Changes In mtDNA Content Research Articles

Relationship of Mitochondrial DNA Oxidation and Content with Metabolic Syndrome and Cardiovascular Risk in Obesity Phenotypes.

Obesity, chronic inflammation, and oxidative stress can influence mitochondrial DNA (mtDNA) content. Our objective was to evaluate the oxidation level and content of mtDNA and its relationship with metabolic parameters in metabolically healthy obese (MHO) compared to metabolically unhealthy obese (MUO) and normal weight (NW) controls. We studied 94 NW, 95 MHO, and 97 MUO individuals between 18 and 80 years old. Relative mtDNA content and mtDNA oxidation level (8-oxoguanine, 8-OxoG) were determined in peripheral blood leukocytes by the SYBR Green method of real-time PCR. One-way ANOVA and Tukey test were used to compare biochemical, clinical, and anthropometric characteristics, as well as mtDNA content and 8-OxoG. A progressive decrease in mtDNA content was observed between NW, MHO, and MUO with significant differences in MUO vs. NW (p: 0.04). An increase in 8-OxoG was observed in MUO patients compared to the other groups (MUO vs. MHO p: 0.01; MUO vs. NW p: 0.04). mtDNA content was directly correlated with HDL-c (p < 0.01) and inversely with waist circumference (p: 0.01) and LDL-c (p: 0.05). mtDNA content decreased, and the oxidation level increased concomitantly with the presence of obesity, the number of MS components, higher coronary risk, and insulin resistance parameters. MHO presented a similar mtDNA oxidation level to NW and mtDNA content to the MUO, placing the MHO individuals as having an intermediate phenotype. Changes in mtDNA content and oxidation were correlated to the lipid profile related to obesity and/or MS presence, probably associated with oxidative stress and chronic low-grade inflammation.

Urinary Levels of Sirtuin-1, π-Glutathione S-Transferase, and Mitochondrial DNA in Maize Farmer Occupationally Exposed to Herbicide.

Epidemiologic studies have suggested an association between agrochemical exposure and risk of renal injury. Farmers face great risks to developing adverse effects. The most appropriate biomarker related to renal injury needs to be developed to encounter earlier detection. We aim to study the association between early renal biomarker and occupational herbicide exposure in maize farmers, Thailand. Sixty-four farmers were recruited and interviewed concerning demographic data, herbicide usage, and protective behavior. Two spot urines before (pre-work task) and after (post-work task) herbicide spraying were collected. To estimate the intensity of exposure, the cumulative herbicide exposure intensity index (cumulative EII) was also calculated from activities on the farm, type of personal protective equipment (PPE) use, as well as duration and frequency of exposure. Four candidate renal biomarkers including π-GST, sirtuin-1, mitochondrial DNA (mtDNA) were measured. Most subjects were male and mostly sprayed three herbicides including glyphosate-based herbicides (GBH), paraquat, and 2,4-dichlorophenoxyacetic acid (2,4-D). A type of activity in farm was mixing and spraying herbicide. Our finding demonstrated no statistical significance of all biomarker levels between pre- and post-work task urine. To compare between single and cocktail use of herbicide, there was no statistical difference in all biomarker levels between pre- and post-work task urine. However, the urinary mtDNA seems to be increased in post-work task urine. Moreover, the cumulative EII was strongly associated with change in mtDNA content in both ND-1 and COX-3 gene. The possibility of urinary mtDNA as a valuable biomarker was promising as a noninvasive benchmark for early detection of the risk of developing renal injury from herbicide exposure.

Elevated Blood Mitochondrial DNA in Early Life Among Uninfected Children Exposed to Human Immunodeficiency Virus and Combination Antiretroviral Therapy in utero.

