Effects of antisense PONs on P-gp activity.

(A) The percentage of dead CEM-VBL100 cells untreated (■) or pretreated with sense (▨) or antisense (▪) PONs, or parental CCRF-CEM cells (░), and then treated with vinblastine (vbl), verapamil plus vinblastine (ver/vbl), or nothing (ctr), using the live/dead viability/cytotoxicity test (Molecular Probes). Histograms represent mean ± SD of 5 different experiments. In antisense-treated cells only, VBL susceptibility was significantly restored, at levels comparable to those in the parental CCRF-CEM line. (B) Flow cytometric analysis of percentage of drug retention in untreated (■) or antisense PON–treated (▨) CEM-VBL100 cells. Cells were incubated with VBL-bodipy (see “Materials and methods”), washed, and allowed to recover in fresh medium for the indicated times. As a positive control for P-gp activity inhibition, untreated CEM-VBL100 cells were incubated with VPL during the efflux time (▪). The percentage of drug retention was calculated as the ratio between the MFC of each sample at the end of the efflux time and the MFC detected at the end of drug treatment, times 100. (C) Confocal microscopy analysis of VBL-bodipy retention in ERM sense (left panel) and antisense (right panel) PON-treated CEM-VBL100 cells in 1 of 5 representative experiments. The insets show magnifications of the cells pointed by the arrowheads, further illustrating the differences in the drug distribution between ERM sense and antisense treatments. Bar = 10 μm.