STI571诱导K562细胞凋亡机制研究

目的　研究格列卫 (Glivec ,STI5 71)诱导P2 10BCR/ABL(+)K5 6 2细胞凋亡机制。方法　采用Annexin Ⅴ /PI双染实验、DNA的PI染色、碘化二己基恶碳菁 (DiOC6 [3])染色、2 ,7 二氯荧光素二乙酸酯 (DCFH DA)染色及DNALadder等方法测定凋亡细胞。利用Westernblot实验分析K5 6 2细胞蛋白酪氨酸磷酸化水平 ,测定Bcl XL、caspase 3蛋白的表达并分别测定线粒体及胞浆部分的细胞色素C(cytoC)蛋白。结果　STI5 71可诱导K5 6 2细胞凋亡 ,出现DNA亚二倍体凋亡峰及DNALadder,线粒体跨膜电位及活性氧物质降低 ,P2 10BCR/ABL酪氨酸磷酸化水平降低 ,Bcl XL、蛋白减少 ,caspase 3蛋白前体活化降解出相对分子质量为 2 0× 10 3 亚单位 ,胞浆部分出现cytoC ,同时线粒体cytoC减少。结论　STI5 71可迅速使P2 10BCR/ABL酪氨酸磷酸化水平下降 ,线粒体cytoC胞浆转位介导的信号通路是STI5 71诱导K5 6 2细胞凋亡的途径之一 ,STI5 71是有效的基因靶向治疗剂     

Chronic myeloid leukemia: pathophysiology,diagnostic parameters,and current treatment concepts

Chronic myeloid leukemia (CML) is a stem cell disease characterized by excessive accumulation of clonal myeloid (precursor) cells in hematopoietic tissues. CML cells display the translocation t(9; 22) that creates the bcr/abl oncogene. The respective oncoprotein (= BCR/ABL) exhibits constitutive tyrosine kinase activity and promotes growth and survival in CML cells. Clinically, CML can be divided into three phases: the chronic phase (CP), the accelerated phase (AP), and the blast phase (BP) that resembles acute leukemia. Progression to AP and BP is associated with occurrence of additional genetic defects that cooperate with bcr/abl in leukemogenesis and lead to resistance against antileukemic drugs. The prognosis in CML is variable depending on the phase of disease, age, and response to therapy. The only curative approach available to date is stem cell transplantation. For those who cannot be transplanted, the BCR/ABL tyrosine kinase inhibitor STI571 (Glivec, Imatinib), interferon-alpha (with or without ARAC), or other cytoreductive drugs are prescribed. Currently available data show that STI571 is a superior compound compared to other drugs in producing complete cytogenetic and molecular responses. However, despite superior initial data and high expectations for an effect on survival, long term results are not available so far, and resistance against STI571 has been reported. Forthcoming strategies are therefore attempting to prevent or counteract STI571 resistance by co-administration of other antileukemic drugs. Whether these strategies will lead to curative drug therapy in CML in the future remains at present unknown.     

BCR/ABL-mediated increased expression of multiple known and novel genes that may contribute to the pathogenesis of chronic myelogenous leukemia

