title: Mitochondrial DNA mutation and depletion increase the susceptibility of human cells to apoptosis.
abstract: Mitochondrial diseases, such as MELAS, MERRF, and CPEO syndromes, are associated with specific point mutations or large-scale deletions of mitochondrial DNA (mtDNA), which impair mitochondrial respiratory functions and result in decreased production of ATP in affected tissues. Recently, mitochondria have been recognized to act as key players in the regulation of cell death. To investigate whether a pathogenic mutation of mtDNA exerts any effect on the process of apoptosis of human cells, we constructed a series of cybrid human cells harboring different proportions of mtDNA with the A3243G or the A8344G transition, or with the 4,977-bp deletion, by cytoplasmic fusion of patients' skin fibroblasts with mtDNA-depleted rho(0) cells of an immortal human osteosarcoma cell line (143B). We observed that the decrease in cell viability upon staurosporine treatment or exposure to ultraviolet (UV) irradiation was more pronounced in the cybrids harboring high levels of mutated mtDNA compared with the control cybrids. Using DNA fragmentation analysis, we found that the cell death induced by treatment with 100 nM staurosporine or by exposure to UV irradiation at 20 J/m(2) was caused by apoptosis, not necrosis. Moreover, we demonstrated activation of caspase 3 by Western blot and enhanced release of cytochrome c after 100 nM staurosporine treatment or 20 J/m(2) UV irradiation of the cybrids harboring high levels of the three mtDNA mutations. Furthermore, as compared with parental osteosarcoma 143B cells, the rho(0) cells were found to be more susceptible to apoptosis, which was accompanied by caspase 3 activation and cytochrome c release. This indicates that mtDNA plays an important role in the regulation of apoptosis in human cells. Taken together, these findings suggest that mutation and depletion of mtDNA increase the susceptibility of human cells to apoptosis triggered by exogenous stimuli such as UV irradiation or staurosporine.
authors.affiliation: Department of Biochemistry, National Yang-Ming University, Taipei, Taiwan 112.
authors.forename: Chun-Yi
authors.initials: CY
authors.lastname: Liu
authors.forename: Cheng-Feng
authors.initials: CF
authors.lastname: Lee
authors.forename: Chiung-Hui
authors.initials: CH
authors.lastname: Hong
authors.forename: Yau-Huei
authors.initials: YH
authors.lastname: Wei
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000528:radiation effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005838:Genotype; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017237:Mitochondrial Encephalomyopathies / Q000235:genetics / Q000503:physiopathology; D009154:Mutation; D019311:Staurosporine / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D004272:DNA, Mitochondrial; D004791:Enzyme Inhibitors; D045304:Cytochromes c; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine