Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes

Pneumonia caused by Mannheimia (Pasteurella) haemolytica is a highly fatal disease of bighorn sheep (Ovis canadensis). Leukotoxin (Lkt), secreted by M. haemolytica, is an important virulence factor of this organism, and is cytolytic to bighorn sheep leukocytes. Previously, we have shown that CD18, the beta subunit of beta2 integrins, serves as the receptor for Lkt on bovine leukocytes. Furthermore, anti-CD18 antibodies inhibit Lkt-induced cytotoxicity of bighorn sheep leukocytes. Therefore, we hypothesized that Lkt utilizes CD18 as its receptor on bighorn sheep leukocytes. Confirmation of bighorn sheep CD18 as a receptor for Lkt requires the demonstration that the recombinant expression of bighorn sheep CD18 in Lkt-nonsusceptible cells renders them susceptible to Lkt. Therefore, we transfected cDNA encoding CD18 of bighorn sheep into a Lkt-nonsusceptible murine cell line. Cell surface expression of bighorn sheep CD18 on the transfectants was tested by flow cytometry with anti-CD18 antibodies. Transfectants stably expressing bighorn sheep CD18 on their surface were subjected to flow cytometric analysis for detection of Lkt binding, and cytotoxicity assays for detection of Lkt-induced cytotoxicity. Leukotoxin bound to the transfectants. More importantly, the transfectants were effectively lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. These results clearly indicate that M. haemolytica Lkt utilizes CD18 as a receptor on bighorn sheep leukocytes. Identification of CD18 as a receptor for Lkt on bighorn sheep leukocytes should enhance our understanding of the pathogenesis of pneumonia, which in turn should help in the development of control measures against this fatal disease of bighorn sheep.     

Down-regulation of glucose-regulated protein (GRP) 78 potentiates cytotoxic effect of celecoxib in human urothelial carcinoma cells

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has been reported to elicit anti-proliferative response in various tumors. In this study, we aim to investigate the antitumor effect of celecoxib on urothelial carcinoma (UC) cells and the role endoplasmic reticulum (ER) stress plays in celecoxib-induced cytotoxicity. The cytotoxic effects were measured by MTT assay and flow cytometry. The cell cycle progression and ER stress-associated molecules were examined by Western blot and flow cytometry. Moreover, the cytotoxic effects of celecoxib combined with glucose-regulated protein (GRP) 78 knockdown (siRNA), (-)-epigallocatechin gallate (EGCG) or MG132 were assessed. We demonstrated that celecoxib markedly reduces the cell viability and causes apoptosis in human UC cells through cell cycle G1 arrest. Celecoxib possessed the ability to activate ER stress-related chaperones (IRE-1α and GRP78), caspase-4, and CCAAT/enhancer binding protein homologous protein (CHOP), which were involved in UC cell apoptosis. Down-regulation of GRP78 by siRNA, co-treatment with EGCG (a GRP78 inhibitor) or with MG132 (a proteasome inhibitor) could enhance celecoxib-induced apoptosis. We concluded that celecoxib induces cell cycle G1 arrest, ER stress, and eventually apoptosis in human UC cells. The down-regulation of ER chaperone GRP78 by siRNA, EGCG, or proteosome inhibitor potentiated the cytotoxicity of celecoxib in UC cells. These findings provide a new treatment strategy against UC.     

Si RNA inhibition of GRP58 associated with decrease in mitomycin C-induced DNA cross-linking and cytotoxicity

