Dario C. Altieri

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo. A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serine-threonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of anti-apoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo. vascular endothelial growth factor angiopoietin microvascular endothelial cells human umbilical vein endothelial cells phosphate-buffered saline Tris-buffered saline green fluorescent protein wortmannin β-galactosidase tumor necrosis factor α During angiogenesis, endothelial cells receive cues from growth factors to initiate mitosis, migration, and organization of endothelial cells into primitive angiotubes and patent vascular networks (1.Risau W. Nature. 1997; 386: 671-674Crossref PubMed Scopus (4925) Google Scholar, 2.Hanahan D. Science. 1997; 277: 48-50Crossref PubMed Scopus (1056) Google Scholar). These processes critically depend on preservation of endothelial cell viability. Disruption of endothelial cell-matrix contacts or interference with extracellular survival signals is sufficient to initiate caspase-dependent apoptosis in endothelium, culminating with rapid involution of vascular structures (3.Brooks P.C. Montgomery A.M. Rosenfeld M. Reisfeld R.A. Hu T. Klier G. Cheresh D.A. Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2199) Google Scholar, 4.O'Reilly M.S. Holmgren L. Chen C. Folkman J. Nat. Med. 1996; 2: 689-692Crossref PubMed Scopus (1155) Google Scholar). Unlike most angiogenic regulators, including fibroblast growth factor or vascular endothelial growth factor (VEGF),1 angiopoietin-1 (Ang-1) does not stimulate endothelial cell growth but rather promotes stabilization of vascular networks and branching morphogenesis in vivo and in vitro (5.Davis S. Aldrich T.H. Jones P.F. Acheson A. Compton D.L. Jain V. Ryan T.E. Bruno J. Radziejewski C. Maisonpierre P.C. Yancopoulos G.D. Cell. 1996; 87: 1161-1169Abstract Full Text Full Text PDF PubMed Scopus (1709) Google Scholar, 6.Koblizek T.I. Weiss C. Yancopoulos G.D. Deutsch U. Risau W. Curr. Biol. 1998; 8: 529-532Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 7.Papapetropoulos A. Garcia-Cardena G. Dengler T.J. Maisonpierre P.C. Yancopoulos G.D. Sessa W.C. Lab. Invest. 1999; 79: 213-223PubMed Google Scholar, 8.Witzenbichler B. Maisonpierre P.C. Jones P. Yancopoulos G.D. Isner J.M. J. Biol. Chem. 1998; 273: 18514-18521Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Little is known about the signaling requirements of these responses, and the mechanism(s) of Ang-1-induced cytoprotection are unknown (7.Papapetropoulos A. Garcia-Cardena G. Dengler T.J. Maisonpierre P.C. Yancopoulos G.D. Sessa W.C. Lab. Invest. 1999; 79: 213-223PubMed Google Scholar, 9.Kontos C.D. Stauffer T.P. Yang W.P. York J.D. Huang L. Blanar M.A. Meyer T. Peters K.G. Mol. Cell. Biol. 1998; 18: 4131-4140Crossref PubMed Scopus (187) Google Scholar). The major goal of this paper was to elucidate a potential link between endothelial cell viability and maintenance of angiogenesis by examining the ability of Ang-1 to activate the anti-apoptotic serine-threonine kinase, Akt (or protein kinase B). Moreover, we examined the relationship between Ang-1, Akt activation, and the expression of the anti-apoptotic genes, bcl-2 and survivin, in cultured microvascular endothelial cells (MVEC). Bovine MVEC (Vec Technologies, Rensselaer, NY) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, l-glutamine, and antibiotics (penicillin and streptomycin). Cells (up to passage 12) were used for the experiments. In experiments examining endogenous survivin expression, human umbilical vein endothelial cells (HUVEC) were used, because the survivin antibody recognized human survivin better than bovine survivin. HUVEC were cultured on gelatin-coated tissue culture flasks in M199 medium containing 20% fetal bovine serum, 50 μg/ml endothelial cell growth supplement (a commercial preparation that contains mainly acidic fibroblast growth factor), 100 μg/ml porcine heparin, 10 units/ml penicillin, and 100 μg/ml streptomycin. Two to three individual donors were pooled at passage one and used up to passage three. Both MVEC and HUVEC cultures had typical cobblestone morphology and stained uniformly for von Willebrand factor, as assessed by indirect immunofluorescence. Angiopoietin-1 and -2 and soluble recombinant Tie 1 and 2 receptors were provided by Regeneron. A recombinant form of Ang-1 was used in all of the experiments. This form of Ang-1 differs from the native Tie 2 ligand in that it possesses a modified NH2-terminal sequence and a mutation in Cys245 that make it easier to produce and purify. Cells were washed twice with PBS and lysed with cell lysis buffer (1% Nonidet P-40, 10% glycerol, 137 mm NaCl, 20 mm Tris-HCl, pH 7.4, 20 mm NaF, 2 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride). 20 μg of protein was separated on SDS-polyacrylamide gel electrophoresis gel and transferred onto a polyvinylidine difluoride membrane (Millipore). After blocking with PBS containing 0.2% Tween 20 containing 5% milk for 1 h, the membrane was incubated with anti-Akt antibody (Santa Cruz Biotechnology), phosphospecific Akt antibody (New England Biolabs). ECL (Amersham Pharmacia Biotech) was used for detection. For activity assays, lysates were precleared with protein G-agarose for 30 min at 4 °C and immunoprecipitated for 2 h with anti-Akt antibodies in the presence of 2 μg/ml bovine serum albumin with or without 16 μg/ml competitor peptides (Santa Cruz Biotechnology). Immunoprecipitates were washed twice with cell lysis buffer, once with water, and once with kinase buffer (20 mm HEPES, pH 7.2, 10 mm MgCl2, 10 mm MnCl2). Immunoprecipitated proteins were incubated in 50 μl of kinase buffer containing 2 μg of histone H2B (Roche Molecular Biochemicals) and [32P]ATP (5 μm, 10 μCi) for 30 min at room temperature. Kinase reactions were stopped by the addition of SDS sample buffer, and samples were subjected to Cerekenov counting. Parallel samples were processed to confirm equal amounts of precipitated Akt. MVEC were plated onto bacteriological dishes in serum-free medium in the presence of either vehicle (TBS containing CHAPS) or Ang-1 (250 ng/ml). Cells were incubated for 18 h, and both floating and adherent cells were collected. To determine the number of subdiploid cells, MVEC were fixed for 1 h in 70% ethanol and stained with a solution containing 500 μg/ml RNase H and 50 μg/ml propidium iodide and analyzed by using an FACS. At least 5000 events were analyzed, and the percentage of cells in the sub-G1 population was calculated. Aggregates of cell debris at the origin of the histogram were excluded from the analysis of sub-G1 cells as indicated in the legends to Figs. 2 and 4.Figure 4Survivin mediates the anti-apoptotic effect of Ang-1. A, TNFα-cycloheximide. B, anoikis. C, morphology. MVEC were transfected with GFP vector, GFP-survivin (survivin), or GFP-C84A survivin (C84A survivin) for 24 h followed by treatment with TNFα (5 ng/ml) plus cycloheximide (5 μg/ml) for 9 h (A,TNF/CHX) or by plating in serum-free medium on bacteriological dishes (B) in the absence or presence of Ang-1 (250 ng/ml). Data are representative of two experiments in duplicate. Surv, survivin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) MVEC were infected with 50–100 multiplicities of infection of herpes simplex viruses expressing β-galactosidase or the dominant negative Δp85 subunit of PI3 kinase as described (10.Fryer H.J.L. Knox R.J. Wolf D.H. Yen L. Strittmatter S.M. O'Leary R.M. Pennica D. Russell D.S. Kalb R.G. J. Neurochem. 2000; 74: 582-595Crossref PubMed Scopus (57) Google Scholar). Alternatively, MVEC were infected with similar multiplicities of infection of adenoviruses containing the β-galactosidase or the hemagglutinin-tagged activation-deficient phosphorylation mutant Akt (AA-Akt). After 4 h, the virus was removed, and the cells were left to recover overnight in complete medium. In preliminary experiments with the β-galactosidase virus, these conditions were optimal for infecting 95% of the cultures. Infected cells were either plated in bacteriological dishes or lysed in lysis buffer for immunoblotting. MVEC were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and serum-starved for 24 h followed by challenge with Ang-1 as described above. Total RNA was extracted from cell pellets with TRI reagent (106 cells/0.2 ml, Molecular Research Center, Inc., Cincinnati, Ohio). For Northern analysis, 10–20 μg of total RNA were separated on 1% agarose gels with formaldehyde, transferred to nylon filters (Hybond-N, Amersham Pharmacia Biotech), UV cross-linked, and hybridized with the corresponding 32P-labeled cDNA probes (survivin, bcl-2, or β-actin) in ExpressHyb hybridization solution (CLONTECH, Palo Alto, CA). After washing, the filter was exposed for autoradiography. pLuc-cyc1.2 (+1 to −268) was generated by polymerase chain reaction with the human survivin promoter sequence as a template and confirmed by DNA sequencing. pLuc-42 was generated by inserting the first 42-base pair fragment of the 3′-end of the human survivin promoter upstream of the luciferase gene and confirmed by sequencing. Transient transfection of MVEC was performed using Lipofectin reagent (Life Technologies, Inc.) as described previously (16.Gerber H. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 13313-13316Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). Briefly, MVEC were seeded in a 12-well plate (1–2 × 105 cells/well) in 1 ml of medium and grown to 50–80% confluence. 50 μl of Opti-MEM I (Life Technologies, Inc.) containing 1 μg of various plasmid DNAs was combined with 50 μl of Opti-MEM I containing 4 μl of Lipofectin reagent. The combined mixture was overlaid onto cells that were preincubated with serum-free medium for 20–30 min. The transfected cells were then incubated at 37 °C for 4–6 h. The DNA-liposome complex was replaced with complete medium, and luciferase activity/β-gal expression (internal control) were measured within 36–48 h post-transfection. MVEC were transfected with the cDNAs for GFP, GFP-survivin (survivin), or GFP-C84A survivin (C84A survivin) for 24 h. Fusion of survivin with GFP does not interfere with its biological activity or localization. The survivin-GFP (Cys84-Ala) construct is a mutation in the Bir1 domain that is targeted to the mitotic spindle but is devoid of anti-apoptosis function. In experiments using GFP-survivin, approximately 30% of the cells were transfected, and apoptosis, under the various conditions tested, was determined by propidium iodide staining and flow cytometry. The percentage of cells with hypodiploid DNA content quantified in the GFP-expressing population is shown in each histogram. Aggregates of cell debris at the origin of the histogram were excluded from the analysis of sub-G1 cells. In some experiments, cells were imaged on an inverted microscope (Zeiss, Axiovert) using DIC optics. Stimulation of MVEC with Ang-1 increased Akt phosphorylation on Ser473 and Thr308 (not shown) and in a reaction suppressed by the PI3 kinase inhibitor, wortmannin (WM; Fig.1 A). Ang-1 also increased Akt activity in a wortmannin-sensitive manner (6.7 ± 0.6, 13.2 ± 1.8, and 6.1 ± 0.7 counts per min of 32P (× 103) incorporated into histone H2B for control; Ang-1- and Ang-1 plus wortmannin-treated cells, n = 3;p < 0.05). To directly test the role of PI3 kinase in Ang-1-stimulated Akt activation, MVEC were infected with a replication-deficient herpes simplex virus encoding β-gal or a dominant negative construct for the p85 subunit of PI3 kinase (Δp85; 10), As seen in Fig. 1 B, Ang-1 increased Akt phosphorylation in β-gal-transduced cells, whereas infection with the virus encoding Δp85 abrogated basal and Ang-1-stimulated Akt phosphorylation analogous to wortmannin. Ang-1 stimulated Akt phosphorylation in a time-dependent manner with maximal activation occurring within 15–30 min and sustained phosphorylation lasting for up to 2 h (Fig. 1 C). Ang-1-stimulated phosphorylation of Akt on Ser473 was antagonized by preincubation of Ang-1 with soluble Tie 2 receptor but not by incubation with soluble Tie 1 receptor bodies (Fig. 1 D). In addition, Ang-1-induced Akt phosphorylation was partially blocked by the physiological antagonist of Ang-1, angiopoeitin-2 (Ang-2; 11). Interestingly, Ang-2 alone weakly activated Akt in MVEC. Our results are consistent with data from heterologous expression systems and transformed endothelial cells documenting that Ang-1 activation of PI3 kinase is important for cell migration and survival (9.Kontos C.D. Stauffer T.P. Yang W.P. York J.D. Huang L. Blanar M.A. Meyer T. Peters K.G. Mol. Cell. Biol. 1998; 18: 4131-4140Crossref PubMed Scopus (187) Google Scholar, 12.Jones N. Master Z. Jones J. Bouchard D. Gunji Y. Sasaki H. Daly R. Alitalo K. Dumont D.J. J. Biol. Chem. 1999; 274: 30896-30905Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Therefore, Ang-1 via the Tie 2 receptor stimulates Akt activation through a PI3 kinase-dependent mechanism. Next, we asked if Ang-1 could influence endothelial cell apoptosis induced by detachment from the matrix, i.e. anoikis (13.Frisch S.M. Ruoslahti E. Curr. Opin. Cell Biol. 1997; 9: 701-706Crossref PubMed Scopus (999) Google Scholar). MVEC in serum-free media was plated onto Petri dishes for 18 h and underwent extensive apoptosis as determined by appearance of a hypodiploid cell population (∼25% versus 2% of control, adherent cultures) by propidium iodide staining and flow cytometry (Fig. 2 A). Incubation of MVEC cultured under these conditions with Ang-1 inhibited apoptosis by 75% in a reaction abrogated by WM (Fig. 2 A). To examine whether Akt was required for Ang-1 cytoprotection, we infected MVEC with adenoviral β-galactosidase or activation-deficient Akt (AA-Akt; 14) and determined the degree of apoptosis by FACS analysis. Transduction of MVEC with AA-Akt abrogated the cytoprotective effect of Ang-1 against anoikis, whereas a control adenovirus encoding β-galactosidase was ineffective. Moreover, Ang-1 stimulated Akt phosphorylation while MVEC were in suspension (Fig. 2 B). WM also prevented Akt phosphorylation on Ser473 induced by Ang-1 in suspended endothelial cells. Collectively, these data indicate that Ang-1 mediates endothelial cell protection through an integrin-independent, PI3 kinase/Akt-dependent pathway. Next, we examined a potential link between Ang-1 and expression of two known anti-apoptotic genes, survivin and bcl-2 (15.Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar, 16.Gerber H. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 13313-13316Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). Treatment of MVEC with Ang-1 rapidly induced a time-dependent increase in survivin mRNA levels (17.Li F. Ambrosini G. Chu E.Y. Plescia J. Tognin S. Marchisio P.C. Altieri D.C. Nature. 1998; 396: 580-584Crossref PubMed Scopus (1756) Google Scholar), which peaked 12 h after stimulation and remained sustained for up to 24 h (Fig. 3 A). In contrast, Ang-1 did not up-regulate bcl-2 mRNA expression in MVEC (Fig. 1 A). Consistent with a receptor-mediated response, preincubation of Ang-1 with soluble Tie 2 receptor abolished Ang-1 induction of survivin RNA in MVEC (Fig.3 B). When MVEC were transfected with a survivin-luciferase promoter construct (18.Li F. Altieri D.C. Biochem. J. 1999; 344: 305-311Crossref PubMed Scopus (290) Google Scholar), Ang-1 stimulated a 3–7-fold up-regulation of survivin transcriptional activity, which persisted for up to 24 h after stimulation (Fig. 3 C). Ang-1 induced the expression of survivin protein in HUVEC, an effect abrogated by WM, or by transduction with adenoviral AA-Akt (Fig. 3 D). In contrast, Ang-1 did not increase the expression of bcl-2 protein expression in MVEC (Fig. 3 D). These data demonstrate that Ang-1 stimulates survivin expression in endothelial cells via a PI3 kinase/Akt-dependent mechanism. To determine whether survivin can mediate the anti-apoptotic function of Ang-1, we transfected MVEC with cDNAs containing GFP fused to wild-type survivin (GFP-survivin) or to a dominant negative Cys84 → Ala survivin mutant (GFP-C84A survivin) and determined cytoprotection in response to apoptosis-inducing stimuli (19.Grossman D. McNiff J.M. Li F. Altieri D.C. Lab. Invest. 1999; 79: 1121-1126PubMed Google Scholar). Treatment with Ang-1 or expression of GFP-survivin, alone or in combination with Ang-1, suppressed the appearance of MVEC with hypodiploid DNA content induced by TNFα-/cycloheximide or by anoikis (Fig. 4, A and B). In contrast, transfection of MVEC with GFP-C84A survivin abrogated the cytoprotective effect of Ang-1 against TNFα-cycloheximide- or anoikis-induced cell death (Fig. 4, A and B). Consistent with the above analysis, Ang-1 alone or in combination with transfected GFP-survivin resulted in healthier morphology of any adherant cells, in contrast to cells transfected with GFP alone or GFP-C84A survivin plus Ang-1 (Fig. 4 C) These data identify survivin as a novel PI3 kinase/Akt-dependent target gene for Ang-1 and demonstrate that survivin is necessary for the anti-apoptotic effect of Ang-1 in endothelial cells. In summary, Ang-1 prevents endothelial cell apoptosis by activating a critical survival messenger, Akt, and by up-regulating a broad spectrum apoptosis inhibitor, survivin. Although Akt activation is required for survivin expression and interference with survivin function by the C84A survivin mutant abolishes Ang-1 cytoprotection, activated Akt may also execute parallel anti-apoptosis pathways through phosphorylation of caspase-9 and/or Bad (20.Cardone M.H. Roy N. Stennicke H.R. Salvesen G.S. Franke T.F. Stanbridge E. Frisch S. Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2748) Google Scholar, 21.Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1194) Google Scholar). Recent studies have suggested that VEGF increases survivin expression in endothelial cells (22.Tran J. Rak J. Sheehan C. Saibil S.D. LaCasse E. Korneluk R.G. Kerbel R.S. Biochem. Biophys. Res. Commun. 1999; 264: 781-788Crossref PubMed Scopus (308) Google Scholar, 23.O'Connor D.S. Schechner J.S. Adida C. Mesri M. Rothermel A.L. Li F. Nath A.K. Pober J.S. Altieri D.C. Am. J. Pathol. 2000; 156 (in press)Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Complementing and extending these findings with VEGF, our study with Ang-1, a non-mitogenic survival factor, may have far-reaching implications for angiogenesis when endothelial cells need to loosen their focal contacts with the underlying matrix prior to emigration, proliferation, and reorganization into patent structures that can accommodate blood flow. In this scenario, inhibition of apoptosis by Ang-1/Akt/survivin may protect the endothelium during this complex transition and maintain a critical anti-apoptotic environment during stabilization of vascular networks. Targeted manipulation of this mechanism may be exploited to improve endothelial cell viability and favor therapeutic angiogenesis in vivo. We thank Dr. K. Walsh for adenoviral constructs, Dr. R. Neve for herpes simplex constructs, Drs. G. D. Yancopoulos and P. C. Maisonpierre for enthusiastic support and angiopoietins, and Dr. J. S. Pober for constructive critiques of the paper.

The interface between apoptosis (programmed cell death) and the cell cycle is essential to preserve homeostasis and genomic integrity. Here, we show that survivin, an inhibitor of apoptosis over-expressed in cancer, physically associates with the cyclin-dependent kinase p34 cdc2 on the mitotic apparatus, and is phosphorylated on Thr 34 by p34 cdc2 -cyclin B1, in vitro and in vivo . Loss of phosphorylation on Thr 34 resulted in dissociation of a survivin-caspase-9 complex on the mitotic apparatus, and caspase-9-dependent apoptosis of cells traversing mitosis. These data identify survivin as a mitotic substrate of p34 cdc2 -cyclin B1 and suggest that survivin phosphorylation on Thr 34 may be required to preserve cell viability at cell division. Manipulation of this pathway may facilitate the elimination of cancer cells at mitosis.

Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single- and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75–130 kilobases (kb) on chromosome 17q25. In Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin. This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants. These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer. Survivin is a new IAP apoptosis inhibitor expressed during development and in human cancer in vivo. The coding strand of the survivin gene was extensively complementary to that of effector cell protease receptor-1 (EPR-1), prompting the present investigation on the origin and functional relationship of these two transcripts. Southern blots of genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes. By pulsed field gel electrophoresis and single- and two-color fluorescence in situ hybridization, these were contained within a contiguous physical interval of 75–130 kilobases (kb) on chromosome 17q25. In Northern blots, a single strand-specific probe identified a 1.3-kb EPR-1 mRNA broadly distributed in normal adult and fetal tissues, structurally distinct from the 1.9-kb Survivin transcript expressed in transformed cell lines. Transient co-transfection of an EPR-1 cDNA potentially acting as a Survivin antisense with a lacZ reporter plasmid resulted in loss of viability of HeLa cells. In contrast, co-transfection of an antisense cDNA of intercellular adhesion molecule-1 or a sense-oriented Survivin cDNA was without effect. In stably transfected HeLa cells, ZnSO4 induction of an EPR-1 mRNA under the control of a metallothionein promoter suppressed the expression of endogenous survivin. This resulted in (i) increased apoptosis as detected by analysis of DNA content and in situ internucleosomal DNA fragmentation and (ii) inhibition of cell proliferation as compared with induced vector control transfectants. These findings suggest the existence of a potential EPR-1/survivin gene cluster and identify survivin as a new target for disrupting cell viability pathways in cancer. Regulated inhibition of programmed cell death (apoptosis) preserves normal homeostasis and tissue and organ morphogenesis (1Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4578) Google Scholar, 2Vaux D.L. Haecker G. Strasser A. Cell. 1994; 76: 777-779Abstract Full Text PDF PubMed Scopus (705) Google Scholar). Aberrations of this process participate in human diseases and may contribute to cancer by abnormally prolonging cell viability with accumulation of transforming mutations (3Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6244) Google Scholar). Recently, several apoptosis inhibitors related to the baculovirus iap gene have been identified in mouse, Drosophila, and human (4Clem R.J. Duckett C.S. Trends Cell Biol. 1997; 7: 337-339Abstract Full Text PDF PubMed Scopus (90) Google Scholar). Intercalated in TNF receptor signaling (5Rothe M. Pan M.-G. Henzel W.J. Merrill-Ayres T. Goeddel D.V. Cell. 1995; 83: 1242-1252Abstract Full Text PDF Scopus (1062) Google Scholar, 6Uren A.G. Pakusch M. Hawkins C.J. Puls K.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4974-4978Crossref PubMed Scopus (449) Google Scholar) and NF-κB-dependent survival (7Chu Z.-L. McKinsey T.A. Gentry J.J. Malim M.H. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10057-10062Crossref PubMed Scopus (830) Google Scholar), IAP proteins contain two/three Cys/His baculovirus IAP repeats plus a carboxyl terminus RING finger and are thought to block an evolutionarily conserved step in apoptosis (6Uren A.G. Pakusch M. Hawkins C.J. Puls K.L. Vaux D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4974-4978Crossref PubMed Scopus (449) Google Scholar, 8Liston P. Roy N. Tamal K. Lefebvre C. Baird S. Cherton-Horvat G. Farahani R. McLean M. Ikeda J.-E. MacKenzie A. Korneluk R.G. Nature. 1996; 379: 349-353Crossref PubMed Scopus (876) Google Scholar, 9Roy N. Mahadevan M.S. McLean M. Shutler G. Yaraghi Z. Farahani R. Baird S. Besner-Johnston A. Lefebvre C. Kang X. Salith M. Aubry H. Tamai K. Guan X. Ioannou P. Crawford T.O. de Jong P.J. Surh L. Ikeda J.-E. Korneluk R.G. MacKenzie A. Cell. 1995; 80: 167-178Abstract Full Text PDF PubMed Scopus (881) Google Scholar, 10Duckett C.S. Nava V.E. Gedrich R.W. Clem R.J. Van Dongen J.L. Gilfillan M.C. Shiels H. Hardwich J.M. Thompson C.B. EMBO J. 1996; 14: 2685-2694Crossref Scopus (526) Google Scholar). At least in the case of XIAP (8Liston P. Roy N. Tamal K. Lefebvre C. Baird S. Cherton-Horvat G. Farahani R. McLean M. Ikeda J.-E. MacKenzie A. Korneluk R.G. Nature. 1996; 379: 349-353Crossref PubMed Scopus (876) Google Scholar), this may involve direct inhibition of the terminal effector caspases −3 and −7 (11Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1732) Google Scholar). A novel member of the IAP gene family, designated Survivin (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar), was recently identified by hybridization screening of human genomic libraries with the cDNA of a factor Xa receptor, effector cell protease receptor-1 (EPR-1) (13Altieri D.C. FASEB J. 1995; 9: 860-865Crossref PubMed Scopus (85) Google Scholar). 1The abbreviations used are: EPR-1, effector cell protease receptor-1; kb, kilobase(s); nt, nucleotide(s). Unlike all other IAP proteins (4Clem R.J. Duckett C.S. Trends Cell Biol. 1997; 7: 337-339Abstract Full Text PDF PubMed Scopus (90) Google Scholar), Survivin contained a single baculovirus IAP repeat and no RING finger and was selectively expressed during development and in all the most common human cancers but not in normal adult tissues in vivo (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). Intriguingly, the Survivin coding strand was extensively complementary to that of EPR-1, thus suggesting a potential functional interaction between these two transcripts (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). In this study, we sought to dissect the molecular relationship between EPR-1 and Survivin (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar, 13Altieri D.C. FASEB J. 1995; 9: 860-865Crossref PubMed Scopus (85) Google Scholar) and its role in apoptosis inhibition. We found that EPR-1 and Survivin are encoded by structurally and topographically distinct messages potentially originating from a gene cluster at 17q25. Secondly, down-regulation of Survivin by forced expression of EPR-1 increased apoptosis and inhibited growth of transformed cells. Peripheral blood mononuclear cells were isolated from heparinized blood collected from normal informed volunteers by differential centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech) at 400 × g for 22 °C and washed in phosphate-buffered saline, pH 7.4. The epithelial carcinoma HeLa cell line was obtained from American Type Culture Collection (Rockville, MD) and maintained in culture in complete growth medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (BioWhittaker) and 2 mml-glutamine, according to the manufacturer's specifications. For fluorescence in situ hybridization, purified DNA from a Survivin P1 genomic clone (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar) was labeled with digoxigenin dUTP (Amersham Pharmacia Biotech) by nick translation, combined with sheared human DNA, and hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood mononuclear cells in 50% formamide, 10% dextran sulfate, and 2× SSC. For two-color staining, biotin-conjugated probe D17Z1, specific for the centromere of chromosome 17, was co-hybridized with the digoxigenin-labeled P1 clone. Specific chromosomal staining was detected by fluoresceinated anti-digoxigenin antibodies and Texas red avidin. Slides were counterstained with propidium iodide or DAPI for one- or two-color labeling, respectively. A total of 80 metaphase cells were analyzed with 69 cells exhibiting specific labeling. Human genomic DNA was extracted from HeLa cells, digested with EcoRI, BamHI,XbaI, or HindIII, separated on a 0.8% agarose gel and transferred to GeneScreen nylon membranes (NEN Life Science Products). After UV cross-linking (Stratagene, San Diego, CA), the membrane was prehybridized with 100 μg/ml of denatured salmon sperm DNA (Promega Corp., Madison, WI) in 5× SSC, 0.5% SDS, 5× Denhardt's solution, and 0.1% sodium pyrophosphate at 65 °C in a roller hybridization oven (Hoefer Scientific, San Francisco, CA). Hybridization was carried out with gel-purified (GeneClean Bio101, Vista, CA), [32P]dCTP (Amersham Pharmacia Biotech) random-primed labeled (Boehringer Mannheim) 1.6-kb EPR-1 cDNA (13Altieri D.C. FASEB J. 1995; 9: 860-865Crossref PubMed Scopus (85) Google Scholar) for 16 h at 65 °C. After two washes in 2× SSC, 1% SDS for 30 min at 65 °C, and 0.2× SSC at 22 °C, radioactive bands were visualized by autoradiography using a Kodak X-Omat AR x-ray film and intensifying screens (DuPont). In other experiments, cultured lymphoblastoid cells were embedded in low melting preparative agarose (Bio-Rad) at the concentration of 2 × 106/220 μl block, and DNA was extracted by standard procedures. After block digestion with MluI or NotI, samples were separated by pulsed field gel electrophoresis on a 1% agarose gel for 20 h at 200 V with a pulse time of 75 s using a Bio-Rad CHEF DRII apparatus. After transfer to nylon membranes and UV cross-linking, hybridization with the EPR-1 cDNA and washes were carried out as described. In another series of experiments, a blot containing aliquots of genomic DNA isolated from several species (CLONTECH, San Francisco, CA) was hybridized with a 3′ 548-nt fragment of the EPR-1 cDNA, as described above. Multiple tissue blots of adult and fetal mRNA (CLONTECH) were prehybridized with 100 μg/ml of denatured salmon sperm DNA (Promega) and hybridized with an EPR-1-single strand-specific probe (see below) in 5× SSPE, 10× Denhardt's, 2% SDS, for 14 h at 60 °C. The membranes were washed twice in 2× SSC, 1% SDS for 30 min at 60 °C and once in 0.2× SSC at 22 °C before exposure for autoradiography. An EPR-1-specific single strand probe was generated by asymmetric polymerase chain reaction amplification of a 301-nt fragment of the EPR-1 cDNA generated by EcoRI (cloning site) andSacII digest and comprising the first 5′ 226 nt of the EPR-1 coding sequence plus 75 nt of the retained regulatory intron (13Altieri D.C. FASEB J. 1995; 9: 860-865Crossref PubMed Scopus (85) Google Scholar). The gel-purified fragment was mixed with 15 pmol dNTP (New England Biolabs, Beverly, MA), 7.5 pmol of dCTP, and 25 μCi of [32P]dCTP (Amersham Pharmacia Biotech) in 20 mm Tris HCl, 50 mm KCl, pH 8.4, 1.5 mm MgCl2, plus 0.2 μg/μl of a SacII reverse EPR-1 primer 5′TGCTGGCCGCTCCTCCCTC3′ and 2.5 units of Taq DNA polymerase (Life Science) in a total volume of 10 μl. 25 cycles of amplification were carried out with denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 1 min. After centrifugation through a Sephadex G-50 spin column (Worthington Biochemical Corp., Freehold, NJ) at 14,000 × g for 5 min, the EPR-1 or Survivin probes were heated at 100 °C for 2 min and immediately added to the various hybridization reactions. A control antisense construct of intercellular adhesion molecule-1 was generated by polymerase chain reaction amplification of the full-length human intercellular adhesion molecule-1 cDNA (14Simmons D. Makgoba M.W. Seed B. Nature. 1988; 331: 624-627Crossref PubMed Scopus (582) Google Scholar) using oligonucleotides 5′-GATCTAGACTCGCTATGGCTCCCAGC-3′ and 5′-CCGCAAGCTTTCAGGGAGGCGTGGCTTG-3′ containingXbaI and HindIII restriction sites, respectively (underlined sequences). The amplified product of 1605 nt was gel purified and directionally cloned in pcDNA3 (Invitrogen, San Diego, CA) for transfection in HeLa cells. A 708-ntSmaI-EcoRI fragment of the EPR-1 cDNA (nt 379–1087) (13Altieri D.C. FASEB J. 1995; 9: 860-865Crossref PubMed Scopus (85) Google Scholar), potentially acting as a Survivin antisense, and a sense-oriented Survivin construct (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar) were also used for these experiments. Subconfluent cultures of HeLa cells in 6-well tissue culture plates were cotransfected with 1 μg of lacZ reporter plasmid and 4 μg of the various sense- and antisense-oriented constructs or the empty pcDNA3 vector using LipofectAMINE (Life Technologies, Inc.). 48 h after transfection, cells were fixed in 2% paraformaldehyde for 1 h and stained for β-galactosidase expression with 0.5 mg/ml 5-bromo-4-chloro-3-indolyl β-d-galactoside (Amersham Pharmacia Biotech), 5 mm potassium ferrocyanide, 5 mm potassium ferricyanide, and 2 mm MgCl2 in phosphate-buffered saline. The blue cells were counted and scored on an inverted microscope. In another series of experiments, HeLa cells (1 × 107) were transfected with 10 μg of control pcDNA3 vector alone or the survivin antisense cDNA plus 50 μg of salmon sperm DNA by electroporation (Gene Pulser, Bio-Rad) with a single electric pulse at 350 V at 960 microfarads. 48 h after transfection, HeLa cells were collected by trypsinization, pooled with nonattached cells, and fixed in 70% ethanol on ice for 30 min. Fixed cells were pelletted by centrifugation and suspended in 10 μg/ml propidium iodide, 100 μg/ml RNase A, and 0.05% Triton X-100 in phosphate-buffered saline, pH 7.4. After a 45-min incubation at 22 °C, samples were analyzed for DNA content by flow cytometry using a FACScan (Becton Dickinson). The 708-nt SmaI-EcoRI fragment of the EPR-1 cDNA (see above) was directionally cloned in the sense orientation in the mammalian cell expression vector pML1 (a gift of Dr. R. Pytela, University of California, San Francisco). The vector is derived from the episomal mammalian expression vector pCEP4 by replacing the cytomegalovirus promoter cassette with the mMT1 promoter, directing Zn2+-dependent expression of recombinant proteins in mammalian cells (15Lukashev M.E. Sheppard D. Pytela R. J. Biol. Chem. 1994; 269: 18311-18314Abstract Full Text PDF PubMed Google Scholar). 10 million HeLa cells were transfected with 10 μg of control pcDNA3 vector or the Survivin antisense by electroporation as described above. 48 h after transfection, cells were diluted, plated onto 100-mm diameter tissue culture dishes, and selected for 4 weeks in complete growth medium containing 0.4 mg/ml hygromycin. Modulation of survivin expression in control cultures or Zn2+-induced antisense transfectants was carried by immunoblotting of detergent-solubilized cell extracts using 25 μg/ml aliquots of the affinity-purified antibody raised against the survivin sequence Ala3–Ile19, as described (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). In control experiments, Zn2+-induced vector control or survivin antisense transfectants were analyzed for modulation of Class I major histocompatibility complex by flow cytometry with monoclonal antibody W6/32. Vector control or Survivin antisense transfectants were treated with 200 μm ZnSO4in 0% fetal bovine serum for 24 h at 37 °C following byin situ determination of apoptosis by internucleosomal DNA fragmentation (TUNEL). Briefly, cells were harvested and centrifuged at 800 × g for 10 min at 4 °C, the pellet was fixed in 10% formalin overnight, dehydrated, and embedded in paraffin blocks, and sections of 3–5 μm were put on high adhesive slides. Samples were treated with 20 μg/ml proteinase K for 15 min at 22 °C, washed in distilled water, quenched of endogenous peroxidase in 2% H2O2 in phosphate-buffered saline, and subsequently mixed with digoxigenin-labeled dUTP in the presence of terminal deoxynucleotidyl transferase followed by peroxidase-conjugated anti-digoxigenin antibody. Nuclear staining in apoptotic cells was detected by 3′, 3′-diaminobenzidine tetrahydrochloride dihydrate, according to the manufacturer's instructions (ApopTag, Oncor, Gaithersburg, MD). For control experiments, the enzyme incubation step was omitted. Morphologic features of apoptotic cells (apoptotic bodies) under the various conditions tested were also analyzed by hematoxylin/eosin staining. For proliferation experiments, vector control or Survivin antisense transfectants at 2 × 104/well were plated in 24-well tissue culture plates (Costar) and induced with 200 μm ZnSO4 in complete growth medium for 16 h at 37 °C, and cell proliferation was determined microscopically at 24 h intervals by direct cell count. Two independent clones of HeLa cell transfectants were used in these experiments with comparable results. In some experiments, analysis of DNA content in induced vector control or Survivin antisense transfectants in complete growth medium was carried out by propidium iodide staining and flow cytometry, as described above. A digoxigenin-labeled P1 genomic clone (∼100 kb) containing all four exons of the survivin gene (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar) specifically labeled a single region on the long arm of a group E chromosome by fluorescencein situ hybridization (Fig. 1 A). In two-color staining with probe D17Z1 specific for the centromere of chromosome 17, the Survivin P1 clone reacted with the long arm of chromosome 17 with band 17q25 (Fig. 1, A and B). Probing human genomic DNA with the EPR-1 cDNA revealed several hybridizing bands (Fig. 2 A). Of these, a ∼7.5-kb XbaI, a 7.6-kb BamHI, and four HindIII fragments of ∼15, 7.5, 6.4, and 3.7 kb, respectively (Fig. 2 A, arrowheads), were not predicted from the complete restriction map of 14,796 nt of thesurvivin gene (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). In contrast, other bands of comparable intensity, including a 5.1-kb XbaI and a 7.1-kbBamHI fragment, or of stronger intensity, including fragments of 17.5-kb HindIII, 10.5-kb XbaI, 8.5-kb BamHI, and ∼25-kb EcoRI (Fig. 2 A), genuinely originated from the survivin gene (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). At variance with this complex hybridization pattern, pulsed field gel electrophoresis of high molecular mass human genomic DNA revealed only single EPR-1-hybridizing bands of ∼75 and ∼130 kb inMluI- or NotI-digested samples, respectively (Fig. 2 B). Finally, the EPR-1 cDNA strongly hybridized with several bands in genomic DNA from various mammalian species, with fainter signals in rabbit or chicken DNA (Fig. 2 C). Consistent with the size of the spliced EPR-1 message (13Altieri D.C. FASEB J. 1995; 9: 860-865Crossref PubMed Scopus (85) Google Scholar), a single strand EPR-1-specific probe detected a prominent ∼1.3-kb EPR-1 mRNA band in most adult and terminally differentiated human tissues (Fig. 3,upper panel). Strong EPR-1 expression was observed in human pancreas, skeletal muscle, heart, and various hematopoietic cell types, including peripheral blood leukocytes, lymph node, and spleen (Fig. 3,upper panel). Consistent with the reactivity of an anti-EPR-1 antibody with fetal tissues (16Adida C. Crotty P.L. McGrath J. Berrebi D. Diebold J. Altieri D.C. Am. J. Pathol. 1998; 152: 43-49PubMed Google Scholar), a 1.3-kb EPR-1 mRNA was also found prominently in fetal kidney and liver and less abundantly in fetal lung and brain (Fig. 3, lower panel). Control hybridization with an actin probe confirmed comparable loading of mRNA in the various fetal samples (Fig. 3). In contrast, a Survivin-specific single strand probe did not react with mRNA isolated from normal adult tissues (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar), whereas it detected a prominent ∼1.9-kb transcript plus a fainter 3.4-kb species in various transformed cell lines (not shown) and in agreement with previous observations (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). Transient co-transfection of HeLa cells with an EPR-1 cDNA potentially acting as a Survivin antisense plus alacZ reporter plasmid produced significant loss of viability in β-galactosidase-expressing cells (Fig. 4). In contrast, co-transfection of pcDNA3 vector alone, a sense-oriented Survivin construct, or a control antisense of intercellular adhesion molecule-1 cDNA did not affect HeLa cell viability under the same experimental conditions (Fig. 4). To determine more precisely the effect of regulated expression of EPR-1 on Survivin inhibition of apoptosis, HeLa cells were stably transfected with an EPR-1 cDNA under the control of an metallothionein-inducible promoter. In these experiments, ZnSO4 induction of EPR-1 mRNA suppressed the expression of endogenous Survivin, as determined by immunoblotting with an anti-Survivin antibody (Fig. 5 A). In contrast and consistent with the expression of Survivin in transformed cell types, a single 16.5-kDa Survivin band was immunoblotted in metallothionein-induced HeLa cells transfected with the pML1 vector alone (Fig. 5 A). In control experiments, metallothionein induction of EPR-1 mRNA did not affect the expression of Class I major histocompatibility complex molecules in HeLa cell transfectants, and no modulation of Survivin expression was observed in the absence of ZnSO4 (not shown). Under these experimental conditions, antisense down-regulation of Survivin resulted in massive apoptosis in growth factor-deprived HeLa cells, as detected by in situinternucleosomal DNA fragmentation by the TUNEL system (Fig. 5 B, panel 1). Specific nuclear staining was observed in 60–70% of metallothionein-induced, serum-starved HeLa cell transfectants, whereas induced vector control cultures did not stain with the digoxigenin-labeled dUTP probe (Fig. 5 B,panel 3). No staining was observed in the absence of terminal deoxynucleotidyl transferase labeling (not shown). Hematoxylin/eosin staining confirmed the presence of numerous apoptotic bodies in ZnSO4-induced Survivin antisense transfectants, as compared with vector control HeLa cells (Fig. 5 B,panels 2 and 4, arrowheads). The effect of antisense down-regulation of Survivin on HeLa cell proliferation was also investigated. As shown in Fig. 6 A, suppression of endogenous Survivin resulted in significant inhibition of cell proliferation, as compared with induced vector control cultures (Fig. 6 A). 3 days after metallothionein induction, the number of vector control HeLa cell transfectants increased by 288% during optimal serum mitogen stimulation, as opposed to a 20% increase in Survivin antisense transfectants, under the same experimental conditions (Fig. 6 A). The increased cell proliferation observed at later time intervals (days 4–5) in induced antisense transfectants may reflect heterogeneity in antisense expression with selective expansion of low expressing cells (Fig. 6 A). The potential ability of Survivin to modulate apoptosis and cell proliferation under optimal concentrations of serum mitogens was further investigated. Analysis of DNA content in transiently transfected HeLa cells revealed a ∼2-fold increase in the fraction of apoptotic cells (sub-G1 peak) in Survivin antisense transfectants as compared with vector control cells, under the same experimental conditions (Fig. 6 B,M1 marker). This was also associated with a ∼15–20% decrease in the G2/M fraction in Survivin antisense transfectants, as compared with vector control cultures (Fig. 6 B, M4 marker). In stable HeLa cell transfectants, zinc induction of Survivin antisense under optimal growth conditions produced a ∼1.4-fold increase in the sub-G1 fraction and a ∼20–36% reduction in the G2/M peak, as compared with induced vector control cultures (n = 2).Figure 5Effect of metallothionein induction of EPR-1 mRNA on Survivin expression and apoptosis. A, aliquots of HeLa cells stably transfected with the empty pML1 vector (Vector) or the EPR-1 cDNA potentially acting as a Survivin antisense (Antisense) were induced with 200 μm ZnSO4, detergent-solubilized, and immunoblotted with the anti-survivin antibody. Molecular weight (×10−3) markers are shown on the left.B, the experimental conditions are as in A, except that serum-starved Survivin antisense transfectants (1 and 2) or vector control cells (3and 4) were stained for internucleosomal DNA fragmentation by the ApopTag method (TUNEL) (1 and 3) or by hematoxylin-eosin (2 and 4).Arrowheads, apoptotic bodies. Magnification, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Inhibition of cell proliferation by Survivin antisense. A, 20,000 vector control HeLa cell transfectants (Vector) or Survivin antisense transfectants were seeded in 24-well plates in complete growth medium, induced with 200 μm ZnSO4, and harvested at the indicated time intervals with determination of cell proliferation by direct cell count. Data are the means ± S.E. of replicates of a representative experiment out of seven independent determinations.B, HeLa cells were transiently transfected by electroporation with control vector pcDNA3 or the survivin antisense cDNA. After a 48-h culture in complete growth medium, cells were analyzed for DNA content by propidium iodide staining and flow cytometry. The M1 marker contains the sub-G1 apoptotic fraction (18.2% in Survivin antisense transfectants versus 9.6% in vector control cells), whereas the M4 marker contains the proliferating G2/M fraction (18% in Survivin antisense transfectants versus21% in vector control cells).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we have shown that EPR-1 (13Altieri D.C. FASEB J. 1995; 9: 860-865Crossref PubMed Scopus (85) Google Scholar) and Survivin (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar) are encoded by structurally and topographically distinct mRNA transcripts potentially originating from a gene cluster at 17q25. Secondly, constitutive or metallothionein induction of EPR-1, potentially acting as a Survivin antisense, down-regulated endogenous Survivin in transformed cells and resulted in increased apoptosis and inhibition of cell proliferation, even in the presence of optimal serum mitogen concentrations. Among the regulators of programmed cell death (apoptosis), IAP proteins have recently attracted considerable attention for their ability to suppress an evolutionarily conserved step in apoptosis (4Clem R.J. Duckett C.S. Trends Cell Biol. 1997; 7: 337-339Abstract Full Text PDF PubMed Scopus (90) Google Scholar), potentially involving direct caspase inhibition (11Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1732) Google Scholar). Deregulation of this pathway may also participate in human diseases, because inactivating mutations of neuronal apoptosis inhibitory protein contributed to spinal muscular atrophy (9Roy N. Mahadevan M.S. McLean M. Shutler G. Yaraghi Z. Farahani R. Baird S. Besner-Johnston A. Lefebvre C. Kang X. Salith M. Aubry H. Tamai K. Guan X. Ioannou P. Crawford T.O. de Jong P.J. Surh L. Ikeda J.-E. Korneluk R.G. MacKenzie A. Cell. 1995; 80: 167-178Abstract Full Text PDF PubMed Scopus (881) Google Scholar), and this molecule was cytoprotective against cerebral ischemia in vivo (17Xu D.G. Crocker S.J. Doucet J.-P. St-Jean M. Tamai K. Hakim A.M. Ikeda J.-E. Liston P. Thompson C.S. Korneluk R.G. MacKenzie A. Robertson G.S. Nat. Med. 1997; 3: 997-1004Crossref PubMed Scopus (224) Google Scholar). More recently, this paradigm has been extended to cancer, with the identification of Survivin as a structurally unique IAP protein selectively expressed during development and in all the most common human cancers but not in normal adult tissues in vivo (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). Intriguingly, thesurvivin gene was identified by hybridization with the EPR-1 cDNA, and its coding sequence was found to be extensively complementary to that of EPR-1 (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar), suggesting the possibility of apoptosis regulation by a potential interaction between these two transcripts, i.e. natural antisense (18Kimmelman D. Kischner M.W. Cell. 1989; 59: 687-696Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 19Farrell C.M. Lukens L.N. J. Biol. Chem. 1995; 270: 3400-3408Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 20Celano P. Berchtold C.M. Kizer D.L. Weeraratna A. Nelkin B.D. Baylin S.B. Casero Jr., R.A. J. Biol. Chem. 1992; 267: 15092-15096Abstract Full Text PDF PubMed Google Scholar, 21Khochbin S. Lawrence J. EMBO J. 1989; 8: 4107-4114Crossref PubMed Scopus (81) Google Scholar). Here, Southern blots of human genomic DNA were consistent with the presence of multiple, evolutionarily conserved, EPR-1/Survivin-related genes, with several hybridizing fragments that could not be recapitulated by the complete physical map of 14,796 nt of thesurvivin gene (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar). Despite this complex hybridization pattern, pulsed field gel electrophoresis and fluorescence in situ hybridization studies suggested the existence of a single EPR-1/Survivin locus spanning 75–130 kb on chromosome 17q25. Although mammalian genes transcribed in both directions have been described (22Adelman J.P. Bond C.T. Douglass J. Herbert E. Science. 1987; 235: 1514-1517Crossref PubMed Scopus (167) Google Scholar,23Kindy M.S. McCormack J.E. Buckler A.J. Levine R.A. Sonenshein G.E. Mol. Cell. Biol. 1987; 7: 2857-2862Crossref PubMed Scopus (44) Google Scholar), these data are more consistent with a model of separate genes encoding EPR-1 and Survivin, potentially arisen from duplication event(s) and clustered in relatively close proximity at 17q25 in a head-to-head configuration (24Graham G.J. J. Theor. Biol. 1995; 175: 71-87Crossref PubMed Scopus (66) Google Scholar). The use of single strand-specific probes further demonstrated that EPR-1 and Survivin originated from two structurally different messages of 1.3 and 1.9 kb, respectively, expressed in a mutually exclusive fashion in adult and fetal tissues (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar, 16Adida C. Crotty P.L. McGrath J. Berrebi D. Diebold J. Altieri D.C. Am. J. Pathol. 1998; 152: 43-49PubMed Google Scholar). This is consistent with the heterogeneity of EPR-1 transcripts detected by conventional, double strand probes with hybridizing bands of 1.9, 3.4, and ∼1.5 kb previously identified in EPR-1+ cells (25Nicholson A.C. Nachman R.L. Altieri D.C. Summers B.D. Ruf W. Edgington T.S. Hajjar D.P. J. Biol. Chem. 1996; 271: 28407-28413Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Although it is currently not known if these two messages actually interact in vivo, we found that metallothionein induction of an EPR-1 mRNA suppressed the expression of endogenous Survivin in transfected cells. Consistent with the anti-apoptosis properties of Survivin (12Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3049) Google Scholar), this resulted in increased apoptosis and significant inhibition of cell proliferation. Although accentuated by serum mitogen withdrawal, HeLa cell apoptosis following antisense down-regulation of Survivin was also observed under optimal growth conditions and was associated with a reduced number of proliferating cells in the G2/M fraction. In these experiments, the use of a noncoding EPR-1 cDNA potentially acting as a Survivin antisense ruled out the possibility that inhibition of Survivin was due to protein interactions. It is also unlikely that ZnSO4 induction of the metallothionein promoter may exert an independent anti-apoptotic function, because this has been attributed to ZnCl2, at concentrations 5–10-fold higher than those used here (26Newmeyer D.D. Farschon D.M. Reed J.C. Cell. 1994; 79: 353-364Abstract Full Text PDF PubMed Scopus (499) Google Scholar). The findings described here may have profound implications for cancer therapy, where antisense-based strategies have been postulated for inhibition of several proto-oncogenes (27Henry S.P. Monteith D. Levin A.A. Anti-Cancer Drug Des. 1997; 12: 395-408PubMed Google Scholar). Specifically, antisense blockade of anti-apoptotic bcl-2 decreased survival of leukemic cells in vitro (28Campos L. Sabido O. Rouault J.P. Guyotat D. Blood. 1994; 84: 595-600Crossref PubMed Google Scholar), reduced tumorigenicity of lymphoma cells in athymic mice (29Reed J.C. Cuddy M. Haldar S. Croce C. Nowell P. Makover D. Bradley K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3660-3664Crossref PubMed Scopus (166) Google Scholar), and provided, at least in some cases, a positive therapeutic response in patients with non-Hodgkin's lymphoma (30Webb A. Cunningham D. Cotter F. Clarke P.A. di Stefano F. Ross P. Corbo M. Dziewanowska Z. Lancet. 1997; 349: 1137-1141Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). In this context and consistent with the data presented here, targeting Survivin may selectively increase the susceptibility of cancer cells to apoptosis-based treatment and reduce their overall growth potential. In addition to inhibiting cell viability pathways distinct and complementary with those of bcl-2 (11Deveraux Q.L. Takahashi R. Salvesen G.S. Reed J. Nature. 1997; 388: 300-304Crossref PubMed Scopus (1732) Google Scholar), suppression of Survivin by an endogenous EPR-1 transcript potentially acting as a natural antisense may overcome the drawbacks of limited specificity and insufficient delivery commonly observed with antisense oligonucleotides (27Henry S.P. Monteith D. Levin A.A. Anti-Cancer Drug Des. 1997; 12: 395-408PubMed Google Scholar). Elucidation of the mechanisms regulating Survivin and EPR-1 gene expression should further facilitate the selective disruption of this novel anti-apoptosis pathway in cancer without affecting viability of normal tissues. We thank Dr. Pytela for providing the pML1 vector.

Neuroblastoma may be aggressive with a dismal outcome or regress spontaneously. Prognostic factors including age, stage, and ploidy, N-myc amplification, and histology are necessary to direct treatment. Gene products controlling apoptosis may modulate neuroblastoma's variable course. 1 Pritchard J Hickman JA Why does stage 4s neuroblastoma regress spontaneously?. Lancet. 1994; 344: 869-870 Abstract PubMed Scopus (104) Google Scholar Although expression of anti-apoptotic bcl-2 has been linked to a more aggressive disease, 2 Castle VP Heidelberger KP Bromberg J Ou X Dole M Nunez G Expression of the apoptosis-suppressing protein bcl-2, in neuroblastoma is associated with unfavourable histology and N-myc amplification. Am J Pathol. 1993; 143: 1543-1550 PubMed Google Scholar and inversely correlated with morphological features of apoptosis, it is unclear whether inhibition or apoptosis is a predictive factor in neuroblastoma or whether it reflects the degree of cell differentiation. 3 Hoehner JC Hedborg F Wiklund HJ Olsen L Pahlman S Cellular death in neuroblastoma: In situ correlation of apoptosis and bcl-2 expression. Int J Cancer. 1995; 62: 19-24 Crossref PubMed Scopus (59) Google Scholar An apoptosis inhibitor, survivin, 4 Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997; 3: 917-921 Crossref PubMed Scopus (3000) Google Scholar has recently been identified. Unlike other members of the bcl-2 and IAP gene families, survivin is selectively expressed in all the most common human cancers but not in normal adult tissues. 4 Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997; 3: 917-921 Crossref PubMed Scopus (3000) Google Scholar We determined the expression of survivin in neuroblastoma and its relation to disease progression.

Molecular chaperones, especially members of the heat shock protein 90 (Hsp90) family, are thought to promote tumor cell survival, but this function is not well understood. Here, we show that mitochondria of tumor cells, but not most normal tissues, contain Hsp90 and its related molecule, TRAP-1. These chaperones interact with Cyclophilin D, an immunophilin that induces mitochondrial cell death, and antagonize its function via protein folding/refolding mechanisms. Disabling this pathway using novel Hsp90 ATPase antagonists directed to mitochondria causes sudden collapse of mitochondrial function and selective tumor cell death. Therefore, Hsp90-directed chaperones are regulators of mitochondrial integrity, and their organelle-specific antagonists may provide a previously undescribed class of potent anticancer agents.

Despite its genetic complexity and multifactoriality, two processes appear almost universally compromised in cancer: the control of cell proliferation and the regulation of cell lifespan. Survivin is a recently described molecule that has been implicated in both processes, and is overexpressed in most human cancers. The exploitation of the survivin signaling pathway might provide important predictive and prognostic clues in cancer diagnosis, and offer new therapeutic alternatives for cancer treatment.

Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP–/– cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis. Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP–/– cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis. Among the regulators of programmed cell death, or apoptosis (1Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar), Bcl-2 proteins (2Cory S. Adams J.M. Nat. Rev. Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3349) Google Scholar) control the release of apoptogenic proteins from mitochondria, notably cytochrome c (3Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar), whereas members of the inhibitor of apoptosis (IAP) 1The abbreviations used are: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; WT, wild type; MEF, mouse embryonic fibroblast; GST, glutathione S-transferase; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; AFC, amino-4-trifluoromethylcoumarin; GFP, green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole. gene family act as endogenous inhibitors of caspases (4Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1581) Google Scholar), the enzymatic effectors of apoptosis (1Hengartner M.O. Nature. 2000; 407: 770-776Crossref PubMed Scopus (6296) Google Scholar). The structural requirements of IAP-caspase(s) complexes have been defined in considerable detail (5Shi Y. Mol. Cell. 2002; 9: 459-470Abstract Full Text Full Text PDF PubMed Scopus (1465) Google Scholar). Survivin is a structurally unique IAP protein that has been implicated in protection from apoptosis and regulation of mitosis (6Altieri D.C. Nat. Rev. Cancer. 2003; 3: 46-54Crossref PubMed Scopus (1110) Google Scholar). A role of survivin in cell division has been linked to assembly/stability of metaphase and anaphase microtubules (7Giodini A. Kallio M. Wall N.R. Gorbsky G.J. Tognin S. Marchisio P.C. Symons M. Altieri D.C. Cancer Res. 2002; 62: 2462-2467PubMed Google Scholar) and spindle checkpoint function (8Lens S.M. Medema R.H. Cell Cycle. 2003; 2: 507-510Crossref PubMed Scopus (70) Google Scholar). In contrast, despite its ability to counteract apoptosis in vitro, and in transgenic animals (6Altieri D.C. Nat. Rev. Cancer. 2003; 3: 46-54Crossref PubMed Scopus (1110) Google Scholar), the mechanism(s) by which survivin inhibits apoptosis has remained elusive. This is important because IAPs, especially XIAP (9Holcik M. Gibson H. Korneluk R.G. Apoptosis. 2001; 6: 253-261Crossref PubMed Scopus (359) Google Scholar) and survivin (6Altieri D.C. Nat. Rev. Cancer. 2003; 3: 46-54Crossref PubMed Scopus (1110) Google Scholar), have emerged as critical regulators of cell survival in tumors and promising targets for rational anti-cancer therapy (10O'Connor D.S. Wall N.R. Porter A.C. Altieri D.C. Cancer Cell. 2002; 2: 43-54Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 11Schimmer A.D. Welsh K. Pinilla C. Wang Z. Krajewska M. Bonneau M.J. Pedersen I.M. Kitada S. Scott F.L. Bailly-Maitre B. Glinsky G. Scudiero D. Sausville E. Salvesen G. Nefzi A. Ostresh J.M. Houghten R.A. Reed J.C. Cancer Cell. 2004; 5: 25-35Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). In this study, we investigated the mechanism(s) of survivin cytoprotection. We found that in response to cell death stimulation, survivin physically associates with XIAP, and this complex promotes enhanced XIAP stability and synergistic inhibition of caspase-9 activation. Cell Culture—Breast carcinoma MCF-7, lymphoblastoid Raji, and kidney embryonic HEK293T cells were from the American Type Culture Collection (ATCC, Manassas, VA). Wild type (WT) or XIAP–/– mouse embryonic fibroblasts (MEF) (12Harlin H. Reffey S.B. Duckett C.S. Lindsten T. Thompson C.B. Mol. Cell. Biol. 2001; 21: 3604-3608Crossref PubMed Scopus (376) Google Scholar) were the gift of Dr. C. Duckett (University of Michigan). Protein-Protein Interactions—Affinity fractionation and immunoprecipitation experiments were carried out as described (10O'Connor D.S. Wall N.R. Porter A.C. Altieri D.C. Cancer Cell. 2002; 2: 43-54Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 13Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar). Full-length survivin, truncated survivin BIR-(1–87), full-length XIAP, cIAP1, cIAP2, or the three isolated XIAP, BIR1-(1–123), BIR2-(123–259) and BIR3-(260–336) were expressed as GST fusion proteins (14Fortugno P. Wall N.R. Giodini A. O'Connor D.S. Plescia J. Padgett K.M. Tognin S. Marchisio P.C. Altieri D.C. J. Cell Sci. 2002; 115: 575-585PubMed Google Scholar). Replication-deficient adenoviruses encoding GFP (pAd-GFP) or survivin (pAd-survivin) were described (15Blanc-Brude O.P. Mesri M. Wall N.R. Plescia J. Dohi T. Altieri D.C. Clin. Cancer Res. 2003; 9: 2683-2692PubMed Google Scholar). Pull-down experiments with recombinant survivin (0.1–0.4 μg) and GST, GST-XIAP, GST-cIAP1, GST-cIAP2 (8 μg), or the individual GST-BIR1, -BIR2, or -BIR3 of XIAP (10 μg) bound to glutathione beads (100 μl) were as described (13Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar). Alternatively, XIAP was translated in vitro in the presence of [35S]methionine (Amersham Sciences), mixed with 5 μg of GST or GST-survivin, and used in pull-down experiments. Modulation of XIAP Ubiquitination—HEK293T cells were transfected with FLAG-XIAP in the presence of survivin or control vector, pulse-labeled with 0.5 mCi of [35S]methionine and [35S]cysteine, chased with unlabeled l-methionine, and immunoprecipitated with an antibody to FLAG (Sigma). Bands were quantified by NIH Image analysis (1.62/3DV11.01/ppc). In vitro ubiquitination reactions were performed for 1 h at 30 °C using 1 μg of survivin-His6 or control ΔN-Traf3-His6 in 40 mm Tris, pH 7.5, 5 mm MgCl2, 1 mm DTT, 10% glycerol, 10 mm phosphocreatine, 100 μg/ml creatine phosphokinase, 0.5 mm ATP, 1 mg/ml methylated ubiquitin, 1 μm ubiquitin aldehyde, 1 μl of in vitro translated 35S-labeled XIAP or 35S-labeled XIAP Lys238 → Arg mutant, with or without HEK293T extracts (30 μg). HEK293T cells were transfected with His6-ubiquitin, pcDNA3-Myc-survivin, and FLAG-XIAP, lysed after 24 h, and mixed with 60 μl of cobalt-chelation resin, and bound proteins were analyzed by immunoblotting. In some experiments, XIAP expression levels and formation of a survivin-XIAP complex were analyzed in MCF-7 cells treated with staurosporine (1–1.5 μm for 12 h) with or without a caspase inhibitor, Z-VAD-fmk (20 μm), or proteasome inhibitor lactacystin (5 μm). Analysis of Caspase Activity and Cell Death—Caspase assays were performed using recombinant caspase-9, XIAP, and survivin by continuously monitoring AFC release from the fluorigenic substrate Ac-LEHD-AFC. Alternatively, recombinant procaspase-9 (4 nm) was incubated with Apaf-1 (4 nm), cytochrome c (600 nm), dATP (200 μm), procaspase-3 (4 nm), and GST-XIAP in the presence of recombinant survivin for 30 min at 30 °C, and reaction mixtures were analyzed for cleavage of Ac-DEVD-AFC. HEK293T cells were transfected with pEGFP (Clontech) and various plasmids (Superfect, Quiagen) and harvested after 24 h, and GFP+ cells were scored for nuclear morphology of apoptosis by 4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescence microscopy. Alternatively, WT or XIAP–/– MEF were transduced with pAd-GFP or pAd-survivin, exposed to staurosporine (0.75 μm) or UVB (1500 J/m2) for 18 h, and analyzed by DAPI staining and fluorescence microscopy. Identification of a Survivin-XIAP Complex during Apoptosis—We fractionated Raji cell extracts by affinity chromatography over an antibody to survivin (14Fortugno P. Wall N.R. Giodini A. O'Connor D.S. Plescia J. Padgett K.M. Tognin S. Marchisio P.C. Altieri D.C. J. Cell Sci. 2002; 115: 575-585PubMed Google Scholar) and analyzed bound proteins by immunoblotting. Survivin co-eluted with the molecular chaperone complex comprising both Hsp90 and Hsp70 (Fig. 1A), in agreement with previous observations (13Fortugno P. Beltrami E. Plescia J. Fontana J. Pradhan D. Marchisio P.C. Sessa W.C. Altieri D.C. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 13791-13796Crossref PubMed Scopus (299) Google Scholar). In addition, the survivin complex contained the IAP family protein XIAP (4Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1581) Google Scholar) in the same fractions (Fig. 1A). We next immunoprecipitated endogenous survivin from MCF-7 cells with or without exposure to the apoptotic stimulus staurosporine and probed the immune complexes by immunoblotting. Immunoprecipitated survivin co-associated with endogenous XIAP, and this interaction was increased by staurosporine treatment (Fig. 1B). Immune complexes precipitated with a control IgG did not contain XIAP (Fig. 1B). Interactions of survivin with homologous IAP proteins, cIAP1 and cIAP2, were also detected but not with Bcl-2, Bcl-XL, or Bax (not shown). To investigate a role of apoptotic stimulation in the formation of a survivin-XIAP complex, we overexpressed survivin in MCF-7 cells using a replication-deficient adenovirus (pAd-survivin) and performed co-immunoprecipitation experiments with or without staurosporine. Exposure of MCF-7 cells to staurosporine increased the association of survivin with XIAP, whereas immunoprecipitates of non-immune IgG did not contain XIAP (Fig. 1C). We next determined whether survivin and XIAP interacted directly. Recombinant survivin bound GST-XIAP in a concentration-dependent manner, whereas no interaction with GST was observed (Fig. 1D). Reciprocally, 35S-labeled in vitro translated XIAP associated with GST-survivin but not GST (Fig. 1D). We next expressed IAP family proteins cIAP1 and cIAP2 (4Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1581) Google Scholar) and tested their association with survivin. Both cIAP1 and cIAP2 bound survivin directly, whereas no interaction with GST was detected (Fig. 1E). A role of homologous BIR sequences in IAP-IAP complex formation was investigated. Recombinant survivin associated with XIAP-BIR1 and XIAP-BIR3 and more weakly with XIAP-BIR2 (Fig. 1F). Reciprocally, a truncated survivin 1–87 mutant containing the single survivin BIR bound full-length XIAP (not shown). Regulation of XIAP Stability by Survivin—MCF-7 cells transduced with pAd-GFP and exposed to staurosporine exhibited a time-dependent loss of XIAP expression (Fig. 2A). In contrast, transduction of MCF-7 cells with pAd-survivin preserved XIAP expression over time in the presence of staurosporine (Fig. 2A). We then performed [35S]l-methionine pulse-chase experiments in HEK293T cells expressing XIAP together with survivin or a control vector. Co-transfection of HEK293T cells with survivin resulted in persistence of XIAP levels over a 6-h interval after chase (Fig. 2B) and prolongation of XIAP half-life from 2 h in vector-transfected cells to 5.5 h (Fig. 2C). Addition of the proteasome inhibitor lactacystin prevented loss of XIAP expression in staurosporine-treated MCF-7 cells, whereas a caspase inhibitor, Z-VAD-fmk, was ineffective (Fig. 2D). Regulation of XIAP Ubiquitination by Survivin—When mixed with ubiquitin, in vitro translated, 35S-labeled XIAP exhibited a ladder of bands typical of polyubiquitinated conjugates (Fig. 3A). In contrast, a XIAP (Lys328 → Arg) mutant lacking a critical lysine residue involved in ubiquitination of this protein exhibited minimal ubiquitin conjugation in vitro (Fig. 3A). Addition of recombinant survivin suppressed XIAP ubiquitin conjugate formation, whereas a control Traf3 protein was ineffective (Fig. 3B). Increasing concentrations of recombinant survivin inhibited the binding of the ubiquitin-conjugating enzyme, UbcH5, to GST-XIAP (Fig. 3C), suggesting that a survivin-XIAP complex may prevent substrate accessibility to the ubiquitination machinery. We then explored whether survivin interfered with XIAP ubiquitination in vivo. Polyubiquitinated XIAP conjugates were readily detectable in HEK293T cells transfected with control pcDNA3 in the presence of a proteasome inhibitor (Fig. 3D). In contrast, expression of survivin attenuated XIAP polyubiquitination in vivo (Fig. 3D), and produced a >4-fold reduction in the relative amounts of XIAP-ubiquitin conjugates, as compared with control cells (Fig. 3E). Cytoprotective Mechanism of the Survivin-XIAP Complex—We next used purified components in vitro to test whether a survivin-XIAP complex synergistically suppressed caspase activity. Whereas survivin alone had essentially no effect on caspase-9 generation, the combination with a suboptimal concentration of XIAP (Fig. 4A, left panel) dose-dependently inhibited caspase-9 generation (Fig. 4A, right panel). Similarly, when combined with a suboptimal concentration of XIAP (Fig. 4B, left panel), survivin inhibited caspase activity in the context of a functional apoptosome in vitro (Fig. 4B, right panel). To determine whether XIAP and survivin synergistically inhibited apoptosis in vivo, we transfected HEK293T cells with survivin and XIAP alone or in combination, and stimulated apoptosis by expressing Bax or Fas, which activate mitochondrial or death receptor apoptosis, respectively. Expression of suboptimal amounts of survivin or XIAP in HEK293T cells individually did not significantly reduce apoptosis (Fig. 4C). In contrast, the combination of XIAP and survivin synergistically suppressed cell death induced by Bax (Fig. 4C, left panel), or Fas (Fig. 4C, right panel). Next, we used XIAP–/– MEF to determine whether a survivin-XIAP complex was required for cytoprotection in vivo. Transduction of WT MEF with pAd-survivin inhibited apoptosis stimulated by staurosporine or UVB (Fig. 4D). In contrast, survivin cytoprotection was completely lost in XIAP–/– MEF (Fig. 4D). In this study, we have shown that IAP family members XIAP and survivin form a heterocomplex in response to cell death stimulation in vivo. This interaction promotes cell survival by enhancing the stability of XIAP against proteasomal destruction and by synergistically antagonizing apoptosome-mediated cell death, in a pathway abolished in XIAP–/– cells. Although the formation of heterocomplexes is a hallmark of Bcl-2 proteins (2Cory S. Adams J.M. Nat. Rev. Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3349) Google Scholar), IAP-IAP interactions have not been described previously. This recognition involves survivin binding sites on all three BIRs of XIAP, albeit with different affinities, and may affect the interaction with other BIR-interacting partners, including XIAP antagonists Smac (3Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar), Omi/HtrA2 (16Yang Q.H. Church-Hajduk R. Ren J. Newton M.L. Du C. Genes Dev. 2003; 17: 1487-1496Crossref PubMed Scopus (272) Google Scholar), XAF (17Liston P. Fong W.G. Kelly N.L. Toji S. Miyazaki T. Conte D. Tamai K. Craig C.G. McBurney M.W. Korneluk R.G. Nat. Cell Biol. 2001; 3: 128-133Crossref PubMed Scopus (394) Google Scholar), survivin co-factors (HBXIP) (18Marusawa H. Matsuzawa S. Welsh K. Zou H. Armstrong R. Tamm I. Reed J.C. EMBO J. 2003; 22: 2729-2740Crossref PubMed Scopus (389) Google Scholar), and XIAP targets (caspase-3, -7, and -9) (4Salvesen G.S. Duckett C.S. Nat. Rev. Mol. Cell. Biol. 2002; 3: 401-410Crossref PubMed Scopus (1581) Google Scholar). Formation of a survivin-XIAP complex resulted in increased stability of XIAP against polyubiquitination and proteasomal degradation in vitro and in vivo, potentially by excluding the ubiquitin-conjugating enzyme, UbcH5. Sudden changes in IAP stability influence cell viability (19Martin S.J. Cell. 2002; 109: 807-809Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and ubiquitin-dependent proteasomal destruction of IAPs enhances cell death (20Yang Y. Fang S. Jensen J.P. Weissman A.M. Ashwell J.D. Science. 2000; 288: 874-877Crossref PubMed Scopus (873) Google Scholar). Here, a stabilized survivin-XIAP complex synergistically suppressed caspase-9 processing/activity, alone or in the context of the apoptosome, and blocked apoptosis in vivo. Although the mechanism(s) of survivin cytoprotection have long remained elusive, these data suggest a model of intermolecular cooperation in which survivin enhances the anti-apoptotic activity of XIAP to suppress the upstream initiation of mitochondrial cell death (15Blanc-Brude O.P. Mesri M. Wall N.R. Plescia J. Dohi T. Altieri D.C. Clin. Cancer Res. 2003; 9: 2683-2692PubMed Google Scholar, 18Marusawa H. Matsuzawa S. Welsh K. Zou H. Armstrong R. Tamm I. Reed J.C. EMBO J. 2003; 22: 2729-2740Crossref PubMed Scopus (389) Google Scholar). Apoptotic stimulation induces the formation of a survivin-XIAP complex in vivo, and future studies will investigate whether this reflects post-translational modifications in survivin (10O'Connor D.S. Wall N.R. Porter A.C. Altieri D.C. Cancer Cell. 2002; 2: 43-54Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) and/or XIAP (21Dan H.C. Sun M. Kaneko S. Feldman R.I. Nicosia S.V. Wang H.G. Tsang B.K. Cheng J.Q. J. Biol. Chem. 2004; 279: 5405-5412Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) enabling protein interaction or, alternatively, redistribution of survivin from a specialized subcellular pool during cell death (14Fortugno P. Wall N.R. Giodini A. O'Connor D.S. Plescia J. Padgett K.M. Tognin S. Marchisio P.C. Altieri D.C. J. Cell Sci. 2002; 115: 575-585PubMed Google Scholar). Targeted antagonists of the IAP-IAP complex may be suitable to disable apoptosis resistance in tumors, where survivin (6Altieri D.C. Nat. Rev. Cancer. 2003; 3: 46-54Crossref PubMed Scopus (1110) Google Scholar), and XIAP (9Holcik M. Gibson H. Korneluk R.G. Apoptosis. 2001; 6: 253-261Crossref PubMed Scopus (359) Google Scholar) are commonly overexpressed. We thank Dr. Colin Duckett for XIAP–/– cells and for critically reading the manuscript, Damon Banks for preliminary experiments, and Judie Valois for manuscript preparation.

Deregulated inhibition of apoptosis (programmed cell death) may facilitate the insurgence of neoplasia, but whether it also influences the outcome of common cancers has remained controversial. In this study, we investigated the expression of a novel inhibitor of apoptosis, survivin, in colorectal cancer and its relationship with tumor cell apoptosis and overall prognosis. By immunohistochemistry, survivin was expressed in 91 of 171 (53.2%) cases of colorectal carcinomas of histological stages 0 to IV. In contrast, normal colon epithelium did not express survivin. Although survivin expression did not correlate with p53 abnormalities (46.5% versus 58.0%; P = 0.18), survivin-positive cases were strongly associated with bcl-2 expression (72.5% versus 27.4%; P < 0.0001) and reduced apoptotic index (0.76% +/- 0.39% versus 1.17% +/- 0.62%; P < 0.0001). Expression of survivin alone in bcl-2-negative (discordant) cases also resulted in reduced apoptotic index (0.82% +/- 0.57% versus 1.16% +/- 0.66%; P = 0.0046). When analyzed for prognostic significance, patients with low apoptotic index (< 0.97%) had worse survival rates than the group with high apoptosis (P < 0.001), and a multivariate Cox proportional hazard model identified reduced apoptosis as an independent predictive factor for overall survival (P < 0.0001). These data demonstrate that apoptosis inhibition by survivin, alone or in cooperation with bcl-2, is an important predictive/prognostic parameter of poor outcome in colorectal carcinoma and identify survivin as a new diagnostic/therapeutic target in cancer.

Mitogenesis, cellular aggregation, and motility follow upon the interaction of fibrinogen with certain defined cell surface receptors. In addition to circulating platelets and vascular endothelium, monocytes express what appears to be a receptor for fibrinogen. Evidence is presented here that the leukocyte adhesion receptor Mac-1 can be specifically induced to bind fibrinogen with characteristics immunochemically and functionally distinct from the established Arg-Gly-Asp-directed fibrinogen receptors. The competence of Mac-1 as a fibrinogen receptor is a general property of cells of monocyte and myeloid lineage acquired after maturational changes of some regions of the alpha subunit of Mac-1 during the process of cell differentiation. This ligand recognition specificity of Mac-1 is lacking for the resting cell. Rather, induction of fibrinogen binding capacity of Mac-1 is due to a cellular response to selected agonists characterized by inducing rapid transients of cytosolic Ca2+. Although different in activation pathways and recognition specificity, Mac-1 exhibits an oligospecific ligand versatility characteristic of other homologous Arg-Gly-Asp-directed adhesion receptors.

The newly described apoptosis inhibitor survivin is expressed in many human cancers and appears to play a critical part in both apoptosis regulation and cell cycle progression. Its potential role in malignant melanoma is unknown. In a panel of 30 malignant melanomas, survivin was strongly expressed in all cases (15 of 15) of metastatic malignant melanomas and 13 of 15 cases of invasive malignant melanomas by immunohistochemistry. In invasive malignant melanomas, survivin was also expressed in the in-situ component of the lesion. Survivin expression was found in all cases (11 of 11) of nevi, but not in melanocytes in sections of normal skin. The apoptosis inhibitor bcl-2 was expressed in 26 of 30 cases, but generally at lower levels than that of infiltrating lymphocytes. The mitotic index, as assessed by MIB-1 staining, was consistently higher in metastatic than invasive malignant melanomas. Assessment of apoptotic index by in situ end-labeling revealed extremely low rates of apoptosis in most malignant melanomas. Survivin expression by western blotting was detected in four human metastatic malignant melanoma cell lines but not in cultured normal human melanocytes. Transfection of both YUSAC-2 and LOX malignant melanoma cells with green fluorescence protein-conjugated survivin anti-sense or green fluorescence protein-conjugated survivin dominant negative mutant (Cys84Ala) [corrected] resulted in increased apoptosis in the absence of other genotoxic stimuli. Two-color flow cytometry confirmed that YUSAC-2 cells transfected with survivin anti-sense expressed less endogenous survivin and exhibited an increased fraction of cells with sub-G1 DNA content. These data demonstrate that apoptosis inhibition by survivin may participate in the onset and progression of malignant melanomas, and suggest that therapeutic targeting of survivin may be beneficial in patients with recurrent or metastatic disease.

From the realization that cell number homoeostasis is fundamental to the biology of all metazoans, and that deregulation of this process leads to human diseases, enormous interest has been devoted over the last two decades to map the requirements of cell death and cell survival. This effort has led to tangible progress, and we can now chart with reasonable accuracy complex signalling circuitries controlling cell-fate decisions. Some of this knowledge has translated into novel therapeutics, and the outcome of these strategies, especially in cancer, is eagerly awaited. However, the function of cell-death modifiers have considerably broadened over the last few years, and these molecules are increasingly recognized as arbiters of cellular homoeostasis, from cell division, to intracellular signalling to cellular adaptation. This panoply of functions is best exemplified by members of the IAP (inhibitor of apoptosis) gene family, molecules originally narrowly defined as endogenous caspase inhibitors, but now firmly positioned at the crossroads of multiple normal and transformed cellular responses.

Leukocyte traffic in immune-inflammatory responses requires regulated adhesion of leukocyte subsets to vascular endothelium. We show that fibrinogen or normal human plasma enhances by 2- to 5-fold the adhesion of cells of myeloid and lymphoid lineage to endothelium. This mechanism is mediated by fibrinogen binding to complementary membrane receptors on leukocytes and endothelial cells. Using an affinity chromatography purification strategy, genetically engineered transfectants, and direct binding studies to the isolated recombinant protein, we identified a novel hematopoietic fibrinogen receptor participating in this adhesion pathway as intercellular adhesion molecule 1 (ICAM-1). Accordingly, a new model can be proposed, in which fibrinogen binding to a variety of vascular cell receptors mediates a specific pathway of cell to cell adhesion by bridging together leukocytes and endothelial cells.

Anticancer agents that selectively kill tumor cells and spare normal tissues are urgently needed. Here, we engineered a cell-permeable peptidomimetic, shepherdin, modeled on the binding interface between the molecular chaperone Hsp90 and the antiapoptotic and mitotic regulator, survivin. Shepherdin makes extensive contacts with the ATP pocket of Hsp90, destabilizes its client proteins, and induces massive death of tumor cells by apoptotic and nonapoptotic mechanisms. Conversely, shepherdin does not reduce the viability of normal cells, and does not affect colony formation of purified hematopoietic progenitors. Systemic administration of shepherdin in vivo is well tolerated, and inhibits human tumor growth in mice without toxicity. Shepherdin could provide a potent and selective anticancer agent in humans.

Recruitment of monocytic myeloid-derived suppressor cells (MDSCs) and differentiation of tumor-associated macrophages (TAMs) are the major factors contributing to tumor progression and metastasis. We demonstrated that differentiation of TAMs in tumor site from monocytic precursors was controlled by downregulation of the activity of the transcription factor STAT3. Decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. Upregulation of CD45 tyrosine phosphatase activity in MDSCs exposed to hypoxia in tumor site was responsible for downregulation of STAT3. This effect was mediated by the disruption of CD45 protein dimerization regulated by sialic acid. Thus, STAT3 has a unique function in the tumor environment in controlling the differentiation of MDSC into TAM, and its regulatory pathway could be a potential target for therapy.

Molecular chaperones may promote cell survival, but how this process is regulated, especially in cancer, is not well understood. Using high throughput proteomics screening, we identified the cell cycle regulator and apoptosis inhibitor survivin as a novel protein associated with the molecular chaperone Hsp60. Acute ablation of Hsp60 by small interfering RNA destabilizes the mitochondrial pool of survivin, induces mitochondrial dysfunction, and activates caspase-dependent apoptosis. This response involves disruption of an Hsp60-p53 complex, which results in p53 stabilization, increased expression of pro-apoptotic Bax, and Bax-dependent apoptosis. In vivo, Hsp60 is abundantly expressed in primary human tumors, as compared with matched normal tissues, and small interfering RNA ablation of Hsp60 in normal cells is well tolerated and does not cause apoptosis. Therefore, Hsp60 orchestrates a broad cell survival program centered on stabilization of mitochondrial survivin and restraining of p53 function, and this process is selectively exploited in cancer. Hsp60 inhibitors may function as attractive anticancer agents by differentially inducing apoptosis in tumor cells.

Inhibitor-of-Apoptosis (IAP) proteins contribute to tumor progression, but the requirements of this pathway are not understood. Here, we show that intermolecular cooperation between XIAP and survivin stimulates tumor cell invasion and promotes metastasis. This pathway is independent of IAP inhibition of cell death. Instead, a survivin-XIAP complex activates NF-κB, which in turn leads to increased fibronectin gene expression, signaling by β1 integrins, and activation of cell motility kinases FAK and Src. Therefore, IAPs are direct metastasis genes, and their antagonists could provide antimetastatic therapies in patients with cancer.

Runx2, a bone-specific transcriptional regulator, is abnormally expressed in highly metastatic prostate cancer cells. Here, we identified the functional activities of Runx2 in facilitating tumor growth and osteolysis. Our studies show that negligible Runx2 is found in normal prostate epithelial and non-metastatic LNCaP prostate cancer cells. In the intra-tibial metastasis model, high Runx2 levels are associated with development of large tumors, increased expression of metastasis-related genes (MMP9, MMP13, VEGF, Osteopontin) and secreted bone-resorbing factors (PTHrP, IL8) promoting osteolytic disease. Runx2 siRNA treatment of PC3 cells decreased cell migration and invasion through Matrigel in vitro, and in vivo shRunx2 expression in PC3 cells blocked their ability to survive in the bone microenvironment. Mechanisms of Runx2 function were identified in co-culture studies showing that PC3 cells promote osteoclastogenesis and inhibit osteoblast activity. The clinical significance of these findings is supported by human tissue microarray studies of prostate tumors at stages of cancer progression, in which Runx2 is expressed in both adenocarcinomas and metastatic tumors. Together these findings indicate that Runx2 is a key regulator of events associated with prostate cancer metastatic bone disease.

ABSTRACT Survivin (also known as BIRC5) is an evolutionarily conserved eukaryotic protein that is essential for cell division and can inhibit cell death. Normally it is only expressed in actively proliferating cells, but is upregulated in most, if not all cancers; consequently, it has received significant attention as a potential oncotherapeutic target. In this Cell Science at a Glance article and accompanying poster, we summarise our knowledge of survivin 21 years on from its initial discovery. We describe the structure, expression and function of survivin, highlight its interactome and conclude by describing anti-survivin strategies being trialled.

Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi.

Molecular therapies are hallmarks of "personalized" medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, "spatiotemporal" mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.

Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer.

Although neutrophils have been linked to the formation of the pre-metastatic niche, the mechanism of their migration to distant, uninvolved tissues has remained elusive. We report that bone marrow neutrophils from mice with early-stage cancer exhibited much more spontaneous migration than that of control neutrophils from tumor-free mice. These cells lacked immunosuppressive activity but had elevated rates of oxidative phosphorylation and glycolysis, and increased production of ATP, relative to that of control neutrophils. Their enhanced spontaneous migration was mediated by autocrine ATP signaling through purinergic receptors. In ectopic tumor models and late stages of cancer, bone marrow neutrophils demonstrated potent immunosuppressive activity. However, these cells had metabolic and migratory activity indistinguishable from that of control neutrophils. A similar pattern of migration was observed for neutrophils and polymorphonuclear myeloid-derived suppressor cells from patients with cancer. These results elucidate the dynamic changes that neutrophils undergo in cancer and demonstrate the mechanism of neutrophils’ contribution to early tumor dissemination. Neutrophils are linked to tumor progression. Gabrilovich and colleagues demonstrate that neutrophils have tumor-stage-dependent alterations in motility, function and metabolism: in early phases, they are highly motile with altered metabolism, whereas at later stages, they become highly suppressive.

