RLIP76 Is a Major Determinant of Radiation Sensitivity

Abstract

RLIP76 (RALBP1) is a glutathione-conjugate transporter that is a critical component of clathrin-coated pit-mediated endocytosis, as well as in stress responses. In cultured cells, it provides protection from stressors including heat, oxidant chemicals, chemotherapeutic agents, UV irradiation, and X-irradiation. Here, we show marked reduction in glutathione conjugate transport capacity and stepwise increase in radiation sensitivity associated with heterozygous or homozygous loss of the RLIP76 gene in mice. Survival after radiation in homozygous knockout animals was significantly shorter than either the heterozygous knockouts or the wild type. Delivery of recombinant RLIP76 to mice lacking RLIP76 via a liposomal delivery system rescued radiation sensitivity. Furthermore, treatment of wild-type mice with RLIP76-containing liposomes conferred resistance to radiation. These findings suggest that inhibiting RLIP76 could be used for sensitization to radiation during cancer therapy and that RLIP76 liposomes could be radioprotective agents useful for treatment of iatrogenic or catastrophic radiation poisoning. (Cancer Res 2005; 65(14): 6022-8)

RLIP76 Protein Knockdown Attenuates Obesity Due to a High-fat Diet

“…This is remarkable because RLIP76 Ϫ/Ϫ mice are known to have very high levels of tissue oxidative stress as compared with RLIP76 ϩ/ϩ mice (45,46), and imply that oxidative stress alone is insufficient to mediate obesity caused by HFD. Instead, the direct logical conclusion from our studies is that RLIP76 activity is necessary to translate the effects of the HFDmediated increased oxidative stress into obesity.…”

“…RLIP76 heterozygous knock-out animals (RLIP76 ϩ/Ϫ ) were generated by Lexicon Genetics (The Woodlands, TX). RLIP76 Ϫ/Ϫ mice derived from RLIP76 ϩ/Ϫ ϫ RLIP76 ϩ/Ϫ mating were genotyped by PCR strategy as described previously (45). Forty-eight (24 RLIP76 ϩ/ϩ and 24 RLIP76 Ϫ/Ϫ ) 12-week-old mice were fed a HFD (60% fat, 20% protein, 20% carbohydrate) or standard diet/ normal chow diet (NC, 10% fat, 20% protein, 70% carbohydrate) for 12 weeks (12 RLIP76 ϩ/ϩ and 12 RLIP76 Ϫ/Ϫ mice) and 24 weeks (12 RLIP76 ϩ/ϩ and 12 RLIP76 Ϫ/Ϫ mice) (Research Diets, New Brunswick, NJ).…”

Mitogenic and drug-resistance mediating effects of PKCα require RLIP76

“…Twelve-week-old C57BL/6 mice born of heterozygous × heterozygous (RLIP76 +/− × RLIP76 +/− ) mating were genotyped by PCR strategy on mouse tail DNA using forward, reverse, and long terminal region (LTR) primers [14]. These mice were commissioned from Lexicon Genetics and were created using Cre-Lox technology as described previously [14].…”

“…These mice were commissioned from Lexicon Genetics and were created using Cre-Lox technology as described previously [14]. Embryonic fibroblast lines were prepared from RLIP76 +/+ , RLIP76 +/− , and RLIP76 −/− mice on the 13th or 14th day of pregnancy according to the method of Johnson et al [20] and maintained in RPMI-1640 medium containing 10% (v/v) heat-inactivated FBS, 1% (v/v) P/S solution, 2 mM L-glutamine, 10 mM Hepes, 1 mM sodium pyruvate, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate and plated in 25-cm 2 tissue culture flasks, and incubated at 37 °C in a humidified atmosphere of 5% CO 2 .…”

“…Increased PKCα-mediated ☆ Abbreviations: RLIP76 (RALBP1), Ral-interacting protein; DOX, doxorubicin; SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; GSH, glutathione; GS-E, glutathione-electrophile conjugate; DNP-SG, dinitrophenyl S-glutathione; MRP, multidrug-resistance associated protein; PMA, phorbol ester (phorbol 12-myristate 13-acetate); PKC, protein-kinase-C. We have recently identified a new target of PKCα, RLIP76 (RALBP1). RLIP76 is a Ralbinding Rho-GAP protein (inhibitor of Rho-signaling) which we have shown to be the predominant cellular mechanism for ATP-dependent effux of glutathione-conjugate (GS-E) and chemotherapy drugs (such as DOX) [6][7][8][9][10][11][12][13][14][15][16][17][18]. Although it can function as a drugresistance transporter, the major physiological role of RLIP76 appears to be the regulation of intracellular levels of lipid-alkenals and alkenal-glutathione conjugates, the formation of which is an early and obligatory event in course of oxidant/radiant stress or signaling [8][9][10]19] The transport activity of RLIP76 functions to regulate cellular levels of glutathionyladducts of lipidoxidation derived reactive oxygen species which are known to exert direct effects in cell proliferation, differentiation, and apoptosis [8][9][10]19].…”