Relationship between Phosphorylation of p38 and Erythroid Differentiation of Human K562 Erythroleukemia Cells / 实用儿科临床杂志

ABSTRACT

Objective To investigate the role of directly constitutive activation of p38 mitogen-activated protein kinases(p38MAPKs)signaling in ?-globin gene expression and fetal hemoglobin(HbF)induction,and provide direct data for the relationship between phosphorylation of p38 and erythroid differentiation of human K562 erythroleukemia cells.Methods The human K562 erythroleukemia cells were transfected with pCDNA 3.1-MKK3(Glu)and pCDNA 3.1-MKK3(Ala)recombinant plasmids by lipofectamineTM 2000.Then,the stable cell lines overexpressing constitutively active p38 and constitutively inhibitive p38 activation were established by the addition of G418 to select single cell G418-resistant clones and identification with reverse transcriptase-polymerase chain reaction(RT-RCR)and Western blot assays,named K562-MKK3(Glu)and K562-MKK3(Ala)cells,respectively.Furthermore,the direct effects of constitutively active p38 on the ?-globin gene expression and HbF induction were analyzed by RT-PCR and benzidine staining,respectively.Results The results of RT-PCR and Western blot showed that there were no evident changes in the mRNA and protein levels of p38 for various cell models,but compared with K562,K562-vect,and K562-MKK3(Ala)cells,the phosphorylation of p38 and expression of ?-globin levels in K562-MKK3(Glu)cells were significantly up-regulated.The results of benzidine staining displayed that the mean percentages of positive cells stained by benzidine in K562,K562-vect,K562-MKK3(Ala),K562-MKK3(Glu)cells,and K562-MKK3(Glu)cells treated with SB203580 were(3.2?1.4)%,(3.7?1.2)%,(2.8?0.9)%,(32.6?5.3)%,and(7.8? 2.3)%(q = 7.56 P