Fushimi K, Iijima M, Gao C, Kondo T, Tsuji T, Hashimoto T, Mihara K, Namba M
Department of Cell Biology, Institute of Cellular and Molecular Biology, Okayama University Medical School, Japan.
Int J Cancer. 1997 Jan 6;70(1):135-40. doi: 10.1002/(sici)1097-0215(19970106)70:1<135::aid-ijc20>3.0.co;2-j.
In vitro cell transformation is a valuable approach for studying the mechanisms of multistep carcinogenesis of human cells. Since immortalization is an essential step for in vitro neoplastic transformation of human cells, this study addresses the question of whether mutant p53 contributes to the immortalization process of human cells. The mutant p53 gene (mp53: codon273Arg-His) was introduced into normal human fibroblasts (OUMS-24 line) and a G418-resistant clone, OUMS-24/P6 line, was obtained. This clone showed an extended life span and chromosome abnormalities, but senesced at the 79th population doubling level (PDL). When these cells were subjected to intermittent X-ray treatment, they became an immortalized cell line (OUMS-24/P6X). Although these immortalized cells showed chromosome abnormalities, they were not tumorigenic. On the other hand, normal OUMS-24 cells into which mp53 had not been introduced were not immortalized by the same X-ray treatment. These results indicate that introduction and expression of mp53 alone were not sufficient for immortalization of human cells, and that mutations of the remaining wild-type p53 or other genes may have been necessary for immortalization. In fact, no expression of the wild-type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in an increase in cdk2 and cdc2 kinase activity. These findings indicate that the p53-p21 cascade may play some role in the immortalization of human cells. On the other hand, there was no significant difference in expression of proteins such as Rb, p16, cdk4, cdk6, cyclin A and cyclin D1 between the normal and immortalized human fibroblasts.
体外细胞转化是研究人类细胞多步骤致癌机制的一种有价值的方法。由于永生化是人类细胞体外肿瘤转化的关键步骤，本研究探讨了突变型p53是否有助于人类细胞的永生化过程。将突变型p53基因（mp53：密码子273Arg-His）导入正常人成纤维细胞（OUMS-24系），获得了一个G418抗性克隆OUMS-24/P6系。该克隆显示出延长的寿命和染色体异常，但在第79代群体倍增水平（PDL）时衰老。当这些细胞接受间歇性X射线处理时，它们变成了一个永生化细胞系（OUMS-24/P6X）。尽管这些永生化细胞显示出染色体异常，但它们没有致瘤性。另一方面，未导入mp53的正常OUMS-24细胞在相同的X射线处理下没有永生化。这些结果表明，单独导入和表达mp53不足以使人类细胞永生化，其余野生型p53或其他基因的突变可能是永生化所必需的。事实上，通过RT-PCR在永生化细胞中未检测到野生型p53的表达。位于p53下游的p21的表达在永生化细胞中显著降低，导致cdk2和cdc2激酶活性增加。这些发现表明，p53-p21级联可能在人类细胞的永生化中发挥一定作用。另一方面，正常人和永生化人成纤维细胞之间Rb、p16、cdk4、cdk6、细胞周期蛋白A和细胞周期蛋白Dl等蛋白质的表达没有显著差异。
