Residual gammaH2AX foci as an indication of lethal DNA lesions.
Residual gammaH2AX foci as an indication of lethal DNA lesions.
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DOI：
10.1186/1471-2407-10-4
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发表时间：
2010-01-05
期刊：
影响因子：
3.8
通讯作者：
Olive PL
中科院分区：
文献类型：
--
作者：
Banáth JP;Klokov D;MacPhail SH;Banuelos CA;Olive PL
有证据表明，暴露于某些DNA损伤剂的肿瘤细胞如果保留显微镜可见的γ H2 AX灶（已知标记双链断裂部位），则更有可能死亡。这似乎是真实的，即使暴露于烷化剂MNNG后，不会导致直接的双链断裂，但确实产生γ H2 AX灶时，受损的DNA进行复制。为了进一步检查这种预测能力，将SiHa人宫颈癌细胞暴露于8种DNA损伤药物（喜树碱、顺铂、多柔比星、依托泊苷、过氧化氢、MNNG、替莫唑胺和替拉扎明），并将30或60分钟处理后24小时保留γ H2 AX病灶的细胞分数与丧失克隆形成性的细胞分数进行比较。为了确定具有残留修复灶的细胞是否是死亡的细胞，用RAD 51-GFP构建体稳定转染SiHa宫颈癌细胞，并使用活细胞分析来跟踪具有RAD 51-GFP灶的辐照细胞的命运。对于所有药物，无论其与DNA相互作用的机制如何，在处理后24小时，在克隆形成存活分数和保留γ H2 AX灶的细胞分数之间观察到接近1：1的相关性。最初的研究确定，在照射后保留RAD 51灶的细胞部分与保留γ H2 AX灶并随后丧失克隆形成性的细胞部分相似。跟踪单个辐照的活细胞证实，辐照后24小时具有RAD 51-GFP病灶的SiHa细胞更可能死亡。DNA损伤诱导的γ H2 AX灶的保留似乎是致死性DNA损伤的指示，因此可以简单地通过对保留γ H2 AX灶的细胞分数进行评分来预测各种DNA损伤剂对肿瘤细胞的杀伤。
Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible γH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce γH2AX foci when damaged DNA undergoes replication. To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained γH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci. For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained γH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained γH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die. Retention of DNA damage-induced γH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain γH2AX foci.
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