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Involvement of Wnt pathway in thyroid cancer around Semipalatinsk Nuclear Test Site
International Congress Series 1258 (2003) 177 – 183
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Involvement of Wnt pathway in thyroid cancer around Semipalatinsk Nuclear Test Site Serik K. Meirmanov a,*, Masahiro Nakashima b, Noboru Takamura c, Masahiro Ito d, Yuri V. Prouglo e, Shunichi Yamashita f,g, Ichiro Sekine a,b a
Department of Molecular Pathology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan b Tissue and Histopathology Section, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan c Department of Public Health, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan d Department of Pathology, National Nagasaki Medical Center, Nagasaki, Japan e Department of Pathology, Semipalatinsk Medical Academy, Semipalatinsk, Kazakhstan f Department of Nature Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan g Department of International Health and Radiation Research, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Abstract. A total of 459 nuclear tests were officially conducted at the Semipalatinsk Nuclear Test Site of Kazakhstan during the period from 1949 to 1989. The exposure to radioactive fallout could affect the population health. The aberrant activation of Wnt pathway elements is linked to thyroid tumorigenesis. The activated Wnt signaling stabilizes h-catenin and results in transactivation of target genes including cyclin D1. To investigate the involvement of the Wnt/h-catenin pathway in thyroid carcinomas around the Semipalatinsk Nuclear Test Site, we compared immunohistochemical results for h-catenin and cyclin D1 expression in chronic thyroiditis, follicular adenoma and papillary carcinoma. This study revealed a higher prevalence of both aberrant h-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the Semipalatinsk Nuclear Test Site than sporadic cases. The analysis of the alteration of the Wnt signaling-related molecules in thyroid cancer around the Semipalatinsk Nuclear Test Site may be important to gain an insight into radiation-induced thyroid tumorigenesis. D 2003 Elsevier B.V. All rights reserved. Keywords: Wnt pathway; h-catenin; Thyroid tumors; Semipalatinsk Nuclear Test Site; Radiation
* Corresponding author. Tel.: +81-95-849-7107; fax: +81-95-849-7108. E-mail address: [email protected] (S.K. Meirmanov). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01198-1
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S.K. Meirmanov et al. / International Congress Series 1258 (2003) 177–183
1. Introduction A total of 459 nuclear explosions, including 87 atmospheric ones, were officially conducted at the Semipalatinsk Nuclear Test Site of Kazakhstan during 1949 – 1989 [1]. Several hundred thousand people living in the northeast Kazakhstan region were exposed to radioactive fallout. The incidence of thyroid cancer was reported to be elevated in the population living close to the Semipalatinsk Nuclear Test Site as compared with the control group [2 –4]. The highly conserved Wnt signaling pathway regulates cell proliferation, differentiation and cell fate decision [5]. A key downstream effector of the Wnt signaling pathway hcatenin exhibits its physiological function as a cell –cell adhesion molecule together with E-cadherin at the cellular submembrane [6]. Activation of the Wnt pathway, caused by genetic mutations, stabilizes the h-catenin protein. The stabilized h-catenin accumulates in the cytoplasm, then subsequently translocates to the nucleus, binds to the T-cell factor/ lymphoid enhancer factor (Tcf/Lef) [7– 9] and activates genes such as cyclin D1 and c-myc [10,11]. A role of h-catenin in the genesis of thyroid cancer is supported by recent observations of a high frequency of h-catenin mutations and nuclear h-catenin immunostaining in poorly differentiated thyroid carcinomas [12]. Elevated nuclear cyclin D1 expression is also found in a subset of thyroid papillary carcinomas [13,14]. We report here a higher prevalence of both cytoplasmic h-catenin expression and cyclin D1 overexpression in papillary carcinoma around the Semipalatinsk Nuclear Test Site as compared with the sporadic cases. 2. Materials and methods 2.1. Materials A total of 40 archival thyroid tissue samples obtained from surgical specimens at the Semipalatinsk regional hospitals from 1986 to 1996 were used in this study (Table 1). Among the samples studied, nine cases of chronic thyroiditis in the Semipalatinsk Nuclear Test Site were used as a non-neoplastic control (Table 1). Histologically, of the 31 primary thyroid tumors, 23 were papillary and 8 were follicular adenomas (Table 1). Eighty-seven Japanese cases of papillary thyroid cancer were also examined as sporadic cancer controls. 