Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression

Pituitary tumor-transforming gene (PTTG) originally isolated from GH-secreting pituitary adenoma cells causes in vitro cell transformation, in vivo tumorigenesis, and induces basic fibroblast growth factor. These functions require an intact C-terminal proline-proline-serine-proline motif. PTTG1 is abundantly expressed in human pituitary tumors and plays a role in the early stages of experimental prolactinoma formation. We now determined direct effects of PTTG1 on hormonal phenotypes of functional pituitary tumor cells. Overexpression of PTTG1 C terminus (amino acids 147-202) containing intact proline-proline-serine-proline motifs in rat prolactin (PRL)- and GH-secreting GH3 cells markedly abrogates PRL mRNA expression by more than 90% (P < 0.001) and hormone levels (P < 0.001) and PRL promoter activity (P < 0.01) compared with control vector cells or to a PTTG1 C terminus mutant (P163A, S165Q, P166L, P170L, P172A, and P173L). Wild-type PTTG1 C-terminal transfectants formed smaller (P < 0.05) sc tumors in rats compared with control or mutated PTTG1 C-terminal transfectants. Estrogen (10 nm) treatment for 48 h partially restored PRL expression in stable wild-type PTTG1 C-terminal transfectants. These results indicate that targeting PTTG1-mediated signaling alters the hormonal phenotype in pituitary cells and disrupted PTTG1 action may be a potential subcellular therapeutic tool for repressing PRL hypersecretion.     

Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis.

Pituitary tumors are commonly encountered, and result from clonal expansion of a single mutated cell. Hypothalamic hormones, local growth factors and circulating sex steroid hormones promote pituitary tumor growth and expansion into large invasive tumors. Estrogen acting directly through its receptor and by stimulation of fibroblast growth factor regulates prolactin synthesis and secretion. Fibroblast growth factor-2 (bFGF) modulates angiogenesis, tumor formation and progression in many tissues, including the anterior pituitary. A pituitary tumor-derived transforming gene (PTTG) has been isolated, which is tumorigenic in vivo, regulates bFGF secretion, and inhibits chromatid separation. The human PTTG family consists of at least three homologous genes, of which PTTG1 is located on chromosome 5q33 and is expressed at low levels in most normal human tissues but is highly expressed in malignant human cell lines and in pituitary tumors. We report here that pituitary pttg is regulated in vivo and in vitro by estrogen. Maximal induction of rat pituitary pttg mRNA in vivo occurred early in pituitary transformation (normal cell to hypertrophic/hyperplastic cell), coincident with bFGF and vascular endothelial growth factor induction and pituitary angiogenesis. We also demonstrate that pttg expression is induced by bFGF, and show concordant pttg and bFGF expression in experimental and human pituitary adenomas. As bFGF and estrogen both induce pttg, and pttg expression coincides with the early lactotrophic hyperplastic response, angiogenesis and prolactinoma development, we propose a previously unknown paracrine growth factor-mediated mechanism for pituitary tumor pathogenesis and potentially other estrogen-regulated tumors.     

Mechanisms for growth factor-induced pituitary tumor transforming gene-1 expression in pituitary folliculostellate TtT/GF cells

PTTG1, a securin protein, also behaves as a transforming gene and is overexpressed in pituitary tumors. Because pituitary folliculostellate (FS) cells regulate pituitary tumor growth factors by paracrine mechanisms, epidermal growth factor (EGF) receptor (EGFR)-mediated PTTG1 expression and cell proliferation was tested in pituitary FS TtT/GF cells. EGFR ligands caused up to 3-fold induction of Pttg1 mRNA expression, enhanced proliferating cell nuclear antigen, and increased entry of G0/1-arrested cells into S-phase. PTTG binding factor mRNA expression was not altered. EGF-induced Pttg1 expression and cell proliferation was abolished by preincubation of TtT/GF cells with EGFR inhibitors AG1478 and gefitinib. Phosphatidylinositol 3 kinase, protein kinase C, and MAPK, but not c-Jun N-terminal kinase and Janus activating kinase signaling regulated EGF-induced Pttg1, as well as proliferating cell nuclear antigen mRNA expression and entry into S-phase. EGF-induced EGFR and ERK1/2 phosphorylation was followed by rapid MAPK kinase/ERK kinase-dependent activation of Elk-1 and c-Fos. EGF-induced Pttg1 expression peaked at the S-G2 transition and declined thereafter. Pttg1 cell cycle dependency was confirmed by suppression of EGF-induced Pttg1 mRNA by blockade of cells in early S-phase. The results show that PTTG1 and its binding protein PTTG binding factor are expressed in pituitary FS TtT/GF cells. EGFR ligands induce PTTG1 and regulate S-phase, mediated by phosphatidylinositol 3 kinase, protein kinase C, and MAPK pathways. PTTG1 is therefore a target for EGFR-mediated paracrine regulation of pituitary cell growth.     

Molecular aspects of pituitary tumors

Pituitary adenomas are common benign neoplasms, accounting for approximately 15% of intracranial tumors. In systematic autopsy, pituitary tumors are found in 25%, of the population, but only one-third of these tumors give rise to clinical manifestations. Why most of these neoplasms remain undiagnosed and pituitary carcinomas are extremely rare? The progress in the studies concerning pituitary tumorigenesis is rather slow and, due to several limitations, including the anatomic inaccessibility of human pituitary gland, the lack of functional human cell lines in culture and the discrepancies between human and animal pituitary oncogenesis (in rodents pituitary hyperplasia is a prerequisite for adenoma development). In humans, the majority of pituitary tumors are monoclonal in origin and derived from single mutated pituicyte, rarely hyperplasia is a prerequisite for adenoma formation. As in the case of other tumors, activating mutations in oncogenes (GNAS1, PTTG) and inactivating mutations in tumor suppressor genes (MEN1, CNC1) lead to pituitary tumors development. However, mutations in classic oncogenes are very rarely associated with these tumors. Moreover, the important role of some hypothalamic hormones, peripheral hormones and their receptors (e.g. GHRH, dopamine D2 receptor, PRL receptor, estrogens, thyroid hormone receptor) and growth factors (e.g. FGF, EGF, TGF) is postulated and partially proved in promotion of pituitary tumorigenesis. Further studies are required to determine which of these events are truly primary changes in pituitary tumorigenesis, what may allow development of gene therapy.     

垂体瘤转化基因1(PTTG1)的表达变化对人前列腺癌细胞LNCaP生长增殖的影响

垂体肿瘤转化基因1(PTTG1)具有促进肿瘤生长和转移的作用.通过上调或下调基因表达的策略,观察PTTG1基因对人前列腺癌细胞株LNCaP细胞生长增殖的影响.利用PCR技术分离出PTTG1全长cDNA,分别正向和反向插入真核表达载体pIRES2-EGFP,重组载体分别命名为正义PTTG1-S/pIRES2-EGFP(即pI-P-S)和反义PTTG1-AS/pIRES2-EGFP(即pI-P-AS),将这两种重组载体稳定转染LNCaP细胞,通过流式细胞仪和MTT法分别检测了细胞周期和细胞增殖的情况.转染正义PTTG1后处于S期和G2期的细胞明显增加,细胞生长增殖能力增强;相反,转染反义PTTG1后处于S期和G2期细胞明显减少,细胞生长增殖能力减弱(P<0.05).结果表明,PTTG1能明显改变人前列腺癌细胞株LNCaP的细胞周期和细胞生长增殖能力,它的异常表达可能参与前列腺癌细胞生长增殖过程.     

