Expression of mutant thyroid hormone nuclear receptors is associated with human renal clear cell carcinoma

Yuji Kamiya,*, Monika Puzianowska-Kuznicka2,3,4,*, Peter McPhie1, Janusz Nauman2, Sheue-yann Cheng and Alicja Nauman3

Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive MSC 4255,
1 Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-4255, USA,
2 Department of Endocrinology, Medical Research Center, Polish Academy of Sciences and
3 Department of Biochemistry, Medical Center of Postgraduate Education, Warsaw, Poland


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thyroid hormone (T3) regulates proliferation and differentiation of cells, via its nuclear receptors (TRs). These processes have been shown to be abnormally regulated during carcinogenesis. We have previously found aberrant expression of TR{alpha} and TRß mRNAs in renal clear cell carcinoma (RCCC), suggesting possible involvement of TRs in the carcinogenesis of RCCC. To understand the molecular actions of TRs in RCCC, cDNAs for TRß1 and TR{alpha}1 were cloned from 22 RCCC tissues and 20 surrounding normal tissues. Mutations were found in seven TRß1 and three TR{alpha}1 cDNAs. Two TRß1 cDNAs had a single mutation, while five TRß1 and three TR{alpha}1 had two or three mutations. Most of the mutations were localized in the hormone-binding domain. Using the TRs prepared by in vitro transcription/translation, we found that these mutations led to a loss of T3 binding activity and/or impairment in binding to thyroid hormone response elements (TREs). Furthermore, nuclear extracts from RCCC tissues also exhibited impairment in binding to TREs. These results indicate that the normal functions of TRs in RCCC tissues were impaired. Together with the aberrant expression patterns, these mutated TRs could contribute to the carcinogenesis of RCCC.

Abbreviations: EMSA, electrophoresis mobility shift assay; HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; NFTs, non-functioning tumors; RCCC, renal clear cell carcinoma; RTH, thyroid hormone resistance syndrome; RXRs, retinoic X receptors; T3, thyroid hormone; TRs, thyroid hormone nuclear receptors; TRE, thyroid hormone response element; VHL, von Hippel–Lindau tumor suppressor gene; w-TR, wild-type TR.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Renal clear cell carcinoma (RCCC) constitutes ~80% of all renal neoplasms and is the cause of death in 10 000 cases per year (1). Most cases are sporadic and the molecular genetics underlying this disease are largely unknown. Only a few genes have been suggested to be involved in the carcinogenesis of RCCC. The best known is the von Hippel–Lindau tumor suppressor gene (VHL), which was found to be mutated in 57% of RCCC, and loss of heterozygosity (LOH) was observed in 98% of tumor samples (2). Recent studies have shown that hypermethylation of the VHL promoter may contribute to its inactivation (3). Other tumor suppressor genes involved in the pathogensis of RCCC are FHIT (4,5), NRC-1 (6) and unknown genes localized in the 3p21 region (7–9).

Thyroid hormones regulate growth, development and differentiation. Their action is mediated by the thyroid hormone nuclear receptors (TRs), which are derived from two genes, TR{alpha} and TRß, located on chromosomes 17 and 3, respectively (10). Each gene gives rise to two receptor isoforms, {alpha}1, {alpha}2, ß1 and ß2, by alternative splicing of the primary transcripts. The gene regulating activity of TRs is mediated by binding to specific DNA sequences, known as thyroid hormone response elements (TREs), located on the promoter regions of thyroid hormone target genes. Recent studies have indicated that the gene regulatory functions of TR are modulated by formation of different heterodimers with other members of the receptor superfamily, notably the retinoic X receptors (RXRs) (10,11), by differential interaction with different types of TREs (10) and by interaction with various co-activators, co-repressors and other cellular proteins (10,12).

Increasing evidence has suggested that aberrant expression and/or mutations in TR genes could be associated with carcinogenesis. A reduction in the expression of mRNA for TRß1 and TRß2 was implicated in inappropriate expression of the glycoprotein hormone {alpha}-subunit gene in non-functioning tumors (NFTs) of the anterior pituitary and was proposed to contribute to uncontrolled tumor growth (13,14). Reduced expression of TRß1 was also found in poorly differentiated fibroblast-like osteosarcoma (15). However, in poorly differentiated hepatocarcinomas, overexpression of TRß1 was correlated with enhanced thyroid hormone (T3)-induced proliferation (16,17). These results suggest that aberrant expression of TRs may be associated with different types of tumors and/or different states of differentiation. Furthermore, LOH of the chromosomal regions where TR genes are located (3p21–p25 for the TRß gene and 17q21 for the TR{alpha} gene) was a frequent event in tumors (18–21). For example, LOH of the TRß gene was observed in most of the small cell lung cancers examined (20,22), in 60% of posterior uveal melanomas (23), 30% of breast (24) and 64% of non-familial renal cell carcinomas (25). Consistent with these observations, microdeletion of both TR{alpha} alleles was found in 20% of gastrointestinal tumors (26). LOH of the TR{alpha} gene was also observed in 79% of breast (27) and prostate cancers (28). In addition to LOH in regions in which TR{alpha} and TRß genes are located, high frequencies of mutant TRs (65% of TR{alpha}1 and 76% of TRß1 in 17 tumors) with impaired function were identified in human hepatocellular carcinoma (17). TR{alpha}1 mutants (13% of 23 tumors) were also found in NFTs of the anterior pituitary (14).

