Defects in 8-oxo-guanine repair pathway cause high frequency of C > A substitutions in neuroblastoma

Neuroblastomas are childhood tumors with frequent fatal relapses after induction treatment, which is related to tumor evolution with additional genomic events. Our whole-genome sequencing data analysis revealed a high frequency of somatic cytosine > adenine (C > A) substitutions in primary neuroblastoma tumors, which was associated with poor survival. We showed that increased levels of C > A substitutions correlate with copy number loss (CNL) of OGG1 or MUTYH. Both genes encode DNA glycosylases that recognize 8-oxo-guanine (8-oxoG) lesions as a first step of 8-oxoG repair. Tumor organoid models with CNL of OGG1 or MUTYH show increased 8-oxoG levels compared to wild-type cells. We used CRISPR-Cas9 genome editing to create knockout clones of MUTYH and OGG1 in neuroblastoma cells. Whole-genome sequencing of single-cell OGG1 and MUTYH knockout clones identified an increased accumulation of C > A substitutions. Mutational signature analysis of these OGG1 and MUTYH knockout clones revealed enrichment for C > A signatures 18 and 36, respectively. Clustering analysis showed that the knockout clones group together with tumors containing OGG1 or MUTYH CNL. In conclusion, we demonstrate that defects in 8-oxoG repair cause accumulation of C > A substitutions in neuroblastoma, which contributes to mutagenesis and tumor evolution.

Neuroblastoma is a childhood tumor of the peripheral sympathetic nervous system that accounts for 15% of childhood cancer mortality (1). Survival rates vary between spontaneous remissions in the low-stage neuroblastoma tumors to less than 50% survival in the high-stage patient group (2). Like other pediatric cancer types, the number of somatic mutations in neuroblastoma is relatively low compared to adult cancers (3). Relapsed neuroblastoma tumors have an increased mutational burden compared to primary tumors and show recurrent alterations in, e.g., the RAS-MAPK pathway (4, 5).Over the last years, substitution patterns and mutational signatures have been investigated in adult and pediatric cancer types (3, 6, 7). These mutational signatures are characteristic combinations of substitutions in the context of neighboring bases (6). For some of these mutational signatures, the underlying mutational process has been described, while it is unknown for others (6, 8). In the majority of pediatric cancer types, cytosine > thymine substitutions are most abundant, which has been linked to spontaneous deamination of 5-methyl-cytosines and mutational signature 1 (3). Several studies reported a higher frequency of cytosine > adenine (C > A) substitutions in neuroblastoma (3, 7). C > A mutations can result from defects in 8-oxo-guanine (8-oxoG) repair (9). 8-oxoG is one of the most abundant DNA lesions generated by reactive oxygen species (10–12). 8-oxoG can base pair with both a cytosine (C) and an adenine (A). During replication, DNA polymerases can therefore insert an incorrect A opposite of 8-oxoG. In the next round of replication, a thymine (T) will be inserted opposite the A, finally resulting in a C > A substitution (13, 14). To combat reactive oxygen species–induced DNA damage, 8-oxoG can be recognized by the DNA glycosylases OGG1 and MUTYH when it forms a base pair with a C or A, respectively. 8-oxoG repair is initiated by excision of 8-oxoG by OGG1 or excision of the wrongly inserted A opposite 8-oxoG by MUTYH (Fig. 1). In addition, NUDT1 prevents incorporation of 8-oxo-dGTP from the free nucleotide pool, by hydrolysis of 8-oxo-dGTP to 8-oxo-GMP, which cannot be incorporated (13, 15, 16).Open in a separate windowFig. 1.Schematic overview of 8-oxoG repair pathway. When a G in the DNA is oxidized to an 8-oxo-guanine [8-oxoG(8)], the replicative DNA polymerases will insert an A opposite of the 8-oxoG during replication. In the next round of replication, a T will be inserted opposite of the A, resulting in a C > A substitution. The DNA glycosylases OGG1 and MUTYH are able to recognize 8-oxoG base pairing with a C or A, respectively. OGG1 excises 8-oxoG from the DNA, while MUTYH excises the A opposite of the 8-oxoG. The DNA is further repaired by the base excision repair pathway to a C:G base pair for OGG1-initiated repair or to a C:8-oxoG for MUTYH-initiated repair. This C:8-oxoG base pair is then again a substrate for OGG1. In addition, NUDT1 prevents the incorporation of 8-oxo-dGTP into the DNA, by hydrolyzing 8-oxo-dGTP, from the free nucleotide pool, to 8-oxo-dGMP.In this study, we identify increased accumulation of C > A substitutions in high-risk neuroblastoma, resulting in a strong contribution of mutational signature 18 and 36 in these tumors. We show that neuroblastoma tumors with high C > A substitution frequencies were enriched for copy number loss (CNL) of OGG1 and MUTYH. To mimic this phenotype, we used CRISPR-CAS9 to engineer defects in the 8-oxoG repair genes OGG1 and MUTYH in neuroblastoma cells, resulting in an increased accumulation of C > A substitutions in single-cell knockout clones and a high contribution of C > A mutational signatures 18 and 36. In clustering analysis, these clones group together with neuroblastoma tumors with OGG1 or MUTYH CNL. Finally, we evaluated a neuroblastoma relapse cohort and identified that 47% of alterations in RAS-MAPK pathway genes were caused by C > A substitutions. Taken together, our study identifies that defects in the 8-oxoG repair pathway cause an accumulation of C > A substitutions in neuroblastoma tumors, potentially leading to increased adaptive capacity and tumor evolution.     

