| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Biochemistry, Chang-Gung University, 259 Wen-hwa 1 Road, Taoyuan, Taiwan, Republic of China
1 First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan, Taiwan, Republic of China
(Requests for offprints should be addressed to K-H Lin; Email: khlin{at}mail.cgu.edu.tw)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 or TRß1 over-expressing HepG2 cell lines were used. Growth of the HepG2-TR stable cell line was inhibited by over 50% following treatment with T3. However, transforming growth factor (TGF)-ß neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3. Flow cytometric analysis indicated that the growth inhibition was apparent at the transition point between the G1 and S phases of the cell cycle. The expression of major cell cycle regulators was used to provide further evidence for the growth inhibition. Cyclin-dependent kinase 2 (cdk2) and cyclin E were down-regulated in HepG2-TR cells. Moreover, p21 protein or mRNA levels were up-regulated by around 5-fold or 7.3-fold respectively following T3 treatment. Furthermore, phospho-retinoblastoma (ppRb) protein was down-regulated by T3. The expression of TGF-ß was studied to delineate the repression mechanism. TGF-ß was stimulated by T3 and its promoter activity was enhanced six- to eight-fold by T3. Furthermore, both T3 and TGF-ß repressed the expression of cdk2, cyclin E and ppRb. On the other hand, TGF-ß neutralizing but not control antibody blocked the repression of cdk2, cyclin E and ppRb by T3. These results demonstrated that T3 might play a key role in liver tumor cell proliferation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Two main types of TRs have been identified, termed TR and TRß, which are encoded on human chromosomes 17 and 3 respectively (Cheng 2000, Aranda & Pascual 2001). Transcripts of each of these genes undergo alternative promoter choice for generating both the TR1 and 2 and the TRß1 and ß2 receptor isoforms (Cheng 2000, Hulbert 2000, Aranda & Pascual 2001).
Regarding previously published results (Lin et al. 2004), transforming growth factor-beta (TGF-ß) was stimulated by T3 at the mRNA level. TGF-ß regulates cell growth and proliferation, and has been shown to block the growth of numerous cell types (De Caestecker 2004). The TGF-ß receptor includes type 1 and type 2 subunits. These subunits comprise serine-threonine kinases that signal through the smad family of transcriptional regulators. T3/T4 have been shown to stimulate the proliferation of eukaryotic cells (Barrera-Hernandez et al. 1999, Aranda & Pascual 2001). Several studies demonstrated that cyclin D1 induction is an early event in T3-induced hepatocyte proliferation (Pibiri et al. 2001, Alisi et al. 2004). These previous studies indicate that this cyclin may be a common target responsible for mitogenic activity of ligands of nuclear receptors. However, the influence of T3 on human liver tumor cell proliferation is currently unknown although a similar observation has been reported in rats (Ledda-Columbano et al. 2000).
The liver has long been recognized as a target organ for thyroid hormones. In fact, Chamba et al.(1996) reported that roughly equal quantities of TR1 and TRß1 protein occur in human hepatocytes (Chamba et al. 1996). HepG2 is a well-differentiated hepatocellular carcinoma cell line without detectable TR protein expression. However, it secretes all 15 plasma proteins and preserves numerous liver-specific functions and thus can serve as an in vitro model (Chang et al. 1983). Consequently, the HepG2 cell line provides a useful model system for studying the influence of T3 on the proliferation of liver tumor cells. The system was recently used to demonstrate that TGF-ß is regulated by T3 (Lin et al. 2004). This work shows that T3 up-regulates the expression of TGF-ß and subsequently suppresses liver tumor cell proliferation.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Human hepatoma cell lines, HepG2-TR1#1, HepG2-TR1#2, HepG2-TRß1 and HepG2-Neo, and rat pituitary tumor GC cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. Three TR over-expressing lines, and the control cell line, HepG2-Neo, have been described previously (Lin et al. 2004). The serum was depleted of T3 (Td) as described by Samuels et al.(1979). Cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2.
Flow cytometry
Flow cytometric analysis was performed as described by Fan et al.(1995). Briefly, cells were harvested via trypsinization and fixed in 75% ethanol for at least 24 h at 4 °C. The cells were then washed with PBS containing 1% BSA (Life Technologies, Inc., Rockville, MD, USA) and incubated with 100 µg/ml RNase A (Sigma, St Louis, MO, USA) and 50 µg/ml propidium iodide (Sigma) for 2 h at room temperature. Finally, the stained cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).
Cell proliferation assay
Cells were plated on 6-cm dishes at 2 x 105cells/dish, with each sample being plated in triplicate. Cells were counted using the Coulter Counter ZM (Coulter Electronics Inc., Luton, Beds, UK).
Immunoblot analysis
Cell lysates were fractionated using SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and the separated proteins were transferred to a nitrocellulose membrane (Pall Life Sciences, Ann Arbor, MI, USA) and subsequently visualized via chemiluminescence using an ECL detection kit (Amersham Inc., Piscataway, NJ, USA) as described previously (Shih et al. 2004). The antibodies used were rabbit polyclonal antibodies to cyclin E, and retinoblastoma (Rb) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or mouse monoclonal antibody to cdk2 (1:1000 dilution in PBS) (Santa Cruz Biotechnology) or TGF-ß (1:500–1000 dilution in PBS) (Serotec Ltd, Oxford, Oxon, UK). TGF-ß1 was purchased from Pepro Techec (London, UK).
Northern blot analysis
Total RNA was extracted from the cells using TRIzol Reagent (Life Technologies) and equal amounts of total RNA (20 µg) were analyzed on a 1.2% agarose-formaldehyde gel as described previously (Lin et al. 2000, 2002). The separated RNA molecules were then transferred to a nylon membrane (Amersham) and subjected to northern blot analysis as described previously (Lin et al. 2004, Shih et al. 2004).
Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR)
Total RNA was extracted from cells using TRIzol, as described above. Subsequently, cDNA was synthesized using the Superscript II kit for RT-PCR (Life Technologies) as described previously (Shih et al. 2004).
Real-time Q-RT-PCR was performed in a 25-µl reaction mixture containing 50 nM forward and reverse primers, 1x Syber Green reaction mix (Applied Biosystems, Werrington, UK), and various quantities of template as described previously (Shih et al. 2004). Fluorescence emitted by Syber Green was detected using the ABI PRISM 7000 sequence detection system (Applied Biosystem), as described previously (Lin et al. 2004, Shih et al. 2004).
Cloning the TGF-ß 5'-flanking region and promoter activity assay
Fragments of the TGF-ß promoter (nucleotides –1362/+101) were amplified via PCR, according to the published nucleotide sequence (Kim et al. 1989a,b), and were then inserted into the pGL3 vector (Promega Corp., Madison, WI, USA). The sequence of the promoter construct was confirmed by automated DNA sequencing. To determine the influence of T3 on the transcriptional activity of the TGF-ß promoter, HepG2-TR1#1 cells (1 x 105 per 35-mm dish) were co-transfected, via a Lipofectamine protocol, using 3 µg pGL3 vector containing TGF-ß promoter sequences (Invitrogen) as described previously (Shih et al. 2004).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
1 and HepG2-TRß1 cell growth by prolonging the G0/G1 phase
The effect of T3 on cell proliferation was assayed in three HepG2 stable cell lines with high expression of TR1 or TRß1 as previously described (Lin et al. 2004). Figure 1
shows that without T3 cells proliferated roughly 2 to 3 times faster than those grown in media containing either 10 or 100 nM T3 in three TR stable lines. However, this T3-repressed effect was not observed in the control cell line, HepG2-Neo, which did not express TR (Fig. 1). Following T3 treatment, the doubling time for HepG2-TR stable lines increased from 1.03 ± 0.12 to 2.49 ± 0.31 days for TR1#1 cells, and from 1.15 ± 0.21 to 2.64 ± 0.25 days for TRß1 cells; thus T3 repressed the growth of HepG2-TR cells by around two- to three-fold. However, T3 did not influence the doubling time in HepG2-Neo cells (1.58 vs 1.67 days, 0 vs 100 nM T3). All data indicate that T3 significantly suppresses the growth of HepG2-TR but not HepG2-Neo cells.
|
illustrates that the addition of T3 increased the percentage of cells in the G0/G1 phases by roughly 1.2-, 1.4-, and 1.35-fold following 12, 24, and 48 h respectively. Simultaneously with the increase in cell number in the G0/G1 phases, the percentage of cells in the S phase was reduced in HepG2-TR1#1 (Table 1). Similar results occurred in HepG2-TRß1 (Table 1) and HepG2-TR1#2 cells (data not shown).
|
The major kinase associated with cyclin E in human cells is cdk2. The formation of the cyclin E–cdk2 complex is an important step in the biochemical pathway that controls cell proliferation during G1. Additionally, cyclin E is one of the key regulators of the G1/S transition in the cell cycle. Over-expression of cyclin E has been noted in several malignancies and is associated with high cell proliferation (Keyomarsi & Herliczek 1997, Nielsen et al. 1998). Therefore, this study used western blot analysis to determine that the increase in the G0/G1 cell population was inversely associated with the level of cyclin E-cdk2 proteins. Treatment of HepG2-TR1#1, -TR1#2 and -TRß1 cells using 100 nM T3 led to a down-regulation of approximately 32 and 52% in the level of cdk2 protein following 24, and 48 h compared with T3-depleted media (Td) in HepG2-TR1#1 cells (Fig. 2A, B). Similarly, 100 nM T3 led to down-regulation of approximately 27 and 31% in the protein level of cyclin E following 24 and 48 h (Fig. 2A, B). A similar effect was observed in the HepG2-TRß1 (Fig. 2C), and HepG2-TR1#2 cell lines (data not shown). Taken together, repression of both components of the cyclin E–cdk2 complex strongly agrees with the previous result of cell cycle blockage in G0/G1.
|
Levels of the negative regulator of cell cycle progression, p21 protein, increase in senescent cells, while p21 over-expression has been demonstrated to block tumor cell growth (Gong et al. 2003). Thus, p21 was investigated as an additional target for controlling cell proliferation. p21 mRNA was strongly induced 1.3-, 3.9-and 7.3-fold in HepG2-TR1#1 cells at 12, 24, and 48 h respectively, following the addition of 10 nM T3 to the media (Fig. 3A, B). Similarly, p21 protein was also significantly induced two- to five-fold by T3 treatment in two TR1 stable cell lines (Fig. 3C, D). However, T3 did not markedly increase p21 mRNA or protein expression in the control cell line, HepG2-Neo (data not shown).
|
Rb tumor suppressor is a critical negative regulator of cellular proliferation. The Rb protein was de-phosphorylated (Fig. 4A) in HepG2-TR1#1 or two other TR stable cell lines (data not shown) following T3 addition, possibly indicating that cyclin E was inactivated by p21. The expression of hyperphosphorylated Rb protein increased significantly following 24 or 48 h T3 treatment when HepG2-TR1 cells were incubated in control conditions (Td) (Fig. 4A). As a further positive control, the phosphorylation status of Rb was investigated in the GC cell line. Consistent with previous studies (Barrera-Hernandez et al. 1999), Rb was hyperphosphorylated in GC cells after T3 treatment for 48 h (Fig. 4B). However, T3 did not significantly change the phosphorylation status of Rb in the control cell line, HepG2-Neo (data not shown). These experimental results indicate that the incubation of HepG2 cells over-expressing TR in media containing T3 represses the hyperphosphorylation of Rb.
|
To better understand how T3 inhibited the proliferation of HepG2-TR1 cells, this study investigated the influence of T3 on the TGF-ß expression. Similar to previously published results (Lin et al. 2004), TGF-ß at the mRNA level was significantly stimulated about 1.5- to 3-fold by T3 in HepG2-TR1#1 cells (Fig. 5A). Additionally, the 12 kDa TGF-ß protein was up-regulated about two- to threefold 24 or 48 h following the addition of T3 (Fig. 5B). To further clarify the influence of T3 on TGF-ß at the transcriptional level, the TGF-ß 5'-flanking region (from –1362 to +101) was cloned into the pGL3 vector and its activity was assayed. T3 was demonstrated to increase the promoter activity by approximately 7.8- and 5.8-fold at 10 and 100 nM concentrations of T3 respectively in the TR1 stable cell line, compared with its activity in control (Td) media without the addition of T3 (Fig. 5C). However, T3 did not considerably increase the promoter activity in the control cell line, HepG2-Neo (Fig. 5C).
|
To clarify the signaling pathways involved in the repression of cyclin E, cdk2 and Rb by T3, this study investigated the involvement of TGF-ß. The data indicate that treating cells with T3 for 48 h represses cdk2 expression by at least 40% at the protein level compared with the control (Td) conditions (Fig. 2A, B
; Fig. 6A, B, C, lane 1 vs 2). Moreover, TGF-ß alone also repressed the expression of cdk2 following 40 min treatment (Fig. 6A, B, C lane 1 vs 3). Notably, either T3 or TGF-ß repressed the expression of cyclin E, cdk2, and rendered the Rb protein in the hypophosphorylated form (Fig. 6A, B, C lane 1 vs 2 and 3) in the HepG2-TR cells. Importantly, the repression of cyclin E, cdk2, and ppRb by T3 was blocked by the addition of TGF-ß neutralizing antibody (nAb) (Fig. 6A, B, C lane 2 vs 6), but not by the non-specific antibody (nsAb) (Fig. 6A, B, C, lane 2 vs 5) in the HepG2-TR1#1, -TR1#2, and -TRß1 stable lines. However, the effects of T3 and TGF-ß were not observed in the Neo cells (Fig. 6D) and did not synergistically repress the expression of cyclin E, cdk2, and ppRb (Fig. 6A, B, C lane 4 vs 2 and 3) in the HepG2-TR cells. Moreover, TGF-ß neutralizing antibody, but not the control antibody can reverse the cell growth inhibition effect of T3 (Fig. 1). Thus, cell proliferation is repressed by T3 through a TGF-ß- mediated mechanism. Additionally, T3 controls the expression and activity of a number of cell cycle regulators via TGF-ß, including p21, pRb and the cyclin E–cdk2 complex.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
. PCNA is an essential auxiliary protein of DNA polymerase , and is synthesized in the early G1 and S phases for processing of DNA replication. PCNA mRNA was repressed following T3 treatment in HepG2-TR stable lines (data not shown). This provides further support for the notion that T3 can suppress proliferation in cells that express TR.
cdk2 has been demonstrated to play a pivotal role in regulating cell cycle progression, is regulated by phosphorylation and can associate with cyclins A, E, D1, and D3. This investigation found that the inhibitory effect of T3 on cell proliferation occurs during the G0/G1 phase of the cell cycle. Interestingly, the cdk2–cyclinE complex is active in the G1 and S phases and is important for the progression from G1 to S (Harwell et al. 2004, Lents & Baldassare 2004). Additionally, cyclin E is pivotal in regulating the restriction point transition in the cell cycle. The role of cyclin E as a cdk2 activator in controlling restriction point transition in the cell cycle makes cyclin E an excellent candidate as a factor for involvement in tumor development. The hepatoma cell system presented here demonstrated reduced expression of both cyclin E and cdk2 following T3 application, although further study is required to determine whether this phenomenon is because of a direct or an indirect effect. Aberrant regulation of cyclin E is a common phenomenon seen in tumor cells and has been reported in tumor tissues isolated from breast cancer patients (Akli et al. 2004, Harwell et al. 2004). An interesting study by Pibiri et al.(2001) demonstrated that, in Wistar rats, hepatocyte proliferation induced by T3 occurred in the absence of AP-1, nuclear factor-
B, and STAT3 activation or any change in the mRNA levels of the immediate early genes c-fos, c-jun, and c-myc. However, this study found that T3 treatment increased cyclin D1 mRNA and protein levels, and moreover this increase occurred much more rapidly than liver regeneration following a two-thirds partial hepatectomy. Regrettably, the expression level of TGF-ß was not examined. In contrast, in this investigation, we did not observe any significant change in the level of cyclin D1 expression following T3 treatment (data not shown). It is possible that T3 influences the late G1 but not the early G1 phase. The cyclin D1–cdk4, cdk-6 complexes are activated in early G1, whereas cyclin E–cdk2 is activated in late G1 (Sherr 1996, Martin-Castellanos & Moreno 1997). Therefore, our observations regarding hepatoma cells differ from those for normal regenerating rat liver cells. Summarizing the results of this and previous studies, T3 appears both to induce proliferation of normal hepatocytes and to suppress the proliferation of hepatoma cells with TR expression.
p21 can bind and inhibit each member of the cdk family. It also directly binds to PCNA and thus inhibits DNA replication. The present work showed that the expression of p21 was stimulated markedly by T3 at both the mRNA and protein levels, and may be, at least partially, responsible for blocking cell proliferation.
TGF-ß is a pleiotropic cytokine that elicits a broad range of cellular responses, including cell growth, differentiation, and apoptosis. One of the biological effects of TGF-ß is to inhibit epithelial cell proliferation by inducing cell cycle arrest (Sporn & Roberts 1992, Massague 1998). The effectors of TGF-ß-induced cell growth inhibition are cyclin-dependent kinase inhibitors; among these inhibitors, p21Cip1 plays a major role in numerous biological contexts (Li et al. 1995). Additionally, Buzzard et al. (2003) demonstrated that T3 and other hormones induced the progressive accumulation of the cell cycle inhibitors p27Kip1 and p21Cip1 in Sertoli cells. However, the underlying mechanisms responsible for suppressing proliferation remain largely unknown (Gong et al. 2003). This study found that T3 acted on the TGF-ß promoter to activate transcription. However, further work is required to determine whether this activation is due of a direct or an indirect effect. Therefore, in these hepatoma cells, the two signaling pathways are linked and TGF-ß works downstream of the T3 pathway.
Rb is a nuclear phosphoprotein that undergoes differential phosphorylation during the cell cycle. Hypophosphorylated Rb protein complexes with E2F to inhibit its trans-activity on target genes and thus halts cell cycle progression. Barrera-Hernandez et al.(1999) reported that T3 increased the phosphorylation of Rb protein and thus stimulated GC cell line proliferation, similar to the results obtained here using these cells. By contrast, in this study T3 caused the accumulation of hypophosphorylated Rb protein, thus suppressing cell cycle progression in hepatoma cells overexpressing TR proteins.
Shimizu et al.(2004) reported that OSI-461 (a potent protein kinase G activator) enhanced the G0/G1 arrest resulting from acyclic retinoid (belonging to the thyroid/steroid super-family), and a combination of these agents synergistically decreased expression of cyclin D1 protein and mRNA, inhibited cyclin D1 promoter activity, reduced the level of hyperphosphorylated forms of the Rb protein and induced cellular levels of the p21Cip1 protein and mRNA in HepG2 cells. These observations mirror some of the results reported here.
Sumitani et al.(1994) reported that androgen significantly stimulates growth of the mouse mammary Shionogi carcinoma SC-3 cells. This androgen-induced growth is partially blocked by T3. TGF-ß also inhibits SC-3 cell growth. Thus, they investigated whether T3 exerted its inhibitory effects on SC-3 cell growth through TGF-ß mRNA expression. This study showed that T3 stimulated the expression of TGF-ß at the mRNA and protein levels in HepG2-TR stable cells. Meanwhile, TGF-ß1 exerted its inhibitory effects through down-regulation of PCNA, cdks, and cyclin E. Although this study did not demonstrate the effect of TGF-ß neutralizing antibody on the growth of HepG2 cells, it did show that TGF-ß antibody neutralizing T3 repressed cdk2.
In conclusion, this work provides evidence that T3 and its receptor mediates the suppression of hepatoma cell proliferation by TGF-ß. The results presented here raise the possibility that T3, via its receptors and TGF-ß, helps to regulate hepatocyte tumor growth and development.
|
Acknowledgements |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alisi A, Spagnuolo S, Napoletano S, Spaziani A & Leoni S 2004 Thyroid hormones regulate DNA-synthesis and cell-cycle proteins by activation of PKCalpha and p42/44 MAPK in chick embryo hepatocytes. Journal of Cellular Physiology 201 259–265.[CrossRef][Web of Science][Medline]
Aranda A & Pascual A 2001 Nuclear hormone receptors and gene expression. Physiological Reviews 81 1269–1304.
Barrera-Hernandez G, Park KS, Dace A, Zhan Q & Cheng SY 1999 Thyroid hormone-induced cell proliferation in GC cells is mediated by changes in G1 cyclin/cyclin-dependent kinase levels and activity. Endocrinology 140 5267–5274.
Buzzard JJ, Wreford NG & Morrison JR 2003 Thyroid hormone, retinoic acid, and testosterone suppress proliferation and induce markers of differentiation in cultured rat sertoli cells. Endocrinology 144 3722–3731.[CrossRef][Web of Science][Medline]
Chamba A, Neuberger J, Strain A, Hopkins J, Sheppard MC & Franklyn JA 1996 Expression and function of thyroid hormone receptor variants in normal and chronically diseased human liver. Journal of Clinical Endocrinology and Metabolism 81 360–367.[Abstract]
Chang C, Lin Y, Ol TW, Chou CK, Lee TS, Liu TJ, P’eng FK, Chen TY & Hu CP 1983 Induction of plasma protein secretion in a newly established human hepatoma cell line. Molecular and Cellular Biology 3 1133–1137.
Cheng SY 2000 Multiple mechanisms for regulation of the transcriptional activity of thyroid hormone receptors. Reviews in Endocrine and Metabolic Disorders 1 9–18.
Chou CK, Ho LT, Ting LP, Hu CP, Su TS, Chang WC, Suen CS, Huang MY & Chang CM 1987 Selective suppression of insulin-induced proliferation of cultured human hepatoma cells by somatostatin. Journal of Clinical Investigation 79 175–178.
De Caestecker M 2004 The transforming growth factor-beta superfamily of receptors. Cytokine and Growth Factor Reviews 15 1–11.[CrossRef][Web of Science][Medline]
Fan XG, Fan XJ, Xia HX, Keeling PW & Kelleher D 1995 Up-regulation of CD44 and ICAM-1 expression on gastric epithelial cells by H. pylori. APMIS: Acta Pathologica, Microbiologica et Immunologica Scandinavica 103 744–748.
Gong J, Ammanamanchi S, Ko TC & Brattain MG 2003 Transforming growth factor beta 1 increases the stability of p21/WAF1/CIP1 protein and inhibits CDK2 kinase activity in human colon carcinoma FET cells. Cancer Research 63 3340–3346.
Harwell RM, Mull BB, Porter DC & Keyomarsi K 2004 Activation of cyclin-dependent kinase 2 by full length and low molecular weight forms of cyclin E in breast cancer cells. Journal of Biological Chemistry 279 12695–12705.
Hulbert AJ 2000 Thyroid hormones and their effects: a new perspective. Biological Reviews of the Cambridge Philosophical Society 75 519–631.[Medline]
Keyomarsi K & Herliczek TW 1997 The role of cyclin E in cell proliferation, development and cancer. Progress in Cell Cycle Research 3 171–191.[Medline]
Kim SJ, Glick A, Sporn MB & Roberts AB 1989a Characterization of the promoter region of the human transforming growth factor-beta 1 gene. Journal of Biological Chemistry 264 402–408.
Kim SJ, Jeang KT, Glick AB, Sporn MB & Roberts AB 1989b Promoter sequences of the human transforming growth factor-beta 1 gene responsive to transforming growth factor-beta 1 autoinduction. Journal of Biological Chemistry 264 7041–7045.
Ledda-Columbano GM, Perra A, Loi R, Shinozuka H & Columbano A 2000 Cell proliferation induced by triiodothyronine in rat liver is associated with nodule regression and reduction of hepatocellular carcinomas. Cancer Research 60 603–609.
Lents NH & Baldassare JJ 2004 CDK2 and cyclin E knockout mice: lessons from breast cancer. Trends in Endocrinology and Metabolism 15 1–3.[CrossRef][Web of Science][Medline]
Li CY, Suardet L & Little JB 1995 Potential role of WAF1/Cip1/p21 as a mediator of TGF-beta cytoinhibitory effect. Journal of Biological Chemistry 270 4971–4974.
Lin KH, Lin YW, Parkison C & Cheng SY 1994 Stimulation of proliferation by 3,3',5-triiodo-L-thyronine in poorly differentiated human hepatocarcinoma cells overexpressing beta 1 thyroid hormone receptor. Cancer Letter 85 189–194.
Lin KH, Shieh HY & Hsu HC 2000 Negative regulation of the antimetastatic gene Nm23-H1 by thyroid hormone receptors. Endocrinology 141 2540–2547.
Lin KH, Wang WJ, Wu YH & Cheng SY 2002 Activation of antimetastatic Nm23-H1 gene expression by estrogen and its alpha-receptor. Endocrinology 143 467–475.
Lin KH, Chen CY, Chen SL, Yen CC, Huang YH, Shih CH, Shen JJ, Yang RC & Wang CS 2004 Regulation of fibronectin by thyroid hormone receptors. Journal of Molecular Endocrinology 33 445–458.
Martin-Castellanos C & Moreno S 1997 Recent advances on cyclins, CDKs and CDK inhibitors. Trends in Cell Biology 7 95–98.[CrossRef][Web of Science][Medline]
Massague J 1998 TGF-beta signal transduction. Annual Reviews of Biochemistry 67 753–791.
Nielsen NH, Arnerlov C, Cajander S & Landberg G 1998 Cyclin E expression and proliferation in breast cancer. Analytical Cellular Pathology 17 177–188.[Web of Science][Medline]
Pibiri M, Ledda-Columbano GM, Cossu C, Simbula G, Menegazzi M, Shinozuka H & Columbano A 2001 Cyclin D1 is an early target in hepatocyte proliferation induced by thyroid hormone (T3). FASEB Journal 15 1006–1013.
Samuels HH, Stanley F & Casanova J 1979 Depletion of L-3,5,3'-triiodothyronine and L-thyroxine in euthyroid calf serum for use in cell culture studies of the action of thyroid hormone. Endocrinology 105 80–85.
Sherr CJ 1996 Cancer cell cycles. Science 274 1672–1677.
Shih CH, Chen SL, Yen CC, Huang YH, Chen CD, Lee YS & Lin KH 2004 Thyroid hormone receptor-dependent transcriptional regulation of fibrinogen and coagulation proteins. Endocrinology 145 2804–2814.
Shimizu M, Suzui M, Deguchi A, Lim JT, Xiao D, Hayes JH, Papadopoulos KP & Weinstein IB 2004 Synergistic effects of acyclic retinoid and OSI-461 on growth inhibition and gene expression in human hepatoma cells. Clinical Cancer Research 10 6710–6721.
Sporn MB & Roberts AB 1992 Transforming growth factor-beta: recent progress and new challenges. Journal of Cell Biology 119 1017–1021.
Sumitani S, Kasayama S & Sato B 1994 Thyroid hormone inhibits androgen-enhanced DNA synthesis in Shionogi carcinoma 115 cells without affecting autocrine growth factor mRNA expression. Journal of Steroid Biochemistry and Molecular Biology 50 5–11.[CrossRef][Web of Science][Medline]
Received in final form 3 October 2005
Accepted 26 October 2005
Made available online as an Accepted Preprint 28 October 2005
This article has been cited by other articles:
|
|
 |
|
  A. Chiloeches, A. Sanchez-Pacheco, B. Gil-Araujo, A. Aranda, and M. Lasa Thyroid Hormone-Mediated Activation of the ERK/Dual Specificity Phosphatase 1 Pathway Augments the Apoptosis of GH4C1 Cells by Down-Regulating Nuclear Factor-{kappa}B Activity Mol. Endocrinol., November 1, 2008; 22(11): 2466 - 2480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-N. Chen, Y.-H. Huang, Y.-C. Lin, C.-T. Yeh, Y. Liang, S.-L. Chen, and K.-H. Lin Thyroid Hormone Promotes Cell Invasion through Activation of Furin Expression in Human Hepatoma Cell Lines Endocrinology, August 1, 2008; 149(8): 3817 - 3831. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Otto and J. Fandrey Thyroid Hormone Induces Hypoxia-Inducible Factor 1{alpha} Gene Expression through Thyroid Hormone Receptor {beta}/Retinoid X Receptor {alpha}-Dependent Activation of Hepatic Leukemia Factor Endocrinology, May 1, 2008; 149(5): 2241 - 2250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.-N. Chen, Y.-H. Huang, C.-T. Yeh, C.-H. Liao, and K.-H. Lin Thyroid Hormone Receptors Suppress Pituitary Tumor Transforming Gene 1 Activity in Hepatoma Cancer Res., March 15, 2008; 68(6): 1697 - 1706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Fava, Y. Ueno, S. Glaser, H. Francis, S. DeMorrow, L. Marucci, M. Marzioni, A. Benedetti, J. Venter, B. Vaculin, et al. Thyroid hormone inhibits biliary growth in bile duct-ligated rats by PLC/IP3/Ca2+-dependent downregulation of SRC/ERK1/2 Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1467 - C1475. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 10 (•) or 100 (
) nM T3 for the indicated time. In some experiments, cells were simultaneously incubated with 100 nM T3 and 800 ng of the TGF-ß neutralizing antibody (Ab, 
) or non-specific control antibody (nsAb, 
). Subsequently, cell number was determined using the Coulter Counter ZM. Data are expressed as means ± S.E. of values from three independent experiments. **P < 0.01, 100 nM T3-treated vs Td-treated (0) (Student’s t-test).