2.2. Immunohistochemistry Paraffin-embedded tissues were deparaffinized in xylene and rehydrated in phosphatebuffered saline. After immersion in 0.3% H2O2/methanol to block endogenous peroxidase activity, sections were preincubated with 10% normal goat serum to prevent nonspecific binding. After antigen retrieval using a microwave treatment in sodium citrate buffer (pH 6.0), tissues were incubated overnight at 4 jC with anti-h-catenin monoclonal antibodies (Transduction Laboratories, Lexington, KY) at a 1:100 dilution, or anti-cyclin D1 monoclonal antibody (Zymed Laboratories, South San Francisco, CA) at a 1:50 dilution. The slides were subsequently incubated with biotinylated goat anti-mouse IgG antibody for 1 h, followed by incubation with avidin-peroxidase for 30 min, and visualized with
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Table 1 Patients’ profiles Diagnosis
Case
Female/Male
Mean age (years)
Chronic thyroiditis Follicular adenoma Papillary carcinoma Sporadic papillary carcinoma
9 8 23 87
9/0 6/2 20/3 74/13
44.9 37.9 46.9 52.1
diaminobenzidine. Control experiments including incubation with nonimmunized mouse serum instead of the primary antisera did not show any staining. 2.3. Evaluation of b-catenin and cyclin D1 expression Two independent observers (S.M. and M.N.) evaluated the immunohistochemical staining. h-catenin immunoreactivity was expressed as the intensity of positive staining ( : no staining; F : weak; +: moderate; ++: strong) in each subcellular compartment such as membrane and cytoplasm. The expression of cyclin D1 protein was evaluated by the intensity of cyclin D1 immunoreactivity in the nuclei ( : no staining; F : weak; +: moderate; ++: strong). 3. Results 3.1. b-catenin expression in thyroid tissues from the Semipalatinsk Nuclear Test Site The results of h-catenin expression are presented in Table 2. Normal thyroid follicular cells showed clear and intense membrane h-catenin staining (Fig. 1A), while the maximum level of cytoplasmic h-catenin expression was found in the majority of papillary carcinomas (Fig. 1B). Eight of nine (88.9%) cases of chronic thyroiditis, serving as non-neoplastic controls, showed strong or moderate h-catenin levels in the membranes. Only three cases (33.3%) of thyroiditis showed moderate or weak cytoplasmic h-catenin expression. One case (11.1%) of thyroiditis was negative for h-catenin staining. Seven of eight (87.5%) follicular adenomas showed strong or moderate h-catenin staining in the membranes, and six Table 2 Immunohistochemical resultsa Diagnosisb Case
h-catenin (%) Membrane
CT FA PC sPC a
9 8 23 87
(100%) 11.1 (100%) 0 (100%) 8.7 (100%) 13.8
Cytoplasm
Cyclin D1(%)
F
+
++
F
+
++
0 12.5 39.1 23.0
33.3 12.5 43.5 41.4
55.6 66.7 75.0 25.0 8.7 0 21.8 6.9
11.1 37.5 0 16.1
22.2 37.5 30.4 46.0
0 100 0 0 37.5 25.0 69.6 13.0 0 31.0 18.4 12.6
F
+
++
0 37.5 34.8 39.1
0 0 52.2 29.9
Based on the following scale: = no staining; F = weak; + = moderate; + + = strong. CT = chronic thyroiditis; FA = follicular adenoma; PC = papillary carcinoma; sPC = sporadic papillary carcinoma. b
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Fig. 1. Immunohistochemical expression of h-catenin and cyclin D1. Normal thyroid follicles showed clear membrane h-catenin staining (A). Papillary carcinoma displayed strong cytoplasmic h-catenin expression with a marked decrease in membrane h-catenin immunoreactivity (B). Normal follicles did not display cyclin D1 expression (C). Strong cyclin D1 immunoreactivity was detected in more than 30% of papillary carcinoma cells (D).
(75.0%) of these samples also displayed weak or moderate cytoplasmic h-catenin staining. Twelve of twenty-three (52.2%) papillary carcinomas showed strong or moderate h-catenin staining in the membranes. The remaining 11 cases (47.8%) showed a marked decrease in membrane h-catenin immunoreactivity. Conversely, all papillary carcinomas displayed strong (16 cases or 69.6%) or moderate (seven cases or 30.4%) cytoplasmic h-catenin expression (Fig. 1B). Thus, the level of cytoplasmic h-catenin expression was significantly increased in the order of papillary carcinoma > follicular adenoma > chronic thyroiditis. 3.2. Cyclin D1 expression and its correlation with cytoplasmic b-catenin No cyclin D1 immunoreactivity was evident in normal follicular cells or chronic thyroiditis (Fig. 1C). In contrast, five of eight (62.5%) follicular adenomas and 20 of 23 (87.0%) carcinoma cases showed a nuclear cyclin D1 expression. Cyclin D1-positive follicular adenomas exhibited moderate (three cases or 37.5%) or weak (two cases or 25.0%) immunoreactivity, while all of the cyclin D1-positive papillary carcinomas showed strong (12 cases or 52.2%) or moderate (eight cases or 34.8%) immunoreactivity (Fig.
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1D). The proportional increase of cyclin D1 expression to the cytoplasmic h-catenin immunoreactivity was clarified by each case comparison of immunochemical results in each case (data not shown). 3.3. b-catenin and cyclin D1 expressions in sporadic papillary cancers In sporadic cancers, 55 of 87 (63.2%) cases showed strong or moderate h-catenin staining in the membranes. Like the Semipalatinsk Nuclear Test Site cases, the remaining 32 cases (36.8%) showed a marked decrease in membrane h-catenin immunoreactivity. On the other hand, 67 of 87 (77.0%) sporadic papillary carcinomas showed strong or moderate h-catenin staining in the cytoplasms, and 20 (23.0%) of these samples also displayed weak or no cytoplasmic h-catenin staining. The level of cytoplasmic h-catenin expression was significantly higher in the Semipalatinsk Nuclear Test Site cases than in sporadic cases. Furthermore, 71 of 87 (81.6%) sporadic cases showed cyclin D1 immunoreactivity. Cylin D1 immunoreactivity was significantly higher in Semipalatinsk Nuclear Test Site cases than sporadic cases (Table 2). 4. Discussion We found that cytoplasmic h-catenin immunoreactivity was very common (23 of 23 cases) in papillary thyroid cancers from near the Semipalatinsk Nuclear Test Site. Both chronic thyroiditis and follicular adenomas showed significantly lower cytoplasmic hcatenin expression. On the other hand, normal thyroid follicular cells showed strong membrane h-catenin immunoreactivity with no cytoplasmic localization. These results suggest a significance of accumulated cytoplasmic h-catenin during thyroid carcinogenesis around the Semipalatinsk Nuclear Test Site. In the present study, a direct comparison with sporadic cases revealed a higher prevalence of cytoplasmic h-catenin expression in papillary carcinomas from the Semipalatinsk Nuclear Test Site. Some investigators reported aberrant h-catenin expression in classical papillary carcinomas [15,16]. A higher level of aberrant cytoplasmic h-catenin expression was observed in papillary cancers from the Semipalatinsk Nuclear Test Site than in sporadic tumors. We consider that alteration of the Wnt signaling pathway could lead to a higher level of h-catenin expression. Indeed, it has recently been reported that elements of the Wnt signaling pathway are expressed in thyroid cells, and that this pathway is functionally active [17]. However, our preliminary study using polymerase chain reaction –single-strand conformation polymorphism could not detect mutations in hcatenin gene exon 3 in our samples (data not shown) as well as in the previous reports on papillary thyroid cancers [12,18]. The role of h-catenin as an oncogene has already been established [12,19]. A few reports indicate the significance of immunoreactive cytoplasmic h-catenin in papillary thyroid cancers. An accumulated h-catenin by the activation of Wnt signaling induces a gene encoding cyclin D1, which plays a role in the initiation of cell transformation [10]. In our recent paper, we have demonstrated that dislocalization of h-catenin correlates with overexpression of cyclin D1 in sporadic papillary cancers [14]. We therefore clarified the correlation between cytoplasmic h-catenin expression and cyclin D1 overexpression in thyroid tissues, including not only papillary cancer but also benign tumor.
