Lck激活STAT5b致细胞恶性转化及SOCS1/3的抑制作用
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摘要
淋巴细胞特异性蛋白酪氨酸激酶(Lymphocyte specific protein tyrosine kinase, Lck)是Src家族成员的蛋白酪氨酸激酶,主要存在于T淋巴细胞内,参与T细胞的发育、分化、活化的信号转导过程。国内外许多研究显示：Lck的异常表达和调节与细胞的癌变密切相关,如人淋巴细胞和非淋巴细胞恶性肿瘤均有Lck异常表达或激活,在转基因小鼠,Lck的表达导致胸腺肿瘤。Lck结构域中催化区含有两个可供调节的酪氨酸残基(tyr394和tyr505), tyr394是一自身磷酸化位点,可通过自身磷酸化作用使Lck激酶活性上调；tyr505的磷酸化则可使Lck激酶活性下调,若以不能被磷酸化的苯丙氨酸置换酪氨酸(Y505F)后,则出现Lck激酶活性的持续升高。但Lck介导肿瘤的发生的机制至今仍未能充分阐明。
细胞因子与相应的受体结合后引起受体分子的二聚化,与受体偶联的JAK激酶相互接近并通过交互的酪氨酸磷酸化作用而活化。活化的JAK催化结合在受体上的STAT蛋白发生磷酸化修饰,STAT蛋白活化后以二聚体的形式进入细胞核内与靶基因结合,调控基因的转录,包括SOCS基因的转录。因此,JAK-STAT信号转导通路参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。
细胞因子信号抑制因子(suppressors of cytokine signaling, SOCS)家族是一类可被多种细胞因子诱导产生,且对细胞因子信号通路具有负反馈调节作用的蛋白分子。SOCS主要通过抑制JAK-STAT通路抑制信号转导,从而对细胞因子、激素、生长因子作用的强度和持续时间进行调控。SOCS主要通过以下三种不同的方式对细胞因子信号通路进行调节：第一,通过其SH2结构域与靶蛋白的磷酸酪氨酸结合,使JAK激酶的N末端失活而抑制信号转导；第二,抑制STAT与受体位点的结合；第三,通过SOCS盒促进所结合蛋白发生蛋白酶体依赖途径的降解。研究表明：SOCS1和SOCS3可能作用于持续性激活的酪氨酸蛋白激酶,或作为JAK/STAT通路的负调控因子,因而可能为肿瘤抑制因子。但SOCS在肿瘤发生中的作用有待进一步的研究。
本研究首先建立了四环素诱导精确控制Lck激酶表达的T-REx(T-REx-293, T-REx-BaF3)细胞系统,转染组成型激活的Lck (Y505F)及STAT5b真核表达质粒,通过表达外源性Lck和STAT5,精确地研究Lck激活对STAT5的影响,且可进行互动免疫沉淀观察细胞内Lck与STAT5b蛋白质的相互作用。
同时应用表达高水平Lck的小鼠淋巴瘤系LSTRA细胞及转染组成型激活Lck (Y505F)后转化的BaF3细胞,从细胞和分子等不同水平,系统观察了细胞增殖、细胞凋亡,信号蛋白间的相互作用、信号通路激活机制及SOCS1、SOCS3作为负性调控因子对JAK/STAT信号通路的影响。本文为阐明Lck蛋白酪氨酸激酶与JAK-STAT-SOCS信号通路在淋巴细胞和非淋巴细胞白血病的作用及机制提供实验依据,为白血病的防治提供一条新的线索。
全文包括两部分：
第一部分：组成型激活Lck (Y505F)致细胞恶性转化
目的：研究组成型激活Lck (Y505F)激酶对细胞的转化作用及机制
方法：利用加入四环素可去除pcDNA5/TO中启动子抑制因子,从而启动插入基因表达的T-REx-293细胞,转染组成型激活的Lck (Y505F)及STAT5b真核表达质粒,通过表达外源性Lck和STAT5,精确地研究Lck激活对STAT5的影响。
用抗Myc抗体和抗STAT5抗体,免疫印迹法检测转染后Lck及STAT5蛋白的表达水平；抗STAT5抗体免疫沉淀法沉淀STAT5,用抗蛋白酪氨酸磷酸化抗体4G10观察Lck (Y505F)对STAT5b酪氨酸磷酸化的影响。
收集短暂转染STAT5b真核表达质粒后24小时Lck (Y505F)转化的T-REx-293细胞,采用细胞外液低渗透压法提取核蛋白；用T4多聚核苷酸激酶将32P-ATP标记寡聚核苷酸；提取的核蛋白与32P标记的MGE一致序列的DNA结合后,进行电泳迁移率变动分析(EMSA法)实验,放射自显影观察Lck(Y505F)对STAT5b与DNA结合力的影响。
以抗Lck抗体,抗STAT5抗体和蛋白A／G—琼脂糖微球,采取抗Lck抗体沉淀STAT5,抗STAT5抗体沉淀Lck的互动免疫沉淀法观察细胞内Lck与STAT5b蛋白质的相互作用。
深入研究Lck (Y505F)对小鼠原B细胞系BaF3细胞生物学的影响。以细胞增殖,细胞凋亡为指标,通过台盼蓝染色用血细胞计数板计算BaF3细胞转染后2小时及24小时的细胞数。计存活细胞及死细胞数,计算细胞存活率及死亡细胞百分率。
收集培养液中去除白介素-3后24小时Lck (Y505F)转化的BaF3细胞,用Alexa Fluor 647-conjugated Annexin V萤光染色,经FACSCantTM流式细胞仪分析,Quest软件采集数据分析细胞AnnexinⅤ阳性率,研究Lck (Y505F)对转化的BaF3细胞凋亡的影响。
收集经电穿孔短暂转染质粒后24小时的BaF3细胞,用酚／氯仿抽提纯化,α-丙醇沉淀基因组DNA。1.2%琼脂糖凝胶电泳,溴化乙锭染色。观察DNA断裂片断。
为进一步研究STAT5b在Lck(Y505F)致细胞转化中的作用,T-REx-BaF3细胞转染WT型STAT5b和突变型STAT5b (Y699F)真核质粒,表达STAT5或主域负性蛋白STAT5b,观察WT型STAT5b和突变型STAT5b (Y699F)对Lck(Y505F)细胞增殖及凋亡的影响。
结果：Lck的表达12小时达高峰并维持至24小时,作为阴性对照,转染载体的T-REx-293细胞无Lck的表达；抗STAT5抗体免疫沉淀STAT5后,蛋白酪氨酸磷酸化抗体4G10的免疫印迹显示：Lck可促进STAT5b酪氨酸磷酸化,从而激活STAT5b。
Lck可促进STAT5b与32P标记的MGE一致序列的DNA的结合,未用32P标记的寡聚DNA无结合及加入STAT5b抗体出现超迁移现象,证明STAT5b与DNA结合具有特异性。结果显示Lck可激活STAT5b,促进STAT5b与DNA结合力增加。
互动免疫沉淀试验结果表明：T-REx-293细胞中共同表达外源性Lck和STAT5b后,用抗STAT5b抗体免疫沉淀后可沉淀Lck蛋白,反之,用抗Lck抗体进行的免疫沉淀可沉淀STAT5b,由此可见,细胞中Lck和STAT5b结合在一起,故Lck和STAT5b在细胞中存在蛋白间的相互作用,Lck可能直接激活STAT5b。这也表明组成型激活的Lck (Y505F)可激活STAT5b。
表达Lck (Y505F)后第一、二、三天BaF3细胞计数量均明显增加(P<0.05及P<0.01),结果表明Lck激活促进BaF3细胞增殖。
BaF3的生长依赖于白介素-3,去除培养液中的白介素-3后,BaF3细胞可发生细胞凋亡。转染载体的BaF3细胞在去除培养液中白介素-3后至第六天几无存活,而转染Lck(Y505F)真核表达质粒的BaF3细胞在去除培养液中白介素-3后,前三天存活率下降,但其降幅明显低于载体对照(P<0.01及P<0.001),第四天后细胞存活率明显增加,至第六天细胞存活率恢复至去除白介素-3的水平,BaF3细胞能独立于白介素-3的刺激生长。
转染Lck的BaF3细胞Annexin V阳性率明显减低(P<0.01)；DNA断裂片断也明显减少。结果表明Lck激活可促进BaF3细胞抗凋亡。
T-REx-BaF3细胞短暂转染WT STAT5b或STAT5b (Y699F)后可表达等量的STAT5b蛋白。与转染载体质粒相比,转染WT型STAT5b质粒可促进BaF3细胞增殖,与之相反,主域负性STAT5b (Y699F)则抑制Lck激活诱导的细胞增殖(p<0.05及P<0.01)。去除培养液中的白介素-3后,与转染载体质粒相比转染WT型STAT5b质粒的BaF3细胞死亡率明显降低(P<0.05),而转染主域负性STAT5b (Y699F)质粒的BaF3细胞死亡率明显增加(P<0.05),结果显示STAT5b在Lck激活诱导的细胞生物学中发挥重要的下游信号分子作用。
结论：组成型激活的Lck (Y505F)诱导STAT5b的酪氨酸磷酸化,激活STAT5b;促进STAT5b与DNA结合力增加；Lck与STAT5b在细胞内相互作用。转染Lck (Y505F)促进BaF3细胞增殖,抵抗去除IL-3诱导的细胞凋亡,而主域负性蛋白STAT5b (Y699F突变)逆转Lck (Y505F)的促细胞增殖及抗凋亡作用。因此,Lck通过STAT5b的激活导致细胞恶性转化。
第二部分SOCS1和SOCS3抑制Lck (Y505F)导致的细胞转化
目的：研究SOCS1和SOCS3对抑制Lck (Y505F)导致的细胞转化的抑制作用
方法：小鼠T细胞系LSTRA淋巴瘤细胞(过度表达Lck激酶),用32P标记的全长cDNA以Northern杂交分析法检测中SOCS1, SOCS3, Bcl-xL, GAPDH的表达；观察用过钒酸钠(酪氨酸激酶激活剂)刺激LSTRA细胞后SOCS1,SOCS3的表达变化；用5-’氮杂胞苷(DNA甲基抑制剂)处理LSTRA和U266细胞,采用RT-PCR法检测5′-氮杂胞苷处理后LSTRA细胞SOCS1 mRNA表达水平。
用电穿孔法将pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3真核表达质粒短暂转染入Lck (Y505F)转化的BaF3细胞。以细胞增殖,细胞凋亡为指标,通过台盼蓝染色用血细胞计数板计算BaF3细胞转染后2小时及24小时的细胞数。计存活细胞及死细胞数,计算细胞存活率及死亡细胞百分率。
收集经电穿孔短暂转染pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3质粒后24小时BaF3细胞,用Alexa Fluor 647-conjugated AnnexinⅤ萤光染色,经FACSCantoTM流式细胞仪分析,Quest软件采集数据分析细胞AnnexinⅤ阳性率,观察SOCS1, SOCS3对细胞凋亡的影响。
收集经电穿孔短暂转染质粒后24小时的BaF3细胞,用酚／氯仿抽提纯化,α-丙醇沉淀基因组DNA。1.2%琼脂糖凝胶电泳,溴化乙锭染色。观察DNA断裂片断,进一步检测SOCS1, SOCS3对细胞凋亡的影响。
收集经电穿孔短暂转染pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3质粒后24小时BaF3细胞,用抗Myc抗体和抗Flag抗体免疫印迹法检测全细胞裂解液中外源性Lck和SOCS蛋白的表达水平。用抗Lck抗体免疫沉淀Lck,测定体外激酶活性(IVK),观察SOCS1, SOCS3对Lck激酶的抑制作用。
收集经电穿孔短暂转染pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3质粒后24小时BaF3细胞,采用细胞外液低渗透压法提取核蛋白；用T4多聚核苷酸激酶将32P-ATP标记寡聚核苷酸；提取的核蛋白与32P标记的MGE一致序列的DNA结合后,进行电泳迁移率变动分析(EMSA法)实验,放射自显影观察SOCS1, SOCS3对Lck (Y505F)转化的BaF3细胞STAT5b与DNA结合力的变化。
免疫印迹检测转染后Lck及STAT蛋白的表达水平,用抗STAT5b抗体和蛋白A／G-琼脂糖微球,免疫沉淀法沉淀STAT5,用抗蛋白酪氨酸磷酸化抗体4G10测定SOCS1, SOCS3对Lck (Y505F)转化的BaF3细胞STAT5b酪氨酸磷酸化的影响。
结果：使用SOCS1和SOCS3全长cDNA探针,没有检测到LSTRA细胞中SOCS1和SOCS3 mRNA的表达。与此结果相似,EL4细胞,表达正常水平Lck的小鼠T细胞系也没有检测到SOCS1和SOCS3基因的转录。而作为阳性对照,白介素-3刺激强烈激活BaF3细胞SOCS1和SOCS3基因的表达。表明SOCS1和SOCS3基因在LSTRA细胞中表达缺失。
过钒酸钠处理LSTRA细胞15min-2h,利用Northern杂交检测SOCS1和SOCS3基因的表达。过钒酸钠刺激LSTRA细胞1-2h后可检测到SOCS3基因的表达,显示LSTRA细胞启动SOCS3基因表达的机制仍然完好无损。而在过钒酸钠刺激的LSTRA细胞,观察的时间内未检测到SOCS1的表达,说明SOCS1基因沉默可能是通过DNA甲基化而发生。用DNA甲基抑制剂5-’氮杂胞苷抑制DNA甲基化的影响,采用RT-PCR分析显示5’-氮杂胞苷处理可重新激活SOCS1基因表达,但未经处理或溶剂对照处理LSTRA细胞无此现象。
因此,通过酪氨酸激酶激活剂过钒酸钠刺激LSTRA细胞及DNA甲基抑制剂5-’氮杂胞苷处理LSTRA的研究表明：DNA甲基化介导SOCS1的基因沉默,而SOCS3表达缺失的机制独立于DNA甲基化。
与载体相比,短暂转染SOCS1和SOCS3真核表达质粒的Lck (Y505F)转化的BaF3细胞增殖明显降低,细胞死亡明显增加(P均<0.001)；Annexin V阳性细胞百分比明显增加(P<0.001)；DNA断裂形成也明显增加。提示SOCS1和SOCS3抑制Lck转化的BaF3细胞增殖及促进细胞凋亡
转染空载体质粒的Lck转化的BaF3细胞中由于Lck (Y505F)自身磷酸化激活致掺入的32P明显增加。短暂转染SOCS1和SOCS3真核表达质粒的Lck(Y505F)转化的BaF3细胞磷酸化明显降低。
Lck转化的BaF3细胞核提取物与STAT5的一致序列DNA体外结合明显增加。与空载体质粒相比,短暂转染SOCS1和SOCS3真核表达质粒的Lck(Y505F)转化的BaF3细胞STAT5b与DNA的结合明显降低。STAT5b免疫沉淀结果显示转染空载体质粒的Lck转化的BaF3细胞中有较高水平的酪氨酸磷酸化。同样,表达SOCS1或SOCS3明显降低STAT5b酪氨酸磷酸化水平。结果提示：表达SOCS1和SOCS3可以降低Lck的转化BaF3细胞中Lck (Y505F)下游的STAT5b活动。
结论：SOCS1和SOCS3 mRNA在LSTRA淋巴瘤细胞中因基因沉默而表达缺失,DNA甲基化介导SOCS1的基因沉默；而SOCS3表达缺失的机制独立于DNA甲基化。SOCS1和SOCS3显著抑制Lck转化的BaF3细胞增殖及促进细胞凋亡：明显降低Lck (Y505F)转化的BaF3细胞中Lck自身磷酸化；抑制Lck(Y505F)转化的BaF3细胞STAT5b与DNA的结合；明显降低STAT5b酪氨酸磷酸化水平。SOCS1和SOCS3可以抑制Lck的转化BaF3细胞中Lck及下游的STAT5b活动。因此,SOCS1和SOCS3逆转Lck (Y505F)介导的BaF3细胞的恶性转化。
Lck is a Src family protein tyrosine kinase and is expressed predominantly in T cells. It is essential for normal T cell development and activation. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and non-lymphoid malignancies. Like other Src family members, Lck kinase activity is negatively regulated by phosphorylation of a highly conserved tyrosine (Tyr505) located near the carboxy terminus of the protein. Intramolecular interaction between phosphorylated Tyr505 and the Src homology 2 (SH2) domain confers a closed conformation and excludes substrate binding to the kinase domain. A point mutation of Tyr505 to Phe locks Lck in an open conformation and results in a constitutively active kinase. The constitutively active Lck kinase is oncogenic and transforms fibroblasts in culture. Nevertheless, the molecular mechanisms of Lck-mediated tumorigenesis have not been fully characterized.
