The Roles Of DNA Repair Genes In Cellular Response To Oxidative Stress

Oxidative stress caused by reagents used in tumor chemo- and radiotherapy is an important secondary reaction leading to cell death additional to direct killing effects of those reagents. Under oxidative stress which causes imbalance of the intracellular oxidation-reduction steady state, reactive oxidative species (ROS) are overwhelmingly produced by the mitochondria and render oxidative lesions to biological macromolecules such as nucleic acids, proteins and lipids which consequently cause apoptosis and necrosis. Oxidative DNA damage, which directly cause mutations and closely related to pathological status including tumorigenesis, ageing and inflammation, are diminished by the cellular DNA repair pathways to maintain the genomic integrity. There are seven DNA repair pathways and only one of which is mainly in charge of the DNA oxidative lesions caused by ROS, the base excision repair (BER). APE1 is the rate-limiting enzyme of BER. The fact that ROS is mainly produced by electron transportation chain (ETC) in proximity to mtDNA, makes mtDNA as a critical target of ROS. Previous reports demonstrated that APE1 translocalizes to mitochondria from nuclei after oxidative stress and mitochondrial function is significantly down regulated in APE1 deficient cell, however, the detailed mechanisms are still unknown. Therefore, it is of importance to investigate the mechanisms of APE1 mitochondrial targeting and regulation of mitochondrial function by APE1 after oxidative stress. Additionally, recent studies indicated that DNA damage response (DDR) pathway involves in the cellular fate decision after oxidative stress. In response to double strand break, DDR pathway normally coordinates DNA repair, apoptosis and cell cycle arrest after sever DNA damage. However, it is thought inactivated in the early phase after oxidative stress. Actually a growing body of evidences indicated that the upstream kinase in DDR pathway can be activated after oxidative stress with the mechanism unknown. Objective1. To investigate the mechanism of APE1 mitochondrial targeting by identifying the mitochondrial targeting sequence (MTS) and the key amino acids to its mitochondrial targeting;2. To investigate the mechanism of mitochondrial regulatory role of APE1, especially regulation of mitochondria related genes expression though a redox dependent activity;3. To explore the impact of APE1 on gene expression post oxidative stress, based on the whole genome expression chip, sheding light on the systematically analysis of the role of APE1 in oxidative stress;4. To investigate the crosstalk between ATM and DNA-PKcs in the early phase of oxidative stress and their different roles in cell fate decision after oxidative stress.Materials and Methods1. Study of APE1 mitochondrial targeting mechanism: The hotspots of APE1, which possess high affinities to Tom receptors, were screened out by an APE1 peptide array. Then construct the truncated or mutated APE1 expression vector fused with EGFP. The subcellular localization of exogenously expressed proteins was traced by laser confocal microscopy and western blots to identify the MTS of APE1 and the critical sites of it.2. Study of mitochondrial regulatory role of APE1 through a redox dependent mechanism: This study was based on the APE1 stable knockdown and mutant knockin cell lines generated by Prof. Gianluca Tell. Firstly,mitochondrial membrane potential was measured in cells with different APE1 status. Secondly, expression of mitochondria-related genes were tested by quantitative RT-PCR, followed by the DNA binding activity and transcriptional activity assay of mitochondria-related nuclear transcriptional factor, NRF-1 by EMSA and ChIP separately. Consequently, siRNA specific to NRF-1 was employed to confirm the mediate role of NRF-1 in mitochondrial functional regulation by APE1.3. Genome-wide analysis of APE1 function in oxidative stress: Based on the whole genome mRNA expression array, both HeLa cell derived APE1 proficient (Scr-1) and deficient (CL.3) cell lines at 6 hour post hydrogen peroxide treatment andγ-ray irradiation were assayed for gene expression differences. The array data were then statistically processed and subjected to analysis through the Ingenuity IPA system.4. Study of the roles of DNA damage response pathway in cellular response to oxidative stress: Gene knockout HCT116 cell lines using gene targeting strategy were