Human Inhibitor Of Apoptosis Proteins Research Articles

Structures of BIRC6-client complexes provide a mechanism of SMAC-mediated release of caspases.

Tight regulation of apoptosis is essential for metazoan development and prevents diseases such as cancer and neurodegeneration. Caspase activation is central to apoptosis, and inhibitor of apoptosis proteins (IAPs) are the principal actors that restrain caspase activity and are therefore attractive therapeutic targets. IAPs, in turn, are regulated by mitochondria-derived proapoptotic factors such as SMAC and HTRA2. Through a series of cryo-electron microscopy structures of full-length human baculoviral IAP repeat-containing protein 6 (BIRC6) bound to SMAC, caspases, and HTRA2, we provide a molecular understanding for BIRC6-mediated caspase inhibition and its release by SMAC. The architecture of BIRC6, together with near-irreversible binding of SMAC, elucidates how the IAP inhibitor SMAC can effectively control a processive ubiquitin ligase to respond to apoptotic stimuli.

A novel inhibitor of apoptosis protein (IAPs) from Crassostrea gigas is involved in anti-apoptosis and innate defense role under Vibrio alginolyticus stimulation

Apoptosis is an essential biological process in vertebrates and invertebrates for the development and maintenance of homeostasis. Inhibition of apoptosis can help some pathogens survive in host cells and escape from immune surveillance. Importantly, it also plays an important role in immune signaling regulation from insects to mammals. In this study, we identified a new inhibitor of the apoptosis protein gene (IAP), CgIAP3 from the Pacific oyster C. gigas, which contained one BIR domain and one RING domain. Blast analysis showed that CgIAP3 share the highest sequence identity of 65.68% with C. virginica IAP8, and clustered with other shellfish IAP in the phylogenetic tree, indicating that CgIAP3 belonged to a member of the IAPs family of shellfish. Homology comparison showed that the BIR domain of CgIAP3 was highly conserved with the BIR3 domain of human IAP. The tissue distribution of CgIAP3 in healthy C. gigas showed that CgIAP3 is ubiquitously expressed in all analyzed tissues, with the highest level of expression in the gills and digestive gland than the adductor muscle and was rapidly up-regulated after V. alginolyticus stimulation. Moreover, cell experiments showed CgIAP3 could inhibit apoptosis by maintaining cell morphology and cell viability. The result of subcellular localization showed that CgIAP3 is widely distributed in cytoplasm and nucleus and only expressed in A549 cells with damaged nucleus. Taken together, we speculated that CgIAP3 might be involved in the innate immune process of the Pacific oyster against V. alginolyticus through maintaining host cell morphology and viability.

Synthesis, in vitro cytotoxic and apoptotic effects, and molecular docking study of novel adamantane derivatives.

[4-(Adamantane-1-carboxamido)-3-oxo-1-thia-4-azaspiro[4.4]nonan-2-yl]acetic acid (4a) and [4-(adamantane-1-carboxamido)-8-nonsubstituted/substituted-3-oxo-1-thia-4-azas-piro[4.5]decane-2-yl]acetic acid (4b-g) derivatives were synthesized; their structures were verified by elemental analysis, infrared spectroscopy, 1 H nuclear magnetic resonance (NMR), 13 C NMR, and massspectroscopy data; and their in vitro cytotoxicity activities were investigated against human hepatocellular carcinoma, human prostate adenocarcinoma, and human lung carcinoma cell lines (HepG2, PC-3, and A549, respectively), and a mouse fibroblast cell line (NIH/3T3). All compounds, exceptcompound 4e, were found as cytotoxic, especially on A549 cells as compared with the other cells (selectivity index = 2.01-11.6). As a further step, the effects of compounds 4a-c on apoptosis induction were tested and the expression of selected apoptosis genes was analyzed. Among the selected compounds, compound 4a induced apoptosis remarkably. Moreover, computational calculations of the binding of compounds 4a-c to the BIR3 domain of the human inhibitor of apoptosis protein revealed ligand-protein interactions at the atomistic level and emphasized the importance of a hydrophobic moiety on the ligands for better binding.

Unraveling novel survivin mRNA transcripts in cancer cells using an in-house developed targeted high-throughput sequencing approach

The human baculoviral IAP repeat containing 5 (BIRC5), also known as survivin, is a conserved member of the inhibitor of apoptosis protein (IAPs) family, which is normally expressed during embryonic and fetal development. Although the expression levels of survivin are low in terminally differentiated cells and/or tissues, they can be found notably increased in certain pathological conditions including malignant tumors. Conventional cloning and sequencing techniques have already confirmed that alternative splicing events of the survivin pre-mRNA result in five distinct alternative transcript variants. In the present study, however, we implemented an innovative, in-house developed, targeted DNA-seq assay to identify novel survivin alternative transcript variants with increased depth and coverage that high-throughput sequencing approaches offer. Bioinformatics analysis of the derived NGS datasets unveiled several novel splice junctions between annotated exons of survivin gene as well as the existence of a novel exon of 117 nt, spanning between the annotated exons 3 and 3B. Validation of the NGS findings with PCR-based assays, using variant-specific primers, led to the identification of fourteen novel survivin alternative splice variants (BIRC5 v.4 - v.17), which demonstrate wide expression profiles in a broad established panel of human cell lines. Although the presented novel findings provide a crystal-clear overview of the survivin mRNAs that are actually generated from the pre-mRNA, future studies should focus on the impending necessity of characterizing the biological function of all novel alternative transcript variants as well as the putative protein isoforms. Such studies will further contribute to our understanding of how the balance between survivin isoforms regulate malignant cell proliferation and apoptosis, providing novel diagnostic, prognostic and predictive biomarkers as well as therapeutic targets.

Livin promotes colon cancer progression by regulation of H2A.XY39ph via JMJD6

Livin is an important member of the human inhibitor of apoptosis proteins (IAPs) family. IAPs are proteins with antiapoptotic abilities, and their functions are different from the Bcl-2 (B-cell lymphoma-2) family proteins. However, the precise role of Livin in colon cancer progression remains unclear. The purpose of this study is to assess the effect of overexpression Livin in colon cancer cells and to examine its molecular mechanism. We demonstrated that Livin induced a colon cancer phenotype, including proliferation and migration, by regulating H2A.XY39ph (histone family 2A variant (H2AX) phosphorylated on the 39th serine site). We elucidated that Livin degraded Jumonji-C domain-containing 6 protein (JMJD6), which was mediated by the proteasome murine double minute 2 (MDM2), thereby regulating H2A.XY39ph. Above all, the overexpression of JMJD6 recovered H2A.XY39ph in colon cancer cells with a high level of Livin, thus inhibiting colon cancer malignancy progression. These results reveal a previously unrecognized role for Livin in regulating the tumor-initiating capacity in colon cancer and provide a novel treatment strategy in cancer via the interruption of H2A.XY39ph function and the interaction between H2A.XY39ph and JMJD6.

Inhibitor of apoptosis proteins in human health and disease.

The inhibitor of apoptosis proteins (IAPs) are best known for their ability to regulate cell survival and death processes. However, in addition to cell death, IAPs also act as innate immune sensors and modulate multiple pathways, such as autophagy and cell division. Many of these IAP functions are non-redundant even though they are based on the same molecular mechanism of action. These distinct functions of IAPs derive from their capacity to target specific substrates for ubiquitination and/or proteolytic cleavage. The unique functions of IAPs also derives from their unique cellular localizations, cell type and tissue-specific expression patterns. The diverse roles of IAPs are reflected by the fact that in humans the IAP family comprises eight distinct members. Genetic evidence from human pathologies also attests to the non-redundant functions of the IAPs since very diverse diseases arise upon aberrant IAP expression. In this review, we give an overview of the known mechanisms of action of the various IAPs, and focus on their specific roles in mediating innate immunity. We also look at the distinct phenotypes related to the dysregulation of the IAPs, and the human pathologies associated with each human IAP.

CIAP2 promotes gallbladder cancer invasion and lymphangiogenesis by activating the NF-κB pathway.

Several studies have produced contradictory findings about the prognostic implications for inhibitor of apoptosis proteins (IAP) in different types of cancer. Cellular inhibitor of apoptosis 2 (cIAP2/BIRC) is one of the most extensively characterized human IAP. To date, no studies have focused on the expression level of cIAP2 in human gallbladder cancer (GBC), and the mechanism of cIAP2 in GBC invasion and lymphangiogenesis remains unclear. Therefore, in the present study, cIAP2 expression in GBC was detected using quantitative real‐time polymerase chain reaction and immunohistochemistry, and the relationship between cIAP2 levels in cancer tissues and the clinicopathological characteristics of patients was analyzed. The biological effect of cIAP2 in GBC cells was tested using the Cell Counting Kit‐8 Assay, Transwell assays and the ability of human dermal lymphatic endothelial cells (HDLEC) to undergo tube formation. The role of cIAP2 in activating the NF‐κB pathway was determined using a dual‐luciferase reporter assay, immunofluorescence staining, western blotting and ELISA. Finally, an animal model was used to further confirm the role of cIAP2 in lymphangiogenesis. We showed that cIAP2 expression was elevated in human GBC tissues and correlated with a negative prognosis for patients. Moreover, cIAP2 was identified as a lymphangiogenic factor of GBC cells and, thus, promoted lymph node metastasis in GBC cells. Our study is the first to suggest that cIAP2 can promote GBC invasion and lymphangiogenesis by activating the NF‐κB pathway.

Livin enhances tumorigenesis by regulating the mitogen-activated protein kinase signaling pathway in human hypopharyngeal squamous cell carcinoma.

Livin, a member of the human inhibitor of apoptosis protein (IAP) family, is expressed at high levels in various human cancer tissues and may have prognostic significance. The aim of the present study was to evaluate the effect of Livin on tumor cell behavior and oncogenic signaling pathways in human hypopharyngeal squamous cell carcinoma (HSCC). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used to determine the mRNA and protein expression levels, respectively. A cell proliferation assay and cell cycle analysis were used to assess the functional effects of small interfering RNA‑mediated Livin knockdown. Livin was overexpressed in fresh HSCC tissues, compared with the adjacent normal mucosa. Livin knockdown led to significantly reduced cell proliferation and cell cycle arrest in the G1 phase of the human HSCC cells. The expression levels of c‑myc, cyclin D1, cyclin D3, cyclin‑dependent kinase (CDK)4 and CDK6 were decreased. The phosphorylation levels of extracellular signal‑regulated kinase1/2, p38, c‑Jun N-terminal kinase and Akt were also decreased by Livin knockdown in the HSCC cells. Taken together, the results of the present study suggested that Livin may enhance tumorigenesis by modulating the mitogen‑activated/Akt signaling pathways in human HSCC.

Pulse Probiotic Administration Induces Repeated Small Intestinal Muc3 Expression in Rats

Upon ingestion, probiotics may act to protect the host through a number of protective mechanisms including modulation of genes involved in intestinal innate mucosal defense such as epithelial cell-derived mucin glycoproteins and inhibitor of apoptosis proteins. To determine the specificity of effect and sustainability of response in vivo, Lactobacillus plantarum 299v (Lp299v), Lactobacillus rhamnosus R0011 (LrR0011), and Bifidobacterium bifidum R0071 (BbR0071) were added repeatedly or intermittently to the drinking water of Sprague-Dawley rats. After killing the rats via CO2 suffocation, Muc2, Muc3, neuronal apoptosis inhibitor protein (NAIP), human inhibitor of apoptosis protein 1/cellular inhibitor of apoptosis 2 (HIAP1/cIAP2), and human inhibitor of apoptosis protein 2/cellular inhibitor of apoptosis 1 (HIAP2/cIAP1) mRNA and protein levels were analyzed via RT-PCR and immunohistochemistry. Live Lp299v, BbR0071, and LrR0011 increased Muc3 protein and mRNA expression in jejunum and ileum. Heat-killed and a nonadherent derivative of Lp299v failed to induce Muc3 expression. Lp299v did induce expression of HIAP2/cIAP1 and NAIP expression. Muc3 mucin expression was elevated for 5 d after oral administration of Lp299v; however, this effect was not sustained despite ongoing daily ingestion of a probiotic. Intermittent pulse ingestion of probiotics, however, was found to repeatedly increase Muc3 expression. We conclude that selected probiotics can induce protective genes of mucosal intestinal epithelial cells, an effect that is reproducible with pulse probiotic administration.

The Role of Inhibitor of Apoptosis Proteins Family Expression In Acute Myeloid Leukemia Patients

AbstractAbstract 2728 Background:IAPs (Inhibitor of Apoptosis Proteins) family acts as an inhibitor of both external and internal apoptosis pathways by binding to specific caspases. These proteins also participate in intracellular signal transduction. Seven human IAPs have been identified: XIAP, cIAP-1, cIAP-2, survivin, livin, NAIP and BRUCE/Apollon. IAPs family can be inhibited by Smac/DIABLO (second mitochondrial derived activator of caspase/direct IAP binding protein with low pI) protein. The role, pathway of action and prognostic significance of IAPs family is not clearly determined in acute myeloid leukemia (AML) patients. Aims:The main objective of this study was to verify whether expressions of selected IAPs as XIAP, cIAP-1, cIAP-2 and survivin proteins have a prognostic impact on response to induction chemotherapy of adult AML patients. Additionally, to analyse the correlation between members of IAPs family and their inhibitor, Smac/DIABLO protein in leukemic blasts. Material and Methods:Intracellular expression of XIAP, cIAP-1, cIAP-2, survivin and Smac/DIABLO proteins were examined in leukemic blasts isolated from bone marrow or peripheral blood of 56 de novo AML patients with median age 57 (range 21–82). All measurements were carried out using multi-colour flow cytometry. In parallel, the isotype controls were performed for all measurements. Protein expression was assessed by percentage of XIAP, cIAP-1, cIAP-2, survivin and Smac/DIABLO positive cells. Expression above 20% of cells was recognized as positive. Results:The median of intracellular expression of XIAP, cIAP-1, cIAP-2, survivin and Smac/DIABLO proteins were 1% (ranged 0,1–16,7%), 3,2% (ranged 0,1–67,0%), 1,7% (ranged 0–41%), 29,9% (ranged 0,3–71,1%) and 65,9% (ranged 3,8–99,9%); respectively.Correlation of cIAP-1 with cIAP-2 and XIAP was observed (p<0.001, p<0.01; respectively). Trend of correlation between cIAP-1 and survivin was also noticed (p=0.07). Similarly to cIAP-1, cIAP-2 was strongly correlated with XIAP (p<0.01). Neither cIAP-2 nor XIAP showed correlation with survivin. Additionally, all of investigated IAPs family members except survivin showed reverse correlation with Smac/DIABLO protein (p<0.01).Thirty three out of 56 AML patients received standard induction chemotherapy with daunorubicine and cytarabine (Ara-C) (3+7), 23/56 received non-intensive chemotherapy. Twenty (60.7%) of intensively treated patients achieved complete remission (CR). We examined influence of presence of cIAPs family members in leukemic blast on response to chemotherapy in intensively treated patients. It was found that probability of CR with absence of cIAP-1, cIAP-2 and survivin was 83%. CR rate with presence of one of IAPs member was 68%. Patients with expressions of three IAPs members did not respond to the induction therapy. Conclusions:Strong correlations between members of IAPs family may bear the evidence for cooperation between these proteins in leukemic blasts. Additionally, reverse correlation between members of IAPs family and their antagonist Smac/DIABLO protein confirm intracellular interaction between them. These data also demonstrate that lack of IAPs family members expressions is associated with higher sensitivity to standard chemotherapy.However, further investigations evaluating the relationship between all known IAPs family members and their inhibitors should be undertaken to better demonstrate its pathways of action as well as prognostic and potentially therapeutic value. Disclosures:Robak:Johnson & Johnson: Research Funding.

Regulation of XIAP Translation and Induction by MDM2 following Irradiation

Increases in protein levels of XIAP in cancer cells have been associated with resistance to apoptosis induced by cellular stress. Herein we demonstrate that the upregulation of XIAP protein levels is regulated by MDM2 at the translational level. MDM2 was found to physically interact with the IRES of the XIAP 5'-UTR, and to positively regulate XIAP IRES activity. This XIAP IRES-dependent translation was significantly increased in MDM2-transfected cells where MDM2 accumulated in the cytoplasm. Cellular stress and DNA damage triggered by irradiation induced the dephosphorylation and cytoplasmic localization of MDM2, which also led to an increase in IRES-dependent XIAP translation. Upregulation of XIAP in MDM2-overexpressing cancer cells in response to irradiation resulted in resistance of these cells to radiation-induced apoptosis.

Human inhibitor of apoptosis proteins: why XIAP is the black sheep of the family.

Several of the inhibitor of apoptosis protein (IAP) family members regulate apoptosis in response to various cellular assaults. Some members are also involved in cell signalling, mitosis and targeting proteins to the ubiquitin-proteasome degradation machinery. The most intensively studied family member, X-linked IAP (XIAP), is a potent inhibitor of caspase activity; hence, it is generally assumed that direct caspase inhibition is an important conserved function of most members of the family. Biochemical and structural studies have precisely mapped the elements of XIAP required for caspase inhibition. Intriguingly, these elements are not conserved among IAPs. Here, we review current knowledge of the caspase-inhibitory potential of the human IAPs and show that XIAP is probably the only bona fide caspase inhibitor, suggesting that the other family members never gained the ability to directly inhibit caspase activity.

Smac/Diablo Antagonizes Ubiquitin Ligase Activity of Inhibitor of Apoptosis Proteins

Inhibitor of apoptosis proteins (IAPs) can block apoptosis through binding to active caspases and antagonizing their function. IAP function can be neutralized by Smac/Diablo, an IAP-binding protein that is released from mitochondria during apoptosis. In addition to their ability to interact with caspases, certain IAPs also display ubiquitin-protein isopeptide ligase activity because of the presence of a RING domain. However, it is not known whether the ubiquitin-protein isopeptide ligase activities of human IAPs contribute to their apoptosis inhibitory activity or whether this IAP property can be modulated through association with Smac/Diablo. Here we demonstrate that the ubiquitin ligase activities of XIAP, and to a lesser extent c-IAP-1 and c-IAP2, are potently repressed through binding to Smac/Diablo. We also show that mutation of the XIAP RING domain rendered this IAP a less effective inhibitor of apoptosis, suggesting that the ubiquitin ligase activity of XIAP contributes to its anti-apoptotic function. These data suggest that Smac/Diablo potentiates apoptosis by simultaneously antagonizing caspase-IAP interactions and repressing IAP ubiquitin ligase activities.

Reduction of tumor necrosis factor-alpha (TNF-α) related nuclear factor-kappaB (NF-κB) translocation but not inhibitor kappa-B (Iκ-B)-degradation by Rho protein inhibition in human endothelial cells

Degradation of inhibitor kappa-B (Iκ-B) followed by translocation of nuclear factor-kappaB (NF-κB) into the nucleus and activation of gene expression is essential in tumor necrosis factor-alpha (TNF-α)-signaling. In order to analyze the role of Rho proteins in TNF-α-induced NF-κB-activation in human umbilical cord vein endothelial cells (HUVEC) we used Clostridium difficile toxin B-10463 (TcdB-10463) which inactivates RhoA/Rac1/Cdc42 by glucosylation and Clostridium botulinum C3-toxin which inhibits RhoA/B/C by ADP-ribosylation. Exposure of HUVEC to 10ng/mL TcdB-10463 or 2.5μg/mL C3-toxin inhibited TNF-α (100ng/mL)-induced expression of a NF-κB-dependent reporter gene. Moreover, preincubation of HUVEC with 10ng/mL TcdB-10463 reduced TNF-α-related expression of interleukin-8 (IL-8), TNF-receptor associated factor-2 (TRAF2), and human inhibitor of apoptosis protein 1 (hIAP1)-mRNA. Blocking of Rho reduced NF-κB DNA-binding as shown by electrophoretic mobility shift assays. TcdB-10463 and C3-toxin blocked TNF-α-related nuclear translocation of NF-κB although Iκ-Bα/β was still degraded. In contrast, TcdB-10463 had no effect on IL-1β-related NF-κB-translocation and activation in HUVEC. Neither 1μM Rho kinase inhibitor Y-27632 nor microfilament depolymerization by 50ng/mL C. botulinum C2-toxin blocked TNF-α-induced degradation of Iκ-B, nuclear NF-κB translocation or expression of a NF-κB-dependent reporter gene. Therefore, TNF-α-related Iκ-B-degradation is Rho-independent in HUVEC, whereas a Rho protein-dependent signal is necessary to induce nuclear transport of NF-κB in these cells pointing to a novel and unique role of Rho in NF-κB-translocation.

Se-methylselenocysteine induces apoptosis through caspase activation and Bax cleavage mediated by calpain in SKOV-3 ovarian cancer cells

Se-methylselenocysteine (Se-MSC) is a potent chemopreventive agent in many test systems and has been shown to inhibit tumor promotion and induce apoptosis, but its mechanism of action is still not well understood. The present study was designed to assess the mechanism of Se-MSC on the induction of apoptosis in SKOV-3 ovarian cancer cells. Se-MSC displayed strong inhibitory effects on cell proliferation and viability of SKOV-3 cells in dose and time dependent manners and induced apoptosis. Investigation of the mechanism of Se-MSC-induced apoptosis revealed that treatment with Se-MSC produced morphological features of apoptosis and DNA fragmentation. This was associated with caspase-3 activation and cleavage of poly(ADP-ribose) polymerase and phospholipase C-γ1 proteins. However, SKOV-3 cells treated with Se-MSC did not demonstrate cytochrome c accumulation in the cytosol during apoptosis induction. Pretreatment of cells with the caspase inhibitors (z-VAD-fmk and DEVD-CHO) prevented Se-MSC-induced apoptosis. These results suggested that Se-MSC induces apoptosis through cytochrome c-independent caspase-3 activation in SKOV-3 cells. In late stage of apoptosis, p18 kDa fragment of Bax was generated with the down-regulation of the expressions of survivin, X-linked inhibitor of apoptosis protein, and human inhibitor of apoptosis protein 1 following Se-MSC treatment, suggesting that the modulation of Bax and IAP (inhibitors of apoptosis) family proteins play some role in Se-MSC-mediated apoptosis. Pre-treatments of z-VAD-fmk and the calpain inhibitor, calpeptin inhibited Bax cleavage. These results suggested that Bax cleavage is mediated by calpain, and calpain activation may be a caspase-dependent one. Taken together, the chemopreventive effects of Se-MSC may be related in part to the caspase-3 activation, the down-regulation of IAP family proteins, and Bax cleavage mediated by caspase-dependent calpain activation.

Human inhibitor of apoptosis protein (IAP) survivin participates in regulation of chromosome segregation and mitotic exit.

A signaling cascade termed the "spindle checkpoint" monitors interactions between the kinetochores of chromosomes and spindle microtubules to prevent precocious separation of sister chromatids. We have investigated the role of human inhibitor of apoptosis protein (IAP) surviving in regulation of cell division. We demonstrate that HeLa and PtK1 cells transfected or microinjected with surviving anti-sense oligonucleotides produce significantly more polyploid and micronucleated progeny cells and show abortive mitosis when treated with spindle poisons. Furthermore, perturbation of surviving function in HeLa and PtK1 cells with anti-surviving antibodies at the beginning of mitosis affects the normal timing of separation of sister chromatids and disturbs the 3F3/2 phosphoepitope-recognized tension sensing mechanism of the spindle checkpoint. This leads to premature separation of sister chromatids, which results in an uneven distribution of chromosomes between the newly formed progeny cells-an event associated with tumor formation in many cell types. Finally, cells injected with anti-surviving antibody exit mitotic block induced with microtubule drugs. Our data suggest that surviving protein may function within the spindle checkpoint pathway.

Livin, a Novel Inhibitor of Apoptosis Protein Family Member

A novel human inhibitor of apoptosis protein (IAP) family member termed Livin was identified, containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. The mRNA for livin was not detectable by Northern blot in most normal adult tissues with the exception of the placenta, but was present in developmental tissues and in several cancer cell lines. Highest levels were observed in two melanoma-derived cell lines, G361 and SK-Mel29. Transfection of livin in HeLa cells resulted in protection from apoptosis induced by expression of FADD, Bax, RIP, RIP3, and DR6. Similar to other IAP family members, the anti-apoptotic activity of Livin was dependent on the BIR domain. Livin was also capable of inhibiting DEVD-like caspase activity triggered by tumor necrosis factor-alpha. In vitro binding studies demonstrated a direct interaction between Livin and the active form of the downstream caspases, caspase-3 and -7, that was dependent on the BIR domain of Livin. In addition, the unprocessed and cleaved forms of caspase-9 co-immunoprecipitated with Livin in vivo, and recombinant Livin could inhibit the activation of caspase-9 induced by Apaf-1, cytochrome c, and dATP. The subcellular distribution of the transfected Livin was analyzed by immunofluorescence. Both Livin and Survivin were expressed in the nucleus and in a filamentous pattern throughout the cytoplasm. In contrast to the apoptotic activity, the COOH-terminal RING domain mediated its subcellular localization patterning. Further studies found that transfection of an antisense construct against livin could trigger apoptosis specifically in cell lines expressing livin mRNA. This was associated with an increase in DNA fragmentation and in DEVD-like caspase activity. Thus, disruption of Livin may provide a strategy to induce apoptosis in certain cancer cells.

Regulation of X Chromosome-Linked Inhibitor of Apoptosis Protein (XIAP) in Kainic Acid Induced Neuronal Cell Death in the Rat Hippocampus.

INTRODUCTION. Inhibitor of apoptosis protein (IAP) family consists of several antiapoptotic proteins conserved among species. The IAPs have a well-conserved motif of approximately 65 residues, called the baculovirus inhibitory repeat (BIR) (1). Baculovirus and drosophila IAPs have two, but most IAPs contain three BIR domains. Most of the IAPs also have a C-terminal RING domain which consists of conserved amino acids with zinc binding capacity. XIAP is one of the five known human IAPs and it binds directly and inhibits the activity of caspases (2). The BIR2 domain in XIAP is sufficient to mediate this inhibition (3). However little is known about the presence and function of XIAP in the nervous system. METHOD. In this study, we have investigated the expression and distribution of XIAP mRNA and protein in rat brain using RNase protection assay in addition to Western blotting and immunohistochemistry with a specific anti-XIAP antibody. Neuronal cell death upon kainic acid stimulation was investigated using TUNEL method and caspase-3 activation. RESULTS. Two transcripts of XIAP mRNA are present in rat brains, suggesting a heterogeneity of XIAP in the carboxyterminal region. Using a specific anti-XIAP antibody, XIAP protein was shown to be expressed in embryonic and adult brains with slightly higher levels in embryonic and neonatal brains compared to adults. There was a widespread distribution of XIAP immunoreactive cells in adult rat brains, among others in hippocampus, cortex and substantia nigra. Using double-labelling, it was shown that both neuronal and glia cells express XIAP. Kainic acid treatment is known to cause widespread neuronal cell death in hippocampus and amygdala. XIAP protein levels were increased in the hippocampus 12th and 24th after kainic acid. The intensity of XIAP staining was increased particularly in the hippocampal CA3 area. In the same area, many TUNEL positive and active caspase-3 positive cells were found. Caspase-3 is activated 24th after kainic acid in vivo and in vitro in cultured embryonic hippocampal neurons upon kainic acid stimulus. In hippocampus, XIAP does not colocalize with markers for cell death raising the possibility that XIAP is expressed in viable cells but not in dying cells. Using cultured embryonic hippocampal neurons, it was shown that XIAP becomes a target for active caspase-3 and that it is degradated into smaller pieces. DISCUSSION. XIAP mRNA is expressed in developing and adult rat brains, and the number of XIAP positive cells and XIAP protein levels were regulated by kainic acid. The largest change in XIAP immunoreactivity was observed in CA3 area, which was particularly vulnerable in this model. Labelling of cells with the TUNEL method and with antibody for

The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner.

Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G(2)/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G(1) phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level. In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G(1) phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein.

Genomic Organization and Primary Characterization ofmiap-3: The Murine Homologue of Human X-Linked IAP

IAPs (inhibitor of apoptosis proteins) are a recently identified family of proteins that function in the cell death pathway to inhibit programmed cell death. We have earlier reported cloning of four human IAPs: NAIP,hiap-1,hiap-2, andxiap.To facilitate studies of these genes, we have undertaken the cloning of their murine homologues. We report here the cloning, mapping, and preliminary characterization (including cellular and tissue distribution profile) of the murine homologue of the human X-linked IAP (xiap). The mouse gene calledmiap-3 (for murine IAP-3; GenBank Accession No. nuc 1 U88990) has a coding region of 1.5 kb that encodes a protein of 55 kDa and has 87 and 94% homology with its human homologue at DNA and amino acid levels, respectively. Northern blot analysis reveals an 8-kbmiap-3 transcript in all tissues examined to date.miap-3 is composed of six exons and five introns spanning approximately 20 kb.miap-3 has been assigned to the A3–A5 region of mouse chromosome X by FISH analysis.