| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 90 === Some of the mitochondrial diseases, such as chronic progressive external ophthalmoplegia syndrome (CPEO) and Kearns-Sayre syndrome (KSS), are associated with large-scale deletions of mitochondrial DNA (mtDNA). Most of these mtDNA mutations are able to cause defects in the respiration and oxidative phosphorylation system of mitochondria in the affected tissues. Such defects have been shown to cause a decrease of ATP production and abnormal increase of intracellular ROS level. Recently, mitochondria have been established to act as a key player in the regulation of cell death. We have hypothesized that human cells harboring mutated mtDNA are more susceptible to apoptosis triggered by different apoptotic stimuli.
To test this hypothesis, we used a series of human cells harboring different proportions of the 4,977-bp deleted mtDNA constructed by cytoplasmic fusion of skin fibroblasts from a CPEO patient with mtDNA-depleted ro cells. We observed that cell viability was decreased with the increase of the proportions of the 4,977 bp-deleted mtDNA in the cybrids 25 hr after 20 J/m2 of UV-irradiation. By DNA ladder analysis and propidium iodide staining, we found that the cell death induced by exposure of 20 J/m2 of UV-irradiation is due to apoptosis, not necrosis. Furthermore, we detected the proteolytic cleavage of pro-caspase 3 by Western blot and the caspase 3 activity by fluorescence spectrophotometry. The results showed that the extent of activation of caspase 3 was significantly increased with the increase of the proportion of the 4,977 bp-deleted mtDNA in the cybrids. In order to study the changes upstream of caspase 3 activation in the UV-induced apoptotic process, we monitored the distribution and release of cytochrome c by immunostaining and Western blot analysis. The results showed that cytochrome c release was induced by UV-irradiation and cytochrome c release from mitochondria occurred much earlier in the cybrids harboring higher proportions of the 4,977 bp-deleted mtDNA than those contained only wild-type mtDNA. Moreover, we observed changes in morphology and distribution of mitochondria and mitochondrial potential depolarization in the cybrids exposed to UV-irradiation by confocal microscopy. Mitochondria moved away from the cell periphery to form aggregates around the nucleus and mitochondrial potential was declined upon UV-irradiation. These changes were more pronounced in the cybrids harboring higher proportions of the 4,977 bp-deleted mtDNA. Lastly, we investigated whether the intracellular ROS triggers the initiation of apoptotic processes and causes difference in the behaviors of cybrids harboring different levels of 4,977 bp-deleted mtDNA. The results showed that the ROS contents of cybrids exposed to UV-irradiation were increased in both wild-type and mutant cybrids, but no significant differences between the wild-type and mutant cybrids were observed at 25 hr after UV-irradiation. Based on these findings, we suggest that large-scale deletion of mtDNA renders human cells more susceptible to apoptosis triggered by UV-irradiation. We conjecture that in mitochondrial myopathies, apoptosis may play an important role in the pathogenesis of the diseases and pathologies such as muscle weakness, muscle wasting and neurological disorders.
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