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(Circulation Research. 2000;87:699.)
© 2000 American Heart Association, Inc.

Cellular Biology

Cyclin A Downregulation and p21^cip1 Upregulation Correlate With GATA-6–Induced Growth Arrest in Glomerular Mesangial Cells

Daisuke Nagata, Etsu Suzuki, Hiroaki Nishimatsu, Masao Yoshizumi, Toshiaki Mano, Kenneth Walsh, Masataka Sata, Masao Kakoki, Atsuo Goto, Masao Omata, Yasunobu Hirata

From the Second Department of Internal Medicine, Faculty of Medicine (D.N., E.S., M.S., M.K., A.G., M.O., Y.H.), Department of Urology, Faculty of Medicine (H.N.), Department of Geriatrics, Faculty of Medicine (M.Y.), University of Tokyo, Japan, and Division of Cardiovascular Research, St. Elizabeth’s Medical Center, Tufts University School of Medicine (T.M., K.W.), Boston, Mass.

Correspondence to Etsu Suzuki, MD, PhD, The Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail suzuki-2im{at}h.u-tokyo.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract—The GATA-6 transcription factor is reported to be expressed in vascular myocytes. Because glomerular mesangial cells (GMCs) and vascular myocytes have similar properties, we examined whether GATA-6 was expressed in cultured GMCs and whether overexpression of GATA-6 induced cell cycle arrest in GMCs, using a recombinant adenovirus that expresses GATA-6 (Ad GATA-6). GATA-6 expression in GMCs was downregulated when quiescent GMCs were stimulated by serum to reenter the cell cycle. [³H]thymidine uptake was inhibited in GMCs infected with Ad GATA-6 in a dose- and time-dependent manner. The expression of cyclin A protein was decreased and that of the cyclin-dependent kinase inhibitor p21^cip1 was increased in GMCs infected with Ad GATA-6. Although the expression of p21^cip1 transcripts did not change remarkably, p21^cip1 protein was stabilized in GMCs infected with Ad GATA-6, suggesting a post-transcriptional regulation of p21^cip1 expression. Northern blot analysis showed that expression of the cyclin A transcript was decreased in Ad GATA-6–infected cells, whereas this decrease of cyclin A was not observed in GMCs derived from p21^cip1 null mice. Our results demonstrate that GATA-6 is endogenously expressed in GMCs and that overexpression of GATA-6 can induce cell cycle arrest. Our results also show that GATA-6–induced cell cycle arrest is associated with inhibition of cyclin A expression and p21^cip1 upregulation. Finally, our results indicate that the GATA-6–induced suppression of cyclin A expression depends on the presence of p21^cip1.

Key Words: cell cycle • transcription factors • kidney • gene transfer • cyclin-dependent kinases

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Glomerular mesangial cells (GMCs), which are interposed among glomerular capillaries, not only support the glomerular capillary loops but also have contractile and phagocytic properties. Like vascular smooth muscle cells (VSMCs), GMCs contain both actin and myosin filaments and contract in response to vasoactive agents such as angiotensin II and vasopressin.^{1 2} It is well established that proliferation of GMCs and increase in the surrounding matrix are implicated in the pathophysiology of some forms of glomerulonephritis. It is also well known that persistence of glomerulonephritis often results in renal hypertension (hypertension associated with renal parenchymal disease), which is the major cause of secondary hypertension, and renal failure. However, little attempt has been made to modulate the growth of GMCs using endogenously expressed genes that may be implicated in maintenance of the differentiated phenotype or quiescence of GMCs.

The GATA transcription factors comprise 6 isoforms. Among them, the GATA-1/-2/-3 genes are mainly expressed in hematopoietic cells, whereas the GATA-4/-5/-6 genes are predominantly expressed in the heart and gastrointestinal tract.³ Gene disruption experiments of each GATA transcription factor have shown that each GATA gene has distinct functions.^{4 5 6 7 8 9} We have recently shown that the GATA-6 gene was expressed in cultured human and rat VSMCs and that its expression is rapidly downregulated when quiescent cultured VSMCs are stimulated by serum mitogens to initiate cell cycle reentry.¹⁰ We have also shown that overexpression of GATA-6 gene induced cell cycle arrest in VSMCs and fibroblasts.¹¹ These results suggest that GATA-6 is a candidate gene that may be implicated in maintenance of the differentiated phenotype in VSMCs and that overexpression of GATA-6 can inhibit proliferation of VSMCs.

Cell cycle progression is regulated by cyclin-dependent kinase (cdk), the activities of which oscillate during the cell cycle. Cdks are regulated by cyclins, positive coactivators, and cdk inhibitors.^{12 13} In mammalian cells, cyclin D–cdk4/cdk6, cyclin E–cdk2, cyclin A–cdk2, and cyclin B–cdc2 are the main cyclin-cdk complexes that regulate the progression of G₁, G₁/S, S, and G₂/M phases, respectively. Cdk inhibitors comprise 2 families, the ink4 and cip/kip families. Cdk inhibitors of the cip/kip family are of particular interest in that they inhibit the activity of a broader spectrum of cdks, including cdk2, cdk4, and cdk6. The cip/kip family is composed of p21^cip1, p27^kip1, and p57^kip2.^{14 15 16}

Given that GMCs and VSMCs have similar properties, it is tempting to speculate that the GATA-6 transcription factor may participate in maintenance of the quiescent phenotype in GMCs. To test the hypothesis, we examined whether the GATA-6 gene was expressed in GMCs and whether overexpression of GATA-6 inhibited GMC proliferation using a replication-defective adenovirus (Ad) expressing GATA-6.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Culture
Rat GMCs were obtained from cultures of isolated rat glomeruli as previously described.¹⁷ GMCs were also isolated from glomeruli of p21^cip1-/- mice. Rat VSMCs were cultured from rat thoracic aortas according to the explant method, as previously described.¹⁷ The HepG2 cell line, which is derived from human hepatoma cells, was cultured in DMEM with 10% FBS.

Plasmids
Details of the subcloning of hemagglutinin (HA) epitope–tagged human GATA-6 cDNA in a mammalian expression vector (pcDNA3HA-hGATA-6), isolation of the human cyclin A cDNA, and preparation of the full-length p21^cip1 cDNA are described elsewhere (see online-only supplementary information; data supplement available at http://www.circresaha.org).

Construction of a Replication-Defective Ad Encoding Human GATA-6
Construction of a replication-defective Ad that expresses HA-tagged human GATA-6 (Ad GATA-6) was performed according to the cosmid cassettes and Ad DNA-terminal protein complex (COS-TPC) method as previously described.¹⁸ A recombinant Ad that expresses glomerular-filtered phosphate (green fluorescent protein) was obtained from Quantum Biotechnologies.

Adenoviral Infection
Subconfluent GMCs were infected with Ad GATA-6 or Ad GFP in low-serum medium (DMEM/0.1% FBS) with multiplicity of infection (MOI) varying from 0 to 50. Cells were incubated in the low-serum medium for 72 hours and then restimulated with growth medium (DMEM/10% FBS) for 8, 16, and 24 hours.

RNA Extraction and Northern Blot Analysis
Total RNA was extracted using Trizol reagent (Gibco-BRL), and poly(A) RNA was purified using SCIGEN mRNA isolation kit (SCIGEN) according to the instructions provided by the manufacturer. Northern blot analysis was performed as previously described.¹⁷

Preparation of Protein Extracts
Protein extracts were prepared as previously described.¹⁹

Western Blot Analysis
Western blot analysis was carried out as previously described.¹⁹ Primary antibodies were used at a dilution of 1:100.

cdk2 Kinase Assays
The assays were performed as previously described¹⁹ ; 75 µg of each protein extract was used for the assays.

Measurement of [³H]thymidine Incorporation
[³H]thymidine incorporation was measured as previously described.¹⁹

Statistical Analyses
Values are mean±SEM. The effects of adenoviral infection on expression of cyclins/cdks/cdk inhibitors, cdk2 kinase activities, and [³H]thymidine uptake were assessed using ANOVA followed by the Student-Newman-Keuls test.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
GATA-6 Is Expressed in Cultured GMCs
We first examined whether GATA-6 was expressed in rat GMCs by Northern blot analysis using a probe corresponding to the zinc finger domain of human GATA-6. A single transcript was detected in GMCs, and the size of the transcript was identical with that observed for GATA-6 in VSMCs (Figure 1). The accumulation of GATA-6 transcripts was downregulated in quiescent GMCs by stimulation with serum mitogens, and the suppression was maximal at 8 hours after stimulation (19% expression compared with the expression level at 0 hours). The GATA-6 expression returned to the basal level 20 hours after stimulation.

View larger version (47K): [in this window] [in a new window]   Figure 1. Expression of GATA-6 transcript in GMCs. GMCs and VSMCs were serum-starved, and GMCs were stimulated with serum mitogen for the indicated periods. Poly(A) RNA (4 µg) extracted from GMCs and VSMCs was loaded in each lane. The membrane was hybridized with the human GATA-6 cDNA probe and then reprobed with the GAPDH cDNA probe.

GATA-6 Induces Growth Arrest in GMCs
To examine whether overexpression of GATA-6 induced growth arrest in GMCs, GMCs were infected with a recombinant Ad expressing HA-tagged human GATA-6 (Ad GATA-6). A recombinant Ad expressing GFP (Ad GFP) was used as the negative control. In GMCs treated with Ad GFP or PBS, stimulation with serum mitogen increased [³H]thymidine incorporation in a time-dependent manner (Figure 2A). Infection with Ad GATA-6, infected at an MOI of 50, completely suppressed a serum mitogen-induced increase in [³H]thymidine uptake to the basal level (P<0.01) (Figure 2A). We also examined the dose dependence of Ad GATA-6–induced inhibition of [³H]thymidine uptake. Ad GATA-6, infected at an MOI varying from 0 to 100, inhibited serum mitogen-induced increase in [³H]thymidine uptake in a dose-dependent manner (Figure 2B). Because the effect of Ad GATA-6 on cell growth reached a plateau at 50 MOI, all subsequent experiments were performed at 50 MOI unless otherwise specified.

View larger version (23K): [in this window] [in a new window]   Figure 2. GATA-6 overexpression inhibits [3H]thymidine uptake in GMCs. A, GATA-6 inhibits [3H]thymidine uptake in a time-dependent manner. GMCs were infected with 50 MOI of Ad GATA-6 (•) or Ad GFP (), or were treated with PBS () in low-serum medium. GMCs were then stimulated with serum mitogen for the indicated periods. Values are mean±SEM (n=6). *P<0.001 vs Ad GATA-6 infection. B, GATA-6 inhibits [3H]thymidine uptake in a dose-dependent manner. GMCs were infected with indicated MOI of Ad GATA-6 (solid columns) or Ad GFP (open columns) in low-serum medium. GMCs were then stimulated with serum mitogen for 16 hours. Values are mean±SEM (n=6). *P<0.01 and **P<0.001 vs Ad GFP infection at each MOI.

GATA-6–Induced Changes in the Expression of the Cell Cycle–Regulatory Factors
We next examined the effects of Ad GATA-6 infection on the protein expression levels of the cell cycle–regulatory factors in GMCs (Figure 3). Expression of HA-tagged GATA-6 protein in Ad GATA-6–infected GMCs was confirmed by Western blot analysis using anti-HA antibody. Expression of cyclin D1 was induced by serum mitogen in a time-dependent manner, and Ad GATA-6 infection did not affect the induction, suggesting that the early G₁ phase progressed normally in Ad GATA-6–infected cells. In contrast, time-dependent induction of cyclin A expression by serum mitogen was substantially inhibited by Ad GATA-6 infection. Consistent with previous observations,²⁰ expression of p21^cip1 was induced by serum mitogen in a time-dependent fashion. However, expression of p21^cip1 was significantly higher in cells infected with Ad GATA-6 compared with those infected with Ad GFP at each time point examined (Ad GFP:Ad GATA-6=1:3.3±0.9, 8 hours after stimulation [n=4, P<0.05]; 1:3.7±0.3, 16 hours after stimulation [n=4, P<0.01]). Expression of cyclin E, cdk2, cdk4, and p27^kip1 did not change remarkably during the time course, and Ad GATA-6 infection did not affect their expression. We next examined whether amounts of cyclin A and p21^cip1 associated with cdk2 were indeed changed. Protein extracts prepared from cells infected with Ad GATA-6 or Ad GFP were immunoprecipitated with anti-cdk2 antibody and immunoblotted with anti–cyclin A antibody or anti-p21^cip1 antibody. As shown in Figure 4, the amount of cdk2-associated cyclin A was lower in Ad GATA-6–infected cells compared with that in Ad GFP–infected cells (63% of control level), whereas the amount of cdk2-associated p21^cip1 was higher in Ad GATA-6–infected cells (340% of control level).

