Effects of Histone Acetylation on Sodium Iodide Symporter Promoter and Expression of Thyroid-Specific Transcription Factors
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《内分泌学杂志》
Dipartimento di Scienze e Tecnologie Biomediche (C.P., F.D., A.V.D., G.T., A.P., G.D.) and Istituto di Genetica (L.P.), Policlinico Universitario di Udine, 33100 Udine
Dipartimento di Scienze Farmacobiologiche (D.R.), Università di Catanzaro, 88100 Catanzaro
Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole (L.C.), Università di Trieste, 34127 Trieste
Dipartimento di Scienze Cliniche (S.F., E.F., E.T., T.M.), Università di Roma "La Sapienza," 00161 Rome, Italy
Abstract
Inhibitors of histone deacetylases (HDACs) activate the sodium iodide symporter (NIS) expression in thyroid tumor cells. In this study, mechanisms accounting for these effects were investigated. Various human thyroid tumor cell lines (ARO, BCPAP, FRO, TPC-1) were treated with the HDAC inhibitors Na butyrate (NaB) and tricostatin A (TSA), and the effects on the expression of NIS and several thyroid-specific transcription factors together with the activity of NIS promoter were evaluated. TSA and NaB increased NIS mRNA levels in all cell lines. Among thyroid-specific transcription factors, only expression of PAX8 in ARO cells was increased. Down-regulation of thyroid-specific transcription factor-1 expression was observed in BCPAP and TPC-1 cell lines. Thyroid-specific transcription factor-2 mRNA was reduced in FRO, BCPAP, and TPC-1 cells. Histone acetylation had no significant effects on HEX expression. Altogether, these data indicate that the increase of NIS expression is not mediated by modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcription factors), treatment with TSA and NaB increased NIS promoter activity. Stimulation of NIS promoter activity was also obtained by overexpressing histone acetylating proteins pCAF and p300 in HeLa cells. Conversely, overexpression of the HDAC 1 enzyme inhibited basal activity of the NIS promoter. Effects of TSA and NaB on NIS expression were also evaluated in nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. In all cell lines TSA and NaB greatly increased NIS mRNA levels. We concluded that control of NIS expression by inhibition of HDAC appears not to be mediated by cell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors.
Introduction
HISTONE ACETYLATION AND deacetylation involve the reversible transfer of the acetyl group of acetyl-coenzyme A to the amino group of lysine residues in histone proteins. This reaction is catalyzed by histone acetyltransferases (HATs) and can be reversed by histone deacetylases (HDACs) (1, 2, 3). Therefore, HAT and HDAC activities determine acetylation levels of histones. Several compounds induce histone hyperacetylation by reducing the rate of histone deacetylation through the inhibition of HDAC (4). A correlation has been established between hyperacetylated histones and transcriptionally active chromatin because acetylation makes chromatin less condensed and more accessible to transcriptional activators (5, 6, 7). Moreover, recent studies indicate a potential role for HDAC inhibitors in promoting differentiation of some tumor cells, suggesting their possible use as anticancer agents (8, 9, 10, 11).
Poorly differentiated thyroid carcinomas have a very poor prognosis (12). One of the reasons for this behavior is the inability to concentrate the radioiodine, which is used as very effective tool for diagnosis and treatment for both tumor remnants and distant metastases (13, 14, 15). The loss of iodide uptake capability in poorly differentiated thyroid carcinomas is mainly due to a reduced/lost expression of the sodium/iodide symporter (NIS) gene (16, 17, 18).
Several authors have reported that HDAC inhibitors stimulate NIS expression in thyroid cancer cells (19, 20). However, the molecular mechanism by which this effect occurs has not been addressed yet. This consideration induced us to study the role of histone acetylation on NIS expression and NIS promoter and expression of several thyroid-specific transcription factors in both thyroid and nonthyroid tumor cells.
Materials and Methods
Cell cultures
Human thyroid carcinoma cell lines ARO, BCPAP, FRO, and TPC-1 were used (21) as well as the nonthyroid cell lines HeLa, MCF-7 (from human breast carcinoma, no. HTB-22; American Type Culture Collection, Manassas, VA), Hep-G2 (from human hepatocellular carcinoma, ATCC no. HB-8065), and SAOS-2 (from human osteogenic sarcoma, ATCC no. HTB-85). The ARO cell line was grown in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Inc., Milan, Italy). BCPAP, FRO, TPC-1, HeLa, Hep-G2, and MCF-7 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (Life Technologies). The SAOS-2 cell line was grown in DMEM-F12 supplemented with 10% fetal bovine serum (Life Technologies). All cell lines were treated with tricostatin A (TSA; 300 nM; Sigma-Aldrich Srl, Milan, Italy) and Na butyrate (NaB; 3 mM; Sigma) for 30 h.
Quantitative and qualitative RT-PCR
Total RNA was extracted with the classical guanidinium isothiocyanate/acid phenol method (22). Two microliters of total RNA were reverse transcribed to single-strand cDNA using oligo (dT) primer and 200 U Superscript II reverse transcriptase (Invitrogen, Milan, Italy) in a final volume of 20 μl at 42 C for 50 min following by heating at 70 C for 15 min.
Quantitative PCR analysis of NIS, PAX8, TTF-1, TTF-2, and HEX mRNA expression was performed on cDNA samples using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). Oligonucleotide primers and probes for genes analyzed were purchased from Applied Biosystems as Assays-on-Demand gene expression products that consist of a 20 x mix of unlabeled PCR primers and TaqMan MGB probe (5-carboxyfluorescein dye labeled). Oligonucleotide primers and probe for the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or -actin were purchased from Applied Biosystems as TaqMan PDAR (VIC dye labeled). A 25-μl reaction mixture containing 2.5 μl of cDNA template, 12.5 μl TaqMan universal PCR master mix (Applied Biosystems), and 1.25 μl primer probe mixture was amplified using the following thermal cycler parameters: incubation at 50 C for 2 min and denaturing at 95 C for 10 min and then 40 cycles of the amplification step (denaturation at 95 C for 15 sec and annealing/extension at 60 C for 1 min).
