Cathepsin G Induces Cell Aggregation of Human Breast Cancer MCF-7 Cells via a 2-Step Mechanism: Catalytic Site-Independent Binding to the Cell Surface and Enzymatic Activity-Dependent Induction of the Cell Aggregation
Figure 6
Characterization of the binding of 125I-labeled CG to the MCF-7 cell surface. (a) Dose dependency of 125I-CG binding to MCF-7 cells. The cells were incubated with 125I-labeled CG in RPMI 1640 medium containing 1% BSA for 60 min on ice. After washing, the cells were disrupted by the addition of 0.1 M NaOH, and the radioactivity of the lysate was determined using a -counter. In the cold inhibition experiment, a 20-fold excess of unlabeled CG was simultaneously added to the medium for competitive binding. Specific binding was determined by subtracting the value obtained for nonspecific binding (cold inhibition) from the total binding. The data are expressed as single-point values. (b) Hill plot analysis of 125I-CG binding to MCF-7 cells. The slope of the Hill plot is the Hill coefficient (), which indicates cooperativity. (c) Time course of 125I-CG binding to MCF-7 cells. 125I-CG was added at a final concentration of 834 nM. (d) Binding activities of 125I-trypsin and 125I-chymotrypsin. (e) Suc-Val-Pro-PheP-(OPh)2 and chymostatin have no effect on CG binding to MCF-7 cells. MCF-7 cells were incubated with 125I-CG (83.4 nM) that was pretreated with serine protease inhibitors (Suc-Val-Pro-PheP-(OPh)2, 100 M; chymostatin, 82.5 nM). The bound 125I-CG is expressed as relative binding comparing the radioactivity of bound intact 125I-CG with that of serine protease-treated 125I-CG. Unless otherwise indicated, similar results were obtained from 2 independent experiments, each with duplicates. The results are shown as mean SD. When the bars are not shown, they are smaller than the size of the symbols.
