Article |
Address correspondence to Jacques Baudier, INSERM EMI-0104, DRDC-TS, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. Tel.: (33) 4-38-78-43 28. Fax: (33) 4-38-78-50-58. email: jbaudier{at}cea.fr
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Abstract |
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Key Words: actin; calcium; cytoskeleton; cell adhesion; S100B
Abbreviation used in this paper: siRNA, small interfering RNA.
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Introduction |
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Results |
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To evaluate the contribution of S100A10 in the annexin 2/S100A10/AHNAK interaction, we examined the effect of the S100B protein on that interaction. Previously, we have shown that S100B, another member of the S100 family, interacts with AHNAK-Cter in a strict calcium-dependent manner (Gentil et al., 2001). Because striking structural similarities exist between S100A10 and Ca2+-bound S100B (McClintock et al., 2002), we examined whether S100B can displace annexin 2/S100A10. First, we controlled that the interaction between the annexin 2/S100A10 complex and GST-AHNAK-Cter occurs in the presence of Ca2+. Next, we observed that in the presence of Ca2+, but not in EGTA (unpublished data), addition of recombinant S100B to the cell extracts fully antagonizes the binding of the annexin 2/S100A10 complex in a dose-dependent and competitive manner (Fig. 2 D). These data suggest that the annexin 2/S100A10- and S100B-binding domains on AHNAK-Cter overlap, and that S100A10 mediates the interaction between AHNAK and annexin 2.
The direct interaction of S100A10 with AHNAK was confirmed in vitro. When expressed in rabbit reticulocyte, S100A10 protein binds to and is pulled down by the recombinant GST-COOH-terminal domain of AHNAK (Fig. 2 E). No binding of rabbit reticulocyte that expressed annexin 2 could be detected (unpublished data). The direct interaction of S100A10 with AHNAK was also confirmed in yeast two-hybrid experiments. A human heart cDNA library was screened using the extreme COOH-terminal domain of AHNAK (CterC; aa 51245643) as a bait. 15 colonies were obtained out of 2 x 106 primary yeast transformants. The interaction specificity was retested after the mating assays on YC-UWLH medium and X-gal filter tests. Out of the 15 library plasmids retested, only two interacted specifically with CterC. The remaining plasmids gave a positive result with lamin and were considered as false positive. The nucleotide sequence of the two positive clones revealed that they correspond to S100A10. The specificity of interaction between S100A10 and CterC is illustrated in Fig. 2 F. Only CterC is competent for interaction with S100A10, and not CterN (aa 46425124), CterdLZ (aa 46424826), or lamin. Our double-hybrid experiment also reveals that S100B is capable of heterodimerizing with S100A10 in a yeast two-hybrid system (Fig. 2 F). Heterodimerization of S100B with other S100 proteins, such as S100A1, S100A6, and S100A11, has been observed previously (Deloulme et al., 2000). In contrast to the results obtained with S100A10, no interaction between S100B and AHNAK peptides could be detected in the yeast two-hybrid system (unpublished data). This is likely attributable to the limitation of yeast two-hybrid analysis to calcium-independent interactions (Deloulme et al., 2003).
AHNAK and the annexin 2/S100A10 complex are recruited to the sites of early cellcell contacts
Confocal scanning immunofluorescence analyses of confluent epithelial MDCK cells reveal that AHNAK and annexin 2 colocalize all over the plasma membrane, including the sites of cellcell contacts (Fig. 3 A). An identical pattern of distribution was observed for S100A10 (unpublished data). We had observed that AHNAK was recruited to the plasma membrane as cell confluence increased and as cells polarized (Fig. 1 A, e). The polarized phenotype of these cells was confirmed by the reorganization of the actin cytoskeleton (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1) and the apicolateral polarization of the tight junction marker ZO-1 (unpublished data). In polarized epithelial cells, cellcell contacts initiate plasma membrane remodelling and the development of cell polarity (Drubin and Nelson, 1996). Therefore, we tested the role of the formation of cellcell contacts in recruiting AHNAK to the plasma membrane. Lowering the Ca2+ concentration in the culture medium of an MDCK monolayer interferes with the stability of intercellular contacts (Grindstaff et al., 1998). Treatment of confluent MDCK cells with low Ca2+ medium resulted in the redistribution of AHNAK and of the annexin 2/S100A10 complex from cell contacts to the cytoplasm, and correlated with a more flattened morphology of the cells (Fig. 3 B, a and b). This membrane dissociation of AHNAK and of the annexin 2/S100A10 complex was reversible (Fig. 3 B, cf). When cellcell contacts were allowed to reform by readdition of calcium into the culture medium, AHNAK and the annexin 2/S100A10 complex were recruited to the sites of cellcell contacts with a similar kinetic of relocation and distribution pattern. Within 30 min of calcium addition, AHNAK and S100A10 started to relocate to the newly forming cellcell contacts (Fig. 3 B, c and d), and to strongly accumulate there after 3 h, as cell acquired a more cuboidal epithelial morphology (Fig. 3 B, e and f). Coimmunoprecipitation experiments on cross-linked MDCK cells during the calcium switch experiment revealed that AHNAK forms a multimeric complex containing actin and annexin 2/S100A10, and that this association is strictly dependent on the localization of AHNAK at the plasma membrane (Fig. 3 C, lane 4). Upon membrane dissociation of AHNAK, only S100A10, and to a lesser extent annexin 2, are recovered within the AHNAK immunoprecipitates (lane 2).
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The annexin 2/S100A10/AHNAK complexes colocalize to intercellular junctions in a cholesterol-dependent mechanism
In addition to the Ca2+-dependent association of annexin 2 with phospholipids, annexin 2 can also associate with the plasma membrane lipid raft microdomains in a cholesterol-dependent manner (Oliferenko et al., 1999; Babiychuk and Draeger, 2000; Babiychuk et al., 2002). Several laboratories have reported that annexin 2 may link lipid rafts with the cortical cytoskeleton (Oliferenko et al., 1999), and that annexin 2 recruits signaling proteins to intercellular junctions in a cholesterol-dependent manner (Hansen et al., 2002). To test a possible association of AHNAK with the annexin 2/S100A10 complex within lipid rafts, we performed flotation experiments in OptiPrepTM gradients in the presence of cold Triton X-100 (Fig. 4 A). In this gradient, the detergent-insoluble lipid rafts will float at the interphase between the 0 and 20% OptiPrepTM layers. In MDCK cells, a significant amount of AHNAK partitioned with annexin 2 and S100A10 into the lipid raft fraction together with caveolin-1, a known cholesterol-binding protein (Fig. 4 A). Not all the annexin 2 cosedimented with AHNAK, suggesting that only a portion of the annexin 2/S100A10 complex interacts with AHNAK and associates with the lipid rafts. We confirmed the association of AHNAK with lipid rafts in whole cells by colocalization of AHNAK with the FITC-labeled ß subunit of cholera toxin, which specifically binds the lipid raft ganglioside GM1 (Harder et al., 1998). Confocal laser scanning microscopy of MCF-7 cells showed an extensive overlap of AHNAK staining with the FITC-labeled cholera toxin ß chain at the plasma membrane, including intercellular junctions (Fig. 4 B). Furthermore, depletion of plasma membrane cholesterol in MDCK and MCF-7 cells with methyl-ß-cyclodextrin released a population of annexin 2 from the plasma membrane and totally abolished the junctional membrane localization of AHNAK, causing their redistribution to the cell cytoplasm (Fig. 4 C).
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Discussion |
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In cultured epithelial cells, AHNAK targeting to the plasma membrane is reversible and triggered by Ca2+-dependent cell adhesion. Disruption of E-cadherinmediated cellcell contacts by low extracellular Ca2+ results in the dissociation of AHNAK from the plasma membrane. After readdition of Ca2+, AHNAK is rapidly re-recruited to the plasma membrane at the sites of newly formed cellcell contacts. In contrast to the tight junction-associated protein ZO-1, AHNAK immunoreactivity will progressively become more widely distributed all over the plasma membrane (unpublished data). In fully polarized MDCK cells, AHNAK immunoreactivity decorates the entire basal, lateral, and apical cell membranes. Such distribution of AHNAK suggests that AHNAK is not a junctional protein. Down-regulation of AHNAK levels in MDCK cells using AHNAK-specific siRNA prevents cortical actin cytoskeleton reorganization. These observations suggest a more general function for AHNAK in organizing cell cytoarchitecture at the plasma membrane.
