DNA repair and related diseases]

DNA-repair is a complex enzymatic process which enables all living cells to withstand the deleterious and mutagenic effects of most genotoxic agents. Defective DNA-repair is caused by mutations involving genes which encode the enzymes responsible for recognition and excision of DNA lesions. Some of these genes have been identified in humans. Several severe human diseases are caused by defective DNA repair affecting the entire genome (e.g. xeroderma pigmentosum and trichotiodystrophy) or only actively transcribed genes (Cockayne's syndrome). Some of these conditions are associated with extremely high rate of cancer.     

DNA damage and repair: from molecular mechanisms to health implications

DNA is subjected to several modifications, resulting from endogenous and exogenous sources. The cell has developed a network of complementary DNA-repair mechanisms, and in the human genome, >130 genes have been found to be involved. Knowledge about the basic mechanisms for DNA repair has revealed an unexpected complexity, with overlapping specificity within the same pathway, as well as extensive functional interactions between proteins involved in repair pathways. Unrepaired or improperly repaired DNA lesions have serious potential consequences for the cell, leading to genomic instability and deregulation of cellular functions. A number of disorders or syndromes, including several cancer predispositions and accelerated aging, are linked to an inherited defect in one of the DNA-repair pathways. Genomic instability, a characteristic of most human malignancies, can also arise from acquired defects in DNA repair, and the specific pathway affected is predictive of types of mutations, tumor drug sensitivity, and treatment outcome. Although DNA repair has received little attention as a determinant of drug sensitivity, emerging knowledge of mutations and polymorphisms in key human DNA-repair genes may provide a rational basis for improved strategies for therapeutic interventions on a number of tumors and degenerative disorders.     

Importance of DNA damage checkpoints in the pathogenesis of human cancers

All forms of life on earth must cope with constant exposure to DNA-damaging agents that may promote cancer development. As a biological barrier, known as DNA damage response (DDR), cells are provided with both DNA repair mechanisms and highly conserved cell cycle checkpoints. The latter are responsible for the control of cell cycle phase progression with ATM, ATR, Chk1, and Chk2 as the main signaling molecules, thus dealing with both endogenous and exogenous sources of DNA damage. As cell cycle checkpoint and also DNA repair genes, such as BRCA1 and BRCA2, are frequently mutated, we here discuss their fundamental roles in the pathogenesis of human cancers. Importantly, as current evidence also suggests a role of MAPK's (mitogen activated protein kinases) in cell cycle checkpoint control, we describe in this review both the ATR/ATM-Chk1/Chk2 signaling pathways as well as the regulation of cell cycle checkpoints by MAPK's as molecular mechanisms in DDR, and how their dysfunction is related to cancer development. Moreover, since damage to DNA might be the common underlying mechanism for the positive outcome of chemotherapy, we also discuss targeting anticancer treatments on cell cycle checkpoints as an important issue emerging in drug discovery.     

DNA双链断裂修复缺陷在神经退行性疾病发生中的作用

DNA双链断裂修复（DNA Double-Strand Break Repair,DSBR）在保持神经元基因组稳定性和细胞存活方面发挥着重要作用。DSBR主要通过同源重组（Homologous Recombination,HR）及非同源末端连接（Non-Homologous End Joining,NHEJ）来完成,这两种修复途径对于维持神经元的正常生理功能至关重要。另外,DSBR异常在多种神经退行性疾病中扮演重要角色,因此,深入剖析DSBR机制对于理解神经退行性疾病的病理发生及研发有效治疗手段具有重要意义。本文综述了常见的DSBR途径,并概述了DSBR异常与几种常见神经退行性疾病发病机制的最新研究进展。     

Developmental mechanisms in the pathogenesis of neurodegenerative diseases

Cellular genes that are mutated in neurodegenerative diseases code for proteins that are expressed throughout neural development. Genetic analysis suggests that these genes are essential for a broad range of normal neurodevelopmental processes. The proteins they code for interact with numerous other cellular proteins that are components of signaling pathways involved in patterning of the neural tube and in regional specification of neuronal subtypes. Further, pathogenetic mutations of these genes can cause progressive, sublethal alterations in the cellular homeostasis of evolving regional neuronal subpopulations, culminating in late-onset cell death. Therefore, as a consequence of the disease mutations, targeted cell populations may retain molecular traces of abnormal interactions with disease-associated proteins by exhibiting changes in a spectrum of normal cellular functions and enhanced vulnerability to a host of environmental stressors. These observations suggest that the normal functions of these disease-associated proteins are to ensure the fidelity and integration of developmental events associated with the progressive elaboration of neuronal subtypes as well as the maintenance of mature neuronal populations during adult life. The ability to identify alterations within vulnerable neuronal precursors present in pre-symptomatic individuals prior to the onset of irrevocable cellular injury may help foster the development of effective therapeutic interventions using evolving pharmacologic, gene and stem cell technologies.     

