Mechanism of cell cycle checkpoint in the cells transfected with sense or antisense gadd45 gene

正反义gadd45基因转染细胞的细胞周期检查点机制

• 批准号: 07680585
• 负责人: KODAMA Seiji
• 金额: $ 1.47万
• 依托单位: Nagasaki University
• 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-07680585/
• 关键词: gadd45; cell cycle checkpoint; radiation hypersensitivity; ataxia telangiectasia; ataxoa telangiectasia; G_1チェックポイント

Gadd (growth arrest and DNA damage-inducible) 45 gene is inducible with ionizing radiation and suggested to be involved in the p53 dependent G1 checkpoint pathway. Ataxia telangiectasia (AT) cells are characterized by hypersensitivity to ionizing radiation-induced cell killing and chromosome aberrations. Recently, it has been shown that AT cells have a defect in p53 dependent G1 checkpoint pathway. To understand the defect of G1 checkpoint in AT cells, we studied the relationship between AT phenotype and abnormality of the expression of the gadd45 gene product (GADD45). We examined the time dependent expression of GADD45 after X-irradiation and confirmed that X-ray-induced expression of the GADD45 was severely delayd in AT5BIVA cells compared to the control RKO cells. The basal expression level also diminished in the AT cells. To know the function of the gadd45 gene in the AT cells, we transfected the inducible human gadd45 gene into the AT5BIVA cells and established the AT cells whose GADD45 expression was inducible by isopropyl thiogalactoside (IPTG) treatment. Using this AT cells, we investigated the effect of GADD45 expression on colony forming ability, cell growth, cell cycle arrest and radiation sensitivity. We found that the increased expression of GADD45 reduced plating efficiency to 60% of control and delayd cell growth, confirming that the gadd45 gene functions as a negative growth regulator. The excess GADD45 expression, however, did not cause G1 or G2 arrest. The hypersensitivity to X-rays in the AT cells was not changed after induction of GADD45. The results indicate that the defect in GADD45 expression is not responsible for the abnormality in cell cycle checkpoint and the radiation hypersensitivity in AT cells.

GADD(生长停滞和DNA损伤诱导)45基因在电离辐射诱导下是可诱导的，可能参与了P53依赖的G1检查点通路。共济失调毛细血管扩张症(AT)细胞对电离辐射诱导的细胞杀伤和染色体异常敏感。最近的研究表明，AT细胞在P53依赖的G1检查点通路中存在缺陷。为了了解AT细胞中G1检查点的缺陷，我们研究了AT表型与GADD45基因产物(GADD45)表达异常的关系。我们检测了X射线照射后GADD45的时间依赖性表达，证实了X射线诱导的AT5BIVA细胞中GADD45的表达比对照RKO细胞严重延迟。AT细胞的基础表达水平也降低。为了了解GADD45基因在AT细胞中的功能，我们将诱导型人GADD45基因导入AT5BIVA细胞，建立了异丙基硫代半乳糖苷(IPTG)诱导表达GADD45的AT细胞。利用该AT细胞，我们研究了GADD45的表达对克隆形成能力、细胞生长、细胞周期停滞和辐射敏感性的影响。我们发现，GADD45基因的表达增加使细胞的培养效率降低到对照的60%，并延迟了细胞的生长，证实了GADD45基因起到了负生长调节的作用。然而，过量的GADD45表达并没有导致G1或G2期停滞。经GADD45诱导后，AT细胞对X射线的敏感性无明显变化。结果表明，GADD45表达缺陷不是AT细胞细胞周期检查点异常和辐射敏感性的重要原因。