Depletion Of Mitochondrial Genome Research Articles

Deciphering the Spectrum of Mitochondrial DNA Mutations in Hepatocellular Carcinoma Using High-Throughput Sequencing.

Accumulation of mitochondrial DNA (mtDNA) mutations has been proposed to contribute to the initiation and progression of tumors. By using high-throughput sequencing strategies, we measured 33 specimens including 11 hepatocellular carcinoma (HCC) tissues, 11 corresponding adjacent tissues, and 11 normal liver tissues. We identified 194 single nucleotide variants (SNVs; including insert and deletion) in 33 liver tissues, and 13 somatic novel mutations were detected, including 7 mutations in the coding region. One of the seven somatic mutations (T7609C, 91.09%) is synonymous, which does not change amino acid coding; the other four somatic mutations (T6115C, 65.74%; G8387A, 12.23%; G13121A, 93.08%; and T14180C, 28.22%) could result in amino acid substitutions, potentially leading to mitochondrial dysfunction. Furthermore, two mutations in tRNA might influence amino acid transportation. Consistent with a previous study, we also found that mtDNA copy number was significantly reduced in HCC tissues. Therefore, we established a mitochondrial genome depletion cell line ρ0 and revealed that mtDNA loss reduced proliferation and migration in HCC cells but promoted their resistance to 5-fluorouracil. Our results suggested that somatic mtDNA mutations may cause mitochondrial dysfunction and affect chemoresistance of HCC cells. These new identified somatic mutations may serve as a reference for future studies of cancer mitochondrial genomes.

Lack of mitochondrial DNA impairs chemical hypoxia-induced autophagy in liver tumor cells through ROS-AMPK-ULK1 signaling dysregulation independently of HIF-1α

Alterations in mitochondrial DNA (mtDNA) and autophagy activation are common events in tumors. Here we have investigated the effect of mitochondrial genome depletion on chemical hypoxia-induced autophagy in liver tumor cells. Human SK-Hep-1 wild-type and mtDNA-depleted (Rho) cells were exposed to the hypoxia mimetic agents CoCl2 and deferoxamine (DFO). Up-regulation of HIF-1α, but not HIF-2α was observed. The expression of several HIF-1α target genes was also found. In human SK-Hep-1 and mouse Hepa 1–6 liver tumor cells, but not in the counterpart Rho derived lines, chemical hypoxia increased the abundance of autophagosomes and autolysosomes. In wild-type and Rho cells, chemical hypoxia induced down-regulation of HIF-1α-dependent autophagy inhibitors Bcl-2 and mTOR, whereas activation of AMPK/ULK1-mediated pro-autophagy pathway occurred only in wild-type cells. Chemical (compound C) and genetic (shRNA) inhibition of AMPK activation resulted in reduced autophagy. ATP levels were similar in both cell types, whereas constitutive and chemical hypoxia-induced reactive oxygen species (ROS) generation was lower in Rho cells. In wild-type cells, the antioxidant N-acetylcysteine blocked CoCl2- and DFO-induced AMPK and autophagy activation, but not endoplasmic reticulum stress induced by CoCl2. Enhanced Bax-α/Bcl-2 ratio and cell death was induced by hypoxia mimetic agents more markedly in wild-type than in Rho cells. Upon blocking autophagy activation with 3-methyladenine, DFO-induced cell death was partially prevented whereas that induced by CoCl2 was increased, but only in wild-type cells. These results suggest that mitochondrial dysfunction associated with the lack of mtDNA impairs the signaling pathways mediated by ROS, controlling autophagy activation in liver tumor cells, which may contributes to cancer development.

Global genetic determinants of mitochondrial DNA copy number.

Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role.

The expression of genes involved in hepatocellular carcinoma chemoresistance is affected by mitochondrial genome depletion.

Deletions and mutations in mitochondrial DNA (mtDNA), which are frequent in human tumors, such as hepatocellular carcinoma (HCC), may contribute to enhancing their malignant phenotype. Here we have investigated the effect of mtDNA depletion in the expression of genes accounting for mechanisms of chemoresistance (MOC) in HCC. Using human HCC SK-Hep-1 cells depleted of mtDNA (Rho), changes in gene expression in response to antitumor drugs previously assayed in HCC treatment were analyzed. In Rho cells, a decreased sensitivity to doxorubicin-, SN-38-, cisplatin (CDDP)-, and sorafenib-induced cell death was found. Both constitutive and drug-induced reactive oxygen species generation were decreased. Owing to activation of the NRF2-mediated pathway, MDR1, MRP1, and MRP2 expression was higher in Rho than in wild-type cells. This difference was maintained after further upregulation induced by treatment with doxorubicin, SN-38, or CDDP. Topoisomerase-IIa expression was also enhanced in Rho cells before and after treatment with these drugs. Moreover, the ability of doxorubicin, SN-38 and CDDP to induce proapoptotic signals was weaker in Rho cells, as evidenced by survivin upregulation and reductions in Bax/Bcl-2 expression ratios. Changes in these genes seem to play a minor role in the enhanced resistance of Rho cells to sorafenib, which may be related to an enhanced intracellular ATP content together with the loss of expression of the specific target of sorafenib, tyrosine kinase receptor Kit. In conclusion, these results suggest that mtDNA depletion may activate MOC able to hinder the efficacy of chemotherapy against HCC.

