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.
NADPH oxidase 4; breast cancer; oncogenesis; catalase
Human mitochondrial DNA (mtDNA) encodes 13 proteins involved in oxidative phosphorylation (OXPHOS). In order to investigate the role of mitochondrial OXPHOS genes in breast tumorigenesis, we have developed a breast epithelial cell line devoid of mtDNA (ρ0 cells). Our analysis revealed that depletion of mtDNA in breast epithelial cells results in in vitro tumorigenic phenotype as well as breast tumorigenesis in a xenograft model. We identified two major gene networks which were differentially regulated between parental and ρ0 epithelial cells. The focal proteins in these networks include (i) FN1 (fibronectin) and (ii) p53. Bioinformatic analyses of FN1 network identified laminin, integrin and 3 of 6 members of peroxiredoxin whose expression were altered in ρ0 epithelial cells. In the p53 network, we identified SMC4 and WRN whose changes in expression suggest that this network may affect chromosomal stability. Consistent with above finding our study revealed an increase in DNA double strand breaks and unique chromosomal rearrangements in ρ0 breast epithelial cells. Additionally, we identified tight junction proteins claudin-1 and claudin-7 in p53 network. To determine the functional relevance of altered gene expression, we focused on detailed analyses of claudin-1 and -7 proteins in breast tumorigenesis. Our study determined that (i) claudin-1 and 7 were indeed downregulated in ρ0 breast epithelial cells, (ii) downregulation of claudin-1 or -7 led to neoplastic transformation of breast epithelial cells, and (iii) claudin-1 and -7 were also downregulated in primary breast tumors. Together, our study suggest that mtDNA encoded OXPHOS genes play a key role in transformation of breast epithelial cells and that multiple pathway involved in mitochondria-to-nucleus retrograde regulation contribute to transformation of breast epithelial cells.
mitochondria; breast tumorigenesis; mtDNA depletion; claudin-1; claudin-7; ρ0 cells; mitochondrial DNA
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.
Breast Cancer; POLG; MtDNA; Mitochondria; Mutation; Mitochondrial
We previously hypothesized a role for mitochondria damage checkpoint (mito-checkpoint) in maintaining the mitochondrial integrity of cells. Consistent with this hypothesis, defects in mitochondria have been demonstrated to cause genetic and epigenetic changes in the nuclear DNA, resistance to cell-death and tumorigenesis. In this paper, we describe that defects in mitochondria arising from the inhibition of mitochondrial oxidative phosphorylation (mtOXPHOS) induce cell cycle arrest, a response similar to the DNA damage checkpoint response.
Materials and Methods:
Primary mouse embryonic fibroblasts obtained from p53 wild-type and p53-deficient mouse embryos (p53 -/-) were treated with inhibitors of electron transport chain and cell cycle analysis, ROS production, mitochondrial content analysis and immunoblotting was performed. The expression of p53R2 was also measured by real time quantitative PCR.
We determined that, while p53 +/+ cells arrest in the cell cycle, p53 -/- cells continued to divide after exposure to mitochondrial inhibitors, showing that p53 plays an important role in the S-phase delay in the cell cycle. p53 is translocated to mitochondria after mtOXPHOS inhibition. Our study also revealed that p53-dependent induction of reactive oxygen species acts as a major signal triggering a mito-checkpoint response. Furthermore our study revealed that loss of p53 results in down regulation of p53R2 that contributes to depletion of mtDNA in primary MEF cells.
Our study suggests that p53 1) functions as mito-checkpoint protein and 2) regulates mtDNA copy number and mitochondrial biogenesis. We describe a conceptual organization of the mito-checkpoint pathway in which identified roles of p53 in mitochondria are incorporated.
