新型溶癌腺病毒抗白血病细胞作用及其机制的实验研究
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摘要
白血病是一种克隆性起源,多能干细胞或早期的祖细胞(髓系或淋系)突变而引起的造血系统恶性肿瘤。白血病细胞恶性增殖,进入血循环、并浸润全身各组织脏器;临床可见不同程度的贫血、出血、感染发热以及肝、脾、淋巴结肿大和骨骼疼痛等浸润症状。目前白血病已经成为我国最常见恶性肿瘤之一,发病率在各种肿瘤中占第六位,而且在年轻人恶性疾病中排名首位。尽管通过传统细胞毒药物联合化疗、维甲酸和砷剂诱导分化治疗、造血干细胞移植和支持治疗等综合方法,已经使急性白血病的完全缓解率达到70-90%,3年无病生存率达40-50%。但仍存在化疗相关毒性、化疗药物的非选择性以及对复发难治白血病的耐药性等诸多问题,促使研究者致力于研究各种新型的、更具靶向性的治疗手段,而基因病毒治疗正是其中新兴的一个研究方向。溶癌腺病毒在肿瘤细胞中复制后可以溶解细胞,并释放出子代病毒颗粒,进一步感染周边的肿瘤细胞,直至完全消灭肿瘤。本研究中,我们构建新型溶癌腺病毒,具有5型和35型嵌合纤毛或5型和11型嵌合纤毛,并且E1B-55kDa缺失,同时可以携带TRAIL或自噬相关基因BECN等效应基因,从而达到基因治疗与病毒治疗相结合的策略。此外,我们进一步设计和研究化疗、基因治疗和病毒治疗三种手段相结合杀伤白血病细胞这一新思路,以期为临床白血病治疗提供新的方法和策略。
本研究分三部分进行:
第一部分:溶癌腺病毒SG235-TRAIL抗白血病细胞的体内外研究;
第二部分:溶癌腺病毒SG235-TRAIL协同高三尖杉酯碱杀伤白血病细胞的研究;
第三部分:溶癌腺病毒SG511-BECN抗白血病细胞的体内外研究。
第一部分:溶癌腺病毒SG235-TRAIL抗白血病细胞的体内外研究
目的:构建新型溶癌腺病毒SG235,其具有5型和35型嵌合纤毛并且E1B-55kDa缺失,同时可以携带TRAIL表达框;通过体内外实验证实和比较SG235及SG235-TRAIL抗白血病细胞效应,并探讨其作用机制。从而为白血病靶向治疗提供基因治疗和病毒治疗相结合的手段。
方法:构建新型溶癌腺病毒SG235、SG235-增强绿色荧光蛋白(enhanced greenfluorescence protein,EGFP)以及SG235-TRAIL,通过荧光倒置显微镜、流式细胞术、病毒滴度检测和western blot法检测E1A的手段,来研究SG235对白血病细胞株、淋巴瘤细胞株、多发性骨髓瘤(multiple myeloma,MM)细胞株、患者原代白血病细胞和白血病细胞集落(leukemic colony forming unit,CFU-L)的感染能力及其复制活性;通过western blot法来明确SG235 E1B-55kDa是否缺失;通过四甲基偶氮唑蓝(3-(4,5-dimethylthiazol-2-yl)-2,5-diphemyltetra-zolium bromide,MTT)法检测各型腺病毒对白血病细胞株的细胞毒作用;通过末端脱氧核苷酸转移酶介导的脱氧尿嘧啶核苷三磷酸缺口末端标记(terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate in situ nick end labeling,TUNEL)法和磷脂酰丝氨酸(phosphatidylserine,PS)转位法来证实SG235、SG235-TRAIL诱导白血病细胞株和患者原代白血病细胞发生凋亡;通过罗丹明123(rhodamine 123,Rh123)染色法来检测SG235-TRAIL感染白血病细胞株后线粒体膜电位改变;采用western blot法来检测各型腺病毒感染白血病细胞株和患者原代白血病细胞后Caspase家族蛋白、B细胞白血病/淋巴瘤因子-2(B cell leukemia/lymphoma,Bcl-2)家族蛋白的改变,并进一步加用Caspase-3抑制剂(z-DEVD-FMK)和Caspase-8抑制剂(z-IETD-FMK)来观察其对SG235-TRAIL感染白血病细胞株后诱导凋亡水平的影响;以各型腺病毒作用Hela细胞,通过结晶紫染色法和western blot法检测Caspase家族蛋白来明确SG235和SG235-TRAIL是否具有杀伤实体瘤细胞株的能力;收集临床确诊白血病患者骨髓液,检测原代白血病细胞的CD46表达情况;通过CFU-L和粒细胞-巨噬细胞集落(giranulocyte-macrophage colony forming unit,GM-CFU)形成能力实验来证实各型腺病毒是否具有选择性杀伤白血病细胞的特性;通过酶标记免疫吸附测定(enzyme-labeled immunosorbent assay,ELISA)法来检测各型腺病毒感染细胞后上清液TRAIL水平;通过观察各型腺病毒对Kasumi-1细胞荷瘤小鼠瘤体生长的影响以及TUNEL染色法来研究各型腺病毒在体内杀伤白血病细胞的作用。
结果:(1)收集不同类型白血病患者原代白血病细胞122例,CD46总体阳性率为78.7%。表明以CD46作为受体的腺病毒载体SG235具备了感染白血病细胞的前提条件;(2)SG235体外实验中能够有效感染白血病、淋巴瘤和MM细胞株,并呈时间依赖性和剂量依赖性;(3)SG235本身能够诱导白血病细胞株发生凋亡,激活Caspase-9、Caspase-3和多聚腺苷二磷酸核糖聚合酶(poly-(adenosinediphosphate -ribose)polymerase,PARP),并具有剂量依赖性;(4)SG235-TRAIL和SG235都对白血病细胞株具有明显细胞毒作用,并具有剂量依赖性,而SG235-TRAIL较SG235细胞毒作用更显著;(5)SG235-TRAIL能够诱导白血病细胞株发生凋亡,且诱导凋亡效应较SG235更强;(6)SG235-TRAIL感染白血病细胞株中Caspase-8及其作用底物Bcl-2同源结构域3结构域凋亡诱导蛋白(Bcl-2homology 3 interacting domain death agonist,BID)激活,Bcl-2相关X蛋白(Bcl-2associated X protein,BAX)上调,线粒体膜电位下降,而Caspase-9和Caspase-3也可见激活;(7)SG235-TRAIL诱导白血病细胞株凋亡的效应可以被Caspase-3抑制剂与Caspase-8抑制剂部分抑制,其中Caspase-3抑制剂作用更为明显;(8)SG235与SG235-TRAIL感染后,Kasumi-1细胞Bcl-2与粒细胞白血病序列-1(myeloid cell leukemia sequence-1,MCL-1)表达下调,而Bcl-2相互作用细胞死亡介导因子(Bcl-2 interacting mediator of cell death,BIM)、Bcl-2同源拮抗物(Bcl-2homologous antagonist/killer,BAK)和Bcl-2样蛋白(Bcl-2 like protein,Bcl-XL)的表达未见明显改变;(9)SG235与SG235-TRAIL对实体瘤细胞株也有杀伤效应;(10)SG235以及携带TRAIL表达框的SG235-TRAIL能够有效感染原代白血病细胞,诱导其发生凋亡,激活Caspase-8、Caspase-3及PARP,并有效杀伤原代白血病细胞,而SG235-TRAIL作用较SG235更显著;(11)SG235与SG235-TRAIL能够有效感染CFU-L,并选择性抻制CFU-L形成,而SG235-TRAIL几乎完全抑制CFU-L的形成,此外,SG235与SG235-TRAIL不抑制或轻度抑制正常GM-CFU形成;(12)SG235-TRAIL感染白血病细胞株后表达TRAIL的水平要明显高于作为载体对照的Ad5/35-TRAIL组,SG235未见明显TRAIL表达。此外,SG235-TRAIL表达TRAIL水平呈时间依赖性;(13)SG235能显著抑制Kasumi-1细胞荷瘤小鼠瘤体生长,而ZD55几乎不影响瘤体生长;(14)SG235-TRAIL较SG235更能显著地抑制Kasumi-1细胞荷瘤小鼠瘤体生长,而且小鼠瘤体内凋亡细胞比例更高,而作为载体对照的Ad5/35-TRAIL没有表现出抗肿瘤效应。
第二部分:溶癌腺病毒SG235-TRAIL协同高三尖杉酯碱杀伤白血病细胞的研究
目的:根据化疗、基因治疗和病毒治疗三种手段相结合杀伤白血病细胞这一新思路,以SG235-TRAIL与HHT联合作用白血病细胞,并通过体外实验证实细胞毒作用并分析两者的协同效应,并进一步探讨其作用机制以及对正常细胞的杀伤效应,以期为临床白血病治疗提供新的方法和策略。
方法:通过MTT法和乳酸脱氢酶(lactate dehydrogenase,LDH)法检测SG235-TRAIL与HHT对白血病细胞株的细胞毒作用,并通过Chou-Talalay联合指数法进行分析;通过PS转位法检测SG235-TRAIL与HHT诱导白血病细胞株凋亡;采用western blot法来检测SG235-TRAIL与HHT作用白血病细胞株后E1A、TRAIL、Caspase家族蛋白和Bcl-2家族蛋白的改变;通过MTT法检测SG235-TRAIL联合HHT对正常骨髓细胞的杀伤效应;体外培养和鉴定间充质干细胞(mesenchymal stem cells,MSCs),并通过结晶紫染色法检测HHT和SG235-TRAIL对MSCs的细胞毒作用。
结果:(1)SG235-TRAIL与HHT在体外联用杀伤白血病细胞株,表现出不同程度的协同效应;(2)SG235-TRAIL与HHT能协同诱导白血病细胞株凋亡;(3)SG235-TRAIL对白血病细胞株的感染能力及其复制水平没有因为HHT的联用而发生改变;(4)SG235-TRAIL与HHT联用作用Kasumi-1细胞,其TRAIL蛋白表达水平高于SG235-TRAIL单用;(5)SG235-TRAIL以激活Caspase-8为主,HHT以激活Caspase-9为主。而SG235-TRAIL与HHT联用能较大程度激活Caspase-8及其作用底物BID、Caspase-9、Caspase-3和PARP,同时下调Bcl-2和Mcl-1的表达;(6)SG235-TRAIL与HHT联用,白血病细胞株Bcl-2与Mcl-1表达下调;(7)SG235-TRAIL联合HHT对正常骨髓细胞的杀伤效应较小,具有靶向性。
第三部分:溶癌腺病毒SG511-BECN抗白血病细胞的体内外研究
目的:构建新型溶癌腺病毒SG511-BECN,其具有5型和11型嵌合纤毛并且E1B-55kDa缺失,同时携带BECN表达框,通过体内外实验证实SG511-BECN通过诱导白血病细胞自噬性死亡而发挥抗白血病细胞效应。从而为白血病靶向治疗提供基因治疗和病毒治疗相结合的手段。
方法:构建新型溶癌腺病毒SG511、SG511-EGFP以及及SG511-BECN,通过荧光倒置显微镜、流式细胞术和western blot法检测beclin-1的手段,来研究SG511对白血病细胞株、淋巴瘤细胞株、MM细胞株、原代白血病细胞和CFU-L的感染能力及其复制活性;通过结晶紫染色法检测SG511-BECN对实体瘤细胞株的细胞毒作用,通过MTT法检测SG511-BECN对恶性血液病细胞株的细胞毒作用;通过吖啶橙染色法、透射电镜和微管相关蛋白1轻链3(light chain 3,LC3)-GFP-K562细胞荧光改变检测SG511-BECN诱导白血病细胞株自噬,并进一步通过western blot法检测SG511-BECN感染K562细胞后LC3和p62表达改变;通过观察各型腺病毒对K562细胞荷瘤小鼠瘤体生长的影响来研究各型腺病毒在体内杀伤白血病细胞的作用。
结果:(1)SG511体外实验中能够有效感染白血病、淋巴瘤和MM细胞株,并呈剂量依赖性和细胞特异性;(2)SG511体外实验中能够有效感染CFU-L;(3)SG511-BECN能有效感染K562细胞,并表达beclin-1蛋白;(4)SG511-BECN和SG511在体外都对实体瘤细胞株、恶性血液病细胞株具有明显细胞毒作用,而SG511-BECN较SG511细胞毒作用更显著;(5)SG511-BECN在体外能够诱导白血病细胞株发生自噬,并伴随LC3-Ⅱ的表达和p62的降解;(6)SG511-BECN和SG511显著地抑制K562细胞荷瘤小鼠瘤体生长,而SG511-BECN较SG511细胞毒作用更显著。
结论:SG235和SG511作为两种新型的具有嵌合纤毛的溶癌腺病毒,能有效感染白血病细胞株和原代白血病细胞,而携带了效应基因的SG235-TRAIL,不仅能发挥溶癌腺病毒的效应,还能诱导白血病细胞发生凋亡;同样,携带了效应基因的SG511-BECN,不仅能发挥溶癌腺病毒的效应,还能诱导白血病细胞发生自噬性死亡。此外,SG235-TRAIL联合HHT能够发挥协同抗白血病效应。
Leukemia is a series of clonal hematopoietic malignancies caused by mutations of stem cells or early progenitor cells(myeloid or lymphoid).Malignant proliferation of leukemic cells makes themselves into the circulation,infiltrating organs and tissues. Varying degrees of anemia,bleeding,fever can be observed as well as hepatomegaly, splenomegaly,bone pain and so on.So far,leukemia has become one of the most common malignancies in China with the incidence accounting sixth place,and leukemia has become the most common malignant disease among young people. Despite the integrated adoption of traditional combined chemotherapy,retinoic acid and arsenic trioxide-induced therapy,hematopoietic stem cell transplantation and supporting therapy has resulted in a complete remission rate of 70-90%and a 3-year disease-free survival rate of 40-50%,there are still chemotherapy-related toxicity, non-selectivity as well as drug resistance of relapsed or refractory leukemia.These questions prompt researchers to study for a variety of novel target therapies,while the gene-viral therapy is a important one among them.Oncolytic adenoviruses replicate in tumor cells and cause cell lysis,and then they release themselves to further infect surrounding tumor cells,until the total eradication of the tumor.In this study,we constructed novel oncolytic adenoviruses of ElB-55kDa deletion with chimeric Ad5/35 fibers or chimeric Ad5/ll fibers.These adenoviruses.can also carry TRAIL gene or BECN gene,so as to achieve the combination of gene therapy and viral therapy.In addition,we further designed chemo-gene-viral therapy in order to provide novel strategy for leukemia clinical practice.
