Methods for detection of MDR1 mRNA expression on acute myelogenous leukemia cells.
Overexpression of the human multidrug resistance gene (MDR1) on acute myelogenous leukemia (AML) correlates with poor prognosis. We evaluated several methods for mRNA estimation to standardize simple and reliable techniques for identifying MDR1 positive leukemia among untreated AMLs in large scale studies. Northern blot detection of MDR1 mRNA suffered from low signal-to-noise ratio under the conventional conditions, that was improved mainly by removing unincorporated radioactivity. The amount of MDR1 transcripts on positive cells was estimated less than 10% of that of constitutive mRNA species. A modified method seemed useful in estimating the total amount of the MDR1 mRNA in a whole leukemic cell population, and suitable to study stock samples or for large prospective clinical trials. RT-PCR was more sensitive in detecting MDR1 mRNA than Northern blot analysis, and the very feature made it virtually impossible to exclude contamination with normal hematopoietic cells. This procedure showed that FAB M3 leukemias were essentially MDR1 negative, and there existed frequently myelodysplastic syndrome subpopulation which had excessive MDR1 transcripts. In situ hybridization of the mRNA with a FITC-labeled phosphorothioate oligonucleotide probe was visualized using flowcytometry or con-focus lightmicroscopy, enabled us to recognize the difference between multidrug resistant K562/ADM and its wild type.
Sato
H
H
Fourth Department of Internal Medicine, Teikyo University School of Medicine, Kanagawa, Japan.
Oonishi
T
T
Wada
C
C
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D006801:Humans; D017403:In Situ Hybridization; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger