Noël Georges, Giocanti Nicole, Fernet Marie, Mégnin-Chanet Frédérique, Favaudon Vincent
Unité 350 INSERM, Institut Curie-Recherche, Bâts, 110-112, Centre Universitaire, 91405 Orsay Cedex, France.
BMC Cell Biol. 2003 Jul 16;4:7. doi: 10.1186/1471-2121-4-7.
The cytotoxicity and the rejoining of DNA double-strand breaks induced by gamma-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair.
PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by gamma-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose) synthesis following gamma-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as gamma-rays and H2O2.
The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to gamma-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose) synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.
在正常和PARP - 1基因敲除的小鼠3T3成纤维细胞中，研究了γ射线、过氧化氢和新制癌菌素诱导的细胞毒性以及DNA双链断裂的重新连接情况，以确定聚（ADP - 核糖）聚合酶（PARP - 1）在DNA双链断裂修复中的作用。
PARP - 1基因敲除的细胞比PARP - 1基因正常的3T3细胞对γ射线和过氧化氢诱导的细胞杀伤更为敏感。然而，在药物浓度低于1.5 nM时，这两种细胞系对新制癌菌素的敏感性没有显著差异。通过逆转录病毒转染完整的PARP - 1 cDNA来恢复PARP - 1基因敲除的3T3细胞中PARP - 1的表达，并未引起新制癌菌素反应的任何变化。此外，新制癌菌素诱导的DNA双链断裂的发生率和重新连接动力学在PARP - 1基因正常和PARP - 1基因敲除的3T3细胞中是相同的。仅在PARP - 1功能正常的细胞中观察到γ射线和过氧化氢后聚（ADP - 核糖）的合成。相反，即使在超致死浓度下，新制癌菌素也无法启动PARP - 1的激活，但它在PARP - 1基因正常和PARP - 1基因敲除的3T3细胞中诱导H2AX组蛋白磷酸化的效率与γ射线和过氧化氢相同。
结果表明，以链断裂重新连接或细胞存活为终点时，PARP - 1不是DNA双链断裂恢复的主要决定因素。尽管PARP - 1和ATM的激活都是细胞对γ射线和过氧化氢反应的主要决定因素，但数据表明，在新制癌菌素诱导的DNA双链断裂的修复途径中，PARP - 1依赖性聚（ADP - 核糖）合成和ATM依赖性H2AX磷酸化并不相互关联。