Structural chromosome abnormalities,increased DNA strand breaks and DNA strand break repair deficiency in dermal fibroblasts from old female human donors

Dermal fibroblasts provide a paradigmatic model of cellular adaptation to long-term exogenous stress and ageing processes driven thereby. Here we addressed whether fibroblast ageing analysed ex vivo entails genome instability. Dermal fibroblasts from human female donors aged 20–67 years were studied in primary culture at low population doubling. Under these conditions, the incidence of replicative senescence and rates of age-correlated telomere shortening were insignificant. Genome-wide gene expression analysis revealed age-related impairment of mitosis, telomere and chromosome maintenance and induction of genes associated with DNA repair and non-homologous end-joining, most notably XRCC4 and ligase 4. We observed an age-correlated drop in proliferative capacity and age-correlated increases in heterochromatin marks, structural chromosome abnormalities (deletions, translocations and chromatid breaks), DNA strand breaks and histone H2AX-phosphorylation. In a third of the cells from old and middle-aged donors repair of X-ray induced DNA strand breaks was impaired despite up-regulation of DNA repair genes. The distinct phenotype of genome instability, increased heterochromatinisation and (in 30% of the cases futile) up-regulation of DNA repair genes was stably maintained over several cell passages indicating that it represents a feature of geroconversion that is distinct from cellular senescence, as it does not encompass a block of proliferation.     

原发性肝癌患者DNA损伤修复酶基因多态性分析

目的探讨DNA损伤修复酶基因多态性与原发性肝癌发生发展的关系。方法 PCR-RFLP法检测150例肝癌患者（肝癌组）和150例健康体检者（对照组）DNA损伤修复酶基因的表型,并进行比较。结果肝癌组XRCC1 Arg399Gln位点野生型的比率明显低于对照组（P〈0.05）;XRCC1（Arg194Trp、Arg280His）、hOGG1Ser326Cys位点多态性型别在两组中出现的频率相近（P〉0.05）;各基因位点不同表型者的尿8-羟基脱氧鸟苷（8-OHdG）水平相比,P均〉0.05。结论 DNA修复酶基因多态性与肝癌的发生发展无明确的相关关系。     

慢性染砷大鼠肝脏线粒体DNA及相关酶活性变化研究

目的 探讨慢性染砷大鼠肝脏线粒体DNA（mtDNA）及线粒体相关酶，特别是mtDNA编码酶活性的变化。方法 Wistar大鼠经饮水给75mg／L三氧化二砷6个月。用分光光度法测定肝脏线粒体呼吸酶活性：碱变性法提取大鼠肝脏mtDNA；以PCR大片段扩增法扩增长片段mtDNA；用HindⅢ、EcoRⅠ、SalⅠ、BamHⅠ、MspⅠ、MboⅠ、StuⅠ7种限制性内切酶对扩增片段进行酶切分析。结果 染砷大鼠肝脏线粒体细胞色素C氧化酶（CytCOX）和ATP酶活性下降；而经mtDNA扩增片段凝胶电泳分析和7种限制性内切酶酶切分析，未发现染砷大鼠肝脏参与编码上述2种线粒体酶的4151bp片段有碱基缺失、重复和突变等异常表现。结论 慢性染砷能够导致肝细胞mtDNA参与编码的CytCOX和ATP酶活性下降，进一步证实了染砷对肝细胞及其线粒体的损害作用。但其活性下降的机制，目前尚不能以砷致mtDNA片段的变化来解释，需进一步进行DNA序列分析研究。     

