Ionizing radiation-induced DNA damage and repair

Ionizing radiation has been shown to induce various types of chromosomal DNA damages. Among these DNA damages, DNA double strand breaks(DSBs) are the most severe damages resulting in cell death or chromosome abnormalities. Proteins associated with DNA repair, such as phosphorylated form of histone H2AX, a histone variant of H2A, and a DNA recombinase RAD51, has been shown to form radiation-induced repair foci at sites containing DNA damage. Reorganization of damaged chromatin by protein modifications or exchange of histones has been shown to play an important role in the formation of radiation induced repair foci.     

Positive Regulation of DNA Double Strand Break Repair Activity during Differentiation of Long Life Span Cells: The Example of Adipogenesis

Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs), and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE) and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ) that relies on the DNA dependent protein kinase (DNA-PK) activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue.     

The influence of radiation and chemotherapy-related DNA strand breaks on carcinogenesis: an evaluation.

PURPOSE: DNA strand breaks are believed to induce carcinogenesis. This study was conducted to analyze induction and repair of irradiation- and chemotherapy-related strand breaks in vitro. METHODS: Friend Leukemia cells were exposed to irradiation and various chemotherapeutic agents at different doses and concentrations. Occurrence of strand breaks was determined fluorometrically, measuring the rate of DNA unwinding immediately after exposure and 24 hours later. RESULTS: The amount of double-stranded DNA decreased significantly for irradiation, doxorubicin, dactinomycin and etoposide (p < or = 0.05, t-test). After 24 hours free of exposure, the persistent damage was detectable for all of these agents but not for irradiated cells, with DNA strand breaks being decreased for etoposide, unchanged for doxorubicin and increased for methotrexate as well as for dactinomycin. CONCLUSIONS: Severe DNA damage is induced by various chemotherapeutic agents and by irradiation. While repair of chemotherapy-related strand breaks may remain incomplete or prolonged for some chemotherapeutic agents, repair of radiation induced strand breaks is faster and more complete. Therefore chemotherapy-related carcinogenesis may partially be explained by prolonged persistence of DNA strand breaks.     

A regulatory role for the cohesin loader NIPBL in nonhomologous end joining during immunoglobulin class switch recombination

