Cell-direct PCR Approach Reveals Dynamic Changes Of Oxidative Mitochondrial DNA Damage,Repair And Cellular Toxicity In Oral Cancer Cells

Objectives The increase of oxidative stress in tumor cells is an important cause for the pathological changes in mitochondria and mitochondrial DNA(mt DNA).However,the interactions between reactive oxidants and mt DNA damage as well as subsequent cellular toxicity and mutagensis are not very clear.In this study,we use improved ss-q PCR method to study and contrast dynamic changes of mt DNA damage,repair and cell toxicity induced by exogenous reactive oxygen species(H2O2)and reactive nitrogen species(SIN-1),respectively.Methods A malignant oral squamous cell carcinoma cell line SCC-25 was used as an experiment model and cell-direct q PCR methods were used to measure mt DNA damage responses induced by exogenous H2O2 and SIN-1.Five concentrations of H2O2(60-960 ?M)and 4 sampling time points(20 min,1 h,24 h,48 h)were used for H2O2 treatment.While,5 concentrations of SIN-1(0.25-1 m M)and 4sampling time points(1,6,24,48 h)were used for SIN-1 treatment.A cell-direct ss-q PCR method coupled with Taq Man probes was used to measure mt DNA damage and repair,a cell-direct dd PCR method used to quantify mt DNA copy number change in treated cells.Meanwhile,cell viability and/or growth inhibition was measured by MTT test,while single cell survival rate was measured suing a10-day clonal test.Results First of all,we developed a cell-direct approach for quantitative analysis of mt DNA damage responses.This approach using cell lysis directly for PCR analysis,avoided DNA extraction and purification,which could be coupled with96-well plated for high throughput analysis.We demonstrated that cell-direct ss-q PCR was effective to analyze mt DNA damage and repair processes,while cell-direct dd PCR was effective for absolute quantification of mt DNA copy number change in treated cells.Secondly,using the cell-direct approach,we discovered a very dynamic process in mt DNA damage responses induced by exogenous H2O2,which included a very sensitive early mt DNA damage increase,a dose separating repairable and non-repairable damage.Interestingly,the non-repairable mt DNA damage was associated with a significant drop in mt DNA copy number and severe growth inhibition,while repairable damage seemed to be associated with significant reduction in 10 day survival in SCC-25 cells.Finally,we demonstrated for the first time that SIN-1could induced a dose-dependent mt DNA damage in SCC-25 cells,but with different features as compared to H2O2,suggesting that SIN-1 induced mt DNA damage through different mechanisms from H2O2.Conclusions The cell-direct method for ss-q PCR and dd PCR provided a powerful new approach for mt DNA damage and copy number analysis.We revealed a very unique and dynamic process in mt DNA damage responses induced by H2O2,and the level of early mt DNA damage dictated different acute and chronic cellular responses in SCC-25 cells.We demonstrated for the first time that SIN-1 caused dose-dependent mt DNA damage but with different features as compared to H2O2,suggesting different mode of action..