Mitochondrial genome instability resulting from SUV3 haploinsufficiency leads to tumorigenesis and shortened lifespan.

Mitochondrial dysfunction has been a hallmark of cancer. However, whether it has a causative role awaits to be elucidated. Here, using an animal model derived from inactivation of SUV3, a mitochondrial helicase, we demonstrated that mSuv3+/- mice harbored increased mitochondrial DNA (mtDNA) mutations and decreased mtDNA copy numbers, leading to tumor development in various sites and shortened lifespan. These phenotypes were transmitted maternally, indicating the etiological role of the mitochondria. Importantly, reduced SUV3 expression was observed in human breast tumor specimens compared with corresponding normal tissues in two independent cohorts. These results demonstrated for the first time that maintaining mtDNA integrity by SUV3 helicase is critical for cancer suppression.

Bioenergetic origins of complexity and disease.

The organizing power of energy flow is hypothesized to be the origin of biological complexity and its decline the basis of "complex" diseases and aging. Energy flow through organic systems creates nucleic acids, which store information, and the annual accumulation of information generates today's complexity. Energy flow through our bodies is mediated by the mitochondria, symbiotic bacteria whose genomes encompass the mitochondrial DNA (mtDNA) and more than 1000 nuclear genes. Inherited and/or epigenomic variation of the mitochondrial genome determines our initial energetic capacity, but the age-related accumulation of somatic cell mtDNA mutations further erodes energy flow, leading to disease. This bioenergetic perspective on disease provides a unifying pathophysiological and genetic mechanism for neuropsychiatric diseases such as Alzheimer and Parkinson Disease, metabolic diseases such as diabetes and obesity, autoimmune diseases, aging, and cancer.

Mitochondrial mutations in cancer.

The metabolism of solid tumors is associated with high lactate production while growing in oxygen (aerobic glycolysis) suggesting that tumors may have defects in mitochondrial function. The mitochondria produce cellular energy by oxidative phosphorylation (OXPHOS), generate reactive oxygen species (ROS) as a by-product, and regulate apoptosis via the mitochondrial permeability transition pore (mtPTP). The mitochondria are assembled from both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) genes. The mtDNA codes for 37 genes essential of OXPHOS, is present in thousands of copies per cell, and has a very high mutations rate. In humans, severe mtDNA mutations result in multisystem disease, while some functional population-specific polymorphisms appear to have permitted humans to adapt to new environments. Mutations in the nDNA-encoded mitochondrial genes for fumarate hydratase and succinate dehydrogenase have been linked to uterine leiomyomas and paragangliomas, and cancer cells have been shown to induce hexokinase II which harnesses OXPHOS adenosine triphosphate (ATP) production to drive glycolysis. Germline mtDNA mutations at nucleotides 10398 and 16189 have been associated with breast cancer and endometrial cancer. Tumor mtDNA somatic mutations range from severe insertion-deletion and chain termination mutations to mild missense mutations. Surprisingly, of the 190 tumor-specific somatic mtDNA mutations reported, 72% are also mtDNA sequence variants found in the general population. These include 52% of the tumor somatic mRNA missense mutations, 83% of the tRNA mutations, 38% of the rRNA mutations, and 85% of the control region mutations. Some associations might reflect mtDNA sequencing errors, but analysis of several of the tumor-specific somatic missense mutations with population counterparts appear legitimate. Therefore, mtDNA mutations in tumors may fall into two main classes: (1) severe mutations that inhibit OXPHOS, increase ROS production and promote tumor cell proliferation and (2) milder mutations that may permit tumors to adapt to new environments. The former may be lost during subsequent tumor oxygenation while the latter may become fixed. Hence, mitochondrial dysfunction does appear to be a factor in cancer etiology, an insight that may suggest new approaches for diagnosis and treatment.

Adeno-associated virus-mediated gene transfer of the heart/muscle adenine nucleotide translocator (ANT) in mouse.

