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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-0969
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 4 2403-2411
Copyright © 2005 by The Endocrine Society

All-Trans-Retinoic Acid Treatment Inhibits the Growth of Retinoic Acid Receptor ß Messenger Ribonucleic Acid Expressing Thyroid Cancer Cell Lines but Does Not Reinduce the Expression of Thyroid-Specific Genes

Rossella Elisei, Agnese Vivaldi, Laura Agate, Raffaele Ciampi, Eleonora Molinaro, Pamela Piampiani, Cristina Romei, Pinuccia Faviana, Fulvio Basolo, Paolo Miccoli, Alessandra Capodanno, Paola Collecchi, Furio Pacini and Aldo Pinchera

Department of Endocrinology and Metabolism, Section of Endocrinology, (R.E., A.V., L.A., R.C., E.M., P.P., C.R., A.P.), Department of Oncology, Division of Pathology III, (P.F., F.B.), Division of Pathology I, (A.C., P.C.), and Department of Surgery (P.M.), University of Pisa, 56124 Pisa, Italy; Department of Internal Medicine, Endocrinology and Metabolism and Biochemistry (F.P.), University of Siena, 53100 Siena, Italy; AmbiSEN Center (A.P.), High Technology Center for the Study of the Environmental Damage of the Endocrine and Nervous Systems, University of Pisa, 56100 Pisa, Italy

Address all correspondence and requests for reprints to: Rossella Elisei, M.D., Department of Endocrinology and Metabolism, University of Pisa, via Paradisa n° 2, 56124 Pisa, Italy. E-mail: relisei{at}endoc.med.unipi.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Conventional chemoteraphy and radiotherapy are ineffective for the treatment of advanced thyroid tumors like poorly differentiated papillary, anaplastic, and medullary thyroid cancer. In the attempt to evaluate the possibility of using retinoic acid (RA) in the treatment of thyroid cancer refractory to conventional therapy, we studied the effect of all-trans-RA treatment on five human thyroid cancer cell lines. We found that WRO and NPA, derived from follicular and poorly differentiated human thyroid carcinoma, respectively, showed a growth inhibition after 25 and 21 d of RA treatment. Both apoptosis and a decrease in DNA synthesis were observed as mechanisms of growth inhibition. In the NPA cell line, a delay of cell-cycle progression has also been observed.

On the contrary, we did not observe any recovery of mRNA expression of thyroid-specific genes and in particular of the sodium iodide symporter gene. The lack of recovery of radioiodide uptake after all-trans-RA treatment confirmed the inability to reexpress sodium iodide symporter mRNA.

The main difference between the all-trans-RA responding cells (WRO and NPA) and the nonresponding cells [ARO, FRO (derived from human anaplastic thyroid tumors) and TT (derived from human medullary thyroid tumor)] was the basal and all-trans-RA induced RA receptor (RAR)ß mRNA expression. Interestingly, 14 thyroid tumors (10 papillary and four anaplastic) showed a significant lower expression of RARß mRNA when compared with normal thyroid tissues. In agreement with this result, only 30% of papillary thyroid carcinomas analyzed were positive for RARß protein expression with a degree of expression that was much lower than that found in normal thyroid tissue.

In conclusion we found that all-trans-RA treatment can determine a significant in vitro growth inhibition especially in differentiated thyroid tumor-derived cell lines but it seems unable to reinduce the expression of thyroid-specific genes and in particular to reinduce the ability to take up iodine. The growth inhibition is likely due to apoptosis in an early phase and to a decrease of DNA synthesis later. In some cases, a delay of the cell-cycle progression also may be responsible for the growth inhibition. The finding of a basal and RA-induced RARß mRNA expression only in cell lines responding to all-trans-RA suggests that the growth inhibition might be mediated by RARß.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID MALIGNANCIES ARE mainly represented by well differentiated papillary and follicular (PTC and FTC) thyroid tumors that are usually well treated by the combination of total thyroidectomy and radioiodine therapy (1). Patients treated with this protocol have an overall 5 yr survival rate of 85–90%. However, about 10–15% of patients develop an aggressive disease with local recurrence and distant metastases as a consequence of their progressive inability to take up radioiodine (2). During the dedifferentiation process, the tumor progressively loses the expression of the differentiation genes usually starting with the loss of the sodium iodide symporter (NIS) and continuing with the loss of thyroglobulin (Tg) and TSH-stimulating hormone receptor (3). Poorly differentiated PTC, anaplastic (ATC), and medullary thyroid cancer (MTC) have an even worse prognosis, and patients not cured by surgery have a very low chance of surviving the disease. Conventional chemotherapy and radiotherapy are in fact ineffective for the treatment of these advanced thyroid tumors (4).

In vitro and in vivo studies are still being performed to investigate the possibility of using retinoic acid (RA) to treat human tumors refractory to conventional therapy (5). RA belongs to the family of retinoids, which are the natural and synthetic derivatives of vitamin A or retinol. RA, the biologically active metabolite of vitamin A, is of central importance for growth, differentiation, and morphogenesis in vertebrates. A successful RA therapy has been obtained for acute promyelocytic leukemia, by which up to 90% remission can be achieved (6). In animal models retinoids prevent or delay the tumor promotion of various cancers like skin, breast, lung, digestive tract, pancreas, liver, bladder, and prostate cancers (7). Because in vitro studies have suggested that RA increases NIS mRNA expression and iodide uptake in some thyroid cancer cell lines (8, 9), RA treatment is under evaluation for its potential utility in thyroid cancer therapy (10, 11).

In the attempt to evaluate the possibility of using RA in the treatment of thyroid cancer refractory to conventional therapy, we studied the effect of RA treatment on five human thyroid cancer cell lines deriving from different human thyroid tumors. To this purpose we evaluated the growth rate, the thyroid-specific genes’ mRNA expression, and the iodide uptake before and after RA administration. The analysis of RA receptors (RAR) {alpha}, ß, {gamma} mRNA expression was also performed to establish any correlation with the response to the RA treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human thyroid tumors and cell lines

Two anaplastic (FRO and ARO), one poor differentiated papillary (NPA), one follicular (WRO), and one medullary (TT) human carcinoma cell lines were used for this study. FRO, ARO, NPA, and WRO were cultured in RPMI 1640 supplemented with 10% FCS (Sigma, Milan, Italy), 1% penicillin/streptomycin solution, 1% sodium pyruvate, and 1% nonessential amino acids, whereas the TT cell line was cultured in Ham’s F-12 (Invitrogen, Milan, Italy) supplemented with 10% FCS.

Ten PTC and the corresponding normal contralateral tissues and four ATC were analyzed for RARß mRNA expression. Tissues were frozen in liquid nitrogen at surgery and stored at –80 C. Ten formalin-fixed and paraffin-embedded PTC and 10 normal thyroid tissues were used for the analysis of RARß protein expression.

RA treatment

All-trans-RA (Sigma) was resuspended in ethanol and used at a final concentration of 1 µM. One thousand cells were seeded in 9.5-cm2 plates, transferred to 58-cm2 plates when reaching confluence, and subsequently transferred to 152-cm2 plates. The day after seeding they were exposed to RA or ethanol, as control, for 24, 48, 72 h, or 1 wk, up to a time corresponding to 12–15 doublings (between 2 wk and 1 month depending on cell type). Cells were shielded from light by aluminum foil to prevent RA degradation.

Determination of cell growth, cell proliferation, and apoptosis

Cell viability was determined by their adherence to plastic dishes, because trypan blue was found to be unreliable in predicting viability of cells in the presence of retinoids (12). Cell growth was determined by cell counting after their harvesting at the indicated time (0 h, 24 h, 48 h, 72 h, 1 wk, or 1 month) of all-trans-RA treatment. All experiments were performed in triplicate.

Cell proliferation was measured using the CellTiter 96 Aqueous One Solution cell proliferation assay (Promega, Madison, WI), based on the cellular conversion of the colorimetric reagent MTS [3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] into soluble formazan by dehydrogenase enzymes produced only by metabolically active, proliferating cells. Treated and untreated cells were grown in triplicates in a 96-well plate. After each treatment, 20 µl of dye solution was added into each well and incubated for 2 h. Absorbance was then recorded at a 490-nm wavelength using an ELISA plate reader (Molecular Devices, Sunnyvale, CA). Cell proliferation was also determined by the Cell Proliferation ELISA BrdU (Roche Applied Science, Milan, Italy), a colorimetric immunoassay for the quantification of cell proliferation, based on the measurement of bromodeoxyuridine (BrdU) incorporation during DNA synthesis.

To quantify the induction of apoptosis, a DNA fragmentation assay was performed using the Cell Death Detection ELISAPLUS (Roche Applied Science), according to the manufacturer’s instructions. The level of DNA fragmentation found at 0 h was set to 1, and the increases were evaluated as fold induction.

The cell cycle of treated and untreated WRO and NPA cells was also analyzed by fluorescence-activated cell sorting (FACS) analysis. Cells were harvested and rinsed twice in 1 ml cold PBS, fixed in 1 ml 70% ethanol overnight at 4 C, rinsed twice in cold PBS, and resuspended in saponin-propidium iodide (0.3% saponin, 25 µg/ml propidium iodide, 0.1 mM EDTA, 125 U/ml ribonuclease A in PBS). Flow cytometry for cell-cycle analysis was performed by FACSort flow cytometry (Becton Dickinson, San Jose, CA). CellQuest software programs (Becton Dickinson) were used for acquisition and analysis of data.

RNA extraction and RT-PCR

Total RNA was extracted from cell lines treated with all-trans-RA for different time periods (i.e. 24 h, 48 h, 72 h, 1 wk, and 1 month) and it was analyzed to detect possible changes in several thyroid-specific genes’ expression. In particular, cDNA was amplified by PCR (RT-PCR) using specific primers for a ubiquitous gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) as control and for NIS, Tg, TSH-stimulating hormone receptor, thyroperoxidase, pendrin, and transcription factors Pax-8 and TTF-1. RAR {alpha}, ß, and {gamma} mRNA expression was also studied in treated and untreated cells. Primers and conditions used for PCR are reported in Table 1Go. PCR products were electrophoresed in a 2% agarose gel and transferred onto a nylon membrane. To assess the specificity of PCR products, each filter was hybridized with internal probes specific for each amplified fragment (Table 1Go) labeled using a chemiluminescent method (ECL-CDP star detection; Amersham Pharmacia Biotech, Milan, Italy).


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  		TABLE 1. Primers and experimental conditions for qualitative RT-PCR amplification of thyroid specific genes and RAR {alpha}, ß, and {gamma}

 
Real time quantitative RT-PCR

To perform the quantitative RT-PCR we used the Real Time Sequence Detection System 7700 (PE Applied Biosystems, Foster City, CA). A standard curve with serial dilution of a normal thyroid cDNA was constructed. Primers and probes specific for the ubiquitously expressed GAPDH and RAR {alpha} and ß were from Applied Biosystems (Monza, Italy; PDAR and Assay-on-Demand Gene Expression). Samples omitting reverse transcriptase and cDNA were included in each run as controls of potential laboratory and/or assay contamination. Each sample was assayed in triplicate according to conditions recommended by Applied Biosystems: 10 ng cDNA was added to a mixture of 1x Universal Master Mix, 900 nM each primer, 200 nM probe in a final volume of 25 µl. Reaction mixtures were incubated for 2 min at 50 C, denatured for 10 min at 95 C, and subjected to 40 cycles of a two-step PCR consisting of a 15-sec denaturation at 95 C and 1 min annealing/extension at 60 C. Results were expressed as fold induction after calculating the relative amounts of RARß and {alpha} in the cells (treated/untreated) and normalizing to the amount of GAPDH mRNA.

Iodide uptake test

Cells were plated in 24-well plates at a density of 1 x 105 cells/well and cultured in their own media. When cells reached confluence, they were washed with 2 ml of 0.9% NaCl solution and then incubated with 500 µl Hank’s balanced salt solution (HBSS, Sigma) incubation buffer [HBSS, 0.5% BSA (Sigma), and 10 mM HEPES] with 0.1 µCi of Na125I and 1 µM of cold NaI for 45 min; cells were then washed twice with 2 ml nonradioactive ice-cold HBSS incubation buffer and solubilized with 1 ml of 0.1 M NaOH, 0.1% sodium dodecyl sulfate, and 2% Na2CO3 lysis buffer. Radioactivity in each sample was counted in a {gamma}-counter. Cell numbers were also determined and iodide uptake was expressed as counts per minute. All experiments were performed in triplicate. The FRTL5 (Fisher rat thyroid L-5) cell line was used as positive control.

Immunohistochemistry

Ten formalin-fixed and paraffin-embedded PTC and 10 normal thyroid tissues were used for RARß protein expression by immunohistochemistry. Sections (5 µm) were cut from blocks, deparaffinized, and rehydrated through graded alcohols. After heating twice in a microwave oven for 5 min at 700 W in citrate buffer (pH 6.0), sections were incubated for 1 h with an anti-RARß (1:25) monoclonal antibody (Lab Vision-NeoMarkers, Fremont, CA). Biotinylated antimouse IgG (Vector Laboratories, Burlingame CA) was applied and followed by detection using avidin biotin peroxidase-complex method. Diaminobenzidine was used as chromogen. Light counterstaining with hematoxylin was used.

Statistical analysis

Statistical analysis was performed by ANOVA test or Student’s t test using StatView 4.5 software (Abacus Concepts Inc., Berkeley, CA). Results were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of RA on cell growth

As shown in Fig. 1Go, cell growth was inhibited both in the follicular WRO (Fig. 1AGo) and poorly differentiated NPA thyroid cancer-derived cell lines (Fig. 1BGo). A statistically significant difference in cell number was found after 25 d of all-trans-RA treatment in WRO (P < 0.0001) corresponding to about 12 doublings and 21 d of all-trans-RA treatment in NPA (P = 0.0007) corresponding to about 15 doublings. This difference persisted even after 1 month of all-trans-RA treatment (P = 0.0047 and P < 0.0001, respectively). On the contrary, all-trans-RA treatment did not show any effect on cell growth of the two human anaplastic (ARO and FRO) and medullary (TT) thyroid cancer-derived cell lines after more than 15 doublings (Fig. 1Go, C–E). MTS cell proliferation test performed after 5, 10, and 15 d of 1 µM RA treatment confirmed a significant decrease of cell proliferation in both responding cell line (WRO and NPA) after 15 d of treatment (P = 0.03) (data not shown). As shown in Fig. 2AGo, the all-trans-RA treatment determined a slow but increasing reduction of both WRO and NPA cell proliferation likely due to the DNA synthesis inhibition, as assessed by the BrdU test.



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  		FIG. 1. Cell growth in five thyroid cancer cell lines treated with all-trans-RA and with ethanol as control. A significant growth inhibition was observed after 25 and 21 d of RA treatment in both WRO (A) and NPA (B) cell lines, respectively. No growth inhibition was observed in ARO (C), FRO (D), and TT (E) cell lines.

 


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  		FIG. 2. Inhibition of WRO and NPA cell proliferation as determined by the test of BrdU incorporation during DNA synthesis. A, Evidence of a slow but increasing inhibition of DNA synthesis during the treatment with all-trans-RA. Cell apoptosis detection in WRO and NPA cell lines as detected by the determination of cytoplasmic histone-associated DNA fragments (nucleosome enrichment) after inducing cell death. B, Evidence of precocious involvement (48 h) of the apoptotic mechanism.

 
The apoptosis test showed a significant nucleosome enrichment after 48 h of all-trans-RA treatment in both WRO, corresponding to 1.6-fold induction, and NPA, corresponding to 1.5-fold induction (P < 0.05 in both cases). After 5 d of all-trans-RA treatment we did not observe apoptosis in the two cell lines (Fig. 2BGo).

The FACS analysis of the cell cycle of NPA cells treated with RA for 24 h, 48 h, 72 h, 5 d, and 21 d showed an evident increase of the number of cells in phases S and G2/M associated with a simultaneous decrease in phases G0/G1 when cells were treated for at least 21 d (Fig. 3Go), while no difference was observed in the cell cycle of treated and untreated WRO cell line, at any time (data not shown). At variance with the results of the nucleosome enrichment test, the FACS analysis with propidium iodide did not show any increase of the percentage of apoptotic cells between the RA-treated and untreated cells, neither in the NPA nor in the WRO cell line.



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  		FIG. 3. NPA cell cycle analysis by FACS with propidium iodide. After 21 d, cells treated with RA (B) showed an increased number of cells in phases S (15%) and G2/M (10%), associated to a simultaneous decrease in phases G0/G1 (55%) with respect to untreated cells (A) that showed 11% of cells in phase S, 8% in phase G2/M and 68% in phase G0/G1.

 
Thyroid-specific gene expression

Untreated thyroid carcinoma cell lines expressed only a few thyroid differentiation genes. In particular, all cell lines, with the exception of FRO, expressed TTF-1; WRO also expressed Pax-8 and TT expressed TTF-1, thyroperoxidase, and an aberrant form of Tg, with a lower molecular weight. FRO cell line expressed only Pax-8. When studying the effect of all-trans-RA treatment, we didn’t observe any change in the mRNA expression of the thyroid-specific genes at any time during all-trans-RA treatment. In particular, no recovery of NIS mRNA expression was seen (Table 2Go).


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  		TABLE 2. Expression of thyroid specific differentiation genes in the thyroid cancer cell lines analyzed, both in basal conditions (B) and after all-trans-RA treatment (T)

 
125Iodide uptake studies

The human thyroid cancer cell lines analyzed in this study (ARO, FRO, NPA, WRO, and TT) did not show the ability to take up iodide in basal conditions. The iodide uptake test was also negative after all-trans-RA treatment at any time and in any cell type (Fig. 4Go).



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  		FIG. 4. Iodide uptake of WRO cells in basal conditions and after RA treatment. No uptaking activity was shown in this cell line as well as in NPA, FRO, ARO, and TT cell lines, neither in basal condition nor after RA treatment (data not shown). Fisher rat thyroid L-5 (FRTL-5) cell line was used as positive control.

 
RAR gene expression

Because WRO and NPA growth were found to be sensitive to all-trans-RA treatment, we investigated RAR {alpha}, ß and {gamma} mRNA expression in basal and all-trans-RA-treated cell lines. As shown in Fig. 5Go, the qualitative RT-PCR analysis showed that RAR{gamma} mRNA was highly expressed in all cell lines, both in basal conditions and after all-trans-RA treatment. RAR{alpha} was expressed both at lower levels and more heterogeneously with respect to RAR{gamma}, in all cell lines both in basal conditions and after all-trans-RA treatment. RARß was expressed only in WRO and NPA for both basal conditions and after all-trans-RA treatment.



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  		FIG. 5. Qualitative analysis of RAR{alpha}, ß, and {gamma} mRNA expression in the five cell lines in basal conditions (–) and after RA treatment (+). RAR{gamma} was highly expressed in all cell lines both before and after RA treatment, as revealed by gel electrophoresis; RAR{alpha} was expressed at lower levels and more heterogeneously in all cell lines both in basal conditions and after RA treatment, as revealed by Southern blot with specific probes; RARß was expressed only in WRO and NPA, both in basal conditions and after RA treatment, as revealed by Southern blot with specific probes. C+, Normal thyroid was used as positive control. RT–, sample omitting reverse transcriptase and cDNA; B, sample omitting cDNA.

 
Quantitative real time RT-PCR was used to confirm that RAR{alpha} was expressed by all cell lines in basal conditions, with a slight but significant increase in NPA and TT cell lines, a slight but significant decrease in ARO and FRO, and no variation in WRO when treated with all-trans-RA (Fig. 6AGo). The quantitative analysis of RARß expression in WRO and NPA cell lines revealed that RARß mRNA expression was significantly increased during all-trans-RA treatment in both cell lines with a peak after 48 h of RA treatment. The level of the induction was higher in WRO, which showed a 26-fold induction of RARß mRNA, than in NPA, which showed a 7-fold induction (P < 0.0001 in both cases) (Fig. 6BGo).



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  		FIG. 6. Quantitative analysis of RAR{alpha} mRNA expression in all cell lines in basal conditions and after RA treatment. A, We observed a slight but significant increase in NPA and TT cell lines, a slight but significant decrease in ARO and FRO, and no variation in WRO. Quantitative analysis of RARß mRNA expression in WRO and NPA cell lines in basal conditions and after RA treatment. B, Evidence of a significant increase of RARß mRNA both in WRO and NPA responding cell lines.

 
Analysis of RARß mRNA expression in 10 PTC, their normal contralateral tissues and four ATC using real time RT-PCR showed a significant decrease of RARß mRNA levels in PTC and ATC vs. normal controls (P < 0.05 in both cases) (Fig. 7Go).



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  		FIG. 7. Levels of RARß mRNA expression detected by quantitative RT-PCR in 10 PTC and their normal contralateral tissues and four ATC. The expression of RARß mRNA was significantly lower in PTC and ATC tumors with respect to normal thyroid tissue.

 
RARß protein expression

As shown in Fig. 8AGo, a positive nuclear staining was found in all normal thyroid tissues. Seven of 10 PTC samples were completely negative for RARß protein expression (Fig. 8BGo). Three PTC showed some areas of positivity and, within the positive areas, about 40% of malignant cells had a well evident positive nuclear staining (Fig. 8CGo).



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  		FIG. 8. Immunohistochemical analysis of RARß protein expression in normal thyroid tissue and in PTC. A, Normal thyroid tissue showing a high level of nuclear RARß protein expression. B, Absence of RARß protein expression in one PTC. C, PTC with areas of nuclear-positive staining for RARß protein.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We studied the effect of all-trans-RA treatment on five human thyroid cancer-derived cell lines (ARO, FRO, NPA, WRO, and TT) and we found that two of them (WRO and NPA) showed a growth inhibition after 21 and 25 d of all-trans-RA treatment. The analysis of the putative causes of growth inhibition suggested an early involvement of apoptosis (48 h after starting the RA treatment) and a late involvement of inhibition of proliferation (starting after 72 h and continuing until the end of the experiment). In the NPA cell line, a delay in cell-cycle progression, as demonstrated by FACS analysis, may contribute to the growth inhibition. This hypothesis is apparently not valid for the WRO cell line, which did not show any delays in cell-cycle progression when analyzed by FACS.

In agreement with other studies (13, 14), we observed the involvement of both apoptosis and decrease of the DNA synthesis as mechanisms of all-trans-RA induced growth inhibition in responding cell lines. However, as shown in Table 3Go, data on the activation of the apoptosis in thyroid carcinoma cell lines treated with RA are scanty. We were unable to confirm the RA induction of apoptosis by FACS analysis. This discrepancy with the results obtained with the nucleosome enrichment test may be due both to a lower sensitivity of the FACS analysis (15) and/or to a lower specificity of the ELISA test (16). As a consequence of these different results, the question of whether the apoptosis is involved as a mechanism responsible for the cell growth inhibition in thyroid carcinoma cells treated with RA remains to be answered.


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  		TABLE 3. Summary of the most important effects exerted by different types of retinoids on human thyroid carcinoma-derived cell lines

 
Up-to-date, controversial data, summarized in Table 3Go, have been reported on the RA-induced growth inhibition in thyroid cell lines (9, 13, 17, 18, 19). Whereas some authors (9, 17, 18, 19) found a RA-induced growth inhibition in FTC-derived cell lines (WRO and FTC-133, respectively), others (13) observed this phenomenon only in ATC-derived cell line (DRO). This discrepancy might depend on the different type of RA employed, as assumed by the fact that some authors used all-trans-RA (17, 18, 19), whereas others used 9-cis- or 13-cis-RA (9, 13), whose interaction with the RAR and RXR receptors is different (20). Another variable that could play a role in determining these discrepancies might be both the duration (7 d vs. 1 month) and the dose (1 vs. 10 µM) of RA treatment. Taken together, these studies intriguingly suggest that RA treatment might determine a growth control in some thyroid cell lines but not in others.

Unfortunately, we did not observe any recovery of mRNA expression of thyroid-specific genes and in particular of NIS gene, either in responding or nonresponding cell lines. Previous studies have been reported showing redifferentiating effects of RA on thyroid cell lines such as the induction of 5'deiodinase activity, NIS, and other differentiation markers expression (17, 19, 21, 22, 23) and iodide uptake (9) (Table 3Go). There is evidence suggesting that the degree of differentiation might play a role in the ability of responding to RA treatment in terms of recovering of the differentiation gene expression (8). In our study, neither cells derived from differentiated thyroid carcinoma (NPA and WRO) nor those derived from undifferentiated or MTC (ARO, FRO and TT) showed NIS, or any other thyroid gene, mRNA induction after all-trans-RA treatment. The lack of recovery of radioiodide uptake after all-trans-RA treatment confirmed the inability to reexpress NIS mRNA. As a matter of fact, with the exception of one study (9), no other evidence of RA induction of iodine uptake in thyroid carcinoma cell lines has been reported so far, not even in cells that recovered the NIS mRNA expression (8).

At variance with our nonresponding cells, NPA and WRO showed a basal and an all-trans-RA significant induction of RARß mRNA expression. On the basis of these results we can hypothesize that RARß expression plays a role in the growth inhibition observed after all-trans-RA treatment. Indeed, a role of RARß in mediating the response to retinoids has been previously demonstrated in human breast cancer-derived cell lines (24) and, at least supposed, in several others (25, 26, 27). The recent observation that RARß mRNA was neither expressed nor induced by 9-cis-RA in WRO cells (13) is only apparently in conflict with our results because the growth of this cell line was not inhibited by 9-cis-RA treatment, in agreement with the hypothesis that the presence of RARß is required for the growth inhibition. The reason for which RARß mRNA expression is present in our WRO cells, but not in WRO used in other laboratories is difficult to explain; one reason might be the wide diffusion of WRO cell line and the possibility that they underwent some changes during the passages.

The involvement of RARß in the growth inhibition of WRO and NPA is also supported by previous demonstrations of an antioncogenic role of RARß that has been found to be missing in several types of human cancer including oral, lung, breast, esophageal cancers and PTC (28, 29, 30, 31, 32, 33) and able to inhibit the cancer progression when exogenously expressed in RARß-negative cancer cell lines (24). Even in our series of human thyroid cancer tissues both the mRNA and protein RARß expression was significantly reduced with respect to normal thyroid. On the basis of our in vitro results and whether the hypothesis of the involvement of RARß is correct, the in vivo treatment of these tumors might at least reduce their growth, even if no recovery of 131I uptake is expected.

In conclusion, we found that all-trans-RA treatment can determine a significant in vitro growth inhibition especially in differentiated thyroid tumor-derived cell lines but it seems unable to reinduce the expression of thyroid-specific genes and in particular to reinduce the ability to take up iodine. The growth inhibition is likely due to apoptosis in an early phase and to a decrease of DNA synthesis later. A delay in cell-cycle progression may be also involved, at least in the NPA cell line. The finding of a basal and RA-induced RARß mRNA expression only in cell lines responding to all-trans-RA suggests that the growth inhibition might be mediated by RARß.


   Acknowledgments
 
We thank Dr James Fagin, Director of the Endocrinology and Metabolism Division of the University of Cincinnati (Cincinnati, OH), for the kind gift of WRO, NPA, FRO, and ARO cell lines.


   Footnotes
 
This study was supported in part by grants from Ministero della Istruzione Universitaria e Ricerca Scientifica (ex 40%) 2003 and Associazione Italiana per la Ricerca sul Cancro 2004.

L.A. and E.M. are Ph.D. students in Endocrine and Metabolic Sciences.

First Published Online December 28, 2004

Abbreviations: ATC, Anaplastic thyroid cancer; BrdU, bromodeoxyuridine; FACS, fluorescence-activated cell sorting; FTC, follicular thyroid cancer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hank’s balanced salt solution; MTC, medullary thyroid cancer; NIS, sodium iodide symporter; PTC, papillary thyroid cancer; RA, retinoic acid; RAR, RA receptor; Tg, thyroglobulin.

Received May 21, 2004.

Accepted December 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schlumberger M, Pacini F 1999 Thyroid tumors. Paris: Nucleon Editions
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