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2 Institute of General Pathology, Catholic University Medical School, 00168 Rome, Italy
3 Laboratory of Molecular Oncology, Picower Institute for Medical Research, Manhasset, NY 10030
Address correspondence to Giampietro Ramponi, Dipartimento di Scienze Biochimiche, viale Morgagni 50, 50134 Firenze, Italy. Tel.: 39-055-413765. Fax: 39-055-4222725. E-mail: ramponi{at}scibio.unifi.it
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Abstract |
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Key Words: reactive oxygen species; integrin-mediated cell adhesion; Rac; LMW-PTP; focal adhesion kinase
The online version of this article includes supplemental material.
* Abbreviations used in this paper: AA, arachidonic acid; BSO, butionine sulphoximide; DCA-DA, 2',7'-dichlorofluorescein diacetate; DPI, diphenyl iodide; FA, focal adhesion; 5'-F-IAA, 5'-iodoacetamidofluorescein; LMW-PTP, low molecular weight PTP; LOX, 5-lipoxygenase; NAC, N-acetyl-cysteine; NDGA, nordihydroguaiaretic acid; PNPP, p-paranitro phenyl-phosphase; PTP, phosphotyrosine protein phosphatase; ROS, reactive oxygen species; SHP, Src homology phosphatase.
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Introduction |
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The downstream effect of ROS production is the more or less reversible oxidation of proteins (Finkel, 2001). Thiols, by virtue of their ability to be reversibly oxidized, are recognized as key targets of oxidative stress. Redox-sensitive proteins, which include protein tyrosine phosphatases (PTPs) as the active site cysteine, are the target of specific oxidation by various oxidants, including H2O2, and this modification can be reversed by intracellular reducing agents (Xu et al., 2002). The reversible oxidation of PTPs family member was first demonstrated for PTP1B (Lee et al., 1998) during EGF signaling and then for low molecular weight PTP (LMW-PTP) (Chiarugi et al., 2001), and Src homology phosphatase (SHP)-2 (Meng et al., 2002) during PDGF stimulation. The inhibition exerted by ROS on PTPs helps the propagation of receptor tyrosine kinase (RTK) signals mediated by protein tyrosine phosphorylation, generally associated with the proliferative stimulus (Chiarugi et al., 2002).
The small GTPase Rac-1 has a key role in the regulation of cell growth and orchestrates cytoskeletal changes and gene expression in response to mitogenic cues from both soluble growth factors and ECM proteins. Although a function for Rac proteins in regulating the formation of ROS during the phagocyte respiratory burst has been long recognized, it is a recent notion that transient expression of constitutively activated forms of the small GTP-binding proteins Ras or Rac-1 in nonphagocytic cells also leads to a significant increase in intracellular ROS, suggesting that the family of Ras-related small GTP-binding proteins may function as general regulators of the intracellular redox state (Sundaresan et al., 1996). More importantly, Rac-1 mediates the transient rise of ROS observed after H-Ras expression or after cell stimulation by either growth factors or cytokines in a variety of cell types (Irani and Goldschmidt-Clermont, 1998). There are several recent studies on the role of Rac in the generation of ROS for both NADPH and arachidonic acid (AA)dependent oxidases, although many details of the signaling pathway are still remaining unclear. These findings indicate that phospholipase A2 and subsequent AA metabolism by 5-lipoxygenase (LOX) act as downstream mediators in a Rac-signaling pathway leading to the generation of ROS (Woo et al., 2000). Moreover, in neutrophils the activity of the NADPH oxidase system is regulated by the small GTP-binding protein Rac-2 (Knaus et al., 1991), whereas in macrophages the NADPH oxidase appears to be regulated by Rac-1 (Abo et al., 1991).
In spite of the growing attention toward the mechanisms of intracellular signaling by adhesion molecules and the modality of signal integration between integrins and RTK receptors, a direct role for ROS in cell response to ECM proteins has not been directly addressed so far. Given the known role of small GTP-binding proteins in the signal transduction of integrin-mediated cell adhesion and in driving the production of intracellular ROS, we reasoned that ROS may be produced during cell adhesion, thus functioning as second messengers.
