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Address correspondence to Pier Paolo Di Fiore, Department of Experimental Oncology, European Institute of Oncology, Via Ripamonti, 435, 20141 Milano, Italy. Tel.: (39) 025-748-9833. Fax: (39) 025-748-9851. E-mail: pdifiore{at}ieo.it
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Abstract |
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Key Words: Eps8; Rac; Sos-1; cytoskeleton; GEF
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Introduction |
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The molecular mechanisms of this cascade are being elucidated. Biochemical and genetic studies have shown how the Son of Sevenless (Sos)-1 GEF transduces the signal from active RTKs to Ras (for review see Bar-Sagi, 1994; Schlessinger, 2000). Sos-1 interacts with the SH3-containing adaptor molecule Grb2. Grb2 in turn displays an SH2 domain responsible for the recruitment of the Grb2Sos-1 complex to active, autophosphorylated RTKs. The relocalization of the complex to the plasma membrane is thought to be sufficient for Sos-1 to catalyze the exchange of guanine nucleotides on Ras, which is also present at the plasma membrane.
How Ras signals to Rac is less understood. Phosphatidylinositol 3 kinase (PI3-K) binds directly to Ras-GTP and it is required for activation of Rac (for review see Rodriguez-Viciana et al., 1997). In hematopoietic cells, the product of PI3-K's catalytic activity, phosphatidylinositol 3,4,5 trisphosphate (PIP3), contributes through direct binding to the activation of a Rac-specific GEF, Vav-1 (Han et al., 1998; Das et al., 2000). In nonhematopoietic cells, the lack of expression of Vav-1 indicates that other GEFs must be involved. Indeed, two recently identified members of the Vav family, Vav2 and Vav3, display ubiquitous expression and have been implicated in RTK-mediated actin remodelling (Schuebel et al., 1998; Liu and Burridge, 2000; Moores et al., 2000; Trenkle et al., 2000).
Sos-1 was also implicated in Ras-to-Rac signaling (Nimnual et al., 1998; Scita et al., 1999). Sos-1 was shown to participate in vivo in a tricomplex with two signaling molecules, Eps8 (Fazioli et al., 1993) and E3b1 (also known as Abi-1) (Shi et al., 1995; Biesova et al., 1997). E3b1 contains an SH3 domain that mediates its binding to Sos-1 (Scita et al., 1999; Fan and Goff, 2000). In addition, E3b1 binds to the SH3 domain of Eps8, thus acting as a scaffold protein which holds together Sos-1 and Eps8 (Biesova et al., 1997; Mongiovi et al., 1999; Scita et al., 1999). The tricomplex Sos-1E3b1Eps8 displays Rac GEF activity in vitro (Scita et al., 1999). Therefore, Sos-1 might be endowed with a dual GEF activity, for Ras and Rac, respectively. At the molecular level, this is mirrored by the presence of two GEF domains in Sos-1: (a) a Cdc25-like domain, responsible for activity on Ras, and (b) a DH-PH tandem domain, a hallmark of GEFs for Rho GTPases, the subfamily to which Rac belongs. However, purified Sos-1 does not display Rac-GEF activity, whereas Ras-GEF activity could be readily detected. Thus, a coherent picture of how Sos-1 regulates Rac activation is still missing. Another unresolved question concerns the mechanism responsible for the proper compartmentalization of Sos-1 to sites where the Rac-based actin polymerizing machinery needs to be active. In this study, we provide evidence that Eps8 is a critical factor in the regulation of both these functions.
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Results |
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Mapping of the effector region of Eps8
We engineered a series of deletion mutants of Eps8 (Fig. 2 A) fused to GFP. A mutant, 586821, lacking the SH3 domain retained ruffling activity in the absence of growth factor treatment (Fig. 2, A and B), indicating that the SH3 domain of Eps8 is dispensable for actin reorganization activity in the absence of growth factors. Thus, an interaction with E3b1, mediated by the SH3 domain of Eps8, is not required for the effector function of the COOH-terminal fragment of Eps8.
