Medullary thyroid carcinoma (MTC) originates from parafollicular C cells. Estrogen receptor β(ERβ) expressionwas detected in normal parafollicular C cells and MTC tumor tissue, but ERα expression in MTC tumors still remains undetermined. The appearance and loss of ERα or ERβ expression has been known to play a role in the development and progression of many human cancers. We performed immunohistochemical studies of ERα, ERβ, and Ki67, a mitotic index, in 11 human MTC tissue samples. ERα was detected in 10 cases (91%), and ERβ expression was observed in 8 cases (72.7%). A majority (8/10) of ERα-positive tumors showing ERβ Ki67 expression was detected in three cases (27.3%). Neither clinical parameters nor tumor node metastasis (TNM) tumor staging was correlated with the positivity for ERs or Ki67. To investigate the biological role of each ER, we used ER-negative MTC TT cells and adenoviral vectors carrying ERα (Ad-ERα), ERβ (Ad-ERβ), estrogen response element (ERE)-Luc (Ad-ERE-Luc), and activator protein 1 (AP1)-Luc (Ad-AP1-Luc). Estrogen stimulated and anti-estrogen, ICI 182 780, suppressed ERE reporter activity in TT cells expressing ERα or ERβ, suggesting that both ERs use the same classical ERE-mediated pathway. Ad-ERα infection stimulated TT cell growth; in contrast, Ad-ERβ infection suppressed their growth. Apoptosis was detected in Ad-ERβ-infected TT cells. Estrogen and anti-estrogen suppressed AP1 activity in Ad-ERα-infected cells, whereas upon Ad-ERβ infection estrogen further stimulated AP1 activity which in turn is suppressed by anti-estrogen, suggesting that each ER acts differently through a non-ERE-mediated pathway. Our results suggest that ERα and ERβ may play different roles in MTC tumor growth and progression.
Medullary thyroid carcinoma (MTC), which comprises 3–10% of all carcinomas in the thyroid gland, may be either sporadic or familial. Sex ratio is 1:1 with no difference between sporadic and familial MTC (Kebebew et al. 2000a,b). The familial form is part of the multiple endocrine neoplasia type 2 (MENII) syndrome. MTC arises from the parafollicular C cells of the thyroid gland. Metastases to distant sites including lungs, liver, and bone usually occur early. Therefore, any form of effective therapy with curative intent must be systemic (Messina et al. 2000). The primary form of treatment for MTC is total thyroidectomy, but recurrent and metastatic diseases are difficult to manage. Although many patients live with a recurrent or residual tumor for up to a decade, the case fatality rate is ultimately 30–50% over 10 years (Marsh et al. 1995). Because MTC responds poorly to both chemotherapy and radiotherapy, targeted therapy through cancer-specific signaling may offer an alternative approach (Petrangolini et al. 2006).
Parafollicular C cells secrete a polypeptide hormone, calcitonin. Previously, it has been demonstrated that calcitonin gene expression is elevated in response to estrogen, suggesting the presence of an estrogen receptor (ER; Naveh-Many et al. 1992). Immunohistochemical studies using monoclonal antibody revealed that ERβ, but not ERα, was detected in normal human thyroid follicular and parafollicular C cells (Taylor & Al-Azzawi 2000). More recently, a study showed that ERβ was expressed in most of the MTC tissues (Blechet et al. 2007). However, immunohistochemistry results (Bur et al. 1993, Hiasa et al. 1993, Yane et al. 1993, Colomer et al. 1996, Lewy-Trenda 2002, Arain et al. 2003, Blechet et al. 2007) demonstrating ERα expression in thyroid tumors including follicular adenoma or carcinoma, papillary carcinoma, medullary carcinoma, and anaplastic carcinoma remain controversial depending on the antibody source.
Thyroid cancer is more prevalent in women than men, and thyroid cancer incidence decreases after menopause (Cady et al. 1979). These facts suggest a possible involvement of estrogens in the carcinogenesis and progression of thyroid cancer. Demonstration of ERα protein (Lee et al. 1998) and ERα mRNA (Yane et al. 1994) in the normal and malignant tissues of the thyroid seems to further support the hypothesis that there is a direct action of estrogens on the thyroid tissues and tumors. Estrogen has long been known to promote the growth of certain human neoplasms, notably tumors of the breast, endometrium, and pituitary. In contrast, ERβ appears to play a different role in carcinogenesis. Many reports have shown that a loss of ERβ expression is associated with the progression of normal prostate epithelium into prostate cancers (Latil et al. 2001, Leav et al. 2001, Fixemer et al. 2003). A similar down-regulation in ERβ expression is also noted in ovarian, breast, or colon tumors (Pujol et al. 1998, Roger et al. 2001).
