Functional analysis of XRCC4 mutations in reported microcephaly and growth defect patients in terms of radiosensitivity

Non-homologous end joining is one of the main pathways for DNA double-strand break (DSB) repair and is also implicated in V(D)J recombination in immune system. Therefore, mutations in non-homologous end-joining (NHEJ) proteins were found to be associated with immunodeficiency in human as well as in model animals. Several human patients with mutations in XRCC4 were reported to exhibit microcephaly and growth defects, but unexpectedly showed normal immune function. Here, to evaluate the functionality of these disease-associated mutations of XRCC4 in terms of radiosensitivity, we generated stable transfectants expressing these mutants in XRCC4-deficient murine M10 cells and measured their radiosensitivity by colony formation assay. V83_S105del, R225X and D254Mfs^*68 were expressed at a similar level to wild-type XRCC4, while W43R, R161Q and R275X were expressed at even higher level than wild-type XRCC4. The expression levels of DNA ligase IV in the transfectants with these mutants were comparable to that in the wild-type XRCC4 transfectant. The V83S_S105del transfectant and, to a lesser extent, D254Mfs^*68 transfectant, showed substantially increased radiosensitivity compared to the wild-type XRCC4 transfectant. The W43R, R161Q, R225X and R275X transfectants showed a slight but statistically significant increase in radiosensitivity compared to the wild-type XRCC4 transfectant. When expressed as fusion proteins with Green fluorescent protein (GFP), R225X, R275X and D254Mfs^*68 localized to the cytoplasm, whereas other mutants localized to the nucleus. These results collectively indicated that the defects of XRCC4 in patients might be mainly due to insufficiency in protein quantity and impaired functionality, underscoring the importance of XRCC4’s DSB repair function in normal development.     

In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells.     

吸烟与温石棉单独及联合作用对人胚肺细胞DNA链断裂的影响

目的研究吸烟与温石棉对细胞的遗传毒性。方法采用单细胞凝胶电泳技术（ＳＣＧＥ），在体外实验中测定了香烟烟雾溶液（ＣＳＳ）与温石棉（ＣＨ）单独及联合作用于人胚肺（ＨＥＬ）细胞时ＤＮＡ链断裂的情况。结果ＣＳＳ与ＣＨ单独与ＨＥＬ细胞作用１小时，均可使ＤＮＡ链断裂明显增多，当二者以３种不同的剂量组合联合作用时，均可协同增加ＤＮＡ链断裂。结论ＣＳＳ和ＣＨ单独作用均为肿瘤启动剂；当二者联合作用时可协同启动肿瘤的发生。     

苯与甲醛致小鼠睾丸细胞DNA损伤联合作用

目的探讨苯与甲醛致小鼠睾丸细胞DNA联合损伤效应。方法采用单细胞凝胶电泳方法，分别检测苯染毒（剂量为50，200，800mg／kg）、甲醛染毒（剂量分别为0．3，1．2，4．8mg／kg）和苯＋甲醛复合染毒后小鼠睾丸细胞DNA损伤情况。结果在受试剂量范围内，小鼠睾丸细胞彗星率随苯或甲醛染毒剂量增加而增加（P〈0．01），其彗星尾长随苯染毒剂量增加而延长（P〈0．01），随甲醛染毒剂量增加而缩短，尤其高甲醛染毒组（P〈0．01）；复合染毒组中彗星尾长随联合染毒中苯染毒剂量增加而延长（P〈0．01），随联合染毒中甲醛染毒剂量增加而缩短（P〈0．01）。苯＋甲醛联合作用经析因素分析存在相加作用（P〈0.01）。结论苯和甲醛均能彼此增强各自所致的睾丸细胞DNA损伤效应。     

社会支持在妇科癌症患者应对适应能力与生活质量间的中介效应

目的 探讨社会支持在妇科癌症患者应对适应能力与生活质量间的中介效应。方法 随机抽取唐山市3所三级甲等医院,再整群纳入于2021年8月—2022年1月在这3所医院住院治疗的310例妇科癌症患者,采用汉化版应对适应过程量表、社会支持评定量表、欧洲癌症研究和治疗组织生活质量量表进行调查。采用SPSS 22.0对数据进行描述性分析、单因素分析、Pearson相关分析和多元线性回归分析,AMOS 22.0和bootstrap方法建立中介模型并对其进行检验。结果 妇科癌症患者的应对适应能力、社会支持均处于中等水平,分别为（42.24±6.33）分、（38.84±7.47）分;生活质量较差,为（57.79±18.89）分。应对适应能力、社会支持、生活质量三个变量间呈正相关（r=0.481,P<0.01;r=0.409,P<0.01;r=0.393,P<0.01）;且社会支持在应对适应能力与生活质量间起部分中介作用,中介效应值为0.120 (95%CI:0.047,0.216),中介效应占总效应的22.35%。结论 社会支持是妇科癌症患者应对适应能力与生活质量间的中介变量,医护人员可...     

木犀草素对脑胶质瘤细胞氨肽酶N活性影响

目的探讨木犀草素对脑胶质瘤细胞氨肽酶N活性影响机制。方法本研究采用酶抑制动力学方法, 研究了木犀草素对氨肽酶N的抑制作用、抑制类型和抑制动力学常数, 并进行了脑胶质瘤U87细胞的凋亡生长抑制试验。结果木犀草素能够诱导了脑胶质瘤U87细胞的凋亡, 并呈现一定的剂量依赖性, 当浓度≥10 μmol时, 与阳性对照bestatin 相比差异有统计意义(P<0.05), 求得半细胞凋亡率IC₅₀为(61.23±2.13)μmol;此外木犀草素是一种可逆的竞争型氨肽酶N抑制剂, 半抑制率IC₅₀为(70.85± 2.05)μmol, 抑制常数Ki为(24.83± 0.14)μmol;失活动力学时间进程分析表明,木犀草素能快速与氨肽酶N发生作用并迅速降低酶的活性。结论木犀草素是一个竞争性的氨肽酶N抑制剂, 使氨肽酶N的活性降低, 对诱导了脑胶质瘤U87细胞的凋亡起到重要作用。     

厄洛替尼联合山奈酚诱导卵巢癌细胞凋亡协同作用

目的 探讨厄洛替尼和山奈酚对表皮生长因子受体酪氨酸激酶(EGFR-TPK)的抑制作用,并阐明两者诱导卵巢癌细胞凋亡的协同作用机制。方法 在EGFR-TPK、SKOV-3人卵巢癌细胞株中加入不同浓度(0.1、0.5、1.0、2.5、5.0 mg/mL)山奈酚与厄洛替尼,采用酶联免疫吸附试验(ELISA)测定山奈酚和厄洛替尼对EGFR-TPK的抑制作用,四甲基偶氮唑盐(MTT)法测定山奈酚和厄洛替尼对SKOV-3人卵巢癌细胞株生长的抑制作用和协同效应,酶标仪法检测山奈酚和厄洛替尼对细胞凋亡蛋白caspase-3活性的影响。结果 山奈酚和厄洛替尼对EGFR-TPK的半抑制率(IC₅₀)分别为(2.33±0.32)和(1.75±0.18) mg/mL,差异无统计学意义(P>0.05);与空白对照组比较,0.1、0.5、1.0、2.5、5.0 mg/mL山奈酚和厄洛替尼组肿瘤细胞转移数量均减少,差异均有统计学意义(P<0.05);山奈酚和厄洛替尼协同能力检测结果显示,0.5 mg/mL厄洛替尼与0.5、2.5和5.0 mg/mL山奈酚联合使用对抑制SKOV-3人卵巢癌细胞生长均具有协同效应,2.5和5.0 mg/mL厄洛替尼与0.5、2.5和5.0 mg/mL山奈酚联合使用对抑制SKOV-3人卵巢癌细胞生长均具有加和效应。结论 山奈酚联合厄洛替尼能够诱导SKOV-3人卵巢癌细胞的凋亡,并对SKOV-3人卵巢癌细胞转移起到抑制作用。