DNA损伤剂表阿霉素诱导survivin表达增高的机制研究
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摘要
Survivin是已鉴定的8个凋亡抑制蛋白(inhibitor of apoptosis protein,IAP)家族成员:X-IAP、c-IAP-1、c-IAP-2、NIAP、ILP2、ML-IAP/Livin、apollon和survivin中分子量最小(16.5kD),最具有基础和临床意义而受广泛关注的明星分子[1]。与其他的IAPs家族成员不同,survivin在近乎所有的人类肿瘤中高表达,而对应的癌旁组织和人体终末分化组织不表达;survivin表达的另一个特点是受细胞周期严格调控,在G1期表达极低,S期为G1期的6倍,G2/M期则增高至40倍!显示其表达具有G2/M期依赖的特异性[2]。更重要的是,通过临床的回顾性分析发现,survivin的高表达是肿瘤进展快和恶性度高的可靠指标,意味着肿瘤治疗抗性,患者存活期缩短,复发率增高;反之,通过其反义分子、RNA干涉、核酶、负显性突变体以及小分子抑制剂等手段降低survivin表达或抑制survivin功能,都可以促进肿瘤细胞凋亡而增强肿瘤对治疗的敏感性。因此,survivin被称为关键癌基因(pivotal cancer gene)[3,4,5]。目前,以拮抗survivin为目的的抗肿瘤药物和基因治疗方案已有多项进入临床前或临床研究[6]。
值得关注的是,在肿瘤治疗的基础研究中发现:多种肿瘤细胞系和肿瘤组织,对于DNA损伤剂类化疗药物诸如顺铂,阿霉素等诱导的survivin表达增高与肿瘤对药物的治疗抗性直接相关[7]!从而影响了这些DNA损伤药物的临床应用效果。然而,旨在直接抑制survivin表达的治疗策略却能增强肿瘤细胞对DNA损伤药物的敏感性。因此,开展survivin抗DNA损伤应激机制的研究工作将对临床用药具有指导意义。为此,我们以DNA损伤药物治疗反应中survivin诱导表达升高的分子机制这一科学问题为研究切入点,采用目前临床常用的DNA损伤剂类化疗药-表阿霉素和HeLa细胞系为主要的实验材料,从转录调控、mRNA水平、蛋白稳定性及细胞定位等角度综合研究survivin在DNA损伤应激反应中的表达调控,以期揭示survivin在肿瘤细胞拮抗DNA损伤应激反应的分子机制,并为以survivin为靶标的肿瘤治疗策略的应用提供理论指导。
1)建立表阿霉素诱导HeLa细胞中survivin表达增高的研究模型。首先,通过免疫印迹技术检测不同浓度梯度和不同作用时间的表阿霉素对肿瘤细胞中survivin蛋白表达水平的调控作用。结果显示,表阿霉素诱导survivin表达上调具有明显的时间和浓度梯度依赖性。细胞周期同步化实验证实survivin在对照组HeLa细胞中严格遵守周期时相依赖的表达规律,同时发现表阿霉素能诱导同步化于G1期的HeLa细胞中survivin表达上调,即表阿霉素能诱导非周期依赖的survivin表达上调。通过台盼蓝活细胞计数显示,表阿霉素诱导HeLa细胞中survivin表达增高同肿瘤细胞的化疗抗性密切相关: survivin knockdown-HeLa细胞中存活细胞减少;survivin overexpression-HeLa细胞中存活细胞增多。
2)以真核基因表达调控的多层次复杂调控模式为依据,探讨表阿霉素影响survivin表达调控的可能环节。survivin近端启动子序列的双荧光素酶活性检测发现,表阿霉素并未增加survivin的转录起始活性;实时定量PCR结果显示表阿霉素对survivin的mRNA水平没有显著影响;而用蛋白合成的非特异性抑制剂放线菌酮检测survivin的半衰期实验表明,表阿霉素增加了survivin蛋白的稳定性。同时证明,外源性survivin也可被表阿霉素上调。上述结果表明:表阿霉素对HeLa细胞中survivin的表达调控发生在蛋白水平,抑制了survivin的降解。
已有的研究工作揭示,p53-survivin构成的转录抑制调控网络在抗DNA损伤药上具有重要意义[8]。考虑到HeLa细胞中p53蛋白被人乳头瘤病毒(human papillomavirus,HPV)表达的原癌蛋白E6所降解,我们通过表达外源野生型p53而增加p53蛋白的表达量。结果显示,p53活性增高尽管可以在一定程度上抑制survivin近端启动子活性,但对survivin的mRNA和蛋白表达水平没有显著影响,可能是p53-survivin转录抑制调控网络与更为精细复杂的时空表达选择机制相关联。
3)免疫荧光检测发现,表阿霉素能使survivin的定位由细胞质转移到细胞核,对GFP-survivin融合蛋白的荧光共聚焦观察进一步验证了此结果。免疫印迹检测显示细胞核内survivin蛋白水平随表阿霉素药物浓度升高而增高,而细胞质内survivin蛋白水平保持不变甚至降低。作用不同时间点,荧光计数定位发现表阿霉素(0.5μg/ml)可以在3小时内使大部分细胞的survivin主要募集至细胞核,并且这种定位效应与细胞周期不相关。为此,进一步探讨了调节survivin核质定位的核孔转运蛋白CRM-1在表阿霉素诱导survivin核内累积中的功能。CO-IP结果显示,表阿霉素对CRM-1与survivin的结合没有显著影响;免疫荧光共定位结果表明,表阿霉素没有改变GFP-survivin和CRM-1的共定位数量,但使其共定位从核膜转移到了细胞核内。提示表阿霉素通过改变CRM-1/survivin复合物的定位,使survivin不能有效的转运到细胞质。
4)原位胞质去除的免疫组化研究发现,GFP-survivin与DNA染色的DAPI荧光共定位,即表阿霉素使survivin结合于染色质结构。在低渗有丝分裂染色体制备实验中,意外地发现表阿霉素可以强烈的阻止HeLa细胞进入M期,而用RNAi抑制survivin则可以使少量的HeLa细胞进入M期,但染色体形态出现异常。彗星尾实验检测发现,过表达survivin的HeLa细胞中药物作用后DNA损伤程度轻,而通过RNAi抑制survivin表达的HeLa细胞中DNA损伤程度重,提示:survivin可能有促进DNA损伤修复的功能。凋亡检测发现,用survivin34A DN抑制survivin表达使HeLa细胞的凋亡比例比空载体对照可增加79.2%。因此,实验结果提示survivin增高在表阿霉素作用12小时表现为减少凋亡诱导,这至少部分是survivin促进DNA损伤修复的结果,但有待于进一步深入揭示其抗DNA损伤的分子机制。
