mtDNA Depletion Research Articles

Mitochondrial DNA replication: clinical syndromes.

Each nucleated cell contains several hundreds of mitochondria, which are unique organelles in being under dual genome control. The mitochondria contain their own DNA, the mtDNA, but most of mitochondrial proteins are encoded by nuclear genes, including all the proteins required for replication, transcription, and repair of mtDNA. MtDNA replication is a continuous process that requires coordinated action of several enzymes that are part of the mtDNA replisome. It also requires constant supply of deoxyribonucleotide triphosphates(dNTPs) and interaction with other mitochondria for mixing and unifying the mitochondrial compartment. MtDNA maintenance defects are a growing list of disorders caused by defects in nuclear genes involved in different aspects of mtDNA replication. As a result of defects in these genes, mtDNA depletion and/or multiple mtDNA deletions develop in affected tissues resulting in variable manifestations that range from adult-onset mild disease to lethal presentation early in life.

Phenotypic spectrum of SLC25A4 mutations.

There is no comprehensive overview concerning the phenotypic variability in patients carrying SLC25A4 mutations available. Therefore, the aim of the present review was to summarise and discuss recent findings concerning the clinical presentation and phenotypic heterogeneity of SLC25A4 mutations. The study was conducted by systematically reviewing the literature using the search terms 'mitochondrial', "myopathy', 'nuclear DNA', 'mitochondrial DNA', in combination with 'SLC25A4' or 'AAC1'. The results indicated that the phenotypic heterogeneity in patients carrying a SLC25A4 mutation is broader than so far anticipated. Patients carrying a SLC25A4 mutation not only manifest as encephalo-myo-cardiomyopathy but also with scoliosis, cataract, depression, headache, hydrocephalus or arterial hypertension. SLC25A4 mutations may result in mtDNA depletion or multiple mitochondrial (mt)DNA deletions. SLC25A4-associated mtDNA depletion presents with the more severe phenotype and the worse outcome than patients with multiple mtDNA deletions. Depletion syndrome due to SLC25A4 mutations is associated with congenital respiratory insufficiency requiring mechanical ventilation with poor prognosis in the majority of the cases. Mutations in the SLC25A4 gene manifest phenotypically with multiorgan abnormalities in addition to encephalo-myo-cardiomyopathy. SLC25A4 mutations, causing mtDNA depletion, present with a more severe phenotype, including respiratory insufficiency and more widespread cerebral disease than mutations causing multiple mtDNA deletions.

Mitochondrial genome analysis in penile carcinoma.

Penile cancer is a rare neoplasm that seems to be linked to socio-economic differences. Mitochondrial genome alterations are common in many tumors types and are reported as regulating oxidative metabolism and impacting tumorigenesis. In this study, we evaluate for the first time the mitochondrial genome in penile carcinoma (PeCa), aiming to evaluate heteroplasmy, mitochondrial DNA (mtDNA) mutational load and mtDNA content in Penile tumors. Using next generation sequencing (NGS), we sequenced the mitochondrial genome of 13 penile tumors and 12 non-neoplastic tissue samples, which allowed us to identify mtDNA variants and heteroplasmy. We further evaluated variant's pathogenicity using Mutpred predictive software and calculated mtDNA content using quantitative PCR. Mitochondrial genome sequencing revealed an increase number of non-synonymous variants in the tumor tissue, along with higher frequency of heteroplasmy and mtDNA depletion in penile tumors, suggesting an increased mitochondrial instability in penile tumors. We also described a list of mitochondrial variants found in penile tumor and normal tissue, including five novel variants found in the tumoral tissue. Our results showed an increased mitochondrial genome instability in penile tumors. We also suggest that mitochondrial DNA copy number (mtDNAcn) and mtDNA variants may act together to imbalance mitochondrial function in PeCa. The better understanding of mitochondrial biology can bring new insights on mechanisms and open a new field for therapy in PeCa.