Immune mechanisms in the pathogenesis of demyelinating diseases

The loss of myelin which characterises many human and experimental demyelinating diseases, among them multiple sclerosis, is thought to be immune mediated, but the precise mechanisms responsible remain unknown despite intense research. Normally, myelin in the central nervous system (CNS) is protected from systemic immune responses by the blood brain barrier, which separates nervous tissue from the peripheral circulation. Here we review evidence suggesting that an understanding of the demyelinating disorders may be helped by considering their immune pathogenesis in two stages. The first is damage to the blood brain barrier; this appears to be cell mediated, and allows infiltration into the CNS of other immune effectors. These include complement and also macrophages, which together may mediate the second stage, injury to the myelin/oligodendrocyte complex.     

DNA damage and repair in human lymphocytes exposed to three anticancer platinum drugs

Cisplatin is a widely used anticancer drug, but its application is limited due to severe side effects. To reduce these effects, many other platinum drugs have been synthesized. In the present work comparative analysis of the toxicity of cisplatin, oxoplatin, and a conjugate (NH(3))(2)Pt(SeO(3)) (Se-Pt) in terms of cell viability, DNA binding, and DNA damage and repair in human lymphocytes was performed using the Trypan blue exclusion test, atomic absorption spectroscopy, and the comet assay, respectively. Cisplatin and oxoplatin did not cause a significant change in the viability of the lymphocytes even at the highest used concentration (750 microM), but the conjugate dramatically diminished viability at 100 microM only about 60% of the lymphocytes were viable (P < 0.05), and at 750 microM, less than 20% (P < 0.001). Se-Pt bound to isolated DNA was about 100 times weaker than the remaining two compounds; the binding of cisplatin was about 30% stronger than oxoplatin. Cisplatin and oxoplatin formed crosslinks with DNA in lymphocytes, whereas the conjugate induced DNA strand breaks. The lesions evoked by cisplatin and oxoplatin were slowly removed, but damage induced by Se-Pt was not repaired after 5 h even at a drug concentration of 10 microM. Severe cytotoxic and genotoxic effects exerted by Se-Pt in normal human lymphocytes preclude its intravenous application in cancer therapy. Teratogenesis Carcinog. Mutagen. 20:119-131, 2000.     

Analysis of DNA damage recovery processes in the adaptive response to inonizing radiation in human lymphocytes

It has been frequently suggested that the adaptive responseto ionizing radiation involves the induction of a chromosomalrepair mechanism. Although several lines of evidence favourthis assumption, direct proof is lacking. We have chosen tostudy this question with the help of the comet assay. Lymphocytesfrom three human donors were given an adapting dose of 0.05Gy 16 h after mitogenic stimulation and a challenging dose of2 Gy 5 h thereafter. While a portion of the cells was removedfrom the cultures for the comet assay, remaining cells wereharvested at 52 h culture time and screened for chromosomalaberrations. In some experiments an analysis of cell proliferationwas additionally carried out by flow cytometry. In the cometassay a reduced level of initial damage and an increased repaircapacity was observed in the adapted + challenged cells; however,this did not result in a reduction of the aberration frequencies.No effect of the adapting dose on cell proliferation was detectable.The analysis of comet distributions revealed that the observedenhanced repair capacity was due to the presence of a subpopulationof slowly repairing cells in the challenged lymphocytes andthe lack of such a subpopulation in the adapted + challengedcells. We assume that the slowly repairing cells were quiescentGo lymphocytes which were removed from the adapted + challengedcell population, probably by apoptotic-like processes. ¹ To whom correspondence should be addressed     

Difference in the capacity to repair DNA damage in human cells chronically infected with the rubella virus

Centrifugation of cell lysates in alkaline sucrose gradients and chromatography on hydroxyapatite columns were used to demonstrate inhibition of reparation of mitomycin C-induced DNA damages at the stage of reunification of single-strand breaks of DNA in human HEp-2 cell cultures chronically infected with rubella virus. At the same time, reparation of single-strand breaks of DNA caused by bleomycin occurs with similar intensity both in chronically infected and noninfected HEp-2 cultures. The experimental results suggest that the chronic course of infection in human cells leads to disorders in reparative synthesis of cellular DNA and/or is due to disconnected effect of reparation enzymes in this system.     

