RESUMO
Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.
Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fase S/fisiologia , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Quinase do Ponto de Checagem 2 , Dano ao DNA/efeitos dos fármacos , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA/efeitos dos fármacos , DNA Mitocondrial/genética , Células HeLa , Humanos , Mitocôndrias/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fase S/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Vitamina K 3/farmacologia , Vitaminas/farmacologiaRESUMO
Scleroderma is a chronic disease characterized by excessive tissue fibrosis. Recent studies indicate that cultured dermal fibroblasts isolated from patients produce excessive amounts of collagen and other extracellular matrix components. In this study, we investigated the mechanism(s) of abnormal extracellular matrix accumulation in the scleroderma biopsies and the healing wounds of Tsk1/+ mice. Full-thickness excisional wounds were made in Tsk1/+ and wild-type mice and were subsequently harvested at days 7, 10, and 14 postinjury. The levels of pro-fibrotic cytokine, transforming growth factor were elevated in the wounds of Tsk1/+ mice. Interestingly, the levels of matrix metalloproteinase were significantly reduced in the granulation tissue of Tsk1/+ mice in comparison with wild-type. Furthermore, immunohistochemical analysis of the wounds indicated that the levels of gamma-glutamyl-epsilon-lysine cross-links were elevated in the granulation tissue of Tsk1/+ mice as well as the fibrotic lesions of scleroderma specimens. Collectively, these findings indicate that elevated collagen synthesis and decreased matrix metalloproteinase levels, in combination with increased isopeptide bond cross-links, contribute to abnormal collagen synthesis and assembly in granulation tissue of Tsk1/+ mice and the fibrotic lesions of scleroderma patients.
