Molecular Mechanism Of Differentiation And Apoptosis Of VPA In T(8,21)AML Leukemia Cell

Purpose:It is the pivotal pathogenesis that t(8;21)AML produces fusion protein AML1-ETO by chromosome translocation to recuit histone deacetylase (HDAC) which represses transcription of AML1 target genes. To investigate the effect of HDAC inhibitor(HDACi) valproic acid (VPA) on biological function of AML1-ETO fusion protein, and to investigate whether or not VPA can reverse the transcription repressing effect of AML1-ETO fusion protein on AML target gene and induce t(8;21) leukemic cells differentiate and apoptosis and repress proliferation.Material and methods: Cells from the t(8,21) cell line kasumi-1were treated with VPA. Trypan blue refusal staining method was used to observe the influence of VPA on the growth of kasumi-1 cells, and light microscope was used to observe the morphological change of these cells, and analysis cell cycle and detecting the expression of myeloid cell differentiation antigen by flow cytometry. We applied DNA gel electrophoresis to observe the apoptosis of the kasumi-1 cells, and the mRNA expression level of regulation genes and apoptosis associated genes of AML1 was detected by RT-PCR.Results: 1. VPA can repress the growth of kasumi-1 cells Treatment the Kasumi-1 cells for 24, 48 and 72 hours with different concentration VPA caused a dose-dependent and time-dependen inhibition of growth by trypan blue refusal assays. The IC50 of VPA was 2.33mmol/L for Kasumi-1cells treated with VPA for 3 days.2. VPA can induce the kasumi-1 cell differentiation The kasumi-1 cells were treated with VPA for 24, 48, 72 and 96 hours. The expression level of the mature granulocyte and monocyte surface antigens CD11b on kasumi-1 cells was detected by flow cytometry. It was showed that VPA induced the expression level of the myeloid cell surface antigens CD11b increase in kasumi-1 cells with a time- and dose- dependent way. It was also showed that the expression rate of CD11b on the kasumi-1 cells was increased from 4.7% to 90.6%(P<0.05) when the kasumi-1 cells were treated with 3mmol/L VPA for 3 days. But when the concentration of VPA was higher than 3mM or the culture time was much more longer with VPA, it could be led most of Kasumi-1 cells to be dead, and in the contrary, the expression level of the myeloid cell antigens CD11b on the kasumi-1 cells were decreased .When the Kasumi-1 cells were incubated with the 1mM VPA for 24 hours, the cell morphology would be changed with some degree, such as karyoplasmic ratio decrease, nucelus disappearance, kidney-shaped nucelus cells increase. All of these cell morphology changes means the differentiation of the Kasumi-1 cells was happened. With the incubation time to be longer and the drug concentration increased, Part of the cells would differentiate into rod shape nucelus.3. VPA can induce the kasumi-1 apoptosis It was noted that Kasumi-1 cell line treated with 3mM VPA for 3 days could lead to typical DNA ladder in apoptosis cells by DNA ladder assays.With the incubation time to be longer and the drug concentration increased, The apoptosis morphological change such as apoptoic body,the cell volume to be smaller,nucelus pycnosis and chromatin aggregation ,could be seen.4. The influence of cell cycle in Kasumi-1 cell line treated with VPA With VPA concentration increase and incubation time longer,cell ratio at G0/G1 phase gradually increase analysed by flow cytometer. Treated with 3mmol/LVPA for 3 days,cell ratio at G0/G1 phase rised from 50.1% to 80.7%(p<0.01), cell ratio at S and G2/M phase deseased.5. the expression influence of GM-CSF incubation with VPA VPA induced AML1 protein downstream regulation gene GM-CSF to increase at Mrna level with dose-and time-dependent manner in Kasumi-1 cell line analysed by semi-quality RT-PCR.Compared with control group,expression of mRNA increase obviously treated with 3mmol/L VPA for 5 days.(p<0.01) 6. the expression influence of Caspase-7 treated with VPA…Incubation with VPA longer,the expression of Caspase-7 increased obviously at mRNA level by semi-quality RT-PCR. The expression of Caspase-7 was higher than control group incubation with 3mmol/L VPA for 3 days(p<0.05) and for 5 days(p<0.01).Incubation with different concentration of VPA, the expression of Caspase-7 at mRNA level presented increase tendency with concentration increase,no significant difference in statistics .Conclusion: HDAC inhibitor VPA could inhibit proliferation of t(8;21)AML leukocyte,arrested the cell cycle at G0/G1 phase;VPA induced cells differentiation and apoptosis,removes transcription inhibition of AML1-ETO fusion protein and increases regulation genes expression of AML1.Our research can offer rationale for clinical therapy and enhance pathogenesy apprehension of acute leukemia deeply.