Recognition and signaling of DNA double-strand breaks in human cells

Abstract: DNA double-strand breaks (DSBs) are the most toxic type of DNA damage. Fortunately, our cells have highly conserved pathways that detect and repair DSBs. Defects in DSB signaling and DSB repair pathways result in genomic instability that predisposes for cancer development. In mammalian cells DSBs signaling involves ATM (ataxia telangiectasia mutated) and DNA-dependent protein kinase (DNA-PK). However, it remains unclear how these enzymes recognize DNA damage and transmit the signal to downstream effectors. The overall aim of the present study was to study DSB signaling and try to use this knowledge to enhance cancer treatment.To validate the application of the enediyne calicheamicin kappa1 (CLM) in DSB studies, we characterized the cleavage of cellular DNA by CLM. We found that CLM specifically targets DNA without interference from proteins or RNA. The DSBs/SSBs ratio in cellular DNA was 1:3, close to the 1:2 ratio observed when CLM cleaved purified plasmid. CLM-induced DSBs were repaired slowly but completely and resulted in a strong DSB response. The high DSBs/SSBs ratio and specificity for DNA makes CLM a superior drug for DSB response studies.We then used CLM together with other agents that produce different number of DSBs and SSBs in cells, to test if ATM was specifically activated by IR-induced DSBs. We found that ATM was not activated at all in response to SSBs. We also found that the number of DSBs induced by different drugs and radiation correlates closely with the ATM activation, whereas no correlation was found with the number of SSBs. Our data indicate that ATM is directly activated by the few DSBs that are introduced by IR.An early event after introduction of DSBs is the phosphorylation of histone H2AX on serine 139 (HAXS139ph). We have developed a flow cytometry-based method optimized for measuring H2AXS139ph in blood cells. We found that all nucleated blood cells are able to phosphorylate H2AX in response to DSBs, but to different levels. There was a pronounced interindividual difference in the H2AXS139ph signal at a given DSB level, indicating that there is an inherent difference in the DSB response among individuals. This method could be used to monitor patients undergoing chemotherapy or to examine variation in response to treatment among cancer patients.In an effort to develop a specific DNA-PK-inhibitor, we have characterized SU11752. Our data showed that SU11752 inhibited DNA-PK by competitive inhibition of ATP-binding. SU11752 inhibited DSB repair and sensitizes cells 5-fold to IR. At concentrations that inhibited DSB repair, cell cycle progression was normal, and ATM kinase activity was not inhibited. Taken together, our data indicate that SU11752 is a selective DNA-PK inhibitor and defines a new class of drugs that may serve as a starting point for the development of specific DNA-PK inhibitors.