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In our series, cyclin D1 immunoreactivity was restricted to both papillary carcinomas and follicular adenomas. The level of cyclin D1 overexpression was significantly higher in papillary carcinomas than in follicular adenomas. Cyclin D1 overexpression has already been well documented in human thyroid cancers, and its frequency in conventional papillary carcinoma ranges between 32.0% and 82.4% [20]. We have reported cyclin D1 overexpression in 82% of sporadic cases [12]. A higher prevalence of cyclin D1 overexpression was observed in our series as well as cytoplasmic h-catenin expression. Additionally, the level of cyclin D1 overexpression was significantly correlated with cytoplasmic h-catenin immunoreactivity in thyroid tissues together with chronic thyroiditis, follicular adenoma and papillary carcinoma. Taken together, these findings show that cyclin D1 upregulation may be caused by aberrant h-catenin expression during malignant transformation of the thyroid gland. In conclusion, this study revealed a higher prevalence of both aberrant h-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the Semipalatinsk Nuclear Test Site as compared with previously reported papillary carcinomas. Accumulation of cytoplasmic h-catenin seems to be closely correlated with the level of cyclin D1 overexpression during thyroid carcinogenesis around the Semipalatinsk Nuclear Test Site. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology (No. 14770101). References [1] M. Yamamoto, T. Tsukatani, Y. Katayama, Residual radioactivity in the soil of the Semipalatinsk nuclear test site in the former USSR, Health Phys. 71 (1996) 142 – 148. [2] S. Yamashita, Proposal for establishment of screening of thyroid diseases around Semipalatinsk, in: M. Hoshi, J. Takada, R. Kim, Y. Nitta (Eds.), Effects of low-level radiation for residents near Semipalatinsk nuclear test site. Proceedings of the Second Hiroshima International Symposium, Hiroshima, July 23 – 25, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 1996, pp. 225 – 234. [3] Z. Zhumadilov, B.I. Gusev, J. Takada, M. Hoshi, A. Kimura, N. Hayakawa, N. Takeichi, Thyroid abnormality trend over time in northeastern regions of Kazakstan, adjacent to the Semipalatinsk nuclear test site: a case review of pathological findings for 7271 patients, J. Radiat. Res. 41 (2000) 35 – 44. [4] B.I. Gusev, R.I. Rosenson, Z.N. Abylkassimova, The Semipalatinsk nuclear test site: a first analysis of solid cancer incidence (selected sites) due to test-related radiation, Radiat. Environ. Biophys. 37 (1998) 209 – 214. [5] K.M. Cadigan, R. Nusse, Wnt signaling: a common theme in animal development, Genes Dev. 11 (1997) 3296 – 3305. [6] P. Polakis, Wnt signaling and cancer, Genes Dev. 14 (2000) 1837 – 1851. [7] B. Rubinfeld, P. Robbins, M. El-Gamil, I. Albert, E. Porfiri, P. Polakis, Stabilization of h-catenin by genetic defects in melanoma cell lines, Science 275 (1997) 1790 – 1792. [8] P.J. Morin, A.B. Sparks, V. Korinek, N. Barker, H. Clevers, B. Vogelstein, K.W. Kinzler, Activation of hcatenin-Tcf signaling in colon cancer by mutations in h-catenin or APC, Science 275 (1997) 1787 – 1790. [9] V. Korinek, N. Barker, P.J. Morin, D. van Wichen, R. de Weger, K.W. Kinzler, B. Vogelstein, H. Clevers, Constitutive transcriptional activation by a h-catenin-Tcf complex in APC / colon carcinoma, Science 275 (1997) 1784 – 1787. [10] O. Tetsu, F. McCormick, h-catenin regulates expression of cyclin D1 in colon carcinoma cells, Nature 398 (1998) 422 – 426.
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[11] T.C. He, A.B. Sparks, C. Rago, H. Hermeking, L. Zawel, L.T. da Costa, P.J. Morin, B. Vogelstein, K.W. Kinzler, Identification of c-myc as a target for the APC pathway, Science 281 (1998) 1509 – 1512. [12] G. Garcia-Rostan, R.L. Camp, A. Herrero, M.L. Carcangiu, D.L. Rimm, G. Tallini, h-catenin dysregulation in thyroid neoplasms—down-regulation, aberrant nuclear expression, and CTNNB1 exon 3 mutations are markers for aggressive tumor phenotypes and poor prognosis, Am. J. Pathol. 158 (2001) 987 – 996. [13] D. Lazzereschi, I. Sambuco, C.C. Scalzo, A. Ranieri, G. Mincione, F. Nardi, G. Colletta, Cyclin D1 and cyclin E expression in malignant thyroid cells and in human thyroid carcinomas, Int. J. Cancer 76 (1998) 806 – 811. [14] K. Ishigaki, H. Namba, M. Nakashima, T. Nakayama, N. Mitsutake, T. Hayashi, S. Maeda, M. Ichinose, T. Kanematsu, S. Yamashita, Aberrant localization of h-catenin correlates with overexpression of its target gene in human papillary thyroid cancer, J. Clin. Endocrinol. Metab. 87 (2002) 3433 – 3440. [15] A.S. Rocha, P. Soares, R. Seruca, V. Maximo, X. Matias-Guiu, J. Cameselle-Teijeiro, M. Sobrinho-Simoes, Abnormalities of the E-cadherin/catenin adhesion complex in classical papillary thyroid carcinoma and in its diffuse sclerosing variant, J. Pathol. 194 (2001) 358 – 366. [16] A. Cerrato, F. Fulciniti, A. Avallone, G. Benincasa, L. Palombini, M. Grieco, Beta- and gamma-catenin expression in thyroid carcinomas, J. Pathol. 185 (1998) 267 – 272. [17] K. Helmbrecht, A. Kispert, R. von Wasielewski, G. Brabant, Identification of a Wnt/h-catenin signaling pathway in human thyroid cells, Endocrinology 142 (2001) 5261 – 5266. [18] N. Miyake, H. Maeta, S. Horie, Y. Kitamura, E. Nanba, K. Kobayashi, T. Terada, Absence of mutations in the h-catenin and adenomatous polyposis coli genes in papillary and follicular thyroid carcinomas, Pathol. Int. 51 (2001) 680 – 685. [19] K. Orford, C.C. Orford, S.W. Byers, Exogenous expression of h-catenin regulates contact inhibition, anchorage-independent growth, anoikis, and radiation-induced cell cycle arrest, J. Cell Biol. 146 (1999) 855 – 867. [20] S. Wang, R.V. Lloyd, M.J. Hutzler, M.S. Safren, N.A. Patwardhan, A. Khan, The role of cell cycle regulatory protein, cyclin D1, in the progression of thyroid cancer, Mod. Pathol. 13 (2000) 882 – 887.
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Involvement of Wnt pathway in thyroid cancer around Semipalatinsk Nuclear Test Site Serik K. Meirmanov a,*, Masahiro Nakashima b, Noboru Takamura c, Masahiro Ito d, Yuri V. Prouglo e, Shunichi Yamashita f,g, Ichiro Sekine a,b a
Department of Molecular Pathology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan b Tissue and Histopathology Section, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan c Department of Public Health, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan d Department of Pathology, National Nagasaki Medical Center, Nagasaki, Japan e Department of Pathology, Semipalatinsk Medical Academy, Semipalatinsk, Kazakhstan f Department of Nature Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan g Department of International Health and Radiation Research, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
Abstract. A total of 459 nuclear tests were officially conducted at the Semipalatinsk Nuclear Test Site of Kazakhstan during the period from 1949 to 1989. The exposure to radioactive fallout could affect the population health. The aberrant activation of Wnt pathway elements is linked to thyroid tumorigenesis. The activated Wnt signaling stabilizes h-catenin and results in transactivation of target genes including cyclin D1. To investigate the involvement of the Wnt/h-catenin pathway in thyroid carcinomas around the Semipalatinsk Nuclear Test Site, we compared immunohistochemical results for h-catenin and cyclin D1 expression in chronic thyroiditis, follicular adenoma and papillary carcinoma. This study revealed a higher prevalence of both aberrant h-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the Semipalatinsk Nuclear Test Site than sporadic cases. The analysis of the alteration of the Wnt signaling-related molecules in thyroid cancer around the Semipalatinsk Nuclear Test Site may be important to gain an insight into radiation-induced thyroid tumorigenesis. D 2003 Elsevier B.V. All rights reserved. Keywords: Wnt pathway; h-catenin; Thyroid tumors; Semipalatinsk Nuclear Test Site; Radiation
* Corresponding author. Tel.: +81-95-849-7107; fax: +81-95-849-7108. E-mail address: [email protected] (S.K. Meirmanov). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0531-5131(03)01198-1
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1. Introduction A total of 459 nuclear explosions, including 87 atmospheric ones, were officially conducted at the Semipalatinsk Nuclear Test Site of Kazakhstan during 1949 – 1989 [1]. Several hundred thousand people living in the northeast Kazakhstan region were exposed to radioactive fallout. The incidence of thyroid cancer was reported to be elevated in the population living close to the Semipalatinsk Nuclear Test Site as compared with the control group [2 –4]. The highly conserved Wnt signaling pathway regulates cell proliferation, differentiation and cell fate decision [5]. A key downstream effector of the Wnt signaling pathway hcatenin exhibits its physiological function as a cell –cell adhesion molecule together with E-cadherin at the cellular submembrane [6]. Activation of the Wnt pathway, caused by genetic mutations, stabilizes the h-catenin protein. The stabilized h-catenin accumulates in the cytoplasm, then subsequently translocates to the nucleus, binds to the T-cell factor/ lymphoid enhancer factor (Tcf/Lef) [7– 9] and activates genes such as cyclin D1 and c-myc [10,11]. A role of h-catenin in the genesis of thyroid cancer is supported by recent observations of a high frequency of h-catenin mutations and nuclear h-catenin immunostaining in poorly differentiated thyroid carcinomas [12]. Elevated nuclear cyclin D1 expression is also found in a subset of thyroid papillary carcinomas [13,14]. We report here a higher prevalence of both cytoplasmic h-catenin expression and cyclin D1 overexpression in papillary carcinoma around the Semipalatinsk Nuclear Test Site as compared with the sporadic cases. 2. Materials and methods 2.1. Materials A total of 40 archival thyroid tissue samples obtained from surgical specimens at the Semipalatinsk regional hospitals from 1986 to 1996 were used in this study (Table 1). Among the samples studied, nine cases of chronic thyroiditis in the Semipalatinsk Nuclear Test Site were used as a non-neoplastic control (Table 1). Histologically, of the 31 primary thyroid tumors, 23 were papillary and 8 were follicular adenomas (Table 1). Eighty-seven Japanese cases of papillary thyroid cancer were also examined as sporadic cancer controls. 2.2. Immunohistochemistry Paraffin-embedded tissues were deparaffinized in xylene and rehydrated in phosphatebuffered saline. After immersion in 0.3% H2O2/methanol to block endogenous peroxidase activity, sections were preincubated with 10% normal goat serum to prevent nonspecific binding. After antigen retrieval using a microwave treatment in sodium citrate buffer (pH 6.0), tissues were incubated overnight at 4 jC with anti-h-catenin monoclonal antibodies (Transduction Laboratories, Lexington, KY) at a 1:100 dilution, or anti-cyclin D1 monoclonal antibody (Zymed Laboratories, South San Francisco, CA) at a 1:50 dilution. The slides were subsequently incubated with biotinylated goat anti-mouse IgG antibody for 1 h, followed by incubation with avidin-peroxidase for 30 min, and visualized with
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Table 1 Patients’ profiles Diagnosis
Case
Female/Male
Mean age (years)
Chronic thyroiditis Follicular adenoma Papillary carcinoma Sporadic papillary carcinoma
9 8 23 87
9/0 6/2 20/3 74/13
44.9 37.9 46.9 52.1
diaminobenzidine. Control experiments including incubation with nonimmunized mouse serum instead of the primary antisera did not show any staining. 2.3. Evaluation of b-catenin and cyclin D1 expression Two independent observers (S.M. and M.N.) evaluated the immunohistochemical staining. h-catenin immunoreactivity was expressed as the intensity of positive staining ( : no staining; F : weak; +: moderate; ++: strong) in each subcellular compartment such as membrane and cytoplasm. The expression of cyclin D1 protein was evaluated by the intensity of cyclin D1 immunoreactivity in the nuclei ( : no staining; F : weak; +: moderate; ++: strong). 3. Results 3.1. b-catenin expression in thyroid tissues from the Semipalatinsk Nuclear Test Site The results of h-catenin expression are presented in Table 2. Normal thyroid follicular cells showed clear and intense membrane h-catenin staining (Fig. 1A), while the maximum level of cytoplasmic h-catenin expression was found in the majority of papillary carcinomas (Fig. 1B). Eight of nine (88.9%) cases of chronic thyroiditis, serving as non-neoplastic controls, showed strong or moderate h-catenin levels in the membranes. Only three cases (33.3%) of thyroiditis showed moderate or weak cytoplasmic h-catenin expression. One case (11.1%) of thyroiditis was negative for h-catenin staining. Seven of eight (87.5%) follicular adenomas showed strong or moderate h-catenin staining in the membranes, and six Table 2 Immunohistochemical resultsa Diagnosisb Case
h-catenin (%) Membrane
CT FA PC sPC a
9 8 23 87
(100%) 11.1 (100%) 0 (100%) 8.7 (100%) 13.8
Cytoplasm
Cyclin D1(%)
F
+
++
F
+
++
0 12.5 39.1 23.0
33.3 12.5 43.5 41.4
55.6 66.7 75.0 25.0 8.7 0 21.8 6.9
11.1 37.5 0 16.1
22.2 37.5 30.4 46.0
0 100 0 0 37.5 25.0 69.6 13.0 0 31.0 18.4 12.6
F
+
++
0 37.5 34.8 39.1
0 0 52.2 29.9
Based on the following scale: = no staining; F = weak; + = moderate; + + = strong. CT = chronic thyroiditis; FA = follicular adenoma; PC = papillary carcinoma; sPC = sporadic papillary carcinoma. b
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Fig. 1. Immunohistochemical expression of h-catenin and cyclin D1. Normal thyroid follicles showed clear membrane h-catenin staining (A). Papillary carcinoma displayed strong cytoplasmic h-catenin expression with a marked decrease in membrane h-catenin immunoreactivity (B). Normal follicles did not display cyclin D1 expression (C). Strong cyclin D1 immunoreactivity was detected in more than 30% of papillary carcinoma cells (D).
