EGFR抑制剂调节DNA双链断裂修复对放射敏感度影响的研究进展

0 引言 肿瘤放射治疗过程中DNA损伤具有多样性,包括碱基和核苷酸水平的损伤以及DNA链断裂等.其中DNA双链断裂(double-strand breaks,DSBs)是最常见的损伤,DSBs的修复是一个很复杂的过程,通过以下两条途径进行修复:非同源末端连接(nonhomologous end-joining,NHEJ)和同源重组修复(homologous recombination,HR).NHEJ途径中不需要模版染色体,DNA断端直接进行连接,是一种不精确的应急修复,主要修复G0、G1和早S期的绝大多数DSBs,也修复少量在其他细胞周期产生的DSBs.而HR途径需要姐妹染色体作为复制修复模版,从而进行精确修复,主要修复晚S期及G2期的DSBs[1].因此,抑制DNA修复通路,包括NHEJ和HR,能增加放射敏感度,提高肿瘤治疗疗效.     

DSB修复蛋白ATM DNA-PKcs与肿瘤放射敏感性关系的研究进展

放射线主要通过导致细胞DNA双链断裂(DSB)而起到杀死肿瘤细胞的作用,但细胞都有不同程度的DSB修复能力,研究证实DSB 修复水平与细胞放射敏感性关系密切.在人类细胞内有2 种DSB 修复途径:一种是以DNA 依赖蛋白激酶(DNA-PK)复合物为主的非同源末端连接(NHEJ)修复,另一种是以毛细血管扩张性共济失调症突变蛋白(ATM)为主的同源重组(HR)修复.在人体内,NHEJ修复是最主要的修复途径,DNA依赖蛋白激酶催化亚单位(DNA-PKcs)是DNA-PK复合物的主要功能单位.DNA-PKcs的激酶活性是NHEJ修复所必须的,其在DSB修复中起核心作用.近年来的研究显示ATM、DNA-PKcs蛋白表达水平与肿瘤放射敏感性有关.该综述将有关ATM、DNA-PKcs的功能、在肿瘤组织中的表达及与肿瘤放射敏感性关系的研究进行简要回顾.     

小鼠DNA双链断裂修复缺陷细胞的γ射线剂量率效应

目的：探讨γ射线照射后小鼠DNA双链断理解修复缺陷细胞（SCID）的剂量率效应和潜在致死性损伤的修复。方法：采用低剂量率和高剂量率以及间隔24h的2次γ射线照射正常细胞（CB．17＋／＋）和SCID细胞，通过成克隆分析法观察被照射细胞的存活分数。结果：应用间隔24h的2次γ射线照射CB．17＋／＋细胞时，其存活分数明显高于相同剂量的单次照射，而SCID细胞二者无明显差异。在高剂量率单次和2次γ射线照射时，SCID细胞均比CB．17＋／＋细胞更敏感。在低剂量率γ射线照射时，SCID细胞亦显示比CB．17＋／＋细胞更敏感。低剂量率γ射线照射 CB．17＋／＋细胞和SCID细胞后，二者的存活分数均明显高于剂量率照射。结论：SCID细胞不具有DNA双链断裂的修复能力。SCID细胞和CB．17＋／＋细胞均具有剂量率效应。     

叠氮胸苷对人胶质瘤细胞辐射后DNA双链损伤修复的影响

目的 观察逆转录酶抑制剂叠氮胸苷（AZT）对人脑胶质瘤U251放射性DNA损伤双链修复（DSB），探讨其放射增敏的机制。方法实验分为4个组：空白组：未行AZT与7一射线处理；放射组：细胞接受2Gyγ射线单次照射；加药组：细胞培养液中加入终浓度为0.8mm／L的AZT，作用24h；药放组：细胞经过0．8mm／L的AZT作用24h后，再行2Gyγ射线单次照射。用中性单细胞凝胶电泳方法检测辐射后DNA双链断裂（尾矩）。结果 空白组与加药组细胞无慧尾，表明AZT本身不会导致DNADSB。而放射组和药放组均出现明显的彗星图象，药放组的初始DSB与放射组相比无显著性差异（P〉0.05）。但前者的修复速度较慢，照射后2h放射组的DSB基本修复完全，而药放组仍有50％的残留。结论 AZT的放射增敏作用机制与其对DNADSB的修复有关。     

DNA双链断裂修复抑制与放射增敏

全文着眼于近期以抑制肿瘤细胞DNA双链断裂(DSBs)修复为放射增敏策略的相关研究:加温、化疗、DSBs修复基因或蛋白抑制剂和表皮生长因子受体抑制剂,以进一步探讨放射增敏的生物学机制,并为临床综合治疗提供依据.     

DNA双链断裂修复通路和卵巢癌的关系

卵巢癌发生发展与DNA损伤累积引起的基因不稳定性和细胞行为异常密切相关。DNA双链断裂修复通路中最为重要的是同源重组和非同源末端连接机制以及最近发现的微同源介导的末端修复形式,能够快速准确地修复DNA双链断裂,对维持基因组稳定性起着至关重要的作用,对卵巢癌的基因诊断、基因治疗以及卵巢癌分子水平研究均有重要意义。     

抑制DNA双键断裂修复对肿瘤细胞的放射增敏作用

DNA双键断裂(DSB)是放射引起DNA损伤的主要机制之一,它的修复途径包括同源重组(HR)和非同源末端连接(NHEJ)两种方式.近年来国内外研究发现化疗药物的应用、温热疗法、抑制修复基因和蛋白的表达及表皮生长因子受体抑制剂等可以抑制DSB的修复,从而增强肿瘤细胞的放射增敏性.     

中性单细胞凝胶电泳测定DNA双链断裂

目的 :检测人类肿瘤细胞系统经 X线照射产生的 DNA双链断裂 (DSB)及其修复过程 ,初步验证双链断裂及其修复与细胞放射敏感性的关系。材料与方法 :采用中性单细胞凝胶电泳定量测定 DSB的实验方法 ("彗星 "法 ) ,测定了细胞 SF2差异较大的 He La和 GL C细胞的 DSB及 DSB修复动力学过程。结果 :不同剂量 X线照射引起的细胞 DSB呈显著的线性关系 ,与细胞 SF2不存在相关性 ,但其修复与 SF2具有一定相关性。结论 :X线导致细胞 DSB的修复能力比 DSB初始断裂数更能反映细胞的放射敏感性     

缺氧条件下DNA双链断裂修复机制变动及其意义

实体肿瘤中相当一部分细胞处于乏氧的微环境中。乏氧是细胞对放化疗抵抗的重要原因之一。DNA双链断裂是最严重的DNA损伤,也是放疗杀伤肿瘤细胞的分子基础。在缺氧的情况下,肿瘤细胞发生一系列适应性变化,双链断裂修复机制也随之受到影响。非同源末端连接修复(NHEJ)和同源重组修复(HRR)是最主要的两个DNA双链断裂修复途径。在缺氧情况下,NHEJ活性得到增强,而HRR活性则受到削弱。这一变化对肿瘤的生物学行为产生影响,同时也为我们提供了提高乏氧肿瘤细胞杀伤率的新的思考角度。     

真核细胞DNA双链断裂修复通路的选择和调节

许多外源及内源性因素均可导致DNA损伤,从而对遗传信息的有效传递甚至生物体的生存造成极大的威胁。真核细胞依靠长期进化出的DNA损伤应激反应(DDR)系统来对抗DNA损伤。DNA双链断裂(DSB)是最为严重的损伤形式之一,DSB修复失败会导致细胞死亡、遗传疾病以及肿瘤发生等严重后果。真核细胞中主要存在2种DSB修复方式:同源重组(HR)和非同源末端连接(NHEJ)。本文介绍了HR和NHEJ通路的执行过程和参与成员,并对当前HR和NHEJ修复通路选择和调节机制的最新研究进展进行综述。     

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair

Purpose: From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. Materials and methods: DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Results: Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37?°C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. Conclusion: It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.     

ＤＮＡ双链断裂修复通路与遗传性乳腺癌的关系

遗传性乳腺癌具有家族聚集、早发、双侧等特点,多为易感基因发生胚系突变所致。ＤＮＡ损伤修复是哺乳动物细胞保证遗传物质稳定性的重要机制。双链断裂是最严重的ＤＮＡ损伤之一,修复过程涉及同源重组和非同源末端连接通路。ＤＮＡ双链断裂修复或信号传导相关基因或蛋白功能缺陷可以诱导染色体不稳定而增加乳腺癌的易感性。与ＤＮＡ修复功能相关的链交联剂和ＰＡＲＰ-１抑制剂为ＢＲＣＡ相关遗传性乳腺癌的治疗提供了新的途径。本文就ＤＮＡ双链断裂修复通路相关基因的突变与遗传性乳腺癌发病的关系作一综述。     

Initiation of DNA double strand break repair: signaling and single-stranded resection dictate the choice between homologous recombination, non-homologous end-joining and alternative end-joining

A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.     

Evaluation of cell survival, DNA double strand breaks, and DNA synthesis during concurrent camptothecin and X-radiation treatments

We evaluated cell survival, DNA double strand breaks (dsbs), and DNA synthesis following camptothecin (CPT) alone or concurrent CPT and X-radiation treatments in exponential-phase cultures of a radioresistant human melanoma cell line. Cell survival was measured by a clonogenic assay. DNA dsbs were measured by pulsed-field gel electrophoresis. DNA synthesis was measured by incorporation of (3)H-thymidine. We found that (i) concurrent CPT and X-radiation interacted additively, unlike previous results with plateau-phase cultures of these cells, which showed synergistic interaction; (ii) there were strong negative correlations (correlation coefficients of at least 0.82) between clonogenic surviving fractions and DNA dsbs following CPT alone or concurrent CPT and radiation treatments; and (iii) concurrent CPT and radiation (10 Gy) treatment did not completely inhibit DNA synthesis, even though addition of radiation to CPT did further decrease DNA synthesis (relative to CPT alone) at CPT concentrations below 20 microM. Our results suggest that during concurrent CPT and radiation treatment residual DNA dsb levels were good indicators of cell killing and that the absence of complete inhibition of DNA synthesis could at least in part explain the additive interaction between CPT and radiation.     

Variants in DNA double-strand break repair and DNA damage-response genes and susceptibility to lung and head and neck cancers

Cigarette smoking is the major risk factor for lung cancer, and together with alcohol for head and neck (H--N) cancer. These genotoxics produced DNA damage and particularly double-strand breaks (DSB) that are removed by various repair pathways. To understand the initiation of these cancers, we performed a genotype analysis to correlate some variants in specific genes in a case-control study of lung and H-N cancers. In a discovery phase, we sequenced DNA samples of 32 healthy Caucasians to describe genetic variants in 30 genes involved in the repair of DSB and in DNA damage response. 625 variants were detected on 29 out of the 30 genes successfully screened by sequencing exons, parts of introns and flanking regions. These included 470 non-exonic variants, from which 33 insertions/deletions, and 155 exonic alterations, corresponding to 59 non synonymous polymorphisms. 223 of these variants were not previously described. In total, 379 variants were successfully genotyped in a case-control study restricted to smokers including 151 lung cases, 251 H-N cases, and 172 controls. To account for multiple testing, we associated to each p-value a proportion of false positives (q-value). Haplotype-analysis suggested potential associations (p < 0.05) between lung cancer and 2 genes (RECQL4 and RAD52), which came with q-value of 8%, and between H-N cancer and 1 gene (DNA-PK) but with q-value of 56%. The 3 genes are key players for regulating the efficiency of DSB repair. Large-scale studies are needed to show if any of these 3 variants are truly associated with an increased risk of cancer.     

Maintenance of genomic integrity after DNA double strand breaks in the human prostate and seminal vesicle epithelium: The best and the worst

Prostate cancer is one of the most frequent cancer types in men, and its incidence is steadily increasing. On the other hand, primary seminal vesicle carcinomas are extremely rare with less than 60 cases reported worldwide. Therefore the difference in cancer incidence has been estimated to be more than a 100,000-fold. This is astonishing, as both tissues share similar epithelial structure and hormonal cues. Clearly, the two epithelia differ substantially in the maintenance of genomic integrity, possibly due to inherent differences in their DNA damage burden and DNA damage signaling. The DNA damage response evoked by DNA double strand breaks may be relevant, as their faulty repair has been implicated in the formation of common genomic rearrangements such as TMPRSS2-ERG fusions during prostate carcinogenesis. Here, we review DNA damaging processes of both tissues with an emphasis on inflammation and androgen signaling. We discuss how benign prostate and seminal vesicle epithelia respond to acute DNA damage, focusing on the canonical DNA double strand break-induced ATM-pathway, p53 and DNA damage induced checkpoints. We propose that the prostate might be more prone to the accumulation of genetic aberrations during epithelial regeneration than seminal vesicles due to a weaker ability to enforce DNA damage checkpoints.     

Fluorodeoxyuridine-induced radiosensitization and inhibition of DNA double strand break repair in human colon cancer cells

The halogenated pyrimidine, fluorodeoxyuridine (FdUrd), has been used in combination with radiation for the treatment of human neoplasms. In an attempt to improve the clinical use of this combination, FdUrd-radiation interactions were studied in vitro using human HT29 colon cancer cells. It was found that FdUrd produced radiosensitization at clinically achievable (1-100 nM) concentrations. Sensitization depended critically on the timing of exposure. When cells were irradiated after a 12-hr exposure to 100 nM FdUrd, marked sensitization was produced (mean inactivation dose (MID) = 2.01 +/- 0.01, compared to control of 4.35 +/- 0.16, p less than .01). No radiosensitization occurred when cells were irradiated 4 hr prior to incubation (MID = 3.95 +/- 0.05, p greater than 0.4). Radiosensitization appeared to result from an inhibition of thymidylate synthase since concentrations of FdUrd which produced radiosensitization depleted intracellular TTP pools and blocked the incorporation of deoxyuridine into DNA. Furthermore, radiosensitization was completely inhibited by co-incubation with thymidine. FdUrd also decreased the repair, but not the formation, of radiation-induced DNA double strand breaks (DSB's). These data are consistent with the hypothesis that FdUrd produces radiosensitization by depleting thymidine pools which leads to a decreased rate of DNA DSB repair. Furthermore, they suggest that in clinical trials FdUrd should be infused at least 8 hr before irradiation.     

Effects of inhibitors of DNA strand break repair on HeLa cell radiosensitivity

The effects of three drugs (hydroxyurea, 1-beta-arabinofuranosylcytosine, and diamide) known to inhibit DNA synthesis on the repair of ionizing radiation-induced DNA single-strand breaks measured by alkaline elution and on cellular radiosensitivity were examined. Inhibition of repair was observed at 10(-2) M hydroxyurea, 10(-4) M 1-beta-D-arabinofuranosylcytosine, and 5 X 10(-5) M diamide, levels causing only 10% cell kill. While the mechanisms by which the drugs inhibit DNA synthesis differ, they are equally effective at inhibiting repair; without drug, cells, after a dose of 10 grays, repair 35% of DNA strand breaks in 3 min and a further 35% in 1 hr; with drug, only 10% is repaired in 3 min, and the deficiency in repair amount remains, even after 60 min. The effect of similar drug treatment on radiation-induced cell killing shows that radiosensitivity is increased; the major effect is reduction in D0 from 1.3 grays to approximately 0.8 grays with smaller effects on Dq. The data are consistent with the hypothesis that radiation produces potential double-strand breaks in DNA which, if not rapidly repaired, are converted into lethal actual double-strand breaks.     

In tumor cells regulation of DNA double strand break repair through EGF receptor involves both NHEJ and HR and is independent of p53 and K-Ras status

Purpose

The purpose of this study was to examine whether the epidermal growth factor receptor (EGFR) may be used as a general target to modulate DNA double strand break (DSB) repair in tumor cells.

Material and methods

Experiments were performed with human tumor cell lines A549, H1299 and HeLa and primate cell line CV1. EGF, ARG and TGFα were used for EGFR activation, cetuximab or erlotinib for inhibition. Overall DSB repair was assessed by γH2AX/53BP1 co-immunostaining and non-homologous end-joining (NHEJ) and homologous recombination (HR) by using NHEJ and HR reporter cells; cell cycle distribution was determined by flow cytometry and protein expression by Western blot.

Results

EGFR activation was found to stimulate overall DSB repair as well as NHEJ regardless of the ligand used. This stimulation was abolished when EGFR signaling was blocked. This regulation was found for all cell lines tested, irrespective of their p53 or K-Ras status. Stimulation and inhibition of EGFR were also found to affect HR.

Conclusions

Regulation of DSB repair by EGFR involves both the NHEJ and HR pathway, and appears to occur in most tumor cell lines regardless of p53 and K-Ras mutation status.