LYN AND FGR PROTEIN-TYROSINE KINASES PREVENT APOPTOSIS DURING RETINOIC ACID-INDUCED GRANULOCYTIC DIFFERENTIATION OF HL-80 CELLS

The human promyelocytic leukemia cell line HL-60 can be induced to dif ferentiate toward neutrophils and subsequently die via apoptosis in vi tro. In this paper, we investigated the roles of protein-tyrosine kina ses (PTKs) in retinoic acid (RA)-induced granulocytic differentiation of HL-60 cells. Accompanying the RA-induced differentiation, activitie s of src family PTKs Lyn and Fgr became detected and reached a plateau 2 days after the stimulation. The immunoblotting using anti-phosphoty rosine antibody (PY-20) showed that the proteins of 56 and 53 kDa were predominantly tyrosine-phosphorylated at day 2. Adsorption and immuno precipitation of the cell lysate by specific antibodies evidenced that these phosphotyrosine-containing proteins are Lyn and Fgr PTKs. The d egree of both activities and tyrosine phosphorylation of these PTKs wa s reduced to be minimal at day 5 when the HL-60 cells start to die by apoptosis. The inhibitors of PTKs, herbimycin A and genistein, were de monstrated to cause premature cell death of HL-60 cells in the presenc e of RA. The death was the consequence of an apoptotic process. The RA -treated HL-60 cells, when incubated with specific c-lyn or c-fgr anti sense oligodeoxynucleotide, also underwent premature death at day 2. T hese data implicate that Lyn and Fgr PTKs prevent programmed cell deat h to promote granulocytic differentiation of HL-60 cells.