Association of stromal type IV collagen and prognosis in neoadjuvant chemotherapy-treated pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has poor prognosis due to its low surgical eligibility and resistance to chemotherapy. Abundant stroma is characteristic of PDAC, and cancer-associated fibroblasts (CAFs) are a major stromal constituent, contributing to chemoresistance. Because neoadjuvant chemotherapy (NAC) is included in PDAC treatment as a standard regimen, the role of CAFs in NAC resistance must be studied. Although type IV collagen (COLIV) is present in the tumor of PDAC, the association between COLIV and disease advancement of NAC-treated PDAC is unclear. METHODS: Using a cohort of NAC-treated patients with PDAC, we examined clinicopathological data and conducted immunohistochemical analysis of COLIV in tissue specimens prepared from surgically resected pancreas. RESULTS AND CONCLUSIONS: Our analysis revealed that ~50% of the cases were positive for COLIV in the stroma and diffuse COLIV staining was an independent poor prognosis factor alongside high serum CA19-9 before NAC treatment (>37 U/mL) and postsurgical residual tumors. Based on these findings, we propose that stromal COLIV staining can be used to predict prognosis in NAC-treated patients with PDAC after surgery. Additionally, these findings suggest a possibility that stromal COLIV staining indicates resistance to anticancer drugs and/or contributes to malignancy in PDAC.

Aberrant RNA processing contributes to the pathogenesis of mitochondrial diseases in trans-mitochondrial mouse model carrying mitochondrial tRNALeu(UUR) with a pathogenic A2748G mutation.

Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.

Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability.

Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.

Cdk5rap1-mediated 2-methylthio-N6-isopentenyladenosine modification is absent from nuclear-derived RNA species.

2-Methylthio-N6-isopentenyl modification of adenosine (ms2i6A) is an evolutionally conserved modification that is found in transfer RNAs (tRNAs). We have recently shown that Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1) specifically converts i6A to ms2i6A at position A37 of four mitochondrial DNA-encoded tRNAs, and that the modification regulates efficient mitochondrial translation and energy metabolism in mammals. Curiously, a previous study reported that ms2i6A is present abundantly in nuclear-derived RNA species such as microRNAs, but not in tRNA fractions. To fully understand the molecular property of ms2i6A, the existence of non-canonical ms2i6A must be carefully validated. In the present study, we examined ms2i6A in total RNA purified from human and murine ρ0 cells, in which mitochondrial DNA-derived tRNAs were completely depleted. The ms2i6A was not detected in these cells at all. We generated a monoclonal antibody against ms2i6A and examined ms2i6A in murine RNAs using the antibody. The anti-ms2i6A antibody only reacted with the tRNA fractions and not in other RNA species. Furthermore, immunocytochemistry analysis using the antibody showed the predominant localization of ms2i6A in mitochondria and co-localization with the mitochondrial elongation factor Tu. Taken together, we propose that ms2i6A is a mitochondrial tRNA-specific modification and is absent from nuclear-encoded RNA species.

Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model.

All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17-/- liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17-/- mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.

Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria.

The genetic information in mammalian mitochondrial DNA is densely packed; there are no introns and only one sizeable noncoding, or control, region containing key cis-elements for its replication and expression. Many molecules of mitochondrial DNA bear a third strand of DNA, known as "7S DNA," which forms a displacement (D-) loop in the control region. Here we show that many other molecules contain RNA as a third strand. The RNA of these R-loops maps to the control region of the mitochondrial DNA and is complementary to 7S DNA. Ribonuclease H1 is essential for mitochondrial DNA replication; it degrades RNA hybridized to DNA, so the R-loop is a potential substrate. In cells with a pathological variant of ribonuclease H1 associated with mitochondrial disease, R-loops are of low abundance, and there is mitochondrial DNA aggregation. These findings implicate ribonuclease H1 and RNA in the physical segregation of mitochondrial DNA, perturbation of which represents a previously unidentified disease mechanism.

Isolated and repeated stroke-like episodes in a middle-aged man with a mitochondrial ND3 T10158C mutation: a case report.

BACKGROUND: Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, is the most common phenotype of mitochondrial disease. It often develops in childhood or adolescence, usually before the age of 40, in a maternally-inherited manner. Mutations in mitochondrial DNA (mtDNA) are frequently responsible for MELAS. CASE PRESENTATION: A 55-year-old man, who had no family or past history of mitochondrial disorders, suddenly developed bilateral visual field constriction and repeated stroke-like episodes. He ultimately presented with cortical blindness, recurrent epilepsy and severe cognitive impairment approximately 6 months after the first episode. Genetic analysis of biopsied biceps brachii muscle, but not of peripheral white blood cells, revealed a T10158C mutation in the mtDNA-encoded gene of NADH dehydrogenase subunit 3 (ND3), which has previously been thought to be associated with severe or fatal mitochondrial disorders that develop during the neonatal period or in infancy. CONCLUSION: A T10158C mutation in the ND3 gene can cause atypical adult-onset stroke-like episodes in a sporadic manner.

Increased negative supercoiling of mtDNA in TOP1mt knockout mice and presence of topoisomerases IIα and IIß in vertebrate mitochondria.

Topoisomerases are critical for replication, DNA packing and repair, as well as for transcription by allowing changes in DNA topology. Cellular DNA is present both in nuclei and mitochondria, and mitochondrial topoisomerase I (Top1mt) is the only DNA topoisomerase specific for mitochondria in vertebrates. Here, we report in detail the generation of TOP1mt knockout mice, and demonstrate that mitochondrial DNA (mtDNA) displays increased negative supercoiling in TOP1mt knockout cells and murine tissues. This finding suggested imbalanced topoisomerase activity in the absence of Top1mt and the activity of other topoisomerases in mitochondria. Accordingly, we found that both Top2α and Top2ß are present and active in mouse and human mitochondria. The presence of Top2α-DNA complexes in the mtDNA D-loop region, at the sites where both ends of 7S DNA are positioned, suggests a structural role for Top2 in addition to its classical topoisomerase activities.

Mitochondrial DNA replication proceeds via a 'bootlace' mechanism involving the incorporation of processed transcripts.

The observation that long tracts of RNA are associated with replicating molecules of mitochondrial DNA (mtDNA) suggests that the mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello, we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing replication intermediates are chased into mature mtDNA, to which they are thus in precursor-product relationship. While a DNA chain terminator rapidly blocks the labeling of mitochondrial replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a 'bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances.

Assessing TFAM Binding to Human Mitochondrial DNA.

Mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA)-binding protein that plays a crucial dual role in the initiation of mitochondrial transcription initiation and mtDNA maintenance. Because TFAM directly interacts with mtDNA, assessing its DNA-binding property can provide useful information. This chapter describes two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay with recombinant TFAM proteins, which both require simple agarose gel electrophoresis. These are used to investigate the effects of mutations, truncation, and posttranslational modifications on this key mtDNA regulatory protein.

Increased RUNX3 expression mediates tumor-promoting ability of human breast cancer-associated fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are a major stromal component of human breast cancers and often promote tumor proliferation, progression and malignancy. We previously established an experimental CAF (exp-CAF) cell line equipped with a potent tumor-promoting ability. It was generated through prolonged incubation of immortalized human mammary fibroblasts with human breast cancer cells in a tumor xenograft mouse model. RESULTS: Herein, we found that the exp-CAFs highly express Runt-related transcription factor 3 (RUNX3), while counterpart fibroblasts do not. In breast cancer patients, the proportion of RUNX3-positive stromal fibroblast-like cells tends to be higher in cancerous regions than in non-cancerous regions. These findings suggest an association of RUNX3 with CAF characteristics in human breast cancers. To investigate the functional role of RUNX3 in CAFs, the exp-CAFs with or without shRNA-directed knockdown of RUNX3 were implanted with breast cancer cells subcutaneously in immunodeficient mice. Comparison of the resulting xenograft tumors revealed that tumor growth was significantly attenuated when RUNX3 expression was suppressed in the fibroblasts. Consistently, Ki-67 and CD31 immunohistochemical staining of the tumor sections indicated reduction of cancer cell proliferation and microvessel formation in the tumors formed with the RUNX3-suppressed exp-CAFs. CONCLUSION: These results suggest that increased RUNX3 expression could contribute to the tumor-promoting ability of CAFs through mediating cancer cell growth and neoangiogenesis in human breast tumors.

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells.

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.

Mitochondria metabolomics reveals a role of ß-nicotinamide mononucleotide metabolism in mitochondrial DNA replication.

