慢性髓系白血病相关PTP家族成员的表达谱芯片筛选和初步功能研究
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摘要
研究背景与目的：
第9号与第22号染色体相互易位形成的Ph染色体是慢性髓系白血病(chronic myeloid leukemia，CML)的肿瘤恶性克隆标志。这种染色体平衡易位导致9q34上的c-Abl原癌基因与22q11上的Bcr(breakpoint cluster region)基因断裂后并置，形成新的融合基因Bcr-Abl，后者转录翻译的P210~(BCR-ABL)蛋白具有异常增高的蛋白酪氨酸激酶(protein tyrosine kinase，PTK)活性，被认为是导致CML发病的根本原因。P210~(BCR-ABL)导致CML发生的分子病理机制涉及多个信号通路，但最原始的信号启动因素则是过高的PTK活性催化P210~(BCR-ABL)自身和底物蛋白上特定位点的酪氨酸残基(tyrosine residue，Tyr)过度磷酸化所致。
在机体内，蛋白Tyr的磷酸化程度是直接调控诸多信号传导途径中信号的触发与终止、放大与衰减的重要因素。Tyr磷酸化水平的异常常会打破生理状态下多个信号网络的平衡，导致许多疾病如肿瘤的发生。近年来研究发现Tyr的磷酸化是一个可逆的动态过程，主要受PTK和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase，PTP)两大酶家族控制，它们互相拮抗与协调，在机体内构建了一个调节Tyr磷酸化状态的网络。我们设想，当PTK活性异常增高的P210~(BCR-ABL)打破了细胞内部PTK-PTP网络的平衡，使细胞发生肿瘤转化的过程中，机体为了维持自身的内部稳定必会作出相应的调节，而基因在mRNA表达水平上的调整又常是机体调节基因功能的最基本、最有效的方式之一。因此我们试图(1)应用P210~(BCR-ABL)特异高效的PTK抑制剂STI571封闭P210~(BCR-ABL)的活性并诱导其凋亡；在此基础上应用表达谱基因芯片对STI571处理前后K562细胞(人CML急变细胞株)部分PTP的表达差异进行分析，并用半定量RT-PCR对芯片结果进行验证，确定下一步研究的候选PTP基因。(2)对候选基因在慢性原代细胞中的表达进行半定量RT-PCR分析，初步确定候选基因的临床相关性；在此基础上应
用RT.pCR克隆该基因全长。DNA，并构建真核表达载体。(3)应用脂质体
转基因技术进一步探讨候选基因的潜在功能如诱导凋亡与分化等。
研究方法:
观察STI571(l .0~FL)不同时间点诱导K562细胞凋亡的规律及相
应各组细胞胞浆总PTP活性的改变。选择最适合的ST1571作用时间处理
K562细胞，应用BiostarH40s型表达谱芯片对处理前后的21个PTP成员
表达差异进行检测。对表达差异显著的PTP基因进行生物信息学分析，确
定下一步研究的目标。根据基因序列号设计引物，应用RT.pCR法克隆候
选PTP的全长。DNA序列并定向亚克隆于真核表达载体peDNA3 .0。在进
一步测序和酶切鉴定所克隆的基因序列正确后，应用脂质体转染技术使候
选基因在K562细胞中过表达。通过凋亡检测(Hoechst33258染色后荧光显
微镜观察形态，Anne劝nV一PI双标后FACS分析)、分化检测(联苯胺染色，
GPA表达率分析)和细胞周期分析，初步探讨候选PTP成员的潜在功能。
实验结果:
1.sTI571(l.oumol几)作用K562细胞12、24和45小时;Annexinv一Pl
双标FACS分析:早期凋亡细胞百分率分别为(11 .31士0.74)%、(27.15士2.13)
%和(20.02士0.66)%，晚期凋亡细胞百分率为(4.86士0.84)%、(18.81士0.93)
%和(32.62士1.09)%;Hoeehst 33258荧光染色检测的凋亡百分率为11.4%、
35.1%和44.8%。DNA电泳发现，STI571作用24和48小时组可检测到明
显的DNA ladder。对各处理时间组细胞的PTP活性进行检测发现对照组、
12、24、48小时组的PTP活性分别为22.5，34.5，44.0和39.spmol/mi功/ug。
通过上述定性与定量检测，我们选择可以明显诱导K562细胞凋亡，但细胞
以早晚期凋亡为主、死细胞少且PTP活性变化最大的作用24小时组进行下
一步芯片研究。应用Biostar H40s型表达谱芯片(含约4000个基因)对
STI571处理前后的基因差异表达谱进行检测，其中包括21个PTP成员。
结果共有377个基因出现了差异表达，其中2个PTP出现表达上调，分别
是FAPI(NMesoo6264，ration=2.417)与SHpl(M77273，ration=5.olZ)。用
半定量RT一PCR对结果进行验证，处理组FAPI/GAPDH的mRNA扩增后条
带的光密度比值为0.245，较对照组0.097上升了2.62倍;未处理组SHPI
未检测到转录本，结果阴性，处理组结果阳性，sHPI/GApDH的InRNA扩
增后条带的光密度比值为0.105。
2.应用染色体G显带分析和I一FlsH技术选择Ph(+)和/或Ber一Abl(+)
的初诊慢性期或急变期患者，应用半定量RT.pcR分析sHPI在cML原代
细胞中的表达情况，结果7例CML一CP中SHPI的表达与正常对照无差异，
但4例CML一BC中SHPI表达下调。应用RT一PCR技术克隆SHPI基因的
全长cDNA序列，定向亚克隆入真核表达载体PcDNA3.o，构建的
peoNA3一sHP一酶切、测序均正确。
3.脂质体法将peDNA3一sHPI转染K562细胞，RT一pCR、Westem·印
迹和PTP活性分析证实SHPI在K562细胞中可以表达并具有PTP活性。
转染48小时后，K562细胞出现凋亡，Annexinv一PI双标FACS分析细胞
凋亡率为16.84%，与转染空载体PcDNA3.0(6 .23%)相比，具有显著统计
学意义，P二0.000。联苯胺染色细胞阳性率14.67%，GPA表达率19.38%，
与转染空?