For each amplification run, standard curve was generated using six serial dilutions (100, 50, 10, 2, 0.4, 0.2 ng) of cDNA mix expressing all the genes analyzed. The cDNA mix was obtained in our laboratory: total RNA was extracted from different tissues and cell types and mix together. Then 250 μg of such a mix was reverse transcribed obtaining 50 aliquots of 5 μg each of cDNA. The reverse transcription (RT) efficiency of the individual cDNAs in the mix have been validated measuring for a standard sample the PCR cycles of a standard gene (used to calculate the amount of transcript in the sample). All amplification reactions were performed in triplicate, and the averages of the threshold cycles were used to interpolate standard curves and calculate the transcript amount in samples using SDS software (version 1.7a; Applied Biosystems). To obtain the mRNA expression quantification of each target gene analyzed, results were calculated as follows: untreated cells were considered as calibrator, mRNA expression was calculated as sample to calibrator ratio normalized with four endogenous controls [GAPDH, -actin, 2-microglobulin, and hypoxantine guanine phosphoribosyl transferase (HPRT-1) mRNAs], giving very similar results. Thus, mean values using the four control genes were calculated. Two negative controls were included in PCR: a samples with PCR mix without cDNA and a sample with PCR mix and RNA before RT reaction (–RT control).
Qualitative analysis of PAX8 expression by RT-PCR was performed by using the following primers: PAX8–1, 5'-GGCCACCAAGTCCCTGAGTC-3'; and PAX8–2, 5'-TCGGGGGTTTCCTGCTTTATG-3'.
Amplification was performed with the following conditions: one cycle at 94 C for 5 min, followed by 30 cycles at 94 C for 1 min, 65 C for 1 min, and 72 C for 2 min, with a final elongation step at 72 C for 7 min. RT-PCR for -actin was used to confirm RNA integrity for each specimen. PCR products were visualized on 2% agarose gel 0.5x Tris-borate EDTA.
Promoter activity studies
The NIS promoter activity was investigated by using a clone, kindly provided by U. Loos (University of Ulm, Ulm, Germany) containing 2.2 kb of 5' genomic sequence for the NIS promoter, containing the minimal promoter linked to the luciferase (LUC) gene as reporter (23, 24). The efficiency of transfection was normalized by using constructs containing either the Rous sarcoma virus (RSV) promoter linked to the chloramphenicol-acetyl-transferase (CAT) gene or the cytomegalovirus (CMV) promoter linked to the -galactosidase gene (-GAL). Expression vectors for pCAF and p300 were provided by A. Gulino (University of Rome, Rome, Italy). Plasmids containing c-fos and Simian virus 40 (SV40) promoters (linked to the CAT and LUC reporter genes, respectively) were provided by G. Manfioletti (University of Trieste, Trieste, Italy). The HDAC1 gene was overexpressed by using the vector pcDNA3-HD1-F, provided by C. Brancolini (University of Udine, Udine, Italy). The calcium phosphate coprecipitation method used for transfections was performed as previously described (25). HeLa cells were plated at 6 x 105 cells/100-mm culture dish 20 h before transfection. Plasmids were used in the following amount per dish: CMV-LUC, 2 μg; RSV-CAT, 2 μg; CMV--GAL, 2 μg; pNIS-LUC, 7 μg; c-fos-CAT, 7 μg; SV40-LUC, 7 μg; pCAF, 2 μg; p300, 2 μg; and pcDNA3-HDI-F, 2 μg. Cells were harvested 44 h after transfection, and cell extracts were prepared by a standard freeze and thaw procedure. CAT and -GAL proteins were measured by ELISA method (Amersham Pharmacia Biotech, Milan, Italy). LUC activity was measured by a standard chemiluminescence procedure (26).
Chromatin immunoprecipitation (ChIP) assay
HeLa cells were transfected as above with 12 μg of NIS-LUC plasmid and treated for 24 h with TSA (300 nM) and NaB (3 mM). Aliquots of transfected cells were taken aside before immunoprecipitation to test the efficiency of transfection. Then cells were washed once with PBS at room temperature and fixed with 1% (vol/vol) formaldehyde in PBS at 37 C for 10 min. Cells were quickly rinsed twice with ice-cold PBS and scraped into 1 ml of ice-cold PBS. After spinning at 700 x g at 4 C, cell pellets were resuspended in 1.2 ml of lysis buffer [1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 50 mM Tris/HCl (pH 8.1), plus protease inhibitor cocktail] and incubated on ice for 10 min. After lysis, the samples were centrifuged for 10 min at 14,000 x g at 4 C, and NaCl and Triton X-100 were added to final concentrations of 150 mM and 1% (vol/vol), respectively. Then the samples were incubated for immunoclearing with Protein A-Sepharose (Sigma) [80 μl of 50% slurry in 10 mM Tris/HCl (pH 8.1), 1 mM EDTA] overnight at 4 C. After incubation, the samples were quickly centrifuged at 9000 x g, and the supernatants were collected. Immunoprecipitation was performed by adding 5 μg of polyclonal antiacetyl-histone H3 antibody (Upstate Biotechnologies Inc., Lake Placid, NY) and incubating the mixture overnight at 4 C; 80 μl of Protein A-Sepharose was then added and the slurry was incubated overnight at 4 C. The Sepharose beads were harvested by centrifugation at 8000 x g and washed sequentially for 10 min in 1 ml each of TSE I [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris/HCl (pH 8.1), 150 mM NaCl], TSE II [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris/HCl (pH 8.1), 500 mM NaCl], TSE III [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris/HCl (pH 8.1)], and Tris/EDTA buffer [10 mM Tris/HCl (pH 8.1), 1 mM EDTA]. Immune complexes were eluted from beads with 100 μl of elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature for 10 min and cross-links were reversed by heating at 56 C overnight with the addition of proteinase K. DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation and was used as a template for PCR.
PCR was performed by using the following primers: hNIS, forward, 5'-GACTCAAGATGTCAGTCAGC-3' that contains a NIS promoter-specific sequence; and LUC Spec, reverse, 5'-GCCTTTCTTTATGTTTTTGGC-3' that contains a luciferase-specific sequence.
Amplification was performed with the following conditions: one cycle at 95 C for 10 min, followed by 30 cycles at 95 C for 30 sec, 52 C for 30 sec, and at 72 C 1 min. PCR products were visualized on 2% agarose gel 0.5x Tris-borate EDTA.
Results
Effects of HDAC inhibitors on NIS gene expression
It has been previously shown that HDAC inhibitors stimulate NIS expression in thyroid cancer cells (19, 20, 27, 28). To test the reproducibility of these findings in our system, ARO and FRO cell lines (from anaplastic carcinoma) and BCPAP and TPC1 cell lines (from papillary carcinoma) were treated with TSA (300 nM) and NaB (3 mM) for 30 h; then total RNA was extracted and NIS expression was evaluated by real-time PCR, using four genes for normalization (GAPDH, -actin, 2-microglobulin, HPRT-1). Results are shown in Fig. 1 and are expressed considering 1 the value of unstimulated cells. As shown, both TSA and NaB treatment greatly induced expression of NIS mRNA in all cell lines tested.
