The Third European Workshop on Cell Death

Salobreña, Spain

February 23rd to 28th 2002

Kristina A. Archer; Francis K. Chan; Michael J. Lenardo.  

Analysis of the Mechanism of Decoy Receptor Regulation of TRAIL Cytotoxicity in Lymphocytes.

National Institutes of Health

The tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL) receptor subfamily consists of four receptors, TR1, TR2, TR3, and TR4 that coordinately regulate the cytotoxic function of TRAIL.  The extracellular region of the four receptors share two conserved cysteine-rich domains (CRD) and a partial CRD.  When TR1 and TR2 bind TRAIL ligand, the cytoplasmic death domains of these receptors aggregate in homotypic trimeric complexes to transduce the death signal, causing caspase-mediated apoptosis.  However, both TR3 and TR4, which are known as decoy receptors, lack functional cytoplasmic death domains and therefore are unable to activate apoptosis.  It has yet to be determined how these decoy receptors regulate the signaling of TR1 and TR2.  Due to the high homology among the TRAIL receptors, it is possible that TR3 and TR4 pre-associate to form mixed complexes with TR1 and TR2.  To investigate whether decoy receptors can cross-associate, we have performed co-immunoprecipitation assays using differentially-tagged receptors.  We have also performed kill assays to assess whether the presence of decoy receptors can protect the cell from TRAIL-induced death.  These experiments have shown that cross-association between decoy and functional TRAIL receptors is possible.

CYCLIN D3 AND C-MYC CONTROL GLUCOCORTICOID-INDUCED CELL CYCLE ARREST BUT NOT APOPTOSIS IN LEUKEMIA CELLS

MICHAEL J. AUSSERLECHNER, PETRA OBEXER, STEPHAN GELEY, AND REINHARD KOFLER

Institute for General and Experimental Pathology, Division of Molecular Pathophysiology, University of Innsbruck, and Tyrolean Cancer Research Institute, Innsbruck, Austria

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoblastic leukemia cells, but the molecular basis of this therapeutically exploited phenomenon is not well understood. To investigate cell cycle effects of GC in the absence of obscuring apoptotic effects, we overexpressed bcl-2 in CCRF-CEM lymphoblastic leukemia cells, which delays apoptosis but does not interfere with the anti-proliferative effects of GC. GC treatment arrested cells in the G1 phase of the cell cycle due to downregulation of cyclin D3 and c-Myc. Cyclin E and Cdk2 protein levels remained high in GC-treated cells, but the kinase complex was inactive, most likely due to increased levels of bound p27Kip1. Conditional expression of either cyclin D3 and/or c-Myc was sufficient to prevent GC-induced G1 arrest, but did not interfere with the induction of apoptosis. Thus, GC seem to regulate apoptosis independent of the cell cycle.

Cheng-Yu Lee, Emily Clough, Claudio Simon, Damali Martin, and Eric H. Baehrecke.

Genetic regulation of programmed cell death during  Drosophila development

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute, College Park, MD 20742 USA

The steroid hormone ecdysone activates caspase-dependent cell death during Drosophila development.  Genes that function in apoptosis are utilized during salivary gland cell death, but the morphology of these cells indicate that they die by autophagy.  While autophagy occurs during the development of diverse animals, little is known about the mechanisms that regulate this form of programmed cell death.  The ecdysone-regulated EcR, ßFTZ-F1, BR-C, E74, and E93 genes are required for cell death, and regulate the transcription of the apoptotic genes rpr, hid, the CED4/Apaf-1 homolog ark, and the caspase dronc.  While EcR, ßFTZ-F1, BR-C, and E74 also function in processes other than cell death, E93 functions more specifically in cell killing.  DNA microarrays were used to identify genes with similar and unique temporal patterns of transcription during steroid-triggered autophagy and radiation-induced apoptosis.  While transcription of hundreds of genes is induced during steroid-triggered cell death, less than 30 are induced during radiation-activated apoptosis.  Significantly, some of the genes that were identified using microarrays were also identified in a cell death genetic screen.  These molecular and genetic screens indicate that a large number of DNA binding proteins, cell remodeling factors, and proteases (caspases and other families) contribute to programmed cell death.

Requirement of Abl caspase cleavage in the apoptotic signaling

Daniela Barilá (1), Alessandra Rufini (1), Ivano Condó (1), Natascia Ventura (1), Karel Dorey (2), Giulio Superti-Furga (2) and Roberto Testi (1)

(1) Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", Via Montpellier,1 00133 Rome, Italy. daniela.barila@uniroma2.it

(2) European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.

c-Abl is a non-receptor tyrosin kinase localized in the cytoplasm and the nucleus of cells. Its functions largely depend on its subcellular localization. In particular Abl activity in the nucleus is usually proapoptotic. However, the mechanisms responsible for Abl recruitment to the apoptotic program are not clear yet.

We have identified Abl as a new caspase substrate during CD95-induced apoptosis in lymphoid cells. Caspase cleavages occur in the C-terminal portion of Abl and leave the kinase domain intact. In particular cleavage at the very C-terminal region of Abl generates a fragment which has lost the nuclear export signal and the actin binding region, but retains the three nuclear localization signals and the DNA binding domain. This fragment rapidly accumulates in the nucleus. Importantly, the expression of wt and caspase cleaved fragment of Abl sensitizes lymphoid cells to CD95-induced apoptosis, in a kinase-dependent manner, while a caspase-resistant mutant of Abl significantly delays apoptosis.

Abl caspase cleavage might lead to Abl accumulation in the nucleus where its activity is required for the execution of cell death. We therefore propose a role for Abl tyrosine kinase in death receptor-mediated apoptosis and provide a new mechanism through which Abl can be recruited to the apoptotic program.

Identification of proteins interacting with the intracellular domain of CD95 Ligand.

Wiebke Baum and Martin Zörnig

 CD95 Ligand (CD95L) is a type II transmembrane protein belonging to the tumour necrosis factor family. The N-terminal 80aa form a cytoplasmic tail which has been implicated in routing of the molecule to intracellular vesicles where the ligand is stored in unstimulated haematopoietic cells (1). Engagement of the CD95 receptor by the extracellular part of CD95L can result in apoptotic death of the receptor-bearing cell. Interestingly, reverse signalling through CD95L influences the proliferative behaviour of primary mouse T-cells indicating that CD95L itself can transduce signals (2). The cytoplasmic part of CD95L contains several typical signalling motives like a casein kinase I phosphorylation site and a proline-rich region with binding sites for SH3- and WW-domains. To gain further insight into such intracellular CD95L signalling we performed a Yeast-Two-Hybrid screen with its cytoplasmic domain as the bait using a mouse T-cell lymphoma cDNA library. We obtained 14 different cDNAs of which 5 encode SH3- or WW-domain containing proteins. For at least one of the potential interaction partners we could show binding to CD95L both in vitro and in vivo. This potential binding partner is involved in T-cell signalling and also binds to the cytoplasmic tail of CD2 (3). The physiological consequences of this interaction are currently investigated in functional proliferation and apoptosis assays.

(1) Bossi, G., and Griffiths, G. M. (1999).Nat Med 5, 90-96; (2) Suzuki I., and Fink, P. J. (1998).J Exp Med. 5, 123-128

(3) Bai Y, Ding Y, Spencer S, Lasky LA, Bromberg JS. (2001) Exp Mol Pathol; 71(2):115-24

MUTANT PROTEOLIPID PROTEIN TRIGGERS APOPTOSIS IN OLIGODENDROCYTES THROUGH AN ER STRESS PATHWAY INVOLVING CASAPSE-12 ACTIVATION.

J.S. Beesley, L.A. Lavy, N.B. Eraydin, R. Siman and J.B. Grinspan*. Dept. of Neurology Research, Children’s Hospital of Philadelphia, Dept. of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

 The myelin-deficient (MD) rat has a point mutation in the proteolipid protein (PLP) gene leading to severe dysmyelination and oligodendrocyte cell death. It has been shown that MD oligodendrocytes die via caspase-3 activation although the pathway is poorly understood. The aim of this work was to investigate the events leading to MD oligodendrocyte cell death. Work in the jimpy mouse, a PLP mutant similar to the MD rat, has suggested that PLP accumulates in the ER. Here, we show that PLP is expressed in a perinuclear region in MD oligodendrocytes and colocalizes with BiP, an ER chaperone protein. Furthermore, BiP protein levels are twofold higher in MD oligodendrocytes than in wild type cells. Since BiP upregulation is indicative of ER stress, this suggests that accumulation of mutant PLP may induce ER stress in MD oligodendrocytes. Recently, caspase-12 and calpain have been suggested to mediate apoptosis in neuronal cells following ER stress. We also show that calpain and caspase-12 are activated in MD oligodendrocytes as detected by cleavage of a-spectrin and procaspase-12 respectively. These data suggest that mutant PLP induces an ER stress response in MD oligodendrocytes leading to apoptosis via calpain and caspase-12. (Supported by NIH grants NS34017 and T32-NS07413).

Death of CD4+ T cell lines caused by Human Immunodeficiency Virus-1 does not depend on caspases or apoptosis

Diane L. Bolton,1,2 Beom-Ik Hahn,1 Eugenia A. Park,3 Laura L. Lehnhoff,4 Felicita Hornung,5 and Michael J. Lenardo1

Laboratory of Immunology1, National Institute of Allergy and Infectious Diseases

National Institutes of Health, Bethesda, MD

2FAES(NIH)/Johns Hopkins University Cooperative Graduate Program in Biomedical Sciences

3Present Address: Department of Biology, Massachusetts Institute of Technology, Boston, MA,

4Present Address: University of Colorado School of Medicine, Denver, CO

5Present Address: Department of Genetics and Microbiology, University of Geneva, Geneva, Switzerland

 A critical aspect of AIDS pathogenesis that remains unclear is the mechanism by which HIV-1 induces death in CD4+ T lymphocytes.  A better understanding of the HIV-1 cytopathic effect may help to elucidate the viral component responsible for cell death and aid the design of preventive treatments against the decline of CD4+ T.  To evaluate the mechanism of this effect, we have studied HIV-1-induced cell death extensively by infecting T cell lines and assessing the level of apoptosis.  Contrary to the prevailing view that apoptosis plays a prominent role, we found that Jurkat and H9 cells dying from HIV-1 infection fail to exhibit the collective hallmarks of apoptosis. In addition, neither peptide caspase inhibitors nor the anti-apoptotic proteins Bcl-xL or v-FLIP could prevent cell death in HIV-1-infected cultures, and Jurkat cell lines deficient in RIP, caspase-8, or FADD were as susceptible as wild-type Jurkat cells to HIV-1 cytopathicity.  These results suggest that the primary mode of direct cytopathicity by laboratory-adapted molecular clones of HIV-1 in cultured cell lines is not via apoptosis but rather via a necrotic or lytic form of death independent of caspase activation. Currently, we are evaluating specific HIV-1 genes for their role in inducing necrotic death.

APRIL is an autocrine growth factor for megakaryocytes

D.Bonci*, M.Hahne°, C. Peschle*, R. De Maria*

* Laboratory of Hematology and Oncology, Istituto Superiore di Sanità,Rome, Italy

° Department of Immulology & Oncology Centro Nacional de Biotecnologia, Campus de Cantoblanco Universidad  Autonoma, E-28049 Madrid, Spain

 APRIL is a new TNF family member implicated in tumor cell proliferation. We investigated the role of APRIL in megakaryocytopoiesis, a developmental hematopoietic process responsible for progenitor cell differentiation to megakaryoblasts and megakaryocytes, leading to platelet formation. APRIL is not expressed in CD34+ progenitor cells from normal donors, but it is massively upregulated during terminal megakaryocytic differentiation. Both the two putative APRIL receptors, TACI and BCMA, are expressed in megakaryoblasts and megakaryocytes. Exogenous APRIL expression in primary cells increases megakaryocytic cell growth, suggesting that APRIL acts as a proliferative factor in megakaryopoiesis. More importantly, recombinant soluble TACI and BCMA receptors were able to block megakaryocyte expansion during intermediate and late proliferative stages through the neutralization of endogenous APRIL, resulting in dramatic decrease of platelet-producing cells. In conclusion, our data provide evidence that APRIL acts as a growth factor for terminal megakaryocytopoiesis and may promote physiological platelet production.

The serine protease inhibitor PI-9/SPI-6 blocks the perforin/granzyme B pathway, which results in escape from CTL-mediated cytotoxicity.

Michael Bots, Joan de Jong, Kees Melief, Rienk Offringa and Jan Paul Medema

Dept. of Immunohematology and Bloodtransfusion, Leiden University Medical Center, Leiden, The Netherlands.

 It is well known that cytotoxic T lymphocytes play a major role in the eradication of tumor cells. Upon recognition of a target, CTLs induce apoptosis via two main mechanisms: crosslinking of the so-called death receptors, and the perforin/granzyme B pathway. Despite the cytotoxic capacity, tumors have developed ways to escape from the actions of these effector cells. Recently, it has been found that tumors exhibit mechanisms to block the death receptor pathway. We now show that tumors can inhibit CTL mediated cytotoxicity via the perforin/GrB pathway as well. The capacity to resist this pathway depends on the expression of the serine protease inhibitor PI-9/SPI-6, which neutralizes the activity of the protease GrB. The expression of PI-9/SPI-6 was detected in several human and murine tumors and did indeed correlate with protection from GrB-mediated killing both in vitro and in vivo. Based on these results, we will investigate the role of PI-9/SPI-6 in more detail.

Identification of mammalian proteins inhibiting apoptosis downstream of Cytochrome c release.

Marie-Louise Brezniceanu and Martin Zörnig

 One of the known apoptotic pathways in mammalian cells involves release of mitochondrial Cytochrome c into the cytosol. Cyt c then together with ATP or dATP induces a conformational change in the adaptator protein Apaf-1 (a homologue of the C. elegans Ced-4 protein), leading to its oligomerization and the recruitment of several pro-Casp-9 molecules. This protein complex assembly called "apoptosome" leads to the activation of Casp-9 which then initiates or amplifies the caspase cascade.

The cell death program can be stalled at several points and we are interested in identifying new proteins inhibiting cell death downstream of Cyt c release. For this purpose we have screened a cDNA library derived from a pool of human breast carcinomas in a yeast-based survival screen, using the S. pombe yeast strain HC4 which contains an inducible Ced-4 expression construct. HC4 yeast cells die when Ced-4 is expressed (1), consequently only the colonies expressing a protective cDNA will survive. We isolated 22 growing yeast colonies of which 21 still showed Ced-4 expression in a Western Blot. Library cDNAs were isolated from these colonies, sequenced and studied for their potential to inhibit cell death in human cell lines. Of the genes inhibiting both yeast and mammalian cell death three were chosen for further investigations: Aven (2), RoRet and Fte-1.

(1) James, C., Gschmeissner, S., Fraser, A., and Evan, G. I. (1997). "CED-4 induces chromatin condensation in Schizosaccharomyces pombe and is inhibited by direct physical association with CED-9." Curr Biol. 7, 246-252.

(2) Chau, B. N., Cheng, E. H., Kerr, D. A., and Hardwick, J. M. (2000). "Aven, a novel inhibitor of caspase activation, binds Bcl-xL and Apaf-1". Mol Cell. 6, 31-40

A new cyclin-like protein as Cdk2 activator in apoptosis pathways

Maurici Brunet, Francesc Granés and Gabriel Gil

Unitat de Biologia Cel.lular i Molecular, Institut Municipal d´Investigació Mèdica-Universitat Pompeu Fabra (IMIM-UPF), Dr Aiguader, 80 08003 Barcelona, Spain

 A new cyclin-like protein as Cdk2 activator in apoptosis pathways

Activation of the cell cycle regulatory protein Cdk2 and degradation of the cdk inhibitor p27Kip have been involved in the pathways leading to apoptosis in postmitotic cells like thymocytes. De novo protein synthesis but not cyclin A or cyclin E appears to be required for this process.

We have found a new cyclin-like gene in the human and mouse genome. Northern blotting experiments of this gene showed higher levels of its mRNA in apoptotic than in normal mouse splenocytes and incubation of BaF3 cell lysates with the purified cyclin-like protein provided evidence of "in vitro" interaction with Cdk1 and Cdk2, resulting in kinase activation. Preliminary transient expression assays with this cyclin-like gene seems to be sufficient to induce cell death in 293T cells.

Our current efforts are directed to test the Cdk2 specificity of this cyclin-like protein, its regulation by Cdk2-dependent apoptotic stimuli in thymocytes and the positioning of its function respect to known biochemical steps in the apoptotic signal transduction pathways.

In situ production of glucocorticoids in the intestine as a regulator of life and death in intestinal T-lymphocytes

Igor Cima, Monica Zerilli, Bernhard Dick* and Thomas Brunner

Institute of Pathology, University of Berne, Switzerland, and

*Division of Nephrology and Hypertension, Inselspital, University of Berne, Switzerland.

 The extent of an immune response must be tightly regulated during its course, in order to ensure an adequate defense against pathogens, and maintain tissue integrity at the same time. Lymphocyte apoptosis represent an important process for the maintenance of this balance. In this context, glucocorticoid hormones (GCs) produced by the adrenal glands are important mediators of apoptosis in immune cells. Our previous results show that cell death and survival of intraepithelial lymphocytes of the intestinal mucosa (IELs) are regulated, among other factors, by the interplay between T-cell activation and the action of endogenously produced GCs. The aim of this study is to investigate whether the intestine itself produces GCs and whether this production plays a role in the regulation of IELs survival. Our results demonstrate that important steroidogenic enzymes such as Cyp11A1 are present in the intestine and are upregulated by a systemic T-cell activation. Furthermore we demonstrate that in vitro cultured intestinal tissues secrete glucocorticoids. We hypothesize thus that in situ produced GCs represent an important factor in the regulation of cell death and survival of intestinal lymphocytes.

The Forkhead transcription factor FKHR-L1 is a critical effector of cell death induced by survival

factor withdrawal

Paul J. Coffer, Kim Birkenkamp, Pascale F. Dijkers, Eric W.F. Lam# and Leo Koenderman

Dept. of Pulmonary Diseases, University Medical Center, Utrecht, The Netherlands

# Section of Virology and Cell Biology, Imperial College School of Medicine, London, UK

 Cytokines of the interleukin(IL)-3/IL-5/GM-CSF family regulate survival, proliferation and differentiation of various hemtopoietic lineages and their precursors. In the absence of cytokines, these cells stop priliferating and undergo programmed cell death (PCD). Survival signals elicited by cytokines include activation of phosphatidylinositol 3-kinase (PI3K) which in turn promotes the activation of Protein Kinase B (PKB). Recently PKB has been demonstrated to phopshorylate and inactivate the Forkhead transcription factor FKHR-L1.

We have found that FKHR-L1 is phosphorylated and activated by IL-3 family cytokines in a PI3K-PKB dependent manner. Importantly unregulated, constitutive activation of FKHR-L1 led to induction of apoptosis in a variety of hematopoietic cell lines. To explore the mechanisms underlying induction of apoptosis by FKHR-L1 we have generated a cytokine-dependent BaF3 cell line in which FKHR-L1 activation can be specifically induced by addition of 4-hydroxytamoxifen (4-OHT). Both cytokine-withdrawal and FKHR-L1 activation by 4-OHT induce apoptosis as measured by annexin-binding and DNA laddering. The onset of cell death is preceded by FKHR-L1 induced transcriptional upregulation of p27kip1 and a concommitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and FKHR-L1 activation correlates with disruption of the mitochondrial membrane and cleavage of effector caspases. These events follow upregulation of the pro-apoptotic Bcl-2 family member Bim, also a direct target of FKHR-L1 mediated transcription. Inducible activation of PKB alone was sufficient to inhibit FKHR-L1 and promote cell survival through maintenance of mitochondrial integrity and resultant inhibition of caspase activation. Inhibiting PI3K results in constitutive FKHR-L1 activation and induction of apoptosis even in the presence of cytokine. However, utilising hematopoietic stem cells isolated from Bim (-/-) mice, inhibition of PI3K was found to have a much reduced effect on the induction of apoptosis. Furthermore, ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the amount of apoptotic cells after cytokine withdrawal. This suggests that repression of Bim transcription by PI3K-PKB is a critical mechanism by which cytokines promote cell survival. Taken together, these data demonstrate that activation of FKHR-L1 alone is sufficient to induce the complete program of apoptotic events normally associated with survival factor withdrawal suggesting that this transcription factor is a critical player in the regulation of PCD. 

Multiple members of the Mitogen activated protein kinase family are necessary for PED/PEA-15 anti-apoptotic function.