Suppressor of cytokine signaling (SOCS) modulates Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling in a negative feedback manner. Upon cytokine stimulation, active JAK and downstream STAT proteins induce the expression of STAT-target genes, including the SOCS genes. Cytokine-induced SOCS1 and SOCS3 proteins bind to JAK directly or the JAK-proximal sites on cytokine receptors to inhibit JAK kinase activity. The physiological importance of SOCS1 and SOCS3 is demonstrated by the lethal phenotypes observed in knockout mice, Thus they contribute to the down-regulation of JAK-STAT signaling and the transient kinetics of JAK-STAT activation by cytokines and growth factors. It suggests that SOCS1 and SOCS3 may also target oncogenic protein tyrosine kinases and function as tumor suppressors.
Constitutive activation of the JAK-STAT pathway is frequently associated with oncogenic protein tyrosine kinases and is.reported in a wide variety of human cancers. A causal relationship between STAT activation and tumorigenesis has also been established in distinct tumor models. These findings raise the possibility that the negative feedback control involving SOCS proteins may be defective in these malignant cells. The observation that fibroblasts lacking SOCS1 are more susceptible to transformation supports this hypothesis. Inhibition of SOCS3 activity in human hepatocellular carcinoma cells also promotes cell migration that contribute to metastasis. Further evidence comes from high frequencies of SOCS gene silencing by DNA hypermethylation in human cancers. SOCS1 gene silencing has been reported in both lymphoid and nonlymphoid malignancies, while SOCS3 gene silencing was observed in head and neck cancer and lung cancer. However, it is not clear whether simultaneous loss of multiple SOCS gene expression occurs in tumor cells.
In this report, we investigate the potential involvement of STAT5b activation in Lck-mediated cellular transformation and further characterize the mechanisms of SOCS1 and SOCS3 dysregulation and their tumor suppressing activity in Lck-transformed cells.
This paper includes two parts.
PartⅠ:
AIM:To investigate the potential involvement of STAT5b activation in Lck-mediated cellular transformation.
METHODS:We establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine (Y505F). To determine the effects of the constitutively active Lck kinase on STAT5b phosphorylation, a STAT5b expression construct was also transiently transfected into both T-REx-293/Lck (Y505F) and the vector control cells.
T-REx-293 cells were stably transfected with pcDNA5/TO without (vec) or with Lck (Y505F). Cells were either left untreated or treated with tetracycline (Tet) for various times. Normalized whole cell lysates were subjected to SDS-PAGE and subsequent immunoblotting with anti-Lck antibody to check Lck expression. T-REx-293/Lck (Y505F) and the vector control cells were transiently transfected with STAT5b expression construct, and then either left untreated or treated with tetracycline for 24 hours. Proteins in normalized whole cell lysates were immunoprecipitated (IP) with anti-STAT5b antibody. The immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine (pTyr) monoclonal antibody 4G10. The membrane was stripped and re-blotted with anti-STAT5 antibody to check STAT5 expression.
We also performed electrophoretic mobility shift assay (EMSA) to study whether Lck (Y505F) activates STAT5b DNA binding activity. T-REx-293/Lck (Y505F) and the vector control cells were transfected with STAT5b expression construct. Nuclear extracts containing the same amount of total proteins were subjected to EMSA with a 32P-labeled STAT5 consensus probe, or with the absence or presence of 100-fold molar excess of unlabeled wild-type or mutant MGE, anti-STAT5b or control antibody. Nuclear extract prepared from IL-3-stimulated BaF3 cells was also analyzed by EMSA for comparison.
In order to figure out whether Lck (Y505F) associates with STAT5b in cells, we performed reciprocal co-immunoprecipitation experiments in T-REx-293/Lck (Y505F) and the vector control cells which were transiently transfected with STAT5b expression construct or the vector control, whole cell lysates were analyzed by immunoblotting with anti-Myc and anti-STAT5 antibodies. Proteins were immunoprecipitated with anti-STAT5b antibody or anti-Lck antibody, immunoprecipitates were subjected to SDS-PAGE and subsequent immunoblotting with anti-Myc or anti-STAT5 antibody respectively.
The effects of active Lck kinase on cell growth and IL-3-withdrawal-induced apoptosis in T-REx-BaF3 cells were further studied by cell counting, flow cytometry analyzing annexin V conjugate to phosphatidylserine from the inner to the outer leaflet of the plasma membrane in apoptotic cells and DNA fragmentation assay.
Finally we transiently transfected T-REx-BaF3/Lck(Y505F) with expression constructs carrying wild-type STAT5b, STAT5b with a point mutation of Tyr699 to Phe, or the vector alone to analyze the effect of exogenous STAT5b on cell proliferation and cell death, the cell number was determined by trypan blue staining.
RESULTS:Tetracycline-regulated expression of Lck (Y505F) strongly induced STAT5b tyrosine phosphorylation, as a negative control, tetracycline alone did not induce STAT5b phosphorylation in the vector control cells.
Tetracycline-regulated expression of Lck (Y505F) specifically induced a distinct DNA binding activity. Lck-induced DNA binding activity could be specifically competed out by unlabeled MGE oligonucleotides, but not by unlabeled MGE oligonucleotides with mutations at consensus DNA binding sites, indicating that the binding was specific. The presence of active STAT5b was further confirmed by supershifting specifically with anti-STAT5b antibody, but not control antibody. These results clearly demonstrate that the constitutively active Lck kinase can activate STAT5b and increase DNA binding activity.
Lck (Y505F) co-immunoprecipitated with STAT5b and STAT5b co-immunoprecipitated with Lck (Y505F), the interaction between Lck (Y505F) and STAT5b suggests that the constitutively active Lck kinase may directly phosphorylate STAT5b.
T-REx-BaF3/Lck (Y505F) grew significantly faster than T-REx-BaF3 control cells at each time point, these results support the notion that the constitutively active Lck kinase promotes cell proliferation. Removal of IL-3 also caused cell death of BaF3 expressing Lck (Y505F) in the first three days, but the viability was significantly higher than the vector control. Nevertheless, T-REx-BaF3/Lck (Y505F) gradually recovered after day 3 and became IL-3-independent. The expression of Lck (Y505F) remained high in these cells after continuous passage in the absence of IL-3. Our findings clearly demonstrate that a constitutively active Lck kinase can support IL-3-independent growth of BaF3 cells. As compared to the vector control cells, expression of Lck (Y505F) significantly reduced the percentage of cells recognized by the annexin V conjugate and greatly reduced the levels of DNA fragmentation shown as a distinct ladder pattern of low molecular weight DNAs. These results further illustrate that the active Lck (Y505F) kinase can protect cells from apoptosis induced by cytokine withdrawal.
The effect of exogenous STAT5b on cell proliferation was also analyzed. As compared to the vector control, transfection of wild-type STAT5b significantly accelerated the growth of BaF3 cells expressing Lck (Y505F). Our data suggest that elevated expression of wild-type STAT5b may cooperate with the active Lck kinase in promoting cell proliferation. In contrast, STAT5b (Y699F) functioned as a dominant-negative protein to attenuate Lck-induced cell proliferation. We also examined the effects of exogenous STAT5b on cell death of T-REx-BaF3/Lck (Y505F) after IL-3 deprivation. In comparison to the vector control, transfection of wild-type STAT5b significantly reduced the death of Lck (Y505F)-expressing BaF3 cells; while transfection of STAT5b (Y699F) augmented the death of Lck (Y505F)-expressing BaF3 cells. All together, these results suggest that STAT5b may function as an important effecter molecule downstream of the constitutively active Lck kinase.
CONCLUSION:In this report, we establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine. Expression of the active Lck kinase induces both tyrosine phosphorylation and DNA binding activity of signal transducer and activator of transcription 5b (STAT5b), a STAT family member activated in a wide variety of tumor cells. The active Lck kinase interacts with STAT5b in cells, suggesting that Lck may directly phosphorylate STAT5b. Expression of the constitutively active Lck mutant in interleukin-3 (IL-3)-dependent BaF3 cells promotes cell proliferation. In addition, the active Lck kinase protects BaF3 cells from IL-3-withdrawal-induced apoptotic death and leads to IL-3-independent growth. These transforming properties of the oncogenic Lck kinase can be further augmented by expression of exogenous wild-type STAT5b, but attenuated by a dominant-negative form of STAT5b. All together, our results suggest the potential involvement of STAT5b in Lck-mediated cellular transformation.
Part II:
AIM:To further characterize the mechanisms of SOCS1 and SOCS3 dysregulation and their tumor suppressing activity in Lck-transformed cells.
METHODS:Using SOCS1 or SOCS3 full-length cDNA probes, we examined SOCS1 and SOCS3 gene expression in LSTRA cells by Northern blot analysis. To verify whether STAT5 is functional in activating other target genes, we examined the expression of Bcl-xL, an anti-apoptotic gene induced by active STAT5.
We also performed pervanadate (a potent tyrosine phosphatase inhibitor that strongly activates tyrosine kinase signal transduction) stimulation experiments in LSTRA cells and used Northern blot to detect SOCS1 and SOCS3 gene expression. LSTRA and U266 cells were either untreated (-) or treated with 5'aza-cytidine (5-AzaC) or vehicle, SOCS1 and SOCS3 mRNA expression levels was determined RT-PCP to confirm that SOCS1 and SOCS3 gene silence was cause by DNA hypermethylation.
The effects of SOCS1 and SOCS3 on Lck-transformed BaF3 cell growth and IL-3-withdrawal-induced apoptosis in T-REx-BaF3 cells were further studied by cell counting, flow cytometry analyzing annexin V conjugate to phosphatidylserine from the inner to the outer leaflet of the plasma membrane in apoptotic cells and DNA fragmentation assay.
Lck-transformed BaF3 cells were transfected with pEF (vector), pEF-FLAG-I/mSOCSl (SOCS1), or pEF-FLAG-Ⅰ/mSOCS3 (SOCS3). Proteins in .lysates were immunoprecipitated with anti-Lck antibody and subjected to in vitro kinase assay (IVK). Anti-Lck immunoprecipitates were subjected to SDS-PAGE and transferred to membrane, followed by autoradiography and immunoblotting with anti-Myc antibody.
We also performed electrophoretic mobility shift assay (EMSA) to check whether exogenous SOCS1 and SOCS3 reduce STAT5b activity in Lck-transformed BaF3 cells. Nuclear extracts each containing 2μg of total proteins isolated from Lck-transformed BaF3 cells transfected with pEF (vector), pEF-FLAG-I/mSOCS1 (SOCS1), or pEF-FLAG-I/mSOCS3 (SOCS3) were subjected to EMSA with a 2P-labeled mammary gland element (MGE) that contains consensus STAT5-binding site.
The effects of exogenous SOCS1 and SOCS3 on STAT5b activity in Lck-transformed BaF3 cells were studied by immunoprecipitation using anti-STAT5b antibody, immunoprecipitates were resolved by SDS-PAGE followed by anti-phosphotyrosine immunoblotting, the blot was stripped and then reprobed with anti-STAT5 antibody to check STAT5 expression.
RESULTS:We failed to detect mRNA from either gene in LSTRA cells, that means SOCS1 and SOCS3 gene expression is undetectable in LSTRA cells. The results also demonstrate the levels of Bcl-xL transcripts is elevated in LSTRA cells, from these observations, we conclude that STAT5-target genes are differently regulated in LSTRA cells. While Bcl-xL expression is induced, two SOCS family members are not expressed.
SOCS3 mRNA can be detected in LSTRA cells after stimulation with pervanadate for 1 and 2 hours. These results demonstrate that the machinery in activating SOCS3 gene expression is still intact in LSTRA cells. In contrast to SOCS3, SOCS1 expression cannot be detected during our time course of pervanadate treatment in LSTRA cells. It raises the possibility that SOCS1 gene may be silenced by DNA hypermethylation. Therefore, the relatively fast SOCS3 induction by pervanadate also suggests that the absence of SOCS3 expression in LSTRA. is independent of DNA hypermethylation. As shown by RT-PCR analysis, SOCS1 gene expression can be reactivated by 5'aza-cytidine treatment, a known DNA methyltransferase inhibitor which reverse the effects from DNA hypermethylation, but not in untreated or vehicle-treated cells. These results point to the existence of two distinct mechanisms underlying the loss of SOCS1 and SOCS3 gene expression in LSTRA cells. So in LSTRA leukemia, SOCS1 and SOCS3 genes are silenced by DNA methylation-dependent and independent mechanisms, respectively.
There was a significant decrease in cell proliferation 24 hours after transfection of either SOCS1 or SOCS3 in comparison to vector-transfected cells. SOCS1 and SOCS3 expression also led to a significant increase in cell death over this same time period. These results suggest that enforced SOCS1 and SOCS3 expression could reverse the transformed phenotypes in Lck transformed BaF3 cells.
We found that the percentage of AnnexinⅤ-positive cells is significantly higher in SOCS1-and SOCS3-transfected cells as compared to vector-transfected cells. Consistent with elevated AnnexinⅤstainning, enforced SOCS1 and SOCS3 expression results in significant DNA fragmentation.
IVK results shows that Lck (Y505F) prepared from vector-transfected Lck-transformed BaF3 cells have high levels of 32P incorporation due to autophosphorylation. In contrast, Lck autophosphorylation was greatly reduced in cells expressing exogenous SOCS1 or SOCS3. We conclude from these experiments that Lck kinase activity is attenuated in cells expressing SOCS1 or SOCS3.
Similar to LSTRA cells, Lck-transformed BaF3 nuclear extracts exhibit elevated DNA binding activity to a STAT5 consensus sequence in an electrophoretic mobility shift assays (EMSA). As compared to the vector control, STAT5b DNA-binding activity is greatly reduced in cells expressing either SOCS1 or SOCS3. It demonstrates that enforced SOCSl or SOCS3 expression can suppress STAT5b DNA binding activity in Lck-transformed BaF3 cells.
STAT5b immunoprecipitates show high levels of tyrosine phosphorylation in vector-transfected cells. Similarly, STAT5b tyrosine phosphorylation is greatly reduced in cells expressing SOCS1 or SOCS3. This result illustrates that expression of SOCS1 or SOCS3 in Lck-transformed BaF3 cells also reduces STAT5b activity, which is downstream of Lck (Y505F).
CONCLUSION:In summary, this is the first report confirming tumor suppressor activity of SOCS1 and SOCS3 toward oncogenic Lck kinase. Both SOCS1 and SOCS3 are not expressed in Lck transformed cells, highlighting the importance of silencing both genes to create an intracellular environment favorable for STAT activation and tumor progression. Nevertheless, loss of SOCS1 and SOCS3 gene expression is mediated by different mechanisms in Lck-transformed cells. Further characterization of SOCS gene expression in other tumor cells will provide important insights in understanding the roles of different SOCS family members in tumor progression.
Our results clearly demonstrate that exogenous SOCS1 and SOCS3 inhibit oncogenic Lck kinase activity and downstream STAT5b functions. We showed previously that STAT5b activation is critical in Lck-mediated oncogenesis. Therefore, attenuation of Lck-STAT5b signaling may contribute to the subsequent biological effects, including reduced cell proliferation and augmented apoptosis, in Lck-transformed cells. Altogether, we conclude that introduction of SOCS1 and SOCS3 can reverse Lck-induced transformation in BaF3 cells by inactivating Lck and the downstream transcription factor STAT5b.
细胞因子与相应的受体结合后引起受体分子的二聚化,与受体偶联的JAK激酶相互接近并通过交互的酪氨酸磷酸化作用而活化。活化的JAK催化结合在受体上的STAT蛋白发生磷酸化修饰,STAT蛋白活化后以二聚体的形式进入细胞核内与靶基因结合,调控基因的转录,包括SOCS基因的转录。因此,JAK-STAT信号转导通路参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。
细胞因子信号抑制因子(suppressors of cytokine signaling, SOCS)家族是一类可被多种细胞因子诱导产生,且对细胞因子信号通路具有负反馈调节作用的蛋白分子。SOCS主要通过抑制JAK-STAT通路抑制信号转导,从而对细胞因子、激素、生长因子作用的强度和持续时间进行调控。SOCS主要通过以下三种不同的方式对细胞因子信号通路进行调节：第一,通过其SH2结构域与靶蛋白的磷酸酪氨酸结合,使JAK激酶的N末端失活而抑制信号转导；第二,抑制STAT与受体位点的结合；第三,通过SOCS盒促进所结合蛋白发生蛋白酶体依赖途径的降解。研究表明：SOCS1和SOCS3可能作用于持续性激活的酪氨酸蛋白激酶,或作为JAK/STAT通路的负调控因子,因而可能为肿瘤抑制因子。但SOCS在肿瘤发生中的作用有待进一步的研究。
本研究首先建立了四环素诱导精确控制Lck激酶表达的T-REx(T-REx-293, T-REx-BaF3)细胞系统,转染组成型激活的Lck (Y505F)及STAT5b真核表达质粒,通过表达外源性Lck和STAT5,精确地研究Lck激活对STAT5的影响,且可进行互动免疫沉淀观察细胞内Lck与STAT5b蛋白质的相互作用。
同时应用表达高水平Lck的小鼠淋巴瘤系LSTRA细胞及转染组成型激活Lck (Y505F)后转化的BaF3细胞,从细胞和分子等不同水平,系统观察了细胞增殖、细胞凋亡,信号蛋白间的相互作用、信号通路激活机制及SOCS1、SOCS3作为负性调控因子对JAK/STAT信号通路的影响。本文为阐明Lck蛋白酪氨酸激酶与JAK-STAT-SOCS信号通路在淋巴细胞和非淋巴细胞白血病的作用及机制提供实验依据,为白血病的防治提供一条新的线索。
全文包括两部分：
第一部分：组成型激活Lck (Y505F)致细胞恶性转化
目的：研究组成型激活Lck (Y505F)激酶对细胞的转化作用及机制
方法：利用加入四环素可去除pcDNA5/TO中启动子抑制因子,从而启动插入基因表达的T-REx-293细胞,转染组成型激活的Lck (Y505F)及STAT5b真核表达质粒,通过表达外源性Lck和STAT5,精确地研究Lck激活对STAT5的影响。
用抗Myc抗体和抗STAT5抗体,免疫印迹法检测转染后Lck及STAT5蛋白的表达水平；抗STAT5抗体免疫沉淀法沉淀STAT5,用抗蛋白酪氨酸磷酸化抗体4G10观察Lck (Y505F)对STAT5b酪氨酸磷酸化的影响。
收集短暂转染STAT5b真核表达质粒后24小时Lck (Y505F)转化的T-REx-293细胞,采用细胞外液低渗透压法提取核蛋白；用T4多聚核苷酸激酶将32P-ATP标记寡聚核苷酸；提取的核蛋白与32P标记的MGE一致序列的DNA结合后,进行电泳迁移率变动分析(EMSA法)实验,放射自显影观察Lck(Y505F)对STAT5b与DNA结合力的影响。
以抗Lck抗体,抗STAT5抗体和蛋白A／G—琼脂糖微球,采取抗Lck抗体沉淀STAT5,抗STAT5抗体沉淀Lck的互动免疫沉淀法观察细胞内Lck与STAT5b蛋白质的相互作用。
深入研究Lck (Y505F)对小鼠原B细胞系BaF3细胞生物学的影响。以细胞增殖,细胞凋亡为指标,通过台盼蓝染色用血细胞计数板计算BaF3细胞转染后2小时及24小时的细胞数。计存活细胞及死细胞数,计算细胞存活率及死亡细胞百分率。
收集培养液中去除白介素-3后24小时Lck (Y505F)转化的BaF3细胞,用Alexa Fluor 647-conjugated Annexin V萤光染色,经FACSCantTM流式细胞仪分析,Quest软件采集数据分析细胞AnnexinⅤ阳性率,研究Lck (Y505F)对转化的BaF3细胞凋亡的影响。
收集经电穿孔短暂转染质粒后24小时的BaF3细胞,用酚／氯仿抽提纯化,α-丙醇沉淀基因组DNA。1.2%琼脂糖凝胶电泳,溴化乙锭染色。观察DNA断裂片断。
为进一步研究STAT5b在Lck(Y505F)致细胞转化中的作用,T-REx-BaF3细胞转染WT型STAT5b和突变型STAT5b (Y699F)真核质粒,表达STAT5或主域负性蛋白STAT5b,观察WT型STAT5b和突变型STAT5b (Y699F)对Lck(Y505F)细胞增殖及凋亡的影响。
结果：Lck的表达12小时达高峰并维持至24小时,作为阴性对照,转染载体的T-REx-293细胞无Lck的表达；抗STAT5抗体免疫沉淀STAT5后,蛋白酪氨酸磷酸化抗体4G10的免疫印迹显示：Lck可促进STAT5b酪氨酸磷酸化,从而激活STAT5b。
Lck可促进STAT5b与32P标记的MGE一致序列的DNA的结合,未用32P标记的寡聚DNA无结合及加入STAT5b抗体出现超迁移现象,证明STAT5b与DNA结合具有特异性。结果显示Lck可激活STAT5b,促进STAT5b与DNA结合力增加。
互动免疫沉淀试验结果表明：T-REx-293细胞中共同表达外源性Lck和STAT5b后,用抗STAT5b抗体免疫沉淀后可沉淀Lck蛋白,反之,用抗Lck抗体进行的免疫沉淀可沉淀STAT5b,由此可见,细胞中Lck和STAT5b结合在一起,故Lck和STAT5b在细胞中存在蛋白间的相互作用,Lck可能直接激活STAT5b。这也表明组成型激活的Lck (Y505F)可激活STAT5b。
表达Lck (Y505F)后第一、二、三天BaF3细胞计数量均明显增加(P<0.05及P<0.01),结果表明Lck激活促进BaF3细胞增殖。
BaF3的生长依赖于白介素-3,去除培养液中的白介素-3后,BaF3细胞可发生细胞凋亡。转染载体的BaF3细胞在去除培养液中白介素-3后至第六天几无存活,而转染Lck(Y505F)真核表达质粒的BaF3细胞在去除培养液中白介素-3后,前三天存活率下降,但其降幅明显低于载体对照(P<0.01及P<0.001),第四天后细胞存活率明显增加,至第六天细胞存活率恢复至去除白介素-3的水平,BaF3细胞能独立于白介素-3的刺激生长。
转染Lck的BaF3细胞Annexin V阳性率明显减低(P<0.01)；DNA断裂片断也明显减少。结果表明Lck激活可促进BaF3细胞抗凋亡。
T-REx-BaF3细胞短暂转染WT STAT5b或STAT5b (Y699F)后可表达等量的STAT5b蛋白。与转染载体质粒相比,转染WT型STAT5b质粒可促进BaF3细胞增殖,与之相反,主域负性STAT5b (Y699F)则抑制Lck激活诱导的细胞增殖(p<0.05及P<0.01)。去除培养液中的白介素-3后,与转染载体质粒相比转染WT型STAT5b质粒的BaF3细胞死亡率明显降低(P<0.05),而转染主域负性STAT5b (Y699F)质粒的BaF3细胞死亡率明显增加(P<0.05),结果显示STAT5b在Lck激活诱导的细胞生物学中发挥重要的下游信号分子作用。
结论：组成型激活的Lck (Y505F)诱导STAT5b的酪氨酸磷酸化,激活STAT5b;促进STAT5b与DNA结合力增加；Lck与STAT5b在细胞内相互作用。转染Lck (Y505F)促进BaF3细胞增殖,抵抗去除IL-3诱导的细胞凋亡,而主域负性蛋白STAT5b (Y699F突变)逆转Lck (Y505F)的促细胞增殖及抗凋亡作用。因此,Lck通过STAT5b的激活导致细胞恶性转化。
第二部分SOCS1和SOCS3抑制Lck (Y505F)导致的细胞转化
目的：研究SOCS1和SOCS3对抑制Lck (Y505F)导致的细胞转化的抑制作用
方法：小鼠T细胞系LSTRA淋巴瘤细胞(过度表达Lck激酶),用32P标记的全长cDNA以Northern杂交分析法检测中SOCS1, SOCS3, Bcl-xL, GAPDH的表达；观察用过钒酸钠(酪氨酸激酶激活剂)刺激LSTRA细胞后SOCS1,SOCS3的表达变化；用5-’氮杂胞苷(DNA甲基抑制剂)处理LSTRA和U266细胞,采用RT-PCR法检测5′-氮杂胞苷处理后LSTRA细胞SOCS1 mRNA表达水平。
用电穿孔法将pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3真核表达质粒短暂转染入Lck (Y505F)转化的BaF3细胞。以细胞增殖,细胞凋亡为指标,通过台盼蓝染色用血细胞计数板计算BaF3细胞转染后2小时及24小时的细胞数。计存活细胞及死细胞数,计算细胞存活率及死亡细胞百分率。
收集经电穿孔短暂转染pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3质粒后24小时BaF3细胞,用Alexa Fluor 647-conjugated AnnexinⅤ萤光染色,经FACSCantoTM流式细胞仪分析,Quest软件采集数据分析细胞AnnexinⅤ阳性率,观察SOCS1, SOCS3对细胞凋亡的影响。
收集经电穿孔短暂转染质粒后24小时的BaF3细胞,用酚／氯仿抽提纯化,α-丙醇沉淀基因组DNA。1.2%琼脂糖凝胶电泳,溴化乙锭染色。观察DNA断裂片断,进一步检测SOCS1, SOCS3对细胞凋亡的影响。
收集经电穿孔短暂转染pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3质粒后24小时BaF3细胞,用抗Myc抗体和抗Flag抗体免疫印迹法检测全细胞裂解液中外源性Lck和SOCS蛋白的表达水平。用抗Lck抗体免疫沉淀Lck,测定体外激酶活性(IVK),观察SOCS1, SOCS3对Lck激酶的抑制作用。
收集经电穿孔短暂转染pEF, pEF-FLAG-Ⅰ/mSOCS1, pEF-FLAG-Ⅰ/mSOCS3质粒后24小时BaF3细胞,采用细胞外液低渗透压法提取核蛋白；用T4多聚核苷酸激酶将32P-ATP标记寡聚核苷酸；提取的核蛋白与32P标记的MGE一致序列的DNA结合后,进行电泳迁移率变动分析(EMSA法)实验,放射自显影观察SOCS1, SOCS3对Lck (Y505F)转化的BaF3细胞STAT5b与DNA结合力的变化。
免疫印迹检测转染后Lck及STAT蛋白的表达水平,用抗STAT5b抗体和蛋白A／G-琼脂糖微球,免疫沉淀法沉淀STAT5,用抗蛋白酪氨酸磷酸化抗体4G10测定SOCS1, SOCS3对Lck (Y505F)转化的BaF3细胞STAT5b酪氨酸磷酸化的影响。
结果：使用SOCS1和SOCS3全长cDNA探针,没有检测到LSTRA细胞中SOCS1和SOCS3 mRNA的表达。与此结果相似,EL4细胞,表达正常水平Lck的小鼠T细胞系也没有检测到SOCS1和SOCS3基因的转录。而作为阳性对照,白介素-3刺激强烈激活BaF3细胞SOCS1和SOCS3基因的表达。表明SOCS1和SOCS3基因在LSTRA细胞中表达缺失。
过钒酸钠处理LSTRA细胞15min-2h,利用Northern杂交检测SOCS1和SOCS3基因的表达。过钒酸钠刺激LSTRA细胞1-2h后可检测到SOCS3基因的表达,显示LSTRA细胞启动SOCS3基因表达的机制仍然完好无损。而在过钒酸钠刺激的LSTRA细胞,观察的时间内未检测到SOCS1的表达,说明SOCS1基因沉默可能是通过DNA甲基化而发生。用DNA甲基抑制剂5-’氮杂胞苷抑制DNA甲基化的影响,采用RT-PCR分析显示5’-氮杂胞苷处理可重新激活SOCS1基因表达,但未经处理或溶剂对照处理LSTRA细胞无此现象。
因此,通过酪氨酸激酶激活剂过钒酸钠刺激LSTRA细胞及DNA甲基抑制剂5-’氮杂胞苷处理LSTRA的研究表明：DNA甲基化介导SOCS1的基因沉默,而SOCS3表达缺失的机制独立于DNA甲基化。
与载体相比,短暂转染SOCS1和SOCS3真核表达质粒的Lck (Y505F)转化的BaF3细胞增殖明显降低,细胞死亡明显增加(P均<0.001)；Annexin V阳性细胞百分比明显增加(P<0.001)；DNA断裂形成也明显增加。提示SOCS1和SOCS3抑制Lck转化的BaF3细胞增殖及促进细胞凋亡
转染空载体质粒的Lck转化的BaF3细胞中由于Lck (Y505F)自身磷酸化激活致掺入的32P明显增加。短暂转染SOCS1和SOCS3真核表达质粒的Lck(Y505F)转化的BaF3细胞磷酸化明显降低。
Lck转化的BaF3细胞核提取物与STAT5的一致序列DNA体外结合明显增加。与空载体质粒相比,短暂转染SOCS1和SOCS3真核表达质粒的Lck(Y505F)转化的BaF3细胞STAT5b与DNA的结合明显降低。STAT5b免疫沉淀结果显示转染空载体质粒的Lck转化的BaF3细胞中有较高水平的酪氨酸磷酸化。同样,表达SOCS1或SOCS3明显降低STAT5b酪氨酸磷酸化水平。结果提示：表达SOCS1和SOCS3可以降低Lck的转化BaF3细胞中Lck (Y505F)下游的STAT5b活动。
结论：SOCS1和SOCS3 mRNA在LSTRA淋巴瘤细胞中因基因沉默而表达缺失,DNA甲基化介导SOCS1的基因沉默；而SOCS3表达缺失的机制独立于DNA甲基化。SOCS1和SOCS3显著抑制Lck转化的BaF3细胞增殖及促进细胞凋亡：明显降低Lck (Y505F)转化的BaF3细胞中Lck自身磷酸化；抑制Lck(Y505F)转化的BaF3细胞STAT5b与DNA的结合；明显降低STAT5b酪氨酸磷酸化水平。SOCS1和SOCS3可以抑制Lck的转化BaF3细胞中Lck及下游的STAT5b活动。因此,SOCS1和SOCS3逆转Lck (Y505F)介导的BaF3细胞的恶性转化。
Lck is a Src family protein tyrosine kinase and is expressed predominantly in T cells. It is essential for normal T cell development and activation. Aberrant expression or activation of Lck kinase has been reported in both lymphoid and non-lymphoid malignancies. Like other Src family members, Lck kinase activity is negatively regulated by phosphorylation of a highly conserved tyrosine (Tyr505) located near the carboxy terminus of the protein. Intramolecular interaction between phosphorylated Tyr505 and the Src homology 2 (SH2) domain confers a closed conformation and excludes substrate binding to the kinase domain. A point mutation of Tyr505 to Phe locks Lck in an open conformation and results in a constitutively active kinase. The constitutively active Lck kinase is oncogenic and transforms fibroblasts in culture. Nevertheless, the molecular mechanisms of Lck-mediated tumorigenesis have not been fully characterized.
Suppressor of cytokine signaling (SOCS) modulates Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling in a negative feedback manner. Upon cytokine stimulation, active JAK and downstream STAT proteins induce the expression of STAT-target genes, including the SOCS genes. Cytokine-induced SOCS1 and SOCS3 proteins bind to JAK directly or the JAK-proximal sites on cytokine receptors to inhibit JAK kinase activity. The physiological importance of SOCS1 and SOCS3 is demonstrated by the lethal phenotypes observed in knockout mice, Thus they contribute to the down-regulation of JAK-STAT signaling and the transient kinetics of JAK-STAT activation by cytokines and growth factors. It suggests that SOCS1 and SOCS3 may also target oncogenic protein tyrosine kinases and function as tumor suppressors.
Constitutive activation of the JAK-STAT pathway is frequently associated with oncogenic protein tyrosine kinases and is.reported in a wide variety of human cancers. A causal relationship between STAT activation and tumorigenesis has also been established in distinct tumor models. These findings raise the possibility that the negative feedback control involving SOCS proteins may be defective in these malignant cells. The observation that fibroblasts lacking SOCS1 are more susceptible to transformation supports this hypothesis. Inhibition of SOCS3 activity in human hepatocellular carcinoma cells also promotes cell migration that contribute to metastasis. Further evidence comes from high frequencies of SOCS gene silencing by DNA hypermethylation in human cancers. SOCS1 gene silencing has been reported in both lymphoid and nonlymphoid malignancies, while SOCS3 gene silencing was observed in head and neck cancer and lung cancer. However, it is not clear whether simultaneous loss of multiple SOCS gene expression occurs in tumor cells.
In this report, we investigate the potential involvement of STAT5b activation in Lck-mediated cellular transformation and further characterize the mechanisms of SOCS1 and SOCS3 dysregulation and their tumor suppressing activity in Lck-transformed cells.
This paper includes two parts.
PartⅠ:
AIM:To investigate the potential involvement of STAT5b activation in Lck-mediated cellular transformation.
METHODS:We establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine (Y505F). To determine the effects of the constitutively active Lck kinase on STAT5b phosphorylation, a STAT5b expression construct was also transiently transfected into both T-REx-293/Lck (Y505F) and the vector control cells.
T-REx-293 cells were stably transfected with pcDNA5/TO without (vec) or with Lck (Y505F). Cells were either left untreated or treated with tetracycline (Tet) for various times. Normalized whole cell lysates were subjected to SDS-PAGE and subsequent immunoblotting with anti-Lck antibody to check Lck expression. T-REx-293/Lck (Y505F) and the vector control cells were transiently transfected with STAT5b expression construct, and then either left untreated or treated with tetracycline for 24 hours. Proteins in normalized whole cell lysates were immunoprecipitated (IP) with anti-STAT5b antibody. The immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with anti-phosphotyrosine (pTyr) monoclonal antibody 4G10. The membrane was stripped and re-blotted with anti-STAT5 antibody to check STAT5 expression.
We also performed electrophoretic mobility shift assay (EMSA) to study whether Lck (Y505F) activates STAT5b DNA binding activity. T-REx-293/Lck (Y505F) and the vector control cells were transfected with STAT5b expression construct. Nuclear extracts containing the same amount of total proteins were subjected to EMSA with a 32P-labeled STAT5 consensus probe, or with the absence or presence of 100-fold molar excess of unlabeled wild-type or mutant MGE, anti-STAT5b or control antibody. Nuclear extract prepared from IL-3-stimulated BaF3 cells was also analyzed by EMSA for comparison.
In order to figure out whether Lck (Y505F) associates with STAT5b in cells, we performed reciprocal co-immunoprecipitation experiments in T-REx-293/Lck (Y505F) and the vector control cells which were transiently transfected with STAT5b expression construct or the vector control, whole cell lysates were analyzed by immunoblotting with anti-Myc and anti-STAT5 antibodies. Proteins were immunoprecipitated with anti-STAT5b antibody or anti-Lck antibody, immunoprecipitates were subjected to SDS-PAGE and subsequent immunoblotting with anti-Myc or anti-STAT5 antibody respectively.
The effects of active Lck kinase on cell growth and IL-3-withdrawal-induced apoptosis in T-REx-BaF3 cells were further studied by cell counting, flow cytometry analyzing annexin V conjugate to phosphatidylserine from the inner to the outer leaflet of the plasma membrane in apoptotic cells and DNA fragmentation assay.
Finally we transiently transfected T-REx-BaF3/Lck(Y505F) with expression constructs carrying wild-type STAT5b, STAT5b with a point mutation of Tyr699 to Phe, or the vector alone to analyze the effect of exogenous STAT5b on cell proliferation and cell death, the cell number was determined by trypan blue staining.
RESULTS:Tetracycline-regulated expression of Lck (Y505F) strongly induced STAT5b tyrosine phosphorylation, as a negative control, tetracycline alone did not induce STAT5b phosphorylation in the vector control cells.
Tetracycline-regulated expression of Lck (Y505F) specifically induced a distinct DNA binding activity. Lck-induced DNA binding activity could be specifically competed out by unlabeled MGE oligonucleotides, but not by unlabeled MGE oligonucleotides with mutations at consensus DNA binding sites, indicating that the binding was specific. The presence of active STAT5b was further confirmed by supershifting specifically with anti-STAT5b antibody, but not control antibody. These results clearly demonstrate that the constitutively active Lck kinase can activate STAT5b and increase DNA binding activity.
Lck (Y505F) co-immunoprecipitated with STAT5b and STAT5b co-immunoprecipitated with Lck (Y505F), the interaction between Lck (Y505F) and STAT5b suggests that the constitutively active Lck kinase may directly phosphorylate STAT5b.
T-REx-BaF3/Lck (Y505F) grew significantly faster than T-REx-BaF3 control cells at each time point, these results support the notion that the constitutively active Lck kinase promotes cell proliferation. Removal of IL-3 also caused cell death of BaF3 expressing Lck (Y505F) in the first three days, but the viability was significantly higher than the vector control. Nevertheless, T-REx-BaF3/Lck (Y505F) gradually recovered after day 3 and became IL-3-independent. The expression of Lck (Y505F) remained high in these cells after continuous passage in the absence of IL-3. Our findings clearly demonstrate that a constitutively active Lck kinase can support IL-3-independent growth of BaF3 cells. As compared to the vector control cells, expression of Lck (Y505F) significantly reduced the percentage of cells recognized by the annexin V conjugate and greatly reduced the levels of DNA fragmentation shown as a distinct ladder pattern of low molecular weight DNAs. These results further illustrate that the active Lck (Y505F) kinase can protect cells from apoptosis induced by cytokine withdrawal.
The effect of exogenous STAT5b on cell proliferation was also analyzed. As compared to the vector control, transfection of wild-type STAT5b significantly accelerated the growth of BaF3 cells expressing Lck (Y505F). Our data suggest that elevated expression of wild-type STAT5b may cooperate with the active Lck kinase in promoting cell proliferation. In contrast, STAT5b (Y699F) functioned as a dominant-negative protein to attenuate Lck-induced cell proliferation. We also examined the effects of exogenous STAT5b on cell death of T-REx-BaF3/Lck (Y505F) after IL-3 deprivation. In comparison to the vector control, transfection of wild-type STAT5b significantly reduced the death of Lck (Y505F)-expressing BaF3 cells; while transfection of STAT5b (Y699F) augmented the death of Lck (Y505F)-expressing BaF3 cells. All together, these results suggest that STAT5b may function as an important effecter molecule downstream of the constitutively active Lck kinase.
CONCLUSION:In this report, we establish a tetracycline-inducible system to study the biochemical and biological effects of a constitutively active Lck mutant with a point mutation at the negative regulatory tyrosine. Expression of the active Lck kinase induces both tyrosine phosphorylation and DNA binding activity of signal transducer and activator of transcription 5b (STAT5b), a STAT family member activated in a wide variety of tumor cells. The active Lck kinase interacts with STAT5b in cells, suggesting that Lck may directly phosphorylate STAT5b. Expression of the constitutively active Lck mutant in interleukin-3 (IL-3)-dependent BaF3 cells promotes cell proliferation. In addition, the active Lck kinase protects BaF3 cells from IL-3-withdrawal-induced apoptotic death and leads to IL-3-independent growth. These transforming properties of the oncogenic Lck kinase can be further augmented by expression of exogenous wild-type STAT5b, but attenuated by a dominant-negative form of STAT5b. All together, our results suggest the potential involvement of STAT5b in Lck-mediated cellular transformation.
Part II:
AIM:To further characterize the mechanisms of SOCS1 and SOCS3 dysregulation and their tumor suppressing activity in Lck-transformed cells.
METHODS:Using SOCS1 or SOCS3 full-length cDNA probes, we examined SOCS1 and SOCS3 gene expression in LSTRA cells by Northern blot analysis. To verify whether STAT5 is functional in activating other target genes, we examined the expression of Bcl-xL, an anti-apoptotic gene induced by active STAT5.
We also performed pervanadate (a potent tyrosine phosphatase inhibitor that strongly activates tyrosine kinase signal transduction) stimulation experiments in LSTRA cells and used Northern blot to detect SOCS1 and SOCS3 gene expression. LSTRA and U266 cells were either untreated (-) or treated with 5'aza-cytidine (5-AzaC) or vehicle, SOCS1 and SOCS3 mRNA expression levels was determined RT-PCP to confirm that SOCS1 and SOCS3 gene silence was cause by DNA hypermethylation.
The effects of SOCS1 and SOCS3 on Lck-transformed BaF3 cell growth and IL-3-withdrawal-induced apoptosis in T-REx-BaF3 cells were further studied by cell counting, flow cytometry analyzing annexin V conjugate to phosphatidylserine from the inner to the outer leaflet of the plasma membrane in apoptotic cells and DNA fragmentation assay.
Lck-transformed BaF3 cells were transfected with pEF (vector), pEF-FLAG-I/mSOCSl (SOCS1), or pEF-FLAG-Ⅰ/mSOCS3 (SOCS3). Proteins in .lysates were immunoprecipitated with anti-Lck antibody and subjected to in vitro kinase assay (IVK). Anti-Lck immunoprecipitates were subjected to SDS-PAGE and transferred to membrane, followed by autoradiography and immunoblotting with anti-Myc antibody.
We also performed electrophoretic mobility shift assay (EMSA) to check whether exogenous SOCS1 and SOCS3 reduce STAT5b activity in Lck-transformed BaF3 cells. Nuclear extracts each containing 2μg of total proteins isolated from Lck-transformed BaF3 cells transfected with pEF (vector), pEF-FLAG-I/mSOCS1 (SOCS1), or pEF-FLAG-I/mSOCS3 (SOCS3) were subjected to EMSA with a 2P-labeled mammary gland element (MGE) that contains consensus STAT5-binding site.
The effects of exogenous SOCS1 and SOCS3 on STAT5b activity in Lck-transformed BaF3 cells were studied by immunoprecipitation using anti-STAT5b antibody, immunoprecipitates were resolved by SDS-PAGE followed by anti-phosphotyrosine immunoblotting, the blot was stripped and then reprobed with anti-STAT5 antibody to check STAT5 expression.
RESULTS:We failed to detect mRNA from either gene in LSTRA cells, that means SOCS1 and SOCS3 gene expression is undetectable in LSTRA cells. The results also demonstrate the levels of Bcl-xL transcripts is elevated in LSTRA cells, from these observations, we conclude that STAT5-target genes are differently regulated in LSTRA cells. While Bcl-xL expression is induced, two SOCS family members are not expressed.
SOCS3 mRNA can be detected in LSTRA cells after stimulation with pervanadate for 1 and 2 hours. These results demonstrate that the machinery in activating SOCS3 gene expression is still intact in LSTRA cells. In contrast to SOCS3, SOCS1 expression cannot be detected during our time course of pervanadate treatment in LSTRA cells. It raises the possibility that SOCS1 gene may be silenced by DNA hypermethylation. Therefore, the relatively fast SOCS3 induction by pervanadate also suggests that the absence of SOCS3 expression in LSTRA. is independent of DNA hypermethylation. As shown by RT-PCR analysis, SOCS1 gene expression can be reactivated by 5'aza-cytidine treatment, a known DNA methyltransferase inhibitor which reverse the effects from DNA hypermethylation, but not in untreated or vehicle-treated cells. These results point to the existence of two distinct mechanisms underlying the loss of SOCS1 and SOCS3 gene expression in LSTRA cells. So in LSTRA leukemia, SOCS1 and SOCS3 genes are silenced by DNA methylation-dependent and independent mechanisms, respectively.
There was a significant decrease in cell proliferation 24 hours after transfection of either SOCS1 or SOCS3 in comparison to vector-transfected cells. SOCS1 and SOCS3 expression also led to a significant increase in cell death over this same time period. These results suggest that enforced SOCS1 and SOCS3 expression could reverse the transformed phenotypes in Lck transformed BaF3 cells.
We found that the percentage of AnnexinⅤ-positive cells is significantly higher in SOCS1-and SOCS3-transfected cells as compared to vector-transfected cells. Consistent with elevated AnnexinⅤstainning, enforced SOCS1 and SOCS3 expression results in significant DNA fragmentation.
IVK results shows that Lck (Y505F) prepared from vector-transfected Lck-transformed BaF3 cells have high levels of 32P incorporation due to autophosphorylation. In contrast, Lck autophosphorylation was greatly reduced in cells expressing exogenous SOCS1 or SOCS3. We conclude from these experiments that Lck kinase activity is attenuated in cells expressing SOCS1 or SOCS3.
Similar to LSTRA cells, Lck-transformed BaF3 nuclear extracts exhibit elevated DNA binding activity to a STAT5 consensus sequence in an electrophoretic mobility shift assays (EMSA). As compared to the vector control, STAT5b DNA-binding activity is greatly reduced in cells expressing either SOCS1 or SOCS3. It demonstrates that enforced SOCSl or SOCS3 expression can suppress STAT5b DNA binding activity in Lck-transformed BaF3 cells.
STAT5b immunoprecipitates show high levels of tyrosine phosphorylation in vector-transfected cells. Similarly, STAT5b tyrosine phosphorylation is greatly reduced in cells expressing SOCS1 or SOCS3. This result illustrates that expression of SOCS1 or SOCS3 in Lck-transformed BaF3 cells also reduces STAT5b activity, which is downstream of Lck (Y505F).
CONCLUSION:In summary, this is the first report confirming tumor suppressor activity of SOCS1 and SOCS3 toward oncogenic Lck kinase. Both SOCS1 and SOCS3 are not expressed in Lck transformed cells, highlighting the importance of silencing both genes to create an intracellular environment favorable for STAT activation and tumor progression. Nevertheless, loss of SOCS1 and SOCS3 gene expression is mediated by different mechanisms in Lck-transformed cells. Further characterization of SOCS gene expression in other tumor cells will provide important insights in understanding the roles of different SOCS family members in tumor progression.
Our results clearly demonstrate that exogenous SOCS1 and SOCS3 inhibit oncogenic Lck kinase activity and downstream STAT5b functions. We showed previously that STAT5b activation is critical in Lck-mediated oncogenesis. Therefore, attenuation of Lck-STAT5b signaling may contribute to the subsequent biological effects, including reduced cell proliferation and augmented apoptosis, in Lck-transformed cells. Altogether, we conclude that introduction of SOCS1 and SOCS3 can reverse Lck-induced transformation in BaF3 cells by inactivating Lck and the downstream transcription factor STAT5b.
引文
1. Martin GS. The hunting of the Src. Nat Rev Mol Cell Biol.2001; 2:467-475.
2. Palacios EH, Weiss A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene.2004; 23:7990-8000.
3. Abraham KM, Levin SD, Marth JD, Forbush KA, Perlmutter RM. Thymic tumorigenesis induced by overexpression of p561ck. Proc Natl Acad Sci USA. 1991;88:3977-3981.
4. Marth JD, Disteche C, Pravtcheva D, et al. Localization of a lymphocyte-specific protein tyrosine kinase gene (lck) at a site of frequent chromosomal abnormalities in human lymphomas. Proc Natl Acad Sci USA.1986; 83:7400-7404.
5. Wright DD, Sefton BM, Kamps MP. Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia. Mol Cell Biol.1994; 14:2429-2437.
6. Majolini MB, Boncristiano M, Baldari CT. Dysregulation of the protein tyrosine kinase Lck in lymphoproliferative disorders and in other neoplasias. Leukemia Lymphoma.1999; 35:245-254.
7. Sefton BM. The lck tyrosine protein kinase. Oncogene.1991; 6:683-686.
8. Kazansky AV, Kabotyanski EB, Wyszomierski SL, Mancini MA, Rosen JM. Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A. J Biol Chem.1999; 274:22484-22492.
9. Kazansky AV, Rosen JM. Signal transducers and activators of transcription 5B potentiates v-Src-mediated transformation of NIH-3T3 cells. Cell Growth Differ. 2001;12:1-7.
10. Boggon TJ, Eck MJ. Structure and regulation of Src family kinases. Oncogene. 2004;23:7918-7927.
11. Croker BA, Kiu H and Nicholson SE:SOCS regulation of the JAK/STAT signalling pathway.Sem Cell Dev Biol.2008;19:414-422.
12. Alexander WS, Starr R, Fenner JE, Scott CL, Handman E, Sprigg NS, Corbin JE, Cornish AL, Darwiche R, Owczarek CM, Kay TW, Nicola NA, Hertzog PJ, Metcalf D and Hilton DJ:SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell.1999;98:597-608.
13. Roberts AW, Robb L, Rakar S, Hartley L, Cluse L, Nicola NA, Metcalf D, Hilton DJ and Alexander WS:Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc Natl Acad Sci USA.2001;98: 9324-9329.
14. Marine JC, McKay C, Wang D, Topham DJ, Parganas E, Nakajima H, Pendeville H, Yasukawa H, Sasaki A, Yoshimura A and Ihle JN:SOCS3 is essential in the regulation of fetal liver erythropoiesis. Cell.1999;98:617-627.
15. Liu E, Cote JF and Vuori K:Negative regulation of FAK signaling by SOCS proteins. EMBO J.2003;22:5036-5046.
16. Marth JD, Cooper JA, King CS, et al. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp561ck). Mol Cell Biol. 1988;8:540-550.
17. Amrein KE, Sefton BM. Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine kinase, p56lck, reveals its oncogenic potential in fibroblasts. Proc Natl Acad Sci USA.1988;85:4247-4251.
18. Yu H, Jove R. The STATs of cancer-new molecular target come of age. Nat Rev Cancer.2004;4:97-105.
19. Bromberg J. Stat proteins and oncogenesis. J Clin Invest.2002;109:139-142.
20. Levy DE, Darnell JE. Stats:transcriptional control and biological impact. Nat Rev Mol Cell Biol.2002;3:651-662.
21. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002:new surprises in the Jak/Stat pathway. Cell.2002;109:121-131.
22. Ihle JN. The Stat family in cytokine signaling. Curr Opin Cell Biol. 2001;13:211-217.
23. Sonoyama J, Matsumura I, Ezoe S, et al. Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of BCR/ABL in K562 cells. J Biol Chem.2002;277:8076-8082.
24. Lund TC, Coleman C, Horvath E, et al. The Src-family kinase Lck can induce STAT3 phosphorylation and DNA binding activity. Cell Signal.1999; 11:789-796.
25. Matsumura I, Nakajima K, Wakao H, et al. Involvement of prolonged Ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line. Mol Cell Biol.1998;18:4282-4290.
26. Kofoed EM, Hwa V, Little B, et al. Growth hormone insensitivity associated with a STAT5b mutation. New Engl J Med.2003;349:1139-1147.
27. Paukku K, Silvennoinen O. STATs as critical mediators of signal transduction and transcription:lessons learned from STAT5. Cytokine Growth Factor Rev. 2004;15:435-455.
28. Grimley PM, Dong F, Rui H. Stat5a and Stat5b:fraternal twins of signal transduction and transcriptional activation. Cytokine Growth Factor Rev.1999; 10:131-157.
29. Herrington J, Smit LS, Schwartz J, Carter-Su C. The role of STAT proteins in growth hormone signaling. Oncogene.2000;19:2585-2597.
30. Nieborowska-Skorska M, Slupianek A, Xue L, et al. Role of signal transducer and activator of transcription 5 in nucleophosmin/anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells. Cancer Res. 2001;61:6517-6523.
31. van Engeland M, Nieland LJW, Ramaekers FCS, Schutte B, Reutelingsperger CPM. Annexin V-Affinity assay:A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry.1998;31:1-9.
32. Herrmann M, Lorenz HM, Voll R, et al. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res.1994;22:5506-5507.
33. Skorski T. Oncogenic tyrosine kinases and the DNA-damage response. Nat Rev Cancer.2002;2:351-360.
34. Xi S, Zhang Q, Gooding WE, Smithgall TE, Grandis JR. Constitutive activation of Stat5b contributes to carcinogenesis in vivo. Cancer Res.2003;63:6763-6771.
35. Leong PL, Xi S, Drenning SD, et al. Differential function of STAT5 isoforms in head and neck cancer growth control. Oncogene.2002; 21:2846-2853.
36. Welte T, Leitenberg D, Dittel BN, et al. STAT5 interaction with the T cell receptor complex and stimulation of T cell proliferation. Science.1999; 283: 222-225.
37. Biola A, Lefebvre P, Perrin-Wolff M, et al. Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1. Mol Endocrinol. 2001;15:1062-1076.
38. Yu C-L, Meyer DJ, Campbell GS, et al. Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science.1995; 269:81-83.
1. Croker BA, Kiu H and Nicholson SE:SOCS regulation of the JAK/STAT signalling pathway. Sem Cell Dev Biol.2008;19:414-422.
2. Alexander WS, Starr R, Fenner JE, Scott CL, Handman E, Sprigg NS, Corbin JE, Cornish AL, Darwiche R, Owczarek CM, Kay TW, Nicola NA, Hertzog PJ, Metcalf D and Hilton DJ:SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell. 1999;98:597-608.
3. Roberts AW, Robb L, Rakar S, Hartley L, Cluse L, Nicola NA, Metcalf D, Hilton DJ and Alexander WS:Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc Natl Acad Sci USA.2001;98:9324-9329.
4. Marine JC, McKay C, Wang D, Topham DJ, Parganas E, Nakajima H, Pendeville H, Yasukawa H, Sasaki A, Yoshimura A and Ihle JN:SOCS3 is essential in the regulation of fetal liver erythropoiesis. Cell.1999;98:617-627.
5. Liu E, Cote JF and Vuori K:Negative regulation of FAK signaling by SOCS proteins. EMBO J.2003;22:5036-5046.
6. Liu L, Gao Y, Qiu H, Miller WT, Poli V and Reich NC:Identification of STAT3 as a specific substrate of breast tumor kinase. Oncogene.2006;25:4904-4912.
7. Niwa Y, Kanda H, Shikauchi Y, Saiura A, Matsubara K, Kitagawa T, Yamamoto J, Kubo T and Yoshikawa H:Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene.2005;24:6406-6417.
8. Rottapel R, Ilangumaran S, Neale C, La Rose J, Ho JM, Nguyen MH, Barber D, Dubreuil P and de Sepulveda P:The tumor suppressor activity of SOCS-1. Oncogene.2002;21:4351-4362.
9. Limnander A, Danial NN and Rothman PB:v-Abl signaling disrupts SOCS-1 function in transformed pre-B cells. Mol Cell.2004;15:329-341.
10. Yu H and Jove R:The STATs of cancer-new molecular target come of age. Nat Rev Cancer.2004;4:97-105.
11. Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE, Harris CC and Herman JG:SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet.2001;28:29-35.
12. Galm O, Yoshikawa H, Esteller M, Osieka R and Herman JG:SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood.2003; 101:2784-2788.
13. Liu TC, Lin SF, Chang JG, Yang MY, Hung SY and Chang CS:Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia. Br J Haematol. 2003;123:654-661.
14. Watanabe D, Ezoe S, Fujimoto M, Kimura A, Saito Y, Nagai H, Tachibana I, Matsumura I, Tanaka T, Kanegane H, Miyawaki T, Emi M, Kanakura Y, Kawase I, Naka T and Kishimoto T:Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines. Br J Haematol. 2004;126:726-735.
15. Fukushima N, Sato N, Sahin F, Su GH, Hruban RH and Goggins M:Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer.2003;89:338-343.
16. Nagai H, Naka T, Terada Y, Komazaki T, Yabe A, Jin E, Kawanami O, Kishimoto T, Konishi N, Nakamura M, Kobayashi Y and Emi M:Hypermethylation associated with inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human hepatoblastomas. J Hum Genet.2003;48:65-69.
17. Fujitake S, Hibi K, Okochi O, Kodera Y, Ito K, Akiyama S and Nakao A:Aberrant methylation of SOCS-1 was observed in younger colorectal cancer patients. J Gastroenterol.2004;39:120-124.
18. Oshimo Y, Kuraoka K, Nakayama H, Kitadai Y, Yoshida K, Chayama K and Yasui W:Epigenetic inactivation of SOCS-1 by CpG island hypermethylation in human gastric carcinoma. Int J Cancer.2004;112:1003-1009.
19. To KF, Chan MW, Leung WK, Ng EK, Yu J, Bai AH, Lo AW, Chu SH, Tong JH, Lo KW, Sung JJ and Chan FK:Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line. Br J Cancer.2004;91:1335-1341.
20. Isomoto H, Mott JL, Kobayashi S, Werneburg NW, Bronk SF, Haan S and Gores GJ:Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing. Gastroenterology.2007;132:384-396.
21. Weber A, Hengge UR, Bardenheuer W, Tischoff I, Sommerer F, Markwarth A, Dietz A, Wittekind C and Tannapfel A:SOCS-3 is frequently methylated in head and neck squamous cell carcinoma and its precursor lesions and causes growth inhibition. Oncogene.2005;24:6699-6708.
22. He B, You L, Uematsu K, Zang K, Xu Z, Lee AY, Costello JF, McCormick F and Jablons DM:SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proc Natl Acad Sci USA.2003; 100:14133-14138.
23. Tischoff I, Hengge UR, Vieth M, Ell C, Stolte M, Weber A, Schmidt WE and Tannapfel A:Methylation of SOCS-3 and SOCS-1 in the carcinogenesis of Barrett's adenocarcinoma. Gut.2007;56:1047-1053.
24. Sutherland KD, Lindeman GJ, Choong DY, Wittlin S, Brentzell L, Phillips W, Campbell IG and Visvader JE:Differential hypermethylation of SOCS genes in ovarian and breast carcinomas. Oncogene.2004;23:7726-7733.
25. Kim LC, Song L and Haura EB:Src kinases as therapeutic targets for cancer. Nat Rev Clin Oncol.2009;6:587-595.
26. Sefton BM:The lck tyrosine protein kinase. Oncogene.1991;6:683-686.
27. Abraham KM, Levin SD, Marth JD, Forbush KA and Perlmutter RM:Thymic tumorigenesis induced by overexpression of p561ck. Proc Natl Acad Sci USA. 1991;88:3977-3981.
28. Majolini MB, Boncristiano M and Baldari CT:Dysregulation of the protein tyrosine kinase LCK in lymphoproliferative disorders and in other neoplasias. Leuk Lymphoma.1999;35:245-254.
29. Wright DD, Sefton BM and Kamps MP:Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia. Mol Cell Biol.1994; 14:2429-2437.
30. Yu CL, Jove R and Burakoff SJ:Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase. J Immunol.1997;159:5206-5210.
31. Shi M, Cooper JC and Yu CL:A constitutively active Lck kinase promotes cell proliferation and resistance to apoptosis through signal transducer and activator of transcription 5b activation. Mol Cancer Res.2006;4:39-45.
32. Cooper JC, Boustead JN and Yu CL:Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS). Cell Signal.2006;18:851-860.
33. Yu CL, Prochownik EV, Imperiale MJ and Jove R:Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways. Mol Cell Biol.1993; 13:2011-2019.
34. Ammanamanchi S and Brattain MG:5-azaC treatment enhances expression of transforming growth factor-beta receptors through down-regulation of Sp3. J Biol Chem.2001; 276:32854-32859.
35. Marth JD, Peet R, Krebs EG and Perlmutter RM:A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell.1985;43:393-404.
36. Socolovsky M, Fallon AEJ, Wang S, Brugnara C and Lodish HF:Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice:a direct role for STAT5 in Bcl-XL induction. Cell.1999;98:81-191.
37. Ruff SJ, Chen K and Cohen S:Peroxovanadate induces tyrosine phosphorylation of multiple signaling proteins in mouse liver and kidney. J Biol Chem. 1997;272:1263-1267.
38. Goodman JI and Watson RE:Altered DNA methylation:a secondary mechanism involved in carcinogenesis. Annu Rev Pharmacol Toxicol.2002;42:501-525.
39.Boggon TJ and Eck MJ:Structure and regulation of Src family kinases. Oncogene. 2004;23:7918-7927.
40. Rodriguez-Tarduchy G, Collins M and Lopez-Rivas A:Regulation of apoptosis in interleukin-3-dependent hemopoietic cells by interleukin-3 and calcium ionophores. EMBO J.1990;9:2997-3002.
41. van Engeland M, Nieland LJ, Ramaekers FC, Schutte B and Reutelingsperger CP: Annexin Ⅴ-affinity assay:a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry.1998;31:1-9.
42. Wyllie AH:Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature.1980;284:555-556.
43. Darnell JE, Jr.:STATs and gene regulation. Science.1997;277:1630-1635.
44. Iwamoto T, Senga T, Naito Y, Matsuda S, Miyake Y, Yoshimura A and Hamaguchi M:The JAK-inhibitor, JAB/SOCS-1 selectively inhibits cytokine-induced, but not v-Src induced JAK-STAT activation. Oncogene.2000; 19:4795-4801.
45. Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HME and Hilton DJ: The SOCS box:a tale of destruction and degradation. Trends Biochem Sci.2002; 27:235-241.
46. Kamizono S, Hanada T, Yasukawa H, Minoguchi S, Kato R, Minoguchi M, Hattori K, Hatakeyama S, Yada M, Morita S, Kitamura T, Kato H, Nakayama K and Yoshimura A:The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J Biol Chem.2001;276:12530-12538.
47. Frantsve J, Schwaller J, Sternberg DW, Kutok J and Gilliland DG:Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation. Mol Cell Biol.2001;21:3547-3557.
1. Hanada T, Yoshimura A:Regulation of cytokine signaling and inflammation. Cytokine and Growth Factor. Reviews.2002; 13:413.
2. Ward AC, Touw I, Yoshimura A:The jak-stat pathway in normal and perturbed hematopoiesis. Blood.2000; 95:19-29.
3. Hara T, Miyajima A:Function and signal transduction mediated by the interleukin 3 receptor system in hematopoiesis. Stem Cells.1996; 14:605-618.
4. Stroud R.M. and Wells J.A., Mechanistic diversity of cytokine receptor signaling across cell membranes, Sci. STKE.2004;7-18.
5. Behrmann I. et al., Janus kinase (Jak) subcellular localization revisited:the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase, J Biol Chem.2004; 279:35486-35493.
6. Darnell J.E et al., Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins, Science.1994;264:1415-1421.
7. Croker BA, Kiu H and Nicholson SE:SOCS regulation of the JAK/STAT signalling pathway. Sem Cell Dev Biol.2008; 19:414-422,.
8. Hanada T, Yoshimura A:Regulation of cytokine signaling and inflammation. Cytokine and Growth Factor. Reviews.2002; 13:413.
9. James C. et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera, Nature.2005; 434:1144-1148.
10. Levy DE, Darnell JE. Stats:transcriptional control and biological impact. Nat Rev Mol Cell Biol.2002;3:651-662.
11. Herrington J, Smit LS, Schwartz J, Carter-Su C. The role of STAT proteins in growth hormone signaling. Oncogene.2000;19:2585-2597.
12. Ihle JN. The Stat family in cytokine signaling. Curr Opin Cell Biol. 2001;13:211-217.
13. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002:new surprises in the Jak/Stat pathway. Cell.2002;109:S121-131.
14. Chen, Z., R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, R. Lahesmaa.. Identification of novel IL-4/Stat6-regulated genes in T lymphocytes. J Immunol. 2003;171:3627-3635.
15. Bromberg J. Stat proteins and oncogenesis. J Clin Invest.2002; 109:1139-42.
16. Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N, Taniguchi T, Fujita T, Kanekura Y, Komiya S, Yoshimura A:A new protein containing an SH2 domain that inhibits JAK kinases. Nature.1997;387:921-924.
17. Kubo M, Hanada T, Yoshimura A:Suppressors of cytokine signaling and immunity. Nature Immunol.2003;4:1169-1176.
18. Yoshimura A, Naka T, Kubo M:SOCS proteins, cytokine signaling and immune regulation. Nat Rev Immunol.2007; 7:454-465
19. Piessevaux J, Lavens D, Peelman F, Tavernier J. The many faces of the SOCS box. Cytokine Growth Factor Rev.2008; 19(5-6):371-381.
20. Kamizono S, Hanada T, Yasukawa H, Minoguchi S, Kato R, Minoguchi M, Hattori K, Morita S, Kitamura T, Kato H, Nakayama K, Yoshimura A:The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J Biol Chem.2001; 276:12530-12538.
21. Alexander, W. S., D. J. Hilton. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol.2004; 22:503-529.
22. Platanias, L. C. Mechanisms of type-I-and type-II-interferon-mediated signalling. Nat. Rev. Immunol..2005; 5:375-386.
23. Yoshimura, A., T. Naka, M. Kubo. SOCS proteins, cytokine signalling, and immune regulation. Nat Rev Immunol.2007; 7:454-465.
24. Jegalian AG, Wu H. Regulation of Socs gene expression by the proto-oncoprotein GFI-1B:two routes for STAT5 target gene induction by erythropoietin. J Biol Chem.2002; 18,277(3):2345-52.
25. Sasaki A, Yasukawa H, Suzuki A, Kamizono S, Syoda T, Kinjyo I, Sasaki M, Johnston JA, Yoshimura A:Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells.1999; 4:339-351.
26. Marine J-C, Topham DJ, McKay C, Wang D, Parganas E, Stravopodis D, Yoshimura A, Ihle JN:SOCS1 Deficiency Causes a Lymphocyte-Dependent Perinatal Lethality. Cell.1999; 98:609-616.
27. Kim JH, Kim JE, Liu HY, Cao W, Chen J. Regulation of interleukin-6-induced hepatic insulin resistance by mammalian target of rapamycin through the STAT3-SOCS3 pathway. J Biol Chem.2008;283(2):708-715.
28. Bullock AN, Debreczeni JE, Edwards AM, Sundstrom M, Knapp S. Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc Natl Acad Sci U S A.2006;103(20):7637-7642.
29. Gregorief A,Pyronnet S,Sonenberg N,et al.Regulation of SOCS-1 expression by translational repression.J Biol Chem.2000;275(28):21596-21604.
30. Krebs DL, Hilton DJ. SOCS Proteins:Negative Regulators of Cytokine Signaling.Stem Cells.2001;19(5):378-387.
31. Chen XP, Losman JA, Rothman P. SOCS proteins regulators of intracellular signaling. Immunity.2000;13(3):287-290.
32. Yoshimura A,Ohkubo T,Kiguchi T,et al.A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. EMBO J.1995;14(12):2816-2826.
33. Hansen JA,Lindberg K,Hilton D J,et al. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins.Mol Endocrinol.1999;13(11):1832-1843.
34. Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, Hilton DJ. The SOCS box:a tale of destruction and degradation. Trends Biochem Sci.2002; 27(5):235-241.
35. Yu H, Jove R. The STATs of cancer-new molecular target come of age. Nat Rev Cancer.2004;4:97-105.
36. Marth JD, Cooper JA, King CS, et al. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp561ck). Mol Cell Biol. 1988;8:540-550.
37. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 2002;109:1139-42.
38. Levy DE, Darnell JE. Stats:transcriptional control and biological impact. Nat Rev Mol Cell Biol.2002;3:651-662.
39. Battle TE, Wierda WG, Rassenti LZ, Zahrieh D, Neuberg D, Kipps TJ, Frank DA. In vivo activation of signal transducer and activator of transcription 1 after CD 154 gene therapy for chronic lymphocytic leukemia is associated with clinical and immunologic response. Clin Cancer Res.2003;9(6):2166-2172.
40. Weisberg E, Barrett R, Liu Q, Stone R, Gray N, Griffin JD. FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist Updat. 2009;12(3):81-89.
41. Spiekermann K, Bagrintseva K, Schwab R, Schmieja K, Hiddemann W. Overexpression and constitutive activation of FLT3 induces STAT5 activation in primary acute myeloid leukemia blast cells. Clin Cancer Res. 2003;9(6):2140-2150.
42. O'Farrell AM, Foran JM, Fiedler W, Serve H, Paquette RL, Cooper MA, Yuen HA, Louie SG, Kim H, Nicholas S, Heinrich MC, Berdel WE, Bello C, Jacobs M, Scigalla P, Manning WC, Kelsey S, Cherrington JM. An innovative phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients. Clin Cancer Res.2003;9(15):5465-5476.
43. Cea M, Cagnetta A, Garuti A, Cirmena G, Rocco I, Moran E, Grillo V, Ballestrero A, Patrone F, Nencioni A. Molecular diagnosis and monitoring of chronic myelogenous leukemia:BCR-Abl and more. J BUON.2009;14(4):565-573.
44. Carron C, Cormier F, Janin A, Lacronique V, Giovannini M, Daniel MT, Bernard O, Ghysdael JTEL-JAK2 transgenic mice develop T-cell leukemia. Blood.2000; 15;95(12):3891-3899.
45. Ferrand A, Kowalski-Chauvel A, Bertrand C, Escrieut C, Mathieu A, Portolan G, Pradayrol L, Fourmy D, Dufresne M, Seva C. A novel mechanism for JAK2 activation by a G protein-coupled receptor, the CCK2R:implication of this signaling pathway in pancreatic tumor models. J Biol Chem. 2005;280(11):10710-10715.
46. Liu E, Cote JF and Vuori K:Negative regulation of FAK signaling by SOCS proteins. EMBO J.2003; 22:5036-5046.
47. Liu L, Gao Y, Qiu H, Miller WT, Poli V and Reich NC:Identification of STAT3 as a specific substrate of breast tumor kinase. Oncogene.2006; 25:4904-4912.
48. Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE, Harris CC and Herman JG:SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet.2001;28:29-35.
49. Galm O, Yoshikawa H, Esteller M, Osieka R and Herman JG:SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood.2003;101:2784-2788.
50. Liu TC, Lin SF, Chang JG, Yang MY, Hung SY and Chang CS:Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia. Br J Haematol.2003; 123:654-661.
51. Watanabe D, Ezoe S, Fujimoto M, Kimura A, Saito Y, Nagai H, Tachibana I, Matsumura I, Tanaka T, Kanegane H, Miyawaki T, Emi M, Kanakura Y, Kawase I, Naka T and Kishimoto T:Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines. Br J Haematol. 2004; 126:726-735.
52. Fukushima N, Sato N, Sahin F, Su GH, Hruban RH and Goggins M:Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer.2003;89:338-343.
53. Nagai H, Naka T, Terada Y, Komazaki T, Yabe A, Jin E, Kawanami O, Kishimoto T, Konishi N, Nakamura M, Kobayashi Y and Emi M:Hypermethylation associated with inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human hepatoblastomas. J Hum Genet.2003;48:65-69.
54. Fujitake S, Hibi K, Okochi O, Kodera Y, Ito K, Akiyama S and Nakao A:Aberrant methylation of SOCS-1 was observed in younger colorectal cancer patients. J Gastroenterol.2004;39:120-124.
55. Oshimo Y, Kuraoka K, Nakayama H, Kitadai Y, Yoshida K, Chayama K and Yasui W:Epigenetic inactivation of SOCS-1 by CpG island hypermethylation in human gastric carcinoma. Int J Cancer.2004;112:1003-1009.
56. To KF, Chan MW, Leung WK, Ng EK, Yu J, Bai AH, Lo AW, Chu SH, Tong JH, Lo KW, Sung JJ and Chan FK:Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line. Br J Cancer.2004; 91:1335-1341.
57. Frantsve J, Schwaller J, Sternberg DW, Kutok J, Gilliland DG. Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation. Mol Cell Biol.2001; 21(10):3547-3557
58. Niwa Y, Kanda H, Shikauchi Y, Saiura A, Matsubara K, Kitagawa T, Yamamoto J, Kubo T and Yoshikawa H:Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene.2005; 24:6406-6417.
59. Isomoto H, Mott JL, Kobayashi S, Werneburg NW, Bronk SF, Haan S and Gores GJ:Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing. Gastroenterology.2007;132:384-396.
60. Weber A, Hengge UR, Bardenheuer W, Tischoff I, Sommerer F, Markwarth A, Dietz A, Wittekind C and Tannapfel A:SOCS-3 is frequently methylated in head and neck squamous cell carcinoma and its precursor lesions and causes growth inhibition. Oncogene.2005;24:6699-6708.
61. He B, You L, Uematsu K, Zang K, Xu Z, Lee AY, Costello JF, McCormick F and Jablons DM:SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proc Natl Acad Sci USA.2003; 100:14133-14138.
