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.

RLIP76 (RALBP1)8

8

RALBP1 is the official human genome name for this protein, which was initially cloned by Jullien-Flores et al. (4) and named RLIP76. The previously cloned rat homolog protein is referred to as RLIP76 by Cantor et al. (3), and the mouse homolog protein was named RIP1 by Park and Weinberg (2). Because these three proteins are highly similar in sequence, and functions, and particularly because RIP1 is a nonspecific term because of unrelated proteins also designated RIP1 (death domain serine/threonine kinase, Reiske Fe-S interacting protein, etc.), we have chosen to refer to the mouse, rat and human homologs collectively as RLIP76. Species are specified where needed in context.

is a multifunctional modular ubiquitous protein present from Drosophila to humans (14).9
9

http://www.ensembl.org human exon report.

It is a Ral-regulated protein that exerts GTPase activity towards Rho family GTPases (4, 5). It functions in clathrin-coated pit–mediated receptor-ligand endocytosis and in membrane tyrosine kinase receptor signaling through binding with the AP2-clathrin adaptor protein and to eps-homology domain proteins through the adaptor protein, POB1 (partner of RalBP1). It anchors in the plasma membrane through antennapedia homeodomain homologous sequences in its NH2-terminal (6) and has two ATPase sites (7). Several splice variants are known; one of which, cytocentrin, binds to centrosomes during mitosis and may function as a molecular motor (8). Membrane-associated RLIP76 functions as a multispecific efflux pump for amphiphilic small molecules including anticancer drugs, Vinca alkaloids and anthracylines, and endogenous metabolites such as glutathione electrophile conjugates (GS-E) of reactive oxygen species formed from membrane lipids during the process of lipid peroxidation (915). Its physiologic role in protection from oxidative and radiant stress seems exerted by regulation of cellular concentration of proapoptotic reactive alkenals such as 4-hydroxynonenal (4-HNE), through the active efflux of GS-E derived from these toxic intermediates. Oxidant or radiant stresses, which are known to increase cellular 4-HNE, cause a rapid increase in cellular RLIP76 protein, which precedes heat shock protein accumulation (12, 13). If the intensity of stress is sublethal, and cells are allowed to recover, they become relatively resistant to subsequent stress challenge and blocking RLIP76 abrogates this resistance. These findings have led to a general hypothesis that regulation of cellular levels of endogenous alkenals is the principal mechanism through which RLIP76 confers resistance to oxidant and radiant stressors. This hypothesis predicts that RLIP76-deficient animals will have lower transport activity for GS-E, have greater levels of alkenals, and will be sensitive to oxidant or radiant stress. In the present communication, we describe studies of the effects of RLIP76 loss on cellular oxidative stress and whole animal radiation sensitivity.

Genotyping and tissue analysis. All animal experiments were carried out in accordance with a protocol approved by the Institutional Animal Care and Use Committee. RLIP76+/− heterozygous knockout animals were generated by Lexicon Genetics (The Woodlands, TX). The 129/SvEv strain embryonic stem (ES) cells (OST 359995) in which RLIP76 was disrupted (at chr17.64966078-65001807) through Cre-Lox technology (16) using the VICTR37 LTR retroviral gene trapping vector. Blastocyst microinjection protocols (17) were used to inject ES cells host embryo from C57BL/6 albino strain to obtain RLIP76+/− mice (see Supplementary Figure for details). Mice (12 weeks old), born of RLIP76+/− × RLIP76+/− mating, were genotyped by PCR strategy. Animals sacrificed by CO2 inhalation were subjected to complete autopsy. SDS-PAGE and Western blot analysis were done in detergent-solubilized crude membrane fraction of tissues. Before homogenization, the tissues were perfused with PBS using a 22-gauge syringe. The 10% (w/v) homogenate of tissues were prepared in 10 mmol/L Tris-HCl (pH 7.4), with 1.4 mmol/L β-mercaptoethanol and centrifuged at 8,000 × g for 45 minutes and the pellet was discarded. The supernatant was collected and centrifuged again at 105,000 × g for 60 minutes. Supernatant was then discarded and the membrane pellet obtained was solubilized in lysis buffer [10 mmol/L Tris-HCl (pH 7.4), containing 0.1 mmol/L phenylmethylsulfonyl fluoride, 0.05 mmol/L butylated hydroxytoluene (BHT), 0.1 mmol/L EDTA, and 1.4 mmol/L β-mercaptoethanol] containing 0.5% polidocanol. The mixture was sonicated thrice for 30 seconds at 50 W and incubated for 4 hours at 4°C with occasional shaking. After incubation, the mixture was centrifuged at 48,000 × g for 1 hour at 4°C and the pellet was discarded. The supernatant was collected and subjected to SDS-PAGE.

