ADPRT抑制剂对不同辐射敏感性人肿瘤细胞株的作用比较

从细胞的克隆形成能力和细胞ＤＮＡ双链断裂及修复几方面分析了两个人卵巢癌细胞株ＨＯＣ８和Ａ２７８０对电离辐射的敏感性并探讨了ＡＤＰ－核糖基转移酶的特异性抑制剂３－氨基苯甲酰胺对二的辐射增敏效应,结果表明Ａ２７８０细胞的辐射敏感性大大高于ＨＯＣ８细胞,其Ｄ０值分别为０．９和２．５Ｇｙ;γ射线所致两株细胞的初始ＤＮＡ双链断裂水平没有显差异,但Ａ２７８０细胞对ＤＮＡ双链断裂的修复能力比ＨＯＣ８细胞低。     

HIV-1Tat蛋白抑制DNA修复和增强细胞辐射敏感性

近年来临床研究发现,艾滋病合并肿瘤患者放疗后产生的正常组织和皮肤毒性反应明显高于普通肿瘤患者.本研究将探讨HIV-1Tat蛋白是否影响细胞对电离辐射敏感性及机理. 两个表达Tat蛋白的细胞系TT2和TE671-Tat均来源于人的横纹肌肉瘤细胞（TE671）并已转染了不同来源的tat基因.使用细胞辐射后克隆形成率检测辐射敏感性,RT-PCR和Western 印迹检测基因表达,彗星电泳和γ-H2AX位点检测DNA双链断裂和修复. TT2和TE671-Tat细胞的辐射敏感性与转染空载体及对照细胞相比明显增加.彗星电泳和γ-H2AX位点检测表明,在表达Tat蛋白的细胞中,辐射诱导DNA双链断裂的修复水平明显降低.通过RT-PCR和Western 印迹检测进一步证实,表达Tat蛋白的细胞中DNA修复蛋白DNA-PKcs的表达被抑制. HIV-1Tat蛋白抑制DNA-PKcs的表达,降低DNA双链断裂的修复,使细胞的电离辐射敏感性增高.本研究为了解AIDS合并肿瘤患者对放射治疗敏感性变化提供了重要信息.     

辐射敏感细胞ku80基因突变及其DNA结合活性研究

RT-PCR克隆辐射敏感细胞及其亲本细胞的ku80基因cDNA,发现辐射敏感细胞的ku80基因与双链断裂DNA末端相互作用的位置存在基因突变,用凝胶阻滞和DNA-蛋白质印迹进一步证实突变ku基因编码蛋白结合双链断裂末端DNA的能力下降,暗示其细胞辐射敏感性可能与Ku蛋白功能异常有关.     

AT细胞DNA双链断裂重接修复效率及其忠实性

用脉冲电场凝胶电泳和双标记基因质粒ＤＮＡ转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞（ＡＴ５ＢＩＶＡ）和正常辐射抗性的人宫颈癌细胞（ＨｅＬａＳ３）ＤＮＡ双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发ＤＮＡ双链断裂的产额和重接修复率,在两株细胞间无差别．而ＡＴ细胞对导入的限制性内切酶ＥｃｏＲＶ产生双链断裂质粒ＤＮＡ的重接修复忠实性显著低于ＨｅｌａＳ３ｔｅ胞,表明ＡＴ细胞易发生ＤＮＡ错误修复,这很可能就是ＡＴ细胞高度辐射敏感性的主要原因。     

