial breast cancer cells after having stimulated the synthesis of approximately 60,000 dalton proteins released into the medium.

Regulation of fatty acid synthetase ribonucleic acid in the human endometrium during the menstrual cycle.

Fatty acid synthetase (FAS) is induced by progesterone in MCF7 and T47D breast cancer cell lines. We studied a possible in vivo regulation of expression of this gene by looking for FAS RNA in human endometrial biopsies at various periods of the menstrual cycle, using a cloned cDNA FAS probe. By Northern blot analysis, we detected the 8-kilobase FAS RNA throughout the cycle in 7 uterine samples. RNA in situ hybridization analysis of frozen sections from 22 endometrial biopsies showed that FAS RNA was present during follicular and luteal phases of the menstrual cycle in stromal and epithelial cells. RNA levels were quantified by counting autoradiographic silver grains using a computer-aided image analyzer. FAS RNA levels were significantly higher in epithelial cells than in fibroblasts (P less than 2 x 10(-5]. Furthermore, in both cell types, mean FAS RNA concentrations were higher in biopsies removed during the luteal phase than the follicular phase of the menstrual cycle (P = 2 x 10(-3) and 9 x 10(-5), respectively). A 2- to 3-fold increase in FAS RNA levels between days 8-14 and days 22-24 was detected in 2 normal patients who had previously undergone 2 successive biopsies. This increase was not observed in 2 patients with low plasma estradiol and progesterone concentrations, indicating a probable dysovulation. We conclude that FAS normally increases in both stromal and epithelial endometrial cells during the luteal phase. This increase is probably due to progesterone, which implies that FAS is induced in normal endometrium, as demonstrated in breast cancer.

Progestin-specific markers in human cell lines: biological and pharmacological applications.

We review the progestin-specific responses (induced proteins, increased enzymatic activity) described in uterus, mammary tumours and human breast cancer cell lines established from pleural effusions. Recent data from our laboratory using the T47D breast cancer cell line are then given. They include: (a) a general methodology for evaluating the specific effects of steroids on the production of [35S]methionine-labelled proteins released into the culture medium; (b) results concerning the specificity of regulation by the progestins of a 48 000 dalton protein secreted by T47D cells; (c) the evidence for androgen-specific proteins in the same cells; (d) A discussion of the criteria required to define the receptor responsible for a particular effect of steroids. Lastly, we consider the general interest of progestin-regulated proteins in cell culture for pharmacology and biology.

Progestin-induced fatty acid synthetase in human mammary tumors: from molecular to clinical studies.

Fatty acid synthetase (FAS) is one of the first well-characterized progestin-induced proteins with available antibodies and cDNA. This paper reviews basic studies on FAS regulation in human breast cancer cell lines and recent data on the possible clinical significance of this new marker of hormone responsiveness in mammary cancer and benign breast diseases.

Retinoid regulation of human cathepsin D gene expression.

Retinoic acid (RA) regulation of human cathepsin D (cath D) gene expression was investigated in this study. RA enhanced cath D mRNA levels in a concentration-dependent manner in MCF-7 human breast carcinoma cells. RA regulation of cath D mRNA levels was predominantly transcriptional because RA also increased cath D gene core promoter activity. Upon further characterization of the core promoter we localized RA responsive region to proximal 112-bp. The proximal 112-bp region of cath D gene promoter harbours several retinoid response element (RARE)-like sequences. In gel shift experiments the sequence between -100 and -74 nucleotides in the CD112 region carrying imperfect direct repeat and a palindrome competed with RARE for binding to RAR/RXRs. These sequences, however, exhibited binding to protein complexes which could not be competed with unlabeled RARE or up-shifted with RAR/RXR-specific antibodies. We conclude that RA predominantly regulates cath D gene expression from the proximal 112-bp of the promoter region, but this regulation appears indirect.

Estrogen regulated proteases and antiproteases in ovarian and breast cancer cells.

