Mitochondrial DNA mutations and breast tumorigenesis

Breast cancer is a heterogeneous disease and genetic factors play an important role in its genesis. Although mutations in tumor suppressors and oncogenes encoded by the nuclear genome are known to play a critical role in breast tumorigenesis, the contribution of the mitochondrial genome to this process is unclear. Like the nuclear genome, the mitochondrial genome also encodes proteins critical for mitochondrion functions such as oxidative phosphorylation (OXPHOS), which is known to be defective in cancer including breast cancer. Mitochondrial DNA (mtDNA) is more susceptible to mutations due to limited repair mechanisms compared to nuclear DNA (nDNA). Thus changes in mitochondrial genes could also contribute to the development of breast cancer. In this review we discuss mtDNA mutations that affect OXPHOS. Continuous acquisition of mtDNA mutations and selection of advantageous mutations ultimately leads to generation of cells that propagate uncontrollably to form tumors. Since irreversible damage to OXPHOS leads to a shift in energy metabolism towards enhanced aerobic glycolysis in most cancers, mutations in mtDNA represent an early event during breast tumorigenesis, and thus may serve as potential biomarkers for early detection and prognosis of breast cancer. Because mtDNA mutations lead to defective OXPHOS, development of agents that target OXPHOS will provide specificity for preventative and therapeutic agents against breast cancer with minimal toxicity.     

Analysis of mitochondrial DNA copy number variation in blood and tissue samples of metastatic breast cancer patients (A pilot study)

Changes in mitochondrial DNA (mt-DNA) copy number in blood/tissue have been linked to increased risk of several cancers; however, studies on their association in breast cancer is still lacking. In this pilot study, we investigated mt-DNA copy number variation in peripheral blood and tissue samples from metastatic breast cancer patients and compared their differences. For the study, peripheral blood samples from non-cancer individuals (control) and breast cancer patients, along with resected tissues from adjacent and tumor sites from same breast cancer patients were collected. Total genomic DNA was isolated and changes in mt-DNA copy number were measured by relative quantification using SYBR green based quantitative real time PCR method. Our results indicated a significant reduction in mt-DNA copy number in blood samples of breast cancer patients compared to control. However, a significantly higher mt-DNA copy number was observed in tumor tissue when compared with paired non tumor tissue. There was no significant difference in mt-DNA copy number between blood and adjacent tumor tissue samples of the breast cancer patients. Overall, our study reports for the first time a comparison of mt-DNA copy number in blood and paired tissue together and suggested that mt-DNA copy number is differentially regulated in blood and tumor tissues in breast cancer.     

The landscape of mitochondrial DNA variation in human colorectal cancer on the background of phylogenetic knowledge

Recently, an increasing number of studies indicate that mutations in mitochondrial genome may contribute to cancer development or metastasis. Hence, it is important to determine whether the mitochondrial DNA might be a good, clinically applicable marker of cancer. This review describes hereditary as well as somatic mutations reported in mitochondrial DNA of colorectal cancer cells. We showed here that the entire mitochondrial genome mutational spectra are different in colorectal cancer and non-tumor cells. We also placed the described mutations on the phylogenetic context, which highlighted the recurrent problem of data quality. Therefore, the most important rules for adequately assessing the quality of mitochondrial DNA sequence analysis in cancer have been summarized. As follows from this review, neither the reliable spectrum of mtDNA somatic mutations nor the association between hereditary mutations and colorectal cancer risk have been resolved. This indicates that only high resolution studies on mtDNA variability, followed by a proper data interpretation employing phylogenetic knowledge may finally verify the utility of mtDNA sequence (if any) in clinical practice.     

Complexities and pitfalls in analyzing and interpreting mitochondrial DNA content in human cancer

Mutations in the human mitochondrial genome have been observed in all types of human cancer, indicating that mutations might contribute to tumorigenesis, metastasis, recurrence, or drug response. This possibility is appealing because of the known shift from oxidative metabolism to glycolysis, known as the Warburg effect, that occurs in malignancy. Mitochondrial DNA (mtDNA) mutations could either be maternally inherited and predispose to cancer (germ line mutations) or occur sporadically in the mtDNA of specific tissues (tissue- or tumor-specific somatic mutations) and contribute to the tumor initiation and progression process. High-throughput sequencing technologies now enable comprehensive detection of mtDNA variation in tissues and bodily fluids, with the potential to be used as an early detection tool that may impact the treatment of cancer. Here, we discuss insights into the roles of mtDNA mutations in carcinogenesis, highlighting the complexities involved in the analysis and interpretation of mitochondrial genomic content, technical challenges in studying their contribution to pathogenesis, and the value of mtDNA mutations in developing early detection, diagnosis, prognosis, and therapeutic strategies for cancer.     

