Apoptin对膀胱移行细胞癌抑制增殖和诱导凋亡作用研究
|
摘要
膀胱移行细胞癌是泌尿系统最常见的恶性肿瘤,发病率居高不下,临床上主要以手术、放化疗和免疫治疗为主,虽能明显的延长患者的生存期,但因其具有多发和易复发的特点,远期疗效及患者生活质量并不令人满意,尤其是中、晚期患者,往往丧失了手术机会。作为一种有望彻底治愈肿瘤的治疗方式,肿瘤基因治疗虽然开展较晚,却拥有广阔的前景。近年来,伴随分子生物学、细胞生物学的发展及各种癌基因、抑癌基因的发现,肿瘤基因治疗获得了极大进步,并取得了可喜的成绩。肿瘤基因治疗的关键是靶向性杀伤,既要有效杀伤肿瘤细胞,又不能杀伤或干扰正常细胞。缺乏肿瘤细胞靶向性是限制基因治疗不能广泛应用于临床的瓶颈。具有特异性肿瘤杀伤作用的基因是肿瘤基因治疗的理想选择。
Apoptin是由鸡贫血病毒VP3基因编码的凋亡功能蛋白,包含21个氨基酸,分子量为13.6KD,不与任何已知蛋白同源。研究表明,Apoptin具有广谱的诱导肿瘤细胞凋亡效应,能诱导卵巢癌、肝癌、恶性淋巴瘤等多种肿瘤细胞凋亡,而对正常淋巴、真皮、上皮、内皮等人正常和原代二倍体细胞无杀伤及毒性作用,尤其是对化疗药物及放射治疗极为敏感的二倍体细胞,如骨髓来源的造血干细胞CD34+和间叶干细胞及淋巴细胞等,均不受Apoptin诱导的凋亡影响,不同途径给药亦未发现Apoptin的毒性效应,这均说明其具有潜在的抗肿瘤应用价值。因此,本研究将此外源性基因引入膀胱癌的基因治疗,构建能表达Apoptin的真核表达质粒,以EGFP为报告基因,脂质体介导瞬时转染膀胱移行细胞癌T24细胞,从体外培养和移植瘤模型两方面研究Apoptin对膀胱癌的治疗作用,并对其与化疗药物顺铂的联合应用可行性做初步探讨。
研究目的
1.探讨Apoptin对体外培养的T24细胞增殖、凋亡、细胞周期的影响。
2.探讨Apoptin对人膀胱癌裸鼠移植瘤的治疗作用。
3.探讨Apoptin与化疗药物顺铂对T24的协同杀伤作用。
研究内容和方法
1. EGFP标记的Apoptin真核表达质粒的构建及鉴定:以变异型Apoptin质粒p3×flag-m-Apoptin-myc为模板,根据NCBI GenBank登录的野生型Apoptin基因序列设计引物,矫正变异碱基,扩增出野生型Apoptin基因片断,并将其定向克隆于以EGFP作为报告基因的载体pEGFP-N1中,构建能表达Apoptin-EGFP融合蛋白的重组质粒pApoptin-EGFP,重组质粒经双酶切、电泳初步确认,送上海生工测序鉴定。
2. Apoptin对膀胱癌T24细胞的诱导凋亡作用:脂质体介导,将重组质粒瞬时转染膀胱癌T24细胞,RT-PCR检测Apoptin mRNA表达,倒置荧光显微镜观察融合蛋白表达,MTT检测转染后不同时相点T24增殖抑制,凋亡光学试剂盒检测转染后T24细胞形态变化和凋亡率,流式细胞仪检测凋亡率和细胞周期。
3. Apoptin对膀胱癌裸鼠皮下移植瘤的治疗作用:皮下注射T24细胞,建立人膀胱移行细胞癌裸鼠移植瘤模型,于肿瘤直径0.5cm大小时,采用多点注射的方法,将脂质体包裹的质粒pApoptin-EGFP导入T24裸鼠移植瘤,设立空质粒组、生理盐水组作为对照组,观察裸鼠及移植肿瘤生长情况;5周后处死荷瘤鼠,绘制移植瘤生长曲线,计算抑瘤率,肿瘤组织石蜡包埋,常规切片,H.E.染色观察组织细胞形态,TUNEL法检测移植瘤组织细胞调亡。
4. Apoptin与顺铂(DDP)对T24细胞的杀伤及协同作用:梯度浓度的顺铂作用于转染pApoptin-EGFP和空质粒的T24细胞,MTT法检测顺铂作用24小时后T24细胞增殖,流式细胞仪检测凋亡,金氏法判断质粒转染与顺铂干预是否具有协同作用。
实验结果
1.通过酶切电泳及测序鉴定,确认已成功扩增出野生型Apoptin基因,与GenBank登陆序列完全一致,在EcoRⅠ和BamH I酶切位点间正确插入pEGFP-N1载体内,方向及编码序列正确,与EGFP编码序列构成完整阅读框,无移码突变,成功构建理论上能表达Apoptin-EGFP融合蛋白的真核表达质粒pApoptin-EGFP。
2.瞬时转染人膀胱癌T24细胞后,RT-PCR检测到Apoptin mRNA表达;荧光显微镜下可见绿色荧光,说明融合蛋白能在真核细胞T24中顺利表达;MTT检测T24细胞增殖情况,结果发现转染apoptin对膀胱移行细胞癌T24细胞生长具有明显抑制作用,24h、48h、72h抑制率分别(19.4±3.76)%,(37.5±4.66)%和(49.5±4.15)%;与空质粒对照组相比,抑制率显著增高。经过凋亡光学试剂盒检测,Apoptin的瞬时转染可引起细胞核高度凝聚,染色质密集、呈边缘化等凋亡特征性表现,经计数,凋亡率约为(21.25±1.98)%,而空质粒组仅为(7.3±1.04)%。流式细胞仪Annexin-V/PI法检测细胞转染率和凋亡,转染后48小时,pApoptin-EGFP组早期调亡率为(18.7±1.13)%, pEGFP-N1组为(4.97±0.57)%,两者具有非常显著差异(P<0.01);晚期调亡细胞Apoptin组为(19.2±1.99)%,较对照组(2.7±1.73)%同样有非常显著的提高,进一步说明Apoptin能显著诱导T24细胞凋亡。流式细胞仪检测24h、48h、72 h三个时相点细胞周期变化,发现瞬时转染空质粒24h可引起S期细胞减少,G1、G2/M期轻度阻滞,随时间推移,以上变化逐渐减弱,72小时基本接近空白对照组,而转染pApoptin-EGFP重组质粒的T24细胞S期细胞比例显著减少,并随时间推移,变化逐渐加大,72小时尤为明显,已由46.8%持续下降到28.5%,G1/G0和G2/M期均有上升,呈现双阻滞现象。
3.成功构建人膀胱移行细胞癌裸鼠移植瘤模型;注射pApoptin-EGFP质粒的荷瘤鼠精神状态较好,饮食正常,体重增加,无消瘦等恶病质表现;肿瘤体积小,形态较规则,生长缓慢,处死剥离时与周围组织无明显粘连,净重平均为0.26±0.08g。生理盐水组和空白质粒组荷瘤鼠精神状态差,不喜活动,食欲差,毛色晦暗,消瘦;肿瘤生长速度较快,形态欠规则,处死剥离时与周围组织明显粘连,净重分别为1.02±0.23g和0.83±0.20g。以生理盐水组作对照,pApoptin-EGFP质粒注射组和空质粒组肿瘤抑制率分别为74.5%和18.6%,两者差异明显。三组移植瘤组织切片H.E染色提示三组细胞核大、深染,组织结构紊乱,符合肿瘤细胞特征,pApoptin-EGFP质粒注射组染色质浓缩、聚集的细胞明显增多;TUNEL染色示pApoptin-EGFP、pEGFP-N1、生理盐水三组凋亡率分别为(23.24±6.12)%,(3.52±1.20)%和(1.76±0.44)%,pApoptin-EGFP注射组与空白质粒注射组、生理盐水注射组相比,凋亡率有非常显著差异。说明Apoptin作为外来基因产物对人膀胱癌T24裸鼠移植瘤具有明显的抑瘤作用,治疗期间未见明显的毒、副作用。
4.随顺铂(DDP)浓度的增加,DDP、pEGFP-N1+DDP、pApoptin-EGFP+DDP三组T24细胞抑制率逐渐增高;除64ug/ml浓度组外每个剂量点的pApoptin-EGFP + DDP组IR值均比DDP、pEGFP-N1+DDP组为高,差异显著,具统计学意义(P<0.05);64ug/ml组因抑制效应已属平台期,故仅呈增高趋势,却无统计学意义;经计算,DDP、pEGFP-N1+DDP和pApoptin-EGFP+DDP三组的IC50分别为10.61ug/ml,7.9ug/ml和2.4ug/ml;经金氏法计算和分析,确认pApoptin-EGFP质粒转染与DDP干预对T24的抑制增殖效应具有协同作用,而转染空质粒与DDP干预的作用仅为简单相加;同法分析可见pApoptin-EGFP和DDP诱导T24细胞凋亡效应同样具有协同作用,而空质粒与DDP诱导凋亡效应仅为简单相加。
结论
1.构建了pApoptin-EGFP重组质粒,该质粒能在膀胱癌T24细胞中顺利表达Apoptin-EGFP融合蛋白。
2.与空质粒相比,Apoptin能明显的抑制T24细胞增殖,诱导T24细胞凋亡,阻滞细胞周期于G1/G0和G2/M期。
3. Apoptin能抑制T24裸鼠移植瘤生长,改善荷瘤鼠一般状态,对膀胱移行细胞癌具有明显的治疗作用,作用方式主要为抑制增殖和促进凋亡,治疗期间未见明显毒副作用。
4. Apoptin重组质粒转染联合化疗药物顺铂可加强对T24细胞的抑制增殖效应和诱导凋亡效应,且两种治疗方式具有协同作用;这种协同作用可能与两者均参与对p73家族蛋白的调节有关,其具体机制有待进一步研究。
Transitional cell carcinoma of bladder(TCCB)is the most common malignant tumor in urinary system, and the morbidity rate is very high. Operation, radiotherapy, chemotherapy and immunotherapy are predominant in the clinical treatment of TCCB. Although these means can visibly prolong the survival time of patients, but long-term efficacy and quality of life are not satisfactory owing to its multiple and high recurrence. Furthermore the patients with TBBC in intermediate and advanced stage would always lose the opportunities of operation. As a promising way to cure carcinoma, gene therapy got a late start but have a bright future. In recent years, with the development of molecular biology and cell biology, various oncogenes and tumor suppressor genes were discovered, gene therapy have made great progress and got gratifying achievement. Tumor targeting is the key point of gene therapy, which would kill tumor cells effectively but not kill or interfere normal cell. Tumor targeting insufficient is the bottle neck of gene therapy for clinical application. Genes with the role of anti-tumor selectively would be an ideal choice for tumor gene therapy.
