Unrepaired Lesions Research Articles

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.

Monitoring bypass of single replication-blocking lesions by damage avoidance in the Escherichia coli chromosome

Although most deoxyribonucleic acid (DNA) lesions are accurately repaired before replication, replication across unrepaired lesions is the main source of point mutations. The lesion tolerance processes, which allow damaged DNA to be replicated, entail two branches, error-prone translesion synthesis (TLS) and error-free damage avoidance (DA). While TLS pathways are reasonably well established, DA pathways are poorly understood. The fate of a replication-blocking lesion is generally explored by means of plasmid-based assays. Although such assays represent efficient tools to analyse TLS, we show here that plasmid-borne lesions are inappropriate models to study DA pathways due to extensive replication fork uncoupling. This observation prompted us to develop a method to graft, site-specifically, a single lesion in the genome of a living cell. With this novel assay, we show that in Escherichia coli DA events massively outweigh TLS events and that in contrast to plasmid, chromosome-borne lesions partially require RecA for tolerance.

A UVR-Induced G2-Phase Checkpoint Response to ssDNA Gaps Produced by Replication Fork Bypass of Unrepaired Lesions Is Defective in Melanoma

UVR is a major environmental risk factor for the development of melanoma. Here we describe a coupled DNA-damage tolerance (DDT) mechanism and G2-phase cell cycle checkpoint induced in response to suberythemal doses of UVR that is commonly defective in melanomas. This coupled response is triggered by a small number of UVR-induced DNA lesions incurred during G1 phase that are not repaired by nucleotide excision repair (NER). These lesions are detected during S phase, but rather than stalling replication, they trigger the DDT-dependent formation of single-stranded DNA (ssDNA) gaps. The ssDNA attracts replication protein A (RPA), which initiates ATR-Chk1 (ataxia telangiectasia and Rad3-related/checkpoint kinase 1) G2-phase checkpoint signaling, and colocalizes with components of the RAD18 and RAD51 postreplication repair pathways. We demonstrate that depletion of RAD18 delays both the resolution of RPA foci and exit from the G2-phase arrest, indicating the involvement of RAD18-dependent postreplication repair in ssDNA gap repair during G2 phase. Moreover, the presence of RAD51 and BRCA1 suggests that an error-free mechanism may also contribute to repair. Loss of the UVR-induced G2-phase checkpoint results in increased UVR signature mutations after exposure to suberythemal UVR. We propose that defects in the UVR-induced G2-phase checkpoint and repair mechanism are likely to contribute to melanoma development.

Abstract 4197: A DNA damage checkpoint response to unrepaired ultraviolet radiation-induced lesions which is defective in melanoma

Abstract Ultraviolet radiation (UVR) is a major environmental risk factor in the development of skin cancer. Here we describe a G2 phase cell cycle checkpoint response to suberythemal doses of UVR that is defective in a majority of melanoma cell lines tested. The majority of cells in the basal layer of the epidermis are quiescent, and exposure to suberythemal doses of UVR produces not only DNA damage but also provides a mitogenic stimulus to drive cells into the cell cycle. The majority of UVR-induced DNA lesions are rapidly repaired by NER mechanisms, but a small number of DNA lesions fail to be repaired during G1 phase and are encountered by the replication fork during S phase. This triggers Werner helicase (Wrn) dependent lesion bypass, allowing replication to continue relatively unhindered, but producing gaps in the newly replicated DNA strand opposite the lesions. These single stranded DNA gaps recruit replication protein A (RPA) and components of the ATR-Chk1 checkpoint signaling responsible for the G2 phase arrest observed. The RPA foci also co-localise components of the error free RAD18-RAD51 post replication repair pathway which are essential for repair of the gaps and checkpoint signaling. This checkpoint mechanism ensures detection and repair of all UVR induced DNA damage before proceeding into mitosis. Loss of this checkpoint response increases the rate of UVR induced mutation. Thus this checkpoint response is critical for maintaining genomic stability in the epidermal melanocytes and keratinocytes, and loss of this checkpoint mechanism may account for the increased UVR signature DNA mutations found in melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4197. doi:10.1158/1538-7445.AM2011-4197

Human Telomeres Are Hypersensitive to UV-Induced DNA Damage and Refractory to Repair

