SUMMARY

Beta-lapachone (b-lap) is an anticancer agent that selectively induces cell death in several human cancer cells. The mechanism of b-lap cytotoxicity is not yet fully understood. For that reason, we have studied b-lap toxicity using Saccharomyces cerevisiae as experimental model system. 

b-lap treatment delayed cell cycle progression at the G1/S transition, incremented phosphorylation of the Rad53p checkpoint kinase and decreased cell survival in the budding yeast Saccharomyces cerevisiae. Furthermore, b-lap induced phosphorylation of histone H2A at serine 129. These checkpoint responses were regulated by Mec1p and Tel1p kinases. Mec1p was required for Rad53p/histone H2A phosphorylation and cell survival following b-lap treatment in asynchronous cultures, but not for the G1/S delay. The tel1b mutation increased sensitivity to b-lap in a mec1 defective strain and compromised checkpoint responses. Both Rad53p phosphorylation and G1/S delay were fully dependent on a functional Mre11p-Rad50p Xrs2p (XMR) complex, and mutants in the XMR complex were hypersensitive to b-lap treatment. Finally, XRS2 and TEL1 worked epistatically regarding b-lap sensitivity and Xrs2p was phosphorylated in a Tel1p-dependent manner after b-lap treatment. 

b-lap treatment also generated reactive oxygen species (ROS) which where efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases (NADH-DH). Dicoumarol treatment did not affect viability or the checkpoint responses triggered by the drug. We have identified a mutant, defective in the mitochondrial NADH-DH Nde2p which was resistant to b-lap toxicity.  b-lap elicited ROS production in this mutant, however affecting neither cell viability nor cell cycle progression. The nde2b mutant exhibited a delayed cell cycle entry in S-phase and hypersensitivity to DNA damaging agents. Therefore, these data indicate that Nde2p is the principal determinant of b-lap toxicity in budding yeast. 

General control of nutrients pathway is activated after b-lap treatment. Furthermore, Gcn2p kinase modulates b-lap toxicity and checkpoint responses to treatment in an XMR dependent manner, indicating that the Gcn2p kinase contributes to DNA damage checkpoint control in budding yeast. Moreover, Gcn2p regulated checkpoint function by mechanisms other than eIF2b phosphorylation, suggesting that Gcn2p may have additional functions besides regulating translation.

Taken together, these findings indicate that b-lap activates an Mre11-Tel1p checkpoint pathway in budding yeast via an Nde2p-dependent manner. The evidence presented here is consistent with Nde2p being the molecular target of b-lap, and highlights an unexpected connection between DNA repair pathways, Gcn2p kinase and mitochondrial function.