Cell cycle; metabolic stress; mitocheckpoint; mitochondrial; mitochondria; mtDNA; p53
Epigenetic modification in the nuclear genome plays a key role in human tumorigenesis. In this paper, we investigated whether changes in the mtDNA copy number frequently reported to vary in a number of human tumors induce methylation changes in the nucleus. We utilized the Restriction Landmark Genomic Scanning (RLGS) to identify genes that undergo changes in their methylation status in response to the depletion and repletion of mtDNA. Our study demonstrates that depletion of mtDNA results in significant changes in methylation pattern of a number of genes. Furthermore, our study suggests that methylation changes are reversed by the restoration of mtDNA in cells otherwise lacking the entire mitochondrial genome. These studies provide the first direct evidence that mitochondria regulate epigenetic modification in the nucleus that may contribute to tumorigenesis.
mitochondria; epigenetic; retrograde; mitochondrial DNA; mtDNA depletion; RLGS; OXPHOS; rho0
Oxidative phosphorylation is an indispensable resource of ATP in tissues with high requirement of energy. If the ATP demand is not met, studies suggest that this will lead to senescence and cell death in the affected tissue. The term reserve respiratory capacity or spare respiratory capacity is used to describe the amount of extra ATP that can be produced by oxidative phosphorylation in case of a sudden increase in energy demand. Depletion of the reserve respiratory capacity has been related to a range of pathologies affecting high energy requiring tissues. During aging of an organism, and as a result of mitochondrial dysfunctions, the efficiency of oxidative phosphorylation declines. Based on examples from the energy requiring tissues such as brain, heart, and skeletal muscle, we propose that the age-related decline of oxidative phosphorylation decreases the reserve respiratory capacity of the affected tissue, sensitizes the cells to surges in ATP demand, and increases the risk of resulting pathologies.
In order to investigate the cell death-inducing effects of rotenone, a plant extract commonly used as a mitochondrial complex I inhibitor, we studied cancer cell lines with different genetic backgrounds. Rotenone inhibits cell growth through the induction of cell death and cell cycle arrest, associated with the development of mitotic catastrophe. The cell death inducer staurosporine potentiates the inhibition of cell growth by rotenone in a dose-dependent synergistic manner. The tumor suppressor p53 is involved in rotenone-induced cell death, since the drug treatment results in increased expression, phosphorylation and nuclear localization of the protein. The evaluation of the effects of rotenone on a p53-deficient cell line revealed that although not required for the promotion of mitotic catastrophe, functional p53 appears to be essential for the extensive cell death that occurs afterwards. Our results suggest that mitotic slippage also occurs subsequently to the rotenone-induced mitotic arrest and cells treated with the drug for a longer period become senescent. Treatment of mtDNA-depleted cells with rotenone induces cell death and cell cycle arrest as in cells containing wild type mtDNA, but not formation of reactive oxygen species. This suggests that the effects of rotenone are not dependent from the production of reactive oxygen species. This work highlights the multiple effects of rotenone in cancer cells related to its action as an anti-mitotic drug.
cell death; rotenone; p53; mitotic catastrophe; cell cycle
Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin-induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL-60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL-60 cells which also were more sensitive and contained autophagic vesicles. Studies in Rho0 cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti-proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance.
Cellular stress (reactive oxygen species, mitochondrial respiration); Cell fate (autophagy, cell death); Cell proliferation; Resazurin (Alamar Blue)
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.
Leigh syndrome; brainstem; magnetic resonance imaging; complex V
Mitochondrial dysfunction has been implicated in premature aging, age-related diseases, and tumor initiation and progression. Alterations of the mitochondrial genome accumulate both in aging tissue and tumors. This paper describes our contemporary view of mechanisms by which alterations of the mitochondrial genome contributes to the development of age- and tumor-related pathological conditions. The mechanisms described encompass altered production of mitochondrial ROS, altered regulation of the nuclear epigenome, affected initiation of apoptosis, and a limiting effect on the production of ribonucleotides and deoxyribonucleotides.
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.
African American; Akt; apoptosis; breast cancer; cybrid; G10398A; metastasis; mitochondria
A common metabolic change in cancer is the acquisition of glycolytic phenotypes. Increased expression of glycolytic enzymes is considered as one contributing factor. The role of mitochondrial defects in acquisition of glycolytic phenotypes has been postulated but remains controversial. Here we show that functional defects in mitochondrial respiration could be induced by oncogenic H-RasQ61L transformation, even though the mitochondrial contents or mass was not reduced in the transformed cells. First, mitochondrial respiration, as measured by mitochondrial oxygen consumption, was suppressed in NIH-3T3 cells transformed with H-RasQ61L. Second, oligomycin or rotenone did not reduce the cellular ATP levels in the H-RasQ61L transformed cells, suggesting a diminished role of mitochondrial respiration in the cellular energy metabolism. Third, inhibition of glycolysis with iodoacetic acid reduced ATP levels at a much faster rate in H-RasQ61L transformed cells than in the vector control cells. The reduction of cellular ATP levels was reversed by exogenously added pyruvate in the vector control cells but not in H-RasQ61L transformed cells. Finally when compared to the HRasQ61L transformed cells, the vector control cells had increased resistance toward glucose deprivation. The increased resistance was dependent on mitochondrial oxidative phosphorylation since rotenone or oligomycin abolished the increased survival of the vector control cells under glucose deprivation. The results also suggest an inability of the H-RasQ61L transformed cells to reactivate mitochondrial respiration under glucose deprivation. Taken together, the data suggest that mitochondrial respiration can be impaired during transformation of NIH-3T3 cells by oncogeneic H-RasQ61L.