We had previously found aberrant expression of TR{alpha} and TRß mRNAs in RCCC (29). Expression of both TR{alpha}1 and TR{alpha}2 mRNAs was reduced, while TRß1 mRNA was overexpressed in 30% and significantly reduced in 70% of tumors examined. We hypothesized that these aberrant expression patterns may reflect mutations in TR genes leading to an alteration in expression. In the present study we have cloned TR cDNAs from tumors obtained from RCCC patients. We found multiple mutations in both the TR{alpha} and TRß genes, resulting in impairment of T3 and DNA binding and loss of transcriptional activity. The functional impairment in these mutants may contribute to the carcinogenesis of RCCC.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents
L-[35S]methionine (10 mCi/ml) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). [{alpha}-32P]dCTP (3000 Ci/mmol, 1 Ci = 37 GBq) was obtained from ICN Biomedicals (Costa Mesa, CA). [125I]T3 (2200 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). pcDNA3.1, a eukaryotic expression vector, and Lipofectamine were purchased from Invitrogen (Carlsbad, CA) and Life Technologies (Grand Island, NY), respectively. The pGEM-T vector systems and TNT coupled reticulocyte system were obtained from Promega (Madison, WI). pBluescript KS(–) was obtained from Strategene (La Jolla, CA). The T7 expression plasmids for w-TRß1 (pCJ3) and w-TR{alpha}1 (pCLC13) and the mammalian plasmids expressing TRß1 (pCLC51) and w-TR{alpha}1 (pCLC61) were prepared as described (30).

Tissues
Tissues were kindly provided by Dr A.Tanski of the Department of Urology, Vouvodoship Hospital (Ostroleka, Poland) with the permission of the Ethical Committee on Human Studies, Medical Center of Postgraduate Education, during unilateral nephrectomies following the diagnosis of kidney cancer. Fragments of the tumors, fragments of the opposite poles of the same kidney (not infiltrated with cancer) and fragments of the kidneys with no cancer were excised, immediately frozen on dry ice and stored at –75°C until needed. In total, 22 tumor tissues, 20 corresponding controls and seven non-cancerous kidney tissues were collected. Upon histological evaluation, the renal clear cell cancer diagnosis and tumor borders were established. Tumors were divided into the three groups depending on the grade of differentiation: G1, well differentiated (four tumors); G2, moderately differentiated (10 tumors); G3, undifferentiated cancers (eight tumors). Additionally, they were divided according to the TNM (tumor, nodules and metastases) classification of malignant tumors (31).

RNA isolation
RNA was isolated according to Chomczynski (32). Up to 100 mg deep frozen kidney tissue was manually homogenized in a glass–teflon homogenizer directly in 1 ml of TRI reagent (Molecular Research Center, Cincinnati, OH). Samples were incubated at room temperature for 5–10 min, supplemented with 0.2 ml of chloroform, mixed by shaking for 15 s, incubated at room temperature for 10 min and then centrifuged at 12 000 g for 15 min at 4°C. The upper, aqueous phase was transferred to another Eppendorf tube, mixed with 0.5 ml of isopropyl alcohol, incubated at room temperature for 15 min and then centrifuged at 12 000 g for 15 min at 4°C. The RNA pellet was washed with 75% ethanol, air dried and resuspended in 40–50 µl of DEPC-treated water. Purity of the RNA was evaluated by formaldehyde–agarose electrophoresis and the concentration determined by spectrophotometric measurements.

Cloning of TR cDNAs from kidney cancer tissues
To clone TR{alpha}1 and TRß1 cDNAs in kidney cancer tissues as well as in normal tissues, RT–PCR was performed using a Superscript One-Step RT-PCR System (Life Technologies, Grand Island, NY) or Ready To Go RT–PCR beads (Amersham Pharmacia Biotech). Occasionally the reactions were supplemented with magnesium to a final concentration of up to 3 mM. To clone TR cDNAs, the following primers were used: for TR{alpha}1, forward primer 5'-GGATGGAATTGTGAATG-3' or 5'-ATGGAACAGAAGCCAAGCAA-3' and reverse primer 5'-GGCCGCCTGAGGCTTTA-3'; for TRß1, forward primer 5'-GATCCAGAATGATTACTAACC-3' and reverse primer 5'-GGAATTATAGGAAGGAA TCC-3' and then internal primers, forward 5'-CTATAACCCCCAACAGTATG-3' or 5'-ATGACAGAAAATGGCCTTAC-3' and reverse, 5'-CTAATCCTCGAACACTTCCA-3'. Total RNA (0.5–1 µg) was used as a template in each RT–PCR reaction. Prior to each RT–PCR, RNA was denatured at 80°C for 5 min, cooled to room temperature and then supplemented with enzyme mix. Reverse transcription was performed at 48 or 42–43°C for 30 min. Reverse transcriptase was inactivated by incubation of the samples at 94°C for 2–3 min. cDNA amplification was performed using the following conditions: 40 cycles of 94°C for 40–50 s, 52°C for 1–1.5 min and 72°C for 2.5 min, followed by 10 min incubation at 72°C. In addition, since the amount of TRß1 was very low after RT–PCR, a second PCR was performed with the above described internal primers. RT–PCR products were electrophoresed on 1% agarose gels, then the gel fragment at the expected TRß1 size was excised, dissolved in 100 µl of dH2O and 5 µl of this solution was used as template in a second PCR: 3 min at 94°C, 25 cycles of 94°C for 40–50 s and 52°C for 1 min, then 72°C for 2 min, followed by 10 min incubation at 72°C.

