1, Iaspp Expressed On Hematopoietic Stem Accounted Cell Self-renewal And Study 2 Antiapoptotic Effect, The Influence Of All Trans Retinoic Acid Of Npm1 Mutations Of Leukemia Cell Line Hl 60 Effect And Its Mechanism Is Discussed In This Paper

Objective:iASPP is an inhibitory member of apoptosis stimulating proteins of p53family (ASPP) and negatively regulates the apoptotic function of p53. We study the effect of iASPP overexpression on the self-renewal an anti-apoptosis of hematopoietic stem cells (HSC) in iAPP transgenic (iASPP Tg) mouse model which was established well previously in our laboratory.Content:As an anti-apoptosis gene, iASPP overexpression in hematopoietic system results in blockage of apoptosis, which may play a direct role in regulating HSC number, even in deregulation of hematopoiesis. Our previous study showed that the expression of iASPP was highly expressed in acute leukemia (AL) patients compared with that in normal controls. Then a transgenic mouse model in which human iASPP was specifically expressed in hematopoietic cells of mouse was further established. In this study, we used the iASPP transgenic mouse model to study the effect of iASPP overexpression on number and function of HSC, and the changes of cell cycle and the status of anti-apoptosis and damage of HSC in iASPP transgenic (iASPP Tg) mice.Methods:(1) The polychromatic flow cytometry was used to analyze the ratio of each hematopoietic stem/progenitor cell (HSPC) populations in bone marrow mononuclear cells (BMMNC) of iASPP Tg and wild type mice;(2) in vivo continuous transplantation experiment and in vitro continuous colony forming experiment were used to study the changes of self-renewal and hematopoietic reconstitution of HSPC in iASPP Tg mice;(3) Annexin V and PI staining and flow cytometry was used to analyze the apoptosis levels of HSC and BMMNC induced by irradiation and VP16treatment in iASPP Tg and wild type mice;(4) Western blot and immunofluorescence assay were used to detect the y-H2AX expression level induced by irradiation;(5) Small interference RNA combined with VP16treatment methods were used to research the anti-apoptosis of BMMNC in iASPP Tg mice and the relationship between anti-apoptosis effect and p53;(6) Affymetrix Gene Chip was used to analyze the overall influence of iASPP overexpression on gene expression and signaling pathway, and provide the clue of the relationship between iASPP overexpression and leukemogenesis mechanism.Results:(1) The polychromatic flow cytometry:iASPP Tg mice had a3-fold increase in the percentage of LSKs cells on average and a correlative increases in all of the percentages of long-term HSC (LT-HSC), short-term HSC (ST-HSC) and multipotent progenitor (MPP) than that of littermate controls;(2) in vivo continuous transplantation experiment and in vitro continuous colony forming experiment:HSC population proportions from different rounds recipients in iASPP Tg mice were always higher than that of wild type mice. In the second and third rounds of colony forming experiment, the cells from Tg mice form much more colonies than that from control mice and the difference was significant in the third round;(3) irradiation and VP16treatment:the apoptosis level of HSC and BMMNC form iASPP Tg mice was significantly lower than that of wild type mice;(4) Damage experiment:both Western blot and Immunofluorescence assay showed more y-H2AX expression was observed in iASPP Tg BMMNC after irradiation, while wild type BMMNC showed less y-H2AX expression;(5) Small interference RNA and VP16treatment:the BMMNCs from iASPP mice displayed significantly lower level of apoptosis than that of wild type mice, however, after silencing of P53by specific siRNA, there was no difference in apoptosis level of BMMNC between these two groups;(6) Affymetrix Gene Chip:iASPP overexpression results many changes of pathways of apoptosis-related, p53-related and tumorigensis-related in iASPP Tg mice.Conclusions:These results provide the first evidence that the anti-apoptosis regulator iASPP can increase hematopoietic stem/progenitor populations and reconstitution capacity. Interestingly, in response to cell damage stimuli, hematopoietic cells can be protected against apoptosis by iASPP, while these genotoxic resistant cells have high risk of mutation accumulation and malignant transformation. Objective:In acute myeloid leukemia (AML) patients, NPM1c+AML has a better prog-nosis than NPM wild type AML. In recent years, clinical trials have reported abroad that all-trans retinoic acid (ATRA) was added in intensive chemotherapy, the prognosis and overall survival of NPM1c+AML patients has improved significantly. The objective of this study is to elucidate the role of NPM1mutA gene in leukemogenesis and the effect of ATRA on NPM1mutA.Methods:The coding sequences (CDS) of NPM1wt and NPM1mutA were amplified by PCR from plasmid GM-NPM1wt and GM-NPM1mutA respectively, and then were cloned into retroviral vector MSCV-IRES-GFP.293T cells were transfected by calcium pHospHate precipitation to produce retrovirus. HL-60cells were infected with the retrovirus expressing MSCV-NPM1wt-IRES-GFP and MSCV-NPMmutA-IRES-GFP, respectively, and GFP positive cells were selected by flow cytometry. The MSCV-NPM1wt-IRES-GFP and MSCV-NPMmutA-IRES-GFP highly expressed stable HL-60monoclones were identified by RT-PCR, allel-specific PCR and Western blot. The impact of NPM1mutA on the proliferation and of HL-60cells treated with or without ATRA was detected by MTT assay. The effect of NPM1mutA on the colony forming ability of HL-60treated with or without ATRA was studied using methylcellulose semi-solid medium. HL-60cells were treated with or without ATRA, and the flow cytometry was used to investigate cell cycle, differentiation and apoptosis.Results:NPM1wt and NPM1mutA monoclonal cells have been successful screened.(1) cell proliferation:NPM1mutA did not obviously change the proliferation ability of HL-60cells compared with empty vector (p>0.05);(2) cell cycle:NPM1mutA induced G0/G1-pHase cell fracion of HL-60cells (p<0.05) and increased S-pHase cell fraction of HL-60cells (p<0.05);(3) colony fromation:NPM1mutA enhanced the colony froming capacity of HL-60cells, but the increase in NPM1mutA group was significantly inhibited compared with empty vector and NPMlwt groups (p<0.05);(4) differentiation: NPM1mutA reduced the differentiation of HL-60cells and resist to the differentiation effect of ATRA (p<0.05);(5) apoptosis:NPM1mutA slightly increased the basic apoptosis level of HL-60cells (p>0.05). But NPM1mutA could significantly increase the apoptosis level of HL-60cells treated with different doses of ATRA (p<0.05). Conclusions:NPM1mutA could significantly enhance the colony forming ability. ATRA treatment could obviously inhibit the colony forming ability of HL-60cells transducted with NPM1mutA, and increase the apoptosis proportion of HL-60cells transducted with NPM1mutA compared with empty vector and NPM1wt groups. Howere, NPM1mutA has obvious resistance to the differentiation ability of ATRA. Consequently, the mechanism of NPM1c+AML treated with ATRA may be that ATRA bring about apoptosis and reduce colony formation ability of NPM1c+cells.