The Different Effects Of TGF-β Receptor Ⅱ Isoforms On HL-60Cells Proliferation And Response To ATRA And As₂O₃

Object：The aim of this study was to evaluate the proliferation of HL-60cellsstably expressing TGF-βRII isoforms （TβRII） and to study the effect of ATRA andAs₂O₃respectively on inducing differentiation and apoptosis of these two types ofcell.Methods:1. Electroporation transfection and antibiotic selection were used toconstruct the HL-60cells respectively stably expressing two kinds of TβRⅡ isoform,which were named HL-60/TβRⅡA and HL-60/TβRⅡB cells. RT-PCR and WesternBlot assay were used to identify the expression of TβRⅡmRNA and protein in theHL-60cells.2. Colony formation,MTT assay and trypan blue exclusion test wereused to evaluate the ability of proliferation of HL-60/TβRⅡA and HL-60/TβRⅡBcells.3. Wright-Giemsa staining and surface expression of CD11b antigen were usedto observed the the effect of ATRA on the differentiation of HL-60/TβRⅡA andHL-60/TβRⅡB cells.4. AO/EB staining and AnnexinV-FITC/PI double staining wereused to observed the effects of As₂O₃on the apoptosis of HL-60/TβRⅡA andHL-60/TβRⅡB cells.Results:1. TβRⅡA and TβRⅡB can be stably expressed in HL-60cells,which is validated by RT-PCR and Western blot.2.Colony formation assay found that the formation rate of HL-60/TβRⅡAcells（50.58±1.46%） was significantly lower than that of HL-60cells（62.58±3.46%）and HL-60/TβRⅡB cells（62.33±4.80%）（P<0.05）. MTT assay and trypan blueexclusion test showed that the proliferation of HL-60/TβRⅡA cells was significantlylower than that of HL-60cells and HL-60/TβRⅡB cells.3. The cellular differentiation of these cells was examined by Wright-Giemsastaining after treated with ATRA. Under96h effect of ATRA, expression of CD11bon HL-60/TβRⅡA cells was significantly higher than those on HL-60cells andHL-60/TβRⅡB cells, presenting dose-dependent effect. At the concentration of1μM for96h, the expression of CD11b on HL-60/TβRⅡA, HL-60and HL-60/TβRⅡB cellwere18.20±3.04,6.97±0.87and6.83±1.14（P<0.05）.4.Apoptosis of HL-60/TβR ⅡA cell was inducible when treated withAs₂O₃atthe concentration of4uM for24h, but that is not the case for either HL-60cells orHL-60/TβR ⅡB cells. The apoptosis rate of HL-60/TβRⅡA cells was significantlyhigher than HL-60cells and HL-60/TβRⅡB cells after treated with As₂O₃at theconcentration of （8-10μM） for24h, demonstrating dose-dependent effect as well. Atthe concentration of10μM for24h, the apoptosis rates of HL-60/TβRⅡA cell, HL-60cell and HL-60/TβRⅡB cell were25.5±3.35,15.23±1.55and11.90±1.21（P<0.05）.Conclusion: the proliferation of HL-60/TβRⅡA cells was significantly lowerthan that of HL-60/TβRⅡB cells. TβRⅡA/HL-60cells were more sensitive thanTβRⅡB/HL-60cells to the differentiation-inducing effect of ATRA and theapoptosis-inducing effect of As₂O₃.