Apoptin, which contains 121 amino acids, is encoded by VP3 gene from chicken anemia virus. Its molecular weight is 13.6 KD and it has no homology with any known proteins. Studies show that apoptin could selectively induce apoptosis in tumor cells such as ovarian cancer, liver cancer, malignant lymphoma, while leaving normal cells such as lymph, derma, epithelium, endothelium and other normal human diploid cells intact, especially the diploid cells which are extremely sensitive for chemotherapy and radiotherapy, such as CD34+ hematopoietic stem cells derived from bone marrow, mesenchymal stem cells and lymphocytes were not interfered by Apoptin either, and no toxicity of apoptin had been found with different administration. All of these indicate its prospective anti-tumor value. Therefore, eukaryotic expression plasmid which contains wild type apoptin gene and report gene of EGFP was constructed, and the recombinant plasmid was transfected into transitional cell carcinoma of bladder cell line T24 by mixed with lipofectamin 2000. The therapeutic effects to T24 were detected in vivo and in vitro.
Object
1. To investigate the role of Apoptin on the proliferation, apoptosis and cell cycle of T24.
2. To investigate the role of apoptin in the athymic mouse human bladder tumor transplantation tumor model.
3. To investigate the synergistic interaction of apoptin and chemotherapy drug cisplatin on T24 cells.
Materials and Methods
1. Construction and identification of eukaryotic expression plasmid pApoptin-EGFP: We designed primers according to the wild-type appoptin cDNA sequence from GenBank. Wild type apoptin gene was prepared by PCR from plasmid p3×flag-m-Apoptin-myc which contains a variant-type apoptin gene, then the wild-type apoptin gene was directional cloned to the vector pEGFP-N1. The recombinant plasmid pApoptin-EGFP was identified by double enzyme digestion and sequence.
2. The apoptosis effect induced by Apoptin in T24 cells: The identified plasmid pApoptin-EGFP was transfected into T24 cells with Lipofectamine 2000. Apoptin mRNA was detected by RT-PCR. The fusion protein apoptin-EGFP was observed by fluorescent microscope. The proliferation inhibition induced by apoptin in T24 cells were detected by MTT. The morphous and apoptosis of transfected T24 cells was observed by using apoptosis optical kit. FCM was used to detected apoptosis and cell cycle of T24 cells.
3. The therapeutic effect of Apoptin on bladder xenograft tumor: Athymic mouse T24 xenograft tumor model was established by inoculating T24 cells subcutaneously. pApoptin-EGFP parceled with liposome was injected into the tumor, when the diameter of xenograft tumor is about 0.5cm, empty plasmid and normal saline were injected as control. Tumor formation, tumor growth, tumor size, athymic mice weight and activity status were observed. The volume of transplant tumor was measured and recorded continually, The mice were sacrificed five weeks later, tumor-inhibition rate was evaluated and the apoptosis rate of cells was detected by in situ TUNEL.
4. Synergistic killing effects of Apoptin and cisplatin (DDP) on T24 cells: Concentration gradient cisplatin was administrated to T24 cells which transfected with pApoptin-EGFP. 24 hours later, the proliferation of T24 cells was detected by MTT and the apoptosis of T24 cells was assayed by flow cytometry.
Conclusion
1. Wild-type apoptin gene was amplified by PCR successfully and the product was correctly cloned to vector pEGFP-N1 between the enzyme site of EcoRⅠand BamHⅠ. An integrated reading frame was constructed consist of apoptin gene and EGFP gene. With no frame shift mutation, the recombinant plasmid pApoptin-EGFP would express apoptin-EGFP fusion protein in theory.
2. Wild-type Apoptin mRNA could be amplified from the transfected T24 cells by RT-PCR method and the fluorescence of EGFP could be observed by using fluorescence microscopy. Both indicated fusion protein could be express in T24 cell. The results of MTT showed that Apoptin could inhibit the proliferation of T24 cells. The inhibition rates were 19.4%, 37.5%, 49.5% respectively at the time-point of 24h, 48h, 72h after transient transfection. Analyzed with student’s t test, the mean of proliferation rate on T24 cells showed significant difference between pApopotin-EGFP group and the control. The morphology of T24 cells showed typical apoptosis changes detected by staining with apoptosis detected kit. The apoptosis rate of T24 was 21.25%, much higher than that of the control at the time-point of 48 hours post transient transfection. Transfected cells tagged with APC and PI were analyzed by FCM, the results indicated that the early apoptotic rate of pApoptin-EGFP group could reach 18.3%, while empty plasmid group only 4.7%, significant difference was found between the two groups. Furthermore, the advanced apoptosis or necrosis of Apoptin group was 17.8% compared to 1.6% in the control group, significant difference could be found either. All these suggested that Apoptin play an important role which could obviously induce apoptosis on bladder tumor cells. FCM was performed to study the cell life cycle at different time-point post transfection. We found that the percentage of S phase is reduced and the G1/G2, G2/M phase arrested 24 hours post the transfection, as the time go on, these changes become weaken and the cell cycle is nearly to the cells without transfection.72 hour later. In contrast, pApoptin-EGFP group got S phase reduced at 24h post transfection and as the time go on, these changes become strength, especially at the 72 hours time-point, the percentage of S phase have been reduced from 46.8% to 28.5%, both G1/G0 and G2/M phase were arrested.
3. T24 xenograft tumor model was established successfully. Compared with the control group, the Athymic mice that injected with pApoptin-EGFP got better status and weight gain, without any cachexia syndrome. The tumor weight of pApoptin-EGFP, pEGFP-N1 and NS groups are 0.26±0.08g, 0.83±0.20g, 1.02±0.23g respectively. There are significant difference between pApoptin-EGFP group and the control, No statistical difference have been found between the pEGFP-N1 group and the NS group. The tumor inhibition ratio of pApoptin-EGFP group was 74.5% and pEGFP-N1 group was 18.6% as contrast. xenograft tumor tissue slice stained by H.E, the tissue was identified to be TCCB. The apoptosis rate of the three group (pApoptin-EGFP, pEGFP-N1, NS) were (23.24±6.12)%, (3.52±1.20)%, (1.76±0.44)% respectively detected by in situ TUNEL. There are significant difference between pApoptin- EGFP group and the control.(P<0.01). It suggested that Apoptin as exogenous gene product could inhibit TCCB in vitro and no side effects had been found in our experiment.
4. The proliferation inhibition ratio of the three groups(DDP、pEGFP-N1+DDP、pApoptin-EGFP+DDP) on T24 cells increased as the concentration of DDP raise. Inhibition ratio of each dose point between pApoptin-EGFP+DDP and the other two groups had been found to be significant different by using statistical test, except 64ug/ml dose point because of the curve of inhibition ratio had been reach platform. The IC50 of each group(DDP、pEGFP-N1+DDP, pApoptin-EGFP+DDP) were 10.61ug/ml, 7.9ug/ml and 2.4ug/ml respectively. Calculated by using Jin-formula, we found pApoptin-EGFP and DDP had synergistic effect on inhibiting the proliferation of T24 cells. In contrast inhibition effects of pEGFP-N1and DDP on T24 cells were only simple addition. Same result was found for the apoptosis induction on T24 cells between the pApoptin-EGFP and DDP.
Conclusions
1. The recombinant plasmid pApoptin-EGFP was constructed successfully which could express the apoptin-EGFP fusion protein in transitional cell carcinoma of bladder cell lines T24.
2. Compared to the empty group, the Apoptin gene could significantly inhibit the T24 cells’proliferation , induce apoptosis and make cell life cycle arrested in G1/G0 and G2/M stage.
3. Apoptin can inhibit the growth of xenograft transitional cell carcinoma of bladder by inhibiting proliferation and inducing apoptosis, no side effects were observed during the treatment.
4. Apoptin recombinant plasmid transfection combined with cisplatin could enhance proliferation inhibition and apoptosis induction effects on T24 cells, and the two therapies have synergies.
