Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cell invasion by activating matrix metalloproteinase genes

Motoaki Hanzawa, Masanobu Shindoh1,4, Fumihiro Higashino1, Motoaki Yasuda, Nobuo Inoue2, Kyoko Hida2, Mitsunobu Ono2, Takao Kohgo1, Motoyasu Nakamura, Ken-ichi Notani2, Hiroshi Fukuda2, Yasunori Totsuka2, Koichi Yoshida3 and Kei Fujinaga3

Department of Dental Radiology,
1 Department of Oral Pathology,
2 Department of Oral Surgery, Hokkaido University School of Dentistry, North 13 West 7 Kita-ku, Sapporo 060-0813 and
3 Department of Molecular Biology, Cancer Reseach Institute, Sapporo Medical University School of Medicine, South 1 West 17 Chuo-ku, Sapporo 060, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Hepatocyte growth factor (HGF) is thought to play a role in cell motility and invasion. Matrix metalloproteinases (MMPs) have been implicated in invasion and metastasis of tumor cells. We have previously reported that the Ets-oncogene family transcription factor E1AF positively regulates transcription of MMP genes in transient expression assays and that overexpression of the E1AF gene confers an invasive phenotype on breast cancer cells. Here we examined the effect of HGF on E1AF and MMP gene expression in terms of the invasive potential of the oral squamous cell carcinoma cell line HSC3. HGF stimulated expression of the E1AF gene. The levels of MMP-1, -3 and -9 mRNAs increased in cells treated with HGF and correlated with E1AF upregulation. In contrast, no obvious upregulation of MMP-1 and -9 mRNA was observed in ASE1AFHSC3 cells transfected with the antisense E1AF expression vector into parental HSC3 cells. The wild-type MMP-9 gene promoter was activated by endogenous E1AF in HSC3 cells, and chloramphenicol acetyltransferase (CAT) activities increased when HGF was added to transfected cells. On the other hand, CAT activity was reduced to almost two-thirds of the wild-type activity when HSC3 cells were transfected with a CAT reporter plasmid driven by a mutant MMP-9 promoter lacking the Ets-binding site, and induction of CAT activity was not observed upon addition of HGF. Analysis of organotypic raft cultures revealed that HSC3 cells invaded and degraded collagen gel actively upon addition of HGF. These results suggest that HGF induces expression of the Ets-related E1AF transcription factor gene whose product in turn activates MMP genes and leads to oral cancer cell invasion.

Abbreviations: CAT, chloramphenicol acetyltransferase; HGF, hepatocyte growth factor; MMP, matrix metalloproteinase.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor invasion is the end result of a complex series of steps involving multiple tumor–host interactions (1,2). During invasion, the carcinoma cells must move through and degrade the extracellular matrix (3). Several growth factors such as EGF, FGF and TGF-ß, have been implicated in stimulating cell motility and invasion (4,5).

Hepatocyte growth factor (HGF) was originally identified, purified and molecularly characterized as a potent mitogen for mature hepatocytes (6). This factor has pleiotropic functions. HGF enhances the proliferation of various types of cells by acting as a mitogen, enhances the motility of epithelial cells (motogen) and induces an organized epithelial tissue-like structure (morphogen) (7). The receptor for HGF is a receptor-type tyrosine kinase encoded by the c-met proto-oncogene (8) which was originally identified as a transforming gene formed by a DNA rearrangement in carcinogen-treated cells (9). HGF has been shown to stimulate the invasive activity of various cancer cells in vitro (1012) and in vivo (13). The mechanism by which HGF/c-MET signaling promotes invasive and metastatic activity is still largely unknown. However, the MET signal transduction pathway has been shown to be linked with src and ras activation, and through these oncogenes c-MET may confer invasive and metastatic phenotypes (14). There have been few reports on which transcription factor mediates HGF/c-MET signaling and activates the downstream target genes.

