HSPA9抑制Apoptin诱导HepG2细胞凋亡
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摘要
VP3／Apoptin是源自鸡贫血病毒的一种小分子蛋白质，能特异性诱导肿瘤细胞凋亡。早期的研究提示，VP3(108位丝氨酸)的磷酸化及核定位是其特异性诱导肿瘤细胞凋亡的首要条件，但其作用机制尚未完全阐明。后来，相继发现了数种与VP3相互作用的蛋白质，如：N-myc interacting protein(NMI)，promyelocytic leukemia protein(PML)，anaphase promoting complex／cyclosome subunit 1(APC／C)，death effectordomain associated factor(DEDAF)，SUMO，Acid ceramidase，CRM1，imprtin-β1，Acidceramidase，Ppil3等，这些蛋白质与VP3特异性诱导肿瘤细胞凋亡密切相关；有研究提示：VP3(108位苏氨酸)的磷酸化并非其特异性诱导肿瘤细胞凋亡的必要条件。最近，Maddika等报道，CDK2是磷酸化修饰VP3(108位苏氨酸)的激酶，VP3与PI3K的p85亚基相互作用别构激活PI3K，活化后的PI3K激活Akt，Akt能与VP3共定位于核内，Akt进一步激活CDK2，活化后的CDK2磷酸化修饰VP3。这些研究对VP3特异性诱导肿瘤细胞凋亡的机制有了新的认识。同时也提示，进一步探讨和鉴定VP3在细胞中与之相互作用的蛋白，通过分析研究这些蛋白质在与VP3相互作用后其功能的改变，对阐明VP3的肿瘤特异性凋亡激活机制具有非常重要的意义。
本研究以His-VP3融合蛋白为诱饵，通过pull-dawn技术，捕获His-VP3在肿瘤细胞HepG2与正常细胞L-02中相互作用的蛋白，应用二维电泳技术分离捕获蛋白质，通过软件分析比较HepG2和L-02细胞中与VP3相互作用蛋白质的差异，从胶上切取这些蛋白质点，用质谱技术鉴定出这些有差异的相互作用蛋白质。
HSPA9是在体外捕获的相互作用蛋白质之一，HSPA9(mortalin)是热休克蛋白70(hsp70)家族成员之一，与细胞的各项生理活动密切相关，其功能主要与延长细胞寿命、胞内物质运输、线粒体产能、分子伴侣、原癌基因转化、使肿瘤细胞低分化和应激反应等相关。HSPA9在肿瘤细胞或肿瘤组织内的表达水平均高于正常细胞或组织。在正常细胞内过表达HSPA9可有效延长细胞寿命；抑制HSPA9的表达可导致肿瘤细胞衰老或凋亡。HSPA9在细胞内可与多种蛋白质发生相互作用，如HSPA9与HSP60(线粒体内分子伴侣蛋白)的相互作用是维持肿瘤细胞持续增殖的条件之一。HSPA9是线粒体内的主要蛋白质之一，负责精确调控线粒体蛋白质的输入和输出，对维持细胞的稳态和线粒体产能至关重要。酵母细胞内敲除HSPA9同源蛋白Ssc1可致细胞死亡，Ssc1功能域缺失或突变可致酵母细胞内的线粒体聚集。HSPA9在细胞内是一种抗凋亡蛋白。所以在我们所鉴定出的相关蛋白质中，选取HSPA9作为进一步研究的靶蛋白。
VP3和HSPA9的相互作用经免疫共沉淀方法在胞内得到证实。进一步的研究发现，在HepG2细胞内过表HSPA9时，观察到VP3的核质分布发生了改变，部分VP3滞留于细胞质中；HepG2细胞内过表达HSPA9检测其对VP3诱导HepG2细胞凋亡的影响，在我们检测的时间点(24h)上，共转染了VP3和HSPA9的HepG2细胞凋亡率显著下降；与之相反，当我们si-RNA降低HSPA9在HepG2细胞内的表达量时，VP3对HepG2细胞的杀伤作用显著增加。以上结果表明：HSPA9是VP3的相互作用蛋白质之一；过表达HSPA9可致部分VP3滞留细胞质中；HSPA9的表达水平可显著抑制VP3诱导肿瘤细胞凋亡的效应。
目前对于VP3特异性诱导肿瘤细胞凋亡的机制尚不十分清楚，已有的研究表明，VP3诱导肿瘤细胞凋亡是通过内源性凋亡途径，不依赖死亡受体途径，其诱导肿瘤细胞凋亡活性的发挥与其核定位密切相关。S．Maddika等证实，经VP3处理的肿瘤细胞，VP3聚集于核内，导致orphan steroid receptor Nur77从细胞核内转移至细胞质中，Nur77与线粒体膜上的抗凋亡蛋白质发生作用，降低线粒体膜电位，激活线粒体凋亡途径。Nur77的核质转移是VP3诱导肿瘤细胞凋亡的重要途径之一。我们的结果显示，在HepG2细胞内过表达HSPA9，使VP3的核质分布发生改变，胞质中滞留的VP3增多，进而降低了VP3诱导HepG2细胞凋亡的效率；当抑制HSPA9的表达时，VP3几乎全部聚集于细胞核内，提高了VP3诱导HepG2细胞凋亡率。这表明：HSPA9抑制VP3诱导肿瘤细胞凋亡的效应是通过改变VP3的核质分布实现的。
胞内过表达或抑制HSPA9，可能从两个方面影响了VP3对HepG2细胞的杀伤作用，一方面，过表达时VP3进入细胞核的量减少，使Nur77从核内转移出胞质量减少，减弱了VP3的毒性作用；以此同时，过量表达的HSPA9增强了线粒体对抗各种凋亡因子的刺激作用。与之相反，在HepG2细胞内抑制HSPA9表达时，减弱了线粒体对抗各种凋亡因子的促凋亡作用，进而增加了VP3对HepG2细胞的毒性作用。这些推论还有待于进一步的实验研究来证实。HSPA9与肿瘤细胞持续增殖和侵袭密切相关，有众多的相互作用蛋白质，在VP3存在的情况下，与HSPA9相互作用蛋白质和HSPA9自身产生怎样的变化，以及这些变化对细胞凋亡会造成何种程度的影响都值得我们做进一步的研究。
Apoptin is a small viral protein that was originally identified in chicken anemia virus.Various studies have shown that Apoptin specifically induces apoptosis in a broad range ofdifferent human cancer cell lines derived from various tumor types but not in their normalcounterpart.Furthermore,apoptosis induced by Apoptin is p53-independent.The molecularmechanism of Apoptin is largely unknown.Previous studies have shown that Apoptininduced tumor cell apoptosis is closely related to tumor-specific phosphorylation andcellular localization.Several Apoptin interacting partners were identified,such as N-mycinteracting protein(NMI),promyelocytic leukemia protein(PML),anaphase promotingcomplex/cyclosome subunit 1(APC/C),death effector domain associated factor(DEDAF),Acid ceramidase,CRM1,imprtin-β1,and Ppil3 et al.These proteins playcrucial roles for Apoptin's selective toxicity.Maddika et al recently reported that,throughits proline-rich sequence(aa 81-86),apoptin interacts with the SH3 domain of the p85regulatory subunit of phosphoinositide 3-kinase(PI3K)and activates PI3K.PI3Kdownstream effector kinase Aktl was also activated and interacted with Apoptin,thenApoptin and activated Akt translocated to the Nucleus.Nuclear Akt activates CDK2 byboth direct and indirect mechanisms,CyclinA-CDK2 complex directly phosphorylatesapoptin at Thr-108 and contributes to the regulation of its subcellular.Identification ofthese Apoptin-interacting proteins extends the knowledge of Apoptin tumor-specifictoxicity,and indicates that searching and identification of Apoptin interacting partners isvery important to further elucidating mechanism of Apoptin tumor-specific toxicity.
