Expression of multidrug resistance-associated protein (MRP) mRNA in blast cells from acute myeloid leukemia (AML) patients.
A specific and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay was developed for measuring the mRNA of the multidrug resistance-associated protein (MRP). A region corresponding to bp 3897-4471 of MRP cDNA is amplified, which encompasses approximately half of the second nucleotide-binding domain (NBD2). In two multidrug resistant (MDR) sublines of the HL-60 human acute myeloid leukemia (AML) cell line which overexpress MRP but not P-glycoprotein, the assay detects elevated levels of MRP mRNA (4- to 8-fold) relative to the drug-sensitive parental cells (designated HL-60/W). Blast cells from 24 patients with AML were also studied for MRP expression using this RT-PCR method. Expression of MRP was normalized for that of beta-actin in the blast cells, which was also determined by RT-PCR. All of these blast cell samples had MRP expression that was detectable after 35 PCR cycles. Eighteen of these patients samples had levels of expression of MRP mRNA equal to or less than that expressed by HL-60/W cells. In six patient blast cell specimens, the expression of MRP mRNA was up to 1.7-fold higher than that of HL-60/W cells. In 21 specimens, the steady-state intracellular accumulation of daunorubicin (1 microgram/ml, 3h) was also determined. The blast cells with MRP mRNA expression higher than HL-60/W had a lower median accumulation of daunorubicin compared to those whose MRP expression was less than HL-60/W, suggesting a functional defect in drug transport in the cells with higher MRP expression; a similar trend toward lower daunorubicin accumulation was also noted in the one-third of samples that displayed the highest expression of MDR1 mRNA (also determined by RT-PCR). These studies illustrate the range of expression of MRP in AML blast cell specimens. The identification of MRP overexpression in MDR AML cell lines and in some AML patient blast cells with low intracellular daunorubicin accumulation warrants further study of MRP as a component of clinical drug resistance in AML.
Ross
D D
DD
University of Maryland Cancer Center, Baltimore 21201, USA.
Doyle
L A
LA
Schiffer
C A
CA
Lee
E J
EJ
Grant
C E
CE
Cole
S P
SP
Deeley
R G
RG
Yang
W
W
Tong
Y
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000903:Antibiotics, Antineoplastic / Q000493:pharmacokinetics; D001483:Base Sequence; D001752:Blast Crisis / Q000378:metabolism* / Q000473:pathology; D003630:Daunorubicin / Q000493:pharmacokinetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005260:Female; D018922:HL-60 Cells / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000903:Antibiotics, Antineoplastic; D003630:Daunorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger