Retrovirus-Mediated Transfer of Anti-MDR1 Ribozymes Fully Restores Chemosensitivity of P-Glycoprotein-Expressing Human Lymphoma Cells

Abstract

Development of multidrug resistance (MDR) is the major obstacle to successful cancer chemotherapy. We have developed Daudi human lymphoma cells that are 20-fold more resistant than the parent cell line to vincristine (VCR) by infecting cells with pHaMDR1/A retroviral vector (Daudi/MDR20). Three DNA sequences of anti-MDR1 hammerhead ribozymes (Rzs), one cleaving codon 196 of MDR1 mRNA (196MDR1-Rz), the second a stem II base-modified (U9 G9, U13 A13, G14 A14, A18 C18) Rz against codon 196 (196 MDR 1- sRz), and the third a stem II base-modified Rz directed against the -6∼ -4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized and cloned into the retroviral vector N2A+tRNAiMet downstream of the RNA polymerase III promoter and adjacent to a tRNA gene sequence, forming the constructs N2A+tRNAiMet-196MDR1-Rz, N2A+tRNAMet-196 MDR1-sRz, and N2A+tRNAiMet-iMDR1-sRz. The three constructs were transfected into GP+envAM 12 cells for packaging the retroviral vectors. The supernatants containing the packaged retrovirus in high titers (1.1-2.5 x 10 5 CFU/ml as determined by infection of NIH 3T3 cells) were used to infect Daudi/ MDR20 cells. The iMDR1-sRz and 196 MDR1-sRz-transduced Daudi/MDR20 cells completely restored chemosensitivity to VCR and doxorubicin, and were accompanied by blocked expression of MDR1 mRNA and P-glycoprotein as well as overexpression of anti-MDR1 Rz. In a cell-free system, the chimeric tRNA-sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules. The stem II base-modified Rz were also more stable and efficient in catalytic activities than the unmodified Rz molecules. The base modification in the Rz stem II structure and the development of chimeric tRNA-Rz molecules were identified to enhance the cleavage efficacy. The combination of these two factors, together with the use of a retroviral vector, appear to have contributed to the complete reversal of MDR.