Rubin A L, Ellison B J
Virus Laboratory, University of California, Berkeley 94720.
Carcinogenesis. 1991 Oct;12(10):1801-6. doi: 10.1093/carcin/12.10.1801.
The NIH3T3 cell line undergoes transformation when the cells are maintained at high densities for extended periods. In an effort to define the physiological status of the cells before and during the transformation process, they were seeded in medium containing 2, 5 or 10% calf serum (CS) and growth was monitored by measurements of cell numbers and by incorporation of [3H]thymidine into DNA. Transformed foci began to appear by 7 days in 2% CS, at least 3 days after the onset of density-dependent growth inhibition, and by 10 days in 5% CS, 6 days after the onset of growth inhibition. The rate of DNA synthesis in both serum concentrations had declined by 7 days to less than 1/15th of the initial rate measured by day 2. Transformation did not occur in cells exposed to 10% CS which, though supporting even higher saturation densities, underwent a 100-fold decline in DNA synthesis after reaching confluence. Transfer and regrowth in 2% CS of cells from all three serum concentration revealed a steep increase in capacity for focus formation of cells previously cultured in 2 and 5% CS after they had reached confluence, but no such increase in cells previously cultured in 10% CS, indicating that the lack of foci in the original 10% CS incubation was due to suppression of transformation and not to masking of foci at very high cell densities. Determination of the fraction of dividing cells by [3H]thymidine autoradiography showed a similar decline prior to the appearance of transformed cells in 2% CS. In addition, a reactivation of mitosis in non-focal cells occurred by day 24, indicating that a much higher proportion of cells escaped the moderate growth constraint than might have been expected by the total number of foci formed on transfer. Our results are consistent with the premise that transformation is an adaptive response to moderate growth constraints, but not with a mutational origin.
当NIH3T3细胞系长时间维持在高密度状态时会发生转化。为了确定细胞在转化过程之前和期间的生理状态，将它们接种在含有2%、5%或10%小牛血清（CS）的培养基中，并通过测量细胞数量以及将[3H]胸腺嘧啶核苷掺入DNA来监测生长情况。在2% CS中，到第7天开始出现转化灶，此时密度依赖性生长抑制开始至少3天了；在5% CS中，到第10天出现转化灶，此时生长抑制开始6天了。两种血清浓度下的DNA合成速率到第7天时均下降至第2天测量的初始速率的不到1/15。暴露于10% CS的细胞未发生转化，尽管该浓度支持更高的饱和密度，但细胞汇合后DNA合成下降了100倍。将来自所有三种血清浓度的细胞转移至2% CS中再生长，结果显示，先前在2%和5% CS中培养的细胞在汇合后形成灶的能力急剧增加，但先前在10% CS中培养的细胞没有这种增加，这表明最初在10% CS培养中缺乏灶是由于转化受到抑制，而不是由于在非常高的细胞密度下灶被掩盖。通过[3H]胸腺嘧啶核苷放射自显影法测定分裂细胞的比例，发现在2% CS中转化细胞出现之前也有类似的下降。此外，在第24天时非灶性细胞的有丝分裂重新激活，这表明逃脱适度生长限制的细胞比例比转移时形成的灶总数所预期的要高得多。我们的结果与转化是对适度生长限制的适应性反应这一前提一致，但与突变起源不一致。
