白血病耐药相关膜蛋白SFPQ的发现及其耐药机制的研究
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摘要
目前化学治疗仍是临床治疗白血病的主要手段,也是造血干细胞移植的基础。近年来尽管有多种新的药物和治疗方案推出,但大多数急性白血病患者最终因治疗失败而死亡。肿瘤细胞对抗癌药物产生抗药性是化疗失败的一个主要原因。导致治疗失败的原因是多方面的,其中多药耐药性(multi-drug resistance,MDR)是最重要的一种。MDR又称多药抗药性(pleiotropic durg resistnace),是指恶性肿瘤细胞对一种化疗药物产生耐药现象后,产生了对多种结构不同、作用靶点和作用机制各异的其他抗癌药物的抗药性。
白血病耐药的产生是多因素的,随诱导药物、细胞种类、分化阶段、及细胞所处微环境的不同而表现出不同的耐药表型。虽然已有的研究揭示了一些肿瘤多药耐药的机制,但目前仍然不能完全解释肿瘤多药耐药现象并有效逆转肿瘤细胞的多药耐药。目前在白血病耐药的临床治疗方面主要以抑制P-糖蛋白功能的耐药逆转剂为主,但其临床效果也不理想,至今无一获得FDA批准上市。虽然急性白血病耐药的发生机制已经被广泛的研究,但耐药机制仍不明了,与白血病耐药相关的细胞膜新靶点的筛选方法仍未得以建立。
事实上白血病患者耐药产生并非单一机制,此外,临床上患者间的个体差异也为治疗带来了困难。可见发现更多的白血病耐药相关的治疗靶点以实现多靶点联合检测与治疗、丰富白血病耐药机理从多角度全面阐明白血病耐药机制以综合制定白血病耐药治疗方案,建立治疗和预后指标是解决上述问题的对策。细胞膜蛋白暴露于细胞外表面,特别容易被抗体等试剂识别,作为靶点特异的检测、诊断和治疗的标志物；此外细胞膜蛋白还特别适于作为较为分散的血液肿瘤的治疗靶点。鉴于此,本文着力于寻找新的白血病耐药相关的细胞膜蛋白,考察其与白血病耐药的相关性并探讨其可能的机制。
为寻找白血病耐药相关的细胞膜靶点,本研究首先应用消减免疫法,以人早幼粒细胞HL60活细胞作为耐受原诱导Balb/c小鼠免疫耐受,然后以阿霉素耐药株HL60/ADR细胞作为免疫原免疫小鼠,得到能特异识别差异表达的耐药株与敏感株HL60细胞膜表面蛋白的杂交瘤细胞库。通过对HL60和HL60/ADR两种靶细胞的选择性筛选和亚克隆培养、抗体的制备与纯化等过程,我们最终获得五株与两种靶细胞结合能力不同的单克隆抗体,这些抗体携带特定的耐药蛋白的标志信息。
为了进一步获得相应抗原的信息,我们通过免疫沉淀和蛋白电泳技术分离敏感和耐药的HL60差异表达的细胞膜蛋白,并通过MALDI-TOF-MS鉴定抗原。最终我们获知单抗5D12的靶抗原为SFPQ,该蛋白高表达于敏感的HL60细胞膜表面,在阿霉素耐药株细胞膜其表达降低。通过siRNA干扰SFPQ,我们发现细胞膜高表达该蛋白的HL60细胞其膜SFPQ表达水平降低,由此确认SFPQ确为单抗5D12的靶抗原。此结果系首次发现SFPQ可表达在肿瘤细胞膜表面。
通过实验我们发现5D12为有生物学活性的细胞膜蛋白SFPQ的特异性抗体。在以5D12作为研究SFPQ的功能的实验中我们发现,细胞膜SFPQ可增强细胞对阿霉素的敏感性。进一步的研究显示,细胞膜SFPQ可能通过抑制细胞增殖和克隆形成能力而减缓细胞生长,从而维持HL60细胞对阿霉素的敏感性。在对影响细胞增殖能力可能机制的研究中我们发现,细胞膜SFPQ通过诱导细胞凋亡和减少细胞S期比例减少DNA复制而抑制细胞增殖。该结果系首次报道HL60细胞膜表面蛋白SFPQ为一耐药相关蛋白。
综上结果,我们认为细胞膜SFPQ可作为人早幼粒细胞白血病阿霉素耐药的新的细胞膜靶点,并且该靶点有可能成为检测和诊断该类白血病阿霉素耐药的新指标,为针对多因素耐药而采取的多靶点治疗和新的耐药机制的研究奠定基础。
Now chemotherapy is the primary treatment of leukemia, and is also the basis for hematopoietic stem cell transplantation. In recent years, though a number of new drugs and treatment programs are applied, most acute leukemia patients died of treatment failure and death eventually. MDR (multi-drug resistance) which is also known as pleiotropic durg resistnace is one of the major obstacles for an effective treatment on tumors. MDR describes a phenomenon of cross-resistance of tumor cells to several structurally unrelated chemotherapeutic agents after exposing to a single cytotoxic drug.