Ras; mitochondrial respiration; glycolysis; Electron transport chain; complex IV; transformation; cellular energy metabolism; ATP
Summary:The program Fluctuation AnaLysis CalculatOR (FALCOR) is a web tool designed for use with Luria–Delbrück fluctuation analysis to calculate the frequency and rate from various mutation assays in bacteria and yeast. Three calculation methods are available through this program: (i) Ma-Sandri-Sarkar Maximum Likelihood Estimator (MSS-MLE) method, (ii) Lea-Coulson method of the median (LC) and (iii) frequency.
Availability: The FALCOR rate calculator is currently accessible at http://www.mitochondria.org/protocols/FALCOR.html. This program is written as a Java™ Applet, requiring a web browser enabled with Sun MicroSystems' Java Virtual Machine.
ATP (energy production) production is not the only function of the mitochondria. Mitochondria perform multiple cellular functions. Among others, these functions include control of cell death, growth, development, integration of signals from mitochondria to nucleus and nucleus to mitochondria, and various metabolic pathways. Although defects in mitochondrial function are most commonly associated with bioenergetic deficiencies, our studies demonstrate that mitochondrial defects lead to genome instability in the nuclear DNA, resistance to apoptosis and induction of NADPH oxidase, a designated producer of reactive oxygen species. These transformations in cellular phenotype are known contributors to the development of tumors in humans. Consistent with the role of mitochondria in carcinogenesis, studies in the past few years have described an increased risk of cancers associated with specific mitochondrial DNA (mtDNA) polymorphism among various different haplogroups in human population. However, molecular mechanisms underlying increased risk of cancer due to specific mtDNA polymorphisms is currently lacking. It is likely that mtDNA polymorphisms in mitochondrial genes involved in electron transport chain and oxidative phosphorylation result in increased oxidative stress and hypermutagenesis of mitochondrial as well as nuclear DNA. We suggest that in studies relating to cancer epidemiology, the significance of a particular mtDNA polymorphism(s) should be analyzed together with other polymorphisms in mtDNA and in nuclear DNA.
Apoptosis; mitochondria; oxidative phosphorylation; polymorphism; reactive oxygen species
Cytogenetic biomarkers are essential for assessing environmental exposure, and reflect adverse human health effects such as cellular damage. Arsenic is a potential clastogen and aneugen. In general, the majority of the studies on clastogenic effects of arsenic are based on frequency of micronuclei (MN) study in peripheral lymphocytes, urothelial and oral epithelial cells. To find out the most suitable cell type, here, we compared cytogenetic damage through MN assay in (a) various populations exposed to arsenic through drinking water retrieved from literature review, as also (b) arsenic-induced Bowen's patients from our own survey.
For literature review, we have searched the Pubmed database for English language journal articles using the following keywords: "arsenic", "micronuclei", "drinking water", and "human" in various combinations. We have selected 13 studies consistent with our inclusion criteria that measured micronuclei in either one or more of the above-mentioned three cell types, in human samples. Compared to urothelial and buccal mucosa cells, the median effect sizes measured by the difference between people with exposed and unexposed, lymphocyte based MN counts were found to be stronger. This general pattern pooled from 10 studies was consistent with our own set of three earlier studies. MN counts were also found to be stronger for lymphocytes even in arsenic-induced Bowen's patients (cases) compared to control individuals having arsenic-induced non-cancerous skin lesions.
Overall, it can be concluded that MN in lymphocytes may be superior to other epithelial cells for studying arsenic-induced cytogenetic damage.
