Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ
Volume 11, Issue 14
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July 15, 2012
Pages 2606 - 2619http://dx.doi.org/10.4161/cc.20862
: DNA double-strand breaks, DNA repair, damage response, glioma, homologous recombination, melanoma, nimustine, nitrosoureas, nonhomologous end-joining, sensitivity
Authors: Teodora Nikolova, Frank Hennekes, Anita Bhatti and Bernd Kaina View affiliations
Chloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto- and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci and displayed co-staining with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G1 but increased significantly in S phase and remained at a high level in G2, where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O6-chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O6-chloroethylguanine-triggered cross-links, which give rise to DSBs in the treatment cell cycle, and that HR, but not NHEJ, is the major route of protection against this group of anticancer drugs. Base excision repair had no significant impact on ACNU-induced cytotoxicity.
Received: April 11, 2012; Accepted: May 23, 2012
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