Ionic and ligand-specific effects on the DNA binding of progesterone receptor bound to the synthetic progestin R5020 and the antiprogestin RU486.

Antiprogestin and other antihormones are valuable therapeutic agents in hormone-responsive cancers. A fundamental mechanism in the action of antiprogestin is its binding to PR,3 an intracellular protein that mediates the action of progesterone by direct interaction at the regulatory sites of responsive genes. To elucidate the mechanism of action of PR bound to agonistic and antagonistic ligands, we determined the binding affinity of rabbit uterine PR bound to R5020 and RU486, respectively, with different DNA sequences. We used 2 recombinant plasmid DNAs, pDHf14 and pDHf2, with 60- and 23-base pair inserts of potential Z-DNA-forming (dA-dC)n.(dG-dT)n sequences, respectively, parental plasmid pDPL6 with no insert, calf thymus DNA, and several synthetic polynucleotides in this study. The concentration of DNA required to elute 50% of the receptor bound to DNA-cellulose (EC50) was used as a measure of the relative binding affinity of the receptor for DNA. EC50 values of plasmids pDHf14 and pDHf2 were 1.2 +/- 0.5 (SD) and 2.5 +/- 0.6 micrograms/ml, respectively, for PR bound to R5020. In contrast, EC50 for control plasmid was 350 micrograms/ml. PR.R5020 had lower affinity for calf thymus DNA and polynucleotides compared with pDHf2 and pDHf14. Receptors complexed with the antiprogestin RU486 had lower affinity for the plasmids; EC50 values were 2.4 +/- 0.4 and 10 +/- 1 microgram/ml, respectively, for pDHf14 and pDHf2. This ligand-specific difference in DNA binding was amplified by the presence of 5 mM Mg2+ or Ca2+. The relative binding affinity of PR.R5020 to pDHf14 was 6- and 7-fold higher than that of PR.RU486, in the presence of 5 mM Mg2+ and Ca2+, respectively. These results show that PR.RU486 has lower binding affinity for specific DNA sequences than PR.R5020, but the binding affinity of both receptors is in a range that cannot preclude competitive interactions at the DNA recognition site. The effects of Mg2+ and Ca2+ on PR binding to DNA further suggest that these cations could affect PR recognition of DNA in a ligand-specific manner.