DNA-PK作为放化疗增敏靶点的研究
摘要
第一部分DNA双链断裂修复蛋白在乳腺和宫颈良恶性病变组织中的表达研究
【目的】研究DNA双链断裂修复蛋白(Ku80、DNA-PKcs和ATM)在乳腺癌和乳腺纤维瘤组织中以及宫颈癌和宫颈上皮内瘤样病变(CIN)中的表达情况,探讨3种蛋白在肿瘤发生发展中的作用及相互关系。
【方法】应用免疫组化SP法检测55例乳腺癌和14例乳腺纤维瘤组织,以及41例宫颈癌和15例CIN组织中Ku80,DNA-PKcs和ATM蛋白的表达情况。
【结果】Ku80、DNA-PKcs和ATM蛋白在乳腺癌患者中的阳性率分别为74.55%、54.55%和52.73%,在乳腺纤维瘤患者中的阳性率分别为92.86%、92.86%和71.43%;DNA-PKcs蛋白在乳腺癌组织中的表达显著低于乳腺纤维瘤组织(X2=6.98,P=0.01),而Ku80和ATM的表达虽在乳腺癌组织中的表达亦低于乳腺纤维瘤组织,但差异无统计学意义(P>0.05)。ATM和DNA-PKcs蛋白的表达在不同病理类型、肿块大小、临床分期和淋巴结转移状态的乳腺癌患者中差异无统计学意义(P>0.05);但Ku80的表达与患者的肿块大小和临床分期相关(P<0.01)。Spearman等级相关分析,在55例乳腺癌患者中,Ku80与DNA-PKcs的表达呈正相关(r=0.36,P=0.01);Ku80与ATM的表达亦呈正相关(r=0.33,P=0.02)。Ku80、DNA-PKcs和ATM蛋白在宫颈癌患者中的阳性率分别为70.73%、68.29%和19.51%,在CIN患者中的阳性率分别为80.00%、73.33%和33.33%;3种蛋白在宫颈癌组织中的表达均低于CIN组织,但差异无统计学意义(P>0.05)。3种蛋白的表达在不同年龄、病理类型、分化程度和临床分期的宫颈癌患者中差异无统计学意义(P>0.05)。Spearman等级相关分析,在56例患者中,Ku80与DNA-PKcs的表达呈正相关(r=0.58,P=0.00);Ku80与ATM的表达亦呈正相关(r=0.27,P=0.04)。
【结论】DNA-PKcs和Ku80有可能成为肿瘤治疗增敏的靶点;DNA-PKcs蛋白可能在乳腺癌的发生发展中起重要作用;Ku80与DNA-PKcs及ATM之间均存在密切关系。
第二部分宫颈癌细胞株和正常间质细胞株受X线照射后的辐射效应
【目的】利用宫颈癌细胞系和正常血管上皮细胞系、正常成纤维细胞系体外模拟宫颈癌实质和间质血管组织,研究其受到不同剂量放射线照射后的反应。
【方法】宫颈腺癌细胞系HeLa,宫颈鳞癌细胞系SiHa、C33A、Caski,及人脐静脉内皮细胞系ECV304、小鼠成纤维细胞系NIH/3T3细胞,分别用6MV的X线(300cGy/min)照射6Gy和10Gy,24h和48h后收集细胞碘化丙锭染色,流式细胞术检测凋亡率和细胞周期变化。
【结果】受到照射后C33A凋亡率最高,而SiHa最抵抗,其余细胞系介于两者之间;各宫颈癌细胞系及NIH/3T3照射10Gy后48h产生的凋亡率与6Gy相似(P>0.05),而ECV304照射10Gy后48h产生的凋亡率(13.04%±1.08%)却比6Gy(6.51%±0.61%)高许多(P<0.05);各细胞系受到照射后,均表现出明显的G2/M期阻滞,且G2/M阻滞细胞百分比随照射剂量增加而增加;受到照射后一般在24h~48h G2/M期阻滞细胞百分比最高。
【结论】高剂量照射提高肿瘤血管的凋亡率,宫颈癌细胞受照射后G2/M期阻滞,且呈剂量依赖性,为高剂量射线治疗宫颈癌、追加剂量继续放疗、选择周期特异性化疗药物、以及针对G2/M检测点基因进行靶向阻断提供了依据。
第三部分DNA双链断裂修复蛋白表达水平与肿瘤细胞放射敏感性的关系研究
【目的】检测肿瘤细胞株中DNA双链断裂(DNA double-strand break,DSB)修复蛋白(Ku80、DNA-PKcs和ATM)的表达水平和放射敏感性参数,探讨3个蛋白预示肿瘤细胞放射敏感性的价值。
【方法】4株人宫颈癌细胞株HeLa、SiHa、C33A和Caski,3株人乳腺癌细胞株MCF-7、MDA-MB-231和MDA-MB-453,及1株人肺癌细胞株A549,Western blot检测8株细胞中Ku80、DNA-PKcs和ATM蛋白的表达水平,流式细胞仪检测10Gy 6MV X线照射48h后的凋亡率,克隆形成实验检测SF2(Surviving fraction after 2Gy)值和α、β值,Pearson线性相关分析蛋白表达水平与照射后凋亡率、SF2值和α值的相关性。
【结果】3种蛋白在同一株细胞中的表达及同一蛋白在不同细胞株的表达均存在明显差异;DNA-PKcs的表达水平与SF2之间存在正相关关系(r=0.72,P =0.04<0.05);Ku80和ATM的表达与SF2值间无明显相关关系(P>0.05);3种蛋白与凋亡率和α值均无相关性(P >0.05)。
【结论】DNA-PKcs蛋白表达越高细胞对放射线越抵抗,其表达水平可能成为肿瘤细胞放射敏感性的指标;DNA-PKcs和Ku80可能可以成为肿瘤放疗增敏的理想靶点。
第四部分siRNA抑制Ku80表达后对宫颈癌HeLa细胞放化疗敏感性的影响
【目的】建立利用小干扰RNA(small interfering RNA,siRNA)抑制Ku80表达的HeLa细胞模型,以此探讨Ku80在放化疗增敏方面的潜在应用价值。