Combination antiretroviral therapy (cART) during pregnancy prevents vertical transmission, but many antiretrovirals cross the placenta and several can affect mitochondria. Exposure to maternal human immunodeficiency virus (HIV) and/or cART could have long-term effects on children who are HIV exposed and uninfected (CHEU). Our objective was to compare blood mitochondrial DNA (mtDNA) content in CHEU and children who are HIV unexposed and uninfected (CHUU), at birth and in early life. Whole-blood mtDNA content at birth and in early life (age 0-3 years) was compared cross-sectionally between CHEU and CHUU. Longitudinal changes in mtDNA content among CHEU was also evaluated. At birth, CHEU status and younger gestational age were associated with higher mtDNA content. These remained independently associated with mtDNA content in multivariable analyses, whether considering all infants, or only those born at term. Longitudinally, CHEU mtDNA levels remained unchanged during the first 6 months of life, and gradually declined thereafter. A separate age- and sex-matched cross-sectional analysis (in 214 CHEU and 214 CHUU) illustrates that the difference in mtDNA between the groups remains detectable throughout the first 3 years of life. The persistently elevated blood mtDNA content observed among CHEU represents a long-term effect, possibly resulting from in utero stresses related to maternal HIV and/or cART. The clinical impact of altered mtDNA levels is unclear.

Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution.

Mitochondria are involved in a number of diverse cellular functions, including energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation, and motility, as well as free radical generation. Mitochondrial DNA (mtDNA) is present at hundreds to thousands of copies per cell in a tissue-specific manner. mtDNA copy number also varies during aging and disease progression and therefore might be considered as a biomarker that mirrors alterations within the human body. Here, we present a new quantitative, highly sensitive droplet digital PCR (ddPCR) method, droplet digital mitochondrial DNA measurement (ddMDM), to measure mtDNA copy number not only from cell populations but also from single cells. Our developed assay can generate data in as little as 3 h, is optimized for 96-well plates, and also allows the direct use of cell lysates without the need for DNA purification or nuclear reference genes. We show that ddMDM is able to detect differences between samples whose mtDNA copy number was close enough as to be indistinguishable by other commonly used mtDNA quantitation methods. By utilizing ddMDM, we show quantitative changes in mtDNA content per cell across a wide variety of physiological contexts including cancer progression, cell cycle progression, human T cell activation, and human aging.

Increased mitochondrial DNA4977-bp deletion in catheterization laboratory workers with long-term low-dose exposure to ionizing radiation.

Ionizing radiation may lead to mitochondrial DNA (mtDNA) mutations and changes in mtDNA content in cells, major driving mechanisms for carcinogenesis, vascular aging and neurodegeneration. The aim of this study was to investigate the possible induction of common mitochondrial deletion (mtDNA4977) and mtDNA copy number (mtDNA-CN) changes in peripheral blood of personnel working in high-volume cardiac catheterization laboratories (Cath Labs). A group of 147 Cath Lab workers (median individual effective dose = 16.8 mSv, for the 41 with lifetime dosimetric record) and 74 unexposed individuals were evaluated. The occupational radiological risk score was computed for each subject on the basis of the length of employment, individual caseload and proximity to the radiation source. mtDNA4977 deletion and mtDNA-CN were assessed by using quantitative real-time polymerase chain reaction. Increased levels of mtDNA4977 deletion were observed in high-exposure Cath Lab workers compared with unexposed individuals ( p < 0.0001). Conversely, mtDNA-CN was significantly greater in the low-exposure workers ( p = 0.003). Occupational radiological risk score was positively correlated with mtDNA4977 deletion (Spearman's r = 0.172, p = 0.03) and inversely correlated with mtDNA-CN (Spearman's r = -0.202, p = 0.01). In multiple regression model, occupational radiological risk score emerged as significant predictor of high levels of mtDNA4977 deletion (ß coefficient = 0.236, p = 0.04). mtDNA4977 deletion is significantly high in Cath Lab personnel. Beyond the well-recognized nuclear DNA, mtDNA damage might deserve attention as a pathogenetic molecular pathway and a potential therapeutic target of ionizing radiation damage.

Altered mitochondrial genome content signals worse pathology and prognosis in prostate cancer.

