Correspondence to: Shin Yonehara, Institute for Virus Research, Kyoto University, Shogoin Kawahara-cho 53, Sakyo-ku, Kyoto 606-8507, Japan. Tel:81-75-751-4783 Fax:81-75-751-4784 E-mail:syonehar{at}virus.kyoto-u.ac.jp.
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Abstract |
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By an expression cloning method using Fas-transgenic Balb3T3 cells, we tried to obtain inhibitory genes against Fas-mediated apoptosis and identified proto-oncogene c-K-ras. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibiting Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, including Raf (mitogen-activated protein kinase kinase kinase [MAPKKK]), phosphatidylinositol 3 (PI-3) kinase, and Ral guanine nucleotide exchange factor (RalGDS), only the constitutively active form of Raf (Raf-CAAX) could inhibit Fas-mediated apoptosis. In addition, the constitutively active form of MAPKK (SDSE-MAPKK) suppressed Fas-mediated apoptosis, and MKP-1, a phosphatase specific for classical MAPK, canceled the protective activity of oncogenic K-Ras (K-RasV12), Raf-CAAX, and SDSE-MAPKK. Furthermore, physiological activation of Ras by basic fibroblast growth factor (bFGF) protected Fas-transgenic Balb3T3 cells from Fas-mediated apoptosis. bFGF protection was also dependent on the activation of the MAPK pathway through Ras. All the results indicate that the activation of MAPK through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis.
Key Words: basic fibroblast growth factor, Fas, mitogen-activated protein kinase, oncogenesis, Ras
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Introduction |
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Apoptosis is a form of cell death fundamental to the embryonic development and maintenance of homeostasis (
Apoptosis is also induced by the stimulation of death receptors, members of the tumor necrosis factor receptor superfamily (
Some types of apoptosis are known to be inhibited by the activation of Akt/protein kinase B (PKB) or mitogen-activated protein kinase (MAPK)/extracellular signal regulatory kinase (ERK). Activation of Akt/PKB was reported to prevent the apoptosis induced by withdrawal of survival factors such as insulin-like growth factor (IGF)-I or NGF in neurons (
The key regulator upstream of both Akt/PKB and MAPK is a small G protein Ras, known as an oncogene product. GTP-bound active Ras recruits its effector molecules, including Raf and phosphatidylinositol 3 (PI-3) kinase, under the plasma membrane and then activates the Raf/MAPK pathway and the PI-3 kinase/Akt pathway, respectively. Here, we report that c-K-Ras suppresses Fas-mediated apoptosis, and oncogenic Ras strongly protects cells against Fas-mediated apoptosis through the activation of the MAPK pathway in Fas-transgenic Balb3T3 cells. In addition, we found that basic FGF (bFGF) but not EGF confers resistance on the fibroblasts against Fas-mediated apoptosis. This protective ability of bFGF was also shown to be mediated by the activation of the Ras/MAPK pathway. Although it was recently reported that oncogenic Ras downregulates the expression of Fas through activation of the PI-3 kinase/Akt pathway (
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Materials and Methods |
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Cell Lines
Mouse embryonic fibroblast Balb3T3 cells were kindly provided by K. Nagata (Kyoto University, Kyoto, Japan). The cells were maintained in DME supplemented with 10% FBS and 100 µg/ml kanamycin at 37°C in 5% CO2. Balb3T3 cells were transfected with the expression vector of mouse Fas driven by human ß-actin promoter (
cDNA Library and Plasmid Constructs
cDNA was prepared by using time saver cDNA synthesis kit (Amersham Pharmacia Biotech) from polyA+ RNA of Balb3T3 cells purified by oligo-dT column (Amersham Pharmacia Biotech), and then subcloned into pME18S expression vector (-lacZ (
p85, Akt, constitutively active Akt (HA-m
4-129 Akt), and RalN28 were kind gifts from J.F. Hancock (University of Queensland Medical School, Brisbane, Australia), W. Ogawa (Kobe University, Kobe, Japan), U. Kikkawa (Kobe University), R. Roth (Stanford University, Stanford, CA), and L.A. Feig (Tufts University, Medford, MA), respectively. cDNAs for constitutively active MAPKK (SDSE-MAPKK) were provided by E. Nishida (Kyoto University), and an expression vector for green fluorescence protein (GFP) was from K. Umesono (Kyoto University).