Preparation of RLIP76 liposomes. cDNA of human RLIP76 was cloned from human bone marrow library and was subcloned into the prokaryotic expression vector creating the pET30-RLIP76 plasmid. This plasmid was transfected into Escherichia coli BL21 (DE3) and protein was induced with 0.4 mmol/L isopropyl-l-thio-B-d-galactopyranoside. Recombinant RLIP76 purified by dinitrophenyl-S-glutathione affinity chromatography, and purity was confirmed by SDS-PAGE, Western blot analyses, amino acid composition analyses, and matrix-assisted laser desorption/ionization-mass spectrometry. To prepare proteoliposomes, purified RLIP76 was dialyzed against reconstitution buffer [10 mmol/L Tris-HCl (pH 7.4), 4 mmol/L MgCl2, 1 mmol/L EGTA, 100 mmol/L KCl, 40 mmol/L sucrose, 2.8 mmol/L β-mercaptoethanol, 0.05 mmol/L BHT, and 0.025% polidocanol]. An aqueous emulsion of soybean asolectin (40 mg/mL) and cholesterol (10 mg/mL) was prepared in the reconstitution buffer by sonication, from which a 100-μL aliquot was added to 0.9 mL of dialyzed purified RLIP76 protein. After sonication of the resulting mixture for 30 seconds at 50 W, 200 mg of SM-2 Bio-beads pre-equilibrated with reconstitution buffer (without polidocanol) were added to initiate vesiculation, and after 4 hours of incubation at 4°C, SM-2 beads were removed by centrifugation at 3,620 × g. Control vesicles (control liposomes) were prepared using an equal amount of crude protein from E. coli not expressing RLIP76. The vesicles (RLIP76 liposomes) were collected and analyzed for microbial contamination and size of the vesicles were determined using electron micrography and 14C-inulin entrapment (14).

Transport studies. For transport studies, we prepared the crude membrane inside-out vesicles (IOV) from heart and liver tissues. The reaction mixture consisted of IOVs protein, 10 mmol/L Tris-HCl (pH 7.4), 40 mmol/L sucrose, 4 mmol/L MgCl2, without or with 4 mmol/L ATP. To start the reaction, appropriate volume of radiolabeled 14C-DOX (specific activity, 8.7 × 104 cpm/nmol) or 3H-DNP-SG (specific activity, 3.9 × 103 cpm/nmol) was added. The uptake was stopped by rapid filtration of the reaction mixture through 96-well nitrocellulose plates (0.45-μm pore size). After filtration, the bottoms of the nitrocellulose membranes were punched out and placed in liquid scintillation fluid for counting. ATP-dependent uptake of either 14C-DOX or 3H-DNP-SG was determined by subtracting the radioactivity of the control without ATP from that of the experimental containing ATP and the transport was calculated in terms of pmol/min/mg IOV protein (15).

Determination of enzyme activities. Reduced glutathione (GSH) levels and enzyme activities for glutathione S-transferase (GST), glutathione peroxidase (GPX), glutathione reductase, glucose-6-phosphate dehydrogenase (G6PD), and γ-glutamyl cysteine synthetase (γ-GCS) were determined in 28,000 × g supernatants of 10 % homogenate; lipid hydroperoxides (LOOH) and thiobarbitauric acid reactive substances (TBARS) were determined in whole crude homogenates using established methods described by us previously (18).

Radiation. Whole body X-irradiation was given using a Varian Clinac Linear accelerator (2100C) at Texas Cancer Center (Arlington, TX) followed by monitoring for survival. Briefly, using the Varian 2100C Linear Accelerator 6-MeV photon beam, the mice were irradiated with doses ranging from 500 to 1,000 cGy. We placed the mice in their cage on top of 1.5 cm of super flab bolus, isolating them to one side of the cage, and centering the field of treatment on them. They were irradiated with half of the dose from the anterior and the other half from the posterior, by rotating the accelerator gantry 180 degrees.