Mitochondrial genome (mtDNA) content is depleted in many cancers. In prostate cancer, there is intra-glandular as well as inter-patient mtDNA copy number variation. In this study, we determine if mtDNA content can be used as a predictor for prostate cancer staging and outcomes. Fresh prostate cancer biopsies from 115 patients were obtained at time of surgery. All cores underwent pathological review, followed by isolation of cancer and normal tissue. DNA was extracted and qPCR performed to quantify the total amount of mtDNA as a ratio to genomic DNA. Differences in mtDNA content were compared for prostate cancer pathology features and disease outcomes. We showed a significantly reduced mtDNA content in prostate cancer compared with normal adjacent prostate tissue (mean difference 1.73-fold, P-value <0.001). Prostate cancer with increased mtDNA content showed unfavorable pathologic characteristics including, higher disease stage (PT2 vs PT3 P-value = 0.018), extracapsular extension (P-value = 0.02) and a trend toward an increased Gleason score (P-value = 0.064). No significant association was observed between changes in mtDNA content and biochemical recurrence (median follow up of 107 months). Contrary to other cancer types, prostate cancer tissue shows no universally depleted mtDNA content. Rather, the change in mtDNA content is highly variable, mirroring known prostate cancer genome heterogeneity. Patients with high mtDNA content have an unfavorable pathology, while a high mtDNA content in normal adjacent prostate tissue is associated with worse prognosis.

A unique combination of micronutrients rejuvenates cognitive performance in aged mice

It is widely believed that diet can influence the onset and severity of cognitive aging, but the optimal combination of micronutrients and molecular and cellular mechanisms remain elusive. The purpose of this study was to compare the effects of eight distinct diets, consisting of various concentrations of selected micronutrients, on learning and memory as well as markers of neuronal plasticity, and metabolic and neuro-immune status of the aged hippocampus. Eighteen-month-old male and female C57BL/6J mice were fed the diets for 16 weeks, followed by learning and memory trials on the active avoidance task. Number of immature neurons were measured by immunohistochemical detection of doublecortin (DCX+) in the granule layer of the dentate gyrus. Amount of mitochondrial DNA (mtDNA) and gene expression of molecular markers of mitochondrial biogenesis (Ppargc1α, Sirt1, Tfam), and neuroinflammation (IL-10, Alox15, Ptgs2, IL-1β, IL-6 and Tnf) were assessed by quantitative real time polymerase chain reaction (qRT-PCR) of hippocampal samples. Tissue levels of selected micronutrients and a number of metabolites were measured by liquid chromatography-mass spectrometry. The diet supplemented with RRR d-alpha tocopheryl acetate, citicholine, 5-methyltetrahydrofolic acid, quercetin and the n-3 fatty acid phosphatidylserine-docosahexaenoic acid, improved performance on the active avoidance learning and memory task compared to all the other less-complex diets. This diet also increased IL-10 expression and attenuated the age-related change in mtDNA content in the hippocampus without affecting metabolite levels. Results suggest cognitive benefits of wholesome diets are partially mediated through combined antioxidant and anti-inflammatory activities of optimized mixtures of micronutrients.

Two transgenic mouse models for β-subunit components of succinate-CoA ligase yielding pleiotropic metabolic alterations.

Succinate-CoA ligase (SUCL) is a heterodimer enzyme composed of Suclg1 α-subunit and a substrate-specific Sucla2 or Suclg2 β-subunit yielding ATP or GTP, respectively. In humans, the deficiency of this enzyme leads to encephalomyopathy with or without methylmalonyl aciduria, in addition to resulting in mitochondrial DNA depletion. We generated mice lacking either one Sucla2 or Suclg2 allele. Sucla2 heterozygote mice exhibited tissue- and age-dependent decreases in Sucla2 expression associated with decreases in ATP-forming activity, but rebound increases in cardiac Suclg2 expression and GTP-forming activity. Bioenergetic parameters including substrate-level phosphorylation (SLP) were not different between wild-type and Sucla2 heterozygote mice unless a submaximal pharmacological inhibition of SUCL was concomitantly present. mtDNA contents were moderately decreased, but blood carnitine esters were significantly elevated. Suclg2 heterozygote mice exhibited decreases in Suclg2 expression but no rebound increases in Sucla2 expression or changes in bioenergetic parameters. Surprisingly, deletion of one Suclg2 allele in Sucla2 heterozygote mice still led to a rebound but protracted increase in Suclg2 expression, yielding double heterozygote mice with no alterations in GTP-forming activity or SLP, but more pronounced changes in mtDNA content and blood carnitine esters, and an increase in succinate dehydrogenase activity. We conclude that a partial reduction in Sucla2 elicits rebound increases in Suclg2 expression, which is sufficiently dominant to overcome even a concomitant deletion of one Suclg2 allele, pleiotropically affecting metabolic pathways associated with SUCL. These results as well as the availability of the transgenic mouse colonies will be of value in understanding SUCL deficiency.