TRß1 or TR{alpha}1 cDNAs derived from RT–PCR were electrophoresed on 1% agarose gels, excised and extracted from the gel with a QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA), and then ligated into vector pGEM-T containing T overhangs (Promega, Madison, WI). JM 109 bacteria were transformed with the ligation mix and blue–white selection was performed. Mini-preps were carried out using the Wizard Plus SV Minipreps DNA Purification System (Promega, Madison, WI) or Plasmid Miniprep Plus kits (A&A Biotechnology, Gdansk, Poland). Restriction analyses were carried out to confirm the correct cloning (TR{alpha}1 with NcoI and TRß1 with BstXI) in the pGEM-T vector. In the preliminary studies we have validated the above cloning procedure by isolating wild-type TRs from nomal kidneys and confirmed the sequences of wild-type TRs by sequencing.

Identification of mutation sites by sequencing of TRs isolated from kidney cancer tissues
To identify mutations in TRs isolated from tissues, the TR cDNAs cloned in the pGEM-T vector were sequenced by automatic sequencing using a BigDye Terminator Cycle Sequencing Kit (Perkin Elmer) using T7 and SP6 as primers. Mutation was confirmed by repeated sequencing of the same clone as well as other clones (up to four) originating from the same tumor. Sequence analysis was examined with Lasergene software (DNAstar, Madison, WI). As a control, wild-type TRß1 was cloned from non-cancerous kidneys and sequenced to validate the cloning and to confirm the accuracy of sequencing reactions.

Cloning of TRß1 and TR{alpha}1 mutants into pBluescript KS(–)
For efficient in vitro transcription/translation of mutant proteins, three TR{alpha}1 mutants (23TR{alpha}1, 2TR{alpha}1 and 6TR{alpha}1) and one TRß1 mutant (18TRß1) were recloned into the pBluscript KS(–) expression vector, which utilizes T7 as the promoter. The TR cDNAs were cut from the pGEM-T vector with the restriction enzymes SpeI and ApaI and cloned into the same sites in the pBluscript KS(–) expression vector, which was confirmed by restiction analyses using BstXI or NcoI for TR{alpha}1 and BstXI or PstI with SpeI for TRß1.

Cloning of the TRß1 and TR{alpha}1 mutants into a eukaryotic expression vector
The same TR{alpha}1 and TRß1 mutants described above, positioned with their 5'-ends next to the SP6 promoter within the pGEM-T vector, were cut from this vector with NotI and ApaI and subcloned into vector pcDNA 3.1(+) (Invitrogen, Carlsbad, CA), previously prepared with the same enzymes. To confirm correct cloning of the inserts, restriction analysis was performed with NcoI (TR{alpha}1 clones) or BstXI (TRß1 clones). The remaining six mutated TRß1 mutants, positioned with their 5'-ends next to the T7 promoter within the pGEM-T vector, were restricted with NcoI, treated with Klenow fragment (to blunt ends), phenol/chloroform and gel purified, then restricted with NotI and cloned into pcDNA 3.1(+), previously prepared with EcoRV and NotI. Restriction analysis was performed with BstXI.

These mammalian expression plasmids were purified using the Qiagen Maxi Kit and resequenced to confirm the mutation sites determined above. The sequencing was carried out using an Applied Biosystems model 377 automatic DNA sequencer according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).

Preparation of nuclear extracts from kidney cancer tissues
The buffers used for isolation were as described by Kane (33). All buffers were supplemented with pepstatin A (final concentration 1 µg/ml), leupeptin (1 µg/ml), aprotinin (2 µg/ml) and PMSF (0.5 mM). Up to 100 mg of the tissue was manually homogenized in a glass–teflon homogenizer in 1 ml of ice-cold STM buffer (0.25 M sucrose, 20 mM Tris–HCl, pH 7.85, 1.1 mM MgCl2) and centrifuged at 1000 g for 10 min at 4°C. The pellet was washed twice in 1 ml of STM buffer with 0.5% Triton X-100, followed by centrifugation under the conditions described above. The final pellet was resuspended in 0.1 ml KSTM + 20% glycerol buffer (0.25 M sucrose, 20 mM Tris–HCl, 1.1 mM MgCl2, 0.4 M KCl, 20% glycerol, 5 mM DTT), sonicated and then incubated on ice for 30 min with vortexing every 5 min to extract the soluble nuclear proteins. After solubilization, the suspension was centrifuged at 12 000 g for 15 min at 4°C. The amount of protein in the supernatant was quantified by spectrophotometry with Bio-Rad Protein Assay buffer at 595 nm and stored in 10 µl aliquots at –75°C.

Electrophoresis mobility shift assay (EMSA)
TR proteins were synthesized by in vitro transcription/translation using the TNT coupled reticulocyte kit according to the manufacturer's instructions (Promega). 32P-labeled TRE-Lys was prepared as previously described (30). The in vitro translated TR proteins were quantified by measuring the intensity of the 35S-labeled protein bands after SDS–PAGE. For EMSA, identical amounts of TRs were incubated with the 32P-labeled TRE in the presence or absence of RXRß. After electrophoresis, TR homodimers and heterodimers were visualized by autoradiography.

To further evaluate whether the DNA-binding activity of TRs was altered in RCCC, gel mobility shift assays were performed with 7.5–10 µg nuclear extract isolated from tumors and their respective controls. The probe was a double-stranded DNA containing TRE-DR4 (5'-GATCGCAGGTCATTTCAGGACAGCGATC-3'). Mutated TRE served as a non-specific competitor (5'-GGCAAATCATTTCAAGACAG-3'). Nuclear extracts were incubated at room temperature for 20 min in binding buffer containing 20 mM Tris–HCl, pH 7.5, 50 mM KCl, 2 mM DTT, 0.1% Triton X-100, 6% glycerol, in the presence of 250 ng dI·dC, 1 ng probe and a 10 times excess of specific or non-specific competitor. For supershift experiments, 1 µg mouse monoclonal anti-TR (ß1 and {alpha}1) antibody C4 (34) was added to the binding reaction.

The mixture was first incubated on ice for 30 min, followed by an additional incubation at room temperature for 20 min. The binding reactions were loaded onto a 4% native gel and electrophoresed at 150 V for 2 h at room temperature. The gel was dried and autoradiographed.

Binding of [3'-125I]T3 to TRs
The in vitro translated TR proteins were incubated with 0.2 nM [3'-125I]T3 in the presence of increasing concentrations of unlabeled T3. The TR-bound [3'-125I]T3 was separated from free [3'-125I]T3 as described by Zhu et al. (30). The binding data were analyzed using equation 1Go (below), based on direct competition between [3'-125I]T3 and unlabeled T3 for a single site on the receptor. The concentration of radioactive complex is given by:

where [R]0 is the total concentration of receptor, [h] and [c] are the concentrations of [3'-125I]T3 and unlabeled T3, respectively, and Kd is the dissociation constant of the hormone receptor complex. The data were fitted directly to equation 1Go using the PC-MLAB program (Civilized Software, Bethesda, MD) to evaluate Kd and [R]0.

Determination of the transactivation activity of TRs
To determine the T3-dependent transactivatioin activity, CV1 cells (6-well plates with 1x105 cells/well) were transfected with mammalian expression plasmids for mutant (mutant TRß1, 0.2–1.6 µg; mutant TR{alpha}1, 0.4–0.6 µg) and/or wild-type TR (TRß1, 0.05–0.2 µg; TR{alpha}1, 0.125–0.5 µg) and 0.8 µg TRE-containing luciferase reporter gene (pTK-Pal-Luc or pTK-Lys-Luc) by the Lipofectamine method according to the manufacturer's instructions. After 16 h the medium was changed to a medium containing 10% thyroid hormone-depleted fetal bovine serum with or without 100 nM T3. After 24 h the cells were harvested and lysed and 100 µl was assayed for luciferase activity according to the manufacturer's instructions (BD PharMingen, San Diego, CA). The transfection efficiency was normalized to the protein concentration of the lysates.

Western blotting
Cell lysates (75 µg) from transient transfection experiments, as described above, were loaded onto a 10% SDS–PAGE gel. After electrophoresis, proteins were transferred to a PVDF membrane. The membrane was gently shaken in 10% non-fat milk in Tris-buffered saline (25 mM Tris, pH 7.4, 150 mM NaCl) for 1 h and subsquently washed three times with Tris-buffered saline. The membrane was incubated with mouse monoclonal antibody J51 (2 µg/ml) (36) or C4 (2 µg/ml) (34) overnight at 4°C. After washing with washing buffer (0.1% Tween 20, 25 mM Tris, pH 7.4, 150 mM NaCl), the membrane was incubated with rabbit anti-mouse Ig conjugated to horseradish peroxidase (1:2000 dilution). TR protein bands were visualized by chemiluminescence using the ECL kit (Amersham Pharmacia Biotech).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Cloning of mutant TRs from patients with RCCC
The discovery of aberrant expression of TRs in RCCC (29) prompted us to clone the TR cDNAs from RCCC tumors. To control for possible artifacts, wild-type TRs (w-TRs) were also similarly cloned from normal kidney tissues. Sequencing of cDNAs shows that 40.9% of all RCCCs tested had at least one TR mutated in the ligand-binding domain. Missense mutations within this domain were identified in seven of 22 TRß1 cDNAs cloned from 22 tumors (31.8%) (Figure 1AGo and Table IGo). The mutations detected were not due to cloning artifacts because TRs were similarly cloned from the control healthy kidney tissues and were found to have the w-TR sequence. Analysis of the frequency of mutations indicates that one TRß1 mutant (3TRß1) was isolated from a grade G1 tumor (25% of all G1 tumors tested), three TRß1 mutants (25TRß1, 8TRß1 and 18TRß1) were from grade G2 tumors (33% of all G2 tumors tested) and three TRß1 mutants (15TRß1, 32TRß1 and 6TRß1) were from grade G3 tumors (42.8% of all G3 tumors tested) (Table IGo).



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Fig. 1. Schematic representation of the locations of mutation sites of TRß1 (A) and TR{alpha}1 (B) mutants isolated from RCCC. The mutation sites were identified by sequencing as described in Materials and methods. The mutation sites and hot-spots are marked.

 

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Table I. TR mutants identified in RCCC
 
Sequencing of TR{alpha}1 cDNAs cloned from 22 RCCCs revealed the presence of missense mutations in three TR{alpha}1 (13.6%) (Figure 1BGo and Table IGo). These three mutants were each cloned from a G1, G2 or G3 tumor (Table IGo). 6TR{alpha}1 was isolated from the same G3 tumor from which 6TRß1 was isolated (Table IGo). Therefore patient 6 had mutations in both the TR{alpha} and TRß genes (Figure 1A and BGo). Because only three TR{alpha}1 mutants were cloned from 22 tumors, it was unclear whether an increased frequency of mutant TR{alpha}1 was associated with differentiation status of the tumor. The three TR{alpha}1 mutants were from tumors with heterozygous mutation (Table IGo).

Examination of the mutations shown in Figure 1Go indicates that single (8TRß1 and 25TRß1) and multiple mutations (3TRß1, 18TRß1, 15TRß1, 6TRß1, 32TRß1 and all TR{alpha}1 mutants) were detected. In addition to the S380F mutation, 32TRß1 had a 26 amino acid deletion at the N-terminus. The majority of mutations were clustered in the hormone-binding domain (domains D + E, see Figure 1Go). This mutation pattern is similar to that observed for TR mutants isolated from human hepatocellular carcinoma (HCC) in that single and multiple mutations were observed (17). This is in contrast to that seen for mutations identified for patients with the genetic disease thyroid hormone resistance syndrome (RTH) (35) in which only a single mutation for each mutant was found in the TRß gene. Analysis of the mutation patterns of RTH patients showed that the mutations are clustered in three regions in the T3-binding domain (36). It is of interest to point out that F451I in 3TRß1, F451S in 8TRß1, L456S in 6TRß1 and M388I in 2TR{alpha}1 and 6TR{alpha}1 occurred in the first `hot-spot'. The Y321H mutation identified in 25TRß1 and K288E in 23TR{alpha}1 were in the second hot-spot and H184Q, S183N and R228H in 23TR{alpha}1 and A225T in 6TR{alpha}1 were in the third hot-spot identified in RTH patients (36). However, novel mutations were also observed in the DNA-binding domain for 3TRß1 (S99R), 15TRß1 (K155E), 2TR{alpha}1 (I116N) and 6TR{alpha}1 (I116N) (Table IGo).

T3-binding activity of TR mutants is impaired
To understand the functional consequences of mutations in the TRs isolated from RCCC, we first evaluated hormone-binding activity of TR mutants. Mutant TR proteins were prepared by in vitro transcription/translation. Except for 32TRß1, which had an apparent molecular mass of 52 kDa due to deletion of 26 amino acids at the N-terminus (Figure 2AGo, lane 8), all other TRß1 mutants had sizes similar to w-TRß1 (Figure 2AGo, lane 1 versus lanes 2–7). The in vitro translated wild-type and mutant TRß1 were full-length receptors with an apparent molecular mass of 55 kDa together with a smaller protein, most likely due to initiation from a downstream ATG (36,37). The full-length in vitro translated TRß1 mutants were recognized by monoclonal antibody J51, whose epitope is located in the second half of the A/B domain of TRß1 (36). The 52 kDa 32TRß1 was recognized by monoclonal anti-TR antibody C4, whose epitope is the C-terminal 457EVFED461 of TRß1 and TR{alpha}1 (data not shown; 34). Taken together, the identity of the cloned TRß1 mutants was confirmed.



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Fig. 2. The molecular sizes of the in vitro translated TRß1 (A) and TR{alpha}1 (B). After in vitro transcription/translation, equal volumes (1 µl) of the programmed lysates were analyzed on a 10% SDS gel. The dry gels were autoradiographed.

 
As shown in Figure 2BGo, the molecular sizes of TR{alpha}1 mutants were similar to that of w-TR{alpha}1 (lane 1 versus lanes 2–4). The in vitro translated wild-type and mutant TR{alpha}1 were full-length receptors with an apparent molecular weight of 49 kDa and a minor truncated protein, derived from initiation at a downstream ATG (38). The in vitro translated TR{alpha}1 mutants were recognized by monoclonal anti-TR antibody C4 (data not shown; 34), further confirming the identity of the cloned TR{alpha}1 mutants.

To determine T3-binding activity, equal amounts of in vitro translated wild-type and mutant TRs were used for binding to [125I]T3. Figure 3Go shows the competitive binding displacement curves for wild-type and mutant TRs. Analyses of the binding data shown in Figure 3AGo indicate that w-TRß1 bound to T3 with a Kd of 0.78 nM (Table IIGo). The mutations in 15TRß1 (K155E and K411E) did not affect its T3-binding activity as 15TRß1 retained the hormone-binding activity of w-TRß1. The mutations in the hormone-binding domains of 25TRß1 (Y321H), 6TRß1 (E299K, H412R and L456S) and 32TRß1 (S380F) reduced T3-binding activity by 35, 60 and 46%, respectively (Table IIGo). The mutations in the hormone-binding domains of 3TRß1 (W219L and F451I), 8TRß1 (F451S) and 18TRß1 (Q252R, A387P and F417L) led to a >99% loss of T3-binding activity. A similar analysis of the data shown in Figure 3BGo indicates that the mutations in 23TR{alpha}1 (S183N, H184Q, R228H and K288E), 2TR{alpha}1 (M388I) and 6TR{alpha}1 (A225T and M388I) resulted in virtually complete loss of T3-binding activity (Table IIGo).



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Fig. 3. Competitive binding of 125I-labeled and unlabeled T3 to wild-type and mutant TRß1 (A) and TR{alpha}1 (B). Equal amounts of in vitro translated TRß1 and TR{alpha}1 were incubated with 0.2 nM [125I]T3 in the absence or presence of increasing concentrations of unlabeled T3. The free and bound [125I]T3 were separated as described in Materials and methods. Data are expressed as the percentage of [125I]T3 bound in the absence of unlabeled T3.

 

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Table II. T3 and DNA binding of wild-type and mutant TRs
 
TR mutants exhibit aberrant DNA-binding activity
The in vitro DNA-binding activity of mutants was evaluated by EMSA using the TRE in the lysozyme gene, which consists of an inverted repeat of the two half-site binding motifs (TRE-Lys). Figure 4AGo, lanes 2 and 3 show the control for binding of w-TRß1 to TRE-Lys as a homodimer and heterodimer with RXRß, respectively. Comparison of the intensities of the homodimeric bands of the bound mutants (Figure 4AGo, lanes 4, 6, 8, 10, 12, 14 and 16) with that of w-TRß1 (lane 2) indicates that except for 3TRß1, which bound to TRE-Lys as strongly as w-TRß1 (Figure 4AGo, lane 4 versus lane 2, and Table IIGo), all other mutants bound to TRE-Lys more weakly than w-TRß1, with a 58–100% loss of binding (Table IIGo). Mutation of K155E in the DNA-binding domain of 15TRß1 led to complete loss of DNA binding (lane 8). Except for 3TRß1, weaker binding was also detected for heterodimeric binding with RXRß as compared to w-TRß1 (20–100% loss of binding; Figure 4AGo lane 3 versus lanes 7, 9, 11, 13, 15 and 17, and Table IIGo). These results indicate that mutations of TRß1 led to alterations in the interaction of TR with TRE.




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Fig. 4. Binding of wild-type or mutant TRß1 (A) and TR{alpha}1 (B) to TRE-Lys. Equal amounts of in vitro translated TRß1 (A) and TR{alpha}1 (B) were incubated with [32P]TRE-Lys in the absense or presence of 1 µl of RXRß. The bound TRs were analyzed by gel electrophoresis as described in Materials and methods. The TRE-Lys-bound homodimers and heterodimers are indicated.

 
Mutations in TR{alpha}1 led to a dramatic reduction in binding to TRE-Lys (Figure 4BGo). Figure 4BGo, lanes 5, 7 and 9 clearly show that binding of the 23TR{alpha}1, 2TR{alpha}1 and 6TR{alpha}1 mutants, respectively, to TRE-Lys as homodimers was hardly detectable (versus lane 3; see also Table IIGo). Binding of these mutants to TRE-Lys as heterodimers with RXRß was also decreased by 60–90% (Figure 4BGo, lane 4 versus lanes 6, 8 and 10, and Table IIGo).

To determine the DNA-binding characteristics of endogenous TRs in cancer tissues, nuclear extracts of RCCC tissues as well as extracts prepared from the healthy opposite kidney poles were evaluated by EMSA assay (Figure 5AGo). The DNA-binding patterns were analyzed in 20 cancer–control pairs. Binding of the TRE to TRs present in nuclear extracts of RCCC in which mutations were found was much weaker than that in the healthy controls. One representative example (a tumor from patient 18) in which no binding was detected is shown in lane 2 (Figure 5AGo, lanes 2 versus 5). No significant changes in DNA binding were found in the tumor nuclear extracts from patient 32 (see Figure 5AGo, lane 8 versus lane 11). The DNA-bound bands shown in lanes 5, 8 and 11 were specific because the intensities of the bands were competitively reduced in the presence of a 10-fold excess of unlabeled TRE (lanes 5, 8 and 11 versus 7, 10 and 13, respectively). No competition was seen in the presence of non-specific competitors (lanes 5, 8 and 11 versus 6, 9 and 12, respectively).



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Fig. 5. Gel mobility shift assays of the RCCC nuclear extracts. (A) Aliquots of 7.5–10 µg nuclear extract isolated from tumor tissue (CA) and their respective controls (N) were incubated for 20 min at room temperature with 1 ng [32P]TRE-DR4, 250 ng dI·dC and a 10-fold excess of specific (S) or non-specific (NS) competitor. The reactions were resolved on 4% native gels and the dry gels autoradiographed. (B) Supershift assays were performed as above, except that the binding reaction was supplemented with mouse monoclonal anti-TRß1 antibodies (mAb C4) instead of non-specific competitor and was performed for 30 min on ice and then for 20 min at room temperature.

 
To confirm that the TRE-bound bands were due to interactions of TRs present in the nuclear extracts with TRE, supershift experiments were performed (Figure 5BGo). As shown [Figure 5BGo, lane 4 for cancer tissue (patient 6) and lane 7 for the normal control], when mAb C4 (anti-TRß1 antibody) (34) was present, the TRE-bound bands were shifted to a more retarded position (compared with lanes 2 and 5 for cancer and normal control, respectively). These results clearly show that the DNA-bound bands were due to interactions of the TRE with TRs in the nuclear extracts.

The T3-dependent transactivation activity of TR mutants is impaired
To further assess the functional consequences of mutations in TRs isolated from RCCC, we determined the transactivation activity of TR mutants by transient transfection assays. Mammalian expression plasmids for the mutants were co-transfected with TRE-containing reporter genes into CV-1 cells. Figure 6AGo compares the transactivation activity of TRß1 mutants using a reporter containing TRE-Lys. In the absence of T3 all mutants were more potent than w-TRß1 in repression of basal transactivation activity (bar 3 versus bars 5, 7, 9, 11, 13, 15 and 17). Except for 32TRß1, the T3-dependent transactivation activities of all other mutants were reduced, ranging from 50% reduction as seen for 25TRß1 (bar 12 versus bar 4) to a total loss of transactivation activity for 8TRß1 (bar 8). A similar reduction in transactivation activity was detected using the reporter TRE-Pal (data not shown).




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Fig. 6. Comparison of the transactivation activities of the wild-type and mutant TRs. Expression plasmids for wild-type and muant TRß1 (A) or TR{alpha}1 (B) were co-transfected with reporter plasmids into CV-1 cells (1x105 cells/well in 6-well plates) as described in Materials and methods. Cell lysates were prepared and luciferase activity determined. Luciferase activity was normalized to the protein concentration of lysates. Data are shown as fold T3-induced luciferase activity (mean ± SD, n = 3). (C and D) The lysates (75 µg) obtained from CV-1 cells transfected with wild-type or mutant TRß1 (A) or TR{alpha}1 (B) expression plasmid were analyzed for receptor protein expression by western blotting using mAb 51 (lanes 1–8) and mAb C4 (lanes 9 and 10) for TRß1 (C) and mAb C4 for TR{alpha}1 (D) as described in Materials and methods.