Background and objective:
Philadelphia chromosome(Ph) is the result of a t(9;22) reciprocal chromosomal translocation and is the malignant clonal marker of chronic myeloid leukemia(CML). At the molecular level, Ph translocation leads to the transposition of c-abl proto-oncogene gene on 9q34, which encodes a protein tyrosine kinase(PTK), to a new position downstream of the gene bcr gene on 22ql 1 and forms a new bcr-ab1 fusion gene which encodes a chimeric protein,
p210BCR-ABL.Compared with that of P145C-ABL,the PTK activity of P210BCR-ABL
is aberrantly regulated, which is considered as the sufficient and necessary factor in the pathogenesis of CML. Although the transforming mechanisms of P210BCR-ABL may involve a number of signal pathways, the fundamental and
primary signal trigger is not others but the aberrant PTK activity of P210BCR-ABL, which catalyzes the phosphorylation of tyrosine residues in specific sites of p210BCR-ABL itself and a host of substrates. The state of tyrosine phosphorylation of protein in vivo is a critical factor controlling the initiation , amplification, attenuation and completion in a great number of signal transduction pathways. The abnormal level of tyrosine phosphorylation may break the balance of many a signal networks, resulting in the development of a serial of diseases including tumors. Current data suggest that the tyrosine phosphorylation is a reversible dynamic procedure which is governed by the coordinated and competing actions of PTK and protein tyrosine phosphatases(PTP). As the balance between PTK and FTP are broken by P210BCR-ABL, whose PTK activity is activated aberrantly, we assume that some responsive regulations must been done by the cells in
order to antagonize the impact of PTK activity of P210BCR-ABL. Among those regulations, the changes at the transcriptional level of many important genes including FTP will be most direct and efficient. So in this article our aims are (1) To induce apoptosis of K562 cells with STI571 and analyze the differential expression of PTP with BioStarH40s expression profile cDNA array. Furthermore, the results of PTP' s differential expression were consolidated by semi-quantitative RT-PCR; (2) To analyze the mRNA level of the PTP candidate in CML with semi-quantitative and clone the full length cDNA sequence of candidate PTP, which then will be subcloned into the mammalian expression vector pcDNA3.0; (3)The potential function of the candidate PTP, such as triggering apoptosis and inducing differentiation, were explored by over-expressing the candidate gene in K562 cells with lipofectin transfection technique.
MateriaIs and methods:
After being cultured with 1.0umol/L STI571 in vitro for 0, 12hrs, 24hrs and 48hrs , the apoptotic percentage and PTP activity of K562 cells were measured. Based on above results, we apply a expression profile cDNA microarray, BioStarH40s, which including about 4,000 genes, to analyze the differential expression of 21 PTP members. Combined the results from BioStarH40s with the corresponding bioinformatics analysis, we focused our attention on one or several target genes. According to their cDNA sequence provided by GeneBank, we designed the upper and the lower primers , cloned the full length cDNA with RT-PCR and subcloned it into the mammalian expression vector pcDNA3.0. The cDNA sequence of the cloned gene was validated with enzyme digestion and DNA sequence as well. In addition, we forced the target PTP gene over-expresse in K562 cells with lipofectin transfection technique, aiming at exploring its/their potential functions with apoptosis assay, differentiation analysis and cell cycle detection. The synergic effect of the gene with STI571 was explored too.
Results:
1. When treated the K562 cells with l.Oumol/L STI571 for 12, 24 and 48hrs, the early apoptotic percentage were 11.31, 27.15 and 20.02% respectively; the
late apoptotic percentage are 4.86, 18.81 and 32.62 % respectively. The apoptotic percentage assayed with Hoechst33258 staining were 11.4, 35.1 and 44.8%. At the
第9号与第22号染色体相互易位形成的Ph染色体是慢性髓系白血病(chronic myeloid leukemia，CML)的肿瘤恶性克隆标志。这种染色体平衡易位导致9q34上的c-Abl原癌基因与22q11上的Bcr(breakpoint cluster region)基因断裂后并置，形成新的融合基因Bcr-Abl，后者转录翻译的P210~(BCR-ABL)蛋白具有异常增高的蛋白酪氨酸激酶(protein tyrosine kinase，PTK)活性，被认为是导致CML发病的根本原因。P210~(BCR-ABL)导致CML发生的分子病理机制涉及多个信号通路，但最原始的信号启动因素则是过高的PTK活性催化P210~(BCR-ABL)自身和底物蛋白上特定位点的酪氨酸残基(tyrosine residue，Tyr)过度磷酸化所致。
在机体内，蛋白Tyr的磷酸化程度是直接调控诸多信号传导途径中信号的触发与终止、放大与衰减的重要因素。Tyr磷酸化水平的异常常会打破生理状态下多个信号网络的平衡，导致许多疾病如肿瘤的发生。近年来研究发现Tyr的磷酸化是一个可逆的动态过程，主要受PTK和蛋白酪氨酸磷酸酶(protein tyrosine phosphatase，PTP)两大酶家族控制，它们互相拮抗与协调，在机体内构建了一个调节Tyr磷酸化状态的网络。我们设想，当PTK活性异常增高的P210~(BCR-ABL)打破了细胞内部PTK-PTP网络的平衡，使细胞发生肿瘤转化的过程中，机体为了维持自身的内部稳定必会作出相应的调节，而基因在mRNA表达水平上的调整又常是机体调节基因功能的最基本、最有效的方式之一。因此我们试图(1)应用P210~(BCR-ABL)特异高效的PTK抑制剂STI571封闭P210~(BCR-ABL)的活性并诱导其凋亡；在此基础上应用表达谱基因芯片对STI571处理前后K562细胞(人CML急变细胞株)部分PTP的表达差异进行分析，并用半定量RT-PCR对芯片结果进行验证，确定下一步研究的候选PTP基因。(2)对候选基因在慢性原代细胞中的表达进行半定量RT-PCR分析，初步确定候选基因的临床相关性；在此基础上应
用RT.pCR克隆该基因全长。DNA，并构建真核表达载体。(3)应用脂质体
转基因技术进一步探讨候选基因的潜在功能如诱导凋亡与分化等。
研究方法:
观察STI571(l .0~FL)不同时间点诱导K562细胞凋亡的规律及相
应各组细胞胞浆总PTP活性的改变。选择最适合的ST1571作用时间处理
K562细胞，应用BiostarH40s型表达谱芯片对处理前后的21个PTP成员
表达差异进行检测。对表达差异显著的PTP基因进行生物信息学分析，确
定下一步研究的目标。根据基因序列号设计引物，应用RT.pCR法克隆候
选PTP的全长。DNA序列并定向亚克隆于真核表达载体peDNA3 .0。在进
一步测序和酶切鉴定所克隆的基因序列正确后，应用脂质体转染技术使候
选基因在K562细胞中过表达。通过凋亡检测(Hoechst33258染色后荧光显
微镜观察形态，Anne劝nV一PI双标后FACS分析)、分化检测(联苯胺染色，
GPA表达率分析)和细胞周期分析，初步探讨候选PTP成员的潜在功能。
实验结果:
1.sTI571(l.oumol几)作用K562细胞12、24和45小时;Annexinv一Pl
双标FACS分析:早期凋亡细胞百分率分别为(11 .31士0.74)%、(27.15士2.13)
%和(20.02士0.66)%，晚期凋亡细胞百分率为(4.86士0.84)%、(18.81士0.93)
%和(32.62士1.09)%;Hoeehst 33258荧光染色检测的凋亡百分率为11.4%、
35.1%和44.8%。DNA电泳发现，STI571作用24和48小时组可检测到明
显的DNA ladder。对各处理时间组细胞的PTP活性进行检测发现对照组、
12、24、48小时组的PTP活性分别为22.5，34.5，44.0和39.spmol/mi功/ug。
通过上述定性与定量检测，我们选择可以明显诱导K562细胞凋亡，但细胞
以早晚期凋亡为主、死细胞少且PTP活性变化最大的作用24小时组进行下
一步芯片研究。应用Biostar H40s型表达谱芯片(含约4000个基因)对
STI571处理前后的基因差异表达谱进行检测，其中包括21个PTP成员。
结果共有377个基因出现了差异表达，其中2个PTP出现表达上调，分别
是FAPI(NMesoo6264，ration=2.417)与SHpl(M77273，ration=5.olZ)。用
半定量RT一PCR对结果进行验证，处理组FAPI/GAPDH的mRNA扩增后条
带的光密度比值为0.245，较对照组0.097上升了2.62倍;未处理组SHPI
未检测到转录本，结果阴性，处理组结果阳性，sHPI/GApDH的InRNA扩
增后条带的光密度比值为0.105。
2.应用染色体G显带分析和I一FlsH技术选择Ph(+)和/或Ber一Abl(+)
的初诊慢性期或急变期患者，应用半定量RT.pcR分析sHPI在cML原代
细胞中的表达情况，结果7例CML一CP中SHPI的表达与正常对照无差异，
但4例CML一BC中SHPI表达下调。应用RT一PCR技术克隆SHPI基因的
全长cDNA序列，定向亚克隆入真核表达载体PcDNA3.o，构建的
peoNA3一sHP一酶切、测序均正确。
3.脂质体法将peDNA3一sHPI转染K562细胞，RT一pCR、Westem·印
迹和PTP活性分析证实SHPI在K562细胞中可以表达并具有PTP活性。
转染48小时后，K562细胞出现凋亡，Annexinv一PI双标FACS分析细胞
凋亡率为16.84%，与转染空载体PcDNA3.0(6 .23%)相比，具有显著统计
学意义，P二0.000。联苯胺染色细胞阳性率14.67%，GPA表达率19.38%，
与转染空?
Background and objective:
Philadelphia chromosome(Ph) is the result of a t(9;22) reciprocal chromosomal translocation and is the malignant clonal marker of chronic myeloid leukemia(CML). At the molecular level, Ph translocation leads to the transposition of c-abl proto-oncogene gene on 9q34, which encodes a protein tyrosine kinase(PTK), to a new position downstream of the gene bcr gene on 22ql 1 and forms a new bcr-ab1 fusion gene which encodes a chimeric protein,
p210BCR-ABL.Compared with that of P145C-ABL,the PTK activity of P210BCR-ABL
is aberrantly regulated, which is considered as the sufficient and necessary factor in the pathogenesis of CML. Although the transforming mechanisms of P210BCR-ABL may involve a number of signal pathways, the fundamental and
primary signal trigger is not others but the aberrant PTK activity of P210BCR-ABL, which catalyzes the phosphorylation of tyrosine residues in specific sites of p210BCR-ABL itself and a host of substrates. The state of tyrosine phosphorylation of protein in vivo is a critical factor controlling the initiation , amplification, attenuation and completion in a great number of signal transduction pathways. The abnormal level of tyrosine phosphorylation may break the balance of many a signal networks, resulting in the development of a serial of diseases including tumors. Current data suggest that the tyrosine phosphorylation is a reversible dynamic procedure which is governed by the coordinated and competing actions of PTK and protein tyrosine phosphatases(PTP). As the balance between PTK and FTP are broken by P210BCR-ABL, whose PTK activity is activated aberrantly, we assume that some responsive regulations must been done by the cells in
order to antagonize the impact of PTK activity of P210BCR-ABL. Among those regulations, the changes at the transcriptional level of many important genes including FTP will be most direct and efficient. So in this article our aims are (1) To induce apoptosis of K562 cells with STI571 and analyze the differential expression of PTP with BioStarH40s expression profile cDNA array. Furthermore, the results of PTP' s differential expression were consolidated by semi-quantitative RT-PCR; (2) To analyze the mRNA level of the PTP candidate in CML with semi-quantitative and clone the full length cDNA sequence of candidate PTP, which then will be subcloned into the mammalian expression vector pcDNA3.0; (3)The potential function of the candidate PTP, such as triggering apoptosis and inducing differentiation, were explored by over-expressing the candidate gene in K562 cells with lipofectin transfection technique.
MateriaIs and methods:
After being cultured with 1.0umol/L STI571 in vitro for 0, 12hrs, 24hrs and 48hrs , the apoptotic percentage and PTP activity of K562 cells were measured. Based on above results, we apply a expression profile cDNA microarray, BioStarH40s, which including about 4,000 genes, to analyze the differential expression of 21 PTP members. Combined the results from BioStarH40s with the corresponding bioinformatics analysis, we focused our attention on one or several target genes. According to their cDNA sequence provided by GeneBank, we designed the upper and the lower primers , cloned the full length cDNA with RT-PCR and subcloned it into the mammalian expression vector pcDNA3.0. The cDNA sequence of the cloned gene was validated with enzyme digestion and DNA sequence as well. In addition, we forced the target PTP gene over-expresse in K562 cells with lipofectin transfection technique, aiming at exploring its/their potential functions with apoptosis assay, differentiation analysis and cell cycle detection. The synergic effect of the gene with STI571 was explored too.
Results:
1. When treated the K562 cells with l.Oumol/L STI571 for 12, 24 and 48hrs, the early apoptotic percentage were 11.31, 27.15 and 20.02% respectively; the
late apoptotic percentage are 4.86, 18.81 and 32.62 % respectively. The apoptotic percentage assayed with Hoechst33258 staining were 11.4, 35.1 and 44.8%. At the
引文
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[2] Deininger MW, Goldman JM, Melo JV. The molecular biology of chronic myeloid leukemia[J]. Blood. 2000; 96: 3343-3356．
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[12] Tauchi T, Feng GS, Shen R, et al. SH2-containing phosphotyrosine phosphatase syp is a target of p210bcr/abl tyrosine kinase[J]. J Biol Chem. 1994; 269: 15381-15387．
[13] Lamontagne KR, Flint AJ, Franza BR, et al. Protein tyrosine phosphatase 1B antagonizes signalling by oncoprotein tyrosine kinase p210 bcr-abl in vivo. Mol. Cell. Biol 1998,18:2965．
[14] Lamontagne KR, Hannon G, Tonks NK. Protein tyrosine phosphatase PTP 1B suppresses p210 bcr-ab1-induced transformation of rat-1 fibroblasts and promotes differentiation of K562 cells. Proc Natl Acad Sci USA 1998, 95:14094．
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[16] 汪家政,范明.蛋白质技术手册[M].科学出版社. 2001: 42-46
[17] Merchant SH, Gonchoroff NJ, Hutchison RE. Apoptotic index by Annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes[J]. Cytometry. 2001 ; 46:28-32．
[18] Cai Z, Lin M, Wuchter C, et al. Apoptotic response to homoharringtonine in human wt p53 leukemic cells is independent of reactive oxygen species generation and implicates Bax translocation, mitochondrial cytochrome c release and caspase activation[J]. Leukemia. 2001 ; 15:567-74．
[19] Nowell P, Hungerford D. A minute chromosome in human chronic granulocytic leukemia[J]. Science. 1960;132:1497．
[20] Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyrosine kinase activity and transformation potency of bcr-abl oncogene products[J]. Science. 1990;247: 1079-1082
[21] Oda T, Heaney C, Hagopian JR, et al. Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia[J]. J Biol Chem. 1994; 269:22925-22928．
[22] Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia[J]. Nature. 1987;328:342-344．
[23] Puil L, Liu J, Gish G, et al. Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway[J]. EMBO J. 1994;13:764-773．
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[16] 汪家政,范明.蛋白质技术手册[M].科学出版社. 2001: 42-46
[17] Merchant SH, Gonchoroff NJ, Hutchison RE. Apoptotic index by Annexin V flow cytometry: adjunct to morphologic and cytogenetic diagnosis of myelodysplastic syndromes[J]. Cytometry. 2001 ; 46:28-32．
[18] Cai Z, Lin M, Wuchter C, et al. Apoptotic response to homoharringtonine in human wt p53 leukemic cells is independent of reactive oxygen species generation and implicates Bax translocation, mitochondrial cytochrome c release and caspase activation[J]. Leukemia. 2001 ; 15:567-74．
[19] Nowell P, Hungerford D. A minute chromosome in human chronic granulocytic leukemia[J]. Science. 1960;132:1497．
[20] Lugo TG, Pendergast AM, Muller AJ, Witte ON. Tyrosine kinase activity and transformation potency of bcr-abl oncogene products[J]. Science. 1990;247: 1079-1082
[21] Oda T, Heaney C, Hagopian JR, et al. Crkl is the major tyrosine-phosphorylated protein in neutrophils from patients with chronic myelogenous leukemia[J]. J Biol Chem. 1994; 269:22925-22928．
