| Background and objective:Chronic myeloid leukemia (CML) is characterized by the reciprocal chromosomal translocation 9:22, which generates the Philadelphia chromosome (Ph). This event transposes the c-abl proto-oncogene on chromosome 9 to a new position downstream of the second exon of the gene bcr on chromosome 22.The Philadelphia chromosome is the result of a translocation—(9;22) (q34;q11)—that fuses the breakpoint cluster region (bcr) gene on chromosome 22 to the abl gene on chromosome 9 to produce a fusion bcr-abl transcript that codes for a chimeric Bcr-Abl protein with constitutive tyrosine kinase activity. As a tyrosine kinase, the P210Bcr-Abl protein can phosphoryiate a variety of proteins involved in cell growth, differentiation, adhesion, and apoptosis of CML cells. Signaling pathways that are activated by Bcr-Abl activity include Ras, phosphatidylinositol 3-kinase/AKT, NF-κB, and Stat-5. These signaling cascades govern physiologic hematopoiesis; however, the constitutively active state of the fusion tyrosine kinase results in deregulated activitycontributing to malignant transformation. Activation of these pathways results in growth factor-independent proliferation and enhanced survival of CML cells. Consequently, the leukemic cells may be resistant to immune surveillance and chemotherapy. As a result of these various alterations, CML cells undergo massive clonal expansion.Nevertheless, control over the state of tyrosine phosphorylation of proteins in vivo is governed by the coordinated and competing actions of both PTKs and protein tyrosine phosphatases (PTPs). PTPs have been identified in eukaryotes, prokaryotes, plants, and viruses and comprise a large family of enzymes that will rival the PTKs in structural diversity and complexity. PTPs may either antagonize or potentiate PTK-induced signaling in vivo and have been implicated in such fundamental physiological processes as growth and proliferation, differentiation, cytoskeletal function, as well as in the etiology and pathogenesis of certain diseases. Therefore, characterization of the PTPs that either potentiate or antagonize P210Bcr'Abl induced signaling and transformation would provide critical insights into the genesis and development of CML. Currently, the tyrosine kinase inhibitor, imatinib mesylate (formerly STI571) is successful in achieving complete hematologic responses in patients with chronic phase CML. Yet, while imatinib mesylate also has activity in advanced phases of the disease, the responses tend not to be durable and patients eventually relapse and become resistant to further treatment with imatinib mesylate. Some reports indicate that resistance to imatinib induced by Bcr-Abl over-expression or by Bcr-Abl mutants does not induce cross-resistance to Arsenic trioxide (AS2O3). Combined treatment with this agent and imatinib is beneficial in cell lines that have not been sensitivity to imatinib monotherapy, with the effect of synergistic growth inhibition. In some imatinib- resistant cell lines, combination treatments that use low doses of imatinib lead to antagonism.As the balance between PTK and PTP are broken by P210Bcr'Abl, whose PTK activity is enlarged, we assume the character of CML cells will be changed by increasing PTP activity. So in this article our aims are: (l)To analyze the mRNA level of the PTPIB in CML cells with semi-quantitative RT-PCR. (2) Cloning the full length cDNA sequence of PTPIB, which will be subcloned into the mammalian expression vector pcDNA3.0, and make PTPIB gene over- expression in K562 cells by lipofectin transfection technique. (3) Observing the potential function of the PTPIB, such as triggering apoptosis and inducing differentiation. (4) To study the influence of AS2O3 to K562 cells transfected with PTPIB or not treated by detecting the activity of PTK. Materials and methods:We analyed the mRNA expression level of PTPIB gene in CML patients with semi-quantitative PT-PCR technique, 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. After transfecting the PTPIB gene into K562 cells with lipofectin transfection technique, we verificated the over-expression of the PTPIB by detecting enzyme activity and semi-quantitative RT-PCR analyzing. Then we explored the potential functions of PTPIB gene with apoptosis assay, differentiation analysis and cell cycle detection. On the base of above experiment, we cultured K562 cells with 1.0 u mol/L AS2O3, and study the influence of this agent to K562 cells by detecting the activity of p210Bcr-Abi and ^ ability of caspase3 xhe synergic effect of the PTPIB gene with AS2O3 was explored too. Results:1. Combined the patient's clinic data with G-banding technique and/or interphase in situ hybridization(I-FISH),we had chosen 8 cases CML patients at the stage of initialdiagnosis or blast crisis for further analysis. With semi-quantitative RT-PCR, we found that the mRNA expression level of PTPIB in CML-CP(5 cases) and in CML-AP/BC(3 cases) show statistic difference with normal control. We can concluded that the mRNA expression level of PTPIB has increased significantly either in CML-CP or in CML-AP/BC. But the mRNA expression level show no difference between CML-CP and CML-AP/BC. We cloned the full length cDNA sequence of PTPIB with RT-PCR and sub-cloned it into the mammalian expression vector pcDNA3.0. According the restrictive enzyme digestion analysis and DNA sequence analysis, we can detect the correctness of the orientation and the sequence ofpcDNA3-PTPlB.2. pcDNA3-PTPIB gene can be forced to over-express in K562 cells with lipofectin transfection technique. RT-PCR, Western-blot and PTP enzyme activity assay were applied to validate the over-expression of PTPIB in K562 cells. By FACS analysis, the rate of apoptotic cells which were detected in the K562 cells treated with pcDNA3-PTPlB transfection is 13.05% and the rate in pcDNA3.0/K562 cells is 11.87% after transfection 48hrs. There is no statistic difference. Positive rate of CD71 and GPA expression in K562 treated with pcDNA3-PTPlB and pcDNA3.0 were 43.15%, 12.88% respectively, showing significant statistic difference. Further cell cycle analysis found G0/G1 arrest in K562 cells after pcDNA3-PTPlB transfection.3. Treated the K562 cells with 1.0 U mol/L AS2O3, we can detect apoptosis in cells, which is following with decreasing of the PTK activity and increasing of the caspase3 activity. Moreover, we found that the combination of pcDNA3-PTPlB transfection and As2O3 treatment had shown synergic effect on the apoptosis in K562 cells. Conclusions:1. The mRNA expression level of PTPIB gene in CML cells is higher than in control cells without P210Bcr"Abl. But the expression level is no difference between inCML-CP and in CML-AP/BC.2. The full length cDNA sequence of PTP1B could be cloned by RT-PCR technique. The lipofectin transfection technique is competent for transferring the pcDNA3-PTP1B into the K562 cells and forcing the target gene over-expression in cells.3. Over-expression of PTP1B in K562 cells is not contribute to apoptosis, but is sufficient for inducing erythroid differentiation.4. 1.0 U mol/L AS2O3 can induce apoptosis in K562 cells by decreasing of the PTK activity of P210Bcr"Abl and increasing of caspase3. The apoptosis may be strengthened by the addition of PTP1B transfection.
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