To gain further insight into the molecular mechanisms of AHNAK function at the plasma membrane, we have searched for AHNAK partners at the plasma membrane. We have identified the annexin 2/S100A10 complex as a major AHNAK-binding protein in epithelial cells. Annexin 2 (also called calpactin I heavy chain, p36) is a member of the annexin family of Ca2+- and phospholipid-binding proteins, which has been implicated in membrane trafficking and organization (for review see Gerke and Moss, 2002). Annexin 2 has been proposed to play a role in the organization of cholesterol-rich membrane microdomains (Oliferenko et al., 1999), the connection of lipid rafts with the under lying actin cytoskeleton (Harder et al., 1997; Gerke and Moss, 2002), and in cholesterol-mediated adherent junction formations (Harder et al., 1997; Corvera et al., 2000). Within cells, annexin 2 can occur either as a monomer or as a heteroterameric complex coupled with S100A10 (also called calpactin I light chain, p11). The heterotetrameric annexin 2/S100A10 complex is the predominant form of annexin 2 present at the plasma membrane in epithelial cells (Harder and Gerke, 1993). The interaction between AHNAK and annexin 2/S100A10 occurs within the COOH-terminal domain of AHNAK. The direct interaction of S100A10 with the extreme COOH-terminal domain of AHNAK observed in vitro and in yeast two-hybrid experiments strongly suggests that S100A10 mediates the interaction between AHNAK and annexin 2. The physical interaction between AHNAK and the annexin 2/S100A10 complex in the cell is supported by several observations. First, a strict correlation exists between accumulation of AHNAK and the annexin 2/S100A10 complex to the plasma membrane during both confluence-mediated (Hansen et al., 2002; Fig. 1 A) and Ca2+-dependent cellcell adhesion (Fig. 3). Second, at the plasma membrane, AHNAK colocalizes with the annexin/S100A10 complex, and a significant amount of proteins associate with membrane rafts in a cholesterol-dependent manner (Fig. 4). Third, annexin 2 depletion by siRNA promotes AHNAK release to the cytoplasm (Fig. 5, B and C). The annexin/S100A10 dependence for AHNAK membrane localization strongly suggests that annexin 2/S100A10 contributes to the recruitment and stabilization of AHNAK at the plasma membrane. Two recent reports have pointed out a role for S100A10 in targeting channel proteins to the plasma membrane (Girard et al., 2002; Van de Graaf et al., 2003). A more general function for the annexin 2/S100A10 in the routing of proteins to the plasma membrane must now be considered.
Membrane-bound AHNAK not only interacts with annexin 2/S100A10, but also is part of a complex containing actin (Fig. 3, C and D). This observation is consistent with a recent paper describing physical interaction between AHNAK and F-actin and G-actin (Hohaus et al., 2002), and strongly suggests that AHNAK and annexin 2/S100A10 are part of a submembranous complex that interacts physically with the cortical actin cytoskeleton. Annexin 2/S100A10- and AHNAK-depleted MCF-7 cells both display decreased cell height. A similar and more drastic effect is observed in polarized epithelial MDCK cells, which retain a mesenchymal morphology upon AHNAK depletion (Fig. 7). Down-regulation of AHNAK prevents the apicolateral actin cytoskeleton reorganization required to support cell height. We propose that AHNAK and the annexin 2/S100A10 complex are involved in the regulation of the actin cytoskeleton organization at the lateral plasma membrane, and thus could be implicated in plasma membrane remodelling and in the establishment of specialized intercellular interaction. Such a role for AHNAK is consistent with its specific expression in most lining epithelium and in endothelial cells of impermeable brain capillaries and peripheral blood vessels, and conversely with its absence in the highly permeable fenestrated endothelium of the kidney glomeruli, the hepatic sinusoid, and the continuous capillaries of lung (Gentil et al., 2003). In adult tissues, AHNAK is also abundantly expressed at the plasma membrane of smooth muscle cells (Gentil et al., 2003). In smooth muscle cells, annexin 2 is thought to function as a cross-linker of lipid rafts with the underlying actin cytoskeleton to regulate sarcolemmal tension (Babiychuk and Draeger, 2000; Babiychuk et al., 2002). In these cells, AHNAK may cooperate with annexin 2 in the coordination of cytoskeletal and membrane rearrangementsa coordination that provides structural support of the membrane to respond to and control stretching forces (Babiychuk and Draeger, 2000; Babiychuk et al., 2002). A similar role for AHNAK has already been suggested in cardiac muscle (Hohaus et al., 2002). Although our paper highlights a structural role of AHNAK, the association of AHNAK with lipid rafts also opens the possibility that AHNAK could be implicated in regulation of signaling pathways that are compartmentalized within these microdomains (Simons and Toomre, 2000; Zajchowski and Robbins, 2002).
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Materials and methods |
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For calcium switch experiments, cells were incubated for 30 min with DME supplemented with 5 mM EGTA and 1 mM MgCl2 to lower extracellular Ca2+ concentration and to perturb strict Ca2+-dependent cellcell adhesion, but to maintain Mg2+-dependent cellmatrix adhesion. Cellcell contacts were allowed to reform for the indicated times by returning cells to complete medium.
Reagents and antibodies
The pAbs directed against AHNAK, KIS, and CQL have been described previously (Gentil et al., 2003). Anti-annexin 2 mAb HH7 was a gift from V. Gerke (Universiy of Münster, Münster, Germany). Anti-annexin 2, anti-annexin 2 light chain (S100A10), anti-annexin IV, anti-annexin VI, and anti-caveolin-1 mAbs were purchased from Transduction Laboratories. Texas redphalloidin was purchased from Molecular Probes, Inc., and anti-actin mAb was purchased from Sigma-Aldrich. FITC-labeled cholera toxin B subunit and the cholesterol chelator methyl-ß-cyclodextrin were purchased from Sigma-Aldrich.
In vitro proteinprotein interaction assay/GST pull-down
GST-AHNAK-Cter and GST-AHNAK-M1 proteins (Gentil et al., 2001) were produced in the Escherichia coli AD494(DE3)pLYsS strain (Novagen) and purified by glutathione-Sepharose affinity chromatography. For metabolic labeling, cells were labeled in methionine-free MEM and 5% FCS supplemented with 50 µCi/ml [35S]methionine/cysteine mix for 12 h. For binding assays, cells were lysed at 4°C in TTBS buffer (40 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.3% Triton X-100) plus protease inhibitors (leupeptin, aprotinin, pepstatin, and AEBSF; 10 µg/ml each), and were centrifuged for 10 min. Cell lysates were precleared by incubation for 10 min with 50 µl GST-Sepharose. 500-µl aliquots of precleared supernatant were supplemented with either 5 mM EDTA/5 mM EGTA or with 0.3 mM CaCl2/10 µM ZnSO4 and mixed with 10 µg purified GST fusion proteins plus 30 µl affinity GST-Sepharose beads equilibrated in the same buffers. After mixing for 15 min at 4°C, the beads were spun down and the supernatant was removed. The beads were washed three times with 1 ml binding buffers. At the last wash, beads were transferred to new tubes and boiled in SDS sample buffer.
Mass spectrometric analysis and protein identification
Proteins recovered within AHNAK immunoprecipitates were excised from Coomassie bluestained gels and washed with 50% acetonitrile. Gel pieces were dried in a vacuum centrifuge and rehydrated in 20 µl of 25 mM NH4HCO3 containing 0.5 µg trypsin (sequencing grade; Promega). After 4 h incubation at 37°C, a 0.5-µl aliquot was removed for MALDI-TOF analysis and spotted onto the MALDI sample probe on top of a dried 0.5-µl mixture of 4:3 saturated -cyano-4-hydroxy-trans-cinnamic acid in acetone/10 mg/ml nitrocellulose in acetone/isopropanol 1:1. Samples were rinsed by placing a 5-µl volume of 0.1% TFA on the matrix surface after the analyte solution had dried completely. After 2 min, the liquid was blown off by pressurized air. MALDI mass spectra of peptide mixtures were obtained using a mass spectrometer (Bruker Biflex; Bruker-Franzen Analityk). Internal calibration was applied to each spectrum using trypsin autodigestion peptides (MH+ 842.50, MH+ 1045.55, and MH+ 2211.11). Protein identification was confirmed by tandem mass spectrometry experiments as described previously (Gentil et al., 2001).
Double hybrid
For plasmid constructions, fusion proteins with LexA DNA-binding domain (LexADBD) were constructed in pLex10. For the pLex-AHNAK Cter construction, BamHIEcoRI AHNAK fragment (aa 46425643) from pDY-C (Nishimoto) was subcloned into pcDNA3.1. The insert was then excised with BamHI and XhoI digestion and cloned into BamHISalI sites of pLex10. pLex-CterN was obtained by deleting AHNAK pstIpstI fragment from pLex-AHNAK Cter. To obtain the pLex-AHNAK-CterC construct, pstIpstI AHNAK fragment (aa 51245643) of pLex-AHNAK Cter was subcloned into the pstI site of plex10. For the pLex-Cter-LZ construct, the pLex-CterN plasmid was deleted by SalIPstI digestion, blunted with Deep vent polymerase, and self ligated. pLexS100B was obtained by amplification of human S100B cDNA using primers containing SmaI and pstI sites immediately flanking the start and the stop codon, respectively, and cloning of the PCR product into SmaIpstI sites of pLex10.
Large-scale yeast transformations using the human heart MatchmakerTM cDNA library constructed in pACT2T plasmid (CLONTECH Laboratories, Inc.) and two-hybrid screens were performed using an L40 yeast strain essentially as described previously (Deloulme et al., 2000). Primary transformants were analyzed on YC-UWLH medium plates. Growing clones were then tested for ß-galactosidase expression (Deloulme et al., 2000). Library plasmids expressing LEU2 from positive transformants were selected using HB101 E. coli, which requires leucine supplementation for growth. cDNAs were retested in a mating assay against the original bait construct and pLexA-lamin as a control using the AMR70 yeast strain as described below. The interactions were also tested by yeast mating essentially as described previously (Deloulme et al., 2000).
Immunoprecipitation and Western blotting
For total cell lysates and coimmunoprecipitation, cells were washed in PBS and lysed on ice in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.05% deoxycholic acid, 1% Triton X-100, 10% glycerol, 2 mM EDTA/EGTA, and protease inhibitor cocktail). Lysates were passed through a 26G needle and centrifuged for 10 min at 2,500 rpm in a tabletop centrifuge. Supernatants were either quantified with the BCA protein micro assay (Pierce Chemical Co.) and boiled in 1x DTT Laemmli buffer (total cell lysates), or incubated with either anti-AHNAK-KIS antibody together with protein ASepharose (Amersham Biosciences), anti-AHNAK-CQL antibody cross-linked onto Sepharose beads, or with protein ASepharose alone, for 1 h rotating at 4°C. The immunoprecipitates were washed three times in lysis buffer, and the beads boiled in 1x Laemmli with 20 mM DTT. Proteins were separated by SDS-PAGE using 5, 8, or 14% polyacrylamide concentrations to resolve AHNAK, actin, or annexin 2/S100A10, respectively. Proteins were blotted onto nitrocellulose membranes.
For cross-linking experiments, cells were washed in PBS, incubated for 5 min with 0.5 mM dithiobis succinimidyl propionate (Pierce Chemical Co.) in PBS, and washed twice in 50 mM glycine in PBS before cell lysis and AHNAK immunoprecipitation. Immunoprecipitated proteins were reduced by boiling for 5 min in 1x Laemmli buffer containing 5% ß-mercaptoethanol, and were analyzed by Western blot.
RNA interference
21-nt siRNA duplexes targeting the 5'-AAGAUCUCCAUGCCUGAUGUG-3' mRNA sequence in the repeated domains of ahnak, and the 5'-AAGUGCAUAUGGGUCUGUCAA-3' mRNA sequence corresponding to the NH2-terminal domain of human annexin 2 were purchased from Dharmacon. The sequence used was submitted to a BLAST search to ensure targeting specificity. The specificity of AHNAK and annexin 2 down-regulation was further checked by Western blotting analysis. MCF-7 or MDCK cells plated at low density (3 x 104/cm2) were transfected with 20 nM of either annexin 2 or AHNAK siRNA duplex, or scrambled siRNA using OligofectamineTM (Life Technologies) on two consecutive days, and maintained for a total of 4 d in DME 10% serum.
Immunofluorescence
Cells were either fixed in 4% PFA for 10 min at RT, followed by a permeabilization with 0.1% Triton X-100 for 10 min or with 70% methanol at -20°C for 10 min. Cells were then washed in TBS and incubated with the primary antibody in TBS containing 3% goat serum overnight at 4°C. After TBS washes, cells were then incubated for 1 h at RT with the secondary antibody in TBS goat serum, and phalloidin when required. The secondary antibodies coupled with Alexa® 488 were purchased from Molecular Probes, Inc., and those coupled with Cy3 and Cy5 were purchased from Jackson ImmunoResearch Laboratories. After the final washes, cells were mounted in fluorescence mounting medium (DakoCytomation) and analyzed with a fluorescent microscope (Axiovert 200M; Carl Zeiss MicroImaging, Inc.) or a confocal microscope (TCS-SP2; Leica).
For plasma membrane permeabilization, cells were treated with 0.5 U streptolysin O (Sigma-Aldrich) in 0.1% BSA-PBS at 4°C and transferred at 37°C for 5 min. Cells were then washed with PBS and incubated with the primary antibody diluted in PBS/5% goat serum for 30 min at 4°C. After PBS washes, cells were fixed with 4% PFA and processed as above.
Cholera toxin labeling of GM1
MCF-7 cells were incubated with 0.5 µg/ml FITC-labeled cholera toxin ß chain in DME for 4 min at 37°C. Cells were then washed twice with DME, fixed with 4% PFA for 10 min at RT, and permeabilized with 0.1% Triton X-100 for 10 min at RT. Cells were then analyzed by immunofluorescence for AHNAK and S100A10.
Flotation gradients
For the isolation of lipid rafts, cells grown in 100-mm tissue culture dishes were washed and scraped in PBS, pelleted, and lysed on ice for 30 min in 100 µl Triton X-100 lysis buffer (400 mM Tris, pH 7.5, 150 mM NaCl, 1 mM DTT, 1% Triton X-100, and protease inhibitor cocktail). After a 10-min spin at 2,500 rpm at 4°C, the supernatant was mixed with 200 µl of a 60% OptiPrepTM (Axis-Shield) solution to obtain a 40% final solution, and transferred to centrifuge tubes. The sample was overlayed with 270 µl of a 35, 30, 25, 20, and 5% solution of OptiPrepTM in Triton lysis buffer. The gradient was centrifuged in an ultracentrifuge (Optima TL; Beckman Coulter) at 45,000 rpm for 4 h at 4°C in a rotor (TLS-55; Beckman Coulter). The fractions were collected and analyzed by Western blotting.
Online supplemental material
Fig. S1 shows a Coomassie blue stain of the proteins coimmunoprecipitated with AHNAK. Fig. S2 shows the rearrangement of the actin cytoskeleton in polarized MDCK cells. Fig. S3 shows the specific down-regulation of AHNAK protein by immunofluorescence in MDCK cells treated with AHNAK siRNA. The online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200307098/DC1.
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Acknowledgments |
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This work was supported by grant form the Association pour la Recherche contre le Cancer (ARC 5643 to C. Delphin) and by a fellowship from la Ligue Nationale contre le Cancer (to B.J. Gentil).
Submitted: 15 July 2003
Accepted: 20 November 2003
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