The mechanisms of cellular resistance are very complicated and might vary by different cell types, induced drugs, and differentiation stages as well as by the cellular microenvironment. The MDR mechanisms in tumor cells have been broadly explored, however, exact mechanism is still unclear and the effective reversal agents are also lacking. Up to now, the main strategy for leukemia drug resistance is used to reversal agents based on P-glycoprotein mechanism. Unfortunately, the clinical applicability of these reversal agents was disappointing and on one has been listed on FDA approval.
MDR is very complex. Indeed, owing to the complexity and individual differences among patients, a single indicator can only predict the partial resistance in patients and fails to effectively fit in curative clinic use at present. Therefore, for the pursuit of new drug-resistance associated proteins will have important value for the comprehensive diagnosis and treatment of leukemia MDR. Cell surface molecules are inherently accessible to intravenously injected biological agents such as antibodies and appear to be particularly attractive as site-directed targets. This accessibility of surface proteins is a good fit for hematological neoplasms. Thus, it is significant for leukemia drug resistance to search for new leukemia drug resistance associated membrane proteins and further explore the roles of these membrane proteins.
To find novel membrane protein targeting leukemia resistance, this study applicated subtractive immunization strategy to induce Balb/c mouse to obtain a hybridoma library which could identify the proteins expressed discrepantly between on HL60 and HL60/ADR cell surface. We finally obtain five monocle onal antibodies binding to the two target cell types differently, after some processes including a selective screening through, subcloning culture, antibody preparation and purification.
To reveal the antigen of McAb, we separated the discrepant proteins by immunoprecipitation and protein gel electrophoresis, and further identified the proteins by MALDI-TOF-MS. Finally, a recognized nuclear and cytoplasm protein-SFPQ (polylpyrimidine tract binding protein associated splicing factor) was found as the antigen of McAb 5D12. The membrane SFPQ overexpressed on the cellular surface of HL60 cells compared with HL60/ADR cells. We found that a reduced expression of SFPQ on HL60 cell surface after an interference SFPQ by specific siRNA. We thus confirm that membrane SFPQ is indeed the specific target antigen for monoclonal antibody 5D12, which is the first report that SFPQ expressed on tumor cell surface.
With the billogical activity, McAb 5D12 is specific for membrane SFPQ of HL60 cell. In this study, we found that SFPQ was overexpressed on HL60 cell membranes compared with that on MDR HL60/ADR cells. Furthermore, using the discrepant antibody 5D12, we confirmed that membrane SFPQ contributed to the sensitive phenotype of HL60 cells by inhibiting cellular proliferation and colonization, as well as by inducing cellular apoptosis, reducing the proportion of HL60 cells in S phase and decreasing DNA replication. This is the first report that SFPQ is a drug-resistance associated membrane protein.
From the results described above we believe that membrane SFPQ is likely not only the potentially clinical indicator of human promyelocytic leukemia drug resistance, but also a basis for the study of new MDR mechanisms.
白血病耐药的产生是多因素的,随诱导药物、细胞种类、分化阶段、及细胞所处微环境的不同而表现出不同的耐药表型。虽然已有的研究揭示了一些肿瘤多药耐药的机制,但目前仍然不能完全解释肿瘤多药耐药现象并有效逆转肿瘤细胞的多药耐药。目前在白血病耐药的临床治疗方面主要以抑制P-糖蛋白功能的耐药逆转剂为主,但其临床效果也不理想,至今无一获得FDA批准上市。虽然急性白血病耐药的发生机制已经被广泛的研究,但耐药机制仍不明了,与白血病耐药相关的细胞膜新靶点的筛选方法仍未得以建立。
事实上白血病患者耐药产生并非单一机制,此外,临床上患者间的个体差异也为治疗带来了困难。可见发现更多的白血病耐药相关的治疗靶点以实现多靶点联合检测与治疗、丰富白血病耐药机理从多角度全面阐明白血病耐药机制以综合制定白血病耐药治疗方案,建立治疗和预后指标是解决上述问题的对策。细胞膜蛋白暴露于细胞外表面,特别容易被抗体等试剂识别,作为靶点特异的检测、诊断和治疗的标志物；此外细胞膜蛋白还特别适于作为较为分散的血液肿瘤的治疗靶点。鉴于此,本文着力于寻找新的白血病耐药相关的细胞膜蛋白,考察其与白血病耐药的相关性并探讨其可能的机制。
为寻找白血病耐药相关的细胞膜靶点,本研究首先应用消减免疫法,以人早幼粒细胞HL60活细胞作为耐受原诱导Balb/c小鼠免疫耐受,然后以阿霉素耐药株HL60/ADR细胞作为免疫原免疫小鼠,得到能特异识别差异表达的耐药株与敏感株HL60细胞膜表面蛋白的杂交瘤细胞库。通过对HL60和HL60/ADR两种靶细胞的选择性筛选和亚克隆培养、抗体的制备与纯化等过程,我们最终获得五株与两种靶细胞结合能力不同的单克隆抗体,这些抗体携带特定的耐药蛋白的标志信息。
为了进一步获得相应抗原的信息,我们通过免疫沉淀和蛋白电泳技术分离敏感和耐药的HL60差异表达的细胞膜蛋白,并通过MALDI-TOF-MS鉴定抗原。最终我们获知单抗5D12的靶抗原为SFPQ,该蛋白高表达于敏感的HL60细胞膜表面,在阿霉素耐药株细胞膜其表达降低。通过siRNA干扰SFPQ,我们发现细胞膜高表达该蛋白的HL60细胞其膜SFPQ表达水平降低,由此确认SFPQ确为单抗5D12的靶抗原。此结果系首次发现SFPQ可表达在肿瘤细胞膜表面。