This study is divided into three parts:
PartⅠ:in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cells;
PartⅡ:study of combination of SG235-TRAIL and homoharringtonine on leukemic cells;
PartⅢ:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on leukemic cells.
PartⅠ:in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cells
Objective:We constructed novel oncolytic adenovirus SG235 of E1B-55kDa deletion with a chimeric Ad5/35 fibers,and the adenovirus can also carry TRAIL cassette.We further confirm SG235 and SG235-TRAIL anti-leukemic cell effect by in vitro and in vivo experiments,and explore the mechanism.By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.
Methods:We first constructed the adenovirus SG235,SG235-EGFP and SG235-TRAIL,and study the infection and replication ability of SG235 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry,viral titer testing and western blot for E1A;confirm the ElB-55kDa deletion of SG235 via western blot; confirm cytotoxicity of different adenoviruses on leukemia cell lines via MTT;prove SG235 and SG235-TRAIL inducing apoptosis on leukemia cell lines and primary leukemia cells via TUNEL and PS translocation;detect the mitochondrial membrane potential change of leukemia cell lines after SG235-TRAIL infection via Rh123 staining;use western blot to detect Caspase family proteins,Bcl-2 family proteins after various types of adenoviruses infection in leukemia cell lines and primary leukemia cells;add Caspase-3 inhibitor(z-DEVD-FMK) and Caspase-8 inhibitor (Z-IETD-FMK) to observe the apoptosis levels induced by SG235-TRAIL;observe SG235 and SG235-TRAIL anti-tumor effect on Hela cell line via crystal violet staining and western blot for Caspase family;collect bone marrow cells from clinically diagnosed leukemia patients to detect the expression of CD46;to determine the selective cytotoxicity of adenoviruses on leukemia cells via CFU-L and GM-CFU forming test;detect the supernatant TRAIL levels after adenoviruses infection via ELISA;observe the various types of adenoviruses on tumor growth of Kasumi-1 xenograft-bearing SCID mice and observe the in vivo anti-leukemic effect of adenoviruses by TUNEL staining.
Results:(1) 122 cases of leukemia cells is collected,the overall CD46-positive rate is 78.7%,indicating that SG235 can effectively infect leukemia cells;(2) SG235 can effectively infect leukemia,lymphoma and MM cell lines in vitro with time-dependent and dose-dependent manners;(3) SG235 can induce apoptosis in leukemia cell lines and activate Caspase-9,Caspase-3 as well as PARP with a dose-dependent manner;(4) both SG235-TRAIL and SG235 have obvious cytotoxicity on leukemia cell lines with a dose-dependent manner,while SG235-TRAIL is superior to SG235;(5) SG235-TRAIL can induce apoptosis in leukemia cell lines and is superior to SG235;(6) the leukemia cell line infected by SG235-TRAIL has Caspase-8 and its substrate BID activated,BAX expression increased,mitochondrial membrane potential lost as well as Caspase-9 and Caspase-3 activated;(7) SG235-TRAIL-induced apoptosis in leukemia cell lines can be inhibited by Caspase-3 and Caspase-8 inhibitors,while Caspase-3 inhibitor is superior to Caspase-8 inhibitor;(8) Bcl-2 and Mcl-1 is down regulated while BIM,BAK and Bcl-XL has no obvious change in Kasumi-1 cell lines after SG235 or SG235-TRAIL infection;(9) SG235 and SG235-TRAIL retain their cytotoxic effect on tumor cell lines;(10) SG235 and SG235-TRAIL can effectively infect primary leukemia cells, induce apoptosis,activate Caspase-8,Caspase-3 and PARP as well as kill primary leukemia cells,while SG235-TRAIL is superior to SG235;(11) SG235 and SG235-TRAIL can efficiently infect CFU-L,and selectively inhibit the formation of CFU-L,while SG235-TRAIL almost completely inhibits the formation of CFU-L,in addition,SG235 and SG235-TRAIL can not or slightly inhibit normal formation of GM-CFU;(12) the level of TRAIL expression is significantly higher after SG235-TRAIL infection than Ad5/35-TRAIL,while SG235 do not express TRAIL obviously;(13) SG235 can significantly inhibit the tumor growth in Kasumi-1 xenograft-bearing SCID mice while ZD55 can't;(14) SG235-TRAIL is superior to SG235 in inhibiting tumor growth in Kasumi-1 xenograft-bearing SCID mice and inducing apoptosis in vivo,while Ad5/35-TRAIL shows no anti-tumor effect.
PartⅡ:study of combination of SG235-TRAIL and homoharringtonine on leukemic cells
Objective:According to the thinking of chemo-gene-viral therapy,we combine SG235-TRAIL and HHT on leukemia cells,and confirm the cytotoxicity in vitro as well as conduct synergy analysis,furthermore,we explore the mechanism,with a view to provide new methods and strategies for clinical practice.
Methods:We confirm the cytotoxic effect of combination of SG235-TRAIL and HHT via MTT and LDH test,then analyze the combination effect via Chou-Talalay method;SG235-TRAIL and HHT-induced apoptosis in leukemia cell lines is confirmed by PS translocation;E1A,TRAIL,Caspase family proteins and Bcl-2 family proteins is examined via western blot;SG235-TRAIL and HHT combination on normal bone marrow cells is tested via MTT;MSCs is cultured and identified,then HHT and SG235-TRAIL combination on MSCs is tested via crystal violet staining.
Results:(1) SG235-TRAIL and HHT show anti-leukemia cell lines effect in vitro with different levels of synergy;(2) SG235-TRAIL and HHT synergistically induce apoptosis in leukemia cell lines;(3) the infection and and replication ability of SG235-TRAIL are not affected by HHT;(4) TRAIL expression level of SG235-TRAIL and HHT combination is more signigicant than SG235-TRAIL alone; (5) SG235-TRAIL mainly activates Caspase-8 and HHT mainly activates Caspase-9, Caspase-8 and its substrate BID,Caspase-9,Caspase-3 and PARP are significantly activated by SG235-TRAIL and HHT combination in leukemia cell lines;(6) Bcl-2 and Mcl-1 are down regulated by SG235-TRAIL and HHT combination in leukemia cell lines;(7) SG235-TRAIL and HHT combination has little effect on normal bone marrow cells.
PartⅢ:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on Ieukemic cells
Objective:We constructed novel oncolytic adenovirus SG511 of ElB-55kDa deletion with chimeric Ad5/11 fibers,and the adenovirus can also carry BECN cassette.We further confirm SG511-BECN anti-leukemic cell effect by inducing autophagic cell death via in vitro and in vivo experiments,and explore the mechanism. By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.
Methods:We first constructed the adenovirus SG511,SG511-EGFP and SG511-BECN,and study the infection and replication ability of SG511 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry and western blot for beclin-1;observe SG511 -BECN anti-tumor effect on tumor cell lines via crystal violet staining,and SG511-BECN anti-tumor effect on hematological malignant cell lines via MTT;detect autophagy induced by SG511-BECN via acridine orange staining, transmission electron microscopy and LC3-GFP-K562 cell fluorescence change; further observe the expression of LC3 and p62 via western blot in SG511-BECN-infected K562 cell line;in vivo study of SG511-BECN on the tumor growth of K562 xenograft-bearing SCID mice.
Results:(1) SG511 can effectively infect leukemia,lymphoma and MM cell lines in vitro with a dose-dependent manner;(2) SG511 can efficiently infect CFU-L; (3) SG511-BECN can effectively infect K562 cell line,and express beclin-1;(4) SG511-BECN is superior to SG511 in killing tumor cell lines and malignant hematological cell lines;(5) SG511-BECN can efficiently induce autophagy in leukemia cell lines with LC3-Ⅱexpression and p62 degradation;(6) SG511-BECN is superior to SG511 in inhibiting tumor growth in K562 xenograft-bearing SCID mice.
Conclusion:SG235 and SG511,as two viruses with novel chimeric fiber,can efficiently infect leukemia cell lines and primary leukemia cells.With TRAIL cassette, SG235-TRAIL can cause tumor cell lysis as well as induce apoptosis;with beclin-1 cassette,SG511-BECN can cause tumor cell lysis as well as induce autophagic cell death.In addition,SG235-TRAIL and HHT can exert their anti-leukemia effect synergistically.
