Dietary modulation of mitochondrial DNA deletions and copy number after chemotherapy in rats

Mitochondrial DNA (mtDNA) is particularly susceptible to mutation by alkylating agents, and mitochondrial damage may contribute to the efficacy and toxicity of these agents. We found that folate supplementation decreased the frequency of the "common deletion" (4.8kb, bases 8103-12,936) in liver from untreated rats and from animals treated with cyclophosphamide but not 5-fluorouracil (5-FU). The relative abundance of mitochondrial DNA was greater after chemotherapy but there was no effect of diet. Rats fed with a purified diet had fewer mitochondrial deletions than those maintained on a cereal-based diet after chemotherapy. These results indicate that diet can modulate the extent of mitochondrial damage after cancer chemotherapy, and that folic acid supplementation may be protective against mitochondrial DNA deletions.     

Number matters: control of mammalian mitochondrial DNA copy number

Regulation of mitochondrial biogenesis is essential for proper cellular functioning. Mitochondrial DNA （mtDNA） depletion and the resulting mitochondrial malfunction have been implicated in cancer, neurodegeneration, diabetes, aging, and many other human diseases. Although it is known that the dynamics of the mammalian mitochondrial genome are not linked with that of the nuclear genome, very little is known about the mechanism of mtDNA propagation. Nevertheless, our understanding of the mode of mtDNA replication has ad- vanced in recent years, though not without some controversies. This review summarizes our current knowledge of mtDNA copy number control in mammalian cells, while focusing on both mtDNA replication and turnover. Although mtDNA copy number is seemingly in excess, we reason that mtDNA copy number control is an important aspect of mitochondrial genetics and biogenesis and is essential for normal cellular function.     

Regional variation in mitochondrial DNA copy number in mouse brain

Mitochondria have their own DNA (mitochondrial DNA [mtDNA]). Although mtDNA copy number is dependent on tissues and its decrease is associated with various neuromuscular diseases, detailed distribution of mtDNA copies in the brain remains uncertain. Using real-time quantitative PCR assay, we examined regional variation in mtDNA copy number in 39 brain regions of male mice. A significant regional difference in mtDNA copy number was observed (P<4.8×10(-35)). High levels of mtDNA copies were found in the ventral tegmental area and substantia nigra, two major nuclei containing dopaminergic neurons. In contrast, cerebellar vermis and lobes had significantly lower copy numbers than other regions. Hippocampal dentate gyrus also had a relatively low mtDNA copy number. This study is the first quantitative analysis of regional variation in mtDNA copy number in mouse brain. Our findings are important for the physiological and pathophysiological studies of mtDNA in the brain.     

Discovery of a large number of previously unrecognized mitochondrial pseudogenes in fish genomes

Nuclear inserted copies of mitochondrial origin (numts) vary widely among eukaryotes, with human and plant genomes harboring the largest repertoires. Numts were previously thought to be absent from fish species, but the recent release of three fish nuclear genome sequences provides the resource to obtain a more comprehensive insight into the extent of mtDNA transfer in fishes. From the sequence analyses of the genomes of Fugu rubripes, Tetraodon nigroviridis, and Danio rerio, we have identified 2, 5, and 10 recent numt integrations, respectively, which integrated into those genomes less than 0.6 million years (Myr) ago. Such results contradict the hypothesis of absence or rarity of numts in fishes, as (i) the ratio of numts to the total size of the nuclear genome in T. nigroviridis was superior to the ratio observed in several higher vertebrate species (e.g., chicken, mouse, and rat), and only surpassed by humans, and (ii) the mtDNA coverage transferred to the nuclear genome of D. rerio is exceeded only by human and mouse, within the whole range of eukaryotic genomes surveyed for numts. Additionally, 335, 336, and 471 old numts (>12.5 Myr) were detected in F. rubripes, T. nigroviridis, and D. rerio, respectively. Surprisingly, old numts are inserted preferentially into known or predicted genes, as inferred for recent numts in human. However, because in fish genomes such integrations are old, they are likely to represent evolutionary successes and they may be considered a potential important evolutionary mechanism for the enhancement of genomic coding regions.     

