Decreased proliferation and cell cycle arrest in neoplastic rat pituitary cells is associated with transforming growth factor-β1-induced expression of p15/INK4B
Decreased proliferation and cell cycle arrest in neoplastic rat pituitary cells is associated with transforming growth factor-β1-induced expression of p15/INK4B
复制标题
DOI：
10.1016/s0303-7207(01)00477-4
复制
发表时间：
2001-05-15
影响因子：
4.1
通讯作者：
Farrell, WE
中科院分区：
文献类型：
--
作者：
Frost, SJ;Simpson, DJ;Farrell, WE
转化生长因子（tgf - β）是调节包括脑垂体在内的多种组织的分化和增殖的细胞因子家族的一员。在正常垂体和肿瘤细胞系中，tgf - β 1均具有抗增殖活性，但其细胞内机制尚未明确。在垂体源性GH细胞系(3)中，G(1)/S转换的关键调节因子p27（Kip1）未表达，这表明该蛋白不是tgf - β 1治疗后抗增殖反应的效应因子。在其他tgf - β应答细胞周期调节因子中，p15（Ink4b）已被证明具有与其他细胞类型的细胞周期阻滞相关的抗增殖作用。因此，我们检测了p15（Ink4b）表达对TGF β -1的响应，以确定这种细胞周期蛋白依赖性激酶抑制剂是否对GH(3)细胞的抗增殖活性负责。tgf - β 1 （0.5-30 ng/ml）处理GH(3)细胞显示出明显的剂量依赖性生长抑制（P < 0.001）。在2 ng/ml tgf - β 1治疗后，观察到最大的生长抑制（66%）。与生长研究同时进行的FAGS分析显示，与对照组相比，治疗与s期细胞比例下降（22-9%）有关，G(1)部分从58%显著增加到75% （P < 0.001）。亚G(1)部分的缺失和G(1)停滞在三个周期内的可逆性表明，这些变化分别不是由于凋亡反应或细胞毒性。半定量RT-PCR和Western blot分析显示，细胞周期蛋白依赖性激酶4 （CDK4）、p16（Ink4a）和p21（Cip1）的表达水平没有变化。然而，p15(Ink4b) mRNA和蛋白水平分别显示10酸8倍诱导。p15（Ink4b）水平的升高伴随着pRb的磷酸化状态向活性低磷酸化形式的转变。此外，对p15（Ink4b）诱导动力学的研究表明，在G(1)中细胞的阻滞之前，p15(Ink4b) mRNA和蛋白的诱导。这些研究表明p15（Ink4b）在GH(3)细胞中是tgf - β 1介导的细胞周期阻滞的功能效应者。然而，我们目前的研究不能确定它是否是唯一的中介。识别介导抗增殖信号反应的细胞内靶标将增加我们对这些通路和在肿瘤发生过程中导致其功能障碍的畸变的理解。(C) 2001爱思唯尔科学爱尔兰有限公司版权所有。
Transforming growth factor beta (TGF-beta) is a member of a family of cytokines that regulate differentiation and proliferation in a wide variety of tissues including the pituitary gland. In both the normal pituitary and tumorous cell lines TGF-beta1 has anti-proliferative activity, however the intracellular mechanisms responsible have not been defined. In the pituitary derived cell line GH(3), p27(Kip1), a key regulator of G(1)/S transition is not expressed, suggesting that this protein is not an effector of the anti-proliferative response following TGF-beta1 treatment. Among other TGF-beta responsive cell cycle regulators p15(Ink4b) has been shown to have anti-proliferative effects associated with cell cycle arrest in other cell types. We therefore examined p15(Ink4b) expression in response to TGF beta -1 to determine if this cyclin dependent kinase inhibitor was responsible For anti-proliferative activity in GH(3) cells. Treatment of GH(3) cells with TGF-beta1 (0.5-30 ng/ml) showed significant dose dependent growth inhibition (P < 0.001) as assessed by viable cell counts. Maximum growth inhibition (66%) was observed following treatment with 2 ng/ml TGF-beta1. FAGS analysis carried out in parallel with the growth studies showed treatment was associated with a decrease in the proportion of cells in S-phase (22-9%) and a significant increase in the G(1) fraction from 58 to 75% relative to controls (P < 0.001), The absence of a sub G(1) fraction and reversibility of the G(1) arrest over three cycles showed that these changes were not due to either an apoptotic response or cytoxicity, respectively. Semi-quantitative RT-PCR and Western blot analysis showed no change in the expression level of cyclin dependent kinase 4 (CDK4), p16(Ink4a) or p21(Cip1). However, p15(Ink4b) mRNA and protein levels showed a 10 acid 8 -fold induction, respectively. Increased levels of p15(Ink4b) were accompanied by a shift in the phosphorylation status of pRb toward its active hypophosphorylated form. Furthermore, studies of the kinetics of p15(Ink4b) induction showed that arrest of cells in G(1) is preceded by induction of p15(Ink4b) mRNA and protein. These investigations would suggest that p15(Ink4b) is a functional effector of TGF-beta1 mediated cell cycle arrest in GH(3) cells. However, our present studies cannot determine if it is the sole mediator. Identification of intracellular target(s) that mediate responses to anti-proliferative signals will increase our understanding of these pathways and aberrations responsible for their dysfunction in tumorigenesis. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.