A role of membrane microparticles (MP) released by vascular cells in endothelial cell (EC) activation was investigated. Flow cytofluorimetric analysis of blood samples from normal volunteers revealed the presence of an heterogeneous MP population, which increased by ∼2-fold after inflammatory stimulation with the chemotactic peptide, N -formyl-Met-Leu-Phe (2,799 ± 360 versus 5241 ± 640, p < 0.001). Blood-derived MP stimulated release of EC cytokines interleukin (IL)-6 (377 ± 68 pg/ml) and MCP-1 (1, 282 ± 79) and up-regulatedde novo expression of tissue factor on the EC surface. This was associated with generation of a factor Xa-dependent procoagulant response (2.28 ± 0.56 nm factor Xa/min/104 cells), in a reaction inhibited by a monoclonal antibody to tissue factor. Fluorescent labeling with antibodies to platelet GPIbα or leukocyte lactoferrin demonstrated that circulating MP originated from both platelets and leukocytes. However, depletion of platelet MP with an antibody to GPIbα did not reduce EC IL-6 release, and, similarly, MP from thrombin-stimulated platelets did not induce IL-6 release from endothelium. EC stimulation with leukocyte MP did not result in activation of the transcription factor NF-κB and was not associated with tyrosine phosphorylation of extracellular signal-regulated protein kinase, ERK1. In contrast, leukocyte MP stimulated a sustained, time-dependent increased tyrosine phosphorylation of ∼46-kDa c-Jun NH2-terminal kinase (JNK1) in EC. These findings demonstrate that circulating leukocyte MP are up-regulated by inflammatory stimulation in vivo and activate a stress signaling pathway in EC, leading to increased procoagulant and proinflammatory activity. This may provide an alternative mechanism of EC activation, potentially contributing to dysregulation of endothelial functions during vascular injury. A role of membrane microparticles (MP) released by vascular cells in endothelial cell (EC) activation was investigated. Flow cytofluorimetric analysis of blood samples from normal volunteers revealed the presence of an heterogeneous MP population, which increased by ∼2-fold after inflammatory stimulation with the chemotactic peptide, N -formyl-Met-Leu-Phe (2,799 ± 360 versus 5241 ± 640, p < 0.001). Blood-derived MP stimulated release of EC cytokines interleukin (IL)-6 (377 ± 68 pg/ml) and MCP-1 (1, 282 ± 79) and up-regulatedde novo expression of tissue factor on the EC surface. This was associated with generation of a factor Xa-dependent procoagulant response (2.28 ± 0.56 nm factor Xa/min/104 cells), in a reaction inhibited by a monoclonal antibody to tissue factor. Fluorescent labeling with antibodies to platelet GPIbα or leukocyte lactoferrin demonstrated that circulating MP originated from both platelets and leukocytes. However, depletion of platelet MP with an antibody to GPIbα did not reduce EC IL-6 release, and, similarly, MP from thrombin-stimulated platelets did not induce IL-6 release from endothelium. EC stimulation with leukocyte MP did not result in activation of the transcription factor NF-κB and was not associated with tyrosine phosphorylation of extracellular signal-regulated protein kinase, ERK1. In contrast, leukocyte MP stimulated a sustained, time-dependent increased tyrosine phosphorylation of ∼46-kDa c-Jun NH2-terminal kinase (JNK1) in EC. These findings demonstrate that circulating leukocyte MP are up-regulated by inflammatory stimulation in vivo and activate a stress signaling pathway in EC, leading to increased procoagulant and proinflammatory activity. This may provide an alternative mechanism of EC activation, potentially contributing to dysregulation of endothelial functions during vascular injury. endothelial cell(s) microparticle(s) platelet-rich plasma tissue factor interleukin tumor necrosis factor polymorphonuclear leukocyte(s) phosphate-buffered saline formyl-methionyl-leucyl-phenylalanine fluorescein isothiocyanate enzyme-linked immunosorbent assay monoclonal antibody Vascular endothelial cells (EC)1 respond to environmental and cellular stimuli with profound changes of adhesive, procoagulant, and inflammatory phenotypes (1Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar, 2Pober J.S. Cotran R.S. Physiol. Rev. 1990; 70: 427-451Crossref PubMed Scopus (1134) Google Scholar, 3Cines D.B. Pollak E.S. Buck C.A. Loscalzo J. Zimmerman G.A. McEver R.P. Pober J.S. Wick T.M. Konkle B.A. Schwartz B.S. Barnathan E.S. McCrae K.R. Hug B.A. Schmidt A.M. Stern D.M. Blood. 1998; 91: 3527-3561PubMed Google Scholar). This process of EC activation results in release of inflammatory and chemotactic cytokines IL-6, IL-8, and MCP-1 (2Pober J.S. Cotran R.S. Physiol. Rev. 1990; 70: 427-451Crossref PubMed Scopus (1134) Google Scholar, 4Rot A. Immunol. Today. 1992; 13: 291-294Abstract Full Text PDF PubMed Scopus (413) Google Scholar, 5Schall T.J. Cytokine. 1991; 3: 165-183Crossref PubMed Scopus (639) Google Scholar, 6Tanaka Y. Adams D.H. Hubscher S. Hirano H. Siebenlist U. Shaw S. Nature. 1993; 361: 79-82Crossref PubMed Scopus (846) Google Scholar), expression of procoagulant tissue factor (TF) (3Cines D.B. Pollak E.S. Buck C.A. Loscalzo J. Zimmerman G.A. McEver R.P. Pober J.S. Wick T.M. Konkle B.A. Schwartz B.S. Barnathan E.S. McCrae K.R. Hug B.A. Schmidt A.M. Stern D.M. Blood. 1998; 91: 3527-3561PubMed Google Scholar, 7Napoleone E. Di Santo A. Lorenzet R. Blood. 1997; 89: 541-549Crossref PubMed Google Scholar), and enhanced leukocyte recruitment via expression of adhesion molecules E-selectin, ICAM-1, and VCAM-1 (1Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar). These responses may originate from signal transduction by released cytokines,i.e. TNFα (8Modur V. Zimmerman G.A. Prescott S.M. McIntyre T.M. J. Biol. Chem. 1996; 271: 13094-13102Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 9Tartaglia L.A. Goeddel D.V. Immunol. Today. 1992; 13: 151-153Abstract Full Text PDF PubMed Scopus (1002) Google Scholar), shear stress during vascular remodeling (10Tardy Y. Resnick N. Nagel T. Gimbrone M.A. Dewey C.F. Arterioscler. Throm. Vasc. Biol. 1997; 17: 3102-3106Crossref PubMed Scopus (223) Google Scholar), or cross-talk with different vascular cells, including leukocytes and platelets (11Evangelista V. Manarini S. Sideri R. Rotondo S. Martelli N. Piccoli A. Totani L. Piccardoni P. Vestweber D. de Gaetano G. Cerletti C. Blood. 1999; 93: 876-885Crossref PubMed Google Scholar). Although of critical importance to preserve immune inflammatory responses and leukocyte trafficking (1Carlos T.M. Harlan J.M. Blood. 1994; 84: 2068-2101Crossref PubMed Google Scholar, 12Butcher E.C. Cell. 1991; 67: 1033-1036Abstract Full Text PDF PubMed Scopus (2522) Google Scholar), dysregulated EC activation may contribute to vascular injury and exacerbate the onset and progression of atherosclerosis in vivo (13Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar). Considerable interest has recently focused on alternative mechanisms of EC activation by vascular cells. Recent work has suggested that released membrane microparticles (MP) from platelets (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar) or leukocytes (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar) may provide such an alternative pathway of EC activation. In these studies, platelet or leukocyte MP stimulated increased expression of various adhesion molecules on EC, up-regulation of inflammatory and chemotactic cytokines, and increased monocyte adhesiveness (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar, 16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). For platelet MP, this pathway was recapitulated by arachidonic acid, consistent with the presence of bioactive lipids in platelet MP, and their ability to influence gene expression in target cells (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar). Although the existence of platelet MP in vivo and their potential contribution to procoagulant and/or anticoagulant responses have long been established (17Tans G. Rosing J. Thomassen M.C. Heeb M.J. Zwaal R.F.A. Griffin J.H. Blood. 1991; 77: 2641-2648Crossref PubMed Google Scholar, 18George J. Thoi L.L. McManus L.M. Reimann T.A. Blood. 1982; 60: 834-840Crossref PubMed Google Scholar, 19Shattil S.J. Cunningham M. Hoxie J.A. Blood. 1987; 70: 307-315Crossref PubMed Google Scholar, 20Abrams C.S. Ellison N. Budzynski A.Z. Shattil S.J. Blood. 1990; 75: 128-138Crossref PubMed Google Scholar), little is known about leukocyte MP or their potential ability to stimulate ECin vivo . In this study, we sought to investigate a potential role of leukocyte MP in EC responses and to identify signaling requirements involved in gene expression. We found that although both platelet and leukocyte MP are present in the normal circulation in vivo , only the leukocyte fraction initiates signal transduction and stimulates inflammatory and procoagulant responses in the endothelium. Polymorphonuclear leukocytes (PMN) were isolated from heparin sodium-anticoagulated blood drawn after informed consent from normal healthy volunteers by differential centrifugation on Ficoll/Hypaque gradient and dextran sedimentation as described (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). Human umbilical vein EC were prepared by collagenase treatment, maintained in medium 199 (BioWhittaker, Walkersville, MD) supplemented with 20% heat-inactivated fetal bovine serum (BioWhittaker), l-glutamine (2 mm), and 1% endothelial cell growth supplement, pH 7.4, and used between passages 2 and 4. Heparin sodium-anticoagulated blood was drawn from normal healthy volunteers after informed consent. Aliquots (1.5 ml) of undiluted blood or samples diluted 1:5 in PBS, pH 7.4, were incubated with the fluorescent labeling dye, quinacrine mustard (mepacrine, 0.1 mm; Sigma), in the presence or in the absence of fMLP (1 μm) for 2 h at 37 °C. Blood samples were centrifuged at 1500 × g for 20 min at 22 °C, and the cell-free supernatant was collected and analyzed by flow cytometry. In some experiments, cell-free supernatants prepared as described above were passed through 100-kDa cut-off nanospins (Millipore, Bedford, MA) according to the manufacturer's specifications. Aliquots of 0.5 ml were analyzed on a Becton-Dickinson (Mountain View, CA) flow cytometer as described (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). To identify the MP population, samples were gated according to their fluorescence (mepacrine) labeling. With the light scatter and fluorescence channels set at logarithmic gain, samples were analyzed for 6-s intervals for forward light scatter, right angle light scatter, and mepacrine fluorescence. The light scatter profile of mepacrine-positive MP typically demonstrated one single low light scatter population, in agreement with previous observations (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). Indistinguishable results were obtained when blood samples were anticoagulated with EDTA or acid citrate dextrose. In some experiments, aliquots of diluted blood were incubated with 5 μg/ml FITC-conjugated anti-GPIbα mAb (the generous gift of Dr. Z. M. Ruggeri, Scripps Research Institute), anti-lactoferrin antibody (ICN Biomedicals, OH) or control mouse IgG (Roche Molecular Biochemicals, IN), for 30 min at 22 °C in the dark. FITC conjugation was carried out according to the manufacturer's specifications (Roche Molecular Biochemicals). Samples were then stimulated or not with 200 nm fMLP and 2.5 mm CaCl2 for 1 h at 22 °C and centrifuged at 1500 × g for 20 min to remove the various cellular fractions, and the resulting supernatants were collected and ultracentrifuged at 100,000 × g for 1 h. Pellets containing MP were washed once with PBS, pH 7.4, suspended in 0.5 ml of PBS, and analyzed by flow cytometry. Samples were analyzed for a total of 2000 events for forward, side, and fluorescein light scatters as described above. In parallel experiments, supernatants collected after incubation with the various unconjugated antibodies as described above were tested for induction of endothelial IL-6 release by ELISA (see below). PMN-derived MP were quantitated by flow cytometry using a fluorescent lipid intercalating dye, PKH26-GL (Sigma). This aliphatic chromophore partitions into lipid bilayers and confers a red fluorescence. PMN (2 × 107 cells/ml) were labeled with PKH26-GL (4 μm) according to the manufacturer's specifications. Labeled cells were suspended in serum-free 199 medium and preincubated with or without 100 ng/ml pertussis toxin (Calbiochem, San Diego, CA) for 2 h at 37 °C. Cells were stimulated with fMLP (10 nm, Sigma) or recombinant human IL-8 (Endogen, Woburn, MA; 100 ng/ml) for a further 2 h at 37 °C in the presence of 2.5 mmCaCl2. The cell-containing supernatants were isolated and analyzed by flow cytometry, as described above. Each sample was analyzed for a total of 10,000 events. A gate was chosen to include particles distinctly positive for red fluorescence. Blood was collected from healthy volunteers into a plastic syringe and anticoagulated with acid citrate dextrose. PRP was prepared by centrifugation at 120 × g for 15 min at 22 °C. Platelet MP were isolated after platelet aggregation induced by 1 unit/ml thrombin (Sigma) and 2.5 mm CaCl2 with gentle shaking (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar). After a 10-min incubation at 37 °C, large platelet aggregates were sedimented at 1500 × g for 20 min, and the MP-containing supernatants were collected, diluted 1:5 in PBS, pH 7.4, and analyzed by flow cytometry, as described above. 200-μl aliquots of unstimulated, fMLP (1 μm), or thrombin (1 unit/ml)-stimulated cell-free supernatants from whole blood or PRP samples were added to quiescent EC monolayers grown to confluency in 48-well plates. After a 12-h incubation at 37 °C, EC supernatants were collected, centrifuged at 200 × g for 10 min, and analyzed for released IL-6 and IL-8 by ELISA (Endogen), as described previously. In other experiments, aliquots of whole blood were incubated in the absence or presence of 20 μg/ml anti-GPIbα mAb, anti-lactoferrin polyclonal antibody, or control 14E11 mAb, for 30 min at 22 °C. After fMLP stimulation, cellular fractions of the samples were removed by centrifugation at 1500 × g for 20 min, and 200-μl aliquots of the resulting supernatants were added to EC monolayers for determination of cytokine release, as described above. In control experiments, cell-free supernatants from fMLP (1 μm)-stimulated PMN (3 × 106/ml) were preincubated with neutralizing anti-TNF mAb (Sigma) or control mAb 14E11 (25 μg/ml) for 30 min at 22 °C before addition to EC for and determination of IL-6 release, as described above. In other experiments, cell-free supernatants from unstimulated or fMLP-stimulated blood samples were collected and sterile-filtered through 100-kDa cut-off filter membrane of nanospin units (Millipore), before addition to EC and determination of released IL-6 and MCP-1. For cytofluorimetric analysis of TF induction in EC, cell-free supernatants from resting or fMLP (1 μm)-stimulated PMN were added to confluent EC monolayers grown in a 6-well plate for 6 h at 37 °C. Unstimulated or MP-stimulated EC were harvested, washed, and blocked with 50% human serum for 30 min on ice followed by washing and staining with 20 μg/ml anti-TF mAb 5G9 (the generous gift of Dr. T. S. Edgington, The Scripps Research Institute, La Jolla, CA) or control mAb 14E11. After washing, EC were incubated with a 1:20 dilution of FITC-conjugated goat anti-mouse IgG and immediately analyzed by flow cytometry. In control experiments, EC were stimulated with 10 ng/ml TNFα and analyzed for TF induction as described above. In parallel experiments, EC in 6-well plates at 90% confluence were incubated with medium alone or with 10 ng/ml TNF or cell-free supernatant from fMLP-stimulated PMN (4 × 107/ml) for 4 h at 37 °C. Total RNA was extracted by RNAzol B method (TEL TEST, Friendswood, TX) and reverse transcribed with 1 × 104 unit/ml SuperScript II RNase H− reverse transcriptase (Life Technologies, Inc.) using oligo(dT) or gene-specific primer (5′-CACTCCTGCCTTTCTACAC-3′). The reverse transcription reaction containing 5 μg of total EC RNA or 50 ng of control RNA, 0.5 μg of oligo(dT) or 2 μm GSP, 1 × 104 unit/ml reverse transcriptase in the presence of 25 mm MgCl2, 10 mm dNTP mix, and 0.1m dithiothreitol was incubated for 50 min at 42 °C. At the end of the incubation, samples were heated for 15 min at 70 °C, chilled on ice, mixed with 1 μl of RNase H for 20 min at 37 °C, and amplified by polymerase chain reaction with oligonucleotides 5′-GTCAGAAGGAACAACACT-3′ (forward) and 5′-CACTCCTGCCTTTCTACAC-3′ (reverse) derived from the sequence of human TF. Thirty-five cycles of amplification were carried out in a Perkin-Elmer 480 thermal cycler with denaturation for 30 s at 94 °C, annealing for 40 s at 55 °C, and extension for 1 min at 72 °C. MgCl2 was used at a final concentration of 1.5 mm. Polymerase chain reaction products were analyzed on 1% agarose gels by ethidium bromide staining. In another series of experiments, EC in a 48-well plate were washed and incubated in the presence or absence of 10 ng/ml TNFα, cell-free supernatant from fMLP-stimulated PMN (4 × 107/ml), or equivalent purified MP prepared as described above for 6 h at 37 °C. After washes, EC were incubated with 100 μl of phenol red-free RPMI 1640 medium containing 150 nm human factor X (Alexis), 5 nm activated factor VII (Alexis), and 2 mm CaCl2 for 20 min at 37 °C. In some experiments, EC monolayers were preincubated with anti-TF mAb 5G9 or control mAb 14E11 (20 μg/ml) for 30 min at 22 °C before addition of factors X and VIIa. Generation of activated factor X (factor Xa) was stopped by addition of 10 mm EDTA to each incubation reaction. Samples were transferred to a 96-well plate, and factor Xa activity was determined by hydrolysis of the chromogenic substrate S-2222 (Chromogenix, Moelndal, Sweden) at a final concentration of 0.2 mm. The optical densities were read at 405 nm using a plate-reader spectrophotometer (Thermomax, Molecular Devices). The amount of factor Xa generated under the various conditions tested was calculated by comparison with a standard curve constructed with serial increasing concentrations of factor Xa (Alexis). Serum starved quiescent subconfluent EC in 6-well plates were stimulated with PMN (4 × 107/ml) supernatants for increasing time intervals at 37 °C. EC were washed and lysed with lysis buffer containing 10 mm Tris, 140 mm NaCl, 1% Triton, 0.5% deoxycholate, 0.05% SDS, 100 mm NaF, 200 μm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, and 1 μg/ml leupeptin. EC extracts were centrifuged at 14,000 × g for 30 min, precleared with protein A-Sepharose and immunoprecipitated with antibodies (1 μg/ml) to extracellular signal-regulated kinase 1 (ERK1, Santa Cruz Biotechnologies, Santa Cruz, CA), c-Jun NH2-terminal kinase 1 (JNK1, Santa Cruz,), or phosphotyrosine proteins Py20 (ICN Pharmaceuticals Inc., Costa Mesa, CA) for 2 h at 4 °C. The immune complexes were electrophoresed on a 10% SDS gel, electroblotted to nylon membranes, and immunoblotted with 1 μg/ml anti-phosphotyrosine antibody Py20 or anti-JNK1 antibody. After washes, reactive bands under the various conditions tested were detected by addition of alkaline phosphatase-conjugated goat anti-mouse IgG (1:2000) and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech). For electrophoretic mobility shift assays, confluent EC monolayers were either left untreated (control) or incubated with PMN (4 × 107/ml) supernatant or TNFα (10 ng/ml) for 90 min at 37 °C. Nuclear extracts were prepared as described previously (21Jamieson C. McCaffrey P.G. Rao A. Sen R. J. Immunol. 1991; 147: 416-420PubMed Google Scholar). Nuclear extracts were normalized for protein concentration, and 10 μg were incubated with 4 μg of poly(dI·dC) (Amersham Pharmacia Biotech) for 20 min at 22 °C and then for another 15 min with a32P-labeled probe in a final volume of 15 μl. The oligonucleotide used in these studies for the κB binding was 5′-AGTTGAGGGGACTTTCCCAGGC-3′. Double-stranded oligonucleotide probe was end-labeled with [γ-32P]ATP (Amersham Pharmacia Biotech) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). DNA-protein complexes were resolved on a 6% nondenaturing polyacrylamide gel containing 7.5% glycerol in 0.25% Tris borate-EDTA buffer, pH 8.3 (1× TBE: 89 mm Tris borate, 89 mm boric acid, 20 mm EDTA). Dried gels were exposed to x-ray film (Kodak Co., Rochester, NY), and relevant bands were visualized by autoradiography. Treatment of freshly isolated PKH26-GL-labeled PMN with inflammatory/chemotactic stimuli fMLP (10 nm) or IL-8 (100 ng/ml) induced release of an heterogeneous membrane MP population, as determined by flow cytometry (Fig. 1), and in agreement with previous observations (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). Pretreatment with 100 ng/ml pertussis toxin (22Fensome A. Whatmore J. Morgan C. Jones D. Cockcroft S. J. Biol. Chem. 1998; 273: 13157-13164Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) did not reduce MP release from fMLP- or IL-8-stimulated PMN suspensions (Fig. 1). Flow cytofluorimetric analysis of whole blood supernatants revealed the constitutive presence of a discrete population with low forward scatter consistent with MP (Fig.2 A , top left quadrant ). Stimulation of blood samples with fMLP resulted in an ∼2-fold increase in the MP population (Fig. 2 A , top right quadrant ), in agreement with previous observations (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). In parallel experiments, fMLP stimulation increased the number of fluorescent MP following mepacrine labeling of whole blood samples (Fig. 2 A , lower left and right quadrants ). Consistent with these in vivo data, cytofluorimetric analysis of cell-free supernatants collected from 23 normal volunteers revealed the constitutive presence of an MP population in nonmanipulated blood samples (events, 2799 ± 360). Stimulation of whole blood samples with fMLP resulted in a ∼2-fold increase in the number of MP detected by flow cytometry (events, 5241 ± 640; p = 0.001; Fig. 2 B ). Incubation of quiescent EC with cell-free supernatants from unstimulated whole blood samples did not result in release of cytokines, IL-6, and MCP-1 (Fig.3 A ). In contrast, cell-free supernatants from fMLP-stimulated whole blood supernatants caused a 2–4-fold increased release of cytokines MCP-1 (1282 ± 79 pg/ml) and IL-6 (377 ± 68 pg/ml), respectively (Fig. 3 A ). A 2-fold increase in IL-8 release was also observed under the same experimental conditions (not shown). Filtration of cell-free supernatants through 100-kDa filters prior to incubation with EC completely abolished the MP-stimulated release of IL-6 and MCP-1 by EC (Fig. 3 A ), in agreement with previous observations (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). Consistent with these findings, filtration of cell-free supernatants resulted in complete depletion of the blood MP population, identified by forward and side scatter parameters and quantitated by flow cytometry (Fig. 3 B ). In control experiments, preincubation of PMN-derived MP with a neutralizing mAb to TNFα did not significantly reduce EC IL-6 release (unstimulated, 28 pg/ml; MP-stimulated plus control mAb 14E11, 422 pg/ml; MP-stimulated plus anti-TNFα mAb, 378 pg/ml). In contrast, the anti-TNFα antibody inhibited by ∼50% EC IL-6 release induced by TNFα stimulation (TNFα alone plus control mAb 14E11, 615 pg/ml; TNFα plus anti-TNFα mAb, 305 pg/ml). A 6-h exposure of EC to supernatants from fMLP-stimulated PMN resulted in moderate but consistent increased surface expression of TF, as determined by flow cytofluorimetric staining with mAb 5G9 (Fig.4 A ). Similar results were obtained using whole blood-containing MP (not shown). EC stimulation with blood-derived MP also resulted in de novo expression of TF mRNA, as determined by appearance of a 352-base pair TF RNA transcript detected by reverse transcriptase-polymerase chain reaction amplification of EC RNA (Fig. 4 A , inset ). In control experiments, EC stimulation with TNFα resulted in maximal increase in TF surface expression and TF RNA (Fig. 4 A ), in agreement with previous observations (23Mackman N. Fowler B.J. Edgington T.S. Morrissey J.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2254-2258Crossref PubMed Scopus (71) Google Scholar). In parallel experiments, EC stimulation with MP-containing PMN supernatant, or purified MP resulted in the generation of 2.28 ± 0.56 and 2.75 ± 0.07 nm of factor Xa/min/104 EC, respectively (Fig.4 B ). This response was abolished in the absence of factor VIIa or by addition of anti-TF mAb 5G9 (0.51 ± 0.07 nm of factor Xa/min/104 EC) (Fig.4 B ). In contrast, EC preincubation with control mAb 14E11 did not reduce EC procoagulant activity (Fig. 4 B ). In control experiments, EC stimulation with 10 ng/ml TNFα resulted in the formation of 6 ± 1.5 nm factor Xa/min/104 EC (not shown), in agreement with previous observations (23Mackman N. Fowler B.J. Edgington T.S. Morrissey J.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2254-2258Crossref PubMed Scopus (71) Google Scholar). To identify the potential cell(s) of origin of released MP in vivo , flow cytofluorimetric experiments were carried out with antibodies to platelet GPIbα or leukocyte lactoferrin. Tryptic digestion and ionization mass spectrometry had previously identified the main ∼85-kDa component in purified MP (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar) as lactoferrin. 2M. Mesri and D. C. Altieri, our unpublished observations. In these experiments, unstimulated blood-derived MP reacted with antibodies to GPIbα and lactoferrin, as compared with control nonbinding antibody (Fig. 5). Furthermore, fMLP stimulation increased by ∼10-fold the reactivity of the MP population with the lactoferrin antibody, whereas no significant differences in the binding of anti-GPIbα mAb were observed with unstimulated or fMLP-stimulated samples (Fig. 5). Preincubation of blood samples with 20 μg/ml anti-GPIbα mAb resulted in a significant reduction in the amount of MP released after fMLP stimulation (Fig.6 A ). In contrast, no differences in fMLP-induced MP release were observed in the presence of control mAb 14E11 or the antibody to lactoferrin (Fig. 6 A ). In parallel experiments, preincubation of cell-free supernatants with antibodies to lactoferrin or GPIbα did not reduce EC IL-6 release, as compared with untreated samples or treated with control mAb 14E11 (Fig.6 B ). A potential differential ability of platelet or leukocyte MP to induce EC IL-6 release was further investigated. Thrombin stimulation of PRP resulted in MP release, as determined by flow cytometry (Fig. 6 C ), in agreement with previous observations. However, thrombin-stimulated platelet MP or unstimulated PRP supernatant did not stimulate EC IL-6 release (Fig. 6 D and data not shown). In contrast, TNFα stimulation of EC resulted in the generation of 599 ± 142 pg/ml IL-6 after a 12-h culture at 37 °C (Fig. 6 D ). EC stimulation with TNFα resulted in strong activation of NF-κB, as compared with unstimulated EC extracts, as determined by electrophoretic mobility shift assay (Fig.7 A ). In contrast, leukocyte MP did not induce NF-κB activation (Fig. 7 A ). Similarly, no significant differences in tyrosine phosphorylation of ERK1 were observed in EC treated with leukocyte MP, as compared with unstimulated or serum-containing cultures (Fig. 7 B ). In contrast, immunoprecipitation and Western blot of EC extracts with anti-phosphotyrosine antibody Py20 revealed a time-dependent and sustained increased tyrosine phosphorylation of a ∼46-kDa band in MP-stimulated EC, as compared with untreated cultures (Fig. 7 B ). The identity of the ∼46-kDa band was further investigated. Immunoprecipitation of EC lysates with an antibody to JNK1 followed by Western blot with Py20 antibody revealed a >2-fold increase in tyrosine phosphorylation of JNK1 in MP-stimulated cultures but not in control untreated EC (Fig.7 C ). In contrast, EC serum treatment did not result in tyrosine phosphorylation of immunoprecipitated JNK1 (Fig.7 C ). In control experiments, Western blot of JNK1 immunoprecipitates demonstrated a comparable amount of JNK1/lane, under the various conditions tested (Fig. 7 C ). In this study, we have shown that leukocyte-derived MP circulate in the bloodstream under normal conditions and are rapidly up-regulated by inflammatory stimulation. Secondly, leukocyte, but not platelet, MP stimulate release of inflammatory/chemotactic cytokines and expression of functional tissue factor in EC, in a pathway associated with sustained phosphorylation of ∼46-kDa JNK1. The notion that vascular cells release MP in response to disparate environmental stimuli is well established and has been experimentally validated for platelets (24Sims P.J. Wiedmer T. Esmon C.T. Weiss H.J. Shattil S.J. J. Biol. Chem. 1989; 264: 17049-17057Abstract Full Text PDF PubMed Google Scholar, 25Wiedmer T. Sims P.J. Blood. 1991; 78: 2880-2886Crossref PubMed Google Scholar), monocytes (26Satta N. Toti F. Feugeas O. Bohbot A. Dachary-Prigent J. Eschwege V. Hedman H. Freyssinet J.-M. J. Immunol. 1994; 153: 3245-3255PubMed Google Scholar), and endothelium (27Leeuwenberg J.F. Smeets E.F. Neefjes J.J. Shaffer M.A. Cinek T. Jeunhomme T.M. Ahern T.J. Buurman W.A. Immunology. 1992; 77: 543-549PubMed Google Scholar). Through their ability to assemble a functional prothrombinase complex, platelet (26Satta N. Toti F. Feugeas O. Bohbot A. Dachary-Prigent J. Eschwege V. Hedman H. Freyssinet J.-M. J. Immunol. 1994; 153: 3245-3255PubMed Google Scholar, 28Sims P.J. Wiedmer T. Immunol. Today. 1991; 12: 338-342Abstract Full Text PDF PubMed Scopus (143) Google Scholar) and monocyte (29Robinson R.A. Worfolk L. Tracy P.B. Blood. 1992; 79: 406-416Crossref PubMed Google Scholar) MP may amplify cellular procoagulant responses, thus potentially contributing to aberrant fibrin deposition in vivo (30Mallat Z. Hugel B. Ohan J. Leseche G. Freyssinet J.-M. Tedgui A. Circulation. 1999; 99: 348-353Crossref PubMed Scopus (596) Google Scholar, 31Nomura S. Suzuki M. Katsura K. Xie G.l. Miyazaki Y. Mikake T. Kido H. Kagawa H. Fukuhara S. Atherosclerosis. 1995; 116: 235-240Abstract Full Text PDF PubMed Scopus (213) Google Scholar). However, it has also been recently proposed that platelet (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar), and leukocyte (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar), MP may function as genuine cellular agonists and stimulate complex EC responses. In this context, platelet MP induced COX-2 and prostacyclin production in EC (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar) and stimulated increased monocyte adherence through up-regulation of adhesion molecule ICAM-1 (15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar). A similar paradigm has been also proposed for leukocyte MP for their ability to stimulate EC expression of adhesion molecules ICAM-1, E-selectin, and VCAM-1 and promote release of cytokines IL-6 and IL-8 (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). In expanding these observations, leukocyte MP released by inflammatory/chemotactic mediators fMLP or IL-8 in a pertussis toxin-insensitive pathway mediate a procoagulant response in EC by increasing the expression of TF mRNA and up-regulating functional TF at the cell surface. This is consistent with the ability of leukocyte MP to directly affect EC gene expression, as reflected by the ∼18-fold up-regulation of IL-6 mRNA, under these experimental conditions (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). Combined with the presence of leukocyte MP in vivo , and their rapid quantitative up-regulation by inflammatory stimuli, these data suggest that leukocyte MP may cooperate with locally released cytokines (2Pober J.S. Cotran R.S. Physiol. Rev. 1990; 70: 427-451Crossref PubMed Scopus (1134) Google Scholar) and leukocyte-EC intercellular signaling (7Napoleone E. Di Santo A. Lorenzet R. Blood. 1997; 89: 541-549Crossref PubMed Google Scholar, 32Lo S.K. Cheung A. Zheng Q. Silverstein R.L. J. Immunol. 1995; 154: 4768-4777PubMed Google Scholar) to stimulate a broad proadhesive, procoagulant, and proinflammatory phenotype in EC. This pathway may be potentially relevant to EC dysfunction during vascular diseases, invariably associated with increased leukocyte recruitment, platelet activation, and fibrin deposition at the site of vascular injury (13Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar). As shown here, this mechanism of EC stimulation appears selective for leukocyte MP, because depletion of platelet MP did not decrease cytokine induction in EC, and thrombin-stimulated platelet MP did not stimulate EC IL-6 release. Although induction of EC ICAM-1 by platelet MP required treatment with phospholipase A2 (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar), a potential role of platelet MP on EC cytokine release has not been previously investigated (15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar). In contrast, EC stimulation by leukocyte MP did not require phospholipase A2 treatment, and similarly, arachidonic acid failed to stimulate IL-6 release,2 whereas it recapitulated EC activation by platelet MP (15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar). Altogether, these data suggest that EC activation by platelet (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar) or leukocyte (16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar) MP may involve separate or only partially overlapping signaling pathways. On the other hand, fMLP stimulation was also associated with platelet MP release in vivo , in a reaction inhibited by an antibody to GPIbα. This suggests that inflammatory challenges, directly or through intercellular collaboration (33Weber C. Springer T.A. J. Clin. Invest. 1997; 100: 2085-2093Crossref PubMed Scopus (326) Google Scholar), may result in both platelet and leukocyte MP release, thus further amplifying EC procoagulant and proinflammatory gene expression (14Barry O.P. Pratico D. Lawson J.A. FitzGerald G.A. J. Clin. Invest. 1997; 99: 2118-2127Crossref PubMed Scopus (405) Google Scholar, 15Barry O.P. Pratico D. Savani R.C. FitzGerald G.A. J. Clin. Invest. 1998; 102: 136-144Crossref PubMed Scopus (466) Google Scholar, 16Mesri M. Altieri D.C. J. Immunol. 1998; 161: 4382-4387PubMed Google Scholar). In investigating potential downstream signals of this EC activation pathway, we found that leukocyte MP did not stimulate NF-κB activation or promote ERK1 tyrosine phosphorylation. In contrast, MP stimulated a prominent and sustained tyrosine phosphorylation of c-Jun NH2-terminal kinase, JNK1, in EC. A member of the mitogen-activated kinase gene family, JNK1 phosphorylation through upstream activators SEK and MEKK has been observed in response to growth factors, cytokines, and stress signals, including ultraviolet lights or alkylating agents (34Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar, 35Yan M. Dal T. Deak J.C. Kyriakis J.M. Zon L.I. Woodgett J.R. Templeton D.J. Nature. 1994; 372: 798-800Crossref PubMed Scopus (658) Google Scholar, 36Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1709) Google Scholar, 37Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2414) Google Scholar). JNK1 phosphorylation has also been implicated in apoptosis of neuronal PC12 cells after nerve growth factor deprivation, TNFα treatment, or ischemia (34Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar, 38Xia Z.G. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5036) Google Scholar) and in stimulatory phosphorylation of c-Jun, a component of the transcription factor, AP-1 (39Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2310) Google Scholar). This suggests that MP may target JNK1 activation in the context of a broad EC stress signaling response, culminating with proinflammatory, proadhesive, and procoagulant gene expression through AP-1 activation. In this context, it is of interest that AP-1 has been implicated in regulated transcription of the TF gene in endothelium (40Bierhaus A. Zhang Y. Deng Y. Mackman N. Quehenberger P. Haase M. Luther T. Muller M. Bohrer H. Greten J. Martin E. Baeuerle P.A. Waldherr R. Kisiel W. Ziegler R. Stern D.M. Nawroth P.P. J. Biol. Chem. 1995; 270: 26419-26432Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), and in platelet-derived growth factor-dependent IL-6 gene transcription in osteoblasts (41Franchimont N. Durant D. Rydziel S. Canalis E. J. Biol. Chem. 1999; 274: 6783-6789Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Whether this pathway may also influence EC apoptosis (42Verheij M. Bose R. Lin X.H. Yao B. Jarvis W.D. Grant S. Birrer M.J. Szabo E. Zon L.I. Kyriakis J.M. Haimovitz-Friedman A. Fuks Z. Kolesnick R.N. Nature. 1996; 380: 75-79Crossref PubMed Scopus (1716) Google Scholar), thus further exacerbating vessel wall dysfunction and procoagulant activity (43Bombeli T. Karsan A. Tait J.F. Harlan J.M. Blood. 1997; 89: 2429-2442Crossref PubMed Google Scholar), is currently unknown and may depend on the differential expression of AP-1 components or activation of other modulatory transcription factors (34Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2955) Google Scholar, 39Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2310) Google Scholar). In summary, we have identified a pathway of EC signaling and gene expression centered on the release of leukocyte MP. Future studies will dissect the molecular requirement(s) of this stress response mechanism and its potential contribution to EC dysfunction in vascular diseases (13Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9990) Google Scholar). We thank Drs. Zaverio Ruggeri and Tom Edgington (The Scripps Research Institute) for the generous gift of antibodies to platelet GPIbα and TF, respectively.

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to ∼16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor α, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor α/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis. Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to ∼16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor α, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor α/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis. Apoptosis, the genetic control of cell death and viability, preserves tissue and organ homeostasis by eliminating senescent or damaged cells.1Vaux DL Korsmeyer SJ Cell death in development.Cell. 1999; 96: 245-254Abstract Full Text Full Text PDF PubMed Scopus (1361) Google Scholar This process involves different gene families of inhibitors and stimulators of cell death and culminates with activation of intracellular cysteine proteases, caspases.2Salvesen GS Dixit VM Caspases: intracellular signaling by proteolysis.Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1934) Google Scholar Aberrations of apoptosis are known to contribute to human diseases, including cancer3Thompson CB Apoptosis in the pathogenesis and treatment of disease.Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar and vascular disorders.4Rudin CM Thompson CB Apoptosis and disease: regulation and clinical relevance of programmed cell death.Annu Rev Med. 1997; 48: 267-281Crossref PubMed Scopus (315) Google Scholar Specifically, aberrantly increased cell death has been shown to influence atherosclerotic plaque instability,5Bjorkerud S Bjorkarud B Apoptosis is abundant in human atherosclerotic lesions, especially in inflammatory cells (macrophages and T cells), and may contribute to the accumulation of gruel and plaque instability.Am J Pathol. 1996; 149: 367-380PubMed Google Scholar congestive heart failure,6Olivetti G Abbi R Quaini F Kajstura J Cheng W Nitahara JA Quaini E Di Loreto C Beltrami CA Krajewski S Reed JC Anversa P Apoptosis in the failing human heart.N Engl J Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1477) Google Scholar coronary disease,7Olivetti G Quaini F Sala R Lagrasta C Corradi D Bonacina E Gambert SR Cigola E Anversa P Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart.J Mol Cell Cardiol. 1996; 28: 2005-2016Abstract Full Text PDF PubMed Scopus (453) Google Scholar and ischemic neuronal loss.8Chen J Nagayama T Jin K Stetler RA Zhu RL Graham SH Simon RP Induction of caspase-3-like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia.J Neurosci. 1998; 18: 4914-4928Crossref PubMed Google Scholar The endothelium is one of the most critical sites for the control of apoptosis in vascular injury and vascular remodeling.9Karsan A Harlan JM Modulation of endothelial cell apoptosis: mechanisms and pathophysiological roles.J Atheroscler Thromb. 1996; 3: 75-80Crossref PubMed Scopus (55) Google Scholar In inflammation, a heterogeneous group of protective genes activated by nuclear factor κB opposes cell death and proinflammatory changes in endothelial cells (EC) induced by cytokines, ie, tumor necrosis factor α (TNFα).10Bach FH Hancock WW Ferran C Protective genes expressed in endothelial cells: a regulatory response to injury.Immunol Today. 1997; 18: 483-486Abstract Full Text PDF PubMed Scopus (191) Google Scholar Inhibition of apoptosis may also be required during vascular remodeling and new blood vessel formation, angiogenesis.11Risau W Mechanisms of angiogenesis.Nature. 1997; 386: 671-674Crossref PubMed Scopus (4805) Google Scholar In this context, EC-specific mitogens, including vascular endothelial cell growth factor (VEGF) or basic fibroblast growth factor (bFGF), transduce survival signals critically maintaining EC viability in vivo.12Benjamin LE Keshet E Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.Proc Natl Acad Sci USA. 1997; 94: 8761-8766Crossref PubMed Scopus (443) Google Scholar, 13Alon T Hemo I Itin A Pe'er J Stone J Keshet E Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.Nat Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1407) Google Scholar, 14Yuan F Chen Y Dellian M Safabakhsh N Ferrara N Jain RK Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody.Proc Natl Acad Sci USA. 1996; 93: 14765-14770Crossref PubMed Scopus (602) Google Scholar However, the downstream effector genes coupling mitogen-dependent survival to the anti-apoptotic machinery in EC have not been completely elucidated. In this study, we sought to investigate a potential role of the cell cycle-regulated apoptosis inhibitor survivin15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar, 16Li F Ambrosini G Chu EY Plescia J Tognin S Marchisio PC Altieri DC Control of apoptosis and mitotic spindle checkpoint by survivin.Nature. 1998; 396: 580-584Crossref PubMed Scopus (1723) Google Scholar on EC viability. We found that mitogen stimulation strongly induced survivin expression in endothelium during vascular remodeling and angiogenesis, in vitro and in vivo, and that this pathway counteracted caspase-3 activity and apoptosis induced by TNFα. Human umbilical vein EC were maintained in M199 medium supplemented with 20% fetal calf serum (FCS), 50 μg/ml endothelial cell growth supplement (ECGS), 100 μg/ml heparin, 100 μg/ml penicillin, and 100 μg/ml streptomycin (all from Life Technologies, Grand Island, NY) in 5% CO2 at 37°C. Bovine aortic EC were isolated and maintained in culture as described.17De Luca LG Johnson DR Whitley MZ Collins T Pober JS cAMP and tumor necrosis factor competitively regulate transcriptional activation through and nuclear factor binding to the cAMP-responsive element/activating transcription factor element of the endothelial leukocyte adhesion molecule-1 (E-selectin) promoter.J Biol Chem. 1994; 269: 19193-19196Abstract Full Text PDF PubMed Google Scholar Subconfluent EC were rendered quiescent by 24 hours' culture in M199 plus 0.1% FCS. Cells were detached with 0.05. trypsin/0.02% EDTA, seeded in C6-well plates (Costar Corp., New Bedford, MA), grown to 70% confluency, and used between passages 2 and 3. Quiescent subconfluent EC were incubated with VEGF (Collaborative Biomedical Products, Bedford, MA; 10–100 ng/ml), basic fibroblast growth factor (bFGF; Calbiochem Corp., La Jolla, CA; 5 ng/ml), 10. FCS, or recombinant interleukin-1 (IL-1; R&D Systems, Minneapolis, MN; 2 ng/ml, 200 U/ml) or TNFα (10 ng/ml, Endogen, Woburn, MA) for 14 hours at 37°C in M199 plus 0.1% FCS. Cells were washed, harvested by trypsin/EDTA, and extracted in 4% sodium dodecyl sulfate plus protease inhibitors. Protein-normalized aliquots of cell extracts were electrophoresed on a 13.5% sodium dodecyl sulfate polyacrylamide gel, transferred to nylon membranes (Millipore Corp.) for 1 hour at 1 A, and immunoblotted with 1 μg/ml of a rabbit antibody to survivin followed by chemiluminescence (Amersham, Arlington Heights, IL).18Grossman D McNiff JM Li F Altieri DC Expression of the apoptosis inhibitor, survivin, in nonmelanoma skin cancer and gene targeting in a keratinocyte cell line.Lab Invest. 1999; 79: 1121-1126PubMed Google Scholar Samples were analyzed for equal protein loading by immunoblotting with a mouse antibody to β-actin. For Northern blot hybridization, serum-deprived EC were stimulated with 100 ng/ml VEGF and harvested at increasing time intervals between 1.5 and 24 hours culture at 37°C. Total RNA was extracted using the TRI Reagent (106 cells/0.2 ml, Molecular Research Center, Cincinnati, OH), and further processed for Northern blot hybridization with a 32Pα-dCTP random-primed labeled survivin cDNA or control β-actin probe, as described.15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar EC were suspended at a density of 3 × 106/ml in a liquefied matrix of rat-tail type I collagen (1.5 mg/ml) and human plasma-derived fibronectin (0.15 mg/ml. in M199, pH 7.5. One milliliter of the EC suspension was transferred into each well of rat-tail type I-coated C6 wells and warmed to 37°C to allow polymerization of the matrix. After a 24 hour incubation at 37°C in M199 plus 20% FCS, 50 μg/ml ECGS, 100 μg/ml heparin, 100 μg/ml penicillin, and 100 μg/ml streptomycin, the three-dimensional culture was placed in OCT and paraffin-embedded for immunohistochemical analysis. Alternatively, two- or three-dimensional EC cultures were homogenized in a tissue grinder and immunoblotted for survivin expression. During the incubation period, EC throughout the gel were observed to elongate and form multicellular tubular structures, as described.19Sierra-Honigman MR Nath AK Murakami C Garcia-Cadena G Papapetropoulos A Sessa WC Madge LA Schechner JS Schwabb MB Polverini PJ Flores-Riveros JR Biological action of leptin as an angiogenic factor.Science. 1998; 281: 1683-1686Crossref PubMed Scopus (1267) Google Scholar Four skin biopsies, containing granulation tissue or normal, non-inflamed skin by hematoxylin-eosin staining, were collected from the archives of Yale-New Haven Hospital. Five-micron sections were prepared from paraffin-embedded tissues, deparaffinized in xylene, and rehydrated in graded alcohol with quenching of endogenous peroxidase in 2% H2O2 in methanol. Immunolocalization of survivin was carried out as described previously,15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar after antigen retrieval by pressure cooking for 5 minutes in 0.01 mol/L citrate buffer, pH 6.0. Binding of the primary antibody was revealed by addition of 3,3′-diaminobenzidine, or, alternatively, 3-amino-9-ethylcarbazol (AEC, Vector), as a substrate. Control experiments were carried out in the absence of primary antibody or in the presence of preimmune rabbit IgG. The cDNA of wild-type survivin15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar was inserted in-frame in the Eco RI site of green fluorescence protein (GFP)-encoding plasmid, pEGFPc1 (Clontech, San Francisco, CA). The correct orientation and reading frame of pEGFPc1 fusion plasmid were confirmed by DNA sequencing. Bovine aortic EC were seeded in C6-well plates at 40 to 50% confluency and transfected with GFP vector or GFP survivin by lipofectin for 6 hours at 37°C. After removal of the DNA-lipid mixture, the EC monolayer was placed in complete growth medium for 35 hours at 37°C and incubated with 5 ng/ml TNFα plus 5 μg/ml cycloheximide for an additional 8 hours at 37°C. Cells (floaters plus attached cells) were fixed in 70% ethanol, stained with 10 μg/ml propidium iodide plus 100 μg/ml RNase A and 0.05% Triton X-100 in phosphate-buffered saline, pH 7.4, and GFP-expressing cells were analyzed for DNA content by flow cytometry. In other experiments, bovine EC transfected with GFP vector or GFP survivin were treated with control medium or 5 to 10 ng/ml TNFα plus 10 μg/ml cycloheximide for 8 hours at 37°C. Cells were harvested and analyzed for caspase-3 activity by hydrolysis of the fluorogenic substrate Ac-DEVD-AMC (N-Acetyl-Asp-Glu-Val-Asp-aldehyde, Pharmingen, San Diego, CA), in the presence or in the absence of the caspase-3 inhibitor Ac-DEVD-CHO. Fluorescence emissions were quantitated on a spectrofluorometer with excitation wavelength of 360 nm and emission wavelength of 460 nm. Expression of ∼16.5-kd endogenous survivin in quiescent, serum-deprived endothelium was minimally detectable by immunoblotting (Figure 1A), in agreement with previous observations.15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar EC stimulation with serum or the specific mitogens VEGF or bFGF resulted in an 8- to 16-fold up-regulation of survivin expression, by immunoblotting (Figure 1A). Survivin induction by VEGF was concentration-dependent and maximal at ∼50 ng/ml (Figure 1B). EC stimulation with cytokines TNFα or IL-1 did not increase survivin expression, which was reduced below background levels of untreated cells (Figure 1A). In control experiments by flow cytometry, TNFα or IL-1 stimulated strong up-regulation of intercellular adhesion molecule-1 in EC, whereas VEGF was ineffective (not shown). By Northern blot hybridization, a main 1.9-kb survivin message and a fainter 3.4-kb survivin transcript were minimally detected in quiescent EC (Figure 1C). VEGF treatment resulted in rapid up-regulation of survivin RNA in EC, in a response that peaked 6 to 10 hours after stimulation and decreased to approach background levels 24 hours after treatment (Figure 1C). Survivin was expressed at very low levels in two-dimensional EC cultures, by immunohistochemistry (Figure 2A). In contrast, formation of three-dimensional vascular tubes in collagen/fibronectin matrix resulted in strong expression of survivin in EC (Figure 2B). No staining of three-dimensional EC cultures was observed with control nonbinding antibody (Figure 2C). By immunoblotting, a prominent ∼16.5-kd survivin band was prominently induced in EC extracts of three-dimensional vascular tubes, as compared with two-dimensional EC cultures (Figure 2D). In four out of four cases, survivin was strongly expressed in the cytoplasm of EC of newly formed capillaries of skin granulation tissue, by immunohistochemistry (Figure 3A). Abundant expression of survivin was also demonstrated in EC of large vessels of granulation tissue at the dermis/hypodermis junction (Figure 3C). By contrast, no staining of granulation tissue was observed in the absence of primary antibody (Figure 3, B and D), or with control preimmune antibody (not shown). Analysis of nonproliferating capillaries of noninflamed normal skin revealed minimally detectable expression of survivin in EC (Figure 3E), as compared with control staining with preimmune IgG (Figure 3F). Treatment with TNFα/cycloheximide induced EC apoptosis and generation of a hypodiploid population by propidium iodide staining and flow cytometry (Figure 4A). Expression of GFP survivin inhibited TNFα-induced apoptosis in EC and reduced the percentage of hypodiploid cells to control levels of untreated cultures (Figure 4A). In contrast, transfection of GFP vector alone did not affect TNFα-induced EC apoptosis (Figure 4A). Moreover, expression of GFP survivin in EC strongly inhibited caspase-3 activity in TNFα-treated EC, as determined by DEVD hydrolysis, whereas GFP vector alone was ineffective (Figure 4B). In control experiments, preincubation of TNFα-treated EC extracts with the caspase-3 inhibitor DEVD-CHO abrogated DEVD hydrolysis (Figure 4B). In this study, we have shown that mitogen stimulation of EC results in strong up-regulation of the cell cycle-regulated apoptosis inhibitor, survivin.15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar, 16Li F Ambrosini G Chu EY Plescia J Tognin S Marchisio PC Altieri DC Control of apoptosis and mitotic spindle checkpoint by survivin.Nature. 1998; 396: 580-584Crossref PubMed Scopus (1723) Google Scholar From minimally detectable levels in quiescent endothelium, survivin became abundantly expressed in EC of three-dimensional vascular tubes in vitro and in newly formed capillaries during angiogenesis in vivo. Recombinant expression of survivin efficiently reduced caspase-3 activity in EC and blocked EC apoptosis induced by TNFα/cycloheximide. Recent experimental evidence has suggested that inhibition of EC apoptosis may be an essential prerequisite to maintain angiogenesis in vivo.11Risau W Mechanisms of angiogenesis.Nature. 1997; 386: 671-674Crossref PubMed Scopus (4805) Google Scholar Accordingly, disruption of αvβ3 integrin-matrix interaction20Brooks PC Montgomery AMP Rosenfeld M Reisfeld RA Hu T Klier G Cheresh DA Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels.Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2170) Google Scholar or interference with VEGF-dependent survival signals12Benjamin LE Keshet E Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.Proc Natl Acad Sci USA. 1997; 94: 8761-8766Crossref PubMed Scopus (443) Google Scholar, 13Alon T Hemo I Itin A Pe'er J Stone J Keshet E Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.Nat Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1407) Google Scholar, 14Yuan F Chen Y Dellian M Safabakhsh N Ferrara N Jain RK Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody.Proc Natl Acad Sci USA. 1996; 93: 14765-14770Crossref PubMed Scopus (602) Google Scholar triggered EC apoptosis and involution of newly formed capillaries in vivo. Despite the up-regulation of anti-apoptotic bcl- 2 and A1 molecules in VEGF-stimulated endothelium,21Gerber HP Dixit V Ferrara N Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells.J Biol Chem. 1998; 273: 13313-13316Crossref PubMed Scopus (831) Google Scholar, 22Nor JE Christensen J Mooney DJ Polverini PJ Vascular endothelial growth factor (VEGF)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of Bcl-2 expression.Am J Pathol. 1999; 154: 375-384Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar alternative mechanisms of cytoprotection have been postulated,23Karsan A Yee E Poirier GG Zhou P Craig R Harlan JM Fibroblast growth factor-2 inhibits endothelial cell apoptosis by Bcl-2- dependent and independent mechanisms.Am J Pathol. 1997; 151: 1775-1784PubMed Google Scholar prompting the search for additional effector genes contributing to VEGF-dependent EC survival. Here, the dramatic up-regulation of survivin in mitogen-stimulated endothelium is consistent with the cell cycle-dependent expression of the survivin gene in G2/M16 and suggests that this pathway may maintain a critical anti-apoptotic threshold at cell division. Consistent with the spontaneous induction of apoptosis resulting from survivin targeting in model cell types,18Grossman D McNiff JM Li F Altieri DC Expression of the apoptosis inhibitor, survivin, in nonmelanoma skin cancer and gene targeting in a keratinocyte cell line.Lab Invest. 1999; 79: 1121-1126PubMed Google Scholar, 24Ambrosini G Adida C Sirugo G Altieri DC Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting.J Biol Chem. 1998; 273: 11177-11182Crossref PubMed Scopus (432) Google Scholar these data suggest that VEGF induction of survivin may provide a critical prerequisite to maintain EC viability during angiogenesis, whereas loss of survivin may facilitate involution of newly formed capillaries in vivo.12Benjamin LE Keshet E Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.Proc Natl Acad Sci USA. 1997; 94: 8761-8766Crossref PubMed Scopus (443) Google Scholar, 13Alon T Hemo I Itin A Pe'er J Stone J Keshet E Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.Nat Med. 1995; 1: 1024-1028Crossref PubMed Scopus (1407) Google Scholar, 14Yuan F Chen Y Dellian M Safabakhsh N Ferrara N Jain RK Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody.Proc Natl Acad Sci USA. 1996; 93: 14765-14770Crossref PubMed Scopus (602) Google Scholar On the other hand, survivin expression in VEGF-stimulated endothelium may not be simply a consequence of cell proliferation, since survivin was not detected in other normal proliferating tissues, including the basal layer of epidermis.18Grossman D McNiff JM Li F Altieri DC Expression of the apoptosis inhibitor, survivin, in nonmelanoma skin cancer and gene targeting in a keratinocyte cell line.Lab Invest. 1999; 79: 1121-1126PubMed Google Scholar This suggests that VEGF induction of survivin may provide a unique paradigm of regulation of this anti-apoptotic pathway in a normal, terminally differentiated cell type. The suppression of caspase-3 activity in survivin-expressing EC shown here is consistent with the general function of IAP proteins as caspase inhibitors, either directly or through interference with caspase-9 processing.25Deveraux QL Reed JC IAP family proteins: suppressors of apoptosis.Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2270) Google Scholar, 26LaCasse EC Baird S Korneluk RG MacKenzie AE The inhibitors of apoptosis (IAPs) and their emerging role in cancer.Oncogene. 1998; 17: 3247-3259Crossref PubMed Scopus (940) Google Scholar In EC, active caspase-3 has been directly implicated in proteolysis of p125FAK 27Frisch SM Vuori K Ruoslahti E Chan-Hui PY Control of adhesion-dependent cell survival by focal adhesion kinase.J Cell Biol. 1996; 134: 793-799Crossref PubMed Scopus (989) Google Scholar, 28Levkau B Herren B Koyama H Ross R Raines EW Caspase-mediated cleavage of focal adhesion kinase pp125FAK and disassembly of focal adhesions in human endothelial cell apoptosis.J Exp Med. 1998; 187: 579-586Crossref PubMed Scopus (225) Google Scholar and p27/p21 cyclin-dependent kinase inhibitors,29Levkau B Koyama H Raines EW Clurman BE Herren B Orth KR Roberts JM Ross R Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cell through activation of Cdk2: role of a caspase cascade.Mol Cell. 1998; 1: 553-563Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar thus disassembling cell-to-matrix interactions and dysregulating cell division control mechanisms. In this context, VEGF induction of survivin is expected to provide a broad anti-apoptotic spectrum, counteracting a variety of death-inducing stimuli converging on caspase-3 activation as the executioner phase of apoptosis.2Salvesen GS Dixit VM Caspases: intracellular signaling by proteolysis.Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1934) Google Scholar It is also intriguing that both bcl-2 and survivin become up-regulated in VEGF-stimulated EC.21Gerber HP Dixit V Ferrara N Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells.J Biol Chem. 1998; 273: 13313-13316Crossref PubMed Scopus (831) Google Scholar This suggests that inhibition of EC apoptosis during angiogenesis may occur simultaneously through parallel and non-overlapping pathways, involving preservation of mitochondrial integrity by bcl-230Adams JM Cory S The Bcl-2 protein family: arbiters of cell survival.Science. 1998; 281: 1322-1326Crossref PubMed Scopus (4789) Google Scholar and suppression of caspase activity by survivin.25Deveraux QL Reed JC IAP family proteins: suppressors of apoptosis.Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2270) Google Scholar, 26LaCasse EC Baird S Korneluk RG MacKenzie AE The inhibitors of apoptosis (IAPs) and their emerging role in cancer.Oncogene. 1998; 17: 3247-3259Crossref PubMed Scopus (940) Google Scholar The findings reported here may have potentially far-reaching therapeutic implications. First, targeted inhibition of apoptosis in endothelium may be exploited to limit tissue damage in vascular diseases.31MacLellan WR Schneider MD Death by design: programmed cell death in cardiovascular biology and disease.Circ Res. 1997; 81: 137-144Crossref PubMed Google Scholar Accordingly, caspase antagonists32Cheng Y Deshmukh M D'Costa A Demaro JA Gidday JM Shah A Sun Y Jacquin MF Johnson Jr, EM Holtzman DM Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury.J Clin Invest. 1998; 101: 1992-1999Crossref PubMed Scopus (480) Google Scholar or overexpression of anti-apoptotic bcl-233Tagami MK Yamagata K Nara Y Fujino H Kubota A Numano F Yamori Y Linnik MD Zahos P Geschwind MD Federoff HJ Expression of bcl-2 from a defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia.Stroke. 1995; 26: 1670-1674Crossref PubMed Scopus (287) Google Scholar afforded increased neuronal viability in ischemia/hypoxia models. In this context, survivin gene transfer may result in improved EC viability during VEGF-stimulated compensatory angiogenesis in ischemic vascular diseases.34Battegay EJ Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects.J Mol Med. 1995; 73: 333-346Crossref PubMed Scopus (495) Google Scholar Conversely, for the selective expression of survivin in cancer,15Ambrosini G Adida C Altieri DC A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.Nat Med. 1997; 3: 917-921Crossref PubMed Scopus (3000) Google Scholar molecular antagonists of this pathway may not only sensitize tumor cells to therapy-induced apoptosis, but also remove a critical EC cytoprotective mechanism exploited during tumor angiogenesis.

The ability of polymorphonuclear leukocytes (PMNs) to modulate endothelial cell (EC) activation was investigated. Adding PMNs to cultured HUVECs resulted in a release of IL-6 (888 +/- 71 pg/ml, a 35-fold increase over release by the two cell types alone) and IL-8 (45.2 +/- 14.5 ng/ml, a 6.4-fold over PMN release alone and a 173-fold increase over EC release alone). In contrast, the release of TNF-alpha, IL-1beta, and platelet-derived growth factor was not affected by the EC-PMN coculture. Neutralizing mAbs to ICAM-1 or beta2 integrins or a physical segregation of PMNs and ECs did not reduce EC stimulation. In contrast, cell-free supernatants of PMNs recapitulated EC activation with an 18-fold up-regulation of EC IL-6 mRNA. The filtration of PMN supernatant or PMN pretreatment with metabolic antagonists or membrane cross-linking agents all suppressed EC activation. By flow cytometry, PMNs released in the supernatant, heterogeneous membrane-derived microparticles containing discrete proteins of 28 to 250 kDa as resolved by SDS-PAGE. PMN microparticle formation was enhanced by inflammatory stimuli, including formyl peptide and phorbol ester, and was time-dependent, reaching a plateau after a 1-h incubation from stimulation. Purified PMN microparticles induced EC IL-6 release in a reaction that was quantitatively indistinguishable from that observed with unfractionated PMN supernatant and unaffected by a neutralizing Ab to soluble IL-6R. These findings demonstrate that membrane microparticles released from stimulated PMNs are competent inflammatory mediators to produce EC activation and cytokine gene induction.

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis. This interaction involves the ATPase domain of Hsp90 and the survivin baculovirus inhibitor of apoptosis repeat. Global suppression of the Hsp90 chaperone function or targeted Abmediated disruption of the survivin-Hsp90 complex results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis, and cell cycle arrest with mitotic defects. These data link the cellular stress response to an antiapoptotic and mitotic checkpoint maintained by survivin. Targeting the survivin-Hsp90 complex may provide a rational approach for cancer therapy.

To the Editor: Transitional-cell carcinoma of the bladder is the fourth most common cancer among American men, with approximately 50,000 new cases and 10,500 deaths from the disease every year. Fif...

Background Colorectal cancer is thought to originate in the expansion of colonic crypt cells as a result of aberrant gene expression caused by transcription factors of the T-cell factor (TCF)/β-catenin family. Survivin is a bifunctional regulator of cell death and cell proliferation expressed during embryonic development but undetectable in healthy adult tissues and re-expressed in many cancers, including colorectal cancer. Methods We investigated gene expression by promoter analysis, mutagenesis, and electrophoretic mobility shift assay in colorectal cancer cells. Survivin expression in human and mouse embryonic intestine was determined by insitu hybridisation and immunohistochemistry. Changes in apoptosis were monitored in cell lines engineered to express stabilising mutations in β catenin. Findings TCF/β catenin stimulated a six-fold to 12-fold increased expression of the survivin gene in colorectal cancer cells. Three TCF-binding elements (TBE) in the survivin promoter were occupied by nuclear factors in colorectal cancer cells, and mutagenesis of the two proximal TBE sites abolished survivin gene expression by 75–79%. Strongly expressed at the bottom of human and mouse embryonic intestinal crypts, expression of survivin was lost in TCF-4 knockout animals, and a TCF-4 dominant negative mutant blocked survivin gene transcription in colorectal cancer cells. Expression of non-destructible β catenin mutants increased survivin expression and protected against ultraviolet-Binduced apoptosis. Interpretation Stimulation of survivin expression by TCF/β catenin might impose a stem cell-like phenotype to colonic crypt epithelium coupling enhanced cell proliferation with resistance to apoptosis, and contribute to the molecular pathogenesis of colorectal cancer.

A checkpoint surveying the entry into mitosis responds to defects in spindle microtubule assembly/stability. This has been used to trigger apoptosis in cancer cells, but how the spindle checkpoint couples to the cell survival machinery has remained elusive. Here, we report that microtubule stabilization engenders a survival pathway that depends on elevated activity of p34cdc2 kinase and increased expression of the apoptosis inhibitor and mitotic regulator, survivin. Pharmacologic, genetic, or molecular ablation of p34cdc2 kinase after microtubule stabilization resulted in massive apoptosis independent of p53, suppression of tumor growth, and indefinite survival without toxicity in mice. By ablating this survival checkpoint, inhibitors of p34cdc2 kinase could safely improve the efficacy of microtubule-stabilizing agents used to treat common cancers.

Survivin is a member of the inhibitor of apoptosis gene family that has been implicated in both apoptosis inhibition and regulation of mitosis. However, the subcellular distribution of survivin has been controversial and variously described as a microtubule-associated protein or chromosomal passenger protein. Here, we show that antibodies directed to the survivin sequence Ala3-Ile19 exclusively recognized a nuclear pool of survivin that segregated with nucleoplasmic proteins, but not with outer nuclear matrix or nuclear matrix proteins. By immunofluorescence,nuclear survivin localized to kinetochores of metaphase chromosomes, and to the central spindle midzone at anaphase. However, antibodies to Cys57-Trp67 identified a cytosolic pool of survivin,which associated with interphase microtubules, centrosomes, spindle poles and mitotic spindle microtubules at metaphase and anaphase. Polyclonal antibodies recognizing survivin epitopes Ala3-Ile19,Met38-Thr48, Pro47-Phe58 and Cys57-Trp67 identified both survivin pools within the same mitotic cell. A ratio of ∼1:6 for nuclear versus cytosolic survivin was obtained by quantitative subcellular fractionation. In synchronized cultures, cytosolic survivin abruptly increased at mitosis, physically associated with p34cdc2, and was phosphorylated by p34cdc2 on Thr34, in vivo. By contrast, nuclear survivin began to accumulate in S phase, was not complexed with p34cdc2 and was not phosphorylated on Thr34. Intracellular loading of a polyclonal antibody to survivin caused microtubule defects and resulted in formation of multipolar mitotic spindles, but did not interfere with cytokinesis. These data demonstrate that although both reported localizations of survivin exist in mitotic cells, the preponderant survivin pool is associated with microtubules and participates in the assembly of a bipolar mitotic spindle.

Evasion of apoptosis is a hallmark of cancer, but the molecular circuitries of this process are not understood.Here we show that survivin, a member of the inhibitor of apoptosis gene family that is overexpressed in cancer, exists in a novel mitochondrial pool in tumor cells.In response to cell death stimulation, mitochondrial survivin is rapidly discharged in the cytosol, where it prevents caspase activation and inhibits apoptosis.Selective targeting of survivin to mitochondria enhances colony formation in soft agar, accelerates tumor growth in immunocompromised animals, and abolishes tumor cell apoptosis in vivo.Therefore, mitochondrial survivin orchestrates a novel pathway of apoptosis inhibition, which contributes to tumor progression.

