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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 7 3447-3449
Copyright © 2003 by The Endocrine Society

COMMENT

Expression of TRß1 mRNAs with Functionally Impaired Mutations Is Rare in Thyroid Papillary Carcinoma

Toru Takano, Akira Miyauchi, Hiroshi Yoshida, Yukiko Nakata, Kanji Kuma and Nobuyuki Amino

Department of Laboratory Medicine (T.T., Y.N., N.A.), Osaka University Graduate School of Medicine, Osaka 565-0871, Japan; and Kuma Hospital (A.M., H.Y., K.K.), Hyogo 650-0011, Japan

Address all correspondence and requests for reprints to: Toru Takano, M.D., Department of Laboratory Medicine, Osaka University Graduate School of Medicine, D2, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: ttakano{at}labo.med.osaka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A previous study reported the expression of functionally impaired thyroid hormone receptor (TR)ß1 mutants in almost all papillary thyroid carcinomas. To confirm this, we analyzed the sequence of the entire coding region of TRß1 cDNAs expressed in 16 papillary carcinomas. Among the 48 clones analyzed, we found no mutations with an amino acid substitution, which represents a clear discrepancy between our findings and those in the previous study. Our findings suggest that the expression of functionally impaired mutants in papillary carcinomas is rare, and they raise a question about the possible role of mutated TRß1 in the tumorigenesis of papillary carcinoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PAPILLARY THYROID CARCINOMA (PTC) is the most common histological type of thyroid cancer. However, the molecular mechanism of its tumorigenesis is not completely understood. In previous studies, several genomic changes, such as rearrangement in the RET gene and mutations in the RAS or P53 gene, were reported to take part in tumorigenesis of PTC, although the frequencies of these respective changes do not usually exceed 40% (1, 2, 3, 4, 5, 6). Puzianowska-Kuznicka et al. (7) reported frequent mutations in the thyroid hormone receptor (TR) genes in PTCs. TRs are transcription factors that regulate various cell functions such as differentiation, proliferation, and apoptosis, and they are cellular homologs of the transcriptionally inactive viral oncogene v-erbA (8, 9, 10, 11). Their report involved analysis of 16 PTCs, indicated that 100% of the analyzed PTCs contained mutated TRß1, and in 93.75% of the analyzed PTC mutations resulted in amino acid substitutions. They concluded that these functionally impaired TR mutants play a crucial role in tumorigenesis of PTC.

This extremely high frequency of mutations in the TRß1 gene prompted us to undertake reevaluation of these findings. In this study, we analyzed the sequences of TRß1 cDNAs from 16 PTCs. Our findings showed a clear discrepancy from those in the previous report. Possible reasons for this discrepancy are discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Extraction of RNA from thyroid tissues and reverse transcription (RT)

This study was approved by the local ethics committee. Tissue samples from 16 PTCs and five normal thyroid tissues in the opposite lobe of carcinomas were obtained by surgery after patients gave informed consent. Tissues were frozen in liquid nitrogen immediately after resection. Total RNA was extracted according to the method of Chomczynski and Sacchi (12). RT was performed using 1 µg of total RNA in an RT mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 0.5 mM deoxynucleoside triphosphate (dNTP) mix (Takara, Shiga, Japan), 200 U Maloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD), 2 U/µl RNase inhibitor (Takara), and 2.5 µM oligo dT (Life Technologies, Inc.) in a total volume of 20 µl at 37 C for 60 min.

Cloning of TRß1 cDNAs from PTCs

To clone TRß1 expressed in PTC, cDNAs were amplified by PCR essentially according to the previous study except for the following two points. First, KOD Plus DNA polymerase (TOYOBO, Osaka, Japan), which assures higher fidelity than does Ex Taq DNA polymerase (TAKARA, Shiga, Japan), was used for amplification (13). Second, because the 3' primer used in the previous study has several mismatched bases to the newly revised TRß1 sequence (GenBank accession no. XM002986), we designed a new pair of primers, TRBF and TRBR, for the PCRs to amplify the entire coding region of the TRß1 gene (Table 1Go). The cDNA amplification was performed as follows: 2 min at 94 C, then 40 cycles of 94 C for 15 sec, 55 C for 30 sec, 68 C for 90 sec in a reaction mixture that consisted of 1 µl cDNA, 0.3 µM of each primer, 1 mM MgCl2, 5 µl of 10x PCR buffer, 200 µM dNTP mix, 1 U KOD Plus DNA polymerase, and nuclease-free water to a final volume of 50 µl. The 10x PCR buffer, dNTP mix, and KOD Plus DNA polymerase were obtained from TOYOBO, and the primers were obtained from QIAGEN (Tokyo, Japan). After precipitation with ethanol, the PCR products were incubated at 72 C for 15 min in a reaction mixture containing 2 µl of 10x Ex Taq buffer (TAKARA), 200 µM dNTP mix (TAKARA), 1 U Ex Taq DNA polymerase to a final volume of 20 µl to make adenine overhangs in both ends of the amplified cDNAs. The PCR products were electrophoresed on 1% agarose gel, specific TRß1 bands were excised, and DNA was isolated for the gel with a QIAquick Gel Extraction Kit (QIAGEN) and ligated into pGEM-T Easy vector containing T overhangs (Promega Corp., Tokyo, Japan) at 4 C for 12 h. JM109 bacteria were transformed with the ligation mix, and blue-white selection was performed.


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  		TABLE 1. Primers used in the sequence analysis

 
Sequencing of the TR clones

Automatic sequencing of TRß1 clones was performed with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Warrington, UK) using purified plasmids. Three TRß1 clones originating from the same cancer tissue were pooled and sequenced. In sequence analysis, we used all six primers shown in Table 1Go to cover the entire coding sequence of the TRß1 gene.

Real-time quantitative RT-PCR

Real-time quantitative RT-PCR (TaqMan RT-PCR) using the ABI PRISM 7700 Sequence Detection System was performed as described previously (14). One microliter of the first strand cDNA was used in the following assay. The two primers and one TaqMan probe used for the quantification of TRß1 mRNA were: [TRBQF (0.5 µM): 5'-CCAGAAGACATTGGACAAGCA-3' (base 1024–1044)], [TRBQR (0.5 µM): 5'-GCAGCTCACAAAACATAGGCA-3' (base 1154–1174)], and [TRB-TM (10 pmol): 5'-FAM-ATCATCACACCAGCAATTACCAGAGTGGTG-TAMRA-3' (base 1108–1137)], respectively. The conditions for the TaqMan PCR were as follows: 95 C for 10 min, and 40 cycles of 95 C for 15 sec and 60 C for 1 min. A recombinant pGEM Easy T-Vector (Promega Corp.) containing the partial TRß1 cDNA was constructed by PCR cloning with the same set of primers used in TaqMan PCR and were used as standard samples.

Statistical analysis

Statistical analysis of differences between the groups was performed using the Mann-Whitney U test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The coding sequences (bases 301-1671) of 48 clones from 16 PTCs were analyzed in both directions. It was found that cytosine in base 1020 was changed to thymine in three clones from three different carcinomas. However, this change did not accompany an amino acid substitution (Table 2Go). Overall, in the 16 PTCs analyzed, we found no TRß1 mutant with an amino acid substitution. By real-time quantitative RT-PCR, TRß1 mRNAs were more abundantly expressed in normal thyroid tissues than in PTCs. In all PTCs, however, more than 8.0 x 103 copies of TRß1 mRNA were expressed in 1 µg total RNA (Fig. 1Go).


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  		TABLE 2. Summary of the sequence analysis of TRß1 cDNA clones in PTCs

 


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  		FIG. 1. Quantitative measurement of TRß1 mRNA in normal thyroid tissues and PTCs. Quantitative analysis of TRß1 mRNA was performed using five normal thyroid tissues and 16 PTCs used in this study as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the previous study, Puzianowska-Kuznicka et al. (7) concluded that TRs, once mutated, could play a role in PTC tumorigenesis, based on the high frequency of PTCs affected by TR mutations and the high number of mutations within a single TR allele. However, our study, which used the same number of PTCs, showed the striking discrepancy from theirs in terms of the frequency of mutants with amino acid substitution. We found only a single base substitution, without an amino acid substitution, in three of 48 clones. This might be an individual variation rather than a sporadic change in tumor, because precisely the same change was observed in three clones derived from three different PTCs.

