Okadaic acid mediates p53 hyperphosphorylation and growth arrest in cells with wild-type p53 but increases aberrant mitoses in cells with non-functional p53
Gavin J. Milczarek1,
Weixing Chen2,
Ashok Gupta2,
Jesse D. Martinez2 and
G.Tim Bowden1,2,3
1 Department of Molecular and Cellular Biology and
2 Department of Radiation Oncology, Arizona Cancer Center, The University of Arizona, 1501 North Cambell Avenue, Tucson, AZ 85724, USA
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Abstract
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The protein phosphatase inhibitor and tumor promoting agent okadaic acid (OA), has been shown previously to induce hyperphosphorylation of p53 protein, which in turn correlated with increased transactivation or apoptotic function. However, how the tumor promotion effects of OA relate to p53 tumor supressor function (or dysfunction) remain unclear. Rat embryonic fibroblasts harboring a temperature-sensitive mouse p53 transgene were treated with 50 nM doses of OA. At the wild-type permissive temperature this treatment resulted in: (i) the hyperphosphorylation of sites within tryptic peptides of the transactivation domain of p53; (ii) an increase in p53 affinity for a p21waf1 promotor oligonucleotide; (iii) an increase in cellular steady state levels of p21waf1 message; (iv) a G2/M cell cycle blockage in addition to the G1/S arrest previously associated with p53; and (v) no increased incidence of apoptosis. On the other hand, OA treatment at the mutated p53 permissive temperature resulted in a relatively high incidence of aberrant mitosis with no upregulation of p21waf1 message. These results suggest that while wild-type p53 blocks the proliferative effects of OA through p21waf1-mediated growth arrest, cells with non-functional p53 cannot arrest and suffer relatively high levels of OA-mediated aberrant mitoses.
Abbreviations: DMSO, dimethyl sulfoxide; OA, okadaic acid.
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Introduction
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Induction of the tumor suppressor activity of p53 through DNA damage or overexpression halts cell cycling through the transcriptional upregulation of the cyclin-dependent kinase inhibitor p21waf1 (1; reviewed in ref. 2). The fact that >50% of human cancers contain inactivated p53 (3) has led to the investigation of how wild-type p53 activity is modulated. p53 is known to undergo post-translational modification via phosphate addition or removal by many different enzymes, including casein kinase I and II, dsDNA-dependent protein kinase, Jun kinase I, mitogen activated protein kinase, cyclin dependent kinase II, protein kinase C and protein phosphatase types 1 and 2a (reviewed in refs 4,5). Accordingly, many studies have focused on phosphorylation and the regulation of p53-mediated function.
One approach to the question of phosphorylation and p53 function involves the use of okadaic acid (OA). A tumor-promoting polyether compound produced by a dinoflagellate and isolated from black sponge, OA is a potent inhibitor of types 1, 2a and 3 serine/threonine protein phosphatases (reviewed in ref. 6). The OA pathway (i.e. protein phosphatase inhibition) is now recognized as a general biochemical process of tumor promotion in several different organs, including skin, liver and stomach (6). Presumably through its effects on phosphorylation, OA is known to have regulatory effects on many gene products ranging from transcription factors to matrix metalloproteases (reviewed in ref. 7). Since OA treatment originally was shown to result in hyperphosphorylation of p53 from primary human fibroblasts, hyperphosphorylation was presumed to inactivate p53 as a means of tumor promotion (8). However, OA treatment now is generally thought to upregulate the tumor suppressor activities of p53 through hyperphosphorylation in both the transcriptional transactivation (serines 4, 6 and 15) and DNA binding domains (serines 313, 390 and possibly 309) (9,10).
Many experiments have shown OA to have differing biochemical and functional effects on p53. For example, at the level of transcription of p53, OA treatment appeared to decrease p53 mRNA levels (11). On the other hand, p53 protein has been reported to be stabilized by OA treatment in some cell types (12) but unaffected in others (10). In terms of DNA binding, p53 hyperphosphorylation mediated by OA treatment shows increased affinity for both the ribosomal gene cluster (RGC) and p53 consensus sequence (p53Con) oligonucleotides (9,12,13). Yet p53 transcriptional transactivation, as measured through the activities of RGC and p53Con luciferase constructs, is decreased by exposure to OA (12). However, in other cell types, OA treatment can lead to increased transactivation of mdm2, p21waf1 and bax promoter constructs (10). Interestingly, in terms of programmed cell death, it has recently been shown that OA-induced, p53-dependent apoptosis correlated with increased overall (sites unknown) levels of p53 phosphorylation, but did not require p53-mediated transactivation (14). While these experiments have revealed many insights into the functional role of p53 phosphorylation, investigation of cellular mechanisms through which OA mediates its effects may help to refine our understanding of the `phosphoregulation' of p53 and its role in pathways of tumor promotion with OA.
Our approach has consisted of comparing the biochemical and functional consequences of OA on a rat embryonic fibroblast cell line harboring a constitutively expressed mouse transgene (p53val135). When A1-5 cells are incubated at 39°C, ~75% of p53val135 assumes a mutant conformation that remains in the cytoplasm and, consequently, does not arrest cell growth. However, by 3 h after being shifted to 32°C, the wild-type p53 conformation and function predominate, leading to increased nuclear localization and, ultimately, cell cycle arrest in G1/S (15). Coincident with this shift to wild-type function, p53 phosphorylation and steady state levels of p21waf1 both show an endogenous `background' increase (i.e. in the absence of OA) at 3 h. However, by 6 h after incubation at 32°C, p53 returns to basal levels of phosphorylation (16). We asked how treatment with OA could affect the in vivo phosphorylation and function of p53 in A1-5 cells containing either the wild-type or non-functional mutant p53. Our data indicate that OA treatment of cells at the wild-type temperature led to hyperphosphorylation of specific p53 phosphorylation sites and that prolonged exposure results in upregulation of p53 DNA binding, an increase in p21waf1 steady state mRNA, and both G1 and G2/M growth suppression that is not related to an increase in apoptosis. In contrast, OA treatment of cells with non-functional p53 did not upregulate p21waf1 and led to relatively high levels of aberrant mitosis.
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Materials and methods
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Cell lines and chemicals
The A1-5 rat fibroblast cell line with the temperature-sensitive p53 transgene and activated ras (17) were used in all experiments. These cells were maintained and re-fed just prior to each experiment with DMEM (+10% bovine calf serum and 5% of a 10 000 U streptomycin/penicillin solution) in 5% CO2 atmosphere at 39°C. For all experiments, cells were plated in 100 mm culture dishes and grown to between 70 and 85% confluence. Time zero corresponded both to a shift to the temperature at which p53 assumes the wild-type conformation (32°C) and to treatment with 50 nM OA in 0.5% dimethyl sufoxide (DMSO) or an equal volume of solvent only. This dose was chosen based on minimal toxicity as shown by Herschman et al. (18).
Phosphopeptide mapping
32P labeling, lysis and immunoprecipitation were performed as previously described (16). Immunoprecipitates were then subjected to SDSPAGE (10% acrylamide gel), transferred to Immobilon-P, digested with trypsin and prepared for electrophoresis as described previously (19). Thin layer cellulose electrophoresis and chromatography were performed as described previously (20) using pH 1.9 and phospho-chromatography buffers. Thin layer chromatography plates were then allowed to air dry for 3 h, wrapped in plastic film and exposed for 2 days on a Molecular Dynamics (Sunnyvale, CA) Phosphorimager cassette.