【方法】构建靶向抑制Ku80的siRNA表达质粒,转染HeLa细胞,筛选稳定表达的转化克隆,Western blot检测Ku80表达变化;克隆形成实验、MTT法和裸鼠皮下瘤形成实验分别检测细胞克隆形成率,及细胞在体外和体内的增殖情况;6MV X线照射6Gy后,流式细胞术检测细胞凋亡率和细胞周期,克隆形成实验检测细胞SF2、D0等值;MTT检测细胞受不同浓度托泊替康、依托泊苷和顺铂作用后细胞增殖率变化。
【结果】构建的质粒转染HeLa细胞获得2个稳定转染克隆,Western blot分析表明阳性克隆Ku80蛋白抑制率达到96.4%,命名为HeLa/Ku80-siRNA; HeLa/Neg-siRNA细胞克隆形成率为0.62±0.02,而HeLa/Ku80-siRNA细胞的克隆形成率为0.46±0.05,明显低于对照细胞(t=5.11,P<0.01);MTT显示细胞培养48h和72h时,HeLa/Ku80-siRNA细胞的增殖率均显著低于对照细胞(P<0.05);裸鼠皮下瘤生长实验示种植25天时,HeLa/Ku80-siRNA细胞种植瘤的平均体积(18.92±3.60)mm3,明显低于对照细胞种植瘤体积(194.88±30.61)mm3,(t=12.69,P<0.01),抑瘤率达到90.06%。HeLa/Ku80-siRNA体外受X线照射后48h及72h的凋亡率高于对照细胞(P<0.05),但3株细胞的细胞周期变化无统计学意义(P>0.05);阳性克隆细胞株的D0和SF2值明显降低,在D10剂量时的增敏比为1.365。Ku80受抑细胞对托泊替康和依托泊苷的敏感性增加(P<0.05)。
【结论】Ku80-siRNA可以抑制Ku80的表达,在体内外抑制HeLa细胞的增殖,并促进HeLa细胞对X线和拓扑异构酶抑制剂的敏感性。
第五部分抑制DNA-PKcs表达促进宫颈癌HeLa细胞放射敏感性的研究
【目的】探讨DSB修复蛋白DNA-PKcs成为宫颈癌放疗增敏靶点的可能性。
【方法】利用靶向抑制DNA-PKcs的小发卡样干扰RNA(small hairpin interfering RNA,shRNA)表达质粒和小分子抑制剂LY294002,分别抑制HeLa细胞DNA-PKcs蛋白表达和活性后,克隆形成实验和流式细胞仪检测HeLa细胞受6MV X线照射后的SF2、α值和凋亡率变化。
【结果】靶向抑制DNA-PKcs的shRNA可以促进宫颈癌HeLa细胞的放射敏感性,其SF2值为0.37,显著低于对照HeLa细胞的0.53;单独接受50μmol/L LY294002作用1h未使HeLa细胞的凋亡率明显增加(P>0.05),但先经LY294002处理再照射6Gy的HeLa细胞在48h和72h的凋亡率比单独照射6Gy的HeLa细胞凋亡率显著增加(48h点:t=3.25,P=0.03;72h点:t=3.01,P=0.04)。
【结论】抑制DNA-PKcs的表达或活性可以促进宫颈癌HeLa细胞的放射敏感性;提示DNA-PKcs可能可以成为宫颈癌放疗增敏的理想靶点。
第六部分协同抑制Ku80和DNA-PKcs对HeLa细胞放射生物学功能的影响
【目的】研究siRNA和LY29400抑制单个或多个DNA双链断裂修复蛋白后HeLa细胞放射敏感性和细胞周期的变化。
【方法】Ku80受抑细胞HeLa/Ku80-siRNA和对照细胞HeLa/Neg-siRNA分别转染可以靶向抑制抑制DNA-PKcs的siRNA或用PI-3-K抑制剂LY294002处理,经6MV X线照射后,克隆形成实验检测细胞放射敏感性变化,流式细胞仪检测细胞周期和凋亡率变化。
【结果】转染DNA-PKcs-siRNA后的HeLa/Ku80-siRNA放射敏感性显著高于HeLa/Ku80-siRNA , SF2分别为0.08±0.01和0.20±0.05 , LY294002作用后HeLa/Ku80-siRNA的SF2达到0.03±0.01,作为对照细胞的HeLa/Neg-siRNA其SF2值为0.51±0.07;各细胞照射6Gy后均出现G2/M期阻滞,转染DNA-PKcs-siRNA的HeLa/Neg-siRNA细胞和LY294002作用的HeLa/Ku80-siRNA、HeLa/Neg-siRNA细胞G2/M期阻滞逐渐缓慢出现,照射后72h仍未达到顶点,而其他细胞G2/M期阻滞均于照射后48h达到顶点。
【结论】在抑制Ku80已达95%的基础上,DSB修复功能可以由其他DSB修复蛋白如DNA-PKcs和ATM代偿,协同抑制上述蛋白可以明显增加HeLa细胞的辐射敏感性;Ku80、DNA-PKcs和ATM在细胞的G2/M期阻滞过程中发挥的作用不同。
PartⅠThe expressions of DSB repair protein in benign and malignant tissues of breast and cervix
[Objective] To study the expressions of DSB repair proteins (including Ku80, DNA-PKcs and ATM) in breast carcinoma, breast fibroma, cervical carcinoma and cervical intra-epithelial neoplasia (CIN), and to explore their roles in neoplasia and tumor progression of breast and cervical carcinoma.