Effects of HDAC inhibitors on expression of thyroid-specific transcription factors
Promoters with thyroid-specific activity (such as that of NIS gene) are activated by one or more thyroid-specific transcription factors (29). Thus, the activating effect of HDAC inhibitors on NIS expression could be mediated by the increase of expression of thyroid-specific transcription factors. To test this possibility, ARO, FRO, BCPAP, and TPC-1 cell lines treated with TSA and NaB were assayed for TTF-1, TTF-2, PAX8, and HEX mRNA levels by real-time PCR.
Results (expressed as fold of modification with respect to the levels observed in untreated cells) are shown in Table 1. To be conservative, we decided to define as bona fide effects of histone acetylation those in which TSA and NaB induced significant modification in the same direction. In fact, distinct HDAC inhibitors act by different mechanisms and, in addition to histone acetylation, may induce other effects (30, 31). In the case of TTF-1, a clear inhibitory effect of histone acetylation was observed only in cell lines deriving from papillary carcinomas (BCPAP and TPC-1). TTF-2 expression was significantly reduced in FRO, BCPAP, and TPC-1 cell lines. No clear effects of histone acetylation can be observed in the case of HEX expression. PAX8 expression was significantly increased by histone acetylation in ARO cells only. A correlation of these data to those demonstrating the histone acetylation increase of NIS mRNA levels in all cell lines investigated (Fig. 1) suggests that changes of the TTF-1, TTF-2, HEX, and PAX8 levels induced by histone acetylation cannot account for the increase of NIS gene expression. The effect of TSA and NaB on PAX8 mRNA levels in ARO cells was the only activating effect of histone acetylation on expression of thyroid-specific transcription factors that we were able to detect. This effect can be related to the basal levels of PAX8 mRNA, which in ARO cells were not detectable by qualitative RT-PCR (Fig. 2). In contrast, no relationship between basal levels of expression and effects of histone acetylation was observed in the case of TTF-1, TTF-2, and HEX genes (data not shown).
Effects of HDAC modulation on NIS promoter
Data reported so far suggest that TSA and NaB activate NIS gene expression without modification of expression of thyroid-specific transcription factors. However, HDAC inhibition may control NIS expression by different mechanisms from expression modification of thyroid-specific transcription factors. To demonstrate that effects of TSA and NaB treatment are not mediated by modification of thyroid-specific transcriptional mechanisms, we used a transfection approach. A construct bearing 2.2 kb of 5' genomic sequence for the human NIS promoter, containing the minimal promoter linked to the LUC gene as reporter, was used to transfect HeLa cells (23, 24), a nonthyroid cell line that does not express TTF-1, TTF-2, PAX8, and HEX genes. Therefore, in this experimental situation, any effect on NIS promoter would not be mediated by the thyroid-specific transcriptional machinery. The construct containing the NIS promoter was transfected in HeLa cells. Then transfected cells were treated with or without TSA (300 nM) and NaB (3 mM) for 16 and 28 h, and the expression of the LUC gene was evaluated. Figure 3A shows that either TSA or NaB treatment increases NIS promoter activity. To show that TSA and NaB treatment modified histone acetylation on the transfected NIS-LUC plasmid, a ChIP assay was used. The ChIP assay has been used by a number of laboratories to probe the acetylation state of chromatin (reviewed in Ref. 34). HeLa cells were transfected with the NIS-LUC plasmid and treated with TSA or NaB. After cross-link, the acetylated H3 histone was immunoprecipitated by using a specific antibody (Upstate Biotechnology). As shown in Fig. 3B, after immunoprecipitation, a specific PCR product was obtained only in NIS promoter-transfected cells that have been treated with TSA or NaB.
These results indicate that acetylated H3 histone on NIS promoter was present only on HDAC inhibitor treatment, thus demonstrating that HDAC treatment modify histone acetylation status of the transfected NIS promoter. Among thyroid-specific transcription factors, PAX8 plays a major role in up-regulation of NIS promoter (35, 36, 37). RT-PCR experiments performed by using mRNA of transfected HeLa cells as template indicate that the increase of NIS promoter activity is not mediated by activation of the endogenous PAX8 expression. In fact, in HeLa cells treated with TSA or NaB, no PAX8 expression was observed (data not shown). Altogether, these results indicate that HDAC inhibition is able to activate NIS gene expression by a direct histone acetylation of the promoter, without modification of expression of TTF-1, TTF-2, HEX, and PAX8.
To support the notion that the basal levels of NIS promoter are controlled by histone acetylation, two cotransfection approaches were used. First, the NIS promoter was cotransfected in HeLa cells together with a plasmid expressing the histone deacetylase 1 (HDAC1) (38). As expected, the overexpression of HDAC1 exerted a strong inhibitory effect on the NIS promoter activity (Fig. 4A). The effect was specific because overexpression of HDAC1 determined unsignificant effects on RSV and c-fos promoters and only a modest inhibitory effects on SV40 promoter. In the second approach, the NIS promoter was cotransfected with expression plasmids for the HATs pCAF or p300 (39). As shown in Fig. 4B, overexpression of either pCAF or p300 greatly increased the NIS promoter activity. The HAT effect was specific because neither pCAF nor p300 was able to increase the RSV promoter activity. Altogether, these experiments indicate that basal levels of NIS promoter activity are subjected to histone acetylation control exerted by ubiquitous cofactors.
Effects of HDAC inhibitors on NIS expression of nonthyroid cell lines
Results obtained in HeLa cells suggest that the effects of HDAC inhibition on NIS expression do not occur only by activation of thyroid-specific mechanisms but also involve activation of ubiquitous mechanisms. The prediction of this hypothesis is that HDAC inhibition also would increase NIS mRNA levels in nonthyroid cell lines. This possibility was tested by using the following human cell lines: MCF-7 from mammary carcinoma, Hep-G2, from liver carcinoma, and SAOS-2, from osteosarcoma. The indicated cell lines were treated with TSA (300 nM) and NaB (3 mM) for 30 h and NIS mRNA levels were measured by RT-PCR. As shown in Fig. 5, in all cell lines both TSA and NaB treatment induced a great increase of NIS mRNA levels. Basal values of NIS mRNA levels are much higher in MCF-7 than Hep-G2 and SAOS-2 cell lines. This finding fits with observations indicating that in MCF-7 cells, NIS gene expression is regulated by various signaling pathways (40, 41).