DNA binding assays
Nuclear extracts were prepared according to published procedures (21). Oligonucleotides used to test p53 for p21waf1 promoter binding included wild-type p21waf1 promoter sequence 5'-GAACATGTCCCAACATGTTG-3'; 3'-CTTGTACAGGGTTGTACAAC-5' and a scrambled wild-type p21waf1 negative control sequence 5'-TATGCCAATGTGTACCGAAC-3'; 3'-ATACGGTTACACATGGCTTG-5'. Twenty micrograms of nuclear extract were incubated with 10 ng 32P-labeled oligonucleotide, and 2 µg poly d(IC) in a 25 µl reaction mixture that contained 72 mM KCl, 13% glycerol, 10.6 mM HEPES (pH 7.9), 0.1 mM EDTA and 0.8 mM MgCl2. Reactions were incubated at 15°C for 1 h, loaded onto a 1x Trisborate, 5% polyacrylamide gel containing 2.5% glycerol (cooled to 4°C and pre-electrophoresed at 200 V for 1 h) and electrophoresed at 4°C and 200 V for 2.5 h. The gels were then dried and exposed overnight on a Phosphorimager cassette.
Western analysis and antibodies
Steady state p53 protein levels were measured by western analysis as described previously (21). Primary immunoglobins included the p53 PAb421 (22) or RA3 2C2 (23) murine `pan-specific' antibodies. Goat anti-mouse secondary antibodies conjugated with horseradish peroxidase (Pierce, Rockford, IL) and ECL detection reagents (Amersham, Piscataway, NJ) then were used to visualize proteins upon exposure to Kodak XAD film.
Northern analysis
RNA was isolated by the single-step guanidinium isothiocyanatephenol method as described previously (24). Ten micrograms of RNA were used for northern hybridization as described previously (21).
Flow cytometry analysis
Cells were washed once in 37°C PBS, harvested with trypsin, and pelleted at 1500 r.p.m.. After aspiration of the trypsin, the cell pellet was dispersed in 1 ml FACS buffer per 1.2x106 cells (FACS buffer: 0.1% sodium citrate, 0.2% NP-40, 0.02 mg/ml RNase, 50 µg/ml propidium iodide) and vigorously pipetted 10 times with a Ranin P1000 pipette. Stained nuclei were then stored up to 24 h on ice in the dark until acquisition with a Becton Dickinson (Franklin Lakes, NJ) FACScan and analysis with CellFIT v2.01 software.
Cytospin analysis and imaging
Al-5 cells were incubated at the appropriate temperature with or without OA for 24 h, after which detached cells were collected and set aside. Attached cells were then trypsinized and pooled with an equal number of detached cells so as to obtain a total of ~100000 cells/ml as determined by hemocytometer counts. Next, 0.1 ml of the pooled cells was loaded into the cytospin apparatus and attached to glass slides for 2 min at 600 r.p.m. with low acceleration. Upon completing the cytospin procedure, cells were air dried, fixed in 100% methanol for 5 min, stained for 7 min in freshly prepared Giemsa dye, rinsed in distilled water, air dried again and digitally acquired and processed using Image-ProPlus v3.0 software.
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Results
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N-terminal p53 peptides remain phosphorylated through OA treatment
Phosphopeptide analysis of p53 in OA-treated and solvent (DMSO)-only control treated cells was performed to determine which tryptic peptide phosphorylation sites might be associated with the regulation of wild-type p53 function in this cell system. As shown in Figure 1
, wild-type p53 (32°C) from the 6 h OA-treated samples was strongly phosphorylated at N-terminal peptides (spots 5, 5a, 5b and 6) compared with only one N-terminal peptide (spot 6) in the 6 h DMSO-only sample. However, in cells with mutant p53 (39°C + OA), no N-terminal peptides appeared to be phosphorylated. Similarly, untreated cells at the mutant permissive temperature also displayed no p53 phosphorylation at the N-terminus (data not shown).