G. Condorelli, A. Trencia,  A. Perfetti, U. Goglia, A. Cassese, G. Vigliotta, E. Autuori, and F. Beguinot

Dipartimento di Biologia e Patologia Cellulare e Molecolare & Centro di Endocrinologia ed Oncologia Sperimentale, C.N.R., Universtità degli Studi di Napoli "Federico II"

 PED is a 15kDa protein possessing a Death effector domain (DED). We have previously demonstrated that, in MCF7 cells, overexpression of PED blocks FasL and TNFa apoptotic effect by the displacement of FADD-FLICE binding through its DED. Futhermore, we demonstrated that PED expression levels negatively correlated with TRAIL-induced apoptosis in human glioma cells. In the present work we have analyzed the action of PED on stress signal-induced cell death. We found that expression of PED in 293 cells, reduced apoptosis induced by serum deprivation or stress. JNK and p38 phosphorylation was impaired in 293 overepressing PED cells. On the contrary, PED expression determined an increased MAPK phosphorylation and activity both in the basal state and following growth factors stimulation, while Akt phosphorylation was not changed. Activation of JNK and p38 pathways by transfection of cJNK1 or MKK6 cDNAs simultaneously with MAPK inhibition determined similar levels of cell death in 293 overepressing PED cells and 293 control cells. Thus, PED is able to inhibit both cytokines and growth factors withdrawal-mediated apoptosis, modulating different pathways.

INTERLEUKIN-4 PROTECS TUMOR CELLS FROM CD178 AND CHEMOTHERAPIC AGENTS VIA UPREGULATION OF ANTIAPOPTOTIC PROTEINS

C. Conticello*, F. Pedini°, G. Stassi#, A. Messina*, C. Peschle°, R. De Maria°*

*  Department of Biomedical Sciences, General Pathology Section, University of Catania, Italy

°  Laboratory of Hematology and Oncology, Istituto Superiore di Sanità, Rome, Italy

# Department of Biotechnologies and Forensic Medicine, Medical Biochemistry Section, University of Palermo, Italy

 A functional subset of T helper cells release TH2 cytokines, whose prototype is interleukin-4 (IL-4). TH2 response against tumors is associated with poor prognosis. Therefore, we investigated the effect of IL-4 on tumor cell survival after treatment with CD178 (FasL/CD95L) or chemoterapeutic agents.

IL-4 treatment significantly reduced CD178-induced apoptosis in prostate (LNCaP), breast (MDA-MD231) and bladder (RT112) tumor cells, while IL-2 had no effect. Similarly, IL-4 promoted the survival of tumor cells exposed to cisplatin, camptothecin, daunorubicin, etoposide and vincristine. Analysis of anti-apoptotic protein expression revealed that IL-4 stimulation resulted in upregulation of cFlip/FLAME-1 and Bcl-xL, which may potentially provide protection from CD178- and chemotherapy-mediated cytotoxicity. To determine whether increased expression of antiapoptotic genes protects LNCaP, MDA-MB231 and RT112 cells, we transduced these cells with a retroviral vector containing either cFlip/FLAME-1 or Bcl-xL. Tumor cells transduced with cFlip/FLAME-1 were protected from CD95 stimulation, while those expressing exogenous Bcl-xL were refractory to both CD95 or chemotherapeutic drug stimulation. Thus, IL-4 protects tumor cells from CD178- and chemotherapy-induced apoptosis through!  the upregulation of anti-apoptotic proteins such as cFlip/FLAME-1 and Bcl-xL. These findings may provide useful information for the development of new therapeutic strategy for cancer.

TRAIL and TRAIL receptor expression in normal and malignant tissues.

Raymond A Daniels and Gavin R Screaton

MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, The John Radcliffe, Oxford, OX3 9DS

 The identification of several apoptosis-inducing molecules/receptors belonging to the TNF/receptor superfamily has led to exciting advances in apoptosis research in recent years. TRAIL/receptors form one such a ligand/receptor system recently identified. TRAIL primarily kills tumour cells but not normal cells. In addition, TRAIL and mAb against TRAIL-R2 also exert anti-tumour activity in vivo without systemic toxicity. A unique characteristic of TRAIL/R’s as compared to other death ligand/receptor systems is that their mRNA is detected in a wide range of tissues. To date little effort has been made to screen tissues to investigate their differential expression patterns. We raised and characterised mAbs reactive with TRAIL/R’s in routinely fixed specimens and used it to investigate tissue distribution in normal and pathological tissues. These studies showed that there is a good correlation between mRNA and surface expression in most of the stained tissues. In addition, pathological specimens showed a generalised up-regulation of TRAIL-R1/2 expression. Functional studies will shed light on whether this leads to increased susceptibility to apoptosis. We examined the in vivo tumoricidal ability of mAb against TRAIL-R2 using selected Burkitt’s lymphoma cell lines. The Ab showed strong tumoricidal activity in vivo. More work needs to be done to elucidate the molecular mechanisms of this susceptibility.

INVOLVEMENT OF CD95- AND TNF DEATH SYSTEMS IN THE INDUCTION OF SPINAL CORD INJURY

Deana Demjen1, Susanne Kleber1, Cecilia Zuliani1, Johannes Schenkel2, Manfred Zimmermann2, Peter Krammer1, Ana Martin-Villalba1

1 Tumorimmunology Program, DKFZ, Heidelberg, Germany

2 Institute for Physiology and Pathophysiology, Heidelberg, Germany

 The tissue damage and functional losses following spinal cord injury (SCI) are consequent to secondary reactive processes caused by the primary trauma. These secondary processes involve apoptotic cell death and inflammation. Key players of the apoptotic program in other pathophysiological conditions, such as brain ischemia, are the cytokines CD95L and TNF-alpha. These cytokines are also involved in the attraction and/or activation of inflammatory cells, and, therefore, maintenance of the inflammatory response. To investigate the role of CD95L and TNF-alpha following SCI, we took advantage of mice expressing a mutated non-functional CD95L (gld), deficient in TNF-alpha (tnf -/-) and deficient in both CD95L and TNF-alpha (gld /tnf -/-). We detected a reduced number of TUNEL-positive cells at the lesion site of gld /tnf -/-, in comparison to wt animals. Moreover, gld /tnf -/- animals had less activated microglia at the lesion site when compared to wt animals. Activated microglia may cause the death of oligodendrocytes and consequent axonal demyelination. Loss of myelin can lead to additional death of the neurons that survived the initial injury. Therefore, the neutralization of the CD95-and TNF-L/R systems can reduce the apoptotic and inflammatory components following SCI.

Regulation of hepatocyte apoptosis in the transgenic model of HCV chronic infection.

Olivier Disson(1), Hervé Lerat(1), Stanley Lemon(2), Urszula Hibner(1)

(1) IGMM, CNRS UMR5535, 1919 route de Mende, 34293 Montpellier cedex 5, France

(2) University of Texas, Medical Branch at Galveston, TX, USA.

 Transgenic mice with liver targeted expression of HCV proteins develop pathologies similar to those found in chronically infected patients and notably, hepatocarcinomas (Lerat et al., Gastroenterology, in press). We have investigated the effect of the transgene expression on the regulation of Fas induced hepatocyte apoptosis both in vivo and ex vivo, in primary hepatocyte cultures prior to tumour developement and show that the apoptotic response is strongly inhibited in the transgenic hepatocytes. The expression of Fas is not altered in the transgenic hepatocytes. The decreased apoptotic response correlates with the decreased caspase 3 activation. Similarly, the cytoplasmic relocalisation of mitochondrial cytochrome c is inhibited in anti-Fas treated HCV+ hepatocytes. Indeed, the molecular defect caused by the HCV in the transduction of the apoptotic stimulus appears to reside at the level of the mitochondrial amplification loop, known to be essential in the Fas induced apoptosis of hepatocytes. Currently we are investigating possible defects in Bid/Bax interactions. The compromised response to Fas stimulation of the HCV positive hepatocytes could protect HCV infected hepatocyte from cytotoxic T cells as well as participate in the development of HCV related hepatocarcinomas.

MITOCHONDRIAL UNCOUPLING ACTIVATES CASPASE-9 AND MIMICS ANTIGEN RECEPTOR-INDUCED APOPTOSIS IN B-CELLS

E Eldering1, S Lens2 I Derks1, L Evers3, MHJ v Oers3, and RAW v Lier1 Depts of Experimental Immunology1 and Hematology3, AMC and Molecular Biology2, NCI, Amsterdam

During B-cell development, various routes to apoptosis can be active. Here, we focus on the pathway of apoptosis leading from the BCR via mitochondrial damage to caspase-3 activation. Apoptosis mediated via the BCR can be fully blocked by the caspase inhibitor zVAD, but cytochrome C release and the initial processing of caspase-3 still occur. Thus, the first steps are mediated by a zVAD-insensitive protease, and its identity has not yet been established.

We noticed that in various B cell lines, uncoupling of oxidative phosphorylation by CCCP initiated apoptosis via cyt C release. Significantly, both BCR triggering or CCCP addition in the presence of zVAD resulted in partial processing of caspase-3. This implicated caspase-9 as it is the only caspase activated by cyt C, and can also be active in unprocessed form. Next, transfection of 293 cells showed that only a combination of caspase-9 and –3 reproduced the caspase-3 activation pattern as seen in B-cells.

Thus, no evidence was obtained for involvement of a BCR-specific protease, e.g. caspase-2 or calpain. Instead, our results strongly indicate that after the signal reaches the mitochondrion, caspase-9 is solely responsible for events further downstream. The mediator(s) leading to mitochondrial activation remain to be identified.

CD20-INDUCED B-CELL DEATH CAN BYPASS MITOCHONDRIA AND CASPASE ACTIVATION

LEvd Kolk1, L Evers1, C Omene2, SMA Lens3 S Lederman2, RAW v Lier4, MHJ v Oers1, and E Eldering4

Depts of Hematology1 and Exp. Immunology4, AMC Amsterdam, Dept Mol Biol3, NCI Amsterdam, and Mol Immunology2, Columbia Univ., NY, USA

The chimeric anti-CD20 monoclonal antibody IDEC-C2B8 (rituximab) has become an important treatment modality in various B-cell lymphomas. Since the capacity of anti-CD20 mAbs to induce apoptosis  may very well contribute to the clinical results, we investigated this cell death pathway using Ramos Burkitt lymphoma cells. Crosslinking of CD20 (CD20XL) induced various hallmarks of apoptosis,  including loss of mitochondrial membrane potential (Dym), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, compared to CD95- or BCR-mediated apoptosis, CD20XL triggered a distinct death pathway. The caspase inhibitor zVAD-fmk could not not block  apoptosis as measured by PS exposure, cell size and membrane integrity. EM images confirmed that such cells underwent cell death. Furthermore, Bcl-2 overexpression blocked cyt-c release and the decrease in Dym, and completely prevented CD95- or BCR-mediated apoptosis; however CD20XL-induced cell death still proceeded. Thus, apoptosis mediated via CD20 does not necessarily require active caspases and cannot be blocked by Bcl-2. We propose that the CD20 signal bifurcates and leads to mitochondrial triggering as well as to a caspase-independent route to cell death, which may overlap with stress responses such as autophagy.

CD95 signaling is initiated in lipid rafts

A. Eramo, L.Ricci Vitiani, M. Sargiacomo, C.Peschle and R. De Maria

Laboratory of Hematology and Oncology, Istituto Superiore di Sanità, Rome, Italy.

 Early signaling generation by many surface receptors occurs in caveolae-like glycosphingolipid- and cholesterol-rich membrane microdomains (lipid rafts), which contain a given set of proteins but can change their size and composition in response to intra- or extra-cellular stimuli. We investigated cell surface CD95 distribution in several hematopoietic cell lines. Detergent insoluble low-density cell fractions (LDTI), corresponding to lipid rafts, and soluble fractions were obtained by sucrose gradient ultracentrifugation. Western blot and confocal microscopy analyses showed that CD95 was present both in raft and non-raft plasma membrane sub-domains. CD95 cross-linking induced a massive recruitment to lipid rafts of the receptors not located in these microdomains, suggesting that early apoptotic signals are generated in lipid rafts. Accordingly, CD95 triggering induced the recruitment of the other Death Inducing Signaling Complex (DISC) components to lipid rafts. In most CD95-sensitive cell lines, FADD and caspase-8 translocation to membrane microdomains was observed as soon as 1 minute after CD95 stimulation, while in other cell types it was delayed up to several hours. Active caspase-8 was consistently detected in lipid rafts until the majority of the cytosolic proenzyme was recruited and degradated. Importantly, kinetics of caspase-8 recruitment and activation in the lipid raft strictly correlated with the induction of the cell death process. Conversely, both inactive and active forms of executor caspases were exclusively observed in the detergent soluble fractions. Taken together, our data demonstrated that early events of CD95 signaling occur in lipid rafts, which are the first site of caspase-8 activation in hematopoietic cells.

TNF-induced death of murine embryonic fibroblasts becomes cathepsin B-dependent upon immortalization

Nicole Fehrenbacher, Mads G. Hansen, Birgit Poulsen, Marianne Boes (*), Marcel Leist (**) and Marja Jäättelä. Apoptosis Laboratory, Danish Cancer Society, Copenhagen Denmark,*Department of Pathology, Harvard Medical School, Boston, MA and ** H.Lundbeck A/S, Valby, Denmark

 Immortalization and transformation sensitize many cells to apoptosis by an unknown mechanism. Employing genetically modified cells and pharmacological protease inhibitors, we show here that the lysosomal cysteine protease, cathepsin B, is required for the immortalization-associated sensitization of murine embryonic fibroblasts (MEFs) to tumor necrosis factor (TNF). Whereas, wild type (wt) and cathepsin B-deficient (cathB-/-) primary MEFs (passage 3-5) displayed nearly indistinguishable sensitivity to TNF, wt immortalized MEFs (iMEFs; passage 10-25) were over 10.000x times more sensitive to TNF-induced apoptosis than primary MEFs or cathB-/- iMEFs. In wt iMEFs, TNF induced the release of active cysteine cathepsins into the cytosol and the activation of effector caspases. Interestingly, pharmaceutical inhibitors of caspases failed to affect the cathepsin release, whereas cysteine cathepsin inhibitors attenuated caspase activation and cell death. TNF-induced activation of effector caspases, but not that of nuclear factor kappa B, was also absent in TNF-resistant cathB-/- iMEFs. Thus, cathepsin B, which is commonly overexpressed in primary tumors, may counteract tumor growth and invasion by its proapoptotic activity.  We are currently studying the role of cathepsin B in the immortalization/transformation process and in iMEF apoptosis induced by other stimuli.

Enhanced Caspase-8 Recruitment to the DISC is Critical for Sensitisation of Human Hepatoma cells to TRAIL-induced Apoptosis by Chemotherapeutic Drugs

Tom M. Ganten, Tobias L. Haas, Heiko Stahl, Martin R. Sprick, Anne Grosse-Wilde and Henning Walczak

Division of Apoptosis Regulation, German Cancer Research Center (DKFZ), Heidelberg, Germany

 TNF-related apoptosis-inducing ligand (TRAIL) exhibits potent anti-tumour activity upon systemic administration in mice. TRAIL, however, does not show any of the deleterious side effects observed with other apoptosis-inducing TNF family members such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL we investigated hepatoma cell lines that exhibit differential sensitivity to TRAIL. Pre-treatment with chemotherapeutic drugs, e.g. 5-fluorouracil (5-FU), rendered the TRAIL-resistant hepatoma cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R1 and -R2, and substantially increased caspase-8 recruitment and activation after sensitisation. Marked downregulation of cFLIP in cell lysates was not mirrored in the DISC. The increased activation of caspase-8 could be explained by upregulation of the death-inducing TRAIL receptors and an increase of the ratio of caspase-8 to FLIP within the DISC. The results suggest that TRAIL-mediated apoptosis is controlled at the DISC level through increased caspase-8 recruitment in these cell lines.

RAR g protects neuroblastoma cells from fenretinide – induced apoptosis but increases cell death with natural retinoids

Bojidar B. Goranov1, Quentin Campbell-Hewson1, Birju Rana2, Penny Lovat1, Chris Redfern2

Departments of Child Health1 and Endocrinology2, University of Newcastle upon Tyne, UK

  Multifunctional synthetic retinoic acid receptor (RAR) – specific retinoids are potent pro-apoptotic agents against neuroblastoma in-vitro, currently assessed in clinical trials. Although extensive research has been carried out with these compounds into RAR – independent pathways (primarily free radical induction), the contribution of the retinoic acid receptors to cell death is still unknown. Findings by our group have shown that RAR g is the main receptor transducing the retinoid signal by all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cisRA), and that the addition of RAR b/g - specific antagonists abolishes fenretinide (RAR b/g retinoid) – induced apoptosis. Therefore, we proposed a model for the role of RAR g in cell death, in which the receptor contributes to apoptosis in the presence of the receptor-independent pathway(s) initiated by multifunctional synthetic retinoids. We aimed to test the following predictions, derived from the model, in neuroblastoma SH SY 5Y cells: (1) Increasing RAR g will increase fenretinide – induced apoptosis, (2) Decreasing RAR g will decrease fenretinide – induced apoptosis, (3) Increasing or decreasing RAR g will not have an effect on cell death when treating with ATRA or 9cisRA (which do not induce free radicals). A tetracycline – inducible expression system was used to alter the levels of the receptor from an expression plasmid containing the full length RAR g gene in sense (over-expression) or antisense (under-expression) orientation. Results with RAR g sense clones have shown that over-expression of the receptor: (1) decreases fenretinide – induced cell death, (2) leads to 20-30 % increase in cell death with ATRA or 9cisRA (3) Cells treated with the above retinoids for at least 3 days begin to display an unusual rounded cell morphology with few neurites. Therefore, these findings are contrary to the proposed model and suggest a more complex role for RAR g in apoptosis. Further work is being carried out to confirm and extend the above findings.

PATTERN OF EXPRESSION OF P210BCR-ABL AND P190BCR-ABL AND RESISTANCE TO DRUG-INDUCED APOPTOSIS  IN PARENTAL AND MULTIDRUG RESISTANT K562 CELLS.

Stefania Grimaudo1, Giuliana Cannizzo2, Eleonora Barbusca2, Matilde Todaro3, Michelangelo Catalano3, Marcello Ciaccio3, Luisa Dusonchet4, Maria Meli4, Natale D’Alessandro4, Guglielmo Mariani1, Vincenzo Abbadessa2 and Manlio Tolomeo2.

1Divisione di Ematologia con Trapianto di Midollo; 2Servizio di Riferimento Regionale AIDS; 3Dipartimento di Biotecnologie Mediche e Medicina Legale-Sezione di Biochimica Medica; 4Istituto di Farmacologia. Policlinico, Universita’ di Palermo.

 K562 is a chronic myelogenous leukemia (CML) cell line expressing BCR-ABL fusion proteins and considered to be resistant to apoptosis induced by several anticancer drugs. Recently, however, it has been observed that etoposide induces apoptosis in K562 cells, even though the phenomenon is evident only at 48-96 hours after treatment. We have studied the effects of daunorubicin (DNR) in K562 and in a multidrug resistant (MDR), P-glycoprotein (P-gp) overexpressing, variant (K562ADR) of this cell line. In K562, DNR 0.25 µg/ml induced 30% and 80% of apoptosis after 48 and 96 hours of treatment, respectively. In K562ADR, a combination of DNR (0.5µg/ml) and cyclosporin A (2µg/ml) able to achieve intracellular and intranuclear levels of DNR similar to those effective in K562, did not induce more than 25% of apoptosis even after 96 hours of treatment. In addition, K562ADR was more resistant than K562 to the apoptotic effects of methotrexate, cisplatin and cytosine arabinoside.The higher resistance of K562ADR to drug-induced apoptosis could not be attributed to changes in the expression of p53, Bcl-2 or Fas. However, at difference of  K562 which expresses both p210BCR-ABL and p190BCR-ABL mRNA, K562ADR expressed only p190BCR-ABL mRNA. K562ADR was endowed with a higher tyrosine kinase activity than K562. Genistein, a specific inhibitor of protein tyrosine kinases, and CGP57148B, a specific inhibitor of ABL protein tyrosine kinases, were able to increase apoptosis induced by DNR in both the cell lines. Importantly, under treatment with genistein or CGP57148B and at similar intracellular concentrations of DNR, the extent of apoptosis was almost equal in the two cell lines. On the contrary, genistein did not increase, and actually decreased, apoptosis by DNR in three BCR-ABL-negative leukemic cell lines (HL60, HUT78 and CCRF-CEM). Thus, it appears that, apart from P-gp, the selection of a MDR CML cell line induced also the exclusive expression of p1 90BCR-ABL and of the associated protein tyrosine kinase activity as a specific mechanism of increased resistance to the apoptotic effects of chemotherapeutic drugs.

dNp73 is induced by p53/TAp73 and exerts a dominant negative feedback loop.

