Jon P. Durkin

cIAP1 and cIAP2 Facilitate Cancer Cell Survival by Functioning as E3 Ligases that Promote RIP1 Ubiquitination

The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.

Loss of XIAP protein expression by RNAi and antisense approaches sensitizes cancer cells to functionally diverse chemotherapeutics

Stable expression of short-hairpin RNAs (shRNAs) directed against the X-linked inhibitor of apoptosis (XIAP) resulted in the generation of three MDA-MB-231 cell lines (XIAP shRNA cells) with reductions in XIAP mRNA and protein levels >85% relative to MDA-MB-231 cells stably transfected with the U6 RNA polymerase III promoter alone (U6 cells). This RNA interference (RNAi) approach dramatically sensitized these cells to killing by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Importantly, loss of XIAP also sensitized the cells to killing by taxanes but had no additional effects on killing by carboplatin and doxorubicin. The increased sensitivity of the XIAP shRNA cells to killing by TRAIL and taxanes correlated with enhanced caspase cleavage and activation, including caspase-8, and robust processing of poly(ADP-ribose) polymerase and BID compared to U6 cells. Additionally, increasing XIAP levels by adenovirus-mediated expression protected both XIAP shRNA and U6 cells from TRAIL killing in a dose-dependent manner. The effects observed by stable RNAi with respect to TRAIL sensitization were also achieved following downregulation of XIAP in Panc-1 cells treated with a second-generation, mixed-backbone antisense oligonucleotide, AEG 35156/GEM640. These data indicate that reducing XIAP protein expression by either RNAi or antisense approaches increases cancer cell susceptibility to functionally diverse chemotherapeutic agents and supports the notion that downregulation of XIAP in vivo may synergize with disease-relevant chemotherapeutic regimes, including TRAIL and taxanes, to increase the effectiveness of antineoplastic agents.

AMPA receptor-mediated regulation of a Gi-protein in cortical neurons

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N -methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors,. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na + through these ion channels. Here we report that the binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brought about changes in a guanine-nucleotide-binding protein (Gαi1) that inhibited pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect was observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and was also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibited forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins were activated. Moreover, both Gβγ blockage and co-precipitation experiments demonstrated that the modulation of the Gi protein arose from the association of Gαi1 with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity.

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood–brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3‐neo coding for the SV40 large T antigen and the neomycin gene. The neomycin‐resistant transfected cells overcame proliferative senescence, and after a 6–8 wk period of crisis produced immortalization‐competent cell colonies. Single‐cell clones of near‐diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV‐HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV‐HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII‐related antigen, uptake of acetylated low‐density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor‐mediated endocytosis, and high activities of the BBB‐specific enzymes alkaline phosphatase and γ‐glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV‐HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV‐HCEC monolayers by 2.5‐fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.—Muruganandam, A., Herx, L. M., Monette, R., Durkin, J. P., Stanimirovic, D. B. Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood–brain barrier. FASEB J. 1187–1197 (1997)

Preclinical Characterization of AEG35156/GEM 640, a Second-Generation Antisense Oligonucleotide Targeting X-Linked Inhibitor of Apoptosis

Purpose: Cancer cells can use X-linked inhibitor of apoptosis (XIAP) to evade apoptotic cues, including chemotherapy. The antitumor potential of AEG35156, a novel second-generation antisense oligonucleotide directed toward XIAP, was assessed in human cancer models when given as a single agent and in combination with clinically relevant chemotherapeutics. Experimental Design: AEG35156 was characterized for its ability to cause dose-dependent reductions of XIAP mRNA and protein in vitro and in vivo, to sensitize cancer cell lines to death stimuli, and to exhibit antitumor activity in multiple human cancer xenograft models as a single agent or in combination with chemotherapy. Results: AEG35156 reduced XIAP mRNA levels with an EC50 of 8 to 32 nmol/L and decreased XIAP protein levels by >80%. Loss of XIAP protein correlated with increased sensitization to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–mediated apoptosis in Panc-1 pancreatic carcinoma cells. AEG35156 exhibited potent antitumor activity relative to control oligonucleotides in three human cancer xenograft models (prostate, colon, and lung) and was capable of inducing complete tumor regression when combined with taxanes. Antitumor effects of AEG35156 correlated with suppression of tumor XIAP levels. Conclusions: AEG35156 reduces XIAP levels and sensitizes tumors to chemotherapy. AEG35156 is presently under clinical assessment in multiple phase I trials in cancer patients as a single agent and in combination with docetaxel in solid tumors or cytarabine/idarubicin in leukemia.

The direct measurement of protein kinase C (PKC) activity in isolated membranes using a selective peptide substrate

A protein kinase C (PKC)-selective peptide substrate was used to develop a method for measuring PKC activity directly and quantitatively in isolated cell membranes without prior detergent extraction and reconstitution of the enzyme with phosphatidylserine and TPA in the presence of excess Ca2+. This simple and rapid method can reliably measure changes in membrane-associated PKC activity induced by various bioactive compounds such as hormones and growth factors. Also, this method, which measures PKC activity in its native membrane-associated state, has the advantage of being able to distinguish between active and inactive PKC associated with cell membranes.