DNA double strand breaks (DSBs) are mainly repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ). These breaks pose severe threats to genome integrity but can also be necessary intermediates of normal cellular processes such as immunoglobulin class switch recombination (CSR). During CSR, DSBs are produced in the G1 phase of the cell cycle and are repaired by the classical NHEJ machinery. By studying B lymphocytes derived from patients with Cornelia de Lange Syndrome, we observed a strong correlation between heterozygous loss-of-function mutations in the gene encoding the cohesin loading protein NIPBL and a shift toward the use of an alternative, microhomology-based end joining during CSR. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Association of NIPBL deficiency and impaired NHEJ was also observed in a plasmid-based end-joining assay and a yeast model system. Our results suggest that NIPBL plays an important and evolutionarily conserved role in NHEJ, in addition to its canonical function in sister chromatid cohesion and its recently suggested function in HR.DNA double strand breaks (DSBs) pose a severe threat to genome integrity, but can also be a necessary part of normal cellular processes, such as meiosis and Ig class switch recombination (CSR). Depending on cell cycle phase and DSB structure, different strategies are used for repair. Homologous recombination (HR) depends on a homologous DNA template for repair, preferentially the identical sister chromatid, and is therefore mainly active during the S and G2 phases. Nonhomologous end joining (NHEJ), however, is active throughout the cell cycle and is the principal pathway during the G1 phase, when there is no immediate close template for homologous repair. The classical NHEJ pathway requires not only the key components of the NHEJ machinery, i.e., Ku70/Ku80, DNA-PKcs, Artemis, XLF (Cernunnos), XRCC4, and DNA ligase IV, but also several DNA damage sensors or adaptors, such as ATM, γH2AX, 53BP1, MDC1, RNF168, and the Mre11–Rad50–NBS1 complex.Cohesin is an evolutionarily conserved multisubunit complex consisting of a heterodimer of two structural maintenance of chromosomes (SMC) proteins, SMC1A and SMC3, one kleisin protein RAD21 (MCD1 or SCC1) and SA (STAG1/2 or SCC3). The SMC proteins fold back on themselves in the hinge region to form long antiparallel coiled-coil arms, with the amino and carboxyl termini coming together to create head domains that contain ATPases. RAD21 bridges the two head domains to facilitate the formation of the proposed ring-like structure of the complex, and it also interacts with the SA subunit. The cohesin complex ensures correct chromosome segregation through cohesion between sister chromatids (Nasmyth and Haering, 2009). In addition to this canonical role, cohesin and its loading complex NIPBL/MAU2 have also been suggested to be important for regulation of gene expression and repair of DSBs through HR, presumably by facilitating proximity between the broken DNA ends and the repair template (Sjögren and Nasmyth, 2001; Vrouwe et al., 2007; Nasmyth and Haering, 2009). Smc1, the yeast SMC1A orthologue, has furthermore been suggested to coordinate the HR and NHEJ processes (Schär et al., 2004).Cornelia de Lange syndrome (CdLS) is a developmental disorder characterized by growth retardation, severe intellectual disability, gastrointestinal abnormalities, malformations, of the upper limbs and characteristic facial dysmorphisms. Heterozygous loss-of-function mutations in NIPBL, encoding the cohesin loader NIPBL, are the major cause of CdLS (Liu and Baynam, 2010). In addition, mutations in the SMC1A, SMC3, PDS5B, RAD21, and HDAC8 encoding genes, all being part of the cohesion pathway, have been found in selected CdLS patients. The multisystem dysfunctions connected to the syndrome implicate defective gene regulation during fetal development and current evidence suggests that CdLS may be caused by alterations in cohesin chromatin-binding dynamics (Liu et al., 2009). In addition, cell lines established from CdLS patients have an increased sensitivity to DNA damage that has been suggested to be caused by defective HR-mediated repair (Vrouwe et al., 2007).Here, we show an increased DNA damage sensitivity, especially after exposure to γ-rays, in B-lymphoblastoid (LCLs) and fibroblast cell lines (FBs) from NIPBL-deficient CdLS patients. However, we also observed that the majority of the patient and control cells studied were in the G1 phase of the cell cycle, where NHEJ is the principle DSB repair mechanism. We therefore investigated whether defective NHEJ could underlie the DNA damage sensitivity observed in the patient cells.     

Human DNA Ligases: A Comprehensive New Look for Cancer Therapy

Living organisms belonging to all three domains of life, viz., eubacteria, archaeabacteria, and eukaryotes encode one or more DNA ligases. DNA ligases are indispensable in various DNA repair and replication processes and a deficiency or an inhibition of their activity can lead to accumulation of DNA damage and strand breaks. DNA damage, specially strand breaks at unsustainable levels can lead to replication block and/or cell death. DNA ligases as potential anticancer targets have been realized only recently. There is enough rationale to suggest that ligases have a tremendous potential for novel therapeutics including anticancer and antibacterial therapy, specially when the world is facing acute problems of drug resistance and chemotherapy failure, with an immediate need for new therapeutic targets. Here, we review the current state of the art in the development of human ligase inhibitors, their structures, molecular mechanisms, physiological effects, and their potential in future cancer therapy. Citing examples, we focus on strategies for improving the activity and specificity of existing and novel inhibitors by using structure‐based rational approaches. In the end, we describe potential new sites on the ligase I protein that can be targeted for the development of novel inhibitors. This is the first comprehensive review to compile all known human ligase inhibitors and to provide a rationale for the further development of ligase inhibitors for cancer therapy.     

