Probing daunorubicin accumulation defects in non-P-glycoprotein expressing multidrug-resistant cell lines using digitonin.
Multidrug resistance (MDR) in tumor cells is frequently associated with reduced cellular cytostatic drug accumulation, caused by the drug efflux protein, P-glycoprotein (Pgp). The action of Pgp in tumor cells can be detected by measuring the increase of daunorubicin accumulation upon blocking Pgp with drugs such as verapamil. A number of MDR cell lines have been described, characterized by decreased drug accumulation without Pgp being present. For such non-Pgp MDR cells no gene probes or functional assays are available to study this phenotype in clinical tumor specimens. We have worked out a method which enables the detection of drug-transport-related decreases in cellular daunorubicin accumulations without the need for the use of specific Pgp blockers. The cells used were SW-1573-, GLC4- and HT1080-sensitive cell lines, which accumulated (corrected for DNA content) 272%, 1,288% and 203% more daunorubicin than the non-Pgp MDR sublines SW-1573/2R120, GLC4/ADR and HT1080/DR4. When the plasma membranes of these MDR lines were permeabilized with 20 microM digitonin an increase to 282%, 1,260% and 239% of 14C-daunorubicin control accumulation was measured (at pH = 7.35). The intracellular pH measured with BCECF was the same in parent and corresponding MDR cells, excluding the role of pH differences in the measured effects. This method provides a tool allowing the detection of cellular mechanisms (including Pgp) which are related to active outward transport of daunorubicin.
Free University Hospital, Department of Medical Oncology, Amsterdam, The Netherlands.
C H
CH
Versantvoort
H J
HJ
Broxterman
N
N
Feller
H
H
Dekker
C M
CM
Kuiper
J
J
Lankelma
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001692:Biological Transport; D002460:Cell Line; D002463:Cell Membrane Permeability; D004273:DNA, Neoplasm / Q000032:analysis*; D003630:Daunorubicin / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology*; D004351:Drug Resistance / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004273:DNA, Neoplasm; D008562:Membrane Glycoproteins; D007770:L-Lactate Dehydrogenase; D004072:Digitonin; D003630:Daunorubicin