In this report, we provide evidence that ROS take a role in integrin signaling. Moreover, the production of ROS during integrin receptor engagement is dramatically more pronounced than during growth factor administration. In suggesting a role for oxidant species in integrin signaling, we propose that the production of ROS during cell adhesion leads to an up-regulation of FAK through the reversible oxidation of the LMW-PTP.
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Results |
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Since cell adhesion is associated with increased generation of intracellular peroxide, we next tested whether integrin redox signaling also has a part in cell oxidative response to soluble growth factors. ROS production elicited by PDGF was evaluated in suspended cells and in cells preadhered from 24 h (the general condition in which growth factorelicited ROS induction has been reported previously; Sundaresan et al., 1995; Bae et al., 1997) and in cells in which adhesion and PDGF stimulation are concomitant. The results indicate that hydrogen peroxide production is much more pronounced in adherent than in suspended cells, in particular when integrin and PDGF receptors stimulation is simultaneous, suggesting a synergistic action of integrins and PDGF receptors for redox signaling (Fig. 1 B). Together, these findings indicate that the redox signaling by PDGF is largely anchorage- and integrin-dependent but also suggest that oxidative responses induced by cell adhesion and soluble growth factors are qualitatively and quantitatively different and are likely mediated by partially distinct mechanisms.
To analyze the reliance of cell adhesiondependent hydrogen peroxide production upon integrin receptor engagement, we seeded presuspended NIH-3T3 cells onto either fibronectin or polylysine pretreated dishes. The production of hydrogen peroxide is reported in Fig. 1 C. The findings indicate that stimulation of integrin receptors is likely responsible for the intracellular production of ROS, since fibronectin treatment is extremely more effective than polylysine in the production of H2O2. The relevance of the engagement of integrin receptors in ROS production was further confirmed by the treatment of suspended cells with anti5-integrinstimulating antibodies (Fig. 1 D). Together, these observations indicate that integrin clustering, rather than physical interaction with a solid substrate, induces intracellular production of hydrogen peroxide during cell adhesion.
To identify the source of intracellular ROS generated in response to cell adhesion, we tried to block the production of oxidants using selective inhibitors (Fig. 2). We used 5 µM diphenyl-iodide (DPI) to block NADPH oxidase, 10 µM nordihydroguaiaretic acid (NDGA) to block LOX, and 5 µM rotenone to inhibit mitochondrial superoxide production (Werner and Werb, 2002). The results exclude the involvement of mitochondrial respiratory chain in the production of ROS during cell adhesion, since rotenone is ineffective in preventing the rise of ROS, and indicate a major involvement of LOX and partially of NADPH oxidase in ROS production, since NDGA and moderately DPI impair the generation of hydrogen peroxide in response to cell adhesion.
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Finally, in order to discover the source of ROS that inactivate/oxidize LMW-PTP, we selectively blocked ROS production by NADPH oxidase with DPI and/or by LOX with NDGA before assaying LMW-PTP activity in immunoprecipitates. Cells were pretreated with the inhibitors for 30 min in suspension, and then cell adhesion on fibronectin was allowed. LMW-PTP immunoprecipitates were assayed for phosphatase activity and the results, shown in Fig. 6 D, confirm that the intracellular oxidase system, which is mainly involved in LMW-PTP inactivation during cell adhesion, is likely LOX, although NADPH oxidase appears to retain a secondary role.
ROS control FAK activation through the redox regulation of LMW-PTP
It has been demonstrated recently that LMW-PTP associates with and dephosphorylates p125FAK, interfering with cell motility and spreading. Moreover, LMW-PTP overexpression significantly decreases the number of FAs in adherent cells (Rigacci et al., 2002). To analyze the role of cell adhesiondependent ROS production during FA formation, we explored the activation of p125FAK through an artificial block of ROS production. Presuspended cells, either kept in suspension or seeded onto fibronectin-coated dishes, were treated with NDGA or DPI. p125FAK activation was assessed by antiphosphotyrosine immunoblot of anti-FAK immunoprecipitates (Fig. 7 A). The blot was then reprobed with anti-p125FAK antibodies for normalization, and densitometric analysis was performed. The results prove that phosphorylation of FAK is dependent on ROS generation since NDGA and DPI leads to an impairment of FAK activation. In addition, we analyzed the effect of antioxidants on the activation of MAPKs and p60 Src tyrosine kinase, other well-known events of downstream FAK activation. Our results (Fig. 7, B and C) confirm the central role of ROS in the transduction signals, i.e., MAPK and Src activation, starting from FAK when cytoskeleton rearrangement takes place.