In an attempt to elucidate the molecular mechanism of apoptosis induced by Apoptin.In this research,prokaryotic native His-Apoptin fusion protein purified with Ni-NTAaffinity chromotography served as a bait for capturing the Apoptin-interacting proteins of inboth tumor cell line(HepG2)and normal cell line(L-02)by employing the pull down assay. The captured proteins were separated by two dimensional gel electrophoresis and analyzedby Imagemaster 2D Platinum 6.0 software.The protein spots were cut from the gel,andthen identified by Mass Spectrum.After that the proteomic information ofApoptin-interacting proteins in both tumor cells and normal cells were obtained.HSPA9 isone of proteins captured from HepG2 cells by pull-down assay.HSPA9(Mortalin/mthsp70/Grp75/PBP74/mot-2)is a heat un-inducible member of hsp70 family ofproteins.It is a 74-kDa protein and has been localized to mitochondria,endoplasmicreticulum,plasma membrane and cytoplasmic vesicles.It is an essential protein thatperforms various functions related to proliferation,functional maintenance and stressresponse.Overexpression of HSPA9 may impart growth or proliferative advantages,prolonging cellular lifespan.Expression study of HSPA9 in tumor cells have revealed thatit is frequently upregulated in tumors.In specially,HSPA9 is the major mitochondrialprotein and it plays a central role in the elaborate translocation system for efficient importand export of proteins.Its role in cell viability and mitochondrial biogenesis wasunderscored by experimental data including the yeast cells knocked out for HSPA9homologue(Sscl)were lethal and the loss of function mutations of HSPA9 causedaggregation of yeast mitochondria..HSPA9 acts as an antiapoptosis protein in tumor cells.
So we selected HSPA9 as a target molecule to explore the relationship betweenHSPA9 and Apoptin specific tumor toxicity.The interaction of HSPA9 with Apoptin inHepG2 cells was further verified by co-immunoprecipitation.After that we forcedexpression or knockdown HSPA9 in HepG2 cells to test the effect of HSPA9 on Apoptintumor-specific toxicity.Our data indicated over expression of HSPA9 in HepG2 cellsresulted in partially cytoplasmic distribution of Apoptin and significantly inhibitedapoptosis of HepG2 cells.In contrast,siRNA was applied to suppress of HSPA9 expression,we observed that the apoptosis rate of HepG2 cells induced by Apoptin was elevated.
Up to now,the mechanism of Apoptin specific toxicity to tumor is still unclear.MarekLos research group reported that Apoptin is independent of death receptors but involves the loss of mitochondfial membrane potential and the release of mitochondrial cell-deathmediators by a Nur77-dependent pathway.Apoptin nucleus localization and thecytoplasmic translocation of Nur77 are crucial for the toxicity of Apoptin.Our dataindicated,over expression of HSPA9 lead to a part of Apoptin cytoplasmic distribution,meanwhile Apoptin induced apoptosis rate was dropped;whereas down-regulation ofHSPA9 expression in HepG2 cells,Apoptin's toxicity to HepG2 cells was enhanced.Itsuggested that HSPA9 inhibit cancer-specific toxicity of Apoptin through retention Apoptinin cytoplasmic.
We speculated that over expression of HSPA9 in HepG2 cells may through two waysinhibit cancer-specific toxicity of Apoptin,on one hand,the more HSPA9 presented incytoplasmic the more Apoptin was stagnated in cytoplasmic,as a result less Nur77 wastanslocated to cytoplasmic.On the other hand,over expression of HSPA9 protectedmitoehondria from the stimulation of proapoptosis factors.Down-regulation of HSPA9levels attenuated mitochondrial stability.It indirectly enhanced activities of Apoptin.Butfurther study is needed.
