DNA repair mechanisms in colorectal carcinogenesis

Colon cancer is among the most common cancers and the third cause of cancer deaths worldwide. If detected at an early stage, treatment might often lead to cure. The present review adduces the so far studied alterations in the expression of genes, as well as polymorphisms of genes engaged in DNA repair systems, with particular emphasis on indirect ones that are correlated with colorectal cancer. Such aberrations could be linked to an increased risk for the development of colorectal cancer and might serve as potential targets in the areas of prevention and therapy.     

DNA mismatch repair defects: role in colorectal carcinogenesis

The inactivation of the DNA mismah repair (MMR) system, which is associated with the predisposition to the hereditary non-polyposis colorectal cancer (HNPCC), has also been documented in nearly 20% of the sporadic colorectal cancers. These tumors are characterized by a high frequency of microsatellite instability (MSI(+) phenotype), resulting from the accumulation of small insertions or deletions that frequently arise during replication of these short repeated sequences. A germline mutation of one of the two major MMR genes (hMSH2 or hMLH1) is found in half to two-thirds of the patients with HNPCC, whereas in sporadic cases hypermethylation of the hMLH1 promoter is the major cause of the MMR defect. Germline mutations in hMSH6 are rare and rather confer predisposition to late-onset familial colorectal cancer, and frequent extracolonic tumors. Yet, the genetic background of a number of HNPCC patients remains unexplained, indicating that other genes participate in MMR and play important roles in cancer susceptibility. The tumor-suppressor genes that are potential targets for the MSI-driven mutations because they contain hypermutable repeated sequences are likely to contribute to the etiology and tissue specificity of the MSI-associated carcinogenesis. Because the prognosis and the chemosensitivity of the MSI(+) colorectal tumors differ from those without instability, the determination of the MSI phenotype is expected to improve the clinical management of patients. This review gives an overview of various aspects of the biochemistry and genetics of the DNA mismah repair system, with particular emphasis in its role in colorectal carcinogenesis.     

Chromatin recruitment of DNA repair proteins: lessons from the fanconi anemia and double-strand break repair pathways

In response to DNA damage, eukaryotic cells must rapidly load DNA repair proteins onto damaged chromatin. Chromatin recruitment often entails ubiquitination of a damage-specific DNA repair protein, interaction with a ubiquitin binding factor, assembly of a multisubunit DNA repair complex, and eventually a deubiquitination event once the DNA repair reaction has been completed. This review focuses on the recent discoveries in the Fanconi Anemia (FA) and DNA double-strand break (DSB) repair pathways, which underscore the importance of regulated chromatin loading in the DNA damage response. Interestingly, these two pathways share several features, suggesting a more general mechanism for DNA-repair regulation.     

Interplay of two major repair pathways in the processing of complex double-strand DNA breaks

Radiation-induced complex double-strand breaks (DSBs) characterised by base lesions, abasic sites or single-strand breaks in close proximity to the break termini, are believed to be a major cause of the biological effects of ionising radiation exposure. It has been hypothesised that complex DSBs pose problems for the repair machinery of the cell. Using a biochemical approach, we have investigated the challenge to two major repair processes: base excision repair and ligation of DSB ends. Double-stranded oligonucleotides were synthesised with 8-oxo-7,8-dihydroguanine (8-oxoG) at defined positions relative to readily ligatable 3'-hydroxy or 5'-phosphate termini. The break termini interfere with removal of 8-oxoG during base excision repair as elucidated from the severely reduced efficiency of 8-oxoG removal by OGG1 with AP endonuclease-1 when in close proximity to break termini. NEIL-1, however, can partially restore processing of complex DSBs in an AP endonuclease-1 independent manner. The influence of 8-oxoG on ligation shows delayed rejoining if 8-oxoG is positioned two to three bases from the 3'-hydroxy or six bases from the 5'-phosphate termini. When two 8-oxoG lesions are positioned across the break junction ligation is severely retarded. This reduced efficiency of repair indicates that complex DSBs are likely to persist longer than simple DSBs in cells, and as a consequence are more significant in contributing to the biological effects of ionising radiation.     

