Expression And Significance Of EDAG-1 Gene In Leukemia Cell Lines

OBJECTIVE EDAG-1 is a novel gene identified from the EST bank of the differentially expressed genes between the 4 months fetal liver and adult liver. We explore the roles of EDAG-1 gene in the pathogenesis of leukemia through analysis of the coding region structure, transcriptional activity of EDAG-1 gene, the relationships between the high expression of EDAG-1 and NFicB activity. METHODS: 15 kinds of leukemia cell lines were selected to observe the expression of EDAG-1 by RT-PCR, EDAG-1 cDNA coding fragment (1.5kb) was made purification to construct corresponding recombinant plasmids, subsequently the plasmids were sequenced to analysis the mutation of the coding region; the rearrangement and amplification of EDAG-1 genome in leukemia cell lines were detected by Southern-Blot, the expression of EDAG-1 mRNA and protein in these cell lines were corroborated with Northern Blot and Western Blot respectively. The transcriptional activity of EDAG-1 was detected by Mammalian MATCHMAKER Two-Hybrid Assay Kit and CAT Enzyme Assay System, whether EDAG-1 was involved in the NF B signal transduction pathway was investigated byMercuryTM Pathway Profiling Luciferase System. NFicB DNA binding activity in leukemia cell lines highly expressed EDAG-1 was analyzed through EMS A. RESULTS: the mRNA and protein of EDAG-1 gene were highly expressed in erythroleukemia lines (K-562,HEL), megakaryoblast leukemia lines (DAMI,MEG-01), and Jurkat (T cell leukemia cell line), while the expression levels of mRNA and protein of EDAG-1 were a little high in lymphoma cell lines (Raji). The coding region fragment amplificated by RT-PCR was ligased to pMD-18T vector, which was transformed into JM109 to construct recombinant plasmid. More than 2 kinds of positive plasmids indientified with PCR, emzyme digestion were sequenced, while no gene mutation, insertion or deletion in coding region was found in these cells; no amplification or rearrangement of genome in these cells was found by Southern Blot; EDAG-1 gene was deleted in HL-60, and was rearranged in Hut 78. EDAG-1 was not a transcriptional factor, while it was involved in NFicB signal pathway, and DNA binding activity of NFicB was enhanced in cells highly expressed EDAG-1. CONCLUSION: EDAG-1 may have relationship with the pathogenesis of erythroleukemia and megakaryoblast leukemia; the coding region of this gene may not play a role in the mechanisms above, EDAG-1 may take part in NFxB signal transduction pathway to lead a cascade of biological responses.