An osteochondral culture model to study mechanisms involved in articular cartilage repair

Although several treatments for cartilage repair have been developed and used in clinical practice the last 20 years, little is known about the mechanisms that are involved in the formation of repair tissue after these treatments. Often, these treatments result in the formation of fibrocartilaginous tissue rather than normal articular cartilage. Because the repair tissue is inferior to articular cartilage in terms of mechanical properties and zonal organization of the extracellular matrix, complaints of the patient may return. The biological and functional outcome of these treatments should thus be improved. For this purpose, an in vitro model allowing investigation of the involved repair mechanisms can be of great value. We present the development of such a model. We used bovine osteochondral biopsies and created a system in which cartilage defects of different depths can be studied. First, our biopsy model was characterized extensively: we studied the viability by means of lactate dehydrogenase (LDH) excretion over time and we investigated expression of cartilage-related genes in osteochondral biopsies and compared it with conventional cartilage-only explants. After 28 days of culture, LDH was detected at low levels and mRNA could be retrieved. The expression of cartilage-related genes decreased over time. This was more evident in cartilage-only explants, indicating that the biopsy model provided a more stable environment. We also characterized the subchondral bone: osteoclasts and osteoblasts were active after 28 days of culture, which was indicated by tartrate acid phosphatase staining and alkaline phosphatase measurements, respectively, and matrix deposition during culture was visualized using calcein labeling. Second, the applicability of the model was further studied by testing two distinct settings: (1) implantation of chondrocytes in defects of different depths; (2) two different seeding strategies of chondrocytes. Differences were observed in terms of volume and integration of newly formed tissue in both settings, suggesting that our model can be used to model distinct conditions or even to mimic clinical treatments. After extensive characterization and testing of our model, we present a representative and reproducible in vitro model that can be used to evaluate new cartilage repair treatments and study mechanisms in a controlled and standardized environment.     

Gene PSO5 of Saccharomyces cerevisiae,involved in repair of oxidative DNA damage,is allelic to RAD16

The pos5-1 mutation renders Saccharomyces cerevisiae cells sensitive to DNA-damaging agents. We have isolated plasmids from a S. cerevisiae genomic library capable of restoring wild-type levels of 254-nm ultraviolet light sensitivity of the pso5-1 mutant. DNA sequence analysis revealed that the complementing activity resides in RAD16, a gene involved in excision repair. Tetrad analysis showed that PSO5, like RAD16, is tightly linked to LYS2 on chromosome II. Moreover, allelism between the pso5-1 and rad16 mutants was demonstrated by the comparison of mutagen sensitivity phenotypes, complementation tests, and by meiotic analysis. The cloned RAD16 gene was capable of restoring wild-type resistance of the pso5-1 mutant to H₂O₂ and photoactivated 3-carbethoxypsoralen, both treatments generating oxidative stress-related DNA damage. This indicates that RAD16/PSO5 might also participate in the repair of oxidative base damage.     

The repair of DNA damage induced in human peripheral lymphocytes with styrene oxide

Isolated human peripheral lymphocytes were treated in vitro with styrene-7, 8-oxide (SO) and the kinetics of the repair of induced DNA damage was assessed by comet assay during further incubation of lymphocytes. Using a modified assay we measured simultaneously the number of single strand breaks in DNA (SSBs) and the sites sensitive to endonuclease III (endo III) that most probably represent abasic sites in DNA molecules. SO induced DNA damage in a dose-dependent manner and both SSBs and endo III sites were removed from the DNA by a repair process with a half time about 2-4 hours. The damage was repaired completely within 12 hours after the treatment.     

Influence of DNA repair gene polymorphisms on the initial repair of MMS-induced DNA damage in human lymphocytes as measured by the alkaline comet assay

We have applied the alkaline comet assay to study the functional impact of gene polymorphisms in base excision repair (APEX1 Asp148Glu, XRCC1 Arg194Trp, XRCC1 Arg399Gln) and homologous recombination repair (XRCC3 Thr241Met, NBS1 Glu185Gln), two pathways that play crucial roles in the repair of DNA damage induced by methylmethane sulphonate (MMS). We also examined the effect of polymorphisms in mismatch repair (MLH1 -93 A/G) and nucleotide excision repair (XPD Lys751Gln) as putative negative controls based on the limited roles of these pathways in MMS-induced repair. Phytohemagglutinin-stimulated peripheral lymphocytes from 52 healthy individuals were treated with MMS and allowed to repair for 0, 15, 40, or 120 min after a 6-min washing step. DNA damage was measured as a pseudo-percentage score (comparable to % tail DNA) converted from a total visual score calculated from the distribution of cells with different degrees of damage (normal, mild, moderate and severe). The repair was faster at the beginning of the observation period than towards the end, and was not complete after 2 hr. Presence of the APEX1 148Asp, XRCC3 241Met or NBS1 185Gln alleles were significantly associated with a high pseudo-percentage score (above median) at early time points, with the APEX1 effect being most prolonged (up to 40 min after washing, odds ratio 5.6, 95% confidence interval 2.0-15.5). No significant effects were seen with the XRCC1 Arg194Trp, XRCC1 Arg399Gln, MLH1 -93A/G and XPD Lys751Gln polymorphisms. Our results provide evidence for the functional nature of the variant alleles studied in the APEX1, XRCC3, and NBS1 genes.     