Congenital DNA repair deficiency results in protection against renal ischemia reperfusion injury in mice

Cockayne syndrome and other segmental progerias with inborn defects in DNA repair mechanisms are thought to be due in part to hypersensitivity to endogenous oxidative DNA damage. The accelerated aging-like symptoms of this disorder include dysmyelination within the central nervous system, progressive sensineuronal hearing loss and retinal degeneration. We tested the effects of congenital nucleotide excision DNA repair deficiency on acute oxidative stress sensitivity in vivo . Surprisingly, we found mouse models of Cockayne syndrome less susceptible than wild type animals to surgically induced renal ischemia reperfusion injury, a multifactorial injury mediated in part by oxidative damage. Renal failure-related mortality was significantly reduced in Csb^−/– mice, kidney function was improved and proliferation was significantly higher in the regenerative phase following ischemic injury. Protection from ischemic damage correlated with improved baseline glucose tolerance and insulin sensitivity and a reduced inflammatory response following injury. Protection was further associated with genetic ablation of a different Cockayne syndrome-associated gene, Csa . Our data provide the first functional in vivo evidence that congenital DNA repair deficiency can induce protection from acute stress in at least one organ. This suggests that while specific types of unrepaired endogenous DNA damage may lead to detrimental effects in certain tissues, they may at the same time elicit beneficial adaptive changes in others and thus contribute to the tissue specificity of disease symptoms.     

DNA repair pathways in human multiple myeloma

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.     

Five repair pathways in one context: chromatin modification during DNA repair.

The eukaryotic cell is faced with more than 10 000 various kinds of DNA lesions per day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death. Therefore, cells have developed 5 major repair pathways in which different kinds of DNA damage can be detected and repaired: homologous recombination, nonhomologous end joining, nucleotide excision repair, base excision repair, and mismatch repair. However, the efficient repair of DNA damage is complicated by the fact that the genomic DNA is packaged through histone and nonhistone proteins into chromatin, a highly condensed structure that hinders DNA accessibility and its subsequent repair. Therefore, the cellular repair machinery has to circumvent this natural barrier to gain access to the damaged site in a timely manner. Repair of DNA lesions in the context of chromatin occurs with the assistance of ATP-dependent chromatin-remodeling enzymes and histone-modifying enzymes, which allow access of the necessary repair factors to the lesion. Here we review recent studies that elucidate the interplay between chromatin modifiers / remodelers and the major DNA repair pathways.     

Oxidatively damaged DNA and its repair in colon carcinogenesis

Inflammation, high fat, high red meat and low fiber consumption have for long been known as the most important etiological factors of sporadic colorectal cancers (CRC). Colon cancer originates from neoplastic transformation in a single layer of epithelial cells occupying colonic crypts, in which migration and apoptosis program becomes disrupted. This results in the formation of polyps and metastatic cancers. Mutational program in sporadic cancers involves APC gene, in which mutations occur most abundantly in the early phase of the process. This is followed by mutations in RAS, TP53, and other genes. Progression of carcinogenic process in the colon is accompanied by augmentation of the oxidative stress, which manifests in the increased level of oxidatively damaged DNA both in the colon epithelium, and in blood leukocytes and urine, already at the earliest stages of disease development. Defence mechanisms are deregulated in CRC patients: (i) antioxidative vitamins level in blood plasma declines with the development of disease; (ii) mRNA level of base excision repair enzymes in blood leukocytes of CRC patients is significantly increased; however, excision rate is regulated separately, being increased for 8-oxoGua, while decreased for lipid peroxidation derived ethenoadducts, ?Ade and ?Cyt; (iii) excision rate of ?Ade and ?Cyt in colon tumors is significantly increased in comparison to asymptomatic colon margin, and ethenoadducts level is decreased. This review highlights mechanisms underlying such deregulation, which is the driving force to colon carcinogenesis.     

Rapid assessment of two major repair activities against DNA double-strand breaks in vertebrate cells

A linearized plasmid DNA, in which tandem repeats of 400bp flank the breakpoints, was transfected into vertebrate cells, and breakpoint junctions of plasmid DNA circularized in the cells were analyzed to assess the repair activities against DNA double-strand break (DSB) by non-homologous end joining and homology-directed repair (i.e., homologous recombinational repair and single-strand annealing). The circularization by non-homologous end joining repair of the breakpoints depended on the expression of DNA-PKcs, while that by homology-directed repair through the repeats depended on the length of the repeats, indicating that these two DSB repair activities can be rapidly assessed by this assay. Predominance in circularization by either non-homologous end joining or homology-directed repair differed among cells examined, and circularization was exclusively undertaken by homology-directed repair in DT40 cells known to show a high homologous recombination rate against gene-targeting vectors. Thus, this assay will be helpful in studies on mechanisms and inter-cellular variations of DSB repair.     

Actions of aprataxin in multiple DNA repair pathways

Mutations in the Aptx gene lead to a neurological disorder known as ataxia oculomotor apraxia-1. The product of Aptx is Aprataxin (Aptx), a DNA-binding protein that resolves abortive DNA ligation intermediates. Aprataxin catalyzes the nucleophilic release of adenylate groups covalently linked to 5' phosphate termini, resulting in termini that can again serve as substrates for DNA ligases. Here we show that Aprataxin acts preferentially on adenylated nicks and double-strand breaks rather than on single-stranded DNA. Moreover, we show that whereas the catalytic activity of Aptx resides within the HIT domain, the C-terminal zinc finger domain provides stabilizing contacts that lock the enzyme onto its high affinity AMP-DNA target site. Both domains are therefore required for efficient AMP-DNA hydrolase activity. Additionally, we find a role for Aprataxin in base excision repair, specifically in the removal of adenylates that arise from abortive ligation reactions that take place at incised abasic sites in DNA. We suggest that Aprataxin may have a general proofreading function in DNA repair, removing DNA adenylates as they arise during single-strand break repair, double-strand break repair, and in base excision repair.     

Comprehensive mapping of the C-terminus of flap endonuclease-1 reveals distinct interaction sites for five proteins that represent different DNA replication and repair pathways

Flap endonuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease that plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair, resolution of DNA secondary structures and stalled DNA replication forks, and apoptotic DNA fragmentation. How FEN-1 is involved in multiple pathways, of which some are seemingly contradictory, is of considerable interest. To date, at least 20 proteins are known to interact with FEN-1; some form distinct complexes that affect one or more FEN-1 activities presumably to direct FEN-1 to a particular DNA metabolic pathway. FEN-1 consists of a nuclease core domain and a C-terminal extension. While the core domain harbors the nuclease activity, the C-terminal extension may be important for protein-protein interactions. Here, we have truncated or mutated the C-terminus of FEN-1 to identify amino acid residues that are critical for interaction with five proteins representing roles in different DNA replication and repair pathways. We found with all five proteins that the C-terminus is important for binding and that each protein uses a subset of amino acid residues. Replacement of one or more residues with an alanine in many cases leads to the complete loss of interaction, which may consequently lead to severe biological defects in mammals.     

Opportunities for translation: Targeting DNA repair pathways in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains one of the poorest prognosis neoplasms. It is typified by high levels of genomic aberrations and copy-number variation, intra-tumoural heterogeneity and resistance to conventional chemotherapy. Improved therapeutic options, ideally targeted against cancer-specific biological mechanisms, are urgently needed. Although induction of DNA damage and/or modulation of DNA damage response pathways are associated with the activity of a number of conventional PDAC chemotherapies, the effectiveness of this approach in the treatment of PDAC has not been comprehensively reviewed. Here, we review chemotherapeutic agents that have shown anti-cancer activity in PDAC and whose mechanisms of action involve modulation of DNA repair pathways. In addition, we highlight novel potential targets within these pathways based on the emerging understanding of PDAC biology and their exploitation as targets in other cancers.     

Analysis of DNA double-strand break repair pathways in mice

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.     