The BCR/ABL chimeric protein plays a central role in the pathogenesis of chronic myelogenous leukemia (CML). Intensive research has elucidated many signal transduction pathways activated by BCR/ABL. However, few studies addressed BCR/ABL-dependent alterations in gene expression that may contribute to the pathobiology of CML. To additionally define such downstream genes, we performed a subtractive hybridization between cord blood (CB) CD34(+) cells transduced with an MSCV-retrovirus vector containing either enhanced green fluorescent protein (eGFP) alone or p210(BCR/ABL)-internal ribosome entry site-eGFP. Thirty-four subtracted clones expressed in p210-eGFP but not eGFP-transduced CD34(+) cells have been confirmed by Northern blot and sequenced. Fifty-nine percent represent novel proteins, and 41% are homologous to known genes. Quantitative real-time PCR analysis confirmed that 14 of 14 genes tested were also overexpressed in additional populations of p210(BCR/ABL)-transduced CB CD34(+) cells, as well as in CD34(+) cells from primary newly diagnosed CML patients versus GFP-transduced CB or samples from normal donors. Western blot analysis showed that the known sequences were also overexpressed at the protein level. Treatment of BCR/ABL(+) cells with the Abl-specific tyrosine kinase inhibitor STI571 decreased expression at the mRNA as well as protein level of some but not all of the gene products. This suggests that increased gene expression is in some cases tyrosine kinase-independent. Some of the overexpressed genes are implicated in cellular processes known to be disturbed in CML, including the mitogen-activated protein kinase or the ubiquitin pathway, whereas overexpression of other genes, including RAN and NUP98, may implicate new cellular pathways involved in CML. Additional characterization of downstream genes activated by BCR/ABL may lead to important new insights in the molecular mechanisms underlying CML and identify potentially novel therapeutic targets for CML.     

BCR/ABL induces multiple abnormalities of cytoskeletal function.

The BCR/ABL oncogene causes human chronic myelogenous leukemia (CML), a myeloproliferative disease characterized by massive expansion of hematopoietic progenitor cells and cells of the granulocyte lineage. When transfected into murine hematopoietic cell lines, BCR/ABL causes cytokine-independence and enhances viability. There is also growing evidence that p210(BCR/ABL) affects cytoskeletal structure. p210(BCR/ABL) binds to actin, and several cytoskeletal proteins are tyrosine phosphorylated by this oncoprotein. Also, at least one aspect of cytoskeletal function is abnormal, in that the affinity of beta1 integrins for fibronectin is altered in CML cells. However, isolated changes in beta1 integrin function would be unlikely to explain the clinical phenotype of CML. We used time-lapse video microscopy to study cell motility and cell morphology on extracellular cell matrix protein-coated surfaces of a series of cell lines before and after transformation by BCR/ABL. BCR/ABL was associated with a striking increase in spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia) and accelerated the rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. Also, while untransformed cells were sessile for long periods, BCR/ABL-transformed cells exhibited persistent motility, except for brief periods during cell division. Using cell lines transformed by a temperature-sensitive mutant of BCR/ABL, these kinetic abnormalities of cytoskeletal function were shown to require BCR/ABL tyrosine kinase activity. Similar abnormalities of cytoskeletal function on fibronectin-coated surfaces were observed when hematopoietic progenitor cells purified by CD34 selection from patients with CML were compared with CD34 positive cells from normal individuals. Interestingly, alpha-interferon treatment was found to slowly revert the abnormal motility phenotype of BCR/ABL-transformed cells towards normal. The increase in spontaneous motility and other defects of cytoskeletal function described here will be useful biological markers of the functional effects of BCR/ABL in hematopoietic cells.     

STI571治疗BCR/ABL融合基因阳性白血病观察

目的 :观察酪氨酸激酶抑制剂STI5 71在BCR/ABL融合基因阳性表达白血病的治疗效果和副作用。方法 :已确诊BCR/ABL 的 8例慢性粒细胞性白血病 (CML)和 1例急性淋巴细胞白血病 (ALL)患者采用STI5 71 Ara -C治疗。结果 :2例CML -慢性期患者均CR至今 ,6例CML -急粒变患者有 2例CR ,其中 1例CR至今已 3 7周 ,1例ALL在第三次血液学CR后经治疗维持CR2 8周后复发。结论 :STI5 71可诱导BCR/ABL 白血病患者完全缓解 ,降低骨髓BCR/ABL 白血病细胞 ,延长CR期 ,药物副作用轻微易耐受 ,药物作用的个体差异性较大     

Treatment of chronic myelogenous leukemia with tyrosine kinase inhibitor]

STI571(imatinib) selectively inhibits the ABL-tyrosine kinase, the activity of which is activated by the formation of chimeric BCR/ABL. Phase I study in USA showed a remarkable effectiveness of STI571 in interferon-refractory chronic myelogenous leukemia with little adverse effects. STI571 will plausibly become the first choice drug prior to stem cell transplantation and interferon in the treatment of this leukemia.     

