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Ionic and Ligand-specific Effects on the DNA Binding of Progesterone Receptor Bound to the Synthetic Progestin R5020 and the Antiprogestin RU486¹

Division of Toxicology, Department of Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 [C-C. C., P. E., M. A. G., T. T.] and Clinical Research Center and Division of Rheumatology, Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08903 [C-C. C., T. J. T.]

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 seuqences, 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 µg/ml, respectively, for PR bound to R5020. In contrast, EC₅₀ for control plasmid was 350 µg/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 µg/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.

¹ This work was supported in part by NIH Grants CA 42439 (T. T.) and AR 39020 (T. J. T.), and by grants from the Foundation of the University of Medicine and Dentistry of New Jersey; Environmental and Occupational Health Sciences Institute, Piscataway, NJ; and the General Research Support Program of the University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School.

³ The abbreviations used are: PR, progesterone receptor; ER, estrogen receptor; TED buffer, 10 mM Tris, 1 mM EDTA, and 1 mM dithiothreitol; TD buffer, 10 mM Tris-HCl and 1 mM dithiothreitol; DCC, dextran-coated charcoal; EC₅₀, effective concentration for 50% elution of the receptor from DNA-cellulose.

² To whom requests for reprints should be addressed.

Received 1/ 2/91. Accepted 5/17/91.