Multidisciplinary approach to implants: a review.

As implant dentistry grows in popularity, greater numbers of general dental practitioners (GDPs) are offering this service to their patients. In order to improve treatment success for both patient and practitioner, a multidisciplinary approach to implant planning and placement should be taken. However, the literature currently holds few articles which offer advice to the GDP on how to take a multidisciplinary approach to implant treatment. The aim of this review is to provide practitioners with evidence-based guidance for taking a multidisciplinary approach to implant treatment. A MEDLINE/PubMed search for articles published between 1980 and mid-2012 was undertaken. The search strategy used different combinations of the following terms: dental implants, osseointegration, medical contraindications, patient habits, radiography, prosthodontics, endodontics, orthodontics, periodontics and surgical factors. The review demonstrated the importance of thorough medical and dental history-taking, and how different facets of each speciality contribute to the outcome of implant treatment. Successful implant treatment is the result of careful planning and integration of various areas of dentistry, not just those of prosthodontics and surgery.

Restoration of XAF1 expression induces apoptosis and inhibits tumor growth in gastric cancer.

XAF1 (XIAP-associated factor 1) is a novel XIAP binding protein that can antagonize XIAP and sensitize cells to other cell death triggers. Our previous results have shown that aberrant hypermethylation of the CpG sites in XAF1 promoter is strongly associated with lower expression of XAF1 in gastric cancers. In our study, we investigated the effect of restoration of XAF1 expression on growth of gastric cancers. We found that the restoration of XAF1 expression suppressed anchorage-dependent and -independent growth and increased sensitivity to TRAIL and drug-induced apoptosis. Stable cell clones expressing XAF1 exhibited delayed tumor initiation in nude mice. Restoration of XAF1 expression mediated by adenovirus vector greatly increased apoptosis in gastric cancer cell lines in a time- and dose-dependent manner and sensitized cancer cells to TRAIL and drugs-induced apoptosis. Adeno-XAF1 transduction induced cell cycle G2/M arrest and upregulated the expression of p21 and downregulated the expression of cyclin B1 and cdc2. Notably, adeno-XAF1 treatment significantly inhibited tumor growth, strongly enhanced the antitumor activity of TRAIL in a gastric cancer xenograft model in vivo, and significantly prolonged the survival time of animals bearing tumor xenografts. Complete eradication of established tumors was achieved on combined treatment with adeno-XAF1 and TRAIL. Our results document that the restoration of XAF1 inhibits gastric tumorigenesis and tumor growth and that XAF1 is a promising candidate for cancer gene therapy.

Increased X-linked inhibitor of apoptosis protein (XIAP) expression exacerbates experimental autoimmune encephalomyelitis (EAE).

Dysregulated apoptotic signaling has been implicated in most forms of cancer and many autoimmune diseases, such as multiple sclerosis (MS). We have previously shown that the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) is elevated in T cells from mice with experimental autoimmune encephalomyelitis (EAE). In MS and EAE, the failure of autoimmune cells to undergo apoptosis is thought to exacerbate clinical symptoms and contribute to disease progression and CNS tissue damage. Antisense-mediated knockdown of XIAP, in vivo, increases the susceptibility of effector T cells to apoptosis, thus attenuating CNS inflammation and thereby alleviating the clinical signs of EAE. We report for the first time, generation of transgenic mice whereby the ubiquitin promoter drives expression of XIAP (ubXIAP), resulting in increased XIAP expression in a variety of tissues, including cells comprising the immune system. Transgenic ubXIAP mice and wild-type (WT) littermates were immunized with myelin oligodendrocyte glycoprotein (MOG35-55) in complete Freund's adjuvant and monitored daily for clinical symptoms of EAE over a 21-day period. The severity of EAE was increased in ubXIAP mice relative to WT-littermates, suggesting that XIAP overexpression enhanced the resistance of T cells to apoptosis. Consistent with this finding, T cells derived from MOG35-55-immunized ubXIAP mice and cultured in the presence of antigen were more resistant to etoposide-mediated apoptosis compared to WT-littermates. This work identifies XIAP is an important apoptotic regulator in EAE and a potential pharmacological target for treating autoimmune diseases such as MS.

Silencing of the XAF1 gene by promoter hypermethylation in cancer cells and reactivation to TRAIL-sensitization by IFN-beta.

BACKGROUND: XIAP-associated factor 1 (XAF1) is a putative tumor suppressor that exerts its proapoptotic effects through both caspase-dependent and -independent means. Loss of XAF1 expression through promoter methylation has been implicated in the process of tumorigenesis in a variety of cancers. In this report, we investigated the role of basal xaf1 promoter methylation in xaf1 expression and assessed the responsiveness of cancer cell lines to XAF1 induction by IFN-beta. METHODS: We used the conventional bisulfite DNA modification and sequencing method to determine the methylation status in the CpG sites of xaf1 promoter in glioblastoma (SF539, SF295), neuroblastoma (SK-N-AS) and cervical carcinoma (HeLa) cells. We analysed the status and incidence of basal xaf1 promoter methylation in xaf1 expression in non-treated cells as well as under a short or long exposure to IFN-beta. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-beta-mediated sensitization to TRAIL-induced cell death. RESULTS: We found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-beta across the four cancer cell lines studied. At the basal level, aberrant promoter methylation was linked to xaf1 gene silencing. After a short exposure, the IFN-beta-mediated reactivation of xaf1 gene expression was related to the degree of basal promoter methylation. However, in spite of continued promoter hypermethylation, we find that IFN-beta induced a transient xaf1 expression, that in turn, was followed by promoter demethylation upon a prolonged exposure. Importantly, we demonstrated for the first time that IFN-beta-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-beta-mediated TRAIL sensitivity. CONCLUSION: Together, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-beta treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer.

The X-linked inhibitor of apoptosis protein enhances survival of murine islet allografts.

Allotransplantation of pancreatic islets represents a promising approach to treat type 1 diabetes. Destruction of beta-cells in islet allografts involves multiple immune mechanisms that lead to activation of caspases and apoptotic cell death. The X-linked inhibitor of apoptosis (XIAP) inhibits apoptosis induced by a variety of triggers, primarily by preventing the activation of caspases. To determine whether XIAP would protect beta-cells from apoptosis, we used a recombinant adenovirus to overexpress XIAP in transformed murine beta-cells and in freshly isolated islets. In vitro cytokine-induced beta-cell death was decreased to baseline levels in XIAP-transduced MIN-6 and NIT-1 cell lines compared with controls. To evaluate the potential of XIAP overexpression to prevent in vivo allogeneic graft rejection, we transduced Balb/c islets ex vivo with XIAP before transplantation into CBA mice with streptozotocin-induced diabetes. We observed that almost all mice receiving allografts of XIAP-expressing islets maintained normoglycemia until the experiment was terminated (45-72 days posttransplant), whereas control mice receiving islets transduced with adenovirus expressing LacZ were hyperglycemic by approximately 17 days posttransplantation due to graft rejection. Immunohistochemistry revealed preservation of beta-cells and clearance of infiltrating immune cells in the XIAP-expressing islet grafts. The in vitro allogeneic response of splenocytes isolated from recipients of XIAP-expressing grafts 8 weeks posttransplant was similar to that seen in nonprimed allogeneic mice, suggesting that XIAP overexpression may lead to the acceptance of islet allografts in diabetic recipients. Long-term protection of islet allografts by XIAP overexpression may enhance the survival of islet transplants in diabetes.

XIAP overexpression in human islets prevents early posttransplant apoptosis and reduces the islet mass needed to treat diabetes.

The Edmonton Protocol for treatment of type 1 diabetes requires islets from two or more donors to achieve euglycemia in a single recipient, primarily because soon after portal infusion, the majority of the transplanted cells undergo apoptosis due to hypoxia and hypoxia reperfusion injury. X-linked inhibitor of apoptosis protein (XIAP) is a potent endogenous inhibitor of apoptosis that is capable of blocking the activation of multiple downstream caspases, and XIAP overexpression has previously been shown to enhance engraftment of a murine beta-cell line. In this study, human islets transduced with a XIAP-expressing recombinant adenovirus were resistant to apoptosis and functionally recovered following in vitro stresses of hypoxia and hypoxia with reoxygenation (models reperfusion injury). Furthermore Ad-XIAP transduction dramatically reduced the number of human islets required to reverse hyperglycemia in chemically diabetic immunodeficient mice. These results suggest that by transiently overexpressing XIAP in the immediate posttransplant period, human islets from a single donor might be used to effectively treat two diabetic recipients.

The inhibitors of apoptosis: there is more to life than Bcl2.

The inhibitor of apoptosis (IAP) genes constitute a highly conserved family found in organisms as diverse as insects and mammals. These genes encode proteins that directly bind and inhibit caspases, and thus play a critical role in deciding cell fate. The IAPs are in turn regulated by endogenous proteins (second mitochondrial activator of caspases and Omi) that are released from the mitochondria during apoptosis. Overexpression of the IAPs, particularly the X-chromosome-linked IAP, has been shown to be protective in a variety of experimental animal models of human neurodegenerative diseases. Furthermore, overexpression of one or more of the IAPs in cancer cell lines and primary tumor samples appears to be a frequent event. IAP gene amplification and translocation events provide genetic evidence that further strengthens the case for classifying the IAPs as oncogenes. Therapeutic strategies that interfere with IAP expression or function are under investigation as an adjuvant to conventional chemotherapy- and radiation-based cancer therapy. This paper reviews the structure and function of the IAP family members and their inhibitors, and surveys the available evidence for IAP dysregulation in cancer.

Motoneuron resistance to apoptotic cell death in vivo correlates with the ratio between X-linked inhibitor of apoptosis proteins (XIAPs) and its inhibitor, XIAP-associated factor 1.

Apoptotic cell death occurs in motoneurons in the neonate but not in the adult after a lesion of a peripheral nerve. To investigate the molecular basis for this difference, we have analyzed the expression and localization of inhibitors of apoptosis proteins (IAPs) and their inhibitors X-linked IAP (XIAP)-associated factor 1 (XAF1), Smac/DIABLO, and Omi/HtrA2 in motoneurons at both ages. Quantitative immunohistochemical and immunoblotting analysis of these proteins in motoneurons revealed an increase in IAP expression [XIAP, neuronal apoptosis inhibitory protein, human IAP1 (HIAP1), and HIAP2] during postnatal development as opposed to XAF1, which decreased during the same period; there was no significant alteration in either Smac/DIABO or Omi/HtrA2. The regulation of IAPs and XAF1 varied after axotomy of the sciatic nerve; in the neonate, there was a significant loss of IAP in the injured motoneurons as opposed to the adult, in which there was only a moderate decrease. By overexpressing exogenous IAPs in neonatal axotomized motoneurons, it was possible to delay motoneuron cell death (Perrelet et al., 2000, 2002). In opposition, the overexpression of exogenous XAF1 in adult motoneurons totally abrogated the natural resistance of these cells to axotomy. The degradation in the adult, induced by XAF1, could be overcome by simultaneously expressing high levels of exogenous XIAP in adult motoneurons. These experiments suggest that it may be the ratio between XAF1 and XIAP that confers the resistance of adult motoneurons to axotomy. In addition, the regulation in the levels of IAPs and XAF1 may be essential in the cell death mechanism of injured motoneurons.

Structural and functional protection of photoreceptors from MNU-induced retinal degeneration by the X-linked inhibitor of apoptosis.

PURPOSE: To evaluate the neuroprotective effects of adenoassociated virus delivery of XIAP in N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in Sprague-Dawley rats. METHODS: Sprague-Dawley rats were injected subretinally with recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control). Six weeks after injection, the animals received an intraperitoneal injection of MNU, a DNA methylating agent, at a dose of 60 mg/kg. Electroretinograms (ERGs) were recorded at 0, 24, 48 and 72 hours and 1 week after MNU. The rats were killed after the ERG was performed and were perfused with 4% paraformaldehyde. Eyes were then enucleated and embedded for cryosectioning. Eye sections were analyzed by TUNEL and histologic techniques. Real-time PCR and Western analysis were performed to confirm the overexpression of XIAP in injected eyes. RESULTS: Real-time PCR and Western analysis confirmed the overexpression of XIAP in virus-injected eyes in comparison to uninjected control eyes. At 24 hours after MNU injection, fewer cells had undergone apoptosis in the XIAP-treated eyes in comparison with GFP-injected or uninjected eyes. Hematoxylin and eosin staining revealed that the uninjected and GFP-injected photoreceptors were destroyed by 72 hours after injection of MNU, whereas the AAV-XIAP-injected eyes showed structural protection of the photoreceptors at all time points throughout the 1-week sampling period. ERGs showed functional protection up to 1 week after MNU injection in the AAV-XIAP-injected eye, whereas no response was observed in the control eye. CONCLUSIONS: The results suggest that XIAP is protective against this potent chemotoxic agent and holds promise as a therapeutic agent in gene therapy approaches to treating retinitis pigmentosa.

Amelogenesis imperfecta--multidisciplinary management from eruption to adulthood. Review and case report.

Amelogenesis imperfecta (AI) is a group of hereditary conditions that affect enamel formation. It is associated with a high morbidity for the patients and may present major restorative and sometimes orthodontic challenges for the dental team. Early recognition followed by appropriate preventive and restorative care is essential in the successful management of AI. A multidisciplinary approach with careful planning from early childhood will maximise the treatment options available for the permanent dentition and optimise the final outcome. In this case, a team consisting of two paediatric dentists, an orthodontist, a restorative dentist, and an oral and maxillofacial surgeon were involved in the management of the patient over a 12-year period. Treatment included preventive advice, interim composite restorations, two phases of orthodontic treatment, orthognathic surgery and placement of cast crowns. The patient is extremely happy with the result.

Degradation of survivin by the X-linked inhibitor of apoptosis (XIAP)-XAF1 complex.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a putative tumor suppressor in which expression is significantly reduced in human cancer cell lines and primary tumors. The proapoptotic effects of XAF1 have been attributed to both caspase-dependent and -independent means. In particular, XAF1 reverses the anti-caspase activity of XIAP, a physiological inhibitor of apoptosis. We further investigated the function of XAF1 by examining its relationship with other IAPs. Immunoprecipitation studies indicate that XAF1 binds to XIAP, cIAP1, cIAP2, Livin, TsIAP, and NAIP but not Survivin, an IAP that prevents mitotic catastrophe and in which antiapoptotic activity is exerted through direct XIAP interaction and stabilization. We found that overexpressed XAF1 down-regulates the protein expression of Survivin. Under these conditions, Survivin expression was restored in the presence of the proteasome inhibitor MG132 or a XIAP RING mutant that is defective in ubiquitin-protein isopeptide ligase (E3) activity, suggesting that XAF1 interaction activates E3 activity of XIAP and targets Survivin by direct ubiquitination. In addition, RNA interference targeting endogenous XIAP protected Survivin degradation by XAF1. Furthermore, interferon-beta-mediated XAF1 induction promoted formation of an endogenous XIAP-XAF1-Survivin complex. This complex facilitated Survivin degradation, which was prevented in XAF1(-/-) stable clones. Altogether, our study demonstrates that XAF1 mediates Survivin down-regulation through a complex containing XIAP, supporting dual roles for XAF1 in apoptosis and mitotic catastrophe.

Influence of two different flap designs on incidence of pain, swelling, trismus, and alveolar osteitis in the week following third molar surgery.

OBJECTIVE: To investigate the influence of flap design on postoperative trismus, pain, and swelling. STUDY DESIGN: Thirty-two patients with bilateral symmetrically impacted mandibular third molars participated in a randomized prospective split mouth study. Two flap designs were used: a buccal envelope flap and a modified triangular flap. Postoperative pain was recorded using a standardized visual analog scale. Postoperative swelling was evaluated using laser scans of models of the patient's cheek taken before and two days after surgery, with the scan area being calculated using MatLab software. RESULTS: There were no statistical differences between the flap designs in terms of severity of postoperative pain or trismus. A statistically significant difference was observed in postoperative swelling at 2 days, with the modified triangular flap design being associated with increased swelling. The envelope flap design was associated with a higher incidence of alveolar osteitis. CONCLUSION: The flap designs used in this study did not adversely affect patients in terms of postoperative pain and trismus, and although greater extraoral swelling was seen with the modified triangular flap design, the clinical relevance is debatable.

XAF1 mediates tumor necrosis factor-alpha-induced apoptosis and X-linked inhibitor of apoptosis cleavage by acting through the mitochondrial pathway.

Tumor necrosis factor-alpha (TNF-alpha) and Fas ligand induce apoptosis by interacting with their corresponding membrane-bound death receptors and activating caspases. Since both systems share several components of the intracellular apoptotic cascade and are expressed by first trimester trophoblasts, it is unknown how these cells remain resistant to Fas ligand while sensitive to TNF-alpha. XAF1 (X-linked inhibitor of apoptosis (XIAP)-associated factor 1) is a proapoptotic protein that antagonizes the caspase-inhibitory activity of XIAP. Here, we demonstrated that XAF1 functions as an alternative pathway for TNF-alpha-induced apoptosis by translocating to the mitochondria and promoting XIAP inactivation. In addition, we showed that the overexpression of XAF1 sensitized first trimester trophoblast cells to Fas-mediated apoptosis. Furthermore, we also determined that the differential expression of XAF1 in first and third trimester trophoblast cells was due to changes in XAF1 gene methylation. Our results establish a novel regulatory pathway controlling trophoblast cell survival and provide a molecular mechanism to explain trophoblast sensitivity to TNF-alpha and the increased number of apoptotic trophoblast cells observed near term. Aberrant XAF1 expression and/or localization may have consequences for normal pregnancy outcome.

Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: a role for the IAPs.

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2.

Functional analysis of the inhibitor of apoptosis (iap) gene carried by the entomopoxvirus of Amsacta moorei.

The entomopoxvirus from Amsacta moorei (AmEPV) contains none of the commonly recognized vertebrate poxvirus apoptotic suppressor genes. However, AmEPV carries a single inhibitor of apoptosis (iap) gene (AMViap) not present in vertebrate poxviruses. The AMViap gene was active when coexpressed with the Drosophila proapoptotic gene hid in Ld652 cells and can rescue cells from apoptosis as shown by increased number of surviving cells and reduced levels of caspase-3-like activity. We also showed that expression of the AMViap gene rescued polyhedron production in Autographa californica M nucleopolyhedrovirus (AcMNPV)Deltap35-infected Sf9 cells during an otherwise abortive infection induced by apoptosis. Surprisingly, deletion of the AMViap gene from the AmEPV genome led to only a modest (10-fold) loss of virion production in infected Ld652 cells, indicating that the AMViap gene is nonessential for virus replication under these conditions. However, infection of Ld652 cells by AmEPV lacking a functional iap gene led to a more rapid induction of cytotoxicity and increased levels of caspase-3-like activity. Similar results were observed and were more pronounced in infected Sf9 and S2 cells. The purified AMVIAP protein also inhibits the enzymatic activities of human caspase-9 and caspase-3 in vitro. Our results indicate that while the AMViap gene was active in controlling apoptosis through the intrinsic pathway, the virus likely encodes additional proteins that also regulate apoptosis.

Amsacta moorei Entomopoxvirus inhibitor of apoptosis suppresses cell death by binding Grim and Hid.

Inhibitor of apoptosis (iap) genes have been identified in the genomes of two independent families of insect viruses, the Baculoviridae and the Entomopoxvirinae. In this report, we examined the functional attributes of the Amsacta moorei entomopoxvirus-encoded IAP protein (AMV-IAP). The binding specificity of the individual baculoviral IAP repeat (BIR) domains of AMV-IAP was investigated by using a random-peptide, phage display library, and sequences similar to the amino termini of proapoptotic Drosophila proteins in the Reaper/Hid/Grim family were identified. Furthermore, the BIR domains of AMV-IAP protein were demonstrated to bind the mammalian IAP inhibitor Smac through the AVPI tetrapeptide sequence, suggesting that the peptide binding pocket and groove found in the insect and mammalian IAPs is conserved in this viral protein. Interaction analysis implicated BIR1 as the high-affinity site for Grim, while BIR2 interacted more strongly with Hid. Both Grim and Hid were demonstrated to interact with AMV-IAP in vivo, and Grim- or Hid-induced cell death was suppressed when AMV-IAP was coexpressed.

XIAP overexpression in islet beta-cells enhances engraftment and minimizes hypoxia-reperfusion injury.

Recent advances in clinical islet transplantation have allowed patients with type 1 diabetes to become insulin independent, but the procedure is limited since islets from two donors per recipient are typically required. This limitation arises because within a few days of the islets being embolized into the portal circulation, at least half of the transplanted beta-cells have undergone apoptotic cell death triggered by hypoxic and chemokine/cytokine-mediated stress. We hypothesized that the survival of beta-cells in the early post-transplant period would be enhanced if naturally occurring inhibitor of apoptosis proteins (IAPs) were transiently overexpressed in the grafts. In the present study, we used a growth-regulatable beta-cell line (betaTC-Tet) as a model for beta-cells within islets, and examined whether adenovirally delivered XIAP (X-linked IAP-a highly potent IAP) could enhance beta-cell survival. In vitro, XIAP-expressing betaTC-Tet cells were markedly resistant to apoptosis in an ischemia-reperfusion injury model system and following exposure to cytokines. When Ad-XIAP transduced betaTC-Tet cells were transplanted subcutaneously into immunodeficient mice, the grafts were able to reverse diabetes in 3 days, vs. 21 days for Ad-betaGal transduced cells. This approach may allow more efficient use of the limited existing supply of human islets.

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant.

BACKGROUND AND AIMS: Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. METHODS: Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. RESULTS: Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. CONCLUSIONS: Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy.

Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9.

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.