used in this study. Firstly, differential activation of ATM and its downstream participants following hydrogen peroxide treatment in DNA-PKcs knockout cell, Ligase 4 knockout cell and their parental cell were tested by phosphorylation-specific western blot. Secondly, ROS productions in different cells were measured using flow cytometry analysis of DCF fluorescent probe. Consequently, differences in apoptosis were assayed by flow cytometry analysis of AnnexinV/7-AAD staining and cell proliferation capacities were assayed by colony formation.Results1. Study of APE1 mitochondrial targeting mechanism: According to the previous results, the hotspots of APE1 which may be the potential MTS are: 13-39, 49-63, 211-240, 238-258, 265-279 and 289-312 amino acids. A series of truncated APE1 expression vectors fused with EGFP at their C terminus were generated. It indicated that with the removal of the N terminal sequence of APE1, the subcellular localization of truncated APE1 is from nuclear to mitochondrial. When most of the N terminal sequence of APE1 was removed, almost all of the exogenously expressed truncated APE1 is localized to mitochondria, especially when there only were 289-318 left. These results indicated that the MTS of APE1 may be positioned in 289-318 amino acids at tis C terminus. Consequently, when mutated the K299 and R301 sites to Alanine, the mitochondrial targeting of APE1 disappeared indicating that K299/R301 are critical to mitochondrial targeting of APE1. Finally, we found that when transfected with vector containing K299A/R301A, cells were more susceptible to ROS.2. Study of mitochondrial regulatory role of APE1 through a redox dependent mechanism: The mitochondrial membrane potential in Hela cell with deficient or mutated APE1 expression were significantly loss after hydrogen peroxide or rotenone treatment, when compared with wildtype Hela cell. The mitochondria related genes, including TFAM, COX5b and Tom20 which also are downstream genes of NRF-1 were downregulated at mRNA level in APE1 deficient or mutated cells by quantitative RT-PCR. EMSA and ChIP assay showed that the DNA binding and transcriptional activity of NRF-1 was also reduced in APE1 deficient or mutated cells. Co-IP assay indicated that there was a direct interaction between APE1 and NRF-1 protein. We consequently seek the correlation of the mitochondrial regulatory role of APE1 and NRF-1 by using the siRNA against to NRF-1. The results showed that the in NRF-1 knocked down cell, APE1 overexpression was not restored the TFAM mRNA expression.3. Genome-wide analysis of APE1 function in oxidative stress: The APE1 deficiency mainly related to cancer and cell death/apoptosis process. When treated with hydrogen peroxide, APE1 deficiency leads to major gene expression alteration, in the meantime, when treated withγ-ray, APE1 deficiency leads much less genes undergoing transcriptional alteration.4. Study of the roles of DNA damage response pathway in cellular response to oxidative stress: Following hydrogen peroxide induced oxidative stress, the phosphorylation of ATM and its downstream are higher in DNA-PKcs deficient cell which has lower ATM protein level than wildtype and Ligase4 deficient cell. Theγ-H2AX foci after ROS treatment formed between 30 min-1 hr post treatment indicating the activation of ATM is DSB independent. Flow cytometry assay of DCF fluorescence intensity demonstrated that the ROS production is significantly higher in DNA-PKcs deficient cell than in Ligase 4 deficient cell or wildtype cell. Apoptosis in DNA-PKcs deficient cell was elevated and survival is reduced when compared to Ligase 4 deficient cell or wildtype cell which was assayed by flow cytometry and colony formation separately.Conclusion1. The MTS of APE1 is positioned at 289-318 aa at its N terminus. The K299 and R301 are two critical sites for mitochondrial targeting of APE1, and it significantly increase the cellular sensitivity to oxidative stress by blocking APE1 mitochondrial targeting.2. APE1 is critical for mitochondrial function after oxidative stress. The redox activity of APE1 is important in this regulatory process by controlling the DNA binding and transcriptional activity of NRF-1.3. DNA-PKcs is a key player in the regulation of ATM activation after oxidative stress. DNA-PKcs is also a key gene in response to oxidative stress regarding its regulatory role of ROS production. DNA-PKcs plays a pivotal role in cell fate decision after oxidative stress.