通过实验我们发现5D12为有生物学活性的细胞膜蛋白SFPQ的特异性抗体。在以5D12作为研究SFPQ的功能的实验中我们发现,细胞膜SFPQ可增强细胞对阿霉素的敏感性。进一步的研究显示,细胞膜SFPQ可能通过抑制细胞增殖和克隆形成能力而减缓细胞生长,从而维持HL60细胞对阿霉素的敏感性。在对影响细胞增殖能力可能机制的研究中我们发现,细胞膜SFPQ通过诱导细胞凋亡和减少细胞S期比例减少DNA复制而抑制细胞增殖。该结果系首次报道HL60细胞膜表面蛋白SFPQ为一耐药相关蛋白。
综上结果,我们认为细胞膜SFPQ可作为人早幼粒细胞白血病阿霉素耐药的新的细胞膜靶点,并且该靶点有可能成为检测和诊断该类白血病阿霉素耐药的新指标,为针对多因素耐药而采取的多靶点治疗和新的耐药机制的研究奠定基础。
Now chemotherapy is the primary treatment of leukemia, and is also the basis for hematopoietic stem cell transplantation. In recent years, though a number of new drugs and treatment programs are applied, most acute leukemia patients died of treatment failure and death eventually. MDR (multi-drug resistance) which is also known as pleiotropic durg resistnace is one of the major obstacles for an effective treatment on tumors. MDR describes a phenomenon of cross-resistance of tumor cells to several structurally unrelated chemotherapeutic agents after exposing to a single cytotoxic drug.
The mechanisms of cellular resistance are very complicated and might vary by different cell types, induced drugs, and differentiation stages as well as by the cellular microenvironment. The MDR mechanisms in tumor cells have been broadly explored, however, exact mechanism is still unclear and the effective reversal agents are also lacking. Up to now, the main strategy for leukemia drug resistance is used to reversal agents based on P-glycoprotein mechanism. Unfortunately, the clinical applicability of these reversal agents was disappointing and on one has been listed on FDA approval.
MDR is very complex. Indeed, owing to the complexity and individual differences among patients, a single indicator can only predict the partial resistance in patients and fails to effectively fit in curative clinic use at present. Therefore, for the pursuit of new drug-resistance associated proteins will have important value for the comprehensive diagnosis and treatment of leukemia MDR. Cell surface molecules are inherently accessible to intravenously injected biological agents such as antibodies and appear to be particularly attractive as site-directed targets. This accessibility of surface proteins is a good fit for hematological neoplasms. Thus, it is significant for leukemia drug resistance to search for new leukemia drug resistance associated membrane proteins and further explore the roles of these membrane proteins.
To find novel membrane protein targeting leukemia resistance, this study applicated subtractive immunization strategy to induce Balb/c mouse to obtain a hybridoma library which could identify the proteins expressed discrepantly between on HL60 and HL60/ADR cell surface. We finally obtain five monocle onal antibodies binding to the two target cell types differently, after some processes including a selective screening through, subcloning culture, antibody preparation and purification.
To reveal the antigen of McAb, we separated the discrepant proteins by immunoprecipitation and protein gel electrophoresis, and further identified the proteins by MALDI-TOF-MS. Finally, a recognized nuclear and cytoplasm protein-SFPQ (polylpyrimidine tract binding protein associated splicing factor) was found as the antigen of McAb 5D12. The membrane SFPQ overexpressed on the cellular surface of HL60 cells compared with HL60/ADR cells. We found that a reduced expression of SFPQ on HL60 cell surface after an interference SFPQ by specific siRNA. We thus confirm that membrane SFPQ is indeed the specific target antigen for monoclonal antibody 5D12, which is the first report that SFPQ expressed on tumor cell surface.
With the billogical activity, McAb 5D12 is specific for membrane SFPQ of HL60 cell. In this study, we found that SFPQ was overexpressed on HL60 cell membranes compared with that on MDR HL60/ADR cells. Furthermore, using the discrepant antibody 5D12, we confirmed that membrane SFPQ contributed to the sensitive phenotype of HL60 cells by inhibiting cellular proliferation and colonization, as well as by inducing cellular apoptosis, reducing the proportion of HL60 cells in S phase and decreasing DNA replication. This is the first report that SFPQ is a drug-resistance associated membrane protein.
From the results described above we believe that membrane SFPQ is likely not only the potentially clinical indicator of human promyelocytic leukemia drug resistance, but also a basis for the study of new MDR mechanisms.
引文
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