The expression of ERs or their biological role in the MTC remains unclear. In this study, we investigated the ERs expression in human MTC tissues and examined the role of ERs using ER-negative MTC TT cells after expression of ERs by adenoviral vectors.
Materials and Methods
Subjects and immunohistochemistry of human MTC tumor
Eleven patients (five men and six women, mean age 48 years) with MTC were included in this study. Ten patients had sporadic MTC and one had MENIIa presenting as bilateral MTC. The diagnosis of an MTC was established by the measurement of serum calcitonin and carcino embryonic antigen (CEA), and confirmed in all cases by positive immunohistochemical staining of tumor tissue for calcitonin (10 cases), CEA (11 cases), or chromogranin-A (10 cases). One patient (case 5) with negative for calcitonin and chromogranin-A had elevated serum CEA and immunohistochemical positivity for CEA. Every MTC were poorly differentiated under light microscopic examination. Metastatic cervical lymph nodes were identified in seven patients (63.6%) and distant metastasis occurred in two patients (16.6%). The study design and protocol was approved by the Internal Review Board of Severance Hospital, Yonsei University College of Medicine.
To detect ERα or ERβ in MTC tumors, 4 μm sections of tumor in paraffin blocks were prepared. After deparaffinization, slides were placed in a jar containing 10 mM sodium citrate buffer (pH 6.0), exposed to microwave radiation (1000 W) for 15 min, and cooled to room temperature. After preincubation with serum blocking solution, slides were incubated with mouse monoclonal anti-human ERα (1:50, sc-8002, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA specific to C-terminal human ERα) or ERβ (1:50, NCL-ERβ, Novo-Castra, specific to C-terminal human ERβ) respectively, overnight in a cold room. After washing with Tris-buffered saline (TBS)/0.025% Tween, staining was performed by biotinylated horse anti-mouse secondary antibodies (5 μg/ml, Vector Laboratories, Burlingame, CA, USA), streptavidin-horseradish peroxidase (RTU, Vector), and diamino benzidine (DAB; DAKO). The concentration of each antibody was titrated using positive and negative control breast cancer tissues to minimize non-specific staining. Counterstaining was performed by malachite green. To investigate a possible correlation of the presence of ERs with a cell proliferative index, Ki67 immunohistochemistry was also performed by mouse monoclonal antibody (1:50, DAKO, Fort Collins, CO, USA). Images were obtained using a Zeiss microscope (Axioskop, Carl Zeiss Inc., Oberkochen, Germany).
Recombinant adenoviral vectors
Adenoviral vectors carrying the human ERα (Ad-ERα) and ERβ (Ad-ERβ) were described previously (Cheng et al. 2004). E1-deleted adenoviral vector (Ad-E) was used as a negative control. Adenoviral reporter vectors, Ad-3ERE-Luc and Ad-AP1-Luc, were created to investigate transcriptional activity of the ERα or ERβ delivered by adenoviral vectors. The sequence containing three consensus estrogen response elements (3 ERE) and minimal TATA promoter was generated by oligonucleotide annealing and ligated to the pGL3-basic plasmid (Promega). A portion of the resulting plasmid containing the upstream synthetic p(A) signal, 3 ERE, a minimal TATA promoter, the firefly luciferase gene, and the downstream SV40 p(A) signal was subcloned into an adenoviral transfer plasmid. The resulting plasmid, pC-3ERE-Luc, was used to generate Ad-3ERE-Luc. Similarly, Ad-AP1-Luc was generated from pC-AP1-Luc, in which the sequence containing seven AP1 sites was switched with three ERE sequences in pC-3ERE-Luc. The sequences of the expression cassettes in the adenoviral vectors were confirmed by automated DNA sequencing.
Cell culture and infection with recombinant adenoviruses
TT cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12+10% FBS supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin and were incubated at 37 °C and 5% CO2.
For infection with adenoviral vectors, cells were first depleted of estrogen for 3 days using phenol red-free DMEM/F12 containing 5% dextran/charcoal-stripped FBS. Transduction efficiency of the adenoviral vectors in cell lines was tested using AdGal. β-galactosidase expression was detected in 95–100% of TT cells at 48 h after infection with AdGal at a multiplicity of infection (MOI) of 10 plaque-forming units (PFU) per cell (data not shown). Therefore, subsequent experiments were performed by similar amounts (10 or 20 PFU/cell) of recombinant adenoviral vectors.
The transcriptional activities of ERα or ERβ were assayed using an artificial estrogen-responsive reporter (Ad-3ERE-luc) or AP1 reporter (Ad-AP1-luc) in a viral vector (Lee et al. 2001). Briefly, 12-well plates of TT cells were infected for 5 h with 10 PFU/cell Ad-3ERE-Luc or Ad-AP1-Luc with Ad-ERα or Ad-ERβ. Fresh medium containing ethanol vehicle, 17β-estradiol (E2, 1 nM, Sigma), or ICI 182 780 (100 nM, Tocris, Ellisville, MO, USA), a specific estrogen antagonist, was added; incubation continued for 24 h; and luciferase activity was assayed.
Detection of ERs expression using immunofluorescence and western blot analysis
TT cells on glass slides 48 h after infection with adenoviral vectors were fixed in ice-cold methanol for 10 min and permeabilized with 0.4% Triton-X 100 in PBS for 20 min on ice. After blocking with 10% horse serum for 30 min, specimens were incubated with mouse monoclonal anti-human ERα or ERβ for 1 h at room temperature. After washing with TBS+0.025% Tween, specimens were incubated with biotinylated horse anti-mouse secondary antibodies (5 μg/ml, Vector Laboratories) for 30 min. Streptavidin-fluorescein isothiocyanate (FITC) (1:100, Vector) and Streptavidin-Texas-Red (1:100, Vector) were used for the detection of ERα and ERβ respectively. Cell images were analyzed using a Zeiss microscope (Axioskop, Carl Zeiss Inc).
Infected TT Cells in 10 cm culture dishes at a density of 5 × 106 cells/dish were washed twice with PBS, and whole cell lysates were prepared with lysis buffer (25% glycerol, 0.5 M NaCl, 1.5 mM MgCl2, 20 mM HEPES (pH 7.9), 1 mM phenylmethylsulfonyl fluoride, 0.2 mM EDTA, 25 mM NaF, and protease inhibitor cocktail tablets (Roche Molecular Biochemicals)). Equal amounts of protein (10 μg) were resolved by SDS–PAGE on 10% gel and transferred to nitrocellulose paper. ERα or ERβ was detected using the antibodies as described above.
The effect of ERs on TT cell growth
The effect of ERα or ERβ on TT cell growth was measured with a non-radioactive cell proliferation assay according to the manufacturer’s protocol (Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay, Promega). Cells were seeded onto 96-well plates at a density of 8 × 103 cells/well and infected the following day with adenoviral vectors at different MOIs (10 and 20 PFU/cell). Medium containing ethanol vehicle, E2 (1 nM), or ICI 182 780 (100 nM) was replaced at 5 h after infection and every two days thereafter. Octaplicate wells were assayed for viable cell density at day 8. Relative density was calculated as absorbance at 490 nm divided by that of the Ad-E-infected, 0 nM E2-treated cells, and expressed as a percentage (mean ± s.d).
Terminal deoxynucleotidyltransferase (TdT)-mediated UTP end labeling (TUNEL) assay
TT cells were infected with adenoviral vectors (10 PFU/cell), treated with 1 nM E2 for 6 days, and then washed twice with PBS and mounted on glass slides. Cells were fixed for 30 min in 4% paraformaldehyde and permeabilized with buffer containing 0.1% sodium acetate and 0.4% Triton X-100 for 10 min on ice. After washing with PBS, a modified TUNEL was performed by the In Situ Cell Death Detection Kit, Fluorescein (Boehringer Mannheim Co). TUNEL-positive cells were counted in ten different fields (× 200) and positivity was calculated.
Values are expressed as mean ± s.d. Data analysis was performed by the Statistics Package for Social Sciences (SPSS system for Windows 12.0, SPSS Inc., Chicago, IL, USA). Differences between groups were analyzed using Student’s t-test, and P < 0.05 was considered statistically significant.
Expression of ERs in human MTC tumor
ERα was detected in 10 out of 11 (90.9%) tumors. Five tumor samples exhibited higher (50% cells positive) levels of ERα expression. ERβ was absent in three (27.3%) tumors. Eight tumor samples were positive for ERβ, and five of them exhibited higher (> 40% cells positive) levels of ERβ positivity. The one without ERα also did not have ERβ expression. Five tumors with higher ERα expression also had higher ERβ expression, and three of these were also positive for Ki67 positivity. There was no difference in ERα and ERβ expression according to gender, TNM tumor staging, or survival (Table 1). Positivity for ERs or Ki67 was not correlated with serum levels of calcitonin and CEA. We also performed immunohistochemical staining of ERs in normal human thyroid tissue as a control and found ERβ expression in parafollicular C cells and thyrocytes. ERβ expression was also detected in parafollicular C cells and thyrocytes surrounding the tumors. However, we did not detect ERα in normal thyrocytes or parafollicular C cells from either normal thyroid gland or normal thyroid tissues adjacent to the tumors (Fig. 1A).
Expression of ERs in TT cells infected with adenoviral vectors
First, we examined the presence of ERα or ERβ in MTC TT cells by immunofluorescence staining and western blot analysis. ERs were not detected in TT cells (data not shown). Secondly, we infected TT cells with Ad-ERα or Ad-ERβ. Ad-E-infected cells served as a negative control. The expression of ERα or ERβ was detected by immunofluorescence in the nuclei of TT cells infected with Ad-ERα or Ad-ERβ, but not in cells infected with Ad-E (Fig. 2A and B). The ER expression was detected in 85–95% of TT cells infected with 10 PFU/cell of adenoviral vectors (Fig. 2A and B). Then, further experiments were performed by equivalent amount of adenoviral vectors.
Transcriptional activity of ERs delivered by adenoviral vectors
We evaluated whether expression of ERs delivered by adenoviral vectors activates an estrogen-responsive reporter gene, by co-infecting Ad-3ERE-Luc into TT cells with Ad-ERα or Ad-ERβ. As shown in Fig. 3, ERE reporter gene activity was not detected with or without E2 in control Ad-E-infected cells. Infection with Ad-ERα or Ad-ERβ increased reporter activity without ligand, presumably due to residual estrogen in the medium. E2 (1 nM) addition activated reporter activity by ten- and fivefold in Ad-ERα- and Ad-ERβ-infected cells respectively. ICI 182 780, an anti-estrogen treatment, suppressed reporter activities. These results suggested that expression of ERα or ERβ delivered by adenoviral vectors is functionally active in the ERE reporter system and that both ERs act similarly in the classical ERE-mediated pathway in TT cells.
Effect of ERα or ERβ on TT cell growth
To investigate whether the introduction of ERα or ERβ in TT cells affects cell growth, we analyzed the proliferation of TT cells infected with two different doses (10 and 20 PFU/cell) of adenoviral vectors. Ten PFU/cell of Ad-ERα stimulated TT cell growth without E2 treatment, and the treatment with E2 (1 nM) for 8 days stimulated cell growth further. With a higher dose (20 PFU/cell) of Ad-ERα, growth stimulation was observed in the E2 treatment. Ad-ERβ infection resulted in the suppression of TT cell growth regardless of E2 treatment. Growth suppression was more prominent with a higher dose (20 PFU/cell) of Ad-ERβ. ICI 182 780 treatment suppressed the growth of TT cell infected with either Ad-ERα or Ad-ERβ, and growth suppression was more prominent in Ad-ERβ than Ad-ERα infection (Fig. 4). A positive TUNEL reaction was obtained in Ad-ERβ infection (19.8%), whereas positivity was negligible in Ad-ERα-infected (3.2%) or Ad-E-infected (< 1%) cells (Fig. 5).
Effect of ERs on AP1 pathway in TT cells
To investigate the possible mechanisms of different effects of ERα or ERβ expression on TT cell growth, we evaluated a non-ERE-mediated pathway in TT cells using the AP1 reporter system (Paech et al. 1997). Co-infection of Ad-AP1-Luc into TT cells with Ad-ERα or Ad-ERβ was performed. As shown in Fig. 6, AP1 reporter gene activity was negligible in control Ad-E-infected cells. Infection with Ad-ERα or Ad-ERβ increased AP1 reporter activity without ligand. After the addition of E2, AP1 activity was decreased in Ad-ERα-infected cells, but increased in Ad-ERβ-infected cells. ICI 182 780 treatment decreased AP1 activity in both Ad-ERα- or Ad-ERβ-infected cells, but its activity was higher in Ad-ERβ-infected cells than those infected with Ad-ERα. These results suggest that non-ERE-mediated signals are distinct in TT cells expressing ERα or ERβ and could also explain the different effects on cellular growth and proliferation.
Previously, ERβ expression has been reported in normal thyroid parafollicular C cells (Taylor & Al-Azzawi 2000) and MTC tumor tissues (Blechet et al. 2007). The presence of mRNA for ERα and ERβ has also been demonstrated in the human MTC tumor tissues (Egawa et al. 2001). However, the role of each ER expression is not well defined after cancerous change. In this study, we have shown an appearance of ERα (10/11) and a loss of ERβ expression (3/11) in the human MTC tissue samples. It has been well demonstrated that expression of ERβ mRNA and protein decreased or ERα/ERβ mRNA ratio increased in tumor versus normal tissues in many cancers including breast, ovary, prostate, and colon (Brandenberger et al. 1998, Foley et al. 2000, Rutherford et al. 2000, Campbell-Thompson et al. 2001, Roger et al. 2001, Fixemer et al. 2003, Bardin et al. 2004). Consistent with these reports, appearance of ERα and loss of ERβ in MTC tumor tissue in our study may result in the increased ratio of ERα/ERβ, suggesting a possible role in tumor growth and progression. Although Ki67, a mitotic index, was detected in both ERα- and ERβ-positive tumors, its expression did not correlate with ERα and ERβ expression because these three Ki67-positive tumors had similar levels of both ERα and ERβ. In addition, the expression of ERs did not correlate with cervical lymphadenopathy or distant metastasis (TNM staging), suggesting other signals (Wang et al. 1997, Mustafa et al. 2004), besides ERs, are involved in tumor growth and progression. Of note is that the number of MTC cases in our study was also limited.
The presence of ERα expression could also open the possibility of using anti-estrogenic compounds for the treatment of MTC. In one report, tamoxifen inhibited the growth of medullary carcinoma DR081 cells in an in vitro culture and in vivo tumor cell xenograft implants (Weber et al. 1990). In another report, tamoxifen treatment failed to show clinical or biochemical improvement of two MTC patients (one man and one woman) after 1–3 months of therapy (Garcia-Pascual et al. 1993). The presence of ERs was not identified in either of these reports. It has been suggested that ERβ is significantly up-regulated in tamoxifen-resistant breast cells and could be involved in tamoxifen resistance (Speirs et al. 1999a,b). Although ERβ was still detected in most ERα-expressing MTC tumor tissues in our study, tamoxifen could be a potential therapeutic agent for the treatment of MTC. The effect of this reagent could be evaluated through therapeutic trial as an adjuvant treatment in both genders (Garcia-Pascual et al. 1993).
To examine the roles of ERα and ERβ in MTC, we used adenovirus-mediated expression of each ER in MTC TT cells devoid of both endogenous receptors. We observed that both ERα and ERβ expressed by adenoviral vector were functional in TT cells, resulting in the activation of ERE-mediated gene transcription in a reporter assay. However, we obtained opposing cellular responses with regard to proliferation and apoptosis. ERα acted as a growth stimulator with estrogen addition, whereas ERβ suppressed cell growth regardless of estrogen treatment in this study. We also investigated whether the different effects of each ER on TT cell growth could be reversed by ICI 182 780, an anti-estrogen, but ICI 182 780 suppressed the growth of TT cells expressing either ERα or ERβ. Growth suppression was more prominent in Ad-ERβ-infected cells. This result suggests the presence of other pathways besides the classical ERE-mediated pathway.
The ER has been known to regulate target genes by at least two major pathways. The classical pathway for ER signaling is mediated by receptor binding to EREs. This mode of ER action has been documented for many genes involved in reproduction as well as genes involved in cellular growth and metabolism (Jakacka et al. 2001, 2002). In addition to this classical pathway of estrogen action, the ER can also alter gene transcription by a mechanism that does not require direct ER binding to an ERE. This non-classical pathway affects gene expression from promoters containing an AP1 site (Paech et al. 1997, Jakacka et al. 2001, O’Brien et al. 2006). We tested this possibility using AP1-Luc, the representative non-ERE-mediated reporter system (Paech et al. 1997). Adenovirus carrying AP1 Luc (Ad-AP1-luc) was created and co-infected into TT cells with Ad-ERα or Ad-ERβ. The effect of estrogen and anti-estrogen on AP1-reporter in TT cells was distinct according to ERα or ERβ expression. Estrogen and anti-estrogen treatment suppressed AP1 activity in Ad-ERα-infected cells, whereas alongside Ad-ERβ infection, estrogen further stimulated AP1 activity which in turn is suppressed by anti-estrogen. Suppressed AP1 activity by ICI 182 780 was still higher in ERβ than ERα expression. This result indicates that signals other than the classical ERE-mediated pathway might regulate genes including apoptotic factors and negative cell cycle regulators. Genes responsible for regulating the different actions by ERα or ERβ still remain to be determined in these TT cell models.
A series of studies revealed that the ERβ presence confers a more favorable prognosis and is also associated with low-grade tumors, low S-phase fraction, negative axillary node status, and a higher disease-free survival rate (Jarvinen et al. 2000, Omoto et al. 2001). In addition, exogenously expressing ERβ not only inhibits proliferation and invasion but also induces apoptosis in many cancer cells (Lazennec et al. 2001, Qiu et al. 2002, Cheng et al. 2004, Paruthiyil et al. 2004). Repression of c-myc, cyclin D1, and cyclin A gene transcription and increasing the expression of p21Cip1 and p27Kip1 were demonstrated by ERβ expression (Paruthiyil et al. 2004). These result in a G2 cell cycle arrest. The activation of caspase-3 has also been demonstrated in ER-negative prostate cancer cells, in which ERβ was reintroduced by adenoviral vector (Cheng et al. 2004). Although we did not find an association between ERβ expression and tumor behavior in human MTC tumor samples, an anti-tumor effect of ERβ expression was observed in the TT cell line. These results indicate a potential anti-neoplastic role of ERβ, which is in good agreement with previous studies (Lazennec et al. 2001, Qiu et al. 2002, Cheng et al. 2004, Paruthiyil et al. 2004), and also suggest different roles of ERα and ERβ in MTC tumor growth. New therapeutic approaches for MTC might include pharmaceutical agents to block ER activity and gene therapy by delivery of ERβ.
Summary of clinical and pathologic characteristics of patients with medullary thyroid carcinoma
|Sex||TNM (staging)||Duration of follow-up (years)||Age at diagnosis||Calcitonin (pg/ml)||CEA (ng/ml)||ERα||ERβ||Ki67|
|A, alive; L, lost to follow-up; ND, not done. Normal range: calcitonin, male: < 90 pg/ml; female: < 70 pg/ml; CEA, 0–2.5 ng/ml. Immunohistochemistry score according to the percentage of positive cells: −, negative; +, 5–15%; ++, 15–40%; +++, 40–60%; ++++, > 60%.|
|1||M||T2N1b M0(IVA)||13 (A)||32||320||27.1||−||−||−|
|2||M||T2N1a M0(III)||7 (A)||35||156||2.3||+||+||−|
|3||F||T2N1a M0(III)||8 (A)||58||459.6||2.55||++||−||−|
|4||F||T3N0 M0(II)||7 (A)||29||6.33||0.6||+++||+++||−|
|7||F||T2N1b M0(IVA)||6 (A)||66||75.1||11.3||++||+||−|
|9||F||T3N1 M0(III)||2 (L)||55||2474||2.34||+++||+++||+|
We thank Su-Il Ji for tissue preparation. This work was supported by the Internal Medicine Research Grant 2006–2007 from the Yonsei University College of Medicine and a New Faculty Grant (2007) from the Yonsei University. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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