Mitochondrial myopathy, associated with muscle weakness and progressive external ophthalmoplegia, is caused by mutations in mitochondria oxidative phosphorylation genes including the heart-muscle isoform of the mitochondrial adenine nucleotide translocator (ANT1). To develop therapies for mitochondrial disease, we have prepared a recombinant adeno-associated viral vector (rAAV) carrying the mouse Ant1 cDNA. This vector has been used to transduce muscle cells and muscle from Ant1 mutant mice, which manifest mitochondrial myopathy. AAV-ANT1 transduction resulted in long-term, stable expression of the Ant1 transgene in muscle precursor cells as well as differentiated muscle fibers. The transgene ANT1 protein was targeted to the mitochondrion, was inserted into the mitochondrial inner membrane, formed a functional ADP/ATP carrier, increased the mitochondrial export of ATP and reversed the histopathological changes associated with the mitochondrial myopathy. Thus, AAV transduction has the potential of providing symptomatic relief for the ophthalmoplegia and ptosis resulting from paralysis of the extraocular eye muscles cause by mutations in the Ant1 gene.

Mitochondria and cancer: Warburg addressed.

Otto Warburg recognized that cancer cells generate excessive lactate in the presence of oxygen (aerobic glycolysis). It now appears that this phenomenon is the product of two factors: a return to the more glycolytic metabolism of the embryo and alterations in oxidative phosphorylation (OXPHOS) to increase mitochondrial reactive oxygen species (ROS) production. Alterations in the Ras-PI3K-Akt signal transduction pathway can result in induction of hexokinase II and its attachment to mitochondrial porin redirecting mitochondrial ATP to phosphorylate glucose and drive glycolysis. Furthermore, partial inhibition of OXPHOS by mitochondrial gene mutations (germ-line or somatic) can reduce electron flux through the electron transport chain, increasing mitochondrial ROS production. The increased ROS mutagenizes nuclear proto-oncogenes (initiation) and drives nuclear replication (promotion), resulting in cancer. Therefore, hexokinase II and mitochondrial ROS may be useful alternate targets for cancer therapeutics.

Accumulation of mitochondrial DNA deletions in the malignant prostate of patients of different ages.

It has been shown that mitochondrial DNA (mtDNA) deletion mutations accumulate with age in many tissues of the body. However, to date no one has shown that these deletions occur in the malignant prostate. Therefore, we hypothesize that such deletions do occur in the prostate and increasingly so with advanced age. To test this hypothesis, DNA was isolated from 34 radical prostatectomy specimens, and the entire mitochondrial genome (16.5kb) was amplified using long range PCR (LXPCR). The LXPCR products were visualized by gel electrophoresis, and the presence of low molecular weight (<16kb) bands was considered evidence of large mtDNA deletions. In order to show that these lower molecular weight LXPCR bands were not simply PCR artifact, we also digested mtDNA from a subset of the same patients and did Southern analysis with a mtDNA probe. Southern blots confirmed the existence of large deletions in every sample tested. Furthermore, several of the specific deletions identified by LXPCR were also seen in the Southern blots. From the LXPCR data, we found that as the age of the specimen increased, so did the average number of low molecular weight bands (i.e. deletions). In particular, one prominent band was seen at 1.2kb and became more consistent with advanced age.

Novel mtDNA mutations and oxidative phosphorylation dysfunction in Russian LHON families.

Leber's hereditary optic neuropathy (LHON) is characterized by maternally transmitted, bilateral, central vision loss in young adults. It is caused by mutations in the mitochondrial DNA (mtDNA) encoded genes that contribute polypeptides to NADH dehydrogenase or complex I. Four mtDNA variants, the nucleotide pair (np) 3460A, 11778A, 14484C, and 14459A mutations, are known as "primary" LHON mutations and are found in most, but not all, of the LHON families reported to date. Here, we report the extensive genetic and biochemical analysis of five Russian families from the Novosibirsk region of Siberia manifesting maternally transmitted optic atrophy consistent with LHON. Three of the five families harbor known LHON primary mutations. Complete sequence analysis of proband mtDNA in the other two families has revealed novel complex I mutations at nps 3635A and 4640C, respectively. These mutations are homoplasmic and have not been reported in the literature. Biochemical analysis of complex I in patient lymphoblasts and transmitochondrial cybrids demonstrated a respiration defect with complex-I-linked substrates, although the specific activity of complex I was not reduced. Overall, our data suggests that the spectrum of mtDNA mutations associated with LHON in Russia is similar to that in Europe and North America and that the np 3635A and 4640C mutations may be additional mtDNA complex I mutations contributing to LHON expression.