Importance of DNA repair in carcinogenesis: evidence from transgenic and gene targeting studies

We have generated transgenic mice by introducing copies of the E. coli O6-methylguanine-DNA methyltransferase gene, ada. Liver extracts from homozygotes demonstrate about three times the control enzyme activity and increase up to about eight-fold can be induced by treatment with zinc, since the metal-responsive metallothionein promoter is attached to the ada gene. Furthermore, studies of liver carcinogenesis in our transgenic mice demonstrated significantly reduced rates of development of hepatocellular tumors after treatment with dimethylnitrosamine or diethylnitrosamine. It is well known that xeroderma pigmentosum (XP) patients are deficient in DNA repair. The availability of XPA (XP group A complementing) knockout mice has enabled us to investigate the functional role of the XPA nucleotide excision repair gene in carcinogenesis in vivo, first using the mouse skin as a model system. XPA-/- mice demonstrated skin ulcers 5-7 days after 7,12-dimethylbenz[a]anthracene (DMBA) treatment and papilloma development within 4 weeks prior to promotion, skin tumor incidence being also much higher than in heterozygous and wild-type mice. Experiments targeting the lung, liver and tongue have also been conducted to answer the question of whether the internal organs of these mice are also susceptible to chemical carcinogens. For lung carcinogenesis, mice were instilled intratracheally with a small dose of benzo[a]pyrene. The pulmonary tumor incidence in XPA-/- mice was significantly higher than in XPA+/- and XPA+/+ mice. XPA-/- mice were also found to be have enhanced sensitivity to aflatoxin B1 regarding liver tumor induction. In addition, administration of 4-nitroquinoline-1-oxide in drinking water for 50 weeks resulted in tongue tumors only in XPA-/- mice. These studies, thus, provided convincing evidence that XPA mice are also sensitive to carcinogenesis in organs other than the skin.     

Natural resistance mechanisms may play a role in protection against chemical carcinogenesis

Summary Mice deprived of B lymphocytes by the chronic administration of anti-IgM antibodies have been shown to possess a heightened natural resistance (NR) to micro-organisms, to parental bone marrow, and to natural killer (NK)-sensitive tumors in vitro and in vivo. Experiments described in this communication indicate that the latent period of primary tumors induced by the injection of methylcholanthrene (MC) is also prolonged in these mice. This observation suggests that NR mechanisms may provide protection against primary chemically induced tumors.Abbreviations used in this paper: MC, methylcholanthrene; NR, natural resistance; NRG, normal rabbit serum pool; NK, natural killer cell     

Co-recessive inheritance: a model for DNA repair, genetic disease and carcinogenesis

A genetic model for some cases of excision-deficient xeroderma pigmentosum (XP) is proposed in which the trait (i.e., XP) is expressed if and only if the individual is homozygous or hemizygous for defective alleles at more than one of a specific set of loci. The model might also apply in some cases of certain other diseases associated with defective DNA repair. The model accounts for several paradoxical aspects of XP, including the large number of complementation groups despite the biochemically limited DNA-repair defect, the co-existence of XP and Cockayne's syndrome in two different complementation groups of XP, siblings with markedly different degrees of severity of XP in one family and transmission of the disease in an X-linked manner in another, the existence of some individuals who appear to have the DNA-repair defect but not clinical XP, and the seeming paradox of a disease associated with a marked defect in a DNA-repair mechanism but not associated with an obvious increase in incidence of internal cancer. The model predicts that a large proportion of the general population is a carrier of one or more of these defective genes for DNA-repair mechanisms. Such genes may be important in the etiology of much of human cancer.     

Vaccination with the Leishmania major ribosomal proteins plus CpG oligodeoxynucleotides induces protection against experimental cutaneous leishmaniasis in mice

In the present work we analyze the antigenicity of Leishmania major ribosomal proteins (LRP) in infected BALB/c mice. We show that BALB/c mice vaccinated with LRP in the presence of CpG oligodeoxynucleotides (CpG-ODN) were protected against the development of dermal pathology and showed a reduction in the parasite load after challenge with L. major. This protection was associated with the induction of an IL-12 dependent specific-IFN-gamma response mediated mainly by CD4(+) T cell, albeit a minor contribution of CD8(+) T cells cannot be ruled out. Induction of Th1 responses against LRP also resulted in a reversion of the Th2 responses associated with susceptibility. A marked reduction of IgG1 antibody titer against parasite antigens besides an impaired IL-4 and IL-10 cytokine production by parasite specific T cells was observed. In addition, we show that the administration of the LRP plus CpG-ODN preparation also conferred protection in the naturally resistant C57BL/6 mice. In this strain protection was associated with a LRP specific IFN-gamma production in lymph nodes draining the challenge site. We believe that these evolutionary conserved proteins, combined with adjuvants that favor Th1 responses, may be relevant components of a pan-Leishmania vaccine.