Loss of expression of death-inducing signaling complex (DISC) components in lung cancer cell lines and the influence of MYC amplification.

We have previously reported that the key apoptosis related gene caspase 8 (CASP8) is frequently silenced in small cell lung cancer (SCLC) tumors and cell lines usually, but not always, by aberrant promoter methylation. Because CASP8 is a key component of the death-inducing signaling complex (DISC) when specific death receptors (including DR4, DR5, FAS) are activated by their specific ligands (TRAIL/FASL), we examined expression of the components of the DISC complex in lung cancer cell lines. MYC family members are frequently amplified (MYC+ve) in SCLC, and MYC is a potent inducer of apoptosis.

The human herpes virus 8-encoded viral FLICE inhibitory protein protects against growth factor withdrawal-induced apoptosis via NF-kappa B activation.

The human herpes virus 8 (HHV8)-encoded viral FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme) inhibitory protein (vFLIP) is believed to protect cells against death receptor-mediated apoptosis. In the present study we demonstrate that expression of HHV8 vFLIP in a growth factor-dependent TF-1 leukemia cell line protects against growth factor withdrawal-induced apoptosis. Unlike vector-expressing cells, those expressing HHV8 vFLIP maintain their mitochondrial membrane potential upon withdrawal from growth factor and also exhibit a block in the activation of caspases.

Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines.

Methylation of the promoter regions of CpG-rich sites in genes is the major mechanism for the silencing of many genes in tumors. Methylation of the key apoptosis-related gene caspase 8 (CASP8) has been reported in some childhood tumors and in neuroendocrine lung tumors. We examined the methylation status of 181 pediatric tumors and found frequent methylation in rhabdomyosarcomas (83%), medulloblastomas (81%), retinoblastomas (59%), and neuroblastomas (52%).

Role of TRAF3 and -6 in the activation of the NF-kappa B and JNK pathways by X-linked ectodermal dysplasia receptor.

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to be highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2). By using a subclone of 293F cells with stable expression of XEDAR, we report that XEDAR activates the NF-kappaB and JNK pathways in an EDA-A2-dependent fashion. Treatment with EDA-A2 leads to the recruitment of TRAF3 and -6 to the aggregated XEDAR complex, suggesting a central role of these adaptors in the proximal aspect of XEDAR signaling.

Expression of VPAC2 receptor and PAC1 receptor splice variants in the trigeminal ganglion of the adult rat.

PACAP and VIP are members of the VIP/secretin/glucagon family of peptides with neurotransmitter, neuroprotective, and neurotrophic functions. PACAP and VIP are known to be upregulated in primary sensory neurons following nerve injury, implying that these neuropeptides could be mediators of sensory transmission in neuropathic pain states. Nerve injury at the level of the trigeminal root is thought to be the prime cause of trigeminal neuralgia.

Differential inactivation of caspase-8 in lung cancers.

Caspase-8 (CASP8) is an apoptosis inducing cysteine protease which is activated through the formation of a death-inducing signaling complex when death receptors are complexed to their specific ligands. Recent reports indicate that CASP8 expression is lost via a combination of promoter methylation and allelic loss in subset of neuroblastomas. We investigated the state of the gene in lung tumors and cell lines.

The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex.

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process.

Inhibition of the death receptor pathway by cFLIP confers partial engraftment of MHC class I-deficient stem cells and reduces tumor clearance in perforin-deficient mice.

NK cells mediate acute rejection of MHC class I-deficient bone marrow cell (BMC) grafts. However, the exact cytotoxic mechanisms of NK cells during acute BMC graft rejection are not well defined. Although the granule exocytosis pathway plays a major role in NK cell-mediated rejection, alternative perforin-independent mechanisms also exist.

Neuroprotective effect of alpha(2) agonist (brimonidine) on adult rat retinal ganglion cells after increased intraocular pressure.

Brimonidine, a selective alpha(2)-adrenoceptor agonist, has recently been shown to be neuroprotective as it significantly improves survival of retinal ganglion cells (RGCs) after calibrated optic nerve injury in rats. In the present study, we examined the effect of brimonidine (alpha(2)-adrenoceptor agonist) on RGC survival after increased intraocular pressure (IOP) in adult rats. RGCs were prelabeled by bilateral tectal injection of 5% Fluoro-Gold (FG).

Preparation and studies with 90Y-labelled particles for use in radiation synovectomy.

90Y-FHMA (Ferric hydroxide macroaggregates) and 90Y-HA (hydroxyapatite) were prepared in >95% yield using 90Y from an in-house 90Sr-90Y generator. Most of the particles ranged from 5 to 20 microm in diameter and retained radiochemical purity > 95% in human serum for at least 7 days at 37 degrees C. Gamma camera imaging of normal rabbits after intraarticulation of the particles showed complete retention of activity within the knee cavity with no measurable radioactivity leaching out of the joints over a 96 hour period.

Vanilloid receptor expression and capsaicin excitation of rat dental primary afferent neurons.

Little is known about the molecular mechanisms that cause excitation of neurons which innervate the teeth. We investigated whether rat dental sensory neurons express the vanilloid (capsaicin) receptor (VR1). Dental sensory neurons were identified by retrograde transport of the fluorescent dye DiIC18 placed in maxillary molars.

Cytotoxicity of Tumor necrosis factor related apoptosis-inducing ligand towards Ewing's sarcoma cell lines.