Further deletions of the COOH terminus of Eps8 (Fig. 2, A and B) showed that the minimal region required to elicit ruffling spanned amino acids 648821. Interestingly, this region is also the minimal region required for colocalization with polymerized actin (Fig. 2 A). Indeed, for all the Eps8 mutants containing this region, there was perfect correlation between ruffling activity and colocalization with phalloidin-stained F-actin (or lack of both events; Fig. 2 A). Notably, the Eps8 effector region, similarly to the known inducer of actin polymerization such as activated Rac and PI3-K, induces prominent peripheral ruffles, but only rarely circular ruffles. These latter structures are dependent on PDGF stimulation, pointing to the requirement of additional signaling events, whose nature remains undetermined.
The ruffle-inducing activity of the Eps8 effector region was recorded in real time. The GFP effector region of Eps8 (586821) rapidly induced and accumulated within dynamic structures along the dorsal area and the leading edge of cells, thus confirming the observations obtained in paraformaldehyde-fixed cells. Furthermore, it relocalized in protruding lamellipodia of spreading and moving cells, suggesting its involvement in migratory processes (supplementary information). Thus, Eps8 is endowed with a potent and constitutive ruffle-inducing activity residing in its 173 COOH-terminal amino acids.
Signaling by the effector region of Eps8
Ras and Rho GTPases establish a signaling network that regulates actin rearrangement (for review see Bar-Sagi and Hall, 2000; Scita et al., 2000), a process in which the participation of PI3-K has also been firmly established (for review see Rodriguez-Viciana et al., 1997). To define the site of action of the effector region of Eps8 in the signaling pathways leading to actin remodeling, we attempted to interfere with its function with a series of molecular and pharmacological inhibitors. In particular, we used dominant negative versions of Ras (RasN17), Cdc42 (Cdc42N17), Rho (RhoN19), PI3-K (p85iSH2), and Rac (586-821). We also used the PI3-Kspecific inhibitor, wortmannin.
Upon microinjection in mouse fibroblasts, all the dominant negative mutants were readily expressed (Fig. 3) and biologically active, as shown by the ability of: (a) RasN17 to inhibit EGF-induced mitogen-activated protein kinase (MAPK) activation (Fig. 3 A); (b) Cdc42N17 to reduce growth factorinduced JNK activity (Fig. 3 B); (c) RhoN19 to inhibit the formation of serum-induced stress fibers (Fig. 3 C); and (d) RacN17 (Fig. 3 D) and p85iSH2 (Fig. 3 E) to inhibit PDGF-mediated actin cytoskeleton reorganization. Wortmannin also abrogated completely PDGF-induced actin remodeling (Fig. 3 F).
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As shown in Fig. 7 B, the effector region of Eps8, when alone, bound to purified Sos-1 at least 10-fold better than full length GST-Eps8. The marginal binding of full length GST-Eps8 might reflect the true ability of the holoprotein to interact with Sos-1 or an artifactual situation. Under this latter scenario, one could postulate that in vivo Eps8 does not bind to Sos-1 unless determinants in its COOH terminus are unmasked: a setting mimicked by the enucleation of the effector region from the holoprotein, or by partial unfolding of the bacterially expressed Eps8 full length molecule. To gain insight into this issue we studied the association in vivo of Sos-1 with either the effector region of Eps8 or with the holoprotein. Native Sos-1 could be efficiently recovered in HA-tagged Eps8 (586821) immunoprecipitates of cells double transfected with Sos-1 and HA-Eps8 (586821) (Fig. 7 C). No E3b1 could be detected in the same immunoprecipitates, even under conditions of E3b1 overexpression (not shown). On the contrary, the in vivo association between Sos-1 and Eps8 was strictly E3b1-dependent. Indeed, native Sos-1 could be readily recovered in Eps8 immunoprecipitates from triple Sos-1E3b1Eps8 transfectants, but not when an Eps8 mutant, unable to bind E3b1 due to a deletion in its SH3 domain (Eps8SH3), was used (Sos-1Eps8
SH3E3b1 transfectants), or when E3b1 was not transfected along with Sos-1 and Eps8 (Sos-1Eps8) (Scita et al., 1999; Fig. 7 C).
