慢性粒细胞白血病耐药机制及治疗策略

慢性粒细胞白血病是一类造血干细胞的恶性克隆性疾病,ph染色体是其特征性细胞遗传学标志,即t(9;22)(q34;ql1),存在BCR/ABL融合基因,现阶段造血干细胞移植是当前最有希望治愈CML的疗法,但受年龄、配型等限制,易发生移植物抗宿主病;复发率较高;传统的化疗、干扰素治疗也有副作用,因此,通过信号传导抑制剂抑制BCR-ABL酪氨酸激酶活性,从而阻止一系列信号传导来治疗CML是一个比较好的治疗方法,伊马替尼是一种酪氨酸激酶抑制剂是治疗慢性粒细胞白血病的靶向治疗药物,治疗疗效显著,但是并不能根治慢性粒细胞白血病,需要长期服药,一些患者出现耐药,导致治疗无效或复发。因此,寻求新的治疗方案至关重要。本文就慢性粒细胞白血病的耐药机制及治疗策略做一综述。     

过表达SERPINB2上调RB1水平抑制白血病K562细胞的生长

慢性粒细胞白血病(慢粒)急变期的治疗是目前的重要问题,临床上亟需要找到新的分子治疗靶点。本研究使用基因集群富集分析软件(gene set enrichment analysis,GSEA)分析基因表达综合数据库(gene expression omnibus,GEO)中慢粒临床样本及小鼠细胞模型的基因表达谱数据,获得与慢粒急变期相关的基因集群,然后从中选择丝氨酸蛋白酶抑制剂家族成员B2(SERPINB2)作为研究对象。应用分子克隆技术在空载质粒p Adtrack-CMV的基础上构建p Adtrack-CMV-SERPINB2重组质粒,使用电穿孔方法将上述质粒分别转入慢粒细胞系K562细胞,运用细胞免疫荧光技术检测SERPINB2在细胞内的表达与分布,采用CCK-8实验和克隆形成实验检测细胞增殖和克隆形成能力,使用流式细胞术检测细胞周期,通过Western blotting实验检测SERPINB2、BCR/ABL以及RB1的表达。结果表明,成功构建了p Adtrack-CMV-SERPINB2并转入K562细胞,细胞免疫荧光实验显示SERPINB2主要在细胞浆中分布。与对照组相比,在K562细胞中过表达SERPINB2可轻微抑制K562细胞的增殖(p0.001),明显降低K562细胞的克隆形成能力(p0.01)并导致G0/G1期的细胞比例增多(p0.001)。Western blotting显示BCR/ABL表达无明显变化,SERPINB2、RB1表达水平增高。综上所述,SERPINB2可通过上调细胞内RB1的水平,使细胞周期更多的停留在G1期,从而对K562细胞的增殖与克隆形成能力产生抑制作用。     

FKBP-RAP-FRB系统转运BCR-ABL入核对K562细胞的增殖抑制效应

BCR-ABL融合蛋白是慢性粒细胞白血病（chronic myeloid leukemia,CML）发病的基础。其中,BCR-ABL只能定位于细胞浆、不能易位至细胞核是其致病的关键因素。因此,转运BCR—ABL入核可能是治疗CML的潜在方法。该研究利用基因重组技术,构建HA-2FKBP-ABD（HF2A）和FLAG-3NLS—FRB＊（FN3R）重组腺病毒,与雷帕霉素类似物（Rapamycin analog）同组成FKBP-RAP-FRB系统,转运K562细胞胞浆中的BCR—ABL癌蛋白至细胞核,并探究其对K562细胞增殖的影响。结果显示,成功构建了高滴度的重组腺病毒,Westernblot证实目的蛋白在K562细胞内成功表达。FKBP—RAP—FRB系统可通过转运BCR—ABLA入核。抑制K562细胞生长和克隆形成的能力。结果揭示,FKBP-RAP—FRB系统转运BCR—ABL入核有望为CML提供新的治疗手段。     