 
The decrease in transactivation activity observed for TRß1 mutants was not due to lower expression of the mutant receptor proteins. Preliminary experiments were carried out using western blot analysis to titrate the amounts of expression plasmid used in the transfections such that the levels of mutant receptor proteins expressed were comparable to that of w-TRß1 (Figure 6CGo). Therefore, the impairment in transactivation activity was due to a loss of hormone binding and/or DNA binding.

Similar to the observations for TRß1 mutants, all three TR{alpha}1 mutants were more potent repressors than w-TR{alpha}1 in the absence of T3 (Figure 6BGo, bar 3 versus bars 5, 7 and 9). Furthermore, the T3-dependent transactivation activities of all three mutants were nearly completely lost (bars 6, 8 and 10). Figure 6DGo indicates that the loss of transactivation activity of TR{alpha}1 mutants was not due to lower expression of TR{alpha}1 mutant proteins because the levels of expression of the TR{alpha}1 mutant proteins were comparable to that of w-TR{alpha}1. Thus, the loss of transactivation activity was the consequence of loss of T3-binding and/or DNA-binding activity (Table IIGo).

Dominant negative action of TR mutants
We further evaluated the dominant negative action of TR mutants by examining their inhibitory effect on the transactivation activity of w-TRs. CV-1 cells were transfected with the w-TR expression plasmid together with a 5- or 10-fold excess of mutant expression plasmid. The transactivation activities of w-TR in the absence or presence of mutant TR were compared and the results are shown in Table IIIGo. 8TRß1 was a strong dominant negative mutant in that 49 and 84% of the transactivation activity of w-TRß1 was inhibited at mutant:w-TRß1 plasmid ratios of 5:1 and 10:1, respectively. 3TRß1 exhibited a dominant negative effect at a T3 concentration of 10 nM. However, at a 10-fold higher T3 concentration the dominant negative effect of 3TRß1 was abrogated. This is consistent with the T3 binding affinities of these two mutants (see Table IIGo). All other TRß1 mutants lacked dominant negative activity (Table IIIGo). The three TR{alpha}1 mutants, 23TR{alpha}1, 2TR{alpha}1 and 6TR{alpha}1, exhibited strong dominant negative activity in that they inhibited 63–79 and 81–86% of the transactivation activity of w-TR{alpha}1 at mutant:w-TR{alpha}1 plasmid ratios of 5:1 and 10:1, respectively (Table IIIGo).


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Table III. Dominant negative potency of mutant TRsa
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The role of TRs in RCCC carcinogenesis is not known. We explored the possibility of involvement of TRs in RCCC carcinogenesis based on the consideration that TR{alpha}1 is a transcriptionally active cellular homolg of v-ErbA, which is a viral oncogene product functioning as a dominant negative receptor in the neogenesis of erythroleukemia and sarcomas (39,40). Furthermore, transgenic mice over-expressing v-ErbA developed liver cancer as well as thyroid abnormalities (41), indicating a critical role of v-ErbA (a mutated TR{alpha}1) in oncogenesis. We also considered involvement of the TRß gene in RCCC carcinogenesis, because of its chromosomal localization on 3p21–p25. Previously, van den Berg et al. reported that unknown genes localized in the 3p21 region could contribute to RCCC carcinogenesis (7–9). The possibility that the TRß and TR{alpha} genes could contribute to RCCC carcinogenesis is strengthened by the aberrant expression of TRs; TR{alpha}1 mRNA was decreased and TRß1 mRNA was overexpressed in 30% and decreased in 70% of tumors (29). Furthermore, abnormal expression of TR proteins was detected; TR{alpha}1 proteins were overexpressed and TRß1 proteins were reduced in tumors (29).

We therefore cloned TR cDNAs from RCCC and evaluated their functions. Sequence analyses of TR cDNAs isolated from RCCC show that the TRß and TR{alpha} genes were mutated in seven of 22 and three of 22 tumors, respectively. These somatic mutations led to loss of the hormone-binding, DNA-binding and transcriptional activities. The extent of functional impairment depends on the sites and number of mutations in the TRs. The molecular basis of the functional impairment of these mutant TRs, however, is difficult to quantify. Most contain several mutated residues; the contributions of these changes may not be additive (42). Furthermore, we have shown that T3-dependent transcriptional activation of TRs is modulated by interactions between all domains in the protein (30). Consequently, it is difficult to predict the results of any of these changes. Interestingly, some of the mutations identified in RCCC occurred in the three `hot-spots' in the hormone-binding domain of TRß1 identified from patients with RTH (36). Thus, 8TRß1 has a single mutation (F451S) in hot-spot 1 and has greatly reduced affinity for T3 (Table IIGo). However, 25TRß1, which has a single mutation (Y321H) in hot-spot 2, exhibits only a minor decrease in its affinity for T3, but had only 50% of the wild-type binding to the TRE. 15TRß1 has two sequence changes, one in the DNA-binding domain (K155E) and the other in the putative dimerization interface (K411E) (43). Consistent with these changes, this mutant retains affinity for T3 but shows no binding to DNA. Two of the mutations in 6TRß1 occur at the dimer interface (H412R) and near hot-spot 1 (L456S), respectively, resulting in a significant loss of binding to DNA and a minor reduction in T3 binding. A significant reduction in T3 binding is seen for 18TRß1, which includes sequence changes in hot-spot 3 (Q252R) and the dimerization domain (F417L). Alterations in 3TRß1 were found in hot-spot 1 (F451I), which is consistent with a large decrease in affinity for T3.

