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 p-acti n promoter. Upon transduction of the ribozymes into MDR cells, vincristine resistance was decreased. These two ribozymes were constructed, which showe d different cleavage activities. In this study, to determine suitable targe t sites for the anti-MDR1 ribozyme, the exon 1b-intron I boundary, the tran slation-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 candidate s, To improve the ribozyme activity, a retroviral vector containing RNA pol ymerase III promoter was used, Stable retrovirus producer cells were genera ted by transfecting the retroviral vector plasmids carrying the ribozyme in to the packaging cell line. Retroviral vector transduction of human leukemi a cell lines expressing MDR1 was accomplished by co-culturing these with vi rus producer cells, Stably transduced cells were selected by G418 and poole d to determine the efficacy of each ribozyme, These ribozyme-transduced cel ls became vincristine-sensitive concomitant with the decreases in MDR1 expr ession, P-gp amount and drug efflux pump function, Among the ribozymes test ed, the anti-MDR1 ribozyme against the translation-initiation site exhibite d the strongest efficacy, This retrovirus-mediated transfer of anti-MDR1 ri bozyme may be applicable to the treatment of MDR cells as a specific means to reverse resistance, (C) 1999 Wiley-Liss, Inc.