Mitochondrial DNA (mtDNA) is particularly susceptible to oxidative damage and mutations because of the high level of reactive oxygen species (ROS) generated and the inefficiency of the mtDNA repair system. The oxidative stress elicited by chronic inflammation increases the number of mtDNA mutations and might correlate with a cancerous status. We postulated that increased oxidative stress in primary AML cells might cause mtDNA damage, which can lead to mtDNA mutations, structural changes, perturbation of mtDNA repair and biogenesis. Many mutation and polymorphisms (a total of 606 mtDNA sequence variants) were identified from 48 matched AML bone marrow and buccal mucosa samples, and blood samples from 57 control subjects. There were profound alterations in the 303 poly C, 16184 poly C, and 514 CA repeats. The intracellular ROS generation of cells can be investigated using the 2′,7′-dichlorfluorescein-diacetate and flow cytometry. The results were expressed as mean fluorescence intensity (MFI). MFI in primary AML cells (4,435±709) was significantly higher than those in control blood cells (1,562±141) (P<0.05). After then, we checked mRNA expression of peroxisome proliferators activated receptor gamma coactivator-1α (PGC-1α), PGC-1-related coactivator (PRC) and nuclear respiratory factor 1 (NRF-1) because these have been identified as an important regulator of intracellular ROS level and crucial factors linking external stimuli to mitochondrial biogenesis. As compared to normal blood cells, about 2.0 fold increase in NRF-1 mRNA expression was noted in primary AML, whereas PGC-1α and PRC mRNA expression were not remarkably changed. The supercoiled and open relaxed forms of mtDNA reflect functional and damaged molecules. Thus, sensitive detection of the relaxed and total mtDNA from the same DNA templates should allow quantitative measurements on mtDNA damage, repair and copy number change in stressed cells (
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