高三尖杉酯碱通过影响信号传导途径和基因调控作用诱导T－淋巴细胞白血病细胞凋亡
|
摘要
[目的]高三尖杉酯碱（Homoharringtonine，HHT）是从我国特有的植物海南粗
榧分离的有效抗癌药物。我国在世界上首先用于急性非淋巴细胞白血病（ANLL）的
临床治疗，疗效显著，HHT对慢性髓细胞性白血病同样有效。近年，国内外对HHT
的作用机制的研究发现HHT有抑制蛋白合成、克隆形成以及诱导细胞分化的作用；
对~3H标记的胸腺嘧啶核苷掺入DNA也有影响。研究还发现HHT能促K562细胞
c-myc基因上调，启动凋亡；在凋亡后期能使c-fos基因表达增加，表明HHT促细
胞凋亡是受基因调控的。HHT在体外能诱导HL-60、K562和Jurkat细胞的细胞凋
亡，但未进一步阐述其促凋亡的机理。迄今，人们对HHT促凋亡作用限于髓系白血
病的研究，而HHT作用于T淋巴细胞白血病的促凋亡及其机理研究尚未见报道。
通过对HHT致白血病细胞凋亡的研究，将有助于深入地了解化疗药物对肿瘤细
胞凋亡及其信号传导途径的调控，为进一步提高HHT的临床疗效，扩大HHT的临
床应用范围提供理论依据和研究思路。
[方法］
1 应用流式细胞仪技术、末端脱氧核糖核酸片段标记和细胞染色方法，研究HHT
浙江大学博士学位论文
促人 T－淋巴细胞白血病细胞株 Molt3（P53“）、MO卜16（P53”）、Ichikawa（P53”’）、
Jurkat（P53”’）和 HL－60（P53－）细胞凋亡、细胞周期的改变及其与抑癌基因 P53的
关系。
2 流式细胞仪检测细胞APO表面抗原表达及抗FAS和抗FAS配体作用后的凋
亡、检测线粒体膜电位的改变、ROS产生和抗氧化剂的作用；比色法检测caspase－3
活性及caspase－3－8、－9抑制剂影响HHT对线粒体膜电位改变和细胞凋亡。
3 半定量逆转录酶－多聚酶链式反应检测 HHT对细胞内 Bax和 Bcl－2 mRNA表达
的调控。
4 通过共聚焦激光扫描显微镜分析，线粒体示踪剂 Mito Tracker、Bax和细胞色素
C单克隆抗体及与带有荧光cy－5的羊抗鼠u抗作用，研究Bax和细胞色素C在HHT
作用前后的细胞内的定位。
［结果］
IHHT能诱导T－白血病细胞凋亡，具时间和剂量依赖性，且与P53存在形式相关，
影响细胞周期，阻滞细胞在 GI期 HHT（Zn旮l、10nyml、25 nyml、50 n旮ml）分
别作用于 Mort3（P53“）、Moltl6（P53“）；Ichikawa（P53”’）、Jurkat（P53”’）细胞
和 HL－60（P53－）24小时，Molt3、Moltl6细胞凋亡率随着剂量加大凋亡率增加；Ichikawa
和 Jurkat细胞在 HHT相同剂量时的凋亡率比 Molt3、Moltl 6f氏午值分别＜0刀5）；
而H卜60细胞经HH T作用后，其凋亡率明显低于M。1G、讪1在16 o值分别＜O刀5），
与 Ichikawa和 Jurkat的凋亡率无明显差异厂值分别＞0刀5）。将不同浓度 HHT分别
作用于 Molt3细胞 6、12、18、24小时，其凋亡率随着时间延长而增加。
2 HH”H不影响FAS受体／配体（FAS－R／FAS－L）对Molt3细胞的凋亡诱导作用
HHT作用MOlt－3细胞后的相对荧光密度显示，HHT无诱导FASK表达增强作用；
细胞经抗人FAS单抗CHll作用后，显示凋亡，但HHT对CHll无协同或增强作用；
FAS配体中和抗体（NOKI、2）对 HHT作用的细胞凋亡无任何阻断作用。
2
一
3 HHT作用后细胞线粒体膜电位（么wm）的改变 荧光染料 JC＊显示线粒体膜
电位的存在和下降。当 MOltS细胞经 HHT 25n咖1处理12h后，线粒体膜电位下降，
荧光发散由 3％增加至 42％，与细胞凋亡的百分率相近，推测 HHT诱导细胞凋亡是
与线粒体膜电位下降有关。
4 HHT作用后细胞内细胞色素 C定位的改变在未经 HHT处理的细胞内，细胞色
素 C位于线粒体内，HHT 25ng／il处理细胞 12h后则弥散地分布在 Annexin－V阳性
的早期凋亡细胞的胞浆内。
5 HHT通过激活 caspasel诱导细胞凋亡 在 HHT10ng／ml和 50pgipl水平，可观
察到Motr3细胞的caspase〕活性随着剂量加大而增强，同时，casPase〕活性也随
着 HHT作用时间延长而随之增加。用 caspase上、8、－9特异性抑制齐 Z－DEVD－FKK。
Z．IETD－FMK ＄［IZ．LEHD．FMK先于 HHT与细胞作用，发现 CSSp＆SC－3、－8、－9抑
制剂均能不同程度地抑制 Molt3细胞的凋亡；经caspasel抑制剂处理后，能部分抑
制 HHT处理后的 Ichikawa细胞凋亡，而 caspase｛、刁抑制剂无抑制作用；结果还
发现么mm几乎不受caspase上、七、习抑制剂影响。说明八pm在casPases作用的
上游；但caspase－8、－9抑＄剂言抑伟casnase3的活性，表明casPase－3在casPase－8、
－9的下游。
6 HHT处理的细胞有活性氧（ROS）产生 抗氧化剂 NAC预处理细胞，可观察到
NAC能减少ROS的产生。然而，抗氧化剂并不影响HHT对线粒体膜电位及CCPCSS
3活性，也不抑制HHT诱导的细胞凋亡；
7 Bcl－2、BAX mRNA表达与 HHT的作用的关系 HHT能上调 Molt3细胞BAX
表达、下调 Bc1E表达；IChik洲。细胞 BAX、BCI工的表达在 HHT作用前后未发生
改变：
8 B盼在HHT诱导细胞凋亡过程的表达和细胞内定位 经HHT 25ng／ffil作用12h
后，BSX即易位到线粒体内。细胞
OmECTrvE
Homoharringtonine (HHT) is a cephalotaxine alkaloid isolated from genus
Cephalotaxus. HHT was first introduced to clinic for treatment of leukemia in
l970' in China and has been proven to have strong ami-leukemia activity It is
used as an effective anti-leukemia drug clinica1ly in treatment of acute myeloid
leukemia as well as chronic myeloid leukemia. In the last few decades, studies
have shown that the inhibitory effects of HHT on leukemia cell groWth include
inhibition of protein synthesis, decrease of clonogenicity and induction of
leukemia cell differentiation. As evidenced by 'H-Thymidine incopofation
assnys, HHT also demonstrated the capacity to inhibit DNA synthesis in
leukemia cells. Recent studies indicated that HHT is caPable of uP-regulating of
c-myc and c-fos, consequently promoting aPoptosis in myeloid leukemia cells
such as K562 cells. Although the HHT's effect on inducing aPoptosis in cell
line models such as HL-60, K562 and Jurkat is obvious, the detailed mechanism
of HHT in regulation of aPoptosis in leukemia is not yet clear To date, little is
knOwn aboul the role that HHT plays in regUlation of aPoptosis in lymphocytic
leukemia. The study on the mechanisms of HHT in regulation of aPoptosis in
leukemia cells will give us insigh into further understanding of the role HHT