T. J. Grob, U. Novak, C. Maisse, D. Barcaroli, A. U.  Lüthi, F. Pirnia, B. Hügli, H. U. Graber, V. De Laurenzi, G. Melino, M. F. Fey, A. Tobler

Departement of Clinical Research and Medical Oncology/Hematology, University and Inselspital Bern, Switzerland; Biochemistry laboratory, IDI-IRCCS, c/o Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy

 Inactivation of the tumor suppressor p53 is the most common defect in cancer cells. p53 is activated in response to genotoxic stress and induces cell cycle arrest and apoptosis. The p73 protein shares strong structural and functional similarities with p53. It has the potential to activate p53 responsive genes and induces apoptosis. A p73 variant lacking the N-terminal transactivation domain (dNp73) is described. We found that dNp73 expression is driven by a second promotor located upstream of exon 3’, supporting the idea of two independently regulated proteins, derived from the same gene. dNp73 is capable of regulating the transactivating p73 (TAp73) and p53 function since it blocks their transactivation activity. Interestingly, expression of the dNp73 is strongly up regulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and p53. The regulation of dNp73 is exerted through a p53 responsive element located on the dN promoter. Our results show the existence of an new feed back loop capable of regulating p53 function. While the mdm2/p53 loop regulates the protein degradation of p53 itself, the new loop dNp73/p53 regulates the function of p53.

Biochemical analysis of murine TRAIL receptors

Anne Grosse-Wilde, Heiko Stahl, Martin R. Sprick, Markus A. Weigand, Tom Ganten, Henning Walczak

Division of Apoptosis Regulation, German Cancer Research Center (DKFZ), Heidelberg, Germany

 TRAIL is a member of the TNF family of cytokines. Currently five receptors are known to bind TRAIL, namely TRAIL-R1 to TRAIL-R4 and OPG. Two of them, TRAIL-R1 and TRAIL-R2 are able to induce apoptosis upon ligation. In the mouse, so far only the ligand and one membrane bound receptor have been described in the TRAIL system. This murine TRAIL receptor was shown to be capable of inducing apoptosis and was suggested to be the murine orthologue of human TRAIL-R2. We biochemically analysed murine TRAIL-binding proteins by 2D-gel and identified murine surface proteins that specifically bind to TRAIL. One of these proteins, a murine TRAIL receptor of a molecular weight of 54 kDa, was identical to the known murine TRAIL-death receptor mTRAIL-R/MK. Our results showed for the first time that the native p54 mTRAIL-R (MK) is expressed on the surface of murine cells. Interestingly, p54 mTRAIL-R is N-glycosylated in its extracellular domain, a feature that it shares with huTRAIL-R1 but not with huTRAIL-R2. Our data also indicate that besides p54 mTRAIL-R at least one additional receptor for TRAIL is present in the mouse. However, at this point it remains to be determined whether this novel TRAIL-binding surface protein may be an apoptosis-inducing or a non-apoptosis-inducing receptor. Taken together our data suggest that, like in the human system, also in the murine system multiple receptors are involved in TRAIL-induced apoptosis.

Mechanism of TGF-ß induced apoptosis in Hepatoma Cells

Kerstin Herzer1, Anne Große-Wilde2, Henning Walczak2 and Peter H. Krammer1

Divisions of Immunogenetics1 and Apoptosis Regulation2, Tumorimmunology Program,

German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

 TGF-ß is a pleiotropic cytokine that is involved in the development of liver cirrhosis and of tumors in different organs. TGF-ß suppresses the immune response and supports immune escape of certain tumors. Moreover, TGF-ß has been shown to induce apoptosis in normal or transformed hepatocytes.

However, the mechanism by which apoptosis is induced is largely unknown. We examined whether death receptor-ligand systems play a role in TGF-ß induced apoptosis of hepatoma cells. We found that death ligands were upregulated in hepatoma cells upon treatment with TGF-ß, while receptor levels remained unchanged.  Our results suggest that death receptor-ligand systems are critically involved in  TGF-ß-induced immune escape and apoptosis in liver pathology. The relevance of these mechanisms in liver diseases will be discussed.

RECOGNITION AND PHAGOCYTOSIS OF CELLS DYING BY CASPASE-INDEPENDENT

MECHANISMS

U. Hirt, M. Leist

Department of Molecular Toxicology, University of Konstanz, Konstanz, Germany; H. Lundbeck A/S, Copenhagen-Valby, Denmark

 Apoptotic cells are rapidly engulfed by phagocytes before their lysis. This phagocytosis depends on the exposure of recognition molecules, like phosphatidylserine (PS) on the surface of apoptotic cells. A variety of physiological and pathological stimuli can cause non-apoptotic cell death. Since the mechanisms/markers, by which such cells are recognized by macrophages are largely unknown, we examined here a potential role of PS. As conventional annexin V staining cannot distinguish between PS on the outer and inner leaflet of lysed cells, we developed a magnetic-based assay that specifically labels PS on the outer surface of cells. Two necrosis models were characterized in detail. In ATP starved cells we found that PS-exposure occurred after cell lysis, but then led to efficient phagocytosis. In Jurkat cells exposed to the Ca2+-ionophore ionomycin, PS translocation and phagocytosis occurred without evident signs of apoptosis, and before membrane lysis. In both models, phagocytosis was blocked by inhibitors of PS-recognition. In conclusion, PS-exposure may also have a pivotal role for phagocytosis in very different paradigms of non-apoptotic cell death.

Involvement of mitochondria, endoplasmatic reticulum and lysosomes in programmed cell death of MCF-7 breast cancer cells.

Maria Høyer-Hansen1, Ida Stenfeldt Mathiasen1, Igor N. Sergeev2, David Andrews3, Brian Leber3 and M. Jäättelä1.

1Apoptosis Laboratory, Danish Cancer Society, Copenhagen, Denmark, 2Department of Chemistry and Biochemistry, South Dakota State University, Brookings, SD, USA, and 3McMaster University, Hamilton, Ontario, Canada

 Caspase-independent programmed cell death (PCD) pathways are still poorly understood. Employing MCF-7 breast cancer cells as a model system we compared caspase-dependent and –independent death pathways induced by tumor necrosis factor (TNF) and vitamin D compounds (VDC), respectively. TNF-induced death was associated with the appearance of large cytosolic clusters of “activated” Bax that partially co-localized with mitochondria and the release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol.  Accordingly, overexpression of Bcl-2 or its variant targeted to mitochondria, but not to endoplasmatic reticulum (ER), conferred protection against TNF. Vitamin D compounds induced the release of Ca2+ from the ER and activated Ca2+–dependent cysteine protease µ-calpain. Furthermore, three structurally different calpain inhibitors partially attenuated death induced by vitamin D compounds suggesting a role for ER and calpains in this model. Also the death induced by vitamin D compounds was associated with large Bax clusters and could be attenuated by Bcl-2, whereas the effect of Bcl-2 variants targeted to mitochondria or ER remains to be studied. Our preliminary results employing TNF and other apoptotic stimuli suggest that in addition to ER and mitochondria also lysosomes can play a crucial role on PCD signaling in MCF-7 cells.

SECRETION OF FASL-BEARING MICROVESICLES AS A NOVEL MECHANISM OF LYMPHOCYTE APOPTOSIS INDUCED BY TUMOR CELLS

Veronica Huber1, Giovanna Andreola1, Chiara Castelli1, Anna Maria Zaccheddu1, Paola Perego2, Paola Deho1, Paola Squarcina1, Agata Cova1, Francesco Lozupone3, Luana Lugini3, Giuseppe Arancia4, Giorgio Parmiani1, Stefano Fais3 and Licia Rivoltini1

1Unit of Immunotherapy of Human Tumors and 2Unit of Preclinical Chemotherapy and Pharmacology, Istituto Nazionale dei Tumori, Via Venezian 1, Milan 20133; 3Laboratory of Immunology and 4Ultrastructures, Istituto Superiore di Sanità, V.le Regina Elena 299, Rome 00161, Italy

 The hypothesis that FasL expression by tumor cells may impair the in vivo efficacy of anti-tumor immune responses, through a mechanism known as 'Fas tumor counterattack', has been recently questioned, becoming the object of an intense debate based on conflicting results. Here we definitely show that FasL is indeed detectable in melanoma cells, but its expression appears to be confined to the intracellular compartment of melanosomes. FasL expressed in such organelles retains its functional activity, since melanoma-derived melanosomes induce Fas-mediated T cell apoptosis in a dose-dependent fashion. We additionally show that melanosomes are eventually released extracellularly as FasL-expressing microvesicles. Released vesicles express FasL, together with melanosomal and lysosomal markers, and trigger Fas-dependent apoptosis of lymphoid cells. Hence our data provide evidence for a novel mechanism potentially operating in Fas tumor counterattack through the secretion of subcellular particles expressing functional FasL. Such vesicles may form a sort of front line hindering lymphocytes and other immunocompetent cells from entering neoplastic lesions and exert their anti-tumor activity. Furthermore, our investigation support the existence of a new pathway of FasL-induced apoptosis based on the release of subcellular organelles bearing membrane FasL, that may operate in different cell types as well.

Combined annexin V and propidium iodide labelling of aspirated graft cell populations for monitoring apoptosis and necrosis in the early transplanted human kidney.

Hughes DA1, Dillon RJ2.  

1Department of Surgery, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK and 2Pembroke College, University of Oxford, Oxford OX1 1DW, UK.

 The commonest causes of poor performance of the early renal transplant are primary graft non-function, cytologically associated with tubule cell necrosis, and acute cellular rejection, associated with cytotoxic T lymphocyte-induced apoptosis of targeted tubule epithelial cells.  However, although cyto-morphological evidence of necrosis is common in early graft dysfunction, apoptotic cells are rarely seen in biopsy samples.  CD14 macrophage activity is known to be increased generally in renal allografts during the first week post-transplant and it is probable that targeted kidney cells are eliminated from the graft at an early stage following the induction of apoptosis.  We have studied annexin V - propidium iodide fluorescence labelling patterns in tubule epithelial cells aspirated from early renal allografts with the intention of establishing a more predictive diagnostic strategy based on identifying the prevailing mode of parenchymal cell death at the time of biopsy.  Characteristic necrosis and apoptosis labelling patterns were observed in pre- and post-anastomosis, acute tubular necrosis, early and late acute rejection and graft infarction samples.  We are now correlating these findings with other physiological and immunological markers to determine whether they can be applied for the diagnosis of graft dysfunction.

Tumor Counterattack: Level and Time-Point of CD95L Expression are not Critical Factors in Mice

Frederik H. Igney, Christian K. Behrens, Bernd Arnold, and Peter H. Krammer

Division of Immunogenetics, Tumorimmunology Program, German Cancer Research Center, Im Neuenheimer Feld 280,

69120 Heidelberg, Germany.

 Tumors express CD95L (APO-1L, FasL) and can kill T cells in vitro. This may enable the tumors to suppress anti-tumor immune responses, a phenomenon called "tumor counterattack". However, CD95L-expressing tumors are rapidly rejected in mice. To clarify this controversial situation we established a mouse model with defined components. We investigated whether the level or the time-point of CD95L expression are the critical factors determining counterattack versus rejection. We generated tumor cell lines expressing  different levels of CD95L (LKC-CD95L). All cell lines grew at the same rate in nude mice. In contrast, growth of a CD95L-negative control cell line (LKC) was much faster. Moreover, we generated a cell line in which CD95L can be induced via the tet-system (LKCR-tetCD95L). In this cell line CD95L-expression can be induced by Doxycyclin (Dox) in a dose-dependent manner. Induction of CD95L in established LKCR-tetCD95L tumors lead to rapid rejection of the tumors. A decrease of the Dox-concentration delayed tumor rejection only minimally. Treatment of mice with a CD95L-negative non-inducible control tumor (LKCR) had no effect.

These results demonstrate that rejection of CD95L-expessing tumors in mice (1) is not an overepxression artefact and (2) does not depend on events at the onset of tumor progression.

Hsp70 depletion induces cathepsin-mediated tumor cell death and eradicates tumor xenografts in immunodeficient mice

Jesper Nylandsted, Wolfgang Wick, Nicole Fehrenbacher, Mads Gyrd Hansen, Ulrich Hirt, Marcel Leist, Michael Weller and Marja Jäättelä

Apoptosis Laboratory, Danish Cancer Society, Copenhagen, Denmark

Laboratory of Molecular Neuro-Oncology, Department of Neurology, University of Tübingen, Medical School, Tübingen, Germany

Faculty of Biology, University of Konstanz, Konstanz, Germany

H. Lundbeck A/S, Valby, Denmark

 Heat shock protein 70 (Hsp70) is an anti-apoptotic chaperone protein highly expressed in many human tumors. Here we show that an adenovirus expressing antisense Hsp70 cDNA (Ad.as.Hsp70) exhibits strong anti-tumor activity in tumor-bearing mice. Locoregional application of Ad.as.Hsp70 almost completely eradicated orthotopic xenografts of glioblastoma and breast carcinoma as well as subcutaneous xenografts of colon carcinoma in immunodeficient mice. Ad.as.Hsp70-induced death of tumor cells was associated with an increase in cytosolic cysteine cathepsin level and activity and a reduction in the level of an anti-apoptotic serine threonine kinase, AKT. Inhibition of cysteine cathepsins but not caspases attenuated Ad.as.Hsp70-induced death and AKT reduction. Furthermore, cathepsin L cleaved AKT in vitro and overexpression of AKT in some tumor cells conferred partial protection against Ad.as.Hsp70. Dying tumor cells were effectively phagocytosed by macrophages in a caspase-independent manner. Thus, Hsp70 appears as a promising target for future cancer therapy.

Specific depletion of Hsp70 mRNA and protein by small interference RNA

Mads Daugaard Jensen, Marja Jäättelä and Mikkel Rohde. Danish Cancer Society, Institute of Cancer Biology, Apoptosis Laboratory, Strandboulevarden 49, 2100 Copenhagen.

We have recently shown that tumorigenic cells die when depleted of Hsp70 by adenoviral transfer of antisense Hsp70 (Ad.asHsp70), whereas non-tumorigenic cells are not affected by this treatment. Ad.asHsp70 reduces Hsp70 protein level, without affecting the level of the constitutively expressed Hsp70 member, Hsc70. As antibodies against Hsp70 crossreact with several highly related gene products (i.e. HSPA1A, HSPA1B, HSPA1L, HSPA6 and HSPA2), we established a quantitative RT-PCR method to be able to study the expression of the individual genes and their contribution to cancer cell survival. Interestingly, cancer cells expressed all these genes and generally their expression was higher in carcinoma cells than in immortalized epithelial cells. Surprisingly, Ad.asHsp70 and control virus-treated cancer cells showed no difference in Hsp70 mRNA levels when analysed either by RT-PCR or Northern blotting. Thus, the Ad.asHsp70-induced reduction in Hsp70 protein level does not appear to involve the breakdown of Hsp70 mRNA. Contrary to the adenoviral transfer of antisense cDNA, small interfering RNAs  (siRNA) directed against HSPA1A and HSPA1B rapidly and specifically reduced the level of these mRNAs. RNAi appears thus as a method of choice for studying the role of individual Hsp70 genes in tumor cell survival.  

MODULATION OF ACETAMINOPHEN INDUCED APOPTOSIS IN

CULTURED MURINE TUBULAR EPITHELIAL CELLS BY BclxL AND

INHIBITORS OF CASPASES

Justo P, Ortiz A, Egido J, Lorz C

Laboratory of Experimental Nephrology and Vascular Pathology, Fundacion Jimenez Diaz, Universidad Autonoma de Madrid

 Paracetamol overdose can cause acute renal failure, and chronic exposure to paracetamol has been similarly linked to chronic renal failure. We have studied the mechanisms of paracetamol-induced renal injury in a murine proximal tubular epithelial MCT cell line. Paracetamol induced cell death with features of apoptosis, in a time- and dose-dependent manner. MCT cells constitutively express FasL, while Fas receptor expression was upregulated in cells treated with paracetamol. However, Fas-induced cell death did not seem to be the mechanism underlying paracetamol toxicity since blocking with anti-FasL antibodies or inhibiting caspase-8 activation did not prevent paracetamol toxicity.  While BclxL protein levels were decreased in paracetamol-treated cells, neither alterations in mitochondria nor caspase-3 activation could be detected. Lactacystin, a proteosome inhibitor, only partially prevented both the decrease in BclxL and apoptosis. In contrast, zVAD-fmk prevented paracetamol-induced features of apoptosis, but did not prevent the decrease in BclxL, and did not  prevent eventual cell death. However, overexpression of a human bclxL transgene did reduce apoptosis induced by paracetamol by 60% at 24 h and also increased long-term cell survival. Our results suggest that induction of apoptosis may underlie the nephrotoxic potential ofparacetamol and identify BclxL as a therapeutic target in nephrotoxicity.

Molecular mechanisms of apoptosis in stroke

Susanne Kleber, Min Li-Weber, Cecilia Zuliani,  Deana Demjen, Peter Krammer and Ana Martin-Villalba

German Cancer Research Center, Heidelberg, Germany.

 Oxygen and nutrient deprivation, as in stroke, results in loss of neurons. Neuronal death is induced by the ischemia-triggered Ca2+ overflow, release of reactive oxygen species (ROS) and of excitatory neurotransmitters. Animals lacking functional CD95 and TNF ligand/receptor (L/R) systems are strongly protected against stroke. Still, the link between the initial ischemic injury and the upregulation and activation of these death systems remains unclear. To address this issue we examined these death pathways after modulation of the REDOX-System in a model of ischemia. Primary glial cells subjected to oxygen glucose deprivation (OGD) showed reduced cell death if treated with an antioxidant, compared to non-treated cells. Also, antioxidant  treatment suppressed the expression of CD95L, but not of TNF, as assesed by immunofluorescence. Glial cells transiently transfected with a Luciferase CD95L promotor construct and exposed to OGD, exhibited a reduced luciferase activity after antioxidant treatment compared to non-treated cells. Thus, activation of the CD95 and TNF ligand/receptor systems following brain ischemia seems to be differentialy regulated.

Identification and expression of the serine protease inhibitor SPICI

Ingrid Kolfschoten, Mirjam Rademaker, Sandra Bres, Rienk Offringa and Jan Paul Medema. Dept. of Immunohematology and Bloodtransfusion, LUMC, Leiden, The Netherlands.

 The serpin SPI-6 has been reported to inhibit the activity of Granzyme B and as a result prevent cytotoxic T cell (CTL)-induced apoptosis. In vitro, however, SPI-6 cannot protect cells against CTL-induced lysis (membrane disruption), which is dependent on perforin. Surprisingly, the colon carcinoma cell line CMT93 does resist CTL-induced lysis in vitro. We have now identified a close homologue of SPI-6 in CMT93, which was named SPICI. The inability of CTLs to lyse these tumor cells might be explained by the activity of SPICI. The reactive site loop of SPICI, which is important for its inhibitory capacity, differs from SPI-6. It is therefore likely that SPICI targets a different protease. Importantly, high levels of mRNA expression of SPICI are not only detected in tumor cell lines, but also in the immune-privileged placenta. We are currently establishing the role of SPICI on the activity of cytotoxic cells.   

c-FLIP splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex (DISC)

Andreas Krueger, Ingo Schmitz, Sven Baumann, Peter H. Krammer and Sabine Kirchhoff

Affiliation of the authors: German Cancer Research Center (DKFZ), Department of Immunogenetics, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

 Upon stimulation CD95 (APO-1/Fas) recruits the adapter molecule FADD/MORT1, procaspase-8 and the cellular FLICE-inhibitory proteins (c-FLIP) into the death-inducing signaling complex (DISC). According to the induced proximity model, procaspase-8 is activated in the DISC in an autoproteolytic manner by two subsequent cleavage steps. c-FLIP proteins exist as a long (c-FLIPL) and a short (c-FLIPS) splice variant both of them capable to protect cells from death receptor-mediated apoptosis. In stably transfected BJAB cells both, c-FLIPS and c-FLIPL, block procaspase-8 activation at the DISC. However, cleavage is blocked at different steps. c-FLIPL allows the first cleavage step of procaspase-8 leading to the generation of the p10 subunit. In contrast, c-FLIPS completely inhibits cleavage of procaspase-8. Interestingly, p43-c-FLIPL lacking the p12 subunit also prevents cleavage of procaspase-8. In contrast a non-processable mutant of c-FLIPL allows the first cleavage of procaspase-8. In conclusion, both c-FLIP proteins prevent caspase-8 activation at different levels of procaspase-8 processing at the DISC. Our results indicate that c-FLIPL induces a conformation of procaspase-8 that allows partial but not complete proteolytic processing, whereas, in contrast, c-FLIPS even prevents partial procaspase-8 activation at the DISC.

TRANSLATION INHIBITION IN APOPTOSIS: CASPASE-DEPENDENT PKR ACTIVATION AND eIF2-a PHOSPHORYLATION

Authors: M. Kalai, X. Saelens, M. Lamkanfi, P. Vandenabeele

affiliations: Department of Molecular Biology, Unit of Molecular Signaling and Cell Death, Flanders Interuniversity Institute for Biotechnology and Ghent University, 9000 Ghent, Belgium

 The protein kinase PKR is a major player in the cellular antiviral response, acting mainly by phosphorylation of the a-subunit of the eukaryotic translation initiation factor 2 (eIF2-a) to block de novo protein synthesis. PKR activation requires binding of dsRNA or PACT/RAX proteins to its regulatory domain. Since several reports have demonstrated that translation is inhibited in apoptosis, we investigated whether PKR and eIF2-a phosphorylation contribute to this process. We show that PKR is proteolysed and that eIF2-a is phosphorylated at the early stages of apoptosis induced by various stimuli. Both events coincide with the onset of caspase activity and are prevented by caspase inhibitors. Using site-directed mutagenesis we show that PKR is specifically proteolysed at Asp251 during cellular apoptosis. This site is cleaved in vitro by recombinant caspase-3, caspase-7 and caspase-8, and not by the proinflammatory caspase-1 and caspase-11. The released kinase domain efficiently phosphorylates eIF2-a at the cognate Ser51 residue and its overexpression in mammalian cells impairs the translation of its own mRNA and of reporter mRNAs. Our results demonstrate a new and caspase-dependent activation mode for PKR, leading to eIF2-a phosphorylation and translation inhibition in apoptosis.

Cloning and characterization of human and mouse BAG3 protein in cellular and stress conditions.

#* Lerose, R., #*Pagliuca, M.G., #*Amelio, T., *Festa, M., *Milella, M.R., Turco, M.C.  and *Leone, A.

*University of Salerno, Department of Pharmaceutical Sciences

#University of Naples “Federico II”, Department of Biochemistry and Medical Biotechnologies.

 The Bag proteins are a family of proteins that contain a conserved aminoacid domain near the carboxyl-terminus region (Bag domain) which differ widely in their amino-terminus domains. Through the Bag domain they bind the ATPase domain of Hsp70, inhibiting its protein refolding and functioning as  negative regulator of Hsp70 activity. We cloned by RT-PCR the human cDNA of Bag3, a member of Bag family proteins. The expression of Bag3 gene is regulated :in HeLa cells, it increases after exposure to stress conditions such as heavy metals (cadmium and zinc) and heat shock (42°C), with time-dependent kinetics of accumulation. Polyclonal antibodies raised against the carboxyl terminus of the human Bag3 protein recognized in vitro and in vivo a polypeptide of a molecular mass of 84 kDa. In control cells this protein had a cytosolic location; following exposure to stress, it mobilized toward a perinuclear compartment, identified by immunofluorescence techniques as RER (Rough Endoplasmic Reticulum). Bag 3 appeares to be regulated in a tissue specific manner: high levels of Bag 3 mRNAs are present in skeletal muscle and heart. With the aim to investigate the involvment of Bag 3 protein in muscle differentiation, we cloned by RT-PCR the full-lenght of murine Bag 3. We used as experimental model the C2C12 cells (murine skeletal muscle cells) that can easly be induced to differentiate, reducing the amount of serum in the medium and adding insulin. The cells achieve differentiation in 3 days and the different stages  have been followed detecting specific muscle markers. Northern blotting experiments revealed no changes in the expression of Bag 3 mRNA during differentiation, but increased levels of the corresponding protein,  confirming that Bag 3 is  expressed in skeletal muscle.

Further Studies of the Molecular Mechanism of KICA-Induced Apoptosis: The role of Bcl-2

Adeline Lesay, Mary Kozma, Hiruni Dhanapala and Huseyin Mehmet

Weston Laboratory, Institute of Reproductive and Developmental Biology, Division of Paediatrics, Obstetrics and Gynaecology, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

 Maple Syrup Urine Disease is an inborn error of metabolism that can result in progressive neurodegeneration. It is caused by a deficiency in the enzyme, branched chain amino acid dehydrogenase, leading to the accumulation of branched chain keto acids, in particular ?-keto isocaproic acid (KICA). Recently, we have shown that KICA triggers apoptosis in neural cells and fibroblasts. In the present study, we have investigated the pathway of KICA-induced apoptosis in more detail using Rat-1 fibroblasts. Bcl-2 overexpressing cells were protected from KICA-induced death, demonstrated both by metabolic (MTT) analysis and by clonogenicity assays. Detailed time-course experiments indicated that the protective effect was probably due to a Bcl-2 dependent delay in the commitment of cells to apoptosis. Although in wild type Rat-1 fibroblasts caspase-3 was activated following KICA treatment, very little cytochrome c release occurred, whereas with staurosporine, a significant proportion of the cells released cytochrome c into the cytosol. This was confirmed by both western blotting of subcellular fractions and by immunofluorescence microscopy. These findings suggest that KICA-induced apoptosis may not be executed through the classical "mitochondrial pathway".

RESISTANCE TO TRAIL INDUCED APOPTOSIS IN PRIMARY KERATINOCYTES: A ROLE FOR EFFECTOR CASPASE INHIBITION

M. Leverkus1§, M.R. Sprick3§, T.Mengling1, E. Serfling2, E.B. Bröcker1, M. Neumann2# and H. Walczak3#

University of Würzburg Medical School

1 Department of Dermatology, 2 Department of Pathology, Josef-Schneider-Str. 2, 97080 Würzburg, Germany,

3 DKFZ Heidelberg,Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

 TRAIL as been shown to exert potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant against TRAIL. In this report, we examined intracellular mechanisms of resistance of primary keratinocytes to TRAIL. The presumed antiapoptotic transcription factor NF-KB was rapidly activated by TRAIL in primary keratinocytes. Moreover, upon inhibition of TRAIL-induced NF-KB activation with the proteasomal inhibitor MG115, primary keratinocytes were dramatically sensitized to TRAIL-induced apoptosis. In contrast, transient transfection of keratinocytes with mutant forms of  IKB-kinase 2 did not modulate TRAIL sensitivity in keratinocytes. At the molecular level, sensitization to TRAIL-induced apoptosis by MG115 was found to be downstream of the native TRAIL DISC and its components FADD, caspase 8, caspase 10 and cFLIP, reflected by rapid cleavage of caspase 3 to a 20 kDa fragment. However, full caspase 3 maturation and the release of cytochrome c and smac/DIABLO from mitochondria required the inhibition of the proteasome. Taken together, our data suggest that modulation of proteasomal function sensitizes keratinocytes to TRAIL-induced apoptosis at or upstream of mitochondria, but downstream of caspase 8 or 10 activation at the DISC, leading to cytochrome c and smac/DIABLO release from the mitochondrium and concomitant full caspase 3 maturation.

Identification of tBid-induced release of mitochondrial proteins. Role of endonuclease G and Omi/HtrA2 in the apoptotic process.

Geert van Loo1, Hans Demol2, Marian van Gurp1, Peter Schotte1, Bart Depuydt1, Bart Hoorelbeke2, Rudi Beyaert1, Kris Gevaert2, Ivan Rodriguez3, Boris Zhivotovsky4, Adolf Ruiz-Carrillo5, Srinivasa M. Srinivasula6, Emad S. Alnemri6, Wim Declercq1, Joel Vandekerckhove2 and Peter Vandenabeele1.

1              Flanders Interuniversity Institute for Biotechnology and Ghent University, Department of Molecular Biology, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

2              Flanders Interuniversity Institute for Biotechnology and Ghent University, Department of Medical Protein Research, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

3              The Rockefeller University, Laboratory of Vertebrate Neurobiology, New York, USA

4                Karolinska Institute, Institute of Environmental Medicine, Unit of Toxicology and Neurotoxicology, Box 210, S-171 77 Stockholm, Sweden

5                Department of Molecular and Cell Biology, C.I.D., C.S.I.C., Biotechnology Reference Center of the Generalitat de Catalunya, Barcelona, Spain

6              Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA

 A crucial event in the process of apoptosis is caspase-dependent generation of truncated Bid (tBid), which induces the release of cytochrome c. In an in vitro reconstitution system we combined purified recombinant tBid with isolated liver mitochondria and identified the released proteins combining matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and post-source decay (MALDI-PSD) analysis. Sixteen, mostly mitochondrial, proteins were identified to be released in a tBid-dependent way, among them endonuclease G and the serine protease Omi/HtrA2. The relocalization from mitochondria to cytosol of these proteins under apoptotic conditions was confirmed in vivo in livers from mice injected with agonistic anti-Fas antibodies and was prevented in livers from Bcl-2 transgenic mice. Our data demonstrate that endonuclease G may be involved in caspase- and CAD-independent DNA fragmentation during apoptosis. The serine protease Omi/HtrA2 is shown to interact with the caspase-inhibitor XIAP when released from mitochondria and to enhance cytosolic caspase activation.

Intracellular processing of APRIL, a TNF ligand family member

M. López Fraga, M. Hahne

 The TNF-related ligands APRIL and BLyS and their cognate receptors BCMA/TACI form a two ligand/two receptor system that has been shown to participate in B- and T-cell stimulation. Tumor necrosis factor (TNF) ligand family members are synthesized as transmembrane proteins, and cleavage of the membrane-anchored proteins from the cell surface is frequently observed. In contrast to BLyS, which is known to be cleaved from the cell surface, we found that APRIL is processed intracellularly by furin convertase. Thus, APRIL shows a unique maturation pathway among the TNF ligand family members, as it not detectable as a membrane-anchored protein at the cell surface, but is processed in the Golgi apparatus prior to its secretion.

Another member of the TNF family, FasL, has been shown to be stored in specialized secretory lysosomes in haematopoietic cells. To address this issue we are presently studying the secretory pathway of APRIL and trying to characterize the secretory vesicles involved in this process. These studies also include comparison with other TNF ligand family members such as BLyS and TWEAK.

GADD153 and Bak, novel molecular targets of fenretinide-induced apoptosis of neuroblastoma and the synergistic response with chemotherapeutic drugs.

Penny  Lovat1, Serafina Oliverio2, Bojidar Goranov3, Andy Pearson1, Mauro Piacentini 2

and Chris Redfern1+3

Departments of Child Health1 and Endocrinology3, University of Newcastle upon Tyne, UK and Department of Biology , University of Tor Vergata, Rome, Italy.

 Fenretinide induces apoptosis of neuroblastoma cells and in vitro acts synergistically with the chemotherapeutic drugs, cisplatin, etoposide and carboplatin. To characterize the mechanism of action of fenretinide, cDNA array filters for apoptosis-related genes were screened to identify genes regulating the induction of this synergistic response. Expression of the stress-induced transcription factor, GADD153 and the pro-apoptotic Bcl2-related protein, Bak were up regulated by fenretinide. Although fenretinide is a partial retinoic acid receptor (RAR)- ‚/Á agonist, RAR ‚/Á antagonists did not block the induction of GADD153 or Bak by fenretinide. Conversely, the induction of GADD153 and Bak was blocked by antioxidants. Furthermore, GADD153 was not induced in fenretinide-resistant cells, in which the induction of reactive oxygen species (ROS) by fenretinide is reduced compared to wild-type cells. Chemotherapeutic agents induced neither GADD153 or Bak. Taken together, these results suggest that fenretinide induces apoptosis via RAR-dependent and –independent pathways in which the RAR-independent pathway is characterized by the ROS-dependent induction of GADD153 and Bak. Fenretinide did not induce p21/WAF1, a p53-regulated gene, whereas the converse was true for the chemotherapeutic drugs cisplatin, etoposide and carboplatin. Therefore, the induction of p53-independent and p53-dependent pathways by fenretinide and chemotherapeutic drugs, respectively may be the mechanism underlying synergy between these agents in which GADD153 and Bak may represent novel molecular targets for the therapeutic drug regime used to treat advanced stage neuroblastoma.

CD95/EZRIN INTERACTION IN THE CD95 APOPTOTIC PATHWAY

Francesco Lozupone, Luana Lugini, Francesca Luciani, Cristina Funaro, and Stefano Fais. 

Tumor Immunology Section, Laboratory of Immunology, Istituto Superiore di Sanità

 Ezrin belongs to the ERM family proteins that link the actin cytoskeleton to the plasma membrane. It was known that ERM interact with some adhesion molecules (e.g. CD44 and ICAMs), as well as PIP2, through their 4.1 domain. We have shown that an important requirement for susceptibility to the CD95 (APO-1/Fas)-mediated apoptosis in lymphocytes is the CD95 linkage to the actin cytoskeleton through Ezrin. In lymphocytes CD95 and Ezrin polarized and co-localized in uropods. Consistently, it was shown that exclusively in Fas-susceptible cells (i.e. lymphoblastoid cells and activated T lymphocytes) CD95 and Ezrin co-immunoprecipitated, suggesting a molecular association, while no association was shown in resting and early activated lymphocytes,  refractary to CD95 mediated apoptosis even fully expressing CD95. Moreover, both actin perturbing agents like Cytochalasin D and treatment with Ezrin antisense oligonucleotides, strongly reduced susceptibility to CD95-mediated apoptosis. Further experiments showed that (i) CD95 binds to Ezrin through its juxta-membrane region, upstream to the CD95 Death Domain and (ii) the Ezrin binding region to CD95 is on its 4.1 N-terminal domain close to the PIP2 binding regions as well.  These data suggest a key role of CD95-cytoskeletal interaction in the regulation of CD95-mediated apoptosis.

GP120-INDUCED EZRIN PHOSPHORYLATION: A ROLE IN THE SUSCEPTIBILITY TO CD95-MEDIATED APOPTOSIS IN HIV-1 INFECTION

Francesca Luciani, Paola Matarrese, Luana Lugini, Annamaria Giammarioli, Francesco Lozupone, Cristina Federici, Walter Malorni, and Stefano Fais.

Laboratory of Immunology, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Roma

 It has been reported that the binding of the envelope protein of HIV-1 (gp120), to the CD4 molecules on the surface of T-lymphocytes, is involved in CD4+T- lymphocytes depletion. during HIV-1 infection.  Our results showed that in cells naturally susceptible to CD95-mediated apoptosis: CD95 polarizes, colocalizes and coimmunoprecipitates with the cytoskeletal protein Ezrin; the integrity of actin cytoskeleton, Ezrin expression and Fas/Ezrin association are requested for cell susceptibility to Fas-mediated apoptosis. These findings strongly suggested a key role of CD95/Ezrin/actin association, in rendering human T lymphocytes susceptible to CD95-mediated apoptosis (Parlato et al., 2000). Moreover, it has been shown that Ezrin activation needs phosphorylation and  evidence has been provided that the CD4 crosslinking on human T-lymphocytes results in Ezrin phosphorylation. Thus, we investigated the effect of gp120 stimuli on Ezrin phosphorylation in human T-lymphocytes. Preliminary results showed that in lymphoblastoid cells, susceptible to Fas-mediated apoptosis, Ezrin is constitutively phosphorylated. Moreover, human T-lymphocytes following gp120+rIL-2 stimuli underwent: (i) cellular polarization with co-localization of both Ezrin and CD95 on the forming uropods; (ii) stable Ezrin phosphorylation together with (iii) increased susceptibility to CD95-mediated apoptosis. These results suggest that the gp120-mediated Ezrin phosphorylation may have a role in the pathogenesis of CD4+T-lymphocytes depletion.

TRAIL-induced apoptosis results in rapid release of Smac and its subsequent degradation via the ubiquitin-protein ligase activity of XIAP

Marion MacFarlane, Wendy Merrison, Shawn Bratton and Gerald M. Cohen.

MRC Toxicology Unit, Hodgkin Building, P.O Box 138, University of Leicester, Leicester, LE1 9HN, UK.

 During apoptosis, Smac/DIABLO, a recently identified IAP (inhibitor-of-apoptosis-protein) binding protein, is released from mitochondria and potentiates apoptosis by relieving IAP inhibition of caspases. We demonstrate that exposure of MCF-7 cells to TRAIL results in rapid Smac release from mitochondria, which occurs before or in parallel with release of cytochrome c. Smac release is inhibited by Bcl-2/Bcl-xL or by a pan-caspase inhibitor, demonstrating that death receptor-induced Smac release is both caspase-dependent and sensitive to modulation by Bcl-2 family members. Once released, Smac is rapidly degraded by the ubiquitin-proteasome pathway, an effect suppressed by co-treatment with a proteasome inhibitor. As the RING finger domain of XIAP possesses ubiquitin-protein ligase activity and XIAP binds tightly to mature Smac, an in vitro ubiquitination assay was performed which revealed that XIAP functions as an E3 ligase in the ubiquitination of Smac. Both the association of XIAP with Smac and the RING finger domain of XIAP were essential for ubiquitination. These results demonstrate that the ubiquitin-protein ligase activity of XIAP may promote the rapid degradation of Smac in apoptotic cells. Thus, in addition to its well-characterised role in blocking apoptosis by inhibiting caspase activity, XIAP may also protect cells by targeting pro-apoptotic molecules for proteasomal degradation.

Prevention of B cell antigen receptor-induced apoptosis by T helper signals occurs downstream of cell cycle regulation.

W.J.M. Mackus1,2, S.M.A. Lens2,3, R.H. Medema3, M.J. Kwakkenbos2, L.M. Evers1, M.H.J. van Oers1, R.A.W. van Lier2, and E. Eldering2.

Dept. of Hematology1 and Lab. Exp. Immunology2, Academic Medical Center, Amsterdam, Dept.of Hematology, University Medical Center, Utrecht, The Netherlands.

 Crosslinking of the B cell antigen receptor (BCR) on germinal center B cells can induce growth arrest and apoptosis, thereby eliminating potentially autoreactive B cells. Using the Burkitt lymphoma cell line Ramos as a model, we studied the commitment to apoptosis following growth arrest, as well as how T helper signals can interfere to block cell death. Both BCR-triggering and direct induction of growth arrest by sodium butyrate caused hypophosphorylation of  the retinoblastoma protein (pRb), followed by apoptosis. Interestingly, although CD40 ligation or TNFa efficiently prevented BCR-induced and sodium butyrate-induced apoptosis, these co-stimuli did not inhibit but rather augmented the growth arrest. Analysis of cell cycle regulators showed that each apoptotic and T helper stimulus distinctly affected cyclins or cyclin-dependent kinase inhibitors, indicating that growth arrest can be uncoupled from apoptosis. BCR-ligation and growth arrest activated the intrinsic or mitochondrial route of apoptosis. CD40 ligation and TNFa prevented release of cytochrome c and activation of caspase-3, which could not be explained by effects on the expression of Bcl-2, Bcl-xL or Bax. Finally, the onset of BCR-induced apoptosis occurred after 10-12 h, and  addition of CD40 mAbs or TNFa  at that point still prevented further execution of apoptosis. We conclude that in mature B cells apoptosis is not an obligatory event following growth arrest. Instead, commitment to apoptosis can be rapidly controlled by T cells via CD40L and TNFa, downstream of the pRb regulated restriction point of the cell cycle, but prior to mitochondrial cytochrome c release.

Human DNp73 regulates a dominant negative feedback loop for TAp73 and p53.

Carine Maisse1, Kathleen Lheveder1, Tobias J Grob2, Urban Novak2, Daniela Barcaroli1, Andreas Tobler2, Eliana Munarriz1, Martin F. Fey2, Gerry Melino1 and Vincenzo De Laurenzi1

1Department of Experimental Medicine, University of Rome, Tor Vergata, Rome, Italy;

2Department of Clinical Research and Medical Oncology/Haematology, University and Inselspital, Berne, Switzerland

 Different isoforms of the p53 analogue p73 have been described, some deriving from alternative splicing at the 3' end of the gene leading to different C-termini (TA isoforms a to z), some, cloned so far only in mice, have a different N-terminus and are thought to derive from the usage of an additional exon (exon 3') located in intron 3. These isoforms, named DN, lack the aminoterminal transactivation domain and act as dominant negatives on the TA isoforms. We have cloned different human DN-p73 isoforms and have characterised their expression pattern and functions. We found that DN isoforms are expressed in a number of different adult and foetal tissues and that, differently from what reported for mouse embryos, TA isoforms are always expressed 10 to a 100 fold more than DN isoforms. In addition, most of the tumour cell lines tested show an altered TA/DN ratio, suggesting that the relative amounts of the two forms, rather than the absolute amount, is  important for the normal cell function. We have also determined the transcription start site of the DN forms and then cloned a 2 kb fragment of genomic sequence upstream of it. This fragment contains a second p73 gene promoter responsible for the transcription of the DN isoforms, and is sufficient to drive expression in the cell lines that express DN. Among the different responsive elements found we have studied the role of: N-Myc, Mad-1 and c-Myb.

