Holinka C F, Anzai Y, Hata H, Kimmel N, Kuramoto H, Gurpide E
Department of Obstetrics, Mount Sinai School of Medicine, New York, New York 10029.
Cancer Res. 1989 Jun 15;49(12):3297-301.
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)
对培养的人子宫内膜癌细胞中激素生长调节的研究受到外源性生长因子需求的限制，这些因子通常通过添加血清来提供。本报告提供了证据表明，在无血清或添加生长因子的情况下，雌二醇可以刺激石川系子宫内膜癌细胞的增殖。雌激素的促有丝分裂作用在两个不同的实验系统中得到证实，一个是附着在哺乳动物组织培养皿基质上的细胞，另一个是在非锚定条件下于软琼脂中形成集落的细胞。将雌二醇添加到不含血清、不含酚红的杜尔贝科改良伊格尔培养基和哈姆F-12培养基的混合物中，并补充L-谷氨酰胺和4-(2-羟乙基)-1-哌嗪乙磺酸[基础培养基：(BM)]，显著增加了附着在培养皿上的细胞的增殖。剂量反应实验显示，在10 nM时雌二醇刺激作用最大；在1 nM和100 nM浓度时也观察到显著反应。10 nM雌二醇的促有丝分裂作用与1%经活性炭处理的胎牛血清相当，且两种作用具有相加性。无血清BM中存在雌二醇导致指数增殖细胞的倍增时间从38小时缩短至29小时。从暴露于[3H]胸腺嘧啶脉冲后测得的标记指数以及在指数增殖细胞中测定的有丝分裂指数，计算出S期和M期的长度分别为11小时和1小时。根据这些数据估计，雌二醇使G1期缩短了约40%，从22小时缩短至13小时。在无血清以及存在1%经活性炭处理的胎牛血清的情况下，雌二醇使接种在含有0.3%琼脂的BM中的细胞的集落形成效率提高了一倍。雌二醇对集落形成的刺激受到培养基成分的影响，因为在基本培养基中未观察到作用。集落形成效率与血清浓度呈正相关，并且在可比血清水平下，基本培养基中的集落形成效率仍显著低于BM中的。观察到的集落形成效率与接种时细胞密度之间的正相关表明存在协同促有丝分裂作用，这可能是由于分泌的生长因子的自分泌和旁分泌作用。这些结果定义了一个模型，用于评估在不存在干扰性外源性生长因子的情况下，人子宫内膜癌细胞中由自分泌有丝分裂原介导的激素生长调节。(摘要截短至400字)
