Establishment Of Mitochondrial DNA Depleted Human Laryngeal Carcinoma ρ°Cell Lines

Objective：In order to investigate the role of mitochondrial DNA (mtDNA) in humanlaryngeal carcinoma cell proliferation and apoptosis, and provide the cell modelin the following study, And provide a method for further study of relationshipof mitochondria and mtDNA with tumor． Mitochondrial DNA depletedlaryngeal carcinoma cell lines was generated.Methods：carcinoma of the larynx cells were maintained in1640cell culture medium,supplemented with10%FBS at37C under a5%CO2atmosphere. To developρ0cells, wild-type cells were chronically exposed to EB in mediumadditionally supplemented with pyruvate and uridine. After2months of culture,The mtDNA status of was determined by PCR analysis. To standardize the cellculture condition, in all comparative studies between O11and O11ρ0cells,Result：We selectively depleted mtDNA from laryngeal cancer cell line byculturing cells with EtBr for60days and. mtDNA levels of ρ0clones wereassessed by PCR. A PCR analysis was used to further confirm the depletion ofmtDNA. could be amplified in O11cells without disposing to EB，whilemtDNA can not be amplified in EB treated O11cells． Every generation timeof ρ0O11cell was3d． After success of the induction and passage for about20generations，cell growth began to lag and generation time was longer than7d． Conclusion：In this study, we successfully established a novel ρ0laryngocarcinoma cellline after60day continuous treatment with EtBr. ρ0status was verified by lackof detectable MtDNA by PCR amplification. The resultant ρ0cells displayeddependence upon uridine and pyruvate for growth, a common feature formtDNA-less cell lines. The results from cell proliferation, cell cycledistribution, and clonogenecity assays showed that ρ0O11cells had a slowergrowth rate than parental cells.