[Quantitative analysis of multidrug resistance phenotype in hematological malignancies].
Cells with multidrug resistance (MDR) phenotype express P-glycoprotein (P-gp) on the cell membrane, which functions as a drug-efflux pump. To quantify the expression of the gene encoding P-gp (multidrug resistance 1; MDR1) and assess P-gp function, we developed competitive reverse transcription-polymerase chain reaction (RT-PCR) assay using heterologous competitor RNA and flow cytometric analysis using rhodamine 123 (Rh123; an artificial substrate for P-gp), respectively. First, we adjusted the assays by analyzing leukemia sublines showing various levels of MDR1 expression. The MDR1 expression in leukemia sublines quantified by competitive RT-PCR assay showed linearity over a wide range, and the results were parallel with those of MDR1 expression measured by Northern blot analysis, the P-gp antigen expression measured by flow cytometric analysis using MRK16, P-gp function analysis by Rh123 dye-efflux assay, and MDR phenotype. Then, we applied these assays to leukemic cells from patients with hematological malignancies. All 69 samples from 64 patients were successfully assayed, and the range of MDR1 expression was from 1.6 to 100 amol/microgram RNA. Since subpopulations of normal lymphocytes show a low degree of P-gp function, strict gating of leukemia cells was mandatory for dye-efflux assay of clinical samples. MDR1 expression in normal lymphocytes was below 8 amol/microgram RNA. By comparison to MDR1 expression quantified by competitive RT-PCR assay with P-gp function assessed by Rh123 dye-efflux assay in gated leukemic cells, more than 8 amol/microgram RNA was regarded as positive MDR1 expression in the leukemic cells themselves. These data suggest that these assays are suitable for evaluating P-gp expression and function in clinical samples when the proper cut-off value is used and may provide insights into the contribution of P-gp to drug resistance in hematological malignancies.
Kobayashi
H
H
Department of Pharmacology, Tokai University School of Medicine, Isehara.
Takemura
Y
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000235:genetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005456:Fluorescent Dyes; D019337:Hematologic Neoplasms / Q000235:genetics / Q000503:physiopathology*; D006801:Humans; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D020112:Rhodamine 123; D012235:Rhodamines
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D012235:Rhodamines; D020112:Rhodamine 123