62. Rottapel R, Ilangumaran S, Neale C, La Rose J, Ho JM, Nguyen MH, Barber D, Dubreuil P and de Sepulveda P:The tumor suppressor activity of SOCS-1. Oncogene.2002; 21:4351-4362.
63. Limnander A, Danial NN and Rothman PB:v-Abl signaling disrupts SOCS-1 function in transformed pre-B cells. Mol Cell.2004; 15:329-341.
64. Cho-Vega JH, Rassidakis GZ, Amin HM, Tsioli P, Spurgers K, Remache YK, Vega F, Goy AH, Gilles F, Medeiros LJSuppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma. Leukemia.2004; 18(11):1872-1878.
2. Palacios EH, Weiss A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene.2004; 23:7990-8000.
3. Abraham KM, Levin SD, Marth JD, Forbush KA, Perlmutter RM. Thymic tumorigenesis induced by overexpression of p561ck. Proc Natl Acad Sci USA. 1991;88:3977-3981.
4. Marth JD, Disteche C, Pravtcheva D, et al. Localization of a lymphocyte-specific protein tyrosine kinase gene (lck) at a site of frequent chromosomal abnormalities in human lymphomas. Proc Natl Acad Sci USA.1986; 83:7400-7404.
5. Wright DD, Sefton BM, Kamps MP. Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia. Mol Cell Biol.1994; 14:2429-2437.
6. Majolini MB, Boncristiano M, Baldari CT. Dysregulation of the protein tyrosine kinase Lck in lymphoproliferative disorders and in other neoplasias. Leukemia Lymphoma.1999; 35:245-254.
7. Sefton BM. The lck tyrosine protein kinase. Oncogene.1991; 6:683-686.
8. Kazansky AV, Kabotyanski EB, Wyszomierski SL, Mancini MA, Rosen JM. Differential effects of prolactin and src/abl kinases on the nuclear translocation of STAT5B and STAT5A. J Biol Chem.1999; 274:22484-22492.
9. Kazansky AV, Rosen JM. Signal transducers and activators of transcription 5B potentiates v-Src-mediated transformation of NIH-3T3 cells. Cell Growth Differ. 2001;12:1-7.
10. Boggon TJ, Eck MJ. Structure and regulation of Src family kinases. Oncogene. 2004;23:7918-7927.
11. Croker BA, Kiu H and Nicholson SE:SOCS regulation of the JAK/STAT signalling pathway.Sem Cell Dev Biol.2008;19:414-422.
12. Alexander WS, Starr R, Fenner JE, Scott CL, Handman E, Sprigg NS, Corbin JE, Cornish AL, Darwiche R, Owczarek CM, Kay TW, Nicola NA, Hertzog PJ, Metcalf D and Hilton DJ:SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell.1999;98:597-608.
13. Roberts AW, Robb L, Rakar S, Hartley L, Cluse L, Nicola NA, Metcalf D, Hilton DJ and Alexander WS:Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc Natl Acad Sci USA.2001;98: 9324-9329.
14. Marine JC, McKay C, Wang D, Topham DJ, Parganas E, Nakajima H, Pendeville H, Yasukawa H, Sasaki A, Yoshimura A and Ihle JN:SOCS3 is essential in the regulation of fetal liver erythropoiesis. Cell.1999;98:617-627.
15. Liu E, Cote JF and Vuori K:Negative regulation of FAK signaling by SOCS proteins. EMBO J.2003;22:5036-5046.
16. Marth JD, Cooper JA, King CS, et al. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp561ck). Mol Cell Biol. 1988;8:540-550.
17. Amrein KE, Sefton BM. Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine kinase, p56lck, reveals its oncogenic potential in fibroblasts. Proc Natl Acad Sci USA.1988;85:4247-4251.
18. Yu H, Jove R. The STATs of cancer-new molecular target come of age. Nat Rev Cancer.2004;4:97-105.
19. Bromberg J. Stat proteins and oncogenesis. J Clin Invest.2002;109:139-142.
20. Levy DE, Darnell JE. Stats:transcriptional control and biological impact. Nat Rev Mol Cell Biol.2002;3:651-662.
21. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002:new surprises in the Jak/Stat pathway. Cell.2002;109:121-131.
22. Ihle JN. The Stat family in cytokine signaling. Curr Opin Cell Biol. 2001;13:211-217.
23. Sonoyama J, Matsumura I, Ezoe S, et al. Functional cooperation among Ras, STAT5, and phosphatidylinositol 3-kinase is required for full oncogenic activities of BCR/ABL in K562 cells. J Biol Chem.2002;277:8076-8082.
24. Lund TC, Coleman C, Horvath E, et al. The Src-family kinase Lck can induce STAT3 phosphorylation and DNA binding activity. Cell Signal.1999; 11:789-796.
25. Matsumura I, Nakajima K, Wakao H, et al. Involvement of prolonged Ras activation in thrombopoietin-induced megakaryocytic differentiation of a human factor-dependent hematopoietic cell line. Mol Cell Biol.1998;18:4282-4290.
26. Kofoed EM, Hwa V, Little B, et al. Growth hormone insensitivity associated with a STAT5b mutation. New Engl J Med.2003;349:1139-1147.
27. Paukku K, Silvennoinen O. STATs as critical mediators of signal transduction and transcription:lessons learned from STAT5. Cytokine Growth Factor Rev. 2004;15:435-455.
28. Grimley PM, Dong F, Rui H. Stat5a and Stat5b:fraternal twins of signal transduction and transcriptional activation. Cytokine Growth Factor Rev.1999; 10:131-157.
29. Herrington J, Smit LS, Schwartz J, Carter-Su C. The role of STAT proteins in growth hormone signaling. Oncogene.2000;19:2585-2597.
30. Nieborowska-Skorska M, Slupianek A, Xue L, et al. Role of signal transducer and activator of transcription 5 in nucleophosmin/anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells. Cancer Res. 2001;61:6517-6523.
31. van Engeland M, Nieland LJW, Ramaekers FCS, Schutte B, Reutelingsperger CPM. Annexin V-Affinity assay:A review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry.1998;31:1-9.
32. Herrmann M, Lorenz HM, Voll R, et al. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Res.1994;22:5506-5507.
33. Skorski T. Oncogenic tyrosine kinases and the DNA-damage response. Nat Rev Cancer.2002;2:351-360.
34. Xi S, Zhang Q, Gooding WE, Smithgall TE, Grandis JR. Constitutive activation of Stat5b contributes to carcinogenesis in vivo. Cancer Res.2003;63:6763-6771.
35. Leong PL, Xi S, Drenning SD, et al. Differential function of STAT5 isoforms in head and neck cancer growth control. Oncogene.2002; 21:2846-2853.
36. Welte T, Leitenberg D, Dittel BN, et al. STAT5 interaction with the T cell receptor complex and stimulation of T cell proliferation. Science.1999; 283: 222-225.
37. Biola A, Lefebvre P, Perrin-Wolff M, et al. Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not AP-1. Mol Endocrinol. 2001;15:1062-1076.
38. Yu C-L, Meyer DJ, Campbell GS, et al. Enhanced DNA-binding activity of a Stat3-related protein in cells transformed by the Src oncoprotein. Science.1995; 269:81-83.
1. Croker BA, Kiu H and Nicholson SE:SOCS regulation of the JAK/STAT signalling pathway. Sem Cell Dev Biol.2008;19:414-422.
2. Alexander WS, Starr R, Fenner JE, Scott CL, Handman E, Sprigg NS, Corbin JE, Cornish AL, Darwiche R, Owczarek CM, Kay TW, Nicola NA, Hertzog PJ, Metcalf D and Hilton DJ:SOCS1 is a critical inhibitor of interferon gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Cell. 1999;98:597-608.
3. Roberts AW, Robb L, Rakar S, Hartley L, Cluse L, Nicola NA, Metcalf D, Hilton DJ and Alexander WS:Placental defects and embryonic lethality in mice lacking suppressor of cytokine signaling 3. Proc Natl Acad Sci USA.2001;98:9324-9329.
4. Marine JC, McKay C, Wang D, Topham DJ, Parganas E, Nakajima H, Pendeville H, Yasukawa H, Sasaki A, Yoshimura A and Ihle JN:SOCS3 is essential in the regulation of fetal liver erythropoiesis. Cell.1999;98:617-627.
5. Liu E, Cote JF and Vuori K:Negative regulation of FAK signaling by SOCS proteins. EMBO J.2003;22:5036-5046.
6. Liu L, Gao Y, Qiu H, Miller WT, Poli V and Reich NC:Identification of STAT3 as a specific substrate of breast tumor kinase. Oncogene.2006;25:4904-4912.
7. Niwa Y, Kanda H, Shikauchi Y, Saiura A, Matsubara K, Kitagawa T, Yamamoto J, Kubo T and Yoshikawa H:Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene.2005;24:6406-6417.
8. Rottapel R, Ilangumaran S, Neale C, La Rose J, Ho JM, Nguyen MH, Barber D, Dubreuil P and de Sepulveda P:The tumor suppressor activity of SOCS-1. Oncogene.2002;21:4351-4362.
9. Limnander A, Danial NN and Rothman PB:v-Abl signaling disrupts SOCS-1 function in transformed pre-B cells. Mol Cell.2004;15:329-341.
10. Yu H and Jove R:The STATs of cancer-new molecular target come of age. Nat Rev Cancer.2004;4:97-105.
11. Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE, Harris CC and Herman JG:SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet.2001;28:29-35.
12. Galm O, Yoshikawa H, Esteller M, Osieka R and Herman JG:SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood.2003; 101:2784-2788.
13. Liu TC, Lin SF, Chang JG, Yang MY, Hung SY and Chang CS:Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia. Br J Haematol. 2003;123:654-661.
14. Watanabe D, Ezoe S, Fujimoto M, Kimura A, Saito Y, Nagai H, Tachibana I, Matsumura I, Tanaka T, Kanegane H, Miyawaki T, Emi M, Kanakura Y, Kawase I, Naka T and Kishimoto T:Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines. Br J Haematol. 2004;126:726-735.
15. Fukushima N, Sato N, Sahin F, Su GH, Hruban RH and Goggins M:Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer.2003;89:338-343.
16. Nagai H, Naka T, Terada Y, Komazaki T, Yabe A, Jin E, Kawanami O, Kishimoto T, Konishi N, Nakamura M, Kobayashi Y and Emi M:Hypermethylation associated with inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human hepatoblastomas. J Hum Genet.2003;48:65-69.
17. Fujitake S, Hibi K, Okochi O, Kodera Y, Ito K, Akiyama S and Nakao A:Aberrant methylation of SOCS-1 was observed in younger colorectal cancer patients. J Gastroenterol.2004;39:120-124.
18. Oshimo Y, Kuraoka K, Nakayama H, Kitadai Y, Yoshida K, Chayama K and Yasui W:Epigenetic inactivation of SOCS-1 by CpG island hypermethylation in human gastric carcinoma. Int J Cancer.2004;112:1003-1009.
19. To KF, Chan MW, Leung WK, Ng EK, Yu J, Bai AH, Lo AW, Chu SH, Tong JH, Lo KW, Sung JJ and Chan FK:Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line. Br J Cancer.2004;91:1335-1341.
20. Isomoto H, Mott JL, Kobayashi S, Werneburg NW, Bronk SF, Haan S and Gores GJ:Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing. Gastroenterology.2007;132:384-396.
21. Weber A, Hengge UR, Bardenheuer W, Tischoff I, Sommerer F, Markwarth A, Dietz A, Wittekind C and Tannapfel A:SOCS-3 is frequently methylated in head and neck squamous cell carcinoma and its precursor lesions and causes growth inhibition. Oncogene.2005;24:6699-6708.
22. He B, You L, Uematsu K, Zang K, Xu Z, Lee AY, Costello JF, McCormick F and Jablons DM:SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proc Natl Acad Sci USA.2003; 100:14133-14138.
23. Tischoff I, Hengge UR, Vieth M, Ell C, Stolte M, Weber A, Schmidt WE and Tannapfel A:Methylation of SOCS-3 and SOCS-1 in the carcinogenesis of Barrett's adenocarcinoma. Gut.2007;56:1047-1053.
24. Sutherland KD, Lindeman GJ, Choong DY, Wittlin S, Brentzell L, Phillips W, Campbell IG and Visvader JE:Differential hypermethylation of SOCS genes in ovarian and breast carcinomas. Oncogene.2004;23:7726-7733.
25. Kim LC, Song L and Haura EB:Src kinases as therapeutic targets for cancer. Nat Rev Clin Oncol.2009;6:587-595.
26. Sefton BM:The lck tyrosine protein kinase. Oncogene.1991;6:683-686.
27. Abraham KM, Levin SD, Marth JD, Forbush KA and Perlmutter RM:Thymic tumorigenesis induced by overexpression of p561ck. Proc Natl Acad Sci USA. 1991;88:3977-3981.
28. Majolini MB, Boncristiano M and Baldari CT:Dysregulation of the protein tyrosine kinase LCK in lymphoproliferative disorders and in other neoplasias. Leuk Lymphoma.1999;35:245-254.
29. Wright DD, Sefton BM and Kamps MP:Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia. Mol Cell Biol.1994; 14:2429-2437.
30. Yu CL, Jove R and Burakoff SJ:Constitutive activation of the Janus kinase-STAT pathway in T lymphoma overexpressing the Lck protein tyrosine kinase. J Immunol.1997;159:5206-5210.
31. Shi M, Cooper JC and Yu CL:A constitutively active Lck kinase promotes cell proliferation and resistance to apoptosis through signal transducer and activator of transcription 5b activation. Mol Cancer Res.2006;4:39-45.
32. Cooper JC, Boustead JN and Yu CL:Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS). Cell Signal.2006;18:851-860.
33. Yu CL, Prochownik EV, Imperiale MJ and Jove R:Attenuation of serum inducibility of immediate early genes by oncoproteins in tyrosine kinase signaling pathways. Mol Cell Biol.1993; 13:2011-2019.
34. Ammanamanchi S and Brattain MG:5-azaC treatment enhances expression of transforming growth factor-beta receptors through down-regulation of Sp3. J Biol Chem.2001; 276:32854-32859.
35. Marth JD, Peet R, Krebs EG and Perlmutter RM:A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell.1985;43:393-404.
36. Socolovsky M, Fallon AEJ, Wang S, Brugnara C and Lodish HF:Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice:a direct role for STAT5 in Bcl-XL induction. Cell.1999;98:81-191.
37. Ruff SJ, Chen K and Cohen S:Peroxovanadate induces tyrosine phosphorylation of multiple signaling proteins in mouse liver and kidney. J Biol Chem. 1997;272:1263-1267.
38. Goodman JI and Watson RE:Altered DNA methylation:a secondary mechanism involved in carcinogenesis. Annu Rev Pharmacol Toxicol.2002;42:501-525.