In conclusion,our data suggested that HSPA9 is one of Apoptin-interaction proteins.Over expression of HSPA9 prevented Apoptin from entering the nucleus and carrying outits apoptotic activity,while down-regulation of HSPA9 expression elevated thecancer-specific toxicity of Apoptin significantly.
本研究以His-VP3融合蛋白为诱饵，通过pull-dawn技术，捕获His-VP3在肿瘤细胞HepG2与正常细胞L-02中相互作用的蛋白，应用二维电泳技术分离捕获蛋白质，通过软件分析比较HepG2和L-02细胞中与VP3相互作用蛋白质的差异，从胶上切取这些蛋白质点，用质谱技术鉴定出这些有差异的相互作用蛋白质。
HSPA9是在体外捕获的相互作用蛋白质之一，HSPA9(mortalin)是热休克蛋白70(hsp70)家族成员之一，与细胞的各项生理活动密切相关，其功能主要与延长细胞寿命、胞内物质运输、线粒体产能、分子伴侣、原癌基因转化、使肿瘤细胞低分化和应激反应等相关。HSPA9在肿瘤细胞或肿瘤组织内的表达水平均高于正常细胞或组织。在正常细胞内过表达HSPA9可有效延长细胞寿命；抑制HSPA9的表达可导致肿瘤细胞衰老或凋亡。HSPA9在细胞内可与多种蛋白质发生相互作用，如HSPA9与HSP60(线粒体内分子伴侣蛋白)的相互作用是维持肿瘤细胞持续增殖的条件之一。HSPA9是线粒体内的主要蛋白质之一，负责精确调控线粒体蛋白质的输入和输出，对维持细胞的稳态和线粒体产能至关重要。酵母细胞内敲除HSPA9同源蛋白Ssc1可致细胞死亡，Ssc1功能域缺失或突变可致酵母细胞内的线粒体聚集。HSPA9在细胞内是一种抗凋亡蛋白。所以在我们所鉴定出的相关蛋白质中，选取HSPA9作为进一步研究的靶蛋白。
VP3和HSPA9的相互作用经免疫共沉淀方法在胞内得到证实。进一步的研究发现，在HepG2细胞内过表HSPA9时，观察到VP3的核质分布发生了改变，部分VP3滞留于细胞质中；HepG2细胞内过表达HSPA9检测其对VP3诱导HepG2细胞凋亡的影响，在我们检测的时间点(24h)上，共转染了VP3和HSPA9的HepG2细胞凋亡率显著下降；与之相反，当我们si-RNA降低HSPA9在HepG2细胞内的表达量时，VP3对HepG2细胞的杀伤作用显著增加。以上结果表明：HSPA9是VP3的相互作用蛋白质之一；过表达HSPA9可致部分VP3滞留细胞质中；HSPA9的表达水平可显著抑制VP3诱导肿瘤细胞凋亡的效应。
目前对于VP3特异性诱导肿瘤细胞凋亡的机制尚不十分清楚，已有的研究表明，VP3诱导肿瘤细胞凋亡是通过内源性凋亡途径，不依赖死亡受体途径，其诱导肿瘤细胞凋亡活性的发挥与其核定位密切相关。S．Maddika等证实，经VP3处理的肿瘤细胞，VP3聚集于核内，导致orphan steroid receptor Nur77从细胞核内转移至细胞质中，Nur77与线粒体膜上的抗凋亡蛋白质发生作用，降低线粒体膜电位，激活线粒体凋亡途径。Nur77的核质转移是VP3诱导肿瘤细胞凋亡的重要途径之一。我们的结果显示，在HepG2细胞内过表达HSPA9，使VP3的核质分布发生改变，胞质中滞留的VP3增多，进而降低了VP3诱导HepG2细胞凋亡的效率；当抑制HSPA9的表达时，VP3几乎全部聚集于细胞核内，提高了VP3诱导HepG2细胞凋亡率。这表明：HSPA9抑制VP3诱导肿瘤细胞凋亡的效应是通过改变VP3的核质分布实现的。
胞内过表达或抑制HSPA9，可能从两个方面影响了VP3对HepG2细胞的杀伤作用，一方面，过表达时VP3进入细胞核的量减少，使Nur77从核内转移出胞质量减少，减弱了VP3的毒性作用；以此同时，过量表达的HSPA9增强了线粒体对抗各种凋亡因子的刺激作用。与之相反，在HepG2细胞内抑制HSPA9表达时，减弱了线粒体对抗各种凋亡因子的促凋亡作用，进而增加了VP3对HepG2细胞的毒性作用。这些推论还有待于进一步的实验研究来证实。HSPA9与肿瘤细胞持续增殖和侵袭密切相关，有众多的相互作用蛋白质，在VP3存在的情况下，与HSPA9相互作用蛋白质和HSPA9自身产生怎样的变化，以及这些变化对细胞凋亡会造成何种程度的影响都值得我们做进一步的研究。
Apoptin is a small viral protein that was originally identified in chicken anemia virus.Various studies have shown that Apoptin specifically induces apoptosis in a broad range ofdifferent human cancer cell lines derived from various tumor types but not in their normalcounterpart.Furthermore,apoptosis induced by Apoptin is p53-independent.The molecularmechanism of Apoptin is largely unknown.Previous studies have shown that Apoptininduced tumor cell apoptosis is closely related to tumor-specific phosphorylation andcellular localization.Several Apoptin interacting partners were identified,such as N-mycinteracting protein(NMI),promyelocytic leukemia protein(PML),anaphase promotingcomplex/cyclosome subunit 1(APC/C),death effector domain associated factor(DEDAF),Acid ceramidase,CRM1,imprtin-β1,and Ppil3 et al.These proteins playcrucial roles for Apoptin's selective toxicity.Maddika et al recently reported that,throughits proline-rich sequence(aa 81-86),apoptin interacts with the SH3 domain of the p85regulatory subunit of phosphoinositide 3-kinase(PI3K)and activates PI3K.PI3Kdownstream effector kinase Aktl was also activated and interacted with Apoptin,thenApoptin and activated Akt translocated to the Nucleus.Nuclear Akt activates CDK2 byboth direct and indirect mechanisms,CyclinA-CDK2 complex directly phosphorylatesapoptin at Thr-108 and contributes to the regulation of its subcellular.Identification ofthese Apoptin-interacting proteins extends the knowledge of Apoptin tumor-specifictoxicity,and indicates that searching and identification of Apoptin interacting partners isvery important to further elucidating mechanism of Apoptin tumor-specific toxicity.
