hOGG1基因低表达对DNA氧化损伤及修复的影响初探

背景与目的:初步探讨采用核酶技术获得的hOGG1基因低表达的细胞对氧化剂诱导的DNA氧化损伤与修复作用的影响。材料与方法:以A549细胞和通过稳定转染hOGG1核酶而获得的hOGG1低表达的A549_R细胞为研究对象,分别以重铬酸钾和过氧化氢为受试物,比较两种细胞DNA损伤与修复的差异。彗星试验检测不同浓度受试物作用下两种细胞的彗星细胞率和DNA迁移长度;改良彗星试验比较两种细胞在去除受试物后孵育0、30、60、120和180min时的修复情况。结果:重铬酸钾和过氧化氢对A549_R细胞的DNA损伤效应敏感,在一些浓度下其彗星细胞率和DNA迁移长度明显高于A549细胞(P<0.05);A549_R细胞的DNA修复能力显著低于A549细胞(P<0.05)。结论:hOGG1基因的低表达可以增加细胞对氧化剂诱导的DNA损伤的敏感性,降低细胞DNA的修复能力。     

端粒酶抑制剂叠氮胸苷对HeLa细胞放射性DNA损伤修复的影响

背景与目的:研究端粒酶抑制剂叠氮胸苷(Azidothymidine,AZT)对人宫颈癌HeLa细胞DNA放射性损伤修复的影响,探讨端粒酶在放射诱导的DNA损伤修复中的作用.材料与方法:实验分为空白组,AZT组(400 μmol/L AZT处理HeLa细胞24 h),放射组(2 Gy 60Co γ射线照射),AZT放疗组(400 μmol/L AZT处理HeLa细胞24 h后,用2 Gy 60Co γ射线照射).照射后于0、5、10、30、60、180及360 min分别收集细胞,用端粒重复序列扩增法(PCR-based telomeric repeat amplification protocol,TRAP)-联合酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)即TRAP-ELISA法检测端粒酶的活性.用单细胞凝胶电泳法检测DNA单链断裂损伤,以彗尾DNA百分含量表示DNA单链断损伤量.结果:HeLa细胞受2 Gy 60Co γ射线照射后10 min,端粒酶活性即开始增加,60 min后增加明显,360 min时达到最高.AZT处理HeLa细胞后,能使端粒酶活性下降约50%,而且能抑制HeLa细胞照射后端粒酶活性的增加(P＜0.05).单细胞凝胶电泳实验表明,2 Gy 60Co γ射线照射HeLa细胞后0～10 min,AZT放疗组与放射组的彗尾DNA百分含量无明显差异(P＞0.05),照后30～360 min AZT放疗组彗尾DNA百分含量均高于放射组(P均＜0.05).结论:AZT能阻抑照射后30～360 min DNA单链断裂的修复,说明端粒酶可能在放射性DNA损伤修复中具有重要作用.     

DNA Repair: Translation to the Clinic

It has been well established that an accumulation of mutations in DNA, whether caused by external sources (e.g. ultraviolet light, radioactivity) or internal sources (e.g. metabolic by-products, such as reactive oxygen species), has the potential to cause a cell to undergo carcinogenesis and increase the risk for the development of cancer. Therefore, it is critically important for a cell to have the capacity to properly respond to and repair DNA damage as it occurs. The DNA damage response (DDR) describes a collection of DNA repair pathways that aid in the protection of genomic integrity by detecting myriad types of DNA damage and initiating the correct DNA repair pathway. In many instances, a deficiency in the DDR, whether inherited or spontaneously assumed, can increase the risk of carcinogenesis and ultimately tumorigenesis through the accumulation of mutations that fail to be properly repaired. Interestingly, although disruption of the DDR can lead to the initial genomic instability that can ultimately cause carcinogenesis, the DDR has also proven to be an invaluable target for anticancer drugs and therapies. Making matters more complicated, the DDR is also involved in the resistance to first-line cancer therapy. In this review, we will consider therapies already in use in the clinic and ongoing research into other avenues of treatment that target DNA repair pathways in cancer.     