39.Boggon TJ and Eck MJ:Structure and regulation of Src family kinases. Oncogene. 2004;23:7918-7927.
40. Rodriguez-Tarduchy G, Collins M and Lopez-Rivas A:Regulation of apoptosis in interleukin-3-dependent hemopoietic cells by interleukin-3 and calcium ionophores. EMBO J.1990;9:2997-3002.
41. van Engeland M, Nieland LJ, Ramaekers FC, Schutte B and Reutelingsperger CP: Annexin Ⅴ-affinity assay:a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry.1998;31:1-9.
42. Wyllie AH:Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature.1980;284:555-556.
43. Darnell JE, Jr.:STATs and gene regulation. Science.1997;277:1630-1635.
44. Iwamoto T, Senga T, Naito Y, Matsuda S, Miyake Y, Yoshimura A and Hamaguchi M:The JAK-inhibitor, JAB/SOCS-1 selectively inhibits cytokine-induced, but not v-Src induced JAK-STAT activation. Oncogene.2000; 19:4795-4801.
45. Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HME and Hilton DJ: The SOCS box:a tale of destruction and degradation. Trends Biochem Sci.2002; 27:235-241.
46. Kamizono S, Hanada T, Yasukawa H, Minoguchi S, Kato R, Minoguchi M, Hattori K, Hatakeyama S, Yada M, Morita S, Kitamura T, Kato H, Nakayama K and Yoshimura A:The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J Biol Chem.2001;276:12530-12538.
47. Frantsve J, Schwaller J, Sternberg DW, Kutok J and Gilliland DG:Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation. Mol Cell Biol.2001;21:3547-3557.
1. Hanada T, Yoshimura A:Regulation of cytokine signaling and inflammation. Cytokine and Growth Factor. Reviews.2002; 13:413.
2. Ward AC, Touw I, Yoshimura A:The jak-stat pathway in normal and perturbed hematopoiesis. Blood.2000; 95:19-29.
3. Hara T, Miyajima A:Function and signal transduction mediated by the interleukin 3 receptor system in hematopoiesis. Stem Cells.1996; 14:605-618.
4. Stroud R.M. and Wells J.A., Mechanistic diversity of cytokine receptor signaling across cell membranes, Sci. STKE.2004;7-18.
5. Behrmann I. et al., Janus kinase (Jak) subcellular localization revisited:the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase, J Biol Chem.2004; 279:35486-35493.
6. Darnell J.E et al., Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins, Science.1994;264:1415-1421.
7. Croker BA, Kiu H and Nicholson SE:SOCS regulation of the JAK/STAT signalling pathway. Sem Cell Dev Biol.2008; 19:414-422,.
8. Hanada T, Yoshimura A:Regulation of cytokine signaling and inflammation. Cytokine and Growth Factor. Reviews.2002; 13:413.
9. James C. et al., A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera, Nature.2005; 434:1144-1148.
10. Levy DE, Darnell JE. Stats:transcriptional control and biological impact. Nat Rev Mol Cell Biol.2002;3:651-662.
11. Herrington J, Smit LS, Schwartz J, Carter-Su C. The role of STAT proteins in growth hormone signaling. Oncogene.2000;19:2585-2597.
12. Ihle JN. The Stat family in cytokine signaling. Curr Opin Cell Biol. 2001;13:211-217.
13. O'Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002:new surprises in the Jak/Stat pathway. Cell.2002;109:S121-131.
14. Chen, Z., R. Lund, T. Aittokallio, M. Kosonen, O. Nevalainen, R. Lahesmaa.. Identification of novel IL-4/Stat6-regulated genes in T lymphocytes. J Immunol. 2003;171:3627-3635.
15. Bromberg J. Stat proteins and oncogenesis. J Clin Invest.2002; 109:1139-42.
16. Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N, Taniguchi T, Fujita T, Kanekura Y, Komiya S, Yoshimura A:A new protein containing an SH2 domain that inhibits JAK kinases. Nature.1997;387:921-924.
17. Kubo M, Hanada T, Yoshimura A:Suppressors of cytokine signaling and immunity. Nature Immunol.2003;4:1169-1176.
18. Yoshimura A, Naka T, Kubo M:SOCS proteins, cytokine signaling and immune regulation. Nat Rev Immunol.2007; 7:454-465
19. Piessevaux J, Lavens D, Peelman F, Tavernier J. The many faces of the SOCS box. Cytokine Growth Factor Rev.2008; 19(5-6):371-381.
20. Kamizono S, Hanada T, Yasukawa H, Minoguchi S, Kato R, Minoguchi M, Hattori K, Morita S, Kitamura T, Kato H, Nakayama K, Yoshimura A:The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2. J Biol Chem.2001; 276:12530-12538.
21. Alexander, W. S., D. J. Hilton. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu Rev Immunol.2004; 22:503-529.
22. Platanias, L. C. Mechanisms of type-I-and type-II-interferon-mediated signalling. Nat. Rev. Immunol..2005; 5:375-386.
23. Yoshimura, A., T. Naka, M. Kubo. SOCS proteins, cytokine signalling, and immune regulation. Nat Rev Immunol.2007; 7:454-465.
24. Jegalian AG, Wu H. Regulation of Socs gene expression by the proto-oncoprotein GFI-1B:two routes for STAT5 target gene induction by erythropoietin. J Biol Chem.2002; 18,277(3):2345-52.
25. Sasaki A, Yasukawa H, Suzuki A, Kamizono S, Syoda T, Kinjyo I, Sasaki M, Johnston JA, Yoshimura A:Cytokine-inducible SH2 protein-3 (CIS3/SOCS3) inhibits Janus tyrosine kinase by binding through the N-terminal kinase inhibitory region as well as SH2 domain. Genes Cells.1999; 4:339-351.
26. Marine J-C, Topham DJ, McKay C, Wang D, Parganas E, Stravopodis D, Yoshimura A, Ihle JN:SOCS1 Deficiency Causes a Lymphocyte-Dependent Perinatal Lethality. Cell.1999; 98:609-616.
27. Kim JH, Kim JE, Liu HY, Cao W, Chen J. Regulation of interleukin-6-induced hepatic insulin resistance by mammalian target of rapamycin through the STAT3-SOCS3 pathway. J Biol Chem.2008;283(2):708-715.
28. Bullock AN, Debreczeni JE, Edwards AM, Sundstrom M, Knapp S. Crystal structure of the SOCS2-elongin C-elongin B complex defines a prototypical SOCS box ubiquitin ligase. Proc Natl Acad Sci U S A.2006;103(20):7637-7642.
29. Gregorief A,Pyronnet S,Sonenberg N,et al.Regulation of SOCS-1 expression by translational repression.J Biol Chem.2000;275(28):21596-21604.
30. Krebs DL, Hilton DJ. SOCS Proteins:Negative Regulators of Cytokine Signaling.Stem Cells.2001;19(5):378-387.
31. Chen XP, Losman JA, Rothman P. SOCS proteins regulators of intracellular signaling. Immunity.2000;13(3):287-290.
32. Yoshimura A,Ohkubo T,Kiguchi T,et al.A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. EMBO J.1995;14(12):2816-2826.
33. Hansen JA,Lindberg K,Hilton D J,et al. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins.Mol Endocrinol.1999;13(11):1832-1843.
34. Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, Hilton DJ. The SOCS box:a tale of destruction and degradation. Trends Biochem Sci.2002; 27(5):235-241.
35. Yu H, Jove R. The STATs of cancer-new molecular target come of age. Nat Rev Cancer.2004;4:97-105.
36. Marth JD, Cooper JA, King CS, et al. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp561ck). Mol Cell Biol. 1988;8:540-550.
37. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 2002;109:1139-42.
38. Levy DE, Darnell JE. Stats:transcriptional control and biological impact. Nat Rev Mol Cell Biol.2002;3:651-662.
39. Battle TE, Wierda WG, Rassenti LZ, Zahrieh D, Neuberg D, Kipps TJ, Frank DA. In vivo activation of signal transducer and activator of transcription 1 after CD 154 gene therapy for chronic lymphocytic leukemia is associated with clinical and immunologic response. Clin Cancer Res.2003;9(6):2166-2172.
40. Weisberg E, Barrett R, Liu Q, Stone R, Gray N, Griffin JD. FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist Updat. 2009;12(3):81-89.
41. Spiekermann K, Bagrintseva K, Schwab R, Schmieja K, Hiddemann W. Overexpression and constitutive activation of FLT3 induces STAT5 activation in primary acute myeloid leukemia blast cells. Clin Cancer Res. 2003;9(6):2140-2150.
42. O'Farrell AM, Foran JM, Fiedler W, Serve H, Paquette RL, Cooper MA, Yuen HA, Louie SG, Kim H, Nicholas S, Heinrich MC, Berdel WE, Bello C, Jacobs M, Scigalla P, Manning WC, Kelsey S, Cherrington JM. An innovative phase I clinical study demonstrates inhibition of FLT3 phosphorylation by SU11248 in acute myeloid leukemia patients. Clin Cancer Res.2003;9(15):5465-5476.
43. Cea M, Cagnetta A, Garuti A, Cirmena G, Rocco I, Moran E, Grillo V, Ballestrero A, Patrone F, Nencioni A. Molecular diagnosis and monitoring of chronic myelogenous leukemia:BCR-Abl and more. J BUON.2009;14(4):565-573.
44. Carron C, Cormier F, Janin A, Lacronique V, Giovannini M, Daniel MT, Bernard O, Ghysdael JTEL-JAK2 transgenic mice develop T-cell leukemia. Blood.2000; 15;95(12):3891-3899.
45. Ferrand A, Kowalski-Chauvel A, Bertrand C, Escrieut C, Mathieu A, Portolan G, Pradayrol L, Fourmy D, Dufresne M, Seva C. A novel mechanism for JAK2 activation by a G protein-coupled receptor, the CCK2R:implication of this signaling pathway in pancreatic tumor models. J Biol Chem. 2005;280(11):10710-10715.
46. Liu E, Cote JF and Vuori K:Negative regulation of FAK signaling by SOCS proteins. EMBO J.2003; 22:5036-5046.
47. Liu L, Gao Y, Qiu H, Miller WT, Poli V and Reich NC:Identification of STAT3 as a specific substrate of breast tumor kinase. Oncogene.2006; 25:4904-4912.
48. Yoshikawa H, Matsubara K, Qian GS, Jackson P, Groopman JD, Manning JE, Harris CC and Herman JG:SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet.2001;28:29-35.
49. Galm O, Yoshikawa H, Esteller M, Osieka R and Herman JG:SOCS-1, a negative regulator of cytokine signaling, is frequently silenced by methylation in multiple myeloma. Blood.2003;101:2784-2788.
50. Liu TC, Lin SF, Chang JG, Yang MY, Hung SY and Chang CS:Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia. Br J Haematol.2003; 123:654-661.
51. Watanabe D, Ezoe S, Fujimoto M, Kimura A, Saito Y, Nagai H, Tachibana I, Matsumura I, Tanaka T, Kanegane H, Miyawaki T, Emi M, Kanakura Y, Kawase I, Naka T and Kishimoto T:Suppressor of cytokine signalling-1 gene silencing in acute myeloid leukaemia and human haematopoietic cell lines. Br J Haematol. 2004; 126:726-735.
52. Fukushima N, Sato N, Sahin F, Su GH, Hruban RH and Goggins M:Aberrant methylation of suppressor of cytokine signalling-1 (SOCS-1) gene in pancreatic ductal neoplasms. Br J Cancer.2003;89:338-343.
53. Nagai H, Naka T, Terada Y, Komazaki T, Yabe A, Jin E, Kawanami O, Kishimoto T, Konishi N, Nakamura M, Kobayashi Y and Emi M:Hypermethylation associated with inactivation of the SOCS-1 gene, a JAK/STAT inhibitor, in human hepatoblastomas. J Hum Genet.2003;48:65-69.
54. Fujitake S, Hibi K, Okochi O, Kodera Y, Ito K, Akiyama S and Nakao A:Aberrant methylation of SOCS-1 was observed in younger colorectal cancer patients. J Gastroenterol.2004;39:120-124.
55. Oshimo Y, Kuraoka K, Nakayama H, Kitadai Y, Yoshida K, Chayama K and Yasui W:Epigenetic inactivation of SOCS-1 by CpG island hypermethylation in human gastric carcinoma. Int J Cancer.2004;112:1003-1009.
56. To KF, Chan MW, Leung WK, Ng EK, Yu J, Bai AH, Lo AW, Chu SH, Tong JH, Lo KW, Sung JJ and Chan FK:Constitutional activation of IL-6-mediated JAK/STAT pathway through hypermethylation of SOCS-1 in human gastric cancer cell line. Br J Cancer.2004; 91:1335-1341.
57. Frantsve J, Schwaller J, Sternberg DW, Kutok J, Gilliland DG. Socs-1 inhibits TEL-JAK2-mediated transformation of hematopoietic cells through inhibition of JAK2 kinase activity and induction of proteasome-mediated degradation. Mol Cell Biol.2001; 21(10):3547-3557
58. Niwa Y, Kanda H, Shikauchi Y, Saiura A, Matsubara K, Kitagawa T, Yamamoto J, Kubo T and Yoshikawa H:Methylation silencing of SOCS-3 promotes cell growth and migration by enhancing JAK/STAT and FAK signalings in human hepatocellular carcinoma. Oncogene.2005; 24:6406-6417.
59. Isomoto H, Mott JL, Kobayashi S, Werneburg NW, Bronk SF, Haan S and Gores GJ:Sustained IL-6/STAT-3 signaling in cholangiocarcinoma cells due to SOCS-3 epigenetic silencing. Gastroenterology.2007;132:384-396.
60. Weber A, Hengge UR, Bardenheuer W, Tischoff I, Sommerer F, Markwarth A, Dietz A, Wittekind C and Tannapfel A:SOCS-3 is frequently methylated in head and neck squamous cell carcinoma and its precursor lesions and causes growth inhibition. Oncogene.2005;24:6699-6708.
61. He B, You L, Uematsu K, Zang K, Xu Z, Lee AY, Costello JF, McCormick F and Jablons DM:SOCS-3 is frequently silenced by hypermethylation and suppresses cell growth in human lung cancer. Proc Natl Acad Sci USA.2003; 100:14133-14138.
62. Rottapel R, Ilangumaran S, Neale C, La Rose J, Ho JM, Nguyen MH, Barber D, Dubreuil P and de Sepulveda P:The tumor suppressor activity of SOCS-1. Oncogene.2002; 21:4351-4362.
63. Limnander A, Danial NN and Rothman PB:v-Abl signaling disrupts SOCS-1 function in transformed pre-B cells. Mol Cell.2004; 15:329-341.
64. Cho-Vega JH, Rassidakis GZ, Amin HM, Tsioli P, Spurgers K, Remache YK, Vega F, Goy AH, Gilles F, Medeiros LJSuppressor of cytokine signaling 3 expression in anaplastic large cell lymphoma. Leukemia.2004; 18(11):1872-1878.