Human progesterone receptor A and B isoforms in CHO 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 progesterone receptor isoforms (hPR-A and hPR-B, respectively) and a progesterone receptor-responsive reporter (MMTV-Luc). In the first step, stable transfection of the hPR-A and hPR-B isoform-encoding cDNAs was performed in the steroid receptor-negative CHO K1 cell line. Individual clones were characterized for hPR-isoform expression with respect to Western immuno-blotting, transcriptional activation and hormone binding. With respect to the latter characteristic, individual hPR-isoforms demonstrated similar dissociation constants (Kd for hPR-A: 0.5 +/- 0.3 and hPR-B: 0.8 +/- 0.3 nM, respectively) irrespective of the amount of receptor isoform expressed (Bmax varying from 4.1 to 33.2 nM). The Kd values observed for individual hPR-isoforms were comparable to those found for human breast tumor MCF-7 cells (Kd for hPR-A + hPR-B: 0.6 +/- 0.3 nM). In the second step, hPR-isoform expressing CHO clones were supertransfected with a MMTV-Luc reporter construct resulting in permanent cell lines useful for testing the activity of natural and synthetic steroids in their ability to modulate gene transcription. Both isoform-specific reporter cell lines responded in a similar ranking order towards different progesterone reference compounds such as Org 2058, progesterone (Prog), R5020, norethisterone (NE), and medroxy progesterone acetate (MPA). Moreover, a good correlation was observed between the relative binding affinity (RBA) and the transcriptional activation potency of these compounds towards the individual hPR-isoforms. The latter correlation could not only be demonstrated for the progestagenic agonist 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-isoforms was related to the degree of stimulation of the reporter gene (MMTV-based) within the cellular CHO context. Therefore, these cell lines can be used for the determination and quantitation of the activity of (anti)progestagenic compounds in vitro but may also be useful to predict the activity of compounds in vivo (see also II Comparison of binding, transactivation and ED50 values of several synthetic (anti) progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats).

Human progesterone receptor A and B isoforms in CHO cells. II. Comparison of binding, transactivation and ED50 values of several synthetic (anti)progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats.

The human progesterone receptor A and B isoforms (hPR-A and hPR-B) were stably transfected in Chinese Hamster Ovary (CHO) cells in the presence or absence of the mouse mamma tumor virus (MMTV) promoter and luciferase (LUC) reporter gene. In this way four stably transfected CHO cell lines, i.e. hPR-A, hPR-B, hPR-A-MMTV-LUC and hPR-B-MMTV-LUC cells, were prepared. hPR-A and -B isoforms were compared by binding and transactivation analysis for 14 progestagens and 7 antiprogestagens. Thereby Org 2058 was used as standard in both agonistic and binding assays and Org 31710 in antagonistic assays. The obtained data were compared with relative binding affinities (RBA) for both hPR-A and -B, which are present in human breast tumor MCF-7 cells, and with biopotency estimations with McPhail tests in rabbits and ovulation inhibition and pregnancy interruption tests in rats. The relative binding affinities of 14 progestagens and 7 antiprogestagens towards hPR-A, hPR-B or hPR-A/B of either CHO or MCF-7 cells were highly correlated with respect to ranking. This was also shown by the high correlation coefficients between the RBA's of hPR-B and hPR-A in CHO cells (r = 0.983) and between those of hPR-B of CHO and hPR A/B of MCF-7 cells (r = 0.957). The transactivation data of the 14 progestagens and 7 antiprogestagens for the hPR-B-MMTV-LUC cells were compared with those for hPR-A-MMTV-LUC cells and showed no differences between both cell lines with exception of the progestagens Org 32704 and 33766 and the antiprogestagen Org 33245. Therefore only the relative agonistic activities (RAA) and relative antagonistic activities (RANTA) of hPR-B-MMTV-LUC cells were compared with RBA values of hPR-B, showing a high similarity in ranking for the tested compounds, and high correlation coefficients of r = 0.91 and r = 0.97, respectively. Remarkably, RBA's were 1.6 fold higher than RAA's and RANTA's. These in vitro RBA, RAA and RANTA values for hPR-B were checked for their pharmacological relevance by in vivo biopotency measurements with the 14 progestagens and 7 antiprogestagens in rabbits and rats. The in vitro binding and transactivation potencies of progestagens appeared to be very predictive for in vivo analysis on endometrium proliferation in rabbits in the McPhail test with correlation coefficients of r = 0.81 and r = 0.87, while ovulation inhibition in rats correlated less well with r = 0.516 and r = 0.65. On the other hand, the antiprogestagenic potencies found with binding and transactivation assays had a good correlation with the potencies in the pregnancy interruption test in rats for all antiprogestagens tested, being r = 0.849 and r = 0.744, respectively. In conclusion, the binding and transactivation potencies for the tested compounds in hPR-A and -B containing cell lines showed in general a good resemblance. The transactivation studies with hPR-B-MMTV-LUC cells indicated that ranking of compounds was fairly identical to binding analysis and could be used for pre-screening of the (anti)-progestagenic bioactivity in the McPhail test in rabbits, the ovulation inhibition test and the pregnancy interruption test in rats. Therefore this transactivation assay can replace binding assays. Moreover, direct pre-screening of agonists, antagonists and partial antagonists is even possible in this in vitro bioassay, making transactivation assays for a particular class of chemical compounds to a valuable pre-screening tool for in vivo studies.