Loss Of Mitochondrial DNA Research Articles

Polymorphic duplicate genes and persistent non-coding sequences reveal heterogeneous patterns of mitochondrial DNA loss in salamanders

BackgroundMitochondria are the site of the citric acid cycle and oxidative phosphorylation (OXPHOS). In metazoans, the mitochondrial genome is a small, circular molecule averaging 16.5 kb in length. Despite evolutionarily conserved gene content, metazoan mitochondrial genomes show a diversity of gene orders most commonly explained by the duplication-random loss (DRL) model. In the DRL model, (1) a sequence of genes is duplicated in tandem, (2) one paralog sustains a loss-of-function mutation, resulting in selection to retain the other copy, and (3) the non-functional paralog is eventually deleted from the genome. Despite its apparent role in generating mitochondrial gene order diversity, little is known about the tempo and mode of random gene loss after duplication events. Here, we determine mitochondrial gene order across the salamander genus Aneides, which was previously shown to include at least two DRL-mediated rearrangement events. We then analyze these gene orders in a phylogenetic context to reveal patterns of DNA loss after mitochondrial gene duplication.ResultsWe identified two separate duplication events that resulted in mitochondrial gene rearrangements in Aneides; one occurred at the base of the clade tens of millions of years ago, while the other occurred much more recently (i.e. within a single species), resulting in gene order polymorphism and paralogs that are readily identifiable. We demonstrate that near-complete removal of duplicate rRNA genes has occurred since the recent duplication event, whereas duplicate protein-coding genes persist as pseudogenes and duplicate tRNAs persist as functionally intact paralogs. In addition, we show that non-coding DNA duplicated at the base of the clade has persisted across species for tens of millions of years.ConclusionsThe evolutionary history of the mitochondrial genome, from its inception as a bacterial endosymbiont, includes massive genomic reduction. Consistent with this overall trend, selection for efficiency of mitochondrial replication and transcription has been hypothesized to favor elimination of extra sequence. Our results, however, suggest that there may be no strong disadvantage to extraneous sequences in salamander mitochondrial genomes, although duplicate rRNA genes may be deleterious.

19 - Importing of peroxiredoxins to distinct mitochondrial compartments: possible impacts on physiology and pathology

Peroxiredoxin (Prx) enzymes are considered major systems for reduction of peroxides in mitochondria. Although the import of Prx from cytosolic ribosomes to distinct mitochondrial subcompartments is driven by a cleavable N-terminal presequence, the mechanisms underlying this process are still elusive. Here, we show by cell fractionation/western blot analysis that yeast mitochondrial Prx (Prx1) displays dual localization: matrix and intermembrane space. Prx1 sorting into the intermembrane space likely involves the release of the precursor protein within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase complex (IMP). Alternatively, during its translocation into the matrix, Prx1 is sequentially cleaved by mitochondrial processing peptidase (MPP) and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 removed eight amino acid residues from the N-terminal region of Prx1, but its physiological role is still elusive. Remarkably, the processing of Prx proteins by Oct1 appears to be evolutionarily conserved, since yeast Oct1 could also cleave the human mitochondrial peroxiredoxin Prx3 when this thiol peroxidase was expressed in yeast. In addition, the alignment of the N-terminal amino acid sequences of Prx3 from mammals indicated the putative cleavage sites by MPP and Oct1 proteases. Remarkably, there are orthologues of IMP and Oct1 proteins in human cells and their malfunctioning was associated with diseases. For instance, loss of Oct1 provokes disturbances in iron metabolism and loss of mitochondrial DNA. Currently, we are investigating the import of mammalian Prx enzymes into distinct mitochondrial subcompartments and the involvement of IMP and Oct1orthologues.

Nucleoside reverse transcriptase inhibitors induced hepatocellular mitochondrial DNA lesions and compensatory enhancement of mitochondrial function and DNA repair

Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of combined antiretroviral therapy (cART) and are widely used in anti-human immunodeficiency virus (HIV) therapy. Long-term administration of NRTIs can result in mitochondrial dysfunction in certain HIV-1–infected patients. However, NRTI–associated liver mitochondrial toxicity is not well known. Herein, the liver autopsy of acquired immune deficiency syndrome (AIDS) patients and the liver tissues of mice with 12 months of NRTI exposure were used to identify NRTI–associated liver toxicity with immunofluorescence, quantitative real-time polymerase chain reaction (qPCR), Amplex red and horseradish peroxidase, and cloning and sequencing. Laser capture microdissection was used to capture hepatocytes from liver tissues. We observed DNA oxidative damage and mitochondrial DNA (mtDNA) loss in the livers of AIDS patients, and cART patients had higher DNA oxidative damage and lower DNA repair function in liver tissues than non-cART patients. We also observed liver oxidative damage, increased DNA repair and mtDNA loss in mice with exposure to four different NRTIs for 12 months, and hepatocytes had no more mtDNA loss than liver tissues. Although NRTIs could induce mitochondrial hydrogen peroxide production, increased mitochondrial oxygen consumption was found with a Clark-type electrode. The captured hepatocytes had greater diversity in their mtDNA D-loop, dehydrogenase subunit1 (ND1) and ND4 than the controls. Long-term NRTI exposure induced single nucleotide variation in hepatocellular mtDNA D-loop, ND1 and ND4. Our findings indicate that NRTIs can induce liver mtDNA lesions, but simultaneously enhance mitochondrial function and mtDNA repair.

Sen1, the homolog of human Senataxin, is critical for cell survival through regulation of redox homeostasis, mitochondrial function, and theTORpathway inSaccharomyces cerevisiae

Mutations in the Senataxin gene, SETX are known to cause the neurodegenerative disorders, ataxia with oculomotor apraxia type 2 (AOA2), and amyotrophic lateral sclerosis 4 (ALS4). However, the mechanism underlying disease pathogenesis is still unclear. The Senataxin N-terminal protein-interaction and C-terminal RNA/DNA helicase domains are conserved in the Saccharomyces cerevisiae homolog, Sen1p. Using genome-wide expression analysis, we first show alterations in key cellular pathways such as: redox, unfolded protein response, and TOR in the yeast sen1 ΔN mutant (N-terminal truncation). This mutant exhibited growth defects on nonfermentable carbon sources, was sensitive to oxidative stress, and showed severe loss of mitochondrial DNA. The growth defect could be partially rescued upon supplementation with reducing agents and antioxidants. Furthermore, the mutant showed higher levels of reactive oxygen species, lower UPR activity, and alterations in mitochondrial membrane potential, increase in vacuole acidity, free calcium ions in the cytosol, and resistance to rapamycin treatment. Notably, the sen1 ∆N mutant showed increased cell death and shortened chronological life span. Given the strong similarity of the yeast and human Sen1 proteins, our study thus provides a mechanism for the progressive neurological disorders associated with mutations in human senataxin.

Pre-B-cell colony-enhancing factor protects against apoptotic neuronal death and mitochondrial damage in ischemia.

We previously demonstrated that Pre-B-cell colony-enhancing factor (PBEF), also known as nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in mammalian NAD+ biosynthesis pathway, plays a brain and neuronal protective role in ischemic stroke. In this study, we further investigated the mechanism of its neuroprotective effect after ischemia in the primary cultured mouse cortical neurons. Using apoptotic cell death assay, fluorescent imaging, molecular biology, mitochondrial biogenesis measurements and Western blotting analysis, our results show that the overexpression of PBEF in neurons can significantly promote neuronal survival, reduce the translocation of apoptosis inducing factor (AIF) from mitochondria to nuclei and inhibit the activation of capase-3 after glutamate-induced excitotoxicity. We further found that the overexpression of PBEF can suppress glutamate-induced mitochondrial fragmentation, the loss of mitochondrial DNA (mtDNA) content and the reduction of PGC-1 and NRF-1 expressions. Furthermore, these beneficial effects by PBEF are dependent on its enzymatic activity of NAD+ synthesis. In summary, our study demonstrated that PBEF ameliorates ischemia-induced neuronal death through inhibiting caspase-dependent and independent apoptotic signaling pathways and suppressing mitochondrial damage and dysfunction. Our study provides novel insights into the mechanisms underlying the neuroprotective effect of PBEF, and helps to identify potential targets for ischemic stroke therapy.

TbLOK1/ATOM19 is a novel subunit of the noncanonical mitochondrial outer membrane protein translocase of Trypanosoma brucei.

TbLOK1 has previously been characterized as a trypanosomatid-specific mitochondrial outer membrane protein whose ablation caused a collapse of the mitochondrial network, disruption of the membrane potential and loss of mitochondrial DNA. Here we show that ablation of TbLOK1 primarily abolishes mitochondrial protein import, both in vivo and in vitro. Co-immunprecipitations together with blue native gel analysis demonstrate that TbLOK1 is a stable and stoichiometric component of the archaic protein translocase of the outer membrane (ATOM), the highly diverged functional analogue of the TOM complex in other organisms. Furthermore, we show that TbLOK1 together with the other ATOM subunits forms a complex functional network where ablation of individual subunits either causes degradation of a specific set of other subunits or their exclusion from the ATOM complex. In summary these results establish that TbLOK1 is an essential novel subunit of the ATOM complex and thus that its primary molecular function is linked to mitochondrial protein import across the outer membrane. The previously described phenotypes can all be explained as consequences of the lack of mitochondrial protein import. We therefore suggest that in line with the nomenclature of the ATOM complex subunits, TbLOK1 should be renamed to ATOM19.