Inhibitors of programmed cell death (apoptosis) may regulate tissue differentiation and aberrantly promote cell survival in neoplasia. A novel apoptosis inhibitor of the IAP gene family, designated survivin, was recently found in all of the most common human cancers but not in normal, terminally differentiated adult tissues. The expression of survivin in embryonic and fetal development was investigated. Immunohistochemistry and in situ hybridization studies demonstrated strong expression of survivin in several apoptosis-regulated fetal tissues, including the stem cell layer of stratified epithelia, endocrine pancreas, and thymic medulla, with a pattern that did not overlap with that of another apoptosis inhibitor, bcl-2. A sequence-specific antibody to survivin immunoblotted a single approximately 16.5-kd survivin band in human fetal lung, liver, heart, kidney, and gastrointestinal tract. In mouse embryo, prominent and nearly ubiquitous distribution of survivin was found at embryonic day (E)11.5, whereas at E15 to -21, survivin expression was restricted to the distal bronchiolar epithelium of the lung and neural-crest-derived cells, including dorsal root ganglion neurons, hypophysis, and the choroid plexus. These data suggest that expression of survivin in embryonic and fetal development may contribute to tissue homeostasis and differentiation independently of bcl-2. Aberrations of this developmental pathway may result in prominent re-expression of survivin in neoplasia and abnormally prolonged cell viability.

Survivin has aroused keen interest in disparate areas of basic and translational research. This stems from the complexity of a 'survivin network', which appears to intersect multiple pathways of cell division, resistance to apoptosis, surveillance checkpoints and adaptation to unfavorable environments. Such intricacy has also engendered different models for survivin function and its implications. As antagonists of survivin are being evaluated in the clinic, a critical reassessment of the pathway, especially with respect to cell division and cell survival, is now a priority. Building a unifying model to reconcile the differing views on survivin will aid in the design and interpretation of molecularly based clinical trials targeting this network in humans.

Initiation of the coagulation protease cascade as it assembles on cell surfaces requires limited proteolytic activation of the zymogen factor X. Not previously suspected to be the ligand of an organizing receptor on cell surfaces, we now describe that factor X specifically associates with cells of monocyte lineage and we identify the high affinity receptor for this zymogen. Following stimulation with ADP (10 microM), or with the ionophore ionomycin (1 microM), isolated human monocytes bind 125I-factor X in a saturable fashion with a dissociation constant (Kd) of 21.8-44.9 nM. Equilibrium binding analyses indicate that the reaction is optimal at room temperature, requires Ca2+ ions, and saturates at 128,500 +/- 21,300 molecules of 125I-factor X specifically associated with the cell surface. Molar excess of unlabeled factor X inhibits and reverses the binding, whereas the homologous gamma-carboxylated coagulation proteins factors II, VII, IX, IXa, and Xa are without effect. Similarly, chelation of divalent ions immediately dissociates bound 125I-factor X. The monoblast cell line U 937 and the monocytic cell line THP-1 when stimulated with ADP or ionomycin, bind 125I-factor X with characteristics similar to monocytes. Receptor identity was explored using antibodies to the leukocyte adhesive receptors Mac-1, LFA-1, and p150.95. Monoclonal antibodies specific for the alpha subunit of Mac-1 (M 1/70, LM 2/1) or for the common beta subunit (TS 1/18, 60.3) bound equally to resting and ADP- or ionomycin-stimulated cells and also completely blocked the binding of 125I-factor X to stimulated monocytes, U 937, or THP-1 cells. To distinguish between modulatory effects of the monoclonal antibodies and direct spatial hindrance binding of 125I-factor X to Mac-1 was analyzed directly. OKM10 anti-alpha subunit of Mac-1 monoclonal antibody immunoprecipitated 125I-factor X chemically cross-linked to its receptor on stimulated cells. In addition, the complement protein fragment C3bi, which is a recognized ligand for Mac-1, competitively inhibited the association of 125I-factor X. These findings indicate that human blood monocytes and less differentiated cells of this lineage possess an inducible receptor specific for factor X; and also support the conclusion that the heterodimeric leukocyte adhesive receptor Mac-1 functions as the specific receptor structure. We suggest that the novel properties of this receptor may be of importance in the organization and regulation of certain coagulation protease cascades on the monocyte surface.

Dysregulation of apoptosis may favor onset and progression of cancer and influence response to therapy. Survivin is an inhibitor of apoptosis that is selectively overexpressed in common human cancers, but not in normal tissues, and that correlates with aggressive disease and unfavorable outcomes.To investigate the potential suitability of survivin detection in urine as a novel predictive/prognostic molecular marker of bladder cancer.Survey of urine specimens from 5 groups: healthy volunteers (n = 17) and patients with nonneoplastic urinary tract disease (n = 30), genitourinary cancer (n = 30), new-onset or recurrent bladder cancer (n = 46), or treated bladder cancer (n = 35), recruited from 2 New England urology clinics.Detectable survivin levels, analyzed by a novel detection system and confirmed by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR), in urine samples of the 5 participant groups.Survivin was detected in the urine samples of all 46 patients with new or recurrent bladder cancer using a novel detection system (31 of 31) and RT-PCR (15 of 15) methods. Survivin was not detected in the urine samples of 32 of 35 patients treated for bladder cancer and having negative cystoscopy results. None of the healthy volunteers or patients with prostate, kidney, vaginal, or cervical cancer had detectable survivin in urine samples. Of the 30 patients with nonneoplastic urinary tract disease, survivin was detected in 3 patients who had bladder abnormalities noted using cystoscopy and in 1 patient with an increased prostate-specific antigen level. Patients with low-grade bladder cancer had significantly lower urine survivin levels than patients with carcinoma in situ (P =.002).Highly sensitive and specific determination of urine survivin appears to provide a simple, noninvasive diagnostic test to identify patients with new or recurrent bladder cancer.

We have constructed a replication-deficient adenovirus encoding a nonphosphorylatable Thr34→Ala mutant of the apoptosis inhibitor survivin (pAd-T34A) to target tumor cell viability in vitro and in vivo. Infection with pAd-T34A caused spontaneous apoptosis in cell lines of breast, cervical, prostate, lung, and colorectal cancer. In contrast, pAd-T34A did not affect cell viability of proliferating normal human cells, including fibroblasts, endothelium, or smooth muscle cells. Infection of tumor cells with pAd-T34A resulted in cytochrome c release from mitochondria, cleavage of approximately 46-kDa upstream caspase-9, processing of caspase-3 to the active subunits of approximately 17 and 19 kDa, and increased caspase-3 catalytic activity. When compared with chemotherapeutic regimens, pAd-T34A was as effective as taxol and considerably more effective than adriamycin in induction of tumor cell apoptosis and enhanced taxol-induced cell death. In three xenograft breast cancer models in immunodeficient mice, pAd-T34A suppressed de novo tumor formation, inhibited by approximately 40% the growth of established tumors, and reduced intraperitoneal tumor dissemination. Tumors injected with pAd-T34A exhibited loss of proliferating cells and massive apoptosis by in situ internucleosomal DNA fragmentation. These data suggest that adenoviral targeting of the survivin pathway may provide a novel approach for selective cancer gene therapy.

A role of apoptosis (programmed cell death) in tumor formation and growth was investigated by targeting the apoptosis inhibitor survivin in vivo. Expression of a phosphorylation-defective survivin mutant (Thr(34)-->Ala) triggered apoptosis in several human melanoma cell lines and enhanced cell death induced by the chemotherapeutic drug cisplatin in vitro. Conditional expression of survivin Thr(34)-->Ala in YUSAC2 melanoma cells prevented tumor formation upon s.c. injection into CB.17 severe combined immunodeficient-beige mice. When induced in established melanoma tumors, survivin Thr(34)-->Ala inhibited tumor growth by 60-70% and caused increased apoptosis and reduced proliferation of melanoma cells in vivo. Manipulation of the antiapoptotic pathway maintained by survivin may be beneficial for cancer therapy.

A novel inhibitor of apoptosis designated survivin has recently been found in many common human cancers but not in normal tissues. A potential distribution of survivin in gastric cancer and its implication for apoptosis inhibition have been investigated. Recombinant survivin expressed in Escherichia coli as a glutathione S-transferase fusion protein was used to raise a novel panel of mouse monoclonal antibodies. In an immunohistochemical analysis of 174 cases of gastric carcinomas (stages I-III), anti-survivin monoclonal antibody 8E2 (IgG1) reacted with 34.5% of cases (60 of 174 cases) with a variable number of tumor cells stained (20-100%). In contrast, no expression of survivin in neighboring normal tissues was observed. When stratified for p53 and bcl-2 expression and apoptotic index, the expression of survivin significantly segregated with p53- and bcl-2-positive cases [56.1 versus 15.2% (P = 0.001) and 69.2 versus 31.6% (P = 0.006), respectively] and with a decreased apoptotic index as compared with that of survivin-negative tumors (0.97 +/- 0.64 versus 0.62 +/- 0.39%, P < 0.001). These data identify a role for survivin in promoting aberrantly increased cell viability in gastric cancer and suggest a potential correlation between accumulated p53 and survivin expression in neoplasia.

Although therapeutically targeting a single signaling pathway that drives tumor development and/or progression has been effective for a number of cancers, in many cases this approach has not been successful. Targeting networks of signaling pathways, instead of isolated pathways, may overcome this problem, which is probably due to the extreme heterogeneity of human tumors. However, the possibility that such networks may be spatially arranged in specialized subcellular compartments is not often considered in pathway-oriented drug discovery and may influence the design of new agents. Hsp90 is a chaperone protein that controls the folding of proteins in multiple signaling networks that drive tumor development and progression. Here, we report the synthesis and properties of Gamitrinibs, a class of small molecules designed to selectively target Hsp90 in human tumor mitochondria. Gamitrinibs were shown to accumulate in the mitochondria of human tumor cell lines and to inhibit Hsp90 activity by acting as ATPase antagonists. Unlike Hsp90 antagonists not targeted to mitochondria, Gamitrinibs exhibited a "mitochondriotoxic" mechanism of action, causing rapid tumor cell death and inhibiting the growth of xenografted human tumor cell lines in mice. Importantly, Gamitrinibs were not toxic to normal cells or tissues and did not affect Hsp90 homeostasis in cellular compartments other than mitochondria. Therefore, combinatorial drug design, whereby inhibitors of signaling networks are targeted to specific subcellular compartments, may generate effective anticancer drugs with novel mechanisms of action.

Abstract Survivin, a member of the inhibitors-of-apoptosis gene family, is expressed in a cell-cycle–dependent manner in all the most common cancers but not in normal differentiated adult tissues.Survivin expression and regulation were examined in acute myeloid leukemia (AML). Survivin was detected by Western blot analysis in all myeloid leukemia cell lines and in 16 of 18 primary AML samples tested. In contrast, normal CD34+ cells and normal peripheral blood mononuclear cells expressed no or very low levels of survivin. Cytokine stimulation increasedsurvivin expression in leukemic cell lines and in primary AML samples. In cultured primary samples, single-cytokine stimulation substantially increased survivin expression in comparison with control cells, and the combination of G-CSF, GM-CSF, and SCF increased survivin levels even further. Conversely, all-trans retinoic acid significantly decreased survivinprotein levels in HL-60, OCI-AML3, and NB-4 cells within 96 hours, parallel to the induction of myelomonocytic differentiation. Using selective pharmacologic inhibitors, the differential involvement of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol-3 kinase (PI3K) pathways were demonstrated in the regulation of survivin expression. The MEK inhibitor PD98059 down-regulated survivin expression in both resting and GM-CSF–stimulated OCI-AML3 cells, whereas the PI3K inhibitor LY294002 inhibited survivin expression only on GM-CSF stimulation. In conclusion, these results demonstrate thatsurvivin is highly expressed and cytokine-regulated in myeloid leukemias and suggest that hematopoietic cytokines exert their antiapoptotic and mitogenic effects, at least in part, by increasing survivin levels.

Monocytes initiate coagulation through regulated surface expression of tissue factor and local assembly of a proteolytic enzymatic complex formed by tissue factor and factor VII/activated factor VII. We now show that, in the absence of these initiating molecules, monocytes and cell lines of monocytic/myeloid differentiation can alternatively initiate coagulation after exposure to ADP. The molecular basis for this procoagulant response consists of two distinct events. First, cell stimulation with ADP induces high-affinity binding of coagulation factor X to the surface-adhesive receptor Mac-1. Locally, Mac-1-concentrated factor X is then rapidly proteolytically cleaved to an active protease with size and activity characteristics of activated factor X, which supports the cell-associated formation of thrombin and the procoagulant response. We conclude that the monocytic/myeloid adhesive receptor Mac-1 has the unexpected, specifically inducible property to organize a molecular assembly culminating in rapid fibrin formation that is independently regulated from tissue factor and factor VII/activated factor VII.

Survivin is an inhibitor of apoptosis overexpressed in various human cancers but undetectable in normal differentiated tissues. A potential expression and prognostic significance of survivin was studied in 222 patients with diffuse large B-cell lymphomas (centroblastic, 96%; immunoblastic, 4%). All patients were enrolled between 1987 and 1993 (median follow-up, 7 years) in the LNH87 protocol of the Groupe d'Etudes des Lymphomes de l'Adulte (GELA) and treated either with the reference ACVBP arm (doxorubicin, cyclophosphamide, vindesine, bleomycin, and prednisone)[AU3A] (n = 79) or other experimental anthracycline-containing regimens (n = 143). The characteristics of these patients were median age of 56 years; serum lactate dehydrogenase (LDH) greater than 1N, 60%; stage III-IV, 55%; performance status, according to the Eastern Cooperative Oncology Group (ECOG) scale, more than 1, 23%; extranodal sites more than 1, 29%; mass more than 10 cm, 44%; bone marrow involvement, 15%. Of the 222 patients studied, 134 (60%) revealed survivin expression in virtually all tumor cells by immunohistochemistry. The overall 5-year survival rate was significantly lower in patients with survivin expression than in those without (40% vs 54%, P =.02). Multivariate analysis incorporating prognostic factors from the International Prognostic Index (IPI) identified survivin expression as an independent predictive parameter on survival (P =.03, relative risk [RR] = 1.6) in addition to LDH (P =.02, RR = 1.6), stage (P =.03, RR = 1.7), and ECOG scale (P =.05, RR = 1.6). A second analysis incorporating IPI as a unique parameter demonstrated that survivin expression (P =.02, RR = 1.6) remained a prognostic factor for survival independently of IPI (P =.001, RR = 1.5). Survivin expression may be considered a new unfavorable prognostic factor of diffuse large B-cell lymphoma. (Blood. 2000;96:1921-1925)

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.

Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesion to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the association of fibrinogen with intercellular adhesion molecule 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen gamma-chain sequence N117NQKIVNL-KEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.

The leukocyte-restricted integrin CD11b/CD18 (alpha M beta 2, Mac-1) is a receptor for fibrinogen on stimulated monocytes and neutrophils. At variance with platelet alpha IIb beta 3 or endothelial cell alpha v beta 3 integrins, CD11b/CD18 interacts with a approximately 30-kDa plasmic fragment D (D30) of fibrinogen that lacks the Arg-Gly-Asp sequences in the A alpha chain and the carboxyl terminus of the gamma chain. Using epitope-mapped antibodies and synthetic peptidyl mimicry, we have now identified a unique linear sequence in fibrinogen that mediates ligand binding to CD11b/CD18. Anti-fibrinogen antibodies directed to the gamma chain region 95-264 inhibited 125I-fibrinogen or 125I-D30 binding to chemoattractant-stimulated neutrophils or monocytic THP-1 cells in a dose-dependent fashion. Partially overlapping synthetic peptides reproducing this gamma chain region were tested for their ability to inhibit fibrinogen binding to leukocytes. A synthetic peptide designated P1, duplicating gamma chain Gly190-Val202, inhibited 125I-fibrinogen binding to stimulated neutrophils or THP-1 cells and blocked adhesion of these cells to immobilized fibrinogen in a dose-dependent fashion. Increasing concentrations of P1 inhibited 125I-fibrinogen binding to isolated CD11b/CD18 in a cell-free system. Consistent with genuine peptidyl mimicry, 125I-P1 bound saturably to THP-1 cells in a reaction inhibited by molar excess of unlabeled peptide, fibrinogen, or D30. Finally, immobilized P1 effectively supported adhesion of THP-1 cells in a CD11b/CD18-dependent manner. These data suggest that the fibrinogen gamma chain region Gly190-Val202 functions as a minimal recognition sequence for the leukocyte integrin CD11b/CD18. Given the participation of fibrinogen:leukocyte interaction in inflammation and atherogenesis, antagonists based on this unique structural motif would effectively interfere with aberrant leukocyte adhesion mechanisms without affecting Arg-Gly-Asp-directed vascular integrins.

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.

A little over 10 years after its discovery in 1997, the small inhibitor of apoptosis (IAP) protein, survivin, continues to generate intense interest and keen attention from disparate segments of basic and disease-related research. Part of this interest reflects the intricate biology of this multifunctional protein that intersects fundamental networks of cellular homeostasis. Part is because of the role of survivin as a cancer gene, which touches nearly every aspect of the disease, from onset to outcome. And part is due to the potential value of survivin for novel cancer diagnostics and therapeutics, which have already reached the clinic, and with some promise. Grappling with emerging new signaling circuits in survivin biology, and their implications in cancer, will further our understanding of this nodal protein, and open fresh opportunities for translational oncology research.

Metabolic reprogramming is an important driver of tumor progression; however, the metabolic regulators of tumor cell motility and metastasis are not understood. Here, we show that tumors maintain energy production under nutrient deprivation through the function of HSP90 chaperones compartmentalized in mitochondria. Using cancer cell lines, we found that mitochondrial HSP90 proteins, including tumor necrosis factor receptor-associated protein-1 (TRAP-1), dampen the activation of the nutrient-sensing AMPK and its substrate UNC-51-like kinase (ULK1), preserve cytoskeletal dynamics, and release the cell motility effector focal adhesion kinase (FAK) from inhibition by the autophagy initiator FIP200. In turn, this results in enhanced tumor cell invasion in low nutrients and metastatic dissemination to bone or liver in disease models in mice. Moreover, we found that phosphorylated ULK1 levels were correlated with shortened overall survival in patients with non-small cell lung cancer. These results demonstrate that mitochondrial HSP90 chaperones, including TRAP-1, overcome metabolic stress and promote tumor cell metastasis by limiting the activation of the nutrient sensor AMPK and preventing autophagy.

Protein folding quality control does not occur randomly in cells, but requires the action of specialized molecular chaperones compartmentalized in subcellular microenvironments and organelles. Fresh experimental evidence has now linked a mitochondrial-specific Heat Shock Protein-90 (Hsp90) homolog, Tumor Necrosis Factor Receptor-Associated Protein-1 (TRAP-1) to pleiotropic signaling circuitries of organelle integrity and cellular homeostasis. TRAP-1-directed compartmentalized protein folding is broadly exploited in cancer and neurodegenerative diseases, presenting new opportunities for therapeutic intervention in humans. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).

With almost 4000 citations in Medline in a little over 10 years, survivin has certainly kept scores of investigators busy worldwide. Tangible progress has been made in revealing the multiple functions of survivin, uncovering their wirings as integrated cellular networks, and mapping their exploitation in virtually every human tumor, in vivo. Considering the normally long and excruciating timeline of oncology drug discovery, it is clearly a resounding success that a better understanding of survivin biology has led to several clinical trials of survivin-based therapeutics in cancer patients. However, the portfolio of survivin antagonists available in the clinic remains small, pressing the need for a less rigid drug development approach to fully unlock the potential of this unique, albeit unconventional oncology drug target.

Mitochondrial apoptosis plays a critical role in tumor maintenance and dictates the response to therapy in vivo; however, the regulators of this process are still largely elusive. Here, we show that the molecular chaperone heat shock protein 60 (Hsp60) directly associates with cyclophilin D (CypD), a component of the mitochondrial permeability transition pore. This interaction occurs in a multichaperone complex comprising Hsp60, Hsp90, and tumor necrosis factor receptor-associated protein-1, selectively assembled in tumor but not in normal mitochondria. Genetic targeting of Hsp60 by siRNA triggers CypD-dependent mitochondrial permeability transition, caspase-dependent apoptosis, and suppression of intracranial glioblastoma growth in vivo. Therefore, Hsp60 is a novel regulator of mitochondrial permeability transition, contributing to a cytoprotective chaperone network that antagonizes CypD-dependent cell death in tumors.

CD11b/CD18 (Mac-1) is a member of the leukocyte integrin family, a group of receptors that have been implicated in various effector functions and cellular collaboration in the immune response. It has been shown previously that CD11b/CD18 on cells of monocyte and myeloid lineage appears to undergo rapid activation and acquire new functional receptor specificities after exposure to selected agonists such as adenosine diphosphate (ADP). We now show that ADP induces a reconformation of the CD11b/CD18 receptor with exposure of new epitopes characteristics of this activated state. By direct binding studies, flow cytometry, and immunoprecipitation experiments, it has been found that the mAb 7E3 reacts with CD11b/CD18 only after ADP-stimulation of the cell suspension. The activated state of CD11b/CD18 induced by ADP and recognized by 7E3 can also be recapitulated by agonists inducing transients in cytosolic Ca2+ such as the chemoattractant FMLP. Moreover, this process of receptor activation does not involve quantitative mobilization of the subcellular storage pool of CD11b/CD18 to the plasma membrane. Because 7E3 also recognizes a qualitative, ADP-mediated activated state of the platelet adhesion receptor GP IIb/IIIa, it is suggested that transients in cytosolic Ca2+ might represent early secondary events for a general pathway of rapid activation of integrin receptors and, as such, represent important signals for cellular interactions in the immune response.

Cell death pathways are likely regulated in specialized subcellular microdomains, but how this occurs is not understood. Here, we show that cyclic AMP-dependent protein kinase A (PKA) phosphorylates the inhibitor of apoptosis (IAP) protein survivin on Ser20 in the cytosol, but not in mitochondria. This phosphorylation event disrupts the binding interface between survivin and its antiapoptotic cofactor, XIAP. Conversely, mitochondrial survivin or a non-PKA phosphorylatable survivin mutant binds XIAP avidly, enhances XIAP stability, synergistically inhibits apoptosis, and accelerates tumor growth, in vivo. Therefore, differential phosphorylation of survivin by PKA in subcellular microdomains regulates tumor cell apoptosis via its interaction with XIAP.

Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors.

Reprogramming of tumour cell metabolism contributes to disease progression and resistance to therapy, but how this process is regulated on the molecular level is unclear. Here we report that heat shock protein 90-directed protein folding in mitochondria controls central metabolic networks in tumour cells, including the electron transport chain, citric acid cycle, fatty acid oxidation, amino acid synthesis and cellular redox status. Specifically, mitochondrial heat shock protein 90, but not cytosolic heat shock protein 90, binds and stabilizes the electron transport chain Complex II subunit succinate dehydrogenase-B, maintaining cellular respiration under low-nutrient conditions, and contributing to hypoxia-inducible factor-1α-mediated tumorigenesis in patients carrying succinate dehydrogenase-B mutations. Thus, heat shock protein 90-directed proteostasis in mitochondria regulates tumour cell metabolism, and may provide a tractable target for cancer therapy.

Pluripotent human embryonic stem (hES) cells require mechanisms to maintain genomic integrity in response to DNA damage that could compromise competency for lineage-commitment, development, and tissue-renewal. The mechanisms that protect the genome in rapidly proliferating hES cells are minimally understood. Human ES cells have an abbreviated cell cycle with a very brief G1 period suggesting that mechanisms mediating responsiveness to DNA damage may deviate from those in somatic cells. Here, we investigated how hES cells react to DNA damage induced by ionizing radiation (IR) and whether genomic insult evokes DNA repair pathways and/or cell death. We find that hES cells respond to DNA damage by rapidly inducing Caspase-3 and -8, phospho-H2AX foci, phosphorylation of p53 on Ser15 and p21 mRNA levels, as well as concomitant cell cycle arrest in G2 based on Ki67 staining and FACS analysis. Unlike normal somatic cells, hES cells and cancer cells robustly express the anti-apoptotic protein Survivin, consistent with the immortal growth phenotype. SiRNA depletion of Survivin diminishes hES survival post-irradiation. Thus, our findings provide insight into pathways and processes that are activated in human embryonic stem cells upon DNA insult to support development and tissue regeneration.

Although technically a member of the Inhibitor of Apoptosis (IAP) gene family, survivin has consistently defied assumptions, refuted predictions and challenged paradigms. Despite its more than 5500 citations currently in Medline, the biology of survivin has remained fascinatingly complex, its exploitation in human disease, most notably cancer, tantalizing, and its regulation of cellular homeostasis unexpectedly far-reaching. An inconvenient outsider that resists schemes and dogmas, survivin continues to hold great promise to unlock fundamental circuitries of cellular functions in health and disease.

Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.

Molecular chaperones of the heat shock protein-90 (Hsp90) family promote cell survival, but the molecular requirements of this pathway in tumor progression are not understood. Here, we show that a mitochondria-localized Hsp90 chaperone, tumor necrosis factor receptor-associated protein-1 (TRAP-1), is abundantly and ubiquitously expressed in human high-grade prostatic intraepithelial neoplasia, Gleason grades 3 through 5 prostatic adenocarcinomas, and metastatic prostate cancer, but largely undetectable in normal prostate or benign prostatic hyperplasia in vivo. Prostate lesions formed in genetic models of the disease, including the transgenic adenocarcinoma of the mouse prostate and mice carrying prostate-specific deletion of the phosphatase tensin homolog tumor suppressor (Pten(pc-/-)), also exhibit high levels of TRAP-1. Expression of TRAP-1 in nontransformed prostatic epithelial BPH-1 cells inhibited cell death, whereas silencing of TRAP-1 in androgen-independent PC3 or DU145 prostate cancer cells by small interfering RNA enhanced apoptosis. Targeting TRAP-1 with a novel class of mitochondria-directed Hsp90 inhibitors, ie, Gamitrinibs, caused rapid and complete killing of androgen-dependent or -independent prostate cancer, but not BPH-1 cells, whereas reintroduction of TRAP-1 in BPH-1 cells conferred sensitivity to Gamitrinib-induced cell death. These data identify TRAP-1 as a novel mitochondrial survival factor differentially expressed in localized and metastatic prostate cancer compared with normal prostate. Targeting this pathway with Gamitrinibs could be explored as novel molecular therapy in patients with advanced prostate cancer.

The role of mitochondria in cancer is controversial. Using a genome-wide shRNA screen, we now show that tumours reprogram a network of mitochondrial dynamics operative in neurons, including syntaphilin (SNPH), kinesin KIF5B and GTPase Miro1/2 to localize mitochondria to the cortical cytoskeleton and power the membrane machinery of cell movements. When expressed in tumours, SNPH inhibits the speed and distance travelled by individual mitochondria, suppresses organelle dynamics, and blocks chemotaxis and metastasis, in vivo. Tumour progression in humans is associated with downregulation or loss of SNPH, which correlates with shortened patient survival, increased mitochondrial trafficking to the cortical cytoskeleton, greater membrane dynamics and heightened cell invasion. Therefore, a SNPH network regulates metastatic competence and may provide a therapeutic target in cancer.

Tumors successfully adapt to constantly changing intra- and extracellular environments, but the wirings of this process are still largely elusive. Here, we show that heat-shock-protein-90-directed protein folding in mitochondria, but not cytosol, maintains energy production in tumor cells. Interference with this process activates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase, inhibition of rapamycin-sensitive mTOR complex 1, induction of autophagy, and expression of an endoplasmic reticulum unfolded protein response. This signaling network confers a survival and proliferative advantage to genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mitochondrial heat shock protein 90s are adaptive regulators of tumor bioenergetics and tractable targets for cancer therapy.

Small molecule inhibitors of phosphatidylinositol-3 kinase (PI3K) have been developed as molecular therapy for cancer, but their efficacy in the clinic is modest, hampered by resistance mechanisms. We studied the effect of PI3K therapy in patient-derived tumor organotypic cultures (from five patient samples), three glioblastoma (GBM) tumor cell lines, and an intracranial model of glioblastoma in immunocompromised mice (n = 4–5 mice per group). Mechanisms of therapy-induced tumor reprogramming were investigated in a global metabolomics screening, analysis of mitochondrial bioenergetics and cell death, and modulation of protein phosphorylation. A high-throughput drug screening was used to identify novel preclinical combination therapies with PI3K inhibitors, and combination synergy experiments were performed. All statistical methods were two-sided. PI3K therapy induces global metabolic reprogramming in tumors and promotes the recruitment of an active pool of the Ser/Thr kinase, Akt2 to mitochondria. In turn, mitochondrial Akt2 phosphorylates Ser31 in cyclophilin D (CypD), a regulator of organelle functions. Akt2-phosphorylated CypD supports mitochondrial bioenergetics and opposes tumor cell death, conferring resistance to PI3K therapy. The combination of a small-molecule antagonist of CypD protein folding currently in preclinical development, Gamitrinib, plus PI3K inhibitors (PI3Ki) reverses this adaptive response, produces synergistic anticancer activity by inducing mitochondrial apoptosis, and extends animal survival in a GBM model (vehicle: median survival = 28.5 days; Gamitrinib+PI3Ki: median survival = 40 days, P = .003), compared with single-agent treatment (PI3Ki: median survival = 32 days, P = .02; Gamitrinib: median survival = 35 days, P = .008 by two-sided unpaired t test). Small-molecule PI3K antagonists promote drug resistance by repurposing mitochondrial functions in bioenergetics and cell survival. Novel combination therapies that target mitochondrial adaptation can dramatically improve on the efficacy of PI3K therapy in the clinic.

To test the anticancer effect of combining two drugs targeting different biological pathways, the popular way to show synergistic effect of drug combination is a heat map or surface plot based on the percent excess the Bliss prediction using the average response measures at each combination dose. Such graphs, however, are inefficient in the drug screening process and it doesn't give a statistical inference on synergistic effect. To make a statistically rigorous and robust conclusion for drug combination effect, we present a two-stage Bliss independence response surface model to estimate an overall interaction index (τ) with 95% confidence interval (CI). By taking into all data points account, the overall τ with 95% CI can be applied to determine if the drug combination effect is synergistic overall. Using some example data, the two-stage model was compared to a couple of classic models following Bliss rule. The data analysis results obtained from our model reflect the pattern shown from other models. The application of overall τ helps investigators to make decision easier and accelerate the preclinical drug screening.

Survivin promotes cell division and suppresses apoptosis in many human cancers, and increased abundance correlates with metastasis and poor prognosis. We showed that a pool of survivin that localized to the mitochondria of certain tumor cell lines enhanced the stability of oxidative phosphorylation complex II, which promoted cellular respiration. Survivin also supported the subcellular trafficking of mitochondria to the cortical cytoskeleton of tumor cells, which was associated with increased membrane ruffling, increased focal adhesion complex turnover, and increased tumor cell migration and invasion in cultured cells, and enhanced metastatic dissemination in vivo. Therefore, we found that mitochondrial respiration enhanced by survivin contributes to cancer metabolism, and relocalized mitochondria may provide a "regional" energy source to fuel tumor cell invasion and metastasis.

Small-molecule inhibitors of the phosphoinositide 3-kinase (PI3K), Akt, and mTOR pathway currently in the clinic produce a paradoxical reactivation of the pathway they are intended to suppress. Furthermore, fresh experimental evidence with PI3K antagonists in melanoma, glioblastoma, and prostate cancer shows that mitochondrial metabolism drives an elaborate process of tumor adaptation culminating with drug resistance and metastatic competency. This is centered on reprogramming of mitochondrial functions to promote improved cell survival and to fuel the machinery of cell motility and invasion. Key players in these responses are molecular chaperones of the Hsp90 family compartmentalized in mitochondria, which suppress apoptosis via phosphorylation of the pore component, Cyclophilin D, and enable the subcellular repositioning of active mitochondria to membrane protrusions implicated in cell motility. An inhibitor of mitochondrial Hsp90s in preclinical development (gamitrinib) prevents adaptive mitochondrial reprogramming and shows potent antitumor activity in vitro and in vivo. Other therapeutic strategies to target mitochondria for cancer therapy include small-molecule inhibitors of mutant isocitrate dehydrogenase (IDH) IDH1 (AG-120) and IDH2 (AG-221), which opened new therapeutic prospects for patients with high-risk acute myelogenous leukemia (AML). A second approach of mitochondrial therapeutics focuses on agents that elevate toxic ROS levels from a leaky electron transport chain; nevertheless, the clinical experience with these compounds, including a quinone derivative, ARQ 501, and a copper chelator, elesclomol (STA-4783) is limited. In light of this evidence, we discuss how best to target a resurgence of mitochondrial bioenergetics for cancer therapy.

Rationale: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease with a poor prognosis and limited therapeutic options. Although the mechanisms contributing to vascular remodeling in PAH are still unclear, several features, including hyperproliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs), have led to the emergence of the cancer-like concept. The molecular chaperone HSP90 (heat shock protein 90) is directly associated with malignant growth and proliferation under stress conditions. In addition to being highly expressed in the cytosol, HSP90 exists in a subcellular pool compartmentalized in the mitochondria (mtHSP90) of tumor cells, but not in normal cells, where it promotes cell survival.Objectives: We hypothesized that mtHSP90 in PAH-PASMCs represents a protective mechanism against stress, promoting their proliferation and resistance to apoptosis.Methods: Expression and localization of HSP90 were analyzed by Western blot, immunofluorescence, and immunogold electron microscopy. In vitro, effects of mtHSP90 inhibition on mitochondrial DNA integrity, bioenergetics, cell proliferation and resistance to apoptosis were assessed. In vivo, the therapeutic potential of Gamitrinib, a mitochondria-targeted HSP90 inhibitor, was tested in fawn-hooded and monocrotaline rats.Measurements and Main Results: We demonstrated that, in response to stress, HSP90 preferentially accumulates in PAH-PASMC mitochondria (dual immunostaining, immunoblot, and immunogold electron microscopy) to ensure cell survival by preserving mitochondrial DNA integrity and bioenergetic functions. Whereas cytosolic HSP90 inhibition displays a lack of absolute specificity for PAH-PASMCs, Gamitrinib decreased mitochondrial DNA content and repair capacity and bioenergetic functions, thus repressing PAH-PASMC proliferation (Ki67 labeling) and resistance to apoptosis (Annexin V assay) without affecting control cells. In vivo, Gamitrinib improves PAH in two experimental rat models (monocrotaline and fawn-hooded rat).Conclusions: Our data show for the first time that accumulation of mtHSP90 is a feature of PAH-PASMCs and a key regulator of mitochondrial homeostasis contributing to vascular remodeling in PAH.

Mitochondria are signaling hubs in eukaryotic cells. Here, we showed that the mitochondrial FUN14 domain-containing protein-1 (FUNDC1), an effector of Parkin-independent mitophagy, also participates in cellular plasticity by sustaining oxidative bioenergetics, buffering ROS production, and supporting cell proliferation. Targeting this pathway in cancer cells suppressed tumor growth but rendered transformed cells more motile and invasive in a manner dependent on ROS-mediated mitochondrial dynamics and mitochondrial repositioning to the cortical cytoskeleton. Global metabolomics and proteomics profiling identified a FUNDC1 interactome at the mitochondrial inner membrane, comprising the AAA+ protease, LonP1, and subunits of oxidative phosphorylation, complex V (ATP synthase). Independently of its previously identified role in mitophagy, FUNDC1 enabled LonP1 proteostasis, which in turn preserved complex V function and decreased ROS generation. Therefore, mitochondrial reprogramming by a FUNDC1-LonP1 axis controls tumor cell plasticity by switching between proliferative and invasive states in cancer.

There is now a consensus that mitochondria are important tumor drivers, sophisticated biological machines that can engender a panoply of key disease traits. How this happens, however, is still mostly elusive. The opinion presented here is that what cancer exploits are not the normal mitochondria of oxygenated and nutrient-replete tissues, but the unfit, damaged, and dysfunctional organelles generated by the hostile environment of tumor growth. These ‘ghost' mitochondria survive quality control and thwart cell death to relay multiple comprehensive ‘danger signals' of metabolic starvation, cellular stress, and reprogrammed gene expression. The result is a new, treacherous cellular phenotype, proliferatively quiescent but highly motile, that enables tumor cell escape from a threatening environment and colonization of distant, more favorable sites (metastasis).

The preservation of tissue and organ homoeostasis depends on the regulated expression of genes controlling apoptosis (programmed cell death). In this study, we have investigated the basal transcriptional requirements of the survivin gene, an IAP (inhibitor of apoptosis) prominently up-regulated in cancer. Analysis of the 5′ flanking region of the human survivin gene revealed the presence of a TATA-less promoter containing a canonical CpG island of ∼ 250 nt, three cell cycle dependent elements, one cell cycle homology region and numerous Sp1 sites. PCR-based analysis of human genomic DNA, digested with methylation-sensitive and -insensitive restriction enzymes, indicated that the CpG island was unmethylated in both normal and neoplastic tissues. Primer extension and S1 nuclease mapping of the human survivin gene identified two main transcription start sites at position -72 and within -57/-61 from the initiating ATG. Transfection of cervical carcinoma HeLa cells with truncated or nested survivin promoter-luciferase constructs revealed the presence of both enhancer and repressor sequences and identified a minimal promoter region within the proximal -230 nt of the human survivin gene. Unbiased mutagenesis analysis of the human survivin promoter revealed that targeting the Sp1 sequences at position -171 and -151 abolished basal transcriptional activity by ∼ 63-82%. Electrophoretic mobility-shift assay with DNA oligonucleotides confirmed formation of a DNA-protein complex between the survivin Sp1 sequences and HeLa cell extracts in a reaction abolished by mutagenesis of the survivin Sp1 sites. These findings identify the basal transcriptional requirements of survivin gene expression.

The preservation of tissue and organ homoeostasis depends on the regulated expression of genes controlling apoptosis (programmed cell death). In this study, we have investigated the basal transcriptional requirements of the survivin gene, an IAP (inhibitor of apoptosis) prominently up-regulated in cancer. Analysis of the 5' flanking region of the human survivin gene revealed the presence of a TATA-less promoter containing a canonical CpG island of approximately 250 nt, three cell cycle dependent elements, one cell cycle homology region and numerous Sp1 sites. PCR-based analysis of human genomic DNA, digested with methylation-sensitive and -insensitive restriction enzymes, indicated that the CpG island was unmethylated in both normal and neoplastic tissues. Primer extension and S1 nuclease mapping of the human survivin gene identified two main transcription start sites at position -72 and within -57/-61 from the initiating ATG. Transfection of cervical carcinoma HeLa cells with truncated or nested survivin promoter-luciferase constructs revealed the presence of both enhancer and repressor sequences and identified a minimal promoter region within the proximal -230 nt of the human survivin gene. Unbiased mutagenesis analysis of the human survivin promoter revealed that targeting the Sp1 sequences at position -171 and -151 abolished basal transcriptional activity by approximately 63-82%. Electrophoretic mobility-shift assay with DNA oligonucleotides confirmed formation of a DNA-protein complex between the survivin Sp1 sequences and HeLa cell extracts in a reaction abolished by mutagenesis of the survivin Sp1 sites. These findings identify the basal transcriptional requirements of survivin gene expression.

Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84→ Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via β1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-α (TNF-α). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-α-induced apoptosis. Evidence is provided that expression of β1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-α-induced apoptosis. In contrast, the β1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-α in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis. Integrins are cell surface heterodimeric transmembrane receptors that, in addition to mediating cell adhesion to extracellular matrix proteins modulate cell survival. This mechanism may be exploited in cancer where evasion from apoptosis invariably contributes to cellular transformation. The molecular mechanisms responsible for matrix-induced survival signals begin to be elucidated. Here we report that the inhibitor of apoptosis survivin is expressed in vitro in human prostate cell lines with the highest levels present in aggressive prostate cancer cells such as PC3 and LNCaP-LN3 as well as in vivo in prostatic adenocarcinoma. We also show that interference with survivin in PC3 prostate cancer cells using a Cys84→ Ala dominant negative mutant or survivin antisense cDNA causes nuclear fragmentation, hypodiploidy, cleavage of a 32-kDa proform caspase-3 to active caspase-3, and proteolysis of the caspase substrate poly(ADP-ribose) polymerase. We demonstrate that in the aggressive PC3 cell line, adhesion to fibronectin via β1 integrins results in up-regulation of survivin and protection from apoptosis induced by tumor necrosis factor-α (TNF-α). In contrast, survivin is not up-regulated by cell adhesion in the non-tumorigenic LNCaP cell line. Dominant negative survivin counteracts the ability of fibronectin to protect cells from undergoing apoptosis, whereas wild-type survivin protects non-adherent cells from TNF-α-induced apoptosis. Evidence is provided that expression of β1A integrin is necessary to protect non-adherent cells transduced with survivin from TNF-α-induced apoptosis. In contrast, the β1C integrin, which contains a variant cytoplasmic domain, is not able to prevent apoptosis induced by TNF-α in non-adherent cells transduced with survivin. Finally, we show that regulation of survivin levels by integrins are mediated by protein kinase B/AKT. These findings indicate that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells and identify integrin signaling as a crucial survival pathway against death receptor-mediated apoptosis. It has become increasingly clear that the interactions between extracellular matrix (ECM) 1The abbreviations used are: ECMextracellular matrixIAPinhibitor of apoptosisERKextracellular signal-regulated kinasemAbmonoclonal antibodyTNF-αtumor necrosis factor-αPARPpoly(ADP ribose) polymeraseCHXcycloheximideAct Dactinomycin DHAhemagglutininPI(3,4)P2phosphatidylinositol 3,4-bisphosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-trisphosphateZbenzyloxycarbonylfmkfluoromethyl ketoneBSAbovine serum albuminntnucleotideEGFepidermal growth factorFACSfluorescence-activated cell sorterGFPgreen fluorescent proteindndominant negative. proteins and integrins, their cognate cell surface receptors, mediate cell adhesion but also engender signals that participate in cell survival (1.Frisch S.M. Ruoslahti E. Curr. Opin. Cell Biol. 1997; 9: 701-706Crossref PubMed Scopus (992) Google Scholar). It has been shown that insufficient or inappropriate cell-ECM interactions cause apoptosis, previously designated anoikis (1.Frisch S.M. Ruoslahti E. Curr. Opin. Cell Biol. 1997; 9: 701-706Crossref PubMed Scopus (992) Google Scholar). This mechanism of integrin-dependent cytoprotection is operative against a variety of death-promoting stimuli, acting through the intrinsic (mitochondrial) or extrinsic (death receptor) apoptotic pathway and involving growth factor withdrawal (2.Zhang Z. Vuori K. Reed J.C. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6161-6165Crossref PubMed Scopus (575) Google Scholar, 3.O'Brien V. Frisch S.M. Juliano R.L. Exp. Cell Res. 1996; 224: 208-213Crossref PubMed Scopus (85) Google Scholar, 4.Lee J.W. Juliano R.L. Mol. Biol. Cell. 2000; 11: 1973-1987Crossref PubMed Scopus (143) Google Scholar, 5.Almeida E.A. Ilic D. Han Q. Hauch C.R. Jin F. Kawakatsu H. Schlaepfer D.D. Damsky C.H. J. Cell Biol. 2000; 149: 741-754Crossref PubMed Scopus (337) Google Scholar), exposure to cytotoxic drugs (6.Damiano J.S. Curr. Cancer Drug Targets. 2002; 2: 37-43Crossref PubMed Scopus (91) Google Scholar, 7.Uhm J.H. Dooley N.P. Kyritsis A.P. Rao J.S. Gladson C.L. Clin. Cancer Res. 1999; 5: 1587-1594PubMed Google Scholar), and ligation of death receptors (8.Aoudjit F. Vuori K. J. Cell Biol. 2001; 152: 633-643Crossref PubMed Scopus (254) Google Scholar, 9.Shain K.H. Landowski T.H. Dalton W.S. J. Immunol. 2002; 168: 2544-2553Crossref PubMed Scopus (101) Google Scholar). Critical signaling intermediates involved in integrin-dependent cell survival have been identified, and integrin-mediated adhesion to the ECM stimulates the production of PI(3,4)P2 and PI(3,4,5)P3 (10.Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (940) Google Scholar, 11.King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar), the association of the p85 PI 3-kinase subunit with focal adhesion kinase (12.Chen H.-C. Guan J.-L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (478) Google Scholar) and AKT activation (10.Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (940) Google Scholar, 11.King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar). In turn, active AKT interferes with the apoptotic machinery by phosphorylating thus sequestering the proapoptotic Bcl-2 family protein BAD, by inactivating members of the forkhead family of transcription factors, and by promoting NF-κB-dependent cytoprotection via transcriptional activation of a plethora of downstream target genes (13.Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar). extracellular matrix inhibitor of apoptosis extracellular signal-regulated kinase monoclonal antibody tumor necrosis factor-α poly(ADP ribose) polymerase cycloheximide actinomycin D hemagglutinin phosphatidylinositol 3,4-bisphosphate phosphatidylinositol 3,4,5-trisphosphate benzyloxycarbonyl fluoromethyl ketone bovine serum albumin nucleotide epidermal growth factor fluorescence-activated cell sorter green fluorescent protein dominant negative. The effector molecules involved in integrin-dependent cell survival have not been completely elucidated. Engagement of fibronectin through α5β1 and αvβ1 and of vitronectin through αvβ3 integrins has been associated with up-regulation of anti-apoptotic Bcl-2 and with Bcl-2-mediated protection from apoptosis induced by serum deprivation (2.Zhang Z. Vuori K. Reed J.C. Ruoslahti E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6161-6165Crossref PubMed Scopus (575) Google Scholar, 14.Matter M.L. Ruoslahti E. J. Biol. Chem. 2001; 276: 27757-27763Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Whether other cytoprotective mechanisms for integrin-dependent cell survival exist in normal or tumor cells has not been determined. In addition to the Bcl-2 family of cell death regulators, a second group of inhibitor of apoptosis (IAP) proteins has been recently identified (15.Deveraux Q.L. Reed J.C. Genes Dev. 1999; 13: 239-252Crossref PubMed Scopus (2285) Google Scholar). Survivin, a member of the IAP family of proteins, is expressed during embryonic and fetal development but undetectable in most normal adult tissues (16.Ambrosini G. Adida C. Altieri D.C. Nat. Med. 1997; 3: 917-921Crossref PubMed Scopus (3025) Google Scholar). However, survivin becomes the fourth most expressed transcript in human cancer (17.Velculescu V.E. Madden S.L. Zhang L. Lash A.E. Yu J. Rago C. Lal A. Wang C.J. Beaudry G.A. Ciriello K.M. Cook B.P. Dufault M.R. Ferguson A.T. Gao Y. He T.C. Hermeking H. Hiraldo S.K. Hwang P.M. Lopez M.A. Luderer H.F. Mathews B. Petroziello J.M. Polyak K. Zawel L. Kinzler K.W. et al.Nat. Genet. 1999; 23: 387-388Crossref PubMed Scopus (619) Google Scholar) where it correlates with a more aggressive and disseminated disease and reduced overall survival (18.Altieri D.C. Trends Mol. Med. 2001; 7: 542-547Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Several studies have shown that survivin expression inhibits cell death induced by various apoptotic stimuli in vitro (19.Tamm I. Wang Y. Sausville E. Scudiero D.A. Vigna N. Oltersdorf T. Reed J.C. Cancer Res. 1998; 58: 5315-5320PubMed Google Scholar) as well as in vivo (18.Altieri D.C. Trends Mol. Med. 2001; 7: 542-547Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 20.Blanc-Brude O.P. Yu j. Simosa H. Conte M.S. Sessa W.C. Altieri D.C. Nat. Med. 2002; 8: 987-994Crossref PubMed Scopus (128) Google Scholar). Adenocarcinoma of the prostate is the most common malignancy in men and the second leading cause of cancer-related deaths in the United States (21.Greenlee R.T. Hill-Harmon M.B. Murray T. Thun M. CA Cancer J. Clin. 2001; 51: 15-36Crossref PubMed Scopus (3332) Google Scholar). Evolution from a localized to an invasive fatal disease is accompanied by progression from an androgen-dependent to an androgen-independent state (22.Sadar M.D. Hussain M. Bruchovsky N. Endocrine-Related Cancer. 1999; 6: 487-502Crossref PubMed Scopus (140) Google Scholar) and by the emergence of apoptosis-resistant cells within primary and/or metastatic tumors. Although several anti-cancer strategies have been shown to exert their cytotoxic effect via activation of an apoptotic program, the limited efficacy of anti-cancer drugs against epithelial neoplasms implies the ability of tumor cells to raise their anti-apoptotic threshold, thus evading cell death. Despite growing evidence regarding the important role of integrins in tumorigenesis and in resistance to chemotherapeutic drugs, the molecular mechanisms underlying integrin-mediated survival signaling in cancer cells are not completely understood. In this study, we demonstrate that cell adhesion to fibronectin via β1 integrins regulates survivin protein levels and provides protection against death-induced stimuli via AKT activation. Conversely, inhibition of survivin function reverses the cytoprotective effect of ECM proteins and results in tumor cell apoptosis. We also show that the β1A integrin cytodomain is essential for cytoprotection against death-inducing stimuli in a survivin-dependent manner. We conclude that the activation of survival pathways mediated by integrins and survivin may contribute to drug resistance in cancer cells. Antibodies and Reagents—The following antibodies were used: a rabbit antibody to survivin (Novus Biologicals, Littleton, CO) was characterized in previous studies (23.Fortugno P. Wall N.R. Giodini A. O'Connor D.S. Plescia J. Padgett K.M. Tognin S. Marchisio P.C. Altieri D.C. J. Cell Sci. 2002; 115: 575-585PubMed Google Scholar); rabbit affinity-purified IgG to ERK1 and 2 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit affinity-purified IgG to XIAP (Santa Cruz Biotechnology); rabbit affinity-purified IgG to caspase-3 (BD Transduction Laboratories, San Diego, CA); rabbit affinity-purified IgG to phospho-(Ser/Thr) AKT substrates (Cell Signaling, Beverly, MA); monoclonal antibody (mAb) to survivin (23.Fortugno P. Wall N.R. Giodini A. O'Connor D.S. Plescia J. Padgett K.M. Tognin S. Marchisio P.C. Altieri D.C. J. Cell Sci. 2002; 115: 575-585PubMed Google Scholar) (60.1, Novus Biologicals); mAb to Bcl-2 (BD Transduction Laboratories); mAb to Poly(ADP-ribose) polymerase (PARP, BD Biosciences); mAb to β-actin (Sigma); 1C10, anti-human endothelial cell 140-kDa protein (Invitrogen); W1B10 mAb against the extracellular domain of chicken β1 integrin (Sigma); and 12CA5 mAb to hemagglutinin (HA, Roche Applied Science). Non-immune rabbit IgG and mouse IgG were purchased from Sigma and used as controls. Fibronectin and vitronectin were purified from human plasma and serum, respectively, as described previously (24.Engvall E. Ruoslahti E. Int. J. Cancer. 1977; 20: 1-5Crossref PubMed Scopus (1429) Google Scholar, 25.Yatohgo T. Izumi M. Kashiwagi H. Hayashi M. Cell Struct. Funct. 1988; 13: 281-292Crossref PubMed Scopus (439) Google Scholar). Reagents used in this study were: Z-VAD-fmk (Enzyme Systems Products, Livermore, CA); cycloheximide (CHX, Sigma); tumor necrosis factor-α (TNF-α) (R&D, Minneapolis, MN); actinomycin D (Act D, Sigma); etoposide (Sigma); propidium iodide (Sigma); RNase A (Roche Applied Science); and bovine serum albumin (BSA, Sigma). Cells—RWPE-1, LNCaP, and PC3 cells were obtained from ATCC. LNCaP-LN3 cells (26.Pettaway C.A. Pathak S. Greene G. Ramirez E. Wilson M.R. Killion J.J. Fidler I.J. Clin. Cancer Res. 1996; 2: 1627-1636PubMed Google Scholar) were a kind gift of Dr. C. A. Pettaway (The University of Texas, M. D. Anderson Cancer Center, Houston, TX). LNCaP, LNCaP-LN3, and PC3 cells were cultured as described previously (27.Zheng D.Q. Woodard A.S. Fornaro M. Tallini G. Languino L.R. Cancer Res. 1999; 59: 1655-1664PubMed Google Scholar). RWPE-1 cells were grown in keratinocyte serum-free medium (Invitrogen) supplemented with 5 ng/ml epidermal growth factor (EGF, Invitrogen) and 0.05 mg/ml bovine pituitary extract (Invitrogen). To obtain PC3 stable cell lines expressing either β1C or β1A integrins under the control of a tetracycline-regulated promoter, chimeric cDNAs consisting of the chicken β1 extracellular and transmembrane domain and either human β1C or human β1A cytodomain sequences were generated by PCR-driven splice overlap extension (28.Horton R.M. Cai Z. Ho S.N. Pease L.R. BioTechniques. 1990; 8: 528-535PubMed Google Scholar). These chimeric constructs allow one to distinguish between endogenous β1 integrin and exogenous β1C and β1A integrin variants in human cells. PCR reactions were performed using an automatic thermal cycler (PerkinElmer Life Sciences). The conditions used for the PCR reactions were as follows: denaturation at 95 °C for 1 min; annealing at 45 °C for 1 min; and extension at 72 °C for 1 min. 30 cycles of amplification were used. The amplification was performed in 1× Vent polymerase buffer (New England Biolabs, Beverly, MA), 200 μm each dNTP (PerkinElmer Life Science), 0.15 μm of each primer, 2 mm MgSO4 (PerkinElmer Life Science), and 0.5 units of Vent polymerase (New England Biolabs). The following synthetic nucleotides were used as primers to amplify chicken β1 subunit from nucleotides (nt) 1787 to 2365 using chicken β1 cDNA template (29.Tamkun J.W. DeSimone D.W. Fonda D. Patell R.S. Buck C. Horwitz A.F. Hynes R.O. Cell. 1986; 46: 271-282Abstract Full Text PDF PubMed Scopus (514) Google Scholar): C1 (nt 1787–1807 of chicken β1) 5′-AACTGCGATCGATCAAACGGT-3′ and CH1 (nt 2357–2371 of human β1 and nt 2351–2365 of chicken β1) 5′-TATCATTAAAAGCTTCCAAATCAATAACAA-3′. Additional primers were designed to amplify either human β1A cytodomain sequences from nucleotides 2357 to 2497 (30.Argraves W.S. Suzuki S. Arai H. Thompson K. Pierschbacher M.D. Ruoslahti E. J. Cell Biol. 1987; 105: 1183-1190Crossref PubMed Scopus (386) Google Scholar) or human β1C cytodomain sequences from nucleotides 2357 to 2613 (31.Languino L.R. Ruoslahti E. J. Biol. Chem. 1992; 267: 7116-7120Abstract Full Text PDF PubMed Google Scholar) using human β1A or human β1C cDNA template, respectively. These primers were: CH2 (nt 2351–2365 of chicken β1 and nt 2357–2371 of human β1) 5′-TTGTTATTGATTTGGAAGCTTTTAATGATA-3′ and H1 (nt 2483–2505 of human β1) 5′-GGACTAGTTTTTCCCTCATACTT-3′. The chimeric primers CH1 and CH2 corresponded to a region that was identical between the species and were complementary to each other. The amplified chicken and human cDNA fragments were mixed and subjected to an additional amplification using C1 as forward primer and H1 as reverse primer. The resulting chimeric cDNA fragments (718-bp chicken/human β1A and the 834-bp chicken/human β1C) were subcloned into pCR 2.1 vector (Invitrogen) following manufacturer's instructions. The 718-bp chicken/human β1A and the 834-bp chicken/human β1C chimeras were excised from pCR2.1 using ClaI and SpeI and subcloned into pTet-Splice expression vector (32.Shockett P. Difilippantonio M. Hellman N. Schatz D.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6522-6526Crossref PubMed Scopus (344) Google Scholar). The resulting constructs were digested with SalI and ClaI to allow ligation of a DNA fragment corresponding to nucleotides 1–1787 of the chicken β1 extracellular domain, which was isolated from pECE-β1 (kindly provided by Dr. Hynes, Massachusetts Institutes of Technology, Boston, MA) using SalI and ClaI restriction enzymes. The chimeric constructs (pTet-chicken/human β1C and pTet-chicken/human β1A) were sequenced by the dideoxynucleotide method to confirm the nature of the chimeric inserts. PC3 cells were electroporated as described previously (33.Fornaro M. Manzotti M. Tallini G. Slear A.E. Bosari S. Ruoslahti E. Languino L.R. Am. J. Pathol. 1998; 153: 1079-1087Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) using 100 μg of pTet-chicken/human β1C, pTet-chicken/human β1A, or pTet-Splice along with 10 μg of pTet-tTA. Neomycin-resistant cells were selected using medium containing 0.2 mg/ml G418 (Invitrogen). G418-resistant clones were isolated and screened for cell surface expression of either chicken/human β1C or chicken/human β1A integrin by FACS using W1B10, a monoclonal antibody against the extracellular domain of chicken β1 integrin, or 12CA5 as a negative control as described previously (34.Fornaro M. Zheng D.Q. Languino L.R. J. Biol. Chem. 1995; 270: 24666-24669Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Analysis of Survivin and Bcl-2 Expression by Immunoblotting—Subconfluent RWPE-1, LNCaP, LNCaP-LN3, and PC3 cells were lysed as described previously (35.Fornaro M. Steger C.A. Bennett A.M. Wu J.J. Languino L.R. Mol. Biol. Cell. 2000; 11: 2235-2249Crossref PubMed Scopus (40) Google Scholar). In some experiments, subconfluent PC3 and LNCaP cells were serum-starved for 24 h, detached using 0.05% trypsin, 0.53 mm EDTA (Invitrogen), and washed three times with serum-free medium. PC3 (4 × 105) and LNCaP (1 × 106) cells were then plated onto 60-mm Petri dishes coated with 10 mg/ml BSA, 5 μg/ml fibronectin, or with 10 μg/ml vitronectin for 8 and 24 h in serum-free medium in the absence or in the presence of either 20 μg/ml Act D or of 10 μg/ml CHX. Cells were then lysed as described above. Protein content in each lysate was quantitated using the BCA protein assay reagent (Pierce). Samples were heated at 100 °C for 5 min and separated by electrophoresis on a 12–15% SDS-polyacrylamide gel and transferred onto Immobilon-P membrane (Millipore, Bedford, MA). Expression of survivin, and Bcl-2 was analyzed by immunoblotting using 1 μg/ml of the polyclonal antibody to survivin and 1 μg/ml mAb to Bcl-2, respectively, as described previously (35.Fornaro M. Steger C.A. Bennett A.M. Wu J.J. Languino L.R. Mol. Biol. Cell. 2000; 11: 2235-2249Crossref PubMed Scopus (40) Google Scholar). Rabbit affinity-purified antibody to ERK1/2 was used to control for protein loading as described previously (35.Fornaro M. Steger C.A. Bennett A.M. Wu J.J. Languino L.R. Mol. Biol. Cell. 2000; 11: 2235-2249Crossref PubMed Scopus (40) Google Scholar). Tissue Specimen Procurement and Immunohistochemistry—Specimens from 53 human radical prostatectomies performed for prostatic adenocarcinoma at the Yale-New Haven Hospital (New Haven, CT) were processed according to Review Board-approved protocols. Hematoxylin and eosin sections from all of the specimens were evaluated microscopically to assess tissue integrity and preservation. Sections from formalin-fixed, paraffin-embedded specimens were immunostained as described previously (33.Fornaro M. Manzotti M. Tallini G. Slear A.E. Bosari S. Ruoslahti E. Languino L.R. Am. J. Pathol. 1998; 153: 1079-1087Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) using either 60.1 mAb to survivin (1:1000 dilution) or mAb 1C10 as control antibody (1:1000 dilution). Plasmids, Transient Transfections, and Determination of Apoptosis—The survivin (C84A) mutant or survivin antisense cDNA was subcloned into pEGFPc1 (36.Li F. Ackermann E.J. Bennett F. Rothermel A.L. Plescia J. Tognin S. Villa A. Marchisio P.C. Altieri D.C. Nat. Cell Biol. 1999; 1: 461-466Crossref PubMed Scopus (554) Google Scholar, 37.Mesri M. Wall N.R. Li J. Kim R.W. Altieri D.C. J. Clin. Invest. 2001; 108: 981-990Crossref PubMed Scopus (360) Google Scholar, 39.Mesri M. Morales-Ruiz M. Ackermann E.J. Bennett C.F. Pober J.S. Sessa W.C. Altieri D.C. Am. J. Pathol. 2001; 158: 1757-1765Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Replication-deficient adenoviral constructs encoding wild-type survivin (pAd-survivin), the survivin T34A mutant (pAd-T34A), or control GFP (pAd-GFP) were generated using the pAd-Easy system as described previously (37.Mesri M. Wall N.R. Li J. Kim R.W. Altieri D.C. J. Clin. Invest. 2001; 108: 981-990Crossref PubMed Scopus (360) Google Scholar). The pCMV6 plasmids containing HA-tagged AKT K179M mutant (dn AKT) or wild type AKT were a kind gift of Dr. Franke (Columbia University, New York, NY) (38.Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-738Abstract Full Text PDF PubMed Scopus (1829) Google Scholar). PC3 cells were plated onto 12-well plates at a density of 2 × 105 cells/well. Cells were transfected with the various GFP constructs using a mixture of 2 μg of plasmid DNA and 6 μl of LipofectAMINE 2000 (Invitrogen) in 1 ml of Opti-MEM I (Invitrogen). After a 7-h culture at 37 °C, the transfection mixture was aspirated and substituted with complete growth medium. Cells (floating plus attached cells) were harvested after 1–4 days from transfection, fixed in 70% ethanol for 1 h on ice, and spun for 5 min at 1,300 rpm. Cells were incubated in phosphate-buffered saline containing 1 mg/ml RNase for 30 min at 37 °C, propidium iodide was added at a final concentration of 8 μg/ml, and samples were analyzed for DNA content by flow cytometry. Alternatively, cells were harvested 1–4 days after transfection, washed in phosphate-buffered saline, pH 7.4, and fixed in 4% paraformaldehyde containing 0.25% Triton X-100 for 10 min at 22 °C. Cell nuclei were stained with 6.5 μg/ml 4,6-diamidino-2-phenylindole (Sigma), 16% polyvinyl alcohol (Air Products and Chemicals, Allentown, PA), and 40% glycerol. GFP-expressing cells were independently scored for morphologic signs of apoptosis (chromatin condensation, DNA fragmentation) using a Zeiss Axiophot microscope. In another set of experiments, PC3 cells (1 × 106) in 100-mm plates were transfected with 15 μg of dn AKT along with 3 μg of pEGFPc1 plasmid (Invitrogen) using LipofectAMINE. Cells were then detached using 0.05% trypsin, 0.53 mm EDTA, washed three times with serum-free medium, and plated (4 × 105) onto 60-mm Petri dishes coated with either 10 mg/ml BSA or with 5 μg/ml fibronectin for 8 h in serum-free medium at 37 °C. Cells were then treated with 150 ng/ml TNF-α plus 3 μg/ml CHX for an additional 42 h at 37 °C. Survivin expression was analyzed by immunoblotting as described above. Caspase Activation—PC3 cells transfected with the various GFP constructs in the presence or in the absence of Z-VAD-fmk (20 μm) were harvested after a 2-day culture at 37 °C and assayed for caspase-3-dependent hydrolysis of the fluorogenic substrate Ac-DEVD-AMC (N-acetyl-Asp-Glu-Val-Asp-aldehyde, BD Biosciences) as described previously (39.Mesri M. Morales-Ruiz M. Ackermann E.J. Bennett C.F. Pober J.S. Sessa W.C. Altieri D.C. Am. J. Pathol. 2001; 158: 1757-1765Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). For analysis of caspase-3-proteolytic cleavage of PARP, PC3 cells (1 × 107/ml), transfected with the various GFP constructs in the presence or in the absence of Z-VAD-fmk (20 μm), were lysed in 40 μl of lysis buffer containing 2% SDS, 50 mm Tris-HCl, and 10% glycerol. Samples were heated at 100 °C for 3 min and separated by electrophoresis on a 12% SDS-polyacrylamide gel and transferred onto Immobilon-P membrane for 60 min at 55 V. The transfer membrane was blocked in phosphate-buffered saline, pH 7.4, containing 5% nonfat dry milk at 4 °C overnight, washed in phosphate-buffered saline, pH 7.4, and separately processed for immunoblotting with a mAb to PARP as described previously (39.Mesri M. Morales-Ruiz M. Ackermann E.J. Bennett C.F. Pober J.S. Sessa W.C. Altieri D.C. Am. J. Pathol. 2001; 158: 1757-1765Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). DNA Fragmentation Assay—PC3 cells (4 × 105) were plated onto 60-mm Petri dishes coated with either 10 mg/ml BSA or with 5 μg/ml fibronectin for 4 h in serum-free medium. Cells were treated with 150 ng/ml TNF-α plus 3 μg/ml CHX or with 500 μm etoposide in the presence or in the absence of Z-VAD fmk (40 μm) for an additional 42 h at 37 °C. DNA fragmentation enzyme-linked immunosorbent assay on floating plus attached cells was performed using the Cell Death Detection enzyme-linked immunosorbent assay kit (Roche Applied Science) according to the manufacturer's instructions. Adenoviral Targeting of Survivin in PC3 Cells—The experimental procedures for the generation and expansion in human embryonic kidney 293 cells of replication-defective adenovirus constructs encoding survivin (pAd-survivin), the phosphorylation-deficient survivin Thr-34→ Ala dn mutant (pAd-T34A), or control pAd-GFP have been described previously (37.Mesri M. Wall N.R. Li J. Kim R.W. Altieri D.C. J. Clin. Invest. 2001; 108: 981-990Crossref PubMed Scopus (360) Google Scholar). With this protocol, there was no generation of replication competent adenoviral particles. PC3 cells (1.5 × 106) in 100-mm plates were infected with pAd-GFP, pAd-survivin, or pAd-T34A at m.o.i of 50–100 for 24 h at 37 °C. Cells were then detached using 0.05% trypsin, 0.53 mm EDTA, washed three times with serum-free medium, and plated (4 × 105) onto 60-mm Petri dishes coated with either 10 mg/ml BSA or with 5 μg/ml fibronectin for 8 h in serum-free medium. Cells were then treated with 150 ng/ml TNF-α plus 3 μg/ml CHX for an additional 42 h at 37 °C. Cells (floating plus attached cells) were fixed in 70% ethanol, and GFP-expressing cells were analyzed for DNA content as described above. PC3-β1C and PC3-β1A stable cell lines were cultured for 72 h in growth medium either in the absence or in the presence of 1 μg/ml tetracycline. Cells were infected with pAd-GFP or pAd-survivin at m.o.i. of 50–100 for 24 h at 37 °C during the last 24 h of the 72-h culture. Cells were harvested using 0.05% trypsin, 0.53 mm EDTA and then plated (4 × 105) onto 60-mm Petri dishes coated with 10 mg/ml BSA for 8 h in serum-free medium. Cells were then treated with 150 ng/ml TNFα plus 3 μg/ml CHX for an additional 20–24 h at 37 °C and then analyzed for DNA content as described above. Statistical Analysis—Group differences were compared using Student's t test. Survivin Targeting Causes Spontaneous Apoptosis in PC3 Cells—The relative levels of survivin expression among different prostate cell lines were compared by immunoblotting. Consistent with previous observations (19.Tamm I. Wang Y. Sausville E. Scudiero D.A. Vigna N. Oltersdorf T. Reed J.C. Cancer Res. 1998; 58: 5315-5320PubMed Google Scholar), survivin expression was detected in all of the prostate cancer cell lines analyzed. The highest levels of survivin were present in highly aggressive prostate cancer cells such as PC3 and LNCaP-LN3 (Fig. 1A). At variance with the results obtained in malignant epithelial cells, survivin expression was very low in non-tumorigenic and noninvasive LNCaP prostate cancer