WTK1细胞在tk位点突变和DNA损伤与修复中的应用

背景与目的:为WTK1细胞在遗传毒理学可同时应用于基因突变和DNA损伤的研究提供实验依据.材料与方法:分别用标准诱变剂甲基磺酸甲酯(MMS)和过氧化氢(H2O2)处理WTKl细胞,采用tk基因突变试验和单细胞凝胶电泳技术(Single Cell GelElectrophoresis,SCGE)对细胞的tk位点突变和过氧化氢诱发的DNA损伤情况进行检测.结果:甲基磺酸甲酯可诱发WTKl细胞tk位点的突变,以诱发染色体畸变为主.过氧化氢诱发了WTKl细胞DNA的损伤,并有剂量反应关系.随着修复孵育时间的延长,彗星细胞尾长和彗星细胞出现率明显下降,与对照组比较,差异有显著性(P＜0.01).结论:WTKl细胞可同时应用于tk基因突变和DNA损伤与修复的研究.采用该细胞株可对化合物进行基因突变和DNA损伤进行研究评价.     

DNA损伤修复基因hOGG1的遗传多态与肝癌易感性研究

目的:探索DNA损伤修复基因hOGG1的遗传多态Ser326Cys与肝细胞肝癌易感性的关系。方法:对96例原发性肝细胞肝癌患者和96例对照外周血DNA进行测序分型。结果:Ser/Cys杂合子个体的OR值为1.5,Cys/Cys纯合子个体的OR值为1.9,表现出剂量效应。结论:DNA修复基因hOGG1的Cys等位基因可能增加肝细胞肝癌的遗传易感性。     

应用DNA损伤与修复测定法研究肿瘤细胞的放射敏感性

 应用硷洗脱法研究了⁶⁰Cor射线及其联合高温对L(5178Y)细胞的DNA链断裂与修复的影响。 结果表明, 43℃加温30min能显著抑制r射线引起DNA断裂后的修复, 照前加温的增敏效果更好。 又应用羟基磷灰石层析法分析HL-60细胞和HL-60(VCR)细胞受照后两者DNA单链断裂程度无显著差异, 而DNA断链的修复能力有非常显著差异, 反映HL-60(VCR)细胞的重接修复能力高, 提示耐药的白血病细胞对辐射的敏感性较低。     

Novel Germline Mutations in DNA Damage Repair in Patients with Malignant Pleural Mesotheliomas

IntroductionAlthough next-generation sequencing (NGS) has brought insight into critical mutations or pathways (e.g., DNA damage sensing and repair) involved in the etiology of many cancers and has directed new screening, prevention, and therapeutic approaches for patients and families, it has only recently been used in malignant pleural mesotheliomas (MPMs).MethodsWe analyzed the blood samples from patients with MPM using the NGS platform MSK-IMPACT to explore cancer-predisposing genes. The loss-of-function variants or pathogenic entries were identified, and clinicopathologic information was collected.ResultsOf 84 patients with MPM, 12% (10 of 84) had pathogenic variants. Clinical characteristics were similar between cohorts, although patients with germline pathogenic variants were more likely to have more than two first-degree family members with cancer than those without germline mutations (40% versus 12%; Fisher’s exact test, p < 0.05). Novel, deleterious variants in mesotheliomas included MutS homolog 3 (1% [one of 84]; 95% confidence interval [CI]: 0%–7%), breast cancer gene 1-associated ring domain 1 (1% [one of 84]; 95% CI: 0%–7%), and RecQ-like helicase 4 (2% [two of 84]; 95% CI: 0%–9%). Pathogenic variants previously reported on germline testing in patients with mesotheliomas were breast cancer gene 1-associated protein 1 (4% [three of 84]; 95% CI: 1%–10%), breast cancer gene 2 (1% [one of 84]; 95% CI: 0%–7%), and MRE11 homolog, double strand break repair nuclease (1% [one of 84]; 95% CI: 0%–7%). One patient (1% [one of 84]; 95% CI: 0%–7%) had a likely pathogenic alteration in SHQ1, H/ACA ribonucleoprotein assembly factor that has not been associated with a heritable susceptibility to cancer.ConclusionsOur study lends further support for the role of aberrations in DNA damage repair genes in the pathogenesis of MPMs and suggests that targeting the members of these pathways for screening and treatment warrants further study.     