A part of the invasive activity is thought to depend on the production of matrix-degrading enzymes such as matrix metalloproteinases (MMPs) (1517). Analyses of the transcription regulatory regions of the MMP genes revealed binding sites for ets transcription factors (1822). We previously isolated a cDNA encoding human E1AF, a member of the ets oncogene family, by its ability to bind the adenovirus E1A enhancer element (23). E1AF stimulates transcription from three different classes of MMP genes in transient expression assays (24). We have reported that an oral squamous cell carcinoma-derived cell line, HSC3, has highly invasive ability, and suggested the possible correlation of MMP-1, -9 and E1AF expression in the invasive ability of the cell line (25). Transfection of the weakly invasive human MCF-7 breast cancer cell line with an E1AF expression plasmid results in induction of invasive and motile activities, accompanied by an increase in 92 kDa type IV collagenase (MMP-9) gene expression (26), and the invasion is inhibited when HSC3 cells, a highly invasive oral cancer cell line, are transfected with an anti-sense E1AF expression plasmid (27). Thus, the ets-related E1AF transcription factor participates in cancer cell invasion.

Here we investigated the role of E1AF, an ets-oncogene family transcription factor, in HGF stimulation of oral squamous carcinoma HSC3 cells, especially the invasion-associated gene regulation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines and culture conditions
The human oral squamous cell carcinoma cell line HSC3 (JCRB, Osaka, Japan) was mainly used in the present study. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Gin1 and MRC5 fibroblasts (RIKEN, Tsukuba, Japan) were maintained in the same medium and used as negative controls of c-met expression.

The antisense E1AF expression vector (pRVSV–ASE1AF) was reconstructed (27), and was transfected to HSC3 cells with LipofectAmine reagent (Gibco BRL, Gaithersburg, MD). A G418-resistant clone, ASE1AFHSC3, was isolated and used for experiments.

Northern blot assay
Total-cell RNAs from the above cell lines were prepared and analyzed. The adherent HSC3 cells were washed three times with PBS and cultured in serum-free DMEM supplemented with either 0, 5, 10 or 20 ng/ml of HGF for 6 h to investigate the HGF dose dependency. HSC3 cells were incubated in DMEM containing 10% FBS for 24 h. The adherent cells were washed three times with PBS and cultured in serum-free DMEM supplemented with 10 ng/ml of HGF for 6 h. After incubation, the cytoplasmic RNA was extracted as reported previously (2527). Total-cell RNA (15 µg/lane) was applied to 1.0% agarose gels containing 2.2 M formaldehyde in MOPS running buffer and transferred onto a nitrocellulose filter. 32P-labeled E1AF cDNA (nucleotides 29–1538), MMP-1, -2, -3, -9, tissue inhibitors of metalloproteinases-1 (TIMP-1) cDNA and c-met cDNA (a gift from Dr T.Nakamura) were used as specific probes. Hybridization was carried out at 42°C for 24–48 h in the presence of 32P-labeled probes. Filters were washed twice with 1x SSC/0.1% SDS at room temperature, twice with 0.2x SSC/0.1% SDS at 65°C and exposed to Kodak X-O Mat X-ray films at –80°C for 48 h.

HSC3 cells and ASE1AFHSC3 cells were harvested and cultured in serum-free DMEM supplemented with 0 or 20 ng/ml HGF for 6 h. Total-cell RNA was extracted and subjected to gel electrophoresis and examined for E1AF and MMP-9 mRNA expression by the method described above. The intensities of these results were measured using BAS-2000 Imaging Analyzer (Fuji-Film Co., Tokyo, Japan).

Chloramphenicol acetyltransferase (CAT) assays
Two micrograms of CAT reporter genes driven by promoters of wild-type MMP-9 or the Ets-binding site deletion mutant MMP-9 (Figure 4AGo) (a gift from Prof. M.Seiki in Kanazawa University) were transfected into HSC3 cells using the SuperFect Transfection Reagent (Qiagen, Santa Clarita). After 24 h, 0 or 10 ng/ml HGF were added to the medium and cells were harvested 4 h later. Cell extracts were prepared and CAT activities were assayed by a standard procedure (24). Each assay was done in triplicate.




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Fig. 4. (A) Schematic representation of the wild-type MMP-9 and mutant MMP-9 CAT reporter plasmids. (B) Results of CAT assay. Endogenous E1AF in HSC3 cells activated wild-type MMP-9 promoters, and it was enhanced 2.5-fold by adding HGF. In contrast, the activity was reduced to two-thirds of the wild-type promoter activity in cells transfected with the Ets-binding site-deleted MMP-9 promoter plasmid, and no induction was observed upon addition of HGF. Values represent the means of three measurements; bars show SD.

 
Gelatin zymography
Gelatinolytic enzymes secreted by cultured cells were identified and quantified by electrophoresis of serum-free conditioned medium in gelatin-embedded polyacrylamide gel as decribed previously (17). The same number of HSC3 cells was divided and incubated in DMEM containing 10% FBS for 24 h. The adherent cells were washed three times with PBS and cultured in serum-free DMEM supplemented with either 0, 10 or 20 ng/ml HGF. After a 24 h incubation, the medium was collected and the non-concentrated supernatants were immediately mixed with the SDS sample buffer without 2-mercaptoethanol.

Electrophoresis was carried out on polyacrylamide gels containing 1 mg/ml of gelatin. Following electrophoresis, the gels were rinsed twice with 50 mM Tris–HCl buffer (pH 7.5) containing 2.5% Triton X-100 for 15 min each time, and incubated at 37°C for 16 h in a buffer composed of 0.15 M NaCl, 10 mM CaCl2 and 50 mM Tris–HCl (pH 7.5). The gels were stained with 0.05% Coomassie blue in 10% ethanol and 10% acetic acid, and destained with 10% ethanol and 10% acetic acid. The enzymatic activity was measured using a densitometer (IBAS, Leizz, Germany).

In vitro invasion assays
Biocoat Matrigel invasion chambers (Becton Dickinson, San Lose) were used in invasion assays (28). HSC3 cells (1x105) were suspended in serum-free DMEM and added to the upper chamber. The lower chamber was filled with serum-free control medium or serum-free medium supplemented with HGF (5, 10, 15, 20 ng/ml). Cells were incubated for 6 h at 37°C in a CO2 incubator. At the end of the incubation, cells on the upper surface of the filter were completely removed by wiping with a cotton swab. Cells were fixed in ethanol and stained with Giemsa solution. Cells that invaded the lower surface of the filter were counted under a light microscope at a magnification of x100. HSC3 cells and ASE1AFHSC3 cells were examined with 0 or 10 ng/ml HGF in the same procedures. Each assay was done in triplicate.

Raft culture assays
Raft culture assays were performed as described previously (25,29), but modified as described below. We used type I or type I+IV collagen matrices (type I and type IV mixed 4:1 v/v) without fibroblast. Three milliliters of the collagen solution was poured into 35 mm plastic dishes and allowed to gel for 30 min at 37°C. HSC3 cells from monolayer cultures were trypsinized, seeded onto the collagen matrix at 3x105 cells per dish and incubated in 2 ml of DMEM/10% FBS. At confluence, the rafts were mounted onto stainless steel grids, and medium was changed once every 2 days. The collagen-matrix rafts were harvested after 9 days, and then 0 or 20 ng/ml HGF were added to the medium. Cells were incubated in the presence of HGF for an additional 24 h. Each raft specimen was fixed with 4% paraformaldehyde for histological study. Specimens were embedded in paraffin, sectioned and stained with hematoxylin and eosin for histological evaluation.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We first examined the expression of c-met encoding the HGF receptor gene in oral squamous carcinoma cell line HSC3. Northern blot assays with a c-met-specific cDNA probe detected a 7 kb c-met mRNA in total-cell RNA samples prepared from HSC3 cells, whereas no obvious signal was observed in MRC5 and Gin 1 fibroblasts (Figure 1Go).



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Fig. 1. Expression of c-met mRNA in HSC3 cells. c-met mRNA is detected by northern blotting using 32P-labeled c-met cDNA as a probe. Arrowheads indicate the 7 kb c-met mRNA. ß-Actin mRNA was used as an internal marker for equal RNA loading. The number indicates fold induction measured by imaging analyzer.

 
Northern blot assays detected a relatively low level of the 2.5 kb E1AF mRNA in HSC3 cells. Treatment of HSC3 cells with HGF induced E1AF mRNA in a dose-dependent manner (Figure 2AGo). We analyzed whether HGF activated the type I (MMP-1) and type IV (MMP-2 and -9) collagenases, and the stromelysin-1 (MMP-3) genes. Northern blot assays detected low base levels of 2.2 kb MMP-1, 2.0 kb MMP-3 and 2.8 kb MMP-9 mRNA in HSC3 total-cell RNA preparations. Treatment of HSC3 cells with HGF resulted in an increase of these mRNAs in a dose-dependent manner, whereas the levels of the 3.1 kb MMP-2 mRNA were not significantly altered. TIMP-1 mRNA was not significantly altered by HGF treatment in this cell line (Figure 2BGo).





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Fig. 2. Northern blot assays showing the levels of E1AF, MMPs and TIMP-1 mRNA in HSC3 cells treated with HGF. (A) Northern-blot analysis of E1AF mRNA. E1AF mRNA was detected by using 32P-labeled E1AF cDNA. Arrowhead indicates the 2.5 kb E1AF mRNA. The levels of E1AF mRNA in the HSC3 cells increased in an HGF dose-dependent manner. (B) Northern-blot analysis of MMP-1, -2, -3, -9, TIMP-1 mRNAs. Arrowheads indicate 2.2 kb MMP-1 mRNA, 3.1 kb MMP-2 mRNA, 2.0 kb MMP-3 mRNA, 2.8 kb MMP-9 mRNA and 0.9 kb TIMP-1 mRNA. The levels of MMP-1, -3 and -9 mRNAs in HSC3 cells increased in an HGF dose-dependent manner, whereas those of the MMP-2 and TIMP-1 mRNAs remained unchanged in response to HGF treatment. (C) Expression of E1AF and MMP-9 mRNA in HSC3 cells and ASE1AF cells with or without HGF stimulation. Expression of E1AF and MMP-9 mRNAs in HSC3 cells was promoted when HGF was added, while expression of MMP-9 mRNA was lower in the steady-state level and no induction by HGF was observed in ASE1AFHSC3 cells. The number indicates fold induction measured by imaging analyzer.

 
We examined the role of E1AF on HGF stimulation using parental HSC3 cells and antisense E1AF transfected ASE1AFHSC3 cells. Total RNA from parental HSC3 and ASE1AFHSC3 cells with or without HGF stimulation were probed with 32P-labeled E1AF and MMP-9 cDNAs. The amount of MMP-9 mRNA was significantly reduced in the ASE1AFHSC3 cells compared with those of the parental HSC3 and no induction of MMP-9 mRNA was observed after stimulation by HGF (Figure 2CGo).

MMP release from HSC3 cells into conditioned media was assayed by gelatin zymography. HGF increased the release of both the proenzyme form and the active form of MMP-9 (the 92 kDa type IV collagenase), whereas MMP-2 (the 72 kDa type IV collagenase) was not significantly altered (Figure 3Go).



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Fig. 3. Zymographic detection of gelatinase activity in cells treated with HGF. Gelatin zymography indicates increased amounts of the proenzyme form and active form of MMP-9 (the 92 kDa type IV collagenase), whereas the activity of MMP-2 (the 72 kDa type IV collagenase) was not significantly altered by HGF treatment. The number indicates fold induction measured by IBAS densitometer.