Heparinless partial cardiopulmonary bypass for the repair of aortic trauma.

OBJECTIVE: We hypothesized that partial cardiopulmonary bypass with a heparin-bonded system would be a technically simple, effective adjunct for reducing paraplegia during repair of traumatic aortic rupture. It avoids the risk of heparin, but, unlike left atrial-arterial bypass, it can heat, cool, oxygenate, and rapidly infuse volume if needed. METHODS: A retrospective review was conducted of patients admitted for aortic trauma from July 1994 to December 1999. Bypass consisted of femoral venous (right atrial) cannulation, a centrifugal pump, and an oxygenator-heater/cooler. Arterial return was to the femoral artery or distal aorta. The entire system was heparin-bonded and no systemic heparin was given. RESULTS: Heparin-bonded partial bypass was established in 50 patients (mean age 43 +/- 17 years). Crossclamp time was 32 +/- 11 minutes (range 14-70 minutes), mean flow 3.0 +/- 0.8 L/min, and bypass time 64 +/- 43 minutes. During repair, 58% of patients received volume through the system (mean 1.1 +/- 1.9 L). Core temperature rose slightly (35.9 degrees C +/- 0.7 degrees C to 36.3 degrees C +/- 0.8 degrees C). Three of the 15 patients who underwent percutaneous femoral arterial and venous cannulation concomitant with their angiograms had vessel injury, with one limb loss, and this procedure was discontinued. Thirty-five patients underwent percutaneous femoral vein and direct distal aortic cannulation without event. The mortality rate for patients supported by bypass was 10%, and all deaths were due to other injuries. There were no new cases of paraplegia and no worsening of intracranial or pulmonary injuries. CONCLUSIONS: Heparin-bonded bypass is technically simple to use and avoids the risk of anticoagulation. Paraplegia was avoided. The ability to correct hypothermia, oxygenate, and rapidly infuse volume may simplify management and improve outcomes.

Dietary change through African American churches: baseline results and program description of the eat for life trial.

BACKGROUND: Eat for Life, a multicomponent intervention to increase fruit and vegetable (F & V) consumption among African Americans, is delivered through African American churches. METHODS: Fourteen churches were randomly assigned to one of three treatment conditions: 1) comparison; 2) culturally-sensitive multicomponent intervention with one phone call; and 3) culturally-sensitive multicomponent intervention with four phone calls. The intervention included an 18-minute video, a project cookbook, printed health education materials, and several "cues" imprinted with the project logo and a 5 A Day message. A key element of the telephone intervention was the use of motivational interviewing, a counseling technique originally developed for addictive behaviors. Major outcomes for the trial included total F & V intake, assessed by food-frequency questionnaires (FFQs) and 24-hour recalls, and serum carotenoids. Psychosocial variables assessed included outcome expectations, barriers to F & V intake, preference for meat meals, neophobia, social support to eat more F & V, self-efficacy to eat more F & V, and nutrition knowledge. RESULTS: Baseline mean F & V intakes across the three FFQs ranged from 3.45 to 4.28 servings per day. Intake based on a single 24-hour recall was 3.0 servings. Variables positively correlated with F & V intake included self-efficacy, outcome expectations, and a belief that F & V contain vitamins. Factors negatively correlated with intake include perceived barriers, meat preference, neophobia, and high-fat cooking practices. The completion rate for the first telephone counseling call was 90%. Completion rates for the remaining three calls ranged from 79% to 86%. CONCLUSION: The recruitment and intervention methods of the Eat for Life study appear promising. The telephone intervention based on motivational interviewing is potentially useful for delivering dietary counseling.

Separation of cytochrome c-dependent caspase activation from thiol-disulfide redox change in cells lacking mitochondrial DNA.