[Methods] Immunohistochemistry was applied to detect the expressions of Ku80, DNA-PKcs, and ATM in 55 cases of breast carcinoma, 14 cases of breast fibroma, 41 cases of cervical carcinoma and 15 cases of CIN.
[Results] The positive percentages of Ku80, DNA-PKcs and ATM were 74.55%、54.55% and 52.73% respectively in breast carcinoma, and were 92.86%、92.86% and 71.43% respectively in breast fibroma; The expression of three proteins in fibroma were all higher than that in carcinoma, but the differences of Ku80 and ATM were no significant (P>0.05), only the expression difference of DNA-PKcs was significant (X2=6.98,P=0.01). The expressions of DNA-PKcs and ATM protein were not associated with the pathology, tumor diameter, clinical stage and the lymph node metastasis of breast carcinoma patients (P>0.05), while Ku80 expression associated with tumor diameter and clinical stage of breast carcinoma patients (P<0.01). In breast carcinoma cases, Ku80 was positively correlated with DNA-PKcs (r=0.36, P=0.01) and ATM (r=0.36, P=0.02). The positive percentages of Ku80, DNA-PKcs and ATM were 70.73%, 68.29% and 19.51% respectively in cervical carcinoma, and were 80.00%, 73.33% and 33.33% respectively in CIN; the expression of three proteins in CIN were all higher than that in cervical carcinoma, but the differences were no significant (P>0.05). The expression of these proteins were not associated with the age of patients, pathology, differentiation, and clinical stage (P>0.05).in all 56 cases, Ku80 was positively correlated with DNA-PKcs (r=0.58, P=0.00) and ATM (r=0.27, P=0.04).
[Conclusions] DNA-PKcs and Ku80 are more possible to be the target of cancer gene-therapy; DNA-PKcs might play the major role in the neoplasia and tumor progression of breast carcinoma; Ku80 had close relationship with DNA-PKcs and ATM in breast and cervical carcinoma.
PartⅡResponse of cervical carcinoma and normal interstitial cell lines after X-ray irradiation
[Objective] Study the response of cervical carcinoma cell lines, normal vascular endothelial cell line and fibroblast cell line which simulate as cervical carcinoma tissues after a serial of doses of X-ray irradiation.
[Methods] Human cervical adencarcinoma cell line HeLa, human cervical squamous carcinoma cell lines SiHa, C33A, Caski, human umbilical vein endothelial cell line ECV304 and mouse fibroblast cell line NIH/3T3 were irradiated with 6 MV X-ray at the absorb doses of 6Gy and 10Gy. After irradiated for 24h and 48h, cells were collected and analyzed by flow cytometry for apoptosis and cell cycle.
[Results] The apoptosis rate of C33A was highest, while the SiHa cell line was most resistant for X-ray; The apoptosis rates of ECV304 after irradiation with 10 Gy (13.04%±1.08%) was higher than 6Gy (6.51%±0.61%), P<0.05, while ones of the other cell lines were similar (P>0.05); Every cell lines were blocked at G2/M phase after irradiation, the cell percentages of G2/M phase increased with the doses of irradiation and got to maximum at 24-48h after irradiation.
[Conclusions] High doses of irradiation could improve the apoptosis of tumor vascular endothelial cell and increase the cell accumulation of G2/M phase, which provide the theory evidences for high dose of irradiation at cervical carcinoma radiotherapy, boosting dose of irradiation, selecting the specific drugs aim directly at G2/M phase, and specifically inhibiting the genes of G2/M check point for gene therapy.
PartⅢCorrelativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells
[Objective] To explore the correlation between the expressions of DSB (DNA double-strand break) repair protein (including Ku80, DNA-PKcs and ATM) and radiosensitivity parameters in human tumor cell lines, and to study which protein could prognosticate the radiosensitivity of tumor cell.
[Methods] SF2 (survival fraction at 2Gy),αandβvalues of eight tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation array,apoptosis rates at 48h after 10Gy of 6MV X-ray irradiation were analyzed by flow cytometry analysis, and Western Blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein, the correlativity between protein expression and SF2,α,βvalue or apoptosis rate was analyzed by Pearson linear correlation analysis.
[Results]The expression of same protein in different cell lines and the expression of three proteins in the same cell line were various; there was a positive correlativity between the expression of DNA-PKcs and SF2 (r=0.72, P=0.04), but no correlativity was found between Ku80 and SF2 or ATM and SF2 (P>0.05). Bothαand apoptosis rate had no correlativity with the expression of DSB repair protein (P>0.05).
[Conclusions] The expression of DNA-PKcs protein may be able to prognosticate the radiosensitivity of tumor cells, and could be an ideal target to enhance the radiosensitivity.