核型分析与FISH技术诊断成人B细胞急性淋巴细胞白血病等臂双着丝粒费城染色体

目的提高对携带复杂费城染色体(Philadelphia chromosome,Ph)的急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)的鉴别诊断和治疗的认识。方法用染色体核型分析技术对1例成人B细胞ALL(B-ALL)患者骨髓细胞做细胞遗传学检查;核型异常细胞用荧光原位杂交(FISH)进行验证。结果首次发现该B-ALL患者异常细胞系携带等臂双着丝粒Ph,并呈现克隆演化趋势;干系克隆细胞携带典型的Ph,旁系克隆细胞的Ph发生多种演化;FISH检测显示BCR/ABL1融合基因的拷贝数逐步增加。结论成人B-ALL中Ph经复制重排形成等臂衍生Ph非常罕见;这些重排使BCR/ABL1拷贝数增加,可能是导致白血病细胞快速增殖及耐药的机制。     

过表达KLF4重组质粒的构建及其在白血病K562细胞中的表达

为了构建过表达锌指转录因子Krüppel样因子4(Krüppel-like factor 4,KLF4)基因的重组质粒pEGFP-KLF4,观察其在白血病K562细胞中的表达,以RT-PCR法扩增人KLF4基因,构建pEGFP-KLF4重组质粒;经酶切及测序鉴定后,将pEGFP-KLF4质粒电穿孔转染白血病K562细胞(K562/pEGFP-KLF4组),设pEGFP-C1空质粒转染K562细胞(K562/pEGFP-C1组)及空白K562细胞作为对照,荧光显微镜观察EGFP-KLF4融合蛋白的表达情况,同时用抗生素G418筛选阳性克隆并扩大培养建立稳定过表达KLF4基因的K562细胞株,随后用RT-PCR检测3组K562细胞中KLF4的m RNA表达水平。实验结果显示,pEGFP-KLF4重组质粒构建成功。该质粒转染K562细胞24 h后能观察到较高强度的绿色荧光;经G418筛选出的阳性克隆扩大培养后建立了稳定过表达KLF4基因的K562细胞株;与对照组相比,K562/pEGFP-KLF4细胞组KLF4的m RNA表达水平明显升高,其过表达率为65.71%(P0.05)。实验中构建的过表达KLF4基因的重组质粒pEGFP-KLF4能在K562细胞中高效表达,为进一步研究其在白血病中的作用奠定了基础。     

PESV 对K562 细胞BCR/ABL 融合基因及凋亡调控因子Bcl-2、Bad 表达的影响

目的:探讨PESV对K562细胞BCR/ABL融合基因及凋亡调控因子bcl-2和bad表达的影响.方法:将体外培养K562细胞,经PESV处理不同时间后,流式细胞术检测细胞凋亡率,荧光定量RT-PCR检测BCR/ABL、Bcl-2、Bad mRNA水平变化.结果:与对照组相比,PESV处理后K562细胞,凋亡率增加,BCR/ABL融合基因表达降低,抗凋亡相关基因Bcl-2 mRNA表达降低,促凋亡基因Bad mRNA表达增加.结论:PESV能降低降低K562细胞BCR/ABL融合基因的表达,可能通过调节Bcl-2和Bad表达,抑制K562细胞增殖,促进其凋亡.     

重组泛素连接酶SH2-U—box／RING的构建与表达

目的构建重组泛素连接酶SH2-U—box、SH2-RING,并克隆进入pFlag—CMV4真核表达载体,为研究靶向降解慢性粒细胞白血病（chronic myelocytic leukemia,CML）患者瘤细胞中过度活化的BCR／ABL,抑制肿瘤细胞的生长提供基础。方法设计引物,扩增接头分子Grb2的SH2结构域以及E3泛素连接酶CHIP的U—box、Cb1的RING结构域,通过重组PCR,将SH2分别与U—box、RING进行融合,融合片段双酶切之后插入真核表达载体pFlag—CMV4,经过酶切鉴定及测序后,转染HEK293T细胞,Western印迹验证重组质粒的表达。结果PCR结果提示SH2-U—box条带大小888bp,SH2一RING大小为633bp,重组质粒酶切鉴定和测序结果均正确,转染后可见融合蛋白的表达。结论成功构建真核重组表达载体pFlag—CMV4-SH2-U—box和pFlag—CMV4-SH2-RING,转染HEK293T细胞后能够正确表达,为后续研究奠定了基础。     