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Discussion |
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Our contribution to the general idea of a key role of ROS during cell proliferation is related to the proposal that ROS, in particular those produced in a Rac-1dependent fashion, play a major role in the propagation of intracellular signals triggered by integrins. Given the recent observation that ROS are required for the growth factorinduced tyrosine phosphorylation of proteins, we investigated whether these oxidant molecules are produced and could play a role in integrin-mediated cell adhesion. First, we have shown that during cell adhesion to extracellular matrix proteins, a rise in intracellular ROS occurs. The increase in ROS generation is large (10-fold) and dependent on integrin receptor engagement (Fig. 1 A) as indicated by the growth of ROS during cell adhesion to fibronectin but not to polylysine and by the response elicited by anti
5-integrin antibodies (Fig. 1, C and D).
An important issue raised by our experiments deals with the relative contribution of growth factors and adhesion molecules to redox signaling. Since ROS elicited by PDGF in suspended fibroblasts are significantly less pronounced than those triggered by PDGF during simultaneous integrin receptor engagement (Fig. 1 B), we conclude that the frequently reported generation of ROS by growth factors (Sundaresan et al., 1995; Bae et al., 1997; Zafari et al., 1998; Finkel, 2001) is likely an anchorage-dependent phenomenon. In addition, the relative contribution to ROS production of integrin receptor engagement and growth factor administration is unbalanced, being higher than oxidants produced during cellECM interaction with respect to those produced in response to growth factors (Fig. 1 A). By extension, we suggest that oxidative signals during fibroblast stimulation by mitogens may be combinatory in nature and reflect an integrated activity of both RTKs and adhesion molecules.
Besides mitochondrial respiratory chain malfunction, there are several potential intracellular sources of ROS, including NADPH oxidase and LOX, which catalyze the synthesis of superoxide anion rapidly converted in hydrogen peroxide (Wientjes and Segal, 1995; Bae et al., 1997; Finkel, 2001; Pani et al., 2001). Herein, we rule out that integrins engage ROS molecules through mitochondrial pathways, and we suggest the involvement of cytosolic oxidases, mainly of LOX and only marginally of NADPH oxidase (Fig. 2). Hence, although several reports have indicated the source of growth factorinduced ROS in NADPH-dependent, membrane-bound oxidases (Bae et al., 1997; Zafari et al., 1998; Chiarugi et al., 2001; Finkel, 2001), we propose that cytosolic lipoxygenases account for most oxidative events triggered by integrins. Although the molecular basis of this difference is yet to be elucidated, the different source of ROS may account for the differences in both intensity and the kinetic observed when redox signals elicited by PDGF and cell adhesion were compared.
Moreover, we report that Rac takes a specific role in the production of ROS after cell adhesion (Fig. 3). It has been reported that ROS production in growth factorstimulated nonphagocytic cells appears to require the participation of Rac (Kim et al., 1997; Cool et al., 1998). Although in part speculative, we propose the idea that PDGF-elicited ROS production is greatly dependent on cell anchorage due to the limited capacity of growth factors to activate Rac-1 in the absence of integrin engagement (del Pozo et al., 2000). In this view, Rac may to play a central role in the integration of the combinatorial oxidative signals owing to RTKs and adhesion molecules.
Many data suggest that hydrogen peroxide acts as a signal-transducing molecule during growth factor signaling (Sundaresan et al., 1995; Bae et al., 1997; Zafari et al., 1998; Chiarugi et al., 2001; Colavitti et al., 2002). Our data emphasize that ROS behave as messenger molecules during integrin receptor signaling and growth factor ones. In fact, both the specific blockage of oxidant production by cytosolic oxidases with DPI or NDGA treatment or the scavenge of the ROS produced after cell adhesion with NAC treatment cause a great inhibition or delay of cell adhesion and spreading (Fig. 4). These data are in keeping with a central role of hydrogen peroxide in the cytoskeleton rearrangement that drives cells toward contacts with ECM proteins and completion of cytoskeletal architecture, since it is also indicated by the kinetic of ROS production, with a peak at 45 min in strict concomitance with the stabilization of FAs.
There is increasing evidence that oxidative stress or redox-dependent protein modifications modulate early events of signal transduction for cell growth and death (Pani et al., 2000b; Finkel, 2001). One of the effects of these redox signals may be the inactivation of PTPs through the oxidation of critical sulfydryl groups (Xu et al., 2002). All PTPs contain an essential cysteine residue (pKa 4.75.4) in the signature active site motif that exists as a thiolate anion at neutral pH (Zhang et al., 1992). The active site cysteine is the target of specific oxidation by various oxidants, including H2O2, and this modification can be reversed by incubation with thiol compounds such as dithiothreitol and reduced glutathione (Caselli et al., 1998; Lee et al., 1998, 2002; Meng et al., 2002). These observations suggest that PTPs might undergo H2O2-dependent inactivation in cells, resulting in a shift in the equilibrium with PTKs toward protein phosphorylation. The reversible oxidation has been demonstrated for PTP1B during EGF signaling and for LMW-PTP and SHP-2 during PDGF stimulation (Bae et al., 1997; Chiarugi et al., 2001; Meng et al., 2002). Among these phosphatases, we selected LMW-PTP for its role in cell adhesion, since it has been reported that this phosphatase is involved in cell motility control and in down-regulation of FA formation (Chiarugi et al., 1998; Chiarugi et al., 2000; Rigacci et al., 2002). We report herein that LMW-PTP overexpression causes a delay in cell adhesion to ECM proteins, confirming a negative function of LMW-PTP on FA development and cytoskeleton organization (Fig. 5). We demonstrated that LMW-PTP is oxidized during cell adhesion and that this oxidation is followed by a transient inactivation of the phosphatase enzymatic activity (Fig. 6, A and B). After the removal of the oxidative burst following adhesion, LMW-PTP totally rescues its catalytic activity, due to intracellular reduced glutathione, in agreement with our previously reported findings on the central role of glutathione in the redox regulation of LMW-PTP after PDGF receptor hydrogen peroxide production (Chiarugi et al., 2001). Finally, LMW-PTP oxidation after integrin receptors engagement is mainly due to the activation of LOX, in keeping with the source of ROS during cell adhesion (Fig. 6). The finding that phosphatase inhibition during cellECM interaction is transient and reversible by intracellular reductants may also have important functional implications. It is in fact an emerging concept that cell adhesion is a dynamic process, closely related to cell motility. Although phosphatase inhibition promotes cell adhesion, phosphatase recovery may be essential to local detachment and directional migration in response to integrin signaling. Although partially speculative, this idea is consistent with previous reports of decreased cell motility in dominant-negative LMW-PTPexpressing cells (Chiarugi et al., 1998; Rigacci et al., 2002) and also with the need of PTEN phosphatase activity for cellular chemotaxis (Iijima and Devreotes, 2002).