PartⅣTo study the effect on radio- and chemo- sensitivity of HeLa cells after Ku80 inhibition
[Objective] The HeLa cell model of Ku80 expression suppressed by siRNA was constructed and utilized to study the role of Ku80 in enhancing the radio- and chemo- sensitivity
[Methods] Ku80-siRNA expression plasmids were constructed and HeLa cells were transfected with these plasmids by lipofectamine. Western blot was applied to measure the expression of Ku80;The proliferation of HeLa cells were determined by clone formation assay, MTT assay and tumor subcutaneously formation studies on nude mice. After irradiation with 6MV X-ray,cells were collected and analyzed by flow cytometry for apoptosis and cell cycle at 24h, 48h and 72h; The radiobiology parameters of three cell lines were acquired by clone formation array. The inhibition rate of cells treated with Topotecan, Etoposide or DDP was detected by MTT assay.
[Results] Two cell clones stable transfected were got, and the inhibition rates of Ku80 protein expression of positive clone got to 96.40%; The clone formation efficiency of HeLa/Ku80-siRNA was 0.46±0.05, lower than the control cells (t=5.11, P<0.01). The proliferation rate of HeLa/Ku80-siRNA were slower than control cells 48h and 72h after transfection (P<0.05). And after transplanted for 25 days, the mean tumor volume of HeLa/Ku80-siRNA was only (18.92±3.60) mm3, while the mean tumor volume of control cells got to (194.88±30.61) mm3, the difference was significant (t=12.69, P<0.01), tumor inhibition rate got to 90.06%. The apoptosis rates of HeLa cells Ku80 inhibited were higher than control cells at 48h and 72h after X-ray radiation (P<0.05), but the differences of cell cycle change after irradiation were not significant (P>0.05); HeLa cells silenced of Ku80 had lower SF2 and D0 than control cells, and their SER (sensitization enhancement ratio) based on D10 got to 1.365. HeLa/Ku80-siRNA was more sensitivity to Topotecan and Etoposide than control cells (P<0.05).
[Conclusion] The HeLa cell models Ku80 expression suppressed were successfully established; the inhibition of Ku80 by siRNA could enhance the radio- and chemo- sensitivity of HeLa, and suppress the cell proliferation.
PartⅤStudy of Enhanced Radiosensitivity in Cervical Carcinoma Cell Line HeLa by Inhibition of DNA-PKcs
[Objective] This study tried to probe potential role of DNA-PKcs in enhancing the radiosensitivity of cervical carcinoma.
[Methods] The DNA-PKcs-targeted shRNA (small hairpin interfering RNA) and a competive DNA-PKcs inhibitor LY294002, were applied to inhibit the DNA-PKcs expression or activity in HeLa cells. Then the SF2,αvalues and apoptosis rates were analyzed by clonogenic survival assay and flow cytometry analysis respectively. [Results] The DNA-PKcs-targeted shRNA could enhance the radiosensitivity of HeLa, and its SF2 was 0.37, lower than the control cells clearly (0.53). The apoptosis rates of HeLa cells only administrated by 50μmol/L LY294002 management for 1 hour did not increase significantly (P>0.05), but LY294002 sensitized HeLa cells to 6 MV X-ray, after HeLa cells were administrated LY294002 and irradiation at 48h and 72h after 6Gy irradiation, the apoptosis rates were higher than those of cells only accepted irradiation (48h: t=3.25, P=0.03; 72h: t=3.01, P=0.04).
[Conclusion] Inhibition of DNA-PKcs expression or activities may sensitize HeLa cells to X-ray. As the results, DNA-PKcs may be an ideal target for enhancing radiosensitivity of cervical carcinoma.
PartⅥThe effect on radiobiology of HeLa after co-inhibition of DNA-PKcs and Ku80
[Objective] To study the radiosensibility and cell cycle distribution of HeLa inhibited of one or several DNA double-strand break (DSB) repair proteins after being treated with small interfering RNA (siRNA) or LY294002.
[Methods] Ku80 silenced cell HeLa/Ku80-siRNA and control cell HeLa/Neg-siRNA were applied, and transfected with DNA-PKcs-targeted siRNA or pretreated with 50μM LY294002, a chemically specific phosphatidylinositol (PI) 3-kinase (DNA-PKcs and ATM are both members of PI 3-kinase gene family) inhibitor, after irradiated with 6MV X-ray, cells were detected by clonogenic survival assay for radiosensibility and analyzed by flow cytometry for cell cycle distribution and apoptosis.
[Results] HeLa/Ku80-siRNA transfected with DNA-PKcs-siRNA was more sensitive to radiation than HeLa/Ku80-siRNA, SF2 were 0.08±0.01 and 0.20±0.05 respectively, the SF2 of HeLa/Ku80-siRNA pretreated with LY294002 even got to 0.03±0.01, while the SF2 of control cell line HeLa/Neg-siRNA was 0.51±0.07; G2/M accumulation was observed at all cell lines after 6Gy X-ray exposure, the percentage of DNA-PKcs-siRNA transfected HeLa/Neg-siRNA and LY294002 treated cells in G2/M phase even didn’t get to highest at 72h post-irradiation, while other cell lines had a shorter G2/M delay, the G2/M accumulation got to vertex at 48h post-irradiation.
[Conclusions] After 95% Ku80 was inhibited, HeLa cells could have other proteins, for example DNA-PKcs or ATM to compensate the DSB repair function, co-inhibition of these proteins resulted in markedly sensitive to X-ray; Ku80, DNA-PKcs and ATM may play different role in cell cycle accumulation after irradiation.
