Retrovirus-mediated transfer of anti-MDR1 hammerhead ribozymes into multidrug-resistant human leukemia cells: screening for effective target sites.
One of the underlying mechanisms of multidrug resistance (MDR) is cellular over-production of P-glycoprotein (P-gp), which acts as a drug efflux pump. P-gp is encoded by a small group of related genes termed MDR; only MDR1 is known to confer drug resistance. To overcome P-gp-mediated drug resistance, we have developed two anti-MDR1 hammerhead ribozymes driven by the beta-actin promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showed different cleavage activities. In this study, to determine suitable target sites for the anti-MDR1 ribozyme, the exon 1b-intron 1 boundary, the translation-initiation site, the intron 1-exon 2 boundary and the exon 2-intron 2 boundary, codons 179 and 196 of the MDR1 gene were selected as candidates. To improve the ribozyme activity, a retroviral vector containing RNA polymerase III promoter was used. Stable retrovirus producer cells were generated by transfecting the retroviral vector plasmids carrying the ribozyme into the packaging cell line. Retroviral vector transduction of human leukemia cell lines expressing MDR1 was accomplished by co-culturing these with virus producer cells. Stably transduced cells were selected by G418 and pooled to determine the efficacy of each ribozyme. These ribozyme-transduced cells became vincristine-sensitive concomitant with the decreases in MDR1 expression, P-gp amount and drug efflux pump function. Among the ribozymes tested, the anti-MDR1 ribozyme against the translation-initiation site exhibited the strongest efficacy. This retrovirus-mediated transfer of anti-MDR1 ribozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance.
Kobayashi
H
H
Department of Pharmacology, Tokai University School of Medicine, Isehara, Kanagawa, Japan. hkobayas@is.icc.u-tokai.ac.jp
Takemura
Y
Y
Wang
F S
FS
Oka
T
T
Ohnuma
T
T
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018920:Coculture Techniques; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005091:Exons; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic; D011994:Recombinant Proteins; D012320:RNA Polymerase III