Effects of combined physical exercise training on DNA damage and repair capacity: role of oxidative stress changes

Regular physical exercise has been shown to be one of the most important lifestyle influences on improving functional performance, decreasing morbidity and all causes of mortality among older people. However, it is known that acute physical exercise may induce an increase in oxidative stress and oxidative damage in several structures, including DNA. Considering this, the purpose of this study was to identify the effects of 16 weeks of combined physical exercise in DNA damage and repair capacity in lymphocytes. In addition, we aimed to investigate the role of oxidative stress involved in those changes. Fifty-seven healthy men (40 to 74 years) were enrolled in this study. The sample was divided into two groups: the experimental group (EG), composed of 31 individuals, submitted to 16 weeks of combined physical exercise training; and the control group (CG), composed of 26 individuals, who did not undergo any specifically orientated physical activity. We observed an improvement of overall physical performance in the EG, after the physical exercise training. A significant decrease in DNA strand breaks and FPG-sensitive sites was found after the physical exercise training, with no significant changes in 8-oxoguanine DNA glycosylase enzyme activity. An increase was observed in antioxidant activity, and a decrease was found in lipid peroxidation levels after physical exercise training. These results suggest that physical exercise training induces protective effects against DNA damage in lymphocytes possibly related to the increase in antioxidant capacity.     

Oxidative DNA damage in human esophageal cancer: clinicopathological analysis of 8‐hydroxydeoxyguanosine and its repair enzyme

Both internal and external oxidative stresses act on DNA and can induce carcinogenesis. 8‐hydroxydeoxyguanosine (8‐OHdG) is an indicator of oxidative stress and it leads to transversion mutations and carcinogenesis. 8‐OHdG is excision‐repaired by 8‐OHdG DNA glycosylase (OGG1). The purpose of this study is to clarify the effect of oxidative DNA damage and repair enzymes on esophageal carcinogenesis. The levels of 8‐OHdG and OGG1 were immunohistochemically evaluated in resected specimens, including squamous cell carcinoma (SCC) in 97 patients with esophageal cancer. Higher levels of 8‐OHdG in normal esophageal epithelium were associated with a higher smoking index (P = 0.0464). The 8‐OHdG level was higher in cancerous areas than in normal epithelia (P = 0.0061), whereas OGG1 expression was weaker in cancerous areas than in normal epithelia (P < 0.0001). An increase of OGG1 expression in normal epithelium was observed as 8‐OHdG levels increased (P = 0.0011). However, this correlation was not observed in cancerous areas. High OGG1 expression in the cytoplasm was related to deeper tumors (P = 0.0023), node metastasis (P = 0.0065) and stage (P = 0.0019). Oxidative DNA damage, which is attributable to smoking as well as disturbances in DNA repair systems, appears to be closely related to esophageal carcinogenesis and its progression.     

抗衰1号对老年小鼠肝线粒体DNA缺失的影响

目的研究抗衰1号对老年Balb/c小鼠肝线粒体DNA(mtDNA)缺失突变的影响.方法老年Balb/c小鼠随机分为老年空白组和抗衰1号组,分别给予生理盐水和抗衰1号4个月.采用聚合酶链反应(PCR)技术和光密度扫描检测两组mtDNA的缺失情况.结果与老年空白对照组比较,抗衰1号能显著减少老年Balb/c小鼠mtDNA的缺失(P＜0.001). 结论抗衰1号可以抑制老年小鼠mtDNA的缺失,提示补肾健脾活血通腑法组方能减少mtDNA的氧化损伤,对mtDNA有保护作用,从而从分子水平提供了本法延缓衰老的可能机理.     

韩宗超

目的 观测过氧化氢诱导的大鼠衰老神经细胞DNA损伤与修复情况。方法 在荧光显微镜下观察不同浓度H2O2处理的3月、8月和26月龄大鼠的脑皮质、海马和基底节经分离的神经细胞及氧化损伤断裂的单链DNA,并做彗星图像分析。结果 在相同浓度H2O2介导下,衰老神经细胞DNA损伤分值较正常神经细胞高,皮质细胞较海马、基底节区细胞DNA损伤分值高,细胞更易受损伤。给予0．5～4．0 h孵育后,表明脑细胞最大修复时间仅为1．0 h,且衰老神经细胞较正常神经细胞的DNA损伤分值高,修复能力下降。当H2O2介导浓度为64 μmol／L时,3月龄鼠脑皮质、海马和基底节神经细胞DNA损伤分值依次为250、213和205,26月龄鼠DNA损伤分值依次为355、340和335。当H2O2介导浓度为128 μmol／L时,26月龄鼠DNA修复率约为10％。结论 彗星实验是一种非常敏感的检测DNA损伤与修复的方法。     

The role of DNA exonucleases in protecting genome stability and their impact on ageing

Exonucleases are key enzymes involved in many aspects of cellular metabolism and maintenance and are essential to genome stability, acting to cleave DNA from free ends. Exonucleases can act as proofreaders during DNA polymerisation in DNA replication, to remove unusual DNA structures that arise from problems with DNA replication fork progression, and they can be directly involved in repairing damaged DNA. Several exonucleases have been recently discovered, with potentially critical roles in genome stability and ageing. Here we discuss how both intrinsic and extrinsic exonuclease activities contribute to the fidelity of DNA polymerases in DNA replication. The action of exonucleases in processing DNA intermediates during normal and aberrant DNA replication is then assessed, as is the importance of exonucleases in repair of double-strand breaks and interstrand crosslinks. Finally we examine how exonucleases are involved in maintenance of mitochondrial genome stability. Throughout the review, we assess how nuclease mutation or loss predisposes to a range of clinical diseases and particularly ageing.     

Age-and caste-dependent decrease in expression of genes maintaining DNA and RNA quality and mitochondrial integrity in the honeybee wing muscle

I report here an investigation of the age- and caste-specific expression patterns of nine honeybee orthologs of genes involved in repair of oxidative and methylation damage of DNA, and possibly RNA, in wing muscle tissue of the honeybee Apis mellifera. mRNA expression levels were measured in a comparative study of queens and ageing workers. Two of these genes, both potentially involved in repair and prevention of oxidative damage, showed higher expression in queens than workers and a distinct downregulation during the ageing trajectory in workers. These were an ortholog of mammalian NTH1 and a gene encoding a fusion protein which seems to be unique for the honeybee, consisting of one domain homologous to mammalian MTH1/Nudix/bacterial mutT and another domain homologous to the mitochondrial ribosomal protein gene S23. Orthologs of aag, apn1, msh6, ogg1, smug1 and two orthologs of human ABH/E. coli alkB, had stable expression levels during the ageing trajectory except high apn1 levels in overwintering workers. To estimate eventual age-dependent mitochondrial maintenance, batches of mitochondrial DNA from young and old workers and young queens were re-sequenced using Solexa/Illumina high-throughput sequencing. The results indicate at least a 50% reduction of intact mitochondrial fragments in foragers compared to young workers, winter bees and queens.     

Low-level caloric restriction rescues proteasome activity and Hsc70 level in liver of aged rats

Proteasome activity is known to decrease with aging in ad libitum (AL) fed rats. Severe caloric restriction (CR) significantly extends the maximum life-span of rats, and counteracts the age-associated decrease in liver proteasome activities. Since few investigations have explored whether lower CR diets might positively counteract the age associated decrease in proteasome activity, we then investigated the effects of a mild CR regimen on animal life-span, proteasome content and function. In addition, we addressed the question whether both CR regimens might also affect the expression of Hsc70 protein, a constitutive chaperone reported to share a role in the function of proteasome complex and in the repair of proteotoxic damage, and whose level decreased during aging. In contrast to severe CR, mild CR had a poor effect on life-span; however, it better counteracted the decrease of proteasome activities. Both regimens, however, maintain Hsc70 in liver of old rats at level comparable to that of young rats. Interestingly, the effects of aging and CRs on liver proteasome enzyme activities did not appear to be associated with parallel changes in the amount of proteasome proteins suggesting that the quality (molecular activity of the enzymes) rather than the quantity are likely to be modified with age. In conclusion, the results presented in this work show that a mild CR can have beneficial effects on liver function of aging rats because is adequate to counteract the decrease of proteasome function and Hsc70 chaperone level.     