【目的】研究DNA双链断裂修复蛋白(Ku80、DNA-PKcs和ATM)在乳腺癌和乳腺纤维瘤组织中以及宫颈癌和宫颈上皮内瘤样病变(CIN)中的表达情况,探讨3种蛋白在肿瘤发生发展中的作用及相互关系。
【方法】应用免疫组化SP法检测55例乳腺癌和14例乳腺纤维瘤组织,以及41例宫颈癌和15例CIN组织中Ku80,DNA-PKcs和ATM蛋白的表达情况。
【结果】Ku80、DNA-PKcs和ATM蛋白在乳腺癌患者中的阳性率分别为74.55%、54.55%和52.73%,在乳腺纤维瘤患者中的阳性率分别为92.86%、92.86%和71.43%;DNA-PKcs蛋白在乳腺癌组织中的表达显著低于乳腺纤维瘤组织(X2=6.98,P=0.01),而Ku80和ATM的表达虽在乳腺癌组织中的表达亦低于乳腺纤维瘤组织,但差异无统计学意义(P>0.05)。ATM和DNA-PKcs蛋白的表达在不同病理类型、肿块大小、临床分期和淋巴结转移状态的乳腺癌患者中差异无统计学意义(P>0.05);但Ku80的表达与患者的肿块大小和临床分期相关(P<0.01)。Spearman等级相关分析,在55例乳腺癌患者中,Ku80与DNA-PKcs的表达呈正相关(r=0.36,P=0.01);Ku80与ATM的表达亦呈正相关(r=0.33,P=0.02)。Ku80、DNA-PKcs和ATM蛋白在宫颈癌患者中的阳性率分别为70.73%、68.29%和19.51%,在CIN患者中的阳性率分别为80.00%、73.33%和33.33%;3种蛋白在宫颈癌组织中的表达均低于CIN组织,但差异无统计学意义(P>0.05)。3种蛋白的表达在不同年龄、病理类型、分化程度和临床分期的宫颈癌患者中差异无统计学意义(P>0.05)。Spearman等级相关分析,在56例患者中,Ku80与DNA-PKcs的表达呈正相关(r=0.58,P=0.00);Ku80与ATM的表达亦呈正相关(r=0.27,P=0.04)。
【结论】DNA-PKcs和Ku80有可能成为肿瘤治疗增敏的靶点;DNA-PKcs蛋白可能在乳腺癌的发生发展中起重要作用;Ku80与DNA-PKcs及ATM之间均存在密切关系。
第二部分宫颈癌细胞株和正常间质细胞株受X线照射后的辐射效应
【目的】利用宫颈癌细胞系和正常血管上皮细胞系、正常成纤维细胞系体外模拟宫颈癌实质和间质血管组织,研究其受到不同剂量放射线照射后的反应。
【方法】宫颈腺癌细胞系HeLa,宫颈鳞癌细胞系SiHa、C33A、Caski,及人脐静脉内皮细胞系ECV304、小鼠成纤维细胞系NIH/3T3细胞,分别用6MV的X线(300cGy/min)照射6Gy和10Gy,24h和48h后收集细胞碘化丙锭染色,流式细胞术检测凋亡率和细胞周期变化。
【结果】受到照射后C33A凋亡率最高,而SiHa最抵抗,其余细胞系介于两者之间;各宫颈癌细胞系及NIH/3T3照射10Gy后48h产生的凋亡率与6Gy相似(P>0.05),而ECV304照射10Gy后48h产生的凋亡率(13.04%±1.08%)却比6Gy(6.51%±0.61%)高许多(P<0.05);各细胞系受到照射后,均表现出明显的G2/M期阻滞,且G2/M阻滞细胞百分比随照射剂量增加而增加;受到照射后一般在24h~48h G2/M期阻滞细胞百分比最高。
【结论】高剂量照射提高肿瘤血管的凋亡率,宫颈癌细胞受照射后G2/M期阻滞,且呈剂量依赖性,为高剂量射线治疗宫颈癌、追加剂量继续放疗、选择周期特异性化疗药物、以及针对G2/M检测点基因进行靶向阻断提供了依据。
第三部分DNA双链断裂修复蛋白表达水平与肿瘤细胞放射敏感性的关系研究
【目的】检测肿瘤细胞株中DNA双链断裂(DNA double-strand break,DSB)修复蛋白(Ku80、DNA-PKcs和ATM)的表达水平和放射敏感性参数,探讨3个蛋白预示肿瘤细胞放射敏感性的价值。
【方法】4株人宫颈癌细胞株HeLa、SiHa、C33A和Caski,3株人乳腺癌细胞株MCF-7、MDA-MB-231和MDA-MB-453,及1株人肺癌细胞株A549,Western blot检测8株细胞中Ku80、DNA-PKcs和ATM蛋白的表达水平,流式细胞仪检测10Gy 6MV X线照射48h后的凋亡率,克隆形成实验检测SF2(Surviving fraction after 2Gy)值和α、β值,Pearson线性相关分析蛋白表达水平与照射后凋亡率、SF2值和α值的相关性。
【结果】3种蛋白在同一株细胞中的表达及同一蛋白在不同细胞株的表达均存在明显差异;DNA-PKcs的表达水平与SF2之间存在正相关关系(r=0.72,P =0.04<0.05);Ku80和ATM的表达与SF2值间无明显相关关系(P>0.05);3种蛋白与凋亡率和α值均无相关性(P >0.05)。
【结论】DNA-PKcs蛋白表达越高细胞对放射线越抵抗,其表达水平可能成为肿瘤细胞放射敏感性的指标;DNA-PKcs和Ku80可能可以成为肿瘤放疗增敏的理想靶点。
第四部分siRNA抑制Ku80表达后对宫颈癌HeLa细胞放化疗敏感性的影响
【目的】建立利用小干扰RNA(small interfering RNA,siRNA)抑制Ku80表达的HeLa细胞模型,以此探讨Ku80在放化疗增敏方面的潜在应用价值。