Mitochondrial DNA alteration and pathological progression in colorectal cancer

Mitochondrion, the major organelle involved in cell energy metabolism, plays important roles in many human diseases. Nowadays, its dysfunction during initation and progression of cancers is calling more and more attention. Since Otto Warburg brought out his hypothesis, mitochondrial DNA (mtDNA) may affect on cancer development by regulating oxidative phosphorylation, many important findings have been discovered. It is widely recognized that research the potential correlations between mitochondrion and mtDNA in cancers are of great importance. And the trends of change of mtDNA content are different in different cancers. Thus leaves us numerous works to do. Although some cancers have already been reported their relation with mtDNA alteration, different experimental methods or evidence may lead to different conclusions. This article presents a brief in summarizing the mitochondrion dysfunction in human disease and the alteration of mtDNA in cancers. Moreover, the recent highlighting achievements on colorectal cancer and mtDNA are also summarized to provide a discussion on possible pathological mechanism and clinical usage.

Lower mitochondrial DNA content relates to high-altitude adaptation in Tibetans

Mitochondrial DNA (mtDNA) is crucial to mitochondria in energy production and other physiological functions. When lowlanders arrive at high altitude, the mitochondrial content tends to decrease. However, the mtDNA content of native highlanders share the same feature as lowlanders remains unknown. It is also interesting to dissect the other changes in blood plasma that might accompany the change of mtDNA content. To address these issues, we recruited 241 Tibetan subjects in Tibet and 220 Han subjects in Shaanxi province. Relative mtDNA copy number and blood biochemical indexes were measured. Results show that relative mtDNA copy number in Tibetans is significantly lower as compared to Han subjects; sex, age, blood glucose, triglyceride and total cholesterol show no influence on mtDNA content, but carbon dioxide combining power is negatively correlated with mtDNA content. These results indicate that an increase in CO2 combining power along with lower mtDNA content may provide adaptive potential.

Leukocyte mitochondrial DNA copy number in bipolar disorder

Evidence supports the role for mitochondrial impairment in the pathophysiology of bipolar disorder (BD). BD has been associated with decreased mitochondrial electron transport chain activity and increased oxidative stress. Also, mitochondrial DNA (mtDNA) encodes mitochondrial electron transport chain proteins and has been associated with altered oxidative stress. Preclinical studies showed that lithium treatment increased mtDNA content, but no study has directly assessed mtDNA content in subjects with BD in vivo. Also, the effects of lithium treatment on mtDNA content have never been evaluated in humans. Leukocyte mtDNA content using real time-PCR was evaluated in subjects with BD (n=23) in a depressive episode (≥18 in the 21-item Hamilton Depression Rating Scale) before and after 6-week lithium treatment versus healthy controls (n=24). mtDNA content showed no significant difference between subjects with BD at baseline and controls (p=0.46); also no difference was observed when comparing before and after lithium treatment. A trend for decreased mtDNA content was specifically observed in BD type I compared to controls and BD type II (p=0.05). Importantly, endpoint mtDNA copy number was significantly correlated with age. In BD subjects who were younger, unmedicated and had a shorter duration of illness, no change was observed in mtDNA copy number. More studies with larger samples are warranted to evaluate mtDNA content changes in BD and its potential role as a treatment target, especially in BD type I and its association with aging.

Reduced mitochondrial DNA copy number in Chinese patients with osteosarcoma

A plethora of somatic mutations and germline variations in mitochondrial DNA (mtDNA) have been increasingly reported in numerous cancer entities including osteosarcoma. However, it remains largely unclear whether mtDNA copy number changes occur during the multistep process of osteosarcoma carcinogenesis. For this purpose, we determined quantitative mtDNA levels in 31 primary osteosarcoma specimens and 5 normal bone tissue samples using a real-time polymerase chain reaction assay. Our data showed that the average mtDNA amount was significantly reduced in osteosarcoma tissues compared with normal bone controls. The copy number of mtDNA was statistically associated with tumor metastasis. There was an approximately 2-fold decrease of mtDNA quantity in tumors with metastasis than that in low-grade tumors without metastasis. Furthermore, change in mtDNA content was linked with somatic mutations in the D-loop regulatory region. Tumors carrying somatic D-loop mutations, at the polycytidine stretch between nucleotide positions 303 and 309 or close to the replication origin sites of the heavy strand, had significantly lowered mtDNA levels in comparison with those without mutations. Taken together, these results provide evidence for the first time that reduced mtDNA content may be critically implicated in the development and/or progression of osteosarcoma. Somatic D-loop mutation is likely one key factor among others leading to altered mtDNA amount in osteosarcoma.