The antipsychotic drug trifluoperazine inhibits DNA repair and sensitizes non small cell lung carcinoma cells to DNA double-strand break induced cell death

Trifluoperazine (TFP), a member of the phenothiazine class of antipsychotic drugs, has been shown to augment the cytotoxicity of the DNA-damaging agent bleomycin. In the present study, we investigated the effect of trifluoperazine on (a) survival of bleomycin-treated human non-small cell lung carcinoma U1810 cells, (b) induction and repair of bleomycin-induced DNA strand breaks, and (c) nonhomologous end-joining (NHEJ), the major DNA double-strand break (DSB) repair pathway in mammalian cells. By using a clonogenic survival assay, we show here that concomitant administration of trifluoperazine at a subtoxic concentration enhances the cytotoxicity of bleomycin. Moreover, trifluoperazine also increases the longevity of bleomycin-induced DNA strand breaks in U1810 cells, as shown by both comet assay and fraction of activity released (FAR)-assay. This action seems to be related to suppression of cellular DNA DSB repair activities because NHEJ-mediated rejoining of DSBs occurs with significantly lower efficiency in the presence of trifluoperazine. We propose that TFP might be capable of inhibiting one or more elements of the DNA DSB repair machinery, thereby increasing the cytotoxicity of bleomycin in lung cancer cells.     

DNA double-strand breaks: prior to but not sufficient in targeting hypermutation

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin (Ig) genes, both of which are associated with DNA double-strand breaks (DSBs). As AID is capable of deaminating deoxy-cytidine (dC) to deoxy-uracil (dU), it might induce nicks (single strand DNA breaks) and also DNA DSBs via a U-DNA glycosylase-mediated base excision repair pathway ('DNA-substrate model'). Alternatively, AID functions like its closest homologue Apobec1 as a catalytic subunit of a RNA editing holoenzyme ('RNA-substrate model'). Although rearranged Vlambda genes are preferred targets of SHM we found that germinal center (GC) B cells of AID-proficient and -deficient Vlambda1-expressing GC B cells display a similar frequency, distribution, and sequence preference of DSBs in rearranged and also in germline Vlambda1 genes. The possible roles of DSBs in relation to AID function and SHM are discussed.     

Atypical retinoids ST1926 and CD437 are S-phase-specific agents causing DNA double-strand breaks: significance for the cytotoxic and antiproliferative activity

Retinoid-related molecules (RRM) are novel agents with tumor-selective cytotoxic/antiproliferative activity, a different mechanism of action from classic retinoids and no cross-resistance with other chemotherapeutics. ST1926 and CD437 are prototypic RRMs, with the former currently undergoing phase I clinical trials. We show here that ST1926, CD437, and active congeners cause DNA damage. Cellular and subcellular COMET assays, H2AX phosphorylation (gamma-H2AX), and scoring of chromosome aberrations indicate that active RRMs produce DNA double-strand breaks (DSB) and chromosomal lesions in NB4, an acute myeloid leukemia (AML) cell line characterized by high sensitivity to RRMs. There is a direct quantitative correlation between the levels of DSBs and the cytotoxic/antiproliferative effects induced by RRMs. NB4.437r blasts, which are selectively resistant to RRMs, do not show any sign of DNA damage after treatment with ST1926, CD437, and analogues. DNA damage is the major mechanism underlying the antileukemic activity of RRMs in NB4 and other AML cell lines. In accordance with the S-phase specificity of the cytotoxic and antiproliferative responses of AML cells to RRMs, increases in DSBs are maximal during the S phase of the cell cycle. Induction of DSBs precedes inhibition of DNA replication and is associated with rapid activation of ataxia telangectasia mutated, ataxia telangectasia RAD3-related, and DNA-dependent protein kinases with subsequent stimulation of the p38 mitogen-activated protein kinase. Inhibition of ataxia telangectasia mutated and DNA-dependent protein kinases reduces phosphorylation of H2AX. Cells defective for homologous recombination are particularly sensitive to ST1926, indicating that this process is important for the protection of cells from the RRM-dependent DNA damage and cytotoxicity. [Mol Cancer Ther 2008;7(9):2941-54].     