卵巢癌组织中dbpA的表达及其对卵巢癌细胞增殖的影响

目的:探讨dbp A蛋白在卵巢癌组织中的表达及其对卵巢癌细胞增殖的影响。方法:通过荧光定量和蛋白质免疫印迹方法检测临床卵巢癌组织和正常癌旁组织中dbp A的表达量;设计合成针对dbp A基因的双链小干扰RNA转染人卵巢癌细胞系SKOV3和A2780细胞,用荧光定量和蛋白质免疫印迹方法检测细胞中dbp A的表达量,MTT法和克隆形成试验检测细胞的增殖能力和克隆形成能力,流式细胞术检测各组细胞周期和细胞凋亡的变化。结果:dbp A在卵巢癌组织、SKOV3和A2780细胞中表达较癌旁正常卵巢组织显著升高。沉默dbp A后,SKOV3和A2780细胞中dbp A蛋白表达量显著降低,SKOV3和A2780细胞增殖能力和克隆形成能力显著下降(P0.05),G0/G1期细胞百分比显著增加(P0.01),S期细胞百分比明显减少(P0.05),细胞凋亡率显著升高(P0.01)。结论:dbp A在卵巢癌组织和卵巢癌细胞SKOV3和A2780中过表达,沉默dbp A基因后可抑制SKOV3和A2780细胞的增殖能力和克隆形成能力。     

表观遗传调控：基于染色质环境的DNA双链断裂修复

DNA双链断裂（DNA double-strand breaks, DSBs）是威胁基因组完整性和细胞存活的最有害的DNA损伤类型。同源重组（homologous recombination,HR）和非同源末端连接（non-homologous end joining,NHEJ）是修复DNA双链断裂的两种主要途径。DSB修复涉及到损伤部位修复蛋白的募集和染色质结构的改变。在DNA双链断裂诱导下,染色质结构的动态变化在时间和空间上受到严格调控,进而对DNA双链断裂修复过程进行精细调节。特定的染色质修饰形成利于修复的染色质状态,有助于DNA双链断裂修复机器的招募、修复途径的选择和DNA损伤检查点的活化;其中修复途径的选择对于基因组稳定性至关重要。修复不当或失败可导致基因组不稳定性,甚至促进肿瘤的发生。本文综述了染色质结构和染色质修饰的动态变化在DSB修复中的重要作用。此外,文章还总结了在癌症治疗中靶向关键染色质调控因子在基因组稳定性维持、肿瘤发生发展以及潜在临床应用价值等方面的进展。     

丙线照射所致人血淋巴细胞与中国仓鼠卵巢成纤维细胞DNA单链断裂及其修复的观察

用微孔滤膜碱洗脱法观察了丙线照射引起人血淋巴细胞(Do为400拉德)及中国仓鼠卵巢成纤维细胞(Do为200拉德)的DNA单链断裂及其修复。在0—3000拉德范围内,两种细胞DNA单链断裂的程度基本一致,照射剂量与单链断裂的对数之间呈直线相关。800、1500及3000拉德照射后,经过0.5—7小时的孵育,中国仓鼠卵巢成纤维细胞DNA单链断裂的修复优于人血淋巴细胞,说明这两种细胞辐射敏感性DNA单链断裂修复能力无关。     

丙线照射所致人血淋巴细胞与中国白鼠卵巢成纤维细胞DNA单链断裂及其修复的观察

用微孔滤膜碱洗脱法观察了丙线照射引起人血淋巴细胞(D_o为400拉德)及中国仓鼠卵巢成纤维细胞(D_o为200拉德)的DNA单链断裂及其修复。在0～3000拉德范围内,两种细胞DNA单链断裂的程度基本一致,照射剂量与单链断裂的对数之间呈直线相关。800、1500及3000拉德照射后,经过0.5～7小时的孵育,中国仓鼠卵巢成纤维细胞DNA单链断裂的修复优于人血淋巴细胞,说明这两种细胞辐射敏感性与DNA单链断裂修复能力无关。     

HO·和γ射线对细胞致死效应的比较

利用双链断裂模型比较研究了羟自由基和γ射线对B16、L0 2、SMMC - 772 1和V79四种细胞的致死效应。结果表明 :1.HO·处理和γ射线照射对四种细胞有都明显的致死效应 ,而且剂量越大致死作用越强。 2 .HO·处理和γ射线照射存活曲线都存在肩区 ,说明两种处理过程都有亚致死损伤的修复。 3.HO·诱导DNA双链断裂一般都需要两次击中而γ射线辐照既有一次击中的成分 ,也有两次击中的成分。 4.四种细胞对HO·的敏感性与对γ射线的辐射敏感性顺序相反 ,说明γ射线对细胞的作用不能简单地解释为自由基的行为。     