Mitochondrial DNA content and mass increase in progression from normal to hyperplastic to cancer endometrium

BackgroundAn increase in mitochondrial DNA (mtDNA) content and mitochondrial biogenesis associated with the activation of PGC-1α signalling pathway was previously reported in type I endometrial cancer. The aim of this study has been to evaluate if mtDNA content and the citrate synthase (CS) activity, an enzyme marker of mitochondrial mass, increase in progression from control endometrium to hyperplasia to type I endometrial carcinoma.ResultsGiven that no statistically significant change in mtDNA content and CS activity in endometrium taken from different phases of the menstrual cycle or in menopause was found, these samples were used as control. Our research shows, for the first time, that mtDNA content and citrate synthase activity increase in hyperplastic endometrium compared to control tissues, even if their levels remain lower compared to cancer tissue. In particular, mtDNA content increases seem to precede increases in CS activity. No statistically significant change in mtDNA content and in CS activity was found in relation to different histopathological conditions such as grade, myometrial invasion and stage.ConclusionMtDNA content and citrate synthase activity increases in pre-malignant lesions could be a potential molecular marker for progression from hyperplasia to carcinoma.

Sulfur dioxide inhalation stimulated mitochondrial biogenesis in rat brains

Sulfur dioxide (SO2) is a common environmental pollutant. Mitochondria play essential roles in energy metabolism, generation of reactive oxygen species, and regulation of apoptosis in response to neuronal brain injury. It is of interest to observe the effect of SO2 on mitochondrial function in brain. In the present study, male Wistar rats were housed in exposure chambers and treated with 3.5, 7 and 14mg/m3 SO2 for 4h/day for 30days, while control rats were exposed to filtered air in the same condition. Mitochondrial membrane potential (MMP) was assessed in cerebral mitochondria using the lipophilic cationic probe JC-1. The amount of ATP was measured by the luciferinluciferase method. Analyses of mitochondrial replication and transcription were performed by real time PCR. The protein levels were detected using Western blotting. Our results showed that cerebral mtDNA content was markedly increased in rats after SO2 exposure. Paralleling the change in mtDNA content, MMP, ATP content, MDA level, CO1 & 4 and ATP6 & 8 expression, and cytochrome c oxidase activity were increased in rat cortex after SO2 inhalation. Moreover, mitochondrial biogenesis was accompanied by increased expression of NRF1 and TFAM, whereas PGC-1α was not changed. We report for the first time increased mitochondrial biogenesis in brain of rats exposed to SO2, which might be an adaptive response to mitochondrial depletion by oxidant damage.

Effect of folic acid supplementation on hepatic antioxidant function and mitochondrial-related gene expression in weanling intrauterine growth retarded piglets

The aim of the present study was to investigate the effect of dietary folic acid supplementation on antioxidant function and mRNA expression levels of genes involved in mitochondrial biogenesis and function in the liver of piglets affected by intrauterine growth retardation (IUGR). Sixteen piglets at normal birth weight and 16 IUGR piglets were fed either a control diet (C) or a folic acid-supplemented diet (FS, C+5mg/kg folic acid) from 14d of age to 35d of age postnatal. Blood and liver samples were collected at the end of the study. The results showed that body weight was lower for IUGR piglets than that of NBW piglets at 14d of age and 35d of age postnatal (P<0.05). Dietary folic acid supplementation increased plasma folic acid concentrations and hepatic GPx activity, but decreased plasma homocysteine levels (P<0.05). Reduced activities of Mn-SOD and T-AOC, mitochondrial DNA (mtDNA) content, and ATP concentration (P<0.05) were observed in the liver of IUGR pigs fed a control diet. However, the changes in mtDNA content and activities of Mn-SOD and T-AOC were negated in IUGR pigs fed a FS diet (P<0.05). Also, dietary folic acid supplementation decreased protein carbonyls and MDA concentration in the liver of IUGR piglets (P<0.05). Expression levels of genes encoding for PPARγ coactivator-1α (PGC-1α), mammalian silencing information regulator-2α (SIRT-1), nuclear respiratory factor-1 (NRF-1), mt transcription factor A (TFAM), mt single-strand DNA-binding protein (mt SSB), mt polymerase r (mt polr), glucokinase, citrate synthase (CS), ATP synthase (ATPS), and cytochrome c oxidase (CcOX) subunit I and V were decreased in IUGR piglets compared with NBW piglets. However, mRNA levels of PGC-1α, SIRT-1, NRF-1, TFAM, and mt polr in the liver of IUGR pigs fed FS diet were not different from that of NBW pigs. Gene mRNA expression abundance of CcOX IV was enhanced by folic acid supplementation (P<0.05) regardless of birth weight. The present study indicates that IUGR impaired hepatic antioxidant function and mRNA expression levels of genes are involved in mitochondrial biogenesis and function. Dietary folic acid supplementation prevented the harmful effect of IUGR on hepatic antioxidant function and mtDNA biogenesis, but had no effect on mRNA expression levels of genes involved in mitochondrial function.

Association between the change regularity of peripheral blood mononuclear cell mitochondrial deoxyribonucleic acid content and human immunodeficiency virus-related lipodystrophy

To investigate the change regularity of peripheral blood mononuclear cell (PBMC) mtDNA (mitochondrial deoxyribonucleic acid) content and its association with HIV-LD (human immunodeficiency virus-related lipodystrophy) in HAART (highly active antiretroviral therapy). At baseline, Months 6 and 24 of therapy, the cryopreserved PBMC were collected from 33 patients on a regular follow-up at our clinic. Among them, 17 had HIV-LD. Then total DNA was extracted and mtDNA content quantified by real-time PCR (polymerase chain reaction). The HIV/AIDS patients had a lower content of PBMC mtDNA (2(-ΔΔCt)) than the healthy controls at baseline (9.578 vs 17.195, P < 0.01). The mtDNA content was lower in the HIV-LD group than that in the no LD (NLD) group at each time point of therapy (13.619 vs 5.775, 6.360 vs 1.387, 7.170 vs 1.266, all P < 0.05). In the HIV-LD group, the half- and 2-year PBMC mtDNA content was markedly lower than those at baseline (both P < 0.05). And the change of mtDNA content (within half a year) was earlier than the onset of clinical HIV-LD at one year later. In the NLD group, the PBMC mtDNA content have an insignificant change after therapy. The mtDNA content decreased significantly in stavudine (d4T)-containing regimen group after treatment (P < 0.01), but showed no significant change in zidovudine (AZT)-containing regimen group after therapy. The decreased content of PBMC mtDNA after HIV infection and during HAART therapy is associated with HIV-LD. Nucleoside reverse transcriptase inhibitor, especially d4T, plays an important role in the progression of HIV-LD.

Changes on mitochondrial DNA content in non-small cell lung cancer

背景与目的已有的研究表明：线粒体DNA（mitochondrial DNA, mtDNA）突变和拷贝数的改变和肿瘤有着密切联系；大多数实体性肿瘤中mtDNA拷贝数有明显降低。本研究旨在探讨线粒体基因组含量的改变和原发性非小细胞肺癌（non-small cell lung cancer, NSCLC）的关系。方法通过实时荧光定量PCR，对肺癌及相对应的癌旁肺组织mtDNA的含量进行精确定量（拷贝数/细胞）。结果肺癌组织mtDNA的平均拷贝数/细胞为395±125，而相对应的正常肺组织为733±196，前者明显低于后者（P < 0.001）。肺癌mtDNA含量的改变与患者性别、年龄、是否吸烟及肿瘤的病理类型无关（P > 0.05）。结论mtDNA含量的改变与NSCLC的发生发展密切相关，同时也可能影响其治疗和预后。

