Screening and Identifying of the Protein Tyrosine Phosphatase Superfamily Members Related to Chronic Myeloid Leukemia and Exploration of Its Potential Function.

Chronic myeloid leukemia (CML) is characterized by formation of a BCR-ABL fusion gene, which encodes a chimeric protein, P210^BCR-ABL. The protein tyrosine kinase(PTK) activity of P210^BCR-ABL is aberrantly regulated and lead to catalyzes the phosphorylation of tyrosine residues in specific sites of P210^BCR-ABL itself and a host of substrates, which is considered as the sufficient and necessary factor in the pathogenesis of CML. Current studies 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 PTP are broken by P210^BCR-ABL in CML, we assume that some responsive regulations must been done by the cells in order to antagonize the impact of PTK activity of P210^BCR-ABL. Among those regulations, the changes at the transcriptional level of many important genes including PTP will be most direct and efficient. In order to screen and identify PTP related to CML and explore its potential function, we firstly induced apoptosis of K562 cells with STI571 and analyze the differential expression of PTP with BioStarH40s expression profile cDNA array. Among the 21 PTP included in the BioStarH40s cDNA microarray, 2 PTP, FAP1(NM_006264, ration=2.417) and SHP1(M77273, ration=5.012) showed differential expression. The results of cDNA microarray confirmed by semi-quantitative RT-PCR suggested that FAP1and SHP1 may involve the apoptotic signal transduction pathway triggered by STI571. Secondly, the mRNA level of SHP1 in CML was analyzed with semi-quantitative RT-PCR. The results detected that the mRNA expression level of SHP1 in CML-CP (7 cases) show no statistic difference with normal control. But in contrast, the mRNA expression had down-regulated in CML-BC(4 cases) and show significant difference with normal control. Furthermore, we cloned the full length cDNA sequence of SHP1 with RT-PCR and sub-cloned it into the mammalian expression vector pcDNA3.0. The orientation and the sequence of pcDNA3-SHP1 were further validated by restrictive enzyme digestion analysis and DNA sequence analysis. Thirdly, the potential functions of the SHP1, such as triggering apoptosis and inducing differentiation, were explored by over-expressing the candidate gene in K562 cells with lipofectin transfection technique. The results showed that over-expression of SHP1 in K562 cells is sufficient for inducing apoptosis and erythroid differentiation that may be strengthened by the additional of STI571 treatment.