Abstract
A major problem in cytostatic treatment of many tumours is the development of multidrug resistance (MDR4). This is most often accompanied by the overexpression of a membrane transport protein, P-glycoprotein, and its encoding mRNA. In order to reverse the resistant phenotype in cell cultures, we constructed a specific hammerhead ribozyme possessing catalytic activity that cleaves the 3'-end of the GUC sequence in codon 880 of the mdr1 mRNA. We demonstrated that the constructed ribozyme is able to cleave a reduced substrate mdr1 mRNA at the GUC position under physiological conditions in a cell-free system. A DNA sequence encoding the ribozyme gene was then incorporated into a mammalian expression vector (pH beta APr-1 neo) and transfected into the human pancreatic carcinoma cell line EPP85-181RDB, which is resistant to daunorubicin and expresses the MDR phenotype. The expressed ribozyme decreased the level of mdr1 mRNA expression, inhibited the formation of P-glycoprotein and reduced the cell's resistance to daunorubicin dramatically; this means that the resistant cells were 1,600-fold more resistant than the parental cell line (EPP85-181P), whereas those cell clones that showed ribozyme expression were only 5.3-fold more resistant than the parental cell line.
Highlights
A GUC site in codon 880 of exon 21 of the mdrl mRNA was selected as the cleavage site of the ribozyme
Synthesis of the ribozyme A 60 bp DNA strand was synthesised on a DNA synthesiser (Applied Biosystem) that contained a T7 RNA polymerase promoter sequence and the DNA sequence of the ribozyme gene
The MDR phenotype was mediated by overexpression-of the mdrl gene, which was demonstrated by reverse transcription (RT)-polymerase chain reaction (PCR), by Northern blot analysis and by immunocytochemistry against the P-glycoprotein using the monoclonal antibody C-219) (Centocor (Dietel, 1991)
Summary
A GUC site in codon 880 of exon 21 of the mdrl mRNA was selected as the cleavage site of the ribozyme. For the in vitro analysis, it consisted of a 43-base-long RNA molecule with the two flanking sequences providing specific binding to the mdrl mRNA and to the central core responsible for the cleavage reaction at the GUC site. A second 19-mer complementary to the 3'-sequence of the 60 bp fragment was synthesised (ribo, AAAATGTTG mTTCGTCCTC) Using these two primers, the 60 bp double-strand DNA molecule was amplified by polymerase chain reaction (PCR). Target RNA and ribozyme molecules were separated by electrophoresis on a 8% polyacrylamide-7 M urea gel. The corresponding bands that contained the ribozyme and the target RNA were eluted from the polyacrylamide gel. The plasmid containing the ribozyme sequence pHPAPr-I neo/mdr-Rb was constructed according to the protocol described by Kashani-Sabet et al (1992)
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