Effect of intercalating drugs on the release of uterine nuclear estrogen receptors

Ethidium bromide has been reported to extract estrogen receptors from uterine nuclei whereas actinomycin D appears to block nuclear receptor 'processing' in MCF-7 cells. We have examined the effect of these drugs on the nuclear estrogen receptor during the [³H]estradiol exchange assay. Uterine nuclear fractions were prepared from ovariectomized rats that had received a 5-μg subcutaneous injection of estradiol 1 h prior to killing. Incubation of nuclear fractions with [³H]estradiol at 37°C resulted in a rapid labeling of nuclear estrogen receptor within 30 min which was followed by a loss of receptor sites that qualitatively resembled nuclear estrogen receptor processing. The addition of actinomycin D or ethidium bromide blocked this loss of nuclear estrogen receptor, resulting in a prolonged elevation of specific nuclear [³H]estradiol binding. Examination of the DNA by polyacrylamide-agarose gel electrophoresis showed extensive fragmentation that could be inhibited by actinomycin D in a dose-dependent manner. These findings suggest that a nuclease(s) present in crude nuclear fractions was responsible for the DNA fragmentation and loss of nuclear estrogen receptor complexes. Nuclear estrogen receptor release and DNA hydrolysis did not occur in highly purified nuclei. Nuclear estrogen receptor lost at 37°C could be recovered in the supernatant fraction as a family of soluble macromolecular complexes. Low salt sucrose gradients of this fraction showed specifically bound [³H]estradiol in an aggregate fraction that sedimented to the bottem of the gradient, and a free 8 S form. Both of these were converted to a 6 S form in gradients containing 0.4 M KCl.