Mitochondria,oxidative stress and aging

In the eighties, Miquel and Fleming suggested that mitochondria play a key role in cellular aging. Mitochondria, and specially mitochondrial DNA (mtDNA), are major targets of free radical attack. At present, it is well established that mitochondrial deficits accumulate upon aging due to oxidative damage. Thus, oxidative lesions to mtDNA accumulate with age in human and rodent tissues. Furthermore, levels of oxidative damage to mtDNA are several times higher than those of nuclear DNA. Mitochondrial size increases whereas mitochondrial membrane potential decreases with age in brain and liver.

Recently, we have shown that treatment with certain antioxidants, such as sulphur-containing antioxidants, vitamins C and E or the Ginkgo biloba extract EGb 761, protects against the age-associated oxidative damage to mtDNA and oxidation of mitochondrial glutathione. Moreover, the extract EGb 761 also prevents changes in mitochondrial morphology and function associated with aging of the brain and liver. Thus, mitochondrial aging may be prevented by antioxidants. Furthermore, late onset administration of certain antioxidants is also able to prevent the impairment in physiological performance, particularly motor co-ordination, that occurs upon aging.     

Mitochondria-targeted Ogg1 and Aconitase-2 Prevent Oxidant-induced Mitochondrial DNA Damage in Alveolar Epithelial Cells

Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm²) or H₂O₂ (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1^−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity.     

Discovery of a Novel Function for Human Rad51: MAINTENANCE OF THE MITOCHONDRIAL GENOME*

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.     

Mitochondrial genome maintenance in health and disease

Human mitochondria harbor an essential, high copy number, 16,569 base pair, circular DNA genome that encodes 13 gene products required for electron transport and oxidative phosphorylation. Mutation of this genome can compromise cellular respiration, ultimately resulting in a variety of progressive metabolic diseases collectively known as ‘mitochondrial diseases’. Mutagenesis of mtDNA and the persistence of mtDNA mutations in cells and tissues is a complex topic, involving the interplay of DNA replication, DNA damage and repair, purifying selection, organelle dynamics, mitophagy, and aging. We briefly review these general elements that affect maintenance of mtDNA, and we focus on nuclear genes encoding the mtDNA replication machinery that can perturb the genetic integrity of the mitochondrial genome.     

Molecular analysis for mitochondrial DNA disorders

In this article, we review the current methodologies used for the molecular diagnosis of mitochondrial DNA defects. Definition of mitochondrial disorders at the molecular level has been difficult because of both clinical and genetic heterogeneity. Direct DNA analysis for common point mutations and large mtDNA deletions is readily performed and can be done routinely. However, a large number of patients who have the clinical manifestations and muscle pathology findings consistent with mitochondrial DNA disorders do not have detectable common mutations. Additional mutation screening methods are required for the detection of rare and previously undescribed mutations in the mitochondrial genome.     

Bcl-2 prevents abnormal mitochondrial proliferation during etoposide-induced apoptosis

At the late stage of etoposide-induced apoptosis in HL-60 cells, marked by condensation of chromatin, mitochondria increase in numbers. There is also a drastic increase in mitochondrial DNA content. This increase in mitochondrial numbers and DNA content is an indicator of mitochondrial proliferation during apoptosis. These proliferating mitochondria exhibit abnormal morphology and are impaired, which is demonstrated by decrease in mitochondrial membrane potential and ATP content. The described apoptosis-induced abnormal mitochondrial proliferation was inhibited by overexpression of Bcl-2 protein, which also diminishes mitochondrial impairment. The increase in mitochondrial DNA levels correlated with elevated expression of one of the regulators of mitochondrial DNA replication, mtSSB. Our data suggest that proliferation of mitochondria may be an integral part of a cascade of apoptotic events.     

Depletion of mitochondrial DNA and enzyme in estrogen-induced hamster kidney tumors: a rodent model of hormonal carcinogenesis

Mitochondrial DNA (mtDNA) encodes for 13 polypeptides critical for normal functioning of the electron transport chain and damage to mtDNA has been associated with aging, and implicated in several disease processes. Although damage to mtDNA is being implicated in mutagenesis and carcinogenesis, there are limited studies demonstrating the role and extent of mtDNA damage in human or rodent cancers. Using serial dilution and competitive polymerase chain reaction analysis, we have quantitated the amount of total mtDNA and analyzed the extent of mtDNA damage in estrogen-induced and estrogen-dependent hamster kidney tumors. The hamster kidney tumor model is a useful and widely investigated rodent model of hormonal carcinogenesis, which shares several characteristics with human breast and uterine cancers, and point to a common mechanistic pathway. Our data indicate a significant decrease in the copy number of total mtDNA and the activity of a nuclear-encoded mitochondrial enzyme citrate synthase in hamster kidney tumors compared to age-matched controls. Since there are several hundred mitochondria in a cell and each mitochondrion has multiple copies of mtDNA, a very small percentage of somatic deletion mutation may not be enough to result in a decreased capacity of the mitochondrial genome. However, a significant increase in deletion mutations or a decrease in the mtDNA copy number can result in a decreased oxidative phosphorylation capacity of the mitochondria and decreased energetics, and thus increased susceptibility to the disease process. Therefore, estrogen-induced hamster kidney tumor model can be a useful rodent model of carcinogenesis to understand the role of mtDNA damage in cancer progression and development.     