We then tested whether the biological and biochemical activity of the Eps8 effector region could activate Sos-1 Racspecific GEF in vivo. First, a Rac-GEF assay was performed using lysates of cells expressing the effector domain of Eps8 or a ruffling-incompetent Eps8 fragment (586733) which cannot bind to Sos-1. Expression of the effector region, but not of the ruffling-incompetent fragment, induced a substantial increase in Rac-specific GEF activity in vivo over the basal control values (Fig. 7 D). This effect was dependent on the presence of Sos-1 (or of another GEF, tightly associated to Sos-1), since no Rac-GEF activity could be detected in the same lysates upon immunodepletion of Sos-1 (Fig. 7 D). Second, we used in vitro assays that can score GEF activities in immunoprecipitates. Cells were transfected either with a construct containing the effector region, Eps8 (586821), or with a control construct, Eps8 (1535), alone or in combination with Sos-1. Rac-GEF activity could be readily detected when the Eps8 (586821) protein was immunoprecipitated from cells coexpressing Eps8 (586821) and Sos-1 (Fig. 7 E). Under these conditions, coimmunoprecipitation between Eps8 (586821) and Sos-1 was detected (Fig. 7 E, bottom). Thus, the Eps8 effector region participates in the regulation of a Rac-specific GEF activity, which depends on the presence of Sos-1 or Sos-1associated GEFs.
Eps8 interaction with F-actin, through its effector domain, directs its localization to site of actin remodeling
The effector domain of Eps8 was also responsible for its localization within F-actincontaining structures (Fig. 2 A). Thus, we looked for interactors with the Eps8 effector region. We used various GST-fused fragments of Eps8 in order to specifically recover proteins from cell lysates. As shown in Fig. 8 A, several cellular proteins were detected by silver staining. We concentrated our attention on those bands that were recovered by the 586821 fragment (which is biologically active and properly localized), but not by the 733821 or by the SH3 fragments (which are biologically inactive and delocalized), and subjected them to MALDI mass spectrometry and NanoElectrospray. Two proteins were unequivocally identified: actin and the myosin II heavy chain (Fig. 8 A). Both associations were confirmed by in vitro binding experiments (Fig. 8 B). Of note, the low stoichiometry of interaction between Eps8 and myosin II heavy chain suggested an indirect interaction, possibly mediated by actin. Therefore, we characterized the Eps8/actin association.
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Finally, we investigated whether the Eps8F-actin association occurs in vivo. Native actin could be specifically coimmunoprecipitated with Eps8, whereas no association could be observed when preimmune sera were used, or in Eps8 immunoprecipitates performed on lysates from Eps8 null cells (Fig. 9 D).
The interaction in vivo between Eps8 and F-actin may dictate the proper intracellular localization of Eps8. In this case, disruption of the actin filaments should lead to Eps8 mislocalization. Treatment of cells with cytochalasin D, an inhibitor of actin polymerization (Goddette and Frieden, 1986; Sampath and Pollard, 1991), caused the disruption of the F-actin meshwork (Fig. 10). This resulted in the disruption of membrane ruffles and the scattered accumulation of short bundles of F-actin aggregates, which colocalized (Fig. 10) with Eps8 or with those Eps8 fragments containing an intact actin binding domain (Fig. 10). Thus, the integrity of the F-actin meshwork is required to direct Eps8 to its proper subcellular localization.
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Discussion |
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Several lines of evidence indicate that the effector region of Eps8 functions as a constitutively active variant of Eps8. First, genetic data demonstrated that the mechanism whereby the Eps8 effector region induces ruffles requires the activation and proper localization of Rac. Moreover, the effector region of Eps8 did not require Ras/PI3-K or Cdc42 to induce actin remodeling. This is in agreement with our previous work, which epistatically positioned Eps8 between Ras/PI3-K and Rac (Scita et al., 1999). Biochemical evidence also supported a role of the Eps8 effector region in the activation of Rac. Its transient expression led to activation in vivo of Rac and PAK65. Moreover, the overexpression of the effector region of Eps8 increased the levels of Rac-specific GEF activities in the cell by a mechanism which was shown to be directly dependent on the presence of Sos-1 (or of Sos-1associated GEFs) and on the interaction between Sos-1 and the Eps8 effector region.
Thus, the effector region of Eps8 appears to signal in a manner indistinguishable from full length Eps8, albeit in a growth factorindependent fashion. Indeed, we showed that the overexpression of the effector region, but not of the full length Eps8 protein, is sufficient to elicit actin cytoskeleton remodeling. Thus, within the context of the full length Eps8 protein, a tight regulation of the effector function must be at play and determinants contained in the NH2-terminal half of Eps8 are predicted to exert an inhibitory role. This mode of action is remarkably reminiscent of that of Wasp and its homologue N-Wasp, which link multiple signaling pathways to actin assembly. N-Wasp interacts with the Arp2/3 complex and activates the ability of the latter to nucleate actin filaments (for review see Mullins, 2000). A COOH-terminal domain of N-Wasp, the VCA domain, is sufficient when enucleated from the full length molecule to bind to the Arp2/3 complex and activate it (Mullins, 2000). Physiologically, however, activation of N-Wasp occurs when the molecule is stimulated by the proper set of upstream signals, including active Cdc42 and PIP2. These molecules bind cooperatively to N-Wasp and force it to adopt an open conformation, which exposes the VCA domain, allowing its effector function (Rohatgi et al., 1999; Higgs and Pollard, 2000; Prehoda et al., 2000).
In analogy to Wasp and N-Wasp, and based on our results, a model can therefore be proposed in which Eps8 that is normally inactive in the cell is activated by upstream signals and therefore rendered competent for activation of Rac. From our data, it appears that the key step in this series of events is the binding of the effector region of Eps8 to Sos-1. Indeed several activities, including Sos-1 binding, induction of Rac-specific GEFs and activation of Rac and Rac-dependent pathways, all cosegregated with the same fragment of Eps8. Thus, it is tempting to speculate that a latent, Rac-specific, GEF activity of Sos-1 is unmasked upon direct binding to the effector region of Eps8. In this scenario, the "activation" of Eps8 might correspond to a conformational change (or functionally equivalent mechanism), which renders its effector region available for direct interaction with Sos-1. A corollary of such a model is that full length Eps8 should not be able to bind to Sos-1 directly. Indeed, we show in this and previous studies (Scita et al., 1999) that association of Eps8 with Sos-1 is strictly dependent on the presence of the scaffolding molecule E3b1 (Fig. 7C). On the contrary, this latter molecule is not required for the interaction between the effector region of Eps8 and Sos-1 (Fig. 7, AC), which are readily coimmunoprecipitated in vivo. To validate this model, we are presently attempting to reconstitute in vitro a Rac-specific GEF complex by using purified E3b1Sos-1Eps8 or purified Sos-1Eps8 effector region.
Our model also provide clues as to how the Sos-1 Racspecific GEF activity is regulated under physiological conditions, within the context of a trimeric complex where E3b1 scaffolds together Eps8 and Sos-1. We note that the affinity of Sos-1 for the effector region of Eps8 is rather low. Thus, the scaffolding function of E3b1 might provide an efficient mechanism of increasing the local concentration of the two binders (Eps8 and Sos-1) for the purpose of achieving a stoichiometry of interaction sufficient for biological output. Under conditions of overexpression of the effector region (but not of full length Eps8, according to our model), this requirement might be bypassed simply by virtue of mass action. Of course, this model does not exclude that other events, in addition to the simple Eps8Sos-1 interaction, are required in vivo to activate the Rac-specific GEF function of Sos-1. One obvious level of regulation is the one exerted by growth factor stimulation, which might, among other effects, relieve the inhibition of Eps8. This could be achieved by a variety of mechanisms, including tyrosine phosphorylation of Eps8 or binding of phosphoinositides to the DH-PH domain of Sos-1 or to the PTB domain of Eps8. We are presently investigating these possibilities.
An additional level of regulation is highlighted by our findings of a physical direct interaction between Eps8 and F-actin, which mirrored the in vivo colocalization of Eps8 with F-actincontaining structures. This function is also contained within the effector region of Eps8. We could not identify determinants within this region that would allow the dissection of the abilities to bind to F-actin or to Sos-1. This might be due to a high degree of overlap between the two surfaces. In this case, single amino acid mutations might be required to identify the residues responsible for each individual interaction. The effector region of Eps8 did not appear to be endowed with intrinsic actin-polymerizing activity. Thus, the most likely functional role of the Eps8F-actin interaction is that of directing Eps8 to sites where actin remodeling has to occur in response to extracellular stimuli. E3b1 and Sos-1 are also localized to F-actincontaining structures after growth factor stimulation of intact cells, and therefore interaction of Eps8 with F-actin might dictate the relocalization of the Rac-activating trimeric complex Eps8E3b1Sos-1 to sites where its activity is required.
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Materials and methods |
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Antibodies (Ab) were: polyclonal anti-Eps8 (Fazioli et al., 1993), anti-E3b1 (Biesova et al., 1997), and anti-EGFR (Di Fiore et al., 1990) sera; rabbit polyclonals antiSos-1, anti-Grb2, and antiERK-1, (Santa Cruz Biotechnology, Inc.); monoclonals anti-myc and antiv-H-Ras (Oncogene Research Products). Monoclonals antihistidine, antiactin, antimyosin II heavy chain, and the chemicals, dimethyl pimelidate dihydrochloride, cytochalasin D, and wortmannin were from Sigma-Aldrich. Purified muscle actin was purchased from Cytoskeleton.
Transfection procedures and indirect immunofluorescence
Fibroblasts, seeded on gelatin, were transfected with the indicated expression vectors using the LIPOfectamine reagent (GIBCO BRL), according to the manufacturer's instructions. Alternatively, nuclei of quiescent fibroblasts were injected with 100 ng/ml of the appropriate expression vectors. 36 h later, cells were processed for immunostaining. At least 100 microinjected cells were analyzed in each experiment for indirect immunofluorescence. Cells were fixed in 4% paraformaldheyde for 10 min, permeabilized in 0.1% Triton X-100 and 2% BSA for 10 min, blocked with 2% BSA for 30 min, and then incubated with primary and secondary antibodies for 45 and 30 min, respectively. Transfected and/or microinjected cells were detected by staining of the protein encoded by the transfected or microinjected cDNA. F-actin was detected by staining with rhodamine-conjugated phalloidin (Sigma-Aldrich), used at a concentration of 6.7 U/ml (0.22 µM). Alexa red and green secondary antibodies (Molecular Probes) were used. Where indicated, cells were treated with PDGF (10 ng/ml) or serum (10%) for 10 min before fixation.
Biochemical and functional assays
Standard procedures of protein analysis (in vitro binding assays, immunoprecipitations, and coimmunoprecipitations) were performed as described (Fazioli et al., 1993). The activities of MAPK, JNK, PAK65, and the levels of RacGTP were also measured as described (Scita et al., 1999). Whenever indicated, treatment was performed with EGF (100 ng/ml) after serum starvation for the indicated lengths of time.
Assays for Rac-GEF activity were performed as described (Scita et al., 1999). Data are the mean ± SE of at least three independent experiments performed in triplicate. Results are expressed as the [3H]GDP released after 20 min relative to time 0 after subtracting the background counts released in control reactions (obtained by incubating [3H]GDP-loaded Rac in exchange buffer).
