Ryabokon Nadezhda I, Goncharova Rose I, Duburs Gunars, Hancock Ronald, Rzeszowska-Wolny Joanna
Department of Experimental and Clinical Radiobiology, M Sklodowska-Curie Memorial Cancer Center and Institute, Wybrzeze Armii Krajowej 15, 44-101, Gliwice, Poland.
Mutat Res. 2008 Jan 1;637(1-2):173-81. doi: 10.1016/j.mrfmmm.2007.08.005. Epub 2007 Aug 19.
ADP-ribose polymers are rapidly synthesized in cell nuclei by the poly(ADP-ribose) polymerases PARP-1 and PARP-2 in response to DNA strand interruptions, using NAD(+) as precursor. The level of induced poly(ADP-ribose) formation is proportional to the level of DNA damage and can be decreased by NAD(+) or PARP deficiency, followed by poor DNA repair and genomic instability. Here we studied the correlation between poly(ADP-ribose) level and DNA strand break repair in lymphoblastoid Raji cells. Poly(ADP-ribose) synthesis was induced by 100 microM H(2)O(2) and intensified by the 1,4-dihydropyridine derivative AV-153. The level of poly(ADP-ribose) in individual cells was analyzed by quantitative in situ immunofluorescence and confirmed in whole-cell extracts by Western blotting, and DNA damage was assessed by alkaline comet assays. Cells showed a approximately 100-fold increase in poly(ADP-ribose) formation during the first 5 min of recovery from H(2)O(2) treatment, followed by a gradual decrease up to 15 min. This synthesis was completely inhibited by the PARP inhibitor NU1025 (100 microM) while the cells treated with AV-153, at non-genotoxic concentrations of 1 nM-10 microM, showed a concentration-dependent increase of poly(ADP-ribose) level up to 130% after the first minute of recovery. The transient increase in poly(ADP-ribose) level was strongly correlated with the speed and efficiency of DNA strand break rejoining (correlation coefficient r > or = 0.92, p<0.05). These results are consistent with the idea that poly(ADP-ribose) formation immediately after genome damage reflects rapid assembly and efficient functioning of repair machinery.
在细胞核中，聚（ADP - 核糖）聚合酶PARP - 1和PARP - 2可利用NAD⁺作为前体，响应DNA链中断迅速合成ADP - 核糖聚合物。诱导的聚（ADP - 核糖）形成水平与DNA损伤程度成正比，而NAD⁺或PARP缺乏会使其降低，进而导致DNA修复不良和基因组不稳定。在此，我们研究了淋巴母细胞样Raji细胞中聚（ADP - 核糖）水平与DNA链断裂修复之间的相关性。用100微摩尔/升的H₂O₂诱导聚（ADP - 核糖）合成，并通过1,4 - 二氢吡啶衍生物AV - 153增强。通过定量原位免疫荧光分析单个细胞中的聚（ADP - 核糖）水平，并通过蛋白质免疫印迹在全细胞提取物中进行确认，同时通过碱性彗星试验评估DNA损伤。细胞在从H₂O₂处理中恢复的最初5分钟内，聚（ADP - 核糖）形成增加约100倍，随后逐渐下降直至15分钟。这种合成被PARP抑制剂NU1025（100微摩尔/升）完全抑制，而用非基因毒性浓度为1纳摩尔/升至10微摩尔/升的AV - 153处理的细胞，在恢复的第一分钟后显示聚（ADP - 核糖）水平呈浓度依赖性增加，最高可达130%。聚（ADP - 核糖）水平的短暂增加与DNA链断裂重新连接的速度和效率密切相关（相关系数r≥0.92，p<0.05）。这些结果与基因组损伤后立即形成的聚（ADP - 核糖）反映修复机制的快速组装和高效运作这一观点一致。