In an attempt to elucidate the molecular mechanism of apoptosis induced by Apoptin.In this research,prokaryotic native His-Apoptin fusion protein purified with Ni-NTAaffinity chromotography served as a bait for capturing the Apoptin-interacting proteins of inboth tumor cell line(HepG2)and normal cell line(L-02)by employing the pull down assay. The captured proteins were separated by two dimensional gel electrophoresis and analyzedby Imagemaster 2D Platinum 6.0 software.The protein spots were cut from the gel,andthen identified by Mass Spectrum.After that the proteomic information ofApoptin-interacting proteins in both tumor cells and normal cells were obtained.HSPA9 isone of proteins captured from HepG2 cells by pull-down assay.HSPA9(Mortalin/mthsp70/Grp75/PBP74/mot-2)is a heat un-inducible member of hsp70 family ofproteins.It is a 74-kDa protein and has been localized to mitochondria,endoplasmicreticulum,plasma membrane and cytoplasmic vesicles.It is an essential protein thatperforms various functions related to proliferation,functional maintenance and stressresponse.Overexpression of HSPA9 may impart growth or proliferative advantages,prolonging cellular lifespan.Expression study of HSPA9 in tumor cells have revealed thatit is frequently upregulated in tumors.In specially,HSPA9 is the major mitochondrialprotein and it plays a central role in the elaborate translocation system for efficient importand export of proteins.Its role in cell viability and mitochondrial biogenesis wasunderscored by experimental data including the yeast cells knocked out for HSPA9homologue(Sscl)were lethal and the loss of function mutations of HSPA9 causedaggregation of yeast mitochondria..HSPA9 acts as an antiapoptosis protein in tumor cells.
So we selected HSPA9 as a target molecule to explore the relationship betweenHSPA9 and Apoptin specific tumor toxicity.The interaction of HSPA9 with Apoptin inHepG2 cells was further verified by co-immunoprecipitation.After that we forcedexpression or knockdown HSPA9 in HepG2 cells to test the effect of HSPA9 on Apoptintumor-specific toxicity.Our data indicated over expression of HSPA9 in HepG2 cellsresulted in partially cytoplasmic distribution of Apoptin and significantly inhibitedapoptosis of HepG2 cells.In contrast,siRNA was applied to suppress of HSPA9 expression,we observed that the apoptosis rate of HepG2 cells induced by Apoptin was elevated.
Up to now,the mechanism of Apoptin specific toxicity to tumor is still unclear.MarekLos research group reported that Apoptin is independent of death receptors but involves the loss of mitochondfial membrane potential and the release of mitochondrial cell-deathmediators by a Nur77-dependent pathway.Apoptin nucleus localization and thecytoplasmic translocation of Nur77 are crucial for the toxicity of Apoptin.Our dataindicated,over expression of HSPA9 lead to a part of Apoptin cytoplasmic distribution,meanwhile Apoptin induced apoptosis rate was dropped;whereas down-regulation ofHSPA9 expression in HepG2 cells,Apoptin's toxicity to HepG2 cells was enhanced.Itsuggested that HSPA9 inhibit cancer-specific toxicity of Apoptin through retention Apoptinin cytoplasmic.
We speculated that over expression of HSPA9 in HepG2 cells may through two waysinhibit cancer-specific toxicity of Apoptin,on one hand,the more HSPA9 presented incytoplasmic the more Apoptin was stagnated in cytoplasmic,as a result less Nur77 wastanslocated to cytoplasmic.On the other hand,over expression of HSPA9 protectedmitoehondria from the stimulation of proapoptosis factors.Down-regulation of HSPA9levels attenuated mitochondrial stability.It indirectly enhanced activities of Apoptin.Butfurther study is needed.
In conclusion,our data suggested that HSPA9 is one of Apoptin-interaction proteins.Over expression of HSPA9 prevented Apoptin from entering the nucleus and carrying outits apoptotic activity,while down-regulation of HSPA9 expression elevated thecancer-specific toxicity of Apoptin significantly.
引文
1．Yuasa N，Taniguchi T，and Yoshida I，Isolation and characterization of an agent inducing anmia in chicks．Avian Diseases 1979，23：366．
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3．Subbareddy Maddika，Evan P．Booy，Dina Johar．et al．Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway．J Cell Sci．2005，118：4485-4493．
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8．Teodoro J G，Heilman D W．，Parker A E．，et al．The viral protein Apoptin associates with the anaphase-promoting complex to induce G2/M arrest and apoptosis in the absence of p53．Gene Dev．2004，18：1952-1957．
9．Danen-van Oorschot A A．，Voskamp P．，Seneel M．C．，et al．Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing．Cell Death Different 2004，11：564573．
10．L．Guelen，H．Paterson，J．Gaken，M．Meyers，F．Farzaneh，M．Tavassoli，TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells，Oncogene 2004，(23)：1153-1165．
11．Poon Ivan K．H．，Oro Cristina，Dias M．M．．Apoptin Nuclear Accumulation Is Modulated by a CRM1-Recognized Nuclear Export Signal that Is Active in Normal but not in Tumor Cells．Cancer Res．2005，65：7059-7064．
12．Liu X，Elojeimy S，EI-Zawahry AM，et al．Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer．Mol．Ther．2006，14：637-646．
13．Huo D．H．，Yi L．N．，Yang J．，Interaction with Ppi13 leads to the cytoplasmic localization of Apoptin in tumor cells，Biochem．Biophys．Res．Commun．2008 (372)：14-18．
14．Cheng Chih-Mei，Huang Shiao-ping，Chang Yung-Fu，et al．The viral death protein Apoptin interacts with Hippi，the protein interactor fo Huntingtin-interacting protein 1 ．Biochem．Biophys．Res．Commun．2003，305：359-364．
15．Lee Yen-Hsien，Cheng Chih-Mei，Chang Yung-Fu，et al．Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity．Biochem．Biophys．Res．Commun．2007，354：391-395．
16．Maddika S，Bay G，H，Kroczak T．J．，et al．Akt is transferred to the nucleus of cells treated with apoptin，and it participates in apoptin-induced cell death．Cell Prolif．2007，40：835-848．
17．Maddika S，Wiechec E，Ande SR，et al．Interaction with P13-kinase contributes to the cytotoxic activity of Apoptin．Oncogene．2007，1-6．
18．Maddika S，Panigrahi S，Wiechec E，et al．Unscheduled Akt-triggered activation of CDK2 as a key effector mechanism of apoptin's anticancer toxicity，Mol．Cell．Biol．2009，(29)：1235-1248．
19．Kaul，S．C．，Wadhwa，R．，Matsuda，et al．Mouse and human chromosomal assignments of mortalin，a novel member of the murine hsp70 family of proteins．1995．FEBS Lett．361，269-272．
20．Kaul S．C．，Deocaris C．C．，Wadhwa R．Three faces of mortalin：housekeeper，guardian and killer．Exp Gerontol 2007，42：263-274．
21．Wadhwa， R．， Takano， S．， Kaur， et al．Upregulation of mortalin/mthsp70/Grp75 contributes to human carcinogenesis．Int．J．Cancer．2006，118：2973-2980．
22．Renu Wadhwa，Syuichi Takano，Kazunari Taira et al．Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells．J Gene Med．2004； 6：439-444．
23．王阿慧，孙军，毕昊等．6His-VP3融合蛋白的表达、纯化及其捕获细胞中相关蛋白的初步探讨．医学分子生物学杂志，2007，4(2)：112-125．
24．Rie Ohtsuka，Yasunobu Abe，Tomomi Fujii，Masahiro Yamamoto，Junji Nishimura，Ryoichi Takayanagi．Koichiro Muta Mortalin is a novel mediator of erythropoietin signaling．European Journal of Haematology．2007； 79：114-125．
25．Wadhwa R，Takano S，Kaur K，Deocaris CC，Pereira-Smith OM，Reddel RR，Kaul SC．Upregulation of mortalin/mthsp70/Grp75 contributes to human carcinogenesis．Int J Cancer 2006； 118：2973-2980．
26．Renu Wadhwa，Syuichi Takano，Kazunari Taira，et al．Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells．J Gene Med．2004； 6：439-444．
27．Bhattacharyya，T．，Karnezis，A．N．，Murphy，S．P．，Hoang，T．，Freeman，B．C．，Phillips，B．，Morimoto，R．I．，．Cloning and subcellular localization of human mitochondrial hsp70．J．Biol．Chem．1995； 270：1705-1710．
28．Renu Wadhwa，Syuichi Takano，Kamaljit Kaur，et al．Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60．Biochem．J．2005； 391：185-190
29．Koehler，C．M．，New developments in mitochondrial assembly．Annu．Rev．Cell Dev．Biol．2004； 20：309-335．
30．Rehling，P．，Brandner，K．，Pfanner，N．．Mitochondrial import and the twin-pore translocase．Nat．Rev．Mol．Cell Biol．2004； 5：519-530．
31．Wiedemann，N．，Frazier，A．E．，Pfanner，N．，The protein import machinery of mitochondria．J．Biol．Chem．2004； 279：14473-14476．
32．Craig，E．A．，Kramer，J．，Kosic-Smithers，J．．SSC1，a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae，is essential for growth．Proc．Natl．Acad．Sci．USA．1987； 84，4156-4160．
33．Kawai，A．，Nishikawa Si，S．，Hirata，A．，Endo，T．．Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells．J．Cell Sci．2001；114，3565-3574．
34．Subbareddy Maddika，Evan P．Booy，Dina Johar．et al．Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway．J Cell Sci．2005，118：4485-4493．
1．Yuasa N，Taniguchi T，and Yoshida I，Isolation and characterization of an agent inducing anmia in chicks．Avian Diseases 1979，23：366．
2．Danen-van Oorschot Astrid A．A．M，Zhang Ying-Hui，Rutger Leliveld S，et al．Importance of Nuclear Localization of Apoptin for Tumor-specific Induction of Apoptosis．J．Biol．Chem．2003，278：27729-27736．
3．Maddika S，Wiechec E，Ande SR，et al．Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin．Oncogene．2007，1-6．
4．Poon Ivan K．H．，Oro Cristina，Dias M．M．．Apoptin Nuclear Accumulation Is Modulated by a CRM1-Recognized Nuclear Export Signal that Is Active in Normal but not in Tumor Cells．Cancer Res．2005，65：7059-7064．
5．Maddika S，Panigrahi S，Wiechec E，et al．Unscheduled Akt-triggered activation of CDK2 as a key effector mechanism of apoptin's anticancer toxicity，Mol．Cell．Biol．2009，(29)：1235-1248．
6．Danen-Van Oorschot A．A．A．M，Fischer D．F，Grimbergen J．M，et al．Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells．Proc．Natl．Acad．Sci．USA 1997，94：5843-5847
7．Leliveld SR，Dame RT，J．L．Rohn，et al．Apoptin's functional N-and C-termini independently bind DNA．FEBS Letters，2004，557：155-158．
8．Noteborn MHM，van der Eb AJ．Apoptin induces tumor-specific apoptosis：potentials for a novel anti-tumor therapy．Biogenic Amines，1999，15：73-91．
9．Pietersen AM，Noteborn HM，Apoptin，Adv Exp Med Biol，2000，465：153-161．
10．Zhang YH，Leliveld SR，Kooistra K，et al．Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion．Exp Cell Res，2003，289：36-46．
11．Zhang YH．Activation of Apoptin，a tumor-specific viral death effector．PhD thesis，Leiden University，Leiden，The Netherlands，2004．
12. Danen-van Oorschot AAM, Zhang YH, Erkeland SJ, et al. The effect of Bcl-2 on Apoptin in "normal" vs transformed human cells. Leukemia, 1999, 13: S75-77
13. Danen-van Oorschot AAM, Fisher DF, Grimbergen JM, et al. Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proc Natl Acad Sci USA, 1997,94:5843-5847
14. Maddika S., Evan P. Booy, Dina Johar. et al. Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. J Cell Sci. 2005,118:4485-4493.
