The Effects Of Notch1 On The K562 Cell

Research backgroundAccounting for 20% of all cases of leukemia, chronic myeloid leukemia is a clonal myeloproliferative expansion of transformed, primitive hematopoietic progenitor cells, with an annual incidence of about one in 100,000 people. The common symptoms are asthenia, weight loss, sweating, and the discovery of an abdominal mass(the spleen).The current therapy of CML mainly includes interferonα, protein tyrosine inhibitor(STI571)and bone marrow transplantation, but some problems such as high expenses, lack of donors ,transplantation risks and drug resistance of STI571 are still not solved. The bcr/abl fusion oncogene - the molecular basis of CML, arises from a reciprocal translocation, t(9;22)(q34;q11), encoding a BCR/ABL(p210)fusion protein with deregulated tyrosine kinase activities. The protein influences many intra-cellular signaling pathways and alters cellular adhesion, which induces inhibition of apoptosis and promotion of proliferation. Therefore, it is very essential to further study the mechanism of CML and the signaling pathways of BCR/ABL fusion protein.The Notch gene got its name from a strain of Drosophila with notched wing blades in 1916. The gene was cloned by Artavanis Tsakonas in 1980's. Notch gene encodes 300 kDa single pass transmembrane receptors, composed of 2753 amino acid residues. Notch genes have been found genetically conserved in many species including humans. Notch signaling is involved in cell specification, proliferation, and apoptosis that affect the development and function of many organs. Pathophysiologic alterations in Notch signaling have been associated with tumorigenesis. Now some studies show that overexpression of active Notchl has effect on the differentiation and development of K562 cells. So the relationship between the signaling pathways of BCR/ABL fusion protein and Notch are of great importance to further explain the mechanism of CML and search for new therapy target.AIMTo construct eukaryotic expression vectors of myc-RBPJ (R218H)- IRES2-EGFP; To investigate the effect of gene Notchl on the growth and proliferation of K562 cells by means of transfecting K562 cells with plasmids pIRES2-EGFP ,myc-RAMIC-IRES2-EGFP and myc-RBPJ ( R218H )-IRES2-EGFP.METHODSRBP(JR218H)in PCMX-N/RBP(JR218H)vector was subcloned into vector PCMV-myc to obtain the fusion gene myc RBPJ(R218H); Then fusion gene myc- RBPJ(R218H)were inserted into pIRES2-EGFP in order to get the eukaryotic expression vector myc-RBPJ ( R218H ) -IRES2-EGFP;With LipofectinamineTM2000, the recombination fluorescence plasmid was transiently transfected into HEK 293 cells, and the expressions of the fusion protein and EGFP were identified by Westernblot and FACS; Then by means of electroporation system, we transfected fluorescence plasmid myc-RAMIC-IRES2-EGFP,myc-RBPJ(R218H)-IRES2- EGFP and control plasmid pIRES2-EGFP into K562 cell line.G418 was used to obtain the positive cells. The proteins myc-RAMIC, myc-RBPJ(R218H)and EGFP expressed in K562 cells was detected by Westernblot; In order to evaluate the activation of Notch signaling pathway in stable transfected K562 cells, the expression of gene HES1 and HES5 was detected by RT-PCR; The effects of Notch1 gene on growth and proliferation of K562 cells were investigated by MTT and clone formation.RESULTS1. Successful construction of the recombinant plasmids PCMV-myc-RBPJ(R218H), and the DNA sequencing result showed that the myc-RBPJ(R218H)fusion gene was the same as designed;2. Enzyme digestion results identified the successful construction of the eukaryotic expression vector myc-RBPJ(R218H)-IRES2-EGFP;3. The proteins myc-RBP(JR218H)and EGFP were identified correctly expressed in HEK293 cells by Westernblot, and FACS showed good efficiency of transient transfection;4. Stable transfected K562 cells with plasmids pIRES2-EGFP,myc-RAMIC- IRES2-EGFP and myc-RBPJ(R218H)-IRES2-EGFP were obtained, and the proteins of myc-RAMIC ,myc-RBPJ(R218H)and EGFP were all identified by Westernblot;5. The expression of gene HES1 , but not HES5, was detected by RT-PCR in K562 cells stable transfected plasmid myc-RAMIC-IRES2-EGFP, which showed the activation of Notch signaling pathway mediated by the exogenous expression of myc-RAMIC.6. MTT and clone formation tests showed that Notch1 inhibit the growth of K562 cells and the proliferative ability was also decreased.CONCLUSIONThe myc- RBPJ(R218H)fusion genes was correctly cloned. The eukaryotic expression vector myc-RBP(JR218H)-IRES2-EGFP was successfully constructed, and can be expressed in mammalian cells; K562 cells stable transfected with plasmids pIRES2-EGFP,myc-RAMIC-IRES2-EGFP and myc-RBPJ(R218H)- IRES2 -EGFP were obtained, and Notch signaling pathways was effectively activated in K562 cells expressed protein myc-RAMIC; Notch1 inhibit the growth and proliferation ability of K562 cells. The results provide bases for further studies on the relationship between CML and Notch signaling pathway.We speculate that Notch1 protein may be a useful target in gene therapy of CML.