转录因子E2F-1与垂体肿瘤转化基因（PTTG）在大鼠PRL瘤中的表达

目的通过检测垂体肿瘤转化基因（PTTG）与腺病毒E2启动子结合因子1（E2F-1）在大鼠催乳素（PRL）瘤中的表达来探讨两者在PRL瘤发生发展过程中的作用。方法 40只大鼠随机分为两组：实验组（E组,n=20）：皮下植入17β-雌二醇的方法诱发大鼠PRL瘤;对照组（C组,n=20）：皮下植入空白硅胶管。雌激素诱导10周后处死大鼠,心脏穿刺取血,4%多聚甲醛体内灌流取出脑垂体,称重,ELISA方法检测两组大鼠血清PRL水平,垂体组织行病理组织学观察,免疫组化SP方法检测两组大鼠垂体组织中PTTG蛋白质、E2F-1蛋白质的表达。结果雌二醇作用10周后,据垂体重量、垂体组织学变化和血清PRL水平证实PRL瘤诱导成功。PRL瘤组中,PTTG蛋白质、E2F-1蛋白质均明显高于对照组,差异具有统计学意义（P〈0.01）;且PTTG蛋白质和E2F-1蛋白质的表达呈明显正相关（γ=0.764,P〈0.01）。结论 PTTG与E2F-1在大鼠PRL瘤中共同过度表达,参与了大鼠PRL瘤的发生发展。     

Cloning and expression of human cDNA encoding human homologue of pituitary tumor transforming gene.

Recently, a potent transforming gene which was exclusively expressed in rat pituitary tumor but not in normal pituitary had been isolated and named as pituitary tumor transforming gene (PTTG). A cDNA clone encoding human homologue of rat PTTG was isolated from human fetal liver cDNA library. It contained an open reading frame of 603 base pairs predicting a protein composed of 201 amino acids with a calculated molecular weight of 26 kDa. The deduced protein showed about 85% homology (78% identity, 7% favored substitution) with the rat PTTG. Northern blot analysis showed that the cDNA hybridized to 1.0 kb mRNA species which was expressed in fetal liver and several cancer cell lines. These results suggest that the presence of the human homologue of rat PTTG gene may not be restricted to pituitary tumor.     

垂体瘤转化基因1研究进展

垂雄瘤转化基因1（PTTG1）,也被称为分离酶抑制蛋白基因,是近几年从大鼠垂体肿瘤中发现的癌基因。它不但可以与分离酶结合,使分离酶失活,从而抑制姐妹染色单体的分离,还具有转录激活活性。已有的染色质免疫共沉淀结合芯片数据显示,PTTG1不仅可以直接调控基因的转录,也可以与其他蛋白,如PTTG1结合因子（PBF）、p53、Spl、上游刺激因子1（USF1）等相互作用来调控下游基因的转录。在NIH3T3细胞中,PTTG1激活c-Mvc的转录,增强NIH3T3细胞在裸鼠体内的成瘤能力。PTTG1也能激活肿瘤细胞中成纤维细胞生长因子2（FGF2）的转录,从而促进肿瘤血管生成。PTTG1结合p53、抑制p21表达、激活周期蛋白D3的能力,提示它在凋亡、细胞周期和衰老方面廿．发挥作用。另外,PTTG1在肿瘤转移和肝癌的发生发展中也发挥着重要作用。我们简要综述了PTTG1的靶基因,及其在肝癌及肿瘤转祷中的研究进展。     

A plurihormonal TSH-producing pituitary tumor of monoclonal origin in a patient with hypothyroidism

OBJECTIVE: A clinicopathological and clonal study of a pituitary tumor was made in a 26-year-old woman with chronic thyroiditis to differentiate TSH-producing adenoma from TSH hyperplasia. METHODS: Tumor specimens were subjected to histopathological study and clonal analysis (HUMARA). RESULTS: Immunohistochemical examination disclosed TSH-beta, PRL, GH, ACTH, FSH-beta, LH-beta, and alpha-subunit production in the adenoma cells. These heterogeneous phenotypes are characteristic of both thyrotroph hyperplasia and plurihormonal TSH-producing adenoma. However, the HUMARA method demonstrated monoclonality of the tumor cells. CONCLUSION: Monoclonality of the tumor cells proved that the pituitary tumor was plurihormonal TSH-producing adenoma, not TSH hyperplasia.     