Release of mitochondrial cytochrome c (cyt c) is an early and common event during apoptosis. Previous studies showed that the loss of cyt c triggered superoxide production by mitochondria and contributed to the oxidation of cellular thiol-disulfide redox state. In this study, we tested whether loss of the functional electron transport chain due to depleting mitochondrial DNA (mtDNA) would affect this redox-signaling mechanism during apoptosis. Results showed that cyt c release and caspase activation in response to staurosporine treatment were preserved in cells lacking mitochondrial DNA (rho0 cells). However, unlike the case with rho+ cells, in which a dramatic oxidation of intracellular glutathione (GSH) occurred after mitochondrial cyt c release, the thiol-disulfide redox state in apoptotic rho0 cells remained largely unchanged. Thus, mitochondrial signaling of caspase activation can be separated from the bioenergetic function, and mitochondrial respiratory chain is the principal source of ROS generation in staurosporine-induced apoptosis.

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.

Traumatic aortic rupture: recent outcome with regard to neurologic deficit.

BACKGROUND: Traumatic aortic rupture is highly lethal, and its surgical treatment is complicated by a high rate of paraplegia. METHODS: The charts of 263 patients with traumatic aortic rupture from vehicular accidents treated between 1971 and 1998 were reviewed. Patients were grouped according to four periods: group 1, 1971 to 1975, (n = 31); group 2, 1976 to 1985, (n = 83); group 3, 1986 to 1994, (n = 82); and group 4, 1994 to 1998 (n = 67). Seventy-one patients died of exsanguination before definitive care. One hundred-ninety two patients had surgical repair with the following techniques: clamp and sew, 6 in group 1, 22 in group 2, 54 in group 3, none in group 4; shunt, 23 in group 1, 39 in group 2, 2 in group 3; cardiopulmonary bypass, 2 in group 1, 1 in group 3. Forty-three patients had partial bypass with the centrifugal pump and heparin-coated circuits in group 4. RESULTS: Operative mortality was 6 of 31 (19%) in group 1, 22 of 61 (36%) in group 2, 15 of 57 (26%) in group 3, and 7 of 43 (16%) in group 4. There was one case of paraplegia in group 1 (4%), ten in group 2 (18%), 11 in group 3 (26%), and none in group 4. This difference of paraplegia between the groups was significant (p<0.002). Significant factors for paraplegia were intraoperative hypotension (p<0.000002), cross-clamp time longer than 30 minutes (p<0.008), pump versus no pump (p<0.008), and younger age group (28+/-11 versus 39+/-17 years) (p<0.03). CONCLUSIONS: There were no statistically significant improvements in mortality rate over the four periods, although, the mortality rate was lowest in the last period when partial bypass with the centrifugal pump was used exclusively. Further, the use of the centrifugal pump with heparin-coated circuits, with femoral vein cannulation into the right atrium and distal aortic perfusion, reduced paraplegia significantly.

Up-regulation of nuclear and mitochondrial genes in the skeletal muscle of mice lacking the heart/muscle isoform of the adenine nucleotide translocator.

Mice deficient in the heart/muscle specific isoform of the adenine nucleotide translocator (ANT1) exhibit many of the hallmarks of human oxidative phosphorylation (OXPHOS) disease, including a dramatic proliferation of skeletal muscle mitochondria. Because many of the genes necessary for mitochondrial biosynthesis, OXPHOS function, and response to OXPHOS disease might be expected to be up-regulated in the Ant1(-/-) mouse, we used differential display reverse transcription-polymerase chain reaction techniques in an effort to identify these genes. 17 genes were identified as up-regulated in Ant1-deficient mice, and they fall into four categories: 1) nuclear and mitochondrial genes encoding OXPHOS components, 2) mitochondrial tRNA and rRNA genes, 3) genes involved in intermediary metabolism, and 4) an eclectic group of other genes. Among the latter genes, we identified the gene encoding anti-apoptotic Mcl-1, the Skd3 gene, and the WS-3 gene, which were previously unknown to be related to mitochondrial function. These results indicate that identification of genes up-regulated in the skeletal muscle of the Ant1-deficient mouse provides a novel method for identifying mammalian genes required for mitochondrial biogenesis.

Mitochondrial diseases in man and mouse.

