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Anti-EGFR therapy is among the most promising molecular targeted therapies against cancer developed in the past decade. However, drug resistance eventually arises in most, if not all, treated patients. Emerging evidence has linked epigenetic changes, such as DNA methylation at CpG islands, to the development of resistance to multiple anticancer drugs. In addition, genes that are differentially methylated have increasingly been appreciated as a source of clinically relevant biomarker candidates. To identify genes that are specifically methylated during the evolution of resistance to anti-EGFR therapeutic agents, we performed a methylation-specific array containing a panel of 56 genes that are commonly known to be regulated through promoter methylation in two parental non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cell lines and their resistant derivatives to either erlotinib or cetuximab. We found that death-associated protein kinase (DAPK) was hypermethylated in drug-resistant derivatives generated from both parental cell lines. Restoration of DAPK into the resistant NSCLC cells by stable transfection re-sensitized the cells to both erlotinib and cetuximab. Conversely, siRNA-mediated knockdown of DAPK induced resistance in the parental sensitive cells. These results demonstrate that DAPK plays important roles in both cetuximab and erlotinib resistance, and that gene silencing through promoter methylation is one of the key mechanisms of developed resistance to anti-EGFR therapeutic agents. In conclusion, DAPK could be a novel target to overcome resistance to anti-EGFR agents to improve the therapeutic benefit, and further evaluation of DAPK methylation as a potential biomarker of drug response is needed.

TNF-related weak inducer of apoptosis (TWEAK) is a new member of the TNF superfamily. It signals through TNFRSF12A, commonly known as Fn14. The TWEAK-Fn14 interaction regulates cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, tissue remodeling and inflammation. Although TWEAK has been reported to be associated with autoimmune diseases, cancers, stroke, and kidney-related disorders, the downstream molecular events of TWEAK-Fn14 signaling are yet not available in any signaling pathway repository. In this paper, we manually compiled from the literature, in particular those reported in human systems, the downstream reactions stimulated by TWEAK-Fn14 interactions. Our manual amassment of the TWEAK-Fn14 pathway has resulted in cataloging of 46 proteins involved in various biochemical reactions and TWEAK-Fn14 induced expression of 28 genes. We have enabled the availability of data in various standard exchange formats from NetPath, a repository for signaling pathways. We believe that this composite molecular interaction pathway will enable identification of new signaling components in TWEAK signaling pathway. This in turn may lead to the identification of potential therapeutic targets in TWEAK-associated disorders.

Mitochondria control essential cellular activities including generation of ATP via oxidative phosphorylation. Mitochondrial DNA (mtDNA) mutations in the regulatory D-loop region and somatic mtDNA mutations are common in primary human cancers. The biological impact of a given mutation may vary, depending on the nature of the mutation and the proportion of mutant mtDNAs carried by the cell. Identification of mtDNA mutations in precancerous lesions supports their early contribution to cell transformation and cancer progression. Introduction of mtDNA mutations in transformed cells has been associated with increased ROS production and tumor growth. Studies reveal that increased and altered mtDNA plays a role in the development of cancer but further work is required to establish the functional significance of specific mitochondrial mutations in cancer and disease progression. This review offers some insight into the extent of mtDNA mutations, their functional consequences in tumorigenesis, mitochondrial therapeutics, and future clinical application.

Strategies to address resistance to platin drugs are greatly needed in human epithelial cancers (e.g., ovarian, head/neck, and lung) where platins are used widely and resistance occurs commonly. We found that upon ΔNp63α overexpression, AKT1 and phospho-AKT1 levels are upregulated in cancer cells. Investigations using gel-shift, chromatin immunoprecipitation and functional reporter assays implicated ΔNp63α in positive regulation of AKT1 transcription. Importantly, we found that ΔNp63α, AKT1, and phospho-AKT levels are greater in 2008CI3 CDDP-resistant ovarian cancer cells than in 2008 CDDP-sensitive cells. siRNA-mediated knockdown of ΔNp63α expression dramatically decreased AKT1 expression, whereas knockdown of either ΔNp63α or AKT1 decreased cell proliferation and increased death of ovarian and head/neck cancer cells. Conversely, enforced expression of ΔNp63α increased cancer cell proliferation and reduced apoptosis. Together, our findings define a novel ΔNp63α-dependent regulatory mechanism for AKT1 expression and its role in chemotherapeutic resistance of ovarian and head/neck cancer cells.