The combination of chemotherapy with HVJ-E containing Rad51 siRNA elicited diverse anti-tumor effects and synergistically suppressed melanoma

Dacarbazine (DTIC) is one of the most popular alkylating agents used for the treatment of malignant melanoma. DTIC induces apoptosis of melanoma cells via double-strand breaks (DSBs). Melanoma cells, however, tend to increase their expression of DNA repair molecules in order to be resistant to DTIC. Here, we show that DTIC increases expression of Rad51, but not Ku70, in a cultured B16-F10 mouse melanoma cell line in dose- and time-dependent manners. On introducing Rad51 short interfering RNA (siRNA) with the hemagglutinating virus of Japan envelope (HVJ-E) to B16-F10 cells, DSBs induced by DTIC treatment were not efficiently repaired and resulted in enhanced apoptotic cell death. Colony formation of B16-F10 cells that received Rad51 siRNA was significantly decreased by DTIC treatment as compared with cells that received scramble siRNA. In melanoma-bearing mice, the combination of three intratumoral injections of HVJ-E containing Rad51 siRNA and five intraperitoneal injections of DTIC at a clinical dose synergistically suppressed the tumors. Moreover, HVJ-E demonstrated anti-tumor immunity by inducing cytotoxic T lymphocytes to B16-F10 cells on administration of DTIC. These results suggest that the combination of chemotherapy with HVJ-E containing therapeutic molecules will provide a promising therapeutic strategy for patients bearing malignant tumors resistant to chemotherapeutic agents.     

Oxidant-induced DNA damage of target cells.

In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks.     

(212)Pb-radioimmunotherapy induces G(2) cell-cycle arrest and delays DNA damage repair in tumor xenografts in a model for disseminated intraperitoneal disease

In preclinical studies, targeted radioimmunotherapy using (212)Pb-TCMC-trastuzumab as an in vivo generator of the high-energy α-particle emitting radionuclide (212)Bi is proving an efficacious modality for the treatment of disseminated peritoneal cancers. To elucidate mechanisms associated with this therapy, mice bearing human colon cancer LS-174T intraperitoneal xenografts were treated with (212)Pb-TCMC-trastuzumab and compared with the nonspecific control (212)Pb-TCMC-HuIgG, unlabeled trastuzumab, and HuIgG, as well as untreated controls. (212)Pb-TCMC-trastuzumab treatment induced significantly more apoptosis and DNA double-strand breaks (DSB) at 24 hours. Rad51 protein expression was downregulated, indicating delayed DNA double-strand damage repair compared with (212)Pb-TCMC-HuIgG, the nonspecific control. (212)Pb-TCMC-trastuzumab treatment also caused G(2)-M arrest, depression of the S phase fraction, and depressed DNA synthesis that persisted beyond 120 hours. In contrast, the effects produced by (212)Pb-TCMC-HuIgG seemed to rebound by 120 hours. In addition, (212)Pb-TCMC-trastuzumab treatment delayed open chromatin structure and expression of p21 until 72 hours, suggesting a correlation between induction of p21 protein and modification in chromatin structure of p21 in response to (212)Pb-TCMC-trastuzumab treatment. Taken together, increased DNA DSBs, impaired DNA damage repair, persistent G(2)-M arrest, and chromatin remodeling were associated with (212)Pb-TCMC-trastuzumab treatment and may explain its increased cell killing efficacy in the LS-174T intraperitoneal xenograft model for disseminated intraperitoneal disease.     

Hedamycin, a DNA alkylator, induces (gamma)H2AX and chromosome aberrations: involvement of phosphatidylinositol 3-kinase-related kinases and DNA replication fork movement