Sialic acid acetylation suppresses the pro-apoptotic activity of GD3

F. Malisan*, L. Franchi*, N. Ventura*, B. Tomassini*, M.R. Rippo*, I. Condo’*, A. Rufini*, C. Nachtigall^, B. Kniep^ and R. Testi*.

*Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy.

^ Institute of Immunology, Technical University of Dresden, Germany.

 GD3 disialoganglioside acts as an intracellular mediator of apoptosis being responsible for the opening of the mitochondrial permeability transition pore, the release of cytochrome c and the activation of caspase-9. 9-O-acetylation of sialic acids, the most common post-synthetic modification of GD3, affects the physicochemical properties of the parent molecule and is expected to modify its functions. We investigated whether sialic acid 9-O-acetylation affects the pro-apoptotic function of GD3.

While most cell lines undergo apoptosis upon overexpression of the GD3 synthase, some are resistant. We observed that HEK293 cells, resistant to GD3 synthase overexpression, convert part of the neo-synthesized GD3 into 9-O-acetyl-GD3. When HEK293 cells are co-transfected with GD3 synthase and a 9-O-acetyl-esterase, to prevent 9-O-acetyl-GD3 accumulation, they undergo massive apoptosis. Mitochondria are a primary target for GD3. Interestingly, 9-O-acetyl-GD3 is completely unable to induce cytochrome c release from isolated mitochondria, unlike GD3.

These data indicate that 9-O-acetylation of sialic acids suppresses the pro-apoptotic activity of GD3 and suggest a new role for sialic acid acetylation in the control of the apoptotic program.

Human chronic myielogenous leukemia (CML) consists in malignancy of pluripotent  haematopoietic cells caused by a deregulated activity of the tyrosine kinase encoded by the chimeric bcr-abl oncogene.

G. Marfé, Di Stefano Carla. and Giancotti Paola. 

Dep. Experimental Medicine and Pathology, Medicine College-University "La Sapienza"-Rome

 Human chronic myielogenous leukemia (CML) consists in malignancy of pluripotent  haematopoietic cells caused by a deregulated activity of the tyrosine kinase encoded by the chimeric bcr-abl oncogene . Many studies have shown that sorbitol is able to induce osmotic shock, which activates apoptosis . In the first experimental phase, we performed time course studies (30 min, 60 min., 90 min and 120 min.). for sorbitol at various  concentrations  in the range 1.0-1.4 M on the K562 cells. DNAs were extracted from the untreated and treated cells and were examined for DNA fragmentation by electrophoresis in agarose gel. The fragmentation of chromosomal DNA into oligonucleosome  was showed. after 30 min the sorbitol treatment. Furthemore, we investigated the role of bax gene and caspase-3 in K562 cells during the course of sorbitol-induced apoptosis  and  we analyzed cDNAs with the specific primers. The expression of bax and caspase-3 genes increased after 30 min  of sorbitol treatment. Our results confirm that the treatment with sorbitol induces apoptosis in K562 cells and they are committed to death after 30 min. Pro-apoptotic agents like sorbitol  could give more insights into CML biology and resistance to apoptosis and possibly give rise alternative treatments for CML.

Vav exchange factor block CD4-mediated signals inducing Bax increase and mitochondrial damage

Barbara Marinari, Loretta Tuosto and Enza Piccolella

Dept. Cell. and Develop. Biol. University of Rome “La Sapienza”, Rome Italy

 In the present study, we have aimed at demonstrating that CD4 may represent a critical turning point that governs the apoptotic and survival programs in T cells, without modifying the physical association with TCR/CD3 complex. To address this issue, we have explored the possibility that the activation of CD4 may transduce apoptotic signals unless these are neutralised by signaling effectors. Our data show that in Jurkat T cells CD4 engagement by Leu3a mAb, results in a rapid and strong increase of Lck kinase activity, subsequent alterations of mitochondrial membrane potential and apoptosis. Critical parameters are co-association of CD4/Lck with TCR/CD3 and upregulation of the proapoptotic protein Bax. Indeed, Leu3a-mediated Lck activation failed to induce apoptotic features in Jurkat cells either defective for TCR/CD3 or overexpressing the antiapoptotic protein Bcl-2. Furthermore, we demonstrate that Leu3a treatment of Jurkat cells overexpressing Vav results in the inhibition of mitochondrial damage and apoptosis accompanied with a significant decrease of Bax expression observed in apoptotic cells. Our evidence that Vav, a signaling molecule that cooperate with CD28 to boost TCR signals signals, counteracts Lck-mediated apoptotic pathways, suggests a novel role for costimulation in protecting T cell from CD4-mediated cell death.

The Inflammosome a new signaling complex involved in inflammatory caspases activation

Fabio Martinon  and Jürg Tschopp

Institute of Biochemistry; University of Lausanne; Switzerland

 In pro-apoptotic and inflammatory signaling pathways, the interaction between the different initiator units such as the death receptor Fas, the various adaptor proteins and caspases is primarily mediated by four structurally related protein–protein domains called death domain (DD),  death effector domain (DED), caspase-recruitment domain (CARD) and  the recently identified pyrin domain (PYD).

Interleukin (IL)-1b and IL-18 are produced via cleavage of their respective pro-forms by caspase-1 (and caspase-11 in mice).  The mechanism leading to the activation of these proinflammatory caspases is unknown.  NALP1 is a recently characterized Pyrin domain-containing protein sharing structural homology with NODs and the apoptosis-regulator Apaf-1.  Here we demonstrate that NALP1 simultaneously recruits and activates caspase-1 via the adaptor protein Pycard, and caspase-5 via its C-terminal CARD domain.  The two NALP-associated caspases form a highly active pro-IL1b processing enzyme. Inhibition of caspase-1 recruitment to NALP1 blocks pro-IL-1b processing induced by LPS.  Thus, NALP1, which is a member of a large protein family conserved from plants to animals, appears to constitute an important arm of innate immunity, linking bacterial infection to the caspase-mediated pro-inflammatory cytokine response.

TG2 ablation reduces neuronal death in a murine model of Huntington's disease

Pier Giorgio Mastroberardino (1), Roberta Nardacci (3), Francesca Bernassola (2), Vincenzo De Laurenzi (2), Gennaro Melino (2), Sandra Moreno (5), Flaminia Pavone (6), Serafina Oliverio (1), Laszlo Fesus (4) and Mauro Piacentini (1, 3).

1) Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

2) Department of Experimental Medicine, IRCCS IDI Biochemistry Unit, University of Rome "Tor Vergata", Rome, Italy.

3) Laboratory of Cell Biology and E.M, IRCCS “Lazzaro Spallanzani” Rome, Italy

4) Department of Biochemistry, University of Debrecen, Hungary.

5) Department of Biology-LIME, University “Roma Tre”, Rome, Italy.

6) Istituto di Psicobiologia e Psicofarmacologia CNR, Rome, Italy.

 By crossing Huntington’s disease (HD) R6/1 transgenic mice with “tissue” transglutaminase knock-out (TG2) mice we demonstrated that TG2-catalyzed crosslinking of proteins plays an important role in the neuronal death characterising this disorder in vivo. In fact, a dramatic reduction in cell death is observed in R6/1, TG2 null when compared with R6/1 transgenic mice. The reduction in cell death is paralleled by a significant improvement in motor performances of R6/1, TG2 null when compared with R6/1 mice. Since TG2 has been hypothesized to be involved in the assembly of intranuclear aggregates in Huntington’s disease, we investigated whether the NII assembly occurred in R6/1, TG2-/- mice. Our results show not only that TG2 is not directly involved in NII assembly in vivo, but that the formation of neuronal intranuclear inclusions is, surprisingly, potentiated in absence of the “tissue” transglutaminase, suggesting a complex role for TG2 in the regulation of the assembly of large nuclear protein complexes. Taken together these findings suggest an important role for the enzyme in the induction of cell death occurring during Huntington’disease pathogenesis.

Induction of Apoptosis or Necrosis in neuronal PC12 cells depending on the redox state of NO

Midori Kayahara1,2, Ursula Felderhoff-Mueser1, Jennifer Pocock3, Mary Kozma1, Martin N. Hughes2, Huseyin Mehmet1.

1 Department of Chemistry, King’s College London, Strand, London WC2R 2LS, U.K.

2 Weston Laboratory, Division of Paediatrics, Obstetrics and Gynaecology, Imperial College of Science, Technology and Medicine, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN,U.K.

3 Department of Neurochemistry, Institute of Neurology, University College London, 1 Wakefield Street, London WC1N 1PJ, U.K.

 Nitric oxide (NO) can exhibit either neurotoxic or neuroprotective effects. These results may be explained by the existence of two additional redox-related species, the nitrosonium cation (NO+) and the nitroxyl anion (NO-). We have studied the effects of the redox related species on nerve growth factor (NGF) dependent PC12 cells. At low concentrations, NO and NO+ donors protected against apoptosis induced by NGF withdrawal. Sodium trioxodinitrate, which releases NO-, on the other hand did not show any protective effect. At higher concentrations NO and NO+ both induced apoptosis, while NO- caused necrosis. Mitochondrial membrane potential was disrupted with kinetics parallel to cell viability studies, where NO+ induced effects with a shorter incubation time than NO. Pretreatment of PC12 cells with antioxidants before exposure to the free radicals reduced the toxicity of NO and NO- but not of NO+. Interestingly, the necrotic effect of NO- was attenuated on addition of potassium ferricyanide or TEMPO, due to the oxidation of NO- to NO. However, the resultant NO subsequently induced apoptosis in these cells, which could be prevented by addition of a broad-range caspase inhibitor, BOC-Asp. These results indicate that nitric oxide can exert a variety of effects on cell survival depending on the concentration and the redox state.

Regulation of Programmed Cell Death by Inhibitor of Apoptotsis Proteins (IAPs)

Rebecca Willson¹, Mark Ditzel¹, Lakshmi Goyal², Julie Agapite², Hermann Steller² and Pascal Meier¹

1) The Breakthrough Toby Robins Breast Cancer Research Centre, Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London SW3 6JB, UK; 2) Howard Hughes Medical Institute, Rockefeller University, New York, NY 1002

Members of the IAP family have been found to function as ‘guardians’ of the apoptotic machinery.  They block the activation of the intrinsic cell-death machinery by binding to and neutralizing pro-apoptotic caspases. Typically, IAPs contain two to three Baculovirus Inhibitory Repeats (BIR) and one RING finger.  BIR domains are important protein-protein interaction motifs that link IAPs to specific caspases.  To date, it is generally believed that the mechanism by which IAPs inhibit caspases is due to a steric blockade prohibiting access of caspases to their substrates.  However, our genetic and molecular studies indicate that this simplistic view is not correct.  We found that the mere physical interaction of IAPs with caspases is necessary but not sufficient to regulate caspases in vivo.  We identified the RING finger of DIAP1 as essential for the ability of DIAP1 to regulate apoptosis.  Flies with a single amino acid substitution in the RING finger domain of DIAP1 completely failed to regulate apoptosis induced by ectopic expression of RPR, HID and DRONC.  Surprisingly, the DIAP1 RING mutants suppress HID but enhance RPR-induced cell-death, indicating that RPR and HID function in different ways to induce apoptosis.  Collectively, our results suggest that the RING finger of IAPs is critically important for the anti-apoptotic activity of IAPs.

FLASH and Bub-1 bind to p73 and regulate its function.

Eliana Munarriz, Daniela Barcaroli, Maria Prencipe, Vincenzo De Laurenzi and Gerry Melino

Department of Experimental Medicine, University of Rome, Tor Vergata, Rome, Italy;

 The tumour suppresser protein p53 has been shown to belong to a family that includes two structurally related proteins, p63 and p73.  The complexity of the family has also been enriched by the alternative spliced forms of p63 and p73.  Although all this proteins share similar transcriptional functions and the ability to induce apoptosis,  each of them appears to play distinct role in development and tumor suppression. This hypothesis is supported by mice functionally deficient for that show no increased susceptibility to spontaneous tumorogenesis and exhibit profound defects including hippocampal dysgenesis, hydrocephalus, chronic infections and inflammation as well as abnormalities in pheromone sensory pathways. In order to identify new interacting proteins regulating p73 functions we performed a yeast two hybrid screening using the C-terminal domain of p73a including the oligomerization domain. After the analysis and classification of the positive clones by nucleotide sequencing we focused our attention on two protein partners of p73a called FLASH and Bub-1. FLASH (or FLICE-associated huge protein) was originally described as a Caspace-8 binding protein required for its activation during Fas- mediated apoptosis. The other, protein Bub-1 is a kinase, essential for the mitotic checkpoint function in response to spindle damage and required for the timing of the exit from mitosis. Ours results show that this proteins are able to interact with p73a in co-immunoprecipitation experiments when they are co-expressed in transient transfections and affect its ability to induce apoptosis.

Inhibition of glucose metabolism sensitizes tumor cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, anti-CD95 antibody and tumor necrosis factor-alpha

Cristina Muñoz-Pinedo, Carmen Ruiz-Ruiz and Abelardo López-Rivas

Instituto de Parasitología y Biomedicina, CSIC; Ventanilla 11, 18001 Granada, Spain

Tumours display a high rate of glucose uptake and glycolysis. We investigated how inhibition of glucose metabolism could affect death receptor-mediated apoptosis in human tumor cells. We show that both substitution of glucose for piruvate and treatment with 2-deoxyglucose, enhanced apoptosis induced by TNF-a, CD95 agonistic antibody and TNF-related apoptosis inducing ligand (TRAIL). Inhibition of glucose metabolism enhanced killing of myeloid leukemia U937, cervical carcinoma HeLa and breast carcinoma MCF-7 cells upon death receptor ligation. Caspase activation, mitochondrial depolarization and cytochrome c release were increased under these conditions. However, Bcl-2 overexpression prevented this death in MCF-7 cells. Human B lymphoblastoid cell line SKW6.4, which is a prototype for mitochondria-independent death receptor-induced apoptosis, was also sensitized to anti-CD95 and TRAIL-induced killing. Finally, we observed that glucose deprivation enhanced death receptor-triggered early activation of apical procaspase-8. These results suggest that the glycolytic pathway may be an important target for therapeutic intervention to sensitize tumor cells to non-toxic soluble death ligands or death ligand-expressing cells of the immune system.

Transcriptional regulation of the Human Bax gene promoter by E47

Pagliuca, Alfredo

 Basic helix loop helix (bHLH) proteins are transcription factors playing a key role in developmental processes of metazoans. Beyond their developmental role, bHLH are implicated in the control of cell cycle and in apoptosis. E47, a member of the E-protein family of bHLH, is a cofactor of tissue-restricted bHLH.E-proteins reduces cell proliferation as measured in colony formation assays. In contrast, dominant negative dnHLH proteins, also known as Id proteins, might interphere with master genes in cell cycle control, such as the Retinoblastoma protein pRb. We demonstrated that ectopic expression of E-proteins reduce cell proliferation by inducing apoptosis in different cell lines. Coexpression of E47 with bcl2 partially rescues this phenomenon. We then searched for genes that might be both transcriptionally activated by E47 and relevant targets of the bcl2 protein. The human bax gene promoter was previously identified and partially characterized as a target of the p53 protein. To test whether the Bax promoter is a relevant target for E47, we used a combination of functional and biochemical assays and found putative E-protein binding sites, named E-boxes,in a cluster in the 5’ UTR of the gene. Our results suggest that E47 is a positive regulator of Bax transcription.

INHIBITION OF ERYTHROID CELL GROWTH AND DIFFERENTIATION BY TWEAK/TWEAK-RECEPTOR SYSTEM

N. Felli, F. Pedini, E. Petrucci, U. Testa, C. Peschle, R. De Maria

Dept. of Hematology-Oncology Istituto Superiore di Sanità, Rome, Italy

 The interaction between CD95 and TRAIL receptors with their ligands contributes to the negative regulation of erythropoiesis, through caspases activation.This proapoptotic pathway is potentiated by interferon-gamma (IFN-gamma), a cytokine involved in anemia secondary to pathological inhibition of red cell production. TWEAK is a new member of the tumor necrosis factor (TNF) family, whose transcripts are present in a wide variety of human tissues. TWEAK is a weak inducer of apoptosis in transformed cells when administered in combination with interferon-gamma (IFN-gamma), and acts as a potent inducer of angiogenesis in vivo. To investigated whether TWEAK play a role in the homeostatic control of red cell production, we analyzed expression and function of TWEAK and TWEAK-receptors. As for CD95 ligand (CD178) and TRAIL, semiquantitative RT-PCR analysis showed that TWEAK is weakly expressed in early stages of erythroid differentiation and upregulated during late stages of erythroblast maturation. Both the proposed receptors for TWEAK (DR3 and Fn14) are expressed by immature erythroid cells. Exposure of erythroid progenitors to recombinant soluble TWEAK resulted in a significant inhibition of both erythroid cell growth and differentiation. This effect was counteracted by addition of zVAD, a wide-spectrum inhibitor of caspases, suggesting that TWEAK may contribute together with CD178 and TRAIL to the physiological regulation of erythropoiesis. Importantly, exposure of erythroid cells to IFN-gamma induced upregulation of TWEAK. Moreover,  IFN-gamma inhibition of erythroid maturation was completely abrogated by Fn14-Fc recombinant protein, indicating that TWEAK mediates anti-erythropoietic effects of IFN-gamma. Taken together, these findings suggest that TWEAK and its receptors contribute to the physiological and pathological regulation of erythropoiesis.

c-Myc-mediated apoptosis in p53-negative human melanoma cells.

Lucy T.C. Peltenburg, Elza C. de Bruin, Dorothea Meersma, Jillian de Wilde and Peter I. Schrier

Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands.

 We showed by transfection that the c-myc oncogene sensitizes the p53-negative human melanoma cell line IGR39D to apoptosis triggered by various stress inducers. We investigated whether sensitization by c-Myc is due to accelerated cytochrome c release. As compared to control IGR39D transfectants, cytosolic cytochrome c levels in c-myc transfectants are slightly elevated after irradiation. Unexpectedly, this occurs relatively late and does not precede the first signs of substrate cleavage, suggesting that cytochrome c release does not play a crucial role in irradiation-induced apoptosis of these c-myc transfectants. The observation that PARP cleavage is not blocked by the addition of 10 mM z-VAD-fmk but can be inhibited by the serine protease inhibitor AEBSF hints at the involvement of a caspase-independent mechanism. It is also remarkable that the basal cytosolic cytochrome c levels in IGR39D and its transfectants are very high compared to several other tumor cell lines and normal melanocytes. Apparently, these proliferating tumor cells are resistant to the presence of cytosolic cytochrome c. This protection might be accomplished by inhibitor of apoptosis proteins (IAPs), since the expression levels of XIAP and Survivin are elevated in IGR39D as compared to normal melanocytes. Further functional analysis will indicate which IAP is responsible for the protection against caspase activation.

The tumor-specific apoptosis inducing protein Apoptin in a transgenic mouse model

Alexandra Pietersen1,2, Maud Seelen2, Stefan Erkeland3, Sjaak Philipsen3, Mathieu Noteborn1,2

1Leiden University Medical Center, 2Leadd BV, Leiden, 3Erasmus University, Rotterdam, The Netherlands

 The viral protein Apoptin induces apoptosis in a large panel of mammalian tumor cells, whereas Apoptin is not able to induce apoptosis in cultured, primary diploid cells. To obtain a more detailed insight in the possible effects of Apoptin in normal cells we generated transgenic mice expressing Apoptin under the regulation of the H2kB promoter.

The mice develop normally and the transgene is transmitted in normal numbers. Apoptin RNA can be detected in multiple organs; protein is detectable in thymus, spleen and lung. Proteasome inhibition of Apoptin-transgenic splenocytes, however, dramatically increases the amount of protein. This instability of Apoptin protein has been confirmed in in-vitro experiments with normal human cells. To determine whether there is Apoptin-toxicity in the transgenics, we analyzed different T and B cell subsets by FACS. The first screening did not show any obvious deviations between wild-type and Apoptin mice. To show that the amount of Apoptin expressed is functional, we are currently transforming these cells, which should activate Apoptin to induce apoptosis. In the near future Apoptin transgenic mice will be crossed with a mouse model with a high lymphoma incidence to examine whether Apoptin can prevent or delay tumor formation.

GENETIC POLYMORPHISMS OF Fas (CD95) AND FasL (CD178) IN HUMAN LONGEVITY: STUDIES ON CENTENARIANS

MARCELLO PINTI 1, LEONARDA TROIANO 1, MILENA NASI 1, LAURA MORETTI 1, ELENA MONTERASTELLI 1, ANNALISA MAZZACANI 1, CHIARA MUSSI 2, CLAUDIO FRANCESCHI 3, GIANFRANCO SALVIOLI 2, ANDREA COSSARIZZA 1

1: Department of Biomedical Sciences, Chair of Immunology, University of Modena and Reggio Emilia, via Campi 287, 41100 Modena, Italy

2: Department of Internal Medicine, Chair of Geriatrics, University of Modena and Reggio Emilia, Ospedale Estense, 41100 Modena, Italy

3: Department of Experimental Pathology, Chair of Immunology, University of Bologna, via San Giacomo 12, 40126 Bologna, Italy

 Apoptosis plays a crucial role in immunosenescence, as also evidenced by the increased expression of Fas in lymphocytes from aged people. However, little is known about the genetic regulation of Fas and its ligand, FasL. We have studied their polymorphisms in 50 centenarians and 86 young donors living in Northern Italy. The first Fas polymorphism, at position -670, has in Caucasian a heterozigosity of 51%; the second, at -1377 position, has the wild type allele (G) with a very high frequency (83%) respect to the mutant allele. Genotype and allele distribution for both polymorphisms were similar in controls and centenarians. Similar results were found as far as two FasL polymorphisms (IVS2nt-124 and IVS3nt169) are concerned. On the whole, our data suggest that Fas and FasL polymorphisms, as well as their haplotypes, are unlikely to be associated with successful human longevity.

Rac is Involved in the Apoptotic Response of N1E-115 Neuroblastoma Cells to Staurosporine, but not to Ceramide

Mary Kozma1, Robert Kozma2, Grisha Pirianov1, Anthony Best2, Louis Lim2 and Huseyin Mehmet1

1Weston Laboratory, Division of Paediatrics, Obstetrics and Gynaecology, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, DuCane Road, London W12 0NN, U.K.

2Department of Neurochemistry, Institute of Neurology, University College London, 1 Wakefield Street., London WC1N 1PJ, U.K.

 In the murine neuroblastoma cell line, N1E-115, apoptotic concentrations of staurosporine (SSP), caused rapid changes in actin-dependent cell morphology, indicative of Rac1/Cdc42H activation, whilst treatment with cell permeable ceramide triggered apoptosis without these actin alterations. Microinjection of dominant negative Rac (T17NRac) into wild type N1E-115 cells inhibited these SSP induced reorganisation of actin and protected from apoptosis, whilst ceramide induced death was unaffected.  Similarly, studies using stably transfected N1E-115 cells indicated that over-expression of wild type Rac1 increased cell sensitivity to apoptosis triggered by SSP or serum-withdrawal, but not to ceramide. Moreover, transfectants expressing Rac inhibitory constructs (T17N Rac and a Rac GTPase activating protein (GAP)) did not confer protection from ceramide induced apoptosis but were protected against serum-withdrawal or SSP-induced apoptosis. Finally, over-expression of either wild type Rac1 or the inhinitory mutant T17N Rac1, both resulted in increased phosphorylation of Jun N-terminal kinase (JNK) suggesting that JNK activation can be dissociated form apoptosis. These data demonstrate that Rac1 is required for apoptosis in selected pathways only and that JNK activity is not correlated with either death or survival signalling through Rac. We conclude that other downstream

The role of apoptosis in chemotherapy response in vivo

Farzaneh Pirnia1 ,Stefan Frese2, Daniel Betticher1, and Markus M Borner1.  1Institute of Medical Oncology, University of Bern, Inselspital; 2Departmant of clinical research, University of Bern, 3010 Bern, Switzerland.

A great problem in oncology research is that we have to rely on highly artificial systems such as in vitro assays to examine the action of new drugs. Our goal is to establish a new culture system to examine the action of anticancer agents. We know from cell cultures that anticancer agents induce apoptosis.  Not much is know whether this is also the case in the human body. Thus we propose here to use an ex vivo culture system, which has been developed in our lab, for this purpose. Tumor tissue is taken at the occasion of an operation.  It is cut into small pieces, which is feeded by diffusion. Then tumor pieces are grown on a support matrix to allow attachment, which is important for the survival of solid tumors.  Then tissues were treated with chemotherapy in absence or presence of ZVAD-fluoromethyl ketone, a general caspase inhibitor. The metabolic activity was measure with Alamar blue and apoptosis was quantified by means of Hoechst nuclear staining and activated caspase-3 staining. Chemotherapy induced apoptosis and decreased metabolic activity within 3 days of treament in ex-vivo tissue culture assay. ZVAD inhibited drug induced nuclear fragmentation and caspase activation. This assay is an interesting research tool for examining the effect of therapeutic interventions on human ex- vivo tissue. This assay has a potential to be used as a predictive test for tumor response in vivo.

mRNA degradation: a general, early apoptotic event induced by a specific pathway distinct from that leading to DNA degradation

M. Julieta del Prete1, Maria S. Robles1, Ana Guío1, Carlos Martínez-A.1,

Manuel Izquierdo1,2 and Jose A. Garcia-Sanz1,4

1)DIO, Centro Nacional de Biotecnología (CNB-CSIC), E-28049, Madrid, Spain and, 2) IBGM-CSIC, Facultad de Medicina Universidad de Valladolid, E-47005 Valladolid, Spain

 The fate of cellular mRNAs was analyzed in several cell lines of lymphoid origin, in which apoptosis had been induced by different mechanisms, including direct CD95 crosslinking, activation-induced cell death, okadaic acid, and growth factor deprivation. Cytoplasmic mRNAs are specifically degraded as part of the early apoptotic response. Degradation is not species-restricted, and is independent of the cell line, the apoptotic stimulus, the intrinsic  half-life of the mRNAs, and the transcriptional status of the gene (constitutive or inducible). mRNA degradation precedes DNA fragmentation and correlates with the appearance of phosphatidylserine in the outer cell membrane. In addition, apoptosis-induced mRNA degradation is an active process that can be dissected from other apoptotic hallmarks (annexin V, DNA and PARP degradation), suggesting that apoptosis-induced mRNA degradation it is controlled by a distinct signalling pathway. Furthermore,  specific mRNA degradation could also be demonstrated in vivo upon antigen-induced thymocyte apoptosis. In conclusion, degradation of cellular mRNA may be considered a hallmark of apoptosis. The implications of these data in both the physiology of apoptosis and profiling experiments will be discussed.

TRAF4 deficiency results in defective neurulation

Catherine H. Regnier, Valerie Kedinger* and Marie-Christine Rio*

INSERM U255, 15 rue de l'Ecole de Medecine, 75006 PARIS, France and *IGBMC/INSERM U184, BP 163, 67404 ILLKIRCH, France.

 Tumor Necrosis Factor (TNF) Receptor-Associated Factors (TRAFs) are intracellular adaptor proteins involved in the early steps of signal transduction pathways induced by inflammatory cytokines like TNF or interleukin-1 (IL-1) which, in turn, lead to the activation of a common set of transcription factors including NF-kB and AP-1. Among the six members of the mammalian TRAF family, five are recruited, directly or indirectly, to various receptors of the TNFR superfamily and IL-1R/Toll family.

The fourth member of the TRAF protein family, TRAF4, was cloned by differential screening of breast-derived metastases. Despite high structural similarity to the other TRAF proteins, TRAF4 is an unusual member of the family since 1) it does not associate directly to any known cytokine receptors; 2) it does not activate NF-kB or AP-1 in vitro; 3) it is specifically overexpressed in breast carcinomas due to TRAF4 gene amplification; 4) it is the most similar to Drosophila dTRAF1. These observations suggest an important evolutionary conserved signaling function for TRAF4. To address the, thus far, elusive physiological role of TRAF4 in mammals, we have generated traf4-deficient mice by gene disruption. The traf4 gene mutation led to partial embryonic lethality and to various developmental defects including altered neurulation.

Modulation of ER stress-induced apoptosis by Bcl-2 variants

Claus Reimertz (1), Donat Kögel (1), David W Andrews (2), Jochen H Prehn (1)

(1) IZKF NWG, University of Muenster, Muenster 48149, Germany

(2) Department of Biochemistry, McMaster University, Hamilton, L8N 3Z5, Canada

 Endogenous Bcl-2 inserts into different intracellular membranes where it may exert distinct anti-apoptotic activities. We stably transfected MCF7 cells with wild-type Bcl-2 (wt) or mutant versions of Bcl-2 exclusively targeted to mitochondria (Bcl-2-mito) or the endoplasmatic reticulum (Bcl-2-ER). Cells overexpressing wt or mutant Bcl-2 versions all showed upregulation of the known ER stress target genes Bip and HERP after treatment with the ER stressors tunicamycin (TU) or thapsigargin (TH). Subsequently, ER-stress-induced cell death was determined by quantifying PI uptake of cells using FACS analysis. In cells treated with low TU concentrations (1 and 3 µM), we observed significant protection of ER-targeted Bcl-2 (rank order of cell death: neo>Bcl-2-mito>Bcl-2-wt>Bcl-2-ER). We did not see significant differences in the degree of protection between the Bcl-2 versions in cell treated at a higher TU concentration (10µM), suggesting the activation of additional cell death pathways. In contrast, protection against TH-induced cell death could not be detected with the ER-targeted Bcl-2 mutant (rank order of cell death with 1 or 3 µM TH: neo=Bcl-2-ER>Bcl-2-wt=Bcl-2-mito). Bcl-2-ER potentiated cell death in cultures exposed to 0,1µM TH. Our data indicate that ER-targeted Bcl-2 potently modulates ER-stress induced apoptosis, and suggests the existence of two distinct ER stress pathways.

Alphaviruses induce apoptosis via a mitchondria-dependent pathway

Céline Rhême1, Denis Grandgirard1,2, Christian Gianinazzi2, Laurent Monney3, Marcel R. Michel2 and Christoph Borner1

1Institute of Molecular Medicine, Albert-Ludwigs-University, D-79106 Freiburg, 2Institute of Infectious Diseases,University of Berne, CH-3010 Berne,

3Center of Neurological Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston MA  02115, USA

 During apoptosis cytochrome c is released from mitochondria into the cytoplasm where it participates in the activation of the executioner caspase-3 via caspase-9. We have previously reported that infection with the alphaviruses Semliki Forest (SFV) and Sindbis (SB) leads to caspase-3 activation and subsequent apoptosis, but the underlying mechanisms remain elusive. Here we show that SFV infection causes the efflux of cytochrome c followed by caspase-9 and caspase-3 activation. This pathway is crucial as dominant-negative caspase-9 expression effectively blocks alphavirus-induced apoptosis. On the other hand, caspase-8, and thus the death-receptor-induced pathway does not seem to be involved as caspase-8 activation occurs at a late event downstream of caspase-9.  Moreover, we show that in certain cells Bcl-2 is unable to block SFV-induced cytochrome c release confirming our previously published results that SFV activates a caspase(s) which proteolytically inactivates Bcl-2. Our results suggest that alphaviruses use a mitochondria-dependent pathway for apoptosis induction that is not necessarily blocked by Bcl-2.

"TISSUE" TRANSGLUTAMINASE COMMITS CELLS TOWARDS APOPTOSIS BY TARGETING MITOCHONDRIA

Carlo Rodolfo (1), Maria Grazia Farrace (1), Lucia Piredda (1), Paola Matarrese (3), Fabiola Ciccosanti (2), Anna Maria Giammarioli (3), Walter Malorni (3) and Mauro Piacentini(1, 2).

(1) Department of Biology, University of Rome "Tor Vergata" Rome, Italy, (2) Cell Biology and E.M. Laboratory "L. Spallanzani"-IRCCS, Rome, Italy, (3) Laboratorio Ultrastrutture, Istituto Superiore di Sanità, Rome, Italy.

 "Tissue" transglutaminase (TG2) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationship existing among TG2 expression, apoptosis and mitochondrial function. Two mammalian cell lines in which TG2 expression was either driven by a constitutive or a Tet-off inducible promoter were used. Time course analyses revealed that, when expressing high TG2 levels, both cell lines are "committed" to apoptosis. Morphological analysis of the cells overexpressing TG2 showed marked differences in the ultrastructure of mitochondria even in the absence of any apoptotic stimuli. These modified ultrastructural characteristics (i.e. major matrix electron density and cristae arrangement) were associated with specific functional features: i) constitutively hyperpolarized mitochondria, ii) increased reactive oxygen intermediates production. iii) depletion of intracellular GSH levels. TG2 has been found to be localised on mitochondria where it interacts and crosslinks Bcl-2 family members and other protein substrates. Taken together, these results indicate that TG2-dependent hyperpolarization of mitochondria is an important early step towards the commitment to apoptosis which is still compatible with cell viability and precedes both PTP opening and release of cytochrome c.

Activation and trafficking of the tumor-specific apoptosis-inducing protein, Apoptin

J. L. Rohn, A. Danen-Van Oorschot, Y.-H. Zhang, R. Leliveld, M. Seelen, C. Molenaar, J. P. Abrahams, H. Tanke and M. H. M. Noteborn, Leadd BV and Leiden University Medical Center, Leiden, The Netherlands

 Apoptin, a protein derived from the chicken anemia virus (CAV), has the remarkable characteristic of inducing apoptosis in tumor or transformed cells while leaving healthy, primary cells unscathed. This difference in behavior is reflected in the localization of Apoptin; namely, in tumor cells, it is present primarily in the nucleus, whereas in normal cells, it is sequestered harmlessly in the cytoplasm. Because Apoptin may utilize a cellular pathway that is not only tumor-specific, but tumor-essential, elucidating the mechanism of its activation, trafficking and execution could yield crucial insights into the nature of cellular transformation. We are using a structure/function analysis strategy involving deletion and point mutants, as well as microinjected recombinant proteins, to explore these issues. These studies show that nuclear localization is necessary but not sufficient for apoptosis, but that a cytoplasmic activation step is required for full apoptosis activity. Intriguingly, Apoptin contains two autonomous death domains in either half of the protein. Inhibitor studies show that, although Apoptin can localize to DNA, de novo protein synthesis is not required for Apoptin-induced apoptosis in tumor cells, nor does de novo protein synthesis enable activity in normal cells. The most recent, exciting data of these ongoing studies will be presented.

A.I.R. (Apoptosis- Induced and Regulator), a novel gene that regulates apoptosis

Maria Fiammetta Romano, Antonello Petrella, Annalisa Lamberti, Rita Bisogni, Salvatore Venuta and Maria Caterina Turco

Dipartimento di Biochimica  e Biotecnologie Mediche, “Federico II” University, Napoli, Italy

Dipartimento di Scienze Farmaceutiche, Università di Salerno, Italy

Dipartimento di Medicina Sperimentale e Clinica, Università di Catanzaro, Italy

 The activity of NF-kB/Rel transcription factors modulates apoptotic response in a variety  of normal and neoplastic cell types. In Jurkat cell transfectants with modulated levels of NF-kB/Rel activity, we  identified, by a differential display approach, a novel gene, whose expression is induced by TNF-a only in cells with low NF-kB/Rel nuclear levels. In database mining, the full- lenght cDNA (3564 bp) showed, in a 109 bp segment, a 100% homology with an est sequence identified in neuroblastoma cells (Garnier et al., J.Neurosci, 1997). We named this gene AIR (Apoptosis- Induced and Regulator). AIR mRNA is detectable, as an approx. 3500 bp band in Northern blot, in Fas- stimulated Jurkat cells and in a number of cell lines of different origin (HeLa epithelial, U937 myeloid, and SH-SY-5Y neuroblastoma cell lines) or human normal primary peripheral blood mononuclear cells (PBMC) treated with the oxidative stress inducer diethylmaleate (DEM). A rabbit polyclonal antiserum raised against three designed epitopes of Air putative protein immunoprecipitated an approx. 20 kD protein from Fas- stimulated Jurkat cell extract. To analyse the involvement of Air protein in the apoptotic process, we tested the effect of 25mer antisense oligonucleotides (ODNs), able to impair Air protein production, on cell apoptosis. In Jurkat cells incubated with either DEM or anti- Fas monoclonal antibody, apoptosis, measured as percentage of hypodiploid cells by propidium iodide staining and flow cytometry, was >50% inhibited in cultures with the antisense, but not in those with a control nonsense, oligo . Accordingly, we found reduced levels of caspase-3 activity in extracts from Fas- stimulated Jurkat cells incubated with the antisense, compared to those of cells incubated without or with the control oligonucleotide. The antisense ODNs significantly inhibited DEM- induced  apoptosis also in SH-SY-5Y cells and in PBMC. These results show that: AIR gene expression is stimulated by apoptosis induction; Air protein regulates apoptosis; modulation of Air protein levels and/or activity can influence apoptosis in human primary cells.

Role of Bcl-2 subcellular localisation in radiation-induced and CD95/Fas/APO-1-induced apoptosis.

Justine Rudner 1, Wilfried Budach 1, Klaus Schulze-Osthoff 2, Michael Bamberg 1, Claus Belka 1

1 Department of Radiation Oncology, University of Tuebingen (Germany), 2 Department of Immunology and Cell Biology, Institute of Experimental Dermatology, University of Muenster (Germany)

 The anti-apoptotic protein Bcl-2 is expressed in membranes of mitochondria and endoplasmic reticulum and mediates resistance against a broad range of apoptotic stimuli. Although several mechanisms of Bcl-2 action have been proposed, its role in different subcellular organelles remains elusive. We analysed the function of Bcl-2 targeted specifically to certain subcellular compartments in Jurkat lymphoma cells. Bcl-2 expression was restricted to the outer mitochondrial membrane by replacing its membrane anchor with the mitochondrial insertion sequence of ActA (Bcl-2/MT) or the ER-specific sequence of cytochrome b5 (Bcl-2/ER). Additionally, cells expressing wildtype Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/DTM) were employed. Apoptosis induced by ionising radiation or by stimulation of the death receptor CD95/Fas/APO-1 and the mitochondrial membrane potential (DYm) was quantified using FACS Calibur flow cytometer. Furthermore activation of different caspases was analysing by western blotting. Neither mitochondrial nor endoplasmic Bcl-2 conferred protection against CD95-induced apoptosis and mitochondrial damage. Interestingly, Bcl-2/WT and Bcl-2/MT as well as Bcl-2/ER strongly inhibited radiation-induced apoptosis, DYm breakdown and caspase activation, whereas Bcl-2/DTM had completely lost its anti-apoptotic effect. Here we show for the first time that not only mitochondrial Bcl-2 but also ER-targeted Bcl-2 interfered with mitochondrial DYm breakdown and caspase-9 activation after irradiation. Our finding therefore indicates the presence of a crosstalk between both organelles in radiation-induced apoptosis.

Mechanisms of P-glycoprotein-mediated inhibition of Fas-induced cell death.

Astrid A. Ruefli, Kellie Tainton, Mark J. Smyth, and Ricky W. Johnstone

Peter MacCallum Cancer Institute, Melbourne, Australia

 We have previously shown that the ATP-dependent efflux pump and drug transporter P-glycoprotein (P-gp) can specifically inhibit Fas-induced caspase-activation and cell death. Although inhibitors of P-gp function could reverse inhibition of both Fas-mediated caspase-activation and cell death, the molecular mechanisms underpinning P-gp-mediated resistance to Fas-induced cell death and caspase activation remain unknown.  Therefore, we were interested in undertaking structure-function assays to determine whether the efflux function of P-gp was necessary to confer resistance to Fas-induced cell death.  In addition, we sought to dissect the point(s) at which P-gp could exert its inhibitory effect on the Fas-receptor signaling pathway.  To study the effects of functionally mutated P-gp on Fas-mediated cell death we produced retrovirally transduced CEM cells that overexpress P-gp in the complete absence of drug selection.  We demonstrate that functional P-gp is required for inhibition of Fas-mediated apoptosis, as mutation of regions necessary for ATPase and/or efflux function abolish inhibition of Fas-induced apoptosis. Importantly, these studies reveal that P-gp does not interfere with the formation of the DISC, as equivalent amounts of pro-caspase-8 were associated with the Fas-receptor.  Rather, we have demonstrated that P-gp inhibits the processing and activation of caspase-8, the most proximal caspase in the Fas-receptor caspase-cascade.  Our data demonstrate that in addition to its function as an efflux pump, P-gp also acts as an anti-apoptotic protein to inhibit Fas-induced cell death and caspase activation.  Therefore, P-gp may act at two levels to confer multidrug resistance, firstly by inhibiting intracellular drug accumulation and secondly, by inhibiting activation of key cell death pathways utilized by chemotherapeutic drugs to induce cell death.

