Adenovirus-mediated expression of UHRF1 reduces the radiosensitivity of cervical cancer HeLa cells to γ-irradiation

Aim:

An in vitro study was carried out to determine the effect of UHRF1 overexpression on radiosensitivity in human cervical cancer HeLa cells using adenovirus-mediated UHRF1 gene transfer (Ad5-UHRF1).

Methods:

Cell survival was evaluated using the clonogenic survival assay and the MTT assay; apoptosis and cell cycle distribution were monitored by flow cytometry. Protein levels were measured by Western blotting. Silencing XRCC4 expression was performed by transfection of small interfering RNA (siRNA).

Results:

Increased expression of UHRF1 by Ad5-UHRF1 significantly reduced the radiosensitivity of HeLa cells. The UHRF1-mediated radioresistance was correlated with increased DNA repair capability and increased expression of the DNA damage repair protein, XRCC4. Knocking down XRCC4 expression in the cells using XRCC4 siRNA markedly reduced the UHRF1-mediated radioresistance.

Conclusion:

These results provide the first evidence for revealing a functional role of UHRF1 in human cervical cancer cells as a negative regulator of radiosensitivity.     

Lymphoid and fibroblastic cell lineages from radiosensitive cancer patients: molecular analysis of DNA double strand break repair by major non-homologous end-joining sub-pathways

Aims: Radiation therapy (RT) is used in the treatment of approximately half of all cancer patients. Although there have been great improvements in tumor localization and the technical accuracy of RT delivery, some RT patients still have idiosyncratic hypersensitivity to ionizing radiation (IR) in their normal tissues. Although much effort has been expended in the search for assays that could detect radiosensitive individuals prior to treatment and facilitate tailored therapy; a suitable and clinically practical predictive assay has yet to be realized. Since DNA double‐strand breaks (DSB) are a major lesion caused by IR, we hypothesized that radiation hypersensitive individuals might be deficient in the repair of such lesions. Methods: To test this hypothesis we quantitatively and functionally characterized DSB repair of the two major non‐homologous end‐joining (NHEJ) sub‐pathways in a pilot study using a plasmid repair reconstitution assay in lymphoblastoid and fibroblast cell lines from radiosensitive cancer patients and controls. Experiments using well‐characterized mammalian DSB repair mutants demonstrated the ability of the assay to distinguish NHEJ sub‐pathways. The proportion of direct end‐joining repair compared with that of microhomology‐directed repair was used as a functional end‐point of DSB repair competence in the different cell lines. Results: We found that the overall level of NHEJ sub‐pathway repair competency was similar in cell lines from radiosensitive patients and controls. Conclusion: These data suggest that this assay in these cell lineages has limited usefulness as a predictive screen for the endogenous DNA DSB repair competency of radiosensitive cancer patients' cells but can usefully characterize major cellular DSB repair phenotypes.     

MnSOD基因单核苷酸多态性对食管癌局部放疗的影响

目的：探讨锰超氧化物歧化酶（manganese superoxide dismutase,MnSOD）基因单核苷酸多态性与食管癌放疗敏感性的关系。方法：以聚合酶链反应-限制性片段长度多态性（PCR-RFLP）方法分析93例食管癌患者外周血白细胞DNA的MnSOD基因第16密码子多态性（Ala16Valrs4880）;通过比较食管癌放疗的局部疗效,分析携带MnSOD基因野生型（T/T）与突变基因型（T/C、C/C）食管癌患者对放疗的敏感性差异。结果：放疗后局部疗效达完全缓解者（CR）者22例、部分缓解者（PR）者63例,无缓解（NR）者8例,其MnSOD基因第16密码子多态性（T/T、T/C＋C/C）分布频率分别为59.1%、40.9%;77.8%、22.2%;62.5%、37.5%,3者之间差异无统计学意（x~2=3.223,P〉O.05）。在食管癌病变长度≤5cm的患者中CR与PR、NR之间MnSOD（T/T、F/C＋C/C）基因型分布频率的差异无统计学意义（x~2=2.658,P〉0.05）;而病变长度〉5cm的患者中携带C等位基因患者的疗效优于携带T/T基因型患者。MnSOD（T/T、T/C＋C/C）基因型患者的中位生存时间和1年生存率分别为17个月和68%;22个月和73%,但T/T与T/C＋C/C基因型之间的差异无统计学意义（P〉0.05）。结论：病变长度〉5 cm患者中,携带T/C或C/C基因型患者放疗对肿瘤的治疗效果优于携带T/T基因型患者。     

选择性环氧化酶-2抑制剂联合放疗对人前列腺癌裸鼠移植瘤作用的实验研究

目的研究选择性环氧化酶-2（COX-2）抑制剂塞来昔布（celecoxib）对前列腺癌的抗肿瘤作用和放疗增敏效应,并探讨其协同抑制作用的机制。方法使用前列腺癌PC-3细胞构建人前列腺癌裸鼠移植瘤模型,将荷瘤裸鼠随机分为对照组、celecoxib组、放疗组和celecoxib联合放疗组4组,每组6只。观察移植瘤的肿瘤生长延缓时间（TGD）和增敏系数（EF）;放射免疫法（ELISA法）测定前列腺素E2（PGE2）的含量,逆转录-聚合酶链反应（RT-PCR法）检测移植瘤组织中COX-2mRNA的表达,并观察荷瘤鼠各重要脏器组织的病理学改变。结果对照组和celecoxib组中移植瘤最大径从8.0mm生长至12.0mm所需的时间分别为（6.18±0.72）d和（7.87±0.76）d,而放疗组和celecoxib联合放疗组分别为（9.16±0.89）d和（12.62±1.28）d,组间差异有统计学意义（P〈0.05）,EF为1.59;对照组与其余3组间的PGE：含量差异均有统计学意义（P〈0．05）,而放疗组与celecoxib联合放疗组间PGE2含量差异无统计学意义（P〉0．05）;celecoxib引起的生长延缓与PGE：含量的下降呈正相关（r=0．807）;4组移植瘤组织中COX一2mRNA表达水平差异均无统计学意义（P〉0．05）;各组荷瘤鼠重要脏器未发现明显毒性病理学改变。结论Celecoxib联合放疗治疗人前列腺癌裸鼠皮下移植瘤具有抗肿瘤作用和放射增敏效应,可发挥协同抑制作用,在一定范围内是安全有效的,这为今后的前列腺癌的研究和临床治疗提供了一定的思路。     

Lymphocyte radiosensitivity in BRCA1 and BRCA2 mutation carriers and implications for breast cancer susceptibility

There is conflicting evidence as to whether individuals who are heterozygous for germ-line BRCA1 or BRCA2 mutations have an altered phenotypic cellular response to irradiation. To investigate this, chromosome breakage and apoptotic response were measured after irradiation in peripheral blood lymphocytes from 26 BRCA1 and 18 BRCA2 mutation carriers without diagnosed breast cancer, and 38 unaffected age, ethnically and sex-matched controls. To assess the role of BRCA1 and BRCA2 in homologous recombination, an S phase enrichment chromosome breakage assay was used. BrdUrd incorporation studies allowed verification of the correct experimental settings. We found that BRCA1 mutation carriers without cancer had increased chromosome breaks as well as breaks and gaps per cell post irradiation using the classical G2 assay (p = 0.01 and 0.004, respectively) and the S phase enrichment assay (p = 0.01 and 0.01, respectively) compared to age-matched unaffected controls. BRCA2 mutation carriers without cancer had increased breaks as well as breaks and gaps per cell post irradiation using the S phase enrichment assay (p = 0.045 and 0.012, respectively). No difference was detected using the G2 assay (p = 0.88 and 0.40 respectively). BRCA1 and BRCA2 mutation carriers had normal cell cycle kinetics and apoptotic response to irradiation compared to age-matched controls. Our results show a demonstrable impairment in irradiation induced DNA repair in women with heterozygous germline BRCA1 and BRCA2 mutations prior to being diagnosed with breast cancer.     

放疗前周围血淋巴细胞敏感性是骨盆放疗所致疾病的预示因素(英文)

目的　探讨前列腺癌 (PC)治疗前体外照射诱导的Go淋巴细胞微核量是否与放疗 (xRT)相关疾病的发生有关。方法　对 3 8例PC周围血淋巴细胞 (PBL)以细胞因子阻止的微核法进行前瞻性观察。患者平均年龄 68.7± 1.0岁。临床分期 :T116例 ,T2 16例 ,T36例。治疗前前列腺癌特异性抗原 (PSA)≤ 4ng·ml-1者 5例 ,4～ 10ng·ml-1者 19例 ,>10ng·ml-1者 14例。所有患者均接受标准的骨盆外xRT(41.4～ 5 0 .4Gy) ,追加剂量 (16.4～ 2 6Gy)。平均随访 3 2 .8± 4.6个月。随访末 ,按RTOG标准对胃肠道 (GI)或泌尿生殖道 (GU)xRT相关疾病的发生率进行评定。结果　发生xRT相关疾病 2 4例为高敏 (HS) ,未发生者 14例为正常敏感性 (NS) ,HS患者均有GI(1～ 3级 )或GU(1～ 2级 )xRT相关疾病。体外照射暴露的血标本 ,与照射剂量相关的PBL微核量在HS和NS患者之间有显著差异 ,特别是当剂量≥ 3Gy时。xRT相关疾病的发生与治疗前PBL微核量相关 ,而与病情无关。结论　与xRT前体外照射剂量相关的PBL微核量是骨盆xRT的PC患者GI或GU疾病发生的一个预示因素     

AT细胞系辐射敏感性与细胞凋亡的相关研究

目的 探讨AT细胞高辐射敏感性与细胞凋亡之间的联系。方法 以液体闪烁测量法测3H-TdR掺入百分率来观察^60Co γ射线照射对AT5BIVA（AT）和GM0639（GM）细胞增殖的影响;实验于照射后0、24、48和72 h,用细胞流式术动态检测AT和GM细胞的自发凋亡率及^60Co γ射线照射诱发的凋亡率。结果 ^60Co γ射线0～6 Gy照射后,AT和GM两种细胞3H-TdR掺入百分率均呈剂量依赖性降低,且AT细胞降低得比GM细胞快,两种细胞3H-TdR掺入百分率与受照射剂量间可拟合成指数方程：SAT=0.9718e^-0.6855D（R^2=0.9950）、SGM=1.0928e^-0.3731D（R^2=0.9777）;AT细胞的自发凋亡率高于GM细胞;2～6 Gy照后24 h,两种细胞由辐射诱发的凋亡率均随照射剂量增大而升高,且在同一剂量点,前者凋亡率高于后者;2 Gy照射后0～72 h,AT和GM细胞的凋亡率均呈时间依赖性增加,且随照后时间延长两者间差异逐步增大,在72 h达到显著水平。结论 AT细胞高辐射敏感性与其较高的自发及辐射诱发的凋亡密切相关。     

食管鳞癌和宫颈鳞癌组织中p53蛋白过度表达及其与放疗效果的关系

目的:探讨p53蛋白过度表达与食管鳞癌和宫颈鳞癌临床放射敏感性的关系.方法:免疫组化法研究52例食管鳞癌和宫颈鳞癌标本中p53蛋白过度表达状况,并分析其与近期放疗效果的关系.结果:p53蛋白在食管鳞癌和宫颈鳞癌中的过度表达分别为52.38%和35.48%,过度表达率在高分化鳞癌中较中低分化者低(P＜0.05),与病期无关(P＞0.05).在宫颈鳞癌,p53蛋白过度表达者放疗效果较差(P＜0.05),食管鳞癌p53蛋白过度表达与放疗效果间无明显关系(P＞0.05).结论:p53蛋白过度表达与食管鳞癌和宫颈鳞癌放射敏感性有关,但不是惟一影响因素.     

Heterogeneity in ataxia-telangiectasia: Classical phenotype associated with intermediate cellular radiosensitivity

We identified a subgroup of ataxia-telangiectasia (AT) patients (2 sibs and 1 unrelated case) characterized by typical clinical manifestations of the disease and cellular radiosensitivity intermediate between classical AT and normal subjects. Our data and a literature review of the intermediate radiosensitivity AT cases show that radioresistant DNA synthesis, cellular radiosensitivity (measured in terms of survival and chromosome break-age), and the clinical hallmarks behave independently. This raises a number of interesting questions about the correlation between radiobiological and clinical features, and about the nature of the AT gene(s).     

小鼠骨髓基质细胞的辐射敏感性及某些药物对其作用的观察

本实验证实,小鼠股骨骨髓(BM)基质由成纤维细胞、网状细胞、吞噬细胞及脂肪细胞构成。在本外,由2根股骨的BM细胞所形成的贴壁细胞层对造血多能干细胞CFU-S支持功能最好。BM基质细胞为辐射敏感性低于造血干细胞;AET和兜衣多糖对BM基质细胞及其支持造血干细胞的功能有明显的辐射防护作用。