Prolonged Retention of Estradiol by Human Breast Cancer Cells in Tissue Culture

Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [³H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [³H]estradiol.The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture: (a) the concentration of [³H]estradiol and [³H]estradiol metabolites in the media washes; (b) the intracellular concentration of [³H]estradiol and [³H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [³H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [³H]estradiol was initially 18 nM, and after 78 hr of wash, it was 2.8 nM. After 48 hr of wash, nearly all specifically bound [³H]estradiol was present in the nucleus. Following incubation of the cells with 5 nM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed throughout the 78 hr monitored. When 10^-6 M tamoxifen or 10^-7M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [³H]estradiol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific and specific [³H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one tenth of the [³H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsivene cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem.