Inactivation of mismatch repair (MMR) is the cause of the common cancer predisposition disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer (HNPCC), as well as 10-40% of sporadic colorectal, endometrial, ovarian, gastric, and urothelial cancers. Elevated mutation rates (mutator phenotype), including simple repeat instability [microsatellite instability (MSI)] are a signature of MMR defects. MicroRNAs (miRs) have been implicated in the control of critical cellular pathways involved in development and cancer. Here we show that overexpression of miR-155 significantly down-regulates the core MMR proteins, hMSH2, hMSH6, and hMLH1, inducing a mutator phenotype and MSI. An inverse correlation between the expression of miR-155 and the expression of MLH1 or MSH2 proteins was found in human colorectal cancer. Finally, a number of MSI tumors with unknown cause of MMR inactivation displayed miR-155 overexpression. These data provide support for miR-155 modulation of MMR as a mechanism of cancer pathogenesis.colorectal cancer | DNA repair | microRNA

Oxidative DNA damage is unavoidably and continuously generated by oxidant byproducts of normal cellular metabolism. The DNA damage repair genes, mutY and mutM, prevent G to T mutations caused by reactive oxygen species in Escherichia coli, but it has remained debatable whether deficiencies in their mammalian counterparts, Myh and Ogg1, are directly involved in tumorigenesis. Here, we demonstrate that deficiencies in Myh and Ogg1 predispose 65.7% of mice to tumors, predominantly lung and ovarian tumors, and lymphomas. Remarkably, subsequent analyses identified G to T mutations in 75% of the lung tumors at an activating hot spot, codon 12, of the K-ras oncogene, but none in their adjacent normal tissues. Moreover, malignant lung tumors were increased with combined heterozygosity of Msh2, a mismatch repair gene involved in oxidative DNA damage repair as well. Thus, oxidative DNA damage appears to play a causal role in tumorigenesis, and codon 12 of K-ras is likely to be an important downstream target in lung tumorigenesis. The multiple oxidative repair genes are required to prevent mutagenesis and tumor formation. The mice described here provide a valuable model for studying the mechanisms of oxidative DNA damage in tumorigenesis and investigating preventive or therapeutic approaches.

The Ov/Br septin gene, which is also a fusion partner of MLL in acute myeloid leukaemia, is a member of a family of novel GTP binding proteins that have been implicated in cytokinesis and exocytosis. In this study, we describe the genomic and transcriptional organization of this gene, detailing seventeen exons distributed over 240 kb of sequence. Extensive database analyses identi®ed orthologous rodent cDNAs that corresponded to new, unidenti®ed 5' splice variants of the Ov/Br septin gene, increasing the total number of such variants to six. We report that splicing events, occurring at noncanonical sites within the body of the 3' terminal exon, remove either 1801 bp or 1849 bp of non-coding sequence and facilitate access to a secondary open reading frame of 44 amino acids maintained near the end of the 3' UTR. These events constitute a novel coding arrangement and represent the ®rst report of such a design being implemented by a eukaryotic gene. The various Ov/Br proteins either dier minimally at their amino and carboxy termini or are equivalent to truncated versions of larger isoforms. Northern analysis with an Ov/Br septin 3' UTR probe reveals three transcripts of 4.4, 4 and 3 kb, the latter being restricted to a sub-set of the tissues tested. Investigation of the identi®ed Ov/Br septin isoforms by RT ± PCR con®rms a complex transcriptional pattern, with several isoforms showing tissue-speci®c distribution. To date, none of the other human septins have demonstrated such transcriptional complexity. Oncogene (2001) 20, 5930 ± 5939.

The septin family of genes has been implicated in a variety of cellular processes including cytokinesis, membrane transport and fusion, exocytosis, and apoptosis. One member of the septin family maps to chromosome 17q25.3, a region commonly deleted in sporadic ovarian and breast tumours, and has also been identified as a fusion partner of MLL in acute myeloid leukaemias. The present study demonstrates that the pattern of expression of multiple splice variants of this septin gene is altered in ovarian tumours and cell lines. In particular, expression of the zeta transcript is detectable in the majority of tumours and cell lines, but not in a range of non-malignant adult and fetal tissues. Zeta expression is accompanied by loss of the ubiquitous beta transcript. Somatic mutations of the gene were not detected in ovarian tumours, but it was demonstrated that beta expression in tumour cell lines can be reactivated by 5-azacytidine treatment, suggesting a role for methylation in the control of expression of this gene.

DNA damage response (DDR) activates a complex signaling network that triggers DNA repair, cell cycle arrest, and/or cell death. Depending on the type and severity of DNA lesion, DDR is controlled by "master" regulators including ATM and ATR protein kinases. Cisplatin, a major chemotherapy drug that cross-links DNA, induces ATR-dependent DDR, resulting in apoptosis. However, it is unclear how ATR is activated. To identify the key regulators of ATR, we analyzed the proteins that associate with ATR after cisplatin treatment by blue native-PAGE and co-immunoprecipitation. The mismatch repair protein hMSH2 was found to be a major ATR-binding protein.Functionally, ATR activation and its recruitment to nuclear foci during cisplatin treatment were attenuated, and DNA damage signaling, involving Chk2, p53, and PUMA-␣, was suppressed in hMSH2-deficient cells. ATR activation induced by the DNA methylating agent N-methyl-N-nitrosourea was also shown to be hMSH2-dependent. Intriguingly, hMSH2-mediated ATR recruitment and activation appeared independent of replication protein A, Rad17, and the Rad9-Hus1-Rad1 protein complex. Together the results support a hMSH2-dependent pathway of ATR activation and downstream Chk2/p53 signaling. DNA damage response (DDR)3 is essential for the maintenance of the integrity of the genome (1-5). As a complex multistep process, DDR involves the recognition of DNA damage, activation of DNA damage-responsive protein kinases, signal amplification by downstream protein kinases, and activation of the effector proteins that trigger various cellular processes. At low levels of DNA damage, activation of DDR results in cell cycle arrest and DNA repair. However, at higher doses, DDR signaling frequently results in cell death by apoptosis (1-5). The phosphoinositide 3-kinase-related protein kinases, ATM (ataxiatelangiectasia mutated) and ATR (ATM-and Rad3-related), are the key regulators of DDR (1-8). Once activated, ATM and ATR regulate an array of substrates including Chk1 and Chk2, which culminate in DNA repair, cell cycle arrest, and/or apoptosis. Although ATM is generally activated by doublestranded DNA breaks, ATR activation can result from different types of DNA lesion including single-stranded breaks, replication stress, base adducts, and DNA cross-links (8, 9). In the canonical model, ATR activation involves the recruitment of the ATR-ATRIP (ATR-interacting protein) and the Rad9-Hus1-Rad1 (9-1-1) protein complexes to the DNA damage site via replication protein A (RPA). As a result, the 9-1-1 complex brings topoisomerase-binding protein-1 (TopBP1, an ATR activator) close to ATR for ATR activation (1,5,8). However, alternative pathways of ATR activation may exist. For example, mismatch repair (MMR) proteins have been implicated in ATR activation under certain experimental conditions (10 -12).The MMR system is important for DNA replication and repair (13-18). In mammalian cells, MMR is composed of five MutS homologues (MSH) and four MutL homologue proteins. The function of MMR is to first recognize D...