Immunohistochemical localization. Buffered formalin-fixed, paraffin-embedded tissues were processed and sectioned (5 μm) by standard techniques. Sections were treated with 1% hydrogen peroxidase in PBS for 15 minutes followed by microwave antigen retrieval at 100°C for 10 minutes in DAKO Target Retrieval Solution (DAKO, Carpinteria, CA) in an H2800 Microwave Processor (Energy Beam Sciences, Agawam, MA). Following sequential 15-minute incubations with 0.1% avidin and 0.01% biotin (Vector Laboratories, Burlingame, CA) to block endogenous avidin and biotin, sections were incubated in 0.05% casein (Sigma, St. Louis, MO)/0.05% Tween 20/PBS for 30 minutes to block nonspecific protein binding. Primary antibody (rabbit anti-human RLIP76) was applied to sections at a 1:150 dilution for 60 minutes. Rabbit preimmune serum was applied as a negative control. Biotinylated F(ab′)2 fragment of swine anti-rabbit immunoglobulins (DAKO) served as secondary antibody, positive antibody binding was detected by Streptavidin-horseradish peroxidase and colorized by 3,3′-diaminobenzidine (DAKO). Sections were counterstained with Mayer's Modified Hematoxylin (Poly Scientific, Bay Shore, NY) before mounting; tissues were observed and photographed with standard light microscopic techniques.

We generated C57B mice which carry heterozygous (+/−) or homozygous (−/−) disruption of the RLIP76 gene (see Supplementary Figure) and established colonies of RLIP76+/+, RLIP76+/−, and RLIP76−/− C57B mice by segregation and mating of animals based on genotyping by PCR on tail DNA (Fig. 1A). Western blot analysis of mouse tissues using anti-RLIP76 antibodies confirmed decreased RLIP76 levels in the RLIP76+/− mouse and its absence in tissues from the RLIP76−/− mouse (Fig. 1B). Consistent with the observed function of RLIP76 as a transporter of GS-E and doxorubicin in cell culture studies (1113, 16, 17), GS-E and doxorubicin transport in membrane vesicles was decreased in a stepwise fashion from the RLIP76+/+, to RLIP76+/−, to RLIP76−/− mice (Fig. 1C and D). A >80% loss of total GS-E and doxorubicin transport activity was seen in the RLIP76−/− mice. The differences in transport rates were significantly lower in the RLIP76+/− mice compared with RLIP76+/+ and in the RLIP76−/− mice compared with either RLIP76+/− or RLIP76+/+ mice. These findings showed that RLIP76 is the predominant GS-E and doxorubicin transporter in mouse tissues.

Figure 1.

Effect of RLIP76 disruption on GS-E and doxorubicin (DOX) transport. A, an agarose gel showing genotyping results on mouse tail DNA by PCR (using forward 5′-TCTTCTGCTCAC-TCGTCCCT-3′, reverse 5′-GTTTCCCACTCAGCTTCCAG-3′, and LTR primer 5′-AAATGGCGTTACTTAAGCTAGCTTGC-3′); the DNA-ladder (std), RLIP76+/+ (200-bp band), RLIP76−/− (150-bp band), and RLIP76+/− (both bands). B, the effect of RLIP76 genotype on heart and liver tissue RLIP76 protein is shown by Western blot analysis, with application of 100 μg crude membrane fraction to SDS-PAGE and using anti-RLIP76 IgG as primary antibody and β-actin internal controls. Effect of RLIP76 genotype on (C) DNP-SG and (D) doxorubicin transport activity (14) in IOVs prepared from heart tissue of RLIP76+/+ (black), RLIP76+/− (gray), and RLIP76−/− (white) animals. Statistical analyses by ANOVA were significant at P < 0.01 for RLIP76+/+ vs. RLIP76+/−, RLIP76+/+ vs. RLIP76−/−, and RLIP76+/− versus RLIP76−/−.

Figure 1.