Genotoxic treatments, such as UV light, camptothecin, and adozelesin, stall DNA replication and subsequently generate DNA strand breaks. Typically, DNA breaks are reflected by an increase in ataxia and Rad-related kinase (ATR)-regulated phosphorylation of H2AX (gammaH2AX) and require replication fork movement. This study examined the potential of the monofunctional DNA alkylating agent hedamycin, a powerful inhibitor of DNA replication, to induce DNA strand breaks, phosphorylated H2AX (gammaH2AX) foci, and chromosome aberrations. Hedamycin treatment of HCT116 carcinoma cells resulted in a rapid induction of DNA strand breaks accompanied by increasing H2AX phosphorylation and focalization. Unlike many other treatments that also stall replication, such as UV, camptothecin, and adozelesin, gammaH2AX formation was not suppressed in ATR-compromised cells but actually increased. Similarly, hedamycin induction of gammaH2AX is not dependent on ataxia telangiectasia mutated or DNA-protein kinase, and pretreatment of cells with the phosphatidylinositol 3-kinase-related kinase inhibitor caffeine did not substantially reduce induction of H2AX phosphorylation by hedamycin. Furthermore, the DNA replication inhibitor aphidicolin only modestly depressed hedamycin-induced gammaH2AX formation, indicating that hedamycin-induced DNA double-strand breaks are not dependent on fork progression. In contrast, camptothecin- and adozelesin-induced gammaH2AX was strongly suppressed by aphidicolin. Moreover, after 24 hours following a short-term hedamycin treatment, cells displayed high levels of breaks in interphase nuclear DNA and misjoined chromosomes in metaphase cells. Finally, focalization of a tightly bound form of Ku80 was observed in interphase cells, consistent with the subsequent appearance of chromosomal aberrations via abnormal nonhomologous end joining. Overall, this study has revealed a disparate type of DNA damage response to stalled replication induced by a bulky DNA adduct inducer, hedamycin, that seems not to be highly dependent on ATR or DNA replication.     

Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells.

Hydrogen peroxide (H2O2)-induced DNA damage and cell death have been attributed to the direct cytotoxicity of H2O2 and other oxidant species generated from H2O2. We examined the possibility that oxidants activate endonucleases leading to DNA damage and cell death in renal tubular epithelial cells, similar to that described for apoptosis. Within minutes, H2O2 caused DNA strand breaks in a dose-dependent manner, followed by cell death. DNA fragmentation was demonstrated both by the release of [3H]thymidine in 27,000-g supernatant as well as the occurrence of low molecular weight DNA fragments on agarose gel electrophoresis, characteristic of endonuclease cleavage. Endonuclease inhibitors, aurintricarboxylic acid, Evans blue, and zinc ion prevented H2O2-induced DNA strand breaks, fragmentation, and cell death. Inhibitors of protein or mRNA synthesis had only minor protection against H2O2-induced DNA damage in contrast to complete protection reported in apoptotic thymocytes. Micrococcal endonuclease induced similar DNA strand breaks in LLC-PK1 cells, and the endonuclease inhibitors prevented the events confirming the ability of endonucleases to induce DNA damage. The protective effect of aurintricarboxylic acid was not due to the prevention of the rise in intracellular free calcium. We conclude that endonuclease activation occurs as an early event leading to DNA damage and cell death in renal tubular epithelial cells exposed to oxidant stress and, in contrast to apoptotic thymocytes, does not require macromolecular synthesis.     

ATM regulates ATR chromatin loading in response to DNA double-strand breaks

DNA double-strand breaks (DSBs) are among the most deleterious lesions that can challenge genomic integrity. Concomitant to the repair of the breaks, a rapid signaling cascade must be coordinated at the lesion site that leads to the activation of cell cycle checkpoints and/or apoptosis. In this context, ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) protein kinases are the earliest signaling molecules that are known to initiate the transduction cascade at damage sites. The current model places ATM and ATR in separate molecular routes that orchestrate distinct pathways of the checkpoint responses. Whereas ATM signals DSBs arising from ionizing radiation (IR) through a Chk2-dependent pathway, ATR is activated in a variety of replication-linked DSBs and leads to activation of the checkpoints in a Chk1 kinase-dependent manner. However, activation of the G2/M checkpoint in response to IR escapes this accepted paradigm because it is dependent on both ATM and ATR but independent of Chk2. Our data provides an explanation for this observation and places ATM activity upstream of ATR recruitment to IR-damaged chromatin. These data provide experimental evidence of an active cross talk between ATM and ATR signaling pathways in response to DNA damage.     

