Reversal of Drug Sensitivity in Multidrug-Resistant Tumor Cells

In order to reverse P-glycoprotein-mediated drug resistance in a spe cific manner, we designed two hammerhead ribozymes which can cleave the GUC sequence in codon 179 and 196 of MORI (PGYÕ)mRNA. The ribozymes were directly synthesized using a set of primers, one containing a bacteriophage T7 RNA polymerase promoter. A target MDRl RNA was created by a reverse transcription polymerase chain reaction using a MOLT-3 human acute leukemia cell line resistant to trimetrexate (TMQ) (MOLT-3/TMQnoo), which displayed MDRl overexpression. In a cell-free system, both ribozymes cleaved a target piece of MDRl RNA into 2 fragments at the specific sites at a physiological pH and temperature. The cleavage reaction was dependent on time, ribozyme:substrate ratio, and magnesium concentration. The 196 MDRl ribozyme was more active than the 179 MDRl ribozyme. The 196 MDRl ribozyme was then cloned into a human expression vector, and MOLT-3/TMQ8(M) cells were transfected. The original MOLT-3/TMQ,,(H) cells were nearly 700-fold resistant to vincristine, whereas the transfectant cells selected in G41S became only 20to 30-fold resistant. The level of resistance and the amount of MDRl RNA expressed appeared to correlate inversely with the amount of ribozyme expression. A disabled 196 MDRl ribozyme was capable of neither specific cleavage in vitro nor decreasing MDRl expression in transfectant cells. These results indicate that it was the ribozyme activity and not antisense activity which was responsible for decreased MDRl RNA. This approach may be applicable to cancer patients as a specific means to reverse tumors with P-glycoprotein-mediated MDR phenotype back to a drug-sensitive one.