The chronological lifespan of eukaryotic organisms is extended by the mutational inactivation of conserved growth-signaling pathways that regulate progression into and through the cell cycle. Here we show that in the budding yeast S. cerevisiae, these and other lifespan-extending conditions, including caloric restriction and osmotic stress, increase the efficiency with which nutrient-depleted cells establish or maintain a cell cycle arrest in G1. Proteins required for efficient G1 arrest and longevity when nutrients are limiting include the DNA replication stress response proteins Mec1 and Rad53. Ectopic expression of CLN3 encoding a G1 cyclin downregulated during nutrient depletion increases the frequency with which nutrient depleted cells arrest growth in S phase instead of G1. Ectopic expression of CLN3 also shortens chronological lifespan in concert with age-dependent increases in genome instability and apoptosis. These findings indicate that replication stress is an important determinant of chronological lifespan in budding yeast. Protection from replication stress by growth-inhibitory effects of caloric restriction, osmotic and other stresses may contribute to hormesis effects on lifespan. Replication stress also likely impacts the longevity of higher eukaryotes, including humans.
In prostate cancer, normal citrate-producing glandular secretory epithelial cells undergo a metabolic transformation to malignant citrate-oxidizing cells. m-Aconitase is the critical step involved in this altered citrate metabolism that is essential to prostate malignancy. The limiting m-aconitase activity in prostate epithelial cells could be the result of a decreased level of m-aconitase enzyme and/or the inhibition of existing m-aconitase. Earlier studies identified zinc as an inhibitor of m-aconitase activity in prostate cells; and that the depletion of zinc in malignant cells is an important factor in this metabolic transformation. However, a possibility remains that an altered expression and level of m-aconitase enzyme might also be involved in this metabolic transformation. To address this issue, the in situ level of m-aconitase enzyme was determined by immunohistochemical analysis of prostate cancer tissue sections and malignant prostate cell lines.
The immunocytochemical procedure successfully identified the presence of m-aconitase localized in the mitochondrial compartment in PC-3, LNCaP, and DU-145 malignant prostate cell lines. The examination of prostate tissue sections from prostate cancer subjects demonstrated that m-aconitase enzyme is present in the glandular epithelium of normal glands, hyperplastic glands, adenocrcinomatous glands, and prostatic intraepithelial neoplastic foci. Quantitative analysis of the relative level of m-aconitase in the glandular epithelium of citrate-producing adenomatous glands versus the citrate-oxidizing adenocarcinomatous glands revealed no significant difference in m-aconitase enzyme levels. This is in contrast to the down-regulation of ZIP1 zinc transporter in the malignant glands versus hyperplastic glands that exists in the same tissue samples.
The results demonstrate the existence of m-aconitase enzyme in the citrate-producing glandular epithelial cells; so that deficient m-aconitase enzyme is not associated with the limiting m-aconitase activity that prevents citrate oxidation in these cells. The level of m-aconitase is maintained in the malignant cells; so that an altered enzyme level is not associated with the increased m-aconitase activity. Consequently, the elevated zinc level that inhibits m-aconitase enzyme is responsible for the impaired citrate oxidation in normal and hyperplastic prostate glandular epithelial cells. Moreover, the down-regulation of ZIP1 zinc transporter and corresponding depletion of zinc results in the increase in the activity of the existing m-aconitase activity in the malignant prostate cells. The studies now define the mechanism for the metabolic transformation that characterizes the essential transition of normal citrate-producing epithelial cells to malignant citrate-oxidizing cells.
The genetic and molecular mechanisms responsible for and associated with the development and progression of prostate malignancy are largely unidentified. The peripheral zone is the major region of the human prostate gland where malignancy develops. The normal peripheral zone glandular epithelium has the unique function of accumulating high levels of zinc. In contrast, the ability to accumulate zinc is lost in the malignant cells. The lost ability of the neoplastic epithelial cells to accumulate zinc is a consistent factor in their development of malignancy. Recent studies identified ZIP1 (SLC39A1) as an important zinc transporter involved in zinc accumulation in prostate cells. Therefore, we investigated the possibility that down-regulation of hZIP1 gene expression might be involved in the inability of malignant prostate cells to accumulate zinc. To address this issue, the expression of hZIP1 and the depletion of zinc in malignant versus non-malignant prostate glands of prostate cancer tissue sections were analyzed. hZIP1 expression was also determined in malignant prostate cell lines.