may play in regulation of aPoptosis in leukemia cel1s and to underiine the
signaling pNay that HHT may interact with duing aPoPt()sis induced by the
chemotheraPelltic drug. The data generated from this Stlldy will also provide
valuable information for designing chemotheraPeutical regimens in clinical
treatment of lymphocytic leukemia and to improve the clinical olltcome of
patients receiving anii-leukemic chemotheraPy
MATERIALS AND METHODS
1 In this study, four T-cell leukemia cell 1ines Mo1t-3, Molt~l6,
Ichikawa,Jurkat and one myeloid cell line HL-60 were used in the experiments
to detect the effects of HHT on induction of aPoptosis and to study the possible
mechanisms of HHT in regulation of aPoptosis of the cells. Wth the excePtion
of Molt-3 and Moltl6 cells, which expressed a wild type of p53, the other two
lines expressed mutant-type p53 in Ichikawa and Jurkat, null p53 expressed in
HL-60. Cell cycle was analyzed by fluorescence-activated Flow Cytometry
(FACS); DNA breakage resulting from HHT treatment was detected by TdT
transferase labeling. The morphologic observation using Giemsa staining was
made on various cells treated by HHT.
2 FACS analysis was also used fOr detection of FAS protein expressed on
cell surface, for evaluation of aPoptosis in the cells after treatment with ani-Fas
and anti-Fas ligand (Fas-L ) aniibodies, for detCction of mitochondrial
membrane potential (MMP ) and the generation of reactive oxygen species
(ROS ) in the cells, as well as the effect of ami-oxidants on genefation of ROS
in the cells tfCated with HHT Caspase-3 activity as well as the effects of the
inhibitors of CasPase-3, 8, 9 on MMP and on aPoPtosis in the cells induced by
HHT was also evaluated.
3 Semi-quanitative RT-PCR was used to evaluate the mRNA expression of
Bax and Bcl-2 in the cells treated with HHT
4 By labelling the cells with Mito Tracker Red CM-H,XROS and staining
subsequently with ani-cytochrome C and anti-Bax anibody the subcellular
localization and re-distribution of Bax and cytochrome C in cells after treatment
Wth HHT was analyzed by Confocal Laser Scan Microscopy
ussUrrs
l The results demonstfated that HHT was caPable of inducing aPoptosis in the
T-cell leukemia cells in time- and dose-dependam manners. The statlls of p53
has an effect on cell cycles of the cells as Gl arrest was observed in the two T-
cell lines with wild-type p53 and Ichikawa cells with mutant p53 after
treatmellt with HHT Twniy-four hours after tfCattheflt of Molt-3, Molt-l6,
Ichikawa, Jurkat and HL-60 cells with HHT at different doses of 2ng/ml,
10ng/ml, 25ng/ml and 50ng/ml, significan aPoptotic cells were detected in the
Mol
榧分离的有效抗癌药物。我国在世界上首先用于急性非淋巴细胞白血病（ANLL）的
临床治疗，疗效显著，HHT对慢性髓细胞性白血病同样有效。近年，国内外对HHT
的作用机制的研究发现HHT有抑制蛋白合成、克隆形成以及诱导细胞分化的作用；
对~3H标记的胸腺嘧啶核苷掺入DNA也有影响。研究还发现HHT能促K562细胞
c-myc基因上调，启动凋亡；在凋亡后期能使c-fos基因表达增加，表明HHT促细
胞凋亡是受基因调控的。HHT在体外能诱导HL-60、K562和Jurkat细胞的细胞凋
亡，但未进一步阐述其促凋亡的机理。迄今，人们对HHT促凋亡作用限于髓系白血
病的研究，而HHT作用于T淋巴细胞白血病的促凋亡及其机理研究尚未见报道。
通过对HHT致白血病细胞凋亡的研究，将有助于深入地了解化疗药物对肿瘤细
胞凋亡及其信号传导途径的调控，为进一步提高HHT的临床疗效，扩大HHT的临
床应用范围提供理论依据和研究思路。
[方法］
1 应用流式细胞仪技术、末端脱氧核糖核酸片段标记和细胞染色方法，研究HHT
浙江大学博士学位论文
促人 T－淋巴细胞白血病细胞株 Molt3（P53“）、MO卜16（P53”）、Ichikawa（P53”’）、
Jurkat（P53”’）和 HL－60（P53－）细胞凋亡、细胞周期的改变及其与抑癌基因 P53的
关系。
2 流式细胞仪检测细胞APO表面抗原表达及抗FAS和抗FAS配体作用后的凋
亡、检测线粒体膜电位的改变、ROS产生和抗氧化剂的作用；比色法检测caspase－3
活性及caspase－3－8、－9抑制剂影响HHT对线粒体膜电位改变和细胞凋亡。
3 半定量逆转录酶－多聚酶链式反应检测 HHT对细胞内 Bax和 Bcl－2 mRNA表达
的调控。
4 通过共聚焦激光扫描显微镜分析，线粒体示踪剂 Mito Tracker、Bax和细胞色素
C单克隆抗体及与带有荧光cy－5的羊抗鼠u抗作用，研究Bax和细胞色素C在HHT
作用前后的细胞内的定位。
［结果］
IHHT能诱导T－白血病细胞凋亡，具时间和剂量依赖性，且与P53存在形式相关，
影响细胞周期，阻滞细胞在 GI期 HHT（Zn旮l、10nyml、25 nyml、50 n旮ml）分
别作用于 Mort3（P53“）、Moltl6（P53“）；Ichikawa（P53”’）、Jurkat（P53”’）细胞
和 HL－60（P53－）24小时，Molt3、Moltl6细胞凋亡率随着剂量加大凋亡率增加；Ichikawa