Effect of RLIP76 disruption on GS-E and doxorubicin (DOX) transport. A, an agarose gel showing genotyping results on mouse tail DNA by PCR (using forward 5′-TCTTCTGCTCAC-TCGTCCCT-3′, reverse 5′-GTTTCCCACTCAGCTTCCAG-3′, and LTR primer 5′-AAATGGCGTTACTTAAGCTAGCTTGC-3′); the DNA-ladder (std), RLIP76+/+ (200-bp band), RLIP76−/− (150-bp band), and RLIP76+/− (both bands). B, the effect of RLIP76 genotype on heart and liver tissue RLIP76 protein is shown by Western blot analysis, with application of 100 μg crude membrane fraction to SDS-PAGE and using anti-RLIP76 IgG as primary antibody and β-actin internal controls. Effect of RLIP76 genotype on (C) DNP-SG and (D) doxorubicin transport activity (14) in IOVs prepared from heart tissue of RLIP76+/+ (black), RLIP76+/− (gray), and RLIP76−/− (white) animals. Statistical analyses by ANOVA were significant at P < 0.01 for RLIP76+/+ vs. RLIP76+/−, RLIP76+/+ vs. RLIP76−/−, and RLIP76+/− versus RLIP76−/−.

Close modal

Loss of RLIP76 conferred sensitivity to X-irradiation in a stepwise fashion (Table 1). At each radiation dose, survival was shortest for RLIP76−/−, followed by RLIP76+/− and RLIP76+/+ (P < 0.01 for all comparisons). The mechanism for radioprotection was examined by comparing variables of oxidative injury (18), glutathione-linked enzymes, and transport activity towards GS-E and doxorubicin between radiated and unirradiated animals of both genders and all three genotypes. LOOH and TBARS were increased significantly in a stepwise manner with progressive loss of RLIP76 gene from RLIP76+/+ to RLIP76−/− (P < 0.01; Table 2). These findings indicated progressive increase in tissue oxidative stress with loss of RLIP76. Whereas tissue GSH content was increased, activities of GSH-linked antioxidant enzymes were generally decreased (Table 2) with progressive loss of RLIP76. These results suggested that RLIP76 functions to up-regulate these enzymes, consistent with known effects of RLIP76 on Rho/Rac pathways and or Hsf-1 (a transcription factor regulating heat shock responses; ref. 19). Thus, increase in ambient LOOH might be explained as a secondary effect of the loss of RLIP76 due to decreased activities of GST, GPX, glutathione reductase, and G6PD, which normally metabolize LOOH and consume GSH. Increased GSH levels observed would thus be secondary to decreased consumption of GSH rather than increased synthesis, because the rate-limiting enzyme for GSH synthesis, γ-GCS, was unchanged or decreased. Analyses of these variables by individual tissues supported this assertion (Fig. 2). The only tissue in which GSH, LOOH, and TBARS were decreased was the liver, where GST, GPX, and γ-GCS were increased. The changes in oxidative stress and antioxidant enzymes were generally concordant between tissues for any given variable, and the change was generally greater in the RLIP76−/− animals as compared with the RLIP76+/− animals. X-irradiation resulted in increase tissue oxidative stress with generally increased LOOH and TBARS in most tissues and a greater degree of increase in RLIP76−/− compared with RLIP76+/− animals (Table 2). Tissue-specific effects of gender, genotype, and radiation on levels of lipid peroxidation products and GSH-linked enzymes were analyzed by two- and three-way interaction ANOVA (presented in Supplementary Table and Data). Significant gender-specific differences were seen in the effects of loss of RLIP76, as well as radiation in each tissue. For each of the antioxidant enzymes, gender-specific differences in the effects of RLIP76 loss or radiation were found, with generally greater changes in males compared with females. Gender-specific differences were most prominent in the spleen, brain, and liver, particularly in levels of G6PD and γ-GCS activities.

Table 1.

Effect of loss of RLIP76 on post-radiation survival in mice

Radiation dose (cGy)Survival (h)
RLIP76+/+RLIP76+/−RLIP76−/−
500 648 ± 53 360 ± 28* 264 ± 28*, 
750 336 ± 30 168 ± 21* 144 ± 12*, 
1,000 138 ± 14 19 ± 4* 8 ± 3*, 
Radiation dose (cGy)Survival (h)
RLIP76+/+RLIP76+/−RLIP76−/−
500 648 ± 53 360 ± 28* 264 ± 28*, 
750 336 ± 30 168 ± 21* 144 ± 12*, 
1,000 138 ± 14 19 ± 4* 8 ± 3*, 

NOTE: Survival time (h) was monitored after whole body X-irradiation (n = 6/group). Median survival was calculated from Kaplan-Meier analyses.