hZIP1 gene expression, ZIP1 transporter protein, and cellular zinc were prominent in normal peripheral zone glandular epithelium and in benign hyperplastic glands (also zinc accumulating glands). In contrast, hZIP1 gene expression and transporter protein were markedly down-regulated and zinc was depleted in adenocarcinomatous glands and in prostate intra-epithelial neoplastic foci (PIN). These changes occur early in malignancy and are sustained during its progression in the peripheral zone. hZIP1 is also expressed in the malignant cell lines LNCaP, PC-3, DU-145; and in the nonmalignant cell lines HPr-1 and BPH-1.
The studies clearly establish that hZIP1 gene expression is down regulated and zinc is depleted in adenocarcinomatous glands. The fact that all the malignant cell lines express hZIP1 indicates that the down-regulation in adenocarcinomatous glands is likely due to in situ gene silencing. These observations, coupled with the numerous and consistent reports of loss of zinc accumulation in malignant cells in prostate cancer, lead to the plausible proposal that down regulation of hZIP1 is a critical early event in the development prostate cancer.
prostate cancer; zinc; ZIP1 zinc transporter; citrate; ZIP1 gene expression
Uracil DNA glycosylase (UDG) plays a major role in repair of uracil formed due to deamination of cytosine. UDG in human cells is present in both the nucleus and mitochondrial compartments. Although, UDG's role in the nucleus is well established its role in mitochondria is less clear.
In order to identify UDG's role in the mitochondria we expressed UGI (uracil glycosylase inhibitor) a natural inhibitor of UDG in the mitochondria. Our studies suggest that inhibition of UDG by UGI in the mitochondria does not lead to either spontaneous or induced mutations in mtDNA. Our studies also suggest that UGI expression has no affect on cellular growth or cytochrome c-oxidase activity.
These results suggest that human cell mitochondria contain alternatives glycosylase (s) that may function as back up DNA repair protein (s) that repair uracil in the mitochondria.
Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype. Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho0) or those with deleted mitochondrial DNA (rho–). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho– and rho0 cells were ∼2- to 3-fold higher compared to untreated control and wild-type cells, respectively. Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS). In contrast, inactivation of mitochondrial activity (rho– and rho0) led to decreased intracellular levels of ROS. We also demonstrate that in rho0 cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome. However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A. Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.
The interaction of nuclear and mitochondrial genes is an essential feature in maintenance of normal cellular function. Of 82 structural subunits that make up the oxidative phosphorylation system in the mitochondria, mitochondrial DNA (mtDNA) encodes 13 subunits and rest of the subunits are encoded by nuclear DNA. Mutations in mitochondrial genes encoding the 13 subunits have been reported in a variety of cancers. However, little is known about the nuclear response to impairment of mitochondrial function in human cells.
We isolated a Rho0 (devoid of mtDNA) derivative of a breast cancer cell line. Our study suggests that depletion of mtDNA results in oxidative stress, causing increased lipid peroxidation in breast cancer cells. Using a cDNA microarray we compared differences in the nuclear gene expression profile between a breast cancer cell line (parental Rho+) and its Rho0 derivative impaired in mitochondrial function. Expression of several nuclear genes involved in cell signaling, cell architecture, energy metabolism, cell growth, apoptosis including general transcription factor TFIIH, v-maf, AML1, was induced in Rho0 cells. Expression of several genes was also down regulated. These include phospholipase C, agouti related protein, PKC gamma, protein tyrosine phosphatase C, phosphodiestarase 1A (cell signaling), PIBF1, cytochrome p450, (metabolism) and cyclin dependent kinase inhibitor p19, and GAP43 (cell growth and differentiation).
Mitochondrial impairment in breast cancer cells results in altered expression of nuclear genes involved in signaling, cellular architecture, metabolism, cell growth and differentiation, and apoptosis. These genes may mediate the cross talk between mitochondria and the nucleus.
