A nuclear binding assay to assess the biological activity of steroid receptors in isolated animal and human tissues.
This paper describes a nuclear binding assay (NB assay) which measures not only the presence of a steroid receptor in a tissue, but also the quantity of that receptor which is biologically active or functional, i.e. able to bind to nuclear acceptor sites. The assay involves the isolation viable cells from tissues and their incubation with an excess of radiolabeled steroid to encourage the activation and nuclear binding of all cellular receptors. The nuclei are isolated under conditions that remove unactivated (unbound) steroid-receptor complexes. This NB assay demonstrates, in both animal and human steroid target tissues, a saturable, tissue- and steroid-specific, and temperature- and time-dependent nuclear binding of radiolabeled steroids. These properties support a receptor-dependent nuclear binding of steroids. This assay is reproducible and requires relatively small amounts of tissue. The patterns of nuclear binding of the progesterone receptor, achieved with the assay in the avian oviduct model system, are shown to correlate with the nuclear binding of progesterone in vivo, the ability of the steroid to alter transcription, and the expression of a specific gene product, the protein avidin. The assay has been used to identify the existence of nonfunctional steroid receptors in endometrial and breast carcinomas. Therefore, this NB assay combined with the standard charcoal/hydroxylapatite methods of quantitating total cellular receptors should provide a means of assessing changes in the regulation of the biological activity of steroid receptors. Further, the assay should be useful to assess the ability of steroid analogs to properly activate their respective receptors for subsequent nuclear binding.
D000818:Animals; D001681:Biological Assay; D001943:Breast Neoplasms / Q000032:analysis; D002467:Cell Nucleus / Q000378:metabolism*; D002645:Chickens; D004717:Endometrium / Q000032:analysis / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D006801:Humans; D007700:Kinetics; D003012:Microbial Collagenase / Q000378:metabolism; D010057:Oviducts / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism / Q000494:pharmacology; D011397:Promegestone / Q000378:metabolism; D011960:Receptors, Estrogen / Q000032:analysis / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000032:analysis / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D011374:Progesterone; D004958:Estradiol; D011397:Promegestone; D003012:Microbial Collagenase