*

P < 0.05, for comparisons versus RLIP76+/+.

P < 0.05, for comparisons versus RLIP76+/−.

Table 2.

Percent change in markers of oxidative stress and defenses due to loss of RLIP76 and X-irradiation

VariableUnirradiated
Irradiated (500 cGy)
+/− versus +/+
−/− versus +/+
−/− versus +/−
+/− versus +/+
−/− versus +/+
−/− versus +/−
MaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemale
LOOH 32 37 94 102 47 48 62 63 110 122 60 63 
TBARS 18 17 68 59 42 35 43 42 94 83 64 56 
GSH 31 48 45 59 10 70 46 58 57 76 20 19 
GST −16 −15 −19 −18 — — — — — — — 11 
GPX −36 −21 −46 −37 −15 −19 −27 −12 −43 −30 −10 — 
GR −18 −16 −30 −23 −15 −9 — — −27 −24 −11 −9 
G6PD −18 −12 −22 −17 — — — 19 — 17 — 33 
γ-GCS — — — −21 — — — 14 — −9 — — 
DNP-SG Transport* −65 −67 −81 −82 −46 −47 −55 −56 −83 −79 −51 −37 
DOX Transport* −51 −55 −85 −82 −69 −59 −40 −45 −77 −79 −53 −53 
VariableUnirradiated
Irradiated (500 cGy)
+/− versus +/+
−/− versus +/+
−/− versus +/−
+/− versus +/+
−/− versus +/+
−/− versus +/−
MaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemaleMaleFemale
LOOH 32 37 94 102 47 48 62 63 110 122 60 63 
TBARS 18 17 68 59 42 35 43 42 94 83 64 56 
GSH 31 48 45 59 10 70 46 58 57 76 20 19 
GST −16 −15 −19 −18 — — — — — — — 11 
GPX −36 −21 −46 −37 −15 −19 −27 −12 −43 −30 −10 — 
GR −18 −16 −30 −23 −15 −9 — — −27 −24 −11 −9 
G6PD −18 −12 −22 −17 — — — 19 — 17 — 33 
γ-GCS — — — −21 — — — 14 — −9 — — 
DNP-SG Transport* −65 −67 −81 −82 −46 −47 −55 −56 −83 −79 −51 −37 
DOX Transport* −51 −55 −85 −82 −69 −59 −40 −45 −77 −79 −53 −53 

NOTE: Statistically significant changes (P < 0.05 by ANOVA) are presented in terms of percent of control (either +/+ or +/−). Values not reaching significance are omitted (—). The experiment had a 2 × 2 × 3 factorial (gender × radiation × genotype, 12 groups, n = 36). Animals were sacrificed 8 days after either 500-cGy whole body X-irradiation or sham irradiation, and organs (brain, heart, kidney, liver, lung, spleen, and intestine) were collected. Methods for each measurement (three animals per group, triplicate determinations) have been described previously (18). For the analysis presented, data from all seven tissues are taken together. Individual tissue analyses for both unirradiated and X-irradiated animals, as well as results of one-, two-, and three-way interaction analyses among gender, genotype, and irradiation are presented in Supplementary Table and Data.

Abbreviations: DOX, doxorubicin; GR, glutathione reductase.

*

Only liver and heart tissues were used for transport studies.

Figure 2.

Effect of RLIP76 disruption on GSH-linked antioxidant enzymes. The activity of GST, GPX, glutathione reductase (GR), G6PD, and γ-GCS were determined in 28,000 × g supernatants as previously described (18) from each tissue of RLIP76+/− (gray) and RLIP76−/− (white), normalized to corresponding values for RLIP76+/+. Significant findings (ANOVA) are shown in Supplementary Table and Data.

Figure 2.

Effect of RLIP76 disruption on GSH-linked antioxidant enzymes. The activity of GST, GPX, glutathione reductase (GR), G6PD, and γ-GCS were determined in 28,000 × g supernatants as previously described (18) from each tissue of RLIP76+/− (gray) and RLIP76−/− (white), normalized to corresponding values for RLIP76+/+. Significant findings (ANOVA) are shown in Supplementary Table and Data.

Close modal

Replacement of RLIP76 in tissues of RLIP76−/− animals was examined in animals i.p. given purified human rec-RLIP76 liposomes, authenticated as previously described (refs. 9, 14; Fig. 3). Whereas Western blot and immunohistochemistry showed no immunologically detectable RLIP76 in RLIP76−/− animals given control liposomes, RLIP76 was clearly detected in all tissues, particularly liver, at 48 hours after a single i.p. dose of RLIP76 liposome. Repeated administration increased RLIP76 accumulation. Time-dependent accumulation followed by disappearance was observed, and results for hepatic tissue are shown. Significant accumulations of RLIP76 were also seen in distal renal tubules cardiac vessel endothelium and most remarkably in cerebellar and cerebral neurons. The neuronal findings are particularly remarkable, indicating permeation of the blood-brain barrier by RLIP76. These findings suggested that RLIP76 liposomes had behavior similar to caveolae (of which RLIP76 is a component), capable of traversing vascular endothelium. These results showed that RLIP76 could be effectively delivered to tissue by i.p. administration and suggested the possible pharmacologic utility of RLIP76 liposomes for central nervous system delivery of drugs or oligonucleotides.

Figure 3.

Delivery of RLIP76 to tissues of RLIP76−/− mice. A, Western blot analyses of RLIP76−/− mouse tissues (200 μg protein) from animals sacrificed 48 hours after one (200) or three doses (at 0, 72, and 120 hours; 600) of RLIP76 liposome (200 μg each) followed by sacrifice 48 hours after the last dose. C, control liposome; R, RLIP76 liposome. Immunohistochemical studies: (B) RLIP76+/+ liver (c, central vein; p, portal area); (C) RLIP76−/−; RLIP76−/− sacrificed at (D) 6 hours, (E) 12 hours, (F) 24 hours, or (G) 48 hours after one 200 μg i.p. dose. Results for renal tissue (H), cardiac tissue (I), cerebellar neurons at low magnification (100×; J), and cerebral neurons at high magnification (400×; K).

Figure 3.

Delivery of RLIP76 to tissues of RLIP76−/− mice. A, Western blot analyses of RLIP76−/− mouse tissues (200 μg protein) from animals sacrificed 48 hours after one (200) or three doses (at 0, 72, and 120 hours; 600) of RLIP76 liposome (200 μg each) followed by sacrifice 48 hours after the last dose. C, control liposome; R, RLIP76 liposome. Immunohistochemical studies: (B) RLIP76+/+ liver (c, central vein; p, portal area); (C) RLIP76−/−; RLIP76−/− sacrificed at (D) 6 hours, (E) 12 hours, (F) 24 hours, or (G) 48 hours after one 200 μg i.p. dose. Results for renal tissue (H), cardiac tissue (I), cerebellar neurons at low magnification (100×; J), and cerebral neurons at high magnification (400×; K).

Close modal

RLIP76−/− animals treated with RLIP76 liposomes (at 72 hours before and 48 and 96 hours after) 500-cGy X-irradiation had a remarkable improvement in duration of survival (median survival, 370 days) compared with RLIP76−/− animals treated with the same regimen of control liposomes (median survival, 12 days; Fig. 4A). Improved duration of survival of both RLIP76+/+ and RLIP76−/− was also observed in additional studies, in which control or RLIP76 liposomes were given 12 hours after 750 cGy (Fig. 4B). Whole body X-irradiation, as well as in RLIP76+/+ animals given RLIP76 72 hours after 1,000-cGy whole body X-irradiation (Fig. 4C). Remarkably, the RLIP76−/− mice supplemented with RLIP76 liposomes had significantly (P < 0.05) improved survival compared with even the RLIP76+/+ mice treated with RLIP76 liposomes. A possible explanation of these findings was found in gene expression array analyses and real-time quantitative PCR analyses of unirradiated RLIP76−/− mouse tissues, showing a marked increase in expression of three heat shock proteins (Hsp1α, Hsp40, and Hsp105; data not shown). It should be noted that RLIP76 binding to the transcription factor Hsf-1 has been shown to suppress heat shock protein transcription in studies by others (19). We speculate that acute administration of RLIP76 liposomes to the RLIP76−/− mice could not rapidly suppress heat shock proteins or other defenses to the baseline levels found in control RLIP76+/+ mice. Taken together, these findings provided compelling evidence supporting the assertion that RLIP76 is a major determinant of radiation resistance and can protect against radiation-induced stress. Detailed studies examining radiation and RLIP76 doses and timing of liposome administration are currently being carried out.

Figure 4.

Effect of RLIP76 liposomes on survival of RLIP76−/− and RLIP76+/+ mice after X-irradiation. Six RLIP76−/− mice were randomized in each group treated with 500-cGy whole body X-irradiation. A, control (▵) or RLIP76 liposome (○; 200 μg protein) were given i.p. at day −3, day +3, and day +5 of radiation. B, results are presented for RLIP76−/− with control liposome (▵), RLIP76−/− with 400 μg RLIP76 liposome (○), RLIP76+/+ with control liposome (◊), or RLIP76+/+ with 400 μg RLIP76 liposomes (□) given 12 hours after 750-cGy X-irradiation (n = 4 per group). C, survival results are presented for RLIP76+/+ with control liposomes (◊) or RLIP76+/+ with 400 μg RLIP76 liposomes (□) given after 1,000-cGy X-irradiation (n = 4 per group).

Figure 4.

Effect of RLIP76 liposomes on survival of RLIP76−/− and RLIP76+/+ mice after X-irradiation. Six RLIP76−/− mice were randomized in each group treated with 500-cGy whole body X-irradiation. A, control (▵) or RLIP76 liposome (○; 200 μg protein) were given i.p. at day −3, day +3, and day +5 of radiation. B, results are presented for RLIP76−/− with control liposome (▵), RLIP76−/− with 400 μg RLIP76 liposome (○), RLIP76+/+ with control liposome (◊), or RLIP76+/+ with 400 μg RLIP76 liposomes (□) given 12 hours after 750-cGy X-irradiation (n = 4 per group). C, survival results are presented for RLIP76+/+ with control liposomes (◊) or RLIP76+/+ with 400 μg RLIP76 liposomes (□) given after 1,000-cGy X-irradiation (n = 4 per group).

Close modal

Our findings show that loss of RLIP76 results in multiple and significant effects on glutathione homeostasis, on the metabolism and disposition of lipid peroxidation byproducts, including GS-E, and on sensitivity to irradiation. Ionizing radiation causes formation of LOOH, which give rise to proapoptotic electrophilic α, β-unsaturated aldehydes, such as 4-HNE, which are conjugated with GSH by GST to form GS-E (20). Because this reaction is reversible, removal of GS-E is important not only to minimize the reverse reaction but also because GS-E strongly inhibits GST and glutathione reductase. By removing GS-E through efflux, RLIP76 serves to regulate cellular levels of α, β-unsaturated aldehydes that can cross-link and denature proteins (20). RLIP76 is quantitatively the major GS-E transporter in cells is evident from previous studies in MRP1 null mice that do not display similar degree of loss of GS-E transport activity.

These findings may affect cancer therapy by providing a single novel target, which could affect both radiation and chemotherapy sensitivity. The effect of RLIP76 liposomes in providing protection from radiation toxicity also has implications for normal tissue protection during radiation therapy, as well as in the treatment of iatrogenic, accidental, or catastrophic radiation poisoning. In the present geopolitical environment, the risks of radiation poisoning as a result of a nuclear accident, or nuclear bombs, mandate the development of improved post-exposure treatment of radiation victims. RLIP76 liposomes may be excellent candidates for development as a radiation protective agent that may have broad applicability, particularly given that these liposomes are capable of delivering sustained levels of RLIP76 in all tissue, even brain. These findings also indicate that RLIP76 liposomes may be useful as vehicles for delivery of drugs, antisense therapies, and other therapies to the brain across the blood-brain barrier.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: USPHS grants CA 77495 (S. Awasthi), CA104661 (S. Awasthi), ES 012171 (Y.C. Awasthi), DK 52825 (P. Dent), CA88906 (P. Dent), CA72995 (P. Dent), and DAMD17-1-03-0262 (P. Dent) and the Cancer Research Foundation of North Texas (S. Awasthi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Deborah Gerrity, Sarah Baker, Patricia Rodler, and Richard Jarrett for their assistance in radiation studies at the Texas Cancer Center.