G1和G2期细胞对X射线引起双链DNA断裂修复的影响

目的:探讨X射线对G_1和G_2期细胞双链DNA断裂损伤修复的动力学影响。方法:采用3%多聚甲醛或70%乙醇固定受X射线放射细胞的M059J、M059K和Hela,用流式细胞仪分选G_1和G_2期Hela细胞,以脉冲场电泳分析双链DNA断裂修复动力学。结果:乙醇固定不影响X射线放射引起的双链DNA断裂修复动力学,可用于固定Hela细胞G_1和G_2期的分选,且G_2期细胞双链DNA断裂修复速率显著慢于G_1期细胞。结论:G_1和G_2期细胞中的双链DNA断裂可能采用不同的修复方式。G_1期细胞主要采用快速的末端连接修复方式,而G_2期细胞主要采用相对较慢的同源性重组修复方式。     

MRN complex function in the repair of chromosomal Rag-mediated DNA double-strand breaks

The Mre11–Rad50–Nbs1 (MRN) complex functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) at postreplicative stages of the cell cycle. During HR, the MRN complex functions directly in the repair of DNA DSBs and in the initiation of DSB responses through activation of the ataxia telangiectasia-mutated (ATM) serine-threonine kinase. Whether MRN functions in DNA damage responses before DNA replication in G0/G1 phase cells has been less clear. In developing G1-phase lymphocytes, DNA DSBs are generated by the Rag endonuclease and repaired during the assembly of antigen receptor genes by the process of V(D)J recombination. Mice and humans deficient in MRN function exhibit lymphoid phenotypes that are suggestive of defects in V(D)J recombination. We show that during V(D)J recombination, MRN deficiency leads to the aberrant joining of Rag DSBs and to the accumulation of unrepaired coding ends, thus establishing a functional role for MRN in the repair of Rag-mediated DNA DSBs. Moreover, these defects in V(D)J recombination are remarkably similar to those observed in ATM-deficient lymphocytes, suggesting that ATM and MRN function in the same DNA DSB response pathways during lymphocyte antigen receptor gene assembly.DNA double-strand breaks (DSBs) are generated by genotoxic agents and as intermediates of several important physiological processes including antigen receptor gene assembly in developing lymphocytes. The cellular response to DNA DSBs relies on the sensing of these breaks and the subsequent initiation of effector pathways that enforce cell cycle checkpoints, promote DSB repair, and mediate cell death when DSBs are not efficiently repaired (1–4). DNA DSBs generated at stages of the cell cycle after DNA replication are repaired primarily by homologous recombination (HR), a process that uses the sister chromatid as a template for precise repair (4). DNA DSBs generated before DNA replication in G0/G1-phase cells are repaired primarily through nonhomologous end joining (NHEJ), which religates broken DNA ends in a manner that can be imprecise (4). Very little overlap exists in the protein machinery that carries out DSB repair by NHEJ and HR.The Mre11, Rad50, and Nbs1 proteins form the Mre11–Rad50–Nbs1 (MRN) complex, which is thought to function primarily in DNA DSB responses in cells that have undergone DNA replication (5, 6). MRN is recruited to the sites of newly generated DNA DSBs where it functions to recruit and activate the ataxia telangiectasia-mutated (ATM) serine/threonine kinase, which is a major initiator of DNA damage responses (7–12). However, ATM may be activated in an MRN-independent fashion in response to some DSBs like those generated at stalled replication forks, suggesting that the requirement for MRN in initiating DSB responses may be context dependent (9). MRN also has several DNA damage response functions that are downstream of ATM activation. In this regard, analyses of Nbs1 mutants have implicated MRN in the regulation of cell cycle checkpoints and the activation of apoptotic pathways in response to DNA DSBs (13, 14). Mre11 has endonuclease and exonuclease activities that are important for DNA end processing, and Mre11 dimers may align and bridge two DNA ends during HR-mediated DSB repair (5, 6, 15). Rad50 also has DNA binding activities that may be involved in tethering sister