Expression and regulation of RAD51 mediate cellular responses to chemotherapeutics.

There is evidence that RAD51 expression associates with resistance to commonly used chemotherapeutics. Our previous work demonstrated that inhibitors of thymidylate synthase (TS) induced RAD51-dependent homologous recombination (HR), and depleting the RAD51 recombinase sensitized cells to TS inhibitors. In this study, the consequences of RAD51 over-expression were studied. Over-expression of wild-type RAD51 (∼6-fold above endogenous RAD51) conferred resistance to TS inhibitors. In contrast, over-expression of a mutant RAD51 (T309A) that is incapable of being phosphorylated rendered cells more chemosensitive. Moreover, over-expression of the T309A mutant acted in a dominant negative manner over endogenous RAD51 by causing the reduced localization of RAD51 foci following treatment with TS inhibitors. To measure the effect of mutant RAD51 on the cellular response to other DNA damaging chemotherapeutics, the topoisomerase poison etoposide was utilized. Cells over-expressing wild-type RAD51 showed reduced DNA strand breaks, while cells over-expressing the mutant RAD51 showed more than twice as many strand breaks, suggesting that the mutant RAD51 was actively inhibiting strand break resolution. To directly demonstrate an effect on HR, wild-type RAD51 and T309A mutant RAD51 were transiently expressed in HeLa cells that contained an HR reporter construct. HR events provoked by DNA breaks induced by the I-SceI endonuclease increased in cells expressing wild-type RAD51 and decreased in cells expressing the T309A mutant. Collectively, the data suggest that interference with the activation of RAD51-mediated HR represents a potentially useful anticancer target for combination therapies.