Antibodies and Reagents
Agonistic antimouse Fas mAb RK-8 (-(4-methyl-coumaryl-7-amide) (Ac-DEVD-MCA) and acetyl-Ile-Glu-Thy-Asp-
-(4-methyl-coumaryl-7-amide) (Ac-IETD-MCA) for caspase-3/7 and caspase-8/6, respectively, were purchased from Peptide Institute. For staining ß-galactosidasepositive cells, 5-bromo-4-chloro-3-indolyl-b-D-(-)-galactopyranoside (X-Gal) was purchased from Wako.
Expression Cloning
Subconfluent FH2 cells in five 10-cm dishes were transfected with pME18S encoding the cDNA library described above by the calcium-phosphate method (
Transient Transfection of Expression Vectors
For transient transfection, FH2 cells were seeded at 1 x 105 cells per well in 6-well plates. Cells were cultured for 1 d and then transfected with various expression vectors (0.4 µg/each vector) by using Lipofectamine plus (GIBCO BRL) according to the manufacturer's protocol.
Assay of Fas-mediated Apoptosis in the Transfected Cells
FH2 cells transfected with expression vectors (0.4 µg/each vector) and 0.4 µg pJ7-lacZ were cultured for 2 d and then transferred to 24-well plates at 2 x 104 cells per well. After cultivation for 1216 h, cells were stimulated with 200 ng/ml RK-8 for 0, 4, or 8 h, and fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min. After the removal of apoptotic cells by washing with PBS, attached cells were stained with PBS containing 1 mg/ml X-Gal, 0.02% NP-40, 5 mM K-ferricyanide, 5 mM K-ferrocyanide, and 2 mM MgCl2 for 1 h at 37°C. The total number of ß-galactosidasepositive cells per well was counted for each independent transfection (n = 3) under a microscope. Cell viability was represented by the percentage of the number of ß-galactosidasepositive cells after treatment with anti-Fas mAb against that before the treatment with anti-Fas mAb.
In Vivo Analysis of Caspase-3 Activation
Cells were transfected with various expression vectors together with an expression vector of Flag-tagged MST1-KD as a substrate for caspase-3. 2 d later, cells were stimulated with 0.2 µg/ml RK-8 and lysed in 50 mM Tris-HCl, pH 7.5, containing 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 40 mM Na2P2O7, 50 mM NaF, 5 mM MgCl2, 100 µM Na3VO4, 10 mM EDTA, and protease inhibitor cocktail (Sigma Chemical Co.) at 4°C. For Western blotting, cell lysates were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore). The membrane was blocked with 5% skim milk in PBST (PBS + 0.05% Tween 20) for at least 1 h, and then incubated with anti-Flag antibody M2 (Kodak) for detection of intact and cleaved MST1-KD.
Cytotoxic Assay by Staining with Amido Black
FH2 cells (1 x 105 cells/well) in 96-well plates were treated with anti-Fas mAb RK-8 and stained with 0.05% amido black in 9% CH3COOH with 0.1 M CH3COONa for 3060 min at room temperature. Cells were washed with water, dried, and dissolved in 100 µl/well of 25 mM NaOH. Cell density was determined by measuring optical absorbance at 560 nm with a microplate reader (Molecular Devices).
Quantification of FLIP mRNA by Reverse Transcription PCR
One step reverse trancription (RT)-PCR with 2 µg total RNA was carried out by using ready-to-go RT-PCR beads (Amersham Pharmacia Biotech) supplemented with extra 1.25 U Pfu turbo (Stratagene). Primers used for mouse cellular FLIP and elongation factor 1 (EF1
) were as follows: mouse FLIPL, 5'-GAG CCA AGA TTT GTG GAA TAC CG-3'; mouse FLIPL, 5'-TCT TCC AAC TGG CTA CCT AAC GAC T-3'; mouse EF1
, 5'-TCC TAC CAC CAA CTC GTC CAA C-3'; and mouse EF1
, 5'-CAG CTT CTT ACC AGA ACG ACG ATC-3'.
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Results |
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Cloning of c-K-Ras as a Gene Inhibiting Fas-mediated Apoptosis
To search for inhibitory genes against Fas-mediated apoptosis, we prepared the cells sensitive to the stimulation of Fas from Balb3T3 cells by transfecting an expression vector of mouse Fas under the control of human ß-actin promoter. FH2 was sensitive to the stimulation of Fas. By using FH2 cells, we carried out expression cloning to obtain cDNA that confers resistance to Fas-mediated apoptosis in FH2 cells as described in Materials and Methods, and finally obtained c-K-Ras. To confirm the protective activity of c-K-Ras, we cotransfected expression vectors encoding c-K-Ras and ß-galactosidase, and quantified ß-galactosidasepositive cells after stimulation of Fas (Fig 1). Treatment with agonistic anti-Fas mAb RK-8 dose-dependently induced apoptosis in FH2 cells, and transient expression of c-K-Ras suppressed this apoptosis (Fig 1). We then observed the morphologies of cells that were transfected with an empty vector or an expression vector of c-K-Ras, and treated with or without anti-Fas mAb for 4 h (Fig 2A, Fig B, Fig E, and Fig F). Control cells treated with anti-Fas mAb showed an apoptotic morphology with a rounded form and were detached from the culture dish (Fig 2 E). In contrast, a significant number of the c-K-Rastransfected cells kept an extended morphology even after the stimulation of Fas (Fig 2 F). These results indicate that the overexpression of c-K-Ras decreases the sensitivity of fibroblasts to Fas-mediated apoptosis.