【方法】构建靶向抑制Ku80的siRNA表达质粒,转染HeLa细胞,筛选稳定表达的转化克隆,Western blot检测Ku80表达变化;克隆形成实验、MTT法和裸鼠皮下瘤形成实验分别检测细胞克隆形成率,及细胞在体外和体内的增殖情况;6MV X线照射6Gy后,流式细胞术检测细胞凋亡率和细胞周期,克隆形成实验检测细胞SF2、D0等值;MTT检测细胞受不同浓度托泊替康、依托泊苷和顺铂作用后细胞增殖率变化。
【结果】构建的质粒转染HeLa细胞获得2个稳定转染克隆,Western blot分析表明阳性克隆Ku80蛋白抑制率达到96.4%,命名为HeLa/Ku80-siRNA; HeLa/Neg-siRNA细胞克隆形成率为0.62±0.02,而HeLa/Ku80-siRNA细胞的克隆形成率为0.46±0.05,明显低于对照细胞(t=5.11,P<0.01);MTT显示细胞培养48h和72h时,HeLa/Ku80-siRNA细胞的增殖率均显著低于对照细胞(P<0.05);裸鼠皮下瘤生长实验示种植25天时,HeLa/Ku80-siRNA细胞种植瘤的平均体积(18.92±3.60)mm3,明显低于对照细胞种植瘤体积(194.88±30.61)mm3,(t=12.69,P<0.01),抑瘤率达到90.06%。HeLa/Ku80-siRNA体外受X线照射后48h及72h的凋亡率高于对照细胞(P<0.05),但3株细胞的细胞周期变化无统计学意义(P>0.05);阳性克隆细胞株的D0和SF2值明显降低,在D10剂量时的增敏比为1.365。Ku80受抑细胞对托泊替康和依托泊苷的敏感性增加(P<0.05)。
【结论】Ku80-siRNA可以抑制Ku80的表达,在体内外抑制HeLa细胞的增殖,并促进HeLa细胞对X线和拓扑异构酶抑制剂的敏感性。
第五部分抑制DNA-PKcs表达促进宫颈癌HeLa细胞放射敏感性的研究
【目的】探讨DSB修复蛋白DNA-PKcs成为宫颈癌放疗增敏靶点的可能性。
【方法】利用靶向抑制DNA-PKcs的小发卡样干扰RNA(small hairpin interfering RNA,shRNA)表达质粒和小分子抑制剂LY294002,分别抑制HeLa细胞DNA-PKcs蛋白表达和活性后,克隆形成实验和流式细胞仪检测HeLa细胞受6MV X线照射后的SF2、α值和凋亡率变化。
【结果】靶向抑制DNA-PKcs的shRNA可以促进宫颈癌HeLa细胞的放射敏感性,其SF2值为0.37,显著低于对照HeLa细胞的0.53;单独接受50μmol/L LY294002作用1h未使HeLa细胞的凋亡率明显增加(P>0.05),但先经LY294002处理再照射6Gy的HeLa细胞在48h和72h的凋亡率比单独照射6Gy的HeLa细胞凋亡率显著增加(48h点:t=3.25,P=0.03;72h点:t=3.01,P=0.04)。
【结论】抑制DNA-PKcs的表达或活性可以促进宫颈癌HeLa细胞的放射敏感性;提示DNA-PKcs可能可以成为宫颈癌放疗增敏的理想靶点。
第六部分协同抑制Ku80和DNA-PKcs对HeLa细胞放射生物学功能的影响
【目的】研究siRNA和LY29400抑制单个或多个DNA双链断裂修复蛋白后HeLa细胞放射敏感性和细胞周期的变化。
【方法】Ku80受抑细胞HeLa/Ku80-siRNA和对照细胞HeLa/Neg-siRNA分别转染可以靶向抑制抑制DNA-PKcs的siRNA或用PI-3-K抑制剂LY294002处理,经6MV X线照射后,克隆形成实验检测细胞放射敏感性变化,流式细胞仪检测细胞周期和凋亡率变化。
【结果】转染DNA-PKcs-siRNA后的HeLa/Ku80-siRNA放射敏感性显著高于HeLa/Ku80-siRNA , SF2分别为0.08±0.01和0.20±0.05 , LY294002作用后HeLa/Ku80-siRNA的SF2达到0.03±0.01,作为对照细胞的HeLa/Neg-siRNA其SF2值为0.51±0.07;各细胞照射6Gy后均出现G2/M期阻滞,转染DNA-PKcs-siRNA的HeLa/Neg-siRNA细胞和LY294002作用的HeLa/Ku80-siRNA、HeLa/Neg-siRNA细胞G2/M期阻滞逐渐缓慢出现,照射后72h仍未达到顶点,而其他细胞G2/M期阻滞均于照射后48h达到顶点。
【结论】在抑制Ku80已达95%的基础上,DSB修复功能可以由其他DSB修复蛋白如DNA-PKcs和ATM代偿,协同抑制上述蛋白可以明显增加HeLa细胞的辐射敏感性;Ku80、DNA-PKcs和ATM在细胞的G2/M期阻滞过程中发挥的作用不同。
PartⅠThe expressions of DSB repair protein in benign and malignant tissues of breast and cervix
[Objective] To study the expressions of DSB repair proteins (including Ku80, DNA-PKcs and ATM) in breast carcinoma, breast fibroma, cervical carcinoma and cervical intra-epithelial neoplasia (CIN), and to explore their roles in neoplasia and tumor progression of breast and cervical carcinoma.
