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Cell sorting experiments link persistent mitochondrial DNA damage with loss of mitochondrial membrane potential and
Janine Hertzog Santos1, L'uba Hunakova, Yiming Chen
1Laboratory 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.
Abstract:
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.
Insights
Oxidative stress causes extensive mitochondrial DNA (mtDNA) damage in human cells. Persistent mtDNA lesions are linked to decreased mitochondrial membrane potential and apoptosis, suggesting a self-perpetuating damage cycle.
Area of Science:
- Cellular biology
- Molecular biology
- Genetics
Background:
- Oxidative stress is implicated in aging and disease.
- Mitochondrial DNA (mtDNA) is vulnerable to oxidative damage.
- Understanding mtDNA lesion formation and repair is crucial.
Purpose of the Study:
- To investigate molecular events of oxidative stress-induced mtDNA damage.
- To examine the repair capacity of mtDNA in normal human fibroblasts (NHF) expressing human telomerase reverse transcriptase (hTERT).
- To elucidate mechanisms behind persistent mtDNA lesions.
Main Methods:
- Exposure of NHF hTERTs to hydrogen peroxide (H(2)O(2)) to induce oxidative stress.
- Quantitative PCR to assess DNA lesion formation and repair.
- Cell sorting based on mitochondrial membrane potential (Delta Psi m).
Main Results:
- NHF hTERTs sustained significant mtDNA damage (approx. 4 lesions/10 kb) after H(2)O(2) exposure.
- Partial mtDNA repair occurred within 6 hours, while nuclear DNA (nDNA) remained resistant.
- Persistent mtDNA damage at 24 hours was observed in cells with low Delta Psi m, which also showed increased H(2)O(2) production and underwent apoptosis.
Conclusions:
- A feed-forward cascade of reactive oxygen species (ROS) generation and mtDNA damage is supported.
- A drop in Delta Psi m may lead to persistent mtDNA lesions via compromised protein import, secondary ROS generation, and reduced repair capacity.