New aspects on retinoic acid induced HL-60 granulocytic differentiation

Sammanfattning: The underlying mechanisms of myeloid cell differentiation are still not entirely elucidated. It is therefore an urgent task to extent the basis for a comprehensive model of granulocytes differentiation. A primary goal for studies has been the identification of differentiation markers. These often correspond to genes that are expressed at defined steps during the differentiation process or in the fully differentiated phenotype. Here is demonstrated that the program of phenotype formation entails dramatic changes in long-term tyrosine phosphorylation of some cytoplasmic proteins during retinoic acid (RA)-induced HL-60 cell differentiation to granulocytes. A significant increase in the phosphotyrosine content of a kinase (Erk2) in the commitment and terminal stages of HL-60 cell differentiation (48-120 h) was related to promotion of cell survival and triggering of apoptosis. In addition, some cytosolic proteins of apoptotic origin were delineated in the differentiating population. The pattern of tyrosine phosphorylations of specific cytosolic proteins in maturing and apoptotic granulocytes should be a powerful tool for further elucidation of differentiation mechanisms. We developed a new approach based on cytosolic protein tagging and nuclear import analysis combined with assessment of specific protein tyrosine phosphorylations by two-dimensional gel electrophoresis. Many of the genes involved in the differentiation of multipotent stem cells to specific cellular lineages are still unknown. Accordingly, we searched for additional tyrosine phosphorylated, RA-induced transcription regulators that are translocated into the nucleus during commitment of HL-60 cells to become granulocyte-like cells. It is shown, that transcription factors required for lineage commitment of differentiating (C/EBPß, c-myb) and for survival of differentiated cells (STATs, NFKB) co-operatively promoted signaling in the HL-60 cell line in response to RA. Identification of the tyrosine-phosphorylated proteins being translocated into the nucleus during HL-60 cell differentiation was our further goaL This way, we show for the first time that human gamma~dystrobrevin is expressed in promyelocytic HL-60 cell line. We detected gamma-dystrobrevin in the cytosol and the nuclei of these cells, and, in the latter location; it was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of gamma-dystrobrevin could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and hwnan neutrophils. Moreover, we found gamma-dystrobrevin in association with actin and myosin light chain. Our results provide new information about potential involvement of gamma-dystrobrevin in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells. In addition, our study confirms and extends knowledge about the genes involved in granulocytic differentiation and growth arrest. This shuiy corroborates the value of cDNA RDA to isolate factors involved in myeloid maturation. In conclusion, the more detailed analysis of granulocytic differentiation of HL-60 cells presented here provides new data that should allow further refinement of models of myeloid differentiation.