Mitochondrial DNA (mtDNA) replication is tightly regulated and necessary for cellular homeostasis; however, its relationship with mitochondrial metabolism remains unclear. Advances in metabolomics integrated with the rapid isolation of mitochondria will allow for remarkable progress in analyzing mitochondrial metabolism. Here, we propose a novel methodology for mitochondria-targeted metabolomics, which employs a quick isolation procedure using a hemolytic toxin from Streptococcus pyogenes streptolysin O (SLO). SLO isolation of mitochondria from cultured HEK293 cells is time- and labor-saving for simultaneous multi-sample processing and has been applied to various other cell lines in this study. Furthermore, our method can detect the time-dependent reduction in mitochondrial ATP in response to a glycolytic inhibitor 2-deoxyglucose, indicating the suitability to prepare metabolite analysis-competent mitochondria. Using this methodology, we searched for specific mitochondrial metabolites associated with mtDNA replication activation, and nucleotides and NAD+ were identified to be prominently altered. Most notably, treatment of ß-nicotinamide mononucleotide (ß-NMN), a precursor of NAD+, to HEK293 cells activated and improved the rate of mtDNA replication by increasing nucleotides in mitochondria and decreasing their degradation products: nucleosides. Our results suggest that ß-NMN metabolism plays a role in supporting mtDNA replication by maintaining the nucleotide pool balance in the mitochondria.

TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication.

Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.

Mitochondrial single-stranded DNA binding protein is required for maintenance of mitochondrial DNA and 7S DNA but is not required for mitochondrial nucleoid organisation.

Single-stranded DNA binding protein (SSB) plays important roles in DNA replication, recombination and repair through binding to single-stranded DNA. The mammalian mitochondrial SSB (mtSSB) is a bacterial type SSB. In vitro, mtSSB was shown to stimulate the activity of the mitochondrial replicative DNA helicase and polymerase, but its in vivo function has not been investigated in detail. Here we studied the role of mtSSB in the maintenance of mitochondrial DNA (mtDNA) in cultured human cells. RNA interference of mtSSB expression in HeLa cells resulted in rapid reduction of the protein and a gradual decline of mtDNA copy number. The rate of mtDNA synthesis showed a moderate decrease upon mtSSB knockdown in HeLa cells. These results confirmed the requirement of mtSSB for mtDNA replication. Many molecules of mammalian mtDNA hold a short third strand, so-called 7S DNA, whose regulation is poorly understood. In contrast to the gradual decrease of mtDNA copy number, 7S DNA was severely reduced upon mtSSB knockdown in HeLa cells. Further, 7S DNA synthesis was significantly affected by mtSSB knockdown in an oseteosarcoma cell line. These data together suggest that mtSSB plays an important role in the maintenance of 7S DNA alongside its role in mtDNA replication. In addition, live-cell staining of mtDNA did not imply alteration in the organisation of mitochondrial nucleoid protein-mtDNA complexes upon mtSSB knockdown in HeLa cells. This result suggests that the presence of 7S DNA is not crucial for the organisation of mitochondrial nucleoids.

The accessory subunit of human DNA polymerase γ is required for mitochondrial DNA maintenance and is able to stabilize the catalytic subunit.

Human DNA polymerase γ (POLG) is a mitochondria-specific replicative DNA polymerase consisting of a single catalytic subunit, POLGα, and a dimeric accessory subunit, POLGß. To gain a deeper understanding of the role of POLGß, we knocked out this protein in cultured human cybrid cells and established numerous knockout clones. POLGß-knockout clones presented a clear phenotype of mitochondrial DNA loss, indicating that POLGß is necessary for mitochondrial DNA replication. Moreover, POLGß-knockout cells showed a severe decrease in POLGα levels and acute suppression of POLGß expression efficiently down-regulated POLGα levels. These results suggest that, in addition to its role as the processivity factor of POLG, POLGß acts as a POLGα stabilizer, an important role for POLGß in mitochondrial DNA maintenance.

Chemical acetylation of mitochondrial transcription factor A occurs on specific lysine residues and affects its ability to change global DNA topology.