Decreased copy number of mitochondrial DNA in Ewing's sarcoma

Somatic mutations and germline variations in mitochondrial DNA (mtDNA) have been widely detected in a range of human malignancies including Ewing's sarcoma (EWS). However, it still remains unknown whether quantitative alterations in mtDNA level occur during the initiation and/or progression of EWS. To test this possibility, we determined mtDNA copy number from 17 cases of EWS tumor tissues and 5 normal bone tissue samples using a quantitative real-time PCR assay. Our results showed that the average mtDNA content was significantly reduced in cancerous specimens as compared to that in normal bone controls. mtDNA copy number was statistically associated with tumor metastasis. There was an over 2-fold decrease in tumors with metastasis than in low-grade ones without metastasis. In addition, change in mtDNA content was related to somatic mutations in the D-loop control region. Tumors harboring D-loop mutations, at the polycytidine stretch between nucleotide positions 303 and 309 or close to the replication origin sites of the heavy strand, exhibited significantly lowered mtDNA levels in comparison with those without D-loop alterations. The mtDNA content reduction may be an important genetic event in the carcinogenesis of EWS. This study provides evidence that somatic D-loop mutation is likely one of the key factors contributing to quantitative changes of mtDNA in EWS.

Mitochondrial DNA content and expression of genes involved in mtDNA transcription, regulation and maintenance during human fetal development

The mitochondrial biogenesis and adequate energy production are important for fetal growth and early postnatal adaptation. The aim of the study was to characterize mitochondrial DNA (mtDNA) content and expression patterns of POLG, TFAM, NRF1, NRF2 and PGC1 family of regulated coactivators ( PGC1A, PGC1B and PRC) involved in the mtDNA transcription, regulation and maintenance in human fetal tissues during second trimester of gestation. Further the mRNA expression profiles of selected cytochrome c oxidase (COX) subunits were analysed. Moreover enzyme activities of COX and CS and protein levels of COX subunits were analysed. DNA, RNA and proteins were isolated from 26 pairs of fetal liver and muscle samples obtained at autopsy after termination of pregnancy for genetic indications unrelated to OXPHOS deficiency between 13th and 28th week of gestation. This work offers a broad view on the mtDNA content changes in two different tissues during the second trimester of gestation and in the corresponding tissues after birth. The important differences in expression of POLG, TFAM, NRF2 genes and family PGC1 coactivators were found between the fetal tissues. The significant tissue-specific changes in expression of selected COX subunits on mRNA level ( COX4 and MTCO2) were observed. Further the considerable differences in enzyme activities of COX and CS are demonstrated between fetal and postnatal phase. In conclusion our study indicates that the fetal developing tissues might differ in the control of mitochondrial biogenesis depending on their energy demand and the age of gestation. Moreover the gene expression is changed mainly on transcriptional level through fetal period.

Expression of β-F1-ATPase and mitochondrial transcription factor A and the change in mitochondrial DNA content in colorectal cancer: clinical data analysis and evidence from an in vitro study

Mitochondria play an important role in regulating apoptosis and thus may be involved in tumor progression. This study was conducted to elucidate the role of mitochondrial dysfunction in colorectal cancer (CRC). Mitochondrial DNA (mtDNA) content was analyzed with real-time polymerase chain reaction in 153 CRC patients who had received surgery at the Taipei Veterans General Hospital from January 1999 to December 2000. The expression of mitochondrial transcription factor A (TFAM) and beta-F1-ATPase were analyzed using immunohistochemistry. HCT116 cells were cultured in 1% O(2) for at least 20 passages. Mitochondrial biogenesis, ATP production, and the apoptotic response to 5-fluorouracil were analyzed in the derived cells. Disease stage was associated with changes in mtDNA content (p < 0.001), expression of TFAM (p = 0.004), and/or beta-F1-ATPase (p < 0.001). CRCs with low expression of TFAM or beta-F1-ATPase had a lower mtDNA content. In the multivariate analysis, disease stage was the most significant prognostic factor [95% confidence interval (CI), 2.82-6.23], followed by beta-F1-ATPase [95% CI, 1.10-4.10]. In patients receiving 5-FU based chemotherapy, the 5-year disease-free survival rate was only 27% in CRC patients with a low beta-F1-ATPase tumor and was significantly lower than that in those with a high beta-F1-ATPase tumor (60%; p = 0.042). In the hypoxia-treated cells, mitochondrial mass increased, mtDNA content decreased, sensitivity to 5-fluorouracil decreased, and beta-F1-ATPase expression decreased. Mitochondrial dysfunction may be associated with poor outcomes in CRC patients.