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. doi: 10.1016/j.taap.2003.12.001.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blotting, Southern
  • Cell Division / drug effects
  • Cell Line, Tumor
  • Cell Respiration / drug effects
  • Citrate (si)-Synthase / metabolism
  • DNA, Mitochondrial / genetics
  • DNA, Mitochondrial / metabolism*
  • Electron Transport Complex IV / metabolism
  • Ethidium / toxicity*
  • Humans
  • Leukemia, T-Cell / pathology
  • Membrane Potentials / drug effects
  • Microscopy, Electron
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Mitochondria / ultrastructure*
  • Mutation
  • Oxygen / metabolism


  • DNA, Mitochondrial
  • Electron Transport Complex IV
  • Citrate (si)-Synthase
  • Ethidium
  • Oxygen