HUMAN LEUKEMIA-CELL LINES BIND BASIC FIBROBLAST GROWTH-FACTOR (FGF) ON FGF RECEPTORS AND HEPARAN SULFATES - DOWN-MODULATION OF FGF RECEPTORS BY PHORBOL ESTER

Basic fibroblast growth factor (bFGF) has been identified as an import ant cytokine for blood cells. To determine whether hematopoietic cells have receptors that recognize bFGF, the ability of human leukemia cel l lines to bind I-125-bFGF was investigated. Specific bFGF-binding sit es were identified on K562 and HL60 cells, but not on U937 cells. DAMI cells bound low amounts of I-125-bFGF specifically. Binding of I-125- bFGF to K562 cell surfaces was reduced in a dose-dependent manner by u nlabeled bFGF or by heparin. Scatchard analysis of binding to K562 cel ls revealed two classes of binding sites: 1,650 high affinity binding sites per cell with a dissociation constant (kd) of 192 pmol/L, and 36 ,600 low affinity sites per cell with a kd of 9.3 nmol/L. Chemical cro sslinking experiments with K562, HL60, and DAMI cells revealed recepto r-growth factor complexes with molecular masses of 140 to 160 kD, simi lar in size to complexes formed by known receptor species. Binding of I-125-bFGF to K562 cells was sensitive to heparinase treatment but not to chondroitinase treatment, suggesting that heparan sulfate proteogl ycans (HSPGs) may be responsible for the low affinity binding sites. T o further investigate whether K562 cells make HSPG, the incorporation of (SO4)-S-35 into proteoglycans was assessed. Metabolically labeled c ell-surface proteoglycans with molecular masses of 180 to 300 kD were identified in K562 cells. These proteoglycans were sensitive to hepari nase, demonstrating that K562 cells synthesize bFGF-binding HSPG. Trea tment of K562 cells with phorbol-12-myristate-13-acetate (PMA) caused a loss of bFGF-binding capacity. This decreased binding capacity refle cted a rapid loss of high affinity receptors. The ability to form bFGF -receptor complexes decreased by 65% to 70% within 1 hour and declined continuously thereafter. The decrease in binding of bFGF was not due to an autocrine downregulation of bFGF receptors, because there was no increase in bFGF after PMA treatment as detected by Western blotting, and suramin, which blocks bFGF binding to receptors, did not prevent the loss of receptors after exposure to PMA. In addition, inhibitors o f either protein synthesis or protease activity did not prevent the lo ss of bFGF receptors in PMA-treated cells. In summary, this work demon strates that leukemia cell lines have receptors that specifically bind bFGF and supports the hypothesis that bFGF acts directly on certain b lood cells to stimulate their proliferation. (C) 1996 by The American Society of Hematology.