Karey K P, Sirbasku D A
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Cancer Res. 1988 Jul 15;48(14):4083-92.
A completely serum-free assay method has been used to compare the mitogenic activities of polypeptide growth factors and estrogens with MCF-7 and T47D human breast cancer cells in culture. The lines were maintained in a viable, slowly dividing condition in Ham's F12 and Dulbecco's modified Eagle's medium (1:1) supplemented with sodium bicarbonate (2.2 g/liter), 15 mM 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid, human transferrin (10 micrograms/ml), and bovine serum albumin (200 micrograms/ml) (designated Tf/BSA). This medium allowed the assay of mitogenic activities as measured by multiple rounds of cell division and permitted comparisons of the biological potencies of growth factors within functional families as well as of dissimilar mitogens. Insulin-like growth factor I (IGF-I) was the most potent mitogen studied, showing ED50 values of 160 pg/ml and 1.7 ng/ml with the MCF-7 and T47D cells, respectively. Insulin-like growth factor II and insulin were less active, with ED50 values of 0.55 and 1.2 ng/ml with MCF-7 cells and 4.3 and 10 ng/ml with the T47D cell line, respectively. Mitogens sharing epidermal growth factor-like functional properties had ED50 values from 35 pg/ml to 2.5 ng/ml, while transforming growth factor type beta and platelet-derived growth factor had no detectable stimulatory effects. Basic fibroblast growth factor had ED50 values of 0.42 ng/ml and 3.7 ng/ml for the MCF-7 and T47D cells, respectively, while acidic fibroblast growth factor was nearly inactive. In phenol red-free Tf/BSA, 17 beta-estradiol caused a 60% increase in MCF-7 cell numbers over controls in 8 days while having no effect on growth of the T47D cell line. From MCF-7 conditioned Tf/BSA medium, IGF-I was identified by biological activity, by radioimmunoassay (approximately equal to 2 pg/ml) and by estimation of molecular weight (8,000) under dissociating conditions. The concentration of IGF-I was not affected by 17 beta-estradiol treatment. The data indicate that induction of acid stable, low molecular weight autocrine growth factors involved more regulation than defined by estrogens alone. The minimal effects of 17 beta-estradiol in Tf/BSA opened several possibilities including the putative roles of other serum-borne hormones, growth factors and regulators in autocrine growth factor induction.
采用一种完全无血清的检测方法，比较了多肽生长因子和雌激素对培养的MCF-7和T47D人乳腺癌细胞的促有丝分裂活性。细胞系在添加了碳酸氢钠（2.2 g/升）、15 mM 4-(2-羟乙基)-1-哌嗪乙烷磺酸、人转铁蛋白（10微克/毫升）和牛血清白蛋白（200微克/毫升）（称为Tf/BSA）的Ham's F12和Dulbecco改良的Eagle培养基（1:1）中维持在存活、缓慢分裂的状态。这种培养基能够通过多轮细胞分裂来检测促有丝分裂活性，并允许比较功能家族内生长因子以及不同有丝分裂原的生物学活性。胰岛素样生长因子I（IGF-I）是所研究的最有效的有丝分裂原，对MCF-7和T47D细胞的半数有效剂量（ED50）值分别为160 pg/毫升和1.7 ng/毫升。胰岛素样生长因子II和胰岛素的活性较低，对MCF-7细胞的ED50值分别为0.55和1.2 ng/毫升，对T47D细胞系的ED50值分别为4.3和10 ng/毫升。具有表皮生长因子样功能特性的有丝分裂原的ED50值在35 pg/毫升至2.5 ng/毫升之间，而转化生长因子β型和血小板衍生生长因子没有可检测到的刺激作用。碱性成纤维细胞生长因子对MCF-7和T47D细胞的ED50值分别为0.42 ng/毫升和3.7 ng/毫升，而酸性成纤维细胞生长因子几乎没有活性。在无酚红的Tf/BSA中，17β-雌二醇在8天内使MCF-7细胞数量比对照组增加了60%，而对T47D细胞系的生长没有影响。从MCF-7条件性Tf/BSA培养基中，通过生物活性、放射免疫测定（约2 pg/毫升）以及在解离条件下对分子量（8000）的估计鉴定出了IGF-I。IGF-I的浓度不受17β-雌二醇处理的影响。数据表明，酸性稳定、低分子量自分泌生长因子的诱导涉及比单独雌激素所定义的更多的调节。17β-雌二醇在Tf/BSA中的最小作用开启了多种可能性，包括其他血清源性激素、生长因子和调节剂在自分泌生长因子诱导中的假定作用。