View larger version (43K): [in this window] [in a new window]   Figure 3. GATA-6–induced changes in the expression of the cell cycle–regulatory factors. GMCs were infected with 50 MOI of Ad GATA-6 (GATA6) or Ad GFP (GFP) in low-serum medium. GMCs were restimulated with serum mitogen for the indicated periods. Each protein extract (50 µg) was immunoblotted with indicated antibodies. The same protein extracts were used for all immunoblot experiments.

View larger version (19K): [in this window] [in a new window]   Figure 4. GATA-6–induced changes in p21cip1 and cyclin A association with cdk2. GMCs were infected with 50 MOI of Ad GATA-6 (GATA-6) or Ad GFP (GFP) in low-serum medium and then restimulated with serum mitogen for 16 hours. Each protein extract (200 µg) was immunoprecipitated (IP) with anti-cdk2 antibody and immunoblotted (Blot) with anti-p21cip1 antibody or anti-cyclin A antibody. Each protein extract (50 µg) was immunoprecipitated and immunoblotted with anti-cdk2 antibody as the internal control. The experiment was repeated twice, and the same result was obtained.

Overexpression of GATA-6 Inhibits cdk2 Kinase Activity
We examined whether Ad GATA-6 infection inhibited cdk2 kinase activity. Anti-cdk2 antibody was used to immunoprecipitate total cdk2, and kinase activity was measured using histone H1 as the substrate. Serum mitogen induced cdk2 kinase activity in a time-dependent manner in control cells infected with Ad GFP. In contrast, a serum mitogen–induced increase in cdk2 kinase activity was inhibited to 41% (16 hours after stimulation [n=4, P<0.01]) in Ad GATA-6–infected cells compared with that in Ad GFP–infected cells (Figure 5). We also examined cyclin A–associated cdk2 kinase activity and found that it was inhibited in Ad GATA-6–infected cells (see online Figure 1A; online-only data supplement available at http://www.circresaha.org). Furthermore, we found that Ad GATA-6–infected cells contained heat-stable inhibitors of cdk2 activity that could be largely immunodepleted with anti-p21^cip1 antibody (see online Figure 1B; online-only data supplement available at http://www.circresaha.org).

View larger version (30K): [in this window] [in a new window]   Figure 5. GATA-6 inhibits cdk2 kinase activity in GMCs. GMCs were infected with 50 MOI of Ad GATA-6 (GATA6) or Ad GFP (GFP) in low-serum medium and then restimulated with serum mitogen for the indicated periods. Each protein extract (50 µg) was used for cdk2 kinase assays. The same amount of each protein extract was immunoprecipitated and immunoblotted (Blot) with anti-cdk2 antibody as the internal control.

Overexpression of GATA-6 Inhibits Accumulation of Cyclin A Transcripts and Stabilizes p21^cip1 Protein
We next examined whether GATA-6–induced increase of p21^cip1 expression and decrease of cyclin A expression occurred at the level of mRNA or protein. We performed Northern blot analysis using full-length human p21^cip1 cDNA and human cyclin A cDNA as probes. A single cyclin A transcript was detected in GMCs. The accumulation of cyclin A transcripts increased 20 hours after stimulation with serum mitogen in Ad GFP–infected cells and remained increased until 32 hours after stimulation, whereas the accumulation of cyclin A transcripts was reduced significantly in Ad GATA-6–infected cells (28±6% compared with Ad GFP infection, 24 hours after stimulation [n=4, P<0.05]) (Figure 6A). In contrast, GMCs appeared to express low levels of p21^cip1 transcripts, because p21^cip1 transcripts were not detected when total RNA was used for the analysis (data not shown). We therefore used poly(A) RNA extracted from GMCs that were stimulated with serum mitogen for 20 hours. In these samples, infection with GATA-6 had little or no effect on p21^cip1 transcript levels (Figure 6B). The p21^cip1 transcript levels in Ad GATA-6–infected GMCs increased 1.2-fold at the most, compared with those in Ad GFP–infected GMCs, suggesting the post-transcriptional regulation of p21^cip1 expression. We therefore examined the stability of p21^cip1 protein in Ad GATA-6–infected cells (Figure 6C). After induction of p21^cip1 protein by stimulation with serum mitogen for 16 hours, 10 µg/mL of cycloheximide (CHX) was added to the medium, and the time course of the expression of p21^cip1 protein was examined. The expression of p21^cip1 in Ad GFP–infected cells decreased rapidly (30% of control level, 30 minutes after incubation with CHX), whereas that in Ad GATA-6–infected cells was relatively stable (85% of control level, 30 minutes after incubation with CHX), suggesting stabilization of p21^cip1 protein in Ad GATA-6–infected cells.