综合上述实验结果,本课题的研究证明:DNA损伤剂表阿霉素诱导HeLa细胞中survivin的表达增高同肿瘤细胞的化疗抗性密切相关。表阿霉素作用于肿瘤细胞后,survivin快速结合于细胞核内的染色质结构,使其不能或减弱了在细胞质内的降解,因此半衰期增加,蛋白水平上调而mRNA水平没有显著改变。本研究首次观察到在表阿霉素引起的DNA损伤反应过程中survivin细胞核转位并结合于染色质结构,以及使M期进入受阻和促进DNA损伤修复。这一现象为深入研究survivin的抗DNA损伤机制提供线索,有助于揭示survivin抗肿瘤治疗的分子机制,为DNA损伤剂类化疗药物的临床应用提供科学指导。
Survivin is the smallest member of eight IAP (inhibitor of apoptosis protein) family: X-IAP、c-IAP-1、c-IAP-2、NIAP、ILP2、ML-IAP/Livin、apollon and survivin ,and is the most attractive for its significance in clinical and basic research. However, survivin was is a unique member of the IAP gene family in that it is expressed in most human cancers, but largely undetectable in most differentiated adult tissues. Another feature of survivin is a cell-cycle dependent expression: survivin was very low in G1 phase, increased in S phase and peaked in G2/M phase. More importantly, in retroclinical research, highly expressed survivin in tumors is an index of cancer rapid development and malignancy, which always means resistance against cancer therapy-induced apoptosis, abbreviated patient survival and high relapse. Otherwise, inhibition of survivin by antisense, RNA interference, ribozyme, dominant negative mutant, small molecular inhibitors can increase sensitivity of cancer to therapy. Hence, survivin is called“pivotal cancer gene”and many projects targeted survivin was in preclinical or clinical stage.
Most attractively, in basic cancer-therapy research, DNA damage reagents such as doxorubicin and cisplatin can induce survivin upregulation in many cancer cells lines and tissures, which causes resistance of cancer to chemotherapy. Otherwise, inhibition of survivin can increase sensitivity of cancer cells to DNA damage stress. Howerever, mechanism of resistance to DNA damage caused by survivin was unclear. There is a scientific problem why survivin is upregulated by DNA damage drugs, and so we adopts the common drugs epirubicin and HeLa cells as experimental materials to explore the mechanism of survivin upregulation in DNA damage stress. At last, it may provide rationale for anticancer strategy targeting survivin.
Firstly, we make a model of survivin regulation: survivin was detected by immunoblot in HeLa cells treated with epirubicin in different time points and concentrations. Immunoblots analysis showed that survivin was induced upregulation in a time and concentration dependent manner. Furthermore, synchronization of HeLa cells confirmed that survivin was strictly regulated in a cell cycle dependent manner. Survivin was also upregulated by epirubicin in synchronization in the G1 phase, that is to say, responsive upregulation of survivin by epirubicin was not cell cycle dependent. In the counting viable cells, inducible upregulation of survivin in HeLa cells was associated with therapy-resistance: viable cells decreased significantly in survivin-knockdown HeLa cells.
