Reversal of different drug-resistant phenotypes by an autocatalytic multitarget multiribozyme directed against the transcripts of the ABC transporters MDR1/P-gp, MRP2, and BCRP.
A "multitarget multiribozyme" (MTMR) was constructed. It consists of three trans-acting hammerhead ribozymes directed against the transcripts of the ABC transporters MDR1/P-gp, BCRP, and MRP2; three cis-acting MDR1/P-gp-specific ribozymes; and three MDR1/P-gp-homologous spacer sequences. The trans-acting hammerhead ribozymes are liberated from the MTMR through autocatalytic self-cleavage by the cis-acting ribozymes. The MTMR was characterized with regard to its kinetic parameters. Comparison of the MTMR-specific kinetic values with those of the corresponding monoribozymes demonstrated that MTMR fragments could cleave their specific substrates without loss of efficiency. The MTMR was applied to three cell models, each overexpressing another ABC transporter, i.e., the gastric carcinoma cell line EPG85-257RDB expresses MDR1/P-gp, the cell variant EPG85-257RNOV synthesizes BCRP, and the ovarian carcinoma line A2780RCIS produces MRP2. In all cellular systems, the MTMR could specifically decrease the expression of the respective ABC transporter at the mRNA level (97% decrease in the MDR1/P-gp mRNA, 80% decrease in the BCRP mRNA, 96% decrease in the MRP2 mRNA) and the protein level. Resistance against the selection drug was reversed completely (100% in EPG85-257RDB) or by 94 (EPG85-257RNOV) or 63% (A2780RCIS). Thus, the MTMR technology provides a novel tool for gene therapeutic applications to reverse different ABC-transporter-dependent drug-resistant phenotypes.
Kowalski
Petra
P
Institute of Pathology, Humboldt University Berlin, Charité Campus Mitte, Schumannstrasse 20/21, D-10117 Berlin, Germany.
Surowiak
Pawel
P
Lage
Hermann
H
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003131:Combined Modality Therapy; D019008:Drug Resistance, Neoplasm; D005260:Female; D020868:Gene Silencing; D006801:Humans; D020128:Inhibitory Concentration 50; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000628:therapy*; D010641:Phenotype; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000970:Antineoplastic Agents; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C000715087:ABCC2 protein, human; C116614:ABCG2 protein, human; C427204:hammerhead ribozyme