View larger version (54K): [in this window] [in a new window]   Figure 6. Effects of GATA-6 infection on cyclin A and p21cip1 transcript levels and stability of p21cip1 protein. A, Effects of GATA-6 infection on the accumulation of cyclin A transcripts. GMCs were infected with 50 MOI of Ad GATA-6 (GATA6) or Ad GFP (GFP) in low-serum medium and then restimulated with serum mitogen for the indicated periods. Total RNA (20 µg) was loaded in each lane. The membrane was hybridized with the full-length human cyclin A cDNA probe and then reprobed with the GAPDH cDNA probe. B, Effects of GATA-6 infection on the accumulation of p21cip1 transcripts. Ad-infected GMCs were restimulated with serum mitogen for 20 hours. Poly(A) RNA (4 µg) was loaded in each lane. The membrane was hybridized with the full-length human p21cip1 cDNA probe and then reprobed with the GAPDH probe. RNA extracted from HepG2 cells was used as positive control in both experiments. C, Effects of GATA-6 infection on the stability of p21cip1 protein. GMCs were infected with 50 MOI of Ad GATA-6 (GATA6) or Ad GFP (GFP) in low-serum medium. After restimulation with serum mitogen for 16 hours, 10 µg/mL of CHX was added in the medium and protein extracts were prepared at the indicated time point. Western blot analysis was performed using 50 µg of each protein extract.

GATA-6–Induced Inhibition of Cell Cycle Progression and the Accumulation of Cyclin A Transcripts Depend on p21^cip1 Expression
We finally examined whether GATA-6–induced inhibition of cell cycle progression and cyclin A gene expression depended on the presence of p21^cip1. GMCs obtained from wild-type and p21^cip1–/– mice were infected with Ad GATA-6 or Ad GFP, and [³H]thymidine uptake was examined 16 hours after stimulation of quiescent GMCs with serum mitogen. Forced expression of GATA-6 significantly inhibited [³H]thymidine uptake in GMCs obtained from wild-type mice, whereas GATA-6 overexpression did not significantly inhibit [³H]thymidine uptake in GMCs obtained from p21^cip1–/– mice (Figure 7A). In accordance with the results, the expression of cyclin A transcripts in wild-type GMCs was inhibited by GATA-6 overexpression (55% expression compared with Ad GFP infection, 24 hours after stimulation), whereas that in p21^cip1–/– GMCs was not remarkably inhibited by GATA-6 overexpression (93% expression compared with Ad GFP infection, 24 hours after stimulation) (Figure 7B), suggesting that GATA-6–induced inhibition of the cyclin A expression depended on p21^cip1 expression.