Mitochondria; Mitochondrial DNA; Nuclear genes; Breast Cancer; Cancer
Mutations in mitochondrial DNA (mtDNA) have been reported in cancer and are involved in the pathogenesis of many mitochondrial diseases. Uracil-DNA glycosylase, encoded by the UNG1 gene in Saccharomyces cerevisiae, repairs uracil in DNA formed due to deamination of cytosine. Our study demonstrates that inactivation of the UNG1 gene leads to at least a 3-fold increased frequency of mutations in mtDNA compared with the wild-type. Using a Ung1p–green fluorescent protein (GFP) fusion construct, we demonstrate that yeast yUng1–GFP protein localizes to both mitochondria and the nucleus, indicating that Ung1p must contain both a mitochondrial localization signal (MLS) and a nuclear localization signal. Our study reveals that the first 16 amino acids at the N-terminus contain the yUng1p MLS. Deletion of 16 amino acids resulted in the yUng1p–GFP fusion protein being transported to the nucleus. We also investigated the intracellular localization of human hUng1p–GFP in yeast. Our data indicate that hUng1p–GFP predominately localizes to the mitochondria. Further analysis identified the N-terminal 16 amino acids as important for localization of hUng1 protein into the mitochondria. Expression of both yeast and human UNG1 cDNA suppressed the frequency of mitochondrial mutation in UNG1-deficient cells. However, expression of yUNG1 in wild-type cells increased the frequency of mutations in mtDNA, suggesting that elevated expression of Ung1p is mutagenic. An increase in the frequency of mitochondrial mutants was also observed when hUNG1 site-directed mutants (Y147C and Y147S) were expressed in mitochondria. Our study suggests that deamination of cytosine is a frequent event in S.cerevisiae mitochondria and both yeast and human Ung1p repairs deaminated cytosine in mitochondria.
The OGG1 gene encodes a highly conserved DNA glycosylase
that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria.
We demonstrate that inactivation of ogg1 leads
to at least a 2-fold increase in production of spontaneous mitochondrial
mutants compared with wild-type. Using green fluorescent protein
(GFP) we show that a GFP–Ogg1 fusion protein is transported
to mitochondria. However, deletion of the first 11 amino acids from
the N-terminus abolishes the transport of the GFP–Ogg1
fusion protein into the mitochondria. This analysis indicates that
the N-terminus of Ogg1 contains the mitochondrial localization signal.
We provide evidence that both yeast and human Ogg1 proteins protect
the mitochondrial genome from spontaneous, as well as induced, oxidative
damage. Genetic analyses revealed that the combined inactivation
of OGG1 and OGG2 [encoding
an isoform of the Ogg1 protein, also known as endonuclease three-like
glycosylase I (Ntg1)] leads to suppression of spontaneously
arising mutations in the mitochondrial genome when compared with
the ogg1 single mutant or the wild-type. Together,
these studies provide in vivo evidence for the
repair of oxidative lesions in the mitochondrial genome by human
and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor
of oxidative mutagenesis in mitochondria.
Chronic Myelomonocytic Leukemia (CMML) is a heterogeneous disease that is not only hard to diagnose and classify, but is also highly resistant to treatment. Available forms of therapy for this disease have not shown significant effects and patients rapidly develop resistance early on in therapy. These factors lead to the very poor prognosis observed with CMML patients, with median survival duration between 12 and 24 months after diagnosis. This study is therefore centered around evaluating the selective efficacy of a natural extract from dandelion roots, in inducing programmed cell death in aggressive and resistant CMML cell lines.
To confirm the induction of programmed cell death in three human CMML cell lines, nuclear condensation and externalization of the phosphatidylserine, two main characteristics of apoptosis, were detected using Hoechst staining and annexin-V binding assay. The induction of another mode of cell death, autophagy, was determined using a monodansylcadaverine (MDC) stain, to detect the formation of autophagy vacuoles. The results from this study indicate that Dandelion Root Extract (DRE) is able to efficiently and selectively induce apoptosis and autophagy in these cell lines in a dose and time dependent manner, with no significant toxicity on non-cancerous peripheral blood mononuclear cells. More importantly, we observed early activation of initiator caspase-8, which led to mitochondrial destabilization and the induction of autophagy, suggesting that DRE acts through the extrinsic pathway of apoptosis. The inability of DRE to induce apoptosis in dominant-negative FADD cells, confirms the mechanism of action of DRE in in vitro models of CMML.
The results from this study indicate that natural products, in particular Dandelion Root Extract, have great potential, as non-toxic and effective alternatives to conventional modes of chemotherapy available today.