Up-regulation of TRAIL Receptors in breast tumor cells upon genotoxic treatment: role of p53 protein

Authors: Carmen Ruiz de Almodóvar, M. Carmen Ruiz-Ruiz and Abelardo López-Rivas

Affiliation: Institute of Parasitology and Biomedicine, CSIC, Granada, Spain

 Genotoxic treatments have been reported to up-regulate the expression of death receptor CD95 in breast cancer cells, thereby sensitising these tumor cells to CD95-mediated apoptosis. TRAIL (APO-2 ligand), a member of the TNF family, is a type II transmembrane protein which provokes apoptosis mainly in tumor cells. TRAIL has four specific receptors of which TRAIL-R1, -R2 and -R4 are type I transmembrane protein whereas -R3 is a glycosylphosphatidyl inositol-linked protein. In this report, we have studied the expression of TRAIL receptors in different breast tumor cell lines upon genotoxic treatment with the anti-tumor drug doxorubicin. TRAIL-R1, TRAIL-R2 and TRAIL-R3 mRNA and proteins were up-regulated in p53wt breast tumor cells but not in p53mut cells following treatment with the drug. To examine the role of p53 in drug-induced elevation of TRAIL receptor expression we used MCF-7 cells stably expressing the human papillomavirus protein E6, that causes p53 degradation. In these cells treatment with doxorubicin failed to induce the up-regulation of TRAIL receptors. Furthermore, in MCF-7 cells over-expressing a temperature sensitive p53 (Val135), culture of cells at the permissive temperature was sufficient to elevate the expression of the various TRAIL receptors. Finally, treatment with doxorubicin sensitised MCF-7 cells to TRAIL-induced apoptosis.

SINERGISTIC KILLING OF HUMAN 

BREAST TUMOR CELLS BY INTERFERON-g AND TUMOR NECROSIS FACTOR-RELATED APOPTOSIS-INDUCING LIGAND.

C. Ruiz-Ruiz and A. López-Rivas.

Institute of Parasitology and Biomedicine, CSIC. Granada. Spain

 Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO-2L) induces apoptosis in a variety of tumor cells upon binding to death receptors TRAIL-R1 and TRAIL-R2. We have studied the sensitisation by IFN-g to TRAIL-induced apoptosis in the breast tumor cell lines MCF-7 and MDA-MB231. IFN-g promoted TRAIL-mediated activation of caspase-8, Bid degradation, Bax translocation, cytochrome c release from mitochondria and activation of caspase-9 in these cell lines. No changes in the expression of TRAIL receptors were observed upon IFN-g treatment. Overexpression of Bcl-2 in MCF-7 cells inhibited IFN-g-induced sensitisation to TRAIL-mediated cell death. Interestingly, TRAIL-induced mitochondria-independent apoptosis in caspase-3-overexpressing MCF-7 cells was also clearly potentiated by IFN-g. Therefore, our results suggest that IFN-g facilitates TRAIL-induced activation of mitochondria-regulated as well as mitochondria-independent apoptotic pathways in breast tumor cells.

Unveiling the Apolipoprotein J signalling network: implications for cell survival, proliferation and oncogenesis. 

Gabriella Pagnan, Giorgia Santilli and Arturo Sala.

The Institute of Child Health, Molecular Haematology and Cancer Biology Unit, London UK; Department of Oncology and Neurosciences, Sect. Of Medical Oncology, Chieti, Italy.

 Apolipoprotein J (clusterin) is a heterodimeric secreted protein that is ubiquitously expressed and induced in response to cellular stress, tissue injury and apoptotic stimuli. Numerous studies have revealed that ApoJ is involved in protecting cells from apoptosis. Recently we have described ApoJ as the product of a gene regulated by the B-MYB oncoprotein in neuroblastoma cells. Here we show that secreted ApoJ mediates survival of neuroblastoma cells via interaction with a LDL-like receptor, since its effect is abrogated by the LDL-receptor antagonist RAP. ApoJ effects on neuroblastoma cell proliferation are associated with induction of MYC protein, possibly through activation of the Beta-catenin/TCF pathway. Hence, ApoJ is a novel neuroblastoma survival factor whose overexpression in advanced stages of the disease may be associated with resistance to therapeutic intervention.

Molecular basis of mitochondrial targeting of Bax

Anna Schinzel, Thomas Kaufmann, Jorge A. Martinalbo and Christoph Borner

Institute of Molecular Medicine, Albert-Ludwigs-University, D-79106 Freiburg

 The pro-apoptotic Bcl-2 family member Bax has been proposed to be a predominantly cytoplasmic protein which translocates to the mitochondrial membrane in response to certain apoptotic stimuli. The crystal structure of Bax has provided some clue to this mechanism since the hydrophobic C-terminal tail of Bax is folded back into the molecule and may thus not be available for membrane targeting in surviving cells. Once unleashed, it is believed that the last 19 amino acids of this tail (C19 tail) are sufficient for mitochondrial targeting but only when they aquire a targeting-competent conformation (which can be mimicked by deleting Ser184 or changing Ser184 into Ala or Val). Here we show that already in surviving cell lines endogenous Bax is equally distributed between the cytoplasm and the mitochondrial membrane and thus requires an additional regulatory mechanism than C-terminal backfolding for its subcellular distribution. Moreover, we present evidence by immunocytochemistry using various Bax mutants and EGFP-fusion constructs that it is not the conformation of the C19 tail but additional four amino acids ahead of this tail which are crucial for mitochondrial targeting.

RELATIVE CONTRIBUTION OF THE MITOCHONDRIA AND DEATH

RECEPTOR SIGNALING PATHWAYS IN CARDIAC APOPTOSIS DURING

ISCHAEMIA VERSUS REPERFUSION INJURY

TM Scarabelli*, A Stephanou*, Richard A Knight and

David S Latchman*.

*Institute of Child Health and Great Ormond Street

Hospital, University College London, London UK

National Heart and Lung Institute, Imperial College,

London, UK

 Apoptosis is a form of cell suicide contributing together with necrosis to the cardiac cell loss following ischaemia/reperfusion injury (IRI). The apoptotic cascade can be initiated either by the intrinsic pathway (IP), which follows mitochondrial damage and results in activation of caspase 9 (C9), or the extrinsic pathway (EP), which is death receptor-mediated and leads to activation of caspase 8 (C8). In the present study, carried out in the isolated rat heart exposed either to ischaemia (I) alone or I followed by reperfusion (R), cleavage of caspase 9 (C9) was observed primarily in endothelial cells (EC). Conversely, cleaved C8 is only increased in CM, where it progressively rises throughout R. Consistent with this finding, the addition of a specific C9 inhibitor to the perfusate before I prevented endothelial apoptosis, whilst the pre-ischaemic infusion of a specific C8 inhibitor affected only myocardial apoptosis. Additionally, we investigated the role of BID in mediating the cross talk between EP and IP. In the rat heart exposed to I/R, BID processing is observed only during R and is mediated by C8. Processing of BID to produce tBID then sustains the mitochondrial injury and perpetuates C9 activation.

Ionizing radiation but not cancer drugs causes G2/M cell cycle arrest and failure to activate the apoptotic pathway in breast carcinoma cells

Klaus Schulze-Osthoff1, Ingo H. Engels1, Thorsten Dunkern2, Bernd Kaina2, Reiner U. Jänicke1

1Department of Immunology and Cell Biology, University of Münster, 2Institute of Toxicology, University of Mainz, Germany

 There is considerable evidence that ionizing radiation (IR) and anticancer drugs mediate apoptosis through the intrinsic pathway via the release of cytochrome c. Here we show that MCF-7 cells, both in the presence and absence of caspase-3, undergo caspase-dependent apoptosis following treatment with anticancer drugs, but not after exposure to IR. Re-expression of caspase-3 restored DNA fragmentation and fodrin cleavage following drug treatment, but it did not alter the radiation-resistant phenotype. In contrast to anticancer drugs, IR failed to induce the intrinsic pathway in MCF-7/casp-3 cells, an event readily observed in IR-treated HeLa cells. Although IR-induced DNA strand breaks were repaired with similar efficiencies in all cell lines, cell cycle analyses revealed a persistent G2/M arrest in MCF-7 but not HeLa cells. Interestingly, the relieve of the G2/M arrest restored apoptosis sensitivity of IR-treated MCF-7 cells. Thus, our data suggests that radiation resistance of MCF-7 cells is not caused by the lack of caspase-3, but the failure of IR to activate the intrinsic death pathway. We propose (1) different signaling pathways are induced by anticancer drugs and IR, and (2) IR-induced G2/M arrest prevents the generation of an apoptotic signal. Current studies are investigating molecular mechanisms of how the G2/M arrest interferes with the mitochondrial pathway.

Missing-self Recognition by Perforin Deficient NK cells.

Robert Wallin °#, Valentina Screpanti *, Alf Grandien * and Hans-Gustaf Ljunggren °#.

* Department of Immunology, Wenner-Gren Institute, University of Stockholm, Stockholm, Sweden; ° Microbiology and Tumor Biology Center, Karolinska Institute,

Stockholm, Sweden; and  # Center for Infectious Medicine, Department of Medicine, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden

 NK cells provide a line of defense against tumors and virus-infected cells that have lost the expression of one or more MHC class I isoforms. It has been previuosly postulated that cytotoxicity mediated through the perforin pathway is solely responsible for the rejection of NK-sensitive tumors. We have recently demonstrated that death receptor-mediated apoptosis has a more prominent role in the clearance of NK-sensitive tumors than previously suggested. Moreover We have demonstrated that NK cells can directly mediate the upreguation of Fas on otherwise Fas-negative targets, and consequently kill them by inducing FasL-induced apoptosis. Here, we investigate whether perforin-deficient NK cells can discriminate between MHC class I expressing and deficient cells, moreover we study the mechanisms for the upregulation of Fas on target cells induced by NK cells. We show that MHC class I deficient but not MHC class I intact bone marrow cells are rejected by NK cells upon transplantation to perforin deficient mice. Moreover, MHC class I protects tumor cells from being rapidly eliminated by NK cells in the lungs of perforin deficient mice. In addition, we demonstrate that the inhibition of Fas translocation to the cell surface of NK cells is mediated by MHC class I-dependent inhibitory signals.

RESPONSE IN RASF IN VITRO AND IN VIVO AND INVESTIGATION OF THE APOPTOSIS RESISTANCE AND PROLIFERATION OF RASF IN VITRO

Christian A. Seemayer1, Veronika Řiho_ková1, Stefan Kuchen1, Michel Neidhart1, Peter Kuenzler1, Elena Neumann2, Martin Pruschy3, Thomas Pap1, Ulf Müller-Ladner2, Renate E. Gay1, Steffen Gay1

1Ctr Exp Rheum, Dept Rheum, Univ Hosp Zürich, Switzerland

2Dept Int Medicine I, Univ Regensburg,  Germany

3Radiooncology, Dept Oncology, Univ Hosp Zürich, Switzerland

 To analyze the function of p53 in rheumatoid arthritis synovial fibroblasts (RASF) in vitro and in vivo and to investigate whether activation of p53 is the cause or consequence in the destructive process mediated by RASF. Moreover, we wanted to determine the apoptosis resistance and the cell proliferation of RASF.

Synovial tissues from  RA and controls as well as isolated RASF were stained with DO7 anti-p53 antibodies using immunohistochemistry or immunofluorescence. To test in vitro the functional response to p53, RASF were irradiated with 10 Gy X-rays and analyzed by immunofluorescence. To analyze the in vivo expression of p53 at sites of invasion, RASF and controls were co-implanted with human articular cartilage in the SCID mouse co-implantation model of RA. The invasion score and the number of p53 expressing cells were evaluated. In addition, RASF and control fibroblasts were treated in vitro with apoptosis inducing agents such as staurosporine and ionizing irradiation. The occurrence of apoptosis was measured time dependently by DAPI staining assessing the nuclear morphology. Furthermore, in cultured RASF the growth curves and proliferation were investigated by propidium iodide staining and consecutive FACS analysis. P53 was expressed in RA synovial tissues in an average of 2% of the cells. Correspondingly, the rate of expression of p53 in RASF in vitro was low, but inducible by ionizing irradiation. P53 negative cultured RASF (<5%) revealed, when invading articular cartilage, a p53 signal in the nucleus in 20% of the cases, suggesting that p53 is induced in some RASF during the invasive process in SCID mice. In terms of the apoptosis resistance no major differences were found between RASF, OASF and normal skin fibroblasts. The cell growth and proliferation were in all investigated types of fibroblasts comparable low, but much lower than in SV40 transformed cells and tumor cells. These data indicate that p53 does not appear to be functionally altered in the majority of RASF. Moreover, it could be induced in vitro and in vivo during the invasive process. This suggests that the activation of p53 is rather a consequence than a cause of the destructive process driven by RASF. From these data we conclude that RASF do not behave as tumor cells with respect to the response of p53, the resistance of apoptosis and the proliferation of cells.

The role of XIAP and DIABLO/smac in apoptosis

Silke J.H., Ekert P.G., Verhagen A.M. and Vaux D.L.

The Walter and Eliza Hall Institute, Post Office, Royal Melbourne Hospital, Victoria, 3050

 The Inhibitor of Apoptosis Proteins (IAPs) regulate programmed cell death in a variety of organisms. XIAP contains 3 motifs of approx. 70 amino acids called Baculoviral IAP Repeats (BIRs). Although the molecular details of the functions of XIAP have been extensively characterised in vitro, studies of the molecular functions in vivo are few. Using an extensive selection of point mutants that interefere with individual functions of XIAP but retain the structure of the wild type protein, we have sought to determine the relative importance of each of the individual functions in regulating cell death in vivo. Using individual mutants that interfere with XIAPs ability to bind to DIABLO/Smac, HtrA2/Omi, caspase-9 and caspase-3 and combinations thereof we have dissected the essential functions of XIAP in inhibibiting cell death. Surprisingly, individual mutants that interefere with XIAPs ability to inhibit DIABLO/smac, HtrA2, caspase-9, caspase-3 are all able to inhibit cell death. This study also demostrates for the first time the importance of IAP antagonism by DIABLO/smac in a UV/etoposide induced death model. These results imply that IAPs are present in sufficient quantaties to antagonise caspase induced cell death and must be removed by antagonists such as DIABLO/smac in order for cell death to proceed.

Breaking tumour tolerance; Fas ligand induces an anti-tumour response to melanoma antigens

Katharina Simon, Awen Gallimore, Emma Jones and Gavin Screaton

Fas ligand (FasL) is a well characterized apoptosis inducer. It is thought to be responsible for immune privilege by killing infiltrating lymphocytes and inflammatory cells. However, when tumour or transplants express FasL, rejection is observed as a consequence of pro-inflammatory functions of FasL. Here we show that FasL elicits tumour immunity in a murine melanoma model. Tolerance to melanocyte differentiation self-antigens is broken in protected mice. We show that melanoma expressing FasL induces maturation of dendritic cells in vitro. Furthermore we will discuss our data on the correlates  of tumour immunity. This study indicates that FasL can be used as an adjuvant in tumour vaccination to elicit an efficient anti-tumour response--

A CD95-DEFICIENT CELL LINE RESISTANT TO DEATH RECEPTORS ACTIVATION AND OTHER APOPTOTIC  SIGNALS

A.A.Sokolovskaya 1, D.Yu. Blokhin 1, A.D.Mikhailov 1,2, T.N.Zabotina 1, J.E.Eriksson 2, A.Yu. Baryshnikov 1

1 N.N.Blokhin Memorial Cancer Research Center of the RAMS, Moscow,115478, Russia.

2 Turky Center for Biotechnology, University of  Turku., P.O. Box 123,  FIN-20521 Turku, Finland.

 A CD95-deficient cell line Jurkat/A4 was generated from the parental wild type (Jurkat/wt) cells by serial treatment with apoptosis-inducing anti-CD95 mAb (clone IPO-4, IgM). Comparative studies showed that the Jurkat/A4 cells acquired a CD95 negative phenotype, but showed expression of DR4 and DR5 that was equal to the parent cell line (Jurkat/A4). The Jurkat/A4 line was highly resistant to induction of apoptosis by an agonistic anti-CD95 mAb, which was to be expected, as CD95 expression abolished. However, the Jurkat/A4 line was also highly resistant to apoptosis induced by TRAIL, which was surprising when considering the presence of DR4 and DR5. Even more surprising was that the cell line was extremely insensitive to anti-cancer drugs (doxorubicin, etoposide, cis-DDP, cycloplatam), X-rays, UV-light, hydrogen peroxide, in comparison with Jurkat/wt cells, in which these treatments induced apoptosis in a time- and dose-dependent manner. Taking together, we have created a multiresistant leukemic cell line, by a selection procedure based on continuous stimulation of Fas(CD95) death receptor. The resistance seems reflect protection on several different levels of apoptotic regulation and not only on the deficiency of CD95 expression. Contribution by defects of other molecular targets which are involved in the execution phase of programmed  cell death (apoptosis) is to be expected. The model can reflect usual clinical situation when selection under pressure of strong immune system of young patients yields highly malignant multiresistant tumors.

Caspase-10 is recruited to and activated at the native TRAIL and CD95  death-inducing signalling complexes in a FADD-dependent manner.

Martin R. Sprick, Eva Rieser, Heiko Stahl, Anne Grosse-Wilde, Markus

Weigand and Henning Walczak*

Div. for Apoptosis Regulation, Tumour Immunology Program, DKFZ, Heidelberg, Germany The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase_8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor crosslinking. Mutations in caspase_10, the other death-effector-domain-containing caspase besides caspase_8, have been identified in patients with autoimmune lymphoproliferative syndrome type II (ALPSII). Resulting defects in TRAIL-induced apoptosis were suggested as causative for ALPSII. However, it has been controversial whether caspase_10 is in fact a constituent of the native DISC. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC and that FADD is necessary for its recruitment to and activation at these two protein complexes. We further demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results suggest that the observed caspase-10 mutations in ALPSII patients may be causative for this disease by inhibiting proper DISC formation and caspase activation not only at the TRAIL DISC, but also at the CD95 DISC.

Bad retains its apoptosis promoting activity despite proteolytic cleavage by caspase-3

Anna Stepczynska 1, Jacob Troppmair 2, Ingo Engels 1, Monika Poppe 3, Klaus Schulze-Osthoff 1

1 Department of Immunology and Cell Biology, University of Münster, D-48149 Münster

2 Institute of Medical Radiation and Cell Research, University of Würzburg, D-97078 Würzburg

3 Department of Apoptosis and Cell Death, University of Münster, D-48149 Münster

 Bcl-2 family member, Bad, promotes death by neutralising the protective effect of Bcl-XL and Bcl-2.  The phosphorylation status of Bad determines whether the molecule is sequestered in cytosol by 14-3-3 protein family members or whether it dimerizes with Bcl-XL or Bcl-2 and thereby releases block on cytochrome c release.  We demonstrate that overexpression of Bad in MCF7 cell line is sufficient to trigger cytochrome c release.  We detect cleavage of Bad during CD95 and staurosporine-induced apoptosis in Jurkat cells and identify the SATD sequence in murine Bad to serve as caspase-3 substrate site.  According to our data cleavage of Bad at this site does not improve Bad death promoting activity.  The truncated Bad (tBad) has no increased ability to trigger cytochrome c release in vitro.  Yet, tBad induces formation of apoptotic nuclei, supporting the view that the ability of Bad to trigger apoptosis relies mainly on the intact BH3 domain.  This conserved domain enab les heterodimerization among Bcl-2 family members and is not subjected to the proteolytic control of caspases.

The C-terminal Activation Domain of the STAT-1 Transcription Factor Enhances Ischaemia/Reperfusion -Induced Apoptosis in Cardiac Myocytes

Anastasis Stephanou§, Tiziano M. Scarabelli, Robert Bell +, Derek Yellon+, Richard A. Knight*, and David S. Latchman. 

Medical Molecular Biology Unit, Institute of Child Health,

University College London, 30 Guilford Street, London, WC1N 1EH,

U.K; Hatter Institute, University College London +;

National Heart and Lung Institute, Royal Brompton Hospital, London*.

ABSTRACT We have previously demonstrated that the STAT-1 transcription factor plays a key role in ischaemia/reperfusion (I/R) -induced apoptosis in cardiac myocytes. In the present study we assessed which region of the STAT-1 molecule mediates apoptosis in cardiac myocytes. A STAT-1 construct (aa 390-750), lacking the N-terminus was able to enhance I/R -induced apoptosis in cardiac myocytes. However, a STAT-1 construct, which lacks 60 amino acids at the C-terminus, was not as effective as the full length STAT-1 in promoting I/R -induced apoptosis in cardiac myocytes. A chimaeric STAT-3/STAT-1 construct in which the C-terminus of STAT-3 was exchanged with the C-terminus of STAT-1 (aa 293-750) was also able to enhance I/R  -induced apoptosis. Furthermore, overexpression of a C-terminal STAT-1 construct (aa 691-750) containing the transcriptional activation domain but not the DNA binding domain strongly enhanced I/R -induced apoptotic cell death. Interestingly, cardiac myocytes isolated from mice expressing a truncated C-terminal STAT-1 were more sensitive to I/R -induced cell death. Finally, isolated hearts from these animals exposed to I/R injury had larger infract size and greater number of TUNEL positive myocytes than control hearts. These studies demonstrate that the C-terminal transactivation domain of STAT-1 is necessary and sufficient for I/R injury- induced apoptosis in cardiac myocytes.