Chemical acetylation is postulated to occur in mitochondria. Mitochondrial transcription factor A (TFAM or mtTFA), a mitochondrial transcription initiation factor as well as the major mitochondrial nucleoid protein coating the entire mitochondrial genome, is proposed to be acetylated in animals and cultured cells. This study investigated the properties of human TFAM, in conjunction with the mechanism and effects of TFAM acetylation in vitro. Using highly purified recombinant human TFAM and 3 kb circular DNA as a downsized mtDNA model, we studied how the global TFAM-DNA interaction is affected/regulated by the quantitative TFAM-DNA relationship and TFAM acetylation. Results showed that the TFAM-DNA ratio strictly affects the TFAM property to unwind circular DNA in the presence of topoisomerase I. Mass spectrometry analysis showed that in vitro chemical acetylation of TFAM with acetyl-coenzyme A occurs preferentially on specific lysine residues, including those reported to be acetylated in exogenously expressed TFAM in cultured human cells, indicating that chemical acetylation plays a crucial role in TFAM acetylation in mitochondria. Intriguingly, the modification significantly decreased TFAM's DNA-unwinding ability, while its DNA-binding ability was largely unaffected. Altogether, we propose TFAM is chemically acetylated in vivo, which could change mitochondrial DNA topology, leading to copy number and gene expression modulation.

Analysis of mitochondrial DNA by two-dimensional agarose gel electrophoresis.

In higher vertebrates, the DNA of mitochondria takes the form of circular molecules of approximately 16 kbp. These circles are arranged in multigenomic nucleoprotein complexes or nucleoids. It is envisaged that nucleoid superstructure makes a critical contribution to the twin processes of replication and segregation of mtDNA. Replication intermediates can be isolated from cells or solid tissues and separated on agarose gels in two dimensions to reveal a wealth of data on mechanisms of DNA replication. Using this technique we have demonstrated that many molecules of replicating mtDNA have extensive regions of RNA: DNA hybrid in higher vertebrates. More recently, we have extracted mitochondrial nucleoprotein and analyzed it by the same method to derive information on the distribution of DNA-binding proteins on mitochondrial DNA. Here we describe the procedures used to isolate intact mitochondrial replication intermediates from liver and cultured cells of higher vertebrates and the process of separating DNA fragments on neutral two-dimensional agarose gels.

Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes.

The mechanism of mitochondrial DNA replication is a subject of intense debate. One model proposes a strand-asynchronous replication in which both strands of the circular genome are replicated semi-independently while the other model proposes both a bidirectional coupled leading- and lagging-strand synthesis mode and a unidirectional mode in which the lagging-strand is initially laid-down as RNA by an unknown mechanism (RITOLS mode). Both the strand-asynchronous and RITOLS model have in common a delayed synthesis of the DNA-lagging strand. Mitochondrial DNA is replicated by a limited set of proteins including DNA polymerase gamma (POLG) and the helicase Twinkle. Here, we report the effects of expression of various catalytically deficient mutants of POLG1 and Twinkle in human cell culture. Both groups of mutants reduced mitochondrial DNA copy number by severe replication stalling. However, the analysis showed that while induction of POLG1 mutants still displayed delayed lagging-strand synthesis, Twinkle-induced stalling resulted in maturated, essentially fully double-stranded DNA intermediates. In the latter case, limited inhibition of POLG with dideoxycytidine restored the delay between leading- and lagging-strand synthesis. The observed cause-effect relationship suggests that Twinkle-induced stalling increases lagging-strand initiation events and/or maturation mimicking conventional strand-coupled replication.

The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA.

The mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNA(Leu(UUR)) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA). Electrophoretic mobility shift assays (EMSA) and SELEX, using mitochondrial protein extracts from cells induced to overexpress mTERF, revealed novel, weaker mTERF-binding sites, clustered in several regions of mtDNA, notably in the major non-coding region (NCR). Such binding in vivo was supported by mtDNA immunoprecipitation. Two-dimensional neutral agarose gel electrophoresis (2DNAGE) and 5' end mapping by ligation-mediated PCR (LM-PCR) identified the region of the canonical mTERF-binding site as a replication pause site. The strength of pausing was modulated by the expression level of mTERF. mTERF overexpression also affected replication pausing in other regions of the genome in which mTERF binding was found. These results indicate a role for TERF in mtDNA replication, in addition to its role in transcription. We suggest that mTERF could provide a system for coordinating the passage of replication and transcription complexes, analogous with replication pause-region binding proteins in other systems, whose main role is to safeguard the integrity of the genome whilst facilitating its efficient expression.