Increased efflux of vincristine, but not of daunorubicin, associated with the murine multidrug resistance protein (MRP)

Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3

“…These findings were similar to the previous studies in the presence of glucose (Slapak et al, 1996). Pretreatment and incubation of the cell lines in 6 gM cytochalasin B (CB), a competitive glucose transport inhibitor (Deves and Krupka, 1978;Wheeler and Hinkle, 1985;Holman and Rees, 1987;Garcia et al, 1992), increased the steady-state VCR accumulation in the PC-V40 cell line to the level of PC4-WT cells (P<0.05) (Figure 4).…”

“…At time zero, phloretin was added to a final concentration of 50 gM. Cells were incubated for 5 min at 37°C; VCR efflux has been demonstrated to be linear for at least 5 min (Slapak et al, 1996). Residual cell-associated radioactivity was determined after centrifugation through silicone oil as described above.…”

“…were washed out of drug and resuspended in PBS with 50 gM phloretin. After 5 min of incubation at 370C, the reaction was terminated by centrifugation through silicone oil and cell-associated radioactivity was determined as described in Figure 1 Increased vincristine efflux has been demonstrated in PC-V40 and PC-V160 cells; efflux was linear for the first 5 min (Slapak et al, 1996). VCR efflux was evaluated in the presence of phloretin (PHL).…”

“…The PC-V40 subline was 42-fold more resistant and PC-V160 was 215-fold more resistant to VCR than the parental cell line. Both sublines demonstrated energy-dependent VCR efflux and overexpressed the multidrug resistance-associated protein (MRP) (Slapak et al, 1996); only PC-V160 cells overexpressed P-glycoprotein (Slapak et al, 1994). Another MEL cell line, C7D (Slapak et al, 1990), and AUXB 1 cells (Maher et al, 1992), an auxotrophic CHO cell line (provided by V Ling, Ontario Cancer Institute, Toronto, Canada), were used as controls for GLUT I and GLUT3 identification in membrane preparations.…”

“…The reactions were terminated by centrifugation (3 min at 10 000 x g) of 200-,ul samples (2 x I05 cells) through silicone oil in microfuge tubes previously prepared with 20 gl of formic acid overlaid with 200 ,ul of silicone oil (D = 1.035-1.045; Nye Lubricants, New Bedford, MA, USA). After freezing the tubes, the tips were severed (containing the frozen formic acid/cell pellet) and the cell-associated radioactivity was determined by scintillation counting (Slapak et al, 1996). The data are expressed as a percentage of cell-associated [3H]dGlc in parental cells after 60 min of incubation from six experiments each performed in duplicate.…”