电离辐射诱导DNA—PKcs缺失小鼠T细胞特异的V（D）J重组恢复

NA依赖的蛋白激酶 (DNA PK)是一种DNA活化的核丝氨酸苏氨酸蛋白激酶。DNA PK由一种与DNA末端结合的调节亚单位异构二聚体Ku蛋白和DNA PK催化亚单位 (DNA PKcs)组成。DNA PK在DNA暴露于电离辐射后诱导的双链损伤修复中起主要作用。为了更好地了解与DNA PKcs缺失相关的DNA修复缺陷的本质。建立了DNA PKcs-/ -小鼠胚胎成纤维细胞株和裸鼠模型 ,调查这些突变的细胞和小鼠对DNA损害的反应。DNA PKcs-/ -细胞对电离辐射超敏感 ,在克隆形成实验中显示较低的生成率。同样 ,DNA PKcs-/ -小鼠也显示极大的放射敏感性 ,新生DNA PKcs-/ -小鼠用亚致死剂量电离辐射处理恢复T细胞受体 (TCR) β重组和T细胞成熟。然而 ,放射辐射并不恢复B细胞发育。DNA PKcs-/ -小鼠最终发生胸腺淋巴瘤。这些结果提示DNA双链断裂 (DSB)修复 ,V(D)J重组和淋巴瘤发生之间的相互关系。提供一种体内模型以阐明DNADSB修复调节、V(D)J重组和淋巴瘤发生分子机制三者之间的关键通路     

Misrejoining of DNA double-strand breaks in primary and transformed human and rodent cells: a comparison between the HPRT region and other genomic locations.

Many studies of radiation response and mutagenesis have been carried out with transformed human or rodent cell lines. To study whether the transfer of results between different cellular systems is justified with regard to the repair of radiation-induced DNA double-strand breaks (DSBs), two assays that measure the joining of correct DSB ends and total rejoining in specific regions of the genome were applied to primary and cancer-derived human cells and a Chinese hamster cell line. The experimental procedure involves Southern hybridization of pulsed-field gel electrophoresis blots and quantitative analysis of specific restriction fragments detected by a single-copy probe. The yield of X-ray-induced DSBs was comparable in all cell lines analyzed, amounting to about 1 x 10(-2) breaks/Mbp/Gy. For joining correct DSB ends following an 80 Gy X-ray exposure all cell lines showed similar kinetics and the same final level of correctly rejoined breaks of about 50%. Analysis of all rejoining events revealed a considerable fraction of unrejoined DSBs (15-20%) after 24 h repair incubation in the tumor cell line, 5-10% unrejoined breaks in CHO cells and complete DSB rejoining in primary human fibroblasts. To study intragenomic heterogeneity of DSB repair, we analyzed the joining of correct and incorrect break ends in regions of different gene density and activity in human cells. A comparison of the region Xq26 spanning the hypoxanthine guanine phosphoribosyl transferase locus with the region 21q21 revealed identical characteristics for the induction and repair of DSBs, suggesting that there are no large variations between Giemsa-light and Giemsa-dark chromosomal bands.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.     

Overexpression of human Ku70/Ku80 in rat cells resulting in reduced DSB repair capacity with appropriate increase in cell radiosensitivity but with no effect on cell recovery.

