FIGURE 7.

Stability of APP-IP or APP-IP-TIMP-2 in the culture of HT1080 cells. A, HT1080 cells were incubated with (●) or without (○) 5 μm APP-IP in serum-free DME/F-12 medium for indicated lengths of time. After incubation, 10 μl of samples taken from culture medium were mixed with 180 μl of MMP-2 solution consisting of 0.38 nm MMP-2, TBS-Ca-Brij, and 0.01% bovine serum albumin, and each mixture was incubated at 37 °C for 15 min. B, cells were incubated with (●) or without (○) 50 nm APP-IP-TIMP-2 for indicated lengths of time. After incubation, samples taken from culture medium were first diluted by 10-fold with TBS-Ca-Brij containing 0.01% bovine serum albumin, and 10 μl of the diluted samples were then mixed with 180 μl of the MMP-2 solution and incubated at 37 °C for 15 min. Then, 10 μl of 1.0 mm 3163v was added to the mixture, and the incubation was further continued for 30 min. The amount of 3163v hydrolyzed by MMP-2 in the absence of the cell culture-derived samples was taken as 100%. The enzyme activity is shown as the relative amount of 3163v hydrolyzed by the enzyme on the ordinate. The concentrations of APP-IP (C) or APP-IP-TIMP-2 (D) remaining in the culture media were estimated using the standard curve of the MMP-2 inhibitory activity of APP-IP or APP-IP-TIMP-2 plotted against known concentrations of each inhibitor. The concentration of each inhibitor before incubation was taken as 100%. The concentration of inhibitor in the cell culture is shown as the relative concentration on the ordinate.