chromatids during HR (16–20). In response to DSBs, ATM phosphorylates Mre11, Rad50, and Nbs1, which could potentially modulate their functions in DSB responses (21–24). Importantly, although Mre11, Nbs1, and Rad50 have distinct functions, these individual components are thought to function only in the context of the MRN holocomplex.Whether the MRN complex functions in the response to and NHEJ-mediated repair of DSBs in G0/G1-phase cells has been much less clear. DNA end joining by NHEJ components purified from human cellular extracts was augmented by MRN in vitro; however, the repair activity of purified Xenopus laevis NHEJ components was not affected by the addition of MRN (25, 26). MRX, the yeast orthologue of MRN, functions during NHEJ in the budding yeast Saccharomyces cerevisiae but not in the fission yeast Schizosaccharomyces pombe (27–29). In mammalian cells, MRN is not recruited to the site of DSBs generated by the I-sceI endonuclease in G1-phase cells, and MRN does not appear to be required for the NHEJ-mediated repair of these DSBs (30, 31).DNA DSBs are generated in all developing lymphocytes during the assembly of the second exon of antigen receptor genes from component V, J, and, in some cases, D gene segments (32). This occurs through the process of V(D)J recombination, which is initiated by the Rag-1 and Rag-2 proteins, which together form an endonuclease, hereafter referred to as Rag (33, 34). Rag introduces DSBs at the borders of two recombining gene segments and their associated Rag recognition sequences, which are termed recombination signals (RSs). The generation of these DSBs is restricted to cells at the G1 phase of the cell cycle as a result of the rapid degradation of Rag-2 upon entry into S phase (35). DNA cleavage by Rag leads to the formation of two hairpin-sealed coding ends and two blunt phosphorylated signal ends. These DNA ends are processed and joined by the NHEJ pathway of DNA DSB repair into a coding joint and signal joint, respectively (36, 37). The critical dependence of V(D)J recombination on NHEJ is indicated by the severe joining defects in NHEJ-deficient cell lines and the profound immunodeficiency observed in mice deficient for NHEJ factors required for the repair of Rag-mediated DNA DSBs (36).Mice with homozygous-null mutations in the Mre11, Nbs1, or Rad50 genes exhibit early embryonic lethality; however, mice and humans with hypomorphic Mre11 or Nbs1 mutations are viable and exhibit mild immunodeficiency, suggesting that the MRN complex could function in the response to or repair of Rag-mediated DSBs generated during V(D)J recombination (9, 38–45). In developing lymphocytes, Rag DSBs activate ATM, which initiates a broad genetic program and functions in the repair of these breaks (46–53). In this regard, MRN could function in the activation of ATM in response to Rag-mediated DSBs and also downstream of ATM in the repair of these DSBs. Consistent with this notion, Nbs1 associates with Rag-mediated DSBs generated at T cell receptor loci in thymocytes and MRX function is required for the joining of signal ends generated by Rag cleavage in yeast (54, 55). However, analyses of V(D)J recombination of extrachromosomal substrates in mammalian nonlymphoid cells deficient in MRN have failed to reveal any significant defects in coding or signal joint formation (56–58). In this paper, we show that MRN-deficient lymphocytes exhibit defects in V(D)J recombination at endogenous antigen receptor loci and chromosomally introduced recombination substrates. These findings establish a function for MRN in the response to Rag-mediated DSBs generated in G1-phase lymphocytes.     

Oxidative stress-induced homologous recombination as a novel mechanism for phenytoin-initiated toxicity