Secondly, eukaryotic gene is regulated in multiple level and complex process, and so experiments of different level was designed to seek key step of survivin control in DNA damage response. Activity of survivin proximal promoter was almost not affected by epirubicin in dual luciferase assay; survivin had no change at mRNA level in real-time PCR after stimulated by epirubicin. Howerer, the protein stability of survivin in HeLa cells detected by cycloheximide is increased by epirubicin. At the same time, exogenous survivin controlled by the promoter in the pEGFP-plasmid was induced upregulation, too. Hence, epirubicin had an effects on survivin expression at protein level, and repressed degradation of survivin.
It is reported that p53-survivin pathway is important for resistance of cancer to chemotherapy. Considering that p53 is degraded by HPV-E6(an oncoprotein from human papillomavirus,HPV) , exogenous p53 was overexpressed in HeLa cells. Accidentally, activated p53 could represse proximal promoter of survivin, but not affected on survivin expression at protein and mRNA level. However, HeLa cells overexpressing p53 caused much death and weakend delay of S phase. We proposed that although p53 repressed transcription of survivin, other mechanism of survivin regulation neutralized transcriptional repression of p53.
Thirdly, immunoflorescence analysis showed that survivin translocated from cytoplasm to nucleus after stimulation by epirubicin. The same results was confirmed in the experiment using GFP- tagged fused protein. Immunoblot analysis in different cell fractions showed survivin was nuclear accumulation: protein of survivin in the nuclei is elevated gradually with the increasingly concentration of epirubicin, while protein of survivin in the cytoplasm was almost unchanged. Counting cells located in cytoplasm or nucleus under the flurescence microscopy at different time points,we found that survivin located in the nuclus in most cells within 3 hours, which was not related to cell cycle. It is reported that CRM-1/survivin complex is required for survivin location. CRM-1 is a transportation protein in the nuclear pore and transport many protein between cytoplasm and nucleus, such as p53, cyclin B, cdc25c. it is wondered if epirubicin had an effects on function of CRM-1.Co-IP analysis using GFP antibody showed that epirubicin had no effects on the binding activity of GFP-survivin and CRM-1. Observation by confocal microscopy showed that quantitative colocalization of survivin and CRM-1 was not affected by epirubicin, but survivin and CRM-1 colocalized in the nucleus. On the contrary, survivin and CRM-1 colocalized in the nuclear envelope in the control cells. Therefore, epirubicin change location of survivin in the cells through the relocalization of CRM-1/survivin complex, and it interfered exportation of survivin into cytoplasm.
Forthly, immunofluorescence analysis in situ detergent extraction showed that GFP-survivin and DAPI colocalized in the nucleus, that is to say, epirubicin made survivin binds to chromatin structure. It is accidentally found that epirubicin powerfully prevent HeLa cells into mitosis in hypotonic Chromosome spreads experiment. when survivin was inhibited by RNAi, some HeLa cells could enter mitosis and manifested abnormal chromosomes. During mitosis, survivin binds to Aurora B,INCENP,Borealin to form chromosome passenger complex (CPC), which binds to centromere and contributes to form spindle. Hence, binding of survivin and chromatin may disable formation or location of CPC, which prevents HeLa cells into mitosis and may be in favour of DNA repair. In comet assay, DNA damage in HeLa cells overexpressing wt survivin is in the lower degree. On the contrary, DNA damage in survivin-knockdown HeLa cells is in the higher degree. This results confirmed that survivin can enhance DNA repair, but the mechanism needs to be explored deeply. Transient transfection with wt survivin weakened sensitivity of HeLa cells to epirubicin. Otherwise, Transient transfection with survivin T34A and survivin RNAi enhanced sensitivity to epirubicin. In apoptosis analysis, inhibition of survivin by transient transfection with survivin T34A increased apoptosis by 79.2%. Hence, it is implied that survivin antagonize DNA damage-induced apoptosis, which is at least partly caused by function of survivin in DNA repair.
In summary, it is found that upregulation of survivin induced by epirubicin in HeLa cells was associated closely with resistance of cancer to chemotherapy. DNA damage reagent epirubicin can make survivin bind to chromatin fastly, which may prevent or weaken degradation of survivin in the cytoplasm. Therefore, haf-time of survivin is increased ,while survivin was not changed at RNA level. In present study, it is first time that we observed binding of survivin to chromatin in DNA damage response, which may involve in prevention of mitotic enter and DNA damage repair. This detail may provide a clue for exploring mechanism of antagonization to DNA damage by survivin, and contribute to uncover molecular mechanism of antitherapy caused by survivin, which provide scientific instructions for utility of clinical DNA damage drug.
值得关注的是,在肿瘤治疗的基础研究中发现:多种肿瘤细胞系和肿瘤组织,对于DNA损伤剂类化疗药物诸如顺铂,阿霉素等诱导的survivin表达增高与肿瘤对药物的治疗抗性直接相关[7]!从而影响了这些DNA损伤药物的临床应用效果。然而,旨在直接抑制survivin表达的治疗策略却能增强肿瘤细胞对DNA损伤药物的敏感性。因此,开展survivin抗DNA损伤应激机制的研究工作将对临床用药具有指导意义。为此,我们以DNA损伤药物治疗反应中survivin诱导表达升高的分子机制这一科学问题为研究切入点,采用目前临床常用的DNA损伤剂类化疗药-表阿霉素和HeLa细胞系为主要的实验材料,从转录调控、mRNA水平、蛋白稳定性及细胞定位等角度综合研究survivin在DNA损伤应激反应中的表达调控,以期揭示survivin在肿瘤细胞拮抗DNA损伤应激反应的分子机制,并为以survivin为靶标的肿瘤治疗策略的应用提供理论指导。
1)建立表阿霉素诱导HeLa细胞中survivin表达增高的研究模型。首先,通过免疫印迹技术检测不同浓度梯度和不同作用时间的表阿霉素对肿瘤细胞中survivin蛋白表达水平的调控作用。结果显示,表阿霉素诱导survivin表达上调具有明显的时间和浓度梯度依赖性。细胞周期同步化实验证实survivin在对照组HeLa细胞中严格遵守周期时相依赖的表达规律,同时发现表阿霉素能诱导同步化于G1期的HeLa细胞中survivin表达上调,即表阿霉素能诱导非周期依赖的survivin表达上调。通过台盼蓝活细胞计数显示,表阿霉素诱导HeLa细胞中survivin表达增高同肿瘤细胞的化疗抗性密切相关: survivin knockdown-HeLa细胞中存活细胞减少;survivin overexpression-HeLa细胞中存活细胞增多。
2)以真核基因表达调控的多层次复杂调控模式为依据,探讨表阿霉素影响survivin表达调控的可能环节。survivin近端启动子序列的双荧光素酶活性检测发现,表阿霉素并未增加survivin的转录起始活性;实时定量PCR结果显示表阿霉素对survivin的mRNA水平没有显著影响;而用蛋白合成的非特异性抑制剂放线菌酮检测survivin的半衰期实验表明,表阿霉素增加了survivin蛋白的稳定性。同时证明,外源性survivin也可被表阿霉素上调。上述结果表明:表阿霉素对HeLa细胞中survivin的表达调控发生在蛋白水平,抑制了survivin的降解。
已有的研究工作揭示,p53-survivin构成的转录抑制调控网络在抗DNA损伤药上具有重要意义[8]。考虑到HeLa细胞中p53蛋白被人乳头瘤病毒(human papillomavirus,HPV)表达的原癌蛋白E6所降解,我们通过表达外源野生型p53而增加p53蛋白的表达量。结果显示,p53活性增高尽管可以在一定程度上抑制survivin近端启动子活性,但对survivin的mRNA和蛋白表达水平没有显著影响,可能是p53-survivin转录抑制调控网络与更为精细复杂的时空表达选择机制相关联。
3)免疫荧光检测发现,表阿霉素能使survivin的定位由细胞质转移到细胞核,对GFP-survivin融合蛋白的荧光共聚焦观察进一步验证了此结果。免疫印迹检测显示细胞核内survivin蛋白水平随表阿霉素药物浓度升高而增高,而细胞质内survivin蛋白水平保持不变甚至降低。作用不同时间点,荧光计数定位发现表阿霉素(0.5μg/ml)可以在3小时内使大部分细胞的survivin主要募集至细胞核,并且这种定位效应与细胞周期不相关。为此,进一步探讨了调节survivin核质定位的核孔转运蛋白CRM-1在表阿霉素诱导survivin核内累积中的功能。CO-IP结果显示,表阿霉素对CRM-1与survivin的结合没有显著影响;免疫荧光共定位结果表明,表阿霉素没有改变GFP-survivin和CRM-1的共定位数量,但使其共定位从核膜转移到了细胞核内。提示表阿霉素通过改变CRM-1/survivin复合物的定位,使survivin不能有效的转运到细胞质。
4)原位胞质去除的免疫组化研究发现,GFP-survivin与DNA染色的DAPI荧光共定位,即表阿霉素使survivin结合于染色质结构。在低渗有丝分裂染色体制备实验中,意外地发现表阿霉素可以强烈的阻止HeLa细胞进入M期,而用RNAi抑制survivin则可以使少量的HeLa细胞进入M期,但染色体形态出现异常。彗星尾实验检测发现,过表达survivin的HeLa细胞中药物作用后DNA损伤程度轻,而通过RNAi抑制survivin表达的HeLa细胞中DNA损伤程度重,提示:survivin可能有促进DNA损伤修复的功能。凋亡检测发现,用survivin34A DN抑制survivin表达使HeLa细胞的凋亡比例比空载体对照可增加79.2%。因此,实验结果提示survivin增高在表阿霉素作用12小时表现为减少凋亡诱导,这至少部分是survivin促进DNA损伤修复的结果,但有待于进一步深入揭示其抗DNA损伤的分子机制。
综合上述实验结果,本课题的研究证明:DNA损伤剂表阿霉素诱导HeLa细胞中survivin的表达增高同肿瘤细胞的化疗抗性密切相关。表阿霉素作用于肿瘤细胞后,survivin快速结合于细胞核内的染色质结构,使其不能或减弱了在细胞质内的降解,因此半衰期增加,蛋白水平上调而mRNA水平没有显著改变。本研究首次观察到在表阿霉素引起的DNA损伤反应过程中survivin细胞核转位并结合于染色质结构,以及使M期进入受阻和促进DNA损伤修复。这一现象为深入研究survivin的抗DNA损伤机制提供线索,有助于揭示survivin抗肿瘤治疗的分子机制,为DNA损伤剂类化疗药物的临床应用提供科学指导。
Survivin is the smallest member of eight IAP (inhibitor of apoptosis protein) family: X-IAP、c-IAP-1、c-IAP-2、NIAP、ILP2、ML-IAP/Livin、apollon and survivin ,and is the most attractive for its significance in clinical and basic research. However, survivin was is a unique member of the IAP gene family in that it is expressed in most human cancers, but largely undetectable in most differentiated adult tissues. Another feature of survivin is a cell-cycle dependent expression: survivin was very low in G1 phase, increased in S phase and peaked in G2/M phase. More importantly, in retroclinical research, highly expressed survivin in tumors is an index of cancer rapid development and malignancy, which always means resistance against cancer therapy-induced apoptosis, abbreviated patient survival and high relapse. Otherwise, inhibition of survivin by antisense, RNA interference, ribozyme, dominant negative mutant, small molecular inhibitors can increase sensitivity of cancer to therapy. Hence, survivin is called“pivotal cancer gene”and many projects targeted survivin was in preclinical or clinical stage.
Most attractively, in basic cancer-therapy research, DNA damage reagents such as doxorubicin and cisplatin can induce survivin upregulation in many cancer cells lines and tissures, which causes resistance of cancer to chemotherapy. Otherwise, inhibition of survivin can increase sensitivity of cancer cells to DNA damage stress. Howerever, mechanism of resistance to DNA damage caused by survivin was unclear. There is a scientific problem why survivin is upregulated by DNA damage drugs, and so we adopts the common drugs epirubicin and HeLa cells as experimental materials to explore the mechanism of survivin upregulation in DNA damage stress. At last, it may provide rationale for anticancer strategy targeting survivin.
Firstly, we make a model of survivin regulation: survivin was detected by immunoblot in HeLa cells treated with epirubicin in different time points and concentrations. Immunoblots analysis showed that survivin was induced upregulation in a time and concentration dependent manner. Furthermore, synchronization of HeLa cells confirmed that survivin was strictly regulated in a cell cycle dependent manner. Survivin was also upregulated by epirubicin in synchronization in the G1 phase, that is to say, responsive upregulation of survivin by epirubicin was not cell cycle dependent. In the counting viable cells, inducible upregulation of survivin in HeLa cells was associated with therapy-resistance: viable cells decreased significantly in survivin-knockdown HeLa cells.
Secondly, eukaryotic gene is regulated in multiple level and complex process, and so experiments of different level was designed to seek key step of survivin control in DNA damage response. Activity of survivin proximal promoter was almost not affected by epirubicin in dual luciferase assay; survivin had no change at mRNA level in real-time PCR after stimulated by epirubicin. Howerer, the protein stability of survivin in HeLa cells detected by cycloheximide is increased by epirubicin. At the same time, exogenous survivin controlled by the promoter in the pEGFP-plasmid was induced upregulation, too. Hence, epirubicin had an effects on survivin expression at protein level, and repressed degradation of survivin.
It is reported that p53-survivin pathway is important for resistance of cancer to chemotherapy. Considering that p53 is degraded by HPV-E6(an oncoprotein from human papillomavirus,HPV) , exogenous p53 was overexpressed in HeLa cells. Accidentally, activated p53 could represse proximal promoter of survivin, but not affected on survivin expression at protein and mRNA level. However, HeLa cells overexpressing p53 caused much death and weakend delay of S phase. We proposed that although p53 repressed transcription of survivin, other mechanism of survivin regulation neutralized transcriptional repression of p53.
