title: Chloroethylnitrosourea-induced cell death and genotoxicity: cell cycle dependence and the role of DNA double-strand breaks, HR and NHEJ.
abstract: 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, which co-stained 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 G(1) but increased significantly in S phase and remained at a high level in G(2), 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 O(6)-chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O(6)-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.
authors: Institute of Toxicology, University Medical Center, Mainz, Germany. nikolova@uni-mainz.de
authors: Teodora
authors: T
authors: Nikolova
authors: Frank
authors: F
authors: Hennekes
authors: Anita
authors: A
authors: Bhatti
authors: Bernd
authors: B
authors: Kaina
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis; D016466:CHO Cells; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D059766:DNA End-Joining Repair / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000378:metabolism; D016193:G1 Phase; D016195:G2 Phase; D006657:Histones / Q000378:metabolism; D059765:Homologous Recombination / Q000187:drug effects*; D000072200:Ku Autoantigen; D009336:Necrosis; D015376:Nimustine / Q000633:toxicity*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism; D016196:S Phase
chemical_list: D034961:Antigens, Nuclear; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D006657:Histones; D015376:Nimustine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D051135:Rad51 Recombinase; D000072200:Ku Autoantigen