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Fig. 1. Peptide map showing the N-terminal p53 species hyperphosphorylated by OA. 32P-labeled p53 was immunoprecipitated, gel purified and subjected to phosphopeptide mapping by electrophoresis and thin layer chromatography. All three thin layer chromatography plates were loaded with 500 c.p.m. 32P-labelled p53 and exposed for 24 h. The peptide numbering system is based on Fuchs et al. (9), in which 2 = serine 312; 3 = serine 389; 4 = serines 312 and 389; 5 = serine 7; 5a = serine 9; 5b = serine 12; 6 = unknown phosphorylation site in the N-terminus. Ori, origin.
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Since in some cases, OA treatment has been known to affect steady state levels of p53 message or protein (11,12), western analyses were performed on 10 µg of total protein from A1-5 cells. In three separate experiments using varied film exposure times, we observed no change in the steady state protein levels of OA-treated samples relative to solvent-only controls at 1, 3, 6, 12 or 24 h intervals (data not shown). Therefore, we concluded that upregulation of p53 protein levels could not account for the increase of p53 phosphorylation or function observed in the experiments that follow.
Because the phosphopeptide mapping showed an OA-related retention of phosphorylation in contrast to the usual endogenous decline (16), we next asked whether p53 DNA binding and transactivation might increase as N-terminal phosphorylation correlates with both of these functions (10).
OA treatment prolongs p53 binding to a p21waf1 promoter oligonucleotide
To assess the effect of OA on the specific DNA binding activity of p53, we performed electrophoretic mobility gel shift assays. Under conditions in which the A1-5 cells are not treated with OA, p53 is known to increase its binding activity to a peak at ~3 h after shifting from the mutant to the wild-type permissive temperature. However, this binding activity then begins to decrease and becomes undetectable by 6 h (16). Using an oligonucleotide harboring either a wild-type or scrambled wild-type p53 consensus binding sequence from the p21waf1 promoter, we observed an increase in DNA binding activity over the endogenous background levels up to and including the 6 h time point (Figure 2
). This binding activity was specific since it could be competed away with non-radioactive (cold) wild-type, but not scrambled, oligonucleotide and with a p53-specific monoclonal antibody (PAb421), as opposed to an antibody to T antigen of the same idiotype (PAb419).

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Fig. 2. OA treatment prolongs p53 binding to a p21waf1 promoter oligonucleotide. Al-5 nuclear extracts were assayed for DNA binding using either a wild-type or scrambled p53 binding sequence from the p21waf1 promoter. n.s., non-specific; po, probe only; wt, cold wild-type sequence; mut, hot scrambled sequence; 419, PAb419 antibody to T antigen; 421, PAb421 antibody to p53.
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p53 target gene p21waf1 steady state mRNA levels increase following OA treatment
Since the p21waf1 promoter DNA binding activity of p53 increased with OA treatment, we investigated whether p21waf1 transcript levels also rose. As shown in Figure 3,
a northern analysis representing the results from four independent experiments revealed statistically significant increases (Table I) in transcript levels over background controls at 3 and especially 6 h. After correction for loading, this increase reproducibly occurred in a wild-type p53-specific manner as only basal levels of p21waf1 message were observed in the mutant containing `0 h' cells with or without OA treatment.

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Fig. 3. p53 target gene p21waf1 steady state mRNA levels increase with OA treatment. RNA was isolated from the A1-5 cell line and denatured for northern analysis with a p21waf1 probe. Shown is a representative experiment from four independent tests, which all indicated an OA-associated increase in p21waf1 message of between 1.5- and 3-fold at the 3 and 6 h time points, respectively. The bottom band depicts the loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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OA leads to G2/M cell cycle arrest that is associated with wild-type p53
A1-5 cells treated with OA for up to 24 h were analyzed by flow cytometry to determine if the upregulation of p53 transactivation activity was associated with a change in cell cycle distribution (Figure 4). While the S population showed a decline in both the OA treated and untreated 24 h samples at 32°C (which is indicative of G1/S restriction normally seen when A1-5 cells are shifted to the wild-type p53 temperature), cells receiving OA were reproducibly blocked in G2/M as well. Despite the fact that by 24 h both wild-type and mutant containing cell lines underwent a change to a more rounded, `mitotic-looking' morphology (no detachment was evident), G2/M blockage appeared to be wild-type specific as both the untreated (not shown) and OA-treated cells at 39°C displayed little change in cell cycle distribution over a 24 h period.