Discussion
Several reports have shown that treatment of thyroid cancer cell lines with HDAC inhibitors increases NIS expression as well as iodine uptake (19, 20, 27, 28, 42). These results may have relevance in treatment of undifferentiated thyroid tumors. In fact, the inability to trap iodine by these tumors makes ineffective the radioiodine therapy (43). In this view, restoration of NIS expression in undifferentiated thyroid tumors is considered a means to improve therapeutic strategy. For this reason, control of NIS expression has been extensively investigated (35, 37, 44, 45, 46, 47, 48, 49). The aim of the present study was to investigate molecular mechanisms by which HDAC inhibitors induce NIS expression. Distinct HDAC inhibitors act by different mechanisms and, in addition to histone acetylation, may induce other effects (30, 31). For these reasons, our strategy was the use of two different HDAC inhibitors and the definition as bona fide effects of histone acetylation those in which TSA and NaB induced modifications in the same direction. Our data indicate that effects of HDAC inhibitors on NIS mRNA levels are not mediated by modification of thyroid-specific transcription factors gene expression. Our transfection experiments were performed in the nonthyroid cell line HeLa. Because these cells do not express TTF-1, TTF-2, PAX8, and HEX, the finding that TSA and NaB increase the activity of NIS promoter reinforces the notion that effect of histone acetylation occurs without the contribute of thyroid-specific transcriptional machinery.
The effect of HDAC inhibitors on NIS expression is specific and not the result of a general phenomenon of histone acetylation, as demonstrated by evaluating expression of other genes (see Table 1). According to this view, Western blots performed with a specific antibody against H3 histone indicate no correlation between NIS expression and the bulk histone acetylation state of the cell (data not shown).
Data on NIS promoter activity were obtained by means of transient transfection, i.e. outside the chromosomal structure. It has been demonstrated, however, that precise nucleosome positioning occurs in transiently transfected promoters (50). Moreover, the histone acetyltransferase activity of pCAF is required for activation of transiently transfected promoters (51). In the thyroid field, it has been demonstrated that the transiently transfected thyroglobulin promoter is sensitive to the HDAC inhibitor depsipeptide (19). In agreement with transfection studies, HDAC inhibition induced NIS expression in the tumoral, nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. Because an increase of NIS gene expression, at least in MCF-7 cells, is paralleled by enhanced radioiodine trapping ability (52), our findings suggest that the strategy of inducing NIS expression by HDAC inhibition can be used to make several types of human tumors sensitive to radioiodine therapy.
In agreement with data produced by using TSA and NaB, we have demonstrated that the histone deacetylating protein HDAC1 inhibits NIS promoter activity. Conversely, the HAT proteins pCAF and p300 stimulate NIS promoter activity. It is generally accepted that HDAC1, pCAF, and p300 are recruited to promoters by interaction with transcription factors (53). HDAC1 could be recruited to NIS promoter by the transcription factor specificity protein (SP1). In fact, it has been demonstrated that SP1 interacts with HDAC1 (54) as well as the NIS promoter (48). Interestingly, it has been shown that the nuclear extracts of thyroid tumors have significantly higher SP1 levels than the corresponding normal tissue (48). Thus, the inhibition of NIS gene expression in thyroid tumors could be due to the increase of HDAC1 recruitment, which, in turn, is induced by the higher SP1 binding to the NIS promoter. In the case of HAT proteins, p300 is homologous to the cAMP response element-binding protein, and both are able to interact with the transcription factor cAMP-responsive element binding protein (55). cAMP-responsive element binding protein binding sites are present in the human NIS promoter (32). Thus, HAT proteins may contribute to the stimulation of NIS gene expression obtained by the cAMP pathway activation (41).
In our experimental conditions, HDAC inhibition induces an increase in PAX8 mRNA levels in ARO but not BCPAP, FRO, and TPC-1 cell lines. Interestingly, ARO cells show the lowest amount of basal PAX8 mRNA levels (not detectable by our qualitative PCR method; see Fig. 2). Based on these data, it could be inferred that histone acetylation controls basal levels of PAX8 gene expression. In ARO cells, a reduced histone acetylation state would not be permissive for PAX8 expression; treatment with HDAC inhibitors will allow the basal transcription of PAX8 gene. In contrast, BCPAP, FRO, and TPC-1 cell lines would have higher levels of histone acetylation, enough to allow basal levels of PAX8 mRNA levels, and, therefore, treatment with HDAC inhibitors has no effects on PAX8 expression.
In a recent investigation, the effect of the HDAC inhibitor depsipeptide on TTF-1 and PAX8 mRNA levels in cells derived from differentiated and undifferentiated thyroid cancers (BHP18–21v and ARO, respectively) was investigated (27). In contrast to our findings, the HDAC inhibitor depsipeptide induced increase of TTF-1 mRNA levels in both cell lines. Reasons for this discrepancy are not obvious. However, at least two methodological differences are evident between the two studies: the use of different compounds as HDAC inhibitors and the use of a nonquantitative RT-PCR method to measure TTF-1 and PAX8 mRNA levels. The down-regulation of thyroid-specific transcription factors (TTF-1 and TTF-2) by HDAC inhibition in a cell line that maintains several markers of the thyroid-differentiated phenotype (BCPAP) is in agreement with several observation indicating that TSA blocks intestinal development in Xenopous laevis (56), prevents expression of differentiation markers in chick oviduct explants (32), suppresses differentiation in tissue culture cell models (33).
In conclusion, our data indicate that the NIS promoter, even outside the chromosomal context, is a major target by which HDAC inhibitors increase NIS expression in both thyroid and nonthyroid cancer cells. Thus, future investigations on the relation between NIS promoter activity and histone acetylation may have relevance for defining innovative therapeutic strategies for the treatment of human tumors.
Footnotes
This work was supported by grants from Ministero dell’Istruzione, dell’Università e della Ricerca (to G.D., S.F., and D.R.).
Abbreviations: CAT, Chloramphenicol-acetyl-transferase; ChIP, chromatin immunoprecipitation; -GAL, -galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAT, histone acetyltransferase; HDAC, histone deacetylase; HEX, hematopoietically expressed homeodomain; HPRT, hypoxantine guanine phosphoribosyl transferase; LUC, luciferase; NaB, Na butyrate; NIS, sodium iodide symporter; PAX, paired box transcription factor; RSV, Rous sarcoma virus; RT, reverse transcription; SDS, sodium dodecyl sulfate; SP1, specificity protein; SV40, Simian virus 40; TSA, tricostatin A; TSE, buffer of Triton X-100, SDS, and EDTA; TTF, thyroid-specific transcription factor.
