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1.  Genomic instability induced by mutant succinate dehydrogenase subunit D (SDHD) is mediated by O2−• and H2O2 
Free radical biology & medicine  2011;52(1):160-166.
SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Over expression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (p < 0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (p < 0.0005). Finally, 95% depletion of glutathione using L-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (p < 0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.
PMCID: PMC3249516  PMID: 22041456
succinate dehydrogenase mutations; mutation frequency; mutation rate; genomic instability; reactive oxygen species
2.  SIRT3 is a Mitochondrial Localized Tumor Suppressor Required for Maintenance of Mitochondrial Integrity and Metabolism During Stress 
Cancer cell  2010;17(1):41-52.
The sirtuin gene family (SIRT) is hypothesized to regulate the aging process and play a role in cellular repair. This work demonstrates that SIRT3−/− mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3−/− MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3−/− MEFs and reverses the tumor permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3−/− mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondrial localized tumor suppressor.
PMCID: PMC3711519  PMID: 20129246
SIRT3; Mitochondria; Carcinogenesis; Tumor suppressor
3.  Sensitivity to Low-Dose/Low-LET Ionizing Radiation in Mammalian Cells Harboring Mutations in Succinate Dehydrogenase Subunit C is Governed by Mitochondria-Derived Reactive Oxygen Species 
Radiation research  2010;175(2):150-158.
It has been hypothesized that ionizing radiation-induced disruptions in mitochondrial O2 metabolism lead to persistent heritable increases in steady-state levels of intracellular superoxide (O2•−) and hydrogen peroxide (H2O2) that contribute to the biological effects of radiation. Hamster fibroblasts (B9 cells) expressing a mutation in the gene coding for the mitochondrial electron transport chain protein succinate dehydrogenase subunit C (SDHC) demonstrate increases in steady-state levels of O2•− and H2O2. When B9 cells were exposed to low-dose/low-LET radiation (5–50 cGy), they displayed significantly increased clonogenic cell killing compared with parental cells. Clones derived from B9 cells overexpressing a wild-type human SDHC (T4, T8) demonstrated significantly increased surviving fractions after exposure to 5–50 cGy relative to B9 vector controls. In addition, pretreatment with polyethylene glycol-conjugated CuZn superoxide dismutase and catalase as well as adenoviral-mediated overexpression of MnSOD and/or mitochondria-targeted catalase resulted in significantly increased survival of B9 cells exposed to 10 cGy ionizing radiation relative to vector controls. Adenoviral-mediated overexpression of either MnSOD or mitochondria-targeted catalase alone was equally as effective as when both were combined. These results show that mammalian cells over expressing mutations in SDHC demonstrate low-dose/low-LET radiation sensitization that is mediated by increased levels of O2•− and H2O2. These results also support the hypothesis that mitochondrial O2•− and H2O2 originating from SDH are capable of playing a role in low-dose ionizing radiation-induced biological responses.
PMCID: PMC3080019  PMID: 21268708
4.  Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin 
PLoS ONE  2011;6(9):e24792.
The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho0) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho0 derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho0 breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2.
PMCID: PMC3174207  PMID: 21935467
5.  Impaired OXPHOS Complex III in Breast Cancer 
PLoS ONE  2011;6(8):e23846.
We measured the mitochondrial oxidative phosphorylation (mtOXPHOS) activities of all five complexes and determined the activity and gene expression in detail of the Complex III subunits in human breast cancer cell lines and primary tumors. Our analysis revealed dramatic differences in activity of complex III between normal and aggressive metastatic breast cancer cell lines. Determination of Complex III subunit gene expression identified over expression and co-regulation of UQCRFS1 (encoding RISP protein) and UQCRH (encoding Hinge protein) in 6 out of 9 human breast tumors. Analyses of UQCRFS1/RISP expression in additional matched normal and breast tumors demonstrated an over expression in 14 out of 40 (35%) breast tumors. UQCRFS1/RISP knockdown in breast tumor cell line led to decreased mitochondrial membrane potential as well as a decrease in matrigel invasion. Furthermore, reduced matrigel invasion was mediated by reduced ROS levels coinciding with decreased expression of NADPH oxidase 2, 3, 4 and 5 involved in ROS production. These studies provide direct evidence for contribution of impaired mtOXPHOS Complex III to breast tumorigenesis.
PMCID: PMC3162009  PMID: 21901141
6.  NADPH oxidase 4 is an oncoprotein localized to mitochondria 
Cancer Biology & Therapy  2010;10(3):223-231.
Reactive oxygen species (ROS) are known to be involved in many physiological and pathological processes. Initially ROS-producing NADPH oxidase (NOX) proteins were thought to be present in phagocytes. However, recent studies have demonstrated that NOX proteins are expressed in many other cell types and tissues. NOX family members' expression and function seems to vary from tissue to tissue. We determined the expression of the NOX family of proteins (NOX1-5) in normal breast tissue and breast tumors. Our study revealed that normal breast tissues express NOX1, 4 and 5 genes. Similar pattern of expression was revealed in a breast epithelial cell line. We found that NOX4 was overexpressed in the majority of breast cancer cell lines and primary breast tumors. NOX4 was also overexpressed in ovarian tumors. Overexpression of NOX4 in normal breast epithelial cells resulted in cellular senescence, resistance to apoptosis, and tumorigenic transformation. Overexpression of NOX4 in already transformed breast tumor cells also showed increased tumorigenicity. Strong evidence suggests that regulation of these processes occurs through NOX4 generation of ROS in the mitochondria. We demonstrate that the NOX4 protein contains a 73 amino acid long mitochondrial localization signal at the N-terminus that is capable of transporting a passenger protein GFP into the mitochondria. Treatment of NOX4 overexpressing cells with catalase resulted in decreased tumorigenic characteristics. Together, this study provides evidence for an oncogenic function for NOX4 protein localized to mitochondria and suggests that NOX4 is a novel source of ROS produced in the mitochondria. This study also identifies a possible treatment of NOX4-induced breast cancer by antioxidant treatment.
