Effect of mtDNA depletion from C6 glioma cells and characteristics of the generated C6ρ0 cells

Abstract

Malignant tumors of the central nervous system (CNS) are among the types of cancer with the poorest prognosis and glioma is the commonest primary CNS tumor. A mitochondrial DNA (mtDNA)-depleted cell line C6ρ0 was generated from C6 glioma cells after long-term exposure to ethidium bromide and 2' ,3'-dideoxycytidine in order to determine the effect of mtDNA damage on cell proliferation and pathological changes in glioma cells. Single cell clones were isolated and identified after 42 days of incubation. Repopulated cybrids were formed when the clonal C6ρ0 cells were fused with rat platelets and no difference was observed in their growth in a selective medium without uridine and pyruvate compared with the growth of the parent C6 cells. Disruption of mtDNA resulted in changes in mitochondrial morphology, decreased cell proliferation, reduced intracellular reactive oxygen species and intracellular ATP, along with decreased mtDNA and mitochondrial membrane potential in C6ρ0 cells compared with the C6 cells. Taken together, C6ρ0 cells without mtDNA were established for the first time and their characteristics were compared with parent cells. This C6ρ0 cell line could be used to explore the contribution of mitochondrial dysfunction and mtDNA mutations in the pathogenesis of glioma.

A conditional knockout rat resource of mitochondrial protein-coding genes via a DdCBE-induced premature stop codon

“…These results were further confirmed using rho zero (ρ0) C6 cells as control, which were generated by treatment of ethidium bromide (EB) and 2′,3′-dideoxycytidine (ddC) (fig. S1, I and J) ( 20 ). It has been reported that mutations in mtProteins generally impair the assembly or stability of OXPHOS complexes ( 21 , 22 ).…”

“…Cell culture, nucleofection, and sorting C6 cells (American Type Culture Collection, CCL-107) were cultured in Dulbecco's modified Eagle's medium (DMEM) (A1896701, Gibco) supplemented with 10% fetal bovine serum (FBS) (10099141C, Gibco) at 37°C with 5% CO 2 and were detected without mycoplasma contamination by PCR test. C6 ρ0 cells were generated using EB and ddC treatment as previously described (20). Briefly, wild-type C6 cells were exposed to 10 μM EB (E8751, Sigma-Aldrich) and 16 μM ddC (D5782, Sigma-Aldrich) in DMEM supplemented with uridine (50 μg/ml; U3003, Sigma) and pyruvate (0.1 mg/ml; 11360070, Gibco), with a daily change of the culture medium.…”

Cancer Stem-Like Phenotype of Mitochondria Dysfunctional Hep3B Hepatocellular Carcinoma Cell Line

“…Various causes of mitochondrial dysfunction in cancer cells are known, such as mtDNA mutations and enzyme defects [ 13 , 14 ]. Mitochondrial dysfunction in cancer cells can be induced by gene editing or by depleting mtDNA using chemicals [ 28 , 29 , 30 ]. In a previous study, we generated mitochondria dysfunctional (ρ 0 ) cells, Hep3B/ρ 0 cells, by mtDNA depletion and showed that these Hep3B/ρ 0 cells exhibited an epithelial-mesenchymal transition, a malignant phenotype.…”