| 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.
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