和 Jurkat细胞在 HHT相同剂量时的凋亡率比 Molt3、Moltl 6f氏午值分别＜0刀5）；
而H卜60细胞经HH T作用后，其凋亡率明显低于M。1G、讪1在16 o值分别＜O刀5），
与 Ichikawa和 Jurkat的凋亡率无明显差异厂值分别＞0刀5）。将不同浓度 HHT分别
作用于 Molt3细胞 6、12、18、24小时，其凋亡率随着时间延长而增加。
2 HH”H不影响FAS受体／配体（FAS－R／FAS－L）对Molt3细胞的凋亡诱导作用
HHT作用MOlt－3细胞后的相对荧光密度显示，HHT无诱导FASK表达增强作用；
细胞经抗人FAS单抗CHll作用后，显示凋亡，但HHT对CHll无协同或增强作用；
FAS配体中和抗体（NOKI、2）对 HHT作用的细胞凋亡无任何阻断作用。
2
一
3 HHT作用后细胞线粒体膜电位（么wm）的改变 荧光染料 JC＊显示线粒体膜
电位的存在和下降。当 MOltS细胞经 HHT 25n咖1处理12h后，线粒体膜电位下降，
荧光发散由 3％增加至 42％，与细胞凋亡的百分率相近，推测 HHT诱导细胞凋亡是
与线粒体膜电位下降有关。
4 HHT作用后细胞内细胞色素 C定位的改变在未经 HHT处理的细胞内，细胞色
素 C位于线粒体内，HHT 25ng／il处理细胞 12h后则弥散地分布在 Annexin－V阳性
的早期凋亡细胞的胞浆内。
5 HHT通过激活 caspasel诱导细胞凋亡 在 HHT10ng／ml和 50pgipl水平，可观
察到Motr3细胞的caspase〕活性随着剂量加大而增强，同时，casPase〕活性也随
着 HHT作用时间延长而随之增加。用 caspase上、8、－9特异性抑制齐 Z－DEVD－FKK。
Z．IETD－FMK ＄［IZ．LEHD．FMK先于 HHT与细胞作用，发现 CSSp＆SC－3、－8、－9抑
制剂均能不同程度地抑制 Molt3细胞的凋亡；经caspasel抑制剂处理后，能部分抑
制 HHT处理后的 Ichikawa细胞凋亡，而 caspase｛、刁抑制剂无抑制作用；结果还
发现么mm几乎不受caspase上、七、习抑制剂影响。说明八pm在casPases作用的
上游；但caspase－8、－9抑＄剂言抑伟casnase3的活性，表明casPase－3在casPase－8、
－9的下游。
6 HHT处理的细胞有活性氧（ROS）产生 抗氧化剂 NAC预处理细胞，可观察到
NAC能减少ROS的产生。然而，抗氧化剂并不影响HHT对线粒体膜电位及CCPCSS
3活性，也不抑制HHT诱导的细胞凋亡；
7 Bcl－2、BAX mRNA表达与 HHT的作用的关系 HHT能上调 Molt3细胞BAX
表达、下调 Bc1E表达；IChik洲。细胞 BAX、BCI工的表达在 HHT作用前后未发生
改变：
8 B盼在HHT诱导细胞凋亡过程的表达和细胞内定位 经HHT 25ng／ffil作用12h
后，BSX即易位到线粒体内。细胞
OmECTrvE
Homoharringtonine (HHT) is a cephalotaxine alkaloid isolated from genus
Cephalotaxus. HHT was first introduced to clinic for treatment of leukemia in
l970' in China and has been proven to have strong ami-leukemia activity It is
used as an effective anti-leukemia drug clinica1ly in treatment of acute myeloid
leukemia as well as chronic myeloid leukemia. In the last few decades, studies
have shown that the inhibitory effects of HHT on leukemia cell groWth include
inhibition of protein synthesis, decrease of clonogenicity and induction of
leukemia cell differentiation. As evidenced by 'H-Thymidine incopofation
assnys, HHT also demonstrated the capacity to inhibit DNA synthesis in
leukemia cells. Recent studies indicated that HHT is caPable of uP-regulating of
c-myc and c-fos, consequently promoting aPoptosis in myeloid leukemia cells
such as K562 cells. Although the HHT's effect on inducing aPoptosis in cell
line models such as HL-60, K562 and Jurkat is obvious, the detailed mechanism
of HHT in regulation of aPoptosis in leukemia is not yet clear To date, little is
knOwn aboul the role that HHT plays in regUlation of aPoptosis in lymphocytic
leukemia. The study on the mechanisms of HHT in regulation of aPoptosis in
leukemia cells will give us insigh into further understanding of the role HHT
may play in regulation of aPoptosis in leukemia cel1s and to underiine the
signaling pNay that HHT may interact with duing aPoPt()sis induced by the
chemotheraPelltic drug. The data generated from this Stlldy will also provide
valuable information for designing chemotheraPeutical regimens in clinical
treatment of lymphocytic leukemia and to improve the clinical olltcome of
patients receiving anii-leukemic chemotheraPy
MATERIALS AND METHODS
1 In this study, four T-cell leukemia cell 1ines Mo1t-3, Molt~l6,
Ichikawa,Jurkat and one myeloid cell line HL-60 were used in the experiments
to detect the effects of HHT on induction of aPoptosis and to study the possible
mechanisms of HHT in regulation of aPoptosis of the cells. Wth the excePtion
of Molt-3 and Moltl6 cells, which expressed a wild type of p53, the other two
lines expressed mutant-type p53 in Ichikawa and Jurkat, null p53 expressed in
HL-60. Cell cycle was analyzed by fluorescence-activated Flow Cytometry
(FACS); DNA breakage resulting from HHT treatment was detected by TdT
transferase labeling. The morphologic observation using Giemsa staining was
made on various cells treated by HHT.
