Endo A, Kano Y, Mihara K, Orita K, Namba M
Department of Cell Biology, Okayama University Medical School, Japan.
J Cancer Res Clin Oncol. 1993;119(9):522-6. doi: 10.1007/BF01686461.
Genetic analysis was carried out in human fibroblasts (KMST-6) immortalized by treatment with 60Co gamma rays in order to determine if any genetic change was involved in the immortal transformation of human cells. Analysis by restriction fragment length polymorphism revealed an alteration in chromosome 13q12-14, in which the retinoblastoma (RB) gene locus (13q14) is located. Then the RB gene itself was examined. Structural abnormalities in the RB gene were detected by Southern blot analysis. Furthermore, abnormal RB protein (pRB) was expressed in immortalized KMST-6 cells, as shown by in vitro phosphorylation, whereas normal KMS-6 cells expressed the intact pRB. These findings indicated that inactivation of the RB gene is one of the key events of the immortalization of human cells.
为了确定人类细胞永生化转化过程中是否涉及任何基因变化，对经60Coγ射线处理后永生化的人成纤维细胞（KMST - 6）进行了基因分析。通过限制性片段长度多态性分析发现13q12 - 14染色体发生改变，视网膜母细胞瘤（RB）基因座（13q14）位于该区域。随后对RB基因本身进行了检测。通过Southern印迹分析检测到RB基因存在结构异常。此外，体外磷酸化实验表明，永生化的KMST - 6细胞中表达异常RB蛋白（pRB），而正常KMS - 6细胞表达完整的pRB。这些发现表明，RB基因失活是人类细胞永生化的关键事件之一。