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Dipartimento di Scienze Farmacobiologiche (D.R.), Università di Catanzaro, 88100 Catanzaro
Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole (L.C.), Università di Trieste, 34127 Trieste
Dipartimento di Scienze Cliniche (S.F., E.F., E.T., T.M.), Università di Roma "La Sapienza," 00161 Rome, Italy
Abstract
Inhibitors of histone deacetylases (HDACs) activate the sodium iodide symporter (NIS) expression in thyroid tumor cells. In this study, mechanisms accounting for these effects were investigated. Various human thyroid tumor cell lines (ARO, BCPAP, FRO, TPC-1) were treated with the HDAC inhibitors Na butyrate (NaB) and tricostatin A (TSA), and the effects on the expression of NIS and several thyroid-specific transcription factors together with the activity of NIS promoter were evaluated. TSA and NaB increased NIS mRNA levels in all cell lines. Among thyroid-specific transcription factors, only expression of PAX8 in ARO cells was increased. Down-regulation of thyroid-specific transcription factor-1 expression was observed in BCPAP and TPC-1 cell lines. Thyroid-specific transcription factor-2 mRNA was reduced in FRO, BCPAP, and TPC-1 cells. Histone acetylation had no significant effects on HEX expression. Altogether, these data indicate that the increase of NIS expression is not mediated by modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcription factors), treatment with TSA and NaB increased NIS promoter activity. Stimulation of NIS promoter activity was also obtained by overexpressing histone acetylating proteins pCAF and p300 in HeLa cells. Conversely, overexpression of the HDAC 1 enzyme inhibited basal activity of the NIS promoter. Effects of TSA and NaB on NIS expression were also evaluated in nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. In all cell lines TSA and NaB greatly increased NIS mRNA levels. We concluded that control of NIS expression by inhibition of HDAC appears not to be mediated by cell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors.
Introduction
HISTONE ACETYLATION AND deacetylation involve the reversible transfer of the acetyl group of acetyl-coenzyme A to the amino group of lysine residues in histone proteins. This reaction is catalyzed by histone acetyltransferases (HATs) and can be reversed by histone deacetylases (HDACs) (1, 2, 3). Therefore, HAT and HDAC activities determine acetylation levels of histones. Several compounds induce histone hyperacetylation by reducing the rate of histone deacetylation through the inhibition of HDAC (4). A correlation has been established between hyperacetylated histones and transcriptionally active chromatin because acetylation makes chromatin less condensed and more accessible to transcriptional activators (5, 6, 7). Moreover, recent studies indicate a potential role for HDAC inhibitors in promoting differentiation of some tumor cells, suggesting their possible use as anticancer agents (8, 9, 10, 11).
Poorly differentiated thyroid carcinomas have a very poor prognosis (12). One of the reasons for this behavior is the inability to concentrate the radioiodine, which is used as very effective tool for diagnosis and treatment for both tumor remnants and distant metastases (13, 14, 15). The loss of iodide uptake capability in poorly differentiated thyroid carcinomas is mainly due to a reduced/lost expression of the sodium/iodide symporter (NIS) gene (16, 17, 18).
Several authors have reported that HDAC inhibitors stimulate NIS expression in thyroid cancer cells (19, 20). However, the molecular mechanism by which this effect occurs has not been addressed yet. This consideration induced us to study the role of histone acetylation on NIS expression and NIS promoter and expression of several thyroid-specific transcription factors in both thyroid and nonthyroid tumor cells.
Materials and Methods
Cell cultures
Human thyroid carcinoma cell lines ARO, BCPAP, FRO, and TPC-1 were used (21) as well as the nonthyroid cell lines HeLa, MCF-7 (from human breast carcinoma, no. HTB-22; American Type Culture Collection, Manassas, VA), Hep-G2 (from human hepatocellular carcinoma, ATCC no. HB-8065), and SAOS-2 (from human osteogenic sarcoma, ATCC no. HTB-85). The ARO cell line was grown in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Inc., Milan, Italy). BCPAP, FRO, TPC-1, HeLa, Hep-G2, and MCF-7 cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (Life Technologies). The SAOS-2 cell line was grown in DMEM-F12 supplemented with 10% fetal bovine serum (Life Technologies). All cell lines were treated with tricostatin A (TSA; 300 nM; Sigma-Aldrich Srl, Milan, Italy) and Na butyrate (NaB; 3 mM; Sigma) for 30 h.
Quantitative and qualitative RT-PCR
Total RNA was extracted with the classical guanidinium isothiocyanate/acid phenol method (22). Two microliters of total RNA were reverse transcribed to single-strand cDNA using oligo (dT) primer and 200 U Superscript II reverse transcriptase (Invitrogen, Milan, Italy) in a final volume of 20 μl at 42 C for 50 min following by heating at 70 C for 15 min.
Quantitative PCR analysis of NIS, PAX8, TTF-1, TTF-2, and HEX mRNA expression was performed on cDNA samples using the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). Oligonucleotide primers and probes for genes analyzed were purchased from Applied Biosystems as Assays-on-Demand gene expression products that consist of a 20 x mix of unlabeled PCR primers and TaqMan MGB probe (5-carboxyfluorescein dye labeled). Oligonucleotide primers and probe for the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or -actin were purchased from Applied Biosystems as TaqMan PDAR (VIC dye labeled). A 25-μl reaction mixture containing 2.5 μl of cDNA template, 12.5 μl TaqMan universal PCR master mix (Applied Biosystems), and 1.25 μl primer probe mixture was amplified using the following thermal cycler parameters: incubation at 50 C for 2 min and denaturing at 95 C for 10 min and then 40 cycles of the amplification step (denaturation at 95 C for 15 sec and annealing/extension at 60 C for 1 min).
