Residual γH2AX foci as an indication of lethal DNA lesions

Background: Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible gamma H2AX 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 gamma H2AX foci when damaged DNA undergoes replication. Methods: 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 gamma H2AX 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. Results: 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 gamma H2AX 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 gamma H2AX 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. Conclusion: Retention of DNA damage-induced gamma H2AX 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 gamma H2AX foci.