Initial DNA Lesions Research Articles

Quantification of Intracellular DNA-Protein Cross-Links with N7-Methyl-2'-Deoxyguanosine and Their Contribution to Cytotoxicity.

The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion in vivo, but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPCMdG), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPCMdG in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPCMdG is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPCMdG contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.

Intensity Modulated Radiation Fields Induce Protective Effects and Reduce Importance of Dose-Rate Effects

In advanced radiotherapy, intensity modulated radiation fields and complex dose-delivery are utilized to prescribe higher doses to tumours. Here, we investigated the impact of modulated radiation fields on radio-sensitivity and cell recovery during dose delivery. We generated experimental survival data after single-dose, split-dose and fractionated irradiation in normal human skin fibroblast cells (AGO1522) and human prostate cancer cells (DU145). The dose was delivered to either 50% of the area of a T25 flask containing the cells (half-field) or 100% of the flask (uniform-field). We also modelled the impact of dose-rate effects and intercellular signalling on cell-killing. Applying the model to the survival data, it is found that (i) in-field cell survival under half-field exposure is higher than uniform-field exposure for the same delivered dose; (ii) the importance of sub-lethal damage repair (SLDR) in AGO1522 cells is reduced under half-field exposure; (iii) the yield of initial DNA lesions measured with half-field exposure is smaller than that with uniform-field exposure. These results suggest that increased cell survival under half-field exposure is predominantly attributed not to rescue effects (increased SLDR) but protective effects (reduced induction of initial DNA lesions). In support of these protective effects, the reduced DNA damage leads to modulation of cell-cycle dynamics, i.e., less G1 arrest 6 h after irradiation. These findings provide a new understanding of the impact of dose-rate effects and protective effects measured after modulated field irradiation.

Reprint of "Oxidant and environmental toxicant-induced effects compromise DNA ligation during base excision DNA repair".

DNA lesions arise from many endogenous and environmental agents, and such lesions can promote deleterious events leading to genomic instability and cell death. Base excision repair (BER) is the main DNA repair pathway responsible for repairing single strand breaks, base lesions and abasic sites in mammalian cells. During BER, DNA substrates and repair intermediates are channeled from one step to the next in a sequential fashion so that release of toxic repair intermediates is minimized. This includes handoff of the product of gap-filling DNA synthesis to the DNA ligation step. The conformational differences in DNA polymerase β (pol β) associated with incorrect or oxidized nucleotide (8-oxodGMP) insertion could impact channeling of the repair intermediate to the final step of BER, i.e., DNA ligation by DNA ligase I or the DNA Ligase III/XRCC1 complex. Thus, modified DNA ligase substrates produced by faulty pol β gap-filling could impair coordination between pol β and DNA ligase. Ligation failure is associated with 5'-AMP addition to the repair intermediate and accumulation of strand breaks that could be more toxic than the initial DNA lesions. Here, we provide an overview of the consequences of ligation failure in the last step of BER. We also discuss DNA-end processing mechanisms that could play roles in reversal of impaired BER.

Theoretical considerations for thresholds in chemical carcinogenesis.

There is increasing evidence for non-linear relationships for gene mutations, chromosomal aberrations and even tumor incidences in response to low doses of genotoxic carcinogens. To attain the biological relevance of such non-linear responses, there is a need to identify the underlying defense mechanisms that allow tolerance to low doses of genotoxicants. This communication discusses presumptive cancer prevention mechanisms that may contribute to thresholds, i.e. points of departure, for each endpoint, from initial DNA lesion to tumor formation. We discuss a sequential order of genome protection during carcinogenesis where genotoxicant scavenging, cellular efflux, DNA repair, elimination of damaged cells by apoptosis, autophagy, silencing by DNA damage-triggered replicative senescence, and finally, elimination of transformed (premalignant) cells by the immune system are thought to be responsible for a threshold in tumor formation. We highlight DNA repair, for which experimental evidence has been recently provided to dictate a role in PoDs. In conclusion, from a theoretical perspective it is reasonable to posit that tolerance to low dose levels exists for each requisite step of tumor formation and these tolerance mechanisms are critical in determining thresholds in chemical carcinogenesis.

Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

Glutathione Depletion and Carbon Ion Radiation Potentiate Clustered DNA Lesions, Cell Death and Prevent Chromosomal Changes in Cancer Cells Progeny

Poor local control and tumor escape are of major concern in head-and-neck cancers treated by conventional radiotherapy or hadrontherapy. Reduced glutathione (GSH) is suspected of playing an important role in mechanisms leading to radioresistance, and its depletion should enable oxidative stress insult, thereby modifying the nature of DNA lesions and the subsequent chromosomal changes that potentially lead to tumor escape.This study aimed to highlight the impact of a GSH-depletion strategy (dimethylfumarate, and l-buthionine sulfoximine association) combined with carbon ion or X-ray irradiation on types of DNA lesions (sparse or clustered) and the subsequent transmission of chromosomal changes to the progeny in a radioresistant cell line (SQ20B) expressing a high endogenous GSH content. Results are compared with those of a radiosensitive cell line (SCC61) displaying a low endogenous GSH level.DNA damage measurements (γH2AX/comet assay) demonstrated that a transient GSH depletion in resistant SQ20B cells potentiated the effects of irradiation by initially increasing sparse DNA breaks and oxidative lesions after X-ray irradiation, while carbon ion irradiation enhanced the complexity of clustered oxidative damage. Moreover, residual DNA double-strand breaks were measured whatever the radiation qualities. The nature of the initial DNA lesions and amount of residual DNA damage were similar to those observed in sensitive SCC61 cells after both types of irradiation. Misrepaired or unrepaired lesions may lead to chromosomal changes, estimated in cell progeny by the cytome assay. Both types of irradiation induced aberrations in nondepleted resistant SQ20B and sensitive SCC61 cells. The GSH-depletion strategy prevented the transmission of aberrations (complex rearrangements and chromosome break or loss) in radioresistant SQ20B only when associated with carbon ion irradiation. A GSH-depleting strategy combined with hadrontherapy may thus have considerable advantage in the care of patients, by minimizing genomic instability and improving the local control.

Overview of Base Excision Repair Biochemistry

Base excision repair (BER) is an evolutionarily conserved pathway, which could be considered the “workhorse” repair mechanism of the cell. In particular, BER corrects most forms of spontaneous hydrolytic decay products in DNA, as well as everyday oxidative and alkylative modifications to bases or the sugar phosphate backbone. The repair response involves five key enzymatic steps that aim to remove the initial DNA lesion and restore the genetic material back to its original state: (i) excision of a damaged or inappropriate base, (ii) incision of the phosphodiester backbone at the resulting abasic site, (iii) termini clean-up to permit unabated repair synthesis and/or nick ligation, (iv) gap-filling to replace the excised nucleotide, and (v) sealing of the final, remaining DNA nick. These repair steps are executed by a collection of enzymes that include DNA glycosylases, apurinic/apyrimidinic endonucleases, phosphatases, phosphodiesterases, kinases, polymerases and ligases. Defects in BER components lead to reduced cell survival, elevated mutation rates, and DNA-damaging agent hypersensitivities. In addition, the pathway plays a significant role in determining cellular responsiveness to relevant clinical anti-cancer agents, such as alkylators (e.g. temozolomide), nucleoside analogs (e.g. 5-fluorouracil), and ionizing radiation. The molecular details of BER and the contribution of the pathway to therapeutic agent resistance are reviewed herein.

Overview of Base Excision Repair Biochemistry

Base excision repair (BER) is an evolutionarily conserved pathway, which could be considered the "workhorse" repair mechanism of the cell. In particular, BER corrects most forms of spontaneous hydrolytic decay products in DNA, as well as everyday oxidative and alkylative modifications to bases or the sugar phosphate backbone. The repair response involves five key enzymatic steps that aim to remove the initial DNA lesion and restore the genetic material back to its original state: (i) excision of a damaged or inappropriate base, (ii) incision of the phosphodiester backbone at the resulting abasic site, (iii) termini clean-up to permit unabated repair synthesis and/or nick ligation, (iv) gap-filling to replace the excised nucleotide, and (v) sealing of the final, remaining DNA nick. These repair steps are executed by a collection of enzymes that include DNA glycosylases, apurinic/apyrimidinic endonucleases, phosphatases, phosphodiesterases, kinases, polymerases and ligases. Defects in BER components lead to reduced cell survival, elevated mutation rates, and DNA-damaging agent hypersensitivities. In addition, the pathway plays a significant role in determining cellular responsiveness to relevant clinical anti-cancer agents, such as alkylators (e.g. temozolomide), nucleoside analogs (e.g. 5-fluorouracil), and ionizing radiation. The molecular details of BER and the contribution of the pathway to therapeutic agent resistance are reviewed herein.