2 FACS analysis was also used fOr detection of FAS protein expressed on
cell surface, for evaluation of aPoptosis in the cells after treatment with ani-Fas
and anti-Fas ligand (Fas-L ) aniibodies, for detCction of mitochondrial
membrane potential (MMP ) and the generation of reactive oxygen species
(ROS ) in the cells, as well as the effect of ami-oxidants on genefation of ROS
in the cells tfCated with HHT Caspase-3 activity as well as the effects of the
inhibitors of CasPase-3, 8, 9 on MMP and on aPoPtosis in the cells induced by
HHT was also evaluated.
3 Semi-quanitative RT-PCR was used to evaluate the mRNA expression of
Bax and Bcl-2 in the cells treated with HHT
4 By labelling the cells with Mito Tracker Red CM-H,XROS and staining
subsequently with ani-cytochrome C and anti-Bax anibody the subcellular
localization and re-distribution of Bax and cytochrome C in cells after treatment
Wth HHT was analyzed by Confocal Laser Scan Microscopy
ussUrrs
l The results demonstfated that HHT was caPable of inducing aPoptosis in the
T-cell leukemia cells in time- and dose-dependam manners. The statlls of p53
has an effect on cell cycles of the cells as Gl arrest was observed in the two T-
cell lines with wild-type p53 and Ichikawa cells with mutant p53 after
treatmellt with HHT Twniy-four hours after tfCattheflt of Molt-3, Molt-l6,
Ichikawa, Jurkat and HL-60 cells with HHT at different doses of 2ng/ml,
10ng/ml, 25ng/ml and 50ng/ml, significan aPoptotic cells were detected in the
Mol
引文
1.Cephalotaxus Research Coordinating. Chin. M.J 1976, 2: 263.
2.Kantarjian H, Talpaz J, Cortes MB, et al. Homoharringtonine and Iow-dose cytosine(Ldara-C)in late chronic phase chronic myelogenous leukemia(CML).Blood 1999, 94: 274b.
3.Visan G, Russo D, Ottaviani E, et al. Effect of homoharringtonine alone and in combination with alpha interferon a cytosine arabinoside on "in vitro" growth and induction of apoptosis in chronic myeloid leukemia and normal hematopoitic progenitors. Leukemia 1997, 11: 624-628
4.Zhou JY, Chen DL,Shen ZS, et al. Effect of homoharringtonine on proliferation and differentiation of human leukemic cells in vitro. Cancer Res 1990, 50:2031.
5．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)：375
6.Junghanss C, Etzorodt D, Jain SK, et al. et al. Homoharringtonine in duced apoptosis in HL-60, K562 and Jurkat cells, but not in Rail.Blood 1999, 94: 192b.
7.Solary E. Apoptosis: molecular mechanisms. C-R-Seances-Soc-Biol-Fil 1998,192:1065-76
8.Ohta S, Ishibashi Y. Moleculaer mechanisms of apoptosis. No-To-Hattatsu 1999,31:122
9.Hoffmann K.The modular nature of apoptotic signaling proteins.Cell Mol Life Sci 1999, 55:1113-28
10.McGahon A,Bissonnette R, Schmitt M,et al. Bcr/abl maintains resistence of chronic myelogenous leukemia cell to apoptotic cell death. Blood 1994,83:1179
11.Kerr JFR,Wyllie AH, Currie AR. Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972,26:239-257
12.Thornberry NA, Lazebnik Y. caspases: enemies within. Science 1998, 281:1312-16
13．李林，夏丽娟，蒋超，等．三尖杉酯碱和高三尖杉酯碱诱导人早幼粒白血病细胞的程序性死亡．药理学报 1994，29：667
14.Krammer DL, Vujcic S, Diegelman P. Polyamine Analogue induction of the P53-P21 WAFl/CIP1-Rb pathway and Gl arrest in human melanoma cells. Cancer Res 1999, 59: 1278
15.Harper JW,Adami GR, Wei N,et al. The p21 cdk-interacting protein Cipl is a potent inhibitor of Gl cyclin-dependent kinases. Cell 1993, 75:805-816
16.Waldman T, kinzler KW, Vogelstein B. P21 is necessary for p53-mediated Gl arrest in human cancer ceil. Cancer Res 1995, 55:5187-5190
17.Lowe SW, Ruley HE, Jacks T. P53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993, 67:205
18.Hockenberry D, Nunez G, Milliman C, et al. bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990,348:334
19.Hsiao MH, Yu AL, Yeargin J, et al. Nonhereditary p53 mutation in T-cell acute lymphoblastic leukemia are associated with the relapse phase. Blood 1994,83:2922-30
20.Dittmer D, Pati S, Zambetti G, Gain of function mutation in P53. Nat Genet 1993, 4:42
21.Rowan S, Ludwig RL, Haupt Y, et al. Specific loss of apoptotic but not cell cycle arrest function in a human tumor derived p53 mutant.EMBO J 1996,15:827-838
22.Munker R, Loert M, Yonehara S, et al. Expressio of the Fas antigen on primary human leukemia cells. Ann Hematol 1995, 70:15-17
23.Kaufmann SH, Earnshaw WC.Induction of apoptosis by cancer chemotherapy. Exp Cell Res 2000, 256:42-9
24.Fulda S, Friesen C ,Debatin KM. Molekular determinants of apoptosis induced by
cytotoxic drugs. Klin Padiatr 1998,210:148-52
25． Friesen C , Herr I, Krammer PH, et al. Involvement of the CD95 (APO-1/FAS) receptor/ ligand system in drug-induced apoptosis in leukemia cells. Nat-Med 1996,2:574-7
26． Peter ME, Krammer PH, Mechanism of CD95(APO-l/FAS)-mediated apoptosis. Curr Opin Immunol 1998, 10: 545-51
27． NagataS, Golstein p. The Fas death factor. Science 1995, 267:1449-55
28． Matsumura H, Schimizu Y, Ohsawa Y, et al. Necrosis death pathway in Fas receptor signaling. J Cell Biol 2000,151:1247-56
29． Westendorf JJ, Lammert JM, Jelinek DF, et al. Expression and function of Fas (Apo-l/CD95) in patient myeloma cells and myeloma cell. Blood 1995, 85: 3566-76
30． Gottliebe RA. Mitochondria: execution central. FEBS Lett 2000, 48:6-12
31． Roemer G. Zamzami N. Susin SA; Mitochondial control of apoptosis. Immunol Today 1997, 18:44-51
32． Douglas R G. John C R. Mitochondria and Apoptosis. Science 1998,281:1309-1312
33． Li P. Nijhawan D. Budihardjo I. et al. Cytochrome c and dATP-dependent formation of Apaf-1/Casppase 9 complex initiates an Apoptotic protease cascade. Cell 1997, 91: 479-489
34． Liu X, Kim CN, Yang J, et al. Induction of Apoptotic program in cell free extracts: requrement for dATP and cytochrome c. Cell 1996, 86: 147-157
35． Garl JM, Rudin C.Cytochrome c induces caspase-depenent apoptosis in intact hematopoitic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling map-kinase and malignant change. Blood 1998, 92:1235-46
36． Hu Y, Benedict MA，Ding L，et al．Role of cytochrome c and dATP／ATP hydrolysis in Apaf-1-mediated caspase-9 activition and apoptosis． EMBO J 1999，18：3586-95
37． Sanchez AJA，Khodjakov A，Schneider E，et al．Anticancer drugs induced increased mitochondrial cytochrome c expression in precede cell death． Cancer Res 2001，63：1038-44
38． Tan S，Sagara Y，Liu y, et al．The regulation of ROS production during programmed cell death．J Cell Biol 1998,141：1423-32
39． Dumont A，Hehner SP,Hofmann TG，et al．Hydrogen peroxide-induced apoptosis is CD95-independent，requires the release of mitocjondrial-derived reactive oxygen species and the activation of NF-kappaB． Oncogene 1999，21：747-57
40． Zheng A，Mantymaa P,Saily M，et al．An association between mitochondrial function and ATRA induced apoptosis in AML cells．Br J Heamatol 1999,105：215-24
41． Cai JY,Jones DP. Mitochondrial redox signaling during apoptosis．J Biol and Biomember 1999，31：327-334
42． Hirpara JL，Clement MV，Pervaiz S，et al．Intracellular oxidification triggered by mitochondrial-drived hydrogen peroxide in an effector mecjanosms for drug-induced apoptosis in tumor cells．J Biol Chem 2001,276：514-21
43． Hockenbery DM，Oltvai ZN，Yin XM，et al．Bcl-2 functions in an antioxidant pathway to prevent apoptosis．Cell 1994，75：241
44． Jacobson MD．Apoptosis：bcl-2-related proteins get connected．Curr Biol 1997， 7：R277
45． Wickremasinghe R G．Hoffbrand A V. Biochemical and genetic control of apoptosis： relavauce to normal hematopoiesis and hematological malignancies．Blood 1999，93： 3587-3600
46． Imawati B，Holt O，Michael L，et al．Biochemical pathway of caspase activation
during apoptosis．Annu Rev Cell Dev Biol 1999，15：269-290
47． Thornberry NA．Lazebnik Y. caspase：Enemies within．Science 1998,281：1312-1316
48． Pandey P,Nakazawa A，Ito Y，et al．Requirment for caspase activation in monocytic differentiation of myoloid leukemia cells．Oncogene 2000，19：3941-7
49． Solary E，Drion N，Bettaicb A，et al．Positive and negative regulation of apoptosis pathways by cytotoxin agents in hematological malignencies．Leukemia 2000， 14；1833-49
50． Villa P, Kaufmann SH，Earnshaw WC．caspases and caspase inhibitor． Trends Biochem Sci 1997，22：388-93
51． Cain K．Brown D G．Langlais C．et al．caspase activation involves the formation of the apoposome，a large caspase activating complex． J Biol Chem 1999，274：22686-92
52． Yang J，Lue X，Bhalla K，et al．Prevention of apoptosis by bcl-2：release of cytochrome c from mitochondria blocked．Science 1997，275：1129-32
53． Srinivasula SM，Ahmad M，Fernandes AT,et al．Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization．Mol Cell 1998，1：949-57
54． Bossy WE，Green DR．caspase induce cytochrome c release from mitocjondria by activating cytosolic factors．J Biol Chem 1999，274：17484-90
55． Muzio M，Chinnaiyan AM，Kischkel FC，et al．FLICE，a novel FADD-homologous ICE／CED-3 like protease，is recruruited to the CD-95(Fas／apo-1) death-inducing signaling complex．Cell 1996，85：817
56． Blodin MP，Goncharov YV, wallach D，et al．Involvement of MACH，a novel MORT 1／FADD-interacting protease．in Fas／Apo-1 and TNF receptor-induced cell death．Cell 1996，85：803
57． Kuwana T，Smith JJ，Muzio M，et al．Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome C． J Biol Chem 1998，
