Abstract
BackgroundAccumulation of mitochondrial DNA (mtDNA) damage could enhance the frequency of mitochondrial mutations and promote a variety of mitochondria-related diseases, including cancer. However, the mechanism(s) involved are not fully understood.MethodsQuantitative extended length PCR was used to compare mtDNA and nDNA damage in human lung H1299 cells expressing WT Bcl2 or vector-only control. mtAPE1 endonuclease activity was analyzed by AP oligonucleotide assay. mtDNA mutation was measured by single molecule PCR. Subcellular localization of Bcl2 and APE1 was analyzed by subcellular fractionation.ResultsBcl2, an anti-apoptotic molecule and oncoprotein, effectively inhibits the endonuclease activity of mitochondrial APE1 (mtAPE1), leading to significant retardation of mtDNA repair and enhanced frequency of mtDNA mutations following exposure of cells to hydrogen peroxide (H2O2) or nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, a carcinogen in cigarette smoke). Inversely, depletion of endogenous Bcl2 by RNA interference increases mtAPE1 endonuclease activity leading to accelerated mtDNA repair and decreased mtDNA mutation. Higher levels of mtAPE1 were observed in human lung cancer cells than in normal human bronchial epithelial cells (i.e. BEAS-2B). Bcl2 partially co-localizes with APE1 in the mitochondria of human lung cancer cells. Bcl2 directly interacts with mtAPE1 via its BH domains. Removal of any of the BH domains from Bcl2 abolishes Bcl2’s capacity to interact with mtAPE1 as well as its inhibitory effects on mtAPE1 activity and mtDNA repair.ConclusionsBased our findings, we propose that Bcl2 suppression of mtDNA repair occurs through direct interaction with mtAPE1 and inhibition of its endonuclease activity in mitochondria, which may contribute to enhanced mtDNA mutations and carcinogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1594-1) contains supplementary material, which is available to authorized users.
Highlights
Accumulation of mitochondrial DNA damage could enhance the frequency of mitochondrial mutations and promote a variety of mitochondria-related diseases, including cancer
We show that Bcl2 suppresses mitochondrial DNA (mtDNA) repair through direct interaction with apyrimidinic endonuclease 1 (APE1) in mitochondria via its BH domains and inhibition of mitochondrial APE1 (mtAPE1) endonuclease activity, leading to increased frequency of mtDNA mutations following exposure of cells to H2O2 or nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK)
Results mtDNA is more sensitive than nuclear DNA (nDNA) to NNK or H2O2induced damage To test whether NNK induces mtDNA damage, we employed quantitative amplification (QPCR) of long DNA fragments as previously described [9]
Summary
Accumulation of mitochondrial DNA (mtDNA) damage could enhance the frequency of mitochondrial mutations and promote a variety of mitochondria-related diseases, including cancer. The mechanism(s) involved are not fully understood Mitochondria contain their own genome (i.e. mitochondrial DNA, mtDNA), which comprises a small, self-replicating DNA molecule present in multiple copies in the mitochondrial matrix [1]. The human mitochondrial genome is a tiny 16.6 kb circle containing only 37 genes. Thirteen of these genes encode proteins, and the remaining 24 consist of 2 ribosomal RNAs (rRNAs) and 22 tRNAs that are used for translation of those 13 polypeptides [2]. In contrast to nuclear DNA (nDNA), mtDNA is uninterruptedly replicated even in terminally differentiated cells. Somatic mutations of mtDNA are potentially more harmful for cell functions compared to somatic damages of nDNA.
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