Telomeric repeats preserve genome integrity by stabilizing chromosomes, a function that appears to be important for both cancer and aging. In view of this critical role in genomic integrity, the telomere's own integrity should be of paramount importance to the cell. Ultraviolet light (UV), the preeminent risk factor in skin cancer development, induces mainly cyclobutane pyrimidine dimers (CPD) which are both mutagenic and lethal. The human telomeric repeat unit (5′TTAGGG/CCCTAA3′) is nearly optimal for acquiring UV-induced CPD, which form at dipyrimidine sites. We developed a ChIP–based technique, immunoprecipitation of DNA damage (IPoD), to simultaneously study DNA damage and repair in the telomere and in the coding regions of p53, 28S rDNA, and mitochondrial DNA. We find that human telomeres in vivo are 7-fold hypersensitive to UV-induced DNA damage. In double-stranded oligonucleotides, this hypersensitivity is a property of both telomeric and non-telomeric repeats; in a series of telomeric repeat oligonucleotides, a phase change conferring UV-sensitivity occurs above 4 repeats. Furthermore, CPD removal in the telomere is almost absent, matching the rate in mitochondria known to lack nucleotide excision repair. Cells containing persistent high levels of telomeric CPDs nevertheless proliferate, and chronic UV irradiation of cells does not accelerate telomere shortening. Telomeres are therefore unique in at least three respects: their biophysical UV sensitivity, their prevention of excision repair, and their tolerance of unrepaired lesions. Utilizing a lesion-tolerance strategy rather than repair would prevent double-strand breaks at closely-opposed excision repair sites on opposite strands of a damage-hypersensitive repeat.

Abstract 1966: In vivo detection of nucleotide lesions preceding mutation fixation

Abstract Background and Purpose: Unrepaired DNA damage likely originates a large fraction of human cancer. However, a direct correlation between DNA damage and specific mutations is lacking. To identify in vivo nucleotide lesions that can act as the precursors of neoplastic mutations, we investigated the origin of specific tumor signature mutations previously reported on codon 122 of the p53 tumor suppressor gene (Trp53) in UVB radiation induced mouse skin tumors. Methods: The dorsal skin of wild-type (WT) and repair defective (Xpc−/− Trp53+/−) mice was shaved and exposed to a single dose of 4.8 kJ/m2 UVB radiation. Skin from unirradiated and irradiated animals was excised surgically at specific time points (0, 24 and 48 h after irradiation) and used for DNA extraction. A novel primer-anchored PCR based damage-detection methodology developed in our laboratory was used to identify and map DNA damage on the transcribed (TS) and non-transcribed strand (NTS) of the Trp53 gene. Results: Twenty-four and 48 h after UVB-radiation, significant levels of unrepaired nucleotide lesions persisted in genomic DNA of Xpc−/− Trp53+/− compared with WT mice. We observed that most of the damage blocked the primer-extension, yielding early stops. Additionally, most of the damage affecting Trp53 codon 122 in Xpc−/− Trp53+/− mice occurred in the NTS and strictly localized to 2 nucleotides. Importantly, a small percentage of the damage observed at these nucleotides allowed DNA polymerase bypass with misincorporation. Significantly, these lesions were detected precisely in the two nucleotides of Trp53 codon 122 that were found mutated in 64% of all tumors arising in this genotype. These results support the model that the tumor mutations at this site arise from error-prone bypass of long-lasting, unrepaired DNA lesions. Conclusions: We demonstrate a significant correlation between the persistence of precisely localized nucleotide lesions and the later establishment of tumor mutations at those specific nucleotides. Using a novel primer-anchored DNA damage methodology, we identified and mapped for the first time in vivo polymerase by-passable nucleotide lesions. Given the mutagenic potential of these lesions, the wide application of this novel technique for the identification of those lesions and the quantification of in vivo DNA damage might have major implications for early detection of cancer precursor lesions and evaluation of prevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1966.