Commentary: Mitochondrial DNA damage and loss in diabetes.

This commentary discusses damage and loss of mitochondrial DNA (mtDNA) in type 2 diabetes mellitus from both the clinical and experimental perspectives. Increasingly, an array of studies in experimental models and patients suggests that the cellular stresses of insulin resistance in type 2 diabetes damage mtDNA, leading to loss of mitochondrial genetic content. As such, mtDNA is emerging as both a valuable monitoring tool and translational preventive target for metabolic disease. Copyright © 2016 John Wiley & Sons, Ltd.

Adding Emulsified Isoflurane to Cardioplegia Solution Produces Cardiac Protection in a Dog Cardiopulmonary Bypass Model.

This study investigated whether caridoplegia solution with Emulsified Isoflurane (EI) could improve cardiaoprotection in a dog CPB model of great similarity to clinical settings. Adult dogs were randomly assigned to receive one of the following cardioplegia solutions: St. Thomas with EI (group ST+EI), St. Thomas with 30% Intralipid (group ST+EL) and St. Thomas alone (group ST). The aorta was cross-clamped for two hours followed by reperfusion for another two hours, during which cardiac output was measured and dosages of positive inotropic agent to maintain normal hemodynamics were recorded. Serum level of cardiac troponin I (cTnI) and CK-MB were measured. Deletion of cardiac mitochondrial DNA was examined at the end of reperfusion. Compared with ST, ST+EI decreased the requirement of dopamine support while animals receiving ST+EI had a significantly larger cardiac output. ST+EI reduced post-CPB release of cTnI and CK-MB. Mitochondrial DNA loss was observed in only one of the tested animals from group ST+EI while it was seen in all the tested animals from group ST+EL and ST. Addition of emulsified isoflurane into cardioplegia solution protects against myocardial ischemia reperfusion injury. This protective effect might be mediated by preserving mitochondrial ultrastructure and DNA integrity.

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria.

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.

Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.

Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud.

Mitochondrial retrograde signaling inhibits the survival during prolong S/G2 arrest in Saccharomyces cerevisiae.

Cell senescence is dependent on the arrest in cell cycle. Here we studied the role of mitochondrial retrograde response signaling in yeast cell survival under a prolonged arrest. We have found that, unlike G1, long-term arrest in mitosis or S phase results in a loss of colony-forming abilities. Consistent with previous observations, loss of mitochondrial DNA significantly increased the survival of arrested cells. We found that this was because the loss increases the duration of G1 phase. Unexpectedly, retrograde signaling, which is typically triggered by a variety of mitochondrial dysfunctions, was found to be a negative regulator of the survival after the release from S-phase arrest induced by the telomere replication defect. Deletion of retrograde response genes decreased the arrest-induced death in such cells, whereas deletion of negative regulator of retrograde signaling MKS1 had the opposite effect. We provide evidence that these effects are due to alleviation of the strength of the S-phase arrest.

Toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three popular DNA intercalating fluorescent dyes.

We have compared the toxicity, mutagenicity and transport in Saccharomyces cerevisiae of three DNA-intercalating fluorescent dyes widely used to stain DNA in gels. Safety data about ethidium bromide (EtBr) are contradictory, and two compounds of undisclosed structure (Redsafe and Gelred) have been proposed as safe alternatives. Our results indicate that all three compounds inhibit yeast growth, with Gelred being the most inhibitory and also the only one causing cell death. EtBr and Gelred, but not Redsafe, induce massive formation of petite (non-respiratory) mutants, but only EtBr induces massive loss of mitochondrial DNA. All three compounds increase reversion of a chromosomal point mutation (lys2-801(amber) ), with Gelred being the most mutagenic and Redsafe the least. These dyes are all cationic and are probably taken by cells through non-selective cation channels. We could measure the glucose-energized transport of EtBr and Gelred inside the cells, while uptake of Redsafe was below our detection limit. We conclude that although all three compounds are toxic and mutagenic in the yeast system, Redsafe is the safest for yeast, probably because of very limited uptake by these cells.

Vitamin A exerts its antiinflammatory activities in colitis through preservation of mitochondrial activity

ObjectivesThe aim of this study was to assess the protective effects of vitamin A in a rat model of colitis to elucidate a possible mechanism of action. MethodsMale rats were fed for 21 d with either a normal diet or high vitamin A diet (5000 IU/d). On day 22, colitis was induced with 2,4,6-trinitrobenzenesulfonic acid (TNBS). Rats were sacrificed after 24 h and colonic tissue was removed for evaluation. ResultsMorphologically, in the supplemented group preservation of tissue architecture, no vasculitis or necroses were detected. Biochemically, decreased myeloperoxidase activity and protection of the mitochondria as evaluated by preserving tissue oxygen consumption, mitochondrial DNA, and expression of cytochrome c, was observed. Vitamin A supplementation also increased the levels of nuclear respiratory factor (NFR)-1 and mitochondrial transcription factor-A (TFAM) in normal colon tissue and in colon tissue under inflammatory conditions. ConclusionThe results indicate that tissue damage in colitis is accompanied by the arrest of mitochondrial respiration, loss of mitochondrial DNA, and the expression of mitochondrial proteins. Vitamin A effectively protects colon mitochondria by upregulation of mitochondrial transcription factors, NFR-1 and TFAM, and prevents inflammatory and necrotic changes in colitis. Vitamin A is therefore a potential therapeutic agent in inflammatory bowel disease.

Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies.

ABSTRACTMitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as ‘petite-positivity’), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.

Clinical, morphological, biochemical, imaging and outcome parameters in 21 individuals with mitochondrial maintenance defect related to FBXL4 mutations.

FBXL4 deficiency is a recently described disorder of mitochondrial maintenance associated with a loss of mitochondrial DNA in cells. To date, the genetic diagnosis of FBXL4 deficiency has been established in 28 individuals. This paper retrospectively reviews proxy-reported clinical and biochemical findings and evaluates brain imaging, morphological and genetic data in 21 of those patients. Neonatal/early-onset severe lactic acidosis, muscular hypotonia, feeding problems and failure to thrive is the characteristic pattern at first presentation. Facial dysmorphic features are present in 67% of cases. Seven children died (mean age 37 months); 11 children were alive (mean age at follow-up 46 months), three children were lost to follow-up. All survivors developed severe psychomotor retardation. Brain imaging was non-specific in neonates but a later-onset, rapidly progressive brain atrophy was noted. Elevated blood lactate and metabolic acidosis were observed in all individuals; creatine kinase was elevated in 45% of measurements. Diagnostic workup in patient tissues and cells revealed a severe combined respiratory chain defect with a general decrease of enzymes associated with mitochondrial energy metabolism and a relative depletion of mitochondrial DNA content. Mutations were detected throughout the FBXL4 gene albeit with no clear delineation of a genotype-phenotype correlation. Treatment with "mitochondrial medications" did not prove effective. In conclusion, a clinical pattern of early-onset encephalopathy, persistent lactic acidosis, profound muscular hypotonia and typical facial dysmorphism should prompt initiation of molecular genetic analysis of FBXL4. Establishment of the diagnosis permits genetic counselling, prevents patients undergoing unhelpful diagnostic procedures and allows for accurate prognosis.

Mitochondrial heat shock protein machinery hsp70/hsp40 is indispensable for proper mitochondrial DNA maintenance and replication.

ABSTRACT Mitochondrial chaperones have multiple functions that are essential for proper functioning of mitochondria. In the human-pathogenic protist Trypanosoma brucei, we demonstrate a novel function of the highly conserved machinery composed of mitochondrial heat shock proteins 70 and 40 (mtHsp70/mtHsp40) and the ATP exchange factor Mge1. The mitochondrial DNA of T. brucei, also known as kinetoplast DNA (kDNA), is represented by a single catenated network composed of thousands of minicircles and dozens of maxicircles packed into an electron-dense kDNA disk. The chaperones mtHsp70 and mtHsp40 and their cofactor Mge1 are uniformly distributed throughout the single mitochondrial network and are all essential for the parasite. Following RNA interference (RNAi)-mediated depletion of each of these proteins, the kDNA network shrinks and eventually disappears. Ultrastructural analysis of cells depleted for mtHsp70 or mtHsp40 revealed that the otherwise compact kDNA network becomes severely compromised, a consequence of decreased maxicircle and minicircle copy numbers. Moreover, we show that the replication of minicircles is impaired, although the lack of these proteins has a bigger impact on the less abundant maxicircles. We provide additional evidence that these chaperones are indispensable for the maintenance and replication of kDNA, in addition to their already known functions in Fe-S cluster synthesis and protein import.

Endangered species: mitochondrial DNA loss as a mechanism of human disease.