Apoptin是由鸡贫血病毒VP3基因编码的凋亡功能蛋白,包含21个氨基酸,分子量为13.6KD,不与任何已知蛋白同源。研究表明,Apoptin具有广谱的诱导肿瘤细胞凋亡效应,能诱导卵巢癌、肝癌、恶性淋巴瘤等多种肿瘤细胞凋亡,而对正常淋巴、真皮、上皮、内皮等人正常和原代二倍体细胞无杀伤及毒性作用,尤其是对化疗药物及放射治疗极为敏感的二倍体细胞,如骨髓来源的造血干细胞CD34+和间叶干细胞及淋巴细胞等,均不受Apoptin诱导的凋亡影响,不同途径给药亦未发现Apoptin的毒性效应,这均说明其具有潜在的抗肿瘤应用价值。因此,本研究将此外源性基因引入膀胱癌的基因治疗,构建能表达Apoptin的真核表达质粒,以EGFP为报告基因,脂质体介导瞬时转染膀胱移行细胞癌T24细胞,从体外培养和移植瘤模型两方面研究Apoptin对膀胱癌的治疗作用,并对其与化疗药物顺铂的联合应用可行性做初步探讨。
研究目的
1.探讨Apoptin对体外培养的T24细胞增殖、凋亡、细胞周期的影响。
2.探讨Apoptin对人膀胱癌裸鼠移植瘤的治疗作用。
3.探讨Apoptin与化疗药物顺铂对T24的协同杀伤作用。
研究内容和方法
1. EGFP标记的Apoptin真核表达质粒的构建及鉴定:以变异型Apoptin质粒p3×flag-m-Apoptin-myc为模板,根据NCBI GenBank登录的野生型Apoptin基因序列设计引物,矫正变异碱基,扩增出野生型Apoptin基因片断,并将其定向克隆于以EGFP作为报告基因的载体pEGFP-N1中,构建能表达Apoptin-EGFP融合蛋白的重组质粒pApoptin-EGFP,重组质粒经双酶切、电泳初步确认,送上海生工测序鉴定。
2. Apoptin对膀胱癌T24细胞的诱导凋亡作用:脂质体介导,将重组质粒瞬时转染膀胱癌T24细胞,RT-PCR检测Apoptin mRNA表达,倒置荧光显微镜观察融合蛋白表达,MTT检测转染后不同时相点T24增殖抑制,凋亡光学试剂盒检测转染后T24细胞形态变化和凋亡率,流式细胞仪检测凋亡率和细胞周期。
3. Apoptin对膀胱癌裸鼠皮下移植瘤的治疗作用:皮下注射T24细胞,建立人膀胱移行细胞癌裸鼠移植瘤模型,于肿瘤直径0.5cm大小时,采用多点注射的方法,将脂质体包裹的质粒pApoptin-EGFP导入T24裸鼠移植瘤,设立空质粒组、生理盐水组作为对照组,观察裸鼠及移植肿瘤生长情况;5周后处死荷瘤鼠,绘制移植瘤生长曲线,计算抑瘤率,肿瘤组织石蜡包埋,常规切片,H.E.染色观察组织细胞形态,TUNEL法检测移植瘤组织细胞调亡。
4. Apoptin与顺铂(DDP)对T24细胞的杀伤及协同作用:梯度浓度的顺铂作用于转染pApoptin-EGFP和空质粒的T24细胞,MTT法检测顺铂作用24小时后T24细胞增殖,流式细胞仪检测凋亡,金氏法判断质粒转染与顺铂干预是否具有协同作用。
实验结果
1.通过酶切电泳及测序鉴定,确认已成功扩增出野生型Apoptin基因,与GenBank登陆序列完全一致,在EcoRⅠ和BamH I酶切位点间正确插入pEGFP-N1载体内,方向及编码序列正确,与EGFP编码序列构成完整阅读框,无移码突变,成功构建理论上能表达Apoptin-EGFP融合蛋白的真核表达质粒pApoptin-EGFP。
2.瞬时转染人膀胱癌T24细胞后,RT-PCR检测到Apoptin mRNA表达;荧光显微镜下可见绿色荧光,说明融合蛋白能在真核细胞T24中顺利表达;MTT检测T24细胞增殖情况,结果发现转染apoptin对膀胱移行细胞癌T24细胞生长具有明显抑制作用,24h、48h、72h抑制率分别(19.4±3.76)%,(37.5±4.66)%和(49.5±4.15)%;与空质粒对照组相比,抑制率显著增高。经过凋亡光学试剂盒检测,Apoptin的瞬时转染可引起细胞核高度凝聚,染色质密集、呈边缘化等凋亡特征性表现,经计数,凋亡率约为(21.25±1.98)%,而空质粒组仅为(7.3±1.04)%。流式细胞仪Annexin-V/PI法检测细胞转染率和凋亡,转染后48小时,pApoptin-EGFP组早期调亡率为(18.7±1.13)%, pEGFP-N1组为(4.97±0.57)%,两者具有非常显著差异(P<0.01);晚期调亡细胞Apoptin组为(19.2±1.99)%,较对照组(2.7±1.73)%同样有非常显著的提高,进一步说明Apoptin能显著诱导T24细胞凋亡。流式细胞仪检测24h、48h、72 h三个时相点细胞周期变化,发现瞬时转染空质粒24h可引起S期细胞减少,G1、G2/M期轻度阻滞,随时间推移,以上变化逐渐减弱,72小时基本接近空白对照组,而转染pApoptin-EGFP重组质粒的T24细胞S期细胞比例显著减少,并随时间推移,变化逐渐加大,72小时尤为明显,已由46.8%持续下降到28.5%,G1/G0和G2/M期均有上升,呈现双阻滞现象。
3.成功构建人膀胱移行细胞癌裸鼠移植瘤模型;注射pApoptin-EGFP质粒的荷瘤鼠精神状态较好,饮食正常,体重增加,无消瘦等恶病质表现;肿瘤体积小,形态较规则,生长缓慢,处死剥离时与周围组织无明显粘连,净重平均为0.26±0.08g。生理盐水组和空白质粒组荷瘤鼠精神状态差,不喜活动,食欲差,毛色晦暗,消瘦;肿瘤生长速度较快,形态欠规则,处死剥离时与周围组织明显粘连,净重分别为1.02±0.23g和0.83±0.20g。以生理盐水组作对照,pApoptin-EGFP质粒注射组和空质粒组肿瘤抑制率分别为74.5%和18.6%,两者差异明显。三组移植瘤组织切片H.E染色提示三组细胞核大、深染,组织结构紊乱,符合肿瘤细胞特征,pApoptin-EGFP质粒注射组染色质浓缩、聚集的细胞明显增多;TUNEL染色示pApoptin-EGFP、pEGFP-N1、生理盐水三组凋亡率分别为(23.24±6.12)%,(3.52±1.20)%和(1.76±0.44)%,pApoptin-EGFP注射组与空白质粒注射组、生理盐水注射组相比,凋亡率有非常显著差异。说明Apoptin作为外来基因产物对人膀胱癌T24裸鼠移植瘤具有明显的抑瘤作用,治疗期间未见明显的毒、副作用。
4.随顺铂(DDP)浓度的增加,DDP、pEGFP-N1+DDP、pApoptin-EGFP+DDP三组T24细胞抑制率逐渐增高;除64ug/ml浓度组外每个剂量点的pApoptin-EGFP + DDP组IR值均比DDP、pEGFP-N1+DDP组为高,差异显著,具统计学意义(P<0.05);64ug/ml组因抑制效应已属平台期,故仅呈增高趋势,却无统计学意义;经计算,DDP、pEGFP-N1+DDP和pApoptin-EGFP+DDP三组的IC50分别为10.61ug/ml,7.9ug/ml和2.4ug/ml;经金氏法计算和分析,确认pApoptin-EGFP质粒转染与DDP干预对T24的抑制增殖效应具有协同作用,而转染空质粒与DDP干预的作用仅为简单相加;同法分析可见pApoptin-EGFP和DDP诱导T24细胞凋亡效应同样具有协同作用,而空质粒与DDP诱导凋亡效应仅为简单相加。
结论
1.构建了pApoptin-EGFP重组质粒,该质粒能在膀胱癌T24细胞中顺利表达Apoptin-EGFP融合蛋白。
2.与空质粒相比,Apoptin能明显的抑制T24细胞增殖,诱导T24细胞凋亡,阻滞细胞周期于G1/G0和G2/M期。
3. Apoptin能抑制T24裸鼠移植瘤生长,改善荷瘤鼠一般状态,对膀胱移行细胞癌具有明显的治疗作用,作用方式主要为抑制增殖和促进凋亡,治疗期间未见明显毒副作用。
4. Apoptin重组质粒转染联合化疗药物顺铂可加强对T24细胞的抑制增殖效应和诱导凋亡效应,且两种治疗方式具有协同作用;这种协同作用可能与两者均参与对p73家族蛋白的调节有关,其具体机制有待进一步研究。
Transitional cell carcinoma of bladder(TCCB)is the most common malignant tumor in urinary system, and the morbidity rate is very high. Operation, radiotherapy, chemotherapy and immunotherapy are predominant in the clinical treatment of TCCB. Although these means can visibly prolong the survival time of patients, but long-term efficacy and quality of life are not satisfactory owing to its multiple and high recurrence. Furthermore the patients with TBBC in intermediate and advanced stage would always lose the opportunities of operation. As a promising way to cure carcinoma, gene therapy got a late start but have a bright future. In recent years, with the development of molecular biology and cell biology, various oncogenes and tumor suppressor genes were discovered, gene therapy have made great progress and got gratifying achievement. Tumor targeting is the key point of gene therapy, which would kill tumor cells effectively but not kill or interfere normal cell. Tumor targeting insufficient is the bottle neck of gene therapy for clinical application. Genes with the role of anti-tumor selectively would be an ideal choice for tumor gene therapy.
Apoptin, which contains 121 amino acids, is encoded by VP3 gene from chicken anemia virus. Its molecular weight is 13.6 KD and it has no homology with any known proteins. Studies show that apoptin could selectively induce apoptosis in tumor cells such as ovarian cancer, liver cancer, malignant lymphoma, while leaving normal cells such as lymph, derma, epithelium, endothelium and other normal human diploid cells intact, especially the diploid cells which are extremely sensitive for chemotherapy and radiotherapy, such as CD34+ hematopoietic stem cells derived from bone marrow, mesenchymal stem cells and lymphocytes were not interfered by Apoptin either, and no toxicity of apoptin had been found with different administration. All of these indicate its prospective anti-tumor value. Therefore, eukaryotic expression plasmid which contains wild type apoptin gene and report gene of EGFP was constructed, and the recombinant plasmid was transfected into transitional cell carcinoma of bladder cell line T24 by mixed with lipofectamin 2000. The therapeutic effects to T24 were detected in vivo and in vitro.
Object
1. To investigate the role of Apoptin on the proliferation, apoptosis and cell cycle of T24.
2. To investigate the role of apoptin in the athymic mouse human bladder tumor transplantation tumor model.
3. To investigate the synergistic interaction of apoptin and chemotherapy drug cisplatin on T24 cells.
Materials and Methods
1. Construction and identification of eukaryotic expression plasmid pApoptin-EGFP: We designed primers according to the wild-type appoptin cDNA sequence from GenBank. Wild type apoptin gene was prepared by PCR from plasmid p3×flag-m-Apoptin-myc which contains a variant-type apoptin gene, then the wild-type apoptin gene was directional cloned to the vector pEGFP-N1. The recombinant plasmid pApoptin-EGFP was identified by double enzyme digestion and sequence.
2. The apoptosis effect induced by Apoptin in T24 cells: The identified plasmid pApoptin-EGFP was transfected into T24 cells with Lipofectamine 2000. Apoptin mRNA was detected by RT-PCR. The fusion protein apoptin-EGFP was observed by fluorescent microscope. The proliferation inhibition induced by apoptin in T24 cells were detected by MTT. The morphous and apoptosis of transfected T24 cells was observed by using apoptosis optical kit. FCM was used to detected apoptosis and cell cycle of T24 cells.
3. The therapeutic effect of Apoptin on bladder xenograft tumor: Athymic mouse T24 xenograft tumor model was established by inoculating T24 cells subcutaneously. pApoptin-EGFP parceled with liposome was injected into the tumor, when the diameter of xenograft tumor is about 0.5cm, empty plasmid and normal saline were injected as control. Tumor formation, tumor growth, tumor size, athymic mice weight and activity status were observed. The volume of transplant tumor was measured and recorded continually, The mice were sacrificed five weeks later, tumor-inhibition rate was evaluated and the apoptosis rate of cells was detected by in situ TUNEL.
4. Synergistic killing effects of Apoptin and cisplatin (DDP) on T24 cells: Concentration gradient cisplatin was administrated to T24 cells which transfected with pApoptin-EGFP. 24 hours later, the proliferation of T24 cells was detected by MTT and the apoptosis of T24 cells was assayed by flow cytometry.
Conclusion
1. Wild-type apoptin gene was amplified by PCR successfully and the product was correctly cloned to vector pEGFP-N1 between the enzyme site of EcoRⅠand BamHⅠ. An integrated reading frame was constructed consist of apoptin gene and EGFP gene. With no frame shift mutation, the recombinant plasmid pApoptin-EGFP would express apoptin-EGFP fusion protein in theory.
2. Wild-type Apoptin mRNA could be amplified from the transfected T24 cells by RT-PCR method and the fluorescence of EGFP could be observed by using fluorescence microscopy. Both indicated fusion protein could be express in T24 cell. The results of MTT showed that Apoptin could inhibit the proliferation of T24 cells. The inhibition rates were 19.4%, 37.5%, 49.5% respectively at the time-point of 24h, 48h, 72h after transient transfection. Analyzed with student’s t test, the mean of proliferation rate on T24 cells showed significant difference between pApopotin-EGFP group and the control. The morphology of T24 cells showed typical apoptosis changes detected by staining with apoptosis detected kit. The apoptosis rate of T24 was 21.25%, much higher than that of the control at the time-point of 48 hours post transient transfection. Transfected cells tagged with APC and PI were analyzed by FCM, the results indicated that the early apoptotic rate of pApoptin-EGFP group could reach 18.3%, while empty plasmid group only 4.7%, significant difference was found between the two groups. Furthermore, the advanced apoptosis or necrosis of Apoptin group was 17.8% compared to 1.6% in the control group, significant difference could be found either. All these suggested that Apoptin play an important role which could obviously induce apoptosis on bladder tumor cells. FCM was performed to study the cell life cycle at different time-point post transfection. We found that the percentage of S phase is reduced and the G1/G2, G2/M phase arrested 24 hours post the transfection, as the time go on, these changes become weaken and the cell cycle is nearly to the cells without transfection.72 hour later. In contrast, pApoptin-EGFP group got S phase reduced at 24h post transfection and as the time go on, these changes become strength, especially at the 72 hours time-point, the percentage of S phase have been reduced from 46.8% to 28.5%, both G1/G0 and G2/M phase were arrested.
3. T24 xenograft tumor model was established successfully. Compared with the control group, the Athymic mice that injected with pApoptin-EGFP got better status and weight gain, without any cachexia syndrome. The tumor weight of pApoptin-EGFP, pEGFP-N1 and NS groups are 0.26±0.08g, 0.83±0.20g, 1.02±0.23g respectively. There are significant difference between pApoptin-EGFP group and the control, No statistical difference have been found between the pEGFP-N1 group and the NS group. The tumor inhibition ratio of pApoptin-EGFP group was 74.5% and pEGFP-N1 group was 18.6% as contrast. xenograft tumor tissue slice stained by H.E, the tissue was identified to be TCCB. The apoptosis rate of the three group (pApoptin-EGFP, pEGFP-N1, NS) were (23.24±6.12)%, (3.52±1.20)%, (1.76±0.44)% respectively detected by in situ TUNEL. There are significant difference between pApoptin- EGFP group and the control.(P<0.01). It suggested that Apoptin as exogenous gene product could inhibit TCCB in vitro and no side effects had been found in our experiment.
4. The proliferation inhibition ratio of the three groups(DDP、pEGFP-N1+DDP、pApoptin-EGFP+DDP) on T24 cells increased as the concentration of DDP raise. Inhibition ratio of each dose point between pApoptin-EGFP+DDP and the other two groups had been found to be significant different by using statistical test, except 64ug/ml dose point because of the curve of inhibition ratio had been reach platform. The IC50 of each group(DDP、pEGFP-N1+DDP, pApoptin-EGFP+DDP) were 10.61ug/ml, 7.9ug/ml and 2.4ug/ml respectively. Calculated by using Jin-formula, we found pApoptin-EGFP and DDP had synergistic effect on inhibiting the proliferation of T24 cells. In contrast inhibition effects of pEGFP-N1and DDP on T24 cells were only simple addition. Same result was found for the apoptosis induction on T24 cells between the pApoptin-EGFP and DDP.
Conclusions
1. The recombinant plasmid pApoptin-EGFP was constructed successfully which could express the apoptin-EGFP fusion protein in transitional cell carcinoma of bladder cell lines T24.
2. Compared to the empty group, the Apoptin gene could significantly inhibit the T24 cells’proliferation , induce apoptosis and make cell life cycle arrested in G1/G0 and G2/M stage.
3. Apoptin can inhibit the growth of xenograft transitional cell carcinoma of bladder by inhibiting proliferation and inducing apoptosis, no side effects were observed during the treatment.
4. Apoptin recombinant plasmid transfection combined with cisplatin could enhance proliferation inhibition and apoptosis induction effects on T24 cells, and the two therapies have synergies.
引文
1.吴在德.外科学[M].北京,人民卫生出版社, 2000, 766.
2. Nagao K, Ohyashiki JH, Ohyashiki K, et al. Expression of hTERT mRNA in a mortal liver cell line during S phase without detectable telomerase activity[J]. Int J Mol Med. 2005 Apr;15(4):683-688.
3. Poole JC, Andrews LG, Tollefsbol TO. Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT) [J]. Gene, 2001,269(1-2):185-197.
4. Yuasa N, Taniguchi T, Yoshida I. Isolation and characterization of an agent inducing anemia in chicks [J ]. Avian Diseases, 1979, 23 :366-385.
5. Meehan BM, Todd D, Creelan JL, et al. Characterization of viral DNAs from cells infected with chicken anaemia agent: Sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments[J]. Arch Virol, 1992, 124:301–319.
6. Noteborn MHM, Koch G Chicken anemia virus infection: Molecular basis of pathogenicity [J]. Avian Pathol, 1995, 24:11–31.
7. Koch G, Van Roozelaar DJ, Verschueren CAJ, et al. Immunogenic and protective properties of chicken anemia virus proteins expressed by baculovirus[J]. Vaccine, 1995, 13:763–770.
8. Noteborn MHM, Todd D, Verschueren CAJ, et al. A single chicken anemia virus protein induces apoptosis. [J]. Virol, 1994, 68,346–351.
9. Noteborn MHM, Koch G. Chicken anemia virus infection: Molecular basis of pathogenicity[J]. Avian Pathol, 1995, 24,11–31.
10. Jeurissen, SHM, Wagenaar F, Pol J.M.A, et al. Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection[J].Virol, 1992, 66, 7383–7388.
11. Danen-Van Oorschot AA, Zhang YH, Leliveld SR, et al. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis[J]. Biol Chem, 2003 Jul; 25; 278(30):27729-36.
12. Destin W Heilman, Jose G Teodoro, Michael R Green. Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitmentof the Anaphase-Promoting Complex/Cyclosome to PML Bodies [J]. Virol, 2006, 80(15): 7535–7545.
13. Coffin RS , Thomas SK, Thomas NSB , et al. Pure populations of transduced Primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry[J]. Gene Therapy, 1998, 5:718～722
14. Zha J, Weiler S, Oh KJ,et al. Posttranslational N- myristoy- lation of Bid as a molecμlar switch for targeting mitochondria and apoptosis[J]. Science, 2000, 290(5497):1761- 1765.
15. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes[J]. Cell, 2003, 114:181-190.
16. Thornberry NA, Lazebnik Y. Caspases: enemies within[J]. Science, 1998 Aug 28; 281(5381):1312-6.
17. Zhuang SM, Shvarts A, van Ormondt H, et al. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells[J]. Cancer Res, 1995 Feb 1;55(3):486-9.
18. Zhang YH, Abrahams PJ, van der Eb AJ, et al. The viral protein Apoptin induces apoptosis in UV-C-irradiated cells from individuals with various hereditary cancer-prone syndromes [J]. Cancer Res, 1999, 59(12):3010-3015.
19. White E. Life, death, and the pursuit of apoptosis[J]. Genes Dev. 1996, Jan;10(1):1-15.
20. Jürgensmeier JM, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria[J]. Proc Natl Acad Sci U S A, 1998, Apr 28; 95(9): 4997- 5002.
21. Liou AKF, Clark RS, Henshall DC, et al. To die or not to die for neurons in ischemia,traumatic brain injury and epilepsy,a review on the stress-activated signalling pathways and apoptotic pathways[J].Prog Neurobial, 2003,69:103-142.
22. Gross A, Mcdonnell JM, Korsmeyer SJ, et al. BCL-2 family members and the mitochondria in apoptosis[J]. Genes Dev,1999,13:1899-1911.
23. Cheng EH,Wei MC, Weiler S, et al. Bcl-2,Bcl-XL sequester BH3domain-only molecules preventing Bax and Bak-mediated mitochondrial apoptosis[J]. Mol Cell, 2001, 8(3):705-711.
24. O'Connor L, Strasser A, O'Reilly LA, et al. Bim: a novel member of the Bcl-2 familythat promotes apoptosis[J]. EMBO J, 1998, Jan15;17(2):384-95.
25. Green DR, Reed JC. Mitochondria and apoptosis[J]. Science, 1998 Aug 28;281(5381):1309-12.
26. Zhuang SM, Landegent JE, Verschueren CA. et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro[J]. Leukemia. 1995, 9 S1, 118–120.
27. Zhuang SM, Shvarts A, Jochemsen AG, et al. Differential sensitivity to Ad5 E1B-21kD and Bcl-2 proteins of Apoptin-induced versus p53-induced apoptosis[J]. Carcinogenesis.1995, 16: 2939–2945.
28. Danen-van Oorschot AA, den Hollander AI, Takayama S, et al. BAG-1 inhibits p53-induced but not apoptin-induced apoptosis[J]. Apoptosis, 1997;2(4):395-402.
29. Schoop RA, Kooistra K, Baatenburg De Jong RJ, et al. Bcl-xL inhibits p53- but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line[J]. Int J Cancer, 2004, 109(1):38-42.
30.国泰,杨谦,高洪雷等,凋亡蛋白对Bcl-2家族几个基因表达影响[J].哈尔滨工业大学学报, 2005, 9:1880-1882.
31. Danen-van Oorschot AA, van Der Eb AJ, Noteborn MH. The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells[J]. J Virol, 2000 Aug; 74(15):7072-8.
32. Gagliardini V, Fernandez PA, Lee RK, et al. Prevention of vertebrate neuronal death by the crmA gene[J]. Science, 1994 Feb 11;263(5148):826-8.
33. Maddika S, Bay GH, Kroczak TJ, et al. Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin-induced cell death[J].Cell Prolif, 2007 Dec;40(6):835-48.
34. Maddika S, Wiechec E, Ande SR, et al. Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin[J].Oncogene, 2007 Dec 3. [Epub ahead of print]
35. Klanrit P, Flinterman MB, Odell EW,et al. Specific isoforms of p73 control the induction of cell death induced by the viral proteins, E1A or apoptin[J]. Cell Cycle, 2008 Jan;7(2):205-15.
36. Irie A, Uchida T, Ishida H, et al .p53 Mutation in bladder cancer patients in Japan and inhibition of growth by in vitro adenovirus-mediated wild-type p53 transduction inbladder cancer cells[J].Mol Urol, 2001 Summer, 5(2):53-8.
37. Bubenik J. Established cell line of urinary bladder carcinoma(T24)containing tumor specific antigen[J]. Int J Cancer, 1973, 11:765-773.
38. Murray AW, Marks D. Can sequencing shed light on cellcycling[J]. Nature, 2001,409:844-846.
39. Eissa S, Ahmed MI, Said H, et al. Cell cycle regulators in bladder cancer relationship to schistosomiasis[J]. IUBMB Life, 2004, 56(9):557-564,
40.何湘君,尚世彤,刘玉京等. Apoptin引起肿瘤细胞G2—M阻滞[J].北京大学学报, 2004, 6(36):263-267.
41. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther, 1999 May;6(5):882-92.
42. Ying-Hui Zhang, S Rutger Leliveld, Klaas Kooistra, et al. Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion[J]. Experimental Cell Research, 2003, 289:36–46.
43. Backendorf C, Visser AE, de Boer AG, et al.Apoptin: therapeutic potential of an early sensor of carcinogenic transformation[J]. Annu Rev Pharmacol Toxicol, 2008, 48:143-69. Review.
44. Danen-van Oorschot AAM, Voskamp P, Seelen MCM, et al. Human death effecter domain-associated factor interacts with the viral apoptosis agonist Apoptin and exert tumor-preferential cell killing[J]. Cell Death Differ, 2004, 11:564-573.
45. Cheng CM, Huang SP, Chang YF, et al. The viral death protein Apoptin interacts with Hippi, the protein interactor of Huntingtin-interacting protein 1[J]. Biochem Biophys Res Commun, 2003 May 30;305(2):359-64.
46. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther, 1999 May;6(5):882-92.
47. Alexandra Pietersen, Stefan Erkeland ,Saskia Rutjes et al .Continouous Apoptin Expression in Transgenic mice Does Not Interfere with Lymphocyte Development and Proliferation[J]. Journal of Medical Molecular Biology, 2002(5):321-330
48. Olijslagers S, Dege AY, Dinsart C, et al. Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin[J]. Cancer Gene Ther, 2001 Dec;8(12):958-65.
49. Van der Eb MM, Pietersen AM, Speetjens FM, et al. Gene therapy with apoptin inducesregression of xenografted human hepatomas[J]. Cancer Gene Ther, 2002 Jan;9(1):53-61.
50. Wong E, Giandomenico CM. Current status of platinum-2 based anti-tumor drugs[J]. Chem Rev, 1999, 99(9):2451-66.
51.吴奎,孟刚,汪渊等.顺铂诱导膀胱癌细胞凋亡及其与凋亡相关蛋白的关系[J].中国药理学通报, 2003, 19(10):1143-72.
52.金正均.合并用药中的相加.中国药理学报[J].1980, 1(2):70.
53. Andrews PA, Howell SB. Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance[J].Cancer Cells, 1990,2:35.
54. Jordan P, Carmo-Fonseca M. Molecular mechanisms involved in cisplatin cytotoxicity[J]. Cell Mol Life Sci, 2000 Aug;57(8-9):1229-35.
55. Yamada M, O’Regan, Brown R, et al. Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins[J]. Nucleic Aci Res, 1997,25(3):491-496.
56. Turchi JJ, Henkels K. Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase[J]. J Biol Chem, 1996 Jun 7;271(23):13861-7.
57. Gong JG, Costanzo A, Yang HQ, et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-inducedDNA damage[J]. Nature, 1999,399:806.
58. White E. DNA damage enables p73[J]. Nature, 1999, 399:734-737.
59. Ishimoto O, Kawahara C, Enjo K, et al. Possible oncogenic potential of Delta Np73: a newly identified isoform of human p73.Cancer Res, 2002 Feb 1;62(3):636-41.
60. Falck J, Mailand N, Sylju?sen RG, et al.The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis[J]. Nature, 2001, 410(6830):842-7
61. Ramadan S, Terrinoni A, Catani MV, et al. p73 induces apoptosis by different mechanisms[J]. Biochem Biophys Res Commun, 2005 Jun 10;331(3):713-7. Review.
62. Grob TJ, Novak U, Maisse C, et al. Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53[J]. Cell Death Differ, 2001 Dec;8(12):1213-23.
63. Olijslagers SJ, Zhang YH, Backendorf C,et al. Additive cytotoxic effect of apoptin and chemotherapeutic agents paclitaxel and etoposide on human tumour cells[J]. Basic Clin Pharmacol Toxicol, 2007 Feb;100(2):127-31.
1. Yuasa N , Taniguchi T , and Yoshida I. Isolation and characterization of an agent inducing anemia in chicks [J]. Avian Diseases , 1979 , 23 :366.
2. Gelderblom H, Kling S, Lurz R, et al. Morphological characterization of chicken anaemia agent(CAA) [J]. Arch. Virol, 1989109, 115–120.
3. Todd D, Creelan JL, Mackie DP, e tal. Purification and biochemical characterization of chicken anaemia agent[J]. J Gen Virol, 1990 Apr;71 ( Pt 4):819-23.
4. Noteborn MH, de Boer GF, van Roozelaar DJ, et al. Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle[J]. J Virol, 1991 Jun;65(6):3131-9.
5. Meehan, BM, Todd D, Creelan JL, et al. Characterization of viral DNAs from cells infected with chicken anaemia agent: Sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments[J]. Arch Virol, 1992,124:301–319.
6. Noteborn MHM, Veldkamp S, Verschueren CAJ, et al. Molecular-biological characteristics of chicken anemia virus[J]. Virus Diseases of Poultry-New and Evolving Pathogens, European Commission VI/4104/94-EN, Brussels,1994. 195–213.
7. Noteborn MHM, Koch G. Chicken anemia virus infection: Molecular basis of pathogenicity[J]. Avian Pathol, 1995, 24,11–31.
8. Jeurissen, SHM, Wagenaar F, Pol JMA, et al. Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection[J]. J.Virol, 1992, 66:7383–7388.
9. Noteborn MHM, Verschueren CA, Zantema A, et al . Identification of the promotor region of chicken anemia virus (CAV) containing a novel enhancer like element [J]. Gene ,1994,150(2):313-318.
10. Koch G, Van Roozelaar DJ, Verschueren CAJ, et al. Immunogenic and protective properties of chicken anemia virus proteins expressed by baculovirus[J]. Vaccine, 1995, 13:763–770.
11. Telford WG, King LE, Fraker PJ. Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry[J].Cytometry, 1992,13:137–143.
12. Noteborn MHM, Todd D, Verschueren CAJ, et al. A single chicken anemia virus protein induces apoptosis[J]. J Virol, 1994, 68:346–351.
13. Todd D, Douglas AJ, Phenix KV, et al. Characterization of chicken anaemia virus. In Proceedings of the International Symposium on Infectious Bursal Disease and Chicken Infectious Anemia (E. F. Kaleta, Ed.), 1994, 349–363.
14. Noteborn MHM, Koch G. Chicken anemia virus infection: Molecular basis of pathogenicity[J]. Avian Pathol, 1995,24:11–31.
15. Zhang YH, Leliveld SR, Kooistra K, et al. Recombinant Apoptin multimers kill tumor cells but are non-toxic and epitope-shielded in a normal-cell-specific fashion[J]. Exp Cell Res, 2003, 289:36-46.
