Human 8-Oxoguanine DNA Glycosylase Suppresses the Oxidative Stress–Induced Apoptosis through a p53-Mediated Signaling Pathway in Human Fibroblasts Cha-Kyung Youn,1,2 Peter I. Song,1,5 Mi-Hwa Kim,1,2 Jin Sook Kim,6 Jin-Won Hyun,7 Sang-Joon Choi,3 Sang Pil Yoon,8 Myung Hee Chung,9 In-Youb Chang,1,4 and Ho Jin You1,2 1Korean DNA Repair Research Center, Departments of 2Pharmacology, 3Obstetrics and Gynecology, and 4Anatomy, Chosun University School of Medicine, Gwangju, South Korea; 5Department of Dermatology, University of Colorado Health Sciences Center, Denver, Colorado; 6Department of Herbal Pharmaceutical Development, Korea Institute of Oriental Medicine, Daejeon, South Korea; 7Department of Biochemistry, College of Medicine, Cheju National University, Jeju-do, South Korea; 8Department of Anatomy, College of Medicine, Seonam University, Jeollabuk-Do, South Korea; and 9Department of Pharmacology, School of Medicine, Seoul National University, Seoul, South Korea Abstract wild-type H1299 at the same experimental condition. Human 8-oxoguanine DNA glycosylase (hOGG1) is the Moreover, the array comparative genome hybridization main defense enzyme against mutagenic effects of analyses revealed that the hOGG1-deficient GM00637 cellular 7,8-dihydro-8-oxoguanine. In this study, we showed more significant changes in the copy number of investigated the biological role of hOGG1 in DNA large regions of their chromosomes in response to H2O2 damage–related apoptosis induced by hydrogen treatment. Therefore, we suggest that although p53is a peroxide (H2O2)–derived oxidative stress. The major modulator of apoptosis, hOGG1 also plays a down-regulated expression of hOGG1 by its small pivotal role in protecting cells against the H2O2-induced interfering RNA prominently triggers the H2O2-induced apoptosis at the upstream of the p53-dependent pathway apoptosis in human fibroblasts GM00637 and human to confer a survival advantage to human fibroblasts and lung carcinoma H1299 cells via the p53-mediated human lung carcinomas through maintaining their apoptotic pathway. However, the apoptotic responses genomic stability. (Mol Cancer Res 2007;5(10):1083–98) were specifically inhibited by hOGG1 overexpression. The p53–small interfering RNA transfection into the Introduction hOGG1-deficient GM00637 markedly inhibited the Various cellular metabolites and exogenous DNA-damaging H O -induced activation of p53-downstream target 2 2 materials routinely induce cellular DNA damage, which, unless proteins such as p21, Noxa, and caspase-3/7, which promptly and properly repaired, can cause simple base or more eventually resulted in the increased cell viability. complex changes including deletions, fusions, translocations, Although the cell viability of hOGG1-knockdown or aneuploidy, resulting in cancer and a variety of genetic H1299 p53null cells was similar to that of the hOGG1 disorders (1). wild-type H1299, after the overexpression of p53the Cellular responses induced by DNA damage include the hOGG1-knockdown H1299 showed the significantly activation of several distinct biochemical pathways (2-5).First, decreased cell viability compared with that of the hOGG1 various DNA repair enzymes are activated to recognize and eliminate the damage.Second, DNA damage stimulates the specific mechanism related to cell cycle checkpoints that arrest cell cycle progression and aid in cellular survival under most circumstances.Third, apoptosis is stimulated by DNA damage Received 12/21/06; revised 6/1/07; accepted 6/6/07. Grant support: Korea Science and Engineering Foundation funded by Korea to eliminate heavily damaged or seriously deregulated cells. Government (Ministry of Science and Technology) grants M1063901001 and Although these DNA damage–induced biochemical pathways M10413010003, and Research Center for Proteinous Materials. function independently under certain circumstances, there are The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in extensive interactions between these reactions.For example, the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. regulatory factors in the checkpoints of cell cycle serve not only Note: P.I. Song and C-K. Youn contributed equally to this work. to delay the cell cycle but also to mediate DNA repair, both Requests for reprints: Ho Jin You, Korean DNA Repair Research Center, Bio Engineering BD 2F, Department of Pharmacology, Chosun University School directly and indirectly (3-6).However, the precise mechanisms of Medicine, 375 Seosuk-Dong, Gwangju 501-759, Republic of Korea. of assessing DNA damage both quantitatively and qualitatively Phone: 82-62-230-6337; Fax: 82-62-230-6586. E-mail: [email protected] Copyright D 2007 American Association for Cancer Research. so as to choose between mediating DNA repair and apoptosis doi:10.1158/1541-7786.MCR-06-0432 are not completely understood. Mol Cancer Res 2007;5(10). October 2007 1083 Downloaded from mcr.