Regulation of Tissue Factor Gene Expression In Human Endometrium by Transcription Factors Sp1 and Sp3

Graciela Krikun, Frederick Schatz, Nigel Mackman, Seth Guller, Rita Demopoulos and Charles J. Lockwood

The Departments of Obstetrics and Gynecology (G.K., F.S., S.G., R.D., C.J.L.), Biochemistry (S.G.), and Pathology (R.D.) New York University School of Medicine New York, New York 10016
The Scripps Research Institute (N.M.) Departments of Immunology & Vascular Biology La Jolla, California 92037


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Prior studies indicate that tissue factor (TF), the primary cellular initiator of hemostasis, is persistently up-regulated in human endometrial stromal cells (HESCs) undergoing progestin-induced decidualization in vivo and in vitro. The mechanism underlying progestin enhancement of TF mRNA and protein levels in these cells involves transcriptional activation of the TF gene. Transient transfections of HESCs with the truncated TF promoters driving the luciferase reporter gene have demonstrated that the region spanning -111 to +14 bp retained differential progestin-enhancing effects. We now demonstrate that RU486 displays inhibitory effects on the progestin-induced TF promoter activity, confirming the involvement of the progesterone receptor. Since the TF minimal promoter (pTF 111 spanning -111 to +14 bp) contains three overlapping Sp1 and three Egr-1 sites, the present study determined whether Sp1 and/or Egr-1 were required for progestin-regulated TF expression. The results indicate that the three Sp1 sites are primarily responsible for both the constitutive and progestational activity of the pTF 111 promoter, whereas the Egr-1 sites have only a minor involvement in both activities. Overexpression of the Sp1 protein resulted in greater than a 6-fold induction in TF promoter activity. In contrast, no enhancement was observed when the Sp3 protein was overexpressed. The concomitant overexpression of Sp1 and Sp3 demonstrated that Sp3 completely blocked the induction of TF promoter activity by Sp1. Moreover, the addition of 10 nM mithramycin, a concentration that inhibits Sp1 binding to target DNA, blocked the progestational induction of TF mRNA expression. Immunohistochemical studies demonstrated increased Sp1 levels in perivascular stromal cells in secretory phase compared with proliferative phase endometria. In contrast, Sp3 expression was greatly decreased in stromal cells of secretory, compared with proliferative phase tissues. The levels of Egr-1 were low in both proliferative and secretory endometria. Immunocytochemistry of E2 vs. E2 + medroxyprogesterone acetate-treated HESCs demonstrated a dramatic reduction in Sp3 expression after progestin treatment, and Northern blots demonstrated progestational increases in Sp1 and reduction in Sp3 mRNA expression compared with controls. Taken together, our results demonstrate that progestin enhancement of TF gene expression in HESCs is mediated principally by Sp1. We propose that progestins regulate HESC TF gene expression in vivo by altering the ratio of Sp1 to Sp3 nuclear factors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tissue factor (TF), the cell membrane-bound glycoprotein responsible for the initiation of hemostasis, is synthesized in several cell types (1, 2, 3). Our prior studies demonstrated that TF is also expressed in human endometrial stromal cells (HESCs) where its expression is greatly enhanced by progestins in vitro and in vivo (4, 5). In contrast to the immediate and transient induction of TF expression in endothelial, epithelial, monocytic, and fibroblastic cells (6, 7, 8, 9), the induction of TF in HESCs by progestins is delayed and prolonged. Subsequent studies in our laboratory demonstrated that progestational up-regulation of TF in HESCs occurs at the transcriptional level (10). Moreover, transfection with truncated TF promoter constructs showed that the region -111 to +14 bp retained both basal and differential progestin effects (10).

