Development and characterization of continuous avian cell lines depleted of mitochondrial DNA

Abstract

Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy.

Role of SUV3 Helicase in Maintaining Mitochondrial Homeostasis in Human Cells

“…One effect of cells devoid of mtDNA is slowed proliferation (61). At the cellular level, using our inducible knockdown system, we monitored the gradual reduction of SUV3 expression (with a robust knockdown efficiency of Ͼ90%) and observed multinucleation and senescence (Fig.…”

Restoration of electron transport without proton pumping in mammalian mitochondria

“…Lower animals, plants and fungi can use alternative ways to reduce and oxidize CoQ, such as NADH-DH/CoQ reductase activity or CoQ oxidase activity, albeit without proton translocation (2,3). Vertebrate cells lacking a functional mtETC owing to the absence of mtDNA (°cells), can be maintained in culture if supplemented with uridine and pyruvate (4,5). NDI1 and AOX are monopeptidic enzymes with NADH DH/CoQ reductase and CoQ/O 2 oxidase activities, respectively, that do not translocate protons.…”

Does Mitochondrial DNA Play a Role in Parkinson's Disease? A Review of Cybrid and Other Supportive Evidence

“…During the 1970s and 1980s it was shown that chronic exposure to cationic DNA mutagens, such as ethidium bromide, can selectively block mtDNA replication (84,160). Selective mtDNA toxicity in this case results from the fact that cationic mutagens concentrate FIG.…”

“…Because before its identification as mtDNA cytoplasmic DNA was referred to as q DNA (30), cell lines completely depleted of endogenous mtDNA are typically called q0 cells. First, it was shown that mtDNA-depleted cells require uridine (84). The accepted reason for this is that one step of the pyrimidine de novo pathway, the dihydorotate dehydrogenase-mediated dehydrogenation of dihydroorotate to orotate, is coupled to the reduction of ubiquinone to ubiquinol (51,84,150,163).…”

“…First, it was shown that mtDNA-depleted cells require uridine (84). The accepted reason for this is that one step of the pyrimidine de novo pathway, the dihydorotate dehydrogenase-mediated dehydrogenation of dihydroorotate to orotate, is coupled to the reduction of ubiquinone to ubiquinol (51,84,150,163). Cells that lack a functional ETC theoretically cannot regenerate ubiquinone, which subsequently blocks de novo pyrimidine synthesis.…”