26:16589-94
58． Slee EA, Harte MT, Kluck RM, et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspase-2． -3,-6,-7,-8,and-10 in a caspase-9-dependent manner. J Cell Biol 1999,144:281-292
59． Kitamura K, Minamie Y, Yamamoto T, et al. Involvement of CD-95-independent caspase-8 avtivation in arsenic trooxide-induced apoptosis. Leukemia 2000, 14:1740-50
60． Hegde R, Srinivasula SM, Ahmad M, et al. Blk, a BH3-containing mouse protein that intereacts with bcl-2 and bcl-2 and bcl-XL, is a potent death agonist. J Biol Chem 1998,14:7783-7786
61． Engele IH, Stepcrynska A, Stroh C, et al. caspase-8/FLICE function as an executioner caspase in anticancer drug-induced apoptosis. Oncogene 2000, 19:4563-73
62． Tewari M, Quan LT, O'Rourke K,et al. Tama/CPP32 β, a mammalian homolog of ced-3, is crmA-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase. Cell 1995, 81:801
63． Tsujimoto Y, Gorham J, Cossman J, et al. The t (14; 18) chromosome translocations involved in B-cell neoplasmas result from mistakes in VDJ joining. Science 985,229; 1390
64． Cleary ML, Sklar J. Nucleotide sequence of at (14; 18 ) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near a transcriptionally active locus on chromosome 18． Proc Natl Acad Sci USA 1985,82: 7439
65． Yang E,Korsmeyer SJ. Molecular thanatosis: a discourse on the BCL-2 family and cell death. Blood 1996,88;386-410
66． Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an inner mitochondrial
membrane protein that blocks programmed cell death．Nature 1990，384：334
67． Holinger EP，Chittenden T，Lutz RJ，et al．Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent actovation of caspases． J Biol Chem 1999． 274：13298-304
68． Nuessler V, Stotzer O，Gullis E，et al．Bcl-2，bax and bcl-xL expression in human sensitive and resistant leukemia cell lines．Leukemia 1999,13：1864-1872
69． Delia D，Arello A，Soligo D，et al．Bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells．Blood 1992，79：1291-1298
70． Fadeel B，Hassan Z，Hellstrom E，et al．Cleavage of bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells．Leukemia 1999， 13：719-728
71． Kroemer G．The proto-oncogene Bcl-2 and its role in regulating apoptosis。Nature Med 1997,3：614-620
72． Armstrong JS，Steinauer KK，French J，et al．Bcl-2 inhibitors apoptosis induced by mitochondrial upcoupling but does not prevent mitochondrial transmembrane depolamization．Exp Cell Res 2001，262：170-9
73． Hsu YT，Wolter KG，Youle RJ．Cytosol-to-membrane redistribution of bax and bcl-xl during apoptosis．Proc Natl Acad Sci USA 1997，94：3668-3672
74． Wang K，Gross A，Waksman G，et al．Mutagenesis of the BH3-domain of Bax identifies residues critical for dimerization and killing．Mol Cell Biol 1998，18：6081-89
75． Zhivotovsky B，Orrenius S，Brustugun OT,et al．Injected cytochrome-c induces apoptosis．Nature 1998，391：449-450
76． Li PF，Dietz R，Harsdorf R．P53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis
blocked by bcl-2 . EMBO J 1999,18：6027-6038
77． Liu X，Kim CM,Yang J，et al．induction of apoptotic program in cell-free extracts： requirement for dATP and cytochrome c．Cell 1996，12：147-157
78． Juergensmeier JM，Xie Z，Deveraux Q，et al.Bax directly induces release of cytochrome c from isolated mitochondria．Proc Natl Acad sci USA 1998，95：4997-5002
79． Shimizu S，Eguchi Y, Kamiike W, et al．Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating propon flux．Proc Natl Acad Sci USA 1998，95：1455-59
80． Marshansky V,Wang X，Bertrand R，et al．Proteansomes modulate balance among proapoptotic and antiapoptotic bcl-2 family members and compromise functioning of the electron tansport chani in leukemia cells．J Immunol 2001，166：3130-42
81． Agarwal ML Taylor WR，Chernov MV,et al.The p53 network．． J Biol chem 1999．
2.Kantarjian H, Talpaz J, Cortes MB, et al. Homoharringtonine and Iow-dose cytosine(Ldara-C)in late chronic phase chronic myelogenous leukemia(CML).Blood 1999, 94: 274b.
3.Visan G, Russo D, Ottaviani E, et al. Effect of homoharringtonine alone and in combination with alpha interferon a cytosine arabinoside on "in vitro" growth and induction of apoptosis in chronic myeloid leukemia and normal hematopoitic progenitors. Leukemia 1997, 11: 624-628
4.Zhou JY, Chen DL,Shen ZS, et al. Effect of homoharringtonine on proliferation and differentiation of human leukemic cells in vitro. Cancer Res 1990, 50:2031.
5．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)：375
6.Junghanss C, Etzorodt D, Jain SK, et al. et al. Homoharringtonine in duced apoptosis in HL-60, K562 and Jurkat cells, but not in Rail.Blood 1999, 94: 192b.