16. Zhuang YH. Activation of Apoptin, a tumor-specific viral death effector. PhD thesis,Leiden University, Leiden,The Netherlands, 2004.
17.章应慧,屈伸, Noteborn MHM.凋亡素:一种诱导肿瘤特异性细胞凋亡的病毒蛋白[J].医学分子生物杂志, 2004, 1(2):63-68.
18. Danen-van Oorschot AAM, Fisher DF, Grimbargen JM. et al.Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells[J]. Proc Natl Acad Sci USA, 1997 May 27, 94(11):5843–5847.
19. Zhang YH, Abrahams PJ, van der Eb AJ,et al. The viral protein Apoptin induces apoptosis in UV-C-irradiated cells from individuals with various hereditary cancer-prone syndromes [J]. Cancer Res, 1999, 59(12):3010-3015.
20. Zhuang SM, Shvarts A, van Ormondt H, et al. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells[J]. Cancer Res, 1995 Feb 1;55(3):486-9.
21. White E. Life, death, and the pursuit of apoptosis[J]. Genes Dev, 1996 Jan 1;10(1):1-15
22. Shen Y, Shenk TE. Viruses and apoptosis[J]. Curr Biol, 1995 5; 105–111.
23. Zhuang SM, Shvarts A, Jochemsen AG, et al. Differential sensitivity to Ad5 E1B-21kD and Bcl-2 proteins of Apoptin-induced versus p53-induced apoptosis[J]. Carcinogenesis,1995, 16:2939–2945.
24. Zhuang SM, Landegent JE, Verschueren CA. et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cellsin vitro[J]. Leukemia, 1995, 9 S1:118–120.
25. Jürgensmeier JM, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria[J].Proc Natl Acad Sci U S A, 1998 Apr 28;95(9): 4997-5002.
26. Liou AKF, Clark RS, Henshall DC, et al. To die or not to die for neurons in ischemia,traumatic brain injury and epilepsy,a review on the stress-activated signalling pathways and apoptotic pathways[J]. Prog Neurobial, 2003,69:103-142.
27. Gross A, Mcdonnell JM, Korsmeyer SJ et al. Bcl-2 family members and the mitochondria in apoptosis[J]. Genes Dev, 1999,13:1899-1911.
28. Cheng EH, Wei MC, Weiler S, et al. Bcl-2,Bcl-XL sequester BH3domain-only molecules preventing Bax and Bak-mediated mitochondrial apoptosis[J]. Mol Cell, 2001,8(3):705-711.
29. O'Connor L, Strasser A, O'Reilly LA, et al. Bim:a novel member of the Bcl-2 family that promotes apoptosis[J]. EMBO J, 1998Jan 15;17(2):384-95.
30. Green DR, Reed JC. Mitochondria and apoptosis[J]. Science, 1998 Aug 28; 281(5381):1309-12.
31.国泰,杨谦,高洪雷等.凋亡蛋白对Bcl-2家族几个基因表达影响[J].哈尔滨工业大学学报, 2005,9:1880-1882.
32. Takayama S, Sato T, Krajewski S, et al. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity[J]. Cell, 1995 Jan 27; 80(2):279-84.
33. Danen-van Oorschot AA, den Hollander AI, Takayama S, et al. BAG-1 inhibits p53-induced but not apoptin-induced apoptosis[J]. Apoptosis, 1997;2(4):395-402.
34. Schoop RA, Kooistra K, Baatenburg De Jong RJ,et al. Bcl-xL inhibits p53- but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line[J]. Int J Cancer, 2004 Mar;109(1):38-42.
35. Thornberry NA, Lazebnik Y. Caspases: enemies within[J]. Science, 1998 Aug 28;281(5381):1312-6.
36. Danen-van Oorschot AA, van Der Eb AJ, Noteborn MH. The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells[J]. J Virol, 2000 Aug;74(15):7072-8.
37. Gagliardini V, Fernandez PA, Lee RK, et al. Prevention of vertebrate neuronal death by the crmA gene[J]. Science, 1994 Feb 11;263(5148):826-8.
38.董萍,咸丰,赵洪礼等. Apoptin基因通过激活caspase23诱导人黑色素瘤细胞A375凋亡[J].细胞与分子免疫学杂志, 2006, 22 (3):293-295.
39. Danen-Van Oorschot AA, Zhang YH, Leliveld SR, et al. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis[J]. J Biol Chem, 2003 Jul 25;278(30):27729-36.
40. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther, 1999 May;6(5):882-92.
41. Ying-Hui Zhang, S. Rutger Leliveld, Klaas Kooistra, et al. Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion[J]. Experimental Cell Research, 2003,289:36–46.
42. Lovestone S, McLoughlin DM, Lovestone S, et al. Protein aggregates and dementia: Is there a common toxicity? [J] J Neurol Neurosurg Psychiatry, 2002 Feb;72(2):152-61.
43. Khan MF, Falk RH. Amyloidosis[J]. Postgrad Med J, 2001 Nov;77(913):686-93.
44. Li SH, Lam S, Cheng AL, et al. Intranuclear huntingtin increases the expression of caspase-1 and induces apoptosis[J]. Hum Mol Genet, 2000 Nov 22;9(19):2859-67.
45. Andersson K, Olofsson A, Nielsen EH, et al. Only amyloidogenic intermediates of transthyretin induce apoptosis[J]. Biochem Biophys Res Commun, 2002 Jun 7;294(2):309-14.
46. Leliveld SR ,Zhang YH ,Rohn JL et al. Apoptin induces tumor-specific apoptosis as a random,globular multimer[J]. J Biol Chem, 2003, 278(11) : 9042- 51.
47. Destin W Heilman, Jose G Teodoro, Michael R. Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies [J]. Virol, 2006,80(15): 7535–7545.
48. Kudo N, Wolff B, Sekimoto T, et al. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp Cell Res, 1998 Aug 1;242(2):540-7.
49. Wolff B, Sanglier JJ, Wang Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA[J]. Chem Biol, 1997Feb;4(2):139-47.
50. Rohn JL., YH Zhang, RI Aalbers, et al. A tumor-specific kinase activity regulates the viral death protein Apoptin[J]. J Biol. Chem, 2002. 277:50820-50827.
51. Lee YH, Cheng CM, Chang YF, et al. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity[J]. Biochem Biophys Res Commun, 2007 Mar 9;354(2):391-5.
52. Bao J, Zervos AS. Isolation and characterization of Nmi, a novel partner of Myc proteins[J]. Oncogenes, 1996, 16:2171-2176.
53. Danen-van Oorschot AAM, Voskamp P, Seelen MCM, et al. Human death effecter domain-associated factor interacts with the viral apoptosis agonist Apoptin and exert tumor-preferential cell killing[J]. Cell Death Differ, 2004, 11:564-573.
54. Cheng CM, Huang SP, Chang YF, et al. The viral death protein Apoptin interacts with Hippi, the protein interactor of Huntingtin-interacting protein 1[J]. Biochem Biophys Res Commun, 2003 May 30;305(2):359-64.
55. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther. 1999 May;6(5):882-92.
56. Alexandra Pietersen, Stefan Erkeland ,Saskia Rutjes, et al . Continouous Apoptin Expression in Transgenic mice Does Not Interfere with Lymphocyte Development and Proliferation[J].医学分子生物学杂志,2005,2(5):321-330
57. Olijslagers S, Dege AY, Dinsart C, et al. Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin[J]. Cancer Gene Ther. 2001 Dec;8(12):958-65.
58. van der Eb MM, Pietersen AM, Speetjens FM, et al. Gene therapy with apoptin induces regression of xenografted human hepatomas[J]. Cancer Gene Ther, 2002 Jan;9(1):53-61.
2. Nagao K, Ohyashiki JH, Ohyashiki K, et al. Expression of hTERT mRNA in a mortal liver cell line during S phase without detectable telomerase activity[J]. Int J Mol Med. 2005 Apr;15(4):683-688.
3. Poole JC, Andrews LG, Tollefsbol TO. Activity, function, and gene regulation of the catalytic subunit of telomerase (hTERT) [J]. Gene, 2001,269(1-2):185-197.
4. Yuasa N, Taniguchi T, Yoshida I. Isolation and characterization of an agent inducing anemia in chicks [J ]. Avian Diseases, 1979, 23 :366-385.
5. Meehan BM, Todd D, Creelan JL, et al. Characterization of viral DNAs from cells infected with chicken anaemia agent: Sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments[J]. Arch Virol, 1992, 124:301–319.
6. Noteborn MHM, Koch G Chicken anemia virus infection: Molecular basis of pathogenicity [J]. Avian Pathol, 1995, 24:11–31.
7. Koch G, Van Roozelaar DJ, Verschueren CAJ, et al. Immunogenic and protective properties of chicken anemia virus proteins expressed by baculovirus[J]. Vaccine, 1995, 13:763–770.
8. Noteborn MHM, Todd D, Verschueren CAJ, et al. A single chicken anemia virus protein induces apoptosis. [J]. Virol, 1994, 68,346–351.
9. Noteborn MHM, Koch G. Chicken anemia virus infection: Molecular basis of pathogenicity[J]. Avian Pathol, 1995, 24,11–31.
10. Jeurissen, SHM, Wagenaar F, Pol J.M.A, et al. Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection[J].Virol, 1992, 66, 7383–7388.
11. Danen-Van Oorschot AA, Zhang YH, Leliveld SR, et al. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis[J]. Biol Chem, 2003 Jul; 25; 278(30):27729-36.
12. Destin W Heilman, Jose G Teodoro, Michael R Green. Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitmentof the Anaphase-Promoting Complex/Cyclosome to PML Bodies [J]. Virol, 2006, 80(15): 7535–7545.
13. Coffin RS , Thomas SK, Thomas NSB , et al. Pure populations of transduced Primary human cells can be produced using GFP expressing herpes virus vectors and flow cytometry[J]. Gene Therapy, 1998, 5:718～722
14. Zha J, Weiler S, Oh KJ,et al. Posttranslational N- myristoy- lation of Bid as a molecμlar switch for targeting mitochondria and apoptosis[J]. Science, 2000, 290(5497):1761- 1765.