The P190, P210, and P230 forms of the BCR/ABL oncogene induce a similar chronic myeloid leukemia-like syndrome in mice but have different lymphoid leukemogenic activity

The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3-dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)-like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)-treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non- 5-FU-treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.     

RQ-PCR在慢性粒细胞白血病细胞BCR-ABL融合基因检测中的应用

目的探讨采用实时荧光定量逆转录聚合酶链反应(RQ-PCR)技术检测慢性粒细胞白血病(CML)细胞的BCRABLp210融合基因的临床价值。方法应用RQ-PCR技术对60例CML患者骨髓细胞BCR-ABLp210融合基因进行定量检测分析。结果 60例BCR-ABLp210融合基因阳性的CML患者中,慢性期33例,加速期13例,急变期14例,且CML急变期患者BCR-ABLp210转录本水平明显高于慢性期和加速期患者。经异基因造血干细胞移植治疗或经伊马替尼治疗6个月后,BCRABLp210转录本水平均较6个月前明显降低。但这两种方法治疗CML患者12个月后的效果比较差异无统计学意义(P0.05),而采用其他酪氨酸激酶抑制剂治疗CML 12个月后也可达到相似的治疗效果。结论 RQ-PCR技术监测CML患者细胞BCR-ABLp210融合基因表达有助于CML的诊断、治疗效果评价及微小残留的监测。     

慢性粒细胞白血病cyclin D2基因的表达研究

目的研究cyclin D2基因表达与慢性粒细胞白血病(CML)特征性的P210^BCR/ABL蛋白酪氨酸激酶活性之间的相关性。方法以RT-PCR、免疫印迹法及流式细胞术检测cyclin D2基因在BCR／ABL K562细胞系中的表达。通过表达抗ABL蛋白酪氨酸激酶区胞内抗体的模型即K562-ib-eGFP细胞，检测cyclin D2表达的变化。结果在P210^BCR/ABL蛋白酪氨酸激酶活性受抑制的K562-ib-eGFP细胞中，cyclin D2的表达(18．90％)明显低于K562细胞组(48．10％)，且K562-ib-eGFP组S期细胞比例(40．40％)也明显低于K562细胞组(64．34％)。结论cyclin D2可能是CML-P210^BCR/ABL蛋白酪氨酸激酶下游相关信号传递分子，其表达增高可能促进了CML细胞的过度增殖。     

RHBDD1基因在慢性髓系白血病患者骨髓细胞中的表达及临床意义

本研究旨在检测RHBDD1（Rhomboid domain containing 1）基因在慢性髓系白血病（CML）患者骨髓细胞中的表达水平并初步探讨其临床意义。采用实时定量PCR的方法检测RHBDD1在正常人和CML患者骨髓单个核细胞中的相对表达水平。结果表明,CML患者RHBDD1的表达水平明显高于正常人;BCR／ABL p210表达阴性的患者RHBDD1的表达水平显著高于BCR／ABLp210阳性的患者;≥50岁的患者RHBDD1表达水平低于〈50岁的患者;RHBDD1的表达水平与患者的性别无明显相关性。结论：RHBDD1基因可能参与了CML的发生与发展,尤其在BCR／ABL p210阴性患者的发病中可能发挥重要作用。     

Allogeneic stem cell transplantation]

Over the last two decades, four major therapeutic approaches have dramatically changed the prognosis in chronic myelogenous leukemia(CML). Those include allogeneic stem cell transplantation, interferon-alpha based regimen, donor-leukocyte infusions, and the revolutionary BCR/ABL tyrosine kinase inhibitor such as STI571. Each modality has exploited and targeted different aspects of CML biology, and is associated with different risk-benefit ratios. In this section, we update the results of both related and unrelated donor transplantation, donor lymphocyte infusions, and non-myeloablative stem cell transplantation in CML in comparison with the other treatment modalities.     

