Estrogen-induced proteins in human breast cancer cells.
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Breast Neoplasms↗
Biomedical subjects
F Vignon
Publications and source records attributed to F Vignon.
Steroidal and nonsteroidal antiestrogens in breast cancer cells in culture.
The mode of action of two types of antiestrogens, tamoxifen and progestins, has been studied in the estrogen responsive cell lines, MCF7 and T47D, established from metastatic human breast cancer. (1) Non steroidal antiestrogens: We present evidence indicating that tamoxifen inhibits the growth of breast cancer cells via an interaction with the estrogen receptor (RE), which leads to a partial activation of the receptor and a dissociated effects on gene expression. At concentrations of less than 4 microM, effects of non steroidal antiestrogens are only observed when RE sites are available. At concentrations greater than 4 microM, an additional (cytotoxic?) effect of tamoxifen is observed which is not mediated by the RE. (2) Progestins: Direct antiestrogenic effect of progestins (R5020, progesterone) on breast cancer cells have been demonstrated. Three series of responses to R5020 are obtained: (a) A decreased cell proliferation (antiestrogenic and progestin specific effect). (b) A decreased production of total proteins in the culture medium (antiestrogenic effect). (c) The increased production of a 48,000 dalton protein which is released into the medium after treatment with several progestins (progesterone, medroxyprogesterone acetate, R5020) but not other steroids (specific progestin effect). These responses appear to be mediated by the progesterone receptor and are not observed in RP negative cell line (BT20). Even though these two types of antiestrogens inhibit cell proliferation via different receptors, a common final mechanism (decreased production of estrogen induced growth factors or increased production of antiestrogen induced inhibitory factor) is not excluded.
Breast Neoplasms↗
The antiproliferative effect of tamoxifen in breast cancer cells: mediation by the estrogen receptor.
The effects of tamoxifen (Tam) and its 4-hydroxylated metabolite (OH-Tam) on the growth of two human breast cancer cell lines ( MCF7 and BT20 ) were evaluated by fluorometric DNA assay. The effects of the antiestrogens were dependent upon their concentrations and the nature of the cells. At concentrations below 4 microM, the degree of inhibition was related to their relative affinities for the estrogen receptor and was totally reversed by estradiol in MCF7 cells. No inhibition was observed in the estrogen receptor negative cell line BT20 . This supports and extends the idea that the antiproliferative effect of Tam at these concentrations is mediated by the estrogen receptor even in the absence of measurable estradiol concentration. At concentrations greater than 4 microM, Tam was cytotoxic on MCF7 and BT20 mammary cell lines within 2 days of treatment. The cytotoxic effect was irreversible and was not prevented by occupation of the estrogen receptor with estradiol, suggesting that it was not mediated by the estrogen receptor. The cytotoxicity of the triphenylethylene drugs, however, has some specificity since it was not observed in a fibroblast rat cell line ( 49F ) or in the two mammary cell lines with similar high concentrations of estradiol and diethylstilbestrol.
Breast Neoplasms↗
Effect of estradiol on nonmalignant human mammary cells in primary culture.
We have studied effects of estradiol on primary cultures of nonmalignant human mammary tissue collected surgically from fibroadenomas or during reduction mammoplasties. After enzymatic digestion, "organoids" made of epithelial cells organized in ductal or alveolar structure were grown in primary cultures (up to 12 days) on different substrata (glass, plastic, collagen-coated plastic, and floating collagen membranes). Transmission and scanning electron microscopy showed that these organoids were responsive to physiological concentrations of estradiol. Condensed chromatin of epithelial cells became dispersed following estrogen treatment. The plasma membrane of epithelial cells at the surface of the organoids was dramatically modified by estradiol, which increased the number and the length of the microvilli, as observed previously in the MCF7 breast cancer cell line (Vic et al., Cancer Res., 42: 667-673, 1982). This effect was not observed with the same concentrations of progesterone, dexamethasone, dihydrotestosterone, or 1 microM tamoxifen or in fibroblasts of the same tissue, demonstrating that epithelial mammary cells are specifically responsive to estradiol. By contrast, no effect of estradiol could be evidenced on the [35S]methionine-labeled proteins released into the medium by the organoids. The estrogen-regulated protein of Mr 52,000 was not found in the medium after purification by concanavalin A-sepharose or immunoprecipitation with specific antibodies to the Mr 52,000 protein from MCF7 cells. We conclude that nonmalignant mammary cells are responsive to estrogens in primary culture.