[22] Gordon MY, Dowding CR, Riley GP, Goldman JM, Greaves MF. Altered adhesive interactions with marrow stroma of haematopoietic progenitor cells in chronic myeloid leukaemia[J]. Nature. 1987;328:342-344．
[23] Puil L, Liu J, Gish G, et al. Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway[J]. EMBO J. 1994;13:764-773．
[24] Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ, Jones RJ. Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia[J]. Blood. 1994;83:2038-2044．
[25] Jiang X, Ng E, Yip C, et al. Primitive interleukin 3 null hematopoietic cells transduced with BCR-ABL show accelerated loss after culture of factor-independence in vitro and leukemogenic activity in vivo[J]. Blood. 2002; 100:373 1-40．
[26] Blasiak J, Gloc E, Pertynski T, Drzewoski J. DNA damage and repair in BCR/ABL-expressing cells after combined action of idarubicin, STI571 and amifostine[J]. Anticancer Drugs. 2002;13:1055-60．
[27] Puil L, Liu J, Gish G, et al. Bcr-Abl oncoproteins bind directly to activators of the Ras signalling pathway[J]. EMBO J. 1994;13:764-773
[28] del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. Interleukin-3-induced phosphorylation of bad through the protein kinase akt[J]. Science. 1998;278:687-689
[29] Amarante Mendes GP, Naekyung Kim C, Liu L, et al. Bcr-Abl exerts its antiapoptotic effect against diverse apoptotic stimuli through blockage of mitochondrial release of cytochrome C and activation of caspase-3[J]. Blood. 1998;91:1700-1705．
[30] Dubrez L, Eymin B, Sordet O, Drain N, Turhan AG, Solary E. BCR-ABL delays apoptosis upstream of procaspase-3 activation[J]. Blood. 1998;91:2415-2422．
[31] Kurzrock R, Kantarjian HM, Druker BJ, Talpaz M. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics[J]. Ann Intern Med. 2003;138:819-30．
[32] Kawaguchi Y, Jinnai I, Nagai K, et al. Effect of a selective Abl tyrosine kinase inhibitor, STI571, on in vitro growth of BCR-ABL-positive acute lymphoblastic leukemia cells[J]. Leukemia. 2001 ;15:590-4．
[33] Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia[J]. N Engl J Med. 2001; 344:1031-7．
[34] Yu C, Krystal G, Varticovksi L, et al. Pharmacologic mitogen-activated protein/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase inhibitors interact synergistically with STI571 to induce apoptosis in Bcr/Abl-expressing human leukemia cells[J]. Cancer Res. 2002 Jan l;62(1) :188-99．
[35] Oetzel C, Jonuleit T, Gotz A, et al. The tyrosine kinase inhibitor CGP 57148 (ST1 571) induces apoptosis in BCR-ABL-positive cells by down-regulating BCL-X[J]. Clin Cancer Res. 2000;6:1958-68．
[36] Qin Y, Chen S, Chang Y, et al. The mechanism of STI571 inducing apoptosis of K562 cells[J]. Zhonghua Xue Ye Xue Za Zhi. 2002;23:289-92．
[37] Fang G, Kim CN, Perkins CL, et al. CGP57148B (STI-571) induces differentiation and apoptosis and sensitizes Bcr-Abl-positive human leukemia cells to apoptosis due to antileukemic drugs[J]. Blood. 2000;96:2246-53．
[38] Porosnicu M, Nimmanapalli R, Nguyen D, et al. Co-treatment with As2O3 enhances selective cytotoxic effects of STI-571 against Brc-Abl-positive acute leukemia cells[J]. Leukemia. 2001;15:772-8．
[39] Keeshan K, Mills KI, Cotter TG, McKenna SL. Elevated Bcr-Abl expression levels are
sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype[J]. Leukemia. 2001;15:1823-33．
[40] Saras, J, Claesson-Welsh L, Heldin CH et al. Cloning and characterization of PTPL1, a protein tyrosine phosphatase with similarities to cytoskeletal-associated proteins[J]. 1994; J. Biol. Chem. 269: 24082-24089．