本研究分三部分进行:
第一部分:溶癌腺病毒SG235-TRAIL抗白血病细胞的体内外研究;
第二部分:溶癌腺病毒SG235-TRAIL协同高三尖杉酯碱杀伤白血病细胞的研究;
第三部分:溶癌腺病毒SG511-BECN抗白血病细胞的体内外研究。
第一部分:溶癌腺病毒SG235-TRAIL抗白血病细胞的体内外研究
目的:构建新型溶癌腺病毒SG235,其具有5型和35型嵌合纤毛并且E1B-55kDa缺失,同时可以携带TRAIL表达框;通过体内外实验证实和比较SG235及SG235-TRAIL抗白血病细胞效应,并探讨其作用机制。从而为白血病靶向治疗提供基因治疗和病毒治疗相结合的手段。
方法:构建新型溶癌腺病毒SG235、SG235-增强绿色荧光蛋白(enhanced greenfluorescence protein,EGFP)以及SG235-TRAIL,通过荧光倒置显微镜、流式细胞术、病毒滴度检测和western blot法检测E1A的手段,来研究SG235对白血病细胞株、淋巴瘤细胞株、多发性骨髓瘤(multiple myeloma,MM)细胞株、患者原代白血病细胞和白血病细胞集落(leukemic colony forming unit,CFU-L)的感染能力及其复制活性;通过western blot法来明确SG235 E1B-55kDa是否缺失;通过四甲基偶氮唑蓝(3-(4,5-dimethylthiazol-2-yl)-2,5-diphemyltetra-zolium bromide,MTT)法检测各型腺病毒对白血病细胞株的细胞毒作用;通过末端脱氧核苷酸转移酶介导的脱氧尿嘧啶核苷三磷酸缺口末端标记(terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate in situ nick end labeling,TUNEL)法和磷脂酰丝氨酸(phosphatidylserine,PS)转位法来证实SG235、SG235-TRAIL诱导白血病细胞株和患者原代白血病细胞发生凋亡;通过罗丹明123(rhodamine 123,Rh123)染色法来检测SG235-TRAIL感染白血病细胞株后线粒体膜电位改变;采用western blot法来检测各型腺病毒感染白血病细胞株和患者原代白血病细胞后Caspase家族蛋白、B细胞白血病/淋巴瘤因子-2(B cell leukemia/lymphoma,Bcl-2)家族蛋白的改变,并进一步加用Caspase-3抑制剂(z-DEVD-FMK)和Caspase-8抑制剂(z-IETD-FMK)来观察其对SG235-TRAIL感染白血病细胞株后诱导凋亡水平的影响;以各型腺病毒作用Hela细胞,通过结晶紫染色法和western blot法检测Caspase家族蛋白来明确SG235和SG235-TRAIL是否具有杀伤实体瘤细胞株的能力;收集临床确诊白血病患者骨髓液,检测原代白血病细胞的CD46表达情况;通过CFU-L和粒细胞-巨噬细胞集落(giranulocyte-macrophage colony forming unit,GM-CFU)形成能力实验来证实各型腺病毒是否具有选择性杀伤白血病细胞的特性;通过酶标记免疫吸附测定(enzyme-labeled immunosorbent assay,ELISA)法来检测各型腺病毒感染细胞后上清液TRAIL水平;通过观察各型腺病毒对Kasumi-1细胞荷瘤小鼠瘤体生长的影响以及TUNEL染色法来研究各型腺病毒在体内杀伤白血病细胞的作用。
结果:(1)收集不同类型白血病患者原代白血病细胞122例,CD46总体阳性率为78.7%。表明以CD46作为受体的腺病毒载体SG235具备了感染白血病细胞的前提条件;(2)SG235体外实验中能够有效感染白血病、淋巴瘤和MM细胞株,并呈时间依赖性和剂量依赖性;(3)SG235本身能够诱导白血病细胞株发生凋亡,激活Caspase-9、Caspase-3和多聚腺苷二磷酸核糖聚合酶(poly-(adenosinediphosphate -ribose)polymerase,PARP),并具有剂量依赖性;(4)SG235-TRAIL和SG235都对白血病细胞株具有明显细胞毒作用,并具有剂量依赖性,而SG235-TRAIL较SG235细胞毒作用更显著;(5)SG235-TRAIL能够诱导白血病细胞株发生凋亡,且诱导凋亡效应较SG235更强;(6)SG235-TRAIL感染白血病细胞株中Caspase-8及其作用底物Bcl-2同源结构域3结构域凋亡诱导蛋白(Bcl-2homology 3 interacting domain death agonist,BID)激活,Bcl-2相关X蛋白(Bcl-2associated X protein,BAX)上调,线粒体膜电位下降,而Caspase-9和Caspase-3也可见激活;(7)SG235-TRAIL诱导白血病细胞株凋亡的效应可以被Caspase-3抑制剂与Caspase-8抑制剂部分抑制,其中Caspase-3抑制剂作用更为明显;(8)SG235与SG235-TRAIL感染后,Kasumi-1细胞Bcl-2与粒细胞白血病序列-1(myeloid cell leukemia sequence-1,MCL-1)表达下调,而Bcl-2相互作用细胞死亡介导因子(Bcl-2 interacting mediator of cell death,BIM)、Bcl-2同源拮抗物(Bcl-2homologous antagonist/killer,BAK)和Bcl-2样蛋白(Bcl-2 like protein,Bcl-XL)的表达未见明显改变;(9)SG235与SG235-TRAIL对实体瘤细胞株也有杀伤效应;(10)SG235以及携带TRAIL表达框的SG235-TRAIL能够有效感染原代白血病细胞,诱导其发生凋亡,激活Caspase-8、Caspase-3及PARP,并有效杀伤原代白血病细胞,而SG235-TRAIL作用较SG235更显著;(11)SG235与SG235-TRAIL能够有效感染CFU-L,并选择性抻制CFU-L形成,而SG235-TRAIL几乎完全抑制CFU-L的形成,此外,SG235与SG235-TRAIL不抑制或轻度抑制正常GM-CFU形成;(12)SG235-TRAIL感染白血病细胞株后表达TRAIL的水平要明显高于作为载体对照的Ad5/35-TRAIL组,SG235未见明显TRAIL表达。此外,SG235-TRAIL表达TRAIL水平呈时间依赖性;(13)SG235能显著抑制Kasumi-1细胞荷瘤小鼠瘤体生长,而ZD55几乎不影响瘤体生长;(14)SG235-TRAIL较SG235更能显著地抑制Kasumi-1细胞荷瘤小鼠瘤体生长,而且小鼠瘤体内凋亡细胞比例更高,而作为载体对照的Ad5/35-TRAIL没有表现出抗肿瘤效应。
第二部分:溶癌腺病毒SG235-TRAIL协同高三尖杉酯碱杀伤白血病细胞的研究
目的:根据化疗、基因治疗和病毒治疗三种手段相结合杀伤白血病细胞这一新思路,以SG235-TRAIL与HHT联合作用白血病细胞,并通过体外实验证实细胞毒作用并分析两者的协同效应,并进一步探讨其作用机制以及对正常细胞的杀伤效应,以期为临床白血病治疗提供新的方法和策略。
方法:通过MTT法和乳酸脱氢酶(lactate dehydrogenase,LDH)法检测SG235-TRAIL与HHT对白血病细胞株的细胞毒作用,并通过Chou-Talalay联合指数法进行分析;通过PS转位法检测SG235-TRAIL与HHT诱导白血病细胞株凋亡;采用western blot法来检测SG235-TRAIL与HHT作用白血病细胞株后E1A、TRAIL、Caspase家族蛋白和Bcl-2家族蛋白的改变;通过MTT法检测SG235-TRAIL联合HHT对正常骨髓细胞的杀伤效应;体外培养和鉴定间充质干细胞(mesenchymal stem cells,MSCs),并通过结晶紫染色法检测HHT和SG235-TRAIL对MSCs的细胞毒作用。
结果:(1)SG235-TRAIL与HHT在体外联用杀伤白血病细胞株,表现出不同程度的协同效应;(2)SG235-TRAIL与HHT能协同诱导白血病细胞株凋亡;(3)SG235-TRAIL对白血病细胞株的感染能力及其复制水平没有因为HHT的联用而发生改变;(4)SG235-TRAIL与HHT联用作用Kasumi-1细胞,其TRAIL蛋白表达水平高于SG235-TRAIL单用;(5)SG235-TRAIL以激活Caspase-8为主,HHT以激活Caspase-9为主。而SG235-TRAIL与HHT联用能较大程度激活Caspase-8及其作用底物BID、Caspase-9、Caspase-3和PARP,同时下调Bcl-2和Mcl-1的表达;(6)SG235-TRAIL与HHT联用,白血病细胞株Bcl-2与Mcl-1表达下调;(7)SG235-TRAIL联合HHT对正常骨髓细胞的杀伤效应较小,具有靶向性。
第三部分:溶癌腺病毒SG511-BECN抗白血病细胞的体内外研究
目的:构建新型溶癌腺病毒SG511-BECN,其具有5型和11型嵌合纤毛并且E1B-55kDa缺失,同时携带BECN表达框,通过体内外实验证实SG511-BECN通过诱导白血病细胞自噬性死亡而发挥抗白血病细胞效应。从而为白血病靶向治疗提供基因治疗和病毒治疗相结合的手段。
方法:构建新型溶癌腺病毒SG511、SG511-EGFP以及及SG511-BECN,通过荧光倒置显微镜、流式细胞术和western blot法检测beclin-1的手段,来研究SG511对白血病细胞株、淋巴瘤细胞株、MM细胞株、原代白血病细胞和CFU-L的感染能力及其复制活性;通过结晶紫染色法检测SG511-BECN对实体瘤细胞株的细胞毒作用,通过MTT法检测SG511-BECN对恶性血液病细胞株的细胞毒作用;通过吖啶橙染色法、透射电镜和微管相关蛋白1轻链3(light chain 3,LC3)-GFP-K562细胞荧光改变检测SG511-BECN诱导白血病细胞株自噬,并进一步通过western blot法检测SG511-BECN感染K562细胞后LC3和p62表达改变;通过观察各型腺病毒对K562细胞荷瘤小鼠瘤体生长的影响来研究各型腺病毒在体内杀伤白血病细胞的作用。
结果:(1)SG511体外实验中能够有效感染白血病、淋巴瘤和MM细胞株,并呈剂量依赖性和细胞特异性;(2)SG511体外实验中能够有效感染CFU-L;(3)SG511-BECN能有效感染K562细胞,并表达beclin-1蛋白;(4)SG511-BECN和SG511在体外都对实体瘤细胞株、恶性血液病细胞株具有明显细胞毒作用,而SG511-BECN较SG511细胞毒作用更显著;(5)SG511-BECN在体外能够诱导白血病细胞株发生自噬,并伴随LC3-Ⅱ的表达和p62的降解;(6)SG511-BECN和SG511显著地抑制K562细胞荷瘤小鼠瘤体生长,而SG511-BECN较SG511细胞毒作用更显著。
结论:SG235和SG511作为两种新型的具有嵌合纤毛的溶癌腺病毒,能有效感染白血病细胞株和原代白血病细胞,而携带了效应基因的SG235-TRAIL,不仅能发挥溶癌腺病毒的效应,还能诱导白血病细胞发生凋亡;同样,携带了效应基因的SG511-BECN,不仅能发挥溶癌腺病毒的效应,还能诱导白血病细胞发生自噬性死亡。此外,SG235-TRAIL联合HHT能够发挥协同抗白血病效应。
Leukemia is a series of clonal hematopoietic malignancies caused by mutations of stem cells or early progenitor cells(myeloid or lymphoid).Malignant proliferation of leukemic cells makes themselves into the circulation,infiltrating organs and tissues. Varying degrees of anemia,bleeding,fever can be observed as well as hepatomegaly, splenomegaly,bone pain and so on.So far,leukemia has become one of the most common malignancies in China with the incidence accounting sixth place,and leukemia has become the most common malignant disease among young people. Despite the integrated adoption of traditional combined chemotherapy,retinoic acid and arsenic trioxide-induced therapy,hematopoietic stem cell transplantation and supporting therapy has resulted in a complete remission rate of 70-90%and a 3-year disease-free survival rate of 40-50%,there are still chemotherapy-related toxicity, non-selectivity as well as drug resistance of relapsed or refractory leukemia.These questions prompt researchers to study for a variety of novel target therapies,while the gene-viral therapy is a important one among them.Oncolytic adenoviruses replicate in tumor cells and cause cell lysis,and then they release themselves to further infect surrounding tumor cells,until the total eradication of the tumor.In this study,we constructed novel oncolytic adenoviruses of ElB-55kDa deletion with chimeric Ad5/35 fibers or chimeric Ad5/ll fibers.These adenoviruses.can also carry TRAIL gene or BECN gene,so as to achieve the combination of gene therapy and viral therapy.In addition,we further designed chemo-gene-viral therapy in order to provide novel strategy for leukemia clinical practice.
This study is divided into three parts:
PartⅠ:in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cells;
PartⅡ:study of combination of SG235-TRAIL and homoharringtonine on leukemic cells;
PartⅢ:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on leukemic cells.
PartⅠ:in vitro and in vivo study of oncolytic adenovirus SG235-TRAIL on leukemic cells
Objective:We constructed novel oncolytic adenovirus SG235 of E1B-55kDa deletion with a chimeric Ad5/35 fibers,and the adenovirus can also carry TRAIL cassette.We further confirm SG235 and SG235-TRAIL anti-leukemic cell effect by in vitro and in vivo experiments,and explore the mechanism.By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.
Methods:We first constructed the adenovirus SG235,SG235-EGFP and SG235-TRAIL,and study the infection and replication ability of SG235 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry,viral titer testing and western blot for E1A;confirm the ElB-55kDa deletion of SG235 via western blot; confirm cytotoxicity of different adenoviruses on leukemia cell lines via MTT;prove SG235 and SG235-TRAIL inducing apoptosis on leukemia cell lines and primary leukemia cells via TUNEL and PS translocation;detect the mitochondrial membrane potential change of leukemia cell lines after SG235-TRAIL infection via Rh123 staining;use western blot to detect Caspase family proteins,Bcl-2 family proteins after various types of adenoviruses infection in leukemia cell lines and primary leukemia cells;add Caspase-3 inhibitor(z-DEVD-FMK) and Caspase-8 inhibitor (Z-IETD-FMK) to observe the apoptosis levels induced by SG235-TRAIL;observe SG235 and SG235-TRAIL anti-tumor effect on Hela cell line via crystal violet staining and western blot for Caspase family;collect bone marrow cells from clinically diagnosed leukemia patients to detect the expression of CD46;to determine the selective cytotoxicity of adenoviruses on leukemia cells via CFU-L and GM-CFU forming test;detect the supernatant TRAIL levels after adenoviruses infection via ELISA;observe the various types of adenoviruses on tumor growth of Kasumi-1 xenograft-bearing SCID mice and observe the in vivo anti-leukemic effect of adenoviruses by TUNEL staining.
Results:(1) 122 cases of leukemia cells is collected,the overall CD46-positive rate is 78.7%,indicating that SG235 can effectively infect leukemia cells;(2) SG235 can effectively infect leukemia,lymphoma and MM cell lines in vitro with time-dependent and dose-dependent manners;(3) SG235 can induce apoptosis in leukemia cell lines and activate Caspase-9,Caspase-3 as well as PARP with a dose-dependent manner;(4) both SG235-TRAIL and SG235 have obvious cytotoxicity on leukemia cell lines with a dose-dependent manner,while SG235-TRAIL is superior to SG235;(5) SG235-TRAIL can induce apoptosis in leukemia cell lines and is superior to SG235;(6) the leukemia cell line infected by SG235-TRAIL has Caspase-8 and its substrate BID activated,BAX expression increased,mitochondrial membrane potential lost as well as Caspase-9 and Caspase-3 activated;(7) SG235-TRAIL-induced apoptosis in leukemia cell lines can be inhibited by Caspase-3 and Caspase-8 inhibitors,while Caspase-3 inhibitor is superior to Caspase-8 inhibitor;(8) Bcl-2 and Mcl-1 is down regulated while BIM,BAK and Bcl-XL has no obvious change in Kasumi-1 cell lines after SG235 or SG235-TRAIL infection;(9) SG235 and SG235-TRAIL retain their cytotoxic effect on tumor cell lines;(10) SG235 and SG235-TRAIL can effectively infect primary leukemia cells, induce apoptosis,activate Caspase-8,Caspase-3 and PARP as well as kill primary leukemia cells,while SG235-TRAIL is superior to SG235;(11) SG235 and SG235-TRAIL can efficiently infect CFU-L,and selectively inhibit the formation of CFU-L,while SG235-TRAIL almost completely inhibits the formation of CFU-L,in addition,SG235 and SG235-TRAIL can not or slightly inhibit normal formation of GM-CFU;(12) the level of TRAIL expression is significantly higher after SG235-TRAIL infection than Ad5/35-TRAIL,while SG235 do not express TRAIL obviously;(13) SG235 can significantly inhibit the tumor growth in Kasumi-1 xenograft-bearing SCID mice while ZD55 can't;(14) SG235-TRAIL is superior to SG235 in inhibiting tumor growth in Kasumi-1 xenograft-bearing SCID mice and inducing apoptosis in vivo,while Ad5/35-TRAIL shows no anti-tumor effect.
