Skip Navigation


Carcinogenesis Advance Access originally published online on October 29, 2007
Carcinogenesis 2008 29(1):157-160; doi:10.1093/carcin/bgm203
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
29/1/157    most recent
bgm203v2
bgm203v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Google Scholar
Right arrow Articles by Yueying, W.
Right arrow Articles by Shih-Hsin, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yueying, W.
Right arrow Articles by Shih-Hsin, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oxfordjournals.org

ECRG1, a novel esophageal gene, cloned and identified from human esophagus and its inhibition effect on tumors

Wang Yueying, Wang Jianbo, Liu Hailin, Tang Huaijing, Guo Liping and Lu Shih-Hsin*

Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College 100021, China

* To whom correspondence should be addressed. Tel: +86 10 8778 8450; Fax: +86 10 6771 2368; Email: shlu{at}public.bta.net.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
ECRG-1 (esophageal cancer-related gene 1) has been previously found to be down-regulated in human esophagus cancer. Transient expression of green fluorescent protein (GFP)-tagged ECRG1 showed plasma membrane localization. Treatment of esophagus cancer cell line (NEC) with ECRG-1 fusion protein and over-expression of ECRG-1 in NEC cells can significantly reduce the in vitro proliferation rate of NEC cells. Treatment of established NEC tumors in the nude mice with ECRG-1 fusion protein leads to decreased tumor weight and volume. Over-expression of ECRG-1 in NEC cells can also inhibit tumor formation in nude mice. Cell-cycle analysis showed that over-expression of ECRG-1 in NEC cells results in G2/M phase arrest. Our findings indicate that ECRG1 may be a candidate tumor suppressor gene for esophageal cancer (EC) involved in cell-cycle control. Since ECRG-1 gene significantly suppresses the growth of NEC cells both in vitro and in vivo, its loss may contribute to the causation and progression of the EC in Lin-xian county, which is a high incidence area of EC in China.

Abbreviations: EC, esophageal cancer; ECRG, esophageal cancer-related gene


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Human esophageal cancer (EC) is one of the most common cancers worldwide and occurs at very high frequencies in certain areas of China, Iran, South Africa, Uruguay, France and Italy. EC ranks sixth in terms of cancer mortality in the world (1). Fifty percent of EC cases in the world occurred in China and China has the highest mortality rate for EC in five continents. In China, there are many high EC incidence and mortality rate areas. Areas with high mortality rate of EC are located in southern parts of the Taihang Mountains on the borders of Henen, Shansi and Hopei provinces; Lin-xian county in Henen province has the highest age-adjusted mortality rates for EC, which were 151/100 000 for male and 115/100 000 female (2).

Molecular biological studies showed that genetic abnormalities in several oncogenes and tumor suppressor genes frequently occurred in EC and EC cell lines (3–5), but the mechanism underlying EC tumorigenesis remains poorly understood (6–14). Most recent studies on this cancer have been focused on cloning and identifying novel esophageal cancer-related genes (ECRGs), which might play important roles in the initiation and progression of EC (15,16). The ECRG1 (GenBank accession no. AF071882 [GenBank] .1) was cloned by using messenger RNA differential display technique to compare gene expression between normal esophageal epithelium and EC cells in patients from families with high incidence of (17). Messenger RNA differential display technique was used to compare gene expression between normal esophageal epithelium and EC cells in patients from families with high incidence of esophageal cancer, We report here the isolation of ECRG-1 (GenBank, accession no. AF071882 [GenBank] .1).

Previous analysis using reverse transcriptase–polymerase chain reaction and northern blot showed that the ECRG-1 gene is expressed in normal esophagus, liver, colon and lung, but its expression is down-regulated in tumor samples, especially in esophageal squamous cell carcinoma and its adjacent normal esophageal squamous epithelium (19). The expression of protein encoded by ECRG1 in Escherichia coli was verified by western blot assay; the polyclonal antibody was generated by immunizing BALB/c mice with recombinant ECRG1 protein (20). Furthermore, it has been shown that ECRG1 protein is able to up-regulate P15INK4b expression and induce G1/S cell-cycle arrest by association with Myc-interacting zinc finger protein-1 in esophageal cell line EC9706 (21).

We screened DNA samples from 80 individuals for mutations and single nucleotide polymorphisms in the coding region of ECRG-1. Three single nucleotide polymorphisms were identified and located in exons 3, 8 and 9, respectively. The single nucleotide polymorphism in exon 8 (869G -> A) located in the Asp conserved region of ECRG1 serine protease catalytic domain results in 290Arg -> Gln amino acid change (22). We performed an analysis of 290Arg/Gln polymorphism on 998 EC patients and 1252 controls in a hospital-based, case–control study. We observed a statistically significant increase in risk of EC associated with the ECRG-1 290Arg/Gln and 290Gln/Gln genotypes compared with the 290Arg/Arg genotype (22).

In this study, we demonstrated that ECRG-1 gene is involved in cell-cycle control and can inhibit the EC cell growth in vitro and esophageal tumor formation in vivo.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell culture
NEC cell line was derived from N-nitrosomethylbenzylamine-treated human fetal esophageal epithelia. Cells were maintained in Dulbecco's modified Eagle's medium with 10% newborn calf serum and incubated at 37°C and 5% CO2. Human liver cancer BEL-7402 cells were grown in M199 with 10% newborn calf serum at 37°C and 5% CO2. Hek 293 cells were grown in Dulbecco's modified Eagle's medium with 10% newborn calf serum at 37°C and 5% CO2.

Subcellular localization of ECRG1 protein
Human ECRG1 cDNA was sub-cloned into EcoRI/BamHI site of pEGFP-N1 or pEGFP-C1 (Clontech, Palo Alto, CA) to make a C-terminus or N-terminus fusion to the enhanced GFP. The pEGFP-N1-ECRG1 and pEGFP-C1-ECRG-1 were confirmed to be in frame by DNA sequencing. BEL-7402 and Hek 293 cells were seeded in 35 mm dish and allowed to grow to ~80% confluency. pEGFP-N1-ECRG-1 (2 µg), pEGFP-C1-ECRG-1 (2 µg) and pEGFP-N1 (2 µg) were transfected into the cells. Forty-eight hours after transfection, cells were observed using a fluorescent microscope (excitation wavelength, 488 nm; emission wavelength, 507 nm).

ECRG1 gene transfection and selection
Human ECRG-1 cDNA was sub-cloned into pMAMneo vector (Clontech). pMAMneo-ECRG-1 plasmids were transfected into NEC cells with liposome. Forty-eight hours after transfection, cells were selected in a medium containing 200 µg/ml of G418 (Gibicol, carlsbad, CA). G418-resistant cells with pMAMneo-ECRG1 are referred to as ECRG1 and cells with pMAMneo were referred to as HLE-M. These transfectants were maintained in RPM1-1640 containing 10% fetal bovine serum and 10–6 M dexamethasone was used to induce the expression of ECRG-1 gene. These transfectants were used to study growth inhibition of NEC cell by ECRG-1 gene in vitro.

MTT assay
NEC cells as controls and ECRG-1 transfected cells, at a density of 5 x 104 cells per ml, were seeded in 96-well plates and maintained in 200 µl of RPM1-1640 media plus 10% fetal calf serum and 10–6 M dexamethasone. After incubation for a desired period at 37°C, 10 µl of mg/ml MTT (Sigma, St Louis, MO) was added to each well. Cells were incubated for another 3 h and supernatant was removed. The crystal formazan product was dissolved by adding 200 µl of dimethyl sulfoxide (Fluka) and shaking thoroughly for 10 min. The optical density at 562 nm was measured using spectrophotometer (Graphicord UV-240, Shimadzu, Kyoto, Japan). The results were expressed as percent of control.

Experiment on inhibition of tumor growth in nude mice
ECRG-1 cDNA was sub-cloned into PCDNA3. NEC cells were transfected with either pCDNA3 or pCDNA3-ECRG-1 and transfected cells were selected in 200 µg/ml of G418 (Gibicol, Carlsbad, CA). Tumors were generated subcutaneously in nude mice (male, 4–5 weeks old, from Cancer Institute, Chinese Academy of Medical Sciences, Beijing, China) by the injection of wild-type NEC cells, cells transfected with plasmid pcDNA3 or reconstructed with plasmid pcDNA3-ECRG1. There were five groups of animals and five animals per group. Two groups were given injections of NEC cells transfected with reconstructed plasmid pcDNA3-ECRG-1; the two control groups were given injection of NEC cells transfected with plasmid pcDNA3, and one group was only given injection of NEC cells as a control. Animals were killed 30 days after injection of NEC cells. The tumors were then excised. The weights and volume of tumors were measured. The tissues were frozen in liquid nitrogen, and stored at –80°C or preserved in neutral buffered formalin.

ECRG-1 cDNA was cloned into pGEX-4T-1 (Amersham Pharmacia Piscataway, NY); recombinant proteins were expressed and purified according to the manufacturer's protocol. Recombinant ECRG-1 was added to NEC cells, and MTT assay was performed to measure the proliferation rate. ECRG1 recombinant protein was injected into nude mice with established tumor to observe its tumor inhibition effect in vivo.