 
The wild-type MMP-9 gene promoter was activated by endogenous E1AF in HSC3 cells, and CAT activity increased 2.5-fold when 10 ng/ml of HGF was added to transfected cells. On the other hand, CAT activity was reduced to almost two-thirds of the wild-type activity when HSC3 cells were transfected with a CAT reporter plasmid driven by the mutant MMP-9 promoter lacking the Ets-binding site, and induction of CAT activity was not observed upon addition of HGF (Figure 4BGo).

To measure the invasive properties of HSC3 cells, we used a hybrid matrix composed of a mixture of basement membrane-like matrix. HGF stimulated the invasive activity of these cells in an HGF dose-dependent manner (Figure 5AGo).



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Fig. 5. (A) Effects of HGF on invasion of HSC3 cells in vitro. Biocoat Matrigel invasion chambers were used in the invasion assay. Values represent the mean of three measurements; bars show SD. HGF stimulated invasion in a dose-dependent manner. (B) Invasion activity of HSC3 cells and ASE1AFHSC3 cells with (10 ng/ml) or without HGF stimulation.

 
However, the steady-state level of invasion activity was lower and no induction of invasive activity was observed in ASE1AFHSC3 cells after adding HGF to the culture medium (Figure 5BGo).

We used organotypic raft cultures, composed of type I or a mixture of type I and IV collagen matrices without fibroblasts, as a simplified invasion model in vitro. After 10 days, the HSC3 cells seeded on the type I collagen raft formed a stratified epithelium and invasive potential was not observed (Figure 6AGo). Upon addition of HGF at the final concentration of 20 ng/ml, HSC3 cells proliferated invasively into collagen gels (Figure 6BGo). Similar to the type I collagen raft, HSC3 cells seeded on the type I+IV collagen rafts formed a stratified epithelium on collagen gels (Figure 6CGo), and upon addition of HGF, the cells aggressively invaded the type I+IV collagen gel (Figure 6DGo).



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Fig. 6. Histological findings for raft-cultured cells. (A and B) HSC3 and type I collagen-raft culture. (A) HSC3 and type I collagen-raft. HSC3 cells show stratified epithelium on collagen gels and invasive potential is not observed. (B) HSC3 and type I collagen plus HGF 20 ng/ml. HSC3 cells show invasive proliferation of epithelial cells into collagen gels. (C and D) HSC3 and type I+IV collagen-raft culture. (C) HSC3 and type I+IV collagen-raft, (D) HSC3 and type I+IV collagen plus 20 ng/ml HGF. HSC3 cells invaded collagen gels after HGF treatment. (Bar = 50 µm.)

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The interaction of tumor cells with the host stromal tissue may have an important effect on the malignant behavior of tumor cells (17,30,31). Several growth factors have been implicated in the tumor–host interaction as factors stimulating the process of tumor invasion (4,5). Motility was shown to be associated with metastasis (3234), and HGF was initially identified as a motility-stimulating factor from human MRC5 fibroblasts (35). Weidner et al. (36) demonstrated that HGF purified from MRC5-conditioned medium promotes migration and invasion of malignant epithelial cells in vitro. Anti-HGF antiserum blocks the stimulation of migration and invasion by fibroblast conditioned medium in vitro (37). HGF is produced by mesenchymal cells and acts predominantly on cells of epithelial origin in an endocrine and/or paracrine fashion (7,35).

HGF/c-MET signaling has been shown to promote the invasive metastatic phenotypes of various cancer cells in vitro (3639). MET was originally isolated as the product of a human oncogene, trp-met, which encodes an altered Met protein possessing constitutive, ligand-independent tyrosine kinase activity and transforming ability (40,41). The coexpression of Met and HGF molecules in the same cell, which generates an autocrine stimulatory loop, is oncogenic (38,42). In addition to transforming cells, deregulated Met signaling in NIH 3T3 cells increases their invasiveness in vitro (14,43) and metastatic potential in vivo (14). The MET signal transduction pathway has been linked with src and ras activation, and through these oncogenes Met is thought to confer a metastatic phenotype (14). However, the molecular mechanism by which HGF/c-MET signaling stimulates the invasion metastasis remains largely unknown (44).