The induction of ICAM-1 in human cerebromicrovascular endothelial cells (HCEC) by ischemia-like conditions promotes enhanced neutrophil/HCEC adhesion.

Ischemic brain injury is exacerbated by leukocyte infiltration and formation of vasogenic edema. In this study we demonstrate that intercellular adhesion molecule-1 (ICAM-1) is dramatically (3 to 15-fold) up-regulated in human cerebromicrovascular endothelial cells (HCEC) by a 16 h exposure to the cytokine, IL-1 beta (50-200 u/ml), the phorbol ester, TPA (1-100 nM), or by simulated in vitro ischemia/reperfusion. These treatments also significantly increased the adhesion of allogeneic neutrophils to HCEC monolayers. Both IL-1 beta- and TPA-induced expression of ICAM-1 and increased neutrophil adhesion to HCEC were inhibited by the transcriptional inhibitor, actinomycin D (AcD; 1-10 micrograms/ml), and by an anti-ICAM-1 antibody (ICAM-1 Ab). By contrast, ischemia-induced neutrophil adhesion was only slightly affected by AcD and ICAM-1 Ab alone, but it was abolished by the combination of anti-ICAM-1 and anti-CD18 antibodies. The increase in surface expression of ICAM-1 and neutrophil adhesion by IL-1 beta, TPA and ischemia were significantly reduced by the cyclo-oxygenase (COX) inhibitors, indomethacin (100-300 microM) and dexamethasone (10-50 microM). These results indicate that ICAM-1 expression in HCEC can lead to enhanced neutrophil adhesion and that COX activation in HCEC likely plays a role in the processes by which leukocyte adhesion and recruitment take place in the brain during inflammation and ischemia in vivo.

Evidence that the early loss of membrane protein kinase C is a necessary step in the excitatory amino acid-induced death of primary cortical neurons

Abstract: A rapid loss of protein kinase C (PKC) activity is a prognostic feature of the lethal damage inflicted on neurons by cerebral ischemia in vivo and by hypoxic and excitotoxic insults in vitro. However, it is not known if this inactivation of PKC is incidental or is an essential part of the neurodegenerative process driven by such insults. To address this issue, the effects of glutamate on PKC activity and neurotoxicity were studied in immature [8 days in vitro (DIV)] and mature (15–20 DIV) embryonic day 18 rat cortical neuronal cultures. Exposing 16 DIV neurons to as little as 20–50 µM glutamate for 15 min was neurotoxic and induced a rapid (∼1–2 h) Ca2+‐dependent inactivation of membrane PKC. By contrast, neurons 8 DIV were resistant to >800 µM glutamate, and no evidence of PKC inactivation was observed. Reverse transcription‐polymerase chain reaction analysis of NMDA and AMPA receptor subtypes and fluorometric intracellular Ca2+ concentration measurements of the effects of NMDA, AMPA, kainate, and metabotropic glutamate receptor activation demonstrated that this striking difference in vulnerability was not due to an absence of functional glutamate receptor on neurons 8 DIV. However, 8 DIV neurons became highly vulnerable to low (<20 µM) concentrations of glutamate when PKC activity was inhibited by 50 nM staurosporine, 1 µM calphostin C, 5 µM chelerythrine, or chronic exposure to 100 nM PMA. A 15‐min coapplication of 50 nM staurosporine with glutamate, NMDA, AMPA, or kainate killed between 50 and 80% of 8 DIV cells within the ensuing 24 h. Moreover, cell death was observed in these cells even when PKC inactivation was delayed up to 4 h after glutamate removal. The evidence indicates that a loss of PKC activity is an essential element of the excitotoxic death of neurons 8 DIV and that cellular event(s) responsible for linking glutamate‐mediated Ca2+ influx to PKC inactivation in vulnerable neurons 16 DIV are undeveloped in resistant cells 8 DIV. These results also suggest that the loss of neuronal PKC activity observed in cerebral ischemia may indeed be an important part of the neurodegenerative process. The 8 DIV/16 DIV cortical cell model may prove to be valuable in discerning those intracellular signaling events critical to glutamate‐mediated neuronal death.

Evaluation of glutathione-sensitive fluorescent dyes in cortical culture

The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione‐S‐transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t‐butoxycarbonyl‐Leu‐Met‐7‐amino‐4‐chloromethylcoumarin (CMAC), 7‐amino‐4‐chloromethylcoumarin (CMAC‐blue), monochlorobimane (MCB), and 5‐chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3‐naphthalenedicarboxaldehyde (NDA) and o‐phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL‐buthionine‐(S,R)‐sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC‐blue, and MCB and partial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocytes between 5–6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC‐blue and MCB. This decrease in staining was not due to a decrease in GST activity, as neurons stained with the GST‐insensitive OPD also exhibited a decline in GSH‐sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co‐localized with GFAP‐positive astrocytes, but not with MAP‐2‐positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve astrocytes in living mixed cultures co‐loaded with the Ca2+‐sensitive dye, calcium green 5N‐AM. GLIA 30:329–341, 2000. Published 2000 Wiley‐Liss, Inc.

Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR11XX, NR10XX, and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.