Analysis of sensitivity to ionizing radiation in mitochondrial mutant cells

My Ph.D. dissertation work has focused on studying the effects of ionizing radiation on mitochondrial mutant cells in humans. Mutations in mitochondrial DNA can impair energy metabolism and lead to reduction in ATP synthesis which has a profound effect on cellular functions including repair of DNA damage. Ionizing radiation produces a variety of lesions in DNA including DNA double strand breaks (DSBs) which are widely regarded as severe, as just one DSB can be cell-lethal. Consequently, a series of integrated cellular processes have evolved to repair DNA double strand breaks. These include nonhomologous end joining (NHEJ) and homologous recombination repair (HR). HR is an ATP-dependent pathway whereas NHEJ is partly ATP-independent. Our understanding of the effects of ionizing radiation on cells deficient in ATP synthesis, and on the mechanisms of energy metabolism associated with DNA repair, are presently very limited. I hypothesized that cells with defective ATP synthesis due to mutations in the mitochondria will be hypersensitive to radiation.;By exposing normal and mitochondrial mutant cells to ionizing radiation and by using chromosomal aberrations as a measure for DNA damage, I initially confirmed my hypothesis. These findings suggest that mitochondrial mutant cells exhibit hypersensitivity towards oxidative and genotoxic stress.;To identify the effects of mutations in the mitochondrial genome on the ATP generation and also on the altered sensitivity of the cells towards ionizing radiation, evaluated the expression of the 13 mitochondrial-encoded protein coding genes in the normal and the mutant cells. A cell survival assay was carried out to assess growth inhibition after exposure. The results showed differences in gene expression among the three cell lines and also provided information about the altered sensitivity of the mutant cells.;In the next part of my project, I evaluated expression changes in genes involved in nonhomologous and homologous recombination repair. After radiation, the normal cell line showed increased expression in repair genes whereas in the mutant cells, the expression of repair genes was reduced. To determine if the mutant cells were undergoing more apoptosis compared to the normal cells, I evaluated expression changes in 84 genes involved in the apoptotic process. The data obtained suggest that the genes involved in apoptosis in the mutant cells are more highly expressed compared to the normal cells after acute radiation exposure.;Taken together, my work provides strong evidence showing that cells with mitochondrial mutations are hypersensitive to ionizing radiation. This work also advocates the need for proper evaluation for mutagen sensitivity in people with mitochondrial mutations.