[Evaluation of multidrug resistance phenotype on medullary specimens from patients with acute leukemia by determination of nuclear efflux of tetrahydropyranyl-doxorubicin. Approach by confocal laser microspectrofluorometry].
Confocal microspectrofluorometry allows the analysis of fluorescent molecules such as anthracylines in isolated living cells. An optical microscope fitted with a phase-contrast 100 X water-immersion objective enables simultaneous observation of the sample, focusing of the laser beam on the selected cell fraction (nucleus) and collection of the fluorescence emitted from the sample. The resulting intranuclear spectra are interpreted according to a quantitative model of the fluorescence spectra of both free and DNA-bound anthracycline. The intranuclear drug concentration can thus be determined. This technique has been applied to blast cells collected in patients with acute leukemia. Leukemic cells are aspirated from bone marrow, separated by Ficoll sedimentation and resuspended in RPMI-1640 containing 10% fetal calf serum and 200 nM tetrahydropyranyl-doxorubicin (THP-DOX). After one hour, 20 cells are analyzed and the mean nuclear drug content is determined (C1). Cells are then resuspended in the same medium but without anthracycline for 3 hours and the mean intranuclear drug concentration is then also determined (C3). From C1 and C3 the retention rate (RR) is calculated. Firstly, the accuracy of the method was checked. In 4 AML patients, two different samples aspirated on the same day were divided into two portions. Thus, two measurements were made on each one (4 values per patient). Coefficients of variation were satisfactory (4, 6, 12, and 12%). Secondly, blast cells collected in patients with AML and ALL at diagnosis or in relapse were studied. P-glycoprotein (P-gp) and CD34 expression was also studied using respectively immunohistochemistry land flow cytometry. Results obtained from the first 21 patients showed that there was no correlation between RR and either P-gp or CD34 expression. This could result from the efflux of THP-DOX by other mechanisms and/or low sensitivity of the staining technique.
Morjani
H
H
Laboratoire de spectroscopie biomoléculaire, UFR de pharmacie, Reims, France.
Pignon
B
B
Vilque
J P
JP
Millot
J M
JM
Lartigue
B
B
Simon
G
G
Etienne
J C
JC
Potron
G
G
Manfait
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D000208:Acute Disease; D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics; D018952:Antigens, CD34 / Q000235:genetics; D001692:Biological Transport; D001853:Bone Marrow / Q000378:metabolism* / Q000473:pathology; D002467:Cell Nucleus / Q000378:metabolism; D002648:Child; D004317:Doxorubicin / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic; D006801:Humans; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000473:pathology; D018613:Microscopy, Confocal / Q000379:methods; D015591:Microspectrophotometry / Q000379:methods; D008875:Middle Aged; D010641:Phenotype; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000473:pathology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D018952:Antigens, CD34; D004317:Doxorubicin; C027260:pirarubicin
Evaluation du phénotype de la résistance pléiotropique sur des prélèvements médullaires de patients présentant une leucémie aiguë par mesure de l'efflux nucléaire de la tétrahydropyranyl-doxorubicine. Approche par microspectrofluorimétrie confocale laser.