Changes in DNA Damage Repair Gene Expression and Cell Cycle Gene Expression Do Not Explain Radioresistance in Tamoxifen-Resistant Breast Cancer

Tamoxifen-induced radioresistance, reported in vitro, might pose a problem for patients who receive neoadjuvant tamoxifen treatment and subsequently receive radiotherapy after surgery. Previous studies suggested that DNA damage repair or cell cycle genes are involved, and could therefore be targeted to preclude the occurrence of cross-resistance. We aimed to characterize the observed cross-resistance by investigating gene expression of DNA damage repair genes and cell cycle genes in estrogen receptor-positive MCF-7 breast cancer cells that were cultured to tamoxifen resistance. RNA sequencing was performed, and expression of genes characteristic for several DNA damage repair pathways was investigated, as well as expression of genes involved in different phases of the cell cycle. The association of differentially expressed genes with outcome after radiotherapy was assessed in silico in a large breast cancer cohort. None of the DNA damage repair pathways showed differential gene expression in tamoxifen-resistant cells compared to wild-type cells. Two DNA damage repair genes were more than two times upregulated (NEIL1 and EME2), and three DNA damage repair genes were more than two times downregulated (PCNA, BRIP1, and BARD1). However, these were not associated with outcome after radiotherapy in the TCGA breast cancer cohort. Genes involved in G₁, G₁/S, G₂, and G₂/M phases were lower expressed in tamoxifen-resistant cells compared to wild-type cells. Individual genes that were more than two times upregulated (MAPK13) or downregulated (E2F2, CKS2, GINS2, PCNA, MCM5, and EIF5A2) were not associated with response to radiotherapy in the patient cohort investigated. We assessed the expression of DNA damage repair genes and cell cycle genes in tamoxifen-resistant breast cancer cells. Though several genes in both pathways were differentially expressed, these could not explain the cross-resistance for irradiation in these cells, since no association to response to radiotherapy in the TCGA breast cancer cohort was found.     

DNA Damage Response and Repair Pathway Alteration and Its Association With Tumor Mutation Burden and Platinum-Based Chemotherapy in SCLC

IntroductionImpairment in DNA damage response and repair (DDR) pathway is known as a predictive biomarker of platinum sensitivity. Recently, DDR alteration is re-emphasized as a predictive biomarker of immune checkpoint inhibitor due to its positive correlation to tumor mutation burden (TMB).MethodsTarget gene sequencing (381 genes) was conducted from 100 extensive disease (ED) and 66 limited disease (LD) patients with SCLC. Detected mutations were classified as double-strand breaks (DSB) (n = 82): homologous recombination (n = 54), non-homologous end joining (n = 19), and Fanconi anemia (n = 32); or single-strand breaks (SSB) (n = 31): mismatch repair (n = 19), base excision repair (n = 7), and nucleotide excision repair (n = 6).ResultsCompared to patients with an intact DDR pathway (n = 70), a higher TMB was observed in patients with homologous recombination (p < 0.001), non-homologous end joining (p = 0.002), mismatch repair (p < 0.001), DSB (p < 0.001), and SSB (p < 0.001). Survival analyses based on TMB level showed no predictive or prognostic values in ED patients. In LD patients, prolonged progression-free survival (hazard ratio [HR] = 0.497, p = 0.015), and overall survival (HR = 0.383, p = 0.010) to concurrent chemoradiotherapy were observed in those with TMB above median. Individual DDR pathway alteration showed no survival benefit in ED patients receiving platinum-based chemotherapy. In LD patients, those with mutations in the Fanconi anemia gene set had shorter progression-free survival (HR = 2.048, p = 0.036) to initial treatment.ConclusionsDDR pathway alterations, both DSB and SSB, in SCLC have a positive correlation with high TMB. However, it has shown limited value in prediction of platinum efficacy.     

Nuclear DNA Damage and Repair in Normal Ovarian Cells Caused by Epothilone B

This study was designed to assess, whether a new chemotherapeutic microtubule inhibitor, Epothilone B(EpoB, Patupilone), can induce DNA damage in normal ovarian cells (MM14.Ov), and to evaluate if such damagecould be repaired. The changes were compared with the effect of paclitaxel (PTX) commonly employed in theclinic. The alkaline comet assay technique and TUNEL assay were used. The kinetics of DNA damage formationand the level of apoptotic cells were determined after treatment with IC50 concentrations of EpoB and PTX. Itwas observed that PTX generated significantly higher apoptotic and genotoxic changes than EpoB. The peakwas observed after 48 h of treatment when the DNA damage had a maximal level. The DNA damage induced byboth tested drugs was almost completely repaired. As EpoB in normal cells causes less damage to DNA it mightbe a promising anticancer drug with potential for the treatment of ovarian tumors.     

MBD4基因的单核苷酸多态与肝细胞肝癌遗传易感性研究

目的:探索DNA损伤修复基因hMBD4的遗传多态Glu346Lys与肝细胞癌(hepatocellularcarcinoma,HCC)易感性的关系。方法:对96例原发性HCC患者和96例对照外周血DNA进行测序分型。结果:我们发现正常人群Glu/Glu、Glu/Lys、Lys/Lys基因型频率分别为40.4%、46.8%和12.8%,HCC组为41.7%、48.8%和9.5%。等位基因Lys频率在病例和对照分别为33.9%和36.2%,两组比较差异无统计学意义,P>0.05。结论:DNA修复基因hMBD4的Lys等位基因可能与HCC的遗传易感性无关。     

Biological Consequences of Radiation-induced DNA Damage: Relevance to Radiotherapy

DNA damage of exposed tumour tissue leading to cell death is one of the detrimental effects of ionising radiation that is exploited, with beneficial consequences, for radiotherapy. The pattern of the discrete energy depositions during passage of the ionising track of radiation defines the spatial distribution of lesions induced in DNA with a fraction of the DNA damage sites containing clusters of lesions, formed over a few nanometres, against a background of endogenously induced individual lesions. These clustered DNA damage sites, which may be considered as a signature of ionising radiation, underlie the deleterious biological consequences of ionising radiation. The concepts developed rely in part on the fact that ionising radiation creates significant levels of clustered DNA damage, including complex double-strand breaks (DSB), to kill tumour cells as clustered damage sites are difficult to repair. This reduced repairability of clustered DNA damage using specific repair pathways is exploitable in radiotherapy for the treatment of cancer. We discuss some potential strategies to enhance radiosensitivity by targeting the repair pathways of radiation-induced clustered damage and complex DNA DSB, through inhibition of specific proteins that are not required in the repair pathways for endogenous damage. The variety and severity of DNA damage from ionising radiation is also influenced by the tumour microenvironment, being especially sensitive to the oxygen status of the cells. For instance, nitric oxide is known to influence the types of damage induced by radiation under hypoxic conditions. A potential strategy based on bioreductive activation of pro-drugs to release nitric oxide is discussed as an approach to deliver nitric oxide to hypoxic tumours during radiotherapy. The ultimate aim of this review is to stimulate thinking on how knowledge of the complexity of radiation-induced DNA damage may contribute to the development of adjuncts to radiotherapy.     

MBD4基因的单核苷酸多态与肝细胞肝癌遗传易感性研究

目的：探索DNA损伤修复基因hMBD4的遗传多态Glu346Lys与肝细胞癌（hepatocellular carcinoma，HCC）易感性的关系。方法：对96例原发性HCC患者和96例对照外周血DNA进行测序分型。结果：我们发现正常人群Glu／Glu、Glu／Lys、Lys／Lys基因型频率分别为40．4％、46．8％和12．8％，HCC组为41.7％、48．8％和9．5％。等住基因Lys频率在病例和对照分别为33．9％和36．2％，两组比较差异无统计学意义，P〉0．05。结论：DNA修复基因hMBD4的Lys等位基因可能与HCC的遗传易感性无关。     

Efficacy of Hsp90 inhibition for induction of apoptosis and inhibition of growth in cervical carcinoma cells in vitro and in vivo

Purpose Heat shock protein 90 (Hsp90) is a conserved chaperone involved in crucial signaling events in normal and malignant cells. Previous research suggests that tumor cells are particularly dependent on Hsp90 for survival as well as malignant progression. Hsp90 inhibitors which are derivates of the natural compound geldanamycin, such as the orally bioavailable 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), are currently being tested in clinical trials and small molecule inhibitors are in development. In this study we investigated the response of a panel of cervical carcinoma cell lines in vitro and in vivo to determine potential factors that might influence the sensitivity towards Hsp90 inhibition. Methods Cell viability, proliferation and drug-induced changes on Hsp90 chaperoned “client” factors were examined with focus on G2/M cell cycle regulators, and a comparison with immortalized and normal keratinocytes was performed. ME180 and CaSki cells were grown as subcutaneous xenografts in mice treated with 6–10 mg/kg 17-DMAG by oral gavage 2×/day on a chronic schedule. Tissue concentrations of 17-DMAG were measured by high performance liquid chromatography. Results Cell death during abnormal mitosis was observed within 48 h after treatment start. ME180 and CaSki showed more cell death at this time point than SiHa and HeLa, and higher levels of pre-treatment Akt activity. IC₅₀ values ranged between 17 and 37 nanoM geldanamycin (MTS). Keratinocytes were at least as sensitive as carcinoma cells. All cell lines responded with an increase of the G2/M fraction. Despite in vitro effectiveness and tissue concentrations of 1 μM, only a limited tumor growth reduction was observed with 17-DMAG given close to the maximum tolerated dose level. Lower levels of Hsp90 protein, a lower Akt activity and signs of tissue hypoxia were observed in xenografts compared to cell cultures. Conclusions We show here that Hsp90 inhibition effectively induces apoptosis and growth arrest in cervical carcinoma cells in vitro. Mitotic catastrophe was identified as one mechanism of cell death. In contrast, a limited efficacy of 17-DMAG was observed in subcutaneous xenograft models. Induction of a heat shock response has previously been implicated in resistance towards Hsp90 inhibition. Additional factors might be (1) an altered abundance and/or activity of primary (Hsp90) and secondary (e.g., Akt) target(s), (2) a narrow therapeutic range of 17-DMAG by oral application and (3) response-modifying factors within the tumor environment. The further development of synthetic Hsp90 inhibitors with increased therapeutic window is warranted.     

Defective Repair of UV-induced DNA Damage in Cultured Primary Skin Fibroblasts from Saudi Thyroid Cancer Patients

This study was conducted to examine the sensitivity of primary skin fibroblasts from Saudi thyroid cancer (TC) ‍patients to ultraviolet (UV) irradiation. Cell survival was studied by a colony forming assay and DNA repair defects ‍with a host cell reactivation (HCR) assay using UV-irradiated Herpes Simplex Virus (HSV). In addition, p53 gene ‍expression was examined in the same TC cells exhibiting enhanced radiosensitivity. Skin fibroblasts from TC patients ‍(n=4) showed significantly enhanced sensitivity to UV radiation. The average UV dose to reduce survival to 37% of ‍the initial survival (D37) value (in Jm-2) for fibroblasts from TC patients was 4.6 (3.7-5.6) compared to 7.3 (6.3-8.3) for ‍healthy individuals (n=3). UV-sensitive xeroderma pigmentosum (XP) cells, which were used as positive control, ‍were found to be extremely sensitive with a D37 value of 0.6 Jm-2. In a host cell reactivation assay, UV-irradiated ‍HSV was tested for its plaque-forming ability (PFA), by plating infected fibroblasts from TC patients (used as host ‍cells) on African Green Monkey (Vero) kidney cells to form plaques. A significant reduction in the PFA of the UVirradiated ‍virus (about three fold) on TC cells compared to fibroblasts from the healthy subjects was seen, suggesting ‍a DNA-repair deficiency in the primary fibroblasts of the TC patients. Furthermore, no significant accumulation in ‍radiation-induced p53 expression was observed in cells from the TC patients. Our results, based on a relatively ‍small group of subjects, indicate that Saudi TC patients primary fibroblasts (non-cancerous in nature) may be ‍carriers of cancer-susceptible gene(s) arising from defective DNA repair/processing. These results warrant a larger ‍study to investigate the role of UV-induced bulky DNA damage in thyroid cancer susceptibility. ‍     

Inhibition of Nuclear PTEN Tyrosine Phosphorylation Enhances Glioma Radiation Sensitivity through Attenuated DNA Repair

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三丁基锡引起人羊膜细胞氧化损伤及DNA损伤的研究

背景与目的： 研究三丁基锡 (tributyltin,TBT)对人羊膜细胞FL (human amnion cells) 氧化损伤和DNA损伤的诱导作用。 材料与方法： 将不同浓度TBT (0、2、4、6、8、10 μmol/L),分别对FL染毒2 h和4 h,各染毒组同时设不加TBT的对照组,染毒后分别用MTT法检测TBT对FL细胞增殖率的影响,用DCFH_DA法检测FL细胞活性氧自由基 (ROS)水平,用彗星实验检测TBT对FL细胞DNA的损伤。 结果： TBT对FL细胞染毒4 h时,其2、8、10 μmol/L浓度组的细胞增殖率较对照组显著下降(P<0.05,P<0.01,P<0.001),且随TBT浓度升高而增殖率呈下降的趋势。TBT 3、4 μmol/L染毒组FL细胞的ROS水平较对照组升高,且4 μmol/L染毒组与对照组比较差异具有统计学意义(P<0.05)。在TBT 2、3、4 μmol/L染毒组随着TBT浓度的升高,FL细胞核尾长、尾相均显著升高(P均<0.05)。 结论： TBT可引起FL细胞的氧化损伤及DNA损伤。