heparitin-sulfate and Lymphoma--Large-B-Cell--Diffuse

Basic fibroblast growth factor (bFGF) has been identified as an important cytokine for blood cells. To determine whether hematopoietic cells have receptors that recognize bFGF, the ability of human leukemia cell lines to bind 125I-bFGF was investigated. Specific bFGF-binding sites were identified on K562 and HL60 cells, but not on U937 cells. DAMI cells bound low amounts of 125I-bFGF specifically. Binding of 125I-bFGF to K562 cell surfaces was reduced in a dose-dependent manner by unlabeled bFGF or by heparin. Scatchard analysis of binding to K562 cells 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 crosslinking experiments with K562, HL60, and DAMI cells revealed receptor-growth factor complexes with molecular masses of 140 to 160 kD, similar in size to complexes formed by known receptor species. Binding of 125I-bFGF to K562 cells was sensitive to heparinase treatment but not to chondroitinase treatment, suggesting that heparan sulfate proteoglycans (HSPGs) may be responsible for the low affinity binding sites. To further investigate whether K562 cells make HSPG, the incorporation of 35SO4 into proteoglycans was assessed. Metabolically labeled cell-surface proteoglycans with molecular masses of 180 to 300 kD were identified in K562 cells. These proteoglycans were sensitive to heparinase, demonstrating that K562 cells synthesize bFGF-binding HSPG. Treatment of K562 cells with phorbol-12-myristate-13-acetate (PMA) caused a loss of bFGF-binding capacity. This decreased binding capacity reflected 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 of either protein synthesis or protease activity did not prevent the loss of bFGF receptors in PMA-treated cells. In summary, this work demonstrates that leukemia cell lines have receptors that specifically bind bFGF and supports the hypothesis that bFGF acts directly on certain blood cells to stimulate their proliferation.

Topics: Animals; Binding Sites; CHO Cells; Cricetinae; Down-Regulation; Fibroblast Growth Factor 2; Heparan Sulfate Proteoglycans; Heparin; Heparitin Sulfate; HL-60 Cells; Humans; Leukemia; Leukemia, Megakaryoblastic, Acute; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma, Large B-Cell, Diffuse; Megakaryocytes; Neoplasm Proteins; Protein Binding; Proteoglycans; Receptors, Fibroblast Growth Factor; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Glycosaminoglycans (heparins, dermatan sulfate, chondroitin sulfate) with different structures and physicochemical properties were evaluated for their capacity to influence proliferation and differentiation of U-937 cell line. The contrasting and specific effects of glycosaminoglycans (depending on their structures and properties) on a leukemia cell line could help explain the regulation of proliferative and/or differentiative processes of hematopoietic cells in order to clarify the control of development and differentiation of hematopoietic progenitor cells by bone marrow extracellular matrix. Heparin from beef intestinal mucosa, heparan sulfate from beef spleen, dermatan sulfate from beef intestinal mucosa, and chondroitin sulfate from bovine trachea were extracted and purified, and their purity, structures, and physicochemical properties were evaluated. Fast-moving heparin was obtained by its selective precipitation as barium salt, and partially desulfated and re-N-sulfated heparin was produced by chemical modifications. Different glycosaminoglycans were tested to evaluate their effects on proliferation and differentiation processes of a monoblastic leukemia cell line (U-937). Heparin and derivatives (from 0.1 to 100 micrograms/ml) inhibit cell proliferation; heparan sulfate does not produce modifications, while chondroitin sulfate and dermatan sulfate (from 0.01 to 100 micrograms/ml) significantly stimulate cell growth. Cell differentiation was evaluated by cytoenzymatic determination of alpha-naphthyl butyrate esterase and by fluorescein-labeled anti-HLA-DR, anti-CD11b, and anti-CD14 antibodies. Nitro blue tetrazolium reduction and phagocytosis were also evaluated. Heparin and derivatives significantly increase U-937 differentiation. Heparin sulfate has no effect, while chondroitin sulfate and, to a lesser extent, dermatan sulfate, induce a strong decrease of differentiative markers. The regulation of U-937 cell properties appears to be related to charge density and to the amount of N-sulfate and N-acetyl groups. In particular, glycosaminoglycans with lower sulfate-to-carboxyl ratios and N-sulfate group percentages (chondroitin sulfate and dermatan sulfate) stimulate proliferation and produce a decrease of differentiative markers; on the contrary, polysaccharides with high charge density and N-sulfate group amounts (heparin and derivatives) inhibit U-937 proliferation and induce terminal differentiation. A previous paper (N. Volpi, L. Bolognani, A. C

Topics: Animals; Carbohydrate Conformation; Carbohydrate Sequence; Cattle; Cell Differentiation; Cell Division; Cell Line; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Dermatan Sulfate; Disaccharides; DNA, Neoplasm; Glycosaminoglycans; Heparin; Heparitin Sulfate; Humans; Intestinal Mucosa; Leukemia; Lymphoma, Large B-Cell, Diffuse; Molecular Sequence Data; Molecular Structure; Structure-Activity Relationship; Thymidine; Tumor Cells, Cultured

The effect of various glycosaminoglycans on the growth of cultured Tawa sarcoma cells (CTS cells) were determined under both fast and slow growth conditions. Hyaluronic acid, chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate (all of which have only one type of uronic acid, glucuronic acid) inhibited the growth of CTS cells during fast growth and accelerated it during slow growth. Both keratan sulfate and keratan polysulfate (containing galactose) inhibited the growth of CTS cells during both growth conditions. Only glycosaminoglycans containing iduronic acid (heparin, heparan sulfate, and dermatan sulfate) accelerated the growth of the cells during fast growth. However, heparin inhibited the growth during slow growth while heparan sulfate and dermatan sulfate accelerated it. Growth regulation seems to require complete structural integrity of the glycosaminoglycans. The component subunits alone lack such activity when not linked together.

Topics: Animals; Cell Division; Chondroitin Sulfates; Culture Media, Conditioned; Dermatan Sulfate; Extracellular Matrix; Glycosaminoglycans; Growth Substances; Heparin; Heparitin Sulfate; Hyaluronic Acid; Keratan Sulfate; Lymphoma, Large B-Cell, Diffuse; Rats; Sarcoma, Experimental; Tumor Cells, Cultured