Adenofibroma↗
Effect of estrogen in breast cancer cells in culture: released proteins and control of cell proliferation.
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Breast Neoplasms↗
Induction of two estrogen-responsive proteins by antiestrogens in R27, a tamoxifen-resistant clone of MCF7 cells.
In an attempt to approach the mechanism by which estrogen and antiestrogen regulate the growth of estrogen receptor-positive breast cancer, we have studied the effects of the antiestrogens tamoxifen (TAM) and 4-hydroxytamoxifen (OH-TAM) on the induction of two estrogen-specific proteins in a variant of MCF7 cells termed R27, cloned for its ability to grow in the presence of TAM. We found that, in the R27 variant, antiestrogens as well as estradiol were able to increase specifically the production of a Mr 52,000 protein which was released into the culture medium. This protein was shown to be identical to the Mr 52,000 glyco-protein induced by estrogen and released by MCF7 cells on the basis of its specific inducibility by physiological concentrations of 17 beta-estradiol and of its resolution in two-dimensional gel analysis. Dose-response analysis showed that, in the R27 variant, TAM and OH-TAM acquired the ability to induce the Mr 52,000 protein at concentrations compatible with their relative affinities for the estrogen receptor, while these antiestrogens were inefficient in the wild MCF7 cells. Whereas the relative increase of the Mr 52,000 protein was similar with TAM, OH-TAM, and 17 beta-estradiol, the general production of Mr 52,000 and of total labeled proteins was less with the antiestrogens than with 17 beta-estradiol. Moreover, OH-TAM displayed a biphasic dose-response curve with inhibitory effects at concentrations above 10 nM, suggesting an additional mechanism. Neither TAM nor OH-TAM had any antiestrogenic effect when added in the presence of 17 beta-estradiol. In the R27 variant, both estradiol and antiestrogens induced the progesterone receptor sites; however, the extent of the stimulation was lower with antiestrogens than with estradiol. This study shows that, in addition to the classical antiestrogen-resistant breast cancer cells, in which the estrogen receptor is absent or inactive, there is another class of antiestrogen-resistant cells in which the drug becomes a full estrogen agonist as evidenced by the induction of the Mr 52,000 estrogen-specific protein, which is not induced in the wild-type cells.
Breast Neoplasms↗
[Acquired remissions of insulin dependence in recent diabetes mellitus. Essential prognostic factors].
24 patients with recent diabetes mellitus (less than three years) were given an intravenous insulin infusion over a short period (84 to 252 hours) in an attempt to achieve a remission of their disease through rigorous normalization of blood glucose concentrations. After this treatment, strict control of diabetes mellitus was achieved with oral mediactions in 15 of the 24 patients. In 17 cases, onset of diabetes mellitus had been sudden and insulin was required immediately (acute cetosic diabetes: ACD); in 7 patients, signs of insulin deficiency had occurred only after seven months of hyperglycemia (secondary insulin-dependent diabetes mellitus: SIDDM). A strong probability of achieving an insulin-induced remission exists only during the first six months of ACD. The success of the insulin infusion is partly dependent upon the residual beta cell function. During the remission, which lasts for more than 12 months in 50% of cases, control of diabetes mellitus is optimal.
Diabetes Mellitus↗
[Estrogen-induced proteins secreted by MCF7 human breast cancer cells stimulate their proliferation].
In the MCF7 human breast cancer cells, estrogens stimulate in vitro cell proliferation and biosynthesis of specific proteins which are released into the culture medium; the most abundant of these is a 52,000 dalton glycoprotein. Conditioned media from E2-treated MCF7 cells stimulate the growth of E2-deprived MCF7 cells, whereas conditioned media from untreated control cells are ineffective. At protein concentration greater than 5 micrograms/ml, an inhibitory effect was also obtained with control media. The mitogenic factors of these conditioned media are non-dialysable, sensitive to temperature and proteases and adsorbed on Con A Sepharose. The cell surface of MCF7 cells, examined by scanning electron microscopy, is differentially affected by these two types of conditioned media. We conclude that estrogens may stimulate the growth of breast cancer cells indirectly via the induction of glycoproteins acting as growth factors.