15. Danen-van Oorschot AAM, van der Eb AJ, Noteborn MHM. The chicken anemia virus-derived protein Apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol, 2000,74: 7072-7078
16. van der Eb MM, Pietersen AM, Speetjens F, et al. Gene therapy with Apoptin induces regression of xenografted human hepatomas. Gene Therapy, 2002, 9:53-61.
17. Ferreira CG, Tolis C, Giaccone G, p53 and chemosensitivity, Ann. Oncol. 1999,10:1011-1021.
18. Zhuang SM, Landegent JE, Verschuerren CA, et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro. Leukemia 9 Suppl 1, S118-120.
19. Murphy AL. Apoptin : nuclear switch triggers cancer cell death. Gene Ther.1999 May;6(5): 713-4.
20. Maddika S, Bay G H, Kroczak T. J.,et al. Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin-induced cell death. Cell Prolif.2007,40: 835-848.
21. Lee Yen-Hsien, Cheng Chih-Mei, Chang Yung-Fu, et al. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity．Biochem．Biophys．Res．Commun．2007，354：391-395．
22．Sun G．J．，Tong X．，Dong Y．，et al．Identification of a protein interacting with apoptin from human leucocyte cDNA library by using yeast two-hybrid screening，ShengWu HuaXue Yu ShengWu WuLi XueBao (Shanghai) 2002，34：369-372．
23．Danen-van Oorschot A A．，Voskamp P．，Seneel M．C．，et al．Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing．Cell Death Different 2004，11：564573．
24．Cheng Chih-Mei，Huang Shiao-ping，Chang Yung-Fu，et al．The viral death protein Apoptin interacts with Hippi， the protein interactor fo Huntingtin-interacting protein 1．Biochem．Biophys．Res．Commun．2003，305：359-364．
25．Teodoro J G，Heilman D W．，Parker A E．，et al．The viral protein Apoptin associates with the anaphase-promoting complex to induce G2/M arrest and apoptosis in the absence of p53．Gene Dev．2004，18：1952-1957．
26．Liu X，Elojeimy S，El-Zawahry AM，et al．Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer．Mol．Ther．2006，14：637-646．
27．Huo D．H．，Yi L．N．，Yang J．，Interaction with Ppil3 leads to the cytoplasmic localization of Apoptin in tumor cells，Biochem．Biophys．Res．Commun．2008(372)：14-18．
28．Zhang YH，Leliveld SR，Kooistra K，et al．Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion．Exp Cell Res．2003，289：36-46．
29．Pietersen AM，van der Eb MM，Rademaker HJ，et al．Specific tumor-cell killing with adenovirus vectors containing the Apoptin gene．Gene Therapy．1999，6： 882-892
30．Marjolijn M van der Eb，Alexandra M Pietersen，et al．Gene therapy with apoptin induxes regression of xenografted human hepatomas．Cancer Gene Therapy．2002，9：53-61．
31．Guelen L，Paterson H，Gaken J，et al．TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells．Oncogene．2004，23：1153-1165．
32．Flinterman M．，Farzaneh F．，Habib N．，et al．Delivery of Therapeutic Proteins as Secretable TAT Fusion Products．Molecular Therapy．2008，1-9．
33．孙军，王宇哲，屈伸等．脱唾液酸糖蛋白受体介导的vp3基因靶向性治疗肝癌的实验研究．医学分子生物学杂志，2004，1：5-9．
34．Lian H．，Jin NY-Li X．，et al．Induction of an effective anti-tumor immune response and tumorregression by combined administration of IL-18 and Apoptin．Cancer Immunol Immunother 2006
35．Xiao Li，Ningyi Jin，Zhiqiang Mi，Hai Lian，Lili Sun，Xuemei Li and Hongling Zheng．Antitumor effects of a recombinant fowlpox virus expressing Apoptin in vivo and in vitro．Int．J．Cancer．2006，119：2948-2957
36．Olijslagers S．J．，Zhang Y．H．，Backendorf C．，et al．Additive Cytotoxic Effect of Apoptin and Chemotherapeutic Agents Paclitaxel and Etoposide on Human Tumour Cells．Basic＆Clinical Pharmacology＆Toxicology．100，127-131．
37．Sun J．，Yan Y．，Wang X．T．，et al．PTD4-apoptin protein therapy inhibits tumor growth in vivo．Int．J．Cancer．2009，124 (12)：2973-2981．
38．Los M．，Panigrahi S．，Rashedi I．et al．Apoptin，a tumor-selective killer．Biochim．Biophys．Acta (2009)，doi：10．10 1 6/j．bbamcr．2009．04．002
2．Danen-Van Oorschot A．A．A．M，Fischer D．F，Grimbergen J．M，et al．Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells．Proc．Natl．Acad．Sci．USA 1997，94：5843-5847
3．Subbareddy Maddika，Evan P．Booy，Dina Johar．et al．Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway．J Cell Sci．2005，118：4485-4493．
4．Rohn Jennifer L，Zhang Ying-Hui，Aalbers Remco I．J．M，et al．A Tumor-specific Kinase Activity Regulates the Viral Death Protein Apoptin．J．Biol．Chem．2002，227(50)：50820-50827．
