ORIGINAL ARTICLE Targeting human 8-oxoguanine DNA glycosylase to mitochondria protects cells from 2-methoxyestradiol-induced-mitochondria-dependent apoptosis A Chatterjee 1 , X Chang 1 , JK Nagpal, S Chang, S Upadhyay, J Califano, B Trink and D Sidransky Head and Neck Cancer Research Division, Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins University, School of Medicine, Baltimore, MD, USA 2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite of 17b-estradiol, is known to induce mitochon- dria-mediated apoptosis through several mechanisms. We sought to study the effect of mitochondrialy targeted hOGG1 (MTS-hOGG1) on HeLa cells exposed to 2-ME. MTS-hOGG1-expressing cells exposed to 2-ME showed increased cellular survival and had significantly less G 2 /M cell cycle arrest compared to vector-only-transfected cells. In addition, 2-ME exposure resulted in an increase in mitochondrial membrane potential, increased apoptosis, accompanied by higher activation of caspase-3, -9, cleavage of Bid to tBid and protein poly(ADP-ribose) polymerase (PARP) cleavage in HeLa cells lacking MTS- hOGG1. Fas inhibitors cerulenin or C75 inhibited 2-ME- induced caspase activation, PARP cleavage, apoptosis and reversed mitochondrial membrane hyperpolarization, thereby recapitulating the increased expression of MTS-hOGG1. Hence, MTS-hOGG1 plays an important protective role against 2-ME-mediated mitochondrial damage by blocking apoptosis induced through the Fas pathway. Oncogene (2008) 27, 3710–3720; doi:10.1038/onc.2008.3; published online 4 February 2008 Keywords: hOGG1; mitochondria; 2-methoxyestradiol; Fas activation Introduction Mitochondrion appears to be the key regulator of programmed cell death. Mitochondrial DNA (mtDNA) is constantly subjected to damage by many cellular processes such as spontaneous depurination or deami- nation and by endogenous and environmental mutagens (Krokan et al., 2000) and also through replication and repair errors (Chi and Kolodner, 1994). Because of the lack of protective histones, the accumulation of muta- tions in mitochondria is approximately 10-fold higher than that in nuclear DNA (Lightowlers et al., 1997; Yakes and Van Houten, 1997; Singh et al., 2001). A wide range of DNA lesions that include single- and double-strand DNA breaks, apurinic–apyrimidinic (AP) sites, DNA-protein cross-links and oxidized DNA bases are a result of predisposition of mtDNA to chemical damage (Dizdaroglu, 1991; Cadet et al., 1997). One of the important mtDNA repair mechanisms is base excision repair (BER), and the detection of various BER enzymes in mitochondria highlights the impor- tance of maintaining mtDNA integrity for normal cellular functioning (Bohr et al., 2002; Shokolenko et al., 2003). Likewise, mtDNA integrity has been targeted for enhanced cancer chemotherapy by over- expressing Escherichia coli exonuclease III or N-methyl- purine DNA glycosylase in the mitochondria of human breast cancer cells (Fishel et al., 2003; Shokolenko et al., 2003). 2-Methoxyestradiol (2-ME) is an endogenous meta- bolite of 17b-estradiol and has been reported to induce mitochondria-mediated apoptosis. Several mechanisms have been implicated including inhibition of mitochon- drial respiration, accumulation of cells in the G 2 /M phase and inhibition of mitochondrial superoxide dismutase (Huang et al., 2000; Lambert et al., 2002, 2004; Qanungo et al., 2002; Djavaheri-Mergny et al., 2003a,b; Pelicano et al., 2003; Hagen et al., 2004). Recent studies by Achanta and Huang (2004) demon- strated that 2-ME treatment in colon cancer cell lines led to a significant increase in 8-oxoG levels (also known as 7,8-dihydro-8-oxoguanine or 8-hydroxyguanine). It has been shown that the presence of 8-oxoG results in the incorporation of deoxyadenosine triphosphate opposite 8-oxoG during the replication yielding G:C to T:A transversions (Wood et al., 1990; Moriya et al., 1991; Cheng et al., 1992; Grollman and Moriya, 1993). Since 8-oxoG constitutes a premutagenic lesion, efficient repair mechanisms are vital to prevent these lesions from becoming permanent mutations (Grollman and Moriya, 1993; Tajiri et al., 1995). To maintain proper genetic integrity and to minimize cancer risk, organisms ranging from E. coli to humans have evolved elaborate mechanisms for repairing 8-oxoG (Michaels and Miller, 1992; Hazra et al., 2001). In humans, 8-oxoG incorpo- rated into DNA is removed by hOgg1, a DNA Received 10 September 2007; revised 30 November 2007; accepted 21 December 2007; published online 4 February 2008 Correspondence: Professor D Sidransky, Head and Neck Cancer Research Division, Department of Otolaryngology-Head and Neck Surgery, 1550 Orleans Street, Johns Hopkins University School of Medicine, Cancer Research Building II, Baltimore, MD 21231, USA. E-mail: dsidrans@jhmi.edu 1 These authors contributed equally to this work. 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