PartⅡ:study of combination of SG235-TRAIL and homoharringtonine on leukemic cells
Objective:According to the thinking of chemo-gene-viral therapy,we combine SG235-TRAIL and HHT on leukemia cells,and confirm the cytotoxicity in vitro as well as conduct synergy analysis,furthermore,we explore the mechanism,with a view to provide new methods and strategies for clinical practice.
Methods:We confirm the cytotoxic effect of combination of SG235-TRAIL and HHT via MTT and LDH test,then analyze the combination effect via Chou-Talalay method;SG235-TRAIL and HHT-induced apoptosis in leukemia cell lines is confirmed by PS translocation;E1A,TRAIL,Caspase family proteins and Bcl-2 family proteins is examined via western blot;SG235-TRAIL and HHT combination on normal bone marrow cells is tested via MTT;MSCs is cultured and identified,then HHT and SG235-TRAIL combination on MSCs is tested via crystal violet staining.
Results:(1) SG235-TRAIL and HHT show anti-leukemia cell lines effect in vitro with different levels of synergy;(2) SG235-TRAIL and HHT synergistically induce apoptosis in leukemia cell lines;(3) the infection and and replication ability of SG235-TRAIL are not affected by HHT;(4) TRAIL expression level of SG235-TRAIL and HHT combination is more signigicant than SG235-TRAIL alone; (5) SG235-TRAIL mainly activates Caspase-8 and HHT mainly activates Caspase-9, Caspase-8 and its substrate BID,Caspase-9,Caspase-3 and PARP are significantly activated by SG235-TRAIL and HHT combination in leukemia cell lines;(6) Bcl-2 and Mcl-1 are down regulated by SG235-TRAIL and HHT combination in leukemia cell lines;(7) SG235-TRAIL and HHT combination has little effect on normal bone marrow cells.
PartⅢ:in vitro and in vivo study of oncolytic adenovirus SG511-BECN on Ieukemic cells
Objective:We constructed novel oncolytic adenovirus SG511 of ElB-55kDa deletion with chimeric Ad5/11 fibers,and the adenovirus can also carry BECN cassette.We further confirm SG511-BECN anti-leukemic cell effect by inducing autophagic cell death via in vitro and in vivo experiments,and explore the mechanism. By this study,we try to provide target therapy for leukemia with the strategy of combined gene-viral therapy.
Methods:We first constructed the adenovirus SG511,SG511-EGFP and SG511-BECN,and study the infection and replication ability of SG511 on leukemia cell lines,lymphoma cell lines,MM cell lines,primary leukemia cells as well as CFU-L via inverted fluorescence microscope,flow cytometry and western blot for beclin-1;observe SG511 -BECN anti-tumor effect on tumor cell lines via crystal violet staining,and SG511-BECN anti-tumor effect on hematological malignant cell lines via MTT;detect autophagy induced by SG511-BECN via acridine orange staining, transmission electron microscopy and LC3-GFP-K562 cell fluorescence change; further observe the expression of LC3 and p62 via western blot in SG511-BECN-infected K562 cell line;in vivo study of SG511-BECN on the tumor growth of K562 xenograft-bearing SCID mice.
Results:(1) SG511 can effectively infect leukemia,lymphoma and MM cell lines in vitro with a dose-dependent manner;(2) SG511 can efficiently infect CFU-L; (3) SG511-BECN can effectively infect K562 cell line,and express beclin-1;(4) SG511-BECN is superior to SG511 in killing tumor cell lines and malignant hematological cell lines;(5) SG511-BECN can efficiently induce autophagy in leukemia cell lines with LC3-Ⅱexpression and p62 degradation;(6) SG511-BECN is superior to SG511 in inhibiting tumor growth in K562 xenograft-bearing SCID mice.
Conclusion:SG235 and SG511,as two viruses with novel chimeric fiber,can efficiently infect leukemia cell lines and primary leukemia cells.With TRAIL cassette, SG235-TRAIL can cause tumor cell lysis as well as induce apoptosis;with beclin-1 cassette,SG511-BECN can cause tumor cell lysis as well as induce autophagic cell death.In addition,SG235-TRAIL and HHT can exert their anti-leukemia effect synergistically.
引文
1.Bradstock KF,Matthews JP,Lowenthal RM,et al.Australasian Leukaemia and Lymphoma Group.A randomized trial of high-versus conventional-dose cytarabine in consolidation chemotherapy for adult de novo acute myeloid leukemia in first remission after induction therapy containing high-dose cytarabine.Blood 2005; 105(2):481-488.
2.Blaese RM,Culver KW,Miller AD,et al.T lymphocyte-directed gene therapy for ADA-SCID:initial trial results after 4 years.Science 1995; 270(5249):475-480.
3.Honjo Y,Inohara H,Akahani S,et al.Expression of cytoplasmic galectin-3 as a prognostic marker in tongue carcinoma.Clin Cancer Res 2000; 6(12):4635-4640.
4.Armstrong AC,Eaton D,Ewing JC.Cellular vaccine therapy for cancer.Expert Rev Vaccines 2002; 1(3):303-316.
5.Liu K.Breakthroughs in cancer gene therapy.Semin Oncol Nurs 2003; 19(3):217-226.
6.Nettelbeck DM.Virotherapeutics:conditionally replicative adenoviruses for viral oncolysis.Anticancer Drugs 2003; 14(8):577-584.
7.Chu RL,Post DE,Khuri FR,et al.Use of replicating oncolytic adenoviruses in combination therapy for cancer.Clin Cancer Res 2004; 10(16):5299-5312.
8.Pin RH,Reinblatt M,Fong Y.Utilizing alpha-fetoprotein expression to enhance oncolytic viral therapy in hepatocellular carcinoma.Ann Surg 2004; 240(4):659-665.
9.Kurihara T,Brough DE,Kovesdi I,et al.Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen.J Clin Invest 2000; 106(6):763-771.
10.Jakubczak JL,Ryan P,Gorziglia M,et al.An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors:dependence on E1A,the E2F-1 promoter,and viral replication for selectivity and efficacy.Cancer Res 2003,63(7):1490-1499.
11.Bischoff JR,Kirn DH,Williams A,et al.An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.Science 1996; 274(5286):342-343.
12.Geoerger B,Grill J,Opolon P,et al.Potentiation of radiation therapy by the oncolytic adenovirus dl 1520 (ONYX-015)in human malignant glioma xenografts.Br J Cancer 2003; 89(3):577-584.
13.Rogulski KR,Freytag SO,Zhang K,et al.In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy.Cancer Res 2000; 60(5):1193-1196.
14.Chen Y,DeWeese T,Dilley J,et al.CV706,a prostate cancer-specific adenovirus variant,in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity.Cancer Res 2001; 61(14):5453-5460.
15.Khuri FR,Nemunaitis J,Ganly I,et al.A controlled trial of intratumoral ONYX-015,a selectively-replicating adenovirus,in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer.Nat Med 2000;6(8):879-885.
16.Pei Z,Chu L,Zou W,et al.An oncolytic adenoviral vector of Smac increases antitumor activity of TRAIL against HCC in human cells and in mice.Hepatology 2004; 39(5):1371-1381.
17.Zhang Z,Zou W,Luo C,et al.An armed oncolytic adenovirus system,ZD55-gene,demonstrating potent antitumoral efficacy.Cell Res 2003; 13(6):481-489.
18.Mentel R,Dopping G,Wegner U,et al.Adenovirus-receptor interaction with human lymphocytes.J Med Virol 1997; 51(3):252-257.
19.Rebel VI,Hartnett S,Denham J,et al.Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells.Stem cells 2000;18(3):176-182.
20.Wang Y,Xue S,Hallden G,et al.Virus-associated RNA I-deleted adenovirus,a potential oncolytic agent targeting EBV-associated tumors.Cancer Res 2005;65(4):1523-1531.
21.Medina D,Sheay W,Goodell L,et al.Adenovirus mediated cytotoxicity of chronic lymphocytic leukemia cells.Blood 1999; 94(10):3499-3508.
22.Yotnda P,Onishi H,Heslop HE,et al.Efficient infection of primitive hematopoietic stem cells by modified adenovirus.Gene Ther 2001; 8(12):930-937.
23.Wuchter C,Krappmann D,Cai Z,et al.In vitro susceptibility to TRAIL-induced apoptosis of acute leukemia cells in the context of TRAIL receptor gene expression and constitutive NF-kappa B activity.Leukemia 2001; 15(6):921-928.
24.Nieda M,Nicol A,Koezuka Y,et al.TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukemia cells.Blood 2001; 97(7):2067-2074.
25.Plasilova M,Zivny J,Jelinek J,et al.TRAIL (Apo2L)suppresses growth of primary human leukemia and myelodysplasia progenitors.Leukemia 2002; 16(1):67-73.
26.Pan Q,Liu B,Liu J,et al.Synergistic induction of tumor cell death by combining cisplatin with an oncolytic adenovirus carrying TRAIL.Mol Cell Biochem 2007;304(1-2):315-323.
27.Zhang Y,Qin X,Zhang Y,et al.Combination of ZD55-MnSOD therapy with 5-FU enhances antitumor efficacy in colorectal cancer.J Cancer Res Clin Oncol 2008; 134(2):219-226.
28.Baaske DM,Heinstein P.Cytotoxicity and cell cycle specificity of homoharringtonine.Antimicrob Agents Chemother 1977; 12(2):298-300.
29.Wilkoff LJ,Dulmadge DA,Laster WR Jr,et al.Effect of homoharringtonine on the viability of murine leukemia P388 cells resistant to either adriamycin,vincristine,or 1-beta-D-arabinofuranosylcytosine.Cancer Chemother Pharmacol 1989; 23(3):145-150.
30.Fanucchi MP,Kong XR,Chou TC.Hexamethylene bisacetamide (HMBA)does not enhance the cytotoxic effects of adriamycin (ADR),1-beta-D-arabinofuranosylcytosine (ARA-C)and homoharringtonine (HT)in HL-60 cells [abstract 1492].Proc Am Assoc Cancer Res 1986; 27:376.
31.Liang C,Feng P,Ku B,et al.Autophagic and tumour suppressor activity of a novel Beclinl-binding protein UVRAG Nat Cell Biol 2006; 8(7):688-699.
32.Miracco C,Cosci E,Oliveri G,et al.Protein and mRNA expression of autophagy gene Beclin 1 in human brain tumors.Int J Oncol 2007; 30(2):429-436.
33.Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature 1999; 402(6762):672-676.
34.Qian W,Liu J,Tong Y,et al.Enhanced antitumor activity by a selective conditionally replicating adenovirus combining with MDA-7/interleukin-24 for B-lymphoblastic leukemia via induction of apoptosis.Leukemia 2008; 22(2):361-369.
35.Guo BC,Xu YH.Bcl-2 over-expression and activation of protein kinase C suppress the trail-induced apoptosis in Jurkat T cells.Cell Res 2001; 11(2):101-106.
36.Lamothe B,Aggarwal BB.Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL)through suppression of caspases-8,7,and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60.J Interferon Cytokine Res 2002; 22(2):269-279.
37.Wang J,Gu JF,Yang SY,et al.Security of dual cancer-specific targeting vector and its cytotoxic effect when harbored.Ai Zheng 2006; 25(4):385-392.
38.Shayakhmetov DM,Eberly AM,Li ZY,et al.Deletion of penton RGD motifs affects the efficiency of both the internalization and the endosome escape of viral particles containing adenovirus serotype 5 or 35 fiber knobs.J Virol 2005; 79(2):1053-1061.
39.Aye MT,Niho Y,Till JE,et al.Study of leukemic cell population in culture.Blood 1974; 44(2):205-219.
40.Kirn D,Martuza RL,Zwiebel J.Replication-selective virotherapy for cancenbiological principles,risk management and future directions.Nat Med 2001; 7(7):781-787.
41.Hawkins LK,Lemoine NR,Kirn D.Oncolytic biotherapy:a novel therapeutic platform.Lancet Oncol 2002; 3(1):17-26.
42.Martuza RL,Malick A,Markert JM,et al.Experimental therapy of human glioma by means of a genetically engineered virus mutant.Science 1991; 252(5007):854-856.
43.Everts B,van der Poel HG.Replication-selective oncolytic viruses in the treatment of cancer.Cancer Gene Ther 2005; 12(2):141-161.
44.Peng KW,Ahmann GJ,Pham L,et al.Systemic therapy of myeloma xenografts by an attenuated measles virus.Blood 2001; 98(7):2002-2007.
45.Ong HT,Timm MM,Greipp PR,et al.Oncolytic measles virus targets high CD46 expression on multiple myeloma cells.Exp Hematol 2006; 34(6):713-720.
46.Peng KW,Donovan KA,Schneider U,et al.Oncolytic measles viruses displaying a single-chain antibody against CD38,a myeloma cell marker.Blood 2003; 101(7):2557-2562.
47.Grote D,Russell SJ,Cornu TI,et al.Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice.Blood 2001; 97(12):3746-3754.
48.Kunzi V,Oberholzer PA,Heinzerling L,et al.Recombinant measles virus induces cytolysis of cutaneous T-cell lymphoma in vitro and in vivo.J Invest Dermatol 2006; 126(11):2525-2532.
49.Heinzerling L,Kunzi V,Oberholzer PA,et al.Oncolytic measles virus in cutaneous T-cell lymphomas mounts antitumor immune responses in vivo and targets interferon-resistant tumor cells.Blood 2005; 106(7):2287-2294.
