| Background: The proliferation and differentiation of hematopoietic cells are strictly controlled by a series of cytokines and their receptors. JAKs (Janus kinases ) and STATs (signal transducers and activators of transcription ) are very important in mediating cellar signal transduction and hematopoiesis regulation. Normal hematopoiesis depends on correct signal pathway and any of abnormality in signal transduction pathway will result in multiple diseases, such as: leukemia, bone marrow proliferative diseases and lymphatic proliferative diseases. In the past decades, study on the signal transduction pathways mediated by cytokines/ growth factors had becomed into the focus for basic applicated biological medcine. Many important themries about signal transduction have been described by now. STATs may convert and transmit downstream signal evoked by interaction between cytokines/ growth factors and their ligands. JAKs also play an important role in various kinds of signal transduction pathways. STATs and JAKs regulate the proliferation and differentiation of cells. Some studies show that the activation of STATs is mediated by JAKs or Src family members. STATs can be phosphorylated on a single tyrosine residue and activated by the JAKs, then phosphorylated STATs become dimerizing and migrate into the nucleus, and regulate gene transcription of Bcl-XL.The BCR/ABL chimeric oncongene, origining from the translocation of chromosome 9 and 22 (t(9;22)(q34;q11)), is a charicteristic molecular mark of chronic myelogenous leukemia(CML). P210 protein, an expressive product of BCR/ABL gene, can constitutively activate several signaling pathways. These pathways include the RAS-Erk2, the PI3Kinase, and the JAK/STAT pathways. It has been shown thatSTATs are activated in various BCR/ABL positive cell lines, especially STAT5 and STAT1.As the medicine intervention or therapy for CML has reached a therapeutic plateau, it has become urgent to look for a new therapeutic medicine of CML. Increasing evidence suggests that the STATs signaling cascade may be one target of these therapies. STATs tyrosine phosphorylation can occur through inappropriate JAK activation or by direct activation of an oncoprotein such as BCR/ABL. Thus, blocking STATs signal pathway, inhibiting STATs activation or interventing STATs-mediated gene transcription, will become a new strategy for CML therapy.Previous investigation in our laboratory suggested indomethacin (EN) could induce CML bone marrow leukemia cells and K562 cells apoptosis. The cleavage and activation of caspase3 and caspase8 play an important role in the course, and the apoptosis of K562 cells induced by EN is cyclooxygenase (COX)-independent. In this study, indomathecin is applied to K562 cells and CML fresh bone marrow leukemia cells, the proliferation-inhibiting effect of CML by EN is observed and JAK/STATs signals are detected and analyzed by various methods. The goal lies on determinating the mechanism of anti-CML cells proliferation by EN.Part I The Proliferation-Inhibiting Effect of CML Cells byIndomethacin and JAK2, STAT1, STAT5Proteins ExpressionObjectives: To determine whether JAK2, STATi, STAT5 are involved in proliferative inhibition of CML cells induced by IN and observe the subcellular distribution of STATi and STAT5. Combining with IN, to determine if GM-CSF or EFN- display any synergistic effect in inhibiting CML cells proliferation and blocking the expression of STATi or STAT5.Methods: MTT assay and trypan blue dye exclusion test were used to assay theproliferation and viability of CML cells. The expression of JAK2, STAT1, STAT5 proteins was detected by Western blot technique, and the subcellular location of STAT1, STAT5 was detected with indirect immunofluorescence technique.Results: Western blot assay showed the expression of STAT1, STAT5 was down-regulated by IN, and up-regulated by GM-CSF or IFN- a respectively.The expression of JAK2 was not influenced by IFN- a or EN, but GM-CSF can stimulate the expression of JAK2 protein No syn...
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