The -111 to +14 region contains three overlapping Sp1/Egr-1 sites (Fig. 1Go). Sp1 is a nuclear activator protein that stimulates eukaryotic transcriptional initiation by aiding in the formation of a functional preinitiation complex consisting of RNA polymerase II, activator-proteins, and the target DNA (11, 12, 13). Activation is further enhanced if multiple Sp1 binding sites are present (14, 15). Four members of the Sp family of proteins have thus far been identified. They include Sp1, Sp2, Sp3, and Sp4 (16, 17, 18, 19). Sp3 and Sp4 proteins bind with similar affinities to the same consensus DNA site as Sp1 (19, 20, 21). Sp1 and Sp3 are ubiquitously expressed, whereas Sp4 is expressed predominantly in the brain (22, 17). Interestingly, Sp3 generally functions as a competitive repressor of Sp1-induced transcription (17). In addition to competition for binding sites among the Sp family of proteins, other zinc-binding proteins, such as Egr-1, bind to GC-rich regions and can partially interfere with Sp binding (23, 24). The Egr-1 transcription factor, also known as NGFI-A, Zif 268, Krox24, and TIS8 (25, 26), is involved in cell proliferation and differentiation (27). It is a nuclear phosphoprotein that is rapidly and transiently induced by serum and growth factors and binds to a specific DNA sequence in a zinc-dependent manner (25).



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Figure 1. TF Promoter Constructs

The minimal TF promoter spanning the region -111 to + 14 bp (pTF 111) was altered as described in Materials and Methods. The resulting promoters contained critical mutations in the three Sp1 sites (pTF Sp1m), the three Egr-1 sites (pTF Egr-1m), and the six overlapping Sp1/Egr-1 (pTF Sp1/Egr-1m) sites as denoted by the bold letters.

 
Sp1 and/or Egr-1 have been shown to regulate the immediate and transient expression of TF in various systems. In HeLa cells, Sp1 is constitutively expressed, whereas Egr-1 was induced in response to phorbol myristate acetate (PMA) or serum (28). Shear stress-induced TF expression by human umbilical vein endothelial cells and human aortic endothelial cells requires Sp1 and Egr-1 (29, 30). Hypoxia enhances transcription and expression of TF in lung via Egr-1 (31). The current study determined the role of the three Sp1 and the three Egr-1 sites contained within the region -111 to +14 bp of the TF gene in progestin-induced transcription in HESCs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RU486 Blocks Progestin-Enhanced TF Gene Transcription
The antiprogestin RU486 was used to assess the requirement for the progesterone receptor (PR) in mediating progestin enhancement of TF transcription. Figure 2Go demonstrates that RU486 abolished progestin-enhanced transcription of both the pTF 278 and pTF 111 promoter constructs. Although RU486 blocks the action of progesterone and glucocorticoid receptors, we have previously shown that glucocorticoids have no effect on the induction of TF expression (32). Therefore, these effects of RU486 confirm the involvement of the PR in our system.



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Figure 2. RU486 Blocks MPA Transcriptional Activation of TF

Cultured HESCs were treated with E2, E2 + MPA, E2 + RU486 (RU), or E2 + MPA + RU as described in Materials and Methods. Transfections were carried out with two TF promoter constructs spanning the regions -278 to +14 bp (P-278) and -111 to +14 bp (P-111). Firefly luciferase activity derived from the TF promoter was normalized for transfection efficiency by comparison with Renilla luciferase activity derived from the cotransfected Renilla vector. (n = 6, triplicates from two separate experiments). *, P < 0.05 comparing E2 + MPA vs. E2.

