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Reversal of Drug Sensitivity in Multidrug Resistant Human Hepatocarcinoma Cells BEL-7402/DOX by a Ribozyme which can Cleave Human MDR1 mRNA
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Abstract:
To reverse drug resistance mediated by the MDR1 gene product P-glycoprotein(P-gp) in tumor cells in a specific manner, a hammerhead ribozymes which can cleave the GUC sequence in codon 196 of MDR1 mRNA was designed and cloned into a recombinant retroviral vector pDOR-neo at BamH I restriction sites and packaged with packaging cell line PA317 cells.The viral supernatant was used to infect the multidrug-resistant human hepatocarcinoma cell line BEL-7402/DOX. After selection with G418,resistant colonies were obtained.Stable expression of retroviruses in both PA317 and infected BEL-7402/DOX cells was confirmed by Northern Blot hybridization. Down-regulation of P-gp and even of MDR1 mRNA was found in BEL-7402/DOX infected with ribozyme construct. The rate of BEL - 7402/DOX infected cell defected by flow cytometric analysis was 8.2 ~ 14.6% while in uninfected cell it was 93 .4 ~ 97.5% . The BEL-7402/ DOX cell infected with ribozyme construct was found back to drug-sensitivity to a series of drugs by MTT colorimetric assay . The results demonstrated that the recombinant retroviral vector expressing ribozyme transfecting human hepatocarcinoma BEL-7402/DOX could inhibit MDR1 gene expression and reverse tumor MDR phenotype back to drug-sensitive condition.
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