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10.1245/ASO.2005.04.010
Annals of Surgical Oncology 12:488-496 (2005)
© 2005 Society of Surgical Oncology
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Original Article

Expression of the Transcription Factors Snail, Slug, and Twist and Their Clinical Significance in Human Breast Cancer

Tracey A. Martin, PhD, Amit Goyal, MB ChB, Gareth Watkins, BSc and Wen G. Jiang, MD

Metastasis & Angiogenesis Research Group, University Department of Surgery, Wales College of Medicine, Cardiff University, Cardiff, CF14 4XN, Heath Park, United Kingdom

Correspondence: Address correspondence and reprint requests to: Tracey A. Martin, PhD; E-mail: martinta1{at}cf.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Slug, Snail, and Twist are transcription factors that regulate the expression of tumor suppressors such as E-cadherin. We examined the distribution and expression of these three molecules together with the methylation of the Twist gene promoter in human breast cancer to elucidate their clinical significance.

Methods: Frozen sections from breast cancer primary tumors (tumor, n = 114; background, n = 30) were immunostained with Slug, Snail, and Twist antibodies. RNA was reverse-transcribed, quantified, and analyzed by quantitative polymerase chain reaction (Q-PCR). Results were expressed as copy number of transcript per 50 ng of RNA (standardized against ß-actin).

Results: Immunohistochemistry revealed that all three molecules were stained in mammary tissues, with an increase in Twist within tumor tissues; this was supported by Q-PCR analysis. Q-PCR analysis showed that Slug was elevated with increasing tumor grade and prognostic indices. Twist was elevated with increasing nodal involvement (tumor-node-metastasis status). Conversely, Snail was reduced in expression corresponding with prognostic indices and tumor grade. Increased levels of Slug were associated with tumors from patients with metastatic disease or disease recurrence, and increased expression of Twist was associated with tumors from patients who had died from breast cancer. It is interesting to note that Snail expression was significantly reduced in patients with a poor outcome and those who had node-positive tumors. In addition, tumors exhibited methylation of the Twist promoter.

Conclusions: These data demonstrate that all three transcription factors have inappropriate expression in breast cancer and that this may play a part in the progression of human breast tumors.

Key Words: Breast cancer • Twist • Snail • Slug


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Snail and Slug belong to the Snail family of genes, which is conserved among species during evolution. They encode transcription factors that are expressed at different stages of development in different tissues.1 These transcription factors encode a zinc finger–type transcription factor that is necessary for gastrulation and mesoderm formation2 and are involved in a broad spectrum of biological functions, such as cell differentiation, cell motility, cell-cycle regulation, and apoptosis.1,3,4 They share highly conserved C2H2-type zinc finger domains, which bind to the E-box and repress the transcription of target genes.3 More recently, they have been implicated in the progression of human tumors via their regulatory action on the epithelial cell adhesion molecule E-cadherin.5–7 Such transcriptional repression mechanisms have emerged as one of the crucial processes for the downregulation of E-cadherin expression during development and tumor progression with both Snail and Slug, acting through an interaction with proximal E-boxes of the E-cadherin promoter.7

Loss of expression of the E-cadherin cell/cell adhesion molecule is important in carcinoma development and progression.8 It has been shown that Slug and Snail are potential repressors of E-cadherin transcription in carcinomas that lack E-cadherin expression,6 and analysis of the expression patterns of Slug, Snail, and E-cadherin in breast cancer cell lines demonstrated that the expression of Slug was strongly correlated with a loss of E-cadherin transcripts. Slug is the more likely in vivo repressor of E-cadherin expression in breast cancer.

In vertebrates, the basic helix-loop-helix protein Twist may be involved in the negative regulation of cellular determination and in the differentiation of several lineages, including myogenesis, osteogenesis, and neurogenesis.9–11 Twist is able to inhibit oncogene- and p53-dependent cell death and is thus known as an antiapoptotic factor. Twist is also known to trigger epithelial-mesenchymal transition (EMT) mechanisms, possibly regulating the E- to N-cadherin switch during EMT.12 Twist has been found to be inappropriately suppressed in 50% of rhabdomyosarcomas.10 Twist is also an activator of GLI1 reporter expression, where defects in the signaling pathway lead to severe birth defects and cancer formation in humans.13 Methylation-specific polymerase chain reaction (PCR) has been used to detect cancer cells from ductal lavage fluid for cyclin D2, RAR-ß , and Twist,14 which was frequently found to be methylated in patients with carcinomas (17 of 20). Upregulation of Twist is associated with cellular resistance to paclitaxel but not with other drugs that have different mechanisms of action.15 This indicates a novel mechanism that leads to resistance to microtubule-disrupting anticancer drugs through upregulation of Twist.

EMT results in the loss of polarity of epithelial cell layers and cell/cell contacts, with a corresponding remodeling of the cell cytoskeleton.16 Evidence has accumulated showing that EMT is an important in vitro correlation of late-stage tumor progression.17–20 In mouse mammary cells, it has been demonstrated that EMT occurs before the acquirement of an invasive, metastatic tumor phenotype.21 Twist has been shown to play a role in metastasis in murine models.22 Ectopic expression of Twist resulted in the loss of E-cadherin–mediated cell/cell adhesion, activation of mesenchymal markers, and induction of cell motility; this suggests that Twist may contribute to metastasis by promoting EMT. Increased Twist expression has also been correlated with invasive lobular cancers associated with E-cadherin loss in human breast cancer.22 Because Snail and Slug are potential regulators of cell adhesion and migration, this study aimed to determine the levels of expression of Snail, Slug, and Twist in human breast cancer tissues and to elucidate whether these levels are clinically significant.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell and Tissue Collection and Preparation
Human breast cancer cell lines (BT-549, BT-474KC, MDA-MB-231, MDA-MB-453, MDA-MB-231, MDA-MB-468, MDA-MB-436, MCF-7, MCF-10a, and ZR-751), putative melanoma cell line MDA-MB-435S, prostate cancer cell line PC-3, and HeLa and human fibroblast cell line MRC-5 (both from ECACC, Wiltshire, UK) were maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum. Breast tissue samples23 (tumor and matched background; Table 1Go) were collected and immediately frozen in liquid nitrogen before processing—a portion of each sample for quantitative PCR (Q-PCR) analysis, a portion for immunohistochemical analysis, and a portion for routine histological examination.


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  		TABLE 1. Clinical information of patient samples analyzed
 
RNA was isolated from tissue samples and cells by using standard RNAzol procedures. For reverse transcription-PCR, complementary DNA (cDNA) was synthesized in a 20-µL reaction mixture with 1 µg of RNA, as described in the protocol (AB Gene Reverse Transcription System; ABGene, Surrey, UK).

Quantitative PCR
The Q-PCR system used the Amplofluor Uniprimer system (Intergen Company, Oxford, UK) and Thermo-Start (ABgene, Epsom, Surrey, UK), as we recently reported.16 Specific primer pairs for Snail, Slug, and Twist (Table 2Go) were designed by the authors by using Beacon Designer software and were manufactured by Invitrogen (Invitrogen Life Technologies, Paisley, Scotland, UK), each amplifying a region that spans at least one intron (primer details are given in supplement 1), thus generating an approximately 100–base pair product from both the control plasmid and cDNA.


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  		TABLE 2. Quantitative polymerase chain reaction primer pairs for analysis of angiogenic marker molecules
 
By using the iCycler IQ system (Bio-Rad), which incorporates a gradient thermocycler and a 96-channel optical unit, the plasmid standards and breast cancer cDNA were simultaneously assayed in duplicate reactions with a standard hot-start Q-PCR master mix. Q-PCR conditions were as follows: enzyme activation at 95° C for 12 minutes for 1 cycle, followed by 60 cycles of denaturing at 95° C for 15 seconds, annealing at 55° C for 40 seconds, and extension at 72° C for 25 seconds. With use of purified plasmids as internal standards, the levels of each tight junction molecule cDNA (copies per 50 ng of RNA) in the breast cancer samples were calculated. Q-PCR for ß-actin was also performed on the same samples to correct for any residual differences in the initial level of RNA in the specimens (in addition to spectrophotometry). The products of Q-PCR were verified on agarose gels (not shown).

Immunohistochemistry
Cryostat sections of frozen tissue were cut at 6 µm, placed on Super Frost Plus (Optech Scientific Instruments, Oxon, UK) slides, air-dried, and fixed in a 50:50 solution of alcohol and acetone. The sections were then air-dried again and stored at –20° C until use. Immediately before immunostaining began, the sections were washed in buffer for 5 minutes and treated with horse serum/buffer solution for 20 minutes as a blocking agent to nonspecific binding. Sections were stained with Snail, Slug, and Twist antibodies (Insight Biotechnology, Wembley, Cambridgeshire, UK). Primary antibodies were used at a 1/50 dilution for 60 minutes and then washed in buffer. The secondary biotinylated antibody at 1/100 dilution (universal secondary, Vectastain Elite ABC; Vector Laboratories Inc., Burlingham, CA) was added (in horse serum/buffer solution) for 30 minutes, followed by numerous washings in buffer. Avidin/biotin complex was added for 30 minutes, again followed with washes in buffer. Diaminobenzidine was used as a chromogen to visualize the antibody/antigen complex. Sections were counterstained in Mayer’s hematoxylin for 1 minute, dehydrated, cleared, mounted in dextropropoxyphene, and screened with an x 25 objective.

Evaluation of Twist Promoter Hypermethylation
Genomic DNA from multiple sections from frozen tissue and human breast cancer cells were extracted by using a standard DNA-extraction protocol. Methylation-specific PCR was performed as previously described.17 One microgram of DNA was denatured in NaOH (to a final concentration of .2 mol/L) for 10 minutes at 37° C, followed by 10 mmol/L of freshly prepared hydroquinone and 3 mol/L of sodium bisulfite (pH 5.0). After incubation at 50° C for 16 hours, the DNA was purified with the Wizard DNA purification kit (Promega UK Ltd., Southampton, UK) and standard ethanol precipitation. Hypermethylation was analyzed by PCR with primers listed in Table 2Go. Methylation-specific PCR conditions were as follows: enzyme activation at 95° C for 5 minutes for 1 cycle, followed by 36 cycles of denaturing at 95° C for 15 seconds, annealing at 58° C for 15 seconds, and extension at 72° C for 30 seconds. The PCR products were separated on standard 10% polyacrylamide gel electrophoresis gels.

Statistical Analysis
Statistical analysis was performed with Minitab version 13.32 (Minitab Inc., State College, PA) by using a two-sample Student’s t-test and the non-parametric Mann-Whitney confidence interval and test, where appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Slug, Snail, and Twist Show Increased Expression in Tumor Tissues
Levels of transcripts of Slug, Snail, and Twist were increased in tumor samples in comparison to background (expressed as transcript copy number per 50 µg of messenger RNA and standardized with ß-actin; Fig. 1Go). Transcript copy numbers for Slug were .54 ± .19 for tumor and .29 ± .11 for background (P = .25); for Snail, they were .18 ± .07 for tumor and .05 ± .02 for background (P = .09); and for Twist, they were 68.1 ± 20.9 for tumor and 52.4 ± 18.8 for background (P = .58), although these data did not reach significance. This pattern agreed with the results from immunohistochemistry: in tumor sections, more cells stained positively for all three regulatory factors (Fig. 2Go). From the sections themselves (Fig. 2A and 2BGo), it is observable that in normal background tissue, the cells surrounding the vessels and ducts stain very intensely for these transcription regulators. In the tumor tissues, although the intensity of staining is less, most cells stain positively (Fig. 2Go).


Figure 1
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  		FIG. 1. Levels of transcripts of Slug, Snail, and Twist in tumor samples in comparison to background (expressed as transcript copy number per 50 µg of messenger RNA and standardized with ß-actin).

 

Figure 2
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  		FIG. 2. Immunostaining of Slug (A), Snail (B), and Twist (C) in background (left panel) and tumor (right panel) sections. Representative cases of increased positive staining in tumor sections are shown at x 4, x 10, and x 20.

 
It is interesting to note that both Slug and Twist had increased expression in node-positive tumors compared with node-negative tumors (slug: node positive, .63 ± .36; node negative, .51 ± .20; P = .78; Twist: node positive, 89 ± 21; node negative, 52 ± 40; P = .40). Snail was reduced in node-positive tumors (node positive, .10 ± .06; node negative, .25 ± .13; P = .32), although these values were not significantly different (Fig. 3Go).


Figure 3
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  		FIG. 3. Levels of transcripts of Slug, Snail, and Twist expression in node-positive (Node +ive) tumors compared with node-negative (Node –ive) tumors.

 
Examination of Predictive Factors Shows That Snail Has a Different Expression From Slug and Twist
All three transcription molecules were assessed by using Q-PCR and compared with a series of predictive factors (Tables 3Go–5GoGo). Slug was sequentially increased with increasing Nottingham Prognostic Index (NPI) status from .51 ± .55 in NPI 1 (NPI <3.5) tumors to .84 ± .59 in NPI 3 tumors (NPI >5.4; Table 3Go). Twist was increased in NPI 2 (NPI 3.5–5.4) tumors (106.8 ± 54.4 vs. 51.5 ± 20.7 in NPI 1) but showed little change in NPI 3 tumors (32.5 ± 18.2). Conversely, Snail expression was reduced with increasing NPI status (NPI 1, .25 ± .13; NPI 2, .13 ± .08; NPI 3, .03 ± .03); however, these changes did not reach significance (P = .2).


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  		TABLE 3. Analyses of messenger RNA from samples showing levels of the three transcription factors tested and NPI status (transcript copy number)
 

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  		TABLE 4. Analyses of messenger RNA from samples showing levels of the three transcription factors tested and tumor grade (transcript copy number)
 

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  		TABLE 5. Analyses of messenger RNA from samples showing levels of the three transcription factors tested and tumor-node-metastasis (TNM) status (transcript copy number)
 
When expression was compared with tumor grade (Table 4Go), a similar change was observed for Slug —i.e., expression increased with increasing tumor grade (grade 1, .39 ± 019; grade 3, .57 ± .24), and Twist again showed no obvious correlation (Table 4Go). Snail once again showed reduced expression with increasing tumor grade (grade 1, .38 ± .25; grade 2, .18 ± .14; grade 3, .09 ± .05), although this did not reach significance (P = .3).

Twist was sequentially increased with increasing tumor-node-metastasis status of tumors (tumor-node-metastasis 1, 56.0 ± 14.3; tumor-node-metastasis 2, 81.1 ± 54.3; tumor-node-metastasis 3, 197.0 ± 165.0). Neither Slug nor Snail showed a correlation with tumor-node-metastasis status (Table 5Go). It is interesting that Twist was progressively increased with increased nodal involvement only, whereas Slug was positively associated with NPI status and grade of tumor and Snail was negatively associated.

Correlation of Snail, Slug, and Twist With Patient Prognosis
Our patient follow-up of 72.2 months allowed us to examine the Q-PCR data with regard to patient outcome. Patients were classified as (1) disease free, (2) with metastatic disease, (3) with local recurrence of breast cancer, and (4) dead as a result of breast cancer. Slug showed the lowest expression in patients who had remained disease free (.31 ± .12) and was highest in patients who had died of breast cancer (1.91 ± 1.29; P = .2). Slug was also increased in patients with metastatic disease (1.00 ± .8), but there was little change of expression in patients with local recurrences (Table 6Go). Twist showed the highest expression in patients who had died of breast cancer (169.0 ± 138.0). Snail was reduced in patients who had metastatic disease (.18 ± .10) and was significantly reduced in those with local recurrences (recurrence, .01 ± .007; alive and well, .24 ± .11; P = .04) but was increased in patients who had died of breast cancer (.26 ± .02; P = .05). When all poor outcomes are considered together, it can be observed that both Slug and Twist were increased in patients with poor outcome (metastatic disease and recurrence of and death from breast cancer), whereas Snail was significantly reduced in patients with a poor outcome compared with those who remained alive and well after 72.2 months (Fig. 4Go).


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  		TABLE 6. Analysis of messenger RNA to investigate correlations between survival status and transcription factor (transcript copy number)
 

Figure 4
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  		FIG. 4. Comparison of transcript levels of Slug, Snail, and Twist considering poor-outcome patients compared with those who remained disease free. Snail was significantly reduced in patients with poor outcome compared with those who remained alive and well after 72.2 months (P = .05).

 
Twist Shows Hypermethylation in Human Breast Cancer
Hypermethylation of the Twist promoter was observed in 50% of the tumors (four of eight) tested (Fig. 5AGo), whereas only one background tissue showed methylation (one of eight). Of the 10 breast cancer cell lines analyzed, only 3 showed Twist promoter methylation: MCF-7 cells showed a strong signal for methylation, and ZR-751 and MDA-MB-231 cells showed only a weak signal (Fig. 5BGo). It is interesting to note that control cells (such as human endothelial cells from vein), the melanoma cell line MDA-MB-435S, and HeLa cells also showed promoter methylation of Twist. The fibroblast cell line MRC-5 was not methylated.


Figure 5
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  		FIG. 5. Agarose gel indicating hypermethylation of the Twist promoter in representative tumors (3 of 8) tested (A). Of the 10 breast cancer cell lines analyzed, only 3 showed Twist promoter methylation (B). N, normal; T, tumor.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this article, we have demonstrated that the regulatory proteins Slug, Snail, and Twist are expressed aberrantly in human breast cancer. Expression of Slug and Twist shows increased levels in tumors, whereas Snail seems reduced.

The members of the Snail family have been implicated in the triggering of EMT during embryonic development,24,25 and EMT induction by Snail in epithelial cells is mediated by direct transcriptional repression of the cell adhesion molecule E-cadherin. Indeed, Snail has been shown to repress E-cadherin expression and to trigger EMT associated with epithelial tumor progression.23–26 Snail expression has been demonstrated in primary human tumors,27,28 with a correlation between Snail expression and a reduction or absence of E-cadherin expression in several tumors. Snail expression inversely correlates with the grade of differentiation of tumors and is expressed in infiltrating ductal carcinomas with lymph node metastases; it has been concluded that Snail is involved in the progression of breast ductal tumors and possibly serves as a marker of metastatic potential.29,30 We have previously reported that there was no correlation among E-cadherin Slug, Snail, or Twist in this dataset.31

Our results show that Snail is expressed by more cells in tumors (assessed by immunohistochemistry), although the intensity of staining is reduced in stromal and epithelial/tumor cells compared with cells in microvessels. This is supported by Q-PCR analysis and by previous work showing that Snail expression is higher in normal cells compared with breast cancer cell lines.32 However, Snail expression was reduced with both NPI status and grade of tumor and was significantly reduced in patients with poor outcomes, particularly those with local recurrences. Further, Snail expression was reduced in node-positive tumors and in both ductal and lobular carcinomas. This somewhat contradicts previous findings28 that did not use Q-PCR and had a small sample size (n = 21) and so concentrated on the distribution of Snail expression. Moreover, it has been demonstrated6 that Snail expression does not correlate as well with loss of E-cadherin in breast cancer as does the other Snail family member, Slug. Snail is, however, a repressor that downregulates the expression of aromatase (oestrogen synthetase) in healthy breast tissue via suppression of the I.3 promoter.30 Aromatase is up-regulated in breast tumors and stimulates cancer growth in both an autocrine and a paracrine manner.31,32 Because Snail expression is reduced in breast cancer tissue, it has been suggested that Snail may have a cancer-protective role in healthy breast tissue.33 Together with our findings shown here, the reduced expression of Snail in breast cancer may have a direct bearing on the use of aromatase suppressors in breast cancer therapy.34

Slug bears a close homology to the Caenorhabditis elegans CES-1 protein35 and is thought to be an evolutionarily conserved transcriptional repressor whose activation promotes the aberrant survival and eventual malignant transformation of mammalian pro-B cells otherwise scheduled for apoptosis. It been shown to be a transcriptional repressor in humans.3 Both Snail and Slug have been shown to repress E-cadherin expression during development and tumor progression through their interaction with proximal E-boxes of the E-cadherin promoter.7 Slug is also thought to be the likely in vivo repressor of E-cadherin in breast carcinoma.6 This is in contrast to the relationship between Slug and Snail expression in human hepatocellular carcinoma, in which Snail is overexpressed and is the likely E-cadherin repressor,5 as it is thought to be in malignant melanomas.36

In our study, we have shown that Slug expression is increased in tumors compared with normal background tissues. Slug is also increased progressively with NPI status and tumor grade, although some reduction in expression was observed corresponding with tumor-node-metastasis status. Slug expression was increased in node-positive tumors and was high in tumors from patients who had died from breast cancer or had metastatic disease. Moreover, Slug expression was reduced in lobular tumors compared with ductal tumors or tumors of other types. These results differ from those observed for Snail, even though both are transcription factors from the same family (Snail is upstream of Slug).37 However, evidence is accumulating to support the idea that the in vivo action of different factors such as Snail and Slug, in E-cadherin repression, for example, can be modulated by their relative concentrations, as well as by specific cellular or tumoral contexts.7 These repression factors may act alone or in concert6 and may have differing functions that are dependent on tumor type.

Twist is able to inhibit oncogene- and p53-dependent cell death, interferes with p53 reporter activation, and impairs the induction of genes subsequent to that in response to DNA damage.10 It is thus known as an antiapoptotic factor. Twist is also known to trigger EMT mechanisms and possibly regulates the E- to N- cadherin switch during EMT.12 Forced N-cadherin expression exerts a dominant effect over E-cadherin function in breast cancer cells.38 N-cadherin expression in normal epithelial cells causes a reduction in E-cadherin expression, and N-cadherin is also able to enhance tumor cell motility and migration.39 Twist has been found to be inappropriately suppressed in 50% of rhabdomyosarcomas.10 Twist is also an activator of GLI1 reporter expression through E-box interaction via the binding of upstream stimulatory factors (USF) proteins. GLI1 encodes a critical transcription activator in the sonic hedgehog signaling pathway, which regulates vertebrate patterning, where defects lead to severe birth defects and cancer formation in humans.13

Here we demonstrated that Twist was increased in tumor tissues (both in intensity of staining with immunohistochemistry and in Q-PCR analysis) and showed a progressive increase with tumor-node-metastasis status and in patients who had died from breast cancer. Twist was also increased in ductal and lobular cancer compared with other types and also in node-positive tumors. Twist also showed some hypermethylation of its promoter. Little work has been performed investigating the role of Twist in human cancer. Both Twist and Snail have been investigated in human gastric cancer: Snail expression was increased and was associated with decreased E-cadherin expression.40 Moreover, Twist overexpression was also seen and correlated with abnormally positive or increased N-cadherin expression in gastric carcinomas. The epigenetic silencing of a small number of genes by promoter hypermethylation in lobular breast cancers has been studied.41 It has been shown that Twist was hypermethylated less often in invasive lobular cancers than in invasive ductal cancers. Our findings show that the Twist promoter was hyper-methylated in only three of eight breast cancers and that this occurred only in ductal carcinomas. This may suggest that promoter methylation is patient specific and is not indicative of prognosis, as anticipated. In mouse mammary tumors, Twist expression can be regulated by Wnt/ß-catenin signaling, and both Twist and Wnt1 can function as inhibitors of lactogenic differentiation, which could contribute to mammary tumorigenesis.42 Our data suggest that Twist may have a considerable effect in the ultimate increase in tumor/nodal involvement in breast cancer and, as such, could have potential as a marker in human breast cancer. Upregulation of Twist has been shown to be associated with cellular resistance to paclitaxel and vincristine in four types of human cancer (nasopharyngeal, bladder, ovarian, and prostate),15 thus suggesting that a therapeutic strategy may be developed to overcome acquired resistance through upregulation of Twist expression in human cancer.

In conclusion, the EMT regulatory proteins Slug and Twist are upregulated in human breast cancer, whereas Snail is downregulated. Such disparate expression levels may contribute to the progression of tumors in breast cancer, and this deserves further investigation.


   ACKNOWLEDGMENTS
 
We thank Cancer Research UK for supporting this work and Dr. Anthony Douglas-Jones for his invaluable advice on histology.

Received for publication April 8, 2004. Accepted for publication January 19, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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