 
Role of Sp1 and Egr-1 in Progestin Enhancement of TF Gene Transcription
To investigate the role of Sp1 and Egr-1 in mediating progestin-enhanced TF transcription, HESCs were transfected with the luciferase-linked minimal wild-type promoter construct (pTF 111) or with promoter constructs in which either the three Sp1 sites (pTF Sp1-m), the three Egr-1 (pTF Egr1-m), or all six Sp1 and Egr-1 sites (pTF Sp1/Egr1-m) were mutated. The effect of hormone treatment as well as a schematic of the various promoter constructs that were used is depicted in Fig. 3Go. This figure demonstrates that promoter activity was significantly enhanced by treatment with E2 + medroxyprogesterone acetate (MPA) compared with E2 for both pTF 111 and pTF Egr1-m. In addition, the activities of pTF Sp1-m and pTF Sp1/Egr1-m were significantly reduced compared with the activity of pTF 111 after incubation with E2 or E2 + MPA (P < 0.0001 in all cases). By contrast, the promoter activity observed with pTF Egr1-m did not differ significantly from that observed with pTF 111 for either E2 or E2 + MPA. These results indicate that in the presence or absence of progestin, mutating the Egr-1 sites alone does not affect the transcriptional activity of the TF promoter. Conversely, the rates of transcription are greatly diminished by mutating the Sp1 sites, demonstrating the requirement for the latter. When the Sp1 sites are mutated, both the constitutive and the progestational induced transcription rates are decreased. Nevertheless, a trend toward higher rates in the presence of progestins is evident. This trend, as well as the overall constitutive rates for construct pTF Sp1-m, are significantly decreased when all the Sp1 and Egr-1 binding sites are mutated in the pTF Sp1/Egr1-m construct (P < 0.03 for E2 and P < 0.02 for E2 + MPA). This result likely reflects the small binding affinity of Sp1 for Egr-1 sites as has been previously described (23, 24). In summary, Fig. 3Go indicates that the three Sp1 sites are primarily responsible for the constitutive and progestational activity of the pTF 111 promoter, whereas the Egr-1 sites have a minor involvement in both activities.



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Figure 3. Transfection of Mutant TF Promoters

Cultured HESCs were treated with E2 or E2 + MPA as described in Materials and Methods. Transfections were carried out with the minimal TF promoter -111 to +14 bp (pTF111) and with promoters mutated in the three Sp1 (pTFSp1-m), the three Egr-1 (pTFEgr1-m), or all six Sp1 and Egr-1 (pTFSp1/Egr1-m). A schematic of these is shown on the left. Levels of luciferase activity were determined and corrected for transfection efficiency as described in Materials and Methods (n = 9 triplicates from three separate experiments). *, P < 0.0001 comparing E2 vs. E2 + MPA for pTF111 and **, P < 0.002 comparing E2 vs. E2 + MPA for pTFEgr1-m.

 
The promoter activity for construct pTF Sp1/Egr1-m was significantly above that found in control cultures transfected with a promoterless PGL2Basic vector (1.56 ± 0.18 vs. 0.66 ± 0.17 for E2, P < 0.02; and 1.78 ± 0.32 vs. 0.66 ± 0.23 for E2 + MPA, P < 0.01).

Overexpression of Sp1 but not Sp3 Induces TF Promoter Activity
Figure 4Go demonstrates that overexpression of the transcription factor Sp1 increases TF promoter activity greater than 6-fold. In contrast, no increase in promoter activity is observed by Sp3 overexpression. Moreover, simultaneous coexpression of Sp1 and Sp3 abolishes the induction observed with Sp1 overexpression alone. These data demonstrate that the balance between levels of Sp1 and Sp3 regulates TF gene expression in HESCs.



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Figure 4. Overexpression of the Transcription Factor Sp1 and/or Sp3

HESCs were cultured and treated in control medium and transfected with pTF 111 (TF) ± the Sp1, the Sp3, or both overexpressing vectors, as described in Materials and Methods (n = 3 triplicates from a typical experiment). *, P < 0.002 compared with HESCs transfected with pTF 111 alone.

 
Effect of Mithramycin on Progestin Induction of TF mRNA Expression
To further investigate the role of Sp1 on the regulation of TF expression, control, E2, or E2+MPA-treated HESCs were incubated with or without 10 nM mithramycin, an inhibitor of Sp1 binding to its corresponding GC box. As can be seen in Fig. 5Go, mithramycin abrogated the progestational induction of TF mRNA levels, further supporting the role of Sp1 in enhanced TF expression by HESCs.



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Figure 5. Effect of Mithramycin on Progestin Induction of TF mRNA Expression

Control (C), E2 (E)-, or E2 + MPA (P)-treated HESCs were incubated with and without 10 nM mithramycin (Mit) as described in Materials and Methods. Northern blots were performed for TF and loading efficiencies assessed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

 
Localization of Sp1, Sp3, and Egr-1 in Cycling Endometria
Immunohistochemical staining for Sp1, Sp3, and Egr-1 was conducted on six proliferative phase and six secretory phase endometria. Three areas per slide were evaluated by two independent observers (G.K. and F.S.). Figure 6Go reveals that both Sp1 and Sp3 are expressed throughout the menstrual cycle in stromal and endothelial cells. Interestingly, an increase in Sp1 staining was observed in secretory-compared with proliferative-phase stromal cells (mean nuclear staining of 77% secretory vs. 38% proliferative; P < 0.01). Particularly intense immunostaining for Sp1 was observed in the nuclei, and weaker staining was observed in the cytoplasm of perivascular stroma surrounding clusters of spiral arterioles. These are precisely the sites where both decidualization and enhanced TF expression commence (4, 5). In contrast, immunostaining for the Sp1-competitor, Sp3, was decreased in secretory vs. proliferative phase tissues (mean nuclear staining of 31% secretory vs. 71% proliferative; P < 0.01). Unlike the results for Sp1 and Sp3, staining for Egr-1 was of a lower magnitude, unchanged by hormone treatment and limited to a much smaller population of stromal cells in all tissues studied. No staining was observed for Sp4 (not shown).



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Figure 6. Immunohistochemistry of Sp1 and Sp3 in Human Endometrium

Immunohistochemical staining for Sp1and Sp3 in proliferative and secretory phase human endometria were carried out in paraffin-fixed sections as described in Materials and Methods. Detailed perivascular areas for Sp1 and Sp3 staining in a secretory endometrium are shown as insets on the upper left corner. Antigens are identified by brown peroxidase staining (x400).

 
Localization of Sp1 and Sp3 in Cultured HESCs
Immunocytochemical staining was used to observe the direct effects of progestins on the expression of Sp1 and Sp3 in primary cultured HESCs (Fig. 7Go). The in vitro expression of Sp1 showed small increases with progestin treatment compared with E2-treated controls. More dramatically, progestin treatment greatly decreased the expression of the Sp3 nuclear factor in these cells. As with the immunohistochemical studies, cultured HESCs showed low levels of Egr-1 staining that were above background and were not affected by progestin treatment (not shown).



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Figure 7. Immunocytochemistry of Sp1 and Sp3 in Primary Cultured HESCs

Immunocytochemical staining for Sp1 and Sp3 in E2- and E2 + MPA-treated HESCs was carried out as described in Materials and Methods. Antigens are identified by brown peroxidase staining (x200).

 
Sp1 and Sp3 Northern Blots
Figure 8Go displays the results of densitometric analysis of five separate blots and demonstrates statistically significant differences in steady state Sp1 and Sp3 mRNA levels in E2 compared with E2 + MPA-treated HESCs. Thus, progestin treatment of the cells resulted in a 2-fold enhancement of Sp1 mRNA levels and a 2-fold decrease in Sp3 mRNA levels. Progestin treatment had no significant effect on Egr-1 mRNA levels (data not shown).



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Figure 8. Northern Blot Analysis of Sp1 and Sp3 in Primary Cultured HESCs

Densitometric analysis from five separate Northern blots depicting the relative intensity of Sp1 or Sp3 adjusted to glyceraldehyde 3-phosphate dehydrogenase (GAP) from HESCs treated with E2 or E2 + MPA. *, P < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The human endometrium undergoes a series of morphological and biochemical changes involving proliferation (proliferative phase), differentiation, and secretion (secretory phase). The latter facilitates blastocyst implantation and invasion, which normally occurs in the absence of potentially catastrophic hemorrhage. These events are, to a large extent, under the control of the ovarian steroids, estrogen and progesterone. Paradoxically, in the absence of implantation, the endometrium must provide a milieu to facilitate the hemorrhagic bleeding associated with menstruation. Prior in vivo and in vitro studies have demonstrated that decidualization is associated with enhanced HESC TF expression while progestin withdrawal is associated with reduced TF levels (4, 5, 32).

Recently, we have shown that progestin-enhanced TF expression in HESCs is transcriptionally regulated and requires a region of the TF promoter -111 to +14 bp upstream of the transcription start site. This region of the TF promoter contains three overlapping Sp1/Egr-1 sites. The present study demonstrates that in HESCs, the three Sp1 sites are primarily responsible for the constitutive and progestational activity of the pTF 111 promoter, whereas the Egr-1 sites have a minor involvement in both activities. Furthermore, our finding that Sp1 expression is enhanced while Sp3 expression is decreased in perivascular stromal cells from secretory-phase endometrium suggests that Sp1/Sp3 ratios are regulated by progestins during the menstrual cycle. An in vitro model utilizing primary cultured HESCs supports these findings. In this system we observed that progestins increased Sp1 and decreased Sp3 mRNA and protein levels. Furthermore, we noted that the Sp1-DNA binding inhibitor, mithramycin, abolished progestational-induction of TF mRNA, further supporting the role of Sp1 on TF gene expression. Interestingly, we observed enhanced perivascular staining for Sp1 in secretory endometria paralleling the expression of TF (4, 5). While Sp1 staining was primarily nuclear, some cytoplasmic staining was observed. This finding suggests that Sp1 protein is being rapidly synthesized in these regions.

There are growing examples of the reciprocal regulation of target genes by Sp3 and Sp1 (17, 33, 34, 35, 36). In these cases, Sp3 has been shown to inhibit Sp1 binding, resulting in altered gene expression. For example, the mitogenic effects of thrombin are mediated by the human thrombin receptor gene (34). Cotransfection with an Sp1 expression vector significantly augmented human thrombin receptor promoter activity, while cotransfection with Sp1 and Sp3 inhibited Sp1-mediated activation (34). Interestingly, a recent report by Hata and colleagues (35) demonstrated that the kinase domain receptor promoter activity in endothelial cells was partially regulated by variations in the Sp1/Sp3 ratio. Overexpression of Sp1 protein increased kinase domain receptor promoter activity 3-fold, whereas simultaneous coexpression of Sp3 attenuated this response. This Sp3 antagonism of Sp1-mediated transcription has also been reported for activation of the human papillomavirus type 16 promoter during epithelial cell differentiation, as well as in epithelial-specific cellular genes encoding keratin 18 and E-cadherin (36). Our results suggest that progestin plays a critical role in altering the ratio of Sp1/Sp3 expression and that this, in turn, controls the expression of TF in HESCs.

Although expression of Egr-1 protein in cycling endometrium was low, mutation of the three Egr-1 sites together with the three Sp1 sites in the TF promoter indicated that these sites played an additional role in the basal and the progestational induction of TF expression in HESCs. Egr-1 is the prototypic member of a family of immediate-early genes including Egr-2, -3, and -4, each of which contains similar DNA-binding domains (37). Like the Sp family of proteins, Egr proteins are involved in differentiation and mitogenesis (37, 38). In addition to binding to their own sites, Sp proteins can also bind to the Egr-1 sites (23, 24). Figure 3Go shows that mutation of the three Sp sites in pTF 111 greatly reduces the basal transcriptional activity of this promoter. The residual progestin effects that are still apparent likely reflect Sp1 binding to the three remaining Egr-1 sites. The minimal expression of Egr-1 protein in HESCs throughout the cycle also suggests that Egr-1 does not effectively compete with Sp1. Taken together, our results provide a mechanism to account for progestin-enhanced TF expression in HESCs through the interaction of Sp nuclear proteins and through a dramatic alteration in the levels of Sp1 and Sp3 nuclear factors in proliferative vs. secretory endometria.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Preparation of Primary Endometrial Stromal Cell Cultures
Experiments in which Northern blot or immunocytochemical analysis were to be conducted used freshly isolated stromal cells grown to confluence in a basal medium as previously described (4). The experimental period was initiated by the addition of 10-8 M estradiol (E2) ± 10-7 M MPA. The medium was changed every 2–4 days. The cells were treated for 10 days.

Promoter Constructs
The TF promoter-plasmid construct containing the 5'-flanking sequence of the TF promoters spanning -278 to +14 bp (pTF 278) or -111 to +14 bp (pTF 111) was cloned into the luciferase reporter vector pGL2 (Promega Corp., Madison, WI) as described (6). Site-directed mutagenesis of the three Sp1 sites, the three Egr-1 sites, or all six Sp1 and Egr-1 sites were performed as described (28) (Fig. 1Go). The Sp1 and Sp3 expression vectors (pSp1 and pSp3) were generously provided by Jonathan M. Horowitz (North Carolina State University, Raleigh, NC) and were constructed as previously described (39).

Transient Transfections
Primary HESCs were seeded at a density of 2 x 105 cells/3-cm polystyrene well and allowed to attach and proliferate to approximately 60% confluence for 24 h. HESCs were then treated with E2 (10-8 M), E2 + MPA (10-7 M), E2 + RU486 (10-6 M), or E2 + MPA + RU486 or vehicle control for 6 days. RU486 was provided by Dr. Indrani Bagchi (Population Council, New York, NY). Subsequent to this treatment, the cultures were transiently transfected for 4 h with 1 µg of the various TF promoters or with the promoterless pGL2 basic vector (Promega Corp.) utilizing the lipofectamine-Plus reagent (Life Technologies, Inc., Gaithersburg, MD). Experiments in which Sp1 and/or Sp3 were overexpressed were carried out with 1 µg pTF 111 in conjunction with 0.2 µg of either pSp1, pSp3, or both. The cells were cotransfected with 0.1 µg of a Renilla-luciferase reporter vector (Promega Corp.) to provide an internal control of transfection efficiency. The medium was then changed back to basal medium containing either control, E2, or E2 + MPA for an additional 48 h. At this time cells were lysed in passive lysis buffer according to the manufacturer (Promega Corp.) and stored at -80 C.

Transfection Reporter Assays
Luciferase activity was monitored with a luminometer using the Dual Luciferase Reporter Assay System (Promega Corp.) to sequentially assay for TF promoter-dependent firefly luciferase and Renilla luciferase from the internal control.

Immunohistochemistry and Immunocytochemisty
Immunohistochemistry was performed in paraffin-fixed sections of endometria. Immunocytochemistry was conducted on HESCs grown to confluence in tissue culture chamber slides (Nunc Inc., Naperville, IL) and treated with hormones as described above. The cells were fixed in a 4% parformaldehyde solution for 10 min. Peroxidase staining was conducted with the ABC elite kit from Vector Laboratories, Inc. (Burlingame, CA) as described (5). Rabbit polyclonal antibodies to Sp1 (Accurate Surgical & Scientific Corp., Westbury, NY), as well as to Sp3, Sp4, and Egr-1 (Santa Cruz Biotechnology, Inc.; Santa Cruz, CA) were used at a concentration of 0.2 µg/ml.

Northern Blots
Northern blots were carried out as previously described (4, 5). The Sp1 cDNA probe was a kind gift from Dr. C. Kingsley. The Sp3 cDNA probe was purchased from the ATCC (Manassas, VA). The TF cDNA probe was previously described (4, 5). Mithramycin (Sigma, St. Louis, MO) treatment of cells was carried out 12 h before RNA isolation.

Statistical Analysis
All data were analyzed with a Mann-Whitney Rank Sum Test, Sigma Stat computer program (Jandell Scientific, San Rafael, CA). A P value < 0.05 was considered significant.


    FOOTNOTES
 
Address requests for reprints to: Dr. Graciela Krikun, New York University School of Medicine, Tisch Hospital, Room 533, 550 First Avenue, New York, New York 10016.

This work was supported in part by NIH Grant RO1 HL-33937–03 (C.J.L.).

Received for publication April 29, 1999. Revision received December 2, 1999. Accepted for publication December 10, 1999.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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