50.Hara T,Kojima A,Fukuda H,et al.Levels of complement regulatory proteins,CD35 (CR1),CD46 (MCP)and CD55 (DAF)in human haematological malignancies.Br J Haematol 1992; 82(2):368-373.
51.Hengartner MO.The biochemistry of apoptosis.Nature 2000; 407(6805):770-776.
52.Green D,Reed JC.Mitochondria and apoptosis.Science 1998; 281(5381):1309-1312.
53.Krammer PH.CD95's deadly mission in the immune system.Nature 2000;407(6805):789-795.
54.Woo M,Hakem R,Soengas MS,et al.Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.Genes Dev 1998; 12(6):806-819.
55.Burkle A.Physiology and pathophysiology of poly (ADP-ribosyl)ation.Bioessays 2001; 23(9):795-806.
56.Tong WM,Cortes U,Wang ZQ.Poly (ADP-ribose)polymerase:a guardian angel protecting the genome and suppressing tumorigenesis.Biochim Biophys Acta 2001; 1552(1):27-37.
57.Cheng J,Hylander BL,Baer MR,et al.Multiple mechanisms underlie resistance of leukemia cells to Apo2 Ligand/TRAIL.Mol Cancer Ther 2006; 5(7):1844-1853.
58.Riccioni R,Pasquini L,Mariani G,et al.TRAIL decoy receptors mediate resistance of acute myeloid leukemia cells to TRAIL.Haematologica 2005; 90(5):612-624.
59.Wu CH,Kao CH,Safa AR.TRAIL recombinant adenovirus triggers robust apoptosis in multidrug-resistant HL-60/Vinc cells preferentially through death receptor DR5.Hum Gene Ther 2008; 19(7):731-743.
60.Kanerva A,Wang M,Bauerschmitz GJ,et al.Gene transfer to ovarian cancer versus normal tissues with fiber-modified adenoviruses.Mol Ther 2002; 5(6):695-704.
61.Hemminki A,Wang M,Desmond RA,et al.Serum and ascites neutralizing antibodies in ovarian cancer patients treated with intraperitoneal adenoviral gene therapy.Hum Gene Ther 2002; 13(12):1505-1514.
62.Bharti AC,Takada Y,Aggarwal BB.PARP cleavage and caspase activity to assess chemosensitivity.Methods Mol Med 2005; 111:69-78.
63.Rahmani M,Davis EM,Bauer C,et al.Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation.J Biol Chem 2005; 280(42):35217-35227.
64.Moulding DA,Giles RV,Spiller DG,et al.Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells.Blood 2000; 96(5):1756-1763.
65.Zhou P,Qian L,Bieszczad CK,et al.Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage.Blood 1998; 92(9):3226-3239.
66.Teicher BA.Assays for in vitro and in vivo synergy.Methods Mol Med 2003; 85:297-321.
67.Chou TC,Motzer RJ,Tong Y,et al.Computerized quantitation of synergism and antagonism of taxol,topotecan,and cisplatin against human teratocarcinoma cell growth:a rational approach to clinical protocol design.J Natl Cancer Inst 1994;86(2):1517-1524.
68.Chou,TC.Drug combinations:from laboratory to practice.J Lab Clin Med 1998;132(1):6-8.
69.Jin J,Jiang DZ,Mai WY,et al.Homoharringtonine in combination with cytarabine and aclarubicin resulted in high complete remission rate after first induction therapy in patients with de novo acute myeloid leukemia.Leukemia 2006; 20(8):1361-1367.
70.Tallarida,RJ.Drug synergism:its detection and applications.J Pharmacol Exp Ther 2001; 298(3):865-872.
71.White DB,Slocum HK,Brun Y et al.A new nonlinear mixture response surface paradigm for the study of synergisms three drug example.Curr Drug Metab 2003:4(5); 399-409.
72.Levasseur LM,Greco WR,RustumYM,et al.Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells.Cancer Chemother Pharmacol 1997;40(6):495-505.
73.Greco WR,Bravo G,Parsons JC.The search for synergy:a critical review from a response surface perspective.Pharmacol Rev 1995;47(2):331-385.
74.Jordan CT,Upchurch D,Szilvassy SJ,et al.The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.Leukemia 2000;14(10):1777-1784.
75.麦文渊,林茂芳。高三尖杉酯碱诱导K562细胞凋亡的实验研究。中华血液学杂志,1998;19(7):365-376.
76.Yinjun L,Jie J,Weilai X,et al.Homoharringtonine mediates myeloid cell apoptosis via upregulation of pro-apoptotic bax and inducing caspase-3-mediated cleavage of poly(ADP-ribose) polymerase(PARP).Am J Hematol.2004 Jul;76(3):199-204.
77.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science 1999;284(5411):143-147.
78.Horwitz EE.Clarification of the nomenclature for MSC:the international society for cellular therapy position statement.Cytotherapy 2005;7(5):393-395.
79.Calvi LM,Adams GB,Weibrecht KW,et al.Osteoblastic cells regulate the haematopoietic stem cell niche.Nature 2003;425(6960):841-846.
80.Zhang J,Niu C,Ye L,et al.Identification of the haematopoietic stem cell niche and control of the niche size.Nature 2003;425(6960):836-841.
81.Dazzi F,Ramasamy R,Glennie S,et al.The role of mesenchymal stem cells in haemopoiesis.Blood Rev 2005;20(3):161-171.
82.Li J,Law HK,Lau YL,et al.Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents.Br J Haematol 2004;127(3):326-334.
83.Ben-Ishay Z,Barak V.Bone marrow stromal dysfunction in mice administered cytosine arabinoside.Eur J Haematol 2001;66(4):230-237.
84.Liang XH,Kleeman LK,Jiang HH,et al.Protection against fatal Sindbis virus encephalitis by beclin,a novel Bcl-2-interacting protein.J Virol 1998;72(11): 8586-8596.
85.Scarlatti F,Bauvy C,Ventruti A,et al.Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin-1.J Biol Chem 2004;279(18):18384-18391.
1.Levine B.Cell biology:autophagy and cancer.Nature 2007;446(7137):745-747.
2.Amaravadi RK,Thompson CB.The roles of therapy-induced autophagy and necrosis in cancer treatment.Clin Cancer Res 2007;13(24):7271-7279.
3.Mathew R,Karantza-Wadsworth V,White E.Role of autophagy in cancer.Nat Rev Cancer 2007;7(12):961-967.
4.Kroemer G,Jaattela M.Lysosomes and autophagy in cell death control.Nat Rev Cancer 2005;5(11):886-897.
5.Maiuri MC,Zalckvar E,Kimchi A,et al.Self-eating and self-killing:cross talk between autophagy and apoptosis.Nat Rev Mol Cell Biol 2007;8(9):741-752.
6.Boya P,Gonzalez-Polo RA,Casares N,et al.Inhibition of macroautophagy triggers apoptosis.Mol Cell Biol 2005;25(3):1025-1040.
7.Galluzzi L,Miguel J,Kepp VO,et al.To die or not to die:that is the autophagic question.Curr Mol Med 2008;8(2):78-91.
8.Tsujimoto Y,Shimizu S.Another way to die:autophagic programmed cell death.Cell Death Differ 2005;suppl 2:1528-1534.
9.Shaw RJ,Cantley LC.Ras,PI(3)K and mTOR signalling controls tumour cell growth.Nature 2006; 441(7092):424-430.
10.Degenhardt K,Mathew R,Beaudoin B,et al.Autophagy promotes tumor cell survival and restricts necrosis,inflammation,and tumorigenesis.Cancer Cell 2006;10(1):51-64.
11.Mathew R,Kongara S,Beaudoin B,et al.Autophagy suppresses tumor progression by limiting chromosomal instability.Genes Dev 2007; 21(11):1367-1381.
12.Karantza-Wadsworth V,Patel S,Kravchuk O,et al.Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis.Genes Dev 2007; 21(13):1621-1635.
13.Pattingre S,Tassa A,Qu X,et al.Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy.Cell 2005; 122(6):927-939.
14.Maiuri MC,Le Toumelin G,Criollo A,et al.Functional and physical interaction between Bcl-X(L)and a BH3-like domain in Beclin-1.EMBO J 2007; 26(10):2527-2539.
15.Erlich S,Mizrachy L,Segev O,et al.Differential interactions between Beclin 1 and Bcl-2 family members.Autophagy 2007; 3(6):561-568.
16.Saeki K,Yuo A,Okuma E,et al.Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells.Cell Death Differ 2000; 7(12):1263-1269.
17.Akar U,Chaves-Reyes A,Barria M,et al.Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells.Autophagy 2008; 4(5):669-679.
18.Daido S,Kanzawa T,Yamamoto A,et al.Pivotal role of the cell death factor BNIP3 in ceramide-induced autophagic cell death in malignant glioma cells.Cancer Res 2004; 64(12):4286-4293.
19.Hamacher-Brady A,Brady NR,Logue SE,et al.Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy.Cell Death Differ 2007;14(1):146-157.
20.Abedin MJ,Wang D,McDonnell MA,et al.Autophagy delays apoptotic death in breast cancer cells following DNA damage.Cell Death Differ 2007;14(3):500-510.
21.Liang C,Feng P,Ku B,et al.Autophagic and tumour suppressor activity of a novel Beclinl-binding protein UVRAG Nat Cell Biol 2006;8(7):688-699.
22.Fimia GM,Stoykova A,Romagnoli A,et al.Ambral is a novel regulator of autophagy and controls nervous system development.Nature 2007;447(7148):1121-1125.
23.Rodriguez-Enriquez S,Kim I,Currin RT,et al.Tracker dyes to probe mitochondrial autophagy(mitophagy) in rat hepatocytes.Autophagy 2006;2(1):39-46.
24.Takahashi Y,Coppola D,Matsushita N,et al.Bif-1 interactswith Beclin 1 through UVRAG and regulates autophagy and tumorigenesis.Nat Cell Biol 2007;9(10):1142-1151.
25.Oberstein A,Jeffrey P,Shi Y.Crystal structure of the BCL-XL-beclin 1 peptide complex:Beclin 1 is a novel BH3-only protein.J Biol Chem 2007;282(17):13123-13132.
26.Feng W,Huang S,Wu H,et al.Molecular basis of Bcl- XL's target recognition versatility revealed by the structure of Bcl-XL in complex with the BH3 domain of Beclin-1.J Mol Biol 2007;372(1):223-235.
27.Maiuri MC,Criollo A,Tasdemir E,et al.BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between beclin 1and Bcl-2/Bcl-X(L).Autophagy 2007;3(4):374-376.
28.Oltersdorf T,Elmore SW,Shoemaker AR,et al.An inhibitor of Bcl-2 family proteins induces regression of solid tumours.Nature 2005;435(7042):677-681.
29.Schweers RL,Zhang J,Randall MS,et al.NIX is required for programmed mitochondrial clearance during reticulocyte maturation.Proc Natl Acad Sci USA 2007;104(49):19500-19505.
30.Zhang H,Bosch-Marce M,Shimoda LA,et al.Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia.J Biol Chem 2008;283(16):10892-10903.
31.Crighton D,Wilkinson S,O'Prey J,et al.DRAM,a p53-induced modulator of autophagy,is critical for apoptosis.Cell 2006;126(1):121-134.
32.Feng Z,Zhang H,Levine AJ,et al.The coordinate regulation of the p53 and mTOR pathways in cells.Proc Natl Acad Sci USA 2005;102(23):8204-8209.
33.Abida WM,Gu W.p53-Dependent and p53-independent activation of autophagy by ARF.Cancer Res 2008;68(2):352-357.
34.Amaravadi RK,Yu D,Lum JJ,et al.Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma.J Clin Invest 2007;117(2):326-336.
35.Tasdemir E,Maiuri MC,Galluzzi L,et al.Regulation of autophagy by cytoplasmic p53.Nat Cell Biol 2008;10(6):676-687.
36.Gozuacik D,Kimchi A.DAPk protein family and cancer.Autophagy 2006;2(2):74-79.
37.Harrison B,Kraus M,Burch L,et al.DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing.J Biol Chem 2008;283(15):9999-10014.
38.Wang QJ,Ding Y,Kohtz DS,et al.Induction of autophagy in axonal dystrophy and degeneration.J Neurosci 2006;26(31):8057-8068.
39.Miracco C,Cosci E,Oliveri G,et al.Protein and mRNA expression of autophagy gene Beclin 1 in human brain tumors.Int J Oncol 2007;30(2):429-436.
40.Qu X,Yu J,Bhagat G,et al.Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.J Clin Invest 2003;112(12):1809-1820.
41.Yue Z,Jin S,Yang C,et al.Beclin 1,an autophagy gene essential for early embryonic development,is a haploinsufficient tumor suppressor.Proc Natl Acad Sci USA 2003;100(25):15077-15082.
42.Marino G,Salvador-Montoliu N,Fueyo A,et al.Tissuespecific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/Autophagin-3.J Biol Chem 2007;282(25):18573-18583.
1.Eskelinen EL.Maturation of autophagic vacuoles in mammalian cells.Autophagy 2005; 1(1):1-10.
2.Chu CT.Autophagic stress in neuronal injury and disease.J Neuropathol Exp Neurol 2006; 65(5):423-432.
3.Williams MA.Quantitative methods in biology:Practical methods in electron microscopy.Amsterdam,New York,Oxford:North-Holland Publishing Company,1977.
