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Cell cycle checkpoint after DNA damage during in vitro hepatocarcinogenesis

体外肝癌发生过程中 DNA 损伤后的细胞周期检查点

基本信息

批准号：
07670229
负责人：
OGAWA Katsuhiro
金额：
$ 0.26万
依托单位：
Asahikawa Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670229/
关键词：
p53 UV H-ras mouse immortal hepatocytes H-ras マウス肝細胞染色体不安定化

项目摘要

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.

本研究通过对正常肝细胞、永生化肝细胞和恶性转化肝细胞DNA损伤后细胞周期检查点的研究，阐明了以下几点.从小鼠肝细胞原代培养过程中形成的肝细胞集落中分离出许多永生化的肝细胞克隆。通过将突变激活的H-ras基因导入这些细胞，也产生了许多恶性转化的细胞系。使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、永生细胞和恶性细胞中的细胞周期检查点。当正常肝细胞暴露于DNA损伤剂时，细胞生长被完全抑制，细胞周期被阻滞在G1期。在此期间，p53的半衰期显着延长，并且p53在细胞核中过表达。用p53反义寡核苷酸抑制p53蛋白表达可使G1期阻滞消失.虽然正常细胞和永生细胞在紫外线损伤后表现出G1期阻滞，但在H-ras转化的细胞中这种现象要不那么明显.在所有恶性肿瘤细胞中均未发现p53基因突变，紫外线损伤后p53基因出现过度表达。这些观察结果表明p53通路的下游可能被H-ras基因敲除。恶性细胞染色体数目的变异比永生细胞大得多，这表明细胞周期检查点的丢失可导致染色体不稳定。