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. 1084 Youn et al. Reactive oxygen species are produced as by-products of The suppression of oxidative DNA repair activity has cellular metabolism as well as through exposure to UV, ionizing potential drawbacks; one is that the incomplete repair might radiation, and environmental carcinogens (7).The reactive result in the accumulation of mutagenic lesions in the cellular oxygen species react with DNA to produce a myriad of cytotoxic DNA.This leads to illness, death of cells, and unstoppable and mutagenic base lesions (7).Among the oxidative lesions, excessive cell division resulting in cancer and acceleration 7,8-dihydro-8-oxoguanine (8-oxoG) is the major base damage of aging process (10, 11).The human OGG1 ( hOGG1) gene produced by reactive oxygen species (3).Unlike normal is found on chromosome 3p26.2, and its allelic deletions in guanine, 8-oxoG has the propensity to mispair with adenine this region frequently occur in a variety of human cancers (9). during DNA replication and thereby gives rise to G:C to T:A It is somatically mutated in some cancer cells and is highly transversion mutations (8).Oxidatively modified bases, such as polymorphic between human population groups (12).The 8-oxoG, are mainly repaired through the base excision repair accumulation of 8-oxoG is likely to increase dramatically in pathway, the first steps being the recognition and excision of patients with various neurodegenerative diseases such as the damaged base by a specific DNA glycosylase.The major Parkinson’s disease (13), Alzheimer’s disease (14), or amyo- mammalian enzyme for removing 8-oxoG from DNA is trophic lateral sclerosis (15), which are associated with the 8-oxoguanine-DNA glycosylase (OGG1), which is a bifunc- progressive loss of cells.The hOGG1 level was also found tional enzyme with both 8-oxoG excision activity and weak AP to be lower in the orbitofrontal gyrus and the entorhinal cortex lyase strand incision activity at the abasic sites (9).Following in Alzheimer’s disease patients than in control cases.The the excision of 8-oxoG by OGG1, the resultant abasic site is accumulation of 8-oxoG increased in a majority of large further processed in sequential steps by several enzymes to motor neurons in the amyotrophic lateral sclerosis cases complete repair (2).OGG1 plays important roles in eukaryotes with decreased hOGG1 expression (14).Furthermore, several by preventing the accumulation of oxidative DNA damage, in reports have shown a correlation between the induction of both the nuclear and mitochondrial genomes, thereby suppress- apoptosis by oxidative stress and the repair of oxidative ing carcinogenesis and cell death (10). DNA lesions (16-18).Thus, mutations and deletions in the FIGURE 1. Knockdown of hOGG1 expres- sion by hOGG1-siRNA transfection sensitizes human fibroblast GM00637 cells to H2O2- induced apoptosis. The human fibroblast GM00637 cells were stably transfected with the control-siRNA and hOGG1-siRNA as described in Materials and Methods. The expression of hOGG1 was then analyzed by reverse transcription-PCR using hOGG1- pecific synthetic primers (A) and by Western blot using anti-hOGG1 polyclonal antibodies (B). Data were normalized with GAPDH and a-tubulin expression, respectively. C. Graph- ical plot based on A and B. Columns, mean; bars, SD. Statistically significant differences in OGG1 expression were determined by the ANOVA with P values given for both the overall (P) and the pair-wise comparisons as indicated by asterisks (*, P < 0.05). D. The OGG1 enzyme activities were analyzed by 8-oxoG nicking assay using the parental-, control-siRNA – , and two of hOGG1-siRNA – transfected cell lysates as described in Mate- rials and Methods. Purified hOGG1 and buffer alone served as positive and negative con- trols, respectively. Arrowhead, percent ratios of DNA cleavage products (13-mer), which are analyzed by the AxioVision Rel.4.6 Imag- ing System (Carl Zeiss International). E. Parental-, control-siRNA – , and hOGG1- siRNA – transfected cells were cultured to 70% to 80% confluence, and treated with the indicated concentration of H2O2 for 1 h. The medium was then changed to a fresh one, and the extent of cell death was assayed by trypan blue exclusion 24 h after the treatment. Cell viability was determined as the percentage of total cell number that remained unstained. Points, means from six separate experiments; bars, SD. Mol Cancer Res 2007;5(10). October 2007 Downloaded from