Mechanistic simulation of radiation damage to DNA and its repair: on the track towards systems radiation biology modelling

The biophysical simulation code PARTRAC enables, by combining track structure calculations with DNA models on diverse genomic scales, prediction of DNA damage yields and patterns for various radiation qualities. To extend its applicability to later endpoints such as mutagenesis or cell killing, a continuative model for repair of radiation-induced double-strand break (DSB) via non-homologous end-joining has complemented the PARTRAC code by about 12 orders of magnitude on a temporal scale. The repair model describes step-by-step by the Monte Carlo method the attachment and dissociation of involved repair enzymes and diffusion motion of DNA ends. The complexity of initial DNA lesion patterns influences the repair kinetics and outcome via additional cleaning steps required for dirty DNA ends. Model parameters have been taken from measured attachment kinetics of repair enzymes and adaptation to DSB rejoining kinetics after gamma irradiation. Application of the DNA repair model to damage patterns following nitrogen ion irradiation and comparison with experimental results reveal the need for further model refinements. Nevertheless, already the present model represents a promising step towards systems modelling of cellular response to radiation.

Evaluation of curcumin and cisplatin-induced DNA damage in PC12 cells by the alkaline comet assay

A very appropriate method for antigenotoxicity evaluation of antioxidants is the comet assay, since this analytical method detects initial DNA lesions that are still subject to repair; in other words, lesions that are very associated to damages resulting from the generation and subsequent action of reactive species. However, a solid evaluation should be developed in order to avoid inexact interpretations. In our study, besides the association of curcumin with cisplatin, curcumin and cisplatin agents were also tested separately. Classical genotoxic compounds, when tested by the comet assay, present an increase in the nucleoid tail; however, the cisplatin treatment has resulted in a decrease of DNA migration. This was an expected effect, as the cross-links between cisplatin and DNA decrease the DNA electrophoretic mobility. A similar effect was observed with the curcumin treatment, which decreased the nucleoid tail. Such effect was not expected and reinforced the necessity of including in the study, separate treatment groups with potentially antigenotoxic substances. The comet assay results have been analyzed using specific software for image analysis, as well as the classical visual analysis, and we have observed that the effect of decrease in DNA electrophoretic mobility was more easily observed when the data were analyzed by the software.

Role of O6-alkylguanine-DNA alkyltransferase in the cytotoxic activity of cloretazine

Cloretazine (VNP40101M; 101M; 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine) is a sulfonylhydrazine prodrug that generates both chloroethylating and carbamoylating species on activation. To explore the molecular mechanisms underlying the broad anticancer activity observed in preclinical studies, cloretazine and chloroethylating-only [i.e., 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine] and carbamoylating-only (i.e., 1,2-bis(methylsulfonyl)-1-[(methylamino)carbonyl]hydrazine) analogues were evaluated in five murine hematopoietic cell lines. These cell lines were separable into two groups by virtue of their sensitivity to 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine; the sensitive group included L1210, P388, and F-MEL leukemias (IC50s, 6-8 micromol/L) and the resistant group consisted of Ba/F3 bone marrow and WEHI-3B leukemia cells (IC50s, 50-70 micromol/L). Resistant cells expressed O6-alkylguanine-DNA alkyltransferase (AGT), whereas sensitive cells did not. A correlation existed between AGT expression and the functional status of p53; AGT- cells possessed defective p53, whereas AGT+ cells contained wild-type p53. Based on recent findings on regulation of AGT gene expression by others, we suspect that silencing of the AGT gene by promoter hypermethylation frequently occurs during tumor progression involving p53 inactivation. O6-Chloroethylguanine is the initial DNA lesion that progresses to lethal interstrand DNA cross-links. Cloretazine exhibited a much higher preference toward the O6-chloroethylation of guanine, as measured by the difference in IC50s to wild-type and AGT-transfected L1210 cells, than 1,3-bis(2-chloroethyl)-1-nitrosourea, which targets the same site in DNA. Preferential toxicity of cloretazine against AGT- tumor cells coupled with decreased toxicity to AGT+ cells in host tissues constitute the therapeutic basis for cloretazine.