Death ligands of the Tumor Necrosis Factor (TNF) family are known to induce apoptosis upon binding to their cognate receptors. However, the clinical utility of these cytokines as anticancer agents has been limited due to unacceptable toxicity. TRAIL is a recently isolated death ligand that possesses selective anti-tumor activity against a number of cancer cell lines without significant systemic toxicity.

Effects of increased intraocular pressure on rat retinal ganglion cells.

The effects of elevated intraocular pressure (IOP) on the morphology of rat retinal ganglion cells (RGCs) was analyzed in this study. After cauterizing two limbal derived episcleral veins, IOP in experimental eyes was elevated 1.5--1.

The ectodermal dysplasia receptor activates the nuclear factor-kappaB, JNK, and cell death pathways and binds to ectodysplasin A.

The ectodermal dysplasia receptor (EDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to play a key role in the process of ectodermal differentiation. We present evidence that EDAR is capable of activating the nuclear factor-kappaB, JNK, and caspase-independent cell death pathways and that these activities are impaired in mutants lacking its death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death domain, it did not interact with the death domain-containing adaptor proteins TRADD and FADD.

Activation of the NF-kappaB pathway by caspase 8 and its homologs.

Caspase 8 is the most proximal caspase in the caspase cascade and has been known for its role in the mediation of cell death by various death receptors belonging to the TNFR family. We have discovered that Caspase 8 can activate the NF-kappaB pathway independent of its activity as a pro-apoptotic protease. This property is localized to its N-terminal prodomain, which contains two homologous death effector domains (DEDs).

TAJ, a novel member of the tumor necrosis factor receptor family, activates the c-Jun N-terminal kinase pathway and mediates caspase-independent cell death.

We have isolated a novel member of the TNFR family, designated TAJ, that is highly expressed during embryonic development. TAJ possesses a unique cytoplasmic domain with no sequence homology to the previously characterized members of the TNFR family. TAJ interacts with the TRAF family members and activates the JNK pathway when overexpressed in mammalian cells.

Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298.

An endothelial nitric oxide synthase (eNOS) polymorphism in exon 7 (894 G/T) resulting in glutamate or aspartate, respectively, at position 298 on the protein is correlated with severity of cardiopulmonary diseases. Because glutamate and aspartate are considered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene. We now show in transfected cells, primary human endothelial cells, and human hearts, that eNOS with aspartate, but not glutamate, at position 298 is cleaved, resulting in the generation of 100-kDa and 35-kDa products.

Modulation of the NF-kappa B pathway by virally encoded death effector domains-containing proteins.

Death Effector Domains (DEDs) have been known to mediate the recruitment of Caspase 8 and its homologs to the aggregated death-inducing signaling complex (DISC), consisting of the death domain (DD)-containing receptors and various signaling proteins. In addition, several viruses were recently shown to encode proteins with DEDs (also called FLICE inhibitory proteins or vFLIPs) which have the ability of blocking cell death induced by DD-containing receptors. We provide evidence that vFLIPs can also modulate the NF-kappaB pathway and physically interact with several signaling proteins, such as the TRAFs, RIP, NIK and the IKKs.

Activation of the c-Jun N-terminal kinase/stress-activated protein kinase pathway by overexpression of caspase-8 and its homologs.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases.

GrpL, a Grb2-related adaptor protein, interacts with SLP-76 to regulate nuclear factor of activated T cell activation.

Propagation of signals from the T cell antigen receptor (TCR) involves a number of adaptor molecules. SH2 domain-containing protein 76 (SLP-76) interacts with the guanine nucleotide exchange factor Vav to activate the nuclear factor of activated cells (NF-AT), and its expression is required for normal T cell development. We report the cloning and characterization of a novel Grb2-like adaptor molecule designated as Grb2-related protein of the lymphoid system (GrpL).

Caspase inhibitors block the retinal ganglion cell death following optic nerve transection.

Retinal ganglion cells die by apoptosis following axotomy. The molecular mechanisms of the retinal ganglion cell death are not well understood. In the present study using RT-PCR and in situ hybridization techniques we demonstrated that levels of mRNA for Bcl-2 and Bcl-x decreased after axotomy.

Endothelial nitric oxide synthase as a potential susceptibility gene in the pathogenesis of emphysema in alpha1-antitrypsin deficiency.

A role for endothelial nitric oxide synthase (NOS3) in the susceptibility of individuals with alpha1-antitrypsin (alpha1AT) deficiency to destructive lung disease was evaluated. Six polymorphic sites were identified within the NOS3 gene (i.e.

OPG/FDCR-1, a TNF receptor family member, is expressed in lymphoid cells and is up-regulated by ligating CD40.

We have cloned a TNFR family member from a follicular dendritic cell (FDC)-like cell line, FDC-1. This molecule, FDC-derived receptor-1 (FDCR-1), is identical to osteoprotegerin (OPG), a soluble cytokine that regulates osteoclast differentiation. Recently, OPG/FDCR-1 has been characterized as a second receptor for receptor activator of NF-kappaB ligand (RANKL)/TNF-related activation-induced cytokine (TRANCE), a primarily T-cell restricted TNF family member that augments dendritic cell (DC) function.

Lipopolysaccharide mediates endothelial apoptosis by a FADD-dependent pathway.

Endothelial cells play a pivotal role in the inflammatory process by coordinating the recruitment of inflammatory cells to sites of tissue injury. Lipopolysaccharide (LPS) activates many of the proinflammatory and procoagulant responses of endothelial cells, and endothelial injury is thought to play a crucial role in the pathogenesis of septic shock due to Gram-negative bacteria. The receptor used by LPS to signal endothelial responses has not been identified.