4.Mayhew TM.Quantitative immunoelectron microscopy:alternative ways of assessing sub-cellular patterns of gold labeling.Methods Mol Biol 2007; 369:309-329.
5.Mayhew TM,Lucocq JM,Griffiths G.Relative labelling index:a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections.J Microsc 2002; 205:153-164.
6.Niemann A,Baltes J,Elsasser HP.Fluorescence properties and staining behavior of monodansylpentane,a structural homologue of the lysosomotropic agent monodansylcadaverine.J Histochem Cytochem 2001; 49(2):177-185.
7.Paglin S,Hollister T,Delohery T,et al.A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles.Cancer Res 2001; 61(2):439-44.
8.Mizushima N,Ohsumi Y,Yoshimori T.Autophagosome formation in mammalian cells.Cell Structure and Function 2002; 27(6):421-429.
9.Huang WP,Scott SV,Kim J,et al.The itinerary of a vesicle component,Aut7p/Cvt5p,terminates in the yeast vacuole via the autophagy/Cvt pathways.J Biol Chem 2000; 275(8):5845-5851.
10.He H,Dang Y,Dai F,et al.Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B.J Biol Chem 2003; 278(31):29278-29287.
11.Mizushima N,Kuma A,Kobayashi Y,et al.Mouse Apg16L,a novel WD-repeat protein,targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate.J Cell Sci 2003; 116:1679-1688.
12.Mizushima N,Yamamoto A,Hatano M,et al.Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.J Cell Biol 2001;152(4):657-668.
13.Yousefi S,Perozzo R,Schmid I,et al.Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.Nat Cell Biol 2006; 8(10):1124-1132.
14.Mizushima N,Yoshimori T.How to interpret LC3 immunoblotting.Autophagy 2007; 3(6):542-545.
15.Bjorkoy G,Lamark T,Brech A,et al.p62/SQSTMl forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.J Cell Biol 2005; 171(4):603-614.
16.Komatsu M,Wang QJ,Holstein GR,et al.Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration.Proc Natl Acad Sci U S A 2007; 104(36):14489-14494.
17.Wang QJ,Ding Y,Kohtz DS,et al.Induction of autophagy in axonal dystrophy and degeneration.J Neurosci 2006; 26(31):8057-8068.
18.Bardag-Gorce F,Francis T,Nan L,et al.Modifications in p62 occur due to proteasome inhibition in alcoholic liver disease.Life Sci 2005; 77(20):2594-2602.
19.Erlich S,Alexandrovich A,Shohami E,et al.Rapamycin is a neuroprotective treatment for traumatic brain injury.Neurobiol Dis 2007; 26(1):86-93.
20.Lavieu G,Scarlatti F,Sala G,et al.Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation.J Biol Chem 2006;281(13):8518-8527.
21.Kamada Y,Funakoshi T,Shintani T,et al.Tor-mediated induction of autophagy via an Apgl protein kinase complex.J Cell Biol 2000; 150(6):1507-1513.
22.Djavaheri-Mergny M,Amelotti M,Mathieu J,et al.NF-kB activation represses tumor necrosis factor-alpha-induced autophagy.J Biol Chem 2006; 281(41): 30373-30382.
23.Liu Z,Lenardo MJ.Reactive oxygen species regulate autophagy through redox-sensitive proteases.Dev Cell 2007; 12(4):484-485.
24.Scarlatti F,Bauvy C,Ventruti A,et al.Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.J Biol Chem 2004; 279(18):18384-18391.
25.Scherz-Shouval R,Shvets E,Fass E,et al.Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.EMBO J 2007; 26(7):1749-1760.
26.Monastyrska I,Klionsky DJ.Autophagy in organelle homeostasis:peroxisome turnover.Mol Aspects Med 2006; 27(5-6):483-494.
27.Nair U,Klionsky DJ.Molecular mechanisms and regulation of specific and nonspecific autophagy pathways in yeast.J Biol Chem 2005; 280(51):41785-41788.
28.Dunn WA,Jr.,Cregg JM,Kiel JAKW,et al.Pexophagy:the selective autophagy of peroxisomes.Autophagy 2005; 1(2):75-83.
29.Birmingham CL,Brumell JH.Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles.Autophagy 2006; 2(3):156-158.
30.Colombo MI,Gutierrez MG,Romano PS.The two faces of autophagy:Coxiella and Mycobacterium.Autophagy 2006; 2(3):162-164.
31.Vergne I,Singh S,Roberts E,et al.Autophagy in immune defense against Mycobacterium tuberculosis.Autophagy 2006; 2(3):175-178.
32.Yoshimori T.Autophagy vs.Group A Streptococcus.Autophagy 2006; 2(3):154-155.
33.Talloczy Z,Virgin HW,IV,Levine B.PKR-dependent autophagic degradation of herpes simplex virus type 1.Autophagy 2006; 2(3):24-29.
34.Guan J,Stromhaug PE,George MD,et al.Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport,pexophagy,and autophagy in Saccharomyces cerevisiae and Pichia pastoris.Mol Biol Cell 2001; 12(12):3821-3838.
35.Proikas-Cezanne T,Ruckerbauer S,Stierhof YD,et al.Human WIPI-1 puncta-formation:A novel assay to assess mammalian autophagy.FEBS Lett 2007;24;581(18):3396-3404.
36.Petiot A,Ogier-Denis E,Blommaart EF,et al.Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control,macroautophagy in HT-29 cells.J Biol Chem 2000; 275(2):992-998.
37.Kovacs AL,Seglen PO.Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis.Biochim Biophys Acta 1981; 676(2):213-220.
38.Kovacs AL,Laszlo L,Kovacs J.Effect of amino acids and cycloheximide on changes caused by vinblastine,leupeptin and methylamine in the autophagic/lysosomal system of mouse hepatocytes in vivo.Exp Cell Res 1985;157(1):83-94.
39.Kovacs J,Fellinger E,Karpati AP,et al.Morphometric evaluation of the turnover of autophagic vacuoles after treatment with Triton X-100 and vinblastine in murine pancreatic acinar and seminal vesicle epithelial cells.Virchows Arch B Cell Pathol Incl Mol Pathol 1987; 53(3):183-190.
40.Kovacs J,Laszlo L,Kovacs AL.Regression of autophagic vacuoles in pancreatic acinar,seminal vesicle epithelial,and liver parenchymal cells:a comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment.Exp Cell Res 1988; 174(1):244-251.
41.Cantino D,Mosso R,Baccino FM.Changes induced by fasting and cycloheximide in the vacuolar apparatus of rat hepatocytes.A morphometric investigation.Boll Soc Ital Biol Sper 1979; 55(18):1884-1889.
42.Kovacs J.Regression of autophagic vacuoles in seminal vesicle cells following cycloheximide treatment.Exp Cell Res 1983; 144(1):231-234.
43.Kovacs J.Morphometric study of the effect of leupeptin,vinblastine,estron acetate and cycloheximide on the autophagic vacuole-lysosomal compartments in mouse seminal vesicle cells.Virchows Arch B Cell Pathol Incl Mol Pathol 1983;42(1):83-93.
44.Kovacs J,Fellinger E,Karpati PA,et al.The turnover of autophagic vacuoles:evaluation by quantitative electron microscopy.Biomed Biochim Acta 1986;45(11-12):1543-1547.
45.Papadopoulos T,Pfeifer U.Regression of rat liver autophagic vacuoles by locally applied cycloheximide.Lab Invest 1986;54(1):100-107.
46.Rumpelt HJ,Albring M,Thoenes W.Prevention of D-galactosamine-induced hepatocellular autophagocytosis by cycloheximide.Virchows Arch B Cell Pathol 1974;16(2):195-203.
47.Rumpelt HJ,Weisbach T.Effect of cycloheximide on glucagon-induced autophagy.Quantitative examinations on hepatocytes in the rat.Am J Pathol 1978;91(1):49-56.
48.Sarkar S,Floto RA,Berger Z,et al.Lithium induces autophagy by inhibiting inositol monophosphatase.J Cell Biol 2005;170(7):1101-1111.
49.Klionsky DJ.Autophagy:Autophagy and cancer.Georgetown,TX:Landes Bioscience,2004.
50.Sarkar S,Perlstein EO,Imarisio S,et al.Small molecules enhance autophagy and reduce toxicity in Huntington's disease models.Nat Chem Biol 2007;3(6):331-338.
51.Zhang L,Yu J,Pan H,et al.Small molecule regulators of autophagy identified by an image-based high-throughput screen.Proc Natl Acad Sci U S A 2007;104(48):19023-19028.
2.Blaese RM,Culver KW,Miller AD,et al.T lymphocyte-directed gene therapy for ADA-SCID:initial trial results after 4 years.Science 1995; 270(5249):475-480.
3.Honjo Y,Inohara H,Akahani S,et al.Expression of cytoplasmic galectin-3 as a prognostic marker in tongue carcinoma.Clin Cancer Res 2000; 6(12):4635-4640.
4.Armstrong AC,Eaton D,Ewing JC.Cellular vaccine therapy for cancer.Expert Rev Vaccines 2002; 1(3):303-316.
5.Liu K.Breakthroughs in cancer gene therapy.Semin Oncol Nurs 2003; 19(3):217-226.
6.Nettelbeck DM.Virotherapeutics:conditionally replicative adenoviruses for viral oncolysis.Anticancer Drugs 2003; 14(8):577-584.
7.Chu RL,Post DE,Khuri FR,et al.Use of replicating oncolytic adenoviruses in combination therapy for cancer.Clin Cancer Res 2004; 10(16):5299-5312.
8.Pin RH,Reinblatt M,Fong Y.Utilizing alpha-fetoprotein expression to enhance oncolytic viral therapy in hepatocellular carcinoma.Ann Surg 2004; 240(4):659-665.
9.Kurihara T,Brough DE,Kovesdi I,et al.Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen.J Clin Invest 2000; 106(6):763-771.
10.Jakubczak JL,Ryan P,Gorziglia M,et al.An oncolytic adenovirus selective for retinoblastoma tumor suppressor protein pathway-defective tumors:dependence on E1A,the E2F-1 promoter,and viral replication for selectivity and efficacy.Cancer Res 2003,63(7):1490-1499.
11.Bischoff JR,Kirn DH,Williams A,et al.An adenovirus mutant that replicates selectively in p53-deficient human tumor cells.Science 1996; 274(5286):342-343.
12.Geoerger B,Grill J,Opolon P,et al.Potentiation of radiation therapy by the oncolytic adenovirus dl 1520 (ONYX-015)in human malignant glioma xenografts.Br J Cancer 2003; 89(3):577-584.
13.Rogulski KR,Freytag SO,Zhang K,et al.In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy.Cancer Res 2000; 60(5):1193-1196.
14.Chen Y,DeWeese T,Dilley J,et al.CV706,a prostate cancer-specific adenovirus variant,in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity.Cancer Res 2001; 61(14):5453-5460.
15.Khuri FR,Nemunaitis J,Ganly I,et al.A controlled trial of intratumoral ONYX-015,a selectively-replicating adenovirus,in combination with cisplatin and 5-fluorouracil in patients with recurrent head and neck cancer.Nat Med 2000;6(8):879-885.
16.Pei Z,Chu L,Zou W,et al.An oncolytic adenoviral vector of Smac increases antitumor activity of TRAIL against HCC in human cells and in mice.Hepatology 2004; 39(5):1371-1381.
17.Zhang Z,Zou W,Luo C,et al.An armed oncolytic adenovirus system,ZD55-gene,demonstrating potent antitumoral efficacy.Cell Res 2003; 13(6):481-489.
18.Mentel R,Dopping G,Wegner U,et al.Adenovirus-receptor interaction with human lymphocytes.J Med Virol 1997; 51(3):252-257.
19.Rebel VI,Hartnett S,Denham J,et al.Maturation and lineage-specific expression of the coxsackie and adenovirus receptor in hematopoietic cells.Stem cells 2000;18(3):176-182.
20.Wang Y,Xue S,Hallden G,et al.Virus-associated RNA I-deleted adenovirus,a potential oncolytic agent targeting EBV-associated tumors.Cancer Res 2005;65(4):1523-1531.
21.Medina D,Sheay W,Goodell L,et al.Adenovirus mediated cytotoxicity of chronic lymphocytic leukemia cells.Blood 1999; 94(10):3499-3508.
22.Yotnda P,Onishi H,Heslop HE,et al.Efficient infection of primitive hematopoietic stem cells by modified adenovirus.Gene Ther 2001; 8(12):930-937.
23.Wuchter C,Krappmann D,Cai Z,et al.In vitro susceptibility to TRAIL-induced apoptosis of acute leukemia cells in the context of TRAIL receptor gene expression and constitutive NF-kappa B activity.Leukemia 2001; 15(6):921-928.
24.Nieda M,Nicol A,Koezuka Y,et al.TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukemia cells.Blood 2001; 97(7):2067-2074.
25.Plasilova M,Zivny J,Jelinek J,et al.TRAIL (Apo2L)suppresses growth of primary human leukemia and myelodysplasia progenitors.Leukemia 2002; 16(1):67-73.
26.Pan Q,Liu B,Liu J,et al.Synergistic induction of tumor cell death by combining cisplatin with an oncolytic adenovirus carrying TRAIL.Mol Cell Biochem 2007;304(1-2):315-323.