Breast Neoplasms↗
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.
Breast Neoplasms↗
[Proteins induced by estrogens. Search for new markers of hormone dependence in breast cancers].
The hormone dependence and the prognosis of human breast cancers are now based on the assays of the oestrogen receptor (RE) and progesterone receptor (PR) in the tumor. However, such prediction is not fully accurate since RE may be non functional in some tumors and the estrogenic regulation in PR is not directly correlated with the effects of estrogens on cell growth. Here, we review other estrogen induced proteins. A special emphasis is made on a 52,000 dalton glycoprotein released into the medium by human breast cancer cells. Studies on the regulation and biological role of this protein should improve our understanding of the mechanisms by which estrogens stimulate the growth and spreading of breast cancers. Such secreted proteins could later provide convenient circulatory markers of hormone dependency.
Breast Neoplasms↗
Increased DNA binding of the estrogen receptor in an estrogen-resistant mammary cancer.
In the C3H mouse mammary adenocarcinoma, estradiol cannot induce the progesterone receptor, and the tumor growth rate is not decreased by ovariectomy. To find an explanation for this estrogen resistance, we have compared the estrogen receptor (ER) from this tumor to the ER of uterus and of the mammary tumors induced in rats by dimethylbenz(a)anthracene. Since the ER concentration of the C3H tumor is low (congruent to 20 fmol/mg protein), we have used iodoestradiol of high specific activity to label the receptor. Several criteria of ER activation were studied. The dissociation rates of estradiol with or without sodium molybdate were similar in all tissues. In metrizamide isopycnic gradients, ER from rat uterus and C3H tumor had a similar density, both in the presence or absence of DNA. The binding of ER to DNA-cellulose was analyzed by incubating to equilibrium a constant amount of ER with a variable amount of DNA, the cellulose concentration being kept constant. The saturation data were plotted according to the method of Scatchard. The apparent affinity for DNA of the cytosol ER was similar for the rat dimethylbenz(a)anthracene tumors and the uterus (Kd congruent to 10 microM) but was significantly higher for the C3H tumor ER (Kd congruent to 2.3 microM). Neither the substitution of estradiol by iodoestradiol, nor the difference in cytosol protein and ER concentrations, nor the nonspecific steroid binding to DNA-cellulose could explain this result. This difference was confirmed when using DNA-agarose or soluble DNA in sucrose gradients. Finally, the salt concentrations necessary to elute ER from DNA-cellulose columns were 0.20 and 0.28 M for uterine and C3H tumor ER, respectively. To conclude, the C3H tumor has a low content of ER which appears to have a higher affinity for DNA than the ER of estrogen-responsive tissue. We suggest that the reason for the inefficiency of ER in the C3H tumor may be related to its increased affinity for nonspecific DNA sites.
9,10-Dimethyl-1,2-benzanthracene↗
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.
Breast Neoplasms↗
Effect of estradiol on the ultrastructure of the MCF7 human breast cancer cells in culture.
We have analyzed the effect of estradiol and of two classes of antiestrogens on the morphology of the MCF7 human breast cancer cell line by scanning and transmission electron microscopy. Estradiol progressively increased the number and the length of microvilli at the cell surface. The density of the microvilli network increased between 2 and 11 days of estrogen treatment, while the cells became more granular and less tightly attached to the surface of the dish. Estradiol also progressively transformed cells into secretory cells containing, at Day 2, large, clear mitochondria and, at Day 4, rough endoplasmic reticulum and Golgi complex. At Day 6, secretory granules (diameter, 0.2 microM), which mainly contained glycoproteins, were first developed in the cytoplasm. By Day 8, they were concentrated at the cell membrane and being liberated into the medium. Larger granules (diameter, 0.8 microM), which probably contained lipids, were obtained later (Day 11). Cell cultures in 10% fetal calf serum not treated by charcoal contained secretory granules. The modifications were induced by physiological concentrations of estradiol but not 5 alpha-dihydrotestosterone. Progesterone (10 nM for 8 days) completely inhibited the effect of estradiol on the microvilli and secretory activity. Tamoxifen or hydroxy-tamoxifen did not induce secretory activity but did alter the cell morphology compared to control cells. The effects of estradiol were observed in other estrogen receptor-positive breast cancer cell lines (ZR 75-1, T 47 D) but not in an estrogen receptor-negative cell line (BT 20). This morphological evidence that estrogens modify the cell surface of breast cancer cells in culture and transform them into "secretory cells" complements evidence that a molecular weight of approximately 50,000 into the culture medium (Cell, 24: 352-362, 1980). (The molecular weight was found first to be 46,000. It seems to be closer to 52,000 in a 10% polyacrylamide gel and by using the NEN-labeled proteins as molecular weight markers.