Identifying inhibitors of mitochondrial signaling during apoptosis

Stephen Tait, Arlette Werner, Evert de Vries, Jannie Borst

Dept of Cellular Biochemistry, Netherlands Cancer Institute, Amsterdam, Holland

 Cellular transition to a cancerous state involves de-regulation of apoptotic pathways.  Consequently, cancer cells are often resistant to apoptosis inducing, anti-cancer regimen.  This project has investigated mechanisms of apoptotic resistance in a T cell lymphoma cell line which displays resistance to CD95/Fas mediated apoptosis.  Moreover, this cell-line is also resistant to g irradation and etoposide induced apoptosis thereby suggesting a common resistance mechanism.  Cell fractionation experiments demonstrated a failure of the resistant cells to release cytochrome C in response to apoptotic stimuli.  This indicates that the point of cross-resistance lies upstream of mitochondrial activation.   An in vitro mitochondrial assay has been developed to map the point of resistance and to identify the mediator/s of this resistant phenotype.  We demonstrate that initiator caspase activity, leading to Bid cleavage and mitochondrial translocation, is unaffected in these cells.  Intriguingly, in vitro addition of activated Bid to mitochondria, in the presence of resistant cell cytosol, fails to cause cytochrome C release.  This suggests that the mechanism of apoptotic resistance may lie at a novel regulatory step downstream of Bid activation.  These results, together with work aimed at identifying the mediator/s of this apoptotic resistance, will be discussed.

Th1and Th2 cytokines control cell survival in thyroid cancer by modulating apoptotic proteins

M. Todaro*, D. Di Liberto*, M. Catalano*, M. Patti*, M. Ciaccio*, R. De Maria°#, G. Stassi*.

*Department of Biotecnologies and Forensic Medicine, Clinical Biochemistry Chair, University of Palermo, Italy

°Laboratory of Hematology and Oncology, Istituto Superiore di Sanità, Rome, Italy

#Department of Biomedical Sciences, General Pathology Section, University of Catania, Italy

 Although CD95 is expressed in essentially all the histological variants of thyroid carcinomas, CD95 cross-linking fails to induce cell death in thyroid tumor cells. Refractoriness to CD95 stimulation in thyroid tumors may be a critical event in tumorigenesis. This mechanism which seems to be involved in the modulation of key molecules responsible for death or survival of target cells, in particular in thyroid tumor, may depend on the cytokines released by the tumor-associated inflammatory infiltrate. Therefore, we investigated the expression of Th1 and Th2 cytokines and the co-localization with pro- and anti-apoptotic factors in tumor tissues. IL-4 and IL-10 were both intensively detected in thyroid tumor microenvironment, while IFN- g was scarcely present. Interestingly, analysis of pro-apoptotic and anti-apoptotic factors showed that thyroid cancer cells expressed high levels of cFLIP and Bcl-xL. By contrast, healthy neighbouring epithelial follicular cells overexpressed caspase-8 and caspase-3. Accordingly, freshly isolated tumor thyrocytes were completely refractory to CD95 stimulation, whereas freshly isolated neighboring cells were susceptible to CD95-induced apoptosis. To investigate the role of the thyroid tumor microenvironment, control thyrocytes were exposed to T_h1 or T_h2 cytokines and analyzed for susceptibility to apoptosis and expression of the apoptosis-related proteins. IL-4 and IL-10 treatment significantly prevented spontaneous and CD95-induced apoptosis in normal thyrocytes exposed to cisplatin, doxorubicin and taxol, while IFN- g promoted caspase up-regulation sensitizing thyrocytes to both CD95 and chemiotherapy-induced apoptosis. Analysis of anti-apoptotic protein expression, in IL-4 and IL-10 normal treated thyrocytes, revealed a potent up-regulation of cFlip and Bcl-xl. It is therefore likely that the resistance to CD95 and chemiotherapy-induced apoptosis observed in these cells is due to high expression of anti-apoptotic genes. These study may provide key molecular bases to the understanding of thyroid cancer pathogenesis and the definition of novel therapeutic strategies.

The neuronal specific Rai (ShcC) adaptor protein inhibits apoptosis triggered by limited neurotrophic support or environmental stress by activating PI3K/Akt

Flavia Troglio1,Giuliana Pelicci1, Alessandra Bodini1, Valentina Pettirossi1,4 Rosa Marina Melillo2, Laura Coda1, Antonio Di Giuseppe3, Massimo Santoro2, and Pier Giuseppe  Pelicci1,5

1Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy; 2Centro di Endocrinologia ed Oncologia Sperimentale del CNR/Dipartimento di Biologia e Patologia Cellulare e Molecolare, 80131 Napoli, Italy; 3Istituto di Medicina Interna e Scienze Oncologiche, Perugia University, 06100 Perugia, Italy

 Rai is a member of the family of the Shc-like cytoplasmic signal transducers. Rai expression is restricted to embryonic and adult neuronal cells and in vivo regulates the number of post-mitotic sympathetic neurons. We report here that Rai is a physiological substrate of the neurotrophic Ret receptor and that it stimulates neuronal cell survival by inhibiting apoptosis induced by limited availability of the Ret ligand (GDNF) or treatment with various toxic insults (trophic factor withdrawal, oxidative stress, and hypoxia). While Rai expression does not influence Ras activity, it enhances survival, following both types of apoptosis induction, by activating the PI3K/Akt signalling pathway. Rai proteins bind constitutively to the p85 subunit of PI3K and, following ligand gpactivation, to the Ret receptor. In neurons treated with low concentrations of GDNF, the pro-survival effect of Rai depends on Rai phosphorylation and Ret activation, while in neurons exposed to the various toxic insults, neither Rai phosphorylation nor Ret activation is needed. Notably, Rai expression is up-regulated under these stress conditions. We propose that Rai functions in the regulation of Ret-dependent survival signals during the development of sympathetic neurons, and that it is part of an adaptative stress response in post-mitotic adult neurons.

Tet-induced TRAIL expression inhibits tumor growth,

E. Ucur*, J. Mattern*, S.Okouoyo*, A. Schroth*, K.M. Debatin§, I.Herr*

* DKFZ, INF 280, 69120 Heidelberg, Germany; § University Children's Hospital, Prittwitzstr.43, 89075 Ulm, Germany

 We utilized JURKAT cells, transfected with TRAIL under tetracycline control (JURKAT-TRAIL-tet-on), for regulated induction of apoptosis in tumor therapy. Our in vitro studies show that tet-induced TRAIL expression in JURKAT cells induces apoptosis in B-lymphoma (BJAB) target cells. Tumor growth of TRAIL-sensitive xenografts was inhibited using the same donor-target system in vivo. In detail, nude mice were xenografted either with BJAB or with TRAIL-resistant neuroblastoma (KELLY) cells. Tumors were inoculated with JURKAT-TRAIL-tet-on cells and expression of TRAIL was switched on by adding tetracycline to the drinking water. Tumor size was measured during a period of 4 weeks. BJAB-xenografts injected with control cells exhibited a twofold increase in tumor size compared to the tumors inoculated with TRAIL expressing JURKAT cells. In contrast, no alteration was observed in tumor size increase in nude mice xenografted with KELLY cells. These data indicate that control of tumor growth may be achieved with TRAIL expressing effector cells as a local delivery system.

Tumour necrosis factor-alpha-induced cell death in colon cancer cells

Vaculová A.1,2, Hofmanová J.1, Sou&#269;ek K.1,2, Kozubík A.1

1 Institute of Biophysics, Academy of Sciences, Královopolská 135, 612 65 Brno, Czech Republic

2 Masaryk University, Faculty of Sciences, Department of Comparative Animal Physiology and General Zoology, Kotlá&#345;ská 2, 611 37 Brno, Czech Republic

 The response of an HT-29 human colon adenocarcinoma cell line to the treatment with tumour necrosis factor-alpha (TNF-alpha) was analysed. These cells were relatively resistant to acute cytotoxic and antiproliferative effects of TNF-alpha that can be potentiated by cycloheximide. The growth and viability of cells were not significantly decreased before 96 and 120 hours of TNF-alpha treatment. At this time, accumulation of cells in the S-phase of the cell cycle, increased amount of cells in subdiploid population, and nuclear chromatin condensation and fragmentation were also detected. However, TNF-alpha induced earlier events associated with the induction of apoptosis - a decrease in pro-caspase-3 immunoreactivity together with the cleavage of poly(ADP-ribose) polymerase (PARP) to 89 kDa fragment, and increased production of reactive oxygen species. We demonstrated that in addition to being an early marker of apoptosis, activation of caspase-3 and degradation of PARP may play a causative role in HT-29 cell death induced by TNF-alpha.

Supported by grants from IGA MH NC/6171-3 and GA of the Czech Republic No.525/01/0419.

Distinct intracellular signaling in TRAIL- and CD95L-mediated apoptosis.

Jurjen H.L. Velthuis, Kasper M.A. Rouschop, Hans J.G.M. de Bont, Gerard J. Mulder and Fred J. Nagelkerke.

Division of toxicology, LACDR, Leiden University, The Netherlands

Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL) is a potent inducer of apoptosis in tumor cells, but not in healthy cells. Similar to CD95L, TRAIL-signaling requires ligand-receptor interaction; also the downstream signaling molecules, such as FADD and caspase-8 seem similar. Using stably TRAIL and CD95L expressing cells, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531s. The mitochondrial damage (loss of mitochondrial membrane potential(MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than with CD95L-expressing cells. The decrease in MMp induced by both ligands was caspase-8-mediated; no difference in caspase-8-activation by TRAIL or CD95L was found. TRAIL but not CD95L induced activation of caspase-10. Bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, while it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparant inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.    

"Calcitonin and bombesin role in apoptosis control"

Authors: Salido Peracaula M., García Villar, C and Vilches Troya J.

Department of Cellular Biology, Faculty of Medicine, University of Cádiz, Cádiz, Spain

 Calcitonin and bombesin inhibit etoposide-induced apoptosis in prostate carcinoma cells. We investigated whether these neuropeptides block the etoposide-induced changes in elemental content. Cells from the PC-3 and Du 145 prostate carcinoma cells lines were exposed to etoposide for 48 hours in the absence or presence of calcitonin and bombesin. After the exposure, the cells were frozen and freeze dried, and their elemental content was analyzed by energy-dispersive X-ray microanalysis in both in the scanning electron microscope and the scanning transmission electron microscope. Etoposide treatment consistently induced an increased in the cellular Na concentration and a decrease in the cellular K concentration, resulting in a marked increase of the Na/K ratio and also an increase in the P/S ratio. Both bombesin and calcitonin inhibited the etoposide-induced changes in the cellular Na/K ratio, and calcitonin, but no bombesin, inhibited the changes in the P/S ratio. No significant elemental changes were found with bombesin or calcitonin alone.

Bmf: A Proapoptotic BH3-Only Protein Regulated by Interaction with the Myosin V Actin Motor Complex, Activated by Anoikis

Andreas Villunger,1 Hamsa Puthalakath,1 Lorraine A. O'Reilly,1 Jennifer G. Beaumont,1 Leigh Coultas,1 Richard E. Cheney,2 David C. S. Huang,1 Andreas Strasser1

 1 The Walter and Eliza Hall Institute of Medical Research, Melbourne, P.O. Royal Melbourne Hospital, 3050 VIC, Australia; 2 University of North Carolina, Chapel Hill, NC 27599-7545, USA

   Bcl-2 family members bearing only the BH3 domain are essential inducers of apoptosis. We identified a BH3-only protein, Bmf, and show that its BH3 domain is required both for binding to prosurvival Bcl-2 proteins and for triggering apoptosis. In healthy cells, Bmf is sequestered to myosin V motors by association with dynein light chain 2. Certain damage signals, such as loss of cell attachment (anoikis), unleash Bmf, allowing it to translocate and bind prosurvival Bcl-2 proteins. Thus, at least two mammalian BH3-only proteins, Bmf and Bim, function to sense intracellular damage by their localization to distinct cytoskeletal structures.

Reverse signalling via death ligands of the Tumor Necrosis Factor superfamily

Authors: Christoph Wasem, Diana Arnold, Leslie Saurer, Nadia Corazza, Christoph Müller and Thomas Brunner, Institute of Pathology, Division of Immunpathology, University of Bern (Switzerland)

 Members of the Tumor Necrosis Factor (TNF) superfamily, such as TNFa, Fas ligand (FasL), TRAIL, CD30L, CD40L, CD27L, OX40L and 4-1BBL have a widespread implication in the regulation of the immune system by delivering costimulatory or apoptosis-inducing signals after binding to their corresponding receptors. However, recent findings indicate that these ligands may also serve as receptors and their cytoplasmic domains may transmit signals into the ligand-bearing cell (reverse signalling). We are investigating the mechanisms and role of reverse signalling by the apoptosis-inducing ligands of the TNF superfamily (TNFa, FasL and TRAIL), since the length of their intracellular parts and the presence of potential signalling motifs differs significantly. Preliminary experiments have revealed an increase in tyrosine phosphorylation after crosslinkig FasL in transiently transfected cells. To better investigate reverse signalling properties of death ligands, we have generated. Myc epitope-tagged death ligands devoid of the extracellular receptor-binding domain to avoid protease cleavage of the ligand and/or receptor interaction and apoptosis induction. Upon transfection and activation of these constructs in different cell lines, we will investigate differences in the signalling events and functional consequences for the cell, e.g. proliferation, cytokine production and cell death.

Single cell analysis of reactive oxygen species during mitochondrial outer membrane permeabilization

Nigel J Waterhouse and Douglas R Green

La Jolla Institute for Allergy and Immunology (USA)

Peter MacCallum Cancer Institute (Austraila)

 A decrease in mitochondrial membrane potential (delta psi) and an increase in reactive oxygen species (ROS) are early events during apoptosis that have been reported to be involved in mitochondrial outer membrane permeabilization (MOMP). ROS scavengers have been shown to block apoptosis and restore clonogenic potential of cells treated with a variety of apoptotic stimuli. We found that mitochondrial ROS, a major byproduct of oxidative phosphorylation, were elevated after treatment of cells with protonophores to reduce delta psi, possibly due to increased electron transport to regenerate the lost potential. This increase in ROS was followed by release of cytochrome c and caspase dependent apoptosis, both of which were blocked by ROS scavengers and Bcl-2. We also observed an increase in ROS during drug-induced apoptosis, however this was downstream of MOMP and caspase activation and was blocked by zVAD-fmk. Neither MOMP nor phosphatidylserine exposure was blocked by ROS scavengers in this system.    These data show that ROS can play an important part of the signaling pathways to apoptosis in some models (most likely those that are a consequence of changes in metabolism), however they are not required for the upstream signaling pathways during drug-induced apoptosis nor are they required for MOMP.

Defining the mechanism of action of Bfl-1, a novel inhibitor of Bid

A.B. Werner, E. de Vries, I. Bontjer, S. Tait and J. Borst

Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, 1066 CX, The Netherlands

 The BH3 domain-only Bcl-2 family member Bid plays an important role in transmitting the death signal from death receptors to the mitochondria. Following caspase-8/granzyme B mediated cleavage the C-terminus of Bid (tBid) translocates to the mitochondria and induces cytochrome c release. In a yeast two hybrid screen, using Bid as bait, we found that the anti-apoptotic Bcl-2 family member Bfl-1/A1 interacts with Bid. In vivo immunoprecipitation studies demonstrate that Bfl-1 associates with both full length Bid and tBid. Cellular expression of Blf-1 conveys protection against numerous apoptotic stimuli, including CD95- and Trail-induced apoptosis. We show that Bfl-1 functions at the mitochondria where it inhibits cytochrome c release in vitro and in vivo. However, our data demonstrate that this inhibition is not mediated by preventing Bid processing nor by inhibiting tBid association with the mitochondria. Contrastingly, we show that Bfl-1 effectively blocks tBid/Bax and tBid/Bak mediated mitochondrial activation. Therefore, we hypothesize that Bfl-1 sequesters tBid at the mitochondria, thereby preventingBax and/or Bak activation and, in doing so, mitochondrial apoptotic activation.

Resistance of activated T cells towards CD95 mediated apoptosis is dependent on de novo protein synthesis

Ingo Schmitz, Heiko Weyd, Andreas Krueger, Peter H. Krammer and Sabine Kirchhoff

Division of Immunogenetics, Tumorimmunology Program, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

 In vitro-stimulation of peripheral T cells (in the following referred to as day 0 T cells) leads to their activation (day 1 T cells), resulting in increased expression of several genes including CD95 and CD95 ligand. However, besides high CD95 surface expression, day 1 T cells are resistant to CD95-mediated apoptosis and show impaired DISC formation. After prolonged culture in the presence of IL-2 activated T cells develop an apoptosis sensitive phenotype (day 6 T cells) and DISC formation is readily observed. It was previously shown that resistant day 1 T cells can be partly sensitized towards CD95 mediated apoptosis by treatment with the translation inhibitor cycloheximide (CHX). We now show that CHX does not influence DISC formation or expression of anti-apoptotic Bcl-2 family members. However, expression of c-FLIP short protein was downregulated, indicating that c-FLIP proteins influence the composition of the DISC and, thus, might contribute in part to the resistant phenotype of day 1 T cells. In summary, the CD95-resistance of d 1 T cells seems to depend on active protein biosynthesis of short-lived anti-apoptotic proteins like c-FLIP.

The N-terminal caspase-generated RasGAP frgmant protects cells by activating the Ras-PI3K-Akt pathway.

Jiang-yan Yang and Christian Widmann.

Division of Immunogenetics, Tumorimmunology Program, German Cancer Research Center (DKFZ),

Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

Affiliations: Insititute of Cellular Biology and Morphology (IBCM), Lausanne University

Activation of caspases 3 and 9 is thought to irreversibly commit a cell to apoptosis. There are however several documented situations where caspases are activated, and caspase substrates cleaved, with no associated apoptotic response. Why the cleavage of caspase substrates leads to cell death in certain cases, but not in others, is unclear. One possibility is that some caspase substrates generate anti-apoptotic signals when cleaved.  We have recently shown that RasGAP is one such protein. Caspases cleave RasGAP into a C-terminal fragment (fragment C) and an N-terminal fragment (fragment N). While fragment C can induce apoptosis when expressed alone, its pro-apoptotic activity is totally blocked in the presence of fragment N. Fragment N also inhibits apoptosis induced by caspase 9. At higher caspase activity, fragment N is further cleaved and turns into a potentiatior of the cell death response. These data support a model where RasGAP functions as a sensor of caspase activity to determine whether a cell should survive or not. Here we have characterized how fragment N protects cells. Our results indicate that Ras, PI3K, and Akt - but not NFKB - mediate the anti-apoptotic functions of fragment N.

Stem Cell Factor upregualtes Bcl-2 family members and protects erythroid precursor cells from chemotherapy-induced apoptosis

Ann Zeuner, Francesca Pedini, Ugo Testa, Cesare Peschle and Ruggero De Maria

Instituto di Patologia Generale, Universita’ di Catania, Italy; Department of Hematology and Oncology, Istituto Superiore di Sanita', Rome, Italy

 Anemia is a major side effect of anticancer chemotherapy, resulting in an overall patient’s worsening that often leads to the discontinuation of effective treatments. Erythroid precursor cells at an early differentiation stage represent the most sensitive target for chemotherapeutic agents, undergoing apoptosis primarily through caspase 3 activation. Treatment of eryhroid precursor cells with Stem Cell Factor (SCF), a cytokine that binds the c-kit receptor on early hematopoietic cells, results in a strong protection from apoptosis induced by different categories of chemotherapeutic agents. SCF-treated cells are able to regain full proliferative activity and to complete the normal differentiative process following removal of the toxic stimulus. The extent of caspase activation in cells pretreated with SCF and exposed to antineoplastic drugs is significantly lower compared with cells kept in normal erythropoietin-containing medium. As SCF-treated cells show an upregulation of Bcl-XL and Bcl-2 both at the RNA and protein level, we transduced erythroid progenitor cells with Bcl-XL cDNA and tested their sensitivity to chemotherapeutic agents. Erythroblasts overexpressing Bcl-XL display a significant resistance to cytotoxic drugs, suggesting that upregulation of Bcl-2 family members may be responsible for SCF-mediated antiapoptotic effect. These observations suggest a potential clinical use of SCF (which is generally well tolerated in vivo) to limit the depletion of erythroid progenitors induced by antineoplastic therapy.