期刊
TOXICOLOGY AND APPLIED PHARMACOLOGY
卷 196, 期 1, 页码 68-79出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.taap.2003.12.001
关键词
ethidium bromide; depleted mitochondrial DNA; MOLT-4 p(0) petite mutants
资金
- NCI NIH HHS [CA 23108, 1K24 CA84081] Funding Source: Medline
Mitochondrial DNA-deficient (p(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 mitochondria( transmembrane potential (Delta psi(m),). Flow cytometry using JC-1 indicated that MOLT-4 rho(0) had a lower Delta psi(m) than MOLT-4. Sodium fluoride (an inhibitor of the glycolytic pathway) at a concentration of 20 mM further reduced the Atom in MOLT-4-rho(0) cells. This data suggested that a glycolytic pathway product, possibly ATP, was required for the maintenance of Delta psi(m) in MOLT-4 rho(0) cells. (C) 2004 Elsevier Inc. All rights reserved.
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