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Effect of bone morphogenetic proteins on leucemic NB4 and HL60 cell lines

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15 ; 17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor-a gene (RARα ). APL, characterized by the differentiation arrest of granulopoiesis at the promyelocytic stage, is the first human malignancy that can be efficiently treated with a cell differentiation inducer all-trans retinoic acid (ATRA)1. In in vitro model of APL, human promyelocytic NB4 cell line has the t(15 ; 17) translocation and differentiate in response to ATRA, whereas HL60 cell line lacks this chromosomal translocation but is still able to differentiation with ATRA treatment. Despite the great success of ATRA in both remission induction and maintenance therapy in APL, a fair percentage (30-40%) of patients still have a relapse after initial remission and develop resistance to ATRA treatment2. Mechanism of the resistance to ATRA-induced differentiation in those patients is still not fully understood. Bone morphogenetic proteins (BMPs), members of transforming growth factor β superfamily, are multifunctional cytokines involved in the regulation of proliferation, differentiation, morphogenesis and apoptosis in a variety of cell types including hematopoetic lineages. Since several studies suggested that ATRA and BMPs interact in the regulation of cell differentiation and apoptosis, we investigated the effects of BMP2, BMP4 and BMP6 treatment in the ATRA-induced differentiation of human promyelocytic NB4 and HL60 cell lines. The goal of our study was to explore the possible role of BMPs in the promyelocytic cell resistance to ATRA-induced differentiation. NB4 and HL60 cells were treated with ATRA alone (1 μ M and 10 μ M respectively) or in combination with the various doses of BMP2, BMP4 or BMP6 (from 5 pg/mL to 250 pg/mL) for up to 6 days. Cell maturation was evaluated on the basis of cellular morphology and changes in cell surface antigens. Briefly, cell cytospins stained with May-Grienwald-Giemsa (MGG) were analyzed for cell morphology by means of light microscopy. Analysis of cell surface myeloid-specific antigens CD11b and CD11c was performed by flow cytometry at several time points (days 3 to 6). RNA extracted from cultured cells were reversely transcribed ; cDNA was amplified by quantitative PCR, using TaqMan assays (Applied Biosystems) for inhibitor of DNA binding (ID) genes ID1 and ID2, and expressed as the relative amounts of RNA for target genes normalized to GPDH. Treatment of NB4 and HL60 cells with ATRA induced myeloid differentiation, as evidenced by increase in the surface expression of CD11b and CD11c markers, with the most prominent effect after 5 day-treatment in NB4 cells (94%) and 6 day-treatment in HL60 cells (86%) (Figures 1a and 1b). BMPs alone did not increase the expression of CD11b/CD11c markers, but the combined treatment with ATRA and BMP2, BMP4 or BMP6 partially blocked cell differentiation seen in response to ATRA alone (Figures 2 and 3). Ability of BMPs to suppress the effect of ATRA was greater for HL60 than for NB4 cells, which may be at least partially explained by the presence of PML/RARα oncogene in NB4 cells. MGG staining of cell cytospins showed ATRA-induced morphological maturation and differentiation of both cell lines, whereas more immature and undifferentiated cells were observed on slides treated with the combination of ATRA and BMPs (Figures 4a and 4b). Suppression of differentiation seen in combined treatment with ATRA and BMPs was paralleled by increased expression of ID1 gene in NB4 cells and ID2 gene in HL60 cells (Figures 5a and 5b). The greatest effect was seen for ID2 gene, which was induced by more than 50 fold with the combination of BMP6 and ATRA compared to ATRA alone. Our findings suggest that BMP2, BMP4 and BMP6 have the ability to suppress differentiation of NB4 and HL60 cells upon ATRA treatment. We proposed that the mechanisms of this effect include upregulation of ID genes by BMPs. ID genes, known to be directly induced by BMP signal3 , were recently shown to have oncogenic properties in different human malignancies, by inhibiting cell differentiation and support tumourigenesis. Further investigation is needed to explore the mechanism of BMP2, BMP4 and BMP6 activity in promyelocytic cells and their possible role in resistance to ATRA seen in APL patients after the initial remission.

BMP; NB4; HL60; acute promyelocytic leukemia

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