Title: Oxidative stress-induced NF-kB activation is defective in cystic fibrosis lung cells through enhanced proteasome-mediated p21^WAF1/CIP1 degradation 

 Authors: Emilie Boncoeur,  Olivier Tabary,  Elise Bonvin, Annick Clément, Alexandra Henrion-Caude, Jacky Jacquot.

 Address: Inserm U 719, Hôpital Saint-Antoine, UPMC ParisVI, 75571, Paris, France 

 Abstract: Lung in patients with cystic fibrosis (CF) is characterized by structural damage and altered repair due to oxidative stress. To gain insights into the oxidative stress-related damage in CF lung, we studied the effects of hyperoxia (95 % O₂) in CF and normal mouse lung and cell culture models. In normal lung epithelium, protection against hyperoxic injury has been shown to be related to an increased expression of the cell cycle inhibitor p21^WAF1/CIP1 thus confering a survival advantage to damaged cells and to increased NF-kB activity by involving a cytoplasm-to-nucleus translocation after degradation of the inhibitor protein IkB alpha by the proteasome machinery. Here we demonstrate in CF in vivo and in vitro models that hyperoxia did not induce neither increased p21^WAF1/CIP1 expression nor lung NF-kB activation IkB alpha degradation, nor induction of caspase 3 and subsequent apoptosis but, unexpectedly caused enhanced proteasome activity, in contrast to that observed in normal in vivo and in vitro controls. Interestingly, ectopic expression of p21^WAF1/CIP1 in hyperoxia-exposed CF lung epithelial cells or treatment with the selective proteasome inhibitor MG132 restored the NF-kB activation and IkB alpha degradation which was associated with an increased caspase-3 activity and apoptotic cell death. Our data suggest that the use of proteasome inhibitors or related substances modulating p21^WAF1/CIP1 degradation, promoting the activation of NF-kB in hyperoxic conditions could be a valuable approach to protect lung epithelial cells from oxidative stress in CF patients. 

Abstract 2

 Title: Specific involvement of human SERCA3f calcium pump in Endoplasmic Reticulum stress and apoptosis 

 Authors: C CHAABANE, E CORVAZIER, R BREDOUX, S DALLY, J ENOUF and R BOBE 

 Address: INSERM U. 689, Centre de Recherche Cardiovasculaire Lariboisière, IFR 139, Hôpital Lariboisière, 8 Rue Guy Patin, 75475 Paris Cedex 10, France 

 Abstract: Endoplasmic Reticulum (ER) has recently been involved in signalling pathway that leads to apoptosis in a calcium dependent manner. The aim of this study was to determine whether SERCAs (Sarco/Endoplasmic Reticulum Ca2+ ATPases), that control the calcium refilling of the ER may play distinct role in this process. Recent reports have shown that the human SERCA3 family accounts six members named hSERCA3a-SERCA3f (h3a-3f) that only differ in their C-terminal parts (3’ end alternative splicing). Here, h3b and h3f stably transfected HEK-293 cell lines were compared for cellular adhesion and apoptosis signalling. First, we observed that both h3b and h3f overexpression led to a decrease in cell attachment. However, detached cell viability assessed by using trypan blue and FITC- conjugated Annexin-V and PI staining, showed that only h3f overexpression was associated to a decrease of cell viability. Comparative Western blot analysis of h3b and h3f-transfected cell lysats for PA  RP (Poly-ADP-Ribose-Polymerase) and PMCA4b (Plasma Membrane Ca2+ ATPase 4b) revealed major induction of caspase-3 activation in h3f that was further confirmed using caspase-3 inhibitor Z-DEVD-fmk. We also observed the involvement of caspase-9 suggesting the role of mitochondrial apoptotic signalling pathway. ER stress increases the processing of XBP-1 (X Box-Binding-Protein-1) leading to enhancement of the chaperone protein GRP78 (Glucose-Regulated-Protein 78). Accordingly, we showed that overexpression of h3f only resulted in an increase of both ER stress modified XBP-1 mRNA and GRP78 expression. In conclusion, our data suggest that overexpression of h3f but not h3b can enhance apoptosis in a caspase-3 and –9-dependent manner that involves ER stress.   

Abstract 3

 Title: Upregulation of Heme oxygenase-1 confers resistant to uropathogenic Escherichia coli toxins-induced apoptosis via carbon monoxide 

 Authors: Ming Chen, Roshan Tofighi, Wenjie Bao, Sandra Ceccatelli, Gianni Celsi. 

