Flow cytometric assessment of the cellular pharmacokinetics of fluorescent drugs.
Development of multidrug resistance (MDR) in cancer cells decrease net doxorubicin uptake as a result of either increased efflux, or decreased intracellular sequestration, or decreased membrane permeability. Kinetic parameters of drug uptake can distinguish among these forms of altered transport. Cellular uptake of fluorescent drugs was monitored by a flow cytometric assay using a rapid-injection system and analyzed with a three-compartment model in which rapid diffusion from extracellular fluid into the cell was followed by uptake into a nonexchangeable pool. In agreement with our recent studies of 14C-doxorubicin distribution (Dordal et al.: J Pharmacol Exp Ther 271:1286-1290, 1994), sequestration of doxorubicin was decreased 2.7-fold in P-glycoprotein-expressing SU-4R lymphoma cells compared to drug-sensitive SU-4 cells (14.0 +/- 4.8 vs. 5.0 +/- 0.9 nl s-1) without a change in membrane permeability or evidence of active efflux. In contrast, sequestration of the highly fluorescent dye rhodamine 123 was decreased 20-fold (17.1 +/- 8.3 vs. 0.9 +/- 0.8 nl s-1). Resistant cells were significantly less permeable to rhodamine than sensitive cells (3.8 +/- 1.2 vs. 10.2 +/- 2.6 x 10(5) cm2 s-1), and rhodamine efflux was increased by 24%. Thus, SU-4R cells exhibit multiple alterations that cause decreased intracellular drug concentrations, of which decreased sequestration is quantitatively the most significant.
Dordal
M S
MS
Clinical Pharmacology Center, Robert H. Lurie Cancer Center, Northwestern Memorial Hospital, Chicago, Illinois, USA.
Ho
A C
AC
Jackson-Stone
M
M
Fu
Y F
YF
Goolsby
C L
CL
Winter
J N
JN
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D001692:Biological Transport; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability; D003198:Computer Simulation; D004058:Diffusion; D004317:Doxorubicin / Q000378:metabolism*; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008223:Lymphoma / Q000473:pathology; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000378:metabolism; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D012235:Rhodamines; D020112:Rhodamine 123; D004317:Doxorubicin