Treatment plan and informed consent]

Since STI571, a BCR-ABL tyrosine kinase inhibitor, has made a great therapeutic advance in the management of CML, we have to reconsider the treatment protocol for chronic phase CML. Interferon-alpha (IFN-alpha) will be replaced with STI571 therapy. However, some patients are reported to become refractory to STI571, and it is unclear whether STI571 therapy alone may be sufficient to induce long-term survival in CML. There are also important progress in the field of allogeneic hematopoietic stem cell transplantation (SCT); i.e. minitransplant(non-myeloablative SCT) and cord blood stem cell transplantation. Currently, newly-diagnosed CML patients in chronic phase should be initially treated with STI571. If the patients are appropriate candidates for allogeneic SCT and have HLA-indentical sibling donors, allogeneic SCT should be conducted within one year. The other patients should also receive related or unrelated allogeneic SCT if Ph suppression is insufficient with STI571 therapy for several months. The patients who are not candidates for allogeneic SCT may be treated with IFN-alpha and/or Hydrea(or cytosine arabinoside) in addition to STI571 if they become refractory to STI571. Since each therapeutic modality has different risk and benefits, informed consent is very important to determine the treatment plan for individual patients.     

Crosstalk between BCR/ABL oncoprotein and CXCR4 signaling through a Src family kinase in human leukemia cells

Stromal-derived factor (SDF)-1 and its G protein-coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein-coupled receptor signaling and function of mammalian precursors.     

TEC启动子介导BCR/ABL基因在BaF3细胞中的表达

转P210bcr/abl基因小鼠是研究慢性髓系白血病的理想模型,但广泛表达的启动子容易引起转基因小鼠的胚胎致死,所以应用在造血细胞中特异表达的启动子是成功建立转P210bcr/abl基因小鼠模型的关键。本研究探讨TEC启动子介导BCR/ABL基因在BF3细胞中的表达。从小鼠的基因组中克隆TEC启动子的-364至+22区域,并替换掉IRES2-eGFP载体的CMVie启动子,同时在该载体的EcorⅠ酶切位点插入7.2 kb的P210bcr/abl基因,再将构建好的载体分别转染BaF3细胞和293细胞,观察eGFP基因与P210bcr/abl基因在2种细胞中的表达情况。结果通过荧光显微镜和流式细胞仪检测发现,eGFP基因在BaF3细胞成功表达,表达率在转染后6、24、72 h分别为7.10%、23.35%和64.61%,而在293细胞中未见eG FP基因表达。RT-PCR在BaF3细胞中扩增出BCR/ABL mRNA中372 bp的片段,而在293细胞中未扩增出任何片段。结论:TEC启动子的-364至+22区域能介导相关基因在造血细胞中高效特异性表达,适合于构建转P210bcr/abl基因小鼠模型。     

Introduction: chronic myelogenous leukemia leading advanced clinical oncology]

Our understanding of the pathological molecular events underlying chronic myelogenous leukemia(CML) has rapidly grown over the past 2 decades. At the same time, new therapeutic modalities for CML which can cure or prolong survival in this formerly incurable disease, including interferon alpha administration and allogeneic hematopoietic stem cell transplantation, have been established. Now, a new molecule-specific drug, a ABL tyrosine kinase inhibitor(STI571), are developing, which is offering new hope for expanded treatment options for patients with CML. Clinical investigators and medical doctors will have responsibility in helping newly diagnosed patients with CML to properly elect treatment planning among the increased options available now. This specific number of JJ CO will be hoped to provide information to many medical oncologists beyond the hematology speciality.     

