【作者】 杨明华；

【导师】 曹励之；

【作者基本信息】 中南大学 ， 儿科学， 2010， 博士

【摘要】 白血病是儿童最常见的造血系统恶性肿瘤,化疗是当前主要的治疗方法。白血病化疗失败的主要原因之一是多药耐药(multidrug resistance, MDR)的发生,尤其在儿童急性髓细胞白血病(AML)中更为突出。而且多药耐药的发生严重限制患儿长期无事件生存率。因此,揭示MDR的发病机制,寻找新的治疗十预靶点,对于防治儿童白血病具有重要意义。WASP家族富含脯氨酸同源蛋白1(WASP-family verprolin homologous protein 1, WAVE1)为肌动蛋白调节蛋白,在肌动蛋白聚合和细胞骨架重排中发挥重要作用。项目组前期研究中发现WAVE1是白血病细胞中一新型抗凋亡蛋白,并与白血病K562／A02细胞系的多药耐药发生密切相关。本研究首先收集AML患儿的BMMCs,通过实时定量PCR和蛋白印迹方法(Western blotting)对52例AML患儿骨髓单个核细胞(BMMCs)中WAVE1、P-糖蛋白(P-glycoprotein, P-gp)、多药耐药相关蛋白1(Multiple resistance-associated protein-1, MRP1)、肺耐药蛋白(lung-resistance related protein, LRP)和乳腺癌耐药蛋白(breast cancer resistance protein, BCRP)的表达情况进行了检测。研究结果显示复发／难治组AML的WAVE1及耐药相关基因表达水平显著高于缓解组患儿,而且同一患儿初治及复发时的WAVE1基因表达水平明显高于缓解期,表明WAVE1表达水平与AML的预后和疾病活动程度相关。进一步利用白血病HL60,K562及其耐药细胞系,分析了WAVE1对P-gp、MRP1和BCRP表达的影响。研究发现WAVE1能够增加K562细胞系P-gp的表达,HL60细胞系MRP1和BCRP的表达。相反,采用RNA干扰技术降低K562／A02和HL60／ADR耐药细胞系中WAVE1的蛋白表达,能显著下调这些细胞系中耐药相关基因P-gp, MRP1和BCRP的表达水平。为进一步探讨WAVE1影响P-gp等耐药相关蛋白表达的机制,本研究以WAVE1蛋白为诱饵,采用抗WAVE1抗体免疫沉淀白血病细胞中的WAVE1结合蛋白,然后利用SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术对含有WAVE1结合蛋白的复合物进行分离,最后采用电喷雾四极杆飞行时间质谱(ESI-Q-TOF)结合数据库查询鉴定白血病细胞中WAVE1的结合蛋白,提示WAVE1与埃兹蛋白(Ezrin)存在结合。同时通过质谱分析,发现和鉴定了白血病细胞中43个可能与WAVE1具有直接结合的蛋白质,这些蛋白主要包含了肿瘤相关蛋白、细胞骨架蛋白、受体、代谢相关酶、免疫相关蛋白、膜及离子通道蛋白和信号通路蛋白。为进一步证实WAVE1-Ezrin-P-gp之间的相互作用,本研究通过活细胞免疫荧光技术证明了WAVE1和Ezrin存在空间位置上的共分布。通过基因转染和干扰技术,发现K562细胞转染WAVE1基因后可使Ezrin及P-gp表达升高,同时降低了K562细胞对阿霉素(ADM)的敏感性；而在高表达WAVE1的同时,沉默Ezrin基因的表达,P-gp水平并不能明显增加,而且K562细胞对ADM的耐药性也没有明显改变,表明WAVE1对P-gp表达的影响主要是通过Ezrin介导。在降低Ezrin表达的同时,白血病细胞系中WAVE1表达并不能明显降低,进一步提示Ezrin为WAVE1的下游效应蛋白,而不是WAVE1上游调控蛋白。因此,WAVE1对白血病细胞耐药性的影响主要是通过Ezrin来完成的。最后,本研究通过免疫磁珠分选出白血病细胞系和AML患儿BMMCs中的CD34阳性(CD34+)和CD34阴性(CD34-)细胞,通过Western blotting分析发现CD34+细胞的WAVE1表达水平明显高于CD34-细胞,提示WAVE1可能参与了造血干细胞的分化成熟与白血病干细胞的恶性克隆。综上所述,细胞骨架调节蛋白WAVE1参与了儿童AML发病及多药耐药的形成,WAVE1可能通过多个途径影响AML耐药的形成,其中WAVE1-Ezrin-P-gp信号通路为其重要的作用机制。同时,WAVE1可能与白血病干细胞的增值、分化相关。本研究揭示了WAVE1调控儿童AML多药耐药的作用机制,为寻找逆转白血病的多药耐药靶点提供了新线索和实验依据。更多还原

