Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity.
We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H-vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.
Broxterman
H J
HJ
Department of Medical Oncology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands.
Sonneveld
P
P
Feller
N
N
Ossenkoppele
G J
GJ
Währer
D C
DC
Eekman
C A
CA
Schoester
M
M
Lankelma
J
J
Pinedo
H M
HM
Löwenberg
B
B
Schuurhuis
G J
GJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000208:Acute Disease; D000328:Adult; D001692:Biological Transport / Q000187:drug effects; D015925:Cryopreservation; D003630:Daunorubicin / Q000378:metabolism / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D006801:Humans; D007938:Leukemia / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D009363:Neoplasm Proteins / Q000378:metabolism*; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D015203:Reproducibility of Results; D020112:Rhodamine 123; D012235:Rhodamines; D012680:Sensitivity and Specificity; D014700:Verapamil / Q000494:pharmacology; D014750:Vincristine / Q000378:metabolism / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; D012235:Rhodamines; D020112:Rhodamine 123; D014750:Vincristine; D014700:Verapamil; D003630:Daunorubicin