Rubin H, Yao A, Chow M
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3206, USA.
Proc Natl Acad Sci U S A. 1995 May 23;92(11):4843-7. doi: 10.1073/pnas.92.11.4843.
Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in the death of some cells in a manner suggestive of apoptosis. Successive rounds of prolonged incubation at confluence of the surviving cells produce increasing neoplastic transformation in the form of increments in saturation density and transformed focus formation. Cells from the postconfluent cultures are given a recovery period of various lengths to remove the direct inhibitory effect of confluence before their growth properties are studied. It is found that with each round of confluence the exponential growth rate of the cells at low densities gets lower and the size of isolated colonies of the same cells shows a similar progressive reduction. The decreased growth rate of cells from the third round of confluence persists for > 60 generations of growth at low density. The proportion of colonies containing giant cells is much higher after a 2-day recovery from confluence than after a 7-day recovery. Retardation of growth at low density and increased saturation density appear to be two sides of the same coin: both occur in the entire population of cells and precede the formation of transformed foci. We propose that the slowdown in growth and the formation of giant cells result from heritable damage to the cells, which in turn drives their transformation. Similar results have been reported for the survivors of x-irradiation and of treatment with chemical carcinogens and are associated with the aging process in animals. We suggest that these changes result from free radical damage to membrane lipids with particular damage to lysosomes. Proteases and nucleases would then be released to progressively modify the growth behavior and genetic stability of the cells toward autonomous proliferation.
在汇合生长限制条件下对NIH 3T3细胞进行长时间培养，会导致一些细胞以凋亡的方式死亡。对存活细胞进行连续几轮汇合后的长时间培养，会以饱和密度增加和转化灶形成增加的形式产生越来越多的肿瘤转化。在研究汇合后培养的细胞的生长特性之前，给予它们不同长度的恢复期，以消除汇合的直接抑制作用。结果发现，随着每一轮汇合，低密度下细胞的指数生长速率会降低，相同细胞的孤立集落大小也会呈现类似的逐渐减小。来自第三轮汇合的细胞的生长速率降低在低密度下持续生长超过60代。汇合后2天恢复后的含巨细胞集落比例比7天恢复后的要高得多。低密度下的生长迟缓以及饱和密度增加似乎是同一枚硬币的两面：两者都发生在整个细胞群体中，并且在转化灶形成之前就已出现。我们认为，生长减缓以及巨细胞的形成是由于细胞的遗传性损伤导致的，而这反过来又驱动了它们的转化。对于X射线照射和化学致癌物处理后的幸存者也报道了类似的结果，并且这些结果与动物的衰老过程有关。我们认为，这些变化是由于自由基对膜脂的损伤，特别是对溶酶体的损伤所致。然后蛋白酶和核酸酶会被释放出来，逐渐改变细胞的生长行为和遗传稳定性，使其向自主增殖方向发展。
