SYNERGISTIC ENHANCEMENT OF PRB-MEDIATED RU486 AND R5020 AGONIST ACTIVITIES THROUGH CYCLIC ADENOSINE-3',5'-MONOPHOSPHATE REPRESENTS A DELAYED PRIMARY RESPONSE

Activators of protein kinase A have been shown to affect the transacti vation potential of progestins and antiprogestins, To analyze the mech anisms and factors involved, we have created HeLa and CV1 cell clones stably expressing isoform B of progesterone receptor, In the HeLa cell background, the progesterone antagonist RU486 significantly induces p rogesterone-regulatable reporter genes, and this agonistic effect is s ynergistically enhanced by elevating cAMP or through overexpression of protein kinase A catalytic subunit. In contrast, in CV1 cells contain ing functional progesterone receptors no agonist activity of RU486 cou ld be detected, suggesting the involvement of cell specifically expres sed factors. In a PRB-positive HeLa cell clone containing stably integ rated copies of a thymidine kinase-luciferase reporter gene with two p rogesterone response elements, we observed a complete loss of RU486 an tagonist potential upon cotreatment with cAMP for 25 h while partial a ntagonist potential was maintained in a 5-h experiment, This result sh ows that, particularly in the presence of protein kinase A activators, the duration of hormone treatment is a crucial parameter in the evalu ation of antagonist properties of antiprogestins. A detailed analysis of the kinetics of the hormone effects on transcription revealed that the onset of cAMP/RU486 synergism is delayed relative to the responses induced by RU486 or R5020 alone. Moreover, partial inhibition of prot ein synthesis by cycloheximide completely abolished cAMP/RU486 synergi sm while R5020 and RU486 responses were not inhibited, Together, these data indicate that cAMP/RU486 synergism is a delayed primary response requiring the intermediate induction of an essential factor.