Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells

Spadafora, Domenico; Kozhukhar, Natalya; Chouljenko, Vladimir N.; Kousoulas, Konstantin G.; Alexeyev, Mikhail

doi:10.1371/journal.pone.0154684

Cited by 30 publications

(42 citation statements)

References 39 publications

(45 reference statements)

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“…1c). To rule out potential artefacts caused by the expression of mitochondrial pseudogenes integrated in the nuclear genome, we performed dsRNA staining in mtDNA-depleted HeLa cells obtained by either expressing the herpes simplex virus 1 (HSV-1) protein UL12.5M185 or human uracil– N -glycosylase (mUNG1)10. A lack of J2 signal confirmed that the dsRNA identified in our experiments can be wholly attributed to the mitochondrial genome (Extended Data Fig.…”

supporting

confidence: 64%

Mitochondrial double-stranded RNA triggers antiviral signalling in humans

Dhir

Borowski

et al. 2018

Nature

460

530

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Mitochondria are descendants of endosymbiotic bacteria and retain essential prokaryotic features such as a compact circular genome. Consequently, in mammals, mitochondrial DNA is subjected to bidirectional transcription that generates overlapping transcripts, which are capable of forming long double-stranded RNA structures. However, to our knowledge, mitochondrial double-stranded RNA has not been previously characterized in vivo. Here we describe the presence of a highly unstable native mitochondrial double-stranded RNA species at single-cell level and identify key roles for the degradosome components mitochondrial RNA helicase SUV3 and polynucleotide phosphorylase PNPase in restricting the levels of mitochondrial double-stranded RNA. Loss of either enzyme results in massive accumulation of mitochondrial double-stranded RNA that escapes into the cytoplasm in a PNPase-dependent manner. This process engages an MDA5-driven antiviral signalling pathway that triggers a type I interferon response. Consistent with these data, patients carrying hypomorphic mutations in the gene PNPT1, which encodes PNPase, display mitochondrial double-stranded RNA accumulation coupled with upregulation of interferon-stimulated genes and other markers of immune activation. The localization of PNPase to the mitochondrial inter-membrane space and matrix suggests that it has a dual role in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This in turn prevents the activation of potent innate immune defence mechanisms that have evolved to protect vertebrates against microbial and viral attack.

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supporting

confidence: 64%

Mitochondrial double-stranded RNA triggers antiviral signalling in humans

Dhir

Borowski

et al. 2018

Nature

460

530

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“…The mitochondrial morphology changed and the mtDNA depletion induced mitochondrial fragmentation like that described in other Rho-0 cell lines generated by other methods [ 54 , 55 ]. The data obtained on mitochondrial respiration indicated that the 3a6 Rho-0 cells generated in this work have greatly impaired respiration, like that of a typical Rho-0 cell line [ 54 , 55 ]. All these characteristics indicate that hMSCs cultured with d4t are really Rho-0 cells.…”

Section: Discussionsupporting

confidence: 57%

Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines

et al. 2016

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IntroductionThe generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr.MethodologyTwo immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis.ResultsThe results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic capacities were lower.ConclusionAmong the drugs and conditions tested, the use of d4t was the best option for producing Rho-0 cells from hMSCs. Rho-0 cells are useful for studying the role of mitochondria in hMSC differentiation.

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“…A549 Rho0 cells were generated by continuous exposure for 3 month to ethidium bromide (50 ng/mL) as described previously [17]. Duplex PCR was used to validate the phenotype of A549 Rho0 cells [18]. Parental A549 and A549 Rho 0 cells were stained with MitoTracker Red or MitoTracker Deep Red at increasing concentrations.…”

Section: Methodsmentioning

confidence: 99%

Cisplatin-resistant A549 non-small cell lung cancer cells can be identified by increased mitochondrial mass and are sensitive to pemetrexed treatment

et al. 2019

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BackgroundCisplatin plus pemetrexed combination therapy is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC). However, advanced NSCLC has a 5-year survival rate of below 10%, which is mainly due to therapy resistance. We previously showed that the NSCLC cell line A549 harbors different subpopulations including a mesenchymal-like subpopulation characterized by increased chemo- and radiotherapy resistance. Recently, therapy resistance in hematological and solid tumors has been associated with increased mitochondrial activity. Thus, the aim of this study was to investigate the role of the mitochondrial activity in NSCLC chemotherapy resistance.MethodsBased on MitoTracker staining, subpopulations characterized by the highest 10% (Mito-High) or lowest 10% (Mito-Low) mitochondrial mass content were sorted by FACS (Fluorescence-Activated Cell Sorting) from paraclonal cultures of the NSCLC A549 cell line . Mitochondrial DNA copy numbers were quantified by real-time PCR whereas basal cellular respiration was measured by high-resolution respirometry. Cisplatin and pemetrexed response were quantified by proliferation and colony formation assay.ResultsPemetrexed treatment of parental A549 cells increased mitochondrial mass over time. FACS-sorted paraclonal Mito-High cells featured increased mitochondrial mass and mitochondrial DNA copy number compared to the Mito-Low cells. Paraclonal Mito-High cells featured an increased proliferation rate and were significantly more resistant to cisplatin treatment than Mito-Low cells. Interestingly, cisplatin-resistant, paraclonal Mito-High cells were significantly more sensitive to pemetrexed treatment than Mito-Low cells. We provide a working model explaining the molecular mechanism underlying the increased cisplatin- and decreased pemetrexed resistance of a distinct subpopulation characterized by high mitochondrial mass.ConclusionsThis study revealed that cisplatin resistant A549 lung cancer cells can be identified by their increased levels of mitochondrial mass. However, Mito-High cells feature an increased sensitivity to pemetrexed treatment. Thus, pemetrexed and cisplatin target reciprocal lung cancer subpopulations, which could explain the increased efficacy of the combination therapy in the clinical setting.

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Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells

Cited by 30 publications

References 39 publications

Mitochondrial double-stranded RNA triggers antiviral signalling in humans

Mitochondrial double-stranded RNA triggers antiviral signalling in humans

Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines

Cisplatin-resistant A549 non-small cell lung cancer cells can be identified by increased mitochondrial mass and are sensitive to pemetrexed treatment

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