Promyelocytic leukaemia protein links DNA damage response and repair to hepatitis B virus‐related hepatocarcinogenesis

DNA damage response and repair pathways are important barriers to carcinogenesis. Here, we show that promyelocytic leukaemia (PML, also known as TRIM19), involved in sensing DNA damage and executing homologous recombination repair, is down‐regulated in non‐tumour liver cells surrounding hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC). No PML mutation or deletion was found in HBV‐infected liver or HCC cells. Immunohistochemical analysis of liver biopsies from patients with breast or liver cancer and HBV reactivation after chemotherapy revealed PML up‐regulation and HBV exacerbation in normal liver tissue in response to DNA damage (functional PML), PML down‐regulation in HCC peritumour cells associated with high HBsAg accumulation and low HBV replication activity (suppressive PML), and heterogeneous nuclear PML expression in HCC cells that lost HBV DNA and HBsAg and were non‐reactive to DNA damage (dysregulated PML). Loss of PML in HBsAg‐transgenic mice promoted chromosome breaks in liver cells and accelerated the accumulation of body and liver fat and the development of a liver steatosis–dysplasia–adenoma–carcinoma sequence in an inflammation‐independent and male‐predominant manner, compared to PML knock‐out or HBsAg‐transgenic mice during the same time period. These results indicate that PML deficiency facilitates genomic instability and promotes HBsAg‐related hepatocarcinogenesis, which also involves androgen and lipid metabolism. These findings uncover a novel PML link between HBV‐related tumourigenesis, DNA repair, and metabolism. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

DNA repair pathways involved in anaphase bridge formation

Cancer cells frequently exhibit gross chromosomal alterations such as translocations, deletions, or gene amplifications an important source of chromosomal instability in malignant cells. One of the better-documented examples is the formation of anaphase bridges-chromosomes pulled in opposite directions by the spindle apparatus. Anaphase bridges are associated with DNA double strand breaks (DSBs). While the majority of DSBs are repaired correctly, to restore the original chromosome structure, incorrect fusion events also occur leading to bridging. To identify the cellular repair pathways used to form these aberrant structures, we tested a requirement for either of the two major DSB repair pathways in mammalian cells: homologous recombination (HR) and nonhomologous end joining (NHEJ). Our observations show that neither pathway is essential, but NHEJ helps prevent bridges. When NHEJ is compromised, the cell appears to use HR to repair the break, resulting in increased anaphase bridge formation. Moreover, intrinsic NHEJ activity of different cell lines appears to have a positive trend with induction of bridges from DNA damage.     

DNA damage in human sperm is related to urinary levels of phthalate monoester and oxidative metabolites

BACKGROUND: The ubiquitous use of phthalate esters in plastics, personal care products and food packaging materials results in widespread general population exposure. In this report, we extend our preliminary study on the relationship between urinary concentrations of phthalate metabolites and sperm DNA damage among a larger sample of men and include measurements of mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP), two oxidative metabolites of di-(2-ethylhexyl) phthalate (DEHP). METHODS: Among 379 men from an infertility clinic, urinary concentrations of phthalate metabolites were measured using isotope-dilution high-performance liquid chromatography-tandem mass spectrometry. Sperm DNA damage measurements, assessed with the neutral comet assay, included comet extent (CE), percentage of DNA in tail (Tail%) and tail distributed moment (TDM). RESULTS: Monoethyl phthalate (MEP), a metabolite of diethyl phthalate, was associated with increased DNA damage, confirming our previous findings. Mono-(2-ethylhexyl) phthalate (MEHP), a metabolite of DEHP, was associated with DNA damage after adjustment for the oxidative DEHP metabolites. After adjustment for MEHHP, for an interquartile range increase in urinary MEHP, CE increased 17.3% [95% confidence interval (CI) = 8.7-25.7%], TDM increased 14.3% (95% CI = 6.8-21.7%) and Tail% increased 17.5% (95% CI = 3.5-31.5%). CONCLUSIONS: Sperm DNA damage was associated with MEP and with MEHP after adjusting for DEHP oxidative metabolites, which may serve as phenotypic markers of DEHP metabolism to 'less toxic' metabolites. The urinary levels of phthalate metabolites among these men were similar to those reported for the US general population, suggesting that exposure to some phthalates may affect the population distribution of sperm DNA damage.