Genetic changes in the TR{alpha}1 mutants are equally complex. 2TR{alpha}1 and 6TR{alpha}1 show similar behaviour, presumably due to the same structural changes, a large reduction in affinity for the hormone and loss of homodimer binding to TRE-Lys (I116N is in the last helix of the DNA-binding domain). The very low affinity for hormone shown by 23TR{alpha}1 is readily explained by the four mutations found in its ligand-binding domain (three in hot-spot 3 and one in hot-spot 2). However, the origin of its reduced capacity to bind to DNA is far from clear and awaits elucidation.

TR{alpha}1 and TRß1 with multiple mutations were also found in HCC (17). All mutated TR{alpha}1 identified in HCC with known mutation sites (eight of 12 HCC evaluated) were found to have between two and four mutations. For TRß1 mutants, 50% from HCC were found to have between two and four mutations. This is in contrast to RTH patients, in which no multiple mutation of the TRß gene was found and, moreover, no mutation was detected in the TR{alpha} gene. The role of multiple mutations of TRs in carcinogenesis is yet to be elucidated.

Comparison of the mutation sites in TR{alpha}1 and TRß1 mutants between RCCC and HCC (17) shows that there was only one common mutation site in TR{alpha}1, at A225, and no common mutation site was detected in TRß1 mutants between these two cancers. In RCCC, A225 of TR{alpha}1 was mutated to T, whereas in HCC A225 was mutated to G. However, in spite of different mutations of TRs in these two cancers, functional impairment of mutant TRs derived from these two cancers was similar in that mutation led to loss of the hormone-binding, DNA-binding and transcriptional activity. Moreover, TR mutants frequently exhibited dominant negative activity (17,44,45). These findings suggest that impairment of TR functions could play a critical role in the tumorigenesis of these two cancers.

At present, however, the precise roles of mutant TRs in carcinogenesis are not clear. Based on the functional impairment of TRß1 and TR{alpha}1 mutants demonstrated in the present study, it is reasonable to postulate that the transcriptional regulation of TR-mediated genes involved in cell differentiation, proliferation (46–48) and apoptosis (49–51) is abnormally affected in RCCC expressing these mutants. In addition, functional impairment of TRß1 and TR{alpha}1 mutants might also affect the protein–protein interactions in the network of cellular proto-oncogenes and tumor suppressors. TRs have recently been found to cross-talk with other signalling pathways. For example, TRß1 was shown to physically associate with the tumor suppressor protein p53 and repress p53-mediated transcriptional activity. Conversely, p53 reduces the ability of TRß1 to bind to DNA and to activate transcription (52,53). TRs also stimulate expression of the c-fos and c-jun proto-oncogenes, increase expression of the c-Fos and c-Jun proteins and activate AP1 transcriptional activity in a T3-independent pathway. In turn, both c-Fos and c-Jun inhibit T3-dependent transcription activation (54–56). TRs have been shown to directly activate expression of the mdm2 oncogene, which subsequently induces rapid degradation of p53 (57,58) and inhibits retinoblastoma tumor suppressor. Thus, functional impairment due to mutations and the aberrant expression of TRs in RCCC could result in the disarray of the normal regulatory control of cell proliferation, differentiation and apoptosis. Thus, dedifferentiated cells could again become sensitive to proliferation signals, while cells with damaged DNA are possibly not eliminated by means of apoptosis. The present study demonstrates that the percentage of mutated TRß1 was lowest in well differentiated (G1) and highest in poorly differentiated and fast growing (G3) tumors (see Table IGo). This finding is consistent with the notion that regulatory control of these processes is severely affected in poorly differentiated tumors. The functional consequences of mutations may be further accentuated by the dominant negative action of mutants. Indeed, our data clearly indicate that 3TRß1, 8TRß1, 23TR{alpha}1, 2TR{alpha}1 and 6TR{alpha}1 exhibit potent dominant negative activity. In addition to the loss of and/or interference with the normal functions of TRs, TR mutants may act via a gain-of-function mechanism. This mode of action of mutant genes is not unprecedented, as it has been clearly documented in p53 mutants (59). The gain-of-function of TR mutants may provide a growth advantage early in the progression of neoplastic cells, may affect genes which prevent differentiation and/or apoptosis of cells and may promote genomic instability. Any of these actions may contribute to the development of RCCC. These possibilities will await validation in future studies.


    Notes
 
4 To whom correspondence should be addressed Email: monika{at}amwaw.edu.pl Back

* The first two authors contributed equally to this work. Back


    Acknowledgments
 
The authors would like to thank Dr A.Tanski from the Department of Urology, Voivodoship Hospital, Ostroleka, Poland, for his kind donation and histological evaluation of RCCC tissues and Ms Li Zhao for technical assistance. This work was supported by the State Committee for Research, grants 4PO5B04115 and 0513/PO5/98/15, and by the CMKP, grant 501-2-1-01/99. This work was also partly supported by grant 4PO5B12708 from the National Research Council and by grant 501-2-1-01-01/99 from the Medical Center of Postgraduate Education. Y.K. is supported in part by a Japan Society for the Promotion of Science Research Fellowship for Japanese Biomedical and Behavioral Researchers at the National Institutes of Health, Bethesda, MD, USA.


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 Results
 Discussion
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Received July 6, 2001; revised September 5, 2001; accepted September 14, 2001.