Oxidative stress-related alteration of the copy number of mitochondrial DNA in human leukocytes

The role of oxidative stress in the regulation of the copy number of mitochondrial DNA (mtDNA) in human leukocytes is unclear. In this study, we investigated the redox factors in plasma that may contribute to the alteration of mtDNA copy number in human leukocytes. A total of 156 healthy subjects of 25-80 years of age who exhibited no significant difference in the distribution of subpopulations of leukocytes in blood were recruited. Small-molecular-weight antioxidants and thiobarbituric acid reactive substances (TBARS) in plasma and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4,977bp deletion of mtDNA in leukocytes were determined. The mtDNA copy number in leukocytes was determined by real-time PCR. The results showed that the copy number of mtDNA in leukocytes was changed with age in a biphasic manner that fits in a positively quadratic regression model (P = 0.001). Retinol (P = 0.005), non-protein thiols (P = 0.001) and ferritin (P = 0.004) in plasma and total glutathione in erythrocytes (P = 0.046) were the significant redox factors that correlated with the mtDNA copy number in leukocytes in a positive manner. By contrast, alpha-tocopherol levels in plasma (P = 0.001) and erythrocytes (P = 0.033) were negatively correlated with the mtDNA copy number in leukocytes. Three oxidative indices including the incidence of 4,977 bp deletion of mtDNA (P = 0.016) and 8-OHdG content in leukocytes (P = 0.003) and TBARS in plasma (P = 0.001) were all positively correlated with the copy number of mtDNA in leukocytes. Taken these findings together, we suggest that the copy number of mtDNA in leukocytes is affected by oxidative stress in blood circulation elicited by the alteration of plasma antioxidants/prooxidants and oxidative damage to DNA.     

Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH

Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detection of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 microg usually necessary. Using a discrete series of DNA fragments, we defined the parameters adapted to the most faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantitative characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficients of 0.96 and 0.99 were observed for regions of low-copy-level variations and all regions, respectively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reproducibility and to the correct detection of one-copy gain or loss in >90% of the analysed data, even for pseudotriploid tumour genomes.     

Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age

Deletions in mitochondrial DNA (mtDNA) accumulate with age in humans without overt mitochondriopathies, but relatively limited attention has been devoted to the measurement of the total number of mtDNA molecules per cell during ageing. We have developed a precise assay that determines mtDNA levels relative to nuclear DNA using a PCR-based procedure. Quantification was performed by reference to a single recombinant plasmid standard containing a copy of each target DNA sequence (mitochondrial and nuclear). Copy number of mtDNA was determined by amplifying a short region of the cytochrome b gene (although other regions of mtDNA were demonstrably useful). Nuclear DNA content was determined by amplification of a segment of the single copy β-globin gene. The copy number of mtDNA per diploid nuclear genome in myocardium was 6970 ± 920, significantly higher than that in skeletal muscle, 3650 ± 620 (P = 0.006). In both human skeletal muscle and myocardium, there was no significant change in mtDNA copy number with age (from neonates to subjects older than 80 years). This PCR-based assay not only enables accurate determination of mtDNA relative to nuclear DNA but also has the potential to quantify accurately any DNA sequence in relation to any other.     

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E?21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E?4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E?3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.     

Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients

Somatic mutations and large-scale depletion in mitochondrial DNA (mtDNA) have been extensively detected in various human cancers. However, it still remains unclear whether the alterations in mtDNA content are related to the clinicopathological parameters and patient prognosis in breast cancer. In the present study, we analyzed the copy number of mtDNA in 59 cases of invasive breast tumors and paired nontumorous tissues using quantitative real-time PCR. Our data showed that the level of mtDNA was significantly decreased in tumor tissues as compared to the adjacent nontumorous counterparts (P = 0.001). The reduced copy number in mtDNA was associated with an older onset age (>or=50 years old, P = 0.035) as well as a higher histological grade (P = 0.012). Survival analysis measured by the Kaplan-Meier curves and the log-rank test indicated that patients with reduced mtDNA content had significantly poorer disease-free survival (DFS, P = 0.0079) and overall survival (OS, P = 0.011) rate. In addition, tumors harboring mutations in displacement (D)-loop region, particularly at the polycytidine stretch (T/N ratio = 64.3 +/- 8.2%) or close to the replication origins of the heavy-strand (T/N ratio = 68.7 +/- 5.5%), had a significantly lower copy number of mtDNA than the ones without D-loop alterations. Together, our results suggested that reduced copy number of mtDNA may be involved in breast neoplastic transformation or progression and mtDNA content might be potentially used as a tool to predict prognosis. Somatic mutation in the D-loop region probably is one of key contributing factors leading to decreased mtDNA level in breast tumors.     

Release of replication termination controls mitochondrial DNA copy number after depletion with 2',3'-dideoxycytidine

Although cellular mitochondrial DNA (mtDNA) copy number varies widely among cell lines and tissues, little is known about the mechanism of mtDNA copy number control. Most nascent replication strands from the leading, heavy-strand origin (O_H) are prematurely terminated, defining the 3′ boundary of the displacement loop (D-loop). We have depleted mouse LA9 cell mtDNA to ~20% of normal levels by treating with 2′,3′-dideoxycytidine (ddC) and subsequently allowed recovery to normal levels of mtDNA. A quantitative ligation-mediated PCR assay was used to determine the levels of both terminated and extended nascent O_H strands during mtDNA depletion and repopulation. Depleting mtDNA leads to a release of replication termination until mtDNA copy number approaches a normal level. Detectable total nascent strands per mtDNA genome remain below normal. Therefore, it is likely that the level of replication termination plays a significant role in copy number regulation in this system. However, termination of D-loop strand synthesis is persistent, indicating formation of the D-loop structure has a purpose that is required under conditions of rapid recovery of depleted mtDNA.     

Detection of large deletions of mitochondrial DNA in tissues of mice exposed to X-rays

Large mtDNA deletions in mouse brain and spleen cells, induced by X-radiation at doses of 2 and 5 Gy were studied within four weeks after the exposure of animals to X-rays. Variations in the content of extra-cellular deleted mtDNA were examined in the blood plasma of mice irradiated with 5 Gy in the same postir-radiation times. Ionizing radiation was shown to effectively induce large mtDNA deletions at the doses chosen. The level of deletion mtDNA was dependent on dose and postirradiation time.     

Generation, function and diagnostic value of mitochondrial DNA copy number alterations in human cancers

Mitochondria are key organelles in eukaryotic cells principally responsible for multiple cellular functions. In addition to a plethora of somatic mutations as well as polymorphic sequence variations in mitochondrial DNA (mtDNA), the identification of increased or reduced mtDNA copy number has been increasingly reported in a broad range of primary human cancers, underscoring that accumulation of mtDNA content alterations may be a pivotal factor in eliciting persistent mitochondrial deficient activities and eventually contributing to cancer pathogenesis and progression. However, the detailed roles of altered mtDNA amount in driving the tumorigenic process remain largely unknown. This review outlines mtDNA content changes present in various types of common human malignancies and briefly describes the possible causes and their potential connections to the carcinogenic process. The present state of our knowledge regarding how altered mtDNA quantitative levels could be utilized as a diagnostic biomarker for identifying genetically predisposed population that should undergo intensive screening and early surveillance program is also discussed. Taken together, these findings strongly indicate that mtDNA copy number alterations may exert a crucial role in the pathogenic mechanisms of tumor development. Continued insights into the functional significance of altered mtDNA quantities in the etiology of human cancers will hopefully help in establishing novel potential targets for anti-tumor drugs and intervention therapies.     

A case-control study of peripheral blood mitochondrial DNA copy number and risk of renal cell carcinoma

Background

Low mitochondrial DNA (mtDNA) copy number is a common feature of renal cell carcinoma (RCC), and may influence tumor development. Results from a recent case-control study suggest that low mtDNA copy number in peripheral blood may be a marker for increased RCC risk. In an attempt to replicate that finding, we measured mtDNA copy number in peripheral blood DNA from a U.S. population-based case-control study of RCC.

Methodology/Principal Findings

Relative mtDNA copy number was measured in triplicate by a quantitative real-time PCR assay using DNA extracted from peripheral whole blood. Cases (n = 603) had significantly lower mtDNA copy number than controls (n = 603; medians 0.85, 0.91 respectively; P = 0.0001). In multiple logistic regression analyses, the lowest quartile of mtDNA copy number was associated with a 60% increase in RCC risk relative to the highest quartile (OR = 1.6, 95% CI = 1.1–2.2; P _trend = 0.009). This association remained in analyses restricted to cases treated by surgery alone (OR _Q1 = 1.4, 95% CI = 1.0–2.1) and to localized tumors (2.0, 1.3–2.8).

Conclusions/Significance

Our findings from this investigation, to our knowledge the largest of its kind, offer important confirmatory evidence that low mtDNA copy number is associated with increased RCC risk. Additional research is needed to assess whether the association is replicable in prospective studies.     

Statistical evidence for the correlation of DNA deletions in prokaryotic genomes with direct repeats

In the present work a computer analysis of deletion localization in the sequence of the E. coli lacI gene has been carried out by the statistical weight method. Reliable statistical correlation of the deletions location sites with the arrangement of the most perfect direct repeats revealing the shortest distance between repeated fragments has been shown. At the same time statistical analysis did not reveal reliable connection of deletions localization regions with the expected sites of gyrase recognition, sites and other recombination sites. A conclusion has been drawn, that the mechanism of deletions emergence on the basis of repeats appears to be predominant.