 Address: Dept. of Pediatrics, B 62, Karolinska University Hospital-Huddinge, 141 86, Stockholm, Sweden 

 Abstract:   Background Pyelonephritis is a risk factor for renal tubular epithelial cell damage in children. E.coli is the main organism causing UTI. We have recently demonstrated that renal proximal tubular cells undergo iNOS-mediated death after exposed to uropathogenic E.coli toxins. We further examined by which upregulation of heme oxygenase-1 (HO-1) triggered a survival signaling against iNOS-mediated cell death. Methods Uropathogenic E.coli strain ARD6 (O6) strain and transformants of non-pathogenic strains expressing hemolysin were used were used. LLC-PK1 cells were cultured in a carbon oxide (CO) chamber with or without CO-flow (1000 ppm). Soluble toxins from E. coli ARD6 were added at a final dose of 100 ml/ml. Cell viability and morphology were assayed using the trypan blue exclusion test and phase contrast microscopy. Protein expression was detected by Western blotting. Results Activation of HO-1 by NO donor, SNP could prevent iNOS-mediated cell death in E.coli (O6) toxins-inf  ected renal proximal tubular cells, and CO 1000 p.p.m also reduced cell death to the same level as SNP, as examined with cell viability test, phase contrast images, Annexin V / PI staining and PARP cleavage. p21 protein was not affected by O6-toxins alone, it was significantly upregulated in the presence of SNP or CO. p53 protein was not changed by SNP, although p53 protein was upregulated by O6-toxins, indicating that induction of p21 and p53 in our model are most likely two independent phenomenons. Furthermore, we found that in children with pyelonephritis all the E.coli strains expressing hemolysin induced apoptosis. In E.coli strains not expressing hemolysin, only 45% of thems could induce apoptosis. In conclusion, generation of CO elicited by HO-1 could promote survival signalling in renal cells. Hemolysin is one of the secreted toxins that are involved in inducing apoptosis during UTI. Conclusions our data suggest that generation of CO elicited by HO-1 could promote su  rvival signalling in renal cells. CO, therefore, might be a beneficial modality in renal cell response, perhaps in preventing cell shedding in UTI. Hemolysin is one of several secreted toxins that are involved in inducing apoptosis during UTI. 

Abstract 4

 Title: Inhibition of myosin regulatory light chain phosphorylation induces programmed cell death in lung epithelial cells 

 Authors: D. El Mehdi (1), C. Pouzet (2), B. Lardeux (3), A. Grodet (2), G. Feldmann (4), A. Druilhe (1), and M. Pretolani (1) 

 Address: 1 Institut National de la Santé et de la Recherche Médicale (INSERM) U700, Faculté de Médecine Xavier Bichat, Paris, France. 2 INSERM-Institut Fédératif de Recherche 02, Plateforme de morphologie, Faculté de Médecine Xavier Bichat, France. 3 INSERM U539, Faculté de Médecine, Nantes, France. 4 INSERM U481, Faculté de Médecine Xavier Bichat, Paris, France 

 Abstract:   Phosphorylation of myosin regulatory light chain (MRLC) by myosin light chain kinase  (MLCK) and Rho-kinase (ROCK) promotes apoptotic cell death and the associated morphological changes, such as blebbing. However, the role of phosphorylated (p)-MRLC in the cascade of signaling events leading to apoptosis remains controversial. To better delineate this role, we examined the localization of p-MRLC and the effects of ML-7 and Y27632, inhibitors of MLCK and ROCK, respectively, on changes in cell morphology and permeability and on DNA fragmentation, phosphatidylserine exposure and caspase activity induced by apoptotic stimuli in  the alveolar epithelial cell line, A549. Consistently with its role in driving membrane blebbing, the expression of p-MRLC was upregulated at the cell periphery during apoptosis, whereas it localized into the cytoplasm in non-apoptotic cells. Cell incubation with ML-7, but not Y27632, suppressed MRLC phosphorylation, promoted apoptosis and augmented that   induced by FAS activation or cisplatin. Apoptosis was accompanied by cell surrounding and detachment and by delocalization of beta-1 integrin and E-cadherin into large vacuoles, reminiscent of autophagy ultrastructures. Cell death was prevented by a pan-caspase inhibitor, but not by strategies aimed at reinforcing integrin-mediated anchorage, indicating that death was not secondary to cell detachment. Finally, cell incubation with the autophagy blocker, 3-methyl adenine, prevented cell detachment and donwregulated apoptosis. Collectively, these data suggest that constitutive MLCK activation leads to cytoplasmic MRLC phosphorylation that prevents type I (apoptotsis) and II (autophagy) programmed cell death. p-MRLC may thus exert opposite regulatory functions depending on its spatial distribution. 

Abstract 5

 Title: INHIBITION OF NEUTROPHIL APOPTOSIS BY TOLL-LIKE RECEPTOR AGONISTS IN WHOLE BLOOD: INVOLVEMENT OF THE PHOSPHOINOSITIDE 3-KINASE/AKT AND NF-kB SIGNALING PATHWAYS, LEADING TO INCREASED LEVELS OF MCL-1, A1 AND PHOSPHO BAD. 

 Authors: S. François, J. El Benna, P.M.C. Dang, M.A. Gougerot-Pocidalo, C. Elbim 

 Address: Service d’Immunologie-Hématologie et INSERM U683, CHU X. Bichat, Paris. 

 Abstract: Polymorphonuclear neutrophils (PMN) are key components of the first line of defense against microbial pathogens. PMN directly recognize microbial products via pattern recognition receptors such as Toll Like Receptors (TLR). PMN are usually short-lived immune cells, but the prolongation of their life span is critical in their efficiency against pathogens. Many inflammatory mediators, including cytokines, regulate cell survival by interfering with apoptosis. However, regulation of PMN survival remains to be fully elucidated. In particular, the impact on PMN apoptosis of PAMPS recognized by the different TLRs has rarely been investigated. We therefore investigated the effect of TLR agonists on human PMN apoptosis in whole blood in order to minimize PMN activation and to mimic physiological conditions. LPS (TLR4), PGN (TLR2), R-848 (TLR7/8) and CpG-DNA (TLR9) were equally effective at delaying spontaneous apoptosis of PMN, while PamCSK4 (TLR1/2), MALP-2 (TLR2/6), flagelli  n (TLR5) and loxoribine (TLR7) were less effective or inactive. TLR agonists found to delay apoptosis also extended the functional lifespan of PMN. Analysis of signaling pathways revealed that the antiapoptotic effect of TLR agonists required NF-kB and PI3-K activation. Furthermore, analysis of intact cells by flow cytometry showed that TLR agonists delaying PMN apoptosis increased phosphorylation of Akt, a major target of PI3-K. This effect was associated with a PI3-K-dependent increase in Hsp27 phosphorylation, which has been reported to play a key role in PMN survival. Finally, the TLR-induced delay in PMN apoptosis was associated with increased levels of the antiapoptotic proteins Mcl-1 and A1. These effects were reversed by PI3-K and NF-kB inhibitors, respectively. TLR activation also led to PI3-K-dependent phosphorylation of the proapoptotic protein Bad. Our results strongly suggest a role of NF-kB and PI3-K in TLR-induced PMN survival, leading to modulation of Bcl-2 f  amily molecules. The prolonged functional life span induced by TLR agonists may represent a crucial enhancement of PMN defenses against microbial pathogens. 

Abstract 6

 Title: Defects in the clearance of dying cells contribute to the development of human systemic lupus erythematosus (SLE) 

 Authors: U.S. Gaipl (presenter), L. Munoz, A. Sheriff, R.E. Voll, J.R. Kalden, and M. Herrmann 

 Address: Institute for Clinical Immunology Department of Internal Medicine III Friedrich-Alexander University of Erlangen-Nuremberg Glueckstr. 4a 91054 Erlangen Germany 

 Abstract:  Defects in the clearance process of dying cells have been shown to play a crucial role in the development of autoimmune diseases. We described the accumulation of cellular debris in germinal centers of the lymph nodes in humans with SLE. The next aim was to investigate whether the impaired clearance observed in certain SLE patients is an intrinsic defect. We examined the differentiation of macrophages from CD34 positive stem cells from patients with SLE or normal healthy donors (NHD), respectively. NHD and SLE-derived stem cells showed similar proliferation in vitro. However, the differentiation into macrophages was reduced in some SLE stem cell cultures. Most of the SLE stem cell-derived macrophages showed a reduced uptake activity of beads and dying cells. Which defects of extrinsic factors are involved in the decreased clearance? We tested sera of SLE patients and NHD in regard to their capability to degrade necrotic cell-derived chromatin. We found a significant correlation between the percentage of degraded nuclei and the total classical complement activity of the sera. Most of the sera with a high activity reduction of DNase I, which was the case in many SLE sera, showed a reduced degradation capacity of chromatin. We conclude that the clearance defects in some SLE patients are in part intrinsic. An additional protection from chromatin can be achieved by the C1q and DNaseI dependent clearance of degraded chromatin. Altered mechanisms for clearance of dying cells represent a central pathogenic process in the development and acceleration of autoimmune diseases like SLE. 

Abstract 7

 Title: Insulin-like Growth Factor-I Inhibits Fas-induced Apoptosis in Human Neutrophils through the Phosphatidylinositol-3 Kinase Pathway, Independently of Akt 

 Authors: RON KOOIJMAN, EDDY HIMPE, CELINE DEGAILLER and ASTRID COPPENS 

 Address: Laboratory for Neuroendocrine Immunology  Department of Pharmacology (FARC) Medical School, Free University of Brussels (VUB) Laarbeeklaan 103 B-1090 Jette Belgium 

 Abstract: Programmed cell death (apoptosis) of neutrophils and subsequent recognition and phagocytosis by macrophages is a crucial mechanism for resolution of inflammation. We previously showed that insulin-like growth factor-I (IGF-I) inhibits spontaneous neutrophil apoptosis as assessed by light microscopy. The effects of IGF-I on apoptosis went along with inhibiting effects on DNA fragmentation and phosphatidylserine translocation.  In the present study, we addressed the effects of IGF-I in the presence of pro- and anti-apoptotic stimuli. We show that IGF-I also inhibits neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11. Flow cytometric analysis revealed that IGF-I did not affect the cell surface expression of Fas, indicating that IGF-I interacts with Fas-signaling or down-stream components of the apoptotic machinery. We found that IGF-I did not affect caspase-8 activation, whereas it inhibited depolarisation of the mitochondrial membrane.  The anti-apoptotic effect of IGF-I is comparable in magnitude with those of the acknowledged anti-apoptotic cytokines interferon-gamma (IFNgamma) and granulocyte-macrophage-colony stimulating factor (GM-CSF). Furthermore, IGF-I also inhibits Fas-induced apoptosis in the presence of effective concentrations of IFNgamma and GM-CSF, indicating that IGF-I may also affect neutrophil survival during inflammatory reactions in the presence of other anti-apoptotic factors. Inhibitor studies using U0126 as an inhibitor of the extracellular signal-regulated kinase (ERK) kinase (MEK) and Wortmannin as a phosphatidylinositol-3 kinase (PI3K) inhibitor indicate that the PI3K pathway, but not the MEK-ERK pathway mediates the effects of IGF-I. Furthermore, we found that, in contrast to GM-CSF, IGF-I did not induce phosphorylation of Akt, a down-stream target of PI3K, and that the Akt inhibitor triciribine abrogated the effect of GM-CSF but not that of IGF-I. These results indicate that additi  onal effects of GM-CSF and IGF-I may be explained by the existence of different signalling pathways and/or anti-apoptotic mechanisms for GM-CSF and IGF-I. Other targets of PI3K through which IGF-I could inhibit neutrophil apoptosis are PKCdelta, TEC kinases and p70S6K. Using the PKC inhibitors GF109203X and rottlerin, we show that PKCdelta is not involved. The role of TEC kinases is under investigation.   

Abstract 8

 Title: EFFECT OF MACROPHAGE-CONDITIONED MEDIA ON HUMAN PREADIPOCYTE DIFFERENTIATION   

 Authors: Lacasa D, Taleb S, Sellam P*, Bouillot JL*, Guerre-Millo M, Basdevant A & Clement K 

 Address: INSERM, équipe "Avenir", Paris ; Université Paris 6, EA 3502, Paris, F-75004 France ; CHRU Pitié Salpetrière, Département de Nutrition,*Service de Chirurgie Hôtel-dieu, Centre de Recherche en Nutrition Humaine, F-75004 Paris, France. 

 Abstract:  Obesity is now considered as a chronic low-grade inflammatory state. The white adipose tissue itself produces a variety of inflammation-related proteins, including several cytokines (TNF alpha, TGF beta, interferon, IL1, IL6, IL10 and IL8) and chimiokines (MIP1a, MCP1). We have recently shown that the expression of pro-inflammatory factors is increased in the adipose tissue of obese subjects and reduced after very low calorie diet-induced weight loss (Clement et al, FASEB J, (2004) 18:1657-1669). The cellular source of inflammation-related molecules within the adipose tissue is mostly the non-adipose cell fraction, which comprises infiltrated macrophages (Cancello et al, Diabetes, (2005) 54:2277-2286). The effect of macrophages-secreted products on adipose tissue biology is currently unknown. Our working hypothesis is that macrophage infiltration tends to limit fat expansion through a paracrine action on adipose cell metabolism and/or adipocyte differentiation. To te  st the latter hypothesis, conditioned medium was obtained from blood-derived human monocytes differentiated into macrophages in vitro. In some experiments, macrophages were activated by LPS treatment (100 ng/ml). Human preadipocytes isolated from subcutaneous adipose tissue biopsies were made to differentiate in presence of macrophage-conditioned media. A marked alteration of adipogenesis was observed, as assessed by low cellular lipid accumulation and reduced expression of adipogenic transcription factors, PPARgamma2 and C/EBPalpha, and their target genes. The expression of two adipose secreted hormones, namely leptin and adiponectine, was also altered. Moreover, human preadipocytes treated by macrophage-conditioned media exhibited increased proliferation with a more pronounced effect with activated macrophage-media.  These data suggest a dual effect of macrophage-produced factors to inhibit the differentiation and to induce the proliferation of pre-adipose cells. Thus, although the presence of infiltrated macrophages could represent an adaptive response to limit fat accretion, it could also be a major determinant of chronicity and resistance to weight loss in obese individuals, by increasing the number of preadipocyte within the adipose tissue. 

Abstract 9

 Title: Morphologic recognition of early apoptosis in human breast cancer cells by fractal analysis and co-occurrence matrix statistics. 

 Authors: Gabriele A. Losa and Christian Castelli 

 Address: Institute for Scientific Interdisciplinary Studies P.O.Box 1132 6600 Locarno Switzerland 

 Abstract: An analytical strategy combining fractal geometry and grey-level co-occurrence matrix (GLCM) statistics  was devised to investigate ultrastructural changes in oestrogen-insensitive SK-BR3 human  breast cancer cells undergoing apoptosis in vitro. Apoptosis was induced by 1 micromolar calcimycin and assessed by measuring conventional cellular parameters during the culture period. SK-BR3 cells entered the early stage of apoptosis within 24 h of treatment with calcimycin, which induced  detectable changes in nuclear components, as documented by increased values of most GLCM parameters  and by the general reduction of the fractal dimensions. In these affected cells, morphonuclear traits were accompanied by the reduction of distinct gangliosides and loss of unidentifiable glycolipid molecules at the cell surface. All these changes were shown to be involved in apoptosis  before the detection of conventional markers, which were only measurable during the active phases of apoptotic cell death. In overtly apoptotic cells treated with 1 micromolar calcimycin for 72 h, most nuclear components undervent dramatic ultrastructural changes, including marginalisation and condensation of chromatin, as reflected in a significant reduction of their fractal dimensions. The morphological reorganisation of nuclei,attributable to a loss of structural complexity, occurred  early in apoptosis and was quantified only by both fractal and GLCM analyses.    

Abstract 10

 Title: THE ROLE OF FAS AND PERFORIN IN HUMAN AIDC 

 Authors: V. Mateo, M. Menager, G. de Saint Basile, A. Fischer and F. Rieux-Laucat 

 Address: INSERM U429, Pavillon Kirmisson porte 17 Hopital Necker-Enfant Malades 149 Rue de Sèvres 75015 Paris France 

 Abstract:  Autoimmune Lymphoproliferative Syndrome (ALPS) is a pediatric condition characterized by non-malignant lymphoproliferation and accumulation of polyclonal TCR-alpha/beta+/CD4-/CD8- double negative (DN) T-lymphocytes. Overt autoimmune manifestations are hemolytic anemia, thrombocytopenia and neutropenia. Most patients carry germ-line (or less frequently somatic) dominant mutations in the fas gene. The role of Fas-FasL interaction in maintaining lymphocyte homeostasis was initially illustrated in mutant mice bearing germ-line recessive mutation in Fas (lpr) or FasL (gld), which also present with chronic lymphoproliferation and DN T-cells. Both mice have a defective activation-induced cell death (AICD); a mechanism of peripheral T-cell tolerance triggered by Fas-FasL interaction on repeatedly stimulated T-cells. We herein study AICD in Fas defective ALPS individuals to assess the role of fas-fasl interaction in human AICD. Surprisingly, AICD was detected in primary activated T-cells from Fas defective patients, with a magnitude similar to that of control cells. Inhibition experiments showed that the granzyme-perforin pathway was responsible for the AICD observed in ALPS lymphocytes. Such pathway is also observed in AICD of wild type cells, but to a limited extend. This works showed that over expression of the granzyme-perforin pathway is a potential consequence of Fas deficiency in human cells and may explain some phenotypical difference observed between ALPS and lpr or gld models. 

Abstract 11

 Title: NF-kappaB prolongs the life span of ATRA-treated APL cells via inhibition of reactive oxygen species production 

 Authors: Julie Mathieu (1), Stéphane Giraudier (2), Michel Lanotte (1) and Françoise Besançon (1) 

 Address: 1 INSERM U685, Centre Hayem, Hôpital St Louis, 1 avenue Claude Vellefaux, 75475 Paris, France;  2 INSERM U362, Institut Gustave Roussy, 39 rue Camille-Desmoulins, 94805 Villejuif, France.   

 Abstract:  All-trans retinoic acid (ATRA) significantly improves the survival of patients with acute promyelocytic leukemia (APL) by inducing granulocytic differentiation of leukemia cells. Since an activation of the transcription factor NF-kappaB occurs during ATRA-induced maturation of APL cells, a mechanistic link between these two processes was investigated. Using an in vitro model for APL, we report that ectopic overexpression of a repressor of NF-kappaB activation did not affect granulocytic differentiation. Importantly, NF-kappaB inhibition markedly resulted in a decreased viability of the differentiated cells which correlated with increased apoptosis. Apoptosis was accompanied by a sustained activation of the c-Jun N-terminal kinase (JNK). Inhibition of JNK by the specific inhibitor SP600125 or by transfection of a dominant-negative mutant of JNK1 reduced the percentage of apoptotic cells, thus showing that JNK activation constitutes a death signal. Furthermore, impairm  ent of NF-kappaB activation resulted in increased levels of reactive oxygen species (ROS) upon ATRA treatment. ROS accumulation was suppressed by the antioxidant butylated hydroxyanisol, which also abolished ATRA-induced JNK activation and apoptosis. Altogether, our results demonstrate an anti-apoptotic effect of NF-kappaB activation during ATRA-induced differentiation of NB4 cells and identify repression of ROS-mediated JNK activation as a mechanism for this effect. Our observations also suggest that NF-kappaB signalling may contribute to an accumulation of mature APL cells and participate in the development of ATRA syndrome.   

Abstract 12

 Title: RADIO-INDUCED LIFESPAN ALTERATION OF DROSOPHILA MELANOGASTER MUTANTS WITH DEFECTS OF DNA REPAIR AND APOPTOSIS PATHWAYS 

 Authors: Moskalev A. 

 Address: 167982, Syktyvkar, Kommunisticheskaja St. 28, RUSSIA 

 Abstract: It was investigated the influence of low doze gamma-irradiation at pre-imago stages on the life span of Drosophila imago. Shown, that the life span in unaffected strains with apoptosis deregulations (mutations grim, hid, reaper, Dcp-1, dArk, th), DNA repair defects (rad54, mus210, mus209, mei-9, mei-41) and red-ox system weakening (Sod) lower than in wild type strain Canton-S. Revealed, that low doze chronic irradiation (60 cGy per generation) led to significant increasing of the life span (in strains with the mutations grim, hid, reaper, Dcp-1, dArk, th, rad54, mus210, mus209, Sod). In some cases the level of the life span exceeded that in intact strain Canton-S (Sod, th, Dcp-1, dArk). In the sensitive to apoptosis induction strains (mutants on apoptosis and red-ox system), possibly, this is due to induced elimination of the radiosensitive cells that will be subjected to accelerated aging.  The life span in homozygous and hemizygous flies with the mutation of the gene mei-41 (homolog of the human ATM) after irradiation was decreased with comparison to the control, but in heterozygous was increased. Obviously this is because of the major role of ATM/mei-41 in sensing of DNA defects after irradiation. Thus, it is demonstrated the recessive effect of mei-41 mutation in the control of radio-induced life span alteration. It is shown, that lines with various genotypes react in the different way to an irradiation. The results are discussed from the point of view of the role of apoptosis and genomic instability in radio-induced aging. 

