title: 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 μmol/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/l 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.
authors.affiliation: Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Vlárska 5, 833 34 Bratislava, Slovakia.
authors.forename: Mário
authors.initials: M
authors.lastname: Sereš
authors.forename: Peter
authors.initials: P
authors.lastname: Ditte
authors.forename: Albert
authors.initials: A
authors.lastname: Breier
authors.forename: Zdena
authors.initials: Z
authors.lastname: Sulová
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D037281:Calnexin / Q000235:genetics / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007939:Leukemia L1210 / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology*; D014162:Transfection; D014750:Vincristine / Q000494:pharmacology
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D012333:RNA, Messenger; D037281:Calnexin; D014750:Vincristine; D019284:Thapsigargin