For protein identification by MALDI-MS analysis, total cellular proteins were incubated in the presence of various immobilized GST fusion proteins. Specifically bound proteins were subjected to SDS-PAGE followed by detection with silver staining. The protein bands of interest were excised from one-dimensional polyacrylamide gels. After reduction and alkylation, proteins were digested "in gel" for 6 h at 37°C with sequencing-grade trypsin (Boehringer). Samples for MALDI analysis were prepared by using the "fast evaporation" method (Mann and Talbo, 1996). Mass spectra were recorded on a Voyager-DE STR BioSpectrometry workstation (PerSeptive Biosystems). Matrix-related ions and trypsin autolysis products were used for internal calibration. The peptide mass accuracy was >30 ppm (on average). The ProFound-Peptide Mapping software (Rockefeller University edition, v4.10.5) was used to search a nonredundant protein sequence database (searchable at the National Center for Biotechnology Information) with a list of peptide masses. The samples for MS/MS analysis were loaded onto a Poros R2 (PerSeptive Biosystems) microcolumm, desalted, and eluted into nanoelectrospray needles (Protana). Nanoelectrospray MS/MS analysis was performed on a quadrupole time-of-fight mass spectrometer (QSTAR; PerkinElmer). The resulting "peptide sequence tags" were used to search the nonredundant protein sequence database.
For cosedimentation assays with F-actin, monomeric human nonmuscle G-actin (1 mg/ml) was induced to polymerize into F-actin at 37°C for 60 min in F-buffer (5 mM Tris/HCl, pH 7.8, 1 mM ATP, 0.5 mM DTT, 0.2 mM CaCl2, 0.2 mM MgCl2, and 100 mM KCl) (Van Etten et al., 1994). Recombinant, purified GST fusion proteins (2 µM) were subsequently incubated with increasing concentration of F-actin in F-buffer for 45 min at 37°C. The mixtures were centrifuged in a TLA-100 rotor for 60 min at 100,000 g. Equal amounts of starting material, supernatants, and pellets were solubilized in loading buffer, boiled, and subjected to SDS-PAGE. Proteins were detected by immunoblotting using anti-GST and antiactin antibodies. The rate and the extent of actin polymerization in the presence of the Eps8 effector region was determined from the increase in fluorescence of pyrene actin that occurs when pyrene-labeled actin polymerizes, as described (Van Etten et al., 1994).
For Sos-1 purification, total cellular lysates from 293T cells, transfected with HA epitopetagged Sos-1, were immunopurified onto an affinity column obtained by cross-linking anti-HA antibodies to immobilized protein G with dimethyl pimelidate dihydrochloride. After binding, the column was washed with the 10-bed volumes of lysis buffer containing 0.2% SDS. Bound Sos-1 was eluted with the peptide MYDVPDYAS, corresponding to the HA epitope tag. Sos-1 was >90% pure, as assessed by calculating the percentage of Sos-1 with respect to the total amount of protein. This was determined by subjecting serial dilutions of the purified material to SDS-PAGE followed by staining with Coomassie brilliant blue and compared with a standard curve of BSA. Similar results were obtained by comparing the intensity of the band corresponding to Sos-1 with the bands corresponding to contaminants and/or to Sos-1 degradation products.
Online supplemental material
Video 1 and Figure S1. Quiescent mouse embryo fibroblasts, microinjected with expression vectors for GFP-Eps8 (648-821) were kept at 37°C in a CO2 incubator mounted on an inverted microscope (Olympus). Images were captured every 15 min with a coded CCD camera (Hamamatsu; speed, 6 frames/s at 15 min intervals). GFP epifluorescence was detected with a FITC filter set (Chromo Technology). The large fluorescent "blob" (visible in the top half in the final frames) probably represents a GFP-Eps8 (648821)transfected cell undergoing apoptosis after a round of cell division. A set of six consecutive images captured at the indicated time is also shown. The video and figure are available at http://www.jcb.org/content/vol154/issue5.
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Footnotes |
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G. Scita and P. Tenca contributed equally to this work.
* Abbreviations used in this paper: Ab, antibody; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; PIP3, phosphatidylinositol 3,4,5 trisphosphate; PI3-K, phosphatidylinositol 3 kinase; RTK, receptor tyrosine kinase; Sos, Son of Sevenless.
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Acknowledgments |
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This work was supported by grants from Associazione Italiana Ricerca sul Cancro, the Armenise-Harward Foundation, and the Il Consiglio Nazionale delle Ricerche (CNR; Progetto Biotecnologie).
Submitted: 30 March 2001
Revised: 25 July 2001
Accepted: 26 July 2001
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