Mitochondrial genome depletion in human liver cells abolishes bile acid-induced apoptosis: Role of the Akt/mTOR survival pathway and Bcl-2 family proteins

Acute accumulation of bile acids in hepatocytes may cause cell death. However, during long-term exposure due to prolonged cholestasis, hepatocytes may develop a certain degree of chemoresistance to these compounds. Because mitochondrial adaptation to persistent oxidative stress may be involved in this process, here we have investigated the effects of complete mitochondrial genome depletion on the response to bile acid-induced hepatocellular injury. A subline (Rho) of human hepatoma SK-Hep-1 cells totally depleted of mitochondrial DNA (mtDNA) was obtained, and bile acid-induced concentration-dependent activation of apoptosis/necrosis and survival signaling pathways was studied. In the absence of changes in intracellular ATP content, Rho cells were highly resistant to bile acid-induced apoptosis and partially resistant to bile acid-induced necrosis. In Rho cells, both basal and bile acid-induced generation of reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, was decreased. Bile acid-induced proapoptotic signals were also decreased, as evidenced by a reduction in the expression ratios Bax-α/Bcl-2, Bcl-xS/Bcl-2, and Bcl-xS/Bcl-xL. This was mainly due to a downregulation of Bax-α and Bcl-xS. Moreover, in these cells the Akt/mTOR pathway was constitutively activated in a ROS-independent manner and remained similarly activated in the presence of bile acid treatment. In contrast, ERK1/2 activation was constitutively reduced and was not activated by incubation with bile acids. In conclusion, these results suggest that impaired mitochondrial function associated with mtDNA alterations, which may occur in liver cells during prolonged cholestasis, may activate mechanisms of cell survival accounting for an enhanced resistance of hepatocytes to bile acid-induced apoptosis.

Mitochondrial genome depletion dysregulates bile acid‐ and paracetamol‐induced expression of the transporters Mdr1, Mrp1 and Mrp4 in liver cells

Mitochondria are involved in the toxicity of several compounds, retro-control of gene expression and apoptosis activation. The effect of mitochondrial genome (mtDNA) depletion on changes in ABC transporter protein expression in response to bile acids and paracetamol was investigated. Hepa 1-6 mouse hepatoma cells with 70% decrease in 16S/18S rRNA ratio (Rho cells) were obtained by long-term treatment with ethidium bromide. Spontaneous apoptosis and reactive oxygen species (ROS) generation were decreased in Rho cells. Following glycochenodeoxycholic acid (GCDCA) or paracetamol, Rho cells generated less ROS and were more resistant to cell death. Apoptosis induced by GCDCA and Fas was also reduced. The basal expression of Mdr1 was significantly enhanced, but this was not further stimulated by GCDCA or paracetamol, as observed in wild-type (WT) cells. Basal expression of Mrp1 and Mrp4 was similar in WT and Rho cells, whereas they were up-regulated only in WT cells after GCDCA or paracetamol, along with the transcription factors Shp and Nrf2, but not Fxr or Pxr. Increased expression of Nrf2 was accompanied by its enhanced nuclear translocation. Glycoursodeoxycholic acid failed to cause any of the effects observed for GCDCA or paracetamol. The Nrf2-mediated pathway is partly independent of ROS production. Nuclear translocation of Nrf2 is insufficient to up-regulate Mdr1, Mrp1 and Mrp4, which requires the participation of other regulatory element(s) whose activation in response to GCDCA and paracetamol is impaired in Rho cells and hence probably sensitive to ROS.

Inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis

Mitochondrial dysfunction is a hallmark of cancer cells. Consistent with this phenotype mutations in mitochondrial genome have been reported in all cancers examined to date. However, it is not clear whether mitochondrial genomic status in human cells affects nuclear genome stability and whether proteins involved in inter-genomic cross talk are involved in tumorigenesis. Using cell culture model and cybrid cell technology, we provide evidence that mitochondrial genetic status impacts nuclear genome stability in human cells. In particular our studies demonstrate 1) that depletion of mitochondrial genome (rho 0) leads to chromosomal instability (CIN) reported to be present in variety of human tumors and 2) rho 0 cells show transformed phenotype. Our study also demonstrates that mitochondrial genetic status plays a key role in regulation of a multifunctional protein APE1 (also known as Ref1 or HAP1) involved in transcription and DNA repair in the nucleus and the mitochondria. Interestingly we found that altered expression of APE1 in rho 0 cells and tumorigenic phenotype can be reversed by exogenous transfer of wild type mitochondria in rho 0 cells. Furthermore, we demonstrate that APE1 expression is altered in variety of primary tumors. Taken together, these studies suggest that inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis and that APE1 mediates this process.