Classical membrane progesterone receptors in murine mammary carcinomas: agonistic effects of progestins and RU-486 mediating rapid non-genomic effects
In this article, we demonstrate the expression of functional progesterone binding sites at the cell membrane in murine mammary carcinomas that are stimulated by progestins and inhibited by antiprogestins. Using confocal immunofluorescence, ligand binding and cell compartment-specific western blots, we were able to identify the presence of the classical progesterone receptors. Medroxyprogesterone acetate (MPA) and RU-486 (1 × 10−11 and 1 × 10−8 M) behaved as agonists activating extracellular signal-regulated kinases (ERKs) and progestin-regulated proteins, except for Cyclin D1 and Tissue factor which failed to increase with 1 × 10−8 M RU-486, an experimental condition that allows PR to bind DNA. These results predicted a full agonist effect at low concentrations of RU-486. Accordingly, at concentrations lower than 1 × 10−11 M, RU-486 increased cell proliferation in vitro. This effect was abolished by incubation with the ERK kinase inhibitor PD 98059 or by OH-tamoxifen. In vivo, at a daily dose of 1.2 μg/kg body weight RU-486 increased tumor growth, whereas at 12 mg/kg induces tumor regression. Our results indicate that low concentrations of MPA and RU-486 induce similar agonistic non-genomic effects, whereas RU-486 at higher concentrations may inhibit cell proliferation by genomic-induced effects. This suggests that RU-486 should be therapeutically administered at doses high enough to guarantee its genomic inhibitory effect.
KeywordsMembrane progesterone receptors Mammary carcinomas Progesterone receptor isoforms: membrane-initiated steroid signaling Antiprogestins Breast cancer treatment Non-genomic effects Progestins
Steroid-stripped fetal calf serum
Membrane initiated steroid signaling
Membrane progesterone receptors
PR isoform A
PR isoform B
This work was supported by Fundación Sales, ANPCyT (PICT 07-932 and PICT 05-38302), CONICET (PIP 5351). Drs. Gutkind, Amornphimoltham and Molinolo are supported by the Intramural Research Program of the Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD. We are very grateful to Dr. Elisa Bal de Kier Joffe for providing LM3 cells, to Julieta Bolado and to Pablo Do Campo for excellent technical assistance, and to Laboratorios Craveri, Buenos Aires for providing MPA depot and to Bayer Schering Pharma AG for the ZK230211. We also wish to thank Avon Foundation for an AACR travel award to MC Bottino and the UICC for an ICRETT fellowship to MC Bottino.
Conflict of interest statement
The authors declare that they have no competing interests.
Effects of low concentrations of RU-486 on the proliferation of human breast cancer cells. a T47D cells plated in 96 well plates were starved for 24 h and then treated with RU-486 or MPA for 48 h. 3H-thymidine was added for the last 24 h. 3H-thymidine labeling index was calculated as experimental cpm/control cpm. A representative experiment of three is shown and octuplicates were used for each experimental group; ***: p < 0.001 and *: p < 0.05 between experimental and control group. b Representative western blots of pERKs, pAKT, total ERK and AKT illustrating the increase in ERK and AKT activation in T47D cells treated for 10 min with RU-486. (JPG 261 kb)
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