Ribozyme-mediated cleavage of the MDR-1 transcript restores chemosensitivity in previously resistant cancer cells.

Abstract

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.

Ribozymes: Possible New Tools for Treatment of Cancer and Retroviral Diseases?

“…Therefore, it is reasonable to assume that at simi lar doses RZs will be more efficient in eliminating their target molecule than ODNs. This hypothesis has been experimen tally confirmed [9,10].…”

Berberine modulates expression of mdr1 gene product and the responses of digestive track cancer cells to Paclitaxel

“…Previously, it has been reported that the retention of rhodamine 123 correlated well with the function of pgp-170 (Ludescher et al, 1992;Kiehntopf et al, 1994;Petriz and Garcia-Lopez, 1997;Salvioli et al, 1997). Therefore, retention of rhodamine 123 can be used as an indicator of pgp-170 function.…”

“…rhodamine 123 in tumour cells with overexpressed pgp-170 was significantly reduced as compared to the MDR antagonist-treated tumour cells (Watt et al, 1990;Chaudhary and Roninson, 1991;Kiehntopf et al, 1994). The other Paclitaxel resistance type includes TRAG-1-5 (Paclitaxel resistance-associated gene 1-5) (Duan et al, 1997), p53 expression (Catherine et al, 1995) (Vikhanskaya et al, 1998), alteration of β-tubulin isoform (Dumontet et al, 1996), changes in the level of intracellular glutathione (Liebmann et al, 1993), or cell growth morphology (Frankel et al, 1997).…”

Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo

“…For the exogenous application, ribozymes are delivered into cells by microinjection (10) or transfection (21). In the latter case, CaCl 2 or cationic liposomes have been mostly used as transfection reagents.…”