27.Zhang Y,Qin X,Zhang Y,et al.Combination of ZD55-MnSOD therapy with 5-FU enhances antitumor efficacy in colorectal cancer.J Cancer Res Clin Oncol 2008; 134(2):219-226.
28.Baaske DM,Heinstein P.Cytotoxicity and cell cycle specificity of homoharringtonine.Antimicrob Agents Chemother 1977; 12(2):298-300.
29.Wilkoff LJ,Dulmadge DA,Laster WR Jr,et al.Effect of homoharringtonine on the viability of murine leukemia P388 cells resistant to either adriamycin,vincristine,or 1-beta-D-arabinofuranosylcytosine.Cancer Chemother Pharmacol 1989; 23(3):145-150.
30.Fanucchi MP,Kong XR,Chou TC.Hexamethylene bisacetamide (HMBA)does not enhance the cytotoxic effects of adriamycin (ADR),1-beta-D-arabinofuranosylcytosine (ARA-C)and homoharringtonine (HT)in HL-60 cells [abstract 1492].Proc Am Assoc Cancer Res 1986; 27:376.
31.Liang C,Feng P,Ku B,et al.Autophagic and tumour suppressor activity of a novel Beclinl-binding protein UVRAG Nat Cell Biol 2006; 8(7):688-699.
32.Miracco C,Cosci E,Oliveri G,et al.Protein and mRNA expression of autophagy gene Beclin 1 in human brain tumors.Int J Oncol 2007; 30(2):429-436.
33.Liang XH,Jackson S,Seaman M,et al.Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature 1999; 402(6762):672-676.
34.Qian W,Liu J,Tong Y,et al.Enhanced antitumor activity by a selective conditionally replicating adenovirus combining with MDA-7/interleukin-24 for B-lymphoblastic leukemia via induction of apoptosis.Leukemia 2008; 22(2):361-369.
35.Guo BC,Xu YH.Bcl-2 over-expression and activation of protein kinase C suppress the trail-induced apoptosis in Jurkat T cells.Cell Res 2001; 11(2):101-106.
36.Lamothe B,Aggarwal BB.Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL)through suppression of caspases-8,7,and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60.J Interferon Cytokine Res 2002; 22(2):269-279.
37.Wang J,Gu JF,Yang SY,et al.Security of dual cancer-specific targeting vector and its cytotoxic effect when harbored.Ai Zheng 2006; 25(4):385-392.
38.Shayakhmetov DM,Eberly AM,Li ZY,et al.Deletion of penton RGD motifs affects the efficiency of both the internalization and the endosome escape of viral particles containing adenovirus serotype 5 or 35 fiber knobs.J Virol 2005; 79(2):1053-1061.
39.Aye MT,Niho Y,Till JE,et al.Study of leukemic cell population in culture.Blood 1974; 44(2):205-219.
40.Kirn D,Martuza RL,Zwiebel J.Replication-selective virotherapy for cancenbiological principles,risk management and future directions.Nat Med 2001; 7(7):781-787.
41.Hawkins LK,Lemoine NR,Kirn D.Oncolytic biotherapy:a novel therapeutic platform.Lancet Oncol 2002; 3(1):17-26.
42.Martuza RL,Malick A,Markert JM,et al.Experimental therapy of human glioma by means of a genetically engineered virus mutant.Science 1991; 252(5007):854-856.
43.Everts B,van der Poel HG.Replication-selective oncolytic viruses in the treatment of cancer.Cancer Gene Ther 2005; 12(2):141-161.
44.Peng KW,Ahmann GJ,Pham L,et al.Systemic therapy of myeloma xenografts by an attenuated measles virus.Blood 2001; 98(7):2002-2007.
45.Ong HT,Timm MM,Greipp PR,et al.Oncolytic measles virus targets high CD46 expression on multiple myeloma cells.Exp Hematol 2006; 34(6):713-720.
46.Peng KW,Donovan KA,Schneider U,et al.Oncolytic measles viruses displaying a single-chain antibody against CD38,a myeloma cell marker.Blood 2003; 101(7):2557-2562.
47.Grote D,Russell SJ,Cornu TI,et al.Live attenuated measles virus induces regression of human lymphoma xenografts in immunodeficient mice.Blood 2001; 97(12):3746-3754.
48.Kunzi V,Oberholzer PA,Heinzerling L,et al.Recombinant measles virus induces cytolysis of cutaneous T-cell lymphoma in vitro and in vivo.J Invest Dermatol 2006; 126(11):2525-2532.
49.Heinzerling L,Kunzi V,Oberholzer PA,et al.Oncolytic measles virus in cutaneous T-cell lymphomas mounts antitumor immune responses in vivo and targets interferon-resistant tumor cells.Blood 2005; 106(7):2287-2294.
50.Hara T,Kojima A,Fukuda H,et al.Levels of complement regulatory proteins,CD35 (CR1),CD46 (MCP)and CD55 (DAF)in human haematological malignancies.Br J Haematol 1992; 82(2):368-373.
51.Hengartner MO.The biochemistry of apoptosis.Nature 2000; 407(6805):770-776.
52.Green D,Reed JC.Mitochondria and apoptosis.Science 1998; 281(5381):1309-1312.
53.Krammer PH.CD95's deadly mission in the immune system.Nature 2000;407(6805):789-795.
54.Woo M,Hakem R,Soengas MS,et al.Essential contribution of caspase 3/CPP32 to apoptosis and its associated nuclear changes.Genes Dev 1998; 12(6):806-819.
55.Burkle A.Physiology and pathophysiology of poly (ADP-ribosyl)ation.Bioessays 2001; 23(9):795-806.
56.Tong WM,Cortes U,Wang ZQ.Poly (ADP-ribose)polymerase:a guardian angel protecting the genome and suppressing tumorigenesis.Biochim Biophys Acta 2001; 1552(1):27-37.
57.Cheng J,Hylander BL,Baer MR,et al.Multiple mechanisms underlie resistance of leukemia cells to Apo2 Ligand/TRAIL.Mol Cancer Ther 2006; 5(7):1844-1853.
58.Riccioni R,Pasquini L,Mariani G,et al.TRAIL decoy receptors mediate resistance of acute myeloid leukemia cells to TRAIL.Haematologica 2005; 90(5):612-624.
59.Wu CH,Kao CH,Safa AR.TRAIL recombinant adenovirus triggers robust apoptosis in multidrug-resistant HL-60/Vinc cells preferentially through death receptor DR5.Hum Gene Ther 2008; 19(7):731-743.
60.Kanerva A,Wang M,Bauerschmitz GJ,et al.Gene transfer to ovarian cancer versus normal tissues with fiber-modified adenoviruses.Mol Ther 2002; 5(6):695-704.
61.Hemminki A,Wang M,Desmond RA,et al.Serum and ascites neutralizing antibodies in ovarian cancer patients treated with intraperitoneal adenoviral gene therapy.Hum Gene Ther 2002; 13(12):1505-1514.
62.Bharti AC,Takada Y,Aggarwal BB.PARP cleavage and caspase activity to assess chemosensitivity.Methods Mol Med 2005; 111:69-78.
63.Rahmani M,Davis EM,Bauer C,et al.Apoptosis induced by the kinase inhibitor BAY 43-9006 in human leukemia cells involves down-regulation of Mcl-1 through inhibition of translation.J Biol Chem 2005; 280(42):35217-35227.
64.Moulding DA,Giles RV,Spiller DG,et al.Apoptosis is rapidly triggered by antisense depletion of MCL-1 in differentiating U937 cells.Blood 2000; 96(5):1756-1763.
65.Zhou P,Qian L,Bieszczad CK,et al.Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage.Blood 1998; 92(9):3226-3239.
66.Teicher BA.Assays for in vitro and in vivo synergy.Methods Mol Med 2003; 85:297-321.
67.Chou TC,Motzer RJ,Tong Y,et al.Computerized quantitation of synergism and antagonism of taxol,topotecan,and cisplatin against human teratocarcinoma cell growth:a rational approach to clinical protocol design.J Natl Cancer Inst 1994;86(2):1517-1524.
68.Chou,TC.Drug combinations:from laboratory to practice.J Lab Clin Med 1998;132(1):6-8.
69.Jin J,Jiang DZ,Mai WY,et al.Homoharringtonine in combination with cytarabine and aclarubicin resulted in high complete remission rate after first induction therapy in patients with de novo acute myeloid leukemia.Leukemia 2006; 20(8):1361-1367.
70.Tallarida,RJ.Drug synergism:its detection and applications.J Pharmacol Exp Ther 2001; 298(3):865-872.
71.White DB,Slocum HK,Brun Y et al.A new nonlinear mixture response surface paradigm for the study of synergisms three drug example.Curr Drug Metab 2003:4(5); 399-409.
72.Levasseur LM,Greco WR,RustumYM,et al.Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells.Cancer Chemother Pharmacol 1997;40(6):495-505.
73.Greco WR,Bravo G,Parsons JC.The search for synergy:a critical review from a response surface perspective.Pharmacol Rev 1995;47(2):331-385.
74.Jordan CT,Upchurch D,Szilvassy SJ,et al.The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells.Leukemia 2000;14(10):1777-1784.
75.麦文渊,林茂芳。高三尖杉酯碱诱导K562细胞凋亡的实验研究。中华血液学杂志,1998;19(7):365-376.
76.Yinjun L,Jie J,Weilai X,et al.Homoharringtonine mediates myeloid cell apoptosis via upregulation of pro-apoptotic bax and inducing caspase-3-mediated cleavage of poly(ADP-ribose) polymerase(PARP).Am J Hematol.2004 Jul;76(3):199-204.
77.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science 1999;284(5411):143-147.
78.Horwitz EE.Clarification of the nomenclature for MSC:the international society for cellular therapy position statement.Cytotherapy 2005;7(5):393-395.
79.Calvi LM,Adams GB,Weibrecht KW,et al.Osteoblastic cells regulate the haematopoietic stem cell niche.Nature 2003;425(6960):841-846.
80.Zhang J,Niu C,Ye L,et al.Identification of the haematopoietic stem cell niche and control of the niche size.Nature 2003;425(6960):836-841.
81.Dazzi F,Ramasamy R,Glennie S,et al.The role of mesenchymal stem cells in haemopoiesis.Blood Rev 2005;20(3):161-171.
82.Li J,Law HK,Lau YL,et al.Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents.Br J Haematol 2004;127(3):326-334.
83.Ben-Ishay Z,Barak V.Bone marrow stromal dysfunction in mice administered cytosine arabinoside.Eur J Haematol 2001;66(4):230-237.
84.Liang XH,Kleeman LK,Jiang HH,et al.Protection against fatal Sindbis virus encephalitis by beclin,a novel Bcl-2-interacting protein.J Virol 1998;72(11): 8586-8596.
85.Scarlatti F,Bauvy C,Ventruti A,et al.Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin-1.J Biol Chem 2004;279(18):18384-18391.
1.Levine B.Cell biology:autophagy and cancer.Nature 2007;446(7137):745-747.
2.Amaravadi RK,Thompson CB.The roles of therapy-induced autophagy and necrosis in cancer treatment.Clin Cancer Res 2007;13(24):7271-7279.
3.Mathew R,Karantza-Wadsworth V,White E.Role of autophagy in cancer.Nat Rev Cancer 2007;7(12):961-967.
4.Kroemer G,Jaattela M.Lysosomes and autophagy in cell death control.Nat Rev Cancer 2005;5(11):886-897.
5.Maiuri MC,Zalckvar E,Kimchi A,et al.Self-eating and self-killing:cross talk between autophagy and apoptosis.Nat Rev Mol Cell Biol 2007;8(9):741-752.
6.Boya P,Gonzalez-Polo RA,Casares N,et al.Inhibition of macroautophagy triggers apoptosis.Mol Cell Biol 2005;25(3):1025-1040.
7.Galluzzi L,Miguel J,Kepp VO,et al.To die or not to die:that is the autophagic question.Curr Mol Med 2008;8(2):78-91.
8.Tsujimoto Y,Shimizu S.Another way to die:autophagic programmed cell death.Cell Death Differ 2005;suppl 2:1528-1534.
9.Shaw RJ,Cantley LC.Ras,PI(3)K and mTOR signalling controls tumour cell growth.Nature 2006; 441(7092):424-430.
10.Degenhardt K,Mathew R,Beaudoin B,et al.Autophagy promotes tumor cell survival and restricts necrosis,inflammation,and tumorigenesis.Cancer Cell 2006;10(1):51-64.
11.Mathew R,Kongara S,Beaudoin B,et al.Autophagy suppresses tumor progression by limiting chromosomal instability.Genes Dev 2007; 21(11):1367-1381.
12.Karantza-Wadsworth V,Patel S,Kravchuk O,et al.Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis.Genes Dev 2007; 21(13):1621-1635.
13.Pattingre S,Tassa A,Qu X,et al.Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy.Cell 2005; 122(6):927-939.
14.Maiuri MC,Le Toumelin G,Criollo A,et al.Functional and physical interaction between Bcl-X(L)and a BH3-like domain in Beclin-1.EMBO J 2007; 26(10):2527-2539.
15.Erlich S,Mizrachy L,Segev O,et al.Differential interactions between Beclin 1 and Bcl-2 family members.Autophagy 2007; 3(6):561-568.
16.Saeki K,Yuo A,Okuma E,et al.Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells.Cell Death Differ 2000; 7(12):1263-1269.
17.Akar U,Chaves-Reyes A,Barria M,et al.Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells.Autophagy 2008; 4(5):669-679.
