| BackgroundChronic myeloid leukemia (CML) is a hematopoietic neoplastic disorder characterized by accumulation of mature and immature granulocytes due to uncontrolled growth and resistance to apoptosis. The dysregulated activity of the Bcr/abl oncoprotein tyrosine kinase, which is encoded by the p210bcr-abl fusion gene generated from the chromosomal translocation of t(9; 22) and present in approximately 95% of patients with CML, has been shown to be responsible for these malignant phenotypes, but the molecular mechanism responsible for these malignant phenotypes is poorly understood. The tyrosine phosphatase Shp-2, which plays a pivotal role in RTK and cytokine signaling of normal hematopoiesis, is constitutively tyrosyl-phosphorylated in Bcr/abl-transformed cells, and associates with p210bcr/abl oncoprotein upon p210bcr/abl transformation. However, the definite role of Shp-2 in p210bcr/abl-induced leukemogenesis is unknown. The possible mechanisms of leukemogenesis were explored in this study by Western Blot, growth inhibition assay of leukemic cells in suspension culture with Shp-2 antisense oligonucleotide, immunoprecipitation.Materials and methods1. Materials1.1. Cell line: The erythroleukemic cell line K562 express p210bcr/abl was used.1.2. Chronic myeloid leukemia (CML) cell samples: 25 adult CML cell specimens were obtained from patients with CML before any treatment.1.3. Normal hematopoietic tissues: Including 8 samples of bone marrows and 10 samples of PBMNCs from health donors after informed consent.2. Methods2.1. K562 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, and 100ng/mL penicillin-streptomycin.2.2. Isolation of mononuclear cells: Ficoll-Hypaque density-gradient centrifugation.2.3. The expression of Shp-2: The extraction of cells are separated by 8-10% SDS-PAGE gel electrophoresis, then transferred to PVDF membrane, blocked and subsequently reacted with primary antibodies and second antibodies, eventually visualized with ECL.2.4. Treatment of leukemia cells in suspension culture with Shp-2 antisense oligonucleotides: Analysis of Shp-2 expression with Western blot by downregulating Shp-2 expression on CML leukemic cell and normal BM hematopoietic cells with Shp-2 antisense oligonucleotides.2.5. Analysis of p210bcr/abl in leukemia cells: K562 leukemic cells (0.5 × 10~6 cells/ml) were cultured in the presence of STI571 at 1μM for up to 48 hours. Cells were harvested at different time points for analysis of p210bcr/abl oncoproteins.Statistical AnalysisResults were expressed as means± 1SD. All statistical analyses were made with a two-sided Student's t test. P < 0.05 was considered to be statistically significant.ResultsOverexpression of the mshp-2 protein was observed in most primary leukemia samples from adult CML patients , when compared with normal hematopoietic cells. The mean ratios of mshp-2 protein/β-actin in primary leukemia samples is 5.7-fold higher than in normal bone marrow hematopoietic cell samples.The activities of bothAkt kinase and Erk 1/2 kinases were strikingly reduced after downregulation of the mshp-2 protein with Shp-2 AS by measuring activities of both Akt kinase and Erk 1/2 kinase in CML cell lines as well as primary leukemia cells from CML patients before any treatment using Western blot with antibodies against Akt, phospho-Akt, Erk and phospho-Erk kinases. The mshp-2 protein level as well as proliferating cells(S phase cells) significantly decreased, whereas, apoptotic cells increased after we treated K562 leukemic cells with the p210bcr/abl specific inhibitor STI571 at 1μM for up to 48 hours.Conclusions1. Shp-2 is constitutively tyrosyl-phosphorylated in p210bcr/abl-expressed cells.2. Shp-2 protein is a critical downstream effector of p210bcr/abl oncoprotein in adult CML.3. The proliferative capacity of chronic myeloid leukemia cells is regulated by the mshp-2 through activating PI3k/Akt and Ras/Erk signaling pathways.
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