Open AccessDetection of Multidrug Resistance ( MDR 1) Gene RNA Expression in Human Tumors by a Sensitive Ribonuclease Protection Assay
Author(s) -
Ueda Kazumitsu,
Yamano Yoshiaki,
Kioka Noriyuki,
Kakehi Yoshiyuki,
Yoshida Osamu,
Gottesman Michael M.,
Pastan Ira,
Komano Tohru
Publication year - 1989
Publication title -
japanese journal of cancer research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.035
H-Index - 141
eISSN - 1349-7006
pISSN - 0910-5050
DOI - 10.1111/j.1349-7006.1989.tb02269.x
Subject(s) - microbiology and biotechnology , biology , exon , gene , multiple drug resistance , rna , rnase p , messenger rna , gene expression , coding region , genomic dna , genetics , drug resistance
The human MDR 1 gene encoding P‐glycoprotein, an energy‐dependent drug‐efflux pump, was initially isolated from a multidrug‐resistant KB carcinoma cell. When a 3 kb genomlc sequence isolated from normal human tissue including the major downstream promoter and the first and second exons of the MDR 1 gene was compared to the equivalent fragment from KB cells, the MDR 1 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 MDR 1 gene, it creates a single base mismatch between the 5′ KB genomic fragment previously used for RNase protection analysis of MDR 1 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 MDR 1 mRNA levels in 12 renal carcinomas and 4 colon adenocarcinomas. By this RNase protection assay, MDR 1 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 MDR 1 mRNA levels and mRNA initiation sites in clinical samples.