1
Mirey G, Balakireva M, L'Hoste S, Rosse C, Voegeling S, Camonis J. A Ral guanine exchange factor-Ral pathway is conserved in Drosophila melanogaster and sheds new light on the connectivity of the Ral, Ras, and Rap pathways.
Mol Cell Biol
2003
;
23
:
1112
–24.
2
Park SH, Weinberg RA. A putative effector of Ral has homology to Rho/Rac GTPase activating proteins.
Oncogene
1995
;
11
:
2349
–55.
3
Cantor SB, Urano T, Feig LA. Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases.
Mol Cell Biol
1995
;
15
:
4578
–84.
4
Jullien-Flores V, Dorseuil O, Romero F, et al. Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity.
J Biol Chem
1995
;
270
:
22473
–7.
5
Ikeda M, Ishida O, Hinoi T, Kishida S, Kikuchi A. Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral.
J Biol Chem
1998
;
273
:
814
–21.
6
Yadav S, Singhal SS, Singhal J, et al. Identification of membrane anchoring domains of RLIP76 using deletion mutants analyses.
Biochemistry
2004
;
43
:
16243
–53.
7
Awasthi S, Cheng J, Singhal SS, et al. Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences.
Biochemistry
2001
;
40
:
4159
–68.
8
Quaroni A, Paul EC. Cytocentrin is a Ral-binding protein involved in the assembly and function of the mitotic apparatus.
J Cell Sci
1999
;
112
:
707
–18.
9
Stuckler D, Singhal J, Singhal SS, Yadav S, Awasthi YC, Awasthi S. RLIP76 transports vinorelbine and mediates drug resistance in non-small cell lung cancer.
Cancer Res
2005
;
65
:
991
–8.
10
Yadav S, Zajac E, Singhal SS, et al. POB1 over-expression inhibits RLIP76 mediated transport of glutathione-conjugates, drugs and promotes apoptosis.
Biochem Biophys Res Commun
2005
;
328
:
1003
–9.
11
Sharma R, Singhal SS, Wickramarachchi D, Awasthi YC, Awasthi S. RLIP76 (RALBP1)-mediated transport of leukotriene C4 (LTC4) in cancer cells: implications in drug resistance.
Int J Cancer
2004
;
112
:
934
–42.
12
Cheng JZ, Sharma R, Yang Y, et al. Accelerated metabolism and exclusion of 4-hydroxynonenal through induction of RLIP76 and hGST5.8 is an early adaptive response of cells to heat and oxidative stress.
J Biol Chem
2001
;
276
:
41213
–23.
13
Yang Y. Sharma A, Sharma R, et al. Cells preconditioned with mild, transient UVA irradiation acquire resistance to oxidative stress and UVA-induced apoptosis: role of 4-hydroxynonenal in UVA-mediated signaling for apoptosis.
J Biol Chem
2003
;
278
:
41380
–8.
14
Awasthi S, Cheng J, Singhal SS, et al. Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin.
Biochemistry
2000
;
39
:
9327
–34.
15
Awasthi S, Singhal SS, Srivastava SK, et al. Adenosine triphosphate-dependent transport of doxorubicin, daunomyicn, and vinblastine in human tissues by a mechanism distinct from the P-glycoprotein.
J Clin Invest
1994
;
93
:
958
–65.
16
Sauer B. Inducible gene targeting in mice using the Cre/lox system.
Methods Enzymol
1998
;
14
:
381
–92.
17
Joyner AL. Gene targeting: a practical approach. Oxford (England): Oxford University Press; 2000.
18
Khan MF, Srivastava SK, Singhal SS, et al. Iron-induced lipid peroxidation in rat liver is accompanied by preferential induction of glutathione S-transferase 8-8 isozyme.
Toxicol Appl Pharmacol
1995
;
131
:
63
–72.
19
Hu Y, Mivechi NF. HSF-1 interacts with Ral-binding protein 1 in a stress-responsive, multiprotein complex with HSP90 in vivo.
J Biol Chem
2003
;
278
:
17299
–306.
20
Esterbauer H, Zollner H. Methods for determination of aldehydic lipid peroxidation products.
Free Radic Biol Med
1989
;
7
:
197
–203.