Expression And Effects Of ABD Protein On The K562 And HL60 Cell

Background: Chronic myeloid leukaemia (CML) is a rare disease with an incidence of 1 or 2 cases per 105 people every year. It is classified into 3 stages: the chronic stage, the accelerate stage, the blast crisis. The median survival duration is 3-4 years, 6-9 years, 3-6 years respectively.CML is a malignant haemopoietic stem cell disorder. 1845, CML was identified by Craigie and Bennet.1960, Nowell and Hunger-ford found the abnormal chromosome in the CML patients in Philadelphia, the Ph chromosome (Ph) was named then. 1973, Rowley conformed that Ph was characterized by the t (9; 22) (q34; q11) reciprocal chromosomal translocation. 1983 ,researchers confirmed that Ph is cytogenetic hall- mark ,a shortened chromosome 22 that arises from a reciprocal translocation, t(9;22) (q34;q11), which gives origin to a hybrid bcr-abl oncogene, encoding a BCR-ABL(p210) fusion protein with elevated tyrosine kinase activity, and its functional consequence is the Bcr-Abl oncoprotein. Most clinic and laboratary researches provided direct evidences that BCR-ABL is probabaly the main cause of CML, and this fusion gene and BCR–ABL (p210) fusion protein are the main target in the therapy for CML.Currently, there are many methods to treat CML, including chemotherapy, interferon, STi571, stem cell transplant and so on. Conventional chemotherapy can usually control the overall tumor burden during the chronic phase, with resolution of symptoms. However, such treatment could not prevent disease progression or kill the malignant cells. Interferon-αhas been used in CML therapy for many years, however the hematology remission rate is only 70-80 percent . Survival time of cases only can be extended for 72 months. Allogeneic bone marrow transplatantion (allo-BMT) is the only treatment which has been shown to be curative potential in CML. However, some fatal complications often develop, such as graft -versus-host disease (GVHD) in 30%-35% percent of patients. In addition, the requirements for patient's age and histocompatibility (only 30 percent patients can find a matched sibling donor) limit its applicability. The majority of patients are not eligible for this treatment. Autologous bone marrow transplantation (ABMT) has a lower accidence of complications than allo-BMT, and may become a standard treatment for those patients ineligible for sibling domor allogeneic BMT.With the advancement of the construction and function of bcr-abl fusion protein reseaches, scientists have found that blocking some domain's fuction of the BCR-ABL protein may be an effective method for inhibiting CML cell growth. Maybe this method is hopeful to cure CML in the future.ABD (actin binding domain), is a C-terminal domain of BCR-ABL protein. It is required for CML pathogenesis. Therefore, inhibition of ABD represents a potential strategy for inhibiting BCR-ABL oncogencity. It also can helpful to find a new clinical therapeutic medicine or a new target of CML.Aim: To study the ABD's fouctions of BCR-ABL protein on cell cycle and apoptosis of K562 cell and HL60 cell, and to provide the experimental basis for new therapy of CML. Methods: The ABD gene was amplified from PGD210 plasmid. In order to obtain PTD-ABD fusion gene, the ABD and PTD gene were successively inserted into pET32a vector. The recombination plasmids were transformed into E.coli BL21 and the expression of the fusion protein was induced by IPTG. Fusion protein was purificated from the surpernatant of cell lysates via Ni-NTA affinity chromatography. The purified fusion protein was loaded to cultured K562 and HL60 cells. Whether the fusion protein entered into K562 cells and Hela cells was detected by western blot and immunofluorescence. Growth curve of K562 and HL60 cells treated by PTD-ABD fusion protein was determined by MTT assay. The apoptosis of PTD-ABD fusion protein on K562 and HL60 cells was investigated by flow cytometry.Results: The ABD gene was effectively amplified by the method of PCR. The DNA sequencing result showed that the constructed plasmid containing PTD-ABD fusion gene was the same as that designed. The fusion protein was successfully expressed in prokaryotic plasmid, PTD-ABD protein was shifted into both adherent cell and suspension cell successfully.PTD-ABD fusion protein can significantly inhibit the growth of HL60 cell,and could accelerate the apoptosis of HL60 cell.Conclusion: The ABD gene has been successfully amplified and expressed, and stuy to the effects of PTD-ABD fusion protein on K562 and HL60 cells was done. The results of the experiment provide a basis to find a new therapeutic target of CML.