ORIGINAL ARTICLE Histone H2AX is a critical factor for cellular protection against DNA alkylating agents JA Meador, M Zhao, Y Su, G Narayan, CR Geard and AS Balajee Department of Radiation Oncology, College of Physicians and Surgeons, Center for Radiological Research, Columbia University Medical Center, New York, NY, USA Histone H2A variant H2AX is a dose-dependent suppressor of oncogenic chromosome translocations. H2AX partici- pates in DNA double-strand break repair, but its role in other DNA repair pathways is not known. In this study, role of H2AX in cellular response to alkylation DNA damage was investigated. Cellular sensitivity to two monofunctional alkylating agents (methyl methane sulfonate and N-methyl- N 0 -nitro-N-nitrosoguanidine (MNNG)) was dependent on H2AX dosage, and H2AX null cells were more sensitive than heterozygous cells. In contrast to wild-type cells, H2AX-deficient cells displayed extensive apoptotic death due to a lack of cell-cycle arrest at G 2 /M phase. Lack of G 2 /M checkpoint in H2AX null cells correlated well with increased mitotic irregularities involving anaphase bridges and gross chromosomal instability. Observation of elevated poly(ADP) ribose polymerase 1 (PARP-1) clea- vage suggests that MNNG-induced apoptosis occurs by PARP-1-dependent manner in H2AX-deficient cells. Con- sistent with this, increased activities of PARP and poly(ADP) ribose (PAR) polymer synthesis were detected in both H2AX heterozygous and null cells. Further, we demonstrate that the increased PAR synthesis and apoptotic death induced by MNNG in H2AX-deficient cells are due to impaired activation of mitogen-activated protein kinase pathway. Collectively, our novel study demonstrates that H2AX, similar to PARP-1, confers cellular protection against alkylation-induced DNA damage. Therefore, tar- geting either PARP-1 or histone H2AX may provide an effective way of maximizing the chemotherapeutic value of alkylating agents for cancer treatment. Oncogene (2008) 27, 5662–5671; doi:10.1038/onc.2008.187; published online 9 June 2008 Keywords: histone H2AX; poly(ADP) ribose polymer- ase l; base excision repair; alkylation DNA damage; chromosomal instability; mitogen-activated protein kinase Introduction H2AX, a variant form of histone H2A, is rapidly phosphorylated at serine 139 in response to DNA double-strand breaks (DSB) originating from exogenous DNA damage (Rogakou et al., 1998, 1999) replication fork collision (Ward and Chen, 2001; Furuta et al., 2003), shortened telomeres (Takai etal., 2003; d’Adda di Fagagna et al., 2003), apoptosis (Rogakou et al., 2000) and transcription inhibition (Mischo et al., 2005). Phosphorylated histone H2AX, designated as g-H2AX, forms distinct nuclear foci at or in the vicinity of DSB sites (Rogakou et al., 1999). g-H2AX foci colocalize with many DNA damage-signaling compo- nents, including ataxia telangiectasia-mutated (ATM), BRCA1, 53BP1, MDC1, Rad51 and Mre11/Nbs1/ Rad50 complex (Fernandez-Capetillo et al., 2004). g- H2AX is important for recruitment and retention of DSB repair factors (Fernandez-Capetillo et al., 2002; Celeste etal., 2003b) such as Nbs1 (Nijmegen breakage syndrome gene product) and 53BP1 (53 binding protein 1) during the late hours after ionizing radiation (IR). H2AX phosphorylation in response to DSB is mediated by three kinases (Wang etal., 2005) belonging to a super family of phosphatidylinositol kinase-like kinases. These include ATM, ATM- and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-PK). Although ATM and DNA-PK are involved in H2AX phosphor- ylation in an overlapping manner after IR-induced DSB (Stiff et al., 2004), H2AX phosphorylation is mainly mediated by ATR after replicative stress (Ward and Chen, 2001). Two evolutionarily conserved pathways, nonhomo- logous end joining (NHEJ) and homologous recombi- nation (HR), are involved in the repair of DSB in eukaryotes. Although g-H2AX is dispensable for DSB recognition (Celeste et al., 2003b), available data indicate its importance in the modulation of both NHEJ and HR pathways. Elimination of the serine residue at 129 of H2A at the C-terminal region led to NHEJ impairment in yeast (Downs et al., 2000). Also, importance of ser129 of H2A in DNA replication- mediated DSB repair has been demonstrated in Sac- charomyces cerevisiae (Redon et al., 2003). Mice deficient in H2AX are characterized by male-specific infertility, growth retardation and diminished levels of immunoglobulin isotypes (Bassing et al., 2002; Celeste Received 2 January 2008; revised 22 April 2008; accepted 4 May 2008; published online 9 June 2008 Correspondence: Dr AS Balajee, Department of Radiation Oncology, College of Physicians and Surgeons, Center for Radiological Research, Columbia University Medical Center, VC-11, Room 239, 168th Street, 630 West, New York, NY 10032, USA. E-mail: ab836@columbia.edu Oncogene (2008) 27, 5662–5671 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc