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Laboratorio de Biología Celular y Molecular (R.A.M., A.M.M.), Millennium Institute of Fundamental and Applied Biology, Universidad Nacional Andrés Bello, Santiago, Casilla 52164, Chile; Departamento de Fisiopatología (J.C.V., C.G.), Departamento de Histología y Embriología (F.N., A.A.), Departamento de Biología Molecular (M.D.A.G.), Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Casilla 160-C, Chile; Departamento de Endocrinología (S.K., A.C., M.P., G.I.O.), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago 114-D, Chile
Address all correspondence and requests for reprints to: Rodolfo A. Medina, Ph.D., Laboratorio de Biología Celular y Molecular, Millennium Institute of Fundamental and Applied Biology, Universidad Nacional Andrés Bello, Republica 217, Piso 4, Santiago, Casilla 52164, Chile. E-mail: rmedina{at}unab.cl.
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Abstract |
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Introduction |
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and ERß) originating from separate genes and two progesterone receptors (PRs), PRA and PRB, which arise from differential promoter usage (1, 2, 3). During the follicular stage of the menstrual cycle, estrogens stimulate the growth of the ductal system, and then in the luteal phase, after ovulation, high levels of progesterone combine with lower estrogen levels to mediate further growth and differentiation in preparation for the possibility of pregnancy. Along with this endogenous presence of estrogen and progesterone, exogenous combinations of these hormones are found in oral contraceptives and in hormone replacement therapy (HRT). In women receiving estrogen replacement therapy, there is a slight increase in breast cancer; however, when progesterone is added in conjunction with estrogen (combined therapy), the incidence of breast cancer increases dramatically (4, 5). The risk of breast cancer also increases with combined hormone contraceptive pill use (3). These clinical observations, coupled with laboratory findings, indicate that when estrogen and progesterone are given as combination therapy, there is an increase in the incidence of breast cancer. As with any proliferating cells, including cancer cells, an increased energy supply is required to sustain the proliferation process. We postulate herein that the increased requirement for energy is supplied by an increase in cellular glucose uptake. Mammalian cells depend on glucose as a major substrate for energy production. Glucose is transported into the cell via a family of facilitative hexose transporters (GLUT), which are present in most cell types (6, 7, 8). Because of the ubiquitousness of these transporters, their differential expression is involved in various disease states including cancer. Tumor cells exhibit increased glycolytic activity and accumulate greater quantities of lactate, compared with normal tissues (9, 10). A possible reason for this observation is that the majority of cancers and isolated cancer cell lines overexpress the GLUT family members that are present in the respective tissue of origin under noncancerous conditions (11, 12, 13). Moreover, because of the requirement of energy to feed uncontrolled proliferation, cancer cells often express GLUTs, which under normal conditions would not be present in these tissues. This overexpression is predominantly associated with the likelihood of metastasis and hence poor patient prognosis (14).
The effects of estrogen and progesterone on GLUT protein and RNA expression, and glucose uptake, and their possible relation to breast cancer have not been previously studied. To this end, we chose to use a well-characterized human breast cancer cell line, ZR-75-1, which was isolated from an infiltrating ductal carcinoma (15). This cell line stably expresses both forms of the estrogen receptor and both isoforms of the PR and proliferates in response to estrogen, an effect which is antagonized by antiestrogen treatment as observed in breast cancer cells in the clinical setting (16).
Previous studies, dating back over 100 yr (17), have demonstrated a role for ovarian hormones in breast cancer genesis. We postulate that once a cell becomes cancerous the up-regulation of GLUTs and glucose uptake by hormone treatment provides a selective advantage to the cancer cell, supplying additional energy to the burgeoning tumor. In support of this theory, we present data that shows that in the ZR-75 cell line, which expresses the GLUT1–4 family members, estrogen, progesterone, and combined therapy differentially regulate GLUT1–4 protein and RNA expression. These changes in expression are correlated with changes in glucose transport. In line with clinical observations demonstrating an increased breast cancer incidence with combined HRT, we also show that it is the combination of estrogen (17ß-estradiol) and progesterone that causes the greatest increase in GLUT expression and glucose transport.
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Materials and Methods |
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Western blotting
Cells were harvested from 10 cm2 petri dishes in cold PBS and the pellet resuspendend in lysis buffer [0.4 M KCl; 20 mM HEPES (pH 7.4); 1 mM dithiothreitol; 20% glycerol]. After sonication on ice, the lysate was centrifuged at 14,000 x g for 20 min at 4 C to separate membrane (pellet) and cytosolic (supernatant) fractions. The crude membrane fraction was resuspended in the lysis buffer mentioned above and protein concentration determined by Bradford assay and confirmed by Ponceau S staining of the membrane after wet-blot transfer. For GLUT1–3 detection, 100 µg crude membrane extract was loaded in each lane (because of low detection levels, 200 µg was used for GLUT4), separated by PAGE in the presence of sodium dodecylsulfate, transferred to nitrocellulose membranes, and incubated overnight with anti-GLUT1–4 affinity-purified antibodies (1:1000; Alpha Diagnostic, San Antonio, TX). Goat antirabbit IgG secondary antibody coupled to hydrogen peroxidase (1:3000, Bio-Rad Laboratories, Hercules, CA) was applied for 1 h at room temperature. The reaction was developed with chemiluminescence using enhanced chemiluminescence Western blot analysis system (NEN Life Science Products, Boston, MA; Western lightning, PerkinElmer, Norwalk, CT). Semiquantitative densitometry of the bands was performed using NIH Scion Image 1.62c software package for Macintosh. Positive controls used: GLUT1, skeletal muscle; GLUT2, liver; GLUT3, spermatozoid; GLUT4, heart [neither the GLUT4-positive control (data not shown) nor the ZR-75 samples provided a clean blot].
Immunocytochemistry
Immunocytochemistry studies were done as previously described (18). Briefly, after 24 h of hormonal treatment, the cells were fixed with 4% formaldehyde (in PBS) for 30 min at room temperature. Cells were then permeabilized in PBS containing 1% BSA and 0.1% Triton X-100 for 10 min at room temperature. The cells were incubated with the anti-GLUT1–4 antibodies (1:500, Diagnostic) overnight at room temperature. Cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse IgG (1:200, Roche Molecular Biochemicals, Indianapolis, IN) for 2 h, mounted, and analyzed by fluorescence microscopy.
Flow cytometry
The method for flow cytometry is described in detail elsewhere (19). Briefly, after 24 h hormonal treatment, cells were washed in PBS and harvested in Hanks’/EDTA. The cells were spun down in a 15-ml Falcon tube and resuspended in 5% FBS containing DMEM and maintained at room temperature for 1 h. Cells were permeabilized by adding 2 ml of 70% ethanol drop-wise to the cells while vortexing. Cells were incubated on ice for 10 min. Four milliliters cold PBS were added and cells were briefly centrifuged. Cells were washed in PBS containing 2% FBS and centrifuged as before. The cells were then resuspended in PBS/2% FBS containing GLUT antibody and incubated on ice for 20 min. Cells were washed three times by the addition of 1 ml of PBS/2% FBS. Cells were resuspended in PBS/2% FBS containing antirabbit FITC-labeled secondary antibody (1:200, Immunotech, Marseille, France) and incubated on ice for a further 20 min. Cells were washed twice in PBS/2% FBS and resuspended in 0.5 ml PBS containing 1% formaldehyde. Analysis was carried out using a flow cytometer (Beckman Coulter, Fullerton, CA) with the standard argon ion laser tuned to 488 nm. The mean fluorescence intensity was obtained from the recorded data. Results are displayed graphically as overlying histograms demonstrating the shift of the mean FITC staining value in comparison with the ethanol vehicle.
RT-PCR
Total RNA was isolated from ZR-75 cells using the Chomczynski method (20). The cDNA was generated using reverse transcriptase (Superscript II, Invitrogen, Carlsbad, CA). Using previously published methods and GLUT1–4 primers (21), semiquantitative PCR reactions were performed from cDNA generated from ethanol vehicle-, estrogen- (10 nM), progesterone- (10 nM), and combined estrogen-progesterone (10 nM each)-treated samples, using Taq polymerase (Invitrogen). Cycle curves for all sets of PCR primers were performed. The number of cycles performed for each primer set was in the linear range of the curve. As an internal control primers amplifying a region of glucose-6-phosphate dehydrogenase were used.
3H-2-deoxy-D-glucose (DOG) uptake
Two types of uptake experiments were carried out. In the time-course experiment, uptake was measured over time using a constant concentration of the substrate, DOG. For the concentration dependence experiment, uptake was measured at a fixed time (30 sec) at different concentrations of DOG. The transport experiments were performed using cell monolayers growing attached to the bottom of 12-well tissue culture plates. The standard transport medium contained 15 mM HEPES buffer (pH 7.6), 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and the substrate DOG at the concentrations indicated in the figures. Monolayers were washed twice with substrate-free transport medium to eliminate all traces of culture medium and then incubated for 1 h at room temperature in the same solution to deplete the cells of intracellular glucose. Uptake experiments were initiated by replacing the medium with 0.2 ml transport medium containing 1.0 µCi DOG, followed by incubation at room temperature during the time required. Uptake was terminated by adding 2 ml ice-cold stopping solution [15 mM HEPES buffer (pH 7.6); 135 mM NaCl; 5 mM KCl; 0.8 mM MgSO4; 1.8 mM CaCl2; 0.2 mM HgCl2]. The monolayers were then rinsed twice with 2 ml stop solution and lysed in 0.2 ml lysis solution [10 mM Tris-HCl (pH 8.0); 0.2% SDS]. The samples were added to 2 ml scintillation cocktail for radioactivity determination. Statistical analysis was performed using the t test method. Statistical significance was set at P < 0.05.
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Results |
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and 2). In line with other studies (22, 23), it is the GLUT1 isoform that possesses the highest levels of expression (Figs. 1 and 2). This is followed by GLUT4 and GLUT2, with GLUT3 (Figs. 1 and 2) having the lowest levels of expression.
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). In both cases, increased GLUT1 expression resulted in a marked increase in the width and intensity of the anti-GLUT1 immunoreactive band migrating at 40–50 kDa (Fig. 1A). Semiquantitative densitometric analysis of this band revealed that progesterone treatment resulted in a 3.5-fold increase in GLUT1 expression and that combined progesterone-17ß-estradiol treatment further increased GLUT1 expression to 5.4 times the untreated control (Fig. 1, A and E). The specificity of this effect was confirmed in experiments that showed that hormonal treatment had no effect on GLUT2 expression in ZR-75 cells (Fig. 1B). There was no change in the intensity of the anti-GLUT2 band from treated cells, compared with the untreated controls (Fig. 1F). The low expression levels of GLUT3 detected by Western blotting and immunocytochemistry hinder any conclusive interpretation of hormonal regulation in respect to this transporter (Fig. 1C); however, it does appear that progesterone and combines 17ß-estradiol and progesterone produce up-regulation of GLUT3. The GLUT4 Western blot shows that progesterone produces up-regulation of this transporter (Fig. 1D) (a result confirmed by immunocytochemistry; see below). However, because of the compatibility problems with this antibody in Western blotting, we were inclined to have more confidence in the immunocytochemistry results in which this antibody worked well.
Immunocytochemistry was used to confirm the results of the immunoblotting. This analysis showed increased expression of GLUT1, GLUT3, and GLUT4 (Fig. 2). Unfortunately, the GLUT2 antibody proved incompatible with immunocytochemistry. 17ß-Estradiol treatment did not result in a marked increase in anti-GLUT1 immunoreactivity (Fig. 2), confirming the results of the immunoblotting. On the other hand, there was a marked increase in the intensity of the anti-GLUT1 immunofluorescence in cells treated with progesterone (Fig. 2) and the combination 17ß-estradiol and progesterone (Fig. 2). In both cases, all cells showed increased anti-GLUT1 immunoreactivity associated with the cell’s cytoplasm and plasma membrane. Although expressed at very low levels, the immunocytochemistry studies indicated that GLUT3 expression is up-regulated by progesterone and combination treatments (Fig. 2). These studies also revealed that GLUT4 expression is increased by 17ß-estradiol, to a greater degree by progesterone and that the combination 17ß-estradiol plus progesterone produced the highest level of expression (Fig. 2).
To further confirm the above data, the cells were submitted to flow cytometry analysis to directly quantify the changes in glucose transporter expression. This analysis confirmed the immunoblotting and immunocytochemistry analyses by showing that 17ß-estradiol treatment failed to affect the shape and the position of the GLUT1 peak (Fig. 3A). On the other hand, cells treated with progesterone (Fig. 3B) or the combination of 17ß-estradiol plus progesterone (Fig. 3C) showed a shift of the GLUT1 peak to the right along the x-axis. The absence of changes in the shape of the GLUT1 peak suggests the presence of a homogeneous population of cells showing increased GLUT1 expression as a result of the hormonal treatments, which is consistent with the results of the immunofluorescence.
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). We acknowledge that the RT-PCR technique is semiquantitative; however, the fact that regulation of GLUT1 RNA is in accordance with the GLUT1 protein expression results obtained by Western blot, immunocytochemistry and flow cytometry confirms our conclusions. The RT-PCR experiments confirmed that the hormonal treatment failed to affect GLUT2 and GLUT4 mRNA levels (Fig. 4). Although minimal levels of GLUT3 protein were observed in this cell line (Figs. 1C and 2), both progesterone and combined treatment produced a highly reproducible up-regulation of GLUT3 mRNA levels (Fig. 4). No induction of GLUT1–4 mRNA levels was observed in the presence of 17ß-estradiol (Fig. 4).
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A time-course analysis of DOG uptake using substrate concentrations of 0.1, 5, and 15 mM DOG revealed that the uptake rate was linear for at least 60 sec at each tested concentration, which indicates that these rates represent real transport rates (Fig. 5). At 0.1 mM DOG, time-course analysis showed that the rate of DOG transport under control conditions was 0.08 nmol/min x 106 cells (Fig. 5A). 17ß-estradiol (DOG transport rate of 0.11 nmol/min x 106 cells) (Fig. 5A), progesterone (DOG transport rate of 0.10 nmol/min x 106 cells) (Fig. 5B), and combined 17ß-estradiol and progesterone treatments (DOG transport rate of 0.11 nmol/min x 106 cells) (Fig. 5C) all produced slight increases in the rate of DOG incorporation. Although the low expression levels of GLUT3 protein hinder the interpretation of these results, it appeared evident that the observed increase in GLUT3 mediated transport was brought about by two unrelated processes. GLUT3 protein and RNA was increased by progesterone and combined treatment and thus may account for the increase in uptake. At 5 mM DOG, time-course analysis showed that the rate of DOG incorporation under control conditions was 1.0 nmol/min x 106 cells (Fig. 5D). The rate of DOG uptake was not affected by 17ß-estradiol (1.1 nmol/min x 106 cells) (Fig. 5D) On the other hand, progesterone produced a slight increase in the rate of DOG incorporation (to 1.3 nmol/min x 106 cells) (Fig. 5E) and combined 17ß-estradiol plus progesterone produced a clear increase in the rate of DOG incorporation (to 1.6 nmol/min x 106 cells) (Fig. 5F). At 15 mM DOG, time-course analysis shows that the rate of DOG incorporation under control conditions was 2.2 nmol/min x 106 cells (Fig. 5G). 17ß-Estradiol (DOG transport rate of 2.5 nmol/min x 106 cells) (Fig. 5G), progesterone (DOG transport rate of 2.3 nmol/min x 106 cells) (Fig. 5H), or combined 17ß-estradiol and progesterone treatment (DOG transport rate of 2.4 nmol/min x 106 cells) (Fig. 5I) did not alter the rate of DOG transport. These results match the GLUT2 data obtained by immunoblotting in which we saw no changes in GLUT2 expression.
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). However, the apparent total maximal velocity (Vmax) was increased by hormonal treatment from 11 nmol/min x 106 cells in the control cells to 24 nmol/min x 106 cells in 17ß-estradiol plus progesterone-treated cells (Fig. 6A), confirming that hormone treatment increases the transport capacity of the ZR-75-1 cells.
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, B and C). This could be interpreted as indicating the presence of three different transporters, each having a different affinity for the substrate, involved in the transport of DOG in the control as well as the hormone-treated ZR-75-1 cells. Moreover, a detailed observation of the data revealed that hormone treatment differentially affected each functional component. The higher affinity component was clearly identifiable in the untreated control cells and had an apparent, uncorrected Km of 0.4 mM and a Vmax of 2.4 nmol/min x 106 cells (Fig. 6B). The transport Km is close to that expected for GLUT3. This component was less identifiable in the treated cells because of the predominance of the intermediate affinity component (Fig. 6C), confounding the determination of the kinetic parameters.
The intermediate affinity component had an apparent, uncorrected Km of 3.8 mM, and a Vmax of 4 nmol/min x 106 cells (Fig. 6B). This component showed a clear increase in transport Vmax (to 9.1 nmol/min x 106 cells) in the hormone-treated cells, without changes in the transport Km (apparent uncorrected Km of 4.1 mM) (Fig. 6C). The transport Km of the intermediate affinity component corresponded to the expected properties of GLUT1 (and GLUT4), and therefore the increased activity of this component in the hormone-treated cells was consistent with the observed up-regulation of GLUT1 protein expression detected by immunoblotting, immunofluorescence, and flow cytometry.
The lower affinity component had functional properties similar to those of the low-affinity glucose transporter GLUT2 (Fig. 6, B and C), with uncorrected apparent Kms of 21.6 and 15.9 mM for the transport of DOG in the control and the hormone-treated cells, respectively. Moreover, there was no increase in the apparent transport Vmax in the hormone-treated cells (12.2 nmol/min x 106 cells), compared with the untreated control cells (apparent transport Vmax of 11.4 nmol/min x 106 cells). The lack of a functional effect of the hormone treatment on the activity of the lower affinity component is therefore consistent with the lack of an effect on GLUT2 expression detected by immunoblotting and immunocytochemistry.
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Discussion |
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Any proliferating cell, including cancer, requires an increased energy supply to sustain the proliferation process. As we have observed, increased glucose uptake is associated with increased GLUT expression in response to progesterone and combined 17ß-estradiol and progesterone treatment. Therefore, we postulate that hormonally induced increases in glucose transport confers a survival advantage, in the form of extra energy, required by the cancer cell. This theory is in line with the clinical observation in which combined HRT increases the incidence of breast cancer (4). Indirect evidence to support our theory is provided in a study showing that differentiating (nonproliferating) breast cancer cells exhibit decreased glycolysis that is associated with a reduction in GLUT1 expression and glucose transport (13).
GLUT expression has been previously studied in other breast cancer cell lines (14, 22, 24) and biopsies (reviewed in Ref. 14). In these biopsy studies, GLUT1 was expressed in the plasma membrane and cytoplasm of all primary tumors and in lymph node metastases, with corresponding immunostaining of GLUT1 in surrounding normal mammary epithelium being either negative or expressing significantly lower levels. GLUT2 was expressed mainly in the tumor cell cytoplasm with the same intensity as normal tissue (24). These observations suggest that GLUT1 is the main transporter responsible for glucose transport in breast cancer. In the clinic, 17ß-estradiol treatment causes little effect in breast cancer incidence, but the coadministration of 17ß-estradiol and progesterone greatly increases its incidence (4). Our results show that 17ß-estradiol treatment also causes minimal effects on GLUT1 expression and GLUT1-mediated glucose transport in ZR-75 cells. Moreover, treating these cells with 17ß-estradiol and progesterone simultaneously not only causes a dramatic increase in GLUT1 expression but is also associated with a synergistic increase in GLUT1-mediated glucose transport.
If we assume that GLUT1 is the main transporter responsible for DOG uptake at 5 mM DOG, then these results match those obtained with immunoblotting, immunocytochemistry and flow cytometry. Because GLUT1 and GLUT4 have Kms in the same range, it is not possible to differentiate the individual contribution of these transporters to the uptake kinetics. However, although it is likely that both GLUT1 and GLUT4 are contributing to DOG transport in these cells, the immunoblotting and immunocytochemistry data support an important role for GLUT1 as the main glucose transporter affected by hormone treatment. Having said this, it is more than likely that the other GLUTs expressed in these cells are also contributing to glucose transport. We also showed that progesterone causes an increase in GLUT1 expression and GLUT1-mediated glucose transport. Unfortunately, data on the action of progestins alone in breast cancer patients are both very limited and contradictory. Some progestins inhibit, and others stimulate; some studies show no effect or demonstrate a dual action (25). Our data unequivocally show that progesterone treatment, on its own, causes an increase in GLUT1 expression and GLUT1-mediated glucose transport in ZR-75 breast cancer cells. This suggests that, at least in this cell line, progesterone has the potential to promote a proliferative-inducing environment.
This study raises many interesting questions. Because we observed that increased expression is associated with increased transport, we can infer that at least some of the increased GLUT protein is being expressed at the plasma membrane. Currently membrane fractionation studies are ongoing to determine whether hormonal treatments are inducing changes in GLUT distribution between the plasma membrane and cytosol. We have demonstrated changes in GLUT protein expression after 24 h, which suggests that hormonal regulation is occurring by genomic regulation, most probably through 17ß-estradiol or progesterone receptor binding to DNA. In support of this, RT-PCR studies demonstrate the increase in GLUT1 RNA content after 9 h of progesterone and combined 17ß-estradiol and progesterone treatment. Therefore, in relation to GLUT1, we see an increase in RNA levels that are mirrored at the protein level, resulting in an increased uptake in glucose transport. We postulated that this increase in GLUT1 provides an advantage to the growing cancer cell, a theory that is backed up by the observation that GLUT1 is overexpressed in cancers for the majority of 17ß-estradiol and progesterone target tissues, e.g. breast, endometrium, cervix, ovary (14, 26).
Following the same correlation pattern, an absence of effect in GLUT2 protein or RNA levels corresponded to an absence of alteration in glucose transport at the Km corresponding to GLUT2. Furthermore, overexpression of GLUT2 has not been reported in cancers originating from steroid hormone target tissues (14). Although, we demonstrated that ZR-75 cells express extremely low levels of GLUT3 protein, we observed a dramatic increase in the GLUT3 RNA content in response to progesterone and combined 17ß-estradiol and progesterone treatment. Changes observed at the protein level corresponded to changes in glucose uptake. These observations raise the interesting possibility that a small increase in GLUT3 protein and corresponding glucose transport may confer a survival advantage to the proliferating cancer cell. In support of this theory, GLUT3 is overexpressed in tumor biopsies of breast (23) and ovarian (27) cancers. Glucose uptake at the Km corresponding to GLUT3 was also increased in the presence of 17ß-estradiol, a result that was not anticipated because no changes in GLUT3 expression were observed at the protein or RNA level. This result suggests that the observed increase in glucose transport maybe brought about by an increase in protein translocation to the plasma membrane or by other posttranslational modifications. Interestingly, although the GLUT 4 uptake component cannot be separated from GLUT1, GLUT4 did not show a correlation between protein and RNA levels. The immunocytochemistry technique used detected both cytoplasmic and membrane GLUT4 demonstrating that an increase in total protein occurred. We therefore speculated that the nonconformity between protein and RNA levels are due to changes in protein stability and/or other posttranslational effects. This observation does not rule out the possibility that translocation of GLUT4 is occurring in response to hormone treatment. These results demonstrate the complexity and the multifaceted nature by which GLUT expression and glucose uptake are regulated by steroid hormones in ZR-75 breast cancer cells. Experiments are currently ongoing to ascertain whether our observations also apply in normal human mammary primary cell cultures.
In summary, we present an in vitro model in which to study the effects of estrogen and progesterone treatment in breast cancer. We have shown that in ZR-75 cells progesterone and especially the combination of estrogen and progesterone cause an increase in GLUT expression associated with a subsequent increase in glucose transport. These results may provide a mechanism by which estrogen and progesterone present in the luteal phase of the menstrual cycle supply the energy required for the growth and differentiation of the mammary ductal epithelium in preparation for lactation. Furthermore, we speculate that an increase GLUT expression in the presence of combined estrogen and progesterone treatment presents favorable proliferating conditions to the burgeoning breast cancer cell and thus increases breast cancer incidence. Using this model, we have identified that GLUTs and glucose transport are differentially regulated by estrogen and progesterone and therefore may be an important target for conventional pharmaceutical therapy and gene therapy in the treatment of breast cancer.
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Footnotes |
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Abbreviations: DOG, 3H-2-Deoxy-D-glucose; ER, estrogen receptor; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HRT, hormone replacement therapy; Km, Michaelis constant; PR, progesterone receptor; Vmax, maximal velocity.
Received March 5, 2003.
Accepted for publication June 13, 2003.
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