Diminished drug transport and augmented radiation sensitivity caused by loss of RLIP76

Abstract 

This study was undertaken to characterize the consequences of Ral-interacting protein (RLIP76)-loss with respect to drug resistance, transport, radiation resistance, and alternative transport mechanisms in mouse embryonic fibroblasts (MEFs). MEFs were derived from RLIP76^+/+, RLIP76^+/− and RLIP76^−/− mice. The transport of doxorubicin (DOX), colchicine (COL), leukotriene C₄ and dinitrophenyl S-glutathione (DNP-SG) was analyzed in inside-out vesicles (IOVs) prepared from MEFs. We used immuno-titration of transport activity to determine the contribution of RLIP76, MRP1, and p-glycoprotein (Pgp) towards total transport activity. Loss of RLIP76 alleles resulted in significant sensitization to radiation, DOX, cisplatin, and vinorelbine (VRL). In IOVs prepared from MEFs, we observed a stepwise loss of transport activity. Loss of RLIP76 confers sensitivity to xenobiotics and radiation due to the loss of a common transport mechanism for glutathione–electrophile conjugates and xenobiotics.

Abbreviations: RLIP76 (RalBP1), Ral-interacting protein, GSH, glutathione, GS-E, glutathione–electrophile conjugates, DNP-SG, dinitrophenyl S-glutathione, COL, colchicine, DOX, doxorubicin, 4HNE, 4-hydroxynonenal, IOVs, inside-out vesicles, LTC₄, leukotriene C₄, MEFs, mouse embryonic fibroblasts, MRP, multi-drug resistance associated protein, Pgp, p-glycoprotein, VRL, vinorelbine, RLIP76^+/+, wild-type, RLIP76^+/−, heterozygous, RLIP76^−/−, homozygous

Keywords: Ral-interacting protein, Drug resistance, Glutathione conjugate, Transport, Embryonic fibroblasts