18.Daido S,Kanzawa T,Yamamoto A,et al.Pivotal role of the cell death factor BNIP3 in ceramide-induced autophagic cell death in malignant glioma cells.Cancer Res 2004; 64(12):4286-4293.
19.Hamacher-Brady A,Brady NR,Logue SE,et al.Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy.Cell Death Differ 2007;14(1):146-157.
20.Abedin MJ,Wang D,McDonnell MA,et al.Autophagy delays apoptotic death in breast cancer cells following DNA damage.Cell Death Differ 2007;14(3):500-510.
21.Liang C,Feng P,Ku B,et al.Autophagic and tumour suppressor activity of a novel Beclinl-binding protein UVRAG Nat Cell Biol 2006;8(7):688-699.
22.Fimia GM,Stoykova A,Romagnoli A,et al.Ambral is a novel regulator of autophagy and controls nervous system development.Nature 2007;447(7148):1121-1125.
23.Rodriguez-Enriquez S,Kim I,Currin RT,et al.Tracker dyes to probe mitochondrial autophagy(mitophagy) in rat hepatocytes.Autophagy 2006;2(1):39-46.
24.Takahashi Y,Coppola D,Matsushita N,et al.Bif-1 interactswith Beclin 1 through UVRAG and regulates autophagy and tumorigenesis.Nat Cell Biol 2007;9(10):1142-1151.
25.Oberstein A,Jeffrey P,Shi Y.Crystal structure of the BCL-XL-beclin 1 peptide complex:Beclin 1 is a novel BH3-only protein.J Biol Chem 2007;282(17):13123-13132.
26.Feng W,Huang S,Wu H,et al.Molecular basis of Bcl- XL's target recognition versatility revealed by the structure of Bcl-XL in complex with the BH3 domain of Beclin-1.J Mol Biol 2007;372(1):223-235.
27.Maiuri MC,Criollo A,Tasdemir E,et al.BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between beclin 1and Bcl-2/Bcl-X(L).Autophagy 2007;3(4):374-376.
28.Oltersdorf T,Elmore SW,Shoemaker AR,et al.An inhibitor of Bcl-2 family proteins induces regression of solid tumours.Nature 2005;435(7042):677-681.
29.Schweers RL,Zhang J,Randall MS,et al.NIX is required for programmed mitochondrial clearance during reticulocyte maturation.Proc Natl Acad Sci USA 2007;104(49):19500-19505.
30.Zhang H,Bosch-Marce M,Shimoda LA,et al.Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia.J Biol Chem 2008;283(16):10892-10903.
31.Crighton D,Wilkinson S,O'Prey J,et al.DRAM,a p53-induced modulator of autophagy,is critical for apoptosis.Cell 2006;126(1):121-134.
32.Feng Z,Zhang H,Levine AJ,et al.The coordinate regulation of the p53 and mTOR pathways in cells.Proc Natl Acad Sci USA 2005;102(23):8204-8209.
33.Abida WM,Gu W.p53-Dependent and p53-independent activation of autophagy by ARF.Cancer Res 2008;68(2):352-357.
34.Amaravadi RK,Yu D,Lum JJ,et al.Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma.J Clin Invest 2007;117(2):326-336.
35.Tasdemir E,Maiuri MC,Galluzzi L,et al.Regulation of autophagy by cytoplasmic p53.Nat Cell Biol 2008;10(6):676-687.
36.Gozuacik D,Kimchi A.DAPk protein family and cancer.Autophagy 2006;2(2):74-79.
37.Harrison B,Kraus M,Burch L,et al.DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing.J Biol Chem 2008;283(15):9999-10014.
38.Wang QJ,Ding Y,Kohtz DS,et al.Induction of autophagy in axonal dystrophy and degeneration.J Neurosci 2006;26(31):8057-8068.
39.Miracco C,Cosci E,Oliveri G,et al.Protein and mRNA expression of autophagy gene Beclin 1 in human brain tumors.Int J Oncol 2007;30(2):429-436.
40.Qu X,Yu J,Bhagat G,et al.Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.J Clin Invest 2003;112(12):1809-1820.
41.Yue Z,Jin S,Yang C,et al.Beclin 1,an autophagy gene essential for early embryonic development,is a haploinsufficient tumor suppressor.Proc Natl Acad Sci USA 2003;100(25):15077-15082.
42.Marino G,Salvador-Montoliu N,Fueyo A,et al.Tissuespecific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/Autophagin-3.J Biol Chem 2007;282(25):18573-18583.
1.Eskelinen EL.Maturation of autophagic vacuoles in mammalian cells.Autophagy 2005; 1(1):1-10.
2.Chu CT.Autophagic stress in neuronal injury and disease.J Neuropathol Exp Neurol 2006; 65(5):423-432.
3.Williams MA.Quantitative methods in biology:Practical methods in electron microscopy.Amsterdam,New York,Oxford:North-Holland Publishing Company,1977.
4.Mayhew TM.Quantitative immunoelectron microscopy:alternative ways of assessing sub-cellular patterns of gold labeling.Methods Mol Biol 2007; 369:309-329.
5.Mayhew TM,Lucocq JM,Griffiths G.Relative labelling index:a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections.J Microsc 2002; 205:153-164.
6.Niemann A,Baltes J,Elsasser HP.Fluorescence properties and staining behavior of monodansylpentane,a structural homologue of the lysosomotropic agent monodansylcadaverine.J Histochem Cytochem 2001; 49(2):177-185.
7.Paglin S,Hollister T,Delohery T,et al.A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles.Cancer Res 2001; 61(2):439-44.
8.Mizushima N,Ohsumi Y,Yoshimori T.Autophagosome formation in mammalian cells.Cell Structure and Function 2002; 27(6):421-429.
9.Huang WP,Scott SV,Kim J,et al.The itinerary of a vesicle component,Aut7p/Cvt5p,terminates in the yeast vacuole via the autophagy/Cvt pathways.J Biol Chem 2000; 275(8):5845-5851.
10.He H,Dang Y,Dai F,et al.Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B.J Biol Chem 2003; 278(31):29278-29287.
11.Mizushima N,Kuma A,Kobayashi Y,et al.Mouse Apg16L,a novel WD-repeat protein,targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate.J Cell Sci 2003; 116:1679-1688.
12.Mizushima N,Yamamoto A,Hatano M,et al.Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.J Cell Biol 2001;152(4):657-668.
13.Yousefi S,Perozzo R,Schmid I,et al.Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.Nat Cell Biol 2006; 8(10):1124-1132.
14.Mizushima N,Yoshimori T.How to interpret LC3 immunoblotting.Autophagy 2007; 3(6):542-545.
15.Bjorkoy G,Lamark T,Brech A,et al.p62/SQSTMl forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death.J Cell Biol 2005; 171(4):603-614.
16.Komatsu M,Wang QJ,Holstein GR,et al.Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration.Proc Natl Acad Sci U S A 2007; 104(36):14489-14494.
17.Wang QJ,Ding Y,Kohtz DS,et al.Induction of autophagy in axonal dystrophy and degeneration.J Neurosci 2006; 26(31):8057-8068.
18.Bardag-Gorce F,Francis T,Nan L,et al.Modifications in p62 occur due to proteasome inhibition in alcoholic liver disease.Life Sci 2005; 77(20):2594-2602.
19.Erlich S,Alexandrovich A,Shohami E,et al.Rapamycin is a neuroprotective treatment for traumatic brain injury.Neurobiol Dis 2007; 26(1):86-93.
20.Lavieu G,Scarlatti F,Sala G,et al.Regulation of autophagy by sphingosine kinase 1 and its role in cell survival during nutrient starvation.J Biol Chem 2006;281(13):8518-8527.
21.Kamada Y,Funakoshi T,Shintani T,et al.Tor-mediated induction of autophagy via an Apgl protein kinase complex.J Cell Biol 2000; 150(6):1507-1513.
22.Djavaheri-Mergny M,Amelotti M,Mathieu J,et al.NF-kB activation represses tumor necrosis factor-alpha-induced autophagy.J Biol Chem 2006; 281(41): 30373-30382.
23.Liu Z,Lenardo MJ.Reactive oxygen species regulate autophagy through redox-sensitive proteases.Dev Cell 2007; 12(4):484-485.
24.Scarlatti F,Bauvy C,Ventruti A,et al.Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.J Biol Chem 2004; 279(18):18384-18391.
25.Scherz-Shouval R,Shvets E,Fass E,et al.Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.EMBO J 2007; 26(7):1749-1760.
26.Monastyrska I,Klionsky DJ.Autophagy in organelle homeostasis:peroxisome turnover.Mol Aspects Med 2006; 27(5-6):483-494.
27.Nair U,Klionsky DJ.Molecular mechanisms and regulation of specific and nonspecific autophagy pathways in yeast.J Biol Chem 2005; 280(51):41785-41788.
28.Dunn WA,Jr.,Cregg JM,Kiel JAKW,et al.Pexophagy:the selective autophagy of peroxisomes.Autophagy 2005; 1(2):75-83.
29.Birmingham CL,Brumell JH.Autophagy recognizes intracellular Salmonella enterica serovar Typhimurium in damaged vacuoles.Autophagy 2006; 2(3):156-158.
30.Colombo MI,Gutierrez MG,Romano PS.The two faces of autophagy:Coxiella and Mycobacterium.Autophagy 2006; 2(3):162-164.
31.Vergne I,Singh S,Roberts E,et al.Autophagy in immune defense against Mycobacterium tuberculosis.Autophagy 2006; 2(3):175-178.
32.Yoshimori T.Autophagy vs.Group A Streptococcus.Autophagy 2006; 2(3):154-155.
33.Talloczy Z,Virgin HW,IV,Levine B.PKR-dependent autophagic degradation of herpes simplex virus type 1.Autophagy 2006; 2(3):24-29.
34.Guan J,Stromhaug PE,George MD,et al.Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport,pexophagy,and autophagy in Saccharomyces cerevisiae and Pichia pastoris.Mol Biol Cell 2001; 12(12):3821-3838.
35.Proikas-Cezanne T,Ruckerbauer S,Stierhof YD,et al.Human WIPI-1 puncta-formation:A novel assay to assess mammalian autophagy.FEBS Lett 2007;24;581(18):3396-3404.
36.Petiot A,Ogier-Denis E,Blommaart EF,et al.Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control,macroautophagy in HT-29 cells.J Biol Chem 2000; 275(2):992-998.
37.Kovacs AL,Seglen PO.Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis.Biochim Biophys Acta 1981; 676(2):213-220.
38.Kovacs AL,Laszlo L,Kovacs J.Effect of amino acids and cycloheximide on changes caused by vinblastine,leupeptin and methylamine in the autophagic/lysosomal system of mouse hepatocytes in vivo.Exp Cell Res 1985;157(1):83-94.
39.Kovacs J,Fellinger E,Karpati AP,et al.Morphometric evaluation of the turnover of autophagic vacuoles after treatment with Triton X-100 and vinblastine in murine pancreatic acinar and seminal vesicle epithelial cells.Virchows Arch B Cell Pathol Incl Mol Pathol 1987; 53(3):183-190.
40.Kovacs J,Laszlo L,Kovacs AL.Regression of autophagic vacuoles in pancreatic acinar,seminal vesicle epithelial,and liver parenchymal cells:a comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment.Exp Cell Res 1988; 174(1):244-251.
41.Cantino D,Mosso R,Baccino FM.Changes induced by fasting and cycloheximide in the vacuolar apparatus of rat hepatocytes.A morphometric investigation.Boll Soc Ital Biol Sper 1979; 55(18):1884-1889.
42.Kovacs J.Regression of autophagic vacuoles in seminal vesicle cells following cycloheximide treatment.Exp Cell Res 1983; 144(1):231-234.
43.Kovacs J.Morphometric study of the effect of leupeptin,vinblastine,estron acetate and cycloheximide on the autophagic vacuole-lysosomal compartments in mouse seminal vesicle cells.Virchows Arch B Cell Pathol Incl Mol Pathol 1983;42(1):83-93.
44.Kovacs J,Fellinger E,Karpati PA,et al.The turnover of autophagic vacuoles:evaluation by quantitative electron microscopy.Biomed Biochim Acta 1986;45(11-12):1543-1547.
45.Papadopoulos T,Pfeifer U.Regression of rat liver autophagic vacuoles by locally applied cycloheximide.Lab Invest 1986;54(1):100-107.
46.Rumpelt HJ,Albring M,Thoenes W.Prevention of D-galactosamine-induced hepatocellular autophagocytosis by cycloheximide.Virchows Arch B Cell Pathol 1974;16(2):195-203.
47.Rumpelt HJ,Weisbach T.Effect of cycloheximide on glucagon-induced autophagy.Quantitative examinations on hepatocytes in the rat.Am J Pathol 1978;91(1):49-56.
48.Sarkar S,Floto RA,Berger Z,et al.Lithium induces autophagy by inhibiting inositol monophosphatase.J Cell Biol 2005;170(7):1101-1111.
49.Klionsky DJ.Autophagy:Autophagy and cancer.Georgetown,TX:Landes Bioscience,2004.
50.Sarkar S,Perlstein EO,Imarisio S,et al.Small molecules enhance autophagy and reduce toxicity in Huntington's disease models.Nat Chem Biol 2007;3(6):331-338.
51.Zhang L,Yu J,Pan H,et al.Small molecule regulators of autophagy identified by an image-based high-throughput screen.Proc Natl Acad Sci U S A 2007;104(48):19023-19028.
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