Mitochondrial Dysfunctions Regulated Radioresistance through Mitochondria-to-Nucleus Retrograde Signaling Pathway of NF-κB/PI3K/AKT2/mTOR.

We investigated the relationship between significantly different genes of the mitochondria-to-nucleus retrograde signaling pathway (RTG) in H1299 ρ0 cells (mtDNA depleted cell) and compared their radiosensitivity to that of parental ρ+ cells, to determine the possible intervention targets of radiosensitization. ρ0 cells were depleted of mitochondrial DNA by chronic culturing in ethidium bromide at low concentration. Radiosensitivity was analyzed using clonogenic assay. Western blot was used to analyze the cell cycle-related proteins, serine/threonine kinase ataxia telangiectasia mutant (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and cyclin B1 (CCNB1). The γ-H2AX foci were detected using confocal fluorescence microscopy. RNA samples were hybridized using the Agilent human genome expression microarray. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for Gene Ontology (GO) Consortium and pathway annotations of differentially expressed genes, respectively. The H1299 ρ0 cells were found to be more radioresistant than ρ+ cells. The ATP production of H1299 ρ0 cells was lower than that of the ρ+ cells before or after irradiation. Both H1299 ρ0 and ρ+ cells had higher ROS levels after irradiation, however, the radiation-induced ROS production in ρ0 cells was significantly lower than in ρ+ cells. In addition, the percentage of apoptosis in H1299 ρ0 cells was lower than in ρ+ cells after 6 Gy irradiation. As for the cell cycle and DNA damage response-related proteins ATM, ATR and CCNB1, the expression levels in ρ0 cells were significantly higher than in ρ+ cells, and there were less γ-H2AX foci in the ρ0 than ρ+ cells after irradiation. Furthermore, the results of the human genome expression microarray demonstrated that the phosphorylated protein levels of the NF-κB/PI3K/AKT2/mTOR signaling pathway were increased after 6 Gy irradiation and were decreased after treatment with the AKT2-specific inhibitor MK-2206 combined with radiation in H1299 ρ0 cells. MK-2206 treatment also led to an increase in pro-apoptotic proteins. In conclusion, these results demonstrate that mtDNA depletion might activate the mitochondria-to-nucleus retrograde signaling pathway of NF-κB/PI3K/AKT2/mTOR and induce radioresistance in H1299 ρ0 cells by evoking mitochondrial dysfunctions.

Mitochondrial DNA content reduction induces aerobic glycolysis and reversible resistance to drug-induced apoptosis in SW480 colorectal cancer cells

Mutations and reductions in mitochondrial DNA (mtDNA), which are frequent in human tumors, may contribute to enhancing their malignant phenotypes. However, the effects of mtDNA abnormalities in colorectal cancer remain largely unknown. In this study, mtDNA-reduced cell model was established by partial depletion of mtDNA in SW480 cells and the effects of mtDNA reduction in colorectal cancer cells were investigated. We found that mtDNA-reduced cells had enhanced glucose uptake and generated markedly higher level of lactate. These changes were accompanied by only a slight reduction in ATP production compared with the parent cells. Furthermore, the activity of the glycolytic enzymes, hexokinase (HK) and phosphofructokinase (PFK), was increased in mtDNA-reduced cells. These results suggested a switch to aerobic glycolysis in mtDNA-reduced cells, which helped the cells to gain a survival advantage. Notably, when mtDNA content was restored, metabolism returned to normal. In addition, the mtDNA-reduced cells were highly resistant to 5-fluorouracil- and oxaliplatin-induced apoptosis and this drug resistance was reversible following recovery of the mtDNA content. We also found that the Akt/mTOR pathway was activated in the mtDNA-reduced cells. This pathway might play a significant role in drug resistance in the mtDNA-reduced cells as drug susceptibility was restored when this pathway was inhibited. Taken together, our results supported the notion that mtDNA reduction induced aerobic glycolysis and a reversible apoptosis-resistant phenotype in SW480 cells, and that the Akt/mTOR pathway might be involved in the drugs-induced apoptosis resistance.

Mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation links the tricarboxylic acid (TCA) cycle with methionine metabolism and nuclear DNA methylation

Mitochondrial function affects many aspects of cellular physiology, and, most recently, its role in epigenetics has been reported. Mechanistically, how mitochondrial function alters DNA methylation patterns in the nucleus remains ill defined. Using a cell culture model of induced mitochondrial DNA (mtDNA) depletion, in this study we show that progressive mitochondrial dysfunction leads to an early transcriptional and metabolic program centered on the metabolism of various amino acids, including those involved in the methionine cycle. We find that this program also increases DNA methylation, which occurs primarily in the genes that are differentially expressed. Maintenance of mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation in the context of mtDNA loss rescues methionine salvage and polyamine synthesis and prevents changes in DNA methylation and gene expression but does not affect serine/folate metabolism or transsulfuration. This work provides a novel mechanistic link between mitochondrial function and epigenetic regulation of gene expression that involves polyamine and methionine metabolism responding to changes in the tricarboxylic acid (TCA) cycle. Given the implications of these findings, future studies across different physiological contexts and in vivo are warranted.

Endonuclease G promotes mitochondrial genome cleavage and replication.

Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis.

Ubiquitin System

Ever since the discovery of ubiquitin in 1975[...].

Retrospective natural history of thymidine kinase 2 deficiency

BackgroundThymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease...

The presence of rNTPs decreases the speed of mitochondrial DNA replication.

Ribonucleotides (rNMPs) are frequently incorporated during replication or repair by DNA polymerases and failure to remove them leads to instability of nuclear DNA (nDNA). Conversely, rNMPs appear to be relatively well-tolerated in mitochondrial DNA (mtDNA), although the mechanisms behind the tolerance remain unclear. We here show that the human mitochondrial DNA polymerase gamma (Pol γ) bypasses single rNMPs with an unprecedentedly high fidelity and efficiency. In addition, Pol γ exhibits a strikingly low frequency of rNMP incorporation, a property, which we find is independent of its exonuclease activity. However, the physiological levels of free rNTPs partially inhibit DNA synthesis by Pol γ and render the polymerase more sensitive to imbalanced dNTP pools. The characteristics of Pol γ reported here could have implications for forms of mtDNA depletion syndrome (MDS) that are associated with imbalanced cellular dNTP pools. Our results show that at the rNTP/dNTP ratios that are expected to prevail in such disease states, Pol γ enters a polymerase/exonuclease idling mode that leads to mtDNA replication stalling. This could ultimately lead to mtDNA depletion and, consequently, to mitochondrial disease phenotypes such as those observed in MDS.

Dysregulation of autophagy in rat liver with mitochondrial DNA depletion induced by the nucleoside analogue zidovudine.

The nucleoside reverse transcriptase inhibitor zidovudine (AZT), used in HIV infection treatment, induces mitochondrial DNA (mtDNA) depletion. A cause-effect relationship between mtDNA status alterations and autophagy has been reported. Both events are common in several liver diseases, including hepatocellular carcinoma. Here, we have studied autophagy activation in rat liver with mtDNA depletion induced by AZT administration in drinking water for 35days. AZT at a concentration of 1mg/ml, but not 0.5mg/ml in the drinking water, decreased mtDNA levels in rat liver and extrahepatic tissues. In liver, mtDNA-encoded cytochrome c oxidase 1 protein levels were decreased. Although serum biomarkers of liver and kidney toxicity remained unaltered, β-hydroxybutyrate levels were increased in liver of AZT-treated rats. Moreover, autophagy was dysregulated at two levels: (i) decreased induction signalling of this process as indicated by increases in autophagy inhibitors activity (AKT/mTOR), and absence of changes (Beclin-1, Atg5, Atg7) or decreases (AMPK/ULK1) in the expression/activity of pro-autophagy proteins; and (ii) reduced autophagosome degradation as indicated by decreases in the lysosome abundance (LAMP2 marker) and the transcription factor TFEB controlling lysosome biogenesis. This resulted in increased autophagosome abundance (LC3-II marker) and accumulation of the protein selectively degraded by autophagy p62, and the transcription factor Nrf2 in liver of AZT-treated rats. Nrf2 was activated as indicated by the up-regulation of antioxidant target genes Nqo1 and Hmox-1. In conclusion, rat liver with AZT-induced mtDNA depletion presented dysregulations in autophagosome formation and degradation balance, which results in accumulation of these structures in parenchymal liver cells, favouring hepatocarcinogenesis.

Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria

Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.

The mitochondrially targeted antioxidant MitoQ protects the intestinal barrier by ameliorating mitochondrial DNA damage via the Nrf2/ARE signaling pathway

Disruption of the mucosal barrier following intestinal ischemia reperfusion (I/R) is life threatening in clinical practice. Mitochondrial dysfunction and oxidative stress significantly contribute to the early phase of I/R injury and amplify the inflammatory response. MitoQ is a mitochondrially targeted antioxidant that exerts protective effects following I/R injury. In the present study, we aimed to determine whether and how MitoQ protects intestinal epithelial cells (IECs) from I/R injury. In both in vivo and in vitro studies, we found that MitoQ pretreatment downregulated I/R-induced oxidative stress and stabilized the intestinal barrier, as evidenced by MitoQ-treated I/R mice exhibiting attenuated intestinal hyperpermeability, inflammatory response, epithelial apoptosis, and tight junction damage compared to controls. Mechanistically, I/R elevated mitochondrial 8-hydroxyguanine content, reduced mitochondrial DNA (mtDNA) copy number and mRNA transcription levels, and induced mitochondrial disruption in IECs. However, MitoQ pretreatment dramatically inhibited these deleterious effects. mtDNA depletion alone was sufficient to induce apoptosis and mitochondrial dysfunction of IECs. Mitochondrial transcription factor A (TFAM), a key activator of mitochondrial transcription, was significantly reduced during I/R injury, a phenomenon that was prevented by MitoQ treatment. Furthermore, we observed that thee protective properties of MitoQ were affected by upregulation of cellular antioxidant genes, including HO-1, NQO-1, and γ-GCLC. Transfection with Nrf2 siRNA in IECs exposed to hypoxia/reperfusion conditions partially blocked the effects of MitoQ on mtDNA damage and mitochondrial oxidative stress. In conclusion, our data suggest that MitoQ exerts protective effect on I/R-induced intestinal barrier dysfunction.

Elimination of Mitochondrial DNA from Mammalian Cells

AbstractTo cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil‐N‐glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC). Support protocols detail approaches for (1) genotyping ρ° cells of human, mouse, and rat origin by PCR; (2) quantitation of mtDNA by quantitative PCR (qPCR); and (3) preparation of calibrator plasmids for mtDNA quantitation. © 2018 by John Wiley & Sons, Inc.

First description of a novel mitochondrial mutation in the MT-TI gene associated with multiple mitochondrial DNA deletion and depletion in family with severe dilated mitochondrial cardiomyopathy

Mitochondria are essential for early cardiac development and impaired mitochondrial function was described associated with heart diseases such as hypertrophic or dilated mitochondrial cardiomyopathy.In this study, we report a family including two individuals with severe dilated mitochondrial cardiomyopathy. The whole mitochondrial genome screening showed the presence of several variations and a novel homoplasmic mutation m.4318-4322delC in the MT-TI gene shared by the two patients and their mother and leading to a disruption of the tRNAIle secondary structure. In addition, a mitochondrial depletion was present in blood leucocyte of the two affected brother whereas a de novo heteroplasmic multiple deletion in the major arc of mtDNA was present in blood leucocyte and mucosa of only one of them. These deletions in the major arc of the mtDNA resulted to the loss of several protein-encoding genes and also some tRNA genes. The mtDNA deletion and depletion could result to an impairment of the oxidative phosphorylation and energy metabolism in the respiratory chain in the studied patients.Our report is the first description of a family with severe lethal dilated mitochondrial cardiomyopathy and presenting several mtDNA abnormalities including punctual mutation, deletion and depletion.

