Kinetic characterization of ribozymes directed against the cisplatin resistance-associated ABC transporter cMOAT/MRP2/ABCC2.
The enhanced expression of the human ABC transporter, cMOAT (MRP2/ABCC2), is associated with resistance of tumor cells against platinum-containing compounds, such as cisplatin. Therefore, cMOAT represents an interesting candidate factor for modulation of antineoplastic drug resistance. Two different hammerhead ribozymes, which exhibit high catalytic cleavage activities towards specific RNA sequences encoding cMOAT, were designed. Cleavage sites of these ribozymes are the GUC sites in codons 704 and 708 of the open reading frame in the cMOAT-specific mRNA molecule. Hammerhead ribozymes were in vitro synthesized using bacteriophage T7 RNA polymerase and oligonucleotide primers whereby one primer contains a T7 RNA polymerase promoter sequence. cMOAT-encoding substrate RNA molecules were created by a reverse transcription polymerase chain reaction using RNA prepared from the cisplatin-resistant human ovarian carcinoma cell line A2780RCIS overexpressing the cMOAT-encoding transcript. In a cell-free system, both anti-cMOAT ribozymes cleaved their substrate in a highly efficient manner at a physiologic pH and temperature. The cleavage reaction was dependent on time and ribozyme:substrate ratio for determining specific kinetic parameters.
Materna
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Institute of Pathology, Humboldt University Berlin, Germany.
Holm
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PS
Dietel
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Lage
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D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D027321:Anion Transport Proteins; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017123:Bacteriophage T7 / Q000201:enzymology; D002277:Carcinoma / Q000235:genetics; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002945:Cisplatin / Q000627:therapeutic use*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics* / Q000502:physiology; D005260:Female; D006801:Humans; D007700:Kinetics; D000089762:Multidrug Resistance-Associated Protein 2; D010051:Ovarian Neoplasms / Q000235:genetics; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000138:chemical synthesis*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C000715087:ABCC2 protein, human; D018528:ATP-Binding Cassette Transporters; D027321:Anion Transport Proteins; D000970:Antineoplastic Agents; D002352:Carrier Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; D012321:DNA-Directed RNA Polymerases; D002945:Cisplatin