Retrovirus-mediated transfer of anti-MDR1 ribozymes fully restores chemosensitivity of P-glycoprotein-expressing human lymphoma cells

Development of multidrug resistance (MDR) is the major obstacle to successf ul cancer chemotherapy, We have developed Daudi human lymphoma cells that a re 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 o f MDR1 mRNA (196RIDR1-Rz), the second a stem II base-modified (U-9 --> G(9) , U-13 --> A(13), G(14) --> A(14), A(18) --> C-18) Rz against codon 196 (19 6MDR1-sRz), and the third a stem II base-modified Rz directed against the - 6 similar to -4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized and cloned into the retroviral vector N 2A+tRNA(i)(Met) downstream of the RNA polymerase III promoter and adjacent to a tRNA gene sequence, forming the constructs N2A + tRNA(i)(Met)-196MDR1- Rz, N2A+tRNA(i)(Met)-196MDR1-sRz, and N2A+tRNA(i)(Met)-iMDR1-sRz. The three constructs were transfected into GP+envAM 12 cells for packaging the retro viral 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 196MDR1-sRz-tran sduced Daudi/MDR20 cells completely restored chemosensitivity to VCR and do xorubicin, and were accompanied by blocked expression of MDR1 mRNA and P-gl ycoprotein 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 c atalytic activities than the corresponding naked Rz molecules. The stem II base-modified Rz were also more stable and efficient in catalytic activitie s than the unmodified Rz molecules. The base modification in the Rz stem II structure and the development of chimeric tRNA-Rz molecules were identifie d to enhance the cleavage efficacy. The combination of these two factors, t ogether with the use of a retroviral vector, appear to have contributed to the complete reversal of MDR.