Background: The major established etiologic risk factor for bladder cancer is cigarette smoking and one of the major antineoplastic agents used for the treatment of advanced bladder cancer is cisplatin. A number of reports have suggested that cancer patients who smoke while receiving treatment have lower rates of response and decreased efficacy of cancer therapies., Methodology/principal Findings: In this study, we investigated the effect of cigarette smoke condensate (CSC) vapor on cisplatin toxicity in urothelial cell lines SV-HUC-1 and SCaBER cells. We showed that chronic exposure to CSC vapor induced cisplatin resistance in both cell lines. In addition, we found that the expression of mitochondrial-resident protein adenylate kinase-3 (AK3) is decreased by CSC vapor. We further observed that chronic CSC vapor-exposed cells displayed decreased cellular sensitivity to cisplatin, decreased mitochondrial membrane potential (ΔΨm) and increased basal cellular ROS levels compared to unexposed cells. Re-expression of AK3 in CSC vapor-exposed cells restored cellular sensitivity to cisplatin. Finally, CSC vapor increased the growth of the tumors and also curtail the response of tumor cells to cisplatin chemotherapy in vivo., Conclusions/significance: The current study provides evidence that chronic CSC vapor exposure affects AK3 expression and renders the cells resistant to cisplatin.

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NF-κΒ in presence of cisplatin. We find that NF-κΒ promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NF-κΒ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NF-κΒ with siRNA-mediated silencing NF-κΒ expression attenuates chemotherapy induced degradation of ΔNp63α . These data demonstrate that NF-κΒ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NF-κΒ may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.

The p53 family gene p63 plays an instrumental role in cellular stress responses including responses to DNA damage. In addition to encoding a full-length transcriptional activator, p63 also encodes several dominant inhibitory isoforms including the isoform DeltaNp63alpha, the function of which is not fully understood. DeltaNp63alpha is degraded in response to DNA damage, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying regulation of DeltaNp63alpha expression in response to chemotherapeutic agents or tumor necrosis factor-alpha. We found that DeltaNp63alpha interacts with IkappaB kinase (IKK), a multisubunit protein kinase that consists of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. The IKKbeta kinase promotes ubiquitin-mediated proteasomal degradation of DeltaNp63alpha, whereas a kinase-deficient mutant IKKbeta-K44A fails to do so. Cytokine- or chemotherapy-induced stimulation of IKKbeta caused degradation of DeltaNp63alpha and augmented transactivation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, IKKbeta inhibition attenuated cytokine- or chemotherapy-induced degradation of DeltaNp63alpha. Our findings show that IKKbeta plays an essential role in regulating DeltaNp63alpha in response to extrinsic stimuli. IKK activation represents one mechanism by which levels of DeltaNp63alpha can be reduced, thereby rendering cells susceptible to cell death in the face of cellular stress or DNA damage.

Regulation of DeltaNp63alpha can be achieved at the transcriptional and post-translational levels, with the latter being greatly influenced by external stimuli such as DNA damaging agents. In this present study, we demonstrated that DeltaNp63alpha physical interacts with Yes-associated protein 1 (Yap1). Ectopic expression of Yap1 decreased the stability and the half-like of DeltaNp63alpha and resulted in ubiquitin-dependent degradation of DeltaNp63alpha. Further, the cisplatin mediated degradation of DeltaNp63alpha is attenuated with the downregulation of Yap1. Thus, our results strongly suggest that Yap1 plays a role in the regulation of endogenous DeltaNp63alpha levels and is likely to contribute to the regulation of DeltaNp63alpha, in physiological conditions.

DeltaNp63alpha, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, including cisplatin. But little is understood about the post-translational protein stability of DeltaNp63alpha. In this present study we demonstrate for the first time that DeltaNp63alpha physically interacts with U-box-type E4 ubiquitin ligase UFD2a. UFD2a stabilizes DeltaNp63alpha, and ubiquitylation of DeltaNp63alpha is attenuated by UFD2a both in the presence and absence of cisplatin. Ectopic expression of UFD2a increased the half-life of DeltaNp63alpha in association with a significant enhancement of the repressive transcriptional activity of DeltaNp63alpha. Downregulation of endogenous UFD2a by RNAi resulted in degradation of DeltaNp63alpha. Taken together, our current study provides an insight onto the regulation of DeltaNp63alpha protein levels in response to cisplatin and also suggests that UFD2a might play an important role in the regulation of cisplatin mediated cell death mediated by p63.

We showed that TAp63gamma regulates hOGG1. Using chromatin immunoprecipitation (ChIP), we found that TAp63gamma binds to the hOGG1 promoter. Reintroduction of wild-type TAp63gamma into HEK 293 cells, induced transcription of hOGG1 promoter, leading to increase in RNA and protein. Using RNAi studies, we observed that TAp63gamma-RNAi resulted in reduced hOGG1 RNA and protein in HeLa cells. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage. Taken together, our results indicate that hOGG1 is a direct target of TAp63gamma, suggesting a role for TAp63gamma in oxidative damage and repair.

Mitochondrial genomic mutations are found in a variety of human cancers; however, the frequency of mitochondrial DNA (mtDNA) mutations in coding regions remains poorly defined, and the functional effects of mitochondrial mutations found in primary human cancers are not well described. Using MitoChip, we sequenced the whole mitochondrial genome in 83 head and neck squamous cell carcinomas. Forty-one of 83 (49%) tumors contained mtDNA mutations. Mutations occurred within noncoding (D-loop) and coding regions. A nonrandom distribution of mutations was found throughout the mitochondrial enzyme complex components. Sequencing of margins with dysplasia demonstrated an identical nonconservative mitochondrial mutation (A76T in ND4L) as the tumor, suggesting a role of mtDNA mutation in tumor progression. Analysis of p53 status showed that mtDNA mutations correlated positively with p53 mutations (P < 0.002). To characterize biological function of the mtDNA mutations, we cloned NADH dehydrogenase subunit 2 (ND2) mutants based on primary tumor mutations. Expression of the nuclear-transcribed, mitochondrial-targeted ND2 mutants resulted in increased anchorage-dependent and -independent growth, which was accompanied by increased reactive oxygen species production and an aerobic glycolytic metabolic phenotype with hypoxia-inducible factor (HIF)-1alpha induction that is reversible by ascorbate. Cancer-specific mitochondrial mutations may contribute to development of a malignant phenotype by direct genotoxic effects from increased reactive oxygen species production as well as induction of aerobic glycolysis and growth promotion.

Based on the oncogenic role of phosphatidylinositol glycan (PIG) class U in human tumors, we explored the role of two additional subunits of the glycosylphosphatidylinositol (GPI) transamidase complex in human breast cancer. We found that PIG class T (PIG-T) and GPI anchor attachment 1 (GPAA1) were overexpressed in breast cancer cell lines and primary tumors. Forced expression of PIG-T and GPAA1 transformed NIH3T3 cells in vitro and increased tumorigenicity and invasion of these cells in vivo. Suppression of PIG-T expression in breast cancer cell lines led to inhibition of anchorage-independent growth. Moreover, we found that PIG-T and GPAA1 expression levels positively correlated with paxillin phosphorylation in invasive breast cancer cell lines. Furthermore, suppression of PIG-T and GPAA1 expression led to a decrease in paxillin phosphorylation with a concomitant decrease in invasion ability. These results suggest that the GPI transamidase complex is composed of a group of proto-oncogenes that individually or as a group contribute to breast cancer growth. This aberrant growth is mediated, at least partially, by phosphorylation of paxillin, contributing to invasion and progression of breast cancer.

Background: Oxidative damage to mitochondrial DNA has been implicated as a causative factor in a wide variety of degenerative diseases, aging and cancer. The modified guanine, 7,8-dihydro-8-oxoguanine (also known as 8-hydroxyguanine) is one of the major oxidized bases generated in DNA by reactive oxygen species and has gained most of the attention in recent years as a marker of oxidative DNA injury and its suspected role in the initiation of carcinogenesis. 8-hydroxyguanine is removed by hOgg1, a DNA glycosylase/AP lyase involved in the base excision repair pathway., Methods: We over-expressed wild type and R229Q mutant hOGG1 in the nucleus and mitochondria of cells lacking mitochondrial hOGG1 expression through an expression vector containing nuclear and mitochondrial targeting sequence respectively. We used quantitative real time PCR to analyze mtDNA integrity after exposure to oxidative damaging agents, in cells transfected with or without mitochondrially-targeted mutant hogg1., Result: Over-expression of wild type hOgg1 in both nucleus and mitochondria resulted in increased cellular survival when compared to vector or mutant over-expression of hOGG1. Interestingly, mitochondrially-targeted mutant hogg1 resulted in more cell death than nuclear targeted mutant hogg1 upon exposure of cells to oxidative damage. Additional we examined mitochondrial DNA integrity after oxidative damage exposure using real-time quantitative PCR. The presence of mutant hogg1 in the mitochondria resulted in reduced mitochondrial DNA integrity when compared to the wild type. Our work indicates that the R229Q hOGG1 mutation failed to protect cells from oxidative damage and that such mutations in cancer may be more detrimental to cellular survival when present in the mitochondria than in the nucleus., Conclusion: These findings suggest that deficiencies in hOGG1, especially in the mitochondria may lead to reduced mitochondrial DNA integrity, consequently resulting in decreased cell viability.

We showed that the PEA3 transcriptional factor interacted with LKB1, a serine/threonine kinase, which is somatically mutated in lung cancer. This interaction occurred through the ETS domain of PEA3 and the kinase domain of LKB1. Mutation of LKB1 in lung cancer cells stabilized PEA3. Reintroduction of wild-type (WT) LKB1 into cells induced down-regulation of PEA3 and subsequently resulted in reduced cyclooxygenase-2 RNA and protein expression, whereas germ-line and somatic LKB1 mutants were defective in this activity. LKB1 phosphorylated PEA3 and promoted its degradation through a proteasome-mediated mechanism. Cells expressing mutant LKB1 possessed greater invasive potential compared with cells expressing WT LKB1. Increased invasion of cells with mutant LKB1 was partly due to PEA3 expression, as RNA interference inhibition of PEA3 resulted in dramatic decrease of Matrigel invasion. However, forced expression of PEA3 resulted in down-regulation of epithelial markers and induction of mesenchymal markers. These results suggest that PEA3 stabilization due to LKB1 inactivation could lead to epithelial/mesenchymal transition and greater lung cancer invasion potential.

