title: Reduced UDP-glucose Levels Are Associated with P-glycoprotein Over-expression in L1210 Cells and Limit Glucosylceramide Synthase Activity.
abstract: OBJECTIVE
  P-glycoprotein (Pgp) expression in neoplastic cells is known to reduce cell sensitivity to several cytotoxic Pgp substrates. A member of the ABC transporter family, Pgp, represents the most frequently described membrane efflux pump and its expression in neoplastic cells is responsible for multi-drug resistance. Several lines of evidence indicate that the expression and increased function of both Pgp and glucosylceramide synthase (GCS, an enzyme responsible for ceramide pathway de-activation in the regulation of apoptosis progression) enhance the resistance of Pgp-positive cells. Previously, we described a reduction in the uridine diphosphate (UDP)-glucose contents of mouse leukemia cells (R) expressing Pgp due to vincristine selection compared to parental L1210 cells (S). The reduced availability of UDP-glucose as a glucose donor in R cell glycosylation reactions could limit GCS-catalyzed ceramide glycosylation. Consequently, the over-expression of Pgp in Pgp-positive L1210 cells may be associated with reduced ceramide glycosylation.
  
  
  METHODS
  To test this idea, we measured the expression and activities of Pgp and GCS, UDP-glucose levels, cellular uptake of C12-NBD-ceramide (a fluorescent analogue of ceramide) and ceramide-induced cell death in S and R cells. T-cells, another Pgp-positive variant of L1210 cells that express Pgp due to their transfection with a gene encoding human Pgp were also used in this study.
  
  
  RESULTS
  We detected significantly reduced levels of C12-NBD-ceramide glycosylation and reduced UDP-glucose contents in Pgp-positive R and T-cells compared to S cells. C12-NBD-ceramide uptake assays revealed nearly identical dynamics of uptake time-dependency curves. The Pgp-positive L1210 variants (R and T) are more sensitive than Pgp-negative S cells to ceramide-induced cell damage, as measured by an fluorescein isothiocyanate-labeled annexin V and propidium iodide apoptosis necrosis kit. Short chain C2-ceramide was more effective at inducing cell damage than ceramide analogues with longer chains.
  
  
  CONCLUSIONS
  These evidence indicates that the down-regulation of UDP-glucose contents in Pgp-positive L1210 cells is responsible for their collateral sensitivity to ceramide-induced apoptosis.
authors.affiliation: Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovak Republic.
authors.forename: Katarina
authors.initials: K
authors.lastname: Turáková
authors.affiliation: Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic.
authors.forename: Lucia
authors.initials: L
authors.lastname: Pavlíková
authors.affiliation: Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovak Republic Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic.
authors.forename: Lucia
authors.initials: L
authors.lastname: Messingerová
authors.affiliation: Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovak Republic.
authors.forename: Boris
authors.initials: B
authors.lastname: Lakatoš
authors.affiliation: Institute of Biochemistry, Nutrition and Health Protection, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Slovak Republic Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic albert.breier@stuba.sk zdena.sulova@savba.sk.
authors.forename: Albert
authors.initials: A
authors.lastname: Breier
authors.affiliation: Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic albert.breier@stuba.sk zdena.sulova@savba.sk.
authors.forename: Zdena
authors.initials: Z
authors.lastname: Sulová
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000008:administration & dosage; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002518:Ceramides / Q000008:administration & dosage; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005964:Glucosyltransferases / Q000096:biosynthesis*; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D014532:Uridine Diphosphate Glucose / Q000096:biosynthesis
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C120266:C12-NBD-ceramide; D002518:Ceramides; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D009327:4-Chloro-7-nitrobenzofurazan; D014532:Uridine Diphosphate Glucose
keywords: L1210 cells; P-glycoprotein; UDP-glucose; ceramide induced cell death; glucosylceramide synthase