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

Gadd (growth arrest and DNA damage-inducible) 45基因可被电离辐射诱导，并被认为参与了p53依赖性G1检查点通路。共济失调毛细血管扩张（AT）细胞的特点是对电离辐射诱导的细胞杀伤和染色体畸变高度敏感。最近，有研究表明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，并证实与对照RKO细胞相比，x射线诱导的GADD45在AT5BIVA细胞中的表达严重延迟。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，我们将可诱导的人gadd45基因转染到AT5BIVA细胞中，建立了经异丙基硫半乳糖苷（IPTG）处理后可诱导表达gadd45的AT细胞。利用这种AT细胞，我们研究了GADD45表达对集落形成能力、细胞生长、细胞周期阻滞和辐射敏感性的影响。我们发现GADD45表达的增加使镀效率降低到对照组的60%，并延缓了细胞生长，证实了GADD45基因作为负生长调节剂的功能。然而，过量的GADD45表达并未引起G1或G2阻滞。GADD45诱导后AT细胞对x射线的超敏反应未发生改变。结果表明，GADD45表达缺陷与AT细胞周期检查点异常和辐射超敏反应无关。

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.