A model for initial DNA lesion recognition by NER and MMR based on local conformational flexibility

Initial recognition of DNA damage is the crucial but poorly understood first step in DNA repair by the human nucleotide excision repair (NER) and mismatch repair (MMR) systems. Failure by NER or MMR to recognize DNA damage threatens the genetic integrity of the organism and may play a role in carcinogenesis. Both NER and MMR recognize and repair a wide variety of structurally dissimilar lesions against the background of normal DNA. Previous studies have suggested that detection of thermodynamic destabilization of DNA caused by covalent damage and base mismatches is a potential mechanism by which repair pathways with broad specificity such as NER and MMR recognize their substrates. However, both NER and MMR respectively, repair a wide variety of stabilizing and destabilizing covalent DNA lesions and base pair mismatches. A common feature of lesions that are both thermodynamically stabilizing and destabilizing is the alteration of the local DNA flexibility (dynamics). In this review we describe the experimental evidence for altered dynamics from NMR and thermodynamic studies on normal and damaged DNA molecules with respect to recognition by NER and MMR. Based on these data, we propose a model for initial detection of lesions by both NER and MMR that occurs through an indirect readout mechanism of alternative DNA conformations induced by covalent damage and base mismatches.

The activation-induced deaminase functions in a postcleavage step of the somatic hypermutation process.

Activation of B cells by antigen fuels two distinct molecular modifications of immunoglobulin (Ig) genes. Class-switch recombination (CSR) replaces the Igμ heavy chain constant region with a downstream constant region gene, thereby altering the effector function of the resulting antibodies. Somatic hypermutation (SHM) introduces point mutations into the variable regions of Ig genes, thereby changing the affinity of antibody for antigen. Mechanistic overlap between the two reactions has been suggested by the finding that both require the activation-induced cytidine deaminase (AID). It has been proposed that AID initiates both CSR and SHM by activating a common nuclease. Here we provide evidence that cells lacking AID, or expressing a dominant negative form of the protein, are still able to incur DNA lesions in SHM target sequences. The results indicate that an intact cytidine deaminase motif is required for AID function, and that AID acts downstream of the initial DNA lesions in SHM.

Mutagenic Effects of Heavy Charged Particles

Heavy charged particles constitute the most important radiation risk in space. Since mutations can give rise to cancer the investigation of their induction may be helpful for risk quantification. This paper describes experiments in a number of mammalian cells performed with a large range of linear energy transfer (LET). RBE values are always found to increase with LET up to about 100 keV/microm and are similar to those suggested by ICRP. Molecular analyses demonstrate that heavy charged particles not only lead to sometimes very large deletions but also to smaller changes which might represent point mutations. Recent evidence shows that repair phenomena play an important role in the processing of initial DNA lesions to mutagenic alterations.

A comparison of DNA repair using the comet assay in tobacco seedlings after exposure to alkylating agents or ionizing radiation

We employed single cell gel electrophoresis to analyze the kinetics of DNA repair in nuclei isolated from tobacco plants exposed to ethyl methanesulfonate (EMS), N-ethyl- N-nitrosourea (ENU) and γ-radiation. DNA repair was measured as the reduction of the tail moment values as a function of time after the mutagen treatment ended. DNA damage in leaf nuclei of EMS-or ENU-treated tobacco plants persisted over a 72 h recovery period. However, a reduction of the SCGE tail moment values in nuclei isolated from leaves was observed over a 4-week period of recovery. Newly emerged leaves expressed a lower level of DNA damage due to more efficient repair and/or dilution of initial DNA lesions during cell division. After 24 h recovery, leaf nuclei from cells exposed to 20 or 40 Gy of γ-radiation expressed complete DNA repair. These data indicate that DNA lesions induced by alkylating agents are not readily repaired and persist beyond 4 weeks. Enzymes necessary to repair γ-induced DNA lesions are fully functional in non-replicating leaf cells and single and double strand breaks are rapidly repaired.

Mechanisms of the origin of chromosomal aberrations

This manuscript gives a selective survey of some aspects of the origin of chromosomal aberrations (CA). The following topics are discussed: heterogeneity regarding the induction of initial DNA lesions and their repair among chromosomes or regions of the same chromosome, ratio between symmetrical and asymmetrical exchanges, influence of DNA repeats, chromatin condensation and cell-cycle checkpoints on the formation of chromosome alterations.

Radiation Mutagenesis: The Initial DNA Lesions Responsible

This paper is an attempt to initiate a discussion about the causes of radiation-induced mutagenesis. It considers the evidence for the involvement of singly base-damaged sites as sources of this mutagenesis. Several sets of published data are examined: These include endogenous levels of oxidized bases and their production by natural oxidative processes, and yields of mutants produced by hydrogen peroxide and by high-LET radiation. These considerations lead to the conclusion that, at the levels at which they are produced by ionizing radiation, DNA base-damaged sites are not a cause of radiation-induced mutagenesis. In analogy with previous work we invoke the involvement of multiply damaged sites (such as double-strand breaks) produced by radiation as the causative lesions.

