Detection of multidrug resistance (MDR1) gene RNA expression in human tumors by a sensitive ribonuclease protection assay.
The human MDR1 gene encoding P-glycoprotein, an energy-dependent drug-efflux pump, was initially isolated from a multidrug-resistant KB carcinoma cell. When a 3 kb genomic sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR1 gene was compared to the equivalent fragment from KB cells, the MDR1 gene from KB carcinoma cells was found to have a point mutation in the first exon. Although this mutation does not affect the downstream promoter sequence or the coding sequence of the MDR1 gene, it creates a single base mismatch between the 5' KB genomic fragment previously used for RNase protection analysis of MDR1 RNA expression in normal tissues and thereby reduces the sensitivity of this assay. Using the DNA fragment from normal tissues rather than KB cells, we have reanalyzed MDR1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR1 RNA levels are as high in these tumors as in the multidrug-resistant cell line, KB-8-5. The ribonuclease protection assay indicated that the major downstream promoter was mainly used in these clinical samples including two samples of RNA from metastatic renal cancer. This assay appears to be a very sensitive and specific assay for detecting MDR1 mRNA levels and mRNA initiation sites in clinical samples.
Ueda
K
K
Department of Agricultural Chemistry, Kyoto University.
Yamano
Y
Y
Kioka
N
N
Kakehi
Y
Y
Yoshida
O
O
Gottesman
M M
MM
Pastan
I
I
Komano
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004351:Drug Resistance / Q000235:genetics*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000032:analysis*; D012260:Ribonucleases; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D012313:RNA; D004247:DNA; D012260:Ribonucleases