Abstract 2978: Nucleotide excision repair contributes to hexavalent chromium-induced double strand breaks and chromosomal damage

Abstract Certain hexavalent chromium (Cr(VI)) compounds are known human lung carcinogens. Cr(VI) causes several types of DNA damage including bulky adducts and crosslinks. Cr(VI)-induced tumors exhibit chromosomal instability (CIN), however, very little is known about mechanisms of these effects and the role that DNA repair plays in this process. Nucleotide excision repair (NER) is known to be involved in the repair of bulky DNA damage. Unrepaired or misrepaired lesions can lead to chromosomal aberrations (CAs). We investigated the role of NER leading to CAs using Chinese hamster ovary (CHO) parental (AA8) and NER-deficient cells (UV-5). We found that UV-5 cells displayed attenuated Cr(VI)-induced chromosome damage. In general, DNA lesions need to be converted to DNA double strand breaks (DSBs) in order to give rise to CAs. Therefore, we used the neutral Comet assay to measure the induction and repair of DSBs after Cr(VI)-induced damage. We found that both parental and NER-deficient cell lines displayed low levels of DSBs immediately after Cr(VI) treatment. Interestingly, DSB formation peaked after a 24h recovery period, and NER-deficient cells exhibited lower overall levels of DSBs than repair-proficient cells. All damage in both cell lines was repaired by 48h following Cr(VI) exposure. Taken together, our results suggest that Cr(VI)-induced DSBs are not directly formed by Cr(VI) exposure. Instead, these lesions develop as a consequence of repair of, or replication past, Cr(VI)-induced lesions. Importantly, our data suggests that NER contributes to Cr(VI)-induced genomic instability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2978.

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.

Caenorhabditis elegans APN-1 plays a vital role in maintaining genome stability

We previously showed that Caenorhabditis elegans APN-1, the only metazoan apurinic/apyrimidinc (AP) endonuclease belonging to the endonuclease IV family, can functionally rescue the DNA repair defects of Saccharomyces cerevisiae mutants completely lacking AP endonuclease/3′-diesterase activities. While this complementation study provided the first evidence that APN-1 possesses the ability to act on DNA lesions that are processed by AP endonucleases/3′-diesterase activities, no former studies were conducted to examine its biological importance in vivo. Herein, we show that C. elegans knockdown for apn-1 by RNAi displayed phenotypes that are directly linked with a defect in maintaining the integrity of the genome. apn-1(RNAi) animals exhibited a 5-fold increase in the frequency of mutations at a gfp-lacZ reporter and showed sensitivities to DNA damaging agents such as methyl methane sulfonate and hydrogen peroxide that produce AP site lesions and strand breaks with blocked 3′-ends. The apn-1(RNAi) worms also displayed a delay in the division of the P1 blastomere, a defect that is consistent with the accumulation of unrepaired lesions. Longevity was only compromised, if the apn-1(RNAi) animals were challenged with the DNA damaging agents. We showed that apn-1(RNAi) knockdown suppressed formation of apoptotic corpses in the germline caused by an overburden of AP sites generated from uracil DNA glycosylase mediated removal of misincorporated uracil. Finally, we showed that depletion of APN-1 by RNAi partially rescued the lethality resulting from uracil misincorporation, suggesting that APN-1 is an important AP endonuclease for repair of misincorporated uracil.

BCR/ABL Inhibits Mismatch Repair to Protect from Apoptosis and Induce Point Mutations

BCR/ABL kinase-positive chronic myelogenous leukemia (CML) cells display genomic instability leading to point mutations in various genes including bcr/abl and p53, eventually causing resistance to imatinib and malignant progression of the disease. Mismatch repair (MMR) is responsible for detecting misincorporated nucleotides, resulting in excision repair before point mutations occur and/or induction of apoptosis to avoid propagation of cells carrying excessive DNA lesions. To assess MMR activity in CML, we used an in vivo assay using the plasmid substrate containing enhanced green fluorescent protein (EGFP) gene corrupted by T:G mismatch in the start codon; therefore, MMR restores EGFP expression. The efficacy of MMR was reduced approximately 2-fold in BCR/ABL-positive cell lines and CD34(+) CML cells compared with normal counterparts. MMR was also challenged by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which generates O(6)-methylguanine and O(4)-methylthymine recognized by MMR system. Impaired MMR activity in leukemia cells was associated with better survival, accumulation of p53 but not of p73, and lack of activation of caspase 3 after MNNG treatment. In contrast, parental cells displayed accumulation of p53, p73, and activation of caspase 3, resulting in cell death. Ouabain-resistance test detecting mutations in the Na(+)/K(+) ATPase was used to investigate the effect of BCR/ABL kinase-mediated inhibition of MMR on mutagenesis. BCR/ABL-positive cells surviving the treatment with MNNG displayed approximately 15-fold higher mutation frequency than parental counterparts and predominantly G:C-->A:T and A:T-->G:C mutator phenotype typical for MNNG-induced unrepaired lesions. In conclusion, these results suggest that BCR/ABL kinase abrogates MMR activity to inhibit apoptosis and induce mutator phenotype.

RecA‐mediated excision repair: a novel mechanism for repairing DNA lesions at sites of arrested DNA synthesis

In Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF-dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double-stranded DNA sequences. Monomodified single-stranded plasmids exhibit low survival in non-SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10-fold in a RecA-dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA-dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism - RecA-mediated excision repair - in which RecA-dependent pairing of the mono-modified single-stranded template with a complementary sequence allows its repair by the UvrABC excinuclease.

Towards biology-oriented treatment planning in hadrontherapy

By representing damage induction by ionising particles and its repair by the cell, the probabilistic two-stage model provides a detailed description of the main processes involved in cell killing by radiation. To link this model with issues of interest in hadron radiotherapy, a simple Bragg peak model is used. Energy-loss, its straggling and the attenuation of the primary particle fluence are represented in a simplified way, based on semi-phenomenological formulas and energy-loss tables. An effective version of the radiobiological model, considering residual (unrepaired) lesions only, is joined with the simple physical model to estimate cell survival along ions' penetration depth. The predicted survival ratios for CHO cells irradiated by carbon ions are presented, showing very good agreement with experimental data.

Deletion of the chromatin remodeling gene SPT10 sensitizes yeast cells to a subclass of DNA‐damaging agents

The Saccharomyces cerevisiae SPT10 protein possesses a DNA-binding domain that is fused to a putative histone acetyltransferase domain. It binds specifically to upstream-activating sequence elements in the core histone promoters and plays a direct role in histone gene regulation. SPT10 is also required for cell-cycle-specific K56 acetylation at histone genes, allowing the recruitment of the nucleosome remodeling factor Snf5 and subsequent regulation of gene transcription. We reisolated the SPT10 gene in a functional genome-wide screen designed to identify haploid yeast mutants that are hypersensitive to the antitumor drug bleomycin, which acts by damaging DNA. In addition to bleomycin, we show that spt10Delta mutants are also hypersensitive to a limited set of genotoxic agents that create DNA strand breaks, but not to 254-nm ultraviolet light or 4-nitroquinoline-1-oxide, which generate helix distortion. The hypersensitivities of the spt10Delta mutant to the genotoxic agents are rescued by a single copy plasmid carrying the SPT10 gene. We further showed that spt10Delta mutants displayed a modest twofold increase spontaneous mutant frequency, as compared to the parent. Following exposure to bleomycin, these mutants accumulate unrepaired lesions, e.g., DNA strand breaks with blocked 3'-ends in the chromosomal DNA. This defect is not due to the altered expression level or the enzymatic activities of a key DNA repair enzyme, APN1, which is known to repair DNA strand breaks with blocked ends. We propose that SPT10 mediates repair of a subset of DNA lesions by acetylating histones to promote recruitment of DNA repair enzymes.

Evaluation of Right Ventricular Fibrosis in Adult Congenital Heart Disease Using Gadolinium-enhanced Magnetic Resonance Imaging: Initial Experience in Patients with Right Ventricular Loading Conditions

Gadolinium-enhanced cardiac magnetic resonance imaging has been used to show myocardial fibrosis, a finding that appears as late gadolinium enhancement. Its role in the evaluation of right ventricular fibrosis in congenital heart disease is unclear. The purpose of this study was to demonstrate late gadolinium enhancement of the right ventricle in adult and adolescent congenital heart disease and to investigate the relationship between this enhancement and clinical and pathophysiological data. In total, 24 patients, 16 patients with congenital heart disease and right ventricular loading conditions and 8 controls, underwent gadolinium-enhanced viability imaging. Diagnoses varied and included repaired, palliated, and unrepaired lesions. The presence and extent of right ventricular late gadolinium enhancement was compared with patient clinical and hemodynamic data. Exact Wilcoxon tests, Fisher's exact tests, and Spearman's rank correlation were used to compare variables. Nine of 16 patients (56%) were found to have right ventricular late gadolinium enhancement, ranging from 5% to 80% of right ventricular myocardium affected (mean 36.1%, SD 29.7). The combination of right ventricular systolic pressure >or=98 mm Hg and systemic oxygen saturation <or=93% strongly suggested the presence of right ventricular late gadolinium enhancement (positive predictive value 100%), but no single variable or combination of variables could reliably predict its absence (negative predictive values <or=75%). Extent of right ventricular late gadolinium enhancement did not correlate with degree of either hypoxia or right ventricular hypertension. Gadolinium-enhanced cardiac magnetic resonance demonstrates right ventricular late gadolinium enhancement in some patients with congenital heart disease and right ventricular loading conditions. Clinical variables were associated with the presence of fibrosis but did not reliably predict severity. Myocardial preservation is likely a multifactorial process that may affect the right and left ventricles differently.

