Thrombospondin binds to the surface of human osteosarcoma cells and mediates platelet-osteosarcoma cell interaction.
We have previously shown that thrombospondin (TSP) is synthesized and secreted by human MG-63 osteosarcoma cells. In this study, the secretion and cell surface expression of TSP by two different human osteosarcoma cell lines (MG-63 and TE-85) as well as the involvement of TSP in the platelet-aggregating activity of these tumor cells were studied. Using a sandwich enzyme-linked immunosorbent assay, MG-63 cells secreted 3-fold as much TSP as TE-85 cells at 48 h (0.17 +/- 0.01 (SD) versus 0.06 +/- 0.006 micrograms/10(6) cells, P = 0.007). Binding of exogenous 125I-TSP to MG-63 and TE-85 cells in monolayer indicated that binding was time and concentration dependent, saturable, and inhibited by excess cold TSP. However, despite a similar affinity, MG-63 cells had 10-fold more TSP-binding sites than TE-85 cells (402,394 +/- 130,346 versus 36,748 +/- 7,708 TSP-binding sites/cell; P = 0.002). Similar binding differences of 125I-TSP were observed with both osteosarcoma cell lines in suspension. A fluorescence-activated cell-sorting analysis was used in conjunction with an anti-TSP polyclonal antibody, and binding of endogenous TSP to MG-63 and TE-85 cells in suspension was investigated. Addition of an anti-TSP antibody to MG-63 and TE-85 cells in suspension increased the mean fluorescence intensity 50-fold when compared to an irrelevant antibody. Moreover, the fluorescence intensity of MG-63 cells with an anti-TSP polyclonal antibody was increased by 40% when compared to TE-85 cells. Since TSP was expressed on the surface of osteosarcoma cells, the involvement of this glycoprotein in the platelet-aggregating activity of MG-63 and TE-85 cells was therefore investigated using an anti-TSP polyclonal antibody and two monoclonal antibodies (P10 and MA-II), the epitopes of which lie within the Mr 140,000 non-heparin-binding fragment and the Mr 25,000 heparin-binding fragment of TSP, respectively. Preincubation of MG-63 cells (1 x 10(6) cells/ml) with either an anti-TSP polyclonal antibody (100 micrograms/ml) or monoclonal antibody P10 (15 micrograms/ml) inhibited by 80% other platelet-aggregating activity of these tumor cells, while anti-TSP monoclonal antibody MA-II (15 micrograms/ml) had no effect. In sharp contrast, the anti-TSP polyclonal antibody (100 micrograms/ml) only exhibited a slight inhibitory effect on platelet aggregation induced by TE-85 cells when using a low concentration of tumor cells (0.6 x 10(6) cells/ml).(ABSTRACT TRUNCATED AT 400 WORDS)
Clezardin
P
P
INSERM U. 234, Hôpital Edouard Herriot, Lyon, France.
Serre
C M
CM
Trzeciak
M C
MC
Drouin
J
J
Delmas
P D
PD
D000906:Antibodies; D001792:Blood Platelets / Q000502:physiology* / Q000648:ultrastructure; D018955:CD36 Antigens; D002448:Cell Adhesion / Q000187:drug effects; D002450:Cell Communication / Q000187:drug effects; D002460:Cell Line; D004492:Edetic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D012516:Osteosarcoma / Q000503:physiopathology* / Q000648:ultrastructure; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D016024:Receptors, Cytoadhesin / Q000502:physiology*; D019699:Thrombospondins
D000906:Antibodies; D018955:CD36 Antigens; D010980:Platelet Membrane Glycoproteins; D016024:Receptors, Cytoadhesin; D019699:Thrombospondins; D004492:Edetic Acid