Flow cytometry analysis
NEC cells were washed with phosphate-buffered saline, then fixed with ice-cold 70% ethanol and stored at –20°C overnight. Fixed cells were then washed with phosphate-buffered saline and treated with ethidium bromide (400 µg/ml) in 1% Triton X-100 and ribonuclease A (0.5 mg/ml) and analyzed on a Coulter Epics Elite apparatus (Beckman Coulter, Fullerton, CA) to determine the cell-cycle distribution of the cells.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
The subcellular localization of ECRG1 protein
To determine the localization of ECRG1, we expressed GFP-ECRG1 fusion proteins in mammalian cells. BEL-7402 and Hek 293 cells transfected with CRG-1 pEGFP-N1-ECRG-1 or ECRG1 pEGFP-C1-ECRG-1 showed clear GFP signal on the cell membrane. Cells transfected with pEGFP-N1 displayed diffuse GFP signal throughout the cell (Figure 1). The results suggested that ECRG-1 proteins are localized on the cell membrane.


Figure 1
View larger version (39K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Subcellular localization of ECRG1 fusion protein expressed in Bel-7402 cells and Hek 293 cells. Bel-7402 cells and Hek 293 cells were transfected with (A) PEGFP-N1-ECRG1; (B) PEGFP-C1-ECRG1; (C) and (D) PEGFP-N1 vectors.

 
The suppression of tumor growth by ECGR1 gene in vitro and in vivo
Inhibitions of the growth in NEC cells transfected with ECRG1 gene.
To avoid unwanted physiological and toxic effects of ECRG-1 expression, pMAMneo vector containing dexamethasone-inducible MMTV-LTR promoter was used to regulate the ECRG-1 expression in NEC cells. MTT assay was performed to examine the inhibition of NEC cell growth by ECRG-1 expression. The results showed that proliferation rate of NEC cells transfected with ECRG-1 gene was significantly lower than that of NEC cells transfected with empty vector and wild-type NEC cells (Figure 2).


Figure 2
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. The inhibitions on growth of NEC cells transfected with ECRG-1 gene. (Small filled diamond), NEC cells; (small filled rectangle), NEC-neo1; (filled triangle), NEC-neo2; (big filled diamond), NEC-ECRG-1-A; (big filled rectangle), NEC-ECRG-1-B.

 
Inhibition of the tumor growth in nude mice.
NEC cells were transfected with pCDNA3-ECRG-1 or empty vector, stable cell lines were established. NEC cells were injected into nude mice. The tumorigenicity of NEC cells transfected with ECRG-1 gene was decreased (Figure 3A and B), and the weight and volume of tumors were drastically reduced compared with that of tumors formed by wild-type NEC cells and NEC cells with empty vector (Figure 3C).


Figure 3
View larger version (62K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Tumor growth of NEC cells transfected with the ECRG-1 gene in nude mice. (A) Tumorigenicity of NEC cells transfected with ECRG-1 gene in nude mice; (B) sizing of tumors obtained; (C) summary of weight and size of tumors.

 
Our findings indicate that ECRG-1 gene significantly inhibits the growth of NEC cells both in vitro and in vivo.

Inhibition of the tumor cell growth in vitro and tumor growth in vivo with fusion protein encoded by ECRG1 gene
There were significant differences in proliferation rate between NEC cells treated with fusion protein encoded by ECRG-1 gene and control NEC cells (P < 0.001). The growth rate of NEC cells incubated with fusion protein drastically declined by ~70% in comparison with that of NEC control cells (P > 0.05). The growth rate of the NEC tumor treated with recombinant ECRG-1 in nude mice was decreased significantly compared with the control (P < 0.05) (supplementary Table S1 is available at Carcinogenesis Online).

Over-expression of ECRG-1 in NEC cells induced G/M cell-cycle arrest
In vitro cultured NEC cells transfected with ECRG-1 gene were analyzed by flow cytometry. As shown in Figure 4C, 41.6% of the NEC cells transfected with ECRG-1 gene were found in the G2/M stage, only 9.5% of NEC cells Figure 4A and 5.6% of NEC cells Figure 4B with empty vector were at G2/M phase ECRG-1. The flow cytometry analysis showed that over-expression of ECRG1 in NEC cells is able to induce G2/M cell-cycle arrest.


Figure 4
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Alterations of cell-cycle profiles in NEC cells transfected with the ECRG1 gene. (A) Cell-cycle profiles in NEC cells; (B) cell-cycle profiles in NEC cells transfected with plasmid PcDNA3; (C) cell-cycle profiles in NEC cells transfected with ECRG1 gene and injected into nude mice; (D) summary of effect of ECRG1 gene on the cell cycle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
Tumorigenesis is a multistep process that may involve oncogene activation and suppressor gene inactivation (23). Cancerous cells arise through the alterations of the oncogene and tumor suppressor genes, including gene rearrangements, amplifications or deletions. Such genetic changes often affect proteins that are central to cellular growth control, and alteration of these proteins can initiate and/or promote tumorigenesis. The identification and isolation of genes whose expression is markedly altered in cells of one type versus another has provided clues for understanding many biological processes, including neoplastic transformation. Using differential display analysis to compare the messenger RNA expression patterns of esophageal cancerous tissues and normal esophageal epithelia, a novel ECRG, designated ECRG-1, was identified and cloned. The open reading frame of ECRG-1 gene was revealed to be 1176 bp (19). The ECRG1 gene shows no remarkable sequence similarity to known genes.

Bioinformatical analysis demonstrated that the protein encoded by ECRG-1 gene is a member of serine protease family and a putative transmembrane protein. Our GFP localization results confirmed that ECRG-1 is a transmembrane protein. The bioinformatics analysis results also showed that the protein encoded by ECRG-1 is ~40% homologous to the human serine protease. The secondary structure prediction results showed that ECRG-1 protein is mainly composed of a-helix with 30% amino acid, random coil with 46% amino acid and 18% amino acid for the extend strand. The result of prediction of ECRG-1 protein hydrophilicity indicated that the a-superhelix is composed of 21 N-termination amino acids (amino acids 4–23) and the hydrophilicity index is 0.61. ECRG-1 protein has serine phosphorylation site and glycosylation site (data not shown). The predicted serine protease domain is located within the cytoplasmic part of ECRG-1, suggesting that it may be involved in some transmembrane signaling pathway. However, the predicted structure and function of the protein must be further verified by experiment.

In previous studies, we expressed ECRG-1 gene in prokaryotic and eukaryotic cells. The expressions of protein were verified by western blot assay using polyclonal antibody obtained from immunized BALB/c mice (20). In this paper, the in vitro and in vivo assays indicated that recombinant ECRG1 protein inhibited proliferation of tumor cells. The results showed that recombinant ECRG-1 protein may be used in therapy for EC.

The alteration of cell cycle is involved in carcinogenesis. Molecular studies showed that p53 and p16 gene play very important role in regulation of the cell cycle. p53 gene mediates arrest at G1/S checkpoint and accumulates in response to genetic damage that occurs after exposure to chemotherapeutic agents, gamma irradiation or ultraviolet irradiation (24–25). GAAD45, a p53-regulated stress protein, plays an important role in the cell cycle G2/M checkpoint. p16 gene functions in both G/S and G2/M checkpoint response.

In this study, the results showed that ECRG-1 gene and the fusion protein encoded by ECRG-1 inhibited the growth of tumor cells both in vitro and in vivo. In all, 41.6% of the NEC cells transfected with ECRG-1 gene were found in the G2/M stage, suggesting that over-expression of ECRG-1 gene can induce cell-cycle arrest at G2/M phase. It is possible that inhibition of tumor cell growth in vitro and in vivo by either ECRG-1 over-expression or recombinant ECRG1 protein is through the induction of G2/M phase cell-cycle arrest. p16 or GADD45 may be responsible for mediating the G2/M phase arrest induced by ECRG-1. More work is needed to work out the detailed mechanism.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Table S1 can be found at http://carcin.oxfordjournals.org/.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
State Key Basic Research Program, China (G1998051204); British Technology Group, UK (G142291).


   Acknowledgments
 
We thank R.Day for the critical reading of the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

  1. Parkin DM, et al. Estimate of the worldwide frequency of sixteen major cancers in 1980. Int. J. Cancer (1988) 41:184–197.[Web of Science][Medline]
  2. The Coordinating Group for Research on the Etiology of Esophageal Cancer of North China. The epidemiology of esophageal cancer in north China and preliminary results in the investigation of its etiological factors. Sci. Sin. (1975) 18:131–148.[Medline]
  3. Lu SH, et al. Amplification of the EGF receptor and C-myc genes in human esophageal cancer. Int. J. Cancer (1988) 42:502.[Web of Science][Medline]
  4. Montesano R, et al. Genetic alterations in esophageal cancer and their relevance to etiology and pathogenesis: a review. Int. J. Cancer (1996) 69:225–235.[CrossRef][Web of Science][Medline]
  5. Kuroki T, et al. Allele loss and promoter hypermethylation of VHL, RAR-beta, RASSFIA and FHIT tumor suppressor gene on chromosome 3p in esophageal squamous cell carcinoma. Cancer Res. (2003) 63:3724–3728.[Abstract/Free Full Text]
  6. Jiang W, et al. Amplification and expression of the human cyclin D gene in esophageal cancer. Cancer Res. (1992) 52:2980–2983.[Abstract/Free Full Text]
  7. Liang YY, et al. p53 mutations in esophageal tumors from high-incidence areas of China. Int. J. Cancer (1995) 61:611–614.[Web of Science][Medline]
  8. Baron PL, et al. p53 overexpression in squamous cell carcinoma of the esophagus. Ann. Surg. Oncol. (1997) 4:37–45.[CrossRef][Web of Science][Medline]
  9. Chan WC, et al. p16 tumor suppressor gene mutation in Chinese esophageal carcinomas in Hong Kong. Cancer Lett. (1997) 115:201–206.[CrossRef][Web of Science][Medline]
  10. González MV, et al. Mutation analysis of the p53, APC, and p16 genes in the Barrett's oesophagus, dysplasia, and adenocarcinoma. J. Clin. Pathol. (1997) 50:212–217.[Abstract/Free Full Text]
  11. Jiang W, et al. Rapid detection of ras oncogenes in human tumors: application to colon, esophageal and gastric cancer. Oncogene (1988) 4:923–928.[Web of Science]
  12. Jin SQ, et al. Deletions of MTS1/p16 gene in human esophageal carcinoma. Chin. J. Oncol (1998) 20:9–11.
  13. Li HC, et al. Mutation and expression of Rb gene in human esophageal cancer. Chin. J. Oncol (1993) 16:412–415.
  14. Li HC, et al. Mutation of tumor suppressor genes APC and MCC in human esophageal cancer. Chin J Oncol (1995) 17:9–12.
  15. Yang ZQ, et al. Identification of a novel gene, GASC1, within an amplicon at 9p23–24 frequently detected in esophageal cancer cell lines. Cancer Res. (2000) 60:4735–4739.[Abstract/Free Full Text]
  16. Luo A, et al. Discovery of Ca2t-relevant and differentiation–associated genes downregulated in esophageal squamous cell carcinoma using cDNA microarray. Oncogene (2004) 23:1291–1299.[CrossRef][Web of Science][Medline]
  17. Su T, et al. Cloning and identification of cDNA fragments related to human esophageal cancer. Chin. J. Oncol. (1998) 20:254–257.
  18. Netzel-Arnett S, et al. Membrane anchored serine proteases: a rapid expanding group of cell surface proteolytic enzymes with potential roles in cancer. Cancer Metastasis Rev. (2003) 22:237–258.[CrossRef][Web of Science][Medline]
  19. Liu H, et al. Cloning of new related to human esophageal carcinoma and its expression in the human esophageal carcinoma. Proc. Am. Assoc. Cancer Res. (1999) 40. 36, 239.
  20. Yueying Wang, et al. Expression in E.coli of protein encoded by human esophageal cancer related gene-1. Chin. J. Oncol. (2000) 22:198–201.
  21. Zhao N, et al. Induction of G1 cell cycle arrest and P15INK4b expression by ECRG1 through interaction with Miz-1. J. Cell. Biochem. (2004) 92:65–76.[CrossRef][Web of Science][Medline]
  22. Li Yuanyuan, et al. Identification of a novel polymorphism Arg290Gln of esophageal cancer related gene 1(ECRG1) and its related risk to esophageal squamous cell carcinoma. Carcinogenesis (2006) 27:798–802.[Abstract/Free Full Text]
  23. Lu SH. Alterations of oncogenes and tumor suppressor genes in esophageal cancer in China. Mutat. Res. (2000) 462:343–353.[CrossRef][Web of Science][Medline]
  24. Hartwell LH, et al. Cell cycle control and cancer. Science (1994) 266:1821–1828.[Abstract/Free Full Text]
  25. Canman CE, et al. DNA damage responses: p53 induction, cell cycle perturbations, and apoptosis. Cold Spring Harb. Symp. Quant. Biol. (1994) 59:277–286.[Abstract/Free Full Text]
Received January 26, 2007; revised August 28, 2007; accepted August 31, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrowOA All Versions of this Article:
29/1/157    most recent
bgm203v2
bgm203v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Google Scholar
Right arrow Articles by Yueying, W.
Right arrow Articles by Shih-Hsin, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yueying, W.
Right arrow Articles by Shih-Hsin, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?