Replication fork reactivation downstream of a blocked nascent leading strand

Unrepaired lesions in the DNA template pose a threat to accurate replication. Several pathways exist in Escherichia coli to reactivate a blocked replication fork. The process of recombination-dependent restart of broken forks is well understood, but the consequence of replication through strand-specific lesions is less well known. Here we show that replication can be restarted and leading-strand synthesis re-initiated downstream of an unrepaired block to leading-strand progression, even when the 3'-OH of the nascent leading strand is unavailable. We demonstrate that the loading by a replication restart system of a single hexamer of the replication fork helicase, DnaB, on the lagging-strand template is sufficient to coordinate priming by the DnaG primase of both the leading and lagging strands. These observations provide a mechanism for damage bypass during fork reactivation, demonstrate how daughter-strand gaps are generated opposite leading-strand lesions during the replication of ultraviolet-light-irradiated DNA, and help to explain the remarkable speed at which even a heavily damaged DNA template is replicated.

Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells

Damage recognition by repair/checkpoint factors is the critical first step of the DNA damage response. DNA double strand breaks (DSBs) activate checkpoint signaling and are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. However, in vivo kinetics of the individual factor responses and the mechanism of pathway choice are not well understood. We report cell cycle and time course analyses of checkpoint activation by ataxia-telangiectasia mutated and damage site recruitment of the repair factors in response to laser-induced DSBs. We found that MRN acts as a DNA damage marker, continuously localizing at unrepaired damage sites. Damage recognition by NHEJ factors precedes that of HR factors. HR factor recruitment is not influenced by NHEJ factor assembly and occurs throughout interphase. Damage site retention of NHEJ factors is transient, whereas HR factors persist at unrepaired lesions, revealing unique roles of the two pathways in mammalian cells.

The Role of Base Excision Repair in the Sensitivity and Resistance to Temozolomide-Mediated Cell Death

DNA-alkylating agents have a central role in the curative therapy of many human tumors; yet, resistance to these agents limits their effectiveness. The efficacy of the alkylating agent temozolomide has been attributed to the induction of O6-MeG, a DNA lesion repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT). Resistance to temozolomide has been ascribed to elevated levels of MGMT and/or reduced mismatch repair. However, >80% of the DNA lesions induced by temozolomide are N-methylated bases that are recognized by DNA glycosylases and not by MGMT, and so resistance to temozolomide may also be due, in part, to robust base excision repair (BER). We used isogenic cells deficient in the BER enzymes DNA polymerase-beta (pol-beta) and alkyladenine DNA glycosylase (Aag) to determine the role of BER in the cytotoxic effect of temozolomide. Pol-beta-deficient cells were significantly more susceptible to killing by temozolomide than wild-type or Aag-deficient cells, a hypersensitivity likely caused by accumulation of BER intermediates. RNA interference-mediated pol-beta suppression was sufficient to increase temozolomide efficacy, whereas a deficiency in pol-iota or pol-lambda did not increase temozolomide-mediated cytotoxicity. Overexpression of Aag (the initiating BER enzyme) triggered a further increase in temozolomide-induced cytotoxicity. Enhanced Aag expression, coupled with pol-beta knockdown, increased temozolomide efficacy up to 4-fold. Furthermore, loss of pol-beta coupled with temozolomide treatment triggered the phosphorylation of H2AX, indicating the activation of the DNA damage response pathway as a result of unrepaired lesions. Thus, the BER pathway is a major contributor to cellular resistance to temozolomide and its efficacy depends on specific BER gene expression and activity.