The anti-cancer drug mitomycin C is metabolically activated to bind and cross-link DNA. The cross-linking contributes significantly to the cytotoxicity. The complex chemical structure of mitomycin C allows its metabolism by several known (cytosolic NAD(P)H:quinone oxidoreductase and microsomal NADPH:cytochrome P450 reductase) and unknown enzymes. The identification of new enzymes/proteins that metabolize mitomycin C and like drugs is an area of significant research interest since these studies have direct implications in drug development and clinical usage. In the present studies, we have investigated a role of cytosolic glucose regulatory protein GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. The control and GRP58 siRNA were transfected in human colon carcinoma HCT116 cells in culture. The transfection of GRP58 siRNA but not control siRNA significantly inhibited GRP58 in human colon carcinoma HCT116 cells. The inhibition of GRP58 led to decrease in mitomycin C-induced DNA cross-linking and cytotoxicity. These results establish a role of GRP58 in mitomycin C-induced DNA cross-linking and cytotoxicity. Site-directed mutagenesis of cysteines to serines in thioredoxin domains of GRP58 and cross-linking assays revealed that both N- and C-terminal thioredoxin domains are required for GRP58-mediated mitomycin C-induced DNA cross-linking. These results suggest that GRP58 might be an important target enzyme for further studies on mitomycin C and similar drug therapy.     

Gastrin releasing peptide (GRP): effects on basal and stimulated insulin and glucagon secretion in the mouse

GRP is a pancreatic neuropeptide and may be of importance for the neural control of insulin and glucagon secretion. In this study, we investigated the effects of GRP on basal and stimulated insulin and glucagon secretion in the mouse. Intravenous injections of GRP at dose levels exceeding 2.12 nmol/kg were found to rapidly increase basal plasma levels of both insulin and glucagon. Furthermore, at a low dose level without effect on basal plasma insulin levels, GRP was found to potentiate the insulin response to both glucose (by 40%; p less than 0.05) and to the cholinergic agonist carbachol (by 57%; p less than 0.01). Also, GRP was at this dose level found to potentiate the glucagon response to carbachol (p less than 0.01). Glucose abolished GRP-induced glucagon secretion. Moreover, methylatropine given at a dose level that totally abolishes carbachol-induced insulin secretion inhibited GRP-induced insulin secretion by 39% (p less than 0.05) and GRP-induced glucagon secretion by 25% (p less than 0.01). L-Propranolol at a dose level that totally abolishes beta-adrenergically-induced insulin secretion inhibited GRP-induced insulin secretion by 52% (p less than 0.01) and GRP-induced glucagon secretion by 15% (p less than 0.05). In summary, we have shown that GRP stimulates basal and potentiates stimulated insulin and glucagon secretion in mice, and that the stimulatory effects of GRP on insulin and glucagon secretion are partially inhibited by muscarinic blockade by methylatropine or by beta-adrenoceptor blockade by propranolol. We conclude that GRP activates potently both insulin and glucagon secretion in the mouse by mechanisms that are partially related to the muscarinic and the beta-adrenergic receptors.     

Specific binding of platelet-activating factor (PAF) by human peripheral blood mononuclear leukocytes

Binding of platelet-activating factor (PAF) to human peripheral blood mononuclear leukocytes was time-dependent, reversible, and saturable. [3H]PAF binding to the cells was inhibited dose-dependently by unlabeled PAF and PAF receptor antagonists: L-659,989, triazolam, and alprazolam. Scatchard analysis of saturation binding data indicated one class of receptors for PAF with KD = 5.7 nM and Bmax = 18 fmol/10(6) cells (11,100 receptors/cell). PAF (10 nM) increased intracellular free calcium concentration in human lymphocytes and this effect was inhibited by L-659,989 dose-dependently. Our data suggest that human peripheral blood mononuclear leukocytes have specific receptors for PAF.     

Pharmacology and selectivity of various natural and synthetic bombesin related peptide agonists for human and rat bombesin receptors differs