Human mitochondrial DNA (mtDNA) is a small maternally inherited DNA, typically present in hundreds of copies in a single human cell. Thus, despite its small size, the mitochondrial genome plays a crucial role in the metabolic homeostasis of the cell. Our understanding of mtDNA genotype-phenotype relationships is derived largely from studies of the classical mitochondrial neuromuscular diseases, in which mutations of mtDNA lead to compromised mitochondrial bioenergetic function, with devastating pathological consequences. Emerging research suggests that loss, rather than mutation, of mtDNA plays a major role across a range of prevalent human diseases, including diabetes mellitus, cardiovascular disease, and aging. Here, we examine the 'rules' of mitochondrial genetics and function, the clinical settings in which loss of mtDNA is an emerging pathogenic mechanism, and explore mtDNA damage and its consequences for the organellar network and cell at large. As extranuclear genetic material arrayed throughout the cell to support metabolism, mtDNA is increasingly implicated in a host of disease conditions, opening a range of exciting questions regarding mtDNA and its role in cellular homeostasis.

Endangered species mitochondrial DNA loss as a mechanism of human disease

The biologically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor in over 160 enzyme activities involved in a number of metabolic pathways, including neurotransmitter synthesis and degradation. In humans, PLP is recycled from food and from degraded PLP-dependent enzymes in a salvage pathway requiring the action of pyridoxal kinase, pyridoxine 5'-phosphate oxidase and phosphatases. Once pyridoxal 5'-phosphate is made, it is targeted to the dozens different apoenzymes that need it as a cofactor. The regulation of the salvage pathway and the mechanism of addition of PLP to the apoenzymes are poorly understood and represent a very challenging research field. Severe neurological disorders, such as convulsions and epileptic encephalopathy, result from a reduced availability of pyridoxal 5'-phosphate in the cell, due to inborn errors in the enzymes of the salvage pathway or other metabolisms and to interactions of drugs with PLP or pyridoxal kinase. Multifactorial neurological pathologies, such as autism, schizophrenia, Alzheimer's disease, Parkinson's disease and epilepsy have also been correlated to inadequate intracellular levels of PLP.

Highlights of This Issue

Doxorubicin is among the most effective anticancer drugs. Yet, a significant number of patients develop cardiotoxicity, for which identification of predicting factors remains a clinical challenge. Here, Khiati and colleagues demonstrate that mitochondrial topoisomerase, Top1mt, is critical to avoid cardiotoxicity. Top1mt knockout mice are hypersensitive to doxorubicin, and die from cardiac failure with mitochondrial DNA (mtDNA) loss and severe mitochondrial and cardiomyocyte abnormalities. The study demonstrates the importance of mtDNA homeostasis for doxorubicin tolerance and suggests the relevance of testing genomic variants for TOP1mt and others mitochondrial genes in doxorubicin-treated patients.Recent progress of immunotherapy has led to the development of many novel immune modulators and vaccines. With the potential number of different combinations, there is an urgent need to revisit the feasibility of the traditional phase 1 dose escalation design in early cancer vaccine development. Rahma and colleagues reviewed over 200 phase I/II cancer vaccine studies showing a lack of correlation between dose escalation and toxicities or cellular immune response. In addition, the authors described a novel design to identify the safe and immune active dose in cancer vaccines without the need to enroll a large number of patients.Circulating tumor cells (CTCs) expressing EpCAM is an independent prognostic factor recurrence in HCC patients. To comprehensively investigate the clinical significance of these cells in HCC patients undergoing different treatment, Guo and colleagues established a novel optimized negative enrichment and qRT-PCR-based CTC detection platform with high sensitivity, specificity, reproducibility, and a small sample volume requirement and discovered that EpCAM mRNA-positive CTCs serve as a novel indicator for the risk of recurrence or progression in HCC patients and a promising surrogate tool for surveillance of the therapeutic response instead of radiologic scans with the benefits of increased convenience and safety.MET amplification is one of the mechanisms underlying resistance to EGFR TKIs in NSCLC. Noninvasive imaging of proliferation with 3′-deoxy-3′-[18F]-fluorothymidine ([18F]FLT) and PET was used to detect MET-mediated resistance in NSCLC and to monitor its reversal by MET inhibitors. Iommelli and colleagues showed that the persistently high uptake of [18F]FLT in tumors after treatment with EGFR TKIs identified resistance, whereas the significant reduction of tracer uptake in response to treatment with MET inhibitors indicated the reversal of MET-mediated resistance. The major clinical implication of the study is to provide an adaptive imaging biomarker for personalized targeted therapy in NSCLC patients.

Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion.

The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis.