RIBOZYME-MEDIATED CLEAVAGE OF THE MDR-1 TRANSCRIPT RESTORES CHEMOSENSITIVITY IN PREVIOUSLY RESISTANT CANCER-CELLS

被引:65
作者
KIEHNTOPF, M
BRACH, MA
LICHT, T
PETSCHAUER, S
KARAWAJEW, L
KIRSCHNING, C
HERRMANN, F
机构
[1] FREE UNIV BERLIN,KLINIKUM RUDOLF VIRCHOW,ROBERT ROSSLE CANC CTR,DEPT MED ONCOL & APPL MOLEC BIOL,W-1000 BERLIN,GERMANY
[2] MAX DELBRUCK CTR MOLEC MED,BERLIN,GERMANY
[3] NCI,DCBDC,MOLEC BIOL LAB,BETHESDA,MD
关键词
CHEMOSENSITIVITY; MDR-1; RIBOZYME;
D O I
10.1002/j.1460-2075.1994.tb06787.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
How cancer cells become resistant to chemotherapy is not completely understood, but it is believed that resistance is usually associated with overexpression of drug resistance genes. Drug resistance mediated by the MDR-1 gene is the first well characterized form of drug resistance in human cancer. MDR-1 encodes a phosphoglycoprotein, P-GP, that serves as an energy-dependent drug efflux pump, reducing intracellular drug accumulation and thereby cytotoxicity. We have used ribozymes to reverse the multiple drug resistance phenotype. A hammerhead ribozyme recognizing the GUC sequence at position -6 to -4 close to the translation start site of the 4.5 kb MDR-1 mRNA was prepared by in vitro transcription (MDR-1-RZiv) or chemical synthesis (MDR-1-RZs). Both MDR-1-RZiv and MDR-1-RZs specifically cleaved the MDR-1 mRNA into two parts of the expected size under physiological conditions in an extracellular system with MDR-1-RZiv being more effective. Site-specific cleavage was dependent on time, temperature and [MgCl2]. To examine the in vivo potential of MDR-1-RZ, MDR-1-RZiv and MDR-1-RZs were transfected into a human pleural mesothelioma cell line and into one adriamycin-resistant and one vindesine-resistant subline thereof by liposome-mediated transfer. Incorporation of ribozymes resulted in significantly reduced expression of the MDR-1 gene, with MDR-1-RZs being more potent than MDR-1-RZiv in vitro. MDR-1-RZ reduces P-GP overexpression at the protein level. Liposome-mediated transfer of MDR-1-RZiv or MDR-1-RZs reversed the multiple drug resistance phenotype and restored sensitivity towards chemotherapeutic drugs.
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页码:4645 / 4652
页数:8
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