Effect of thapsigargin on P-glycoprotein-negative and P-glycoprotein- positive L1210 mouse leukaemia cells

Abstract

Expression of drug-transporting P-glycoprotein (P-gp, an integral protein of the plasma membrane) in neoplastic cells confers multidrug resistance and also involves alteration of cell sensitivity to inhibitors of the sarco/endoplasmic reticulum calcium pump thapsigargin (Th). Mouse leukaemia L1210 cell sublines that overexpress P-gp due to selection with vincristine (R) or stable transfection with a gene encoding human P-gp (T) were less sensitive to Th than the parental cell line (S). Th at a concentration of 0.1 μmol/l did not induce alterations in the amount of P-gp mRNA in R or T cells (S cells did not contain any measurable amount of this transcript as assessed by RT-PCR) or in the amount of calnexin (CNX) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in all three cell sublines. However, when using a concentration of 10 μmol/l, Th decreases the amounts of CNX, GAPDH (in S, R and T cells) and P-gp (in R and T cells) mRNAs. In contrast to R and T cells (which contain abundant P-gp), S cells did not contain any P-gp detectable by the c219 antibody on a Western blot. Th at a concentration of 0.1 umol/l induced a reduction in the amount of P-gp present in R and T cells, particularly in isoforms with higher molecular weights (i.e., mature fully glycosylated isoforms). Similar results were observed when Th was used at a concentration of 10 μmol/l. R and T cells contained lower levels of CNX than S cells. While Th at a lower concentration did not alter the levels of CNX in S, R or T cells, a higher concentration of this substance induced a measurable decrease in the amount of CNX. S, R and T cells did not differ with respect to GAPDH content, but Th induced a reduction in the amount of this protein in all cell sublines. More pronounced results were observed when Th was applied at a concentration of 10 μmol/1 comparing with a concentration of 0.1 mmol/l. These changes may be involved together with the Th efflux activity of P-gp in Th-resistance associated with the P-gp-mediated multidrug resistance of R and T cells.