The mammalian bombesin (Bn)-receptor family [gastrin-releasing peptide-receptor (GRPR-receptor), neuromedin B-receptor (NMB receptor)], their natural ligands, GRP/NMB, as well as the related orphan receptor, BRS-3, are widely distributed, and frequently overexpressed by tumors. There is increased interest in agonists for this receptor family to explore their roles in physiological/pathophysiological processes, and for receptor-imaging/cytotoxicity in tumors. However, there is minimal data on human pharmacology of Bn receptor agonists and most results are based on nonhuman receptor studies, particular rodent-receptors, which with other receptors frequently differ from human-receptors. To address this issue we compared hNMB-/GRP-receptor affinities and potencies/efficacies of cell activation (assessing phospholipase C activity) for 24 putative Bn-agonists (12 natural, 12 synthetic) in four different cells with these receptors, containing native receptors or receptors expressed at physiological densities, and compared the results to native rat GRP-receptor containing cells (AR42J-cells) or rat NMB receptor cells (C6-glioblastoma cells). There were close correlations (r = 0.92-99, p < 0.0001) between their affinities/potencies for the two hGRP- or hNMB-receptor cells. Twelve analogs had high affinities (≤1 nM) for hGRP receptor with 15 selective for it (greatest = GRP, NMC), eight had high affinity/potencies for hNMB receptors and four were selective for it. Only synthetic Bn analogs containing β-alanine¹¹ had high affinity for hBRS-3, but also had high affinities/potencies for all GRP-/hNMB-receptor cells. There was no correlation between affinities for human GRP receptors and rat GRP receptors (r = 0.131, p = 0.54), but hNMB receptor results correlated with rat NMB receptor (r = 0.71, p < 0.0001). These results elucidate the human and rat GRP-receptor pharmacophore for agonists differs markedly, whereas they do not for NMB receptors, therefore potential GRP-receptor agonists for human studies (such as Bn receptor-imaging/cytotoxicity) must be assessed on human Bn receptors. The current study provides affinities/potencies on a large number of potential agonists that might be useful for human studies.     

Reprint of "Improved spectral resolution in COSY (1)H NMR spectra of proteins via double quantum filtering"

Polyglutamine (polyQ) diseases are inherited neurodegenerative diseases characterized by the aggregation of proteins containing expanded polyQ tract. It has been shown that expanded polyQ tract-containing proteins impair the functions of other cellular proteins. However, quantitative changes of cellular proteins in cells expressing expanded polyQ tract-containing proteins have not been performed. Here, we performed proteomic analysis of cells expressing expanded polyQ tract-containing proteins, and showed that GRP78, the endoplasmic reticulum (ER) chaperone, was significantly decreased in the cells expressing enhanced green fluorescent protein with a pathological-length polyQ tract (EGFP-polyQ97), but not with a non-pathological-length polyQ tract (EGFP-polyQ24). In addition, we revealed that down-regulation of GRP78 expression resulted in increase of the aggregation of EGFP-polyQ97. Conversely, the aggregation of EGFP-polyQ97 was suppressed by the overexpression of GRP78 in the cells. Furthermore, it seemed that the decreased GRP78 expression in the cells expressing EGFP-polyQ97 was due to the enhanced protein degradation of GRP78 through the ubiquitin-proteasome pathway. These findings indicated that GRP78, which has an inhibitory effect on the aggregation of proteins containing expanded polyQ tract, may be an effective target for the treatment of polyQ diseases.     

Overexpression of the 78-kDa glucose-regulated protein/immunoglobulin-binding protein (GRP78/BiP) inhibits tissue factor procoagulant activity

Previous studies have demonstrated that overexpression of GRP78/BiP, an endoplasmic reticulum (ER)-resident molecular chaperone, in mammalian cells inhibits the secretion of specific coagulation factors. However, the effects of GRP78/BiP on activation of the coagulation cascade leading to thrombin generation are not known. In this study, we examined whether GRP78/BiP overexpression mediates cell surface thrombin generation in a human bladder cancer cell line T24/83 having prothrombotic characteristics. We report here that cells overexpressing GRP78/BiP exhibited significant decreases in cell surface-mediated thrombin generation, prothrombin consumption and the formation of thrombin-inhibitor complexes, compared with wild-type or vector-transfected cells. This effect was attributed to the ability of GRP78/BiP to inhibit cell surface tissue factor (TF) procoagulant activity (PCA) because conversion of factor X to Xa and factor VII to VIIa were significantly lower on the surface of GRP78/BiP-overexpressing cells. The additional findings that (i) cell surface factor Xa generation was inhibited in the absence of factor VIIa and (ii) TF PCA was inhibited by a neutralizing antibody to human TF suggests that thrombin generation is mediated exclusively by TF. GRP78/BiP overexpression did not decrease cell surface levels of TF, suggesting that the inhibition in TF PCA does not result from retention of TF in the ER by GRP78/BiP. The additional observations that both adenovirus-mediated and stable GRP78/BiP overexpression attenuated TF PCA stimulated by ionomycin or hydrogen peroxide suggest that GRP78/BiP indirectly alters TF PCA through a mechanism involving cellular Ca(2+) and/or oxidative stress. Similar results were also observed in human aortic smooth muscle cells transfected with the GRP78/BiP adenovirus. Taken together, these findings demonstrate that overexpression of GRP78/BiP decreases thrombin generation by inhibiting cell surface TF PCA, thereby suppressing the prothrombotic potential of cells.     

Natural killer cell-mediated cytotoxicity does not depend on recognition of mannose 6-phosphate residues

Interaction of mannose 6-phosphate-specific receptors with their ligands has been suggested to be essential for natural killer cell (NK)-mediated cytotoxicity. Indeed, mannose 6-phosphate-specific receptors and ligands bearing mannose 6-phosphate residues are demonstrable on human peripheral blood leukocytes with NK activity as well as on K-562 NK target cells, allowing at least in principle such an interaction. It can also be shown that NK activity of human peripheral blood leukocytes is inhibited by mannose 6-phosphate. The following observations, however, exclude an essential role of the mannose 6-phosphate receptor-ligand system in NK cell-mediated cytotoxicity. 1) NK cytotoxicity is sensitive to a broad range of structurally unrelated sugar phosphates. 2) NK activity is normal in patients with I cell disease (mucolipidosis II), which due to a genetic defect are unable to synthesize the ligands for the mannose 6-phosphate-specific receptor. 3) NK cytotoxicity is not inhibited by an antiserum against the mannose 6-phosphate receptor, which blocks the receptor function.     

Triggering of murine NK cells by CD40 and CD86 (B7-2)

NK cell-mediated cytotoxicity is regulated by both triggering and inhibitory signals. The interaction between MHC class I molecules expressed on target cells and specific MHC class I-binding receptors expressed by NK cells generally leads to inhibition of lysis. We have shown recently that CD80 (B7-1) in mice and CD40 in humans trigger NK cell-mediated cytotoxicity in vitro. In the present study, we show that murine CD40 and CD86 (B7-2) trigger murine NK cell-mediated cytotoxicity in vitro when expressed on tumor cells. Preincubation of the transfected cell lines with anti-CD40 F(ab')2 fragments or cytolytic T lymphocyte-associated Ag-4-Ig (CTLA-4-Ig) before the cytotoxic assay abolished the triggering effect. Furthermore, radiolabeled CD40- and B7-2-expressing cells were rapidly eliminated in vivo in an NK cell-dependent manner. NK cells from CD40 ligand (CD40L)-/- or CD28-/- mice were triggered by tumor cells transfected with CD40 and B7-2, respectively, and these transfectants were rapidly eliminated in vivo when inoculated into CD40L-/- and CD28-/- mice. This suggests that the CD40 and B7-2 molecules can interact with receptors on NK cells other than CD40L and CD28, respectively, and that these may account for some of the reactivities observed in the present study. Collectively, these data demonstrate that 1) costimulatory molecules, other than B7-1, can modulate NK cell responses in vitro, 2) they can also affect NK cell-dependent responses in vivo, and 3) parts of these reactions are independent of CD28 and CD40L.     

Modulation of gastrin-releasing peptide (GRP) receptors in insulin secreting cells

Gastrin-releasing peptide (GRP) receptors are present in pancreatic islets, though their regulation is unknown except for homologous desensitization. The modulation of binding of GRP to mouse pancreatic islets and INS-1 cells was studied. At 60 min (steady-state), total binding of [(125)I-Tyr(15)] GRP was 1.62 per cent of total radioactivity per 50 islets; non-specific binding (presence of 1 mM unlabelled GRP(1-27)) was 0.05 to 0.61 per cent of total radioactivity. A preincubation with 1000 nM cholecystokinin (CCK(8)) or with 1000 nM glucose-dependent insulinotropic peptide (GIP) augmented the number of GRP binding sites but not their affinity. [(125)I-Tyr(15)]GRP binding to INS-1 cells was saturable (90 min) and specific with respect to compounds that are not chemically related to GRP (e.g. calcitonin gene-regulated peptide-CGRP and atrial natriuretic peptide-ANP). Displacement studies showed one binding site with a K(d) of 0.39 nM and a B(max) of 13.2 fmoles mg(-1) protein. When the cells were pretreated for 24 h with 10 nM GIP or CCK(8), only GIP but not CCK(8) increased the B(max) of the GRP binding site. The affinity (K(d)) was not changed by either compound. This effect of GIP pretreatment was not affected by downregulating PKC by TPA (phorbol ester; long-term pretreatment). These data indicate that: (1) specific binding sites for GRP are present in mouse pancreatic islets and INS-1 cells; (2) the GRP binding is upregulated by GIP in both islets and INS-1 cells and additionally by CCK(8 ), albeit only in islets; and (3) PKC does not seem to be involved in the up-regulation process. Thus a positive interplay between both the incretins GIP and CCK(8) and the neurotransmitter GRP is obvious.     

Binding of GRP(14-27) but not bombesin or GRP(1-27) to hypothalamic magnocellular elements: an immunohistochemical study

Bombesin, gastrin-releasing peptide (1-27), and gastrin-releasing peptide (14-27) abolished the specific immunocytochemical staining revealed by antiserum directed to the C-terminus of gastrin releasing peptide (GRP) and bombesin (BN) in rat hypothalamus. When the antiserum was preabsorbed with GRP(14-27), a strong reaction appeared in hypothalamic magnocellular neurons. This staining of magnocellular elements was produced by lower concentrations of GRP(14-27) than were needed to block immunocytochemical staining revealed by the antiserum in other hypothalamic locations. The distribution of GRP(14-27)-induced immunostaining was similar to that of neurophysin. Since only GRP(14-27) but not GRP(1-27) or bombesin was found to bind to magnocellular cells, it was concluded that binding was due to the N-terminus of GRP(14-27), which resembles the structure of oxytocin and vasopressin. In agreement with this, oxytocin and vasopressin were found to prevent the binding of GRP(14-27) to magnocellular cells. The similarity in localization and the effect of oxytocin and vasopressin suggest that GRP(14-27) may bind to neurophysin at low concentrations. The results suggest that enhancement of staining after preabsorption of antisera with antigens must be interpreted with care. Enhancement can occur at antigen concentrations lower than those required to block the immunostaining. These results fail to support the premise that antigen-induced enhancement of staining is due to antigen binding to specific receptors and subsequent detection of the receptor-bound antigen with the antiserum.     

Involvement of mitochondria in endoplasmic reticulum stress-induced apoptotic cell death pathway triggered by the prion peptide PrP(106-126)

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrP^Sc. Recently, we have shown that PrP_106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP_106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP_106–126 induces ER stress in both cell lines, given that ER Ca²⁺ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP_106–126-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP_106–126-induced cell death, demonstrating the involvement of Ca²⁺ release through ER ryanodine receptors. However, in PrP_106–126-treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders.     

Cutting edge: CD96 (tactile) promotes NK cell-target cell adhesion by interacting with the poliovirus receptor (CD155)

The poliovirus receptor (PVR) belongs to a large family of Ig molecules called nectins and nectin-like proteins, which mediate cell-cell adhesion, cell migration, and serve as entry receptors for viruses. It has been recently shown that human NK cells recognize PVR through the receptor DNAM-1, which triggers NK cell stimulation in association with beta(2) integrin. In this study, we show that NK cells recognize PVR through an additional receptor, CD96, or T cell-activated increased late expression (Tactile). CD96 promotes NK cell adhesion to target cells expressing PVR, stimulates cytotoxicity of activated NK cells, and mediates acquisition of PVR from target cells. Thus, NK cells have evolved a dual receptor system that recognizes nectins and nectin-like molecules on target cells and mediates NK cell adhesion and triggering of effector functions. As PVR is highly expressed in certain tumors, this receptor system may be critical for NK cell recognition of tumors.     

Effects of bombesin on human small cell lung cancer cells: evidence for a subset of bombesin non-responsive cell lines

The effects of bombesin on three human small cell lung carcinoma cell (SCLC) lines (NCI-H69, NCI-H128, and NCI-H345) have been examined and compared to the effects of the peptide on the mouse fibroblast cell line Swiss 3T3, and the rat pituitary tumor cell line GH3W5. While all three SCLC lines expressed messenger RNA encoding pro-gastrin releasing peptide (GRP), only the NCI-H345 cells expressed detectable membrane receptors for GRP and responded to nanomolar concentrations of bombesin as shown by 125I-GRP binding, total inositol phosphate accumulation, and increased clonal growth in soft agarose. These data show that some SCLC lines are insensitive to bombesin and do not express detectable membrane receptors for GRP.     

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.     

Human Thy-1 (CD90) on activated endothelial cells is a counterreceptor for the leukocyte integrin Mac-1 (CD11b/CD18)

Leukocyte recruitment in response to inflammatory signals is in part governed by interactions between endothelial cell receptors belonging to the Ig superfamily and leukocyte integrins. In our previous work, the human Ig superfamily glycoprotein Thy-1 (CD90) was identified as an activation-associated cell adhesion molecule on human dermal microvascular endothelial cells. Furthermore, the interaction of Thy-1 with a corresponding ligand on monocytes and polymorphonuclear cells was shown to be involved in the adhesion of these leukocytes to activated Thy-1-expressing endothelial cells. In this study, we have identified the specific interaction between human Thy-1 and the leukocyte integrin Mac-1 (CD11b/CD18; alphaMbeta2) both in cellular systems and in purified form. Monocytes and polymorphonuclear cells were shown to adhere to transfectants expressing human Thy-1 as well as to primary Thy-1-expressing human dermal microvascular endothelial cells. Furthermore, leukocyte adhesion to activated endothelium as well as the subsequent transendothelial migration was mediated by the interaction between Thy-1 and Mac-1. This additional pathway in leukocyte-endothelium interaction may play an important role in the regulation of leukocyte recruitment to sites of inflammation.     

Targeting the Unfolded Protein Response in Glioblastoma Cells with the Fusion Protein EGF-SubA

Rapidly growing tumors require efficient means to allow them to adapt to fluctuating microenvironments consisting of hypoxia, nutrient deprivation, and acidosis. The unfolded protein response (UPR) represents a defense mechanism allowing cells to respond to these adverse conditions. The chaperone protein GRP78 serves as a master UPR regulator that is aberrantly expressed in a variety of cancers, including glioma. Therefore, cancer cells may be particularly reliant upon the adaptive mechanisms offered by the UPR and targeting GRP78 may represent a unique therapeutic strategy. Here we report that diffuse expression of GRP78 protein is present in Grade III-IV, but not Grade I-II glioma. To determine the role GRP78 plays in glioblastoma tumorigenesis, we explored the anti-tumor activity of the novel fusion protein EGF-SubA, which combines EGF with the cytotoxin SubA that has been recently shown to selectively cleave GRP78. EGF-SubA demonstrated potent tumor-specific proteolytic activity and cytotoxicity in glioblastoma lines and potentiated the anti-tumor activity of both temozolomide and ionizing radiation. To determine if the tumor microenvironment influences EGF-SubA activity, we maintained cells in acidic conditions that led to both UPR activation and increased EGF-SubA induced cytotoxicity. EGF-SubA was well tolerated in mice and led to a significant tumor growth delay in a glioma xenograft mouse model. The UPR is emerging as an important adaptive pathway contributing to glioma tumorigenesis. Targeting its primary mediator, the chaperone protein GRP78, through specific, proteolytic cleavage with the immunotoxin EGF-SubA represents a novel and promising multi-targeted approach to cancer therapy.