| Background and objective:Philadelphia chromosome(Ph) is the result of a t(9;22) reciprocal chromosomal translocation and is the malignant clonal marker of chronic myeloid leukemia(CML). At the molecular level, Ph translocation leads to the transposition of c-abl proto-oncogene gene on 9q34, which encodes a protein tyrosine kinase(PTK), to a new position downstream of the gene bcr gene on 22ql 1 and forms a new bcr-ab1 fusion gene which encodes a chimeric protein,p210BCR-ABL.Compared with that of P145C-ABL,the PTK activity of P210BCR-ABLis aberrantly regulated, which is considered as the sufficient and necessary factor in the pathogenesis of CML. Although the transforming mechanisms of P210BCR-ABL may involve a number of signal pathways, the fundamental andprimary signal trigger is not others but the aberrant PTK activity of P210BCR-ABL, which catalyzes the phosphorylation of tyrosine residues in specific sites of p210BCR-ABL itself and a host of substrates. The state of tyrosine phosphorylation of protein in vivo is a critical factor controlling the initiation , amplification, attenuation and completion in a great number of signal transduction pathways. The abnormal level of tyrosine phosphorylation may break the balance of many a signal networks, resulting in the development of a serial of diseases including tumors. Current data suggest that the tyrosine phosphorylation is a reversible dynamic procedure which is governed by the coordinated and competing actions of PTK and protein tyrosine phosphatases(PTP). As the balance between PTK and FTP are broken by P210BCR-ABL, whose PTK activity is activated aberrantly, we assume that some responsive regulations must been done by the cells inorder to antagonize the impact of PTK activity of P210BCR-ABL. Among those regulations, the changes at the transcriptional level of many important genes including FTP will be most direct and efficient. So in this article our aims are (1) To induce apoptosis of K562 cells with STI571 and analyze the differential expression of PTP with BioStarH40s expression profile cDNA array. Furthermore, the results of PTP' s differential expression were consolidated by semi-quantitative RT-PCR; (2) To analyze the mRNA level of the PTP candidate in CML with semi-quantitative and clone the full length cDNA sequence of candidate PTP, which then will be subcloned into the mammalian expression vector pcDNA3.0; (3)The potential function of the candidate PTP, such as triggering apoptosis and inducing differentiation, were explored by over-expressing the candidate gene in K562 cells with lipofectin transfection technique.MateriaIs and methods:After being cultured with 1.0umol/L STI571 in vitro for 0, 12hrs, 24hrs and 48hrs , the apoptotic percentage and PTP activity of K562 cells were measured. Based on above results, we apply a expression profile cDNA microarray, BioStarH40s, which including about 4,000 genes, to analyze the differential expression of 21 PTP members. Combined the results from BioStarH40s with the corresponding bioinformatics analysis, we focused our attention on one or several target genes. According to their cDNA sequence provided by GeneBank, we designed the upper and the lower primers , cloned the full length cDNA with RT-PCR and subcloned it into the mammalian expression vector pcDNA3.0. The cDNA sequence of the cloned gene was validated with enzyme digestion and DNA sequence as well. In addition, we forced the target PTP gene over-expresse in K562 cells with lipofectin transfection technique, aiming at exploring its/their potential functions with apoptosis assay, differentiation analysis and cell cycle detection. The synergic effect of the gene with STI571 was explored too.Results:1. When treated the K562 cells with l.Oumol/L STI571 for 12, 24 and 48hrs, the early apoptotic percentage were 11.31, 27.15 and 20.02% respectively; thelate apoptotic percentage are 4.86, 18.81 and 32.62 % respectively. The apoptotic percentage assayed with Hoechst33258 staining were 11.4, 35.1 and 44.8%. At the...
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