5．Danen-van Oorschot Astrid A．A．M，Zhang Ying-Hui，Rutger Leliveld S，et al．Importance of Nuclear Localization of Apoptin for Tumor-specific Induction ofApoptosis．J．Biol．Chem．2003，278：27729-27736．
6．Sun G．J．，Tong X．，Dong Y．，et al．Identification of a protein interacting with apoptin from human leucocyte cDNA library by using yeast two-hybrid screening，ShengWu HuaXue Yu ShengWu WuLi XueBao (Shanghai) 2002，34：369-372．
7．Janssen K，Hofinann TG，Jans DA，et al．Apoptin is modified by SUMO conjugation and targeted to promyelocytic leukemia protein nuclear bodies．Oncogene．2006； 1-10．
8．Teodoro J G，Heilman D W．，Parker A E．，et al．The viral protein Apoptin associates with the anaphase-promoting complex to induce G2/M arrest and apoptosis in the absence of p53．Gene Dev．2004，18：1952-1957．
9．Danen-van Oorschot A A．，Voskamp P．，Seneel M．C．，et al．Human death effector domain-associated factor interacts with the viral apoptosis agonist Apoptin and exerts tumor-preferential cell killing．Cell Death Different 2004，11：564573．
10．L．Guelen，H．Paterson，J．Gaken，M．Meyers，F．Farzaneh，M．Tavassoli，TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells，Oncogene 2004，(23)：1153-1165．
11．Poon Ivan K．H．，Oro Cristina，Dias M．M．．Apoptin Nuclear Accumulation Is Modulated by a CRM1-Recognized Nuclear Export Signal that Is Active in Normal but not in Tumor Cells．Cancer Res．2005，65：7059-7064．
12．Liu X，Elojeimy S，EI-Zawahry AM，et al．Modulation of ceramide metabolism enhances viral protein apoptin's cytotoxicity in prostate cancer．Mol．Ther．2006，14：637-646．
13．Huo D．H．，Yi L．N．，Yang J．，Interaction with Ppi13 leads to the cytoplasmic localization of Apoptin in tumor cells，Biochem．Biophys．Res．Commun．2008 (372)：14-18．
14．Cheng Chih-Mei，Huang Shiao-ping，Chang Yung-Fu，et al．The viral death protein Apoptin interacts with Hippi，the protein interactor fo Huntingtin-interacting protein 1 ．Biochem．Biophys．Res．Commun．2003，305：359-364．
15．Lee Yen-Hsien，Cheng Chih-Mei，Chang Yung-Fu，et al．Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity．Biochem．Biophys．Res．Commun．2007，354：391-395．
16．Maddika S，Bay G，H，Kroczak T．J．，et al．Akt is transferred to the nucleus of cells treated with apoptin，and it participates in apoptin-induced cell death．Cell Prolif．2007，40：835-848．
17．Maddika S，Wiechec E，Ande SR，et al．Interaction with P13-kinase contributes to the cytotoxic activity of Apoptin．Oncogene．2007，1-6．
18．Maddika S，Panigrahi S，Wiechec E，et al．Unscheduled Akt-triggered activation of CDK2 as a key effector mechanism of apoptin's anticancer toxicity，Mol．Cell．Biol．2009，(29)：1235-1248．
19．Kaul，S．C．，Wadhwa，R．，Matsuda，et al．Mouse and human chromosomal assignments of mortalin，a novel member of the murine hsp70 family of proteins．1995．FEBS Lett．361，269-272．
20．Kaul S．C．，Deocaris C．C．，Wadhwa R．Three faces of mortalin：housekeeper，guardian and killer．Exp Gerontol 2007，42：263-274．
21．Wadhwa， R．， Takano， S．， Kaur， et al．Upregulation of mortalin/mthsp70/Grp75 contributes to human carcinogenesis．Int．J．Cancer．2006，118：2973-2980．
22．Renu Wadhwa，Syuichi Takano，Kazunari Taira et al．Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells．J Gene Med．2004； 6：439-444．
23．王阿慧，孙军，毕昊等．6His-VP3融合蛋白的表达、纯化及其捕获细胞中相关蛋白的初步探讨．医学分子生物学杂志，2007，4(2)：112-125．
24．Rie Ohtsuka，Yasunobu Abe，Tomomi Fujii，Masahiro Yamamoto，Junji Nishimura，Ryoichi Takayanagi．Koichiro Muta Mortalin is a novel mediator of erythropoietin signaling．European Journal of Haematology．2007； 79：114-125．
25．Wadhwa R，Takano S，Kaur K，Deocaris CC，Pereira-Smith OM，Reddel RR，Kaul SC．Upregulation of mortalin/mthsp70/Grp75 contributes to human carcinogenesis．Int J Cancer 2006； 118：2973-2980．
26．Renu Wadhwa，Syuichi Takano，Kazunari Taira，et al．Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells．J Gene Med．2004； 6：439-444．
27．Bhattacharyya，T．，Karnezis，A．N．，Murphy，S．P．，Hoang，T．，Freeman，B．C．，Phillips，B．，Morimoto，R．I．，．Cloning and subcellular localization of human mitochondrial hsp70．J．Biol．Chem．1995； 270：1705-1710．
28．Renu Wadhwa，Syuichi Takano，Kamaljit Kaur，et al．Identification and characterization of molecular interactions between mortalin/mtHsp70 and HSP60．Biochem．J．2005； 391：185-190