幽门螺杆菌cagA克隆表达

克隆表达4株幽门螺杆菌的cagA基因,以方便地获得大鼠CagA蛋白和重组表达质粒,为临床诊断CagA阳性幽门螺杆菌感染,以及进一步研究不同类型CagA功能及其与疾病关系提供材料。PCR扩增幽门螺杆菌的cagA基因,克隆至PinPoint^TMXa-1T载体,酶切鉴定连接方向,IPTG诱导正向连接克隆表达CagA融合蛋白并进行SDS－PAGE和Western blots鉴定。结果显示PCR扩增得到3.5-3.8kb的CagA基因,PCR及酶切鉴定得到正向连接的重组克隆,SDS－PAGE及Western blots证实正向连接的重组克隆表达CagA融合蛋白。构建了4种cagA的重组表达质粒,通过转化同一宿主菌可研究不同CagA的功能和致病性差异;通过亲和层析纯化融合蛋白可获大量CagA蛋白,用于血清学诊断CagA阳性幽门螺杆菌感染,及不同抗原性CagA与疾病之间的关系。     

海洋破囊壶菌△4-脂肪酸脱饱和酶基因在酿酒母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1.6 kb的△4-脂肪酸脱饱和酶基因(FAD4).将FAD4酶切后连接到Hond Ⅲ/XbaⅠ处理过的pYES2.0载体,构建重组表达质粒pYFAD4.转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子.添加外源脂肪酸C22:5底物,半乳糖诱导表达.气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪含量的41.13%),△4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达.     

CBX4与HOXA5在慢性粒细胞白血病中的表达及相互作用研究

Polycomb group complex（PcG）作为发挥转录抑制作用的重要表观遗传调控复合物,参与发育、衰老以及肿瘤发生等重要病生理过程。PcG成员众多,分为PRC1与PRC2两种复合物,各组分间功能既协同,又不失特性。PRC1中的CBX4独特的结构域使其功能尤为特殊。近年发现,作为一类造血干细胞恶性克隆性疾病,白血病中常伴有PcG基因的异常表达或者突变。本研究通过qPCR发现,在慢性粒细胞白血病（chronic myeloid leukemia, CML）患者外周血白细胞中存在CBX4的表达明显下调,而PcG经典靶基因HOX家族中的HOXA5则表现为上调。给予伊马替尼（Imatinib）治疗后,二者均向相反方向恢复至正常人的表达水平,并且CBX4的表达水平与CML的经典分子标志物BCR ABL1融合基因有较好的相关性。上述结果提示,CBX4可以作为CML潜在的预后标志物。为了进一步揭示CBX4与HOXA5是否存在相互作用关系,本文通过双荧光素酶实验证实,CBX4能通过HOXA5的启动子而负调控其表达。本研究发现,CBX4与HOXA5在CML中存在负相关的异常表达,且证明CBX4可作为HOXA5的负调控因子。     

Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance

The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.     

Influence of BCR/ABL fusion proteins on the course of Ph leukemias

The hallmark of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) is the presence of the Philadelphia chromosome as a result of the t(9;22) translocation. This gene rearrangement results in the production of a novel oncoprotein, BCR/ABL, a constitutively active tyrosine kinase. There is compelling evidence that the malignant transformation by BCR/ABL is critically dependent on its Abl tyrosine kinase activity. Also the bcr part of the hybrid gene takes part in realization of the malignant phenotype. We supposed that additional mutations accumulate in this region of the BCR/ABL oncogene during the development of the malignant blast crisis in CML patients. In ALL patients having p210 fusion protein the mutations were supposed to be preexisting. Sequencing of PCR product of the BCR/ABL gene (Dbl, PH region) showed that along with single-nucleotide substitutions other mutations, mostly deletions, had occurred. In an ALL patient a deletion of the 5th exon was detected. The size of the deletions varied from 36 to 220 amino acids. For one case of blast crisis of CML changes in the character of actin organization were observed. Taking into account the functional role of these domains in the cell an etiological role of such mutations on the disease phenotype and leukemia progression is plausible.     

BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.     

The BCR/ABL transgene causes abnormal NK cell differentiation and can be found in circulating NK cells of advanced phase chronic myelogenous leukemia patients.