Thirdly, immunoflorescence analysis showed that survivin translocated from cytoplasm to nucleus after stimulation by epirubicin. The same results was confirmed in the experiment using GFP- tagged fused protein. Immunoblot analysis in different cell fractions showed survivin was nuclear accumulation: protein of survivin in the nuclei is elevated gradually with the increasingly concentration of epirubicin, while protein of survivin in the cytoplasm was almost unchanged. Counting cells located in cytoplasm or nucleus under the flurescence microscopy at different time points,we found that survivin located in the nuclus in most cells within 3 hours, which was not related to cell cycle. It is reported that CRM-1/survivin complex is required for survivin location. CRM-1 is a transportation protein in the nuclear pore and transport many protein between cytoplasm and nucleus, such as p53, cyclin B, cdc25c. it is wondered if epirubicin had an effects on function of CRM-1.Co-IP analysis using GFP antibody showed that epirubicin had no effects on the binding activity of GFP-survivin and CRM-1. Observation by confocal microscopy showed that quantitative colocalization of survivin and CRM-1 was not affected by epirubicin, but survivin and CRM-1 colocalized in the nucleus. On the contrary, survivin and CRM-1 colocalized in the nuclear envelope in the control cells. Therefore, epirubicin change location of survivin in the cells through the relocalization of CRM-1/survivin complex, and it interfered exportation of survivin into cytoplasm.
Forthly, immunofluorescence analysis in situ detergent extraction showed that GFP-survivin and DAPI colocalized in the nucleus, that is to say, epirubicin made survivin binds to chromatin structure. It is accidentally found that epirubicin powerfully prevent HeLa cells into mitosis in hypotonic Chromosome spreads experiment. when survivin was inhibited by RNAi, some HeLa cells could enter mitosis and manifested abnormal chromosomes. During mitosis, survivin binds to Aurora B,INCENP,Borealin to form chromosome passenger complex (CPC), which binds to centromere and contributes to form spindle. Hence, binding of survivin and chromatin may disable formation or location of CPC, which prevents HeLa cells into mitosis and may be in favour of DNA repair. In comet assay, DNA damage in HeLa cells overexpressing wt survivin is in the lower degree. On the contrary, DNA damage in survivin-knockdown HeLa cells is in the higher degree. This results confirmed that survivin can enhance DNA repair, but the mechanism needs to be explored deeply. Transient transfection with wt survivin weakened sensitivity of HeLa cells to epirubicin. Otherwise, Transient transfection with survivin T34A and survivin RNAi enhanced sensitivity to epirubicin. In apoptosis analysis, inhibition of survivin by transient transfection with survivin T34A increased apoptosis by 79.2%. Hence, it is implied that survivin antagonize DNA damage-induced apoptosis, which is at least partly caused by function of survivin in DNA repair.
In summary, it is found that upregulation of survivin induced by epirubicin in HeLa cells was associated closely with resistance of cancer to chemotherapy. DNA damage reagent epirubicin can make survivin bind to chromatin fastly, which may prevent or weaken degradation of survivin in the cytoplasm. Therefore, haf-time of survivin is increased ,while survivin was not changed at RNA level. In present study, it is first time that we observed binding of survivin to chromatin in DNA damage response, which may involve in prevention of mitotic enter and DNA damage repair. This detail may provide a clue for exploring mechanism of antagonization to DNA damage by survivin, and contribute to uncover molecular mechanism of antitherapy caused by survivin, which provide scientific instructions for utility of clinical DNA damage drug.
引文
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9. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002;277:3247–57.
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33. Dohi, T., Xia, F. & Altieri, D. C. Compartmentalized phosphorylation of IAP by protein kinase A regulates cytoprotection. Mol. Cell 2007.27, 17–28.
34. Dohi, T., Beltrami, E., Wall, N. R., Plescia, J. & Altieri, D. C. Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J. Clin. Invest. 2004.114, 1117–1127.
35. Mahotka C, Wenzel M, Springer E, Gabbert HE, Gerharz CD. Survivin-deltaEx3 and survivin-2B: Two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. Cancer Res 1999.59:6097–6102.
36. Mahotka C, Liebmann J, Wenzel M, Suschek CV, Schmitt M, Gabbert HE, Gerharz CD. Differential subcellular localization of functionally divergent survivin splice variants. Cell Death Differ 2002.9(12):1334–1342.
37. Noton EA, Colnaghi R, Tate S, Starck C, Carvalho A, Ko Ferrigno P, Wheatley SP. Molecular analysis of survivin isoforms: Evidence that alternatively spliced variants do not play a role in mitosis. J Biol Chem 2006.281:1286–1295.
38. Caldas H, Honsey LE, Altura RA. Survivin 2alpha: A novel Survivin splice variant expressedin human malignancies. Mol Cancer 2005. 4(1):11.
39. Vong QP, Cao K, Li HY, Iglesias PA, Zheng Y. Chromosome alignment and segregation regulated by ubiquitination of survivin. Science 2005.310(5753):1499– 1504.
40. Wheatley SP, Henzing AJ, Dodson H, Khaled W, Earnshaw WC. 2004. Aurora-B phosphorylation in vitro identifies a residue of Survivin that is essential for its localization and binding to INCENP in vivo. J Biol Chem 279(7):5655–5660.
2. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998.396:580–584.
