Study On The Expression Of Interleukin-3 Receptor And Transcript Factors In NB4 And HL-60 Cells Differentiation And Apoptosis Induced By ATRA, AS₂O₃

The self-renewal and directed differentiation of hematopoietic stem cell is regulated by a variety of hematopoietic growth factors, such as interleukin-1 (IL-1), interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte-macrophage colony stimulating factor(GM-CSF). IL-3 can stimulite the proliferation of pluripotent hematopoietic stem cells and induce the differentiation of myeloid stem cells and myeloid progenitor cells, and the mature cells are loss of response to IL-3 gradually. The proliferation and differentiation of mature cells depend on the other cytokines, for example, GM-CSF can stimulite granulocytes and mononuclear cells proliferation and differentiation, while IL-3 and IL-5 stimulite eosinophils and basophils proliferation and differentiation.IL-3 Receptor system are composed of a cytokine-specificαsubunit (IL-3Rα, GM-CSFRα, IL-5Rα) andβcommon subunit (βc) which is shared with IL-3, IL-5 and GM-CSF.Acute myeloid leukemia is characterized by blocking hematopoietic cells differentiation and proliferation uncontrolled.Previous studies showed that abnormal expression of IL-3Rαexisted in acute myeloid leukemia,and that expression of IL-Rαin acute myelogenous leukemia is associated with enhanced proliferation, increased cellularity and poor prognosis. Our previous studies found that the proliferation of NB4 cell was inhibited, accompanied IL-3RαmRNA dencreased significantly during NB4 cell differentiation by ATRA, the expression level of IL-3RαmRNA was gradually restored, and the expression levels of GM-CSFRα, Hβc mRNA increased gradually. However,it have not been reported that the expression level of IL-3R system during NB4, HL-60 cells induced by AS₂O₃.Recent studies indicate that proliferation and differentiation of hematopoietic stem cells is regulated by transcript factors. The transcript factors such as PU.1, CEBPα, GATA1 involved in the differentiation of myeloid cells. Clinical studies also have shown that the mutations and dysfunction of the transcript factors are frequentily exited in human acute myeloid leukemia. Previous studies that the expression of transcript factor were detected by RNA hybridization during NB4 and HL-60 cells differentiation induced by ATRA, the expression levels of CEBPαwere decreased significantly in NB4 and HL-60 cells, the expression levels of GATA1 were increased in NB4 cells by ATRA, and GATA1 was not detected in the HL -60 cell. However it have been reported that the expression level of transcript factors during NB4, HL-60 cells induced by AS₂O₃.In this study, we used real-time RT-PCR to detect the expression changes of IL-3R system (GM-CSFRα, IL-3Rα, Hβc) and transcript factors (PU.1, C/EBP family, GATA1, GATA2) during NB4 and HL-60 cells differentiation and apoptosis induced by ATRA, AS₂O₃,and study that the GM-CSF,IL-3 affect the differentiation and apoptosis of NB4 cell.Results:1. ATRA induced NB4 cell differentiation in the dose of 1μM accompanied the expressions of IL-3Rα, Hβc mRNA increased. while ATRA induced HL-60 cell differentiation in the same dose,and accompanied the expressions of GM-CSFRα, Hβc mRNA increased. AS₂O₃ induced NB4 cell differentiation and apoptosis in the dose of 1μM, and accompanied the expressions of IL-3Rα, Hβc mRNA increased,but it is not more significantly than NB4 cell differentiation induced by ATRA. AS₂O₃ can not induce NB4 cell differentiation and apoptosis in the same dose, and the expressions of GM-CSFRα, IL-3Rα, Hβc mRNA are also not changed.2. The expression levels of PU.1, CEBPe mRNA increased, and the expression levels of CEBPα, CEBPγ, GATA1,GATA2 mRNA decreased during NB4 cell differentiation induced by ATRA.The expression levels of PU.1, CEBPαincreaced, and the expression levels of CEBPγ,GATA1, GATA2 mRNA decreased during HL-60 cell differentiation induced by ATRA.The expression levels of CEBPε, GATA1 mRNA increased, and the expression levels of CEBPα, CEBPγmRNA decreased during NB4 cell differentiation and apoptosis induced by AS₂O₃. The expression levels of CEBPαmRNA increased,and the expression levels of CEBPε, CEBPγ, GATA1 mRNA decreased in HL-60 cell treated by AS₂O₃. 3. GM-CSF and IL-3 can not stimulate NB4 cell proliferation and induce NB4 cell differentiation alone, but GM-CSF cooperated with AS₂O₃ can induce NB4 cell differentiation. IL-3 have no effect on NB4 cell differentiation induced by AS2O3. Conclusion:1. IL-3 receptor system (GM-CSFRα, IL-3Rα, Hβc) involved in NB4 cell differentiation and apoptosis induced by ATRA and AS₂O₃ and in HL-60 cell differentiation induced by ATRA.2. Transcript factors such as PU.1, CEBPα, CEBPγ, CEBPε, GATA1, GATA2 involved in NB4 cell differentiation and apoptosis induced by ATRA and AS₂O₃ and in HL-60 cell differentiation induced by ATRA.3. GM-CSF and IL-3 can not stimulite NB4 cell proliferation and induce NB4 cell differentiation alone, but GM-CSF cooperated with AS2O3 can induce NB4 cell differentiation. IL-3 have no effect on NB4 cell differentiation induced by AS₂O₃.