Modulating mitochondrial DNA base excision repair to alter cell cycle progression in mammalian cell lines

Previous work from our lab has focused on targeting base excision repair enzymes to the mitochondria to augment mitochondrial DNA (mtDNA) repair and cellular viability. The role of mtDNA damage and repair in cell death, specifically apoptosis, has been described in our lab and in others. However, other events occur prior to the initiation of apoptosis in a cell. For example, the arrest of the cell cycle, which is the multistage process by which a cell divides and replicates, can occur prior to cell death in the event of nuclear DNA damage. Due to the importance of mtDNA in ATP production and the importance of ATP to fuel a cell cycle progression, we asked whether mtDNA damage could cause a cell cycle arrest. Using quantitative alkaline Southern blots, we found that two reactive oxygen species generators, 1-methyl-4-phenyl-pyridinium (MPP+) and menadione, produced detectable mtDNA damage in the RCSN-3 cells, a dopaminergic rat cell line. Interestingly, RCSN-3 cells responded to MPP+, albeit at extremely high doses, and menadione by initiating a cell cycle arrest. In addition, similar experiments were conducted in a non-related mammalian cell line to determine if this effect was cell-type dependent. HeLa cells, a human cervical cancer cell line, were utilized to draw comparisons. In HeLa cells, menadione exposure resulted in mtDNA damage that correlated with a cell cycle arrest. To determine if mtDNA damage was causatively linked to observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein. The protein contains 11 amino acids from the HIV-Tat protein to enable protein transduction and the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase (MnSOD) to target the repair protein to the mitochondria. The experiments were performed in HeLa cells to modulate specifically repair of mtDNA and to visualize changes in cell cycle progression. Results demonstrated that the transduction of MTS-hOGG1-Tat into HeLa cells alleviates a cell cycle block following an oxidative insult. Finally, the mechanism of the mtDNA-regulated cell cycle arrest was determined. These experiments utilized the hOGG1 fusion protein to determine if the mechanism of the mtDNA-regulated cell cycle arrest was an ATP-dependent response or if a cell signaling pathway was mediating the effect. The results showed that the mtDNA-regulated cell cycle arrest is an ATP-independent, Chk2-associated cell cycle block. In addition, enhancing mtDNA repair with the hOGG1 fusion protein was shown to decrease the level of Chk2 phosphorylation following menadione exposure in HeLa cells. In conclusion, we found that mtDNA is a regulator of cell cycle progression in mammalian cells and that modulating mtDNA repair could alter cell cycle progression, suggesting applications toward cancer therapy or human development.