[Methods] Immunohistochemistry was applied to detect the expressions of Ku80, DNA-PKcs, and ATM in 55 cases of breast carcinoma, 14 cases of breast fibroma, 41 cases of cervical carcinoma and 15 cases of CIN.
[Results] The positive percentages of Ku80, DNA-PKcs and ATM were 74.55%、54.55% and 52.73% respectively in breast carcinoma, and were 92.86%、92.86% and 71.43% respectively in breast fibroma; The expression of three proteins in fibroma were all higher than that in carcinoma, but the differences of Ku80 and ATM were no significant (P>0.05), only the expression difference of DNA-PKcs was significant (X2=6.98,P=0.01). The expressions of DNA-PKcs and ATM protein were not associated with the pathology, tumor diameter, clinical stage and the lymph node metastasis of breast carcinoma patients (P>0.05), while Ku80 expression associated with tumor diameter and clinical stage of breast carcinoma patients (P<0.01). In breast carcinoma cases, Ku80 was positively correlated with DNA-PKcs (r=0.36, P=0.01) and ATM (r=0.36, P=0.02). The positive percentages of Ku80, DNA-PKcs and ATM were 70.73%, 68.29% and 19.51% respectively in cervical carcinoma, and were 80.00%, 73.33% and 33.33% respectively in CIN; the expression of three proteins in CIN were all higher than that in cervical carcinoma, but the differences were no significant (P>0.05). The expression of these proteins were not associated with the age of patients, pathology, differentiation, and clinical stage (P>0.05).in all 56 cases, Ku80 was positively correlated with DNA-PKcs (r=0.58, P=0.00) and ATM (r=0.27, P=0.04).
[Conclusions] DNA-PKcs and Ku80 are more possible to be the target of cancer gene-therapy; DNA-PKcs might play the major role in the neoplasia and tumor progression of breast carcinoma; Ku80 had close relationship with DNA-PKcs and ATM in breast and cervical carcinoma.
PartⅡResponse of cervical carcinoma and normal interstitial cell lines after X-ray irradiation
[Objective] Study the response of cervical carcinoma cell lines, normal vascular endothelial cell line and fibroblast cell line which simulate as cervical carcinoma tissues after a serial of doses of X-ray irradiation.
[Methods] Human cervical adencarcinoma cell line HeLa, human cervical squamous carcinoma cell lines SiHa, C33A, Caski, human umbilical vein endothelial cell line ECV304 and mouse fibroblast cell line NIH/3T3 were irradiated with 6 MV X-ray at the absorb doses of 6Gy and 10Gy. After irradiated for 24h and 48h, cells were collected and analyzed by flow cytometry for apoptosis and cell cycle.
[Results] The apoptosis rate of C33A was highest, while the SiHa cell line was most resistant for X-ray; The apoptosis rates of ECV304 after irradiation with 10 Gy (13.04%±1.08%) was higher than 6Gy (6.51%±0.61%), P<0.05, while ones of the other cell lines were similar (P>0.05); Every cell lines were blocked at G2/M phase after irradiation, the cell percentages of G2/M phase increased with the doses of irradiation and got to maximum at 24-48h after irradiation.
[Conclusions] High doses of irradiation could improve the apoptosis of tumor vascular endothelial cell and increase the cell accumulation of G2/M phase, which provide the theory evidences for high dose of irradiation at cervical carcinoma radiotherapy, boosting dose of irradiation, selecting the specific drugs aim directly at G2/M phase, and specifically inhibiting the genes of G2/M check point for gene therapy.
PartⅢCorrelativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells
[Objective] To explore the correlation between the expressions of DSB (DNA double-strand break) repair protein (including Ku80, DNA-PKcs and ATM) and radiosensitivity parameters in human tumor cell lines, and to study which protein could prognosticate the radiosensitivity of tumor cell.
[Methods] SF2 (survival fraction at 2Gy),αandβvalues of eight tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation array,apoptosis rates at 48h after 10Gy of 6MV X-ray irradiation were analyzed by flow cytometry analysis, and Western Blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein, the correlativity between protein expression and SF2,α,βvalue or apoptosis rate was analyzed by Pearson linear correlation analysis.
[Results]The expression of same protein in different cell lines and the expression of three proteins in the same cell line were various; there was a positive correlativity between the expression of DNA-PKcs and SF2 (r=0.72, P=0.04), but no correlativity was found between Ku80 and SF2 or ATM and SF2 (P>0.05). Bothαand apoptosis rate had no correlativity with the expression of DSB repair protein (P>0.05).
[Conclusions] The expression of DNA-PKcs protein may be able to prognosticate the radiosensitivity of tumor cells, and could be an ideal target to enhance the radiosensitivity.
