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)

Targeted depletion of the RALBP1-encoded 76-kDa splice variant, RLIP76, causes marked and sustained regression of human xenografts of lung, colon, prostate, and kidney cancers without toxicity in nude mouse models. We proposed that the remarkable efficacy and broad spectrum of RLIP76-targeted therapy is because its glutathione-conjugate (GS-E) transport activity is required for clathrin-dependent endocytosis (CDE), which regulates all ligand-receptor signaling, and that RLIP76 is required not only for survival of cancer cells but also for their very existence. We studied RLIP76 mutant proteins and the functional consequences of their expression into RLIP76 À/À MEFs, identified key residues for GS-E binding in RLIP76, established the requirement of RLIP76-mediated GS-E transport for CDE, and showed a direct correlation between GS-E transport activities with CDE. Depletion of RLIP76 nearly completely blocked signaling downstream of EGF in a CDE-dependent manner and Wnt5a signaling in a CDE-independent manner. The seminal prediction of this hypothesis-RLIP76 À/À mice will be deficient in chemical neoplasia-was confirmed. Benzo[a]pyrene, dimethylbenzanthracene, and phorbol esters are ineffective in causing neoplasia in RLIP76 À/À . PMA-induced skin carcinogenesis in RLIP76 þ/þ mouse was suppressed completely by depletion of either PKCa or RLIP76by siRNA or antisense and could be restored by topical application of RLIP76 protein in RLIP76 À/À mouse skin. Likewise, chemical pulmonary carcinogenesis was absent in female and nearly absent in male RLIP76À/À mice. In RLIP76 À/À mice, p53, p38, and JNK activation did not occur in response to either carcinogen. Our findings show a fundamental role of RLIP76 in chemical carcinogenesis.

Doxorubicin (DOX) transport activity of Ral-interacting protein (RLIP76) in non-small cell lung cancer (NSCLC) is approximately twice that of in small cell lung cancer (SCLC). Since protein-kinase-C (PKC)a mediated phosphorylation of RLIP76 causes doubling of the specific activity of RLIP76, and NSCLC cells are known to have greater PKCa activity, we examined the contribution of PKC mediated phosphorylation of RLIP76 towards intrinsic DOX-resistance in human NSCLC. Expression of a deletion mutant RLIP76 delPKCa-sites followed by depletion of the wild-type RLIP76 using a siRNA targeted at one of the deleted regions resulted in generation of cells expressing only the mutant protein, which could not be phosphorylated by PKCa. DOX-transport activity of the mutant RLIP76 purified from NSCLC and SCLC was similar and comparable to that of RLIP76 purified from the wild-type SCLC. However, this activity was significantly lower than that of RLIP76 purified from the wild-type NSCLC. After siRNA mediated depletion of PKCa, DOX-transport activities of RLIP76 purified from SCLC and NSCLC were indistinguishable. Depletion of PKCa inhibited the growth of NSCLC more than SCLC cells (70 ± 3% vs. 43 ± 5%, respectively). PKCa-depletion lowered the IC 50 of NSCLC cell lines for DOX to the same level as that observed for SCLC. RLIP76 À/À mouse embryonic fibroblasts (MEFs) were significantly more sensitive to DOX as compared with RLIP76 +/+ MEFs (IC 50 25 vs. 125 nM, respectively). However, PKCa-depletion did not affect DOX-cytotoxicity towards RLIP76 À/À MEFs, as opposed to RLIP76 +/+ MEFs which were sensitized by 2.2-fold. These results demonstrate that RLIP76 is a primary determinant of DOX-resistance, and that PKCa mediated accumulation defect and DOX-resistance in NSCLC is primarily due to differential phosphorylation of RLIP76 in SCLC and NSCLC.

RLIP76 (RALBP1) is a multifunctional transporter involved in signaling and transmembrane movement of solute allocrites, which include glutathione conjugates and several natural product antineoplastic agents [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334; (2001) Biochemistry 40, 4159-4168]. Our previous studies suggested that the membrane-anchoring domain resides in the N-terminus of RLIP76, despite the lack of identifiable membrane-spanning domains. Amino acid sequence analysis indicated that this region of RLIP76 contains sequences that are similar to those of vector peptides. We, therefore, have studied the effect of a series of deletion mutant proteins on hydrophobicity and transport activity. RLIP76 or one of its derived deletion mutants was expressed in Escherichia coli, and bacteria were lysed and extracted in buffer without or with the nonionic detergent polidocanol. The ratio of RLIP76 in the detergent/aqueous extracts was found to be 2.5 for the wild-type protein, but decreased to 0.7 in the mutant in which amino acids 154-219 were deleted. Deletion of only one segment of this region (amino acids 171-185) alone resulted in a significant decrease in this ratio to 1.0. For the mutants with deletions within the region from amino acid 154 to 219, loss of hydrophobicity correlated with less incorporation of mutants into artificial liposomes, and decreased transport activity toward doxorubicin and dinitrophenyl-S-glutathione. In contrast, deletion of one of the two ATP-binding sites (at amino acids 65-80 or 415-448) or both sites did not affect hydrophobicity but reduced or abrogated transport activity. NSCLC (H358) stably transfected with del171-185 and del154-219 showed that loss of these regions results in a decrease in the extent of membrane association of RLIP76. Confocal laser immunohistochemistry colocalized amino acids 171-185 with her2/neu on the cell surface. Depletion of wild-type RLIP76 using si-RNA directed to this region in cells transfected with del171-185 resulted in the loss of cell surface expression. These finding demonstrate that amino acids 171-185 constitute a cell surface epitope which is necessary for optimal transport of anthracycline and glutathione conjugates by RLIP76, and that this peptide could be a novel target for antineoplastic therapy.