Figure 4.

pBR expression is not sufficient to produce efflux inhibition by PK11195. (A) Jurkat lymphocytic leukemia cells were confirmed as pBR RNA-deficient in pBR-specific RT-PCR assays in which AML and MM cell lines served as positive controls (inset) and in ligand-binding assays in which DOX40 MM cells served as positive controls. Specific ³H-PK11195 binding was calculated by subtracting ³H-PK11195 counts per minute in the presence of 1000-fold excess unlabeled PK11195 from counts per minute in the absence of competitor. Lentiviral pBR transduction and flow sorting for GFP expression were used to produce J-pBR cells that stably expressed high levels of pBR as measured in ³H-PK11195 binding assays of whole-cell and mitochondrial lysates and as compared with positive control cells (DOX40) and negative controls (Jurkat, J-GFP, J-Neo). (B) Like parental Jurkat cells (white bars) and control J-GFP cells (gray bars), J-pBR cells (black bars) showed no MIT efflux that could be blocked by PK11195 (PK) or by known efflux inhibitors CSA, MK-571 (MK), or Ko143 (KO). Seventy-five micromolar PK11195 sensitized J-pBR cells to MIT significantly more than control J-GFP cells were affected, demonstrating functional pBR expression in J-pBR cells. (C) Lentiviral transduction was used to create K-pBR cells from Pgp-expressing, efflux-competent KG1a parental cells, as demonstrated in whole-cell PK11195 binding assays. K-pBR cells showed no more MIT efflux than control K-GFP cells when PK and CSA were used as efflux inhibitors. Cytotoxicity and efflux data and ³H-PK11195 binding data are presented as described for Figures 1 and 2, with summary data from 3 to 5 assays shown as means plus or minus SEM.