慢性粒细胞性白血病患者骨髓源bcr/abl融合基因阳性Flk1+CD31-CD34-干细胞体外抗STI571机制的初步研究

为进一步了解STI571对慢性粒细胞性白血病（CML）患者体内bcr/abl融合基因阳性的原始及定向白血病干/祖细胞分化及增殖的影响,更深入阐明部分CML患者在经历一段血液学甚至是细胞遗传学水平上的完全缓解后复发及对STI571的耐药机制,利用从CML患者骨髓分离到的具有血管母细胞特性、bcr/abl融合基因阳性、免疫表型为Flk1＋CD31-CD34-细胞,体外检测了STI571对其在造血集落培养基中的分化及处于分化阶段时的增殖抑制作用.结果显示：浓度为5 μmol/L STI571,维持作用96小时（病人体内维持96小时的STI571浓度只可能达到1-2 μmol/L）,即可有效抑制定向造血祖细胞的增殖.没有充分的证据显示,相对原始的bcr/abl融合基因阳性、免疫表型为Flk1^＋CD31^-CD34^-细胞的分化及增殖受到明显的抑制作用.结论：CML患者体内的原始白血病干/祖细胞对STI571具有一定的抗性,临床上所观察到的CML患者在运用STI571一段时间后出现正常的造血恢复现象,可能仅仅是因为STI571杀死或抑制了定向恶性白血病祖细胞的增殖,但随着时间的推移,在经历了短暂的血液学甚至是细胞遗传学水平上的缓解后,对STI571耐药的原始白血病干/祖细胞终究会再次导致CML的复发.     

FTY720, a new alternative for treating blast crisis chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphocytic leukemia

Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL+ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190(BCR/ABL) myeloid and lymphoid cell lines and CML-BC(CD34+) and Ph1 ALL(CD34+/CD19+) progenitors but not of normal CD34+ and CD34+/CD19+ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190(BCR/ABL)-driven [including p210/p190(BCR/ABL)(T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.     

CD27 signaling on chronic myelogenous leukemia stem cells activates Wnt target genes and promotes disease progression

Chronic myelogenous leukemia (CML) results from a chromosomal translocation in hematopoietic stem or early progenitor cells that gives rise to the oncogenic BCR/ABL fusion protein. Clinically, CML has a chronic phase that eventually evolves into an accelerated stage and blast crisis. A CML-specific immune response is thought to contribute to the control of disease. Whether the immune system can also promote disease progression is not known. In the present study, we investigated the possibility that the TNF receptor family member CD27 is present on leukemia stem cells (LSCs) and mediates effects of the immune system on CML. In a mouse model of CML, BCR/ABL+ LSCs and leukemia progenitor cells were found to express CD27. Binding of CD27 by its ligand, CD70, increased expression of Wnt target genes in LSCs by enhancing nuclear localization of active β-catenin and TRAF2- and NCK-interacting kinase (TNIK). This resulted in increased proliferation and differentiation of LSCs. Blocking CD27 signaling in LSCs delayed disease progression and prolonged survival. Furthermore, CD27 was expressed on CML stem/progenitor cells in the bone marrow of CML patients, and CD27 signaling promoted growth of BCR/ABL+ human leukemia cells by activating the Wnt pathway. Since expression of CD70 is limited to activated lymphocytes and dendritic cells, our results reveal a mechanism by which adaptive immunity contributes to leukemia progression. In addition, targeting CD27 on LSCs may represent an attractive therapeutic approach to blocking the Wnt/β-catenin pathway in CML.     

Tyrosine kinase inhibitors: a cure for chronic myeloid leukemia?

Chronic myeloid leukemia (CML), a malignant transformation of hematopoietic cells, accounts for one-fifth of all leukemias and will be diagnosed in 4,400 individuals in the United States this year. CML has three phases: chronic, accelerated, and blastic. Interferon, hydroxyurea, busulfan, and bone marrow and stem cell transplantation are used to treat CML, but individuals who are in the accelerated phases or blast crisis usually respond poorly to treatment. A new tyrosine kinase inhibitor, STI 571, is being evaluated in clinical trials. STI 571 is highly bioavailable in oral form and produced minimal toxicities in phase I studies. Further research is needed to determine the rate of response and survival data, but STI 571 holds great promise in the treatment of CML.