The effect of chronic physical exercise on succinic dehydrogenase activity in the heart muscle of old rats

Succinic dehydrogenase (SDH) activity, preferentially evidenced by cytochemical methods, has been measured by computer-assisted morphometry in the heart muscle of old sedentary and age-matched animals chronically undergone physical exercise (20 min, twice a day, 5 days a week). The area of the SDH-positive mitochondria (MA) and the overall area of the cytochemical precipitates due to SDH activity (PA) were semiautomatically measured and the ratios PA/MA as well as MA/overall myocardial tissue area analysed (MA/TA) were the parameters taken into account. No significant difference was found between the two groups investigated as regards PA/MA, whereas the MA/TA value is significantly increased in the animals undergone physical training. The present findings document that chronic physical exercise significantly increases the overall mitochondrial area involved in energy provision in the old myocardial tissue. Considering that myocardial function highly relies on mitochondrial metabolism, our results support a beneficial effect of chronic physical exercise on the old heart muscle.     

Mutational analysis of the DNA mismatch repair gene hMLH1 in myeloid leukaemias

Mutations of the DNA mismatch repair (MMR) gene hMLH1 have recently been linked to the development of some hereditary and sporadic cancers which frequently display widespread microsatellite instability (MSI). Conflicting results regarding the extent of MSI in myeloid leukaemias prompted us to perform mutational analysis of all 19 exons of the hMLH1 gene by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) and sequence analysis in a total of 133 patients with acute and chronic myeloid leukaemia. Apart from one exonic and one intronic polymorphism, no mutations were detected in any of the samples indicating that the major MMR gene hMLH1 is not involved in the pathogenesis or progression of myeloid malignancies.     

In human hepatocellular carcinoma in cirrhosis proliferating cell nuclear antigen (PCNA) is involved in cell proliferation and cooperates with P21 in DNA repair

BACKGROUND: Proliferating cell nuclear antigen (PCNA) is a nuclear protein involved in DNA-synthesis and repair. During DNA-synthesis and repair the only active PCNA fraction is tightly bound to DNA. Similarly, during DNA-repair, a fraction of p21 colocalizes with PCNA in a detergent-insoluble form. AIM: The aim of the study was to analyze to what extent the presence of DNA-bound PCNA and p21 correlates with cell proliferation and DNA-repair in hepatocellular carcinoma (HCC). METHODS: Twenty-six HCCs and surrounding cirrhosis were studied. The DNA-bound and detergent-soluble fractions of PCNA and p21 were analyzed by immunoblotting. P53 and Ki67-Labeling Index (Ki67-LI) were evaluated by immunocytochemistry. RESULTS: Soluble fractions of PCNA and p21 were found in all samples. One out of 26 cirrhotic samples displayed a DNA-bound fraction of PCNA while no case expressed DNA-bound p21. Fourteen HCCs showed a DNA-bound PCNA fraction. A highly significant correlation was found between Ki67-LI and DNA-bound PCNA but not with detergent-soluble PCNA. DNA-bound p21 and PCNA, indicating ongoing DNA repair activity, were present in 6 of these 14 HCCs and correlated with a high histological grade and high Ki67-LI. CONCLUSIONS: Our results suggest that in HCC PCNA participates both in DNA synthesis and repair and that highly proliferating HCCs may display a sustained DNA-repair.     

Effects of treadmill exercise and training frequency on anabolic signaling pathways in the skeletal muscle of aged rats

Physical exercise is the most effective intervention against sarcopenia of aging; however, the cellular and molecular mechanisms mediating training-induced adaptations are not yet completely understood. Furthermore, it is unclear whether exercise training initiated late in life affects myocyte anabolic signaling in a dose-dependent manner. Hence, we sought to investigate the effects of treadmill exercise and training frequency on anabolic pathways, including insulin signaling, in the skeletal muscle of old rats. Aged (14-16-month-old) male Wistar rats were trained on a treadmill for 3 (EX3) or 5 days/week (EX5) during 8 weeks and compared with age-matched sedentary controls (SED). Four-month-old rats were used as young controls (YC). Protein expression levels of insulin receptor (IR), insulin receptor substrate 1 (IRS-1), activated (phosphorylated) mammalian target of rapamycin (p-mTOR) and glucose transporter GLUT4 were determined in quadriceps muscle extracts via immunoblotting. Mitochondrial cytochrome c oxidase (COX) activity was assessed by histochemical staining, while electron microscopy was employed to quantify the sarcomere volume (V_src). Body weight (BW) increased, whereas muscle weight (MW) and V_src decreased with age. EX5, but not EX3 increased MW and V_src, without affecting BW. The expression of IR and GLUT4 was higher in SED rats relative to the YC group. Conversely, protein levels of IRS-1 and p-mTOR as well as COX activity were reduced in advanced age. Compared with SED rats, EX3 animals displayed reduced IR expression and increased IRS-1 levels and COX activity. The expression of GLUT 4 and p-mTOR was unaffected by EX3. EX5 up-regulated IRS-1 and p-mTOR expression and COX activity, while decreasing GLUT4 levels, with no effect on IR expression. In summary, substantial impairments in muscle anabolic pathways, including insulin signaling, were detected in aged sedentary rats. These changes were ameliorated by exercise training, concomitant with improvements in muscle trophism. Benefits were more evident in rats trained for 5 days/week, suggesting that physical exercise initiated late in life affects anabolic signaling in a dose-dependent manner.     

