In situ localization of the human multidrug-resistance gene mRNA using thymine-thymine dimerized single-stranded cDNA.
In order to detect the mRNA transcribed from the multidrug-resistance gene (MDR1), thymine-thymine (T-T) dimerized single-stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T-T dimerized single-stranded DNA (300-400 bases) so that they could penetrate well into the cytoplasm. The hybridized T-T DNA was detected immunohistochemically using rabbit anti-T-T DNA antibody (Ab) and peroxidase-labeled goat anti-rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug-resistant cell lines K562/ADM, CEM/VLB, 2780AD, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene.
Sugawara
I
I
Department of Pathology, University of Tokyo, Japan.
Koji
T
T
Ueda
K
K
Pastan
I
I
Gottesman
M M
MM
Nakane
P K
PK
Mori
S
S
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D004247:DNA / Q000235:genetics; D015342:DNA Probes / Q000276:immunology; D004277:DNA, Single-Stranded; D004351:Drug Resistance; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D011740:Pyrimidine Dimers / Q000276:immunology; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D015342:DNA Probes; D004277:DNA, Single-Stranded; D008562:Membrane Glycoproteins; D011740:Pyrimidine Dimers; D012333:RNA, Messenger; D004247:DNA