Reversion of multidrug resistance in the P-glycoprotein-positive human pancreatic cell line (EPP85-181RDB) by introduction of a hammerhead ribozyme.
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.
Holm
P S
PS
Institute of Pathology, Kiel, Germany.
Scanlon
K J
KJ
Dietel
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002461:Cell Line, Transformed; D002999:Clone Cells; D003062:Codon; D003630:Daunorubicin / Q000494:pharmacology; D004351:Drug Resistance; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002352:Carrier Proteins; D003062:Codon; D008562:Membrane Glycoproteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D003630:Daunorubicin