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Endocrinology Vol. 145, No. 10 4660-4666
Copyright © 2004 by The Endocrine Society

Control of Phosphatase and Tensin Homolog (PTEN) Gene Expression in Normal and Neoplastic Thyroid Cells

Gianluca Tell, Alex Pines, Franco Arturi, Laura Cesaratto, Eileen Adamson, Cinzia Puppin, Ivan Presta, Diego Russo, Sebastiano Filetti and Giuseppe Damante

Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine (G.T., C.P., G.D.), 33100 Udine, Italy; Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole (A.P., L.C.) and Centro di Eccellenza di Biocristallografia (G.T.), Università di Trieste, Trieste, Italy; Dipartimento di Medicina Sperimentale e Clinica G. Salvatore e Dipartimento di Scienze Farmacobiologiche, Università di Catanzaro (F.A., I.P., D.R.), Catanzaro, Italy; The Burnham Institute (E.A.), La Jolla, California 92037; and Dipartimento di Scienze Cliniche, Università di Roma La Sapienza (S.F.), Rome, Italy

Address all correspondence and requests for reprints to: Dr. Giuseppe Damante, Dipartimento di Scienze e Tecnologie Biomediche, Piazzale Kolbe 1, 33100 Udine, Italy. E-mail: gdamante{at}makek.dstb.uniud.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The lipid phosphatase, phosphatase and tensin homolog (PTEN), is a key element in controlling cell growth and survival and has a well established role as tumor suppressor protein in many neoplasia. Several data indicate that silencing of PTEN gene expression may be relevant in follicular thyroid cell transformation. Thus, in the present study regulation of PTEN gene expression in thyroid cells was investigated. Cotransfection experiments indicated that in normal FRTL-5 rat thyroid cells, PTEN promoter activity was increased by overexpression of the transcription factor early growth response protein-1 (Egr-1). Moreover, Western blot experiments indicated that when Egr-1 expression was up-regulated by treating FRTL-5 cells with H2O2, an increase in PTEN expression was also observed. TSH induced opposite modifications on PTEN and Egr-1 protein levels. Moreover, acute or chronic TSH stimulation determined distinct effects. In fact, acute TSH stimulation (30 and 60 min) induced a decrease in PTEN, but an increase in Egr-1 protein levels. These effects were cAMP dependent; in fact, they were mimicked by forskolin. A chronic TSH treatment (5 d) stimulated PTEN protein expression, whereas Egr-1 protein was down-regulated. In contrast to normal thyroid cells, when the thyroid tumor cell lines ARO and BCPAP were exposed to H2O2, neither Egr-1 nor PTEN protein levels were increased. Acute stimulation of ARO and BCPAP cells with forskolin increased Egr-1, but not PTEN, protein levels. Therefore, thyroid tumor cell lines show alteration of PTEN gene expression regulation. RT-PCR experiments performed on human thyroid tumors showed that the absence of Egr-1 mRNA is always paralleled by the absence of PTEN mRNA. Thus, modification of the Egr-1-dependent mechanisms may play a role in the silencing of PTEN gene expression occurring during thyroid cell transformation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PHOSPHATASE AND TENSIN homolog (PTEN; also called MMAC or TEP1), is a 47-kDa phosphoprotein possessing phosphatase activity through which phosphatidylinositol-3,4,5-trisphosphate (PIP3) levels are regulated (1). By means of its phosphatase activity, PTEN negatively controls the phosphoinositide 3-kinase/Akt signaling pathway involved in the regulation of cell growth and survival in several cell systems (2). A reduced PTEN function determines a marked increase in PIP3 and activation of Akt survival signaling pathways, leading to inhibition of apoptosis and hyperplasia and contributing to tumor formation (1). Inactivating mutations or loss of heterozygosity (LOH) of PTEN gene have been described in a variety of sporadic human neoplasms, including prostate, breast, cervical cancers, and malignant melanoma (3, 4, 5, 6). Moreover, germline loss of function mutations of the PTEN gene have been detected in Cowden disease, a dominant predisposition to tumors that is characterized by multiple hamartomatous lesions, especially of the skin and mucous membranes, and increased risk for breast, thyroid, and endometrial cancer (7). Germline mutations of the PTEN gene are found in the Bannayan-Riley-Ruvalcaba syndrome, which is characterized by lipomatosis, macrocephaly, hemangiomatosis, and speckled penis, and Proteus and Proteus-like syndromes, diseases not associated with the risk of neoplasia (7). Because of the high risk for thyroid malignancy conferred by PTEN mutations, the role of PTEN in thyroid tumorigenesis has been extensively investigated. In thyroid tumors, somatic mutations of the PTEN gene are quite rare (8), whereas LOH at 10q23 is present in 20–60% of thyroid malignancies, with a higher percentage in the more aggressive histotypes (9). In addition, a loss/reduction of PTEN expression has been observed in neoplasms, with an inverse relationship with aggressiveness (10). Several reports indicate that the defective PTEN protein expression seen in thyroid cancers is not often coupled to mutations in the PTEN gene (11, 12). Thus, the aim of this study was clarification of the molecular events involved in the regulation of PTEN expression occurring in thyroid tumors. We focused our attention on the role played by the early growth response protein-1 (Egr-1; called also Zif-268 and Krox-24), a transcription factor induced by several extracellular stimuli (13) that has been recently shown to activate the PTEN gene promoter (14). We found that the H2O2/Egr-1 and TSH/cAMP pathways control PTEN expression in normal rat thyroid cells and that perturbation of these signaling systems occurs in thyroid tumor cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and plasmids
All materials were purchased from Sigma-Aldrich Corp. (St. Louis, MO) unless otherwise specified. The construct containing the PTEN minimal promoter (called min PTEN-luc) has been previously described (14). In this construct, the fragments –1031 to –779, relative to the translation start site, of the PTEN gene promoter were cloned in the plasmid pGL3B-P10, 5' to the luciferase (LUC) gene, which is used as the reporter gene. The Egr-1 expression vector has been previously described (14). Cytomegalovirus (CMV)-ß-galactosidase (ßGAL) plasmid contains the CMV promoter linked to the ßGAL gene.

Cell cultures and transfections
FRTL-5 cells were maintained in F-12 Coon’s modified medium with 5% calf serum and hormone mixture as previously described (15). ARO and BCPAP cell lines were grown in DMEM containing 10% fetal bovine serum. The calcium phosphate coprecipitation method was used for transfections as previously described (16). FRTL-5 cells were plated at 1.5 x 106 cells/100-mm culture dish. Forty-eight hours before transfection and 3 h before the addition of the DNA-calcium phosphate precipitates, the medium was changed to DMEM containing 5% calf serum and growth factors. The plasmids were used in the following amounts: min PTEN-luc, 6 µg; Egr-1 expression vector, 2 µg; and CMV-ßGAL, 2 µg. Cells were harvested 42–44 h after transfection, and cell extracts were prepared by a standard freeze-thaw procedure. ßGAL activity was measured by an ELISA method (Amersham Biosciences, Milan, Italy). LUC activity was measured by a chemiluminescence procedure (16). ßGAL activity values were used to normalize LUC activity values for transfection efficiency.

Cell stimulations were performed on 70–80% confluent cells. Two hours before each stimulation, the medium was replaced by fresh medium. After H2O2 stimulation (5 min), the medium with H2O2 was completely removed, and the cells were washed with PBS and harvested for subsequent subcellular fractionation.

Western blot analysis
Twenty micrograms of either nuclear or cytoplasmic extracts, obtained from the indicated thyroid cell lines and incubated under different conditions, were electrophoresed on 12% SDS-PAGE. Proteins were then transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Membranes were saturated by incubation, at 4 C overnight, with 5% nonfat dry milk in PBS/0.1% Tween 20 and then incubated with the polyclonal anti-Egr-1 sc-110 antibody or with the monoclonal anti-PTEN antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 3 h. After three washes with PBS/0.1% Tween 20, membranes were incubated with an antirabbit (in the case of Egr-1) or antimouse (in the case of PTEN) immunoglobulin coupled to peroxidase (Sigma-Aldrich Corp.). After 60 min of incubation at room temperature, the membranes were washed three times with PBS/0.1% Tween 20, and the blots were developed using the enhanced chemiluminescence procedure (Amersham Biosciences). Normalizations were performed with the polyclonal antiactin antibody (Sigma-Aldrich Corp.). Blots were quantified using a Gel Doc 2000 videodensitometer (Bio-Rad Laboratories, Hercules, CA).

PTEN/Egr-1 RT-PCR
Eleven follicular thyroid adenomas, 22 differentiated thyroid carcinomas (nine follicular and 13 papillary), and nine non-nodular normal thyroid tissues were collected and analyzed. Informed consent was obtained from all patients. Total RNA extraction and cDNA synthesis were performed as previously described (17), and RT-PCR amplification was performed using 3 µl cDNA as previously described (18). The PCR conditions were as follows: a first step of predenaturation at 95 C for 10 min, denaturation at 95 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec (33 total cycles). Primer oligonucleotides for the PTEN gene were: 5'-ACCAGTGGCACTGTTGTTTCAC-3' and 5'-TTCCTCTGGTCCTGGTATGAAG-3'. The amplification yielded a 286-bp DNA product corresponding to fragment 1598–1883 of the PTEN gene according to the sequence reported in GenBank (NM_000314). To exclude contamination of the PCR product with the PTENP1 pseudogene, another couple of primers were used to amplify a region corresponding to fragment 2841–3108 of the PTEN gene. Subsequent digestion with RsaI cut this fragment only in the PTEN gene, as described by Frisk et al. (12). Primers oligonucleotides for the Egr-1 gene were 5'-TCCCGGCTCCTCGACCTAC-3' and 5-'AAGGTTGCTGTCATGTCCGAAAG-3'. The amplification yielded a 176-bp DNA product corresponding to fragment 1698–1874 of the Egr-1 gene according to the sequence reported in GenBank (NM_001964). To check the quality of the RT-PCR procedure, expression of the transcript of ß-actin and glyceraldehyde-3-phosphate dehydrogenase GAPDH, control genes ubiquitously expressed, was also analyzed on all samples (17). Ten of 50 µl of the amplification products were run on 1.5% (wt/vol) Tris-borate-EDTA agarose gel containing ethidium bromide and analyzed to confirm a positive or a negative outcome. The positivity/negativity association between Egr-1 and PTEN mRNAs was analyzed using PRISM software (GraphPad, Inc., San Diego, CA) with a two-sided {chi}2 contingency table test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of Egr-1 in FRTL-5 cells increases PTEN promoter activity
A PTEN promoter element, whose activity is up-regulated by Egr-1 in transient transfection experiments, was recently identified (14). Thus, the effects of Egr-1 on PTEN gene expression in follicular thyroid cells were investigated using the transfection approach. FRTL-5 cells were cotransfected with a construct in which the PTEN minimal promoter was linked to the LUC gene (14) and with an Egr-1 expression vector. Because the Egr-1 gene is ubiquitously expressed, the Egr-1 expression vector was used to increase Egr-1 protein levels. Figure 1Go shows that the presence of the Egr-1 expression vector significantly enhanced LUC gene expression, indicating that overexpression of Egr-1 in FRTL-5 cells induces an increase in PTEN promoter activity. These results suggest that in thyroid cells, the PTEN gene is subjected to Egr-1 control at the transcriptional level.



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  		FIG. 1. Effect of Egr-1 overexpression on PTEN promoter activity. FRTL-5 cells were transfected with a plasmid containing the PTEN minimal promoter in the presence or absence of an Egr-1 expression vector. Each bar represents the mean ± SD of three independent transfections. P values were determined by t test.

 
Effect of H2O2 and TSH/cAMP on Egr-1 and PTEN proteins levels
It is well known that Egr-1 protein levels as well as its transcriptional activity are induced by oxidative stress (13). Thus, if the endogenous PTEN gene is subjected to Egr-1 control, oxidative stress should up-regulate PTEN expression. To test this possibility in thyroid cells, FRTL-5 cells were treated with H2O2 (1 and 5 mM) for 5 min, and Egr-1 and PTEN protein levels were evaluated by Western blot analysis. These conditions (high doses of H2O2 for short times) were chosen under the hypothesis that functionally linked events are kinetically coregulated. The Western blot analysis was performed using both nuclear and cytoplasmic cell extracts. Figure 2Go shows that H2O2 treatment induced a concomitant increase in Egr-1 and PTEN protein levels. This increase was inhibited by cycloheximide treatment of the cells and was suggestive of a protein neosynthesis-dependent mechanism (data not shown). As expected, Egr-1 expression was confined to the nucleus, whereas PTEN was present in both nucleus and cytoplasm. In this latter district, the stimulatory effect on PTEN protein levels was already maximal at an H2O2 concentration of 1 mM. Therefore, in subsequent experiments Egr-1 expression was evaluated in nuclear extracts, whereas PTEN expression was evaluated in cytoplasmic extracts. The results shown in Fig. 2Go strengthen the hypothesis that Egr-1 plays a role in the control of PTEN gene expression in thyroid cells.



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  		FIG. 2. Oxidative stress promotes Egr-1 and PTEN up-regulation in FRTL-5 normal thyroid cells. FRTL-5 cells were incubated with increasing amounts of H2O2 for 5 min at 37 C. Then cells were harvested for cytoplasmic and nuclear extract preparation. Twenty micrograms of each extract were separated on 12% SDS-PAGE and analyzed for the presence of the Egr-1 transcription factor and PTEN proteins by correspondent antibodies from Santa Cruz Biotechnology, Inc. Actin was always measured, as a loading control, using an antiactin polyclonal antibody. A representative image of three concordant experiments is shown.

 
TSH is the main regulator of thyroid cell function. Thus, we tested whether the H2O2 effect was influenced by the presence of the hormone. Egr-1 and PTEN protein levels were evaluated in nuclear and cytoplasmic extracts, respectively, from FRTL-5 grown in either the absence (5H) or the presence (6H) of TSH for 5 d, then stimulated by H2O2. The results are shown in Fig. 3Go. The stimulatory effect of H2O2 was detectable in both the absence (FRTL-5 5H) and the presence (FRTL-5 6H) of TSH. Densitometric scanning of signals indicated that the extent of the H2O2-induced stimulation between 5H and 6H cell was similar (data not shown). However, the presence of the hormone determined opposite effects on basal levels of PTEN and Egr-1 proteins. In fact, with respect to 5H cells, 6H cells had increased basal PTEN protein levels, but reduced basal Egr-1 protein levels (Fig. 3Go). Therefore, the presence of TSH induced distinct effects on PTEN (up-regulation) and Egr-1 (down-regulation) protein levels.



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  		FIG. 3. Chronic TSH treatment inhibits Egr-1 expression and up-regulates PTEN in FRTL-5 normal thyroid cells. FRTL-5 cells were cultured in the absence of TSH (5H) for 5 d for starvation or in the continuous presence of TSH (1 mU/ml; 6H), then exposed to 1 mM H2O2 for the indicated times. The cells were processed to obtain cytoplasmic and nuclear extracts as described in Materials and Methods. Nuclear and cytoplasmic extracts were used to evaluate Egr-1 and PTEN protein levels, respectively. Western blot analysis was performed with anti-Egr-1 polyclonal antibody and anti-PTEN monoclonal antibody from Santa Cruz Biotechnology, Inc. Actin was always measured, as a loading control, using an antiactin monoclonal antibody. A representative image of two concordant experiments is shown.

 
In the experiments shown in Fig. 3Go, cells subjected, or not, to chronic TSH treatment were challenged with acute stimulation of H2O2. Because it is known that acute and chronic TSH stimulations may have distinct effects on thyroid cells (19), the acute TSH effect on PTEN and Egr-1 protein levels was also evaluated. FRTL-5 cells were kept in the absence of TSH for 5 d, then stimulated with 1 mU/ml TSH for 30, 60, and 360 min. The results are shown in Fig. 4Go. It is clear that acute TSH stimulation reduces PTEN, but increases Egr-1 protein levels. The effects of TSH appear to be mediated by a cAMP-dependent mechanism. In fact, stimulation with forskolin (10 µM) induced modification of PTEN and Egr-1 protein levels similar to those induced by TSH (Fig. 4Go). Thus, PTEN expression appears to be regulated in a complex manner by both Egr-1-dependent (induced by H2O2) and TSH-dependent (cAMP-mediated) mechanisms.



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  		FIG. 4. Effects of acute stimulation with TSH or forskolin on PTEN and Egr-1 protein levels in FRTL-5 cells. FRTL-5 cells were cultured in the absence (5H) or presence of (6H) TSH (1 mU/ml) for 5 d. Cells kept in 5H were then exposed to TSH (1 mU/ml) or forskolin (10 µM) for the indicated times. Nuclear and cytoplasmic extracts were prepared and used to evaluate Egr-1 and PTEN protein levels, respectively. Twenty micrograms of each extract were separated on 12% SDS-PAGE and analyzed for the presence of the Egr-1 transcription factor and PTEN proteins by Western blot. Actin was always measured, as a loading control, using an antiactin monoclonal antibody. A representative image of two concordant experiments is shown.

 
Egr-1 and PTEN expression in human thyroid tumors and cell lines
To shed light on the mechanism responsible for PTEN down-regulation in thyroid tumors, we examined whether the H2O2/Egr-1-dependent regulation of PTEN expression was modified in thyroid cancer cells. Therefore, the human thyroid tumor cell lines ARO (from anaplastic carcinoma) and BCPAP (from papillary carcinoma) (20) were treated with H2O2, and Egr-1 and PTEN protein levels were evaluated by Western blot analysis. As shown in Fig. 5Go, in both ARO and BCPAP cells, H2O2 was not able to induce either Egr-1 or PTEN protein levels. These data are in agreement with Vasudevan et al. (21), who reported the absence of PTEN protein expression in ARO cells. We also tested whether a cAMP-dependent mechanism on PTEN and Egr-1 expression was active in thyroid tumor cell lines. The data are reported in Fig. 6Go. ARO and BCPAP cells were stimulated with 10 µM forskolin for 30, 60, and 360 min, then PTEN and Egr-1 protein levels were evaluated by Western blot analysis. In both cell lines, PTEN protein levels were not modified by forskolin (Fig. 6Go). Note that an overexposed image is shown to detect PTEN protein levels. In contrast, forskolin treatment increased Egr-1 protein levels in both cell lines (Fig. 6Go). Our results indicate that neither H2O2- nor cAMP-dependent stimulation was able to increase PTEN expression in ARO and BCPAP cell lines, suggesting that both regulatory molecular mechanisms are altered in thyroid neoplasms. In the case of H2O2, the lack of PTEN response is probably due to the abolition of mechanisms that control Egr-1 expression.



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  		FIG. 5. Different human tumor cells lose the ability to respond to H2O2 treatment in terms of Egr-1 and PTEN activation. FRTL-5 and two human thyroid cell lines (i.e. ARO and BCPAP) were treated with H2O2 for 5 min, then nuclear and cytoplasmic extracts were obtained. Nuclear and cytoplasmic extracts were used to evaluate Egr-1 and PTEN protein levels, respectively. Twenty micrograms of each extract were separated on 12% SDS-PAGE and analyzed for the presence of the Egr-1 transcription factor and PTEN proteins. Actin was always measured, as a loading control, using an antiactin monoclonal antibody. Values obtained from densitometric analysis of Western blot experiments, normalized vs. actin, are reported as histograms. Bars indicate the mean ± SD of three independent experiments.

 


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  		FIG. 6. Effects of acute stimulation with forskolin on PTEN and Egr-1 protein levels in ARO and BCPAP cell lines. ARO and BCPAP cells were exposed to forskolin (10 µM) for the indicated times. Nuclear and cytoplasmic extracts were prepared and used to evaluate Egr-1 and PTEN protein levels, respectively. Twenty micrograms of each extract were separated on 12% SDS-PAGE and analyzed for the presence of Egr-1 and PTEN proteins by Western blot. Actin was always measured, as a loading control, using an antiactin monoclonal antibody. A representative image of two concordant experiments is shown.

 
To test whether a reduction of Egr-1 expression is relevant for the silencing of PTEN gene expression observed in several thyroid tumors in vivo (12), Egr-1 and PTEN mRNA were analyzed in different types of thyroid neoplasms by RT-PCR and correlated to each other. The presence of contamination with cDNA of the PTENP1 pseudogene was excluded using an assay with RT-PCR and restriction cleavage described by Frisk et al. (12) (data not shown). As shown in Fig. 7Go and Table 1Go, most cases of thyroid tumors that were negative for Egr-1 mRNA were also negative for PTEN transcript. In only one follicular adenoma was the presence of Egr-1 mRNA associated with negativity for PTEN transcript. In contrast, PTEN mRNA was never seen in the absence of Egr-1 mRNA. A two-sided {chi}2 test, performed with all analyzed cases, indicates that the positivity/negativity association between Egr-1 and PTEN mRNAs is significant (P < 0.001). These results suggest that in thyroid tumors, alteration of Egr-1 levels may play a role in the silencing of PTEN gene expression.



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  		FIG. 7. Analysis of Egr-1 and PTEN mRNA expression in human thyroid tissues. Total RNA was purified from human normal and tumoral thyroid tissues, and the presence of Egr-1 and PTEN mRNA was evaluated by RT-PCR. Actin and GAPDH genes were amplified as control genes. Sample gels with bands corresponding to Egr-1, PTEN, and GAPDH amplification products are shown. The expected bands are indicated by arrows. Lanes 1 and 8, Normal thyroid tissues; lanes 2 and 3, papillary thyroid carcinomas; lanes 4 and 5, follicular thyroid carcinomas; lanes 6 and 7, follicular thyroid adenomas.

 

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  		TABLE 1. PTEN and Egr-1 expression in human thyroid tissues

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the last few years, the lipid phosphatase PTEN has emerged as a key protein in the control of cell proliferation. In fact, PTEN down-regulates the Akt signaling pathway by inactivating PIP3 and causes G1 cell cycle arrest and/or apoptosis, possibly in a cell type-dependent manner (1). Genetic alterations of PTEN, first described as the causative event of Cowden disease, have been reported in other hereditary syndromes as well as sporadic primary and metastatic human malignancies (7). Thus, a major role of PTEN as a tumor suppressor is well established. Many studies have also elucidated the molecular mechanism by which PTEN silencing may favor tumor cell growth. Again, a major role is played by pathways downstream of Akt, which may be expected to modulate apoptosis or elicit G1 arrest (22). However, PTEN protein phosphatase activity and Akt-independent pathways may also participate in the abnormal regulation of cell survival pathways occurring in tumor cells (22).

Several findings indicate that the PTEN gene also plays a relevant role as a tumor suppressor in thyroid tumorigenesis. In fact, its functional inactivation has been described in a significant fraction of thyroid tumors, particularly in the more undifferentiated histotypes (23). Different mechanisms may be responsible for PTEN impaired function:1) inactivating mutations due to structural alterations (as in Cowden disease) or LOH at the PTEN locus (8, 24), 2) loss or reduction of PTEN protein expression (11, 12); and 3) abnormal cytosol-nucleus translocation (10). In a recent study (11) reduced PTEN expression was reported in approximately 40% of thyroid tumors, with a correlation in the decrease at protein and mRNA levels, suggesting that a major alteration occurs at the transcriptional level of the PTEN gene. Moreover, germline mutations affecting the function of the PTEN gene promoter have been found in several Cowden disease patients (25). However, despite the pathogenic relevance of abnormal transcriptional control of the PTEN gene in thyroid tumorigenesis, the regulatory mechanisms of PTEN expression in thyroid cell type are mostly unknown. A major limitation is the lack of information on the PTEN gene promoter. Its recent cloning and characterization (14) have opened the way to more detailed studies of the alterations underlying the reduced transcription of the PTEN gene in tumor cell types, where p53 is often mutant and cannot contribute to the known ability of p53 to induce PTEN transcription (26).

The major aim of this study was to investigate the mechanisms of PTEN gene expression regulation in thyroid cells. Our findings provide evidence indicating that in follicular thyroid cells, PTEN gene expression is regulated by either Egr-1-dependent or TSH-dependent mechanisms. Moreover, our data indicate that deregulation of these mechanisms may play a role in the down-regulation of PTEN gene expression observed in thyroid tumors. The present findings were obtained using nonhuman FRTL-5 cells and comparing their behavior to that of human thyroid tumor cell lines (ARO and BCPAP). It should be pointed out, however, that FRTL-5 cells have many characteristics of normal human thyrocytes, including TSH-dependent growth and expression of all markers of differentiation (27). For this reason these cells have been and are currently exploited as a model of normal thyroid cells, especially for investigating, as in the case of our study, the regulation of certain proteins. Moreover, a down-regulation of Egr-1 by TSH similar to what we observed in chronically treated FRTL-5 cells has been reported in human adenoma cells by Deleu et al. (28) and Tominaga et al. (29) in Wistar rat thyroid cells.

Egr-1 is an early growth response protein induced by several extracellular stimuli, including UV light and H2O2 (13). Hydrogen peroxide is a well known inducer of Egr-1 gene expression in different cell systems (30) (Cesaratto, L., and G. Tell, unpublished observations). Accordingly, our present data demonstrate that treatment of FRTL-5 cells with H2O2 increases Egr-1 and PTEN protein levels even after 5 min of H2O2 exposure. The quickness of induction is not surprising because it is known that different stimuli are able to very rapidly (within minutes) increase Egr-1 protein levels (13, 31). Although we cannot exclude the possibility that H2O2 stimulation could alter the kinetics of protein degradation, the inhibitory effect of cycloheximide treatment on Egr-1- and PTEN-induced up-regulation (data not shown) suggests a protein neosynthesis-dependent process for the demonstrated stimulation. Interestingly, the same effect was not obtained in thyroid tumor cell lines ARO and BCPAP, suggesting that in thyroid tumors molecular mechanisms able to up-regulate Egr-1 (and, in turn, PTEN) are abolished. Because TSH down-regulated Egr-1 protein levels, the presence of a cAMP-dependent pathway responsible for the inducible expression of PTEN in an Egr-1-dependent manner can be ruled out. The best candidates for this role are, instead, MAPK and/or protein kinase C, both of which have been demonstrated to play a major role in controlling the inducible expression of Egr-1 upon oxidative injury (13).

From a practical point of view, knowledge of the molecular mechanisms regulating PTEN gene expression may have a relevance to the planning of innovative therapeutic approaches. In fact, it has been demonstrated that PTEN expression in thyroid cancer cell lines results in growth arrest and cell death (11, 32). Similar results were obtained in other tumor cell types, including breast, glioma, bladder, and colorectal (33, 34, 35, 36). Furthermore, PTEN has been shown to increase tumor responsiveness to chemotherapy and radiation (34, 37, 38), suggesting that activation of expression of the endogenous PTEN gene may be proposed, alone or in combination, as an antineoplastic tool in many human malignancies. Additional clarification of the transcriptional control of PTEN gene expression will provide useful information for defining innovative therapeutic strategies for recovering PTEN tumor suppressor activity.


   Footnotes
 
G.T. and A.P. equally contributed to this work.

This work was supported by the Italian Ministry of Health (to S.F.) and grants from the Ministero Italiano per l’Università e la Ricerca Scientifica [to D.R., G.T. (FIRB RBNE0155LB), and G.D.] and from Regione Friuli Venezia Giulia (to G.D.).

Abbreviations: CMV, Cytomegalovirus; Egr-1, early growth response protein-1; ßGAL, ß-galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LOH, loss of heterozygosity; LUC, luciferase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PTEN, phosphatase and tensin homolog.

Received March 4, 2004.

Accepted for publication June 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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