Although the mechanism(s) of phenytoin-initiated toxicity is unknown, phenytoin can be enzymatically bioactivated to a reactive intermediate leading to increased formation of reactive oxygen species, which can damage essential macromolecules, including DNA. The oxidation of DNA can induce DNA double-strand breaks (DSBs), which may be repaired through homologous recombination. Increased levels of DSBs may induce hyper-recombination, leading to deleterious genetic changes. We hypothesize that these genetic changes mediate phenytoin-initiated toxicity. To investigate this hypothesis we used a Chinese hamster ovary cell line containing a neo direct repeat recombination substrate to determine whether phenytoin-initiated DNA oxidation increases homologous recombination. Cells were treated with 0 to 800 microM phenytoin for 5 or 24 h, and homologous recombination frequencies and recombinant product structures were determined. Phenytoin-initiated DNA oxidation was determined by measuring the formation of 8-hydroxy-2'-deoxyguanosine. We demonstrate that phenytoin increases both DNA oxidation and homologous recombination in a concentration- and time-dependent manner. All recombination products analyzed arose via gene conversion without associated crossover. Our data demonstrate that phenytoin-initiated DNA damage can induce homologous recombination, which may be a novel mechanism mediating phenytoin-initiated toxicity.     

A Mechanistic Approach for Modulation of Arsenic Toxicity in Human Lymphocytes by Curcumin, an Active Constituent of Medicinal Herb Curcuma longa Linn

Chronic exposure of humans to high concentrations of arsenic in drinking water is associated with skin lesions, peripheral vascular disease, hypertension, blackfoot disease and a high risk of cancer. Arsenic induces single strand breaks, DNA-protein crosslinks and apurinic sites in DNA, which are prerequisites for induction of cancer. Amelioration of such damages with natural compounds could be an effective strategy to combat arsenic toxicity. Curcumin is the active ingredient of turmeric, a common household spice, which is a rich source of polyphenols and this compound has been extensively studied as a chemopreventive agent against many types of cancer. The present study investigates whether curcumin could counteract the DNA damage caused by arsenic as assessed by single cell gel electrophoresis (SCGE) using peripheral blood lymphocytes, from healthy donors. It was observed that DNA damage induced by arsenic could be efficiently reduced by curcumin and the effect was more pronounced when lymphocytes were pre-incubated with curcumin prior to arsenic insult. Arsenic caused DNA damage by generation of reactive oxygen species (ROS) and enhancement of lipid peroxidation levels. Curcumin counteracted the damage by quenching ROS, decreasing the level of lipid peroxidation and increasing the level of phase II detoxification enzymes like catalase, superoxide dismutase and glutathione peroxidase. Curcumin also enhanced the DNA repair activity against arsenic induced damage. The expression of polymerase, a repair enzyme, was found to be highly elevated when arsenite induced damaged cells were allowed to repair in presence of curcumin. Results indicate that curcumin has significant role in confronting the deleterious effect caused by arsenic, which could be an economic mode of arsenic mitigation among rural population in West Bengal, India.     

Apoptotic and genotoxic effects of low-intensity ultrasound on healthy and leukemic human peripheral mononuclear blood cells

Purpose

To scrutinize the apoptotic and genotoxic effects of low-intensity ultrasound and an ultrasound contrast agent (SonoVue; Bracco Diagnostics Inc., EU) on human peripheral mononuclear blood cells (PMBCs).

Methods

PMBCs were subjected to a low-intensity ultrasound field (1-MHz frequency; spatial peak temporal average intensity 0.18 W/cm2) followed by analysis for apoptosis and DNA damage (single-strand breaks + double-strand breaks). The comet assay was then repeated after 2 h to examine the ability of cells to repair DNA breaks.

Results

The results demonstrated that low-intensity ultrasound was capable of selectively inducing apoptosis in leukemic PMBCs, but not in healthy cells. The introduction of ultrasound contrast agent SonoVue resulted in an increase in apoptosis in both groups. DNA analysis after ultrasound exposure indicated that ultrasound triggered DNA damage in leukemic PMBCs (66.05 ± 13.36%), while the damage was minimal (7.01 ± 0.89%) in control PMBCs. However, both cell lines demonstrated an ability to repair DNA single- and double-strand breaks 2 h after sonication.

Conclusions

The study demonstrated that low-intensity ultrasound selectively induced apoptosis in cancer PMBCs. Ultrasound-induced DNA damage was observed primarily in leukemic PMBCs. Nevertheless, both cell lines were able to repair ultrasound-mediated DNA strand breaks.