Cathepsin D (cath-D), an estrogen-regulated protease appears mostly to increase the number of tumor cells rather than their invasion or motility through the extracellular matrix. Estradiol is mitogenic but in vitro it also inhibits invasion and motility. In this review, we discuss the mechanism of this inhibition and the hormonal regulation of other proteases and protease inhibitors possibly involved in the control of tumor cell invasion by estrogens.

The PKCθ pathway participates in the aberrant accumulation of Fra-1 protein in invasive ER-negative breast cancer cells.

Fra-1 is aberrantly expressed in a large number of cancer cells and tissues, and emerging evidence suggests an important role for this Fos family protein in both oncogenesis and the progression or maintenance of many tumour types. Here, we show that the concentration of Fra-1 is high in invasive oestrogen receptor (ER)-negative (ER-) breast cancer cell lines, regardless of their Ras pathway status. All of the ER- cells express high levels of activated PKCθ, and the inhibition of PKCθ activity using RNA interference or the expression of a dominant-negative mutant results in a dramatic reduction in Fra-1 abundance. Conversely, the ectopic expression of constitutively active PKCθ leads to Fra-1 phosphorylation and accumulation in poorly invasive ER+ cells. This accumulation is due to the stabilisation of the Fra-1 protein through PKCθ signalling, whereas other members of the PKC family are ineffective. Both Ste20-related proline-alanine-rich kinase (SPAK) and ERK1/2, whose activities are upregulated by PKCθ, participate in PKCθ-driven Fra-1 stabilisation. Interestingly, their relative contributions appear to be different depending on the cell line studied. ERK1/2 signalling has a major role in ER- MDA-MB-231 cells, whereas Fra-1 accumulation occurs mainly through SPAK signalling in ER- BT549 cells. Fra-1 mutational analysis shows that the phosphorylation of S265, T223 and T230 is critical for PKCθ-driven Fra-1 stabilisation. Phosphorylation of the protein was confirmed using specific antisera against Fra-1 phosphorylated on T223 or S265. In addition, Fra-1 participates in PKCθ-induced cell invasion and is necessary for PKCθ-induced cell migration. In summary, we identified PKCθ signalling as an important regulator of Fra-1 accumulation in ER- breast cancer cells. Moreover, our results suggest that PKCθ could participate in progression of some breast cancers and could be a new therapeutic target.

Differential effect of forms A and B of human progesterone receptor on estradiol-dependent transcription.

In addition to stimulation of the target gene fatty-acid synthetase, the synthetic progestin R5020 strongly inhibited estradiol-induced pS2 and cathepsin D mRNA levels in MCF7 human breast cancer cells as shown by Northern blot analysis. Inhibition was half-maximal with 30 pM R5020, and the antiprogestin RU486 had only a weak effect. Two human progesterone receptor isoforms have been described; isoform A is a truncated form of isoform B and lacks the 164 N-terminal amino acids. We hypothesized that the two isoforms could have a differential capacity to transrepress estrogen-induced responses. Therefore, in MDA-MB231 cells containing no progesterone and estrogen receptors, we transiently transfected progesterone receptor expression vectors coding for form B (hPR1 or hPR0) or form A (hPR2) along with the estrogen receptor expression vector HEO. We show that R5020 inhibited estradiol-induced transcription of the pS2-CAT reporter plasmid only in cells selectively expressing isoform B. The same results were obtained when progesterone receptor isoforms were overexpressed in MCF7, Ishikawa, HeLa, or NIH-3T3 cells. Transrepression was dependent on the promoter context since the extent of inhibition by isoform B was higher when evaluated with pS2 or cathepsin D nonpalindromic estrogen-responsive element-mediated transcription than with the perfect palindromic form of the vitellogenin gene. Isoform A was inefficient regardless of the reporter construct used. Inhibition varied with the isoform ratio, and isoform B had a dominant effect, with > 70% inhibition measured in cells transfected with the same amount of both progesterone receptor isoforms. Progestin repressed only one of the two transcription activation functions of the estrogen receptor, AF-2, which corresponds to the hormone-binding domain. We conclude that differential expression of progesterone receptor isoforms could be responsible for a tissue-specific inhibition of estrogen target genes by progestins.

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D.

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.