Differential nuclear receptor and gene expression in human endometrial adenocarcinoma cells: Regulation by a novel selective progesterone receptor modulator

Document Type

Article (peer-reviewed)

Publication Date

2011

Abstract

Introduction: Selective progesterone receptor (PR) modulators (SPRMs) are PR ligands with agonist or antagonist effects on progesterone-responsive tissues in women, e.g. breast and endometrium. Specific SPRMs have stimulatory, inhibitory or mixed progesterone-like activities, permitting the design of new molecules with potent therapeutic efficacy for safe contraception or hormone therapy with a high therapeutic index. The SPRM 17-acetoxy-11-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione (UPA) binds to PR-A and B forms and in clinical studies inhibits follicle development and ovulation. Objective: This study was designed to characterize the short and long-term effects of UPA on human endometrial cell- and steroid-specific gene expression and cell cycle. Methods: To further test the hypothesis that UPA is safe for women and could be protective of the endometrium, we used an experimental human endometrial adenocarcinoma cell culture paradigm. RNA and protein expression levels for steroid receptors, relevant pathway components and cell cycle-dependent proteins were determined by (A) gene microarray, quantitative real-time PCR analyses and (B) Western analyses using site-specific immunoreagents, respectively. Cells were isolated for cell cycle analyses performed by (C) multiplex flow cytometry. Over a 1-to-15-wk period, in parallel, the responses of continuously UPA-exposed (100nM) cells were compared to those of vehicle-matched control cells. Following acute treatment with either UPA (100nM) or estradiol (E2; 1nM) to determine estrogen responsiveness, RNA extracts, cytoplasmic, nuclear and whole cell protein lysates were characterized. Results: Over a 15-week (wk) UPA or matched control culture period, microarray analyses of replicate total RNA samples revealed that gene expression was significantly altered. After acute UPA treatment, control cells demonstrated up-regulation of 1433 human genes and down regulation of 1464, whereas long-term UPA-exposed cells showed significant regulation of 926 (up) and 1183 (down) genes. Surprisingly, only 27 human genes were similarly up-regulated and 54 genes down regulated and shared between the two conditions. Interestingly, when E2 was added to control endometrial cells, 1503 (up) and 2048 (down) genes were noted; long-term UPA with acute E2 showed 647 (up) and 645 (down). Only 30 up-regulated and 52 down regulated genes were shared between E2-stimulated control and long-term UPA-treated human endometrial cancer cells. Over an 8-wk period, whole cell total protein levels for PR, androgen receptor (AR) and estrogen receptors (ERalpha and beta) were unchanged by UPA. Importantly, over time with UPA treatment, specific subcellular compartmentalization of these receptors was altered. UPA increased the cytoplasm/nucleus ratio of PR. In UPA-treated cells, E2 responses were significantly affected for AR, ER, PR and cyclins D1 and B1. UPA increased cells in G0G1 and decreased cells in S and G2/M. In the 15-wk study, AR, ERalpha, ERbeta; and PR mRNA levels were significantly reduced compared to vehicle and time matched-controls. Conclusion: Taken together, these data are consistent with UPA-specific mediated changes in subcellular steroid receptor distribution and estrogen responsiveness and specific gene regulation of cellular pathways. Our findings are suggestive that protection against abnormal growth may be an additional health benefit.

DOI

10.1093/biolreprod/85.s1.359

Language

English

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