垂体瘤转化基因(PTTG1)可抵抗UV诱导的细胞凋亡作用

垂体瘤转化基因（PTTG1）在很多肿瘤中呈高水平表达.越来越多的研究表明,PTTG1与细胞增殖、细胞转化有关.但PTTG1在凋亡中的作用仍不清楚.通过在细胞中下调和过表达PTTG1,观察PTTG1在UV照射诱导凋亡中的作用.结果发现, RNAi-介导下调HeLa细胞PTTG1表达可增加对UV诱导凋亡的敏感性,而过表达PTTG1则降低对UV诱导凋亡的敏感性.此外,UV照射能降低PTTG1蛋白的表达水平,并且表现为明显的剂量和时间关系.这些研究结果显示,PTTG1在UV照射诱导的凋亡中发挥重要的抗凋亡作用.这为研究PTTG1在肿瘤发生、发展中的作用机制提供了新的实验证据.     

The important anti-apoptotic role and its regulation mechanism of PTTG1 in UV-induced apoptosis

Pituitary tumor transforming gene (PTTG1) is widely detected in many tumors. Increasing evidence reveals that PTTG1 is associated with cell proliferation, cellular transformation and apoptosis. However, the functions of PTTG1, especially its role in DNA damage-induced apoptosis, remain largely unclear. In this report, we used UV irradiation to induce apoptosis in HeLa cells to examine the role of PTTG1 in UV-induced apoptosis by RNAi-mediated knockdown and overexpression of PTTG1. RNAi-mediated knockdown of PTTG1 expression increased and overexpression of PTTG1 decreased the UV-induced apoptosis. Furthermore, UV irradiation decreased PTTG1 mRNA and protein expression. These effects were found to be mediated by JNK pathway. Therefore, PTTG1 had an important anti-apoptotic role in UV-induced apoptosis and this role was mediated by JNK pathway. These results may provide important information for understanding the exact role and the regulation mechanism of PTTG1 in UV-induced apoptosis.     

雄激素通过雄激素受体途径调控垂体肿瘤转化基因(PTTG1)的表达

雄激素能上调大鼠前列腺中垂体肿瘤转化基因1(PTTG1)的表达,在前列腺癌标本中,发现PTTG1的表达明显高于正常组织.因此,用雄激素依赖的前列腺癌细胞LNCaP来研究雄激素调控PTTG1表达的分子机制.在LNCaP细胞中,通过雄激素刺激后,PTTG1的表达明显升高.根据序列分析以及5-缺失体实验(deletion)和突变实验(mutation),发现PTTG1的启动子上游-950到-933的序列上有1个雄激素受体反应元件(ARE),染色体免疫共沉淀实验(CHIP)也证实了雄激素能促进雄激素受体与PTTG1启动子上ARE结合,从而在转录水平调控PTTG1的表达.     

Papel de pituitary tumour-transforming gene (PTTG) en los adenomas hipofisarios

The pathogenesis of pituitary tumours is far to be understood. Pituitary transforming tumour gene (PTTG), a gen that induces aneuploidy, genetic instability, cellular proliferation and to stimulate angiogenesis, has been involved in neoplasic transformation and shown overexpressed in many neoplasm as lung, breast, endometrium, thyroid and colon malignant tumours. On the other hand, PTTG has been inconsistently studied in pituitary tumours. The majority of studies have been performed in animals and there is a great variability in the methods used in its determination. The goal of this review is to resume the role of PTTG in tumourogenesis and critically to revise the studies published in humans in order to advance in the knowledge of the pathogenesis of pituitary adenomas and to find clinical useful predictors of the behavior of these tumours.