(75.0%) of these samples also displayed weak or moderate cytoplasmic h-catenin staining. Twelve of twenty-three (52.2%) papillary carcinomas showed strong or moderate h-catenin staining in the membranes. The remaining 11 cases (47.8%) showed a marked decrease in membrane h-catenin immunoreactivity. Conversely, all papillary carcinomas displayed strong (16 cases or 69.6%) or moderate (seven cases or 30.4%) cytoplasmic h-catenin expression (Fig. 1B). Thus, the level of cytoplasmic h-catenin expression was significantly increased in the order of papillary carcinoma > follicular adenoma > chronic thyroiditis. 3.2. Cyclin D1 expression and its correlation with cytoplasmic b-catenin No cyclin D1 immunoreactivity was evident in normal follicular cells or chronic thyroiditis (Fig. 1C). In contrast, five of eight (62.5%) follicular adenomas and 20 of 23 (87.0%) carcinoma cases showed a nuclear cyclin D1 expression. Cyclin D1-positive follicular adenomas exhibited moderate (three cases or 37.5%) or weak (two cases or 25.0%) immunoreactivity, while all of the cyclin D1-positive papillary carcinomas showed strong (12 cases or 52.2%) or moderate (eight cases or 34.8%) immunoreactivity (Fig.
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181
1D). The proportional increase of cyclin D1 expression to the cytoplasmic h-catenin immunoreactivity was clarified by each case comparison of immunochemical results in each case (data not shown). 3.3. b-catenin and cyclin D1 expressions in sporadic papillary cancers In sporadic cancers, 55 of 87 (63.2%) cases showed strong or moderate h-catenin staining in the membranes. Like the Semipalatinsk Nuclear Test Site cases, the remaining 32 cases (36.8%) showed a marked decrease in membrane h-catenin immunoreactivity. On the other hand, 67 of 87 (77.0%) sporadic papillary carcinomas showed strong or moderate h-catenin staining in the cytoplasms, and 20 (23.0%) of these samples also displayed weak or no cytoplasmic h-catenin staining. The level of cytoplasmic h-catenin expression was significantly higher in the Semipalatinsk Nuclear Test Site cases than in sporadic cases. Furthermore, 71 of 87 (81.6%) sporadic cases showed cyclin D1 immunoreactivity. Cylin D1 immunoreactivity was significantly higher in Semipalatinsk Nuclear Test Site cases than sporadic cases (Table 2). 4. Discussion We found that cytoplasmic h-catenin immunoreactivity was very common (23 of 23 cases) in papillary thyroid cancers from near the Semipalatinsk Nuclear Test Site. Both chronic thyroiditis and follicular adenomas showed significantly lower cytoplasmic hcatenin expression. On the other hand, normal thyroid follicular cells showed strong membrane h-catenin immunoreactivity with no cytoplasmic localization. These results suggest a significance of accumulated cytoplasmic h-catenin during thyroid carcinogenesis around the Semipalatinsk Nuclear Test Site. In the present study, a direct comparison with sporadic cases revealed a higher prevalence of cytoplasmic h-catenin expression in papillary carcinomas from the Semipalatinsk Nuclear Test Site. Some investigators reported aberrant h-catenin expression in classical papillary carcinomas [15,16]. A higher level of aberrant cytoplasmic h-catenin expression was observed in papillary cancers from the Semipalatinsk Nuclear Test Site than in sporadic tumors. We consider that alteration of the Wnt signaling pathway could lead to a higher level of h-catenin expression. Indeed, it has recently been reported that elements of the Wnt signaling pathway are expressed in thyroid cells, and that this pathway is functionally active [17]. However, our preliminary study using polymerase chain reaction –single-strand conformation polymorphism could not detect mutations in hcatenin gene exon 3 in our samples (data not shown) as well as in the previous reports on papillary thyroid cancers [12,18]. The role of h-catenin as an oncogene has already been established [12,19]. A few reports indicate the significance of immunoreactive cytoplasmic h-catenin in papillary thyroid cancers. An accumulated h-catenin by the activation of Wnt signaling induces a gene encoding cyclin D1, which plays a role in the initiation of cell transformation [10]. In our recent paper, we have demonstrated that dislocalization of h-catenin correlates with overexpression of cyclin D1 in sporadic papillary cancers [14]. We therefore clarified the correlation between cytoplasmic h-catenin expression and cyclin D1 overexpression in thyroid tissues, including not only papillary cancer but also benign tumor.