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Activated K-Ras Inhibits Fas-mediated Apoptosis
To analyze whether the inhibitory effect of c-K-Ras on Fas-mediated apoptosis is reflected by the effects of active (GTP-bound) or inactive (guanosine diphosphatebound) Ras, FH2 cells were transfected with an expression vector of Ras mutant together with that of ß-galactosidase, constitutively active Ras (K-RasV12), or dominant-inhibitory Ras (K-RasN17). We counted the ß-galactosidasepositive cells transfected with K-RasV12 or K-RasN17 before and after the treatment with anti-Fas mAb (Fig 3 A) and found that K-RasV12 strongly inhibited, whereas K-RasN17 enhanced, Fas-mediated apoptosis. Fig 2 G shows that most of the cells transfected with K-RasV12 displayed an intact morphology after 4 h of stimulation with anti-Fas mAb. In contrast, almost all the cells transfected with K-RasN17 were completely detached from the culture dish (Fig 2 H). These results show that Fas-mediated apoptosis is suppressed by activated K-Ras and enhanced by dominant-inhibitory K-Ras in FH2 cells.
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Activated K-Ras Inhibits Activation of Caspases
Caspases play a central role in apoptosis by cleaving intracellular proteins, including DNA fragmentation factor (DFF) 45/Inhibitor of caspase-activated DNase (ICAD), poly (ADP-ribose) polymerase (PARP), and protein kinase MST (
Activation of the MAPK Pathway Inhibits Fas-mediated Apoptosis
GTP-bound active Ras was reported to transduce various signals by activating multiple intracellular target molecules, including Raf, PI-3 kinase, and Ral guanine nucleotide exchange factor (RalGDS) (
Raf is an activator of MAPKK, which is an activator of MAPK. To confirm whether the activation of the MAPK pathway is involved in the inhibition of Fas-mediated apoptosis, FH2 cells were transfected with Raf-CAAX (
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Then we examined whether the RalGDS pathway is involved in Ras-dependent protection, and dominant-inhibitory mutant of Ral, RalN28, transfected with K-RasV12 into FH2 cells could not cancel Ras-dependent protection against Fas-mediated apoptosis (data not shown). Recently, H-Rasdependent activation of PI-3 kinase, which is an activator of Akt/PKB, was reported to prevent Fas-mediated apoptosis by downregulating the expression level of Fas (p85 (
p85, which was confirmed by Western blotting (Fig 4 D), did not prevent the protective effect of K-RasV12 against Fas-mediated apoptosis in FH2 cells, although the inhibitory effect of
p85 on phosphorylation of Akt/PKB by K-RasV12 was confirmed (Fig 4 D). Moreover, overexpression of constitutively active Akt/PKB (
To analyze whether K-RasV12, Raf-CAAX, and SDSE-MAPKK regulate Fas-expression in FH2 cells that express exogenous Fas under the control of human ß-actin promoter, we analyzed the expression levels of Fas by flow cytometry on the cells that were cotransfected with K-RasV12, Raf-CAAX, or SDSE-MAPKK together with GFP expression vector. Control cells highly expressed Fas (Fig 5 A), and the expression levels of Fas on GFP-intensive cells were as high as those on GFP-negative cells (Fig 5 B). These results indicate that K-RasV12, Raf-CAAX, and SDSE-MAPKK do not influence the Fas-expression enforced by human ß-actin promoter in FH2 cells, because GFP-intensive cells were considered to highly express K-RasV12, Raf-CAAX, or SDSE-MAPKK. In addition, the expression level of endogenous Fas in parental Balb3T3 cells was also unaffected by overexpressed K-RasV12, Raf-CAAX, or SDSE-MAPKK (Fig 5 C). These results indicate that activation of the MAPK pathway by K-Ras does not regulate the expression level of Fas.
