Cell cycle checkpoint after DNA damage during in vitro hepatocarcinogenesis

对正常肝细胞、永生肝细胞和恶性转化肝细胞DNA损伤后的细胞周期检查点进行了研究，澄清了以下几点：从小鼠肝细胞原代培养过程中形成的肝细胞菌落中分离出许多不朽的肝细胞克隆。通过将突变激活的H-ras基因引入这些细胞，也产生了许多恶性转化细胞系。利用这些细胞以及原代肝细胞培养物，可以比较具有相同背景的正常细胞、不死细胞和恶性细胞的细胞周期检查点。正常肝细胞暴露于DNA损伤剂后，细胞生长完全受到抑制，细胞周期停留在G1期。在此期间，p53的半衰期明显延长，p53在细胞核中过表达。利用p53反义寡核苷酸抑制p53蛋白可消除G1阻滞。虽然正常细胞和不死细胞在紫外线损伤后出现G1阻滞，但在h -转化细胞中却不那么明显。在所有恶性细胞中p53均未发生突变，紫外线损伤后p53出现过表达。这些观察结果表明，p53通路的下游可能被h -ras敲除。恶性细胞的染色体数目变异比非恶性细胞大得多，这表明细胞周期检查点的缺失可能导致染色体不稳定。

Cell cycle checkpoint was investigated after DNA damage in normal, immortal and malignantly-transformed hepatocytes, and following points were clarified.1. Many immortal hepatocyte clones were isolated from hepatocyte colonies developing during primary cultures of mouse hepatocytes. By introducing the mutationally-activated H-ras gene to these cells, many malignantly-transformed cell lines were also produced. Using these cells as well as primary hepatocyte cultures, it became possible to compare cell cycle checkpoint in normal, immortal and malignant cells carrying the same background.2. When normal hepatocytes were exposed to DNA damaging agents, cell growth was completely inhibited, and cell cycle was arrested at G1 phase. During this period, half life of p53 was markedly extended, and p53 was overexpressed in the nuclei. Suppression of p53 protein using p53 antisense oligonucleotide resulted in abrogation of G1 arrest.3. Although normal and immortal cells showed G1 arrest after UV damage, it was much less evident in H-rastransformed cells.4. There was no p53 mutations in any of malignant cells, and overexpression of p53 occurred after UV damage. These observations indicate that downstream of the p53 pathway may be knockout by H-ras.5. Malignant cells show much greater variation of chromosomal numbers than immortal cells, suggesting that loss of cell cycle checkpoint can result in chromosomal instability.