HUMAN PROGESTERONE-RECEPTOR-A AND PROGESTERONE-RECEPTOR-B ISOFORMS INCHO CELLS - I - STABLE TRANSFECTION OF RECEPTOR AND RECEPTOR-RESPONSIVE REPORTER GENES - TRANSCRIPTION MODULATION BY (ANTI)PROGESTAGENS

A hormone-dependent transcription modulation system was established on the basis of a two-step transfection procedure of the human progester one receptor isoforms (hPR-A and hPR-B, respectively) and a progestero ne receptor-responsive reporter (MMTV-Luc). In the first step, stable transfection of the hPR-A and hPR-B isoform-encoding cDNAs was perform ed in the steroid receptor-negative CHO K1 cell Line. Individual clone s were characterized for hPR-isoform expression with respect to Wester n immuno-blotting, transcriptional activation and hormone binding. Wit h respect to the latter characteristic, individual hPR-isoforms demons trated similar dissociation constants (K-d for hPR-A: 0.5 +/- 0.3 and hPR-B; 0.8 +/- 0.3 nM, respectively) irrespective of the amount of rec eptor isoform expressed (B-max varying from 4.1 to 33.2 nM). The K-d v alues observed for individual hPR-isoforms were comparable to those fo und for human breast tumor MCF-7 cells (K-d for hPR-A + hPR-B: 0.6 +/- 0.3 nM). In the second step, hPR-isoform expressing CHO clones were s upertransfected with a MMTV-Luc reporter construct resulting in perman ent cell lines useful for testing the activity of natural and syntheti c steroids in their ability to modulate gene transcription. Both isofo rm-specific reporter cell lines responded in a similar ranking order t owards different progesterone reference compounds such as Org 2058, pr ogesterone (Prog), R5020, norethisterone (NE), and medroxy progesteron e acetate (MPA). Moreover, a good correlation was observed between the relative binding affinity (RBA) and the transcriptional activation po tency of these compounds towards the individual hPR-isoforms. The latt er correlation could not only be demonstrated for the progestagenic ag onist reference compounds but was also observed for the progestagenic antagonist reference compounds like Org 33628, Org 31710, RU 38486 and ZK 98299. The major difference observed between the individual PR-iso forms was related to the degree of stimulation of the reporter gene (M MTV-based) within the cellular CHO context. Therefore, these cell, lin es can be used for the determination and quantitation of the activity of (anti)progestagenic compounds in vitro but may also be useful to pr edict the activity of compounds in vivo (see also II Comparison of bin ding, transactivation and ED50 values of several synthetic (anti) prog estagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and ra ts). (C) 1998 Elsevier Science Ltd. All rights reserved.