Background: The noninvasive identification of bladder tumors may improve disease control and prevent disease progression. Aberrant promoter methylation (i.e., hypermethylation) is a major mechanism for silencing tumor suppressor genes and other cancer-associated genes in many human cancers, including bladder cancer., Methods: A quantitative fluorogenic real-time polymerase chain reaction (PCR) assay was used to examine primary tumor DNA and urine sediment DNA from 15 patients with bladder cancer and 25 control subjects for promoter hypermethylation of nine genes (APC, ARF, CDH1, GSTP1, MGMT, CDKN2A, RARbeta2, RASSF1A, and TIMP3) to identify potential biomarkers for bladder cancer. We then used these markers to examine urine sediment DNA samples from an additional 160 patients with bladder cancers of various stages and grades and from an additional 69 age-matched control subjects. Data were analyzed on the basis of a prediction model and were internally validated using a jacknife procedure. All statistical tests were two-sided., Results: For all 15 patients with paired DNA samples, the promoter methylation pattern in urine matched that in the primary tumors. Four genes displayed 100% specificity. Of the 175 bladder cancer patients, 121 (69%, 95% confidence interval [CI] = 62% to 76%) displayed promoter methylation in at least one of these genes (CDKN2A, ARF, MGMT, and GSTP1), whereas all control subjects were negative for such methylation (100% specificity, 95% CI = 96% to 100%). A logistic prediction model using the methylation levels of all remaining five genes was developed and internally validated for subjects who were negative on the four-gene panel. This combined, two-stage predictor produced an internally validated ROC curve with an overall sensitivity of 82% (95% CI = 75 % to 87%) and specificity of 96% (95% CI = 90% to 99%)., Conclusion: Testing a small panel of genes with the quantitative methylation-specific PCR assay in urine sediment DNA is a powerful noninvasive approach for the detection of bladder cancer. Larger independent confirmatory cohorts with longitudinal follow-up will be required in future studies to define the impact of this technology on early detection, prognosis, and disease monitoring before clinical application.

The MtArg8 reversion assay, which measures point mutation in mtDNA, indicates that in budding yeast Saccharomyces cerevisiae, DNA polymerase zeta and Rev1 proteins participate in the mitochondrial DNA mutagenesis. Supporting this evidence, both polymerase zeta and Rev1p were found to be localized in the mitochondria. This is the first report demonstrating that the DNA polymerase zeta and Rev1 proteins function in the mitochondria.

Constituents of tobacco can cause DNA adduct formation and are implicated in head and neck squamous cell cancer (HNSC) development. We investigated the capacity of HNSC cell lines to repair mitochondrial DNA (mtDNA) damage induced by a DNA adduct-forming agent. HNSC cell lines underwent 4-nitroquinoline 1-oxide (4NQO) exposure with subsequent rescue with normal media. Real-time quantitative PCR for nuclear DNA (nDNA) and mtDNA was performed. mtDNA to nDNA ratios were calculated and standardized to mock-treated cells to assess mtDNA repair ability. Two of three tested cancer cell lines exposed to 4NQO exhibited consistent decreases in mtDNA/nDNA ratios throughout the different repair timepoints. At 24 h mtDNA/nDNA ratios of JHU-O19 and JHU-O22 decreased to 63% and 60% of controls, respectively. Conversely, a control keratinocyte cell line exhibited overall increases in mtDNA/nDNA ratios compared to baseline suggesting intact DNA repair mechanisms. By using a DNA adduct formation and repair model featuring 4NQO and HNSC cell lines, we have implicated faulty mtDNA repair as having a potential role in HNSC.

Recent evidence indicates that in vitro p53 augments base excision repair (BER) activities in mammalian cells. To understand the role of p53 in BER, we analyzed the repair activity of hOgg1 in isogenic cell lines HCT116p53+/+ and HCT116p53-/-. We found that hOgg1 activity was significantly decreased in HCT116p53-/- cells as compared with HCT116p53+/+ cells, indicating a functional role for p53 in the regulation of hOGG1. Using gel-shift assays, we showed that p53 binds to its putative cis-elements within the hOGG1 promoter. In addition we demonstrated that supplementing p53 in HCT116p53-/- cells enhanced the transcription of hOGG1. To further strengthen our findings, we used p53-RNAi to study the effects of decreased p53 levels on hOgg1 activity. We observed that p53-RNAi resulted in decreased hOGG1 expression both at the mRNA and protein levels. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage and reduced hOgg1 activity as evidenced by the 8-oxoG incision assay. Taken together, our results indicate that loss of p53 function can lead to decreased hOgg1 repair activity.

