Role of p53, sdil, cdk, mdm2 genes for immortalization of human fibroblasts.

永生化是体外肿瘤转化人类细胞的重要步骤。为探讨突变型p53基因是否参与人细胞的永生化过程，将突变型p53基因（mp53，密码子275 Arg-His）导入正常人成纤维细胞（OUMS-24），获得了G418抗性克隆OUMS-24/p6。该克隆表达mp53，寿命延长，但在第79个群体倍增水平（PDL）时衰老。当这些细胞用2戈伊的X射线或4-硝基喹啉1氧化物处理时，它们变成永生化的。虽然永生化细胞显示染色体异常，但它们不具有致瘤性。另一方面，在未导入mp53的正常OUMS-24细胞中，通过同样的处理，不能使其永生化，在永生化细胞系中，位于p53下游的p21/cip 1/waf 1/sdi 1的表达显著减少，导致cdk 2和细胞周期蛋白A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。而Rb、p16、cdk 4、cdk 6、cyclin A和cyclin D等蛋白质的表达在正常人成纤维细胞和永生化人成纤维细胞中无明显差异，提示单纯的53基因突变不足以使人成纤维细胞永生化，p53以外的一些未知基因可能参与了人成纤维细胞的永生化过程。

Immortalization is an essential step for in vitro neoplastic transformation of human cells. This study address the question of whether mutant p53 contributes to the immortalization process of human cells.The mutant p53 gene (mp53 ; codon 275Arg-His) was introduced into normal human fibroblasts (OUMS-24), and a G418-resistant clone, OUMS-24/p6, was obtained. This clone expressed mp53 and showed an extended life span, but it senesced at 79th population-doubling level (PDL). When these cells were treated with 2 Gy of x-rays or 4-nitroquinoline 1 oxide, they became immortalized. Although the 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 treatment.Expression of p21/cip1/waf1/sdi1, which is located downstream of p53, was remarkably reduced in the immortalized cell lines, resulting in an increase in cdk2 and cyclin A 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 different expression of proteins such as Rb, p16, cdk4, cdk6, cyclin A and cyclinD between the normal and immortalized human fibroblasts.Taken togehter, our results indicate that mutation of the 53 gene alone was not sufficient for immortalization and some unknown genes other than p53 may be involved in the immortalization process of human cells.