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Pretreatment with bFGF Inhibits Fas-mediated Apoptosis
To examine whether physiological activation of MAPK is sufficient to inhibit Fas-mediated apoptosis, FH2 cells were pretreated with several growth factors, including EGF, IGF, and bFGF, which are known to activate MAPK, and then stimulated with anti-Fas mAb. After pretreatment with bFGF for >12 h, FH2 cells showed a resistant phenotype to Fas-mediated apoptosis, although the cells pretreated with either EGF or IGF were as sensitive as nontreated cells (Fig 6 A). Then we compared the kinetics of the phosphorylation of MAPK after the treatment with EGF, IGF, and bFGF. bFGF treatment induced a strong and sustained phosphorylation of MAPK (Fig 6 B). EGF treatment induced a relatively transient phosphorylation of MAPK (Fig 6 B). These results suggest that strong and constitutive activation of MAPK is necessary to inhibit Fas-mediated apoptosis.
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We then investigated Fas-triggered activation of caspases in FH2 cells pretreated with or without bFGF by using fluorescence tetrapeptides, IETD-MCA and DEVD-MCA, as specific substrates for caspase-8/6 and caspase-3/7, respectively. Protease activity of caspases specific for both IETD and DEVD in control cells increased markedly after a 2-h stimulation of Fas (Fig 6c and Fig d). However, in bFGF-treated cells, the protease activity for IETD was completely suppressed even after a 4-h stimulation of Fas (Fig 6 C). The protease activity for DEVD was also distinctly suppressed by the pretreatment with bFGF (Fig 6 D), although it slightly increased from 3 h after the stimulation of Fas. These results show that bFGF suppressed Fas-triggered apoptotic signaling at a point upstream of caspases the same as oncogenic K-Ras.
bFGF Prevents Fas-mediated Apoptosis by Activating the Ras/MAPK Pathway
To investigate whether the activation of MAPK is involved in the inhibition of Fas-mediated apoptosis by bFGF, FH2 cells were transfected with an expression vector of K-RasN17, MKP-1, or p85, and then treated with bFGF followed by the stimulation with anti-Fas mAb. Fig 7 A shows that K-RasN17 and MKP-1 prevented the protective effect of bFGF against Fas-mediated apoptosis, although
p85 did not have any inhibitory effect on the activity of bFGF. These results indicate that bFGF inhibits Fas-mediated apoptosis in FH2 cells through activation of MAPK.
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We analyzed expression levels of endogenous and stably expressed exogenous Fas on Balb3T3 cells and FH2 cells, respectively, before and after the treatment with bFGF. bFGF treatment did not downregulate expression levels of Fas on either Balb3T3 cells (Fig 7 B) or FH2 cells (data not shown). These results show that the protective effect of bFGF on Fas-mediated apoptosis is mediated by the Ras/MAPK pathway without downregulating Fas expression.
It was reported previously that c-FLIP, which can inhibit Fas-induced apoptosis ( was observed seven cycles earlier than c-FLIP (Fig 7 C). These data suggest that c-FLIP expression was upregulated by the bFGF treatment about twice, but the expression level of c-FLIP was low.
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Discussion |
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We report here that transient expression of oncogenic K-Ras inhibits Fas-mediated apoptosis in Fas-transgenic Balb3T3 cells through the activation of the Ras/MAPK pathway. Although the cells transfected with K-RasV12 were strongly resistant to the stimulation of Fas, prolonged stimulation >8 h caused apoptosis in some of these cells (Fig 2 I). We observed that most of the surviving cells transfected with K-RasV12, even after the prolonged stimulation of Fas, show developed filamentous structures in the cytoplasm, which may indicate strong expression of transfected K-RasV12 (Fig 2I and Fig J). These results suggest that strong expression of oncogenic K-Ras can completely prevent untransformed cells from undergoing Fas-mediated apoptosis, and explain how tumor cells escape from immune surveillance by cytotoxic T cells during the multistep progression of oncogenesis, because cytotoxic T cells utilize FasFas ligand system to kill tumor cells (
Among partial loss-of-function mutants of Ras, RasS35 and RasG37 were reported to activate only Raf and RalGDS, respectively. Both K-RasS35 and K-RasG37 partially protected FH2 cells from Fas-mediated apoptosis (Fig 3 B). However, dominant-inhibitory RalN28 could not cancel Ras-dependent protection against Fas-mediated apoptosis (data not shown). Not only dominant-inhibitory Ral but also dominant-inhibitory PI-3 kinase subunit p85, which inhibited Ras-dependent activation of PI-3 kinase (Fig 4 D), could not disrupt the protective activity of K-Ras against Fas-mediated apoptosis (Fig 4 A). In contrast, MKP-1, a phosphatase specific for activated classical MAPK, could cancel the protective activity of K-RasV12, Raf-CAAX, and SDSE-MAPKK (Fig 4 A). Thus, activation of MAPK is essential for K-Rasdependent protection against Fas-mediated apoptosis in FH2 cells. However, the results indicating that the protective activity of Raf-CAAX and SDSE-MAPKK was slightly lower than that of K-RasV12 (Fig 4a and Fig b) suggest that another signaling pathway activated by K-Ras may contribute to Ras-dependent protection against Fas-mediated apoptosis.