It has been reported that LMW-PTP is able to down-regulate the formation of FA structures by dephosphorylating p125FAK (Rigacci et al., 2002). FAK is a nonreceptor protein tyrosine kinase involved in signal transduction from integrin-enriched FA sites, mediating cell contact with the extracellular matrix. Multiple proteinprotein interaction sites allow FAK to associate with adapters and structural proteins, allowing for the modulation of MAPKs, stress-activated protein kinases, and small GTPases activity. FAK-enhanced signals have been shown to mediate the survival of anchorage-dependent cells and are critical for efficient cell migration in response to growth factor receptor and integrin stimulation (Brakebusch et al., 2002; Hauck et al., 2002). Herein we reported that ROS produced after cell adhesion behave as positive regulators for FAK activation, since the blockage of their synthesis greatly reduced the activation of the kinase. Actually, the ROS produced by 5-LOX are mainly responsible for FAK activation, leaving NADPH oxidase with a marginal role (Fig. 7 A). The key role of cell adhesiondependent ROS increase is further stressed by the analysis of FAK downstream pathways. In fact, both MAPK and Src kinase are dramatically down-regulated when cells are treated with antioxidants (Fig. 7, B and C). Finally, we reported that the oxidation of LMW-PTP during cell adhesion is accompanied by a disruption of the interaction between the phosphatase and FAK (Fig. 8). The peculiarity of LMW-PTP redox regulation is the ability to rescue its catalytic activity by virtue of an intramolecular disulphur bond between two vicinal cysteines (Caselli et al., 1998; Chiarugi et al., 2001). These two cysteines are both in the catalytic site: Cys12 forms the transient cysteinyl-phosphate intermediate, whereas Cys17 cooperates with Arg18 for the binding of the phosphate moiety of the substrate (Cirri et al., 1993). We suppose that the oxidation of both these cysteines to form an intramolecular disulfide affects the ability of the protein to bind the substrate. Hence, during oxidative conditions LMW-PTP is not only oxidized and enzymatically inactivated but it is no more able to bind its natural substrate. With respect to the simple enzyme inhibition, this additional mechanism could further delay FAK dephosphorylation upon enzyme recovery or, more importantly, may prevent phosphotyrosine residues to be occupied and functionally sequestered by the inactive phosphatase, thus permitting these phosphorylated residues to signal through binding of SH2 domaincontaining proteins, i.e., Src kinase, Grb2 adaptor, and so on. Whether this phenomenon is restricted to the LMW-PTPFAK interaction or applies to other phosphatase-substrate pairs is still to be determined.
On the basis of our data, we propose a model of redox regulation of FA formation in which ROS play a key role in the transduction of the signals engaged by cell adhesion through inhibiting LMW-PTP and allowing the activation of p125 FAK. The requirement for ROS in the integrin signaling cascade upstream of FAK may account for the inhibition of cell adhesion and spreading observed in antioxidant-treated cells; however, a direct effect of oxidants on different targets cannot be excluded at the moment.
Finally, we stress that our findings open new avenues for pharmacological intervention in anchorage-independent cell transformation. In fact, malignant cells are often anchorage-independent for their growth, and this property directly correlates with their metastatic potential (Brakebusch et al., 2002). Loss of anchorage dependence is frequently due to a deregulated activation of the signaling pathway normally triggered by integrins. In particular, excess ROS production associated with cell transformation by H-Ras (Irani and Goldschmidt-Clermont, 1998) or c-Myc (Tanaka et al., 2002) could release costimulatory signals which are normally triggered by cellECM interaction. In line with this view, Rat-1 cells overexpressing either active Rac-1 or oncogenic R12 Ras display anchorage-independent growth, which is dramatically inhibited by ROS scavengers (unpublished data).
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Materials and methods |
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Cell culture and protein overexpression
NIH-3T3 cells were cultured in DME with 10% FCS in 5% CO2 humidified atmosphere. 10 µg of pRcCMV-wtLMW-PTP (Chiarugi et al., 1995) were stably transfected in NIH-3T3 cells using the calcium phosphate method. For cell infections, retroviral constructs expressing the RacQL and RacN17 cDNA in the pLPC/Puro backbone were generated and transfected by calcium phosphate coprecipitation in the 293T Phoenix packaging cell line (a gift from Dr. Scott Lowe, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY). Supernatants were collected every 46 h and overlayed onto NIH-3T3 cells. After 24 h of infection, cells were left to recover for 48 h before analyses.
Cell adhesion
106 cells were serum starved for 24 h before detaching with 0.25% trypsin for 1 min. Trypsin was blocked with 0.2 mg/ml soybean trypsin inhibitor, and cells were resuspended in 2 ml/10-cm dish of fresh medium, maintained in suspension for 30 min at 37°C, and then directly seeded onto precoated dishes treated overnight with 10 µg/ml human fibronectin or 10 µg/ml poly-D-lysine in PBS. Control cells were kept in suspension by plating them onto dishes pretreated with 1 mg/ml of BSA in culture medium, thus preventing adhesion to the dish.