It has been shown that extracellular matrix-degrading enzymes play a particularly important role in cancer cell invasion and metastasis as well as in increase of cell motility.

MMPs are thought to play an important role in cancer invasion and metastasis (21,45,46). The production and enzyme activities of MMPs are elevated in invasive tumor tissues and cell lines (45,47,48). Ectopic expression of several MMP genes confers an invasive phenotype. Conversely, downregulation of MMP genes by anti-sense DNA causes invasive cancer cells to become less invasive in vitro (28,49).

We isolated a cDNA encoding human E1AF, a new member of the Ets-oncogene family (23). CAT assays revealed that E1AF can upregulate promoter activities of the MMP-1, -3 and -9 genes, and that the activation levels are as high as those induced by the AP-1 transcription factor (24). We recently reported that invasiveness of the oral squamous cell carcinoma cell line correlates well with E1AF and metalloproteinase gene expression levels (25). The MMP-1 and -9 proteins were detected immunohistochemically in invasive HSC3 cells, whereas they were undetectable in the non-invasive cell lines. Ectopic expression of the E1AF gene results in induction of invasive activities, accompanied by an increase in MMP-9 gene expression (26), and antisense E1AF transfection restrains cancer cell invasion by reducing MMP activities (27).

This study was intended to determine the identities of the invasion-associated genes possibly underlying the HGF stimuli in oral squamous carcinoma HSC3 cells.

Northern blot assays demonstrated that levels of E1AF, MMP-1, -3 and -9 mRNAs in HSC3 cells increased in response to HGF treatment in a dose-dependent manner, while no induction was observed in antisense E1AF-transfected ASE1AFHSC3 cells. CAT assays revealed that the MMP-9 promoter activity was increased in response to HGF, while induction was weaker in cells transfected with an Ets-binding-site-deleted MMP-9 promoter and no induction was observed upon treatment of cells with HGF. Other transcription factors such as AP1 may be involved in MMP-9 gene transcription in addition to the Ets-transcription factor. However, our results indicate that E1AF is closely involved in HGF/c-Met-stimulated upregulation of MMP genes. Gelatin zymography confirmed the increase in the proenzyme and the active form of MMP-9 caused by HGF treatment.

These results were confirmed by the invasion assay and in vitro three-dimensional raft culture. The number of invading HSC3 cells increased in response to HGF treatment in a dose-dependent manner. On the other hand, the number of invading ASE1AFHSC3 cells was fewer and no response was observed upon HGF stimulation. HSC3 cells seeded on type I and type I+IV collagen rafts formed a stratified epithelium on collagen gels, but did not display invasive potential, whereas they proliferated invasively into collagen gels in the presence of HGF. HGF induced production of MMP-1, -3 and -9 proteins in HSC3 cells, which led to the invasion of these cells into collagen gels.

Our results indicate that HGF induces expression of E1AF and imparts an invasive property to oral cancer cells by activating MMP genes. Although the possibility that other Ets family members might respond to HGF stimulation cannot be excluded, E1AF may mediate HGF-induced activation of MMP genes in oral squamous cell carcinoma-derived HSC3 cells.


    Notes
 
4 To whom correspondence should be addressed Email: mshindoh{at}den.hokudai.ac.jp Back


    Acknowledgments
 
We thank Prof. T.Nakamura of Osaka University for providing c-met cDNA, Prof. M.Seiki of Tokyo University and Prof. H.Sato of Kanazawa University for MMP-1, -2, -3, -9, TIMP-1 cDNAs and CAT reportor plasmids for wild-type and mutant MMP-9 promoters. We are grateful to Mr M.K.Barrymore for his help in revising the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research and a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan, and by a Grant-in-Aid from the Uehara Memorial Foundation.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received June 14, 1999; revised February 28, 2000; accepted March 6, 2000.