Estimation of total oestrogen receptors in DMBA-induced rat mammary tumors by exchange of nuclear bound ligand at low temperature: a comparison with rat uterus
article
A nuclear exchange assay for the estimation of oestrogen receptors in dimethylbenzanthracene (DMBA)-induced rat mammary tumors is described. In brief, the method consists of translocation of receptor sites to the nuclei with oestradiol-17β (E2); isolation of Triton-washed nuclear preparations; exchange of ligand by incubation of nuclear pellets with radioactive E2 for 1 h at 0°C and extraction of E2-receptor complexes with 0.4 M KCl. The optimum temperature for nuclear exchange appeared to be 0°C when a 1-h incubation period was used. When nuclear exchange incubations were carried out at temperatures of 15°C or higher, the steroid-receptor complex appeared to be unstable. This instability is not the result of trypsin-like proteolytic activity, nor of dissociation of the steroid-receptor complex during the assay procedure. Exchange of E2 at 0°C was also demonstrated for uterine nuclear pellets. Quantitative aspects of the nuclear exchange assay were studied in ovariectomized rats. No differences were found between in vivo and in vitro labelling of receptor sites. Comparison of extraction of nuclei-bound E2 with ethanol and extraction of steroid-receptor complexes with 0.4 M KCl showed that less radioactivity was extracted from the nuclear preparations with 0.4 M KCl. Ethanol extraction released much aspecifically bound E2 from the nuclear preparations.
TNO Identifier
354949
Source
Journal of Steroid Biochemistry, 13(5), pp. 557-564.
Pages
557-564
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