| 15050409 |
Armand R, Channon JY, Kintner J, White KA, Miselis KA, Perez RP, Lewis LD: The effects of ethidium bromide induced loss of mitochondrial DNA on mitochondrial phenotype and ultrastructure in a human leukemia T-cell line (MOLT-4 cells). Toxicol Appl Pharmacol. 2004 Apr 1;196(1):68-79.
Mitochondrial DNA-deficient (rho (0)) cells were generated following a 26-day incubation of MOLT-4 lymphoblastoid T cells in ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide). The absence of mitochondrial DNA (mtDNA) in the resultant MOLT-4 rho (0) cells was confirmed by Southern analysis and quantitative polymerase chain reaction (PCR). MOLT-4 rho (0) cells proliferated more slowly than parental cells (wild type) and produced significantly more lactate (approximately fourfold increase; P < 0.001) with concomitantly reduced oxygen consumption (12.3% vs. 100%; P < 0.001) compared with the wild type. MOLT-4 rho (0) cells also showed reduced cytochrome c oxidase activity and a reduced cytochrome c oxidase/citrate synthase activity ratio compared to parental wild-type MOLT-4 cells (P < 10 (-11)). Electron microscopy showed elongated mitochondria with parallel cristae in MOLT-4 cells although the mitochondria in MOLT-4 rho (0) cells appeared enlarged, some were vacuolated with either an absent or a grossly distorted cristae pattern. Vital staining with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) was used to image mitochondria in intact cells and study mitochondrial transmembrane potential (Deltapsi (m)). Flow cytometry using JC-1 indicated that MOLT-4 rho (0) had a lower Deltapsi (m) than MOLT-4. Sodium fluoride (an inhibitor of the glycolytic pathway) at a concentration of 20 mM further reduced the Deltapsi (m) in MOLT-4-rho (0) cells. This data suggested that a glycolytic pathway product, possibly ATP, was required for the maintenance of Deltapsi (m) in MOLT-4 rho (0) cells. |
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