The effect of an overexpression of human Ku70/80 was studied using cells of the rat cell lines Rat-1 and R7080, the latter being transfected with the human cDNAs for Ku70 and Ku80. The overexpression was found to result in a 20% reduction of the DNA-PK activity. The kinetics of DSB repair, which was studied after exposure of the cells to 30 Gy of X rays, was biphasic and had identical half-times for Rat-1 and R7080 cells (tfast = 7 min and tslow = 135 min). However, there was a significant difference between the cell lines in the fractions of DSBs repaired with slow and fast kinetics. In R7080 cells, about twice as many DSBs were repaired with slow kinetics compared to Rat-1 cells (34% compared to 16%). A similar difference was found in the number of residual DSBs (3.6% compared to 2.0%). R7080 cells also showed a reduced capacity to repair chromosome damage as detected by the PCC technique. Concerning cell killing, R7080 cells were clearly more radiosensitive than Rat-1 cells (D0.1 = 6.4 compared to 10.5 Gy), and this increase in sensitivity correlated well with the increase in residual DSBs. The two cell lines, however, did not vary in cell recovery. For sublethal as well as potentially lethal damage, Rat-1 and R7080 cells showed identical recovery ratios. These data demonstrate that the overexpression of human Ku70/Ku80 led to a reduced capacity for DSB repair with an associated increase in cell sensitivity but with no effect on cell recovery.     

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0-30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the gammaH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.     

Inhibition of poly(ADP-ribose)polymerase does not affect the recombination events in CHO xrs6 and wild type cells

Activation of poly (ADP-ribose) polymerase -1 (PARP-1) is an early DNA damage response event that, together with phosphorylation of p53, prompts various cellular functions important in the maintenance of the genome stability. In mammalian cells, DSB are repaired by nonhomologous end-joining (NHEJ) and by homologous recombination (HR). To investigate the role of PARP-1 in HR, CHO-K1 wild type and xrs-6 mutant cell line were transfected with pLrec plasmids which carry two nonfunctional copies of the β-galactosidase (lacZ) gene in a tandem array. In result of HR they can give rise to a functional copy of β-galactosidase. To test whether PARP-1 affects the frequency of spontaneous and induced recombination repair, we treated CHO-K1 and xrs6 clones carrying chromosomally integrated pLrec with the PARP-1 inhibitor 3-aminobenzamide (3AB). Our results show that the spontaneous homologous intrachromosomal recombination frequency between the two lacZ copies was almost two orders of magnitude higher in xrs6 cells than in CHO-K1 cells, but that it was not affected by 3AB treatment. Induction of DNA damage by irradiation or electroporation of restriction enzymes did not significantly increase the recombination frequency. Furthermore, in both the cell lines, the effect of PARP-1 inhibition on DSB repair was examined using the neutral comet assay. There was no effect of 3AB treatment on DSB rejoining after 10 Gy irradiation. The results presented support the conclusion that PARP-1 is not directly involved in HR.     

Two-lesion kinetic model of double-strand break rejoining and cell killing

Radiobiological models, such as the lethal and potentially lethal (LPL) model and the repair-misrepair (RMR) model, have been reasonably successful at explaining the cell killing effects of radiation. However, the models have been less successful at relating cell killing to the formation, repair and misrepair of double-strand breaks (DSBs), which are widely accepted as the main type of DNA damage responsible for radiation-induced cell killing. A fully satisfactory model should be capable of predicting cell killing for a wide range of exposure conditions using a single set of model parameters. Moreover, these same parameters should give realistic estimates for the initial DSB yield, the DSB rejoining rate, and the residual number of unrepaired DSBs after all repair is complete. To better link biochemical processing of the DSB to cell killing, a two-lesion kinetic (TLK) model is proposed. In the TLK model, the family of all possible DSBs is subdivided into simple and complex DSBs, and each kind of DSB may have its own repair characteristics. A unique aspect of the TLK model is that break ends associated with both kinds of DSBs are allowed to interact in pairwise fashion to form irreversible lethal and nonlethal damages. To test the performance of the TLK model, nonlinear optimization methods are used to calibrate the model based on data for the survival of CHO cells for an extensive set of single-dose and split-dose exposure conditions. Then some of the postulated mechanisms of action are tested by comparing measured and predicted estimates of the initial DSB yield and the rate of DSB rejoining. The predictions of the TLK model for CHO cell survival and the initial DSB yield and rejoining rate are all shown to be in good agreement with the measured data. Studies suggest a yield of about 25 DSBs Gy(-1) cell(-1). About 20 DSBs Gy(-1) cell(-1) are rejoined quickly (15-min repair half-time), and 4 to 6 DSBs Gy(-1) cell(-1) are rejoined very slowly (10- to 15-h repair half-time). Both the slowly and fast-rejoining DSBs make substantial contributions to the killing of CHO cells by radiation. Although the TLK model provides a much more satisfactory formalism to relate biochemical processing of DSBs to cell killing than did the earlier kinetic models, some small differences among the measured and predicted CHO cell survival and DSB rejoining data suggest that additional factors and processes not considered in the present work may affect biochemical processing of DSBs and hence cell killing.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells

DNA double-strand breaks (DSB) represent a major disruption in the integrity of the genome. DSB can be generated when a replication fork encounters a DNA lesion. Recombinational repair is known to resolve such replication fork-associated DSB, but the molecular mechanism of this repair process is poorly understood in mammalian cells. In the present study, we investigated the molecular mechanism by which recombination resolves camptothecin (CPT)-induced DSB at DNA replication forks. The frequency of homologous recombination (HR) was measured using V79/SPD8 cells which contain a duplication in the endogenous hprt gene that is resolved by HR. We demonstrate that DSB associated with replication forks induce HR at the hprt gene in early S phase. Further analysis revealed that these HR events involve an exchange mechanism. Both the irs1SF and V3-3 cell lines, which are deficient in HR and non-homologous end joining (NHEJ), respectively, were found to be more sensitive than wild-type cells to DSB associated with replication forks. The irs1SF cell line was more sensitive in this respect than V3-3 cells, an observation consistent with the hypothesis that DSB associated with replication forks are repaired primarily by HR. The frequency of formation of DSB associated with replication forks was not affected in HR and NHEJ deficient cells, indicating that the loss of repair, rather than the formation of DSB associated with replication forks is responsible for the increased sensitivity of the mutant strains. We propose that the presence of DSB associated with replication forks rapidly induces HR via an exchange mechanism and that HR plays a more prominent role in the repair of such DSB than does NHEJ.     

Non-homologous end joining is the responsible pathway for the repair of fludarabine-induced DNA double strand breaks in mammalian cells

Fludarabine (FLU), an analogue of adenosine, interferes with DNA synthesis and inhibits the chain elongation leading to replication arrest and DNA double strand break (DSB) formation. Mammalian cells use two main pathways of DSB repair to maintain genomic stability: homologous recombination (HR) and non-homologous end joining (NHEJ). The aim of the present work was to evaluate the repair pathways employed in the restoration of DSB formed following replication arrest induced by FLU in mammalian cells. Replication inhibition was induced in human lymphocytes and fibroblasts by FLU. DSB occurred in a dose-dependent manner on early/middle S-phase cells, as detected by gammaH2AX foci formation. To test whether conservative HR participates in FLU-induced DSB repair, we measured the kinetics of Rad51 nuclear foci formation in human fibroblasts. There was no significant induction of Rad51 foci after FLU treatment. To further confirm these results, we analyzed the frequency of sister chromatid exchanges (SCE) in both human cells. We did not find increased frequencies of SCE after FLU treatment. To assess the participation of NHEJ pathway in the repair of FLU-induced damage, we used two chemical inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), vanillin and wortmannin. Human fibroblasts pretreated with DNA-PKcs inhibitors showed increased levels of chromosome breakages and became more sensitive to cell death. An active role of NHEJ pathway was also suggested from the analysis of Chinese hamster cell lines. XR-C1 (DNA-PKcs-deficient) and XR-V15B (Ku80-deficient) cells showed hypersensitivity to FLU as evidenced by the increased frequency of chromosome aberrations, decreased mitotic index and impaired survival rates. In contrast, CL-V4B (Rad51C-deficient) and V-C8 (Brca2-deficient) cell lines displayed a FLU-resistant phenotype. Together, our results suggest a major role for NHEJ repair in the preservation of genome integrity against FLU-induced DSB in mammalian cells.