3. Dario C Altieri. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene, 2003,8581–8589.
4. FENGZHI LI. Survivin Study: What Is the Next Wave? JOURNAL OF CELLULAR PHYSIOLOGY, 2003,197:8–29.
5. Altieri DC. 2001. The molecular basis and potential role of survivin in cancer diagnosis and therapy. Trends Mol Med 7:542–547.
6. Altieri, D.C. Targeted therapy by disabling crossroad signaling networks: the survivin paradigm. Mol. Cancer Ther. 2006.5, 478–482.
7. Wall NR, O’Connor DS, Plescia J, Pommier Y, Altieri DC. Suppression of survivin phosphorylation on Thr34 by flavopiridol enhances tumor cell apoptosis. Cancer Res 2003;63:230 –5.
8. MUXIANG ZHOU, LUBING GU, FENGZHI LI, YERUN ZHU, WILLIAM G. WOODS, and HARRY W. FINDLEY. DNA Damage Induces a Novel p53-Survivin Signaling Pathway Regulating Cell Cycle and Apoptosis in Acute Lymphoblastic Leukemia Cells JPET 2002. 303:124–131.
9. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002;277:3247–57.
10. Pierre Olivier, Este, Hang Gyeong Chin, and Sriharsa Pradhan, Molecular Mechanisms of Transactivation and Doxorubicin-mediated Repression of survivin Gene in Cancer Cells. 2007. 282. 4. 2615–2625.
11. Ghosh, J. C., Dohi, T., Raskett, C. M., Kowalik, T. F. ,Altieri, D. C. Activated checkpoint kinase 2 provides a survival signal for tumor cells. Cancer Res. 2006.66, 11576–11579.
12. Pawan Kumar, Ila K. Coltas, Bhavna Kumar, Douglas B. Chepeha, Carol R. Bradford, and Peter J. Polverini, Bcl-2 Protects Endothelial Cells againstγ- Radiation via a Raf-MEK-ERK-Survivin Signaling Pathway: That Is Independent of Cytochrome c Release. Cancer Res 2007; 67: (3).1193-1202
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16. Susanne MA Lens, Gerben Vader and Rene′ H Medema. The case for Survivin as mitotic regulator. Current Opinion in Cell Biology, 2006.18:1–7.
17. Rodriguez JA, Span SW, Ferreira CG, Kruyt FA, Giaccone G. CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin. Exp Cell Res 2002. 275:44–53.
18. Colnaghi R, Connell CM, Barrett RM, Wheatley SP. Separating the anti-apoptotic and mitotic roles of survivin. J Biol Chem 2006;281:33450–6.
19. Li F, Yang J, Ramnath N, Javle MM, Tan D. Nuclear or cytoplasmic expression of survivin: what is the significance? Int J Cancer 2005;114:509–12.
20. Stauber, R. H., Mann, W. & Knauer, S. K. Nuclear and cytoplasmic survivin: molecular mechanism, prognostic, and therapeutic potential. Cancer Res. 2007.67,5999–6002.
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21. Rodriguez JA, Span SW, Ferreira CG, Kruyt FA, Giaccone G. CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin. Exp Cell Res 2002. 275:44–53.
22. Stauber, R. H., Mann, W. & Knauer, S. K. Nuclear and cytoplasmic survivin: molecular mechanism, prognostic, and therapeutic potential. Cancer Res. 2007.67,5999–6002.
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5. Hoffman WH, Biade S, Zilfou JT, Chen J, Murphy M. Transcriptional repression of the anti-apoptotic survivin gene by wild type p53. J Biol Chem 2002;277:3247–57.
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8. Ghosh, J. C., Dohi, T., Raskett, C. M., Kowalik, T. F. ,Altieri, D. C. Activated checkpoint kinase 2 provides a survival signal for tumor cells. Cancer Res. 2006.66, 11576–11579.
9. Pawan Kumar, Ila K. Coltas, Bhavna Kumar, Douglas B. Chepeha, Carol R. Bradford, and Peter J. Polverini, Bcl-2 Protects Endothelial Cells againstγ-Radiation via a Raf-MEK- ERK-Survivin Signaling Pathway: That Is Independent of Cytochrome c Release. Cancer Res 2007; 67: (3).1193-1202
1. Dario C Altieri. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene, 2003,8581–8589.
2. Fortugno, P. et al. Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function. J. Cell Sci. 2002.115, 575–585.
3. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998.396:580–584.
4. Achim Temme, Michael Rieger, Friedemann Reber, Dirk Lindemann, Bernd Weigle, Petra Diestelkoetter-Bachert, Gerhard Ehninger, Masaaki Tatsuka, Yasuhiko Terada, and Ernst Peter Rieber .Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell . MOL BIOL CELL 2003.14, 78–92.
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6. Dohi, T. et al. An IAP–IAP complex inhibits apoptosis. J. Biol. Chem. 2004.279, 34087–34090.