Abstract The group of leukocyte integrins CD11a-c/CD18 coordinate disparate adhesion reactions in the immune system through a regulated process of ligand recognition. The participation of the receptor divalent ion binding site(s) in this mechanism of ligand binding has been investigated. As compared with other divalent cations, Mn2+ ions have the unique property to dramatically stimulate the adhesive functions of the leukocyte integrin CD11b/CD18 (Mac-1), expressed on myelo-monocytic cells. This is reflected in a three- to fivefold increased early monocyte adhesion (less than 20 min) to resting, unperturbed endothelial cells, and increased association of CD11b/CD18 with its soluble ligands fibrinogen and factor X. CD11b/CD18 ligand recognition in the presence of Mn2+ ions is specific, time and concentration dependent, and inhibited by anti-CD11b mAb. At variance with Ca(2+)-containing reactions where CD11b/CD18 functions as an inducible receptor activated by adenine nucleotides or chemoattractants, Mn2+ ions induce per se a constitutive maximal ligand binding capacity of CD11b/CD18, that is not further modulated by cell stimulation. Rather than quantitative changes in surface density, Mn2+ ions increase the affinity of CD11b/CD18 for its complementary ligands up to 10-fold, as judged by Scatchard plot analysis of receptor:ligand interaction under these conditions. Furthermore, monocyte exposure to Mn2+ ions induces the expression of activation-dependent neo-antigenic epitopes on CD11b/CD18, selectively recognized by mAb 7E3. These data suggest that in addition to cell-activating stimuli, favorable engagement of divalent ion binding site(s) can provide an alternative pathway to rapidly regulate the receptor affinity of leukocyte integrins.

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, which has been implicated in both preservation of cell viability and regulation of mitosis in cancer cells. Here, we show that HeLa cells microinjected with a polyclonal antibody to survivin exhibited delayed progression in prometaphase (31.5 +/- 6.9 min) and metaphase (126.8 +/- 73.8 min), as compared with control injected cells (prometaphase, 21.5 +/- 3.3 min; metaphase, 18.9 +/- 4.5 min; P < 0.01). Cells injected with the antibody to survivin displayed short mitotic spindles severely depleted of microtubules and occasionally underwent apoptosis without exiting the mitotic block or thereafter. Forced expression of survivin in HeLa cells profoundly influenced microtubule dynamics with reduction of pole-to-pole distance at metaphase (8.57 +/- 0.21 microm versus 10.58 +/- 0.19 microm; P < 0.0001) and stabilization of microtubules against nocodazole-induced depolymerization in vivo. These data demonstrate that survivin functions at cell division to control microtubule stability and assembly of a normal mitotic spindle. This pathway may facilitate checkpoint evasion and promote resistance to chemotherapy in cancer.

Evasion of apoptosis is a hallmark of cancer, but the molecular circuitries of this process are not understood. Here we show that survivin, a member of the inhibitor of apoptosis gene family that is overexpressed in cancer, exists in a novel mitochondrial pool in tumor cells. In response to cell death stimulation, mitochondrial survivin is rapidly discharged in the cytosol, where it prevents caspase activation and inhibits apoptosis. Selective targeting of survivin to mitochondria enhances colony formation in soft agar, accelerates tumor growth in immunocompromised animals, and abolishes tumor cell apoptosis in vivo. Therefore, mitochondrial survivin orchestrates a novel pathway of apoptosis inhibition, which contributes to tumor progression.

Survivin is a member of the inhibitor of apoptosis (TAP) gene family that exhibits differential expression in nearly all human cancers but not in most normal tissues. Recent progress identified a multifunctional survivin pathway positioned at the interface between mitotic progression and apoptosis inhibition, and required to preserve viability of dividing tumor cells (Altieri, 2001; Andersen and Thor, 2002; Jaattela, 1999). The unique properties of survivin have recently found concrete applications for cancer detection, diagnosis, and outcome prediction. In addition, targeting the survivin pathway may offer new therapeutic prospects to lower a general survival threshold in cancer cells. This chapter will focus on the current developments in the field of survivin and its role in apoptosis regulation and mitotic progression. Current perspectives on exploiting the survivin pathway for cancer diagnosis and treatment will be highlighted.

Survivin is a member of the Inhibitor of Apoptosis gene family that has been implicated in cell division and suppression of apoptosis. Here, we show that preferential ablation of the nuclear pool of survivin by RNA interference produces a mitotic arrest followed by re-entry into the cell cycle and polyploidy. Survivin ablation causes multiple centrosomal defects, aberrant multipolar spindle formation, and chromatin missegregation, and these phenotypes are exacerbated by loss of the cell cycle regulator, p21^Waf1/Cip1 in p21^-/- cells. The mitotic checkpoint activated by loss of survivin is mediated by induction of p53 and associated with increased expression of its downstream target, p21^Waf1/Cip1. Accordingly, p53^-/- cells exhibit reduced mitotic arrest and enhanced polyploidy upon survivin ablation as compared with their p53^+/+ counterparts. Partial reduction of the cytosolic pool of survivin by RNA interference sensitizes cells to ultraviolet B-mediated apoptosis and results in enhanced caspase-9 proteolytic cleavage, whereas complete ablation of cytosolic survivin causes loss of mitochondrial membrane potential and spontaneous apoptosis. These data demonstrate that survivin has separable checkpoint functions at multiple phases of mitosis and in the control of mitochondrial-dependent apoptosis.

Survivin is a member of the chromosomal passenger complex implicated in kinetochore attachment, bipolar spindle formation, and cytokinesis. However, the mechanism by which survivin modulates these processes is unknown. Here, we show by time-lapse imaging of cells expressing either green fluorescent protein (GFP)-α-tubulin or the microtubule plus-end binding protein GFP-EB1 that depletion of survivin by small interfering RNAs (siRNAs) increased both the number of microtubules nucleated by centrosomes and the incidence of microtubule catastrophe, the transition from microtubule growth to shrinking. In contrast, survivin overexpression reduced centrosomal microtubule nucleation and suppressed both microtubule dynamics in mitotic spindles and bidirectional growth of microtubules in midbodies during cytokinesis. siRNA depletion or pharmacologic inhibition of another chromosomal passenger protein Aurora B, had no effect on microtubule dynamics or nucleation in interphase or mitotic cells even though mitosis was impaired. We propose a model in which survivin modulates several mitotic events, including spindle and interphase microtubule organization, the spindle assembly checkpoint and cytokinesis through its ability to modulate microtubule nucleation and dynamics. This pathway may affect the microtubule-dependent generation of aneuploidy and defects in cell polarity in cancer cells, where survivin is commonly up-regulated.

The (Y subunits of leukocyte CDll/CDlS integrins contain an -200-amino acid "inserted" domain (I-domain) that may be important for multivalent adhesive recognitions.A recombinant form of the I-domain of CDllbl CDl8 was generated and analyzed directly for interaction with complementary integrin ligands.CDllb I-domain bound the activation-dependent monoclonal antibody 7E3, and the functionally blocking anti-CDllb monoclonal antibodies 0-9, 60.1, and LM2/1, but not 0-1 or M1/70.Fibrinogen or soluble intercellular adhesion molecule-1 associated with CDllb I-domain in a concentration-dependent manner.Binding of laSI-fibrinogen to recombinant CDllb I-domain was saturable, governed by a Kd of "0.22 2 0.06 p ~, and fully inhibited by molar excess of unlabeled fibrinogen, or by the P1 peptide (KY)GWTVFQKRLDGSV (IC, -2 .5 6 pd, duplicating the fibrinogen y chain sequence Gly'Bo-Va1202.In contrast, 'zsI-factor X binding to CDllb I-domain was only partially inhibited ( 5 0 4 0 % ) by a molar excess of unlabeled factor X, and entirely unaffected by functionally blocking anti-CDllb monoclonal antibodies or by factor X-derived synthetic peptidyl antagonists.W e conclude that the I-domain of CDllb participates in qualitative mechanisms of receptor activation and contains the binding site(s) for the CDllb/CDlS ligands fibrinogen and intercellular adhesion molecule-1, while it is only minimally implicated in the recognition of factor X. Leukocyte pz integrins comprise three heterodimeric transmembrane receptors CDlldCD18 (LFA-l), CDllbKD18 (Macl ) , a n d C D l l d C D 1 8 ( ~1 5 0 , 951, generated by the assembly of the common p subunit CD18 with the three respective a chains C D l l a (a,), C D l l b (aM), and C D l l c (a,) (1-3).Broadly distributed on all leukocytes of lymphoid and myelomonocytic lineage,pz integrins mediate crucial adhesive recognitions through their ability to bind multiple and unrelated ligands (3).This is typically exemplified by CDllbKD18, whose ligand recognition repertoire includes low affinity opsonins, such as C3bi (31, high affinity adhesion-promoting molecules, such as fibrinogen (4-7) and factor X (81, and cell-associated counter-receptor(s), such as ICA"1' (9).In an effort to better characterize the structurefunction requirements of multiple ligand recognition, considerable interest has been recently focused on an -200-amino acid HL-43773.

A series of 110 cases of oral squamous cell carcinoma (SCC) together with six lymph node and one distant metastatic lesions was analysed for expression of survivin, a recent apoptosis inhibitor, by immunohistochemistry and Western blotting. In total, 91 cases (82.7%) of carcinoma and all metastasis (seven cases, 100%) were positive for survivin expression, with weighted survivin scores ranging from 1 to 4. In contrast, normal oral epithelium did not express survivin. There was no significant correlation between survivin expression and age, sex, tumour size, the presence of lymph node and distant metastases. Survivin expression was increased in poorly differentiated tumours, even if differences were not statistically significant. In contrast, when analysed for prognostic significance, patients with low survivin expression had statistically significant better survival rates than the group with high survivin expression (P<0.05). These data suggest that survivin expression may identify cases of oral SCC with more aggressive and invasive phenotype.

Coagulation proteases were tested in a rat model of acute inflammation. Subplantar injection of Factor Xa (10-30 microg) produced a time- and dose-dependent edema in the rat paw, and potentiated carrageenin-induced edema. In contrast, the homologous protease Factor IXa was ineffective. This inflammatory response was recapitulated by the Factor Xa sequence L83FTRKL88(G), which mediates ligand binding to effector cell protease receptor-1 (EPR-1), while a control scrambled peptide did not induce edema in vivo. Conversely, injection of the EPR-1-derived peptide S123PGKPGNQNSKNEPP137 (corresponding to the receptor binding site for Factor Xa) inhibited carrageenin-induced rat paw edema, while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was without effect. EPR-1-Factor Xa-induced inflammation was characterized by fast onset and prominent perivascular accumulation of activated and degranulated mast cells, was inhibited by the histamine/serotonin antagonists cyproheptadine and methysergide, but was unaffected by the thrombin-specific inhibitor, Hirulog. These findings suggest that through its interaction with EPR-1, Factor Xa may function as a mediator of acute inflammation in vivo. This pathway may amplify both coagulation and inflammatory cascades, thus contributing to the pathogenesis of tissue injury in vivo.

Survivin is an inhibitor of apoptosis (programmed cell death) overexpressed in various human cancers, but undetectable in normal differentiated tissues. A potential distribution and prognostic significance of survivin in patients with de novo acute myeloid leukaemia (AML) was investigated. By immunofluoresence of bone‐marrow specimens and peripheral blood mononuclear cells, survivin was detected in 75 out of 125 interpretable AML cases (60%), with reactivity in 50–90% of AML cells. Survivin expression correlated with a lower white blood cell count (WBC) ( P = 0·008 by the Mann–Whitney test) and was associated, in the 55 cases of FAB M0/M1/M2, with leukaemic granulocytic maturation (one out of five M/L0, 11 out of 22 M/L1 and 23 out of 28M/L2; P = 0·007 by the Fisher test). In 69 patients treated with the Acute Leukaemia French Association (ALFA) 9000 protocol, survivin expression was significantly associated with a lower WBC ( P = 0·03 by the Mann–Whitney test) and favourable/intermediate cytogenetics ( P = 0·03 by the Fisher test). There was no significant difference in complete remission rate or overall survival between survivin‐positive and survivin‐negative AML patients ( P = 0·15 by the log‐rank test). However, survivin expression became an independent negative prognostic factor for survival when adjusted with the Cox model for established prognostic factors in AML (cytogenetics, age and WBC) or for the ALFA 9000 treatment arm (RR = 2·8 and P = 0·026, by the likelihood‐ratio test). These data suggest that survivin expression may be considered as a new unfavourable prognostic factor of de novo AML and suggest a role for apoptosis inhibition in influencing disease outcome.

Binding of fibrinogen to intercellular adhesion molecule 1 (ICAM-1) enhances leukocyte adhesion to endothelium by acting as a bridging molecule between the two cell types. Here, a panel of four monoclonal antibodies (mAbs) to ICAM-1 was used to dissect the structure-function requirements of this recognition. All four mAbs bound to ICAM-1 transfectants and immunoprecipitated and immunoblotted ICAM-1 from detergent-solubilized JY lymphocyte extracts. Functionally, mAbs 1G12 and 2D5 inhibited binding of 125I-fibrinogen to ICAM-1-transfectants and abrogated the enhancing effect of fibrinogen on mononuclear cell adhesion to endothelium and transendothelial migration. In contrast, mAbs 3D6 and 6E6 did not affect ICAM-1 recognition of fibrinogen. With respect to other ligands, mAbs 1G12 and 2D5 completely inhibited attachment of Plasmodium falciparum-infected erythrocytes to immobilized recombinant ICAM-1-Fc, whereas they had no effect on LFA-1-dependent T cell binding to ICAM-1-Fc. Conversely, mAbs 3D6 and 6E6 completely abolished LFA-1 binding to ICAM-1-Fc. Epitope assignment using ICAM-1 chimeras and receptor mutants revealed that the fibrinogen-blocking mAbs 1G12 and 2D5 reacted with domain 1 of ICAM-1, and their binding was disrupted by 97 and 70% by mutations of D26 and P70, respectively, whereas mAbs 3D6 and 6E6 bound to domain 2 of ICAM-1. By recognizing a site distinct from that of β2 integrins Mac-1 or LFA-1, fibrinogen binding to ICAM-1 may provide an alternative pathway of intercellular adhesion and/or modulate integrin-dependent adherence during inflammation and vascular injury. Binding of fibrinogen to intercellular adhesion molecule 1 (ICAM-1) enhances leukocyte adhesion to endothelium by acting as a bridging molecule between the two cell types. Here, a panel of four monoclonal antibodies (mAbs) to ICAM-1 was used to dissect the structure-function requirements of this recognition. All four mAbs bound to ICAM-1 transfectants and immunoprecipitated and immunoblotted ICAM-1 from detergent-solubilized JY lymphocyte extracts. Functionally, mAbs 1G12 and 2D5 inhibited binding of 125I-fibrinogen to ICAM-1-transfectants and abrogated the enhancing effect of fibrinogen on mononuclear cell adhesion to endothelium and transendothelial migration. In contrast, mAbs 3D6 and 6E6 did not affect ICAM-1 recognition of fibrinogen. With respect to other ligands, mAbs 1G12 and 2D5 completely inhibited attachment of Plasmodium falciparum-infected erythrocytes to immobilized recombinant ICAM-1-Fc, whereas they had no effect on LFA-1-dependent T cell binding to ICAM-1-Fc. Conversely, mAbs 3D6 and 6E6 completely abolished LFA-1 binding to ICAM-1-Fc. Epitope assignment using ICAM-1 chimeras and receptor mutants revealed that the fibrinogen-blocking mAbs 1G12 and 2D5 reacted with domain 1 of ICAM-1, and their binding was disrupted by 97 and 70% by mutations of D26 and P70, respectively, whereas mAbs 3D6 and 6E6 bound to domain 2 of ICAM-1. By recognizing a site distinct from that of β2 integrins Mac-1 or LFA-1, fibrinogen binding to ICAM-1 may provide an alternative pathway of intercellular adhesion and/or modulate integrin-dependent adherence during inflammation and vascular injury.

The interaction of fibrinogen with monocytes was studied. After stimulation with ADP (10 microM) or thrombin (1 U/ml), platelet-free suspensions of human monocytes bind 125I-fibrinogen with two different affinities in a specific and Ca2+-dependent reaction with saturation at 5.80-7.35 X 10(-7) M of added protein. The binding of fibrinogen to specific receptors on monocytes induces the procoagulant activity of these cells. Thrombasthenic cells or normal monocytes preincubated with a monoclonal antibody to the platelet glycoprotein IIb/IIIa complex (10E5) do not bind fibrinogen and have no procoagulant activity. Metabolic studies with [35S]methionine revealed that cultured monocytes actually synthesize a surface antigen precipitated by 10E5 antibody as a major band with 92,000 relative molecular weight. Our data indicate that monocytes express receptors for fibrinogen only in part related to the platelet glycoprotein IIb/IIIa complex. Furthermore, the binding of fibrinogen to monocytes enhances the cooperation of these cells in hemostasis.

Abstract The coupling of cell proliferation to cell death is thought to function as a pivotal crossroad, essential to preserve normal homeostasis and to eliminate dangerous cells before they divide. Survivin is a prototype molecule at this crossroad, intercalated in protection against mitochondrial cell death and orchestrating various aspects of cell division. Dramatically exploited in cancer and an unfavorable gene signature for disease outcome, the survivin pathway has now provided tangible opportunities for targeted, rational cancer therapy. © 2004 Wiley‐Liss, Inc.

Survivin is a member of the inhibitor of apoptosis gene family that is expressed in most human cancers and may facilitate evasion from apoptosis and aberrant mitotic progression. Here, exposure of breast carcinoma MCF-7 or cervical carcinoma HeLa cells to anticancer agents, including Adriamycin, Taxol, or UVB resulted in a 4-5-fold increased survivin expression. Changes in survivin levels after anticancer treatment did not involve modulation of survivin mRNA expression and were independent of de novo gene transcription. Conversely, inhibition of survivin phosphorylation on Thr(34) by the cyclin-dependent kinase inhibitor flavopiridol resulted in loss of survivin expression, and nonphosphorylatable survivin Thr(34)-->Ala exhibited accelerated clearance as compared with wild-type survivin. Sequential ablation of survivin phosphorylation on Thr(34) enhanced tumor cell apoptosis induced by anticancer agents independently of p53 and suppressed tumor growth without toxicity in a breast cancer xenograft model in vivo. These data suggest that Thr(34) phosphorylation critically regulates survivin levels in tumor cells and that sequential ablation of p34(cdc2) kinase activity may remove the survivin viability checkpoint and enhance apoptosis in tumor cells.

Basal-type, or triple-negative, breast cancer (lacking estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 expression) is a high-risk disease for which no molecular therapies are currently available. We studied genetic signatures of basal breast cancer potentially suitable for therapeutic intervention.We analyzed protein expression of the Notch-1 intracellular domain and survivin by immunohistochemistry in a series of basal breast cancer patients. A hierarchical clustering and overall survival analysis was carried out on a microarray mRNA database of 232 breast cancer patients. Fifteen published mRNA datasets containing estrogen receptor-negative or estrogen receptor-positive samples were subjected to meta-analysis for co-segregated gene expression. Experiments of plasmid transfection and gene silencing were carried out in estrogen receptor-negative MDA-MB-231 breast cancer cells.The developmental signaling regulator Notch-1 was highly expressed in breast cancer, compared with normal tissue, and was segregated with basal disease. Higher Notch-1 levels correlated with progressively abbreviated overall survival, and with increased expression of survivin, a tumor-associated cell death and mitotic regulator implicated in stem cell viability. Analysis of Pearson's correlation coefficient indicated that Notch-1 and survivin co-segregated in basal breast cancer. Notch-1 stimulation in MDA-MB-231 cells increased survivin expression, whereas silencing Notch reduced survivin levels.A Notch-1-survivin functional gene signature is a hallmark of basal breast cancer, and may contribute to disease pathogenesis. Antagonists of Notch and survivin currently in the clinic may be tested as novel molecular therapy for these recurrence-prone patients.

Despite progress in the management of breast cancer, the molecular underpinnings of clinically aggressive subtypes of the disease are not well-understood. Here, we show that activation of Notch developmental signaling in estrogen receptor (ER)-negative breast cancer cells results in direct transcriptional up-regulation of the apoptosis inhibitor and cell cycle regulator survivin. This response is associated with increased expression of survivin at mitosis, enhanced cell proliferation, and heightened viability at cell division. Conversely, targeting Notch signaling with a peptidyl gamma-secretase inhibitor suppressed survivin levels, induced apoptosis, abolished colony formation in soft agar, and inhibited localized and metastatic tumor growth in mice, without organ or systemic toxicity. In contrast, ER+ breast cancer cells, or various normal cell types, were insensitive to Notch stimulation. Therefore, ER- breast cancer cells become dependent on Notch-survivin signaling for their maintenance, in vivo. Therapeutic targeting of this pathway may be explored for individualized treatment of patients with clinically aggressive, ER- breast cancer.

Despite the constant exposure to genomic insults that may lead to malignancy, cancer is surprisingly a relatively rare occurrence, and this is largely credited to an elaborate network of endogenous tumor suppression. Many effectors of tumor suppression have been identified, and their functions when activated in damaged cells have in large part been elucidated. What is less clear is whether there are common target gene(s) of tumor suppression, whose expression must be ablated in order to block transformation and preserve cellular homeostasis. Fresh experimental evidence suggests that silencing of the mitotic regulator and cell death inhibitor, survivin, is a universal requirement for successful tumor suppression in humans.

Embedded in the concept of targeted cancer therapy is the expectation that disabling a single oncogenic pathway will eliminate the tumor cells and leave the normal tissues unscathed. Although validated by clinical responses in certain malignancies, challenges exist to generalize this approach to most tumors, as multiple genetic lesions, chromosomal instability, insensitivity of the cancer stem cell compartment, and emergence of drug resistance complicate the identification and therapeutic exploitation of a single, driving oncogenic pathway. Instead, broader therapeutic prospects may be offered by targeting crossroad signaling networks that are selectively exploited in cancer and oversee multiple aspects of tumor cell maintenance. One such pathway is centered on survivin, a cancer gene that intersects cell proliferation, cell survival, and the cellular stress response. Several clinical trials targeting survivin with a collection of approaches from immunotherapy to small-molecule antagonists are currently under way. By simultaneously disabling multiple signaling circuitries, targeting survivin may provide a novel perspective in rational cancer therapy selective for specific cancer mechanisms but broadly applicable to disparate tumors regardless of their genetic makeup.