Recombinant Human Jagged1 Expression And Its Role In HL60 Cells

The Notch signaling cascade is remarkably conserved from Drosophila to man,and the complexity of this cascade is higher in mammals.Receptor-ligand interaction between two neighbouring cells leads to successive proteolytic cleavages of Notch ,resulting in the release of the intracellular domain(NICD) of the receptor.NICD translocates to the nucleus where it converts the transcription factor CBF1/Su(H)/LAG1(CSL) from a repressor to a transcriptional activator. Notch signaling is involved in a variety of cell differentiation,proliferation and apoptosis that affect the development and functions of many organs.Furthermore, pathophysiologic alterations in Notch signaling have been associated closely with tumorigenesis.The first case showing the Notch involvement in human malignancies is the identification of a rare chromosomal translocation event, t(7;9)(q34;q34.3), as a causative factor in the development of T-cell acute lymphoblastic leukemia (T-ALL). In contrast to T-ALL, a definite role for Notch signaling in the development of acute myeloblastic leukemia (AML) is less clear.Activation of Notch signaling is mediated through interactions between Notch ligands and Notch receptors, and we got ligand protein through Pichia pastoris expression and prokaryotic expression. The prokaryotic expression system has no post-translational modification system and the exogenous proteins always emerge in the inclusion body, complicating the course of renaturation and purification. As a eukaryote, Pichia pastoris has many of the advantages of higher eukaryotic expression systems such as protein processing, protein folding, and posttranslational modification, while being as easy to manipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier, and less expensive to use than other eukaryotic expression systems such as baculovirus or mammalian tissue culture, and generally gives higher expression levels. Therefore, we chose the Pichia pastoris expression system firstly.Several experiments were included as follows:1. Expression recombinant human Jagged1 in Pichia pastoris. The extracellular domain of human Jagged1 gene was cloned from normal human bone marrow cells. After DNA sequencing, the extracellular domain of Jagged1 gene was inserted into PIC-Fc constructed previously, which is Pichia pastoris expression vector containing only the Fc fragment of human IgG1. The constructed plasmid was transformed into yeast GS115 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of protein was induced by addition of methanol. Then analyzed protein expression by SDS-PAGE.2. Expression recombinant hJagged1 in E.coli BL21. We constructed expression vectors GEX-Jagged1. Then 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 the glutathione affinity chromatography.3. Verified the bioactivity of recombinant hJagged1. We detected the recombinant hJagged1 bioactivity though reporter assay, and further comfirmed by RT-PCR analysis with primers specifically targeting the Notch downstream molecules Hes1 and Hes5.4. The effects of recombinant hJagged1 to HL60 cells. To investigate the relationship between Notch signaling and AML, we observed the role of Notch signaling in the human promyelocytic leukemia cell line, HL60. Recombinant hJagged1 was used to enhance Notch signaling, while GSI was used to block Notch signaling. Cell numbers were counted with the trypan blue exclusion assay. For morphological observations, treated and untreated cells were stained by Wrigh-Giemsa staining, and examined under a light microscope. Cell cycle, apoptosis and myeloid differentiation were measured by flow cytometry. To elucidate the molecular mechanism, we examined the expression of P21, P27, c-Myc and the phosphorylation of pRB using RT-PCR or Western blot.Results:1,We got recombinant hJagged1-Fc and human IgG1 Fc proteins in Pichia pastoris; Got a soluble recombinant protein GST-Jagged1 in E.coli BL21;2,Verified that the recombinant GST-Jag1 protein was of biological activity and could activate Notch signaling by reporter assay, RT-PCR and Western blot;3,Recombinant Notch ligand Jagged1 had not any effect on the growth or differentiation of HL60 cells. Althoughγ-secretase inhibitor (GSI) had not any effect on the differentiation of HL60 cells, GSI inhibited the growth of HL60 cells significantly. Furthermore, GSI inhibited cell cycle and could induce apoptosis of HL60 cells, which correlated closely with the expression of P21, P27, c-Myc and the phosphorylation of pRB.