NK cells from the blood of chronic myelogenous leukemia (CML) patients are progressively decreased in number as the disease progresses from chronic phase to blast crisis. We hypothesize that BCR/ABL may be directly responsible by interfering with NK cell differentiation. CD34(+)HLA-DR(+) cells from CML patients were studied for their capacity to differentiate into NK cells. The NK cell cloning frequency was significantly decreased from CML CD34(+)HLA-DR(+) cells compared with cells from normal donors, yet CD34(+)HLA-DR(+) cells gave rise to BCR/ABL(+) NK cells in some patients. This finding prompted us to further investigate circulating NK cells from the blood of CML patients. CD56(+)CD3(-) NK cells were sorted from CML patients and examined by fluorescence in situ hybridization (FISH). In contrast to chronic phase CML, significant numbers of NK cells from advanced phase CML patients were BCR/ABL(+), whereas T cells were always BCR/ABL(-) regardless of the disease stage. To test the effects of BCR/ABL as the sole genetic abnormality, BCR/ABL was transduced into umbilical cord blood CD34(+) cells, and NK development was studied. p210-enhanced green fluorescence protein-transduced cells gave rise to significantly decreased numbers of NK cells compared with enhanced green fluorescence protein transduction alone. In addition, the extrinsic addition of BCR/ABL-transduced autologous CD34(+) cells suppressed the NK cell differentiation of normal umbilical cord blood CD34(+)CD38(-) cells. This study provides the first evidence that BCR/ABL is responsible for the altered differentiation of NK cells and that the NK cell lineage can be involved with the malignant clone in advanced stage CML.     

Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH)

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.     

Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 microM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.     

Transferred BCR/ABL DNA from K562 Extracellular Vesicles Causes Chronic Myeloid Leukemia in Immunodeficient Mice

Our previous study showed that besides mRNAs and microRNAs, there are DNA fragments within extracellular vesicles (EVs). The BCR/ABL hybrid gene, involved in the pathogenesis of chronic myeloid leukemia (CML), could be transferred from K562 EVs to neutrophils and decrease their phagocytic activity in vitro. Our present study provides evidence that BCR/ABL DNAs transferred from EVs have pathophysiological significance in vivo. Two months after injection of K562 EVs into the tail vein of Sprague-Dawley (SD) rats, they showed some characteristics of CML, e.g., feeble, febrile, and thin, with splenomegaly and neutrophilia but with reduced neutrophil phagocytic activity. These findings were also observed in immunodeficient NOD/SCID mice treated with K562 EVs; BCR/ABL mRNA and protein were found in their neutrophils. The administration of actinomycin D, an inhibitor of de novo mRNA synthesis, prevented the abnormalities caused by K562 EVs in NOD/SCID mice related to CML, including neutrophilia and bone marrow hyperplasia. As a specific inhibitor of tyrosine kinases, imatinib blocked the activity of tyrosine kinases and the expression of phospho-Crkl, induced by the de novo BCR/ABL protein caused by K562 EVs bearing BCR/ABL DNA. Our current study shows the pathophysiological significance of transferred tumor gene from EVs in vivo, which may represent an important mechanism for tumorigenesis, tumor progression, and metastasis.     

Bcr/Abl interferes with the Fanconi anemia/BRCA pathway: implications in the chromosomal instability of chronic myeloid leukemia cells

Chronic myeloid leukemia (CML) is a malignant clonal disorder of the hematopoietic system caused by the expression of the BCR/ABL fusion oncogene. Although it is well known that CML cells are genetically unstable, the mechanisms accounting for this genomic instability are still poorly understood. Because the Fanconi anemia (FA) pathway is believed to control several mechanisms of DNA repair, we investigated whether this pathway was disrupted in CML cells. Our data show that CML cells have a defective capacity to generate FANCD2 nuclear foci, either in dividing cells or after DNA damage. Similarly, human cord blood CD34(+) cells transduced with BCR/ABL retroviral vectors showed impaired FANCD2 foci formation, whereas FANCD2 monoubiquitination in these cells was unaffected. Soon after the transduction of CD34(+) cells with BCR/ABL retroviral vectors a high proportion of cells with supernumerary centrosomes was observed. Similarly, BCR/ABL induced a high proportion of chromosomal abnormalities, while mediated a cell survival advantage after exposure to DNA cross-linking agents. Significantly, both the impaired formation of FANCD2 nuclear foci, and also the predisposition of BCR/ABL cells to develop centrosomal and chromosomal aberrations were reverted by the ectopic expression of BRCA1. Taken together, our data show for the first time a disruption of the FA/BRCA pathway in BCR/ABL cells, suggesting that this defective pathway should play an important role in the genomic instability of CML by the co-occurrence of centrosomal amplification and DNA repair deficiencies.