Reversal of drug sensitivity in multidrug-resistant tumor cells by an MDR1 (PGY1) ribozyme.
In order to reverse P-glycoprotein-mediated drug resistance in a specific manner, we designed two hammerhead ribozymes which can cleave the GUC sequence in codon 179 and 196 of MDR1 (PGY1) mRNA. The ribozymes were directly synthesized using a set of primers, one containing a bacteriophage T7 RNA polymerase promoter. A target MDR1 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/TMQ800), which displayed MDR1 overexpression. In a cell-free system, both ribozymes cleaved a target piece of MDR1 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 MDR1 ribozyme was more active than the 179 MDR1 ribozyme. The 196 MDR1 ribozyme was then cloned into a human expression vector, and MOLT-3/TMQ800 cells were transfected. The original MOLT-3/TMQ800 cells were nearly 700-fold resistant to vincristine, whereas the transfectant cells selected in G418 became only 20- to 30-fold resistant. The level of resistance and the amount of MDR1 RNA expressed appeared to correlate inversely with the amount of ribozyme expression. A disabled 196 MDR1 ribozyme was capable of neither specific cleavage in vitro nor decreasing MDR1 expression in transfectant cells. These results indicate that it was the ribozyme activity and not antisense activity which was responsible for decreased MDR1 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.
Kobayashi
H
H
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
Dorai
T
T
Holland
J F
JF
Ohnuma
T
T
D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics* / Q000502:physiology; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D008969:Molecular Sequence Data; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000503:physiopathology; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000494:pharmacology* / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D014407:Tumor Cells, Cultured; D014750:Vincristine / Q000494:pharmacology
D003062:Codon; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014750:Vincristine