Abstract
From human neuroblastoma-derived SILA cells we have established a rho-0 cell line that is deficient in both respiration and mitochondrial DNA. Lactate dehydrogenase activity, lactate production, and growth in the medium without glucose indicate that these cells shift from aerobic to anaerobic metabolism. Electron microscopic observations revealed abnormal mitochondria with unique cristae structures. Staining with MitoTracker dye showed that the mitochondrial transmembrane potential was reduced by 30-40% from the parent cell levels. These cells were markedly susceptible to H(2)O(2) and died apparently by a necrotic mechanism, a process blocked by deferoxamine in the parent cells but not rho-0 cells. Analysis by inductively coupled plasma-mass spectrometry revealed an approximately 3-fold accumulation of iron in the rho-0 cells at confluence (n = 4-6, three clones, *p < 0.05). Iron and four other metals were all elevated in the cells of one of the rho-0 clones and were similar to control levels in the control cybrid cells, which were replenished with normal mitochondrial DNA. Their sensitivity to H(2)O(2) was also similar to that of the parent cells. These results indicate that a newly established neuronal related rho-0 cell line is highly susceptible to active oxygen species and that these toxicity effects appear to be related to an accumulation of transition metals, which probably occurs through the respiratory impairment.
Highlights
From human neuroblastoma-derived SILA cells we have established a -0 cell line that is deficient in both respiration and mitochondrial DNA
SILA cells were markedly resistant to Ethidium Bromide (EtBr) toxicity and were able to grow even at the highest concentration
Targeting 3 ng and 30 pg of the total DNA isolated from SILA cells, portions of both the D-loop region of mtDNA and -actin gene were amplified by PCR
Summary
O2 consumption, LDH activity, lactate production, ATP level, and total GSH level of SILA and three representative clones of S-0 lines 1– 6, 4 –2, and 6H10 (mean Ϯ S.D., n ϭ 3– 4) were measured as described under “Experimental Procedures.” *** indicates significance at p Ͻ 0.001 in ANOVA with Bonferroni’s correction.
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