View larger version (40K): [in this window] [in a new window]   Figure 7. Effects of GATA-6 infection on [3H]thymidine uptake and level of cyclin A transcripts in p21cip1-/- GMCs. A, GMCs derived from wild-type and p21cip1-/- mice were infected with 15 MOI of Ad GATA-6 (GATA6) or Ad GFP (GFP) in low-serum medium. GMCs were then restimulated with serum mitogen for 16 hours. [3H]thymidine was added during the last 2 hours of incubation periods. *P<0.001 vs Ad GFP infection. B, GMCs were infected with Ad GATA-6 or Ad GFP in the same way as in panel A. GMCs were stimulated with serum mitogen for the indicated periods, and total RNA was extracted for Northern blot analysis. Total RNA (20 µg) was loaded in each lane. The membrane was hybridized with the full-length human cyclin A cDNA probe and then reprobed with the GAPDH cDNA probe.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
GMCs share characteristics with VSMCs, and abnormal growth of GMCs has been implicated in the pathogenesis of glomerulonephritis. In this study, we first confirmed that GATA-6 was an endogenous gene expressed in GMCs and then examined the effects of GATA-6 overexpression on the growth of GMCs using a recombinant Ad that expressed human GATA-6.

Several data presented in this study suggested that upregulation of p21^cip1 was implicated in growth arrest induced by GATA-6 overexpression in GMCs. We have shown previously that GATA-6 induced growth arrest in VSMCs as well as in fibroblasts and that it was associated with p21^cip1 upregulation.¹¹ Thus, upregulation of p21^cip1 appears to be a general mechanism by which GATA-6 induces cell cycle arrest. Several factors, including MyoD, CCAAT/enhancer binding protein , vitamin D₃ receptor, Gax, and the tumor suppressor BRCA1 reportedly induced p21^cip1 expression, and the induction of p21^cip1 correlates with growth arrest or differentiation.^{21 22 23 24 25} Most of these factors induce p21^cip1 upregulation by stimulating transcription of the p21^cip1 gene. In contrast, transcriptional regulation did not appear to play a major role in GATA-6–induced upregulation of p21^cip1, because the accumulation of p21^cip1 transcripts did not change remarkably compared with the changes in the amounts of p21^cip1 protein. Furthermore, p21^cip1 protein was stabilized in GMCs infected with GATA-6. These data suggest that the induction of p21^cip1 by GATA-6 is largely regulated at a post-transcriptional level. Along these lines, it is noteworthy that CCAAT/enhancer binding protein stabilizes p21^cip1 protein.²²

We have also demonstrated, for the first time, that expression of cyclin A, but not of cyclin D1 or cyclin E, was specifically inhibited by GATA-6 infection. In contrast with p21^cip1, the level of cyclin A transcripts was remarkably influenced by GATA-6 infection. The serum mitogen–induced accumulation of cyclin A transcripts was significantly inhibited by infection with Ad GATA-6, suggesting that the suppression of cyclin A occurred at the transcriptional level. However, because it was possible that GATA-6–induced inhibition of cyclin A expression might be related to cell cycle arrest induced by p21^cip1 upregulation, we examined cyclin A expression in the absence of p21^cip1. Our results demonstrated that GATA-6 overexpression did not inhibit cell growth or the accumulation of cyclin A transcripts in p21^cip1–/– GMCs, suggesting that GATA-6–induced suppression of cyclin A expression depended on p21^cip1 expression. The results also suggested that GATA-6–induced inhibition of cyclin A expression might be a secondary effect of cell cycle arrest induced by p21^cip1 upregulation rather than a direct effect of GATA-6 on the cyclin A expression (see online-only supplementary information; data supplement available at http://www.circresaha.org). However, it should be emphasized that GATA-6–induced suppression of cyclin A expression appears to be a specific effect of GATA-6 rather than a nonspecific result of cell cycle arrest, because expression levels of other cell cycle–regulatory factors were not affected by GATA-6 overexpression.

Several studies have shown that it is possible to modulate the proliferation of GMCs by modifying the cell cycle–regulatory genes.^{26 27 28} Here, we have shown that GATA-6 is an endogenous factor in GMCs that can modulate the proliferation of these cells. GATA-6–induced growth arrest of GMCs was associated with inhibition of cyclin A expression and p21^cip1 upregulation. Therefore, modulation of GATA-6 gene expression in kidney may be a useful strategy to treat the progression of mesangial proliferative glomerulonephritis.

	Acknowledgments

 
This study was supported in part by Grants-in-Aid 09281206 and 10218202 from the Ministry of Education, Culture and Science of Japan (to Y.H.). We thank Etsuko Taira and Marie Morita for technical assistance.

Received May 15, 2000; revision received August 21, 2000; accepted August 21, 2000.

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