OA treatment does not increase the level of apoptosis in wild-type p53 cells, but does increase levels of aberrant mitoses in mutant p53 cells
To determine if the G2/M build up we observed following our flow cytometry experiments was due in part to apoptosis, cells were treated as in previous experiments and subjected to cytospin collection and Giemsa staining for fragmented nuclei. As indicated in Table II, OA treated wild-type p53 cell samples scored no higher than untreated cells in terms of apoptotic bodies. However, mutant p53 cells treated with OA showed a higher incidence of `aberrant mitosis' as inferred through uniformly stained, randomly distributed chromosomes within their nuclei, as seen in Figure 5
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Fig 5. OA treatment does not increase the level of apoptosis in wild-type p53 cells, but does increase levels of aberrant mitoses in mutant p53 cells. The arrows indicate cells undergoing aberrant mitosis.
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Discussion
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Despite the fact that many aspects of p53 phosphorylation have become better understood in recent years, a clear picture of how OA-induced phosphorylation affects p53 function has yet to emerge. Combining the use of OA with the A1-5 cell line offers some unique advantages. First, experiments can be done without the complications resulting from DNA damage-induced p53. Secondly, unlike in vitro or insect cell systems, differences in p53 function resulting from OA treatment can be assayed in a previously characterized, intact mammalian cell environment that may model basic mechanisms of tumor promotion found in vivo. For example, OA treatment leads to a highly phosphorylated transactivation domain of p53 expressed in A1-5 cells in a manner analogous to the N-terminal phosphorylation seen when p53 activity and expression is increased by DNA damage commonly associated with tumor cells (25). Nonetheless, data gleaned from the use of A1-5 cells should be interpreted cautiously given the complexity of OA's effects on multiple pathways and proteins as discussed in more detail below.
Building upon several other recent studies supporting the functional importance of p53 N-terminal phosphorylation at multiple sites (10,13,26,27), our data suggest a physiological pathway through which phosphorylation could upregulate wild-type p53-mediated growth suppression in vivo. This pathway would involve specifically N-terminal phosphorylation of p53, the upregulation of p53 DNA binding to the p21waf1 promoter, and higher steady-state levels of p21waf1 mRNA. Since it has been noted previously that p21waf1 can inhibit many different cyclincyclin-dependent kinase complexes (28) and that p53 can mediate both G2/M and G1 cell cycle blockage in cell lines containing p53val135 (15,29), we decided to utilize flow cytometry to link p53 phosphorylation and elevated levels of p21waf1 with a functional end point, growth suppression. Although OA is known to have pleiotropic cellular affects, the G2/M cell cycle blockage resulting from OA treatment appears to be wild-type p53 dependent as no such block takes place in the mutant p53 bearing cells, which continue to grow but show comparatively high incidences of aberrant mitoses and apoptosis.
One obvious implication of these data is the association of one or more endogenous protein phosphatase activities with the downregulation of p53. Mechanistically, protein phosphatase type 1 has a crucial role in cell cycle progression and is required for mitotic exit in yeast, fruit flies and mammalian systems (30). Similarly, protein phosphatase type 2A is thought to be an important regulator of cell cycle progression in M and G1/S phases (7). Functionally, OA inhibitable serine/threonine protein phosphatases may play a role in the regulation of p53 activity as they do for another tumor suppressor, the retinoblastoma protein (Rb) (31,32). In this case, Rb is known to be activated by dephosphorylation through protein phosphatase type 1 so as to delay the onset of S-phase through the binding of the E2F transcription factor (30,31). Connections between protein phosphatases and tumor suppression also exist. For example, it has been shown that the tyrosine protein phosphatase, LAR, is associated with tumor suppression activity (33). With these examples in mind, it is of interest to note that as yet undetermined sites within the N-terminus of p53 can be dephosphorylated by protein phosphatase 2A in vitro (34). Therefore, elucidating the physiological significance of dephosphorylation of particular amino acids is likely to increase our understanding of p53 regulation.
Another implication of these data revolves around the question of the role of p53 in the OA tumor promotion pathway. OA is now recognized as a general tumor promoter in various murine organs, including skin, glandular stomach and liver (35), but not much is known at the present time about its relevance to human cancers. This is despite the fact that OA is known to be a causative agent of diarrhetic shellfish poisoning, and microcystin, an OA-class compound, is common in ditch and other waste water (6).
Since p53 first was shown to be hyperphosphorylated through OA treatment (8), it has been presumed that this would lead to functional inactivation. However, the data presented herein and by others argue that the opposite may in fact occur, i.e. hyperphosphorylation wrought by OA may upregulate p53 function. Yet, exactly which function or functions it upregulates appears to be complex. As recent experiments have demonstrated, depending on cell lineage and on other factors, some injured cells undergo cell cycle arrest while others undergo apoptosis upon expression of wild-type p53 (36). Moreover, upregulation of either function would, on the surface, seem to be at odds with the tumor promotion activity of OA.
One possible point of reconciliation could involve an `indirect apoptotic mechanism' of OA mediated tumor promotion as was suggested by the recent work of Yan et al. (14). In other words, if OA treatment were to activate a p53-dependent apoptotic response, then the outgrowth of cells harboring dysfunctional p53 would be selectively fostered. Accordingly, cells with inactive p53 may be encouraged to divide aberrantly through the pleiotropic effects of OA on cell growth [e.g. the upregulation of the transcription factor AP-1 (7)], leading to the increased genetic instability characteristic of aneuoploid cancer cells (37). This instability in turn may be compensated for by a non-p53 dependent apoptotic pathway. Our data showing an increase of aberrant mitoses and apoptosis in cells with non-functional p53 would seem to support this model. For, as we have demonstrated, OA treated cells with wild-type p53 undergo some aberrant mitoses, but the vast majority arrest in either G1 or G2. In contrast, OA-treated cells with non-functional p53 do not appear to check growth, resulting in much higher levels of aberrant mitoses. However, these cells also appear to undergo a p53 independent pathway of apoptosis. Although complex, this observation is in accordance with experiments showing that apoptosis in some cells may not involve p53 (36), or alternatively, that overexpression of mutant p53 and/or bcl-2 proteins may render some cells resistant to apoptosis altogether (38).
Taken together, these observations suggest that OA may be selecting for cells that have defects in both the ability to undergo growth arrest and apoptosis while upregulating certain growth inducing genes. Experiments designed to determine the levels of gene transcripts associated with cell growth, combined with the introduction of either p53-dependent or -independent cell death expression vectors into OA treated cells harboring mutant p53, will help to distinguish between these possibilities.
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Table 2. Incidence of apoptotic, aberrant mitotic or normal looking cells with either wild-type or mutant p53 after cytospin processing and Giemsa staining
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Fig.4. OA leads to cell cycle blockage in G2/M specifically in cells containing wild-type p53. Cell nuclei were stained with propidium iodide and profiled for DNA content by flow cytometery as described in Materials and methods.
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Acknowledgments
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The authors express their gratitude for the gifts of OA from Hirota Fujiki, Research Institute Director, Saitama Cancer Center, Japan. This work was support by NIH grants CA40584 awarded to G.T.B. and CA64842 awarded to J.D.M., a Cancer Center Core Grant CA23074 and a Toxicology Center Grant NIEHS ES06694.
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Notes
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3 To whom correspondence should be addressed Email: bowdenlab{at}azcc.arizona.edu 
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Received February 3, 1998;
revised February 17, 1999;
accepted March 1, 1999.