Reverse transcription of retroviral RNA genomes produce a doublestranded linear cDNA molecule. A host degradation system prevents a majority of the cDNA molecules from completing the obligatory genomic integration necessary for pathogenesis. We demonstrate that the human TFIIH complex proteins XPB (ERCC3) and XPD (ERCC2) play a principal role in the degradation of retroviral cDNA. DNA repair-deficient XPB and XPD mutant cell lines exhibited an increase in transduction efficiency by both HIVand Moloney murine leukemia virus-based retroviral vectors. Replicating Moloney murine leukemia virus viral production was greater in XPB or XPD mutant cells but not XPA mutant cells. Quantitative PCR showed an increase in total cDNA molecules, integrated provirus, and 2LTR circles in XPB and XPD mutant cells. In the presence of a reverse transcription inhibitor, the HIV cDNA appeared more stable in mutant XPB or XPD cells. These studies implicate the nuclear DNA repair proteins XPB and XPD in a cellular defense against retroviral infection.A fter entry into a host cell, retroviruses must copy their genomic RNA to a linear cDNA. A preintegration complex (PIC) is formed that contains the retroviral cDNA and viral and host proteins. Lentiviral PICs are able to traverse an intact nuclear membrane. Once inside the nucleus, the viral integrase protein catalyzes covalent joining of the cDNA into the host chromosome, yielding a provirus. A functional provirus is necessary to continue the viral life cycle. Alternative fates for the viral cDNA include formation of 1LTR circles, 2LTR circles, or degradation. Circle formation has long been taken as a measure of successful nuclear import of the PIC, because these products are not observed in the cytoplasm. The mechanism of cDNA degradation has not yet been elucidated.Many recent studies implicate roles for host DNA repair proteins in the retroviral life cycle (1-3). XPB (ERCC3) and XPD (ERCC2) are DNA helicases with opposing polarity that function as integral components of the TFIIH protein complex. TFIIH is required for basal transcription and nucleotide excision repair (NER) (4). The helicase activity of TFIIH is required to separate DNA strands at a promoter during transcription or at a site of DNA damage during NER. Both XPB and XPD are conserved and are essential in eukaryotes, precluding the establishment of null cell lines (5). Hypomorphic mutations of either XPB or XPD may lead to three recessive diseases with varying severity: trichothiodystrophy (TTD), xeroderma pigmentosum (XP), or associated XP and Cockayne syndrome (6-8). TTD-associated mutations appear to affect mainly transcription activity, whereas mutations associated with XP affect NER activity (9, 10). Although Ͼ20 mutations of XPD have been described, only three mutations of XPB have been observed in the human population, suggesting that mutations of XPB may be incompatible with survival (11).Here we demonstrate that transduction by HIV or Moloney murine leukemia virus (MMLV)-based retroviral vectors was substantially ...

The morbilliviruses form a serologically cross-reactive closely related genus in the family Paramyxoviridae with each species having a different host range. Before 1988, only four species were recognized ; namely the human measles virus (MV), rinderpest virus (RPV), which infects large ruminants and artiodactyls, peste-des-petits ruminants virus (PPRV), which infects small ruminants, and canine distemper virus (CDV), which affects all terrestrial carnivores (Appel et al.,

Nitric oxide-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are an emergent class of pharmaceutical derivatives with promising utility as cancer chemopreventive agents. Aspirin and sulindac have been shown to be effective in selecting for cells with reduced microsatellite instability (MSI) that is inherent in mismatch repair (MMR)-deficient hereditary nonpolyposis colorectal cancer (HNPCC) cells. The effect of NO-NSAIDs on MSI in MMR-deficient HNPCC cells is unknown. Here, we have examined genetically defined MMRdeficient murine embryo fibroblasts, murine colonocytes, and isogenic human HNPCC tumor cell lines treated with acetylsalicylic acid (aspirin; ASA) and three isomeric derivatives of NO-aspirin (NO-ASA). The MSI profiles were determined and compared with the Bethesda Criteria. We found that the ASA-and NO-ASA-treated MMR-deficient cell lines displayed a dose-dependent suppression of MSI that appeared as early as 8 weeks and gradually increased to include up to 67% of the microsatellite sequences examined after 19 to 20 weeks of continuous treatment. Residual resistance to microsatellite stabilization was largely confined to mononucleotide repeat sequences. Control (MMR-proficient) cells showed no changes in microsatellite status with or without treatment. The relative dose-dependent stabilization selection was: ortho-NO-ASA % para-NO-ASA > meta-NO-ASA J ASA. Moreover, the doses required for stabilization by the orthoand para-NO-ASA were 300-to 3,000-fold lower than ASA. These results suggest that NO-ASA derivatives may be more effective at suppressing MSI in MMR-deficient cell lines than ASA and should be considered for chemopreventive trials with HNPCC carriers. [Cancer Res 2007;67(22):10966-75]