X线照射对鼻咽癌细胞hMSH2和hMLH1表达的影响

目的研究X线照射对鼻咽癌细胞中错配修复基因hMSH2和hMLH1表达的影响,探讨放射损伤后肿瘤细胞的DNA错配修复机制。方法将鼻咽癌CNE-1细胞分为实验组和对照组,对2组细胞进行X线分次外照射。实验组每次照射剂量为2Gy,总剂量为10Gy。对照组每次照射剂量为0Gy。应用RT-PCR、免疫细胞化学及Western blot方法,检测照射终止后不同时间点细胞hMSH2和hMLH1的表达。结果实验组细胞在照射后hMSH2mRNA和蛋白的表达均显著强于对照组(P<0.01);hMLH1mRNA及蛋白表达与对照组比较无显著差异(P>0.05)。结论放射可以诱导鼻咽癌细胞hMSH2表达;hMSH2对放射造成的DNA损伤可能有修复作用,这可能是临床上鼻咽癌放疗敏感性降低的原因之一。     

Celecoxib对放射所致DNA损伤修复与凋亡的影响

目的探讨选择性COX-2抑制剂celecoxib影响肺腺癌A549细胞凋亡及DNA损伤修复的放疗增敏机制。方法 A549细胞分为对照组(相同体积的DMSO)、药物组(celecoxib)、照射组(6MV X射线6 Gy)、联合组(celecoxib+6Gy X射线(药物作用24 h后))。CCK-8法检测celecoxib对肺腺癌A549细胞的IC50。RT-PCR、Western blot法检测细胞DNA-PKcs、Ku80基因mRNA及蛋白表达。流式细胞仪检测各组细胞凋亡率。结果 Celecoxib抑制A549细胞增殖作用呈剂量和时间依赖性,48 h的IC50值为58.74μmol/L。联合组DNA-PKcs、Ku80基因mRNA及蛋白表达量明显低于对照组、药物组、照射组(P<0.01)。联合组细胞凋亡率明显高于对照组、药物组和照射组(P<0.01)。结论 Celecoxib通过抑制放射所致DNA损伤修复及促进凋亡,从而达到放疗增敏作用。     

“彗星”分析法检测鼻咽癌细胞DNA放射损伤与修复

目的　探讨“彗星”分析法(CometAssay)检测鼻咽癌细胞DNA放射与修复反应的可能性。　方法　采用碱性“彗星”分析法的微胶电泳技术,定量检测评价人高、低分化鼻咽癌细胞系CNE1和CNE2Z细胞在接受X射线照射后,其DNA单链断裂(SingleStrandBreaks,SSBs)的程度,及其与放射剂量的关系和DNA的SSBs在不同检测时间的自身修复情况。　结果　用碱性“彗星”分析法可准确地检测出2Gy单一放射剂量所诱导的两系细胞DNA的SSBs;可见DNA损伤程度在不同照射剂量其结果不同,随剂量递增DNA的损伤加重,与放射剂量呈良好线性相关。15Gy诱导两系细胞的DNA损伤后,10min即可定量检测出受损DNA的确切恢复变化,在照射30min内,其修复较迅速,其损伤程度可降至初时的一半以下,其后修复缓慢,约需持续2h才能复至无照射细胞水平。　结论　“彗星”分析法检测结果能准确反映人鼻咽癌细胞DNA的定量损伤程度及其与放射剂量的关系,以及损伤后自身修复能力在受测时间中的定量变化情况     

沉默CDKN1A基因对鼻咽癌放射抗拒性CNE-2R细胞放射敏感性的影响

目的：探讨CDKN1A基因的表达与鼻咽癌放射敏感性的关系。方法构建慢病毒表达载体LV-CDKN1A-RNAi并转染鼻咽癌放射抗拒性CNE-2R细胞,设转染LV-CDKN1A-RNAi慢病毒的CNE-2R细胞为实验组,转染阴性对照慢病毒的CNE-2R细胞为阴性对照组,未转染的CNE-2R细胞为空白对照组。用CCK-8法、细胞克隆形成实验及流式细胞术分别检测各组细胞增殖、放射敏感性及细胞周期的变化。结果成功构建了CDKN1A基因沉默的CNE-2R细胞,CCK-8法检测显示实验组CNE-2R细胞在照射6 Gy后生长受到抑制,且随时间延长其抑制作用更为明显。细胞克隆形成实验显示实验组CNE-2R细胞放射敏感性增强（放射增敏比为SER=1.24）。流式细胞术检测显示实验组与对照组细胞相比, G0/G1期和G2/M期细胞分布在X射线照射6 Gy前后明显改变（P约0.05）。结论 CDKN1A基因沉默能增强鼻咽癌放射抗拒性CNE-2R细胞的放射敏感性,CDKN1A基因的表达可能与鼻咽癌放射敏感性相关,有望成为鼻咽癌治疗的新靶点。     

siRNA沉默Annexin A2基因表达对鼻咽癌细胞放射敏感性的影响

目的 探讨siRNA沉默Annexin A2基因表达对鼻咽癌CNE-2(R743)细胞放射敏感性的影响。方法 化学合成Annexin A2基因的siRNA经HiPerFect转染入R743细胞。RT-PCR和蛋白印迹法检测转染前后Annexin A2的mRNA和蛋白表达,克隆形成实验分析转染前后对细胞放射敏感性的影响,流式细胞仪和TUNEL实验分别检测转染前后经X线照射后细胞周期、细胞凋亡情况。结果 RT-PCR和蛋白印迹法结果显示转染组细胞Annexin A2基因及蛋白表达下调。克隆形成实验分析结果表明转染组D₀、D_q、SF₂值均低于单纯照射组和转染对照组,其相应放射增敏比(D₀值比)分别为1.30和1.27。X线照射后转染组细胞G₂+M期比例增加并高于单纯照射组和转染对照组(32.46%、9.42%、9.17%,P=0.000、0.000),转染组凋亡率也高于单纯照射组和转染对照组(35.20%、10.87%、11.33%,P=0.000、0.000)。结论 siRNA沉默Annexin A2基因表达可增强R743细胞放射敏感性,可能与DNA损伤修复、细胞周期时相分布变化有关。     

不同时间点电离辐射对胶质瘤C6细胞垂体瘤转化基因影响的实验研究

目的 探讨不同时间点电离辐射对胶质瘤c6细胞垂体瘤转化基因(PTTG)表达的影响.方法 胶质瘤C6细胞用2 Gy剂量X射线照射,逆转录聚合酶链式反应(RT-PCR)检测照射前、照射后4 h、8 h、12 h、24 h、48 h、72 hPTrG mRNA的表达,采用免疫细胞化学检测FITG蛋白表达.结果 FITG mRNA及其蛋白的表达水平在X射线照射后4 h即开始下降,12 h下降最明显,24 h开始回升,72 h恢复正常.结论 电离辐射可以显著影响胶质瘤细胞PTTG的转录和翻译;机体内存在对电离辐射导致的PTTG损伤的修复系统.     

放射诱导多倍体乳腺癌细胞衰老和自噬

目的探讨衰老和自噬在放射诱导的多倍体乳腺癌细胞中的作用。方法在6 MV X射线模式下用7 Gy剂量照射乳腺癌MDA⁃MB⁃231细胞,分别于第3天(Day 3组)、5天(Day 5组)、7天(Day 7组)、11天(Day 11组)和19天(Day 19组)观察细胞形态变化,采用流式细胞术检测细胞倍性,β⁃半乳糖苷酶检测细胞衰老情况,Western blot检测衰老和自噬相关蛋白的表达。应用GEPIA工具分析PLK1在乳腺组织及乳腺癌组织中的表达差异。结果7 Gy剂量照射后,Day 3组、Day 5组、Day 7组MDA⁃MB⁃231细胞体积变大,多倍体细胞亚群(DNA含量>4 N)比例均明显高于未经放射处理的对照组MDA⁃MB⁃231细胞(均P<0.0001),同时发生细胞衰老现象。与未经放射处理的对照组相比,Day 3组和Day 5组中DNA损伤修复蛋白PARP,DNA合成相关蛋白Rb、E2F⁃1、E2F⁃2表达下调,自噬相关蛋白LC3B/LC3A比值显著升高;核膜完整性相关蛋白Lamin B1、DNA损伤修复蛋白PARP表达下调,DNA损伤应答相关蛋白PLK1表达上调,差异均有统计学意义(均P<0.05)。与乳腺组织相比,PLK1在乳腺癌组织中高表达(P<0.05)。结论放射诱导乳腺癌细胞发生衰老现象可能是多倍体细胞形成的有利条件,衰老和自噬现象也可能有助于多倍体细胞自我修复去倍化增殖。     

