Ochieng J, Tait L, Russo J
Department of Pathology, Michigan Cancer Foundation, Detroit 48201.
In Vitro Cell Dev Biol. 1990 Apr;26(4):318-24. doi: 10.1007/BF02623821.
Normal human breast epithelial cells obtained from a reduction mammoplasty (S130) have been maintained in culture for up to a year in Ham's F12:Dulbecco's medium, with 5% equine serum and a low calcium concentration (0.04 mM). These cells undergo senescence and terminal differentiation if they are switched to high Ca2+ medium (1.05 mM). To clarify the mechanism by which Ca2+ regulates the growth of these cells, we studied the role of tubulin assembly-disassembly and the morphologic changes subsequent to high Ca2+ switch. An early Passage (9) of S130 breast epithelial cells growing in low Ca2+ medium was analyzed. Of a total of 785 counted cells, 720 (92%) were rounded and 65 (8%) were flat, elongated, and fibroblastlike. When the cells were switched to high Ca2+ medium, out of 553 cells, only 111 (20%) were rounded and the remaining 442 (80%) were elongated and fibroblastlike. Immunocytochemical localization of tubulin, using the immunogold silver enhancement technique, showed that the majority of low Ca2(+)-grown cells did not display a network of tubulin fibers, whereas high Ca2(+)-grown cells revealed extensive cytoplasmic network of polymerized tubulin, which seemed to stretch out the cells. Experiments designed to determine the mechanisms of tubulin polymerization in these cells revealed that: a) Cells grown in high Ca2+ medium containing 0.1 mM colchicine had a reduced proportion of elongated cells; b) treatment of the cells with the calcium ionophore A23187 in low calcium medium resulted in an increase in the number of elongated cells which had more polymerized tubulin; and d) treatment of the cells with cyclic-AMP in low Ca2+ medium had no observable effect on cell morphology. These results indicate that high levels of Ca2+ either favor tubulin polymerization or stabilize the polymerized state.
从缩乳手术中获取的正常人类乳腺上皮细胞（S130），在含有5%马血清和低钙浓度（0.04 mM）的Ham's F12:Dulbecco培养基中培养长达一年。如果将这些细胞转换至高钙培养基（1.05 mM），它们会经历衰老和终末分化。为了阐明钙离子调节这些细胞生长的机制，我们研究了微管蛋白组装 - 解聚的作用以及高钙转换后随之而来的形态学变化。分析了在低钙培养基中生长的早期传代（第9代）S130乳腺上皮细胞。在总共计数的785个细胞中，720个（92%）呈圆形，65个（8%）扁平、细长且成纤维细胞样。当细胞转换至高钙培养基时，在553个细胞中，只有111个（20%）呈圆形，其余442个（80%）细长且成纤维细胞样。使用免疫金银增强技术对微管蛋白进行免疫细胞化学定位显示，大多数在低钙条件下生长的细胞未显示微管蛋白纤维网络，而在高钙条件下生长的细胞则显示出广泛的聚合微管蛋白细胞质网络，这似乎使细胞伸展。旨在确定这些细胞中微管蛋白聚合机制的实验表明：a）在含有0.1 mM秋水仙碱的高钙培养基中生长的细胞，细长细胞的比例降低；b）在低钙培养基中用钙离子载体A23187处理细胞，导致细长细胞数量增加，且这些细胞有更多的聚合微管蛋白；d）在低钙培养基中用环磷酸腺苷处理细胞，对细胞形态没有可观察到的影响。这些结果表明，高水平的钙离子要么有利于微管蛋白聚合，要么稳定聚合状态。