Mitochondrial DNA (mtDNA) alterations are associated with various cancer types, suggesting that the mitochondrial genome may be a critical contributing factor in carcinogenesis. mtDNA alterations have been suggested as a potentially sensitive and specific biomarker for several cancer types. We examined mtDNA content in 25 pairs of normal and tumor breast tissue samples, 37 papillary thyroid carcinoma (PTC), 21 benign thyroid neoplasms and in 20 paired normal and PTC samples. Our results showed that mtDNA content was reduced in 80% of the breast tumors relative to their corresponding normal. mtDNA was increased in papillary thyroid carcinomas, however, when compared to the corresponding normal DNA taken from the same individual. Also, mtDNA content was increased in none-paired PTC samples compared to the normal controls. Our findings indicate that changes in mtDNA content during carcinogenesis may be regulated in a tumor specific manner. Additionally, changes in mtDNA levels did not correlate with tumor grade and metastasis, suggesting that these alterations may occur in the early stages of tumorigenesis. Our findings suggest that mtDNA content can be used as a molecular diagnostic tool to help identify genetic abnormalities in human tumors.

In humans, the oxidatively induced DNA lesion 8-hydroxyguanine (8-oxoG) is removed from DNA by hOgg1, a DNA glycosylase/AP lyase that specifically incises 8-oxoG opposite cytosine. We analysed the expression of hOGG1 mRNA in 18 lung cancer and three normal cell lines. Although hOGG1 was overexpressed in most cell lines, 2/18 (11.1%) showed a lower hOGG1 mRNA and protein expression (approximately 80% decrease) relative to normal cell lines. Liquid chromatography/mass spectrometry analysis showed increased levels of 8-oxoG in the two cell lines with the lowest hOGG1 mRNA expression. We examined the ability of nuclear and mitochondrial extracts to incise 8-oxoG lesion in cell lines H1650 and H226 expressing lower hOGG1 mRNA and H1915 and H1975 with higher than normal hOGG1 mRNA expression. Both nuclear and mitochondrial extracts from H1915 and H1975 cells were proficient in 8-oxoG removal. However, both cell lines with the lowest hOGG1 mRNA expression exhibited a severe reduction in 8-oxoG incision in both nuclear and mitochondrial extracts. Under-expression of hOGG1 mRNA and hOgg1 protein was associated with a decrease in mitochondrial DNA repair in response to oxidative damaging agents. These results provide evidence for defective incision of 8-oxoG in both nuclear and mitochondria of H1650 and H226 lung cancer cell lines. These results may implicate 8-oxoG repair defects in certain lung cancers.

Introduction: Phosphatidylinositol 3-kinases (PI3Ks) are a group of lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival, and motility. Even though PIK3CA amplification and somatic mutation have been reported previously in various kinds of human cancers, the genetic change in PIK3CA in human breast cancer has not been clearly identified., Methods: Fifteen breast cancer cell lines and 92 primary breast tumors (33 with matched normal tissue) were used to check somatic mutation and gene copy number of PIK3CA. For the somatic mutation study, we specifically checked exons 1, 9, and 20, which have been reported to be hot spots in colon cancer. For the analysis of the gene copy number, we used quantitative real-time PCR and fluorescence in situ hybridization. We also treated several breast cancer cells with the PIK3CA inhibitor LY294002 and compared the apoptosis status in cells with and without PIK3CA mutation., Results: We identified a 20.6% (19 of 92) and 33.3% (5 of 15) PIK3CA somatic mutation frequency in primary breast tumors and cell lines, respectively. We also found that 8.7% (8 of 92) of the tumors harbored a gain of PIK3CA gene copy number. Only four cases in this study contained both an increase in the gene copy number and a somatic mutation. In addition, mutation of PIK3CA correlated with the status of Akt phosphorylation in some breast cancer cells and inhibition of PIK3CA-induced increased apoptosis in breast cancer cells with PIK3CA mutation., Conclusion: Somatic mutation rather than a gain of gene copy number of PIK3CA is the frequent genetic alteration that contributes to human breast cancer progression. The frequent and clustered mutations within PIK3CA make it an attractive molecular marker for early detection and a promising therapeutic target in breast cancer.

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 (rho(0)) or those with deleted mitochondrial DNA (rho(-)). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho(-) and rho(0) cells were approximately 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 rho(0)) led to decreased intracellular levels of ROS. We also demonstrate that in rho(0) 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.