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).
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.
Armand
Ray
R
Section of Clinical Pharmacology, Department of Medicine, Dartmouth Medical School and Dartmouth Hitchcock Medical Center, Lebanon, NH 03756, USA.
Channon
Jacqueline Y
JY
Kintner
Jennifer
J
White
Kristina A
KA
Miselis
Kristin A
KA
Perez
Raymond P
RP
Lewis
Lionel D
LD
D015139:Blotting, Southern; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D019069:Cell Respiration / Q000187:drug effects; D002950:Citrate (si)-Synthase / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism; D004996:Ethidium / Q000633:toxicity*; D006801:Humans; D015458:Leukemia, T-Cell / Q000473:pathology; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D009154:Mutation; D010100:Oxygen / Q000378:metabolism
D004272:DNA, Mitochondrial; D003576:Electron Transport Complex IV; D002950:Citrate (si)-Synthase; D004996:Ethidium; D010100:Oxygen