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Mitochondrial hOGG1-2a overexpression sensitizes HeLaS3 cells to oxidative stress, accompanied by mitochondrial
Weizhi Wang1, Lanyun Yan1, Fengxian Wu1
1Laboratory of Stress Response Biology, Graduate School of Science, Kyoto University, Kyoto, Japan.
Abstract:
Mitochondria are essential organelles responsible for cellular ATP production and contain their own mitochondrial DNA (mtDNA), which encodes key components of oxidative phosphorylation. Because mitochondria continuously generate reactive oxygen species (ROS), mtDNA is particularly susceptible to oxidative damage. Although DNA repair enzymes are present in mitochondria, the regulation of mtDNA repair and its impact on cellular responses to oxidative stress remain incompletely understood. Human 8-oxoguanine DNA glycosylase 1 (hOGG1) is a key enzyme in the base excision repair (BER) pathway, and the mitochondrial isoform hOGG1-2a contributes to the maintenance of mtDNA integrity. In this study, HeLaS3 cell lines stably overexpressing hOGG1-2a were established to examine responses to oxidative stress. hOGG1-2a overexpression was associated with reduced survival following H2O2 treatment, γ-ray exposure, heat shock, and ultraviolet C (UVC) irradiation. Apoptotic cell death increased after oxidative stress. Mitochondrial membrane potential assessed by JC-1 staining was significantly reduced in hOGG1-2a-overexpressing cells. Long-range PCR analysis revealed reduced mtDNA amplification efficiency, and oxidative stress was accompanied by a greater reduction of the mitochondrial enzyme Aconitase 2. These cells exhibited elevated basal ATP levels and altered ATP responses under oxidative stress conditions. In addition, mitochondrial superoxide-associated fluorescence detected by MitoSOX™ was significantly increased. Combined long-range PCR and Sanger sequencing indicated reduced mtDNA amplification after H2O2 exposure without a marked increase in point mutations. Collectively, these findings suggest that hOGG1-2a overexpression sensitizes cells to oxidative stress and is associated with mitochondrial redox dysregulation, reduced mitochondrial membrane potential, altered ATP responses, and reduced mtDNA amplifiability during prolonged stress.
Insights
Overexpressing human 8-oxoguanine DNA glycosylase 1 (hOGG1-2a) in mitochondria sensitizes cells to oxidative stress, leading to reduced survival and mitochondrial dysfunction. This highlights hOGG1-2a
Area of Science:
- Mitochondrial biology
- DNA repair mechanisms
- Cellular oxidative stress response
Background:
- Mitochondria are vital for ATP production and possess their own DNA (mtDNA), crucial for oxidative phosphorylation.
- Mitochondrial DNA (mtDNA) is vulnerable to reactive oxygen species (ROS)-induced damage.
- The role of mitochondrial DNA repair, particularly human 8-oxoguanine DNA glycosylase 1 (hOGG1) isoform 2a (hOGG1-2a), in maintaining mtDNA integrity and cellular response to oxidative stress is not fully understood.
Purpose of the Study:
- To investigate the cellular responses to oxidative stress in HeLaS3 cell lines engineered to overexpress the mitochondrial hOGG1-2a isoform.
- To elucidate the impact of hOGG1-2a overexpression on cell survival, apoptosis, mitochondrial function, and mtDNA integrity under various stress conditions.
Main Methods:
- Establishment of HeLaS3 cell lines with stable hOGG1-2a overexpression.
- Exposure of cells to oxidative stressors including H₂O₂, γ-ray, heat shock, and UVC irradiation.
- Assessment of cell survival, apoptosis, mitochondrial membrane potential (JC-1 staining), mtDNA amplification efficiency (long-range PCR), mitochondrial enzyme levels (Aconitase 2), ATP production, and mitochondrial superoxide levels (MitoSOX™).
Main Results:
- hOGG1-2a overexpression reduced cell survival across multiple oxidative stress conditions.
- Increased apoptotic cell death, decreased mitochondrial membrane potential, and elevated mitochondrial superoxide levels were observed.
- Reduced mtDNA amplification efficiency and altered ATP production/response were noted in hOGG1-2a overexpressing cells, without significant increases in mtDNA point mutations.
Conclusions:
- Overexpression of mitochondrial hOGG1-2a sensitizes cells to oxidative stress, impairing cellular defense mechanisms.
- hOGG1-2a overexpression leads to mitochondrial redox dysregulation, compromised membrane potential, and altered bioenergetics.
- The findings suggest a complex role for hOGG1-2a in mtDNA maintenance, where its overexpression may paradoxically reduce mtDNA amplifiability under prolonged stress.
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