15. Micheau O, Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes[J]. Cell, 2003, 114:181-190.
16. Thornberry NA, Lazebnik Y. Caspases: enemies within[J]. Science, 1998 Aug 28; 281(5381):1312-6.
17. Zhuang SM, Shvarts A, van Ormondt H, et al. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells[J]. Cancer Res, 1995 Feb 1;55(3):486-9.
18. Zhang YH, Abrahams PJ, van der Eb AJ, et al. The viral protein Apoptin induces apoptosis in UV-C-irradiated cells from individuals with various hereditary cancer-prone syndromes [J]. Cancer Res, 1999, 59(12):3010-3015.
19. White E. Life, death, and the pursuit of apoptosis[J]. Genes Dev. 1996, Jan;10(1):1-15.
20. Jürgensmeier JM, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria[J]. Proc Natl Acad Sci U S A, 1998, Apr 28; 95(9): 4997- 5002.
21. Liou AKF, Clark RS, Henshall DC, et al. To die or not to die for neurons in ischemia,traumatic brain injury and epilepsy,a review on the stress-activated signalling pathways and apoptotic pathways[J].Prog Neurobial, 2003,69:103-142.
22. Gross A, Mcdonnell JM, Korsmeyer SJ, et al. BCL-2 family members and the mitochondria in apoptosis[J]. Genes Dev,1999,13:1899-1911.
23. Cheng EH,Wei MC, Weiler S, et al. Bcl-2,Bcl-XL sequester BH3domain-only molecules preventing Bax and Bak-mediated mitochondrial apoptosis[J]. Mol Cell, 2001, 8(3):705-711.
24. O'Connor L, Strasser A, O'Reilly LA, et al. Bim: a novel member of the Bcl-2 familythat promotes apoptosis[J]. EMBO J, 1998, Jan15;17(2):384-95.
25. Green DR, Reed JC. Mitochondria and apoptosis[J]. Science, 1998 Aug 28;281(5381):1309-12.
26. Zhuang SM, Landegent JE, Verschueren CA. et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro[J]. Leukemia. 1995, 9 S1, 118–120.
27. Zhuang SM, Shvarts A, Jochemsen AG, et al. Differential sensitivity to Ad5 E1B-21kD and Bcl-2 proteins of Apoptin-induced versus p53-induced apoptosis[J]. Carcinogenesis.1995, 16: 2939–2945.
28. Danen-van Oorschot AA, den Hollander AI, Takayama S, et al. BAG-1 inhibits p53-induced but not apoptin-induced apoptosis[J]. Apoptosis, 1997;2(4):395-402.
29. Schoop RA, Kooistra K, Baatenburg De Jong RJ, et al. Bcl-xL inhibits p53- but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line[J]. Int J Cancer, 2004, 109(1):38-42.
30.国泰,杨谦,高洪雷等,凋亡蛋白对Bcl-2家族几个基因表达影响[J].哈尔滨工业大学学报, 2005, 9:1880-1882.
31. Danen-van Oorschot AA, van Der Eb AJ, Noteborn MH. The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells[J]. J Virol, 2000 Aug; 74(15):7072-8.
32. Gagliardini V, Fernandez PA, Lee RK, et al. Prevention of vertebrate neuronal death by the crmA gene[J]. Science, 1994 Feb 11;263(5148):826-8.
33. Maddika S, Bay GH, Kroczak TJ, et al. Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin-induced cell death[J].Cell Prolif, 2007 Dec;40(6):835-48.
34. Maddika S, Wiechec E, Ande SR, et al. Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin[J].Oncogene, 2007 Dec 3. [Epub ahead of print]
35. Klanrit P, Flinterman MB, Odell EW,et al. Specific isoforms of p73 control the induction of cell death induced by the viral proteins, E1A or apoptin[J]. Cell Cycle, 2008 Jan;7(2):205-15.
36. Irie A, Uchida T, Ishida H, et al .p53 Mutation in bladder cancer patients in Japan and inhibition of growth by in vitro adenovirus-mediated wild-type p53 transduction inbladder cancer cells[J].Mol Urol, 2001 Summer, 5(2):53-8.
37. Bubenik J. Established cell line of urinary bladder carcinoma(T24)containing tumor specific antigen[J]. Int J Cancer, 1973, 11:765-773.
38. Murray AW, Marks D. Can sequencing shed light on cellcycling[J]. Nature, 2001,409:844-846.
39. Eissa S, Ahmed MI, Said H, et al. Cell cycle regulators in bladder cancer relationship to schistosomiasis[J]. IUBMB Life, 2004, 56(9):557-564,
40.何湘君,尚世彤,刘玉京等. Apoptin引起肿瘤细胞G2—M阻滞[J].北京大学学报, 2004, 6(36):263-267.
41. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther, 1999 May;6(5):882-92.
42. Ying-Hui Zhang, S Rutger Leliveld, Klaas Kooistra, et al. Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion[J]. Experimental Cell Research, 2003, 289:36–46.
43. Backendorf C, Visser AE, de Boer AG, et al.Apoptin: therapeutic potential of an early sensor of carcinogenic transformation[J]. Annu Rev Pharmacol Toxicol, 2008, 48:143-69. Review.
44. Danen-van Oorschot AAM, Voskamp P, Seelen MCM, et al. Human death effecter domain-associated factor interacts with the viral apoptosis agonist Apoptin and exert tumor-preferential cell killing[J]. Cell Death Differ, 2004, 11:564-573.
45. Cheng CM, Huang SP, Chang YF, et al. The viral death protein Apoptin interacts with Hippi, the protein interactor of Huntingtin-interacting protein 1[J]. Biochem Biophys Res Commun, 2003 May 30;305(2):359-64.
46. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther, 1999 May;6(5):882-92.
47. Alexandra Pietersen, Stefan Erkeland ,Saskia Rutjes et al .Continouous Apoptin Expression in Transgenic mice Does Not Interfere with Lymphocyte Development and Proliferation[J]. Journal of Medical Molecular Biology, 2002(5):321-330
48. Olijslagers S, Dege AY, Dinsart C, et al. Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin[J]. Cancer Gene Ther, 2001 Dec;8(12):958-65.
49. Van der Eb MM, Pietersen AM, Speetjens FM, et al. Gene therapy with apoptin inducesregression of xenografted human hepatomas[J]. Cancer Gene Ther, 2002 Jan;9(1):53-61.
50. Wong E, Giandomenico CM. Current status of platinum-2 based anti-tumor drugs[J]. Chem Rev, 1999, 99(9):2451-66.
51.吴奎,孟刚,汪渊等.顺铂诱导膀胱癌细胞凋亡及其与凋亡相关蛋白的关系[J].中国药理学通报, 2003, 19(10):1143-72.
52.金正均.合并用药中的相加.中国药理学报[J].1980, 1(2):70.
53. Andrews PA, Howell SB. Cellular pharmacology of cisplatin: perspectives on mechanisms of acquired resistance[J].Cancer Cells, 1990,2:35.
54. Jordan P, Carmo-Fonseca M. Molecular mechanisms involved in cisplatin cytotoxicity[J]. Cell Mol Life Sci, 2000 Aug;57(8-9):1229-35.
55. Yamada M, O’Regan, Brown R, et al. Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins[J]. Nucleic Aci Res, 1997,25(3):491-496.
56. Turchi JJ, Henkels K. Human Ku autoantigen binds cisplatin-damaged DNA but fails to stimulate human DNA-activated protein kinase[J]. J Biol Chem, 1996 Jun 7;271(23):13861-7.
57. Gong JG, Costanzo A, Yang HQ, et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-inducedDNA damage[J]. Nature, 1999,399:806.
58. White E. DNA damage enables p73[J]. Nature, 1999, 399:734-737.
59. Ishimoto O, Kawahara C, Enjo K, et al. Possible oncogenic potential of Delta Np73: a newly identified isoform of human p73.Cancer Res, 2002 Feb 1;62(3):636-41.
60. Falck J, Mailand N, Sylju?sen RG, et al.The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis[J]. Nature, 2001, 410(6830):842-7
61. Ramadan S, Terrinoni A, Catani MV, et al. p73 induces apoptosis by different mechanisms[J]. Biochem Biophys Res Commun, 2005 Jun 10;331(3):713-7. Review.
62. Grob TJ, Novak U, Maisse C, et al. Human delta Np73 regulates a dominant negative feedback loop for TAp73 and p53[J]. Cell Death Differ, 2001 Dec;8(12):1213-23.
63. Olijslagers SJ, Zhang YH, Backendorf C,et al. Additive cytotoxic effect of apoptin and chemotherapeutic agents paclitaxel and etoposide on human tumour cells[J]. Basic Clin Pharmacol Toxicol, 2007 Feb;100(2):127-31.
1. Yuasa N , Taniguchi T , and Yoshida I. Isolation and characterization of an agent inducing anemia in chicks [J]. Avian Diseases , 1979 , 23 :366.
2. Gelderblom H, Kling S, Lurz R, et al. Morphological characterization of chicken anaemia agent(CAA) [J]. Arch. Virol, 1989109, 115–120.
3. Todd D, Creelan JL, Mackie DP, e tal. Purification and biochemical characterization of chicken anaemia agent[J]. J Gen Virol, 1990 Apr;71 ( Pt 4):819-23.
4. Noteborn MH, de Boer GF, van Roozelaar DJ, et al. Characterization of cloned chicken anemia virus DNA that contains all elements for the infectious replication cycle[J]. J Virol, 1991 Jun;65(6):3131-9.
5. Meehan, BM, Todd D, Creelan JL, et al. Characterization of viral DNAs from cells infected with chicken anaemia agent: Sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments[J]. Arch Virol, 1992,124:301–319.
6. Noteborn MHM, Veldkamp S, Verschueren CAJ, et al. Molecular-biological characteristics of chicken anemia virus[J]. Virus Diseases of Poultry-New and Evolving Pathogens, European Commission VI/4104/94-EN, Brussels,1994. 195–213.
