Fig. 7.

Comparison of effects of CM572 and siramesine upon acute or chronic exposure. The cytotoxic effects of irreversibly binding CM572 and reversibly binding siramesine were compared using acute and chronic treatment conditions. Treatment conditions were as described in Materials and Methods and in the Fig. 6 legend. Solid bars: Cells were exposed to the indicated concentration of sigma-2 ligand continuously for 24 hours. Hashed bars: Cells were exposed to the indicated sigma-2 ligand for 60 minutes, followed by extensive washing to remove unbound ligand, and then incubated for 24 hours. Cell viability was assessed by MTT assay at the end of the 24-hour period. As previously shown in Fig. 6, the cytotoxic effect of CM572 is more or less comparable when the 60-minute acute exposure is compared with the 24-hour continuous exposure (P = 0.06 and P = 0.3, t test for difference between acute and 24-hour treatment with 10 and 100 μM CM572, respectively). By contrast, at each dose of siramesine, only minimal cytotoxicity was observed after the 24-hour period when the 60-minute acute exposure was compared with the 24-hour continuous exposure (****P = 0.000003 and P = 0.000006, t test for difference between acute and 24-hour treatment with 15 and 20 μM siramesine, respectively). This finding indicates that the CM572-induced cytotoxicity after acute treatment is due to irreversible binding, not to irreversible commitment to apoptosis. Data are the averages of three experiments for CM572 and five experiments for siramesine, ± S.D. Each experiment was performed using five wells for each condition.