Abstract 13

 Title: TRAIL INDUCES A RIP (RECEPTOR-INTERACTING-PROTEIN)-DEPENDENT AND CASPASE-DEPENDENT NECROSIS-LIKE CELL DEATH UNDER ACIDIC EXTRACELLULAR CONDITIONS (pH 6.5)   

 Authors: Meurette O., Huc L., Rebillard A., Le Moigne G., Lagadic-Gossmann D. and Dimanche-Boitrel M-T. 

 Address: INSERM Unit 620, University of Rennes; 1, Faculty of Pharmacy 2 Av, du Pr. Leon Bernard  35043; Rennes cedex, France. 

Abstract 14

 Title: Intracellular calcium limits nuclear translocation of clusterin in COLO 205 colorectal cancer cells. 

 Authors: Pajak Beata, Orzechowski Arkadiusz 

 Address: Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, ul. Nowoursynowska 159, 02-776 Warsaw, Poland 

 Abstract:   Background: Clusterin is the protein with pro- or antiapoptotic activities regulated by calcium homeostasis. So far, three isoforms of clusterin were identified, even though, ultimately 50 kDa intracellular clusterin (iClu) is widely known to be apoptogenic. The calcium-dependent cellular retention of iClu correlates positively with cell survival, whereas in calcium-deprived cells this protein translocates to nucleus and promotes cell death. Aim of the study: Our previous results suggest that viability of COLO 205 colorectal cancer cells is reduced by calcium chelators. The aim of the study was to verify if the observed calcium-dependent sensitization to apoptosis is associated with clusterin activity. Results: 1) In COLO 205 cells Western-blot analyses identified either form of clusterin: precursor (60 kDa), intracellular (50 kDa) and secretory (80 kDa). 2) The nuclear presence of 50 kDa iClu was provoked by topoisomerase II inhibitor etoposide. At 24^nd hour of treatment etoposide (50 or m100 M) caused both nuclear translocation of clusterin and extensive cell death. 3) Neither calcimycin A23187 (1 mM, 5 mM, 6 hours) nor the extracellular calcium chelator EGTA (2 mM, 5 mM, 6 hours) seem to modulate the clusterin activity. 4) The intra- and extracellular calcium chelator EDTA (2 mM, 5 mM, 6 hours) stimulated nuclear translocation of iClu. Discussion: It appears, that in COLO 205 cell line the intracellular calcium plays the crucial role in cell survival. The EDTA but not EGTA profoundly decreased cell survival (MTT test). At the same time and in identical conditions the clusterin protein was translocated to the nucleus. We conclude, that in the intracellular calcium deprived COLO 205 cells nuclear translocation of clusterin occurs.. Perspectives: Better understanding of clusterin functions with respect to isoform and the molecular mechanisms that control its activity give prospects to overcome the resistance of cancer cells to cell death. The combined treatment of cytotoxic factors (death ligands, ioniozing radiation, chemotherapeutics) and suitable intracellular calcium modulators might help to overcome “immune escape” of cancer cells. In order to reduce the risk of the side effects observed during chemotherapy they could also validate the use of lower doses of cytostatics . 

Abstract 15

 Title: Proteinase-3 induces procaspase-3 activation in the absence of apoptosis: Potential role of this compartmentalized activation of membrane-associated procaspase-3 in neutrophils 

 Authors: Magali Pederzoli, Chahrazade Kantari, Valérie Gausson, Sandra Moriceau, and Véronique Witko-Sarsat 

 Address: INSERM U507, Hôpital Necker, 161, rue de Sèvres 75015 Paris 

 Abstract: We provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human (HMC1) and rat (RBL) mast cell lines were transfected either with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of i) phosphatidylserine externalization ii) mitochondria cytochrome-c release iii) upstream caspase-8 or caspase-9 activation iv) DNA fragmentation. In vitro, PR3 cleaved procaspase-3 into an active 22 kDa fragment. In neutrophils, the 22 kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. This 22 kDa fragment was not pres  ent when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22 kDa-caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization, further showed that PR3 and procaspase-3 could colocalize in an extra-granular compartment. In conclusion, our results strongly suggested that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophils survival. This work was supported by ARP (MP), and VLM (CK). 

Abstract 16

 Title: APOPTOSIS AND EXPRESSION OF IL1A AND VEGF FOLLOWING PHOTODYNAMIC TREATMENT IN VITRO 

 Authors: A. Sadauskaite1, D. Dabkeviciene1, E. Leman1, V. Jurgelevicius2, V. Kirveliene1 

 Address: 1Vilnius University, Department of Biochemistry and Biophysics, Vilnius, Lithuania 2Lithuanian National Veterinary Laboratory 

 Abstract:  Photodynamic treatment (PDT) mediated by a promising photosensitizer mTHPC (Foscan) in combination with an antitumour drug was used for treatment of the human epidermoid carcinoma cell line A-431 in vitro. Two cytostatics with different mechanisms of action were used: a microtubule-inhibitor Taxotere (Tax) and a DNA-intercalator Doxorubicin (Dox). Under applied conditions, PDT in combination with the cytostatic was found to be significantly more cytotoxic in comparison with either single treatment, and the character of the combination was additive. Enhanced apoptotic cell death after the combined treatment in comparison with either single treatment was detected by recording caspase-3 activity. Caspase-8 was not active under the studied conditions.  Gene expression pattern was analyzed at 3.5 h post-treatment by cDNA macro array technique and verified by real-time RT-PCR. In summary, significant overexpression of genes of vascular endothelial growth factor A (VEGF) and, especially, a proinflammatory protein, interleukin 1, alpha (IL1A), was detected following mTHPC-mediated PDT and both combined treatments.  Investigation of dose effect and time course analysis was carried out by real time RT-PCR. In the case of PDT, increased duration of light exposure resulted in decreased cell viability (from 75% to 20%  at 24 h post-treatment) and significantly increased expression of IL1A (from 3- to 7-fold increase at 4 h post-treatment regarding to untreated cells) and less significantly increased expression of VEGF (from 2- to 3-fold). Neither Tax, nor Dox inreased the amount of mRNA of IL1A or VEGF. The highest amount of mRNA in all cells treated with PDT, single or combined, was detected at 8 h post-treatment: IL1A, 14-fold, and VEGF, 10-fold increase regarding to untreated cells. Our results should point to a hazardous tumour response to a regimen, which at the first sight seemed effective. On the other hands, our results suggest that experiments in vitro could produce valuable data for estimation of tissue response including potential risk of secondary tumor proliferation and spreadin  g after treatment.   

Abstract 17

 Title: Inhibition of adenine nucleotide translocator pore function and protection against apoptosis in vivo by an HIV protease inhibitor anion channel. 

 Authors: Agathe Tarze, Morgane Le Bras, Aurélien Deniaud, Christophe Lemaire, and Catherine Brenner 

 Address: Université de Versailles / St Quentin CNRS FRE2445 45 avenue des Etats-Unis 78035 Versailles 

 Abstract:   Inhibitors of HIV protease have been shown to have antiapoptotic effects in vitro, yet whether these effects are seen in vivo remains controversial. In this study, we have evaluated the impact of the HIV protease inhibitor (PI) nelfinavir, boosted with ritonavir, in models of nonviral disease associated with excessive apoptosis. In  mice with Fas-induced fatal hepatitis, Staphylococcal enterotoxin B–induced shock, and middle cerebral artery occlusion–induced stroke, we demonstrate that PIs significantly reduce apoptosis and improve histology, function, and/or behavioral recovery in each of these models. Further, we demonstrate that both in vitro and in vivo, PIs block apoptosis through the preservation of mitochondrial integrity and that in vitro PIs act to prevent pore function of the adenine nucleotide translocator (ANT) subunit of the mitochondrial permeability transition pore complex. 

Abstract 18

 Title: CHONDROCYTE APOPTOSIS IN ARTICULAR CARTILAGE: COMPARISON OF DIFFERENT TECHNIQUES   

 Authors: CM Thomas (presenter), C Whittles, MJ Perry, MA Adams and M Sharif 

 Address: Department of Anatomy, University of Bristol, Southwell Street, Bristol, BS2 8EJ United Kingdom 

 Abstract:  Introduction:   Apoptosis of articular cartilage chondrocytes may play an important role in the pathogenesis of osteoarthritis (OA) and other joint diseases. This study aims to evaluate three of the main techniques for determining chondrocyte death by apoptosis including the established physical method of detection. Methods:  Articular cartilage from porcine knee joints (n=5) aged between 4 and 6 months was used. Full depth cartilage from the femoral condyle was removed from underlying bone within 1 hour of sacrifice and 5 micrometre thick paraffin embedded sections obtained. Three different methods were used to determine cell death by apoptosis: morphology using light microscopy, Terminal Deoxynucleotide Transferase Mediated UTP Nick End Labelling (TUNEL) and caspase-3 immunohistochemistry. Using haematoxylin and eosin staining, apoptotic cells were classed as those with morphology indicative of apoptosis including membrane blebbing, nuclear condensation and cell shrinkage1. TUNEL is a widely used method for apoptosis quantification detecting DNA cleavage in apoptotic cells. Caspase-3 in its active form is one of the key mediators of apoptosis in its execution phase. Results: Mean percentage (SD) apoptosis values obtained using the 3 methods are presented in the table below.

Cartilage zone

Microscopy

Caspase-3

TUNEL

Superficial

1.83 (1.16)

6.74  (5.47)

36.57  (2.85)

Middle

0.62  (0.59)

10.23(8.36)

21.21 (12.91)

Deep

0.64  (0.66)

13.55  (4.91)

28.71  (13.89)

Average of zones

1.09  (0.57)

10.42.  (5.25)

27.19  (10.31)

Discussion: Results show that there are clear differences in the detection of apoptosis between the 3 methods used. Some of these differences may be attributable to the fact that each method is sensitive to different stages of the apoptosis process. Apoptosis determined by the direct morphological method (microscopy) is in closer agreement with that estimated by caspase-3 immunohistochemistry than TUNEL. Therefore, estimating the rate of cell death by apoptosis by staining for active caspase-3 may be more reliable than the widely used TUNEL technique. These methodological issues need to be resolved before we can accurately quantify the extent of apoptosis and speculate upon a role for this process in OA.  

 Reference: 1. Roach HI, Aigner T et al. (2004) Apoptosis. 9, pp.265-77. 

Abstract 19

 Title: HUMAN CD4+CD25+ REGULATORY T CELLS INHIBIT MONOCYTE LPS-INDUCED ACTIVATION TROUGH A FAS-LIGAND MECHANISM 

 Authors: F Venet (1), AL Debard (1), J Bohé (2), J Bienvenu (1), A Lepape (2), G Monneret (3). 

 Address: (1) Immunology laboratory, Lyon-Sud University Hospital. (2) Intensive care units, Lyon-Sud University Hospital. (3) Immunology Laboratory, Hôpital Neurologique, Lyon.

 Abstract: Introduction:  Septic shock pathophysiology is now described as a 2 phase-mechanism. The first inflammatory phase is rapidly counterbalanced by an anti-inflammatory response leading to a delayed state of immunosuppression largely responsible for high mortality (i.e. 50 %). The elevation of circulating CD4+CD25+ regulatory T cells (Treg) has been described as a feature of immunosuppression. The purpose of this study was to assess the effect of Treg on monocytes (key cells of septic shock pathophysiology) after LPS challenge (mimicking in vivo condition).  Methods and results: In a model of human purified cells (magnetic separation), we first measured the effect of Treg on monocyte surface molecules modulated during sepsis: CD14, TLR4, HLA-DR. We observed a strong inhibitory effect on CD14 increase after LPS challenge (no effect on TLR4 and HLA-DR).

n = 67

Monocytes

Monocytes + LPS

Monocytes + LPS + Treg

MFI CD14

(Mean ± SEM)

20 ± 1 *

32 ± 2

16 ± 1 *

* : p < 0.0001 vs monocytes + LPS

Transwell experiments demonstrated that the effect of Treg was mediated by a soluble factor. Among molecules produced by Treg and potentially responsible for this effect, only IL-10 was present in measurable concentrations. However, recombinant IL-10 failed to decrease monocyte CD14 expression and anti-IL-10 antibodies failed to block Treg effect. Interestingly, Annexin-V and DIOC6 stainings demonstrated that the loss of monocyte CD14 was due to monocyte apoptosis. Among blocking antibodies directed against soluble pro-apoptotic factors (FasL, TNF-alpha, TRAIL), solely anti-FasL antibody almost abrogated Treg inhibiting effect.

% Inhibition of CD14 increase in response to LPS(Mean ± SEM)

Without Ab

+ anti FasL Ab

126 ± 22 %

40 ± 13 % p < 0.05

Discussion: We propose a role for Treg in monocyte apoptosis observed during septic shock through the production of soluble FasL. To our knowledge, this is the first report of FasL release by human Treg. Apoptotic mechanisms are largely responsible for sepsis-induced immunosuppression. The understanding and the definition of the different mediators involved in pathophysiology of septic shock may thus provide new therapeutic options for this hitherto deadly disease. 

Abstract 20

 Title: Survivin expression  in pituitary tumours.

 Authors: R.Wasko1, A.Jankowska2, J.Waligorska-Stachura1, M.Andrusiewicz2, M.Jaskula1, J.Sowinski1 . 

 Address: 1 Department of Endocrinology, Metabolism and Internal Diseases University of Medical Sciences in Poznan 2 Department of Radiobiology and Cell Biology University of Medical Sciences in Poznan 

 Abstract: Survivin is a member of the inhibitor of apoptosis protein ( IAP ) family. Its anti-apoptotic function seems to be related to the ability to directly or indirectly inhibit caspases. It inhibits activity of caspase – 3, caspase – 7 and caspase – 9. Survivin has been also suggested to regulate cell division during mitosis. Moreover, it is only of IAPs that is connected to the cell cycle being upregulated in the G2/M phase. Survivin is strongly expressed in embryonic and fetal organs and it is downregulated in normal adult tissues; it becomes re-expressed, however, in a variety of humans cancers.  The aim of the study was to determine the survivin expression in different types of pituitary adenomas. Methods: Tissue samples were obtained during neurosurgical removal of the tumour from 16 patients with diagnosed: acromegaly in nine cases, non-functioning pituitary tumours in six cases and prolactinoma in one case. After RNA extraction and cDNA synthesis, the amplification of specific survivin’s gene fragment was performed. As a control, the total RNA isolated from a tumour cell line HELA was used. Results: The findings of our study demonstrated the presence of an active survivin gene in all sixteen analysed pituitary tumours.

Conclusions: Based on these findings, we conclude that the estimation of survivin expression in human pituitary tumours may help to predict tumour growth and prognosis. 

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