Breast Neoplasms↗
Growth regulation of two rat adenocarcinoma cell lines by dexamethasone and progesterone.
We have studied the effect of steroids on cell proliferation in two continuous cell lines derived from rat mammary tumors induced by 7, 12-dimethyl-benz (a) anthracene (DMBA) and N-nitrosomethylurea (NMU). These cell lines contain high concentrations of glucocorticoid and androgen receptors but no estrogen and progesterone receptors as previously shown (1). The cell proliferation was evaluated by measuring [3H] thymidine incorporation into DNA, cell number, and DNA content. Dexamethasone was found to markedly stimulate cell proliferation in a dose-dependent manner, suggesting that it was acting via the glucocorticoid receptor. The effect of 5 alpha-dihydrotestosterone (DHT) was weaker since a stimulation of [3H] thymidine incorporation was contrasted by the absence of a constant increase of cell proliferation. Progesterone partially stimulated NMU cell growth and totally inhibited the stimulatory effect of dexamethasone in both cell lines. The synthetic progestin R5020 displayed a similar activity to that of progesterone. These results show that progestins can directly modulate the growth of mammary cancer cells even in the absence of progesterone receptor by interacting on the glucocorticoid receptor. We conclude that progestins act mostly as partial agonist-antagonists of glucocorticoids in these two rat mammary adenocarcinoma cell lines.
Adenocarcinoma↗
Nuclear translocation and interactions of the estrogen receptor in uterus and mammary tumors.
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Animals↗
Effects of plasma estrogen sulfates in mammary cancer cells.
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Animals↗
Hormonal regulation in two rat mammary cancer cell lines: glucocorticoid and androgen receptors.
We have studied the hormonal control of two continuous cell lines derived from rat-mammary tumors induced by two different carcinogens (dimethylbenzanthracene and N-nitrosomethylurea). The steroid receptors were assayed using charcoal or hydroxyapatite and the effect of different hormones on cell growth was evaluated by measuring total DNA and following the growth of transplanted cells in nude mice. Similar results were found for the two cell lines; they were unresponsive to estrogen since they did not contain appreciable amounts of estradiol receptor. Conversely, these two cell lines contained high concentrations of androgen and glucocorticoid receptors, which were both transferable to the nucleus. While dexamethasone stimulated, directly or indirectly, the growth of RBA and NMU cells, the effect of androgens on cell growth is still questioned. These two cell lines offer a potential model to study the mechanism of action of androgens and glucocorticoids in mammary cancer.
9,10-Dimethyl-1,2-benzanthracene↗
Nuclear translocation of the estrogen receptor in autonomous C3H mouse mammary tumors.
The reason for estrogen independence of C3H mouse mammary tumors has been sought in the initial steps of estradiol action. The characteristics of the estrogen receptors were similar to those observed in estrogen-responsive tissues: high affinity and binding specificity, DNA binding and 8S sedimentation constant as shown by sucrose gradient centrifugation. Their concentration averaged 18.5 +/- 3.5 (S.E.) fmol/mg cytosol protein in the cytosol and 3.5 +/- 1.0 fmol/mg cytosol protein in the KCl nuclear extract. The nuclear translocation of the cytosol receptor was investigated with the use of biopsy and in vivo injections of radioactive estradiol. No nuclear translocation of estrogen receptor could be ascertained with the dextran-coated charcoal assay since the free and nonspecifically bound estrogen conjugate(s) were also assayed by this technique. However, when the estrogen-receptor complexes were estimated by more specific methods such as protamine sulfate or hydroxylapatite precipitations, the estrogen receptor translocation into the nucleus was clearly shown. We therefore conclude that the estrogen independence of C3H mammary tumors cannot be explained by a defect in the two initial steps of the mechanism of action of estradiol, namely, cytosol binding and nuclear translocation of receptors.
Animals↗