7.Solary E. Apoptosis: molecular mechanisms. C-R-Seances-Soc-Biol-Fil 1998,192:1065-76
8.Ohta S, Ishibashi Y. Moleculaer mechanisms of apoptosis. No-To-Hattatsu 1999,31:122
9.Hoffmann K.The modular nature of apoptotic signaling proteins.Cell Mol Life Sci 1999, 55:1113-28
10.McGahon A,Bissonnette R, Schmitt M,et al. Bcr/abl maintains resistence of chronic myelogenous leukemia cell to apoptotic cell death. Blood 1994,83:1179
11.Kerr JFR,Wyllie AH, Currie AR. Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972,26:239-257
12.Thornberry NA, Lazebnik Y. caspases: enemies within. Science 1998, 281:1312-16
13．李林，夏丽娟，蒋超，等．三尖杉酯碱和高三尖杉酯碱诱导人早幼粒白血病细胞的程序性死亡．药理学报 1994，29：667
14.Krammer DL, Vujcic S, Diegelman P. Polyamine Analogue induction of the P53-P21 WAFl/CIP1-Rb pathway and Gl arrest in human melanoma cells. Cancer Res 1999, 59: 1278
15.Harper JW,Adami GR, Wei N,et al. The p21 cdk-interacting protein Cipl is a potent inhibitor of Gl cyclin-dependent kinases. Cell 1993, 75:805-816
16.Waldman T, kinzler KW, Vogelstein B. P21 is necessary for p53-mediated Gl arrest in human cancer ceil. Cancer Res 1995, 55:5187-5190
17.Lowe SW, Ruley HE, Jacks T. P53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993, 67:205
18.Hockenberry D, Nunez G, Milliman C, et al. bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990,348:334
19.Hsiao MH, Yu AL, Yeargin J, et al. Nonhereditary p53 mutation in T-cell acute lymphoblastic leukemia are associated with the relapse phase. Blood 1994,83:2922-30
20.Dittmer D, Pati S, Zambetti G, Gain of function mutation in P53. Nat Genet 1993, 4:42
21.Rowan S, Ludwig RL, Haupt Y, et al. Specific loss of apoptotic but not cell cycle arrest function in a human tumor derived p53 mutant.EMBO J 1996,15:827-838
22.Munker R, Loert M, Yonehara S, et al. Expressio of the Fas antigen on primary human leukemia cells. Ann Hematol 1995, 70:15-17
23.Kaufmann SH, Earnshaw WC.Induction of apoptosis by cancer chemotherapy. Exp Cell Res 2000, 256:42-9
24.Fulda S, Friesen C ,Debatin KM. Molekular determinants of apoptosis induced by
cytotoxic drugs. Klin Padiatr 1998,210:148-52
25． Friesen C , Herr I, Krammer PH, et al. Involvement of the CD95 (APO-1/FAS) receptor/ ligand system in drug-induced apoptosis in leukemia cells. Nat-Med 1996,2:574-7
26． Peter ME, Krammer PH, Mechanism of CD95(APO-l/FAS)-mediated apoptosis. Curr Opin Immunol 1998, 10: 545-51
27． NagataS, Golstein p. The Fas death factor. Science 1995, 267:1449-55
28． Matsumura H, Schimizu Y, Ohsawa Y, et al. Necrosis death pathway in Fas receptor signaling. J Cell Biol 2000,151:1247-56
29． Westendorf JJ, Lammert JM, Jelinek DF, et al. Expression and function of Fas (Apo-l/CD95) in patient myeloma cells and myeloma cell. Blood 1995, 85: 3566-76
30． Gottliebe RA. Mitochondria: execution central. FEBS Lett 2000, 48:6-12
31． Roemer G. Zamzami N. Susin SA; Mitochondial control of apoptosis. Immunol Today 1997, 18:44-51
32． Douglas R G. John C R. Mitochondria and Apoptosis. Science 1998,281:1309-1312
33． Li P. Nijhawan D. Budihardjo I. et al. Cytochrome c and dATP-dependent formation of Apaf-1/Casppase 9 complex initiates an Apoptotic protease cascade. Cell 1997, 91: 479-489
34． Liu X, Kim CN, Yang J, et al. Induction of Apoptotic program in cell free extracts: requrement for dATP and cytochrome c. Cell 1996, 86: 147-157
35． Garl JM, Rudin C.Cytochrome c induces caspase-depenent apoptosis in intact hematopoitic cells and overrides apoptosis suppression mediated by bcl-2, growth factor signaling map-kinase and malignant change. Blood 1998, 92:1235-46
36． Hu Y, Benedict MA，Ding L，et al．Role of cytochrome c and dATP／ATP hydrolysis in Apaf-1-mediated caspase-9 activition and apoptosis． EMBO J 1999，18：3586-95
37． Sanchez AJA，Khodjakov A，Schneider E，et al．Anticancer drugs induced increased mitochondrial cytochrome c expression in precede cell death． Cancer Res 2001，63：1038-44
38． Tan S，Sagara Y，Liu y, et al．The regulation of ROS production during programmed cell death．J Cell Biol 1998,141：1423-32
39． Dumont A，Hehner SP,Hofmann TG，et al．Hydrogen peroxide-induced apoptosis is CD95-independent，requires the release of mitocjondrial-derived reactive oxygen species and the activation of NF-kappaB． Oncogene 1999，21：747-57
40． Zheng A，Mantymaa P,Saily M，et al．An association between mitochondrial function and ATRA induced apoptosis in AML cells．Br J Heamatol 1999,105：215-24
41． Cai JY,Jones DP. Mitochondrial redox signaling during apoptosis．J Biol and Biomember 1999，31：327-334
42． Hirpara JL，Clement MV，Pervaiz S，et al．Intracellular oxidification triggered by mitochondrial-drived hydrogen peroxide in an effector mecjanosms for drug-induced apoptosis in tumor cells．J Biol Chem 2001,276：514-21
43． Hockenbery DM，Oltvai ZN，Yin XM，et al．Bcl-2 functions in an antioxidant pathway to prevent apoptosis．Cell 1994，75：241
44． Jacobson MD．Apoptosis：bcl-2-related proteins get connected．Curr Biol 1997， 7：R277
45． Wickremasinghe R G．Hoffbrand A V. Biochemical and genetic control of apoptosis： relavauce to normal hematopoiesis and hematological malignancies．Blood 1999，93： 3587-3600
46． Imawati B，Holt O，Michael L，et al．Biochemical pathway of caspase activation
during apoptosis．Annu Rev Cell Dev Biol 1999，15：269-290
47． Thornberry NA．Lazebnik Y. caspase：Enemies within．Science 1998,281：1312-1316