PartⅣTo study the effect on radio- and chemo- sensitivity of HeLa cells after Ku80 inhibition
[Objective] The HeLa cell model of Ku80 expression suppressed by siRNA was constructed and utilized to study the role of Ku80 in enhancing the radio- and chemo- sensitivity
[Methods] Ku80-siRNA expression plasmids were constructed and HeLa cells were transfected with these plasmids by lipofectamine. Western blot was applied to measure the expression of Ku80;The proliferation of HeLa cells were determined by clone formation assay, MTT assay and tumor subcutaneously formation studies on nude mice. After irradiation with 6MV X-ray,cells were collected and analyzed by flow cytometry for apoptosis and cell cycle at 24h, 48h and 72h; The radiobiology parameters of three cell lines were acquired by clone formation array. The inhibition rate of cells treated with Topotecan, Etoposide or DDP was detected by MTT assay.
[Results] Two cell clones stable transfected were got, and the inhibition rates of Ku80 protein expression of positive clone got to 96.40%; The clone formation efficiency of HeLa/Ku80-siRNA was 0.46±0.05, lower than the control cells (t=5.11, P<0.01). The proliferation rate of HeLa/Ku80-siRNA were slower than control cells 48h and 72h after transfection (P<0.05). And after transplanted for 25 days, the mean tumor volume of HeLa/Ku80-siRNA was only (18.92±3.60) mm3, while the mean tumor volume of control cells got to (194.88±30.61) mm3, the difference was significant (t=12.69, P<0.01), tumor inhibition rate got to 90.06%. The apoptosis rates of HeLa cells Ku80 inhibited were higher than control cells at 48h and 72h after X-ray radiation (P<0.05), but the differences of cell cycle change after irradiation were not significant (P>0.05); HeLa cells silenced of Ku80 had lower SF2 and D0 than control cells, and their SER (sensitization enhancement ratio) based on D10 got to 1.365. HeLa/Ku80-siRNA was more sensitivity to Topotecan and Etoposide than control cells (P<0.05).
[Conclusion] The HeLa cell models Ku80 expression suppressed were successfully established; the inhibition of Ku80 by siRNA could enhance the radio- and chemo- sensitivity of HeLa, and suppress the cell proliferation.
PartⅤStudy of Enhanced Radiosensitivity in Cervical Carcinoma Cell Line HeLa by Inhibition of DNA-PKcs
[Objective] This study tried to probe potential role of DNA-PKcs in enhancing the radiosensitivity of cervical carcinoma.
[Methods] The DNA-PKcs-targeted shRNA (small hairpin interfering RNA) and a competive DNA-PKcs inhibitor LY294002, were applied to inhibit the DNA-PKcs expression or activity in HeLa cells. Then the SF2,αvalues and apoptosis rates were analyzed by clonogenic survival assay and flow cytometry analysis respectively. [Results] The DNA-PKcs-targeted shRNA could enhance the radiosensitivity of HeLa, and its SF2 was 0.37, lower than the control cells clearly (0.53). The apoptosis rates of HeLa cells only administrated by 50μmol/L LY294002 management for 1 hour did not increase significantly (P>0.05), but LY294002 sensitized HeLa cells to 6 MV X-ray, after HeLa cells were administrated LY294002 and irradiation at 48h and 72h after 6Gy irradiation, the apoptosis rates were higher than those of cells only accepted irradiation (48h: t=3.25, P=0.03; 72h: t=3.01, P=0.04).
[Conclusion] Inhibition of DNA-PKcs expression or activities may sensitize HeLa cells to X-ray. As the results, DNA-PKcs may be an ideal target for enhancing radiosensitivity of cervical carcinoma.
PartⅥThe effect on radiobiology of HeLa after co-inhibition of DNA-PKcs and Ku80
[Objective] To study the radiosensibility and cell cycle distribution of HeLa inhibited of one or several DNA double-strand break (DSB) repair proteins after being treated with small interfering RNA (siRNA) or LY294002.
[Methods] Ku80 silenced cell HeLa/Ku80-siRNA and control cell HeLa/Neg-siRNA were applied, and transfected with DNA-PKcs-targeted siRNA or pretreated with 50μM LY294002, a chemically specific phosphatidylinositol (PI) 3-kinase (DNA-PKcs and ATM are both members of PI 3-kinase gene family) inhibitor, after irradiated with 6MV X-ray, cells were detected by clonogenic survival assay for radiosensibility and analyzed by flow cytometry for cell cycle distribution and apoptosis.
[Results] HeLa/Ku80-siRNA transfected with DNA-PKcs-siRNA was more sensitive to radiation than HeLa/Ku80-siRNA, SF2 were 0.08±0.01 and 0.20±0.05 respectively, the SF2 of HeLa/Ku80-siRNA pretreated with LY294002 even got to 0.03±0.01, while the SF2 of control cell line HeLa/Neg-siRNA was 0.51±0.07; G2/M accumulation was observed at all cell lines after 6Gy X-ray exposure, the percentage of DNA-PKcs-siRNA transfected HeLa/Neg-siRNA and LY294002 treated cells in G2/M phase even didn’t get to highest at 72h post-irradiation, while other cell lines had a shorter G2/M delay, the G2/M accumulation got to vertex at 48h post-irradiation.
[Conclusions] After 95% Ku80 was inhibited, HeLa cells could have other proteins, for example DNA-PKcs or ATM to compensate the DSB repair function, co-inhibition of these proteins resulted in markedly sensitive to X-ray; Ku80, DNA-PKcs and ATM may play different role in cell cycle accumulation after irradiation.
引文
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