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1、33埼玉医科大学雑誌 第 31 巻 第 1 号 平成 16 年 1 月原 著Survivin甲状腺濾胞上皮細胞増殖分化対影響鈴木 美穂Role of Survivin on the Proliferation and Differentiation of Thyroid Epithelial Cells Miho Suzuki （Department of Endocrinology and Diabetes, Institute of Internal Medicine, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-049
2、5, Japan）Survivin is one of the IAP (inhibitor of apoptosis proteins), which is expressed during embryonic development, but is not expressed in terminal differentiated adult tissue. Overexpression of survivin is observed in a variety of human tumors including thyroid cancers. We examined the role of
3、 survivin on the proliferation and differentiation of thyroid epithelial cells. Rat thyroid epithelial cell line FRTL-5 was stably transfected with human sense and antisense survivin genes. FRTL-5 cells transfected with a survivin sense gene (S cells) proliferated without TSH. In the presence of TSH
4、, the proliferation of S cells was significantly faster than that of cells transfected with an empty vector (E cells) or cells transfected with a survivin antisense gene (AS cells). The proliferation of AS cells were retarded as compared with that of E cells. Western blot analysis demonstrated that
5、phosphorylation of Rb protein at serine 795 was enhanced in S cells. Activation of E2F-1 was also enhanced in S cells as demonstrated by gel shift assay. Northern blot analysis showed that expression of Pax-8 mRNA was significantly enhanced in S cells, and was significantly decreased in AS cells. Ex
6、pression of thyroglobulin (Tg) and TSH receptor (TSHR) mRNA was decreased in AS cells. There was no significant change in TTF-1 and TTF-2 mRNA expression. These observations suggest that survivin enhances the proliferation of thyroid epithelial cells through activation of E2F-1, and also enhance the
7、 expression of Pax-8, which then affect the expression of thyroid specific proteins. Survivin appears to play an important role in the proliferation as well as differentiation of thyroid epithelial cells. Keywords: Survivin, thyroid cancer, cell cycle, differentiation, Rb protein, E2F-1, Pax-8, Tg,
8、NIS, TSHR J Saitama Med School 2004;31:33-39 (Received November 28, 2003)緒 言Survivin遺伝子，胎生期細胞様癌細胞 増殖能持細胞発現確認1) ，Survivin遺伝子，細胞増殖促進因子働 抑制IAP（Inhibitor of apoptosis protein） family一員知2-5)， BIR （Baculoviral IAP repeat） 一存在6-8)Survivin 発現細胞周期依存，G2/M期紡錘糸 発現多認9-11)survivn癌細胞12-16) 成長期細胞特異的発現， survivin細胞増殖
9、分化何関与 考13)甲状腺細胞，分化過程様特異的遺伝 子蛋白質発現認Pax-8, thyroid transcription factor-1 （TTF-1） , thyroid transcription factor-2 （TTF-2） 転写因子， thyroglobulin （Tg） , thyroid peroxidase （TPO） , TSH （TSHR） , Na-I symporter （NIS）甲状腺特異的蛋白質 遺伝子領域結合， 転写促進遺伝子調節蛋白質知 分化能低下甲状腺腫瘍， 分化発現低下報告 17)我甲状腺癌細胞及正常甲状腺濾胞上皮 細胞株 （FRTL-5） 用，su
10、rvivin遺伝子細胞増 殖能及分化能検討材料及方法細胞培養甲 状 腺 癌 細 胞 株 （8505C, WRO, FRO） ，Hams F12K（SIGMA, Missouri, USA）10胎児血清埼玉医科大学内科学教室 （内分泌糖尿病内科部門） 平成 15 年 11 月 28 日 受付34鈴 木 美 穂鈴 木 美 穂Survivin 甲状腺濾胞上皮細胞増殖分化対影響（JRH BIOSCIENCES, Kansas, USA）加培養液 ，37，5 CO2下培養甲状腺濾 胞上皮細胞株 （FRTL-5）培養，1 mU/ml TSH （SIGMA） , 1g/ml （SIGMA） 加培養 液使用
11、甲状腺癌細胞株，胎児血清除去 24時間後，96 well culture dish（IWAKI, 東京, Japan） 100,000個/ well，培養後 法生細胞数計算同様，FRTL-5 ，TSH存在，非存在培養液使用 法生細胞数計算TransfectionSurvivin gene全長BLAST検索，XbaI BamHI制限酵素配列持以下primer設 定 （FW） 5-CTCTAGACCGCCAGATTTGAA- TCGCGG-3， （RV） 5-CGGATCCTGGTGCAGCCAC- TCTGGGAC-3， polymerase chaine reaction （PCR） 法甲状腺
12、癌細胞survivin遺伝子増幅 回 収 後，pcDNA 3.1（Invitrogen, California, USA）， Survivin gene （open reading frame）発現survivin sense vector及逆向 発現antisense vector作製 vector及empty vector，8505CFRTL-5 TransFast20 （Promega, West Indiana, USA）使用 transfection 行 210610 cm culture dish（IWAKI） 細 胞 培 養 ，10gsense-, antisense-, emp
13、ty-vectortransfection，48時 間 後400g/mlG418 sulfate（Promega） 添加， transfection細胞選択的培養Western blotsurvivintransfection細胞，survivin, 酸化 Rb蛋白発現量Western blot法13 - 14)用検討80達細胞質 抽出，DC試薬（BIO RAD, California, USA）使用蛋白質量測定 50g質SDS 泳動後，第一抗体抗survivin抗体 （R152:43-9.2) Kasof GM, Gomes BC. Livin, a novel inhibitor of a
14、poptosis protein family member. J Biol Chem 2000;276:3238-46.3) Gotz R. Regulation of neuronal cell death and differentiation by NGF and IAP family members. J Neural Transm Suppl 2000;60:247-59. 4) Ogasawara T, Hatano M, Otaki M, Sekita N, Kobayashi K, Miyazaki M, et al. A novel homologue of the TIAP/m-survivin gene. Biochem Biophys Res Commun 2001;282:207-11.5) LaCasse EC, Baird S, Korneluk RG, MacKenzie AE. The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 1998;17:3247-59. 6) Yang E, Korsmeyer SJ. Molecular thanatopsis: a discou