48． Pandey P,Nakazawa A，Ito Y，et al．Requirment for caspase activation in monocytic differentiation of myoloid leukemia cells．Oncogene 2000，19：3941-7
49． Solary E，Drion N，Bettaicb A，et al．Positive and negative regulation of apoptosis pathways by cytotoxin agents in hematological malignencies．Leukemia 2000， 14；1833-49
50． Villa P, Kaufmann SH，Earnshaw WC．caspases and caspase inhibitor． Trends Biochem Sci 1997，22：388-93
51． Cain K．Brown D G．Langlais C．et al．caspase activation involves the formation of the apoposome，a large caspase activating complex． J Biol Chem 1999，274：22686-92
52． Yang J，Lue X，Bhalla K，et al．Prevention of apoptosis by bcl-2：release of cytochrome c from mitochondria blocked．Science 1997，275：1129-32
53． Srinivasula SM，Ahmad M，Fernandes AT,et al．Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization．Mol Cell 1998，1：949-57
54． Bossy WE，Green DR．caspase induce cytochrome c release from mitocjondria by activating cytosolic factors．J Biol Chem 1999，274：17484-90
55． Muzio M，Chinnaiyan AM，Kischkel FC，et al．FLICE，a novel FADD-homologous ICE／CED-3 like protease，is recruruited to the CD-95(Fas／apo-1) death-inducing signaling complex．Cell 1996，85：817
56． Blodin MP，Goncharov YV, wallach D，et al．Involvement of MACH，a novel MORT 1／FADD-interacting protease．in Fas／Apo-1 and TNF receptor-induced cell death．Cell 1996，85：803
57． Kuwana T，Smith JJ，Muzio M，et al．Apoptosis induction by caspase-8 is amplified through the mitochondrial release of cytochrome C． J Biol Chem 1998，
26:16589-94
58． Slee EA, Harte MT, Kluck RM, et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspase-2． -3,-6,-7,-8,and-10 in a caspase-9-dependent manner. J Cell Biol 1999,144:281-292
59． Kitamura K, Minamie Y, Yamamoto T, et al. Involvement of CD-95-independent caspase-8 avtivation in arsenic trooxide-induced apoptosis. Leukemia 2000, 14:1740-50
60． Hegde R, Srinivasula SM, Ahmad M, et al. Blk, a BH3-containing mouse protein that intereacts with bcl-2 and bcl-2 and bcl-XL, is a potent death agonist. J Biol Chem 1998,14:7783-7786
61． Engele IH, Stepcrynska A, Stroh C, et al. caspase-8/FLICE function as an executioner caspase in anticancer drug-induced apoptosis. Oncogene 2000, 19:4563-73
62． Tewari M, Quan LT, O'Rourke K,et al. Tama/CPP32 β, a mammalian homolog of ced-3, is crmA-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase. Cell 1995, 81:801
63． Tsujimoto Y, Gorham J, Cossman J, et al. The t (14; 18) chromosome translocations involved in B-cell neoplasmas result from mistakes in VDJ joining. Science 985,229; 1390
64． Cleary ML, Sklar J. Nucleotide sequence of at (14; 18 ) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near a transcriptionally active locus on chromosome 18． Proc Natl Acad Sci USA 1985,82: 7439
65． Yang E,Korsmeyer SJ. Molecular thanatosis: a discourse on the BCL-2 family and cell death. Blood 1996,88;386-410
66． Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an inner mitochondrial
membrane protein that blocks programmed cell death．Nature 1990，384：334
67． Holinger EP，Chittenden T，Lutz RJ，et al．Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent actovation of caspases． J Biol Chem 1999． 274：13298-304
68． Nuessler V, Stotzer O，Gullis E，et al．Bcl-2，bax and bcl-xL expression in human sensitive and resistant leukemia cell lines．Leukemia 1999,13：1864-1872
69． Delia D，Arello A，Soligo D，et al．Bcl-2 proto-oncogene expression in normal and neoplastic human myeloid cells．Blood 1992，79：1291-1298
70． Fadeel B，Hassan Z，Hellstrom E，et al．Cleavage of bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells．Leukemia 1999， 13：719-728
71． Kroemer G．The proto-oncogene Bcl-2 and its role in regulating apoptosis。Nature Med 1997,3：614-620
72． Armstrong JS，Steinauer KK，French J，et al．Bcl-2 inhibitors apoptosis induced by mitochondrial upcoupling but does not prevent mitochondrial transmembrane depolamization．Exp Cell Res 2001，262：170-9
73． Hsu YT，Wolter KG，Youle RJ．Cytosol-to-membrane redistribution of bax and bcl-xl during apoptosis．Proc Natl Acad Sci USA 1997，94：3668-3672
74． Wang K，Gross A，Waksman G，et al．Mutagenesis of the BH3-domain of Bax identifies residues critical for dimerization and killing．Mol Cell Biol 1998，18：6081-89
75． Zhivotovsky B，Orrenius S，Brustugun OT,et al．Injected cytochrome-c induces apoptosis．Nature 1998，391：449-450
76． Li PF，Dietz R，Harsdorf R．P53 regulates mitochondrial membrane potential through reactive oxygen species and induces cytochrome c-independent apoptosis
blocked by bcl-2 . EMBO J 1999,18：6027-6038
77． Liu X，Kim CM,Yang J，et al．induction of apoptotic program in cell-free extracts： requirement for dATP and cytochrome c．Cell 1996，12：147-157
78． Juergensmeier JM，Xie Z，Deveraux Q，et al.Bax directly induces release of cytochrome c from isolated mitochondria．Proc Natl Acad sci USA 1998，95：4997-5002
79． Shimizu S，Eguchi Y, Kamiike W, et al．Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating propon flux．Proc Natl Acad Sci USA 1998，95：1455-59
80． Marshansky V,Wang X，Bertrand R，et al．Proteansomes modulate balance among proapoptotic and antiapoptotic bcl-2 family members and compromise functioning of the electron tansport chani in leukemia cells．J Immunol 2001，166：3130-42
81． Agarwal ML Taylor WR，Chernov MV,et al.The p53 network．． J Biol chem 1999．