Mitochondrial DNA Ligase Is Dispensable for the Viability of Cultured Cells but Essential for mtDNA Maintenance

Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knock-out observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3-deficient cells is conditional and secondary to the ρ⁰ phenotype.     

一种棉花线粒体DNA的提取方法

线粒体是重要的细胞器,它有自身的基因组。其基因组DNA与细胞核基因组DNA相比,含量较低。棉花当中富含棉酚、丹宁等物质,这对提取DNA有很大的影响。因此我们根据棉花自身的特点,找到了一种提取棉花线粒体DNA经济有效的方法,其质量可以满足限制性酶切、PCR、分子杂交等实验的要求。     

The human mitochondrial replication fork in health and disease

Mitochondria are organelles whose main function is to generate power by oxidative phosphorylation. Some of the essential genes required for this energy production are encoded by the mitochondrial genome, a small circular double stranded DNA molecule. Human mtDNA is replicated by a specialized machinery distinct from the nuclear replisome. Defects in the mitochondrial replication machinery can lead to loss of genetic information by deletion and/or depletion of the mtDNA, which subsequently may cause disturbed oxidative phosphorylation and neuromuscular symptoms in patients. We discuss here the different components of the mitochondrial replication machinery and their role in disease. We also review the mode of mammalian mtDNA replication.     

Mitochondrial dysfunction in human breast cancer cells and their transmitochondrial cybrids

Somatic mitochondrial DNA alterations have been found in all types of cancer. To better understand the role of mitochondria and their involvement in the pathogenic mechanisms of cancer development, the effects of cancer mitochondria were investigated in a defined nuclear background using a transmitochondrial cybrid system. Our results demonstrated that cancer mitochondria confer a significant reduction in cell growth when cells are metabolically stressed in a galactose medium. Activities of the respiratory chain complexes, cellular oxygen consumption, and ATP synthesis rates were found to be much lower in breast cancer cells, than those in normal breast epithelial cells of MCF-10A (10A). These results suggest that there is reduced mitochondrial function in the studied breast cancer cell lines. Similarly reduced mitochondrial function was observed in cybrids containing cancer mitochondria. Novel tRNA mutations were also identified in two breast cancer cell lines, possibly responsible for the observed mitochondrial dysfunction. We conclude that altered mitochondria in cancer cells may play a crucial role in tumor development.     

Mitochondrial DNA Repair Pathways

It has long been held that there is no DNA repair in mitochondria. Early observations suggestedthat the reason for the observed accumulation of DNA damage in mitochondrial DNA is thatDNA lesions are not removed. This is in contrast to the very efficient repair that is seen inthe nuclear DNA. Mitochondrial DNA does not code for any DNA repair proteins, but it hasbeen observed that a number of repair factors can be found in mitochondrial extracts. Mostof these participate in the base excision DNA repair pathway which is responsible for theremoval of simple lesions in DNA. Recent work has shown that there is efficient base excisionrepair in mammalian mitochondria and there are also indications of the presence of morecomplex repair processes. Thus, an active field of mitochondrial DNA repair is emerging. Anunderstanding of the DNA repair processes in mammalian mitochondria is an important currentchallenge and it is likely to lead to clarification of the etiology of the common mutations anddeletions that are found in mitochondria, and which are thought to cause various humandisorders and to play a role in the aging phenotype.     

Mitochondrial mutations may increase oxidative stress: Implications for carcinogenesis and aging?

The sensitivity of mitochondrial DNA to damage by mutagens predisposes mitochondria to injury on exposure of cells to genotoxins or oxidative stress. Damage to the mitochondrial genome causing mutations or loss of mitochondrial gene products, or to some nuclear genes encoding mitochondrial membrane proteins, may accelerate release of reactive species of oxygen. Such aberrant mitochondria may contribute to cellular aging and promotion of cancer.     

Identification of mitochondrial plasmid-like DNAs in Picea abies (L.) Karst.

The Norway spruce (Picea abies (L.) Karst.) mitochondrial DNA has been extracted from embryonal suspensor masses. In addition to a master chromosome, a family of plasmid-like DNAs were identified. These latter shared cross homologies but had no evident sequence homology with the master chromosome. The occurrence of mitochondrial plasmid-like DNAs was investigated in trees from different provenances. A vast majority of trees displayed extrachromosomal DNA elements of variable stoechiometry. For some trees, the sequences homologous to the extrachromosomal DNA elements were found associated with high molecular weight DNA. Received: 9 July 1997 / Revision received: 29 January 1998 / Accepted: 29 November 1998