title: Prolonged retention of estradiol by human breast cancer cells in tissue culture.
abstract: 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(-7) M 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-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.
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D002855:Chromatography, Thin Layer; D046508:Culture Techniques / Q000379:methods*; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D006801:Humans; D011960:Receptors, Estrogen / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology
chemical_list: D011960:Receptors, Estrogen; D013629:Tamoxifen; D004958:Estradiol