title: Saquinavir induces stable and functional expression of the multidrug transporter P-glycoprotein in human CD4 T-lymphoblastoid CEMrev cells.
abstract: BACKGROUND
  The multidrug transporter P-glycoprotein (P-gp) is expressed in HIV-1 target cells, in a range of pharmacological barriers and in AIDS-associated tumours. P-gp substrates include HIV-1 protease inhibitors (PIs) and anticancer drugs, which are efficiently effluxed from multidrug-resistant (MDR) cells.
  
  
  OBJECTIVE
  The aim of this study was to investigate the effect on human CD4 T-lymphoblastoid CEMrev cells of saquinavir and other PIs in terms of P-gp expression and to characterize the functional and biochemical patterns of PI-induced P-gp molecules.
  
  
  METHODS
  CEMrev cells no longer expressing detectable amounts of P-gp were cultured for a prolonged period in the presence of 10 microg/mL saquinavir (CEMsaq10) and tested for P-gp expression and function. Subsequently, CEMsaq10 cells were transferred into medium containing 15 microg/mL saquinavir (CEMsaq15) and cultured for several months. These cell lines were continuously monitored for P-gp expression, function and immunochemical patterns. A similar strategy was adopted to determine whether other PIs, such as ritonavir and indinavir, were able to induce P-gp expression in CEMrev cells.
  
  
  RESULTS
  Compared with the drug-diluent control, the exposure of CEMrev cells to 10 microg/mL saquinavir induced, in a consistent fraction of cells (45-50%), de novo expression of functioning P-gp molecules. The transfer of CEMsaq10 cells to 15 microg/mL saquinavir was associated with a dramatic increase in P-gp expression and function (85-90% of CEMsaq15 cells expressed P-gp and effluxed P-gp dye substrates). These saquinavir-induced P-gp molecules included 75-kDa molecules as well as the classical 170-kDa form of P-gp, suggesting induction of a particular isoform of P-gp termed mini-P-glycoprotein. Conversely, ritonavir and indinavir induced transient P-gp expression in a small percentage of the CEMrev cells.
  
  
  CONCLUSIONS
  Treatment of human CD4 T-lymphoblastoid CEMrev cells with saquinavir caused over-expression of functioning P-gp molecules. This de novo acquired MDR phenotype, which differed from that induced by other PIs, was stable, as expression and activity of P-gp were observed in CEMsaq10 and CEMsaq15 cells during prolonged in vitro culturing, even in drug-free conditions.
authors.affiliation: Laboratorio di Immunologia, Istituto Superiore di Sanità, Rome, Italy. mldupuis@iss.it
authors.forename: M L
authors.initials: ML
authors.lastname: Dupuis
authors.forename: M
authors.initials: M
authors.lastname: Flego
authors.forename: A
authors.initials: A
authors.lastname: Molinari
authors.forename: M
authors.initials: M
authors.lastname: Cianfriglia
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D019380:Anti-HIV Agents / Q000627:therapeutic use*; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D018432:Drug Resistance, Multiple / Q000187:drug effects; D024882:Drug Resistance, Viral / Q000187:drug effects; D015658:HIV Infections / Q000188:drug therapy; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D006801:Humans; D007150:Immunohistochemistry; D019469:Indinavir / Q000494:pharmacology; D010641:Phenotype; D019438:Ritonavir / Q000494:pharmacology; D019258:Saquinavir / Q000494:pharmacology*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D019380:Anti-HIV Agents; D017320:HIV Protease Inhibitors; D019469:Indinavir; D019258:Saquinavir; D019438:Ritonavir