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In our series, cyclin D1 immunoreactivity was restricted to both papillary carcinomas and follicular adenomas. The level of cyclin D1 overexpression was significantly higher in papillary carcinomas than in follicular adenomas. Cyclin D1 overexpression has already been well documented in human thyroid cancers, and its frequency in conventional papillary carcinoma ranges between 32.0% and 82.4% [20]. We have reported cyclin D1 overexpression in 82% of sporadic cases [12]. A higher prevalence of cyclin D1 overexpression was observed in our series as well as cytoplasmic h-catenin expression. Additionally, the level of cyclin D1 overexpression was significantly correlated with cytoplasmic h-catenin immunoreactivity in thyroid tissues together with chronic thyroiditis, follicular adenoma and papillary carcinoma. Taken together, these findings show that cyclin D1 upregulation may be caused by aberrant h-catenin expression during malignant transformation of the thyroid gland. In conclusion, this study revealed a higher prevalence of both aberrant h-catenin expression and cyclin D1 overexpression in papillary thyroid cancers around the Semipalatinsk Nuclear Test Site as compared with previously reported papillary carcinomas. Accumulation of cytoplasmic h-catenin seems to be closely correlated with the level of cyclin D1 overexpression during thyroid carcinogenesis around the Semipalatinsk Nuclear Test Site. Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sports, Culture, Science and Technology (No. 14770101). References [1] M. Yamamoto, T. Tsukatani, Y. Katayama, Residual radioactivity in the soil of the Semipalatinsk nuclear test site in the former USSR, Health Phys. 71 (1996) 142 – 148. [2] S. Yamashita, Proposal for establishment of screening of thyroid diseases around Semipalatinsk, in: M. Hoshi, J. Takada, R. Kim, Y. Nitta (Eds.), Effects of low-level radiation for residents near Semipalatinsk nuclear test site. Proceedings of the Second Hiroshima International Symposium, Hiroshima, July 23 – 25, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, 1996, pp. 225 – 234. [3] Z. Zhumadilov, B.I. Gusev, J. Takada, M. Hoshi, A. Kimura, N. Hayakawa, N. Takeichi, Thyroid abnormality trend over time in northeastern regions of Kazakstan, adjacent to the Semipalatinsk nuclear test site: a case review of pathological findings for 7271 patients, J. Radiat. Res. 41 (2000) 35 – 44. [4] B.I. Gusev, R.I. Rosenson, Z.N. Abylkassimova, The Semipalatinsk nuclear test site: a first analysis of solid cancer incidence (selected sites) due to test-related radiation, Radiat. Environ. Biophys. 37 (1998) 209 – 214. [5] K.M. Cadigan, R. Nusse, Wnt signaling: a common theme in animal development, Genes Dev. 11 (1997) 3296 – 3305. [6] P. Polakis, Wnt signaling and cancer, Genes Dev. 14 (2000) 1837 – 1851. [7] B. Rubinfeld, P. Robbins, M. El-Gamil, I. Albert, E. Porfiri, P. Polakis, Stabilization of h-catenin by genetic defects in melanoma cell lines, Science 275 (1997) 1790 – 1792. [8] P.J. Morin, A.B. Sparks, V. Korinek, N. Barker, H. Clevers, B. Vogelstein, K.W. Kinzler, Activation of hcatenin-Tcf signaling in colon cancer by mutations in h-catenin or APC, Science 275 (1997) 1787 – 1790. [9] V. Korinek, N. Barker, P.J. Morin, D. van Wichen, R. de Weger, K.W. Kinzler, B. Vogelstein, H. Clevers, Constitutive transcriptional activation by a h-catenin-Tcf complex in APC / colon carcinoma, Science 275 (1997) 1784 – 1787. [10] O. Tetsu, F. McCormick, h-catenin regulates expression of cyclin D1 in colon carcinoma cells, Nature 398 (1998) 422 – 426.
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