Derangement of transcription factor profiles during in vitro differentiation of HL60 and NB4 cells.

Sequential up- and down-regulation of a handful of critical transcription factors is required for proper neutrophil differentiation. Malfunction of transcription factors may lead to diseases such as acute myeloid leukemia (AML) and specific granule deficiency. In order to understand the molecular background for normal and malignant granulopoiesis, a good model system is required that faithfully mimics the in vivo transcription factor expression profiles. The two human leukemic cell lines HL60 and NB4 have been widely used as model cell lines for these purposes. Differentiation of HL60 and NB4 cells resulted in asynchronous differentiation to morphologically mature neutrophils over a period of 5-7 days. To obtain cell populations of more even maturity, cells at different stages of in vitro differentiation were purified by immunomagnetic isolation. This resulted in three cell populations that could be classified as promyelocytes, myelocytes/metamyelocytes, and mature neutrophils, respectively. Comparison of transcription factor mRNA profiles from these cell populations with those previously seen in normal human bone marrow, demonstrated that although all of the 14 transcription factors described in vivo, could be detected during in vitro differentiation, vast differences in their expression profiles was observed. These data illustrate the limitations of cell lines as models for normal granulopoiesis.

All-trans retinoic acid-induced expression of bactericidal/permeability-increasing protein (BPI) in human myeloid cells correlates to binding of C/EBPbeta and C/EBPepsilon to the BPI promoter.

Bactericidal/permeability-increasing protein (BPI) neutralizes the proinflammatory effects of lipopolysaccharide and is of potential clinical use in the treatment of fulminant Gram-negative infections. BPI is a cationic protein with antibacterial activity stored in azurophil (primary) granules of neutrophil granulocytes. However, the absence of BPI in patients with specific granule deficiency indicates a transcriptional control of BPI, which is distinct from that of other azurophil granule proteins. Accordingly, we demonstrate in vivo that the BPI mRNA level peaks, together with mRNA for specific granule proteins, during the myelocytic and metamyelocytic stage of granulocytic maturation. The human promyelocytic cell line NB4 expresses several azurophil granule proteins, but expression of BPI is undetectable. We show that treatment of NB4 cells with all-trans retinoic acid (ATRA) induces BPI expression at mRNA and at protein level. The induction is dependent on de novo protein synthesis, as judged by sensitivity to cycloheximide. Previous investigations have indicated a potential role of CCAAT/enhancer-binding protein (C/EBP) transcription factors in the regulation of BPI expression. Here, we show that induction of NB4 cells with ATRA correlates to direct binding of C/EBPbeta and C/EBPepsilon to the proximal BPI promoter, as determined by electrophoretic mobility shift analysis and chromatin immunoprecipitation. The dependency on C/EBPbeta and C/EBPepsilon provides an explanation for delayed BPI mRNA expression, as compared with mRNA of other azurophil granule proteins.