For each amplification run, standard curve was generated using six serial dilutions (100, 50, 10, 2, 0.4, 0.2 ng) of cDNA mix expressing all the genes analyzed. The cDNA mix was obtained in our laboratory: total RNA was extracted from different tissues and cell types and mix together. Then 250 μg of such a mix was reverse transcribed obtaining 50 aliquots of 5 μg each of cDNA. The reverse transcription (RT) efficiency of the individual cDNAs in the mix have been validated measuring for a standard sample the PCR cycles of a standard gene (used to calculate the amount of transcript in the sample). All amplification reactions were performed in triplicate, and the averages of the threshold cycles were used to interpolate standard curves and calculate the transcript amount in samples using SDS software (version 1.7a; Applied Biosystems). To obtain the mRNA expression quantification of each target gene analyzed, results were calculated as follows: untreated cells were considered as calibrator, mRNA expression was calculated as sample to calibrator ratio normalized with four endogenous controls [GAPDH, -actin, 2-microglobulin, and hypoxantine guanine phosphoribosyl transferase (HPRT-1) mRNAs], giving very similar results. Thus, mean values using the four control genes were calculated. Two negative controls were included in PCR: a samples with PCR mix without cDNA and a sample with PCR mix and RNA before RT reaction (–RT control).
Qualitative analysis of PAX8 expression by RT-PCR was performed by using the following primers: PAX8–1, 5'-GGCCACCAAGTCCCTGAGTC-3'; and PAX8–2, 5'-TCGGGGGTTTCCTGCTTTATG-3'.
Amplification was performed with the following conditions: one cycle at 94 C for 5 min, followed by 30 cycles at 94 C for 1 min, 65 C for 1 min, and 72 C for 2 min, with a final elongation step at 72 C for 7 min. RT-PCR for -actin was used to confirm RNA integrity for each specimen. PCR products were visualized on 2% agarose gel 0.5x Tris-borate EDTA.
Promoter activity studies
The NIS promoter activity was investigated by using a clone, kindly provided by U. Loos (University of Ulm, Ulm, Germany) containing 2.2 kb of 5' genomic sequence for the NIS promoter, containing the minimal promoter linked to the luciferase (LUC) gene as reporter (23, 24). The efficiency of transfection was normalized by using constructs containing either the Rous sarcoma virus (RSV) promoter linked to the chloramphenicol-acetyl-transferase (CAT) gene or the cytomegalovirus (CMV) promoter linked to the -galactosidase gene (-GAL). Expression vectors for pCAF and p300 were provided by A. Gulino (University of Rome, Rome, Italy). Plasmids containing c-fos and Simian virus 40 (SV40) promoters (linked to the CAT and LUC reporter genes, respectively) were provided by G. Manfioletti (University of Trieste, Trieste, Italy). The HDAC1 gene was overexpressed by using the vector pcDNA3-HD1-F, provided by C. Brancolini (University of Udine, Udine, Italy). The calcium phosphate coprecipitation method used for transfections was performed as previously described (25). HeLa cells were plated at 6 x 105 cells/100-mm culture dish 20 h before transfection. Plasmids were used in the following amount per dish: CMV-LUC, 2 μg; RSV-CAT, 2 μg; CMV--GAL, 2 μg; pNIS-LUC, 7 μg; c-fos-CAT, 7 μg; SV40-LUC, 7 μg; pCAF, 2 μg; p300, 2 μg; and pcDNA3-HDI-F, 2 μg. Cells were harvested 44 h after transfection, and cell extracts were prepared by a standard freeze and thaw procedure. CAT and -GAL proteins were measured by ELISA method (Amersham Pharmacia Biotech, Milan, Italy). LUC activity was measured by a standard chemiluminescence procedure (26).
Chromatin immunoprecipitation (ChIP) assay
HeLa cells were transfected as above with 12 μg of NIS-LUC plasmid and treated for 24 h with TSA (300 nM) and NaB (3 mM). Aliquots of transfected cells were taken aside before immunoprecipitation to test the efficiency of transfection. Then cells were washed once with PBS at room temperature and fixed with 1% (vol/vol) formaldehyde in PBS at 37 C for 10 min. Cells were quickly rinsed twice with ice-cold PBS and scraped into 1 ml of ice-cold PBS. After spinning at 700 x g at 4 C, cell pellets were resuspended in 1.2 ml of lysis buffer [1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 50 mM Tris/HCl (pH 8.1), plus protease inhibitor cocktail] and incubated on ice for 10 min. After lysis, the samples were centrifuged for 10 min at 14,000 x g at 4 C, and NaCl and Triton X-100 were added to final concentrations of 150 mM and 1% (vol/vol), respectively. Then the samples were incubated for immunoclearing with Protein A-Sepharose (Sigma) [80 μl of 50% slurry in 10 mM Tris/HCl (pH 8.1), 1 mM EDTA] overnight at 4 C. After incubation, the samples were quickly centrifuged at 9000 x g, and the supernatants were collected. Immunoprecipitation was performed by adding 5 μg of polyclonal antiacetyl-histone H3 antibody (Upstate Biotechnologies Inc., Lake Placid, NY) and incubating the mixture overnight at 4 C; 80 μl of Protein A-Sepharose was then added and the slurry was incubated overnight at 4 C. The Sepharose beads were harvested by centrifugation at 8000 x g and washed sequentially for 10 min in 1 ml each of TSE I [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris/HCl (pH 8.1), 150 mM NaCl], TSE II [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris/HCl (pH 8.1), 500 mM NaCl], TSE III [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris/HCl (pH 8.1)], and Tris/EDTA buffer [10 mM Tris/HCl (pH 8.1), 1 mM EDTA]. Immune complexes were eluted from beads with 100 μl of elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature for 10 min and cross-links were reversed by heating at 56 C overnight with the addition of proteinase K. DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation and was used as a template for PCR.
PCR was performed by using the following primers: hNIS, forward, 5'-GACTCAAGATGTCAGTCAGC-3' that contains a NIS promoter-specific sequence; and LUC Spec, reverse, 5'-GCCTTTCTTTATGTTTTTGGC-3' that contains a luciferase-specific sequence.
Amplification was performed with the following conditions: one cycle at 95 C for 10 min, followed by 30 cycles at 95 C for 30 sec, 52 C for 30 sec, and at 72 C 1 min. PCR products were visualized on 2% agarose gel 0.5x Tris-borate EDTA.
Results
Effects of HDAC inhibitors on NIS gene expression
It has been previously shown that HDAC inhibitors stimulate NIS expression in thyroid cancer cells (19, 20, 27, 28). To test the reproducibility of these findings in our system, ARO and FRO cell lines (from anaplastic carcinoma) and BCPAP and TPC1 cell lines (from papillary carcinoma) were treated with TSA (300 nM) and NaB (3 mM) for 30 h; then total RNA was extracted and NIS expression was evaluated by real-time PCR, using four genes for normalization (GAPDH, -actin, 2-microglobulin, HPRT-1). Results are shown in Fig. 1 and are expressed considering 1 the value of unstimulated cells. As shown, both TSA and NaB treatment greatly induced expression of NIS mRNA in all cell lines tested.
Effects of HDAC inhibitors on expression of thyroid-specific transcription factors
Promoters with thyroid-specific activity (such as that of NIS gene) are activated by one or more thyroid-specific transcription factors (29). Thus, the activating effect of HDAC inhibitors on NIS expression could be mediated by the increase of expression of thyroid-specific transcription factors. To test this possibility, ARO, FRO, BCPAP, and TPC-1 cell lines treated with TSA and NaB were assayed for TTF-1, TTF-2, PAX8, and HEX mRNA levels by real-time PCR.
Results (expressed as fold of modification with respect to the levels observed in untreated cells) are shown in Table 1. To be conservative, we decided to define as bona fide effects of histone acetylation those in which TSA and NaB induced significant modification in the same direction. In fact, distinct HDAC inhibitors act by different mechanisms and, in addition to histone acetylation, may induce other effects (30, 31). In the case of TTF-1, a clear inhibitory effect of histone acetylation was observed only in cell lines deriving from papillary carcinomas (BCPAP and TPC-1). TTF-2 expression was significantly reduced in FRO, BCPAP, and TPC-1 cell lines. No clear effects of histone acetylation can be observed in the case of HEX expression. PAX8 expression was significantly increased by histone acetylation in ARO cells only. A correlation of these data to those demonstrating the histone acetylation increase of NIS mRNA levels in all cell lines investigated (Fig. 1) suggests that changes of the TTF-1, TTF-2, HEX, and PAX8 levels induced by histone acetylation cannot account for the increase of NIS gene expression. The effect of TSA and NaB on PAX8 mRNA levels in ARO cells was the only activating effect of histone acetylation on expression of thyroid-specific transcription factors that we were able to detect. This effect can be related to the basal levels of PAX8 mRNA, which in ARO cells were not detectable by qualitative RT-PCR (Fig. 2). In contrast, no relationship between basal levels of expression and effects of histone acetylation was observed in the case of TTF-1, TTF-2, and HEX genes (data not shown).
Effects of HDAC modulation on NIS promoter
Data reported so far suggest that TSA and NaB activate NIS gene expression without modification of expression of thyroid-specific transcription factors. However, HDAC inhibition may control NIS expression by different mechanisms from expression modification of thyroid-specific transcription factors. To demonstrate that effects of TSA and NaB treatment are not mediated by modification of thyroid-specific transcriptional mechanisms, we used a transfection approach. A construct bearing 2.2 kb of 5' genomic sequence for the human NIS promoter, containing the minimal promoter linked to the LUC gene as reporter, was used to transfect HeLa cells (23, 24), a nonthyroid cell line that does not express TTF-1, TTF-2, PAX8, and HEX genes. Therefore, in this experimental situation, any effect on NIS promoter would not be mediated by the thyroid-specific transcriptional machinery. The construct containing the NIS promoter was transfected in HeLa cells. Then transfected cells were treated with or without TSA (300 nM) and NaB (3 mM) for 16 and 28 h, and the expression of the LUC gene was evaluated. Figure 3A shows that either TSA or NaB treatment increases NIS promoter activity. To show that TSA and NaB treatment modified histone acetylation on the transfected NIS-LUC plasmid, a ChIP assay was used. The ChIP assay has been used by a number of laboratories to probe the acetylation state of chromatin (reviewed in Ref. 34). HeLa cells were transfected with the NIS-LUC plasmid and treated with TSA or NaB. After cross-link, the acetylated H3 histone was immunoprecipitated by using a specific antibody (Upstate Biotechnology). As shown in Fig. 3B, after immunoprecipitation, a specific PCR product was obtained only in NIS promoter-transfected cells that have been treated with TSA or NaB.
These results indicate that acetylated H3 histone on NIS promoter was present only on HDAC inhibitor treatment, thus demonstrating that HDAC treatment modify histone acetylation status of the transfected NIS promoter. Among thyroid-specific transcription factors, PAX8 plays a major role in up-regulation of NIS promoter (35, 36, 37). RT-PCR experiments performed by using mRNA of transfected HeLa cells as template indicate that the increase of NIS promoter activity is not mediated by activation of the endogenous PAX8 expression. In fact, in HeLa cells treated with TSA or NaB, no PAX8 expression was observed (data not shown). Altogether, these results indicate that HDAC inhibition is able to activate NIS gene expression by a direct histone acetylation of the promoter, without modification of expression of TTF-1, TTF-2, HEX, and PAX8.
To support the notion that the basal levels of NIS promoter are controlled by histone acetylation, two cotransfection approaches were used. First, the NIS promoter was cotransfected in HeLa cells together with a plasmid expressing the histone deacetylase 1 (HDAC1) (38). As expected, the overexpression of HDAC1 exerted a strong inhibitory effect on the NIS promoter activity (Fig. 4A). The effect was specific because overexpression of HDAC1 determined unsignificant effects on RSV and c-fos promoters and only a modest inhibitory effects on SV40 promoter. In the second approach, the NIS promoter was cotransfected with expression plasmids for the HATs pCAF or p300 (39). As shown in Fig. 4B, overexpression of either pCAF or p300 greatly increased the NIS promoter activity. The HAT effect was specific because neither pCAF nor p300 was able to increase the RSV promoter activity. Altogether, these experiments indicate that basal levels of NIS promoter activity are subjected to histone acetylation control exerted by ubiquitous cofactors.
Effects of HDAC inhibitors on NIS expression of nonthyroid cell lines
Results obtained in HeLa cells suggest that the effects of HDAC inhibition on NIS expression do not occur only by activation of thyroid-specific mechanisms but also involve activation of ubiquitous mechanisms. The prediction of this hypothesis is that HDAC inhibition also would increase NIS mRNA levels in nonthyroid cell lines. This possibility was tested by using the following human cell lines: MCF-7 from mammary carcinoma, Hep-G2, from liver carcinoma, and SAOS-2, from osteosarcoma. The indicated cell lines were treated with TSA (300 nM) and NaB (3 mM) for 30 h and NIS mRNA levels were measured by RT-PCR. As shown in Fig. 5, in all cell lines both TSA and NaB treatment induced a great increase of NIS mRNA levels. Basal values of NIS mRNA levels are much higher in MCF-7 than Hep-G2 and SAOS-2 cell lines. This finding fits with observations indicating that in MCF-7 cells, NIS gene expression is regulated by various signaling pathways (40, 41).
Discussion
Several reports have shown that treatment of thyroid cancer cell lines with HDAC inhibitors increases NIS expression as well as iodine uptake (19, 20, 27, 28, 42). These results may have relevance in treatment of undifferentiated thyroid tumors. In fact, the inability to trap iodine by these tumors makes ineffective the radioiodine therapy (43). In this view, restoration of NIS expression in undifferentiated thyroid tumors is considered a means to improve therapeutic strategy. For this reason, control of NIS expression has been extensively investigated (35, 37, 44, 45, 46, 47, 48, 49). The aim of the present study was to investigate molecular mechanisms by which HDAC inhibitors induce NIS expression. Distinct HDAC inhibitors act by different mechanisms and, in addition to histone acetylation, may induce other effects (30, 31). For these reasons, our strategy was the use of two different HDAC inhibitors and the definition as bona fide effects of histone acetylation those in which TSA and NaB induced modifications in the same direction. Our data indicate that effects of HDAC inhibitors on NIS mRNA levels are not mediated by modification of thyroid-specific transcription factors gene expression. Our transfection experiments were performed in the nonthyroid cell line HeLa. Because these cells do not express TTF-1, TTF-2, PAX8, and HEX, the finding that TSA and NaB increase the activity of NIS promoter reinforces the notion that effect of histone acetylation occurs without the contribute of thyroid-specific transcriptional machinery.
The effect of HDAC inhibitors on NIS expression is specific and not the result of a general phenomenon of histone acetylation, as demonstrated by evaluating expression of other genes (see Table 1). According to this view, Western blots performed with a specific antibody against H3 histone indicate no correlation between NIS expression and the bulk histone acetylation state of the cell (data not shown).
Data on NIS promoter activity were obtained by means of transient transfection, i.e. outside the chromosomal structure. It has been demonstrated, however, that precise nucleosome positioning occurs in transiently transfected promoters (50). Moreover, the histone acetyltransferase activity of pCAF is required for activation of transiently transfected promoters (51). In the thyroid field, it has been demonstrated that the transiently transfected thyroglobulin promoter is sensitive to the HDAC inhibitor depsipeptide (19). In agreement with transfection studies, HDAC inhibition induced NIS expression in the tumoral, nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. Because an increase of NIS gene expression, at least in MCF-7 cells, is paralleled by enhanced radioiodine trapping ability (52), our findings suggest that the strategy of inducing NIS expression by HDAC inhibition can be used to make several types of human tumors sensitive to radioiodine therapy.
In agreement with data produced by using TSA and NaB, we have demonstrated that the histone deacetylating protein HDAC1 inhibits NIS promoter activity. Conversely, the HAT proteins pCAF and p300 stimulate NIS promoter activity. It is generally accepted that HDAC1, pCAF, and p300 are recruited to promoters by interaction with transcription factors (53). HDAC1 could be recruited to NIS promoter by the transcription factor specificity protein (SP1). In fact, it has been demonstrated that SP1 interacts with HDAC1 (54) as well as the NIS promoter (48). Interestingly, it has been shown that the nuclear extracts of thyroid tumors have significantly higher SP1 levels than the corresponding normal tissue (48). Thus, the inhibition of NIS gene expression in thyroid tumors could be due to the increase of HDAC1 recruitment, which, in turn, is induced by the higher SP1 binding to the NIS promoter. In the case of HAT proteins, p300 is homologous to the cAMP response element-binding protein, and both are able to interact with the transcription factor cAMP-responsive element binding protein (55). cAMP-responsive element binding protein binding sites are present in the human NIS promoter (32). Thus, HAT proteins may contribute to the stimulation of NIS gene expression obtained by the cAMP pathway activation (41).
In our experimental conditions, HDAC inhibition induces an increase in PAX8 mRNA levels in ARO but not BCPAP, FRO, and TPC-1 cell lines. Interestingly, ARO cells show the lowest amount of basal PAX8 mRNA levels (not detectable by our qualitative PCR method; see Fig. 2). Based on these data, it could be inferred that histone acetylation controls basal levels of PAX8 gene expression. In ARO cells, a reduced histone acetylation state would not be permissive for PAX8 expression; treatment with HDAC inhibitors will allow the basal transcription of PAX8 gene. In contrast, BCPAP, FRO, and TPC-1 cell lines would have higher levels of histone acetylation, enough to allow basal levels of PAX8 mRNA levels, and, therefore, treatment with HDAC inhibitors has no effects on PAX8 expression.
In a recent investigation, the effect of the HDAC inhibitor depsipeptide on TTF-1 and PAX8 mRNA levels in cells derived from differentiated and undifferentiated thyroid cancers (BHP18–21v and ARO, respectively) was investigated (27). In contrast to our findings, the HDAC inhibitor depsipeptide induced increase of TTF-1 mRNA levels in both cell lines. Reasons for this discrepancy are not obvious. However, at least two methodological differences are evident between the two studies: the use of different compounds as HDAC inhibitors and the use of a nonquantitative RT-PCR method to measure TTF-1 and PAX8 mRNA levels. The down-regulation of thyroid-specific transcription factors (TTF-1 and TTF-2) by HDAC inhibition in a cell line that maintains several markers of the thyroid-differentiated phenotype (BCPAP) is in agreement with several observation indicating that TSA blocks intestinal development in Xenopous laevis (56), prevents expression of differentiation markers in chick oviduct explants (32), suppresses differentiation in tissue culture cell models (33).
In conclusion, our data indicate that the NIS promoter, even outside the chromosomal context, is a major target by which HDAC inhibitors increase NIS expression in both thyroid and nonthyroid cancer cells. Thus, future investigations on the relation between NIS promoter activity and histone acetylation may have relevance for defining innovative therapeutic strategies for the treatment of human tumors.
Footnotes
This work was supported by grants from Ministero dell’Istruzione, dell’Università e della Ricerca (to G.D., S.F., and D.R.).
Abbreviations: CAT, Chloramphenicol-acetyl-transferase; ChIP, chromatin immunoprecipitation; -GAL, -galactosidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAT, histone acetyltransferase; HDAC, histone deacetylase; HEX, hematopoietically expressed homeodomain; HPRT, hypoxantine guanine phosphoribosyl transferase; LUC, luciferase; NaB, Na butyrate; NIS, sodium iodide symporter; PAX, paired box transcription factor; RSV, Rous sarcoma virus; RT, reverse transcription; SDS, sodium dodecyl sulfate; SP1, specificity protein; SV40, Simian virus 40; TSA, tricostatin A; TSE, buffer of Triton X-100, SDS, and EDTA; TTF, thyroid-specific transcription factor.
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