PROLONGED RETENTION OF ESTRADIOL BY HUMAN-BREAST CANCER-CELLS IN TISSUE-CULTURE

Abstract

Prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for 3 h with a physiological concentration of [3H]estradiol (3-5 nM) and then were washed with 3 successive exchanges of medium 3, 17 and 24 or 48 h following incubation with [3H]estradiol. The total wash period was 78 h. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture: the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites and 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 3H was about 50 nM prior to wash and 9 nM following 78 h of wash. The intracellular concentration of specifically bound [3H]estradiol was initially 18 nM, and after 78 h of wash, it was 2.8 nM. After 48 h 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 h monitored. When 10-6 M tamoxifen or 10-7 M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected. Specifically bound [3H]estradiol was essentially eliminated within 24 h. 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 h, cells washed with medium containing 3.5 or 7% bovine serum albumin contained 10% of the [3H]estradiol present in cells washed with medium alone. Medium exchanges alone may not effectively remove estradiol from MCF-7 cells, and estrogen retention by estrogen-responsive 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.