Abstract
T47D cells, cultured in medium containing serum stripped of endogenous steroids, proliferate in response to treatment with the progesterone receptor (PR) agonist, R5020 or the PR agonist/antagonist, RU486, whereas the full PR antagonist, ZK98299 has no proliferative effects. Under estrogenized conditions, all of the PR ligands tested inhibit cell growth [23]. In order to determine whether the levels or phosphorylation state of PR are reflected in the growth patterns of T47D cells, we monitored the effects of these PR ligands on the immunoblotted PR band intensities, the relative intensities, of PR-A and PR-B, and their phosphorylation states that are reflected in their altered mobility during SDS-PAGE. Under conditions where the PR ligands inhibit cell proliferation, each ligand had distinctively different qualitative and quantitative effects on PR. Short term treatment of the cells with R5020 or RU486 induced a characteristic phosphorylation-dependent upshift of both PR-A and PR-B. The phosphorylated PR was stable for up to 4 days after treatment of the cells with RU486, but was down regulated between 6-24 h after treatment with R5020. No replenishment of PR in cells treated with R5020 was detected. ZK98299, at concentrations tested, had no qualitative or quantitative effects on PR. Culturing cells for 8 days in medium containing steroid-depleted serum caused a significant reduction in the PR band intensity without causing a change in the ratio of PR-A and PR-B or their phosphorylation states. This decrease in the PR band intensity was reversed by maintaining the cells in 1 nM estrogen, but was potentiated by RU486 or ZK98299. These observations support the view that decreased PR levels may play a role in the stimulatory effects of R5020 and RU486 when cells are cultured under non-estrogenized conditions.
Similar content being viewed by others
References
Henderson BE, Ross RK, Pike MC: Hormonal chemoprevention of cancer in women. Science 259: 633–638, 1993
McGuire WL: Hormone receptors: Their role in predicting prognosis and response to endocrine therapy. Sem Oncol 5: 428–433, 1978
Bowden RT, Hissom JR, Moore MR: Growth stimulation of T47D human breast cancer cells by the antiprogestin RU486. Endocrinology 124: 2642–2644, 1989
Hissom JR, Bowden RT, Moore MR: Effects of progestins, estrogens, and antihormones on growth and lactate dehydrogenase in the human breast cancer cell line. Endocrinology 125: 418–423, 1989
Sartorius CA, Tung L, Takimoto GS, Horwitz KB: Antagonist-occupied human progesterone receptors bound to DNA are functionally switched to transcriptional agonists by cAMP. J Biol Chem 268: 9262–9266, 1993
Tung L, Mohamed MK, Hoeffler JP, Takimoto GS, Horwitz KB: Antagonist-occupied human progesterone B-receptors activate transcription without binding to progesterone response elements and are dominantly inhibited by A-receptors. Mol Endocrinol 7: 1256–1265, 1993
Gronemeyer H, Benhamou B, Berry M, Bocquel MT, Gofflo D, Garcia T, Lerouge T, Metzer D, Meyer ME, Tora L, Vergezac A, Chambon P: Mechanism of antihormone action. J Steroid Biochem Mol Biol 41: 217–221, 1992
Klein-Hitpass L, Cato ACB, Henderson D, Ryffel GU: Two types of antiprogestins identified by their differential action in transcriptionally active extracts from T47D cells. Nuc Acid Res 19: 1227–1234, 1991
Takimoto GS, Tasset DM, Eppert AC, Horwitz KB: Hormone-induced progesterone receptor phosphorylation consists of sequential DNA-independent DNA-dependent stages: Analysis with zinc finger mutants and the progesterone antagonist ZK98299. Proc Natl Acad Sci USA 89: 3050–3054, 1992
Bagchi MR, Tsai SY, Tsai M-J, O'Malley BW: Ligand and DNA-dependent phosphorylation of human progesterone receptor in vitro. Proc Natl Acad Sci USA 89: 2664–2668, 1992
Delabre K, Guichon-Mantel A, Milgrom E: In vivo evidence against the existence of antiprogestins disrupting receptor binding to DNA. Proc Natl Acad Sci USA 90: 4421–4425, 1993
Reddel RR, Alexander IE, Koga M, Shine J, Sutherland RL: Genetic instability and the development of steroid hormone insensitivity in cultured T47D human breast cancer cells. Cancer Res 48: 4340–4347, 1988
Graham II ML, Dalquist KE, Horwitz KB: Simultaneous measurement of progesterone receptors and DNA indices by flow cytometry: Analysis of breast cancer cell mixtures and genetic instability of the T47D line. Cancer Res 49: 3943–3949, 1989
Sheridan PL, Evan RM, Horwitz KB: Phosphotryptic peptide analysis of human progesterone receptors. New phosphorylated sites formed in nuclei after hormone treatment. J Biol Chem 264: 6520–6528, 1989
Sartorius CA, Melville MY, Hovland AR, Tung L, Takimoto GS, Horwitz KB: A third transactivation function (AF3) of human progesterone receptors located in the unique N-terminal segment of the B-isoform. Mol Endocrinol 8: 1347–1360, 1994
Vegeto E, Shahbaz MM, Wen DX, Goldman ME, O'Malley BW, McDonnell DP: Human progesterone receptor A form is a cell-and promotor-specific repressor of human progesterone receptor B function. Mol Endocrinol 7: 1244–1255, 1993
Wei LL, Sheridan PL, Krett NL, Fransis MD, Toft DO, Edwards DP, Horwitz KB: Immunological analysis of human breast cancer progesterone receptors. II, Structure, phosphorylation and processing. Biochemistry 26: 6262–6272, 1987
Beck CA, Zhang Y, Altman M, Weigel NL, Edwards DP: Stoichiometry and site-specific phosphorylation of human progesterone receptor in native target cells and in the baculovirus expression system. J Biol Chem 271: 19546–19555, 1997
Zhang Y, Beck CA, Poletti A, Clement JP 4th, Prendergast P, Yip TT, Hutchens TW, Edwards DP, Weigel NL: Phosphorylation of human progesterone receptor by cyclindependent kinase 2 on three sites that are authentic basal phosphorylation sites in vivo. Mol Endocrinol 11: 823–832, 1997
Moudgil VK, Anter MJ, Hurd C: Mammalian progesterone receptor shows differential sensitivity to sulfhydryl group modifying agents when bound to agonist and antagonist ligands. J Biol Chem 264: 2203–2211, 1989
Hurd C, Khattree N, Alban P, Nag K, Jhanwar SC, Dinda S, Moudgil VK: Hormonal regulation of the p53 tumor suppressor protein in T47D human breast carcinoma cell line. J Biol Chem 270: 28507–28510, 1995
Hurd C, Khattree N, Dinda S, Alban P, Moudgil VK: Regulation of tumor suppressor proteins, p53 and retinoblastoma, by estrogen and antiestrogens in breast cancer cells. Oncogene 15: 991–995, 1997
Iwasaki K, Underwood B, Herman M, Dinda S, Kodali S, Kloosterboer HJ, Hurd C, Moudgil VK: Effects of antiprogestins on the rate of proliferation of breast cancer cells. Mol Cell Biochem 198: 141–149, 1999
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hurd, C., Nag, K., Khattree, N. et al. Agonist and antagonist-induced qualitative and quantitative alterations of progesterone receptor from breast cancer cells. Mol Cell Biochem 199, 49–56 (1999). https://doi.org/10.1023/A:1006982528297
Issue Date:
DOI: https://doi.org/10.1023/A:1006982528297