O6-methylguanine repair in liver cells in vivo: Comparison between G1- and S-phase of the cell cycle

Summary To compare the formation and persistance of alkylated DNA bases in the G₁- and S-phase compartments in liver in vivo, regnerating rat liver was exposed to [¹⁴C]dimethylnitrosamine (0.57 mg/kg, IP injection) or N-[methyl ¹⁴C]-N-nitrosourea (3.3 mg/kg, intraportal injection) during the G₁ phase of the cell cyle (12 h after partial hepatectomy), or at 24 h after partial hepatectomy with 30% hepatocytes in DNA synthesis, or at 43 h after partial hepatectomy, 4 h after an hydroxyurea block from 14 to 39 h after operation with 80% hepatocytes in DNA synthesis. At 120 min after dimethylnitrosamine and 90 s, 5, 10, or 60 min after the intraportal pulse of N-methyl-N-nitrosourea the molar fractions of 7-methylguanine (7megua), O⁶-methylguanine (O⁶megua), and 3-methyladenine (3mead) and of metabolically labeled guanine were determined from DNA hydrolysates by Sephadex-G10 radiochromatography. After dimethylnitrosamine only minor differences were observed for 7megua formation in the three groups; the 3mead/7megua ratio remained constant irrespective of the number of cells in S phase. In contrast, the O⁶megua/7megua ratio revealed a loss of O⁶megua, the extent of which appeared proportional to the fraction of DNA-synthesizing cells in the liver. The rapid loss of O⁶megua in S-phase cells was confirmed after intraportal administration of N-methyl-N-nitrosourea. During the first 10 min after the methylnitrosourea pulse the O⁶megua/7megua ratio was constant in G₁ cells and dropped from 90 s to 10 min by about 15% in liver containing 30% S-phase cells and by about 40% with 80% cells in DNA synthesis. DNA-synthesizing hepatocytes are apparently endowed with a higher O⁶megua DNA transferase activity than nonproliferating liver cells. The rapid, though exhaustible elimination of O⁶megua during S-phase might result in partial protection of DNA-synthesizing cells from base-mispairing and/or from hypomethylation at G-C sites.Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthdaySupported by grants from the Deutsche Forschungsgemeinschaft and the Dr. Mildred Scheel-Stiftung     

Perspectives on the combination of radiotherapy and targeted therapy with DNA repair inhibitors in the treatment of pancreatic cancer

Pancreatic cancer is highly lethal. Current research that combines radiation with targeted therapy may dramatically improve prognosis. Cancerous cells are characterized by unstable genomes and activation of DNA repair pathways, which are indicated by increased phosphorylation of numerous factors, including H2 AX, ATM, ATR, Chk1, Chk2, DNA-PKcs, Rad51, and Ku70/Ku80 heterodimers. Radiotherapy causes DNA damage. Cancer cells can be made more sensitive to the effects of radiation(radiosensitization) through inhibition of DNA repair pathways. The synergistic effects, of two or more combined non-lethal treatments, led to coadministration of chemotherapy and radiosensitization in BRCA-defective cells and patients, with promising results. ATM/Chk2 and ATR/Chk1 pathways are principal regulators of cell cycle arrest, following DNA doublestrand or single-strand breaks. DNA double-stranded breaks activate DNA-dependent protein kinase, catalytic subunit(DNA-PKcs). It forms a holoenzyme with Ku70/Ku80 heterodimers, called DNA-PK, which catalyzes the joining of nonhomologous ends. This is the primary repair pathway utilized in human cells after exposure to ionizing radiation. Radiosensitization, induced by inhibitors of ATM, ATR, Chk1, Chk2, Wee1, PP2 A, or DNA-PK, has been demonstrated in preclinical pancreatic cancer studies. Clinical trials are underway. Development of agents that inhibit DNA repair pathways to be clinically used in combination with radiotherapy is warranted for the treatment of pancreatic cancer.