【Abstract】 Leukemia, a malignant disease of the bone marrow and blood, is the most common form of cancer in children. Chemotherapy is the main treatment for nearly all type of childhood leukemia. Multidrug resistance (MDR) is one of the primary reasons causing suboptimal chemotherapy in children with acute myeloblastic leukemia (AML). The occurrence of MDR restricts the long-term event-free survival (EFS). It is important to reveal the role of WAVE1 in MDR of AML and to find a new target for therapeutics. WASP-family verprolin-homologous protein 1 (WAVE1) is a member of the actin regulatory protein family and plays an important role in the regulation with actin’s polymerization and cytoskeleton’s reorganization. In our previous study, we demonstrated that WAVE1 was a novel regulator of apoptosis, and was involved in the MDR in K562/A02 leukemia cells.At first, Real-time fluorescence quantitative PCR(RQ-PCR) and Western blotting analysis the mRNA and protein expression levels of WAVE1, P-glycoprotein, MRP 1 (Multiple resistance-associated protein-1), LRP(lung-resistance related protein) and BCRP(breast cancer resistance protein) in bone marrow mononuclear cells (BMMCs) in a cohort of 52 children with AML. We found the expression levels of these genes or proteins in refractory/relapsing group were much higher than complete continuous remission (CCR) group. Moreover, the mRNA and protein expression levels of WAVE1 in BMMCs of AML patients were higher in the phase of newly diagnosed or relapsed than complete continuous remission, suggesting that WAVE1 is correlated to the development of AML. Furthermore, overexpression of WAVE1 in K562 and HL60 cell lines increased the expression levels of P-gp, MRP and BCRP respectively; whereas suppression of WAVE1 expression in K562/A02 and HL60/ADR cells by RNA interference decreased the expression levels of P-gp, MRP1 and BCRP respectively.At second, in order to evaluate the mechanisms of WAVE1-mediated MDR, WAVE1 binding proteins were separated and identified in AML BMMCs and leukemia cell lines respectively. WAVE1 binding proteins were captured by anti-WAVE1 antibody using immunop-recipitation with the total proteins from BMMCs and leukemia cell lines. The captured protein complexes were subjected to SDS-PAGE. Protein bands were cut off from gel and in-gel digested analyzed by ESI-Q-TOF. Ezrin protein were identified by peptide sequence tags (PST) and database searching, and it was confirmed by co-immunoprecipitation and Western blotting analysis. Furthermore,43 proteins were identified in the WAVE1 immunoprecipitates complex by preteomic analysis. These proteins were divided into seven main groups based on their functions: tumor-associated proteins, cytoskeletal proteins, receptors, metabolism-related enzymes, immune-related proteins, membrane and ion channel proteins and signaling pathway proteins.At third, to find the interaction of WAVE1-Ezrin-P-gp in vitro, we use immunofluoresence to observe the co-localization of WAVE1 and Ezrin in cell membrane. Indeed, overexpression of WAVE1 in K562 cell lines increased the expression levels of P-gp and ezrin, whereas suppression of WAVE1 or Ezrin expression by RNA interference decreased the expression levels of P-gp in K562/A02 cell lines and further restored leukemia cells’sensitivity to ADM. In contrast, overexpression of WAVE1 and suppression of ezrin didn’t increase the expression levels of P-gp. Thus, Ezrin may be a downstream acting factor of WAVE1. These results suggested that WAVE1 regulated P-gp via ezrin in leukemia cells.Finally, we use magnetic activated cell sorting (MACS) to isolate CD34+/- cells from AML BMMCs and leukemia cell lines. We found that the expression of WAVE1 in CD34+ cells was significantly higher than CD34- cells by Western blotting. WAVE1 may be involved in the formation and maturation of hematopoietic stem cell (HSC) and leukemia stem cell (LSC).In conclusion, WAVE1 was involved in the regulation of multidrug resistance (MDR) and pathogenesis of acute myeloblastic leukemia. WAVE1 regulated MDR partly via a WAVE1-Ezrin-P-gp dependent way in AML. WAVE1 may be associated with the formation of malignant clones of LSC. Our experimental data revealed a noval function of WAVE1 in leukemia and provided clues to reverse MDR in AML.更多还原