Survivin is an inhibitor of apoptosis protein that also functions during mitosis. It is expressed in all common tumors and tissues with proliferating cells, including thymus. To examine its role in apoptosis and proliferation, we generated two T cell–specific survivin-deficient mouse lines with deletion occurring at different developmental stages. Analysis of early deleting survivin mice showed arrest at the pre–T cell receptor proliferating checkpoint. Loss of survivin at a later stage resulted in normal thymic development, but peripheral T cells were immature and significantly reduced in number. In contrast to in vitro studies, loss of survivin does not lead to increased apoptosis. However, newborn thymocyte homeostatic and mitogen-induced proliferation of survivin-deficient T cells were greatly impaired. These data suggest that survivin is not essential for T cell apoptosis but is crucial for T cell maturation and proliferation, and survivin-mediated homeostatic expansion is an important physiological process of T cell development.
proliferation; apoptosis; T cell development; survivin; IAP
Survivin is a component of the chromosomal passenger complex (CPC) that is essential for accurate chromosome segregation. Interfering with the function of Survivin in mitosis leads to chromosome segregation errors and defective cytokinesis. Survivin contains a Baculovirus IAP Repeat (BIR) and therefore was originally classified as inhibitor of apopotosis protein (IAP), yet its role in apoptosis after cellular stress remains largely unknown. We demonstrate here, that Survivin predominantly suppresses anoikis, a form of programmed cell death induced by loss of cellular adhesion to extracellular matrix. Interestingly, cells ectopically overexpressing EGFP-Survivin showed after loss of cell-matrix-interaction a decreased expression of IκB-α. Subsequent subcellular protein fractionation and immunoprecipitation experiments revealed that XIAP interacts with detergent-soluble Survivin which is known to cooperatively activate NF-κB signaling. Examination of the expression levels of detergent soluble Survivin in colorectal cancer cell lines and in colorectal cancerous tissues revealed that detergent soluble cytoplasmic Survivin levels correlated inversely with anoikis susceptibility in colorectal cancer. Therefore, the detergent soluble cytoplasmic Survivin might be a promising predictive biomarker for lymph node and distant metastases of colorectal cancer. We conclude that an anti-apoptotic function of detergent-soluble Survivin in interphase cells experiencing anoikis is mediated at least via XIAP/IκB-α/NF-κB signaling.
Survivin is a member of the inhibitor of apoptosis protein family and has an essential role in mitosis. Survivin is overexpressed in a large variety of human cancers and represents an attractive target for cancer therapy. Epidermal growth factor receptor and Her/neu-transformed human tumors in particular exhibit high levels of survivin. The survivin protein forms dimers through a conserved region that is critical for subcellular localization and biological functions of the protein. We identified small molecules that target a specific cavity adjacent to the survivin dimerization surfaces. S12, a lead compound identified in the screen, can bind to the survivin protein at the intended target site. Moreover, S12 alters spindle formation, causing mitotic arrest and cell death, and inhibits tumor growth in vitro and in vivo. Cell death occurs in premetaphase stage following mitotic arrest and is not a consequence of general toxicity. Thus, the study validates a novel therapeutic target site in the survivin protein and provides a promising strategy to develop a new class of therapeutic small molecules for the treatment of human cancers.
mitosis; cancer; therapeutic; survivin
Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS) oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.
survivin; apoptosis; antisense; oligonucleotides; cytokinesis
Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.
Survivin is a critical regulator of mitosis, and an inhibitor of apoptosis which is overexpressed in almost all cancers. In the current study, cell cycle profiles of normal proliferating human umbilical vein endothelial cells, prostate cancer, and lung cancer cell lines expressing varying levels of survivin and its splice variants were compared using a novel functional complementation assay. Defects in chromosome segregation and cytokinesis that were observed after depletion of endogenous survivin were not complemented by any of the survivin splice variants: survivin-2B, survivin-3B, survivin-ΔEx3, or survivin-2A when expressed exogenously at a level comparable to endogenous full-length survivin. Survivin variants were not detectable at the endogenous protein level. Cancer cells with higher levels of full-length survivin and survivin-2B expression, exhibited reduced caspase-3 activation following doxorubicin treatment and radiation. Whereas earlier studies focused on function and expression levels of survivin specific to cancer cells, the current study brings forward the essential role of survivin in normal dividing cells. Full-length survivin was found to be associated with Aurora-B kinase in the chromosomal passenger complex and depletion of survivin mimics mitotic phenotypes observed after Aurora-B kinase inhibition, in cancer as well as normal proliferating cells. Thus, our study establishes survivin as a marker of proliferation, rather than a cancer specific marker. Therefore, systemic therapeutic interventions targeting survivin will affect cancer as well as normal proliferating cells.
survivin; survivin splice variants; radiation; mitosis; apoptosis
Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.
antisense; cancer; cytokines; differentiation; infection
We have shown that the ectopic expression of Interferon Regulatory Factor (IRF-1) results in human cancer cell death accompanied by the down-regulation of the Inhibitor of Apoptosis Protein (IAP) survivin and the induction of the cyclin-dependent kinase inhibitor p21WAF1/CIP1. In this report, we investigated the direct role of p21 in the suppression of survivin. We show that IRF-1 down-regulates cyclin B1, cdc-2, cyclin E, E2F1, Cdk2, Cdk4, and results in p21-mediated G1 cell cycle arrest. Interestingly, while p21 directly mediates G1 cell cycle arrest, IRF-1 or other IRF-1 signaling pathways may directly regulate survivin in human cancer cells.
IRF-1; survivin; p21; cell cycle; cancer
Regulation of cell death and cell division are key processes during chondrogenesis and in cartilage homeostasis and pathology. The oncogene survivin is considered to be critical for the coordination of mitosis and maintenance of cell viability during embryonic development and in cancer, and is not detectable in most adult differentiated tissues and cells. We analyzed survivin expression in osteoarthritic cartilage and its function in primary human chondrocytes in vitro.
Survivin expression was analyzed by immunoblotting and quantitative real-time PCR. The localization was visualized by immunofluorescence. Survivin functions in vitro were investigated by transfection of a specific siRNA.
Survivin was expressed in human osteoarthritic cartilage, but was not detectable in macroscopically and microscopically unaffected cartilage of osteoarthritic knee joints. In primary human chondrocyte cultures, survivin was localized to heterogeneous subcellular compartments. Suppression of survivin resulted in inhibition of cell cycle progression and sensitization toward apoptotic stimuli in vitro.
The present study indicates a role for survivin in osteoarthritic cartilage and human chondrocytes. In vitro experiments indicated its involvement in cellular division and viability. Learning more about the functions of survivin in chondrocyte biology might further help toward understanding and modulating the complex processes of cartilage pathology and regeneration.
apoptosis; chondrocyte; osteoarthritis; proliferation; survivin
Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family in that it exhibits antiapoptotic properties and also promotes the cell cycle and mediates mitosis as a chromosome passenger protein. Survivin is highly expressed in neural precursor cells in the brain, yet its function there has not been elucidated.
To examine the role of neural precursor cell survivin, we first showed that survivin is normally expressed in periventricular neurogenic regions in the embryo, becoming restricted postnatally to proliferating and migrating NPCs in the key neurogenic sites, the subventricular zone (SVZ) and the subgranular zone (SGZ). We then used a conditional gene inactivation strategy to delete the survivin gene prenatally in those neurogenic regions. Lack of embryonic NPC survivin results in viable, fertile mice (SurvivinCamcre) with reduced numbers of SVZ NPCs, absent rostral migratory stream, and olfactory bulb hypoplasia. The phenotype can be partially rescued, as intracerebroventricular gene delivery of survivin during embryonic development increases olfactory bulb neurogenesis, detected postnatally. SurvivinCamcre brains have fewer cortical inhibitory interneurons, contributing to enhanced sensitivity to seizures, and profound deficits in memory and learning.
The findings highlight the critical role that survivin plays during neural development, deficiencies of which dramatically impact on postnatal neural function.
It has been previously shown that both survivin and the survivin splice variant survivin-2B are localized in mitochondria. Whereas the mechanism involved in blockade of mitochondria-mediated apoptosis by survivin has been extensively studied, the role of survivin-2B in regulation of apoptosis has not been well defined. In the present study, we report that in addition to mitochondria, survivin-2B is also localized in the microtubule organization center (MTOC) and, in contrast to other survivin isoforms (i.e. survivin and survivin-ΔEx3), behaves as a proapoptotic molecule. We show that forced expression of survivin-2B blocks tubulin polymerization, ablates mitotic cells, and induces mitochondria-dependent apoptosis. The mitochondria-mediated apoptosis induced by survivin-2B was indicated by Smac release from mitochondria, activation of caspases 9 and 3, and loss of mitochondrial potential, while caspase-8 remained inactive. Further analysis of the mechanism for the mitochondria-associated events of apoptosis induced by forced expression of survivin-2B revealed down-regulation of the pro-survival factor Bcl-2 and up-regulation of the pro-apoptotic factor Bax in mitochondria, while the apoptosis-inducing factor (AIF) remains unchanged. Our studies further showed that taxol (paclitaxel) treatment of cancer cells not only up-regulates survivin but also down-regulates survivin-2B and that forced expression of survivin-2B sensitizes cells to taxol-induced cell growth inhibition and cell death, while silencing of endogenous survivin-2B transcripts by survivin-2B-specific siRNA made cells resistant to taxol treatment. These findings advance our current knowledge about survivin-2B and may help to develop novel approaches for cancer treatment.
Survivin, a member of the inhibitor of apoptosis (IAP) gene family, plays an important role in both the regulation of cell cycle and the inhibition of apoptosis, and is frequently overexpressed in many tumor types. In neuroblastomas, the expression of survivin correlates with a more aggressive and histologically unfavorable disease. Survivin is predominantly a cytoplasmic protein that is expressed in a cell cycle-dependent manner, increasing in the G2/M phase of the cell cycle followed by a rapid decline in the G1 phase. Recently, the role of survivin in resistance to chemotherapy has become an area of intensive investigation. In this study, we demonstrate a phase-specific resistance due to survivin in staurosporine (STS)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. G2/M-arrested cultures show an upregulation of survivin expression and are more resistant, whereas G1-phase cells that show decreased levels of survivin are more sensitive to apoptosis. Localization studies revealed differences in the distribution of survivin in two synchronized populations, with G1 cells having weakly positive staining confined to the nucleus, in contrast to G2/M cells that depicted a more uniform and intense expression of survivin throughout the cell. In our experimental system, STS induced apoptosis through the mitochondrial-caspase 9-mediated pathway. Retention of survivin in G1 cells by inhibition of the ubiquitin-proteosome pathway or inhibition of caspase 9 protected the cells against apoptosis. Our data suggest that survivin exerts its antiapoptotic effect by inhibiting caspase 9 activity, an important event in STS-mediated apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the survivin pathway for inducing apoptosis in tumor cells.
Survivin; caspase 9; G2/M; staurosporine; neuroblastoma
Survivin is proposed to play a central role in the progression and resistance to therapy of diverse tumour types. High levels of this molecule in tumour cells also correlate with loss of the TP53 tumour suppressor gene, suggesting a molecular connection between TP53 loss and transcriptional induction of Survivin. Patients with TP53 germline mutations, such as those with Li‐Fraumeni syndrome, are particularly susceptible to sarcomas, including rhabdomyosarcomas. Our study aimed to identify rhabdomyosarcoma tumours that express Survivin, in order to test novel Survivin‐targeted therapies in these tumours.
Tumour microarray slides composed of 63 primary rhabdomyosarcoma tumours were stained with a polyclonal antibody to Survivin to identify tumours expressing Survivin. Subcutaneous tumours were then established in NOD/SCID mice using RH30red cells, a red fluorescent clone of the RH30 human alveolar rhabdomyosarcoma cell line. Tumours were treated by hydrodynamic injection with a cocktail of Survivin‐shRNA‐encoding plasmids for a period of 2 weeks.
Over 80% of primary rhabdomyosarcoma tumours expressed Survivin. Treatment of rhabdomyosarcoma xenografts showed greater than 70% reduction in growth when compared with control injected tumours at study completion (average tumour sizes: 1683 v 304 mm3, p<0.05).
Our findings support a role for Survivin in rhabdomyosarcoma biology and provide preliminary evidence for the therapeutic use of Survivin‐targeted RNA interference for human tumours that express high levels of this molecule.
apoptosis; soft tissue sarcoma; Survivin; RNAi; xenografts
BACKGROUND & AIMS
Defective apoptosis of lamina propria T cells (LPTs) is involved in the pathogenesis of Crohn’s disease. Survivin, a member of the inhibitors of apoptosis family, prevents cell death and regulates cell division. Survivin has been studied extensively in cancer, but little is known about its role in Crohn’s disease.
LPTs were isolated from mucosal samples of patients with Crohn’s disease or ulcerative colitis and healthy individuals (controls). LPTs were activated with interleukin-2 or via CD3, CD2, and CD28 signaling, and cultured at 42°C to induce heat shock. Survivin expression was assessed by immunohistochemistry, confocal microscopy, and immunoblotting; survivin levels were reduced by RNA interference. Cell viability, apoptosis, and proliferation were measured by trypan blue exclusion, annexin-V/7-Aminoactinomycin D staining, and uptake of thymidine, respectively.
LPTs from patients with Crohn’s disease had higher levels of survivin than LPTs from patients with ulcerative colitis or controls. RNA knockdown of survivin in LPTs inhibited their proliferation and promoted apoptosis. Levels of survivin were low in LPTs from patients with ulcerative colitis and controls as a result of ubiquitin-mediated proteasome degradation. In LPTs from patients with Crohn’s disease, survivin bound to the heat shock protein (HSP)90, and therefore was resistant to proteasome degradation. Incubating LPTs with 17-N-allylamino-17-demethoxygeldanamycin, an inhibitor of HSP90, reduced levels of survivin and induced apoptosis.
Levels of survivin are increased in LPTs from patients with Crohn’s disease (compared with ulcerative colitis and controls) because survivin interacts with HSP90 and prevents proteasome degradation. This allows LPTs to avoid apoptosis. Strategies to restore apoptosis to these cells might be developed to treat patients with Crohn’s disease.
Inflammatory Bowel Disease; IAP; 17-AAG; Immune Regulation
The inhibitor of apoptosis protein survivin has been implicated in both cell cycle control and apoptosis resistance. To discriminate between these different roles, we used transgenic expression of survivin in the skin as a model for cell proliferation, differentiation, and apoptosis. Transgenic mice expressing survivin under the control of a keratin-14 promoter developed normally, without histologic abnormalities of the skin or hair, epidermal hyperplasia, or developmental abnormalities of basal or suprabasal epidermis. Keratinocyte proliferation assessed under basal conditions, or after ultraviolet-B (UVB) irradiation, or phorbol ester stimulation was unchanged in survivin transgenic mice. In contrast, survivin expression inhibited UVB-induced apoptosis in vitro and in vivo (i.e., sunburn cell formation), whereas it did not affect Fas-induced cell death. When crossed with p53 knockout mice, transgenic expression of survivin in a p53+/– background substituted for the loss of a second p53 allele and further inhibited UVB-induced apoptosis. These data provide the first in vivo evidence that survivin inhibits apoptosis and suggest that this pathway may oppose the elimination of cancerous cells by p53.
Survivin is an IAP family member that plays an essential role in cellular proliferation as an essential component of the chromosome passenger complex. Survivin is highly expressed in embryos and in proliferating adult tissues, but it is not expressed in most differentiated cells. During tumorigenesis, however, survivin expression is dramatically upregulated. Although many studies have shown that survivin is required for cancer cells, the extent to which survivin contributes to the initiation of tumors is unknown. Here we show that transgenic mice that overexpress survivin in hematopoietic cells are at an increased risk of hematologic tumors. In examining how survivin might contribute to tumorigenesis, we observed that hematopoietic cells engineered to overexpress survivin are less susceptible to apoptosis. We conclude that survivin may promote tumorigenesis by imparting a survival advantage to cells that acquire additional genetic lesions.
Survivin and Plk1 kinase are important mediators of cell survival that are required for chromosome alignment, cytokinesis, and protection from apoptosis. Interference with either survivin or Plk1 activity manifests many similar outcomes: prometaphase delay/arrest, multinucleation, and increased apoptosis. Moreover, the expression of both survivin and Plk1 is deregulated in cancer. Given these similarities, we speculated that these two proteins may cooperate during mitosis and/or in cell death pathways. Here we report that survivin and Plk1 interact during mitosis and that Plk1 phosphorylates survivin at serine 20. Importantly, we find that overexpression of a non-phosphorylatable version, S20A, is unable to correct chromosomes connected to the spindle in a syntelic manner during prometaphase and allows cells harboring these maloriented chromosomes to enter anaphase, evading the spindle tension checkpoint. By contrast, the constitutive phosphomimic, S20D, completes congression and division ahead of schedule and, unlike S20A, is able to support proliferation in the absence of the endogenous protein. Despite the importance of this residue in mitosis, its mutation does not appear to affect the anti-apoptotic activity of survivin in response to TRAIL. Together, these data suggest that phosphorylation of survivin at Ser20 by Plk1 kinase is essential for accurate chromosome alignment and cell proliferation but is dispensable for its anti-apoptotic activity in cancer cells.
Apoptosis; Cell Division; Mitosis; Protein Phosphorylation; RNA Interference (RNAi); Polo-like Kinase 1; Survivin
Survivin is a component of the chromosomal passenger complex (CPC) that plays a role in maintenance of an active spindle checkpoint and in cytokinesis. To study whether these different functions can be attributed to distinct domains within the Survivin protein, we complemented Survivin-depleted cells with a variety of point- and deletion-mutants of Survivin. We show that an intact baculovirus IAP repeat (BIR) domain is required for proper spindle checkpoint functioning, but dispensable for cytokinesis. In line with this, mutants lacking an intact BIR domain localized normally to the central spindle, but their localization to inner centromeres was severely perturbed. Consequently, these mutants failed to recruit Aurora B, Borealin/Dasra B, and BubR1 to centromeres and kinetochores, but they had retained the ability to recruit Aurora B and Borealin/Dasra B to the midzone and midbody. Thus, the C terminus of Survivin is sufficient for central spindle localization and execution of cytokinesis, but the additional presence of a functional BIR domain is essential for centromere targeting and spindle checkpoint function. Importantly, our data show that the function of the CPC at the centromere can be separated from its function at the central spindle and that execution of cytokinesis does not require prior concentration of the CPC at centromeres.
Aurora B regulates chromosome segregation and cytokinesis and is the first protein to be implicated as a regulator of bipolar attachment of spindle microtubules to kinetochores. Evidence from several systems suggests that Aurora B is physically associated with inner centromere protein (INCENP) in mitosis and has genetic interactions with Survivin. It is unclear whether the Aurora B and INCENP interaction is cell cycle regulated and if Survivin physically interacts in this complex. In this study, we cloned the Xenopus Survivin gene, examined its association with Aurora B and INCENP, and determined the effect of its binding on Aurora B kinase activity. We demonstrate that in the Xenopus early embryo, all of the detectable Survivin is in a complex with both Aurora B and INCENP throughout the cell cycle. Survivin and Aurora B bind different domains on INCENP. Aurora B activity is stimulated >10-fold in mitotic extracts; this activation is phosphatase sensitive, and the binding of Survivin is required for full Aurora B activity. We also find the hydrodynamic properties of the Aurora B/Survivin/INCENP complex are cell cycle regulated. Our data indicate that Aurora B kinase activity is regulated by both Survivin binding and cell cycle-dependent phosphorylation.
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a null background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.
Functional β-cell mass deficiency in diabetes results from imbalanced β-cell death and replication, and decreased PAK1 protein levels in human islets from donors with type 2 diabetes implicates a possible role for PAK1 in maintaining β-cell mass. Here, we aim to address the linkage between PAK1 and Survivin, a protein essential for β-cell replication. PAK1 knockout (KO) mouse islets exhibited decreased expression of Survivin protein. MIN6 β-cells with siRNA-mediated suppression of PAK1 also had decreased Survivin protein and exhibited an increased level of ubiquitinated-Survivin. However, no significant changes in Survivin mRNA were found in islets from PAK1 KO mice and PAK1-depleted MIN6 β-cells. The decreased Survivin level in MIN6 cells subjected to hyperglycemic stress was prevented by expression of exogenous PAK1. Moreover, overexpressing Survivin restored proliferation of β-cells that was impaired by the loss of PAK1. These data implicate a role for PAK1 in regulating Survivin protein stability in the β-cell and suggest PAK1 as a potential molecular target for the restoration of β-cell mass.
MIN6; PAK1; Survivin; Ubiquitination; mouse islet; pancreatic β-cell; replication
Survivin is the smallest member of the inhibitor of apoptosis (IAP) gene family. Recently, the zebrafish survivin-1 gene has been cloned, showing remarkable sequence identity and similarity over the BIR domain compared with human and mouse survivin gene. Here we investigated the role of survivin in angiogenesis during zebrafish development. Morpholinos (MOs) targeting the 5' untranslated region (UTR) (SurUTR) and sequences flanking the initiation codon (SurATG) of zebrafish survivin-1 gene were injected into embryos at 1–4 cell stage. Vasculature was examined by microangiography and GFP expression in Tg(fli1:EGFP)y1 embryos. Results: In embryos co-injected with SurUTR and SurATG-MOs, vasculogenesis was intact but angiogenesis was markedly perturbed, especially in the inter-segmental vessels (ISV) and dorsal longitudinal anastomotic vessels (DLAV) of the trunk, the inner optic circle and optic veins of developing eyes and the sub-intestinal vessels. Apoptosis was increased, as shown by TUNEL staining and increase in caspase-3 activity. Efficacy of SurUTR and SurATG-MOs was demonstrated by translation inhibition of co-injected 5'UTR survivin:GFP plasmids. The phenotypes could be recapitulated by splice-site MO targeting the exon2-intron junction of survivin gene and rescued by survivin mRNA. Injection of human vascular endothelial growth factor (VEGF) protein induced ectopic angiogenesis and increased survivin expression, whereas treatment with a VEGF receptor inhibitor markedly reduced angiogenesis and suppressed survivin expression. Conclusion: Survivin is involved in angiogenesis during zebrafish development and may be under VEGF regulation.
The anti-apoptosis protein, survivin, promotes cell survival and mitosis. Recent studies have demonstrated that survivin is expressed in normal gastric mucosa. Using an in vitro model, we examined whether survivin plays a role in the cytoprotection produced in gastric mucosa by mild irritant ethanol (ETOH) against subsequent exposure to concentrated ETOH. Pretreatment of rat gastric epithelial cells with 1% ETOH reduced cell death, in response to subsequent incubation with 5% ETOH, by 94% (P<0.005). This pretreatment also resulted in increased total and phosphorylated survivin protein levels by 180% (P<0.0001) and 540% (P<0.0002), respectively, which required the p34cdc2 cell cycle-dependent kinase. The cytoprotective effect was abrogated upon siRNA knockdown of survivin protein levels. Further, overexpression of exogenous survivin resulted in significant cytoprotection by 62% (P<0.02) in the absence of any pretreatment. We further examined the in vivo relevance of these findings. In fasted rats, administration of 20% ETOH, which we found to be 93% (P<0.0001) cytoprotective against 50% ETOH challenge, resulted in increased total and phosphorylated survivin protein levels by 234% (P<0.001) and 214% (P<0.02), respectively. Administration of 20% ETOH resulted in increased gastric p34cdc2 activity by 146% (P<0.01). Inhibition of p34cdc2 by the potent inhibitor, roscovitine, abolished the increased survivin levels in response to pre-administration of 20% ETOH and reduced the cytoprotection against 50% ETOH challenge by 59% (P<0.01). These results indicate that survivin is a key mediator of cytoprotection against ETOH-induced gastric injury, acting at the epithelial cell level, by a mechanism that is dependent, in part, on p34cdc2.
• Adaptation; • Apoptosis; • Cell death; • Inhibitor; • Phosphorylation
Survivin (BIRC5), a member of the inhibitor of apoptosis protein (IAP) family that inhibits caspases and blocks cell death is highly expressed in cancer and is associated with a poorer clinical outcome. Functioning simultaneously during cell division and apoptosis inhibition, survivin plays a pivotal role in determining cell survival. Survivin has consistently been identified by molecular profiling analysis to be associated with higher tumor grade, more advanced disease, abbreviated survival, accelerated rates of recurrence, and chemotherapy and radiation resistance. Survivin's differential expression in cancer compared to normal tissue and its role as a nodal protein in a number of cellular pathways make it a highly flexible therapeutic target, suitable for small-molecule inhibitiors, molecular antagonists, and vaccination-based therapies. By targeting survivin it is hoped that multiple tumor signaling circuitries may be simultaneously disabled. This effect may be applicable to many tumor histologies irrespective of specific genetic makeup. To date, survivin inhibitors have shown modest activity as single agents, but it is anticipated that when given in combination with cytotoxic chemotherapy or monoclonal antibodies they may exhibit enhanced efficacy. This review discusses the complex circuitry of survivin in human cancers and highlights clinical trials involving novel agents that target this important protein.
The inhibitor of apoptosis (IAP) protein Survivin is a dual mediator of apoptosis resistance and cell cycle progression, and is highly expressed in cancer. We have previously shown that Survivin is upregulated in melanoma compared to normal melanocytes, is required for melanoma cell viability, and that melanocyte expression of Survivin predisposes mice to UV-induced melanoma and metastasis. The mechanism(s) of Survivin upregulation in the course of melanocyte transformation, and its repression in normal melanocytes, however, has not been clearly defined. We show here that p53 and Rb, at basal levels and in the absence of any activating stimuli, are both required to repress survivin transcription in normal human melanocytes. Survivin repression in melanocytes does not involve alterations in protein stability or promoter methylation. p53 and Rb (via E2Fs) regulate Survivin expression by direct binding to the survivin promoter; p53 also affects Survivin expression by activating p21. We demonstrate a novel role for E2F2 in the negative regulation of Survivin expression. In addition, we identify a novel E2F-binding site in the survivin promoter and show that mutation of either the p53- or E2F-binding sites is sufficient to increase promoter activity. These studies suggest that compromise of either p53 or Rb pathways during melanocyte transformation leads to upregulation of Survivin expression in melanoma.
Survivin; melanocyte; p53; Rb; E2F