A novel bioassay for P-glycoprotein functionality using cytochalasin D.
The functional contribution of both P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) to multidrug resistance (MDR) in tumor cells is commonly determined by drug cytotoxicity and/or accumulation/efflux tests. We report on a bioassay developed for the specific detection of functional P-gp levels and the efficacy of related chemosensitizers (CD-P-gp-assay). The assay is based on the flow cytometric measurement of changes in the > or = G2M cell cycle compartment which are due to the induction of polykaryons after exposure of proliferating cells to three defined cytochalasin D (CD) concentrations with and without verapamil. As demonstrated in 13 well-characterized MDR cell models (20 resistant sublines), there is a significant correlation between cytokinesis-blocking CD doses, as well as responsiveness to chemosensitizers and MDR1 gene expression (mRNA and P-gp) allowing discrimination between different levels of P-gp-MDR. CD-P-gp-assay specificity was assessed by testing 23 compounds: 19 known as potent inhibitors of P-gp-MDR, some of them, though to a lesser extent, also of MRP-MDR; 1 inhibiting MRP-but not P-gp-MDR; 3 inactive in both types of MDR. A modulation of CD activity was confined exclusively to both P-gp-expressing cell lines and P-gp chemosensitizers. CD cytoskeletal activity measured by FACS is a specific and sensitive tool with which to detect functional P-gp and related chemosensitizers.
Elbling
L
L
Institute of Tumor Biology-Cancer Research, Department of Applied and Experimental Oncology, Vienna University, Austria. leonilla.elbling@univie.ac.at
Berger
W
W
Weiss
R M
RM
Printz
D
D
Fritsch
G
G
Micksche
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D016466:CHO Cells; D002467:Cell Nucleus; D006224:Cricetinae; D015638:Cytochalasin D / Q000633:toxicity; D004273:DNA, Neoplasm / Q000032:analysis; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005434:Flow Cytometry / Q000379:methods*; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D008938:Mitosis; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D015638:Cytochalasin D; D004273:DNA, Neoplasm; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger; D014700:Verapamil