title: Proliferation and responsiveness to estrogen of human endometrial cancer cells under serum-free culture conditions.
abstract: Studies of hormonal growth regulation in cultured human endometrial cancer cells are limited by the requirement of exogenous growth factors, usually supplied by addition of serum. The present report provides evidence that estradiol can stimulate proliferation of endometrial cancer cells of the Ishikawa line in the absence of serum or added growth factors. Mitogenic effects of estrogen were demonstrated in two different experimental systems, in cells attached to the substratum of mammalian tissue culture dishes, and in cells forming colonies in soft agar under anchorage-independent conditions. Addition of estradiol to a mixture of serum-free, phenol red-free Dulbecco's minimal essential medium and Ham's F-12 medium, supplemented with L-glutamine and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [basal medium: (BM)] significantly increased the proliferation of cells attached to culture dishes. Dose-response experiments revealed maximal estradiol stimulation at 10 nM; significant responses were also observed at 1 nM and at 100 nM concentrations. The mitogenic effect of 10 nM estradiol was comparable to that of 1% charcoal-treated fetal bovine serum and the two effects were additive. The presence of estradiol in serum-free BM resulted in a shortening of the doubling time of exponentially proliferating cells from 38 to 29 h. From the labeling index, measured after exposure to a pulse of [3H]thymidine, and from the mitotic index, both determined in exponentially proliferating cells, the lengths of the S and M phases were calculated to be 11 and 1 h, respectively. From these data it was estimated that estradiol shortened the G1 phase by approximately 40%, from 22 to 13 h. Estradiol doubled the colony formation efficiency of cells plated in BM containing 0.3% agar in the absence of serum as well as in the presence of 1% charcoal-treated fetal bovine serum. The stimulation of colony formation by estradiol was influenced by medium components, since no effects were observed in minimal essential medium. The colony formation efficiency was positively related to the serum concentrations and remained significantly lower in minimal essential medium than in BM at comparable serum levels. The observed positive relationship between colony formation efficiency and cell densities at plating suggests a cooperative mitogenic effect, likely due to autocrine and paracrine action of secreted growth factors. These results define a model to evaluate hormonal growth regulation mediated by autocrine mitogens in human endometrial cancer cells in the absence of interfering exogenous growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Obstetrics, Mount Sinai School of Medicine, New York, New York 10029.
authors.forename: C F
authors.initials: CF
authors.lastname: Holinka
authors.forename: Y
authors.initials: Y
authors.lastname: Anzai
authors.forename: H
authors.initials: H
authors.lastname: Hata
authors.forename: N
authors.initials: N
authors.lastname: Kimmel
authors.forename: H
authors.initials: H
authors.lastname: Kuramoto
authors.forename: E
authors.initials: E
authors.lastname: Gurpide
mesh_terms: D002449:Cell Aggregation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006801:Humans; D014407:Tumor Cells, Cultured / Q000166:cytology* / Q000187:drug effects; D014594:Uterine Neoplasms / Q000473:pathology*
chemical_list: D003470:Culture Media; D004958:Estradiol