Cell adhesion assay
Cells were serum starved for 24 h, and then 3 x 104 cells were seeded onto serum-depleted medium for the indicated times in a 24-well dish precoated with 10 µg/ml human fibronectin. Cells were fixed in 1 ml of 0.25% PFA and photographs were taken with a phasecontrast microscope (Nikon).
Immunoprecipitation and Western blot analysis
106 cells were lysed for 20 min on ice in 500 µl of complete RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM EGTA, 1 mM sodium orthovanadate, 1 mM phenyl-methanesulphonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Lysates were clarified by centrifugation and immunoprecipitated for 4 h at 4°C with 12 µg of the specific antibodies. Immune complexes were collected on protein A Sepharose, separated by SDS-PAGE, and transferred onto nitrocellulose. Immunoblots were incubated in 3% BSA, 10 mM Tris/HCl, pH 7.5, 1 mM EDTA, and 0.1% Tween-20 for 1 h at room temperature, probed first with specific antibodies and then with secondary antibodies. Quantity-One software (Bio-Rad Laboratories) was used to perform quantitative analyses.
In vivo 5'-F-IAA labeling
As reported in Wu et al. (1998), cells were lysed in RIPA buffer at pH 7.5, and 5 µM 5'-F-IAA was added. The lysates were labeled 10 min at 37°C and then were immunoprecipitated with antiLMW-PTP antibodies. The redox state was evidenced by antifluorescein immunoblot.
LMW-PTP activity assay
The tyrosine phosphatase activity was measured as reported previously (Bucciantini et al., 1999). Briefly, cells were lysed in RIPA buffer, and LMW-PTP was immunoprecipitated from lysates. Immunoprecipitates were then resuspended in 100 µl of 0.1 M sodium acetate, pH 5.5, 10 mM EDTA. Phosphatase activity assay was performed adding 100 µl of 10 mM PNPP at 37°C for 1 h. The production of p-nitrophenol was measured colorimetrically at 410 nm. The results were normalized on the basis of LMW-PTP content analyzed by antiLMW-PTP immunoblot.
MAPK and Src activation
1.5 x 105 cells were serum starved for 24 h before detachment. After a 30-min presuspension treatment, cells were seeded on fibronectin-coated dishes for different times and then lysed in RIPA buffer. 20 µg of lysates were used for antiphospho-ERK1/2 or antiphospho-Src immunoblots. The data were normalized by anti-MAPK or anti-Src immunoblot.
Assay of intracellular H2O2
Intracellular production of H2O2 was assayed as described previously (Pani et al., 2000a). 5 min before the end of incubation time, adherent or suspended cells were treated with 5 µg/ml DCF-DA. After PBS washing, adherent cells were lysed in 1 ml of RIPA buffer and analyzed immediately by fluorescence spectrophotometric analysis at 510 nm. Data have been normalized on total protein content.
Determination of Rac-1 activity
NIH-3T3 cells were kept in suspension for 2 h in serum-free medium. After medium renewal, aliquots of 5 x 105 cells were directly lysed or plated onto fibronectin-coated dishes for the indicated times before lysis in RIPA buffer. Rac-GTP was quantified in precleared protein lysates according to Sander et al. (1998). Briefly, lysates were incubated with 510 µg of PAK-CRIB-GST fusion protein absorbed on glutathione-Sepharose beads. Immunoreactive Rac-1 precipitated by PAK-GST was then quantified by anti-Rac Western blot analysis.
Online supplemental material
Western blot, immunoprecipitation, and 5'-IAF labelling are used in Fig. S1 to show the redox regulation of endogenous LMW-PTP. Fig. S1 and the techniques used in Fig. S1 are available at http://www.jcb.org/cgi/content/full/jcb.200211118/DC1.
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Acknowledgments |
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This work was supported by the Italian Association for Cancer Research, the Ministero della Università e Ricerca Scientifica e Tecnologica (MIUR-PRIN 2002 to G. Ramponi), in part by grants from the Consorzio Interuniversitario Biotecnologie (to G. Ramponi) and the Cassa di Risparmio di Firenze (to P. Chiarugi), and by Consiglio Nazionale delle Ricerche, Target project on Biotechnology (to S. Borrello).
Submitted: 26 November 2002
Revised: 18 April 2003
Accepted: 21 April 2003
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