Over the past 10 years, mitochondrial defects have been implicated in a wide variety of degenerative diseases, aging, and cancer. Studies on patients with these diseases have revealed much about the complexities of mitochondrial genetics, which involves an interplay between mutations in the mitochondrial and nuclear genomes. However, the pathophysiology of mitochondrial diseases has remained perplexing. The essential role of mitochondrial oxidative phosphorylation in cellular energy production, the generation of reactive oxygen species, and the initiation of apoptosis has suggested a number of novel mechanisms for mitochondrial pathology. The importance and interrelationship of these functions are now being studied in mouse models of mitochondrial disease.

Coordinate expression of nuclear and mitochondrial genes involved in energy production in carcinoma and oncocytoma.

The expression of mitochondrial and nuclear genes involved in ATP production was examined in renal carcinomas, renal oncocytomas, and a salivary oncocytoma. Renal carcinomas were found to have a reduced mitochondrial DNA (mtDNA) content while oncocytomas had increased mtDNA contents. This parallels morphological changes in mitochondrial number in these tumours. In the carcinomas, mtDNA transcripts were decreased 5- to 10-fold relative to control kidneys, suggesting that mitochondrial transcript levels depend on the mtDNA content. In renal oncocytomas, mtDNA transcripts were slightly reduced in spite of a high mtDNA content. However, in the salivary gland oncocytoma, mtDNA transcripts were increased more than 10-fold in parallel with a 10-fold increase in mtDNA content. The expression of the nuclear DNA oxidative phosphorylation genes, ATPsyn beta and ANT2, was reduced up to 4-fold in renal carcinoma. In contrast, the levels of these two nuclear gene transcripts were induced about 4-fold in renal oncocytoma and up to 30-fold in salivary gland oncocytoma. Moreover, the ANT2 precursors were observed to change in oncocytomas. These data suggest a coordinated regulation of nuclear and mitochondrial gene expression in renal carcinomas and the specific induction of nuclear OXPHOS gene expression in oncocytomas.

Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.

Novel mitochondrial DNA deletion found in a renal cell carcinoma.

Polymerase chain reaction (PCR) was used to analyze a rarely deleted region of mitochondrial DNA (mtDNA) from 39 human renal cell carcinomas (RCC) and matched normal kidney tissue removed during radical nephrectomy. One tumor specimen (E.R.) had a unique PCR product approximately 250 base pairs (bp) smaller than the PCR product found in the normal E.R. kidney. Sequence analysis of the tumor-specific PCR fragment revealed a 264 bp deletion in the first subunit (NDI) of NADH:ubiquinone oxidoreductase (complex I) of the electron transport chain. Southern analysis of the RCCs demonstrated that approximately 50% of the mtDNA molecules in the primary RCC contained a unique 3.2 kb EcoRV restriction fragment found only in E.R. tumor mtDNA. Northern analysis demonstrated preferential transcription of the truncated NDI mRNA. None of the five metastases or any normal tissue from E.R. contained levels of the NDI deletion detectable by PCR. This is the first reported case of an intragenic NDI mtDNA deletion.

Renal amino acid transport in adults with oxidative phosphorylation diseases.

The clinical manifestations of mitochondrial DNA (mtDNA) mutations depend on a variety of factors including ratios of normal to abnormal mtDNA and tissue-specific differences in ATP production by oxidative phosphorylation (OXPHOS). In order to investigate the effects of OXPHOS defects on renal tubule function, we characterized sodium-coupled transport processes in six individuals with OXPHOS diseases. Pathogenic mtDNA mutations were identified in five of these individuals. Sodium coupled transport processes were evaluated by determining fractional excretions of amino acids, glucose, lactate, urate, and phosphate in patients and controls. Four of the six individuals had high fractional excretions of neutral amino acids, indicating abnormal renal tubule reabsorbtion of these amino acids. Abnormalities in fractional excretions of lactate, glucose, urate, and phosphate were less pronounced. These results demonstrate that sodium-coupled transport processes in the kidney are sensitive to OXPHOS impairment. When abnormalities in these processes are encountered, an OXPHOS disease should be included in the differential diagnosis.