[41] Yoshida S, Harada H., Nagai H.et al. Head-to-head juxtaposition of Fas-associated phosphatase-1 (FAP-1) and c-Jun NH2-terminal kinase 3 (JNK3) genes: genomic structure and seven polymorphisms of the FAP-1 gene[J]. J. Hum. Genet. 2002; 47: 614-619．
[42] Sato T, Me S, Kitada S, Reed JC. FAP-1: a protein tyrosine phosphatase that associates with Fas[J]. Science. 1995;268:411-5．
[43] Yanagisawa J, Takahashi M, Kanki H, et al. The molecular interaction of Fas and FAP-1． A tripeptide blocker of human Fas interaction with FAP-1 promotes Fas-induced apoptosis[J]. J Biol Chem. 1997;272:8539-45．
[44] Li Y, Kanki H, Hachiya T, et al. Negative regulation of Fas-mediated apoptosis by FAP-1 in human cancer cells[J]. Int J Cancer. 2000;87:473-9．
[45] Saras J, Engstrom U, Gonez LJ, Heldin CH. Characterization of the interactions between PDZ domains of the protein-tyrosine phosphatase PTPL1 and the carboxyl-terminal tail of Fas[J]. J Biol Chem. 1997;272:20979-81．
[46] Yanagisawa J, Takahashi M, Kanki H, et al. The molecular interaction of Fas and FAP-1． A tripeptide blocker of human Fas interaction with FAP-1 promotes Fas-induced apoptosis[J]. J Biol Chem. 1997;272:8539-45．
[47] Sawa E, Takahashi M, Kamishohara M, et al. Structural modification of Fas C-terminal tripeptide and its effects on the inhibitory activity of Fas/FAP-1 binding[J]. J Med Chem. 1999;42:3289-99．
[48] Ungefroren H, Kruse ML, Trauzold A, et al. FAP-1 in pancreatic cancer cells: functional and mechanistic studies on its inhibitory role in CD95-mediated apoptosis[J]. J Cell Sci. 2001;114:2735-46．
[49] Cuppen E, Nagata S, Wieringa B, Hendriks W. No evidence for involvement of mouse protein-tyrosine phosphatase-BAS-like Fas-associated phosphatase-1 in Fas-mediated apoptosis[J].J Biol Chem. 1997;272:30215-20．
[50] Mori S, Murakami-Mori K, Jewett A, et al. Resistance of AIDS-associated Kaposi's sarcoma cells to Fas-mediated apoptosis[J]. Cancer Res. 1996;56:1874-9．
[51] Meinhold-Heerlein I, Stenner-Liewen F, Liewen H, et al. Expression and potential role of Fas-associated phosphatase-1 in ovarian cancer[J]. Am J Pathol. 2001;158:1335-44．
[52] Broome HE, Dargan CM, Brunner T, Green DR. Sensitivity of S49． 1 cells to anti-CD95 (Fas/Apo-l)-induced apoptosis: effects of CD95, Bcl-2 or Bcl-x transduction[J]. Cell Death Differ. 1998 ;5:200-5．
[53] Yamada Y, Sugahara K, Tsuruda K, et al. Fas-resistance in ATL cell lines not associated with HTLV-I or FAP-1 production[J]. Cancer Lett. 1999 ;147:215-9．
[54] Boekelheide K, Lee J, Shipp EB,et al. Expression of Fas system-related genes in the testis during development and after toxicant exposure[J]. Toxicol Lett. 1998 ;102:503-8．
[55] Bompard G, Puech C, Prebois C, et al. Protein-tyrosine phosphatase PTPL1/FAP-1 triggers apoptosis in human breast cancer cells[J]. J Biol Chem. 2002 ;277:47861-9．
[56] Yi T, Cleveland JL, Ihle JN. Identification of novel protein tyrosine phosphatases of hematopoietic cells by PCR amplification[J]. Blood. 1991; 78: 2222-2228．
[57] Matthews RJ, Bowne DB, FloresE, Thomas M L. Characterization of hematopoietic intracellular protein tyrosine phosphatases: description of a phosphatase containing an SH2 domain and another enriched in proline-, glutamic acid-, serine-, and threonine-rich sequences[J]. Molec. Cell. Biol 1992;12: 2396-2405．
[58] Shen SH.,Bastien L, Posner BI et al. A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases[J]. Nature. 1991;352: 736-739．
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