Differential Repair of the Two Major UV-Induced Photolesions in Trichothiodystrophy Fibroblasts

Defects in nucleotide excision repair have been shown to be associated with the photosensitive form of the disorder trichothiodystrophy (TTD). Most repair-deficient TTD patients are mutated in the XPD gene, a subunit of the transcription factor TFIIH. Knowledge of the kinetics and efficiency of repair of the two major UV-induced photolesions in TTD is critical to understand the role of unrepaired lesions in the process of carcinogenesis and explain the absence of enhanced skin cancer incidence in TTD patients contrarily to the xeroderma pigmentosum D patients. In this study, we used different approaches to quantify repair of UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) at the gene and the genome overall level. In cells of two TTD patients, repair of CPD and 6-4PP was reduced compared with normal human cells, but the reduction was more severe in confluent cells than in exponentially growing cells. Moreover, the impairment of repair was more drastic for CPD than 6-4PP. Most notably, exponentially growing TTD cells displayed complete repair 6-4PP over a broad dose range, albeit at a reduced rate compared with normal cells. Strand-specific analysis of CPD repair in a transcriptional active gene revealed that TTD cells were capable to perform transcription-coupled repair. Taken together, the data suggest that efficient repair of 6-4PP in dividing TTD cells in concert with transcription-coupled repair might account for the absence of increased skin carcinogenesis in TTD patients.

The use of a muscle graft to repair a segmentary nerve defect: An experimental study using the sciatic nerve of rats as model

The use of a devitalized skeletal muscle graft and conventional nerve graft to repair a 5 mm long segmentary sciatic nerve lesion was studied in rats by means of functional, morphometric and spinal cord motor neuron cell response evaluation. Thirty-four rats were used and divided into four groups according to the procedure: (1) sham operation; (2) conventional nerve grafting; (3) muscle grafting; (4) unrepaired lesion. The sciatic functional index (SFI) was evaluated every fortnight up to the 105th postoperative day by measuring three parameters in the rats’ footprint. The animals of Groups 2 and 3 presented initial complete functional loss, followed by slow but steady recovery, with final similar SFIs. The histologic and morphometric studies showed an increased small diameter/thin myelin sheath nerve fiber density distally to the lesion site for both types of graft. An increased population of motor neurons was observed in the anterior horn of the lumbar spinal cord segment with both types of grafts, but not in the control groups. The SFI, histologic and morphometric data did not differ significantly between the two types of graft, thus indicating a similar behavior. The authors conclude that a 5 mm long skeletal muscle graft works as well as a conventional nerve graft.

Mechanisms of DNA Damage, DNA Hypomethylation, and Tumor Progression in the Folate/Methyl-Deficient Rat Model of Hepatocarcinogenesis

Using the folate/methyl-deficient rat model of hepatocarcinogenesis, we obtained evidence that may provide new insights into a major unresolved paradox in DNA methylation and cancer research: the mechanistic basis for genome-wide hypomethylation despite an increase in DNA methyltransferase activity and gene-specific regional hypermethylation. Previous studies revealed that the methyltransferase binds with higher affinity to DNA strand breaks, gaps, abasic sites, and uracil than it does to its cognate hemimethylated CpG sites, consistent with its ancestral function as a DNA repair enzyme. These same DNA lesions are an early occurrence in models of folate and methyl deficiency and are often present in human preneoplastic cells. We hypothesized that the high-affinity binding of the maintenance DNA methyltransferase to unrepaired lesions in DNA could sequester available enzyme away from the replication fork and promote passive replication-dependent demethylation. In support of this possibility, we found that lesion-containing DNA is less efficiently methylated than lesion-free DNA from folate/methyl-deficient rats and that an increase in DNA strand breaks precedes DNA hypomethylation. Despite an adaptive increase in DNA methyltransferase activity, hemimethylated DNA from folate/methyl-deficient rats is progressively replaced by double-stranded unmethylated DNA that is resistant to remethylation with dietary methyl repletion. In promoter regions, the inappropriate binding of the DNA methyltransferase to unrepaired lesions or mispairs may promote local histone deacetylation, methylation, and regional hypermethylation associated with tumor suppressor gene silencing. These insights in an experimental model are consistent with the possibility that DNA lesions may be a necessary prerequisite for the disruption of normal DNA methylation patterns in preneoplastic and neoplastic cells.