No 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)

Ral-binding protein 1 (RALBP1) is a stress-responsive and stress-protective multispecific transporter of glutathione conjugates (GS-E) and xenobiotic toxins. It is frequently overexpressed in malignant cells and plays a prominent antiapoptotic role selectively in cancer cells through its ability to control cellular concentration of proapoptotic oxidized lipid byproducts. In the absence of chemotherapy, depletion or inhibition of RALBP1 causes regression of syngeneic mouse B16 melanoma. Because RALBP1 transports anthracycline and Vinca alkaloid drugs, as well as GS-E, and because it confers resistance to these drugs, we proposed that depletion or inhibition of RALBP1 should cause regression of human solid tumors that overexpress RALBP1 and augment chemotherapy efficacy. Non-small-cell lung cancer (NSCLC) H358 and H520 and colon SW480 cell lines were used. Cytotoxic synergy between anti-RALBP1 immunoglobulin G (IgG), cis-diamminedichloroplatinum (II) [CDDP], and vinorelbine was examined in cell culture and xenografts of NSCLC cells. Effects of RALBP1 depletion by antisense were examined in xenografts of NSCLC H358, NSCLC H520, and colon SW480 cells. RALBP1 depletion by phosphorothioate antisense was confirmed and was associated with rapid, complete, and sustained remissions in established s.c. human lung and colon xenografts. RALBP1 inhibition by anti-RALBP1 IgG was equally as effective as antisense and enhanced CDDP-vinorelbine in lung cancer xenografts. These studies show that RALBP1 is a transporter that serves as a key effector function in cancer cell survival and is a valid target for cancer therapy, and confirm that inhibitory modulation of RALBP1 transport activity at the cell surface is sufficient for antitumor effects.

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.

No abstract

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugate 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes – higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.

Hsf-1 (heat shock factor-1) is a transcription factor that is known to regulate cellular heat shock response through its binding with the multispecific transporter protein, Ralbp1. Results of present studies demonstrate that Hsf-1 causes specific and saturable inhibition of the transport activity of Ralbp1 and that the combination of Hsf-1 and POB1 causes nearly complete inhibition through specific bindings with Ralbp1. Augmentation of cellular levels of Hsf-1 and POB1 caused dramatic apoptosis in non-small cell lung cancer cell line H358 through Ralbp1 inhibition. These findings indicate a novel model for mutual regulation of Hsf-1 and Ralbp1 through Ralbp1-mediated sequestration of Hsf-1 in the cellular cytoskeleton and Hsf-1-mediated inhibition of the transport activity of membranebound Ralbp1.In response to heat stress, human cells respond by activation of Hsf-1 (heat shock factor-1), a transcription factor that binds to NGAAN repeats of the promoter of heat shock genes, augmenting transcription (1-5). Considered the master regulator of the heat shock response (1-3), Hsf-1 binds DNA constitutively, and its binding affinity is based upon its phosphorylation in response to heat shock (1-5). In the unstressed state, Hsf-1 is sequestered in a complex with tubulin, HSP90, and Ralbp1 (6). Stress or constitutively active Ral-GTP binding to Ralbp1 triggers the release of Hsf-1 and its migration to the nucleus, where its transcription factor activity is important for the expression of heat shock proteins (6, 7). Although these studies focused on Ralbp1 present in the cytoplasm bound to the cytoskeleton and nuclear membrane, several previous and subsequent reports have clearly demonstrated the presence of Ralbp1 in nuclear as well as plasma membranes (8 -12). In several recent studies, we have conclusively demonstrated that Ralbp1 is a transmembrane protein with a defined cell surface domain (8 -11) and that it catalyzes in ATP hydrolysis-dependent trans-membrane anti-gradient efflux of toxic xenobiotics as well as endogenous metabolites. The preferred physiological substrates for transport by Ralbp1 are glutathione-electrophile conjugates of electrophilic lipid metabolites that arise from stress or heat shockinduced lipid peroxidation (13). The cell surface domain of Ralbp1 can be targeted by highly specific antibodies that inhibit the transport activity of Ralbp1 and result in dramatic regression of tumor in syngeneic and xenograft models of melanoma, lung cancer, and colon cancer (14, 15). The membrane functionality of Ralbp1 is also evident from its crucial role in endocytosis as a rate-regulatory element (12, 16 -18).An endocytosis-linked protein POB1 that binds Ralbp1 in a similar region as Hsf-1 has been shown to be a specific and saturable inhibitor of the glutathione-electrophile conjugates and doxorubicin (DOX) 2 transport activity of membrane-reconstituted purified Ralbp1 (19). We proposed that just as POB1 could function as an inhibitor of the transport activity of Ralbp1, Hsf-1 could also function as a tr...

Dr-fimbriatedUrinary tract infections (UTI) are among the most common bacterial infections in humans, and Escherichia coli is the predominant etiologic agent (14). About 10 to 20% of women and 12% of men experience symptomatic UTI at some point in their lives (14,19). The characteristic feature of UTI is its marked tendency to recur. About 25% of women with a first episode of UTI have a second episode within 6 months (8). An ascending route of infection is the most common pathogenic mechanism and involves the spontaneous ascent of bacteria from the urethra to the bladder and to the kidneys. Adhesions of uropathogenic E. coli are specific lectin-like structures which are expressed by the bacteria and enable them to attach to and colonize the uroepithelium and to initiate infectious processes (14). Epidemiological studies of clinical isolates from UTI and diarrhea suggest that children and pregnant women are predisposed to infection by the E. coli-expressing Dr family of adhesins. As many as 50% of isolates from children with protracted diarrhea and 25 to 50% of isolates from children with cystitis express Dr adhesins (1, 10, 25), in contrast to 10 to 15% of isolates from adults, especially young healthy women, with cystitis (37, 39). E. coli Dr adhesins are associated with 30% of cases of pyelonephritis in pregnant women, particularly during the third trimester of gestation (26). The expression of Dr adhesins has been associated with a twofold-increased risk of a second episode of UTI (9). The members of the Dr family of adhesins, including Dr, Dr-II, AFA I, AFA III, and F1845, have a similar genetic organization and recognize decay-accelerating factor (DAF or CD55) as their cellular receptor (27,31).DAF is a complement-regulatory protein which protects host tissues from damage by the autologous complement system by inhibiting the formation and accelerating the decay of C3 and C5 convertases (24). DAF has a wide tissue distribution; for example, it is present on epithelial surfaces of the gastrointestinal mucosa, exocrine glands, renal pelvis, ureter, bladder, cervix, and uterine mucosa (22). Using an indirectimmunofluorescence technique, we also demonstrated that the purified Dr fimbria binds to these sites, including the renal epithelium in the urinary tract (28). DAF is a 70-kDa glycoprotein and consists of five domains, i.e., four short consensus repeats (SCR1 to SCR4) followed by a serine-threonine-rich (ST-rich) domain, and is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor (24). The other membrane-bound complement receptors of the regulators of complement activation family are membrane cofactor protein (MCP or CD46), and complement receptors 1 and 2 (CR1 or CD35, CR2 or CD21). Recent reports suggest that these complement receptors are targeted as cellular receptors by several bacteria, viruses, and parasites (2,23,33). E. coli expressing the Dr family of adhesins was the first described example of a pathogen that targets DAF as its cellular receptor (29). Of particular interest...