7. Noteborn MHM, Koch G. Chicken anemia virus infection: Molecular basis of pathogenicity[J]. Avian Pathol, 1995, 24,11–31.
8. Jeurissen, SHM, Wagenaar F, Pol JMA, et al. Chicken anemia virus causes apoptosis of thymocytes after in vivo infection and of cell lines after in vitro infection[J]. J.Virol, 1992, 66:7383–7388.
9. Noteborn MHM, Verschueren CA, Zantema A, et al . Identification of the promotor region of chicken anemia virus (CAV) containing a novel enhancer like element [J]. Gene ,1994,150(2):313-318.
10. Koch G, Van Roozelaar DJ, Verschueren CAJ, et al. Immunogenic and protective properties of chicken anemia virus proteins expressed by baculovirus[J]. Vaccine, 1995, 13:763–770.
11. Telford WG, King LE, Fraker PJ. Comparative evaluation of several DNA binding dyes in the detection of apoptosis-associated chromatin degradation by flow cytometry[J].Cytometry, 1992,13:137–143.
12. Noteborn MHM, Todd D, Verschueren CAJ, et al. A single chicken anemia virus protein induces apoptosis[J]. J Virol, 1994, 68:346–351.
13. Todd D, Douglas AJ, Phenix KV, et al. Characterization of chicken anaemia virus. In Proceedings of the International Symposium on Infectious Bursal Disease and Chicken Infectious Anemia (E. F. Kaleta, Ed.), 1994, 349–363.
14. Noteborn MHM, Koch G. Chicken anemia virus infection: Molecular basis of pathogenicity[J]. Avian Pathol, 1995,24:11–31.
15. Zhang YH, Leliveld SR, Kooistra K, et al. Recombinant Apoptin multimers kill tumor cells but are non-toxic and epitope-shielded in a normal-cell-specific fashion[J]. Exp Cell Res, 2003, 289:36-46.
16. Zhuang YH. Activation of Apoptin, a tumor-specific viral death effector. PhD thesis,Leiden University, Leiden,The Netherlands, 2004.
17.章应慧,屈伸, Noteborn MHM.凋亡素:一种诱导肿瘤特异性细胞凋亡的病毒蛋白[J].医学分子生物杂志, 2004, 1(2):63-68.
18. Danen-van Oorschot AAM, Fisher DF, Grimbargen JM. et al.Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells[J]. Proc Natl Acad Sci USA, 1997 May 27, 94(11):5843–5847.
19. Zhang YH, Abrahams PJ, van der Eb AJ,et al. The viral protein Apoptin induces apoptosis in UV-C-irradiated cells from individuals with various hereditary cancer-prone syndromes [J]. Cancer Res, 1999, 59(12):3010-3015.
20. Zhuang SM, Shvarts A, van Ormondt H, et al. Apoptin, a protein derived from chicken anemia virus, induces p53-independent apoptosis in human osteosarcoma cells[J]. Cancer Res, 1995 Feb 1;55(3):486-9.
21. White E. Life, death, and the pursuit of apoptosis[J]. Genes Dev, 1996 Jan 1;10(1):1-15
22. Shen Y, Shenk TE. Viruses and apoptosis[J]. Curr Biol, 1995 5; 105–111.
23. Zhuang SM, Shvarts A, Jochemsen AG, et al. Differential sensitivity to Ad5 E1B-21kD and Bcl-2 proteins of Apoptin-induced versus p53-induced apoptosis[J]. Carcinogenesis,1995, 16:2939–2945.
24. Zhuang SM, Landegent JE, Verschueren CA. et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cellsin vitro[J]. Leukemia, 1995, 9 S1:118–120.
25. Jürgensmeier JM, Xie Z, Deveraux Q, et al. Bax directly induces release of cytochrome c from isolated mitochondria[J].Proc Natl Acad Sci U S A, 1998 Apr 28;95(9): 4997-5002.
26. Liou AKF, Clark RS, Henshall DC, et al. To die or not to die for neurons in ischemia,traumatic brain injury and epilepsy,a review on the stress-activated signalling pathways and apoptotic pathways[J]. Prog Neurobial, 2003,69:103-142.
27. Gross A, Mcdonnell JM, Korsmeyer SJ et al. Bcl-2 family members and the mitochondria in apoptosis[J]. Genes Dev, 1999,13:1899-1911.
28. Cheng EH, Wei MC, Weiler S, et al. Bcl-2,Bcl-XL sequester BH3domain-only molecules preventing Bax and Bak-mediated mitochondrial apoptosis[J]. Mol Cell, 2001,8(3):705-711.
29. O'Connor L, Strasser A, O'Reilly LA, et al. Bim:a novel member of the Bcl-2 family that promotes apoptosis[J]. EMBO J, 1998Jan 15;17(2):384-95.
30. Green DR, Reed JC. Mitochondria and apoptosis[J]. Science, 1998 Aug 28; 281(5381):1309-12.
31.国泰,杨谦,高洪雷等.凋亡蛋白对Bcl-2家族几个基因表达影响[J].哈尔滨工业大学学报, 2005,9:1880-1882.
32. Takayama S, Sato T, Krajewski S, et al. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity[J]. Cell, 1995 Jan 27; 80(2):279-84.
33. Danen-van Oorschot AA, den Hollander AI, Takayama S, et al. BAG-1 inhibits p53-induced but not apoptin-induced apoptosis[J]. Apoptosis, 1997;2(4):395-402.
34. Schoop RA, Kooistra K, Baatenburg De Jong RJ,et al. Bcl-xL inhibits p53- but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line[J]. Int J Cancer, 2004 Mar;109(1):38-42.
35. Thornberry NA, Lazebnik Y. Caspases: enemies within[J]. Science, 1998 Aug 28;281(5381):1312-6.
36. Danen-van Oorschot AA, van Der Eb AJ, Noteborn MH. The chicken anemia virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells[J]. J Virol, 2000 Aug;74(15):7072-8.
37. Gagliardini V, Fernandez PA, Lee RK, et al. Prevention of vertebrate neuronal death by the crmA gene[J]. Science, 1994 Feb 11;263(5148):826-8.
38.董萍,咸丰,赵洪礼等. Apoptin基因通过激活caspase23诱导人黑色素瘤细胞A375凋亡[J].细胞与分子免疫学杂志, 2006, 22 (3):293-295.
39. Danen-Van Oorschot AA, Zhang YH, Leliveld SR, et al. Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis[J]. J Biol Chem, 2003 Jul 25;278(30):27729-36.
40. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther, 1999 May;6(5):882-92.
41. Ying-Hui Zhang, S. Rutger Leliveld, Klaas Kooistra, et al. Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion[J]. Experimental Cell Research, 2003,289:36–46.
42. Lovestone S, McLoughlin DM, Lovestone S, et al. Protein aggregates and dementia: Is there a common toxicity? [J] J Neurol Neurosurg Psychiatry, 2002 Feb;72(2):152-61.
43. Khan MF, Falk RH. Amyloidosis[J]. Postgrad Med J, 2001 Nov;77(913):686-93.
44. Li SH, Lam S, Cheng AL, et al. Intranuclear huntingtin increases the expression of caspase-1 and induces apoptosis[J]. Hum Mol Genet, 2000 Nov 22;9(19):2859-67.
45. Andersson K, Olofsson A, Nielsen EH, et al. Only amyloidogenic intermediates of transthyretin induce apoptosis[J]. Biochem Biophys Res Commun, 2002 Jun 7;294(2):309-14.
46. Leliveld SR ,Zhang YH ,Rohn JL et al. Apoptin induces tumor-specific apoptosis as a random,globular multimer[J]. J Biol Chem, 2003, 278(11) : 9042- 51.
47. Destin W Heilman, Jose G Teodoro, Michael R. Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies [J]. Virol, 2006,80(15): 7535–7545.
48. Kudo N, Wolff B, Sekimoto T, et al. Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp Cell Res, 1998 Aug 1;242(2):540-7.
49. Wolff B, Sanglier JJ, Wang Y. Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA[J]. Chem Biol, 1997Feb;4(2):139-47.
50. Rohn JL., YH Zhang, RI Aalbers, et al. A tumor-specific kinase activity regulates the viral death protein Apoptin[J]. J Biol. Chem, 2002. 277:50820-50827.
51. Lee YH, Cheng CM, Chang YF, et al. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity[J]. Biochem Biophys Res Commun, 2007 Mar 9;354(2):391-5.
52. Bao J, Zervos AS. Isolation and characterization of Nmi, a novel partner of Myc proteins[J]. Oncogenes, 1996, 16:2171-2176.
53. Danen-van Oorschot AAM, Voskamp P, Seelen MCM, et al. Human death effecter domain-associated factor interacts with the viral apoptosis agonist Apoptin and exert tumor-preferential cell killing[J]. Cell Death Differ, 2004, 11:564-573.
54. Cheng CM, Huang SP, Chang YF, et al. The viral death protein Apoptin interacts with Hippi, the protein interactor of Huntingtin-interacting protein 1[J]. Biochem Biophys Res Commun, 2003 May 30;305(2):359-64.
55. Pietersen AM, van der Eb MM, Rademaker HJ, et al. Specific tumor-cell killing with adenovirus vectors containing the apoptin gene[J]. Gene Ther. 1999 May;6(5):882-92.
56. Alexandra Pietersen, Stefan Erkeland ,Saskia Rutjes, et al . Continouous Apoptin Expression in Transgenic mice Does Not Interfere with Lymphocyte Development and Proliferation[J].医学分子生物学杂志,2005,2(5):321-330
57. Olijslagers S, Dege AY, Dinsart C, et al. Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin[J]. Cancer Gene Ther. 2001 Dec;8(12):958-65.
58. van der Eb MM, Pietersen AM, Speetjens FM, et al. Gene therapy with apoptin induces regression of xenografted human hepatomas[J]. Cancer Gene Ther, 2002 Jan;9(1):53-61.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |