title: A plasma membrane 'vacuum cleaner' for daunorubicin in non-P-glycoprotein multidrug-resistant SW-1573 human non-small cell lung carcinoma cells. A study using fluorescence resonance energy transfer.
abstract: A multidrug resistant (MDR) human non-small cell lung carcinoma cell line, SW-1573/2R120 (2R120), not containing the drug-efflux pump P-glycoprotein (PgP), has been studied for the transport of daunorubicin (DN) across the cellular plasma membrane. Earlier, reduced initial DN-uptake rates and lower cellular DN steady-state concentrations were found for this cell line, when it was compared to the SW-1573 wild-type cell line. This finding was an indication for the presence of another cellular drug-efflux pump. However, we found similar DN-efflux rates in drug-free medium for the two cell lines, while for Pgp-containing MDR SW-1573/2R160 (2R160) cells the efflux rate was increased compared to wild-type cells. In order to elucidate differences in DN transport across the cellular plasma membrane, the association of DN with plasma membranes of intact cells was investigated, using fluorescence-resonance-energy transfer. For this purpose, the plasma-membrane probe 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was chosen since, because of the overlap between the emission spectrum of TMA-DPH and the excitation spectrum of DN, transfer of energy can be achieved from TMA-DPH to DN. Cells were loaded with TMA-DPH and, after addition of 10 microM DN, the TMA-DPH fluorescence was quenched. Rapid initial quenching proved to be similar in the MDR 2R160 (Pgp-containing) cells and in the SW-1573 wild-type cells (21 +/- 1% and 20 +/- 2%, respectively), but was less in the MDR 2R120 cells not containing Pgp (14 +/- 1%). This finding correlated with a lowered amount of DN dissolved in the plasma membrane of 2R120 cells. We interpret these data to be the result of a 'vacuum-cleaner' pumping system other than Pgp which removes DN from a plasma membrane compartment and equilibrates relatively slowly with the interior of the cell.
authors.affiliation: Department of Oncology, Free University Hospital, Amsterdam, The Netherlands.
authors.forename: H S
authors.initials: HS
authors.lastname: Mülder
authors.forename: R
authors.initials: R
authors.lastname: van Grondelle
authors.forename: H V
authors.initials: HV
authors.lastname: Westerhoff
authors.forename: J
authors.initials: J
authors.lastname: Lankelma
mesh_terms: D001692:Biological Transport; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism* / Q000473:pathology; D002462:Cell Membrane / Q000378:metabolism*; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism; D003630:Daunorubicin / Q000378:metabolism*; D004161:Diphenylhexatriene / Q000031:analogs & derivatives / Q000737:chemistry; D004351:Drug Resistance; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D008954:Models, Biological; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology; D014750:Vincristine / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D004161:Diphenylhexatriene; D014750:Vincristine; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D004247:DNA; D014700:Verapamil; D003630:Daunorubicin