It may be possible to explain these results by considering the different conditions of the experiments. For example, it might be assumed that the high iodine intake in the Japanese population could affect the biological characteristics of PTCs. Nevertheless, we identified at least two technical problems in the previous study. First, it was possible to amplify TRß1 cDNA by the first PCR, without nested PCR. We assume that the low amount of TRß1 cDNA after the first PCR described in the previous study was caused by the mismatching between the sequence of the 3' primer used and the newly revised sequence of the TRß1 gene. Second, use of Ex Taq DNA polymerase in sequencing analysis after long PCRs is not appropriate, because Ex Taq DNA polymerase has not shown sufficiently high fidelity for use in this kind of analysis. In fact, when we used Ex Taq DNA polymerase in the PCRs, we found mutations in four of 10 clones from a papillary carcinoma in which we found no mutations when using KOD Plus DNA polymerase (data not shown). Because TRß1 mRNA is less abundant in PTCs than in benign tumors or normal thyroids, additional cycles of amplification in the PCRs are necessary to obtain a sufficient amount of cDNA from PTCs (15). Thus, when the fidelity of the DNA polymerase is not sufficiently high, frequent false mutations may occur in clones from PTCs (13).

Considering these facts, we conclude that before discussion of the relationship between tumorigenesis of PTC and mutations in TRs can take place, intensive reevaluation of the frequency of mutation in TRs, using our method and a group of samples collected in the United States or Poland, is necessary. Furthermore, when results of mutation studies are proved to be reproducible, it is important to confirm the mutations in the genome, which was not done in the previous study.


   Footnotes
 
This work was partially supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants-in-Aid for Scientific Research B, 2001-2, no. 13557227.

Abbreviations: dNTP, Deoxynucleoside triphosphate; PTC, papillary thyroid carcinoma; RT, reverse transcription; TR, thyroid hormone receptor.

Received January 8, 2003.

Accepted April 4, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Grieco M, Santoro M, Berlingieri MT, Melillo RM, Donghi R, Bongarzone I, Pierotti MA, Della Porta G, Fusco A, Vecchio G 1990 PTC is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas. Cell 60:557–563[CrossRef][Medline]
  2. Santoro M, Carlomagno F, Hay ID, Herrmann MA, Grieco M, Melillo R, Pierotti MA, Bongarzone I, Della Porta G, Berger N, Peix JL, Paulin C, Fabien N, Vecchio G, Jenkins RB, Fusco A 1992 Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype. J Clin Invest 89:1517–1522
  3. Namba H, Rubin SA, Fagin JA 1990 Point mutations of ras oncogenes are an early event in thyroid tumorigenesis. Mol Endocrinol 4:1474–1479[Abstract/Free Full Text]
  4. Karga H, Lee JK, Vickery Jr AL, Thor A, Gaz RD, Jameson JL 1991 Ras oncogene mutations in benign and malignant thyroid neoplasms. J Clin Endocrinol Metab 73:832–836[Abstract/Free Full Text]
  5. Fagin JA, Matsuo K, Karmakar A, Chen DL, Tang SH, Koeffler HP 1993 High prevalence of mutations of the p53 gene in poorly differentiated human thyroid carcinomas. J Clin Invest 91:179–184
  6. Zou M, Shi Y, Farid NR 1993 p53 mutations in all stages of thyroid carcinomas. J Clin Endocrinol Metab 77:1054–1058[Abstract]
  7. Puzianowska-Kuznicka M, Krystyniak A, Madej A, Cheng SY, Nauman J 2002 Functionally impaired TR mutants are present in thyroid papillary cancer. J Clin Endocrinol Metab 87:1120–1128[Abstract/Free Full Text]
  8. Damm K 1993 ErbA: tumor suppressor turned oncogene? FASEB J 7:904–909[Abstract]
  9. Chin WW 1994 Molecular mechanisms of thyroid hormone action. Thyroid 4:389–393[Medline]
  10. Puzianowska-Kuznicka M, Damjanovski S, Shi YB 1997 Both thyroid hormone and 9-cis retinoic acid receptors are required to efficiently mediate the effects of thyroid hormone on embryonic development and specific gene regulation in Xenopus laevis. Mol Cell Biol 17:4738–4749[Abstract]
  11. Wolffe AP, Collingwood TN, Li Q, Yee J, Urnov F, Shi YB 2000 Thyroid hormone receptor, v-ErbA, and chromatin. Vitam Horm 58:449–492[CrossRef][Medline]
  12. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  13. Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, Imanaka T 1997 Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR. Appl Environ Microbiol 63:4504–4510[Abstract]
  14. Takano T, Miyauchi A, Yokozawa T, Matsuzuka F, Maeda I, Kuma K, Amino N 1999 Preoperative diagnosis of thyroid papillary and anaplastic carcinomas by real-time quantitative reverse transcription-polymerase chain reaction of oncofetal fibronectin messenger RNA. Cancer Res 59:4542–4545[Abstract/Free Full Text]
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