Chao C C, Thomas T J, Ebede P, Gallo M A, Thomas T
Department of Environmental and Community Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Cancer Res. 1991 Aug 1;51(15):3938-45.
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.
抗孕激素及其他抗激素是激素反应性癌症中有价值的治疗药物。抗孕激素作用的一个基本机制是其与孕激素受体（PR）结合，PR是一种细胞内蛋白质，通过在反应性基因的调控位点直接相互作用来介导孕激素的作用。为阐明与激动剂和拮抗剂配体结合的PR的作用机制，我们分别测定了兔子宫PR与R5020和RU486结合时对不同DNA序列的结合亲和力。在本研究中，我们使用了2种重组质粒DNA，即分别带有60个碱基对和23个碱基对潜在Z-DNA形成序列（dA-dC）n·（dG-dT）n插入片段的pDHf14和pDHf2，无插入片段的亲本质粒pDPL6，小牛胸腺DNA，以及几种合成多核苷酸。将洗脱与DNA纤维素结合的50%受体所需的DNA浓度（EC50）用作衡量受体对DNA相对结合亲和力的指标。对于与R5020结合的PR，质粒pDHf14和pDHf2的EC50值分别为1.2±0.5（标准差）和2.5±0.6μg/ml。相比之下，对照质粒的EC50为350μg/ml。与pDHf2和pDHf14相比，PR-R5020对小牛胸腺DNA和多核苷酸的亲和力较低。与抗孕激素RU486复合的受体对质粒的亲和力较低；对于pDHf14和pDHf2，EC50值分别为2.4±0.4和10±1μg/ml。5 mM Mg2+或Ca2+的存在放大了这种DNA结合中配体特异性的差异。在5 mM Mg2+和Ca2+存在的情况下，PR-R5020对pDHf14的相对结合亲和力分别比PR-RU486高6倍和7倍。这些结果表明，与PR-R5020相比，PR-RU486对特定DNA序列的结合亲和力较低，但两种受体的结合亲和力都处于不能排除在DNA识别位点发生竞争性相互作用的范围内。Mg2+和Ca2+对PR与DNA结合的影响进一步表明，这些阳离子可能以配体特异性的方式影响PR对DNA的识别。
