Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and apoptotic cell death.
In order to understand the molecular events following oxidative stress, which lead to persistence of lesions in the mtDNA, experiments were performed on normal human fibroblast (NHF) expressing human telomerase reverse transcriptase (hTERT). The formation and repair of H(2)O(2)-induced DNA lesions were examined using quantitative PCR. It was found that NHF hTERTs show extensive mtDNA damage ( approximately 4 lesions/10 kb) after exposure to 200 microm H(2)O(2), which is partially repaired during a recovery period of 6 h. At the same time, the nDNA seemed to be completely resistant to damage. Cell sorting experiments revealed persistent mtDNA damage at 24 h only in the fraction of cells with low mitochondrial membrane potential (Delta Psi m). Further analysis also showed increased production of H(2)O(2) by these cells, which subsequently undergo apoptosis. This work supports a hypothesis for a feed-forward cascade of reactive oxygen species generation and mtDNA damage and also suggested a possible mechanism for persistence of lesions in the mtDNA involving a drop in Delta Psi m, compromised protein import, secondary reactive oxygen species generation, and loss of repair capacity.
Santos
Janine Hertzog
JH
Laboratory of Molecular Genetics and Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Hunakova
L'uba
L
Chen
Yiming
Y
Bortner
Carl
C
Van Houten
Bennett
B
D017209:Apoptosis; D002469:Cell Separation; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000187:drug effects* / Q000235:genetics; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects* / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects*; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology
D004272:DNA, Mitochondrial; D006861:Hydrogen Peroxide; D003546:Cysteine Endopeptidases