PMCID: PMC3040835  PMID: 20523116
NADPH oxidase 4; breast cancer; oncogenesis; catalase
7.  Mitochondrial Complex II Dysfunction Can Contribute Significantly to Genomic Instability after Exposure to Ionizing Radiation 
Radiation research  2009;172(6):737-745.
Ionizing radiation induces chronic metabolic oxidative stress and a mutator phenotype in hamster fibroblasts that is mediated by H2O2, but the intracellular source of H2O2 is not well defined. To determine the role of mitochondria in the radiation-induced mutator phenotype, end points of mitochondrial function were determined in unstable (CS-9 and LS-12) and stable (114) hamster fibroblast cell lines derived from GM10115 cells exposed to 10 Gy X rays. Cell lines isolated after irradiation demonstrated a 20–40% loss of mitochondrial membrane potential and an increase in mitochondrial content compared to the parental cell line GM10115. Surprisingly, no differences were observed in steady-state levels of ATP (P > 0.05). Unstable clones demonstrated increased oxygen consumption (two- to threefold; CS-9) and/or increased mitochondrial electron transport chain (ETC) complex II activity (twofold; LS-12). Using Western blot analysis and Blue Native gel electrophoresis, a significant increase in complex II subunit B protein levels was observed in LS-12 cells. Furthermore, immunoprecipitation assays revealed evidence of abnormal complex II assembly in LS-12 cells. Treatment of LS-12 cells with an inhibitor of ETC complex II (thenoyltrifluoroacetone) resulted in significant decreases in the steady-state levels of H2O2 and a 50% reduction in mutation frequency as well as a 16% reduction in CAD gene amplification frequency. These data show that radiation-induced genomic instability was accompanied by evidence of mitochondrial dysfunction leading to increased steady-state levels of H2O2 that contributed to increased mutation frequency and gene amplification. These results support the hypothesis that mitochondrial dysfunction originating from complex II can contribute to radiation-induced genomic instability by increasing steady-state levels of reactive oxygen species.
PMCID: PMC2793528  PMID: 19929420
8.  mtDNA G10398A variant in African-American women with breast cancer provides resistance to apoptosis and promotes metastasis in mice 
Journal of human genetics  2009;54(11):647-654.
We investigated the effect of the mitochondrial DNA (mtDNA) polymorphism G10398A found in African-American women with aggressive breast cancer on apoptosis and tumorigenesis. We generated human cytoplasmic hybrid (cybrid) by repopulation of recipient ρ0 cells (devoid of mtDNA) with donor mtDNA derived from patients with breast cancer harboring the G10398A polymorphism. We investigated a number of functional phenotypes of the G10398A cybrid. The G10398A cybrid showed a slower proliferation rate and progression through the cell cycle, as well as increased complex I activity, increased levels of reactive oxygen species and depolarized mitochondria. The G10398A cybrid also showed resistance to apoptosis triggered by etoposide. Resistance to apoptosis was mediated by Akt activation. In addition, our studies showed that the G10398A cybrid cells form an increased number of anchorage-independent colonies in vitro and metastases in mice. Together our studies suggest that the G10398A variant confers resistance to apoptosis and promotes metastasis.
PMCID: PMC2909846  PMID: 19763141
African American; Akt; apoptosis; breast cancer; cybrid; G10398A; metastasis; mitochondria
9.  Mutations in mitochondrial DNA polymerase γ promote breast tumorigenesis 
Journal of human genetics  2009;54(9):516-524.
Decreased mitochondrial oxidative phosphorylation (OXPHOS) is one of the hallmarks of cancer. To date the identity of nuclear gene(s) responsible for decreased OXPHOS in tumors remains unknown. It is also unclear whether mutations in nuclear gene(s) responsible for decreased OXPHOS affect tumorigenesis. Polymerase γ (POLG) is the only DNA polymerase known to function in human mitochondria. Mutations in POLG are known to cause mtDNA depletion and decreased OXPHOS resulting in mtDNA depletion syndrome (MDS) in humans. We therefore sequenced all coding exons [2-23] and flanking intron/splice junctions of POLG in breast tumors. We found that the POLG gene was mutated in 63% of the breast tumors. We identified a total of 17 mutations across the POLG gene. Mutations were found in all three domains of POLG protein, including T251I (exonuclease domain), P587L (linker region) and E1143G (polymerase domain). We identified two novel mutations that include one silent (A703A) and one missense (R628Q) mutation in the evolutionarily conserved POLG linker region. Additionally, we identified three novel mutations in the intronic region. Our study also revealed that mtDNA was depleted in breast tumors. Consistently, mutant POLG when expressed in breast cancer cells induced depletion of mtDNA, decreased mitochondrial activity, decreased mitochondrial membrane potential, increased levels of reactive oxygen species (ROS), and increased matrigel invasion. Together, our study provides the first comprehensive analysis of the POLG gene mutation in human cancer and suggests a role for POLG in 1) decreased OXPHOS in cancers and 2) in promoting tumorigenicity.
PMCID: PMC2782392  PMID: 19629138
Breast Cancer; POLG; MtDNA; Mitochondria; Mutation; Mitochondrial

Results 1-9 (9)