29．Koehler，C．M．，New developments in mitochondrial assembly．Annu．Rev．Cell Dev．Biol．2004； 20：309-335．
30．Rehling，P．，Brandner，K．，Pfanner，N．．Mitochondrial import and the twin-pore translocase．Nat．Rev．Mol．Cell Biol．2004； 5：519-530．
31．Wiedemann，N．，Frazier，A．E．，Pfanner，N．，The protein import machinery of mitochondria．J．Biol．Chem．2004； 279：14473-14476．
32．Craig，E．A．，Kramer，J．，Kosic-Smithers，J．．SSC1，a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae，is essential for growth．Proc．Natl．Acad．Sci．USA．1987； 84，4156-4160．
33．Kawai，A．，Nishikawa Si，S．，Hirata，A．，Endo，T．．Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells．J．Cell Sci．2001；114，3565-3574．
34．Subbareddy Maddika，Evan P．Booy，Dina Johar．et al．Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway．J Cell Sci．2005，118：4485-4493．
1．Yuasa N，Taniguchi T，and Yoshida I，Isolation and characterization of an agent inducing anmia in chicks．Avian Diseases 1979，23：366．
2．Danen-van Oorschot Astrid A．A．M，Zhang Ying-Hui，Rutger Leliveld S，et al．Importance of Nuclear Localization of Apoptin for Tumor-specific Induction of Apoptosis．J．Biol．Chem．2003，278：27729-27736．
3．Maddika S，Wiechec E，Ande SR，et al．Interaction with PI3-kinase contributes to the cytotoxic activity of Apoptin．Oncogene．2007，1-6．
4．Poon Ivan K．H．，Oro Cristina，Dias M．M．．Apoptin Nuclear Accumulation Is Modulated by a CRM1-Recognized Nuclear Export Signal that Is Active in Normal but not in Tumor Cells．Cancer Res．2005，65：7059-7064．
5．Maddika S，Panigrahi S，Wiechec E，et al．Unscheduled Akt-triggered activation of CDK2 as a key effector mechanism of apoptin's anticancer toxicity，Mol．Cell．Biol．2009，(29)：1235-1248．
6．Danen-Van Oorschot A．A．A．M，Fischer D．F，Grimbergen J．M，et al．Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells．Proc．Natl．Acad．Sci．USA 1997，94：5843-5847
7．Leliveld SR，Dame RT，J．L．Rohn，et al．Apoptin's functional N-and C-termini independently bind DNA．FEBS Letters，2004，557：155-158．
8．Noteborn MHM，van der Eb AJ．Apoptin induces tumor-specific apoptosis：potentials for a novel anti-tumor therapy．Biogenic Amines，1999，15：73-91．
9．Pietersen AM，Noteborn HM，Apoptin，Adv Exp Med Biol，2000，465：153-161．
10．Zhang YH，Leliveld SR，Kooistra K，et al．Recombinant Apoptin multimers kill tumor cells but are nontoxic and epitope-shielded in a normal-cell-specific fashion．Exp Cell Res，2003，289：36-46．
11．Zhang YH．Activation of Apoptin，a tumor-specific viral death effector．PhD thesis，Leiden University，Leiden，The Netherlands，2004．
12. Danen-van Oorschot AAM, Zhang YH, Erkeland SJ, et al. The effect of Bcl-2 on Apoptin in "normal" vs transformed human cells. Leukemia, 1999, 13: S75-77
13. Danen-van Oorschot AAM, Fisher DF, Grimbergen JM, et al. Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells. Proc Natl Acad Sci USA, 1997,94:5843-5847
14. Maddika S., Evan P. Booy, Dina Johar. et al. Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. J Cell Sci. 2005,118:4485-4493.
15. Danen-van Oorschot AAM, van der Eb AJ, Noteborn MHM. The chicken anemia virus-derived protein Apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol, 2000,74: 7072-7078
16. van der Eb MM, Pietersen AM, Speetjens F, et al. Gene therapy with Apoptin induces regression of xenografted human hepatomas. Gene Therapy, 2002, 9:53-61.
17. Ferreira CG, Tolis C, Giaccone G, p53 and chemosensitivity, Ann. Oncol. 1999,10:1011-1021.
18. Zhuang SM, Landegent JE, Verschuerren CA, et al. Apoptin, a protein encoded by chicken anemia virus, induces cell death in various human hematologic malignant cells in vitro. Leukemia 9 Suppl 1, S118-120.
19. Murphy AL. Apoptin : nuclear switch triggers cancer cell death. Gene Ther.1999 May;6(5): 713-4.
20. Maddika S, Bay G H, Kroczak T. J.,et al. Akt is transferred to the nucleus of cells treated with apoptin, and it participates in apoptin-induced cell death. Cell Prolif.2007,40: 835-848.
21. Lee Yen-Hsien, Cheng Chih-Mei, Chang Yung-Fu, et al. Apoptin T108 phosphorylation is not required for its tumor-specific nuclear localization but partially affects its apoptotic activity．Biochem．Biophys．Res．Commun．2007，354：391-395．
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