X射线对人肺癌细胞Pokemon表达影响

目的 研究不同剂量X射线照射及照射后不同时间点对体外培养的人肺癌A₅₄₉细胞系Pokemon基因和蛋白表达的影响。方法 用X射线2、4、6、8 Gy吸收剂量照射体外培养的A₅₄₉细胞系,分别采用荧光实时定量PCR技术和蛋白印记法检测A₅₄₉细胞照射后2、4、8、12、24、48 h的Pokemon mRNA和蛋白表达水平,以未照射组为对照。采用二苯基四氮唑溴盐法分别检测2 Gy照射1、2、3、4、5 d后A₅₄₉细胞增殖能力变化,以未照射组为对照。结果 Pokemon mRNA的表达在2、4、6、8 Gy照射后早期呈降低趋势,但晚期呈升高趋势,大部分时间点差异有统计学意义(t=3.40~154.76,P=0.000~0.041);Pokemon 蛋白的表达较对照组均升高,且各剂量组均在照射后8 h达峰值,在大部分时间点实验组与对照组差异有统计学意义(t=4.18~89.64,P=0.000~0.039)。照射组细胞接种后第3~5天细胞增殖能力显著低于对照组(t=2.34~18.19,P=0.000~0.040)。结论 一定剂量X射线在特定条件下可诱导人肺癌A₅₄₉细胞Pokemon mRNA和蛋白表达升高,可能与A₅₄₉细胞辐射抗性有关。     

X射线照射对HeLa细胞TP和D PD酶表达影响的研究

目的:探讨X射线照射对宫颈腺癌HeLa细胞TP和DPD酶表达的影响,为卡培他滨与放射联合用于宫颈腺癌的治疗提供依据。方法:宫颈腺癌HeLa细胞体外培养,分别行0、2和6 Gy X射线照射,0 Gy为对照组。之后第1、3、5、7和9天收集细胞,采用ELISA及RT-PCR的方法对照射前后HeLa细胞TP和DPD酶的表达进行测定。结果:HeLa细胞被照射后,与对照组相比,2和6 Gy放射组的TP酶蛋白水平均有明显提高,放射后第3天都升高达高峰分别为(1.110±0.115)和(0.845±0.135)ng/mg蛋白,差异有统计学意义,P值均为0.000。放射后HeLa细胞TP酶的mRNA表达,在照射后第3天也迅速升高达到高峰分别为(2.098±0.116)和(1.841±0.104),明显高于未经照射的对照组(0.362±0.059),P值均为0.000。其升高时间与ELISA测定的TP酶蛋白水平的变化情况基本相吻合。而HeLa细胞DPD酶mRNA的表达,在放射组(1.367±0.539、1.211±0.215)与对照组(1.156±0.221)之间显示差异无统计学意义,P=0.085。结论:放射后HeLa细胞TP/DPD比值升高,能增加其对卡培他滨的敏感性,卡培他滨与放疗联合应用于宫颈腺癌的治疗可能获得较好疗效。     

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.     

Changes of hMSH2 and hMLH1 Expression in Nasopharyngeal Carcinoma Cells after X-Radiation

OBJECTIVE To study the effect of X-radiation on expression of hMSH2 and hMLH1 in nasopharyngeal carcinoma （NPC） CNE-1 cells, and explore the mechanism of DNA mismatch repair after radiation. METHODS The cells were divided into experimental and control groups. Experimental cells were exposed to 10 Gy radiation administered as 2 Gy per fraction. Control cells were not radiated. The distribution of cells in the cell cycle was analyzed using flow cytometry. The expression of hMSH2 and hMLH1 was examined by RT-PCR and Western blots. RESULTS The expression of hMSH2 mRNA in experimental cells was significantly greater compared to control cells at 0-3rd weeks and decreased at the 4th week following radiation （P〈0.01）. The expression of hMSH2 protein in experimental cells was up-regulated and significantly greater compared to control cells at the 2nd-4th weeks after radiation （P〈0.01）. There were no significant differences in hMLH1 mRNA and protein expression between experimental and control cells （P 〉0.05）. CONCLUSION Radiation induces hMSH2 expression; hMSH2 has a role in the process of DNA repair, which maybe responsible for reduction of radiosensitivity after radiation.     

X射线照射诱导鼻咽癌细胞mdrl和p53基因的表达及临床意义

目的:探讨X射线照射后鼻咽癌CNE-1细胞产生多药耐药的机制。方法:采用RT-PCR和Westernblot方法,检测X射线照射后CNE-1细胞mdr1和p53基因的表达。结果:照射后细胞mdrlmRNA和P-gp表达均增强,p53蛋白的表达也增强,并与P-gp表达呈正相关性(P<0.05)。结论:X射线照射可诱导CNE-1细胞mdr1和p53基因的表达,两者的相互协调作用可能是照射后肿瘤细胞产生多药耐药的重要机制之一。     

Increased expression of human DNA repair genes, XRCC1, XRCC3 and RAD51, in radioresistant human KB carcinoma cell line N10

The radioresistant N10 and parental KB cell lines were examined for the expression of human DNA repair genes which were related to the repair of radiation-induced DNA damage by northern blot analysis using five kinds of DNA probes (XRCC1, XRCC3, XRCC5, RAD51, RAD52). In the unirradiated condition, N10 cells showed higher expression of XRCC1, XRCC3 and RAD51 mRNA than did KB cells. The X-irradiation induced a time-dependent increase in the mRNA levels of XRCC3 and RAD51 in both cell lines with a maximum at 2 h postirradiation. The XRCC1 mRNA in N10 was maintained at the same level even after irradiation, whereas that in KB was decreased after irradiation. There was no difference in the expression of XRCC5 and RAD52 mRNA between N10 and KB cells in both unirradiated and irradiated conditions. From these findings, it was suggested that XRCC1, XRCC3 and RAD51 contribute to the radioresistance in cell line N10.     

下调VEGF表达影响鼻咽癌细胞放射敏感度的机制

目的　应用RNA干扰技术抑制人鼻咽低分化鳞状上皮细胞癌细胞株CNE-2中血管内皮生长因 子（VEGF）表达,研究阻断VEGF基因表达对鼻咽癌细胞放射敏感度的影响及机制。方法 构建针对 VEGF的siRNA真核表达载体pU-VEGF-siRNA（CNE-2组）、1个阴性对照质粒（CNE-2/Neg-siRNA 组）、经脂质体转染至CNE-2细胞（CNE-2/VEGF-siRNA组）,用平板克隆形成实验检测在6MV-X线 0、2、4、6、8、10 Gy照射后克隆形成能力及通过单击多靶模型、线性二次模型拟合放射生物学参 数,流式细胞检测分析细胞周期和细胞凋亡的变化,用RT-PCR定量分析三组细胞中Cyclin D1、Cyclin E、P16和P53的mRNA表达。结果 经6MV-X线0、2、4、6、8、10 Gy照射后细胞存活率显著下降, CNE-2/VEGF-siRNA组细胞经D0和2 Gy照射后的存活分数明显低于CNE-2组、CNE-2/Neg-siRNA组; CNE-2/VEGF-siRNA组中G1/S期细胞周期阻滞更为明显。在Cyclin D1、Cyclin E、p16和p53基因中, Cyclin D1mRNA表达在放疗后6、12和24 h进行性升高,差异有统计学意义,而其他基因变化差异无统 计学意义,Western blot检测显示Cyclin D1蛋白表达在放疗后24 h明显升高。结论 下调VEGF表达可 增加鼻咽癌细胞的放射敏感度,其机制可能是通过Cyclin D1信号通路途径使细胞周期发生G1/S期阻滞。     

Expression of DNA mismatch repair proteins hMSH2 and hMLH1 and the cyclin G1 inhibitor, p21(waf1/cip1) in pediatric tumors: correlation with response to therapy

The expression of the DNA mismatch repair proteins hMSH2 and hMLH1 and p21(waf1) the cyclin G1 inhibitor, may determine response of adult cancers to anti-cancer drugs, that include alkylating agents and platinum-based drugs. The role of DNA mismatch repair proteins (hMSH2 and hMLH1) and p21(waf1) in pediatric tumor responses to chemotherapy and irradiation is described in the present study of 23 pediatric solid cancers (4 wilms' tumors, 9 neuroblastomas, 3 hepatoblastomas, 3 lymphomas and 4 sarcomas) using immunohistochemical methods. Immunostaining was scored for intensity (0-3) and extent (0-3). Most tumors stained strongly for hMSH2 and weakly or negative for hMLH1. All the hMLH1 negative tumors (1 wilms', 1 hepatoblastoma, 1 sarcoma, 2 lymphomas and 2 neuroblastomas) achieved complete response. p21(waf1) negative and positive tumors achieved relatively similar treatment response. The results suggest that the expression of DNA mismatch repair proteins hMLH1 and hMSH2, and p21(waf1) do not influence individual cancer responses to treatment and the results may reflect the use of multiple drugs and irradiation that cause many different types of DNA damage.     

