The Effect Of NPM1Mutations On The Chemosensitivity Of Leukemia Cells And Its Molecular Mechanisms

Nucleophosmin (NPM1) mutations have been identified as the mostcommon genetic lesions in adult AML patients. The data from clinicalstudies indicate that AML with NPM1mutation is generally associatedwith good response to induction chemotherapy. However, the mechanismsunderlying this chemosensitivity are still unknown. To investigate the effectof NPM1mutations on leukemia cells response to chemotherapeuticalagents, the pEGFPC1-NPM1mA plasmid vector was transfected intoTHP-1cells to establish the stably expressed NPM1mutation A proteinleukemia cells (THP-1mA). The cells transfected with pEGFPC1plasmid(THP-1C1) and the untreated cells (THP-1) were set as control. Optimalconcentration and effect time of daunorubicin (DNR) and cytarabine(Ara-C) were assessed by MTT assay. Flow cytometry was used to detectthe cellular apoptosis. The mRNA and protein expression of Bax and Bcl-2 were analyzed by qPCR and Western blot, respectively. After0.25μMDNR or8.5μM Ara-C treatment for24h, cellular apoptosis of THP-1mAwere significantly increased (p<0.05). Compared with the controls, themRNA and protein expression of Bax in THP-1mA cells were remarkablyincreased (p<0.05), while the expression of Bcl-2significantly decreased(p<0.05). These results demonstrate that NPM1mutations promote cellularapoptosis, which may enhance cells sensitivity to chemotherapeuticalagents. NPM1mutations play an important role in leukemiagenesis. Wehave found that NPM1mutations could promote cells sensitive tochemotherapeutical agents. To explore the mechanisms of leukemiccells carrying NPM1mutations sensitivity to chemotherapeuticalagents, the leukemia cells (THP-1mA) with NPM1mutation A proteinwere established, the mock transfection cells (THP-1C1) and the untreatedcells (THP-1) were set as control. The relative activities of NF-κB were assessed by luciferase reporter assays. The interaction between NPM1mutation protein and NF-κB was evaluated by IP. After TNF-α treatment,the activator of NF-κB, incubation, cellular apoptosis and the expression ofBax and Bcl-2were analyzed. In addition, the mRNA expression ofmiR-29b and its target gene Mcl-1were detected by qPCR. Western blotwas used to analyze the expression of Mcl-1protein. Results showed thatthe activation of NF-κB in THP-1mA cell treated with chemotherapeuticalagents was blocked and its activity maintain at a low level. The interactionbetween NPM1mutation protein and NF-κB took place mainly in thecytoplasm. Upon TNF-α treatment, cellular apoptosis in THP-1mA cellswere significantly decreased (p<0.05); the mRNA and protein expressionof Bax in THP-1mA cells were significantly decreased (p<0.05), while themRNA and protein expression of Bcl-2were remarkably increased(p<0.05). Compared with the controls, the expression of miR-29b in THP-1mA keep at a high level and the protein expression of Mcl-1maintain at alow level; while treated with TNF-α, the expression of miR-29b wassignificantly decreased in the THP-1mA cells and mRNA and protein theexpression of Mcl-1had a remarkably increased (p<0.05). These resultsdemonstrate that NPM1mutations may enhance the sensitivity of leukemiacell to chemotherapeutical agents by reducing NF-κB activity, increasingthe expression of miR-29b and decreasing the expression of Mcl-1.Furthermore, the expressions of Bax were increased while the expressions of Bcl-2were decreased, ultimately the leukemia cells sensitive tochemotherapeutical agents.