Increased dose-rate effect in V79-multicellular aggregates (spheroids). Relation to initial DNA lesions and repair

Survival of exponentially growing V79 monolayer cells was measured after irradiation at low dose-rate (up to 1.1 Gy/h) and at high dose-rate (2.5 Gy/min) and compared to corresponding survival data of V79 spheroid cells. The so-called contact effect, the increased radioresistance of cells irradiated in spheroids, was expressed to a greater extent with low dose-rate exposure. Lesion fixation by hypertonic treatment was more pronounced in spheroid versus monolayer cells and abolished the contact effect. The reduced radiosensitivity of V79 spheroid cells could not be related to a reduced number of initial DNA lesions or a higher capacity to rejoin DNA breaks (measured by neutral elution). These findings suggest that the ratio of lesion repair to fixation/misrepair may differ between cells from spheroids and monolayer culture thus influencing the response of cells to dose-rate changes differentially.

Recombinagenicity of caffeine for Candida albicans

Caffeine at concentrations of 0.5 x 10(-2) M or higher inhibited cell replication and induced gene segregations in Candida albicans cultured on defined complete medium. Both responses increased incrementally with increasing caffeine concentrations, and were more severe during incubation at 37 degrees C than 25 degrees C; at 37 degrees C, caffeine levels above 1.5 x 10(-2) M caused cellular inactivation. Caffeine effects occurred only under conditions permitting cell growth, and their magnitudes were greater for unbudded than budding cells, were influenced by cellular genetic backgrounds, and were unaffected by the presence of adenine in the medium. Evaluations of segregations for recessive auxotrophic markers of a four member linkage group carried heterozygously in a cis arrangement in treated cells established that induced segregants arise through either reciprocal or nonreciprocal recombinations. The frequency distributions of classes of reciprocal and nonreciprocal recombinants for these markers conformed with those previously obtained following induction by ultraviolet radiation, indicating that the probabilities of recombinational events within the chromosomal regions defined by the markers are not biased by the differences in kinds of initial DNA lesions caused by the two recombinagens. A panel of four protoplast fusion hybrids considered deficient for DNA repair because of enhanced susceptibilities to UV induced cellular inactivation and mitotic recombination exhibited corresponding increased sensitivities to caffeine, signifying that DNA damage induced by caffeine is subject to repair. Caffeine did not affect behavior of a variant strain exhibiting high frequency phenotypic switching between minute smooth and large rough colonial forms, and no evidence for mutagenicity of the drug was obtained with systems for detection of forward or reverse mutations. The mechanism of caffeine's recombinagenicity, and the implications of that property for genetic studies of C. albicans are discussed.

Prophage induction and mutagenecity of a series of anti-tumour platinum(II) and platinum(IV) co-ordination complexes

11 platinum compounds with nitrogen donor ligands, previously tested for anti-tumour activity, were studied for induction of prophage lambfa and for mutagenicity in the Ames assay, with various strains of Salmonella. The compounds included cis and trans isomers of Pt(II) and Pt(IV) complexes and were tested with and without metabolic activation. All the cis compounds elicited prophage induction, whereas the trans compounds were inactive. Mutagenicity was found only in strains containing the R factor, indicating that SOS-type repair processes are required for the conversion of initial DNA lesions into mutations. Mutation induction was also influenced by the excision-repair process. The 2 trans compounds were not, or only slightly, mutagenic; all other compounds were mutagenic in at least one strain, exhibiting a 2–20-fold increase over the spontaneous background level. Addition of liver homogenate had no significant effect on the number of mutants. One compound induced exclusively frameshift mutations. The other mutagenic compounds induced frameshift mutations as well as base-pair substitutions. 7 compounds were more mutagenic for the repair-proficient than for the repair-deficient strains; only one showed the opposite effect. This suggests that for mutagenicity testing of platinum compounds, repair-proficient strains are more sensitive indicators. The differences in response of the various strains toward the compounds suggest the formation of different DNA lesions and/or a selective action of repair processes on these lesions. In general, a good qualitative correlation was observed between prophage-inducing capacity, mutagenicity in bacterial and mammalian cells and anti-tumour activity.