We transfected K-RasV12 or K-RasN17 into other Fas-transgenic cells prepared from tumor cell lines such as HeLa and KB cells. Interestingly, Fas-mediated apoptosis in these cells was neither inhibited by transient expression of K-RasV12 nor enhanced by transient expression of K-RasN17 or MKP-1 (data not shown). These cells were relatively resistant to the stimulation with agonistic anti-Fas mAb compared with FH2 cells. These results imply that the protective activity of Ras/Raf/MAPK is specifically observed in untransformed cells or the cells more sensitive to the stimulation of Fas than usual transformed cell lines such as HeLa and KB cells.
We showed here that bFGF treatment desensitized fibroblasts to the stimulation of Fas through activation of the Ras/MAPK pathway, because K-RasN17 and MKP-1 canceled the protective effect of bFGF (Fig 7 A). However, the protective effect of bFGF was not sustained for a long time (Fig 6 A), and caspase-3 was gradually activated after a 3-h stimulation of Fas (Fig 6 D). These results indicate that bFGF can exert its protective ability through the Ras/MAPK pathway, but the activation of endogenous Ras by bFGF may not be sufficient to protect cells against continuous stimulation of Fas. We suppose that a more sustained activation of the Ras/MAPK pathway, such as by high expression of oncogenic K-RasV12 (Fig 2I and Fig J), is necessary for complete protection of cells against Fas-mediated apoptosis.
The protective ability of bFGF against Fas-mediated apoptosis was different from that of EGF (Fig 6 A).
It was shown that oncogenic H-Ras downregulates the expression of endogenous Fas in fibroblast and epithelial cells through the activation of PI-3 kinase (
In bFGF-treated FH2 cells, we detected about twofold upregulation of c-FLIP transcript by RT-PCR when compared with that in control FH2 cells (Fig 7 C). However, we could not detect c-FLIP mRNA in FH2 cells treated or untreated with bFGF by Northern hybridization under the condition where mRNA of caspase-8 and EF1 were detected (data not shown). We suppose that bFGF-induced upregulation of c-FLIP mRNA is not sufficient to protect FH2 cells from Fas-mediated apoptosis, because the quantity of c-FLIP mRNA is much lower than that of caspase-8 mRNA in FH2 cells.
Here we clarified that the Ras/MAPK pathway prevents Fas-mediated apoptosis in untransformed fibroblasts, which may contribute to oncogenesis. However, the protective mechanism of the Ras/MAPK pathway remains to be elucidated and must be clarified in the future.
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Footnotes |
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1 Abbreviations used in this paper: bFGF, basic FGF; EF1, elongation factor 1
; FADD, Fas-associated death domain; FLICE, FADD-like interleukin-1ßconverting enzyme; FLIP, FLICE-inhibitory protein; GFP, green fluorescence protein; IGF, insulin-like growth factor; K-RasV12, oncogenic K-Ras; MAP , mitogen-activated protein; MAPK, MAP kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MKP-1, MAPK phosphatase 1; MST1, mammalian STE-20like protein kinase; MST1-KD, kinase-defective MST1; PE, phycoerythrin; PI-3, phosphatidylinositol 3; PKB, protein kinase B; Raf-CAAX, constitutively active Raf; RalGDS, Ral guanine nucleotide exchange factor; RT, reverse trancription; SDSE-MAPKK, constitutively active MAPKK.
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Acknowledgements |
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We thank K. Nagata for providing Balb3T3 cells, J.F. Hancock for Raf-CAAX, E. Nishida for SDSE-MAPKK, W. Ogawa for p85, U. Kikkawa for Akt, R. Roth and Y. Gotoh for active Akt, L.A. Feig for RalN28, K. Umesono for pCMX-GFP, and K.K. Lee and M. Murakawa for Flag-tagged MST1-KD. We thank K. Sakamaki for helpful comments.
This work was supported in part by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan, and the Ministry of Health and Welfare of Japan, and performed in part through Special Coordination Funds of the Science and Technology Agency of the Japanese Government.
Submitted: 3 August 1999
Revised: 15 November 1999
Accepted: 3 January 2000
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References |
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