Effect Of Nucleophosmin Mutation On Biologic Feature Of Leukemia Cells And Potential Mechanisms

Acute myeloblastic leukemia(AML) is a type of hematological malignancies with emergency, short course and high death rate. The complete remission rate is significantly lower and relapsed quickly in the AML cases that were treated by traditional chemo-therapy and radio-therapy. Transplantation of hemopoietic stem cell is a radical cure of AML, But there are still many problems have to be solved such as immune reaction and high cost treatment. There are many fusion genes have been found, which associated with pathogenesis and progress in leukemia. However, leukemia is a genetically and phenotypically heterogeneous disease, which possessed multitude genes correlated leukemia. It hasn't been reported that a kind of target genes associated with various type AML. Recently, Falini et al reported that the nucleophosmin(NPM) gene is mutated in a high proportion of AML. The NPM mutant pattern was found in AML specimens of all FAB subtypes except M3. Clinical data revealed that NPM mutant pattern was found in adults and childhood AML cases. The incidence rate of NPM mutation increased in older patients. Studies demonstrated that NPM mutation involved leukemia stem/progenitor cell, which no specific genetic abnormalities associated AML were found. This suggested that NPM mutation initiated AML pathogenesis. Clinical reseach revealed that NPM mutation was closely correlated with AML prognosis. NPM mutation not only directed selection of therapeutic methods but also monitored minimal residual disease(MRD). In addition, identification of NPM mutation impacted on the occurent World Health Organization(WHO) classification of AML. This futher confirmed that the role of NPM mutation in pathogenesis, progress and prognosis of AML. Further reseach found that NPM mutation promoted NIH3T3 cells malignant transformation. However, there have been few studies of the effect of NPM mutation on biologic feature of leukemia cells and potential mechanisms.To investigate the effect of NPM mutation on the proliferation and apoptosis of leukemia cells and potential mechanisms, KG-1a cells were chosen as reseach objects. Main results and conclusions as follow:1. CD34 molecule and expression of NPM mutant gene in KG-1a cells was detected. Firstly, CD34 antigen analyses of KG-1a cells were performed with FACS, CD34+ cells accounting for 96.3% was observed in KG-1a cells. Secondly, expession of NPM mutation were detected by RT-PCR and immunohistochemical stain respectively. The results confirmed that no expession of NPM mutation in KG-1a. These finding indicated that KG-1a was a kind of primitive leukemia cells, which was high expression of CD34 antigen and no expression of NPM mutant pattern .2. To observe the effect of mutated NPM on proliferation and apoptosis of KG-1a cells. Firstly, NPM mutant gene was transfected into KG-1a using Lipofectamine 2000. Expession of NPM mutation were detected by RT-PCR and immunohistochemical respectively. The results confirmed that expession of NPM mutation in transfected KG-1a. Secondly, cell cycle analysis were performed with FACS, NPM mutation promoted cell cycle progress. Furthermore, the viable cell count(typan blue stain) revealed that KG-1a acceleratly grow. Lastly, the activity of casepase-3 was assessed by absorption spectrometry. KG-1a cells expressing NPM mutation have decreased the activity of casepase-3. Those results summarized above indicated that NPM mutation not only promoted KG-1a cells malignant proliferation, but also induced anti-apoptosis.3. To explore the molecular pathogenesis of NPM mutation on biologic feature of KG-1a cells, expression of p21 mRNA was determined using RT-PCR. Downregulated expression of p21 mRNA in NPM mutant transfected KG-1a cell was observed. The present data confirmed that NPM mutation promoted KG-1a cells malignant proliferation by downregulated expression of p21. Furthermore, the activity of casepase-9 was assessed by absorption spectrometry. KG-1a cells expressing NPM mutation have decreased the activity of casepase-9. So we could conclude that NPM mutation promote leukemia cells proliferation and apoptosis resistance, which mediated by p21 and casepase-9.