Mitochondrial DNA damage and subsequent activation of Z-DNA binding protein 1 links oxidative stress to inflammation in epithelial cells

This report identifies mitochondrial DNA (mtDNA) as a target and active mediator that links low-level oxidative stress to inflammatory response in pulmonary epithelial cells. Extrusion of mtDNA into the bronchoalveolar lavage fluid occurs as an early event in mice subjected to cigarette smoke injury, concomitantly with the depletion of mtDNA in the lung tissue. In cultured lung epithelial cells, prolonged, low-level oxidative stress damages the mtDNA, without any detectable damage to the nuclear DNA. In turn, cellular depletion of the mtDNA occurs, together with a transient remodeling of cellular bioenergetics and morphology - all without any detectable impairment in overall cell viability. Damaged mtDNA first enters the cytoplasm, where it binds to Z-DNA binding protein 1 (ZBP1) and triggers inflammation via the TANK-binding kinase 1 /interferon regulatory factor 3 signaling pathway. Fragments of the mtDNA are subsequently released into the extracellular space via exosomes. MtDNA-containing exosomes are capable of inducing an inflammatory response in naïve (non-oxidatively stressed) epithelial cells. In vivo, administration of isolated mtDNA into the in lungs of naïve mice induces the production of pro-inflammatory mediators, without histopathologic evidence of tissue injury. We propose that mtDNA-specific damage, and subsequent activation of the ZBP1 pathway, is a mechanism that links prolonged, low-level oxidative stress to autocrine and paracrine inflammation during the early stages of inflammatory lung disease.

MPV17-related mitochondrial DNA maintenance defect: New cases and review of clinical, biochemical, and molecular aspects.

Mitochondrial DNA (mtDNA) maintenance defects are a group of diseases caused by deficiency of proteins involved in mtDNA synthesis, mitochondrial nucleotide supply, or mitochondrial dynamics. One of the mtDNA maintenance proteins is MPV17, which is a mitochondrial inner membrane protein involved in importing deoxynucleotides into the mitochondria. In 2006, pathogenic variants in MPV17 were first reported to cause infantile-onset hepatocerebral mtDNA depletion syndrome and Navajo neurohepatopathy. To date, 75 individuals with MPV17-related mtDNA maintenance defect have been reported with 39 different MPV17 pathogenic variants. In this report, we present an additional 25 affected individuals with nine novel MPV17 pathogenic variants. We summarize the clinical features of all 100 affected individuals and review the total 48 MPV17 pathogenic variants. The vast majority of affected individuals presented with an early-onset encephalohepatopathic disease characterized by hepatic and neurological manifestations, failure to thrive, lactic acidemia, and mtDNA depletion detected mainly in liver tissue. Rarely, MPV17 deficiency can cause a late-onset neuromyopathic disease characterized by myopathy and peripheral neuropathy with no or minimal liver involvement. Approximately half of the MPV17 pathogenic variants are missense. A genotype with biallelic missense variants, in particular homozygous p.R50Q, p.P98L, and p.R41Q, can carry a relatively better prognosis.

Loss of mtDNA activates astrocytes and leads to spongiotic encephalopathy

Mitochondrial dysfunction manifests as different neurological diseases, but the mechanisms underlying the clinical variability remain poorly understood. To clarify whether different brain cells have differential sensitivity to mitochondrial dysfunction, we induced mitochondrial DNA (mtDNA) depletion in either neurons or astrocytes of mice, by inactivating Twinkle (TwKO), the replicative mtDNA helicase. Here we show that astrocytes, the most abundant cerebral cell type, are chronically activated upon mtDNA loss, leading to early-onset spongiotic degeneration of brain parenchyma, microgliosis and secondary neurodegeneration. Neuronal mtDNA loss does not, however, cause symptoms until 8 months of age. Findings in astrocyte-TwKO mimic neuropathology of Alpers syndrome, infantile-onset mitochondrial spongiotic encephalopathy caused by mtDNA maintenance defects. Our evidence indicates that (1) astrocytes are dependent on mtDNA integrity; (2) mitochondrial metabolism contributes to their activation; (3) chronic astrocyte activation has devastating consequences, underlying spongiotic encephalopathy; and that (4) astrocytes are a potential target for interventions.