DNA damage and repair in peripheral blood lymphocytes from healthy individuals and cancer patients: a pilot study on the implications in the clinical response to chemotherapy

Drug resistance is considered the main impediment to successful cancer chemotherapy. The quest for a method useful to predict individual responses to chemotherapy prior to treatment is highly desired. This study was designed to determine the individual influences of doxorubicin and cisplatin on the degree of DNA damage, DNA repair and hMSH2 and the hMLH1 protein expression in peripheral blood lymphocytes (PBL) and their correlations with the clinical response. PBL were obtained from 25 cancer patients (pre- and post-chemotherapy) and from 10 healthy persons, cultured and exposed to doxorubicin or cisplatin. Cells were collected at T0 (immediately after drug treatment) and 24h after damage (T24). The alkaline comet assay was employed to assess the DNA damage and repair function, and immunocytochemistry to study hMLH1 and hMSH2 expression. Clinical response was evaluated after three cycles of chemotherapy. Pre-chemotherapy PBL from cancer patients showed significantly higher levels of basal DNA damage than healthy persons, with appreciable interindividual variations between them. The in vivo administration of antineoplasic drugs was accompanied by significant DNA damage, and an increased in the number of apoptotic cells. Cancer patients with complete response showed a high number of apoptotic cells. The DNA migration increased at T0 and at T24 in cisplatin-treated patients, reflecting a decreased rate of cisplatin adducts repair than that observed in healthy individuals. The ability to repair DNA lesions in doxorubicin-damaged cells was very similar between healthy individuals and cancer patients. Cisplatin-treated patients that died by the disease showed lower DNA migration than the mean value. The expression of hMLH1 and hMSH2 was practically identical between healthy individuals and cancer patients. Nevertheless, chemotherapy induced a depletion mostly of hMLH1. In 83% of cisplatin-treated patients with CR the hMLH1 and hMSH2 expression at T24 was higher than the mean. In this pilot study the alkaline comet assay offered information about the amount of DNA damage and the DNA repair status in PBL from individual patients and this seems useful in predicting the response to chemotherapy.     

Control of radiosensitivity of F9 mouse teratocarcinoma cells by regulation of histone H2AX gene expression using a tetracycline turn-off system

The mouse histone H2AX has unique COOH-terminal serine residues that are phosphorylated in response to double-strand DNA breaks introduced by ionizing radiation. This suggests that H2AX acts to maintain genomic stability. We constructed a tetracycline (tet)-directed turn-off vector and integrated it into F9 mouse teratocarcinoma cells by homologous recombination. In homozygously recombined cells, expression of the histone H2AX gene was repressed to 0.02% of the expression observed in wild-type cells by the addition of doxycycline, an analog of tet. Sensitivity of cells with repressed H2AX expression to X-irradiation was increased 1.95x, indicating that DNA repair was impaired by repression of H2AX. When we s.c. injected tet-regulated F9 cells into the flanks of mice, tumor growth was slightly suppressed by X-irradiation in H2AX-repressed tumors, whereas without X-irradiation, tumor growth did not differ by H2AX status. Thus, H2AX might be a potential molecular target for sensitizing cancer cells to radiotherapy to minimize required irradiation doses.     

Time course of enhanced activity of deoxycytidine kinase and thymidine kinase 1 and 2 in cultured human squamous lung carcinoma cells, SW-1573, induced by gamma-irradiation

Single high dose rate irradiation of 4 Gy in SW-1573 cells, derived from non-small cell lung cancer, led to increased activities of deoxycytidine kinase (dCK) and thymidine kinase 1 and 2 (TK1 and 2). The activity of dCK increased by approximately 30% between 1 and 5 h after irradiation, after which the activity returned to the level of control cells by 8 h after irradiation. TK1 activity also increased by 30-50% between 1 and 6 h after irradiation. The decline to normal levels of dCK concurred with a further increase in the activity of TK1, 8 h after irradiation. TK2 activity was below control levels during the first 4 h after irradiation but rose 3-fold at 8 and 16 h after treatment. The activities of TK1 and TK2 had returned to approximate control levels 24 h after irradiation. The observation that mitochondrial TK2 activity increased to a very high level after irradiation may indicate that the activity of this enzyme is not only important for the damage to mitochondrial DNA, the increased activity may also be instrumental for repair of damage to nuclear DNA. We presume that the increase in activity of TK1 after irradiation is limited to cells in S-phase. Recruitment of cells into S-phase, to replace cells killed by irradiation, is probably too slow to offer an explanation for the enhanced activity of TK1 8 h after irradiation. The increase in activity of both dCK, and TK1 and 2 might be involved in an adaptive response of the cells to radiation by facilitation of DNA repair. The expression of protein kinase C (PKC) decreased during the first 5 h after irradiation. At 5 h after irradiation the level of expression had decreased by >50%. The decrease in PKC expression is concurrent with the increase in dCK activity. This suggests a role of PKC in the signal transduction from DNA damage to the increase in activity of enzymes instrumental in DNA repair.