Chow M, Rubin H
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3206, USA.
Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9793-8. doi: 10.1073/pnas.93.18.9793.
Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in a persistent impairment of proliferation when the cells are subcultured at low density and a greatly increased probability of neoplastic transformation in assays for transformation. These properties, along with the large accumulation of age pigment bodies in the confluent cells, are cardinal cellular characteristics of aging in organisms and validate the system as a model of cellular aging. Two cultures labeled alpha and beta were obtained after prolonged confluence; both were dominated by cells that were both slowed in growth at low population density and enhanced in growth capacity at high density, a marker of neoplastic transformation. An experiment was designed to study the reversibility of these age-related properties by serial subculture at low density of the two uncloned cultures and their progeny clones derived from assuredly single cells. Both uncloned cultures had many transformed cells and a reduced growth rate on subculture. Serial subculture resulted in a gradual increase in growth rates of both populations, but a reversal of transformation only in the alpha population. The clones originating from both populations varied in the degree of growth impairment and neoplastic transformation. None of the alpha clones increased in growth rate on low density passage nor did the transformed clones among them revert to normal growth behavior. The fastest growing beta clone was originally slower than the control clone, but caught up to it after four weekly subcultures. The other beta clones retained their reduced growth rates. Four of the five beta clones, including the fastest grower, were transformed, and none reverted on subculture. We conclude that the apparent reversal of impaired growth and transformation in the uncloned parental alpha population resulted from the selective growth at low density of fast growing nontransformed clones. The reversal of impaired growth in the uncloned parental beta population was also the result of selective growth of fast growing clones, but in this case they were highly transformed so no apparent reversal of transformation occurred. The clonal results indicate that neither the impaired growth nor the neoplastic transformation found in aging cells is reversible. We discuss the possible contribution of epigenetic and genetic processes to these irreversible changes.
在汇合生长限制条件下对NIH 3T3细胞进行长时间培养，当细胞以低密度传代培养时，会导致增殖持续受损，并且在转化试验中发生肿瘤转化的可能性大大增加。这些特性，连同汇合细胞中大量积累的老年色素体，是生物体衰老的主要细胞特征，并验证了该系统作为细胞衰老模型的有效性。长时间汇合后获得了标记为α和β的两种培养物；两者都以在低细胞密度下生长缓慢且在高密度下生长能力增强的细胞为主，这是肿瘤转化的一个标志。设计了一项实验，通过对两种未克隆培养物及其源自确定单细胞的子代克隆进行低密度连续传代培养，来研究这些与年龄相关特性的可逆性。两种未克隆培养物都有许多转化细胞，传代培养时生长速率降低。连续传代培养导致两个群体的生长速率逐渐增加，但仅α群体中的转化现象发生了逆转。源自两个群体的克隆在生长受损程度和肿瘤转化方面存在差异。α克隆在低密度传代时生长速率均未增加，其中的转化克隆也未恢复到正常生长行为。生长最快的β克隆最初比对照克隆慢，但在每周传代四次后赶上了对照克隆。其他β克隆保持其降低的生长速率。五个β克隆中有四个，包括生长最快的那个，发生了转化，传代培养时均未逆转。我们得出结论，未克隆的亲代α群体中生长受损和转化的明显逆转是由于快速生长的未转化克隆在低密度下的选择性生长。未克隆的亲代β群体中生长受损的逆转也是快速生长克隆选择性生长的结果，但在这种情况下它们高度转化，因此没有明显的转化逆转发生。克隆结果表明，衰老细胞中发现的生长受损和肿瘤转化均不可逆。我们讨论了表观遗传和遗传过程对这些不可逆变化可能的贡献。
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