Biomedical subjects

R G Korneluk

Publications and source records attributed to R G Korneluk.

At least 19 recordsLinked to original sources

PubMed2006

Detachment-induced upregulation of XIAP and cIAP2 delays anoikis of intestinal epithelial cells.

Detachment of normal epithelial cells from the extracellular matrix triggers apoptosis, a phenomenon called anoikis. Conversely, carcinoma cells tend to be relatively more anoikis-resistant than their normal counterparts, and this increased resistance represents a critical feature of the malignant phenotype. Mechanisms that control susceptibility and resistance to anoikis are not fully understood. It is now known that detachment of non-malignant epithelial cells triggers both pro- and antiapoptotic signals, and it is the balance between these signals and the duration of detachment that determine further fate of the cells. Detachment-induced antiapoptotic events delay anoikis and if cells reattach relatively soon after detachment they survive. Direct regulators of apoptosis responsible for this delay of anoikis are unknown. We found that detachment of non-malignant intestinal epithelial cells triggers upregulation of inhibitors of apoptosis protein (IAP) family, such as X-chromosome-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis-2 (cIAP2). We demonstrated that this upregulation requires detachment-dependent activation of the transcription factor nuclear factor-kappaB. We further observed that various IAP antagonists accelerate anoikis, indicating that upregulation of the IAPs delays detachment-triggered apoptosis. We conclude that the IAPs are important regulators of the balance between detachment-triggered life and death signals. Perhaps, not by coincidence, these proteins are often upregulated in carcinomas, tumors composed of cells that tend to be anoikis-resistant.

Anoikis↗

PubMed2006

XIAP-mediated neuroprotection in retinal ischemia.

Retinal ischemia results in the loss of vision in a number of ocular diseases including acute glaucoma, diabetic retinopathy, hypertensive retinopathy and retinal vascular occlusion. Recent studies have shown that most of the neuronal death that leads to loss of vision results from apoptosis. XIAP-mediated gene therapy has been shown to protect a number of neuronal types from apoptosis but has never been assessed in retinal neurons following ischemic-induced cell death. We injected an adeno-associated viral vector expressing XIAP or GFP into rat eyes and 6 weeks later, rendered them ischemic by raising intraocular pressure. Functional analysis revealed that XIAP-treated eyes retained larger b-wave amplitudes than GFP-treated eyes up to 4 weeks post-ischemia. The number of cells in the inner nuclear layer (INL) and the thickness of the inner retina were significantly preserved in XIAP-treated eyes compared to GFP-treated eyes. Similarly, there was no significant reduction in optic nerve axon numbers in XIAP-treated eyes. There were also significantly fewer TUNEL (TdT-dUTP terminal nick end labeling) positive cells in the INL of XIAP-treated retinas at 24 h post-ischemia. Thus, XIAP-mediated gene therapy imparts both functional and structural protection to the retina after a transient ischemic episode.

Animals↗

PubMed2003

Attenuation of MPTP-induced neurotoxicity and behavioural impairment in NSE-XIAP transgenic mice.

X-linked IAP protein is a potent inhibitor of cell death. Here, we describe a novel transgenic mouse in which the human XIAP gene is expressed under the control of the neuron-specific enolase promoter (NSE-xiap). We demonstrate that nigrostriatal dopamine neurons of NSE-xiap mice were resistant to the damaging effects of the dopaminergic neurotoxin MPTP. MPTP-induced reduction of striatal dopamine metabolism was also attenuated in NSE-xiap mice. Furthermore, NSE-xiap mice treated with MPTP did not exhibit deficits in exploratory behaviour in an open-field test. Taken together, these findings suggest that strategies to enhance neuronal expression of XIAP may provide therapeutic benefit for the treatment of neurodegeneration in Parkinson's disease.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2002

IAPs are essential for GDNF-mediated neuroprotective effects in injured motor neurons in vivo.

During embryonic development, and in certain neurodegenerative diseases, neurons die by apoptosis. A new family of anti-apoptotic proteins, termed inhibitors of apoptosis (IAP), suppresses apoptosis through the direct inhibition of caspases. The anti-apoptotic activity of IAPs is inhibited by second mitochondria-derived activator of caspase (Smac)/DIABLO and XAF1 (ref. 8). IAPs, as well as neurotrophic factors, can protect degenerating neurons both in vivo and in vitro. However, the downstream targets of neurotrophic factors have not yet been identified. Here, we demonstrate that XIAP and NAIP, but not HIAP2, are directly involved in the intracellular response to glial cell-derived neurotrophic factor (GDNF). In newborn rats, GDNF regulates endogenous levels of XIAP and NAIP in motor neurons after sciatic nerve axotomy. The inhibition of XIAP or NAIP activity prevents GDNF-mediated neuroprotective effects. These results suggest that XIAP and NAIP are essential for intracellular signalling of GDNF in motor neuron survival.

Animals↗

PubMed2001

Thymocyte-targeted overexpression of xiap transgene disrupts T lymphoid apoptosis and maturation.

The X-linked inhibitor of apoptosis (XIAP) and other members of the inhibitor of apoptosis (IAP) family can suppress apoptosis induced by a diverse variety of triggers. Functional studies done to date have focused on tissue culture models and adenovirus overexpression of XIAP and other IAP proteins. Here we report the phenotype of an engineered transgenic mouse overexpressing a human IAP, as well as assessing the long-term consequence of IAP overexpression. We document the relative protein expression levels of the endogenous mouse homologue to XIAP, mouse inhibitor of apoptosis (MIAP 3), within thymocyte and T cell subpopulations. The consequence of lymphoid-targeted overexpression of XIAP in transgenic mice suggests a physiological role for the endogenous protein, MIAP3. Xiap-transgenic mice accumulated thymocytes and/or T cells in primary and secondary lymphoid tissue, T cell maturation was perturbed, and transgenic thymocytes resisted a variety of apoptotic triggers both in vitro and in vivo. These observations imply a possible key function for the intrinsic cellular inhibitor XIAP in maintaining the homeostasis of the immune system.

Animals↗

PubMed2001

Genomic organization of the X-linked inhibitor of apoptosis and identification of a novel testis-specific transcript.

Here we report the genomic organization and mapping of the X-linked inhibitor of apoptosis gene (BIRC4, also known as XIAP and hILP) and the identification of a closely related transcript. BIRC4 is located on Xq25 and is composed of seven exons. The intron/exon structure is highly conserved between the mouse homologue and its human counterpart. Four bands cross-react with a BIRC4 coding region probe on a genomic Southern blot. One of these cross-reactive bands encodes an intronless gene that expresses a 2.2-kb transcript solely in the testis. This testis-specific transcript contains a putative open reading frame (ORF) that is homologous to the carboxy-terminal end of BIRC4; overexpression of this ORF shows protective effects against BAX-induced apoptosis.

Animals↗

PubMed2001

XIAP: apoptotic brake and promising therapeutic target.

The X-linked Inhibitor of Apoptosis, XIAP, is a key member of the newly discovered family of intrinsic inhibitors of apoptosis (IAP) proteins. IAPs block cell death both in vitro and in vivo by virtue of inhibition of distinct caspases. Although other proteins have been identified which inhibit upstream caspases, only the IAPs have been demonstrated to be endogenous repressors of the terminal caspase cascade. In turn, the caspase inhibiting activity of XIAP is negatively regulated by at least two XIAP-interacting proteins, XAF1 and Smac/DIABLO. In addition to the inhibition of caspases, recent discoveries from several laboratories suggest that XIAP is also involved in a number of other biologically significant cellular activities including modulation of receptor-mediated signal transduction and protein ubiquitination. XIAP is also translated by a rare cap-independent mechanism mediated by a specific sequence called IRES (for Internal Ribosome Entry Site) which is found in the XIAP 5(') UTR. XIAP protein is thus synthesized under various conditions of cellular stress such as serum starvation and low dose gamma-irradiation induced apoptosis, conditions that lead to the inhibition of cellular protein synthesis. The multiple biological activities of XIAP, its unique translational and post-translational control and the centrality of the caspase cascade make the control of XIAP expression an exceptionally promising molecular target for modulating apoptosis. Therapeutic benefits can be derived from both the suppression of inappropriate cell death such as in neurodegenerative disorders and ischemic injury or in the activation of latent cell death pathways such as in autoimmune disease and cancer where apoptosis induction is the desired outcome.

Animals↗

PubMed2001

Identification of XAF1 as an antagonist of XIAP anti-Caspase activity.

The inhibitors of apoptosis (IAPs) suppress apoptosis through the inhibition of the caspase cascade and thus are key proteins in the control of cell death. Here we have isolated the protein XIAP-associated factor 1 (XAF1) on the basis of its ability to bind XIAP, a member of the IAP family. XIAP suppresses caspase activation and cell death in vitro, and XAF1 antagonizes these XIAP activities. Expression of XAF1 triggers a redistribution of XIAP from the cytosol to the nucleus. XAF1 is ubiquitously expressed in normal tissues, but is present at low or undetectable levels in many different cancer cell lines. Loss of control over apoptotic signalling is now recognized as a critical event in the development of cancer. Our results indicate that XAF1 may be important in mediating the apoptosis resistance of cancer cells.

Adaptor Proteins, Signal Transducing↗

PubMed2001

XIAP, the guardian angel.

Controlling the activity of caspases is essential for the appropriate execution of cell death and the regulation of cell survival. One cellular inhibitor of apoptosis, XIAP, has emerged as a crucial regulator of caspases, and is itself subject to complex negative regulation.

Animals↗

PubMed2001

NAIP protects the nigrostriatal dopamine pathway in an intrastriatal 6-OHDA rat model of Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder of the basal ganglia, associated with the inappropriate death of dopaminergic neurons of the substantia nigra pars compacta (SNc). Here, we show that adenovirally mediated expression of neuronal apoptosis inhibitor protein (NAIP) ameliorates the loss of nigrostriatal function following intrastriatal 6-OHDA administration by attenuating the death of dopamine neurons and dopaminergic fibres in the striatum. In addition, we also addressed the role of the cysteine protease caspase-3 activity in this adult 6-OHDA model, because a role for caspases has been implicated in the loss of dopamine neurons in PD, and because NAIP is also a reputed inhibitor of caspase-3. Although caspase-3-like proteolysis was induced in the SNc dopamine neurons of juvenile rats lesioned with 6-OHDA and in adult rats following axotomy of the medial forebrain bundle, caspase-3 is not induced in the dopamine neurons of adult 6-OHDA-lesioned animals. Taken together, these results suggest that therapeutic strategies based on NAIP may have potential value for the treatment of PD.

Amphetamine↗

PubMed2001

Human ovarian cancer and cisplatin resistance: possible role of inhibitor of apoptosis proteins.

The inhibitor of apoptosis proteins (IAPs) constitutes a family of highly conserved apoptosis suppressor proteins that were originally identified in baculoviruses. Although IAP homologs have recently been demonstrated to suppress apoptosis in mammalian cells, their expression and role in human ovarian epithelial cancer and chemotherapy resistance are unknown. In the present study we used cisplatin-sensitive and -resistant human ovarian surface epithelial (hOSE) cancer cell lines and adenoviral antisense and sense complementary DNA expression to examine the role of IAP in the regulation of apoptosis in human ovarian cancer cells and chemoresistance. Antisense down-regulation of X-linked inhibitor of apoptosis protein (Xiap), but not human inhibitor of apoptosis protein-2 (Hiap-2), induced apoptosis in cisplatin-sensitive and, to a lesser extent, in -resistant cells. Cisplatin consistently decreased Xiap content and induced apoptosis in the cisplatin-sensitive, but not cisplatin-resistant, cells. Hiap-2 expression was either unaffected or inhibited to a lesser extent. The inhibition of IAP protein expression and induction of apoptosis by cisplatin was time and concentration dependent. Infection of cisplatin-sensitive cells with adenoviral sense Xiap complementary DNA resulted in overexpression of Xiap and markedly attenuated the ability of cisplatin to induce apoptosis. Immunohistochemical localization of the IAPs in hOSE tumors demonstrated the presence of Xiap and Hiap-2, with their levels being highest in proliferative, but not apoptotic, epithelial cells. These studies indicate that Xiap is an important element in the control of ovarian tumor growth and may be a point of regulation for cisplatin in the induction of apoptosis. These results suggest that the ability of cisplatin to down-regulate Xiap content may be an important determinant of chemosensitivity in hOSE cancer.

Aged↗

PubMed2000

Expression and genetic analysis of XIAP-associated factor 1 (XAF1) in cancer cell lines.

X-linked inhibitor of apoptosis protein (XIAP) is a potent modulator of programmed cell death. XIAP specifically binds and inhibits the function of caspase-3, -7, and -9, key effector proteases of apoptosis. We recently isolated, by yeast two-hybrid screening, a novel 34-kDa zinc finger protein, XIAP-associated factor 1 (XAF1). Both the caspase inhibiting and the anti-apoptotic abilities of XIAP were found to be blocked by overexpressed XAF1. Here, we report the isolation and characterization of the human XAF1 gene. The xaf1 gene consists of seven exons spanning 18 kb. Fluorescence in situ hybridization analysis localized the xaf1 locus at 17p13.2, telomeric to the p53 gene. The xaf1 locus was further refined to YAC 746C10, approximately 3 cM distal to TP53. Microsatellite analysis of the xaf1 locus using the NCI 60 cell line panel revealed significantly decreased heterozygosity at all three polymorphic markers tested, suggesting that allelic loss of the xaf1 gene is prevalent in cancer cell lines. Examination of the same NCI cell line panel for xaf1 RNA expression demonstrated that cancer cell lines exhibited very low levels of mRNA relative to normal human liver. In contrast, XIAP mRNA levels were relatively high in the majority of cancer cell lines tested. We propose that a high level of XIAP to XAF1 expression in cancer cells may provide a survival advantage through the relative increase of XIAP anti-apoptotic function.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Translational upregulation of X-linked inhibitor of apoptosis (XIAP) increases resistance to radiation induced cell death.

Inhibitory regulators of apoptosis play a critical role in the responsiveness of tumour cells to cytotoxic agents. The X-linked inhibitor of apoptosis protein (XIAP) is a member of a novel family of Inhibitor of Apoptosis (IAP) proteins. Here we show that acute low dose ionizing irradiation results in the translational upregulation of XIAP that correlates with an increased resistance to radiation in non-small cell lung carcinoma. This upregulation is mediated by an internal ribosome binding mechanism via an IRES element located within a XIAP 5' UTR. Transient overexpression of XIAP rendered human carcinoma cells resistant to low dose gamma-irradiation. By contrast, the antisense targeting of XIAP resulted in increased cell death following irradiation advocating a distinct role for XIAP in radiation resistant phenotype of human cancers. Oncogene (2000) 19, 4174 - 4177

5' Untranslated Regions↗

PubMed2000

The hippocampal neurons of neuronal apoptosis inhibitory protein 1 (NAIP1)-deleted mice display increased vulnerability to kainic acid-induced injury.

The neuronal apoptosis inhibitory protein (NAIP) is a member of a novel family of inhibitor of apoptosis (IAP) proteins. The IAP genes are highly conserved from baculovirus to metazoans and suppress apoptosis induced by a variety of triggers both in vitro and in vivo. Here we describe the generation and characterization of mice with the targeted deletion of NAIP1. We demonstrate that the NAIP1-deleted mice develop normally. However, the survival of pyramidal neurons in the hippocampus after kainic acid-induced limbic seizures is greatly reduced in the NAIP1 knock-out animals. Thus, although NAIP1 is not necessary for normal development of murine central nervous system, the endogenous NAIP1 is required for neuronal survival in pathological conditions.

Animals↗

PubMed2000

Internal ribosome initiation of translation and the control of cell death.

The majority of cellular stresses lead to the inhibition of cap-dependent translation. Some mRNAs, however, are translated by a cap-independent mechanism, mediated by ribosome binding to internal ribosome entry site (IRES) elements located in the 5' untranslated region. Interestingly, IRES elements are found in the mRNAs of several survival factors, oncogenes and proteins crucially involved in the control of apoptosis. These mRNAs are translated under a variety of stress conditions, including hypoxia, serum deprivation, irradiation and apoptosis. Thus, IRES-mediated translational control might have evolved to regulate cellular responses in acute but transient stress conditions that would otherwise lead to cell death.

5' Untranslated Regions↗

PubMed2000

Myotonic dystrophy (DM) protein kinase levels in congenital and adult DM patients.

Myotonic dystrophy is caused by a (CTG)n trinucleotide repeat expansion located in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). To date, the disease mechanism has proven elusive. The mutation would not be expected to affect kinase function and yet the disease is inherited in a dominant fashion. Mutant DMPK transcripts have been demonstrated to be retained in affected cell nuclei which could reduce DMPK protein levels and cause disease by haploinsufficiency. An alternate hypothesis is that the expansion confers a toxic gain of function on the transcript. In previous studies, various 52-55 kDa proteins have been detected using antisera targeted against DMPK and a decline of two of these candidates in disease tissues was reported. Current information now suggests that these proteins are not products of the myotonic dystrophy gene. We have characterised an antiserum which has been confirmed to recognise authentic 71 and 80 kDa isoforms of DMPK. Determination of the kinase levels in disease tissues with controls for patient age and tissue integrity demonstrates a modest overexpression in adult patients. In tissues from severely affected congenital patients only a slight decline is seen. This data argues against DMPK haploinsufficiency as a disease mechanism.

Adult↗

PubMed2000

IAP family proteins delay motoneuron cell death in vivo.

Neuronal apoptosis inhibitory protein (NAIP), and human inhibitors of apoptosis 1 and 2 (HIAP1 and HIAP2) are three members of the mammalian family of antiapoptosis proteins called 'inhibitors of apoptosis' (IAP). These molecules can prevent apoptosis in vitro and the over-expression of NAIP can decrease ischemic damage in the hippocampus. The goal of our experiments was to determine whether administration of NAIP, HIAP1 and HAIP2 could rescue motoneurons following axotomy of a peripheral nerve. In young rats, an adenoviral gene transfer technique was used to deliver and express these proteins in motoneurons; a fluorescent tracer was simultaneously added as a means for quantitatively assessing the rescue of fluorescently labelled motoneurons in serial sections of the lumbar spinal cord. Control experiments using adenoviral vectors (adv) expressing the lacZ gene showed that 14% of the sciatic motoneuron pool could be transfected indicating the existence of a subpopulation of spinal motoneurons susceptible to this class of viral vectors. The administration of an adv-NAIP, adv-HIAP1 and adv-HIAP2 rescued 30-40% of motoneurons at one week after sciatic axotomy. The efficiency of these proteins was similar to that of two neurotrophic factors, ciliary neurotrophic factor and brain-derived neurotrophic factor, administrated by the same viral technique. The effect of the IAP proteins on motoneuron survival decreased with time but was still present after 4 weeks postaxotomy; the duration of the response was dependent upon the viral titre. These experiments demonstrate that IAP family proteins can prevent motoneuron cell death in vivo and may offer a new therapeutic approach for motoneuron diseases.

Adenoviridae↗

PubMed2000

Functional characterization of the X-linked inhibitor of apoptosis (XIAP) internal ribosome entry site element: role of La autoantigen in XIAP translation.

X-linked inhibitor of apoptosis protein (XIAP) is a key regulator of programmed cell death triggered by various apoptotic triggers. Translation of XIAP is controlled by a 162-nucleotide (nt) internal ribosome entry site (IRES) element located in the 5' untranslated region of XIAP mRNA. XIAP IRES mediates efficient translation of XIAP under physiological stress and enhances cell protection against serum deprivation and radiation-induced apoptosis. In the present report we describe the assembly of a sequence-specific RNA-protein complex consisting of at least four cytosolic proteins on the XIAP IRES element. We determine that the core binding sequence is approximately 28 nt long and is located 34 nt upstream of the initiation site. Moreover, we identify the La autoantigen as a protein that specifically binds XIAP IRES in vivo and in vitro. The biological relevance of this interaction is further demonstrated by the inhibition of XIAP IRES-mediated translation in the absence of functional La protein. The results suggest an important role for the La protein in the regulation of XIAP expression, possibly by facilitating ribosome recruitment to the XIAP IRES.

Autoantigens↗