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15. Rodriguez JA, Span SW, Ferreira CG, Kruyt FA, Giaccone G. CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin. Exp Cell Res 2002. 275:44–53.
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17. Stauber RH, Rabenhorst U, Rekik A, Engels K, Bier C, Knauer SK. Nucleocytoplasmic shuttling and the biological activity of mouse survivin are regulated by an active nuclear export signal. Traffic 2006;7:1461–72.
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1. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med, 1997,3: 917-921.
2. Dario C Altieri. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene, 2003,8581–8589.
3. FENGZHI LI. Survivin Study: What Is the Next Wave? JOURNAL OF CELLULAR PHYSIOLOGY, 2003,197:8–29.
4. Altieri DC. 2001. The molecular basis and potential role of survivin in cancer diagnosis and therapy. Trends Mol Med 7:542–547.
5. Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC, Altieri DC. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 1998.396:580–584.
6. Li F, Altieri DC. Transcriptional analysis of human survivin gene expression. Biochem J 1999.344(Pt 2):305–311.
7. Zhao J, Tenev T, Martins LM, Downward J, Lemoine NR. The ubiquitin proteasome pathway regulates survivin degradation in a cell cycle-dependent manner. 2000.J Cell Sci 113(Pt 23):4363–4371.
8. O’Connor DS, Grossman D, Plescia J, Li F, Zhang H, Villa A, Tognin S, Marchisio PC, Altieri DC. Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc Natl Acad Sci USA 2000.97:13103–13107.
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16. Pierre Olivier, Este, Hang Gyeong Chin, and Sriharsa Pradhan, Molecular Mechanisms of Transactivation and Doxorubicin-mediated Repression of survivin Gene in Cancer Cells. 2007. 282. 4. 2615–2625.
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20. Fortugno, P. et al. Regulation of survivin function by Hsp90. Proc. Natl Acad. Sci. USA 2003.100, 13791–13796.
21. Xia, W. et al. Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers. Cancer Res. 2006.66, 1640–1647.
22. Semba, S. et al. Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential. Oncogene 2006.25, 2860–2872.
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26. Li F, Yang J, Ramnath N, Javle MM, Tan D. Nuclear or cytoplasmic expression of survivin: what is the significance? Int J Cancer 2005;114:509–12.
27. Rodriguez JA, Span SW, Ferreira CG, Kruyt FA, Giaccone G. CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin. Exp Cell Res 2002. 275:44–53.
28. Fortugno P, Wall NR, Giodini A, O’Connor DS, Plescia J, Padgett KM, Tognin S, Marchisio PC, Altieri DC. Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function. J Cell Sci 2002. 115:575–585.
29. Knauer SK, Bier C, Habtemichael N, Stauber RH. The Survivin-Crm1 interaction is essential for chromosomal passenger complex localization and function. EMBO Rep 2006;7:1259–65.
30. Colnaghi R, Connell CM, Barrett RM, Wheatley SP. Separating the anti-apoptotic and mitotic roles of survivin. J Biol Chem 2006;281:33450–6.
31. Stauber RH, Rabenhorst U, Rekik A, Engels K, Bier C, Knauer SK. Nucleocytoplasmic shuttling and the biological activity of mouse survivin are regulated by an active nuclear export signal. Traffic 2006;7:1461–72.
32. Dohi, T. et al. An IAP–IAP complex inhibits apoptosis. J. Biol. Chem. 2004.279, 34087–34090.
33. Dohi, T., Xia, F. & Altieri, D. C. Compartmentalized phosphorylation of IAP by protein kinase A regulates cytoprotection. Mol. Cell 2007.27, 17–28.
34. Dohi, T., Beltrami, E., Wall, N. R., Plescia, J. & Altieri, D. C. Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J. Clin. Invest. 2004.114, 1117–1127.
35. Mahotka C, Wenzel M, Springer E, Gabbert HE, Gerharz CD. Survivin-deltaEx3 and survivin-2B: Two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. Cancer Res 1999.59:6097–6102.
36. Mahotka C, Liebmann J, Wenzel M, Suschek CV, Schmitt M, Gabbert HE, Gerharz CD. Differential subcellular localization of functionally divergent survivin splice variants. Cell Death Differ 2002.9(12):1334–1342.
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38. Caldas H, Honsey LE, Altura RA. Survivin 2alpha: A novel Survivin splice variant expressedin human malignancies. Mol Cancer 2005. 4(1):11.
39. Vong QP, Cao K, Li HY, Iglesias PA, Zheng Y. Chromosome alignment and segregation regulated by ubiquitination of survivin. Science 2005.310(5753):1499– 1504.
40. Wheatley SP, Henzing AJ, Dodson H, Khaled W, Earnshaw WC. 2004. Aurora-B phosphorylation in vitro identifies a residue of Survivin that is essential for its localization and binding to INCENP in vivo. J Biol Chem 279(7):5655–5660.