Mitochondrial DNA content of peripheral blood mononuclear cells in ART untreated & stavudine/zidovudine treated HIV-1-infected patients.

Background & objectives:Nucleoside reverse transcriptase inhibitors (NRTIs) are known to cause mitochondrial toxicity. This study was done to estimate mitochondrial DNA (mtDNA) content of peripheral blood mononuclear cells (PBMCs) among human immunodeficiency virus (HIV) infected, NRTI treated and antiretroviral therapy (ART)-naïve patients and evaluate the utility of mtDNA content as a biomarker of mitochondrial toxicity.Methods:mtDNA content in PBMCs of 57 HIV-infected ART untreated and 30 ART treated with stavudine (d4T) or zidovudine (AZT) containing regimen were compared against 24 low-risk healthy controls (LoRHC).Results:There was a significant (P=0.01) reduction in mtDNA content among HIV-infected (104; 80-135) compared to LoRHC (127; 110-167), and it was the same in both the treated (104.8; 88-130) and untreated patients (104.7; 78-142). mtDNA significantly (P=0.014) declined in ART treated patients symptomatic for toxicity (97; 74-111) than the asymptomatic patients (128; 103- 153).Interpretation & conclusions:mtDNA depletion in PBMCs was evident among HIV-infected individuals on ART. Moreover, as mtDNA content was reduced among the patients symptomatic for toxicity than the asymptomatic in both the HIV-infected groups, the current study supports mtDNA content of PBMCs to serve as a biomarker of mitochondrial dysfunction induced by NRTI and HIV. Longitudinal studies with a large sample need to be done to confirm these findings.

Identification of An mtDNA Setpoint Associated with Highest Levels of CD44 Positivity and Chemoresistance in HGC-27 and MKN-45 Gastric Cancer Cell Lines.

Objective Cancer stem cells (CSCs) have important roles in survival and chemoresistance. These cells are commonly recognized with CD44 and CD24 markers. In this study, we aimed to analyze the effects of mtDNA content on cell surface positivity for anti-CD24 and anti-CD44 antibodies and chemoresistance level in AGS, HGC-27 and MKN-45 gastric cancer (GC) cell lines and to determine a setpoint for mtDNA copy for each cell line. Materials and Methods In this experimental study, we initially decreased mtDNA levels in AGS, HGC-27 and MKN-45 by EtBr treatment. This depletion was confirmed with quantitative polymerase chain reaction (qPCR). Changes in cell surface positivity for anti-CD24 and anti-CD44 antibodies in control and mtDNA-depleted AGS, HGC-27 and MKN-45 were then analyzed with flow cytometry. Changes in chemoresistance (5-FU and cisplatin) were analyzed for all cell lines. The relationship between mtDNA content and cell surface positivity for CD24 and CD44 markers was examined. Results The highest CD44 positivity was found in HGC-27 and MKN-45 ρlow cells which had 33-40% mtDNA content of control cells, however, CD24 positivity decreased with mtDNA depletion in all cell lines. The highest chemoresistance levels were found in all ρlow cells. mtDNA-recovered (i.e. reverted) HGC-27 and MKN-45 cells partially maintained their increased chemoresistance while reverted AGS cells did not maintain an increased level of chemoresistance. ConclusionmtDNA depletion triggers chemoresistance in cancer cell lines and is correlated with increase and decrease of CD44 and CD24 positivity respectively in HGC-27 and MKN-45 GC cell lines. A mtDNA content above or below the identified setpoint (33-40% of that in control cells), results in the decrease of CD44 positivity and chemoresistance levels.