Severe congenital neutropenia (SCN) is an inheritable hematopoietic disorder characterized by extremely low level of circulating neutrophils, ineffective intramedulary hematopoiesis with “maturation arrest” at the promyelocytic stage of differentiation, recurrent severe infections, and evolution to acute myelogenous leukemia (AML). Accelerated apoptosis of bone marrow-derived myeloid progenitor cells and cell cycle arrest of CD34+ cells was reported in SCN patients. We also reported that heterozygous mutations in the neutrophil elastase (NE) gene have been identified in approximately 80% of SCN patients. Transient expression of mutant but not normal NE triggered accelerated apoptotic cell death of human promyelocytic HL-60 cells supporting the causative role of mutant NE in pathogenesis of SCN.

Here we present cellular model of SCN based on newly established tet-off HL-60 human promyelocytic cell line with inducible doxycycline-regulated expression of mutant NE with 8-amino acid deletion identified in a patient evolved to develop AML. Induced expression of mutant elastase in tet-off HL-60 cells treated with DMSO led to a block of differentiation or “maturation arrest” at the promyelocytic stage of differentiation with a significantly reduced proportion of differentiated cells (approximately 20% vs 70% in control) and higher proportion (~40%) of primitive undifferentiated cells compared with control DMSO-treated HL-60 cells expressing only normal NE (~10%). Flow cytometry analysis of annexin V-labeled cells repeatedly revealed approximately 2-fold higher rate of apoptosis in tet-off HL-60 cells with induced expression of mutant NE compared with control cells.

FACS analysis of DAPI-labeled tet-off HL-60 cells with induced expression of mutant NE revealed abnormal cell cycle progression with gradual accumulation and apparent arrest of cells in G1-phase of the cell cycle similar to that reported for SCN patients. Interestingly, the apoptotic cell shrinking and swelling as determined by flow cytometry was observed in all phases of the cell cycle suggesting that accelerated apoptosis rather than cell cycle arrest is the primary abnormality caused by expression of mutant NE, and the abnormal cell cycle progression is a consequence of this impaired cell survival. Further analysis revealed a dramatic reduction in mitochondrial membrane potential of tet-off HL-60 cells expressing mutant NE compared with control cells. The reduced mitochondrial membrane potential as determined by FACS was observed as early as 24 hours of induction of mutant NE expression and before the accelerated apoptosis or cell cycle arrest was detected. Western blot analysis demonstrated that caspase-3 was not activated in the cells even after 3 days of mutant NE induction. Further analysis of apoptosis regulators revealed a down-regulation of Bcl-2 expression and up-regulation of Bax in cells with induced expression of mutant elastase. These data suggest that mutant elastase-mediated “maturation arrest” of human promyelocytic cells in patients with SCN and SCN/AML is associated with reduced expression of anti-apoptotic Bcl-2 and upregulation of pro-apoptotic Bax proteins that trigger a dramatic reduction in mitochondrial membrane potential and subsequent caspase-independent apoptosis and cell cycle arrest. Current studies focused on elucidating and characterizing specific molecular interactions mediating the pro-apoptotic effect of mutant neutrophil elastase in SCN.

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