BRD7调控Rb/E2F通路的分子机制研究

BRD7是采用cDNA代表性差异分析法克隆的一个新的Bromodomain基因,过表达BRD7可抑制鼻咽癌细胞的生长和细胞周期进程,同时发现BRD7基因可以调控Rb/E2F通路的活性.该研究旨在进一步探讨BRD7调控Rb/E2F通路的分子机制.通过蛋白质印迹和RT-PCR实验方法发现,BRD7能够降低Rb的磷酸化水平,抑制cyclinD1、cyclinE的蛋白质表达,上调CDK4抑制子P19的mRNA表达,但对CDK4和CDK2的蛋白质表达没有明显影响;通过荧光素酶实验从转录调控水平进一步证实了BRD7能够明显抑制cyclinD1启动子活性;采用反义核酸技术抑制COS7细胞内源性BRD7的表达后,发现cyclinD1、cyclinE、磷酸化Rb的蛋白质表达水平上调,并且可以促进细胞生长.这些结果表明:BRD7参与调控Rb/E2F信号通路中重要靶分子的表达,抑制Rb/E2F通路的活性,从而阻止细胞周期G1-S期进程,抑制鼻咽癌细胞生长.     

BRD7 suppresses the growth of Nasopharyngeal Carcinoma cells (HNE1) through negatively regulating β-catenin and ERK pathways

BRD7 is a novel gene which involved NPC in our lab. Our previous studies showed that BRD7 was expressed at high level in normal nasopharyngeal epithelial tissues, but at low level in nasopharyngeal carcinoma biopsies and cell lines. In these papers, we found that ectopic expression of BRD7 can decrease cell proliferation and capability to form colonies in soft agar. FCM (Flow cytometry) assay indicated that the cell cycle progression from G1 to S phase was inhibited and the expression of cyclinD1 was significantly decreased after being transfected with BRD7 in HNE1 cells (NPC cells). To further investigate the molecular mechanism of BRD7 suppression of NPC cells growth, the cDNA microarray was performed to detect difference in gene expression profile induced by BRD7. The results indicated that 21 genes expression were changed after being transfected with BRD7 and the differentially expressed gene including α-catenin, cyclinD1, E2F3 was confirmed by western-blot. Next, we found that even though no obvious changes of the total expression of β-catenin were observed, the accumulation of β-catenin in nucleus was blocked. In addition, it was found that the expression of β-catenin was up-regulated in the complex composed of β-catenin and α-catenin in HNE1 cells induction of BRD7. So, we concluded that over-expression of BRD7 increased the expression of α-catenin which “hold” β-catenin in the complex and inhibited its accumulating in nucleus. At last, we demonstrated the c-jun, p-MEK, and p-ERK1/2 expression were down-regulated, and the Ap-1 promoter activity was inactive after being transfected with BRD7. We also found that over-expression of BRD7 can inactivate the c-jun and p-ERK1/2 after being treated with EGF in HNE1 cells. These results indicated that BRD7 played a negative role in ERK1/2 pathway. Taken together, our present results provide new insights for BRD7 function to inhibit NPC cells growth through negative regulating β-catenin and ERK1/2 pathways.     

过表达E2F6基因抑制BRD7基因启动子活性

BRD7基因是采用cDNA代表性差异分析法克隆的一个新Bromodomain基因(GenBank 登录号AF152604)。它在鼻咽癌细胞和组织中表达明显下调,过表达BRD7基因可抑制鼻咽癌细胞的生长和细胞周期的进程。前期工作已克隆了BRD7基因启动子区,并将其启动子定位于450bp（-404→+46bp）的区域。为了进一步揭示BRD7基因在鼻咽癌细胞和组织中表达下调的分子机制,生物信息学分析表明BRD7启动子区有E2F6转录因子结合位点,电泳迁移率实验结果表明转录因子E2F6特异性地结合于BRD7启动子区。荧光素酶检测和绿色荧光蛋白表达检测都证实过表达E2F6基因能抑制BRD7基因启动子活性     

ERK1/2 MAP kinases promote cell cycle entry by rapid, kinase-independent disruption of retinoblastoma-lamin A complexes

As orchestrators of essential cellular processes like proliferation, ERK1/2 mitogen-activated protein kinase signals impact on cell cycle regulation. A-type lamins are major constituents of the nuclear matrix that also control the cell cycle machinery by largely unknown mechanisms. In this paper, we disclose a functional liaison between ERK1/2 and lamin A whereby cell cycle progression is regulated. We demonstrate that lamin A serves as a mutually exclusive dock for ERK1/2 and the retinoblastoma (Rb) protein. Our results reveal that, immediately after their postactivation entrance in the nucleus, ERK1/2 dislodge Rb from its interaction with lamin A, thereby facilitating its rapid phosphorylation and consequently promoting E2F activation and cell cycle entry. Interestingly, these effects are independent of ERK1/2 kinase activity. We also show that cellular transformation and tumor cell proliferation are dependent on the balance between lamin A and nuclear ERK1/2 levels, which determines Rb accessibility for phosphorylation/inactivation.     

候选抑瘤基因 BRD7 及家族蛋白的功能研究进展

溴区结构（bromodomain）是近年来发现的广泛分布于多种生物中的一种高度保守的结构域,溴区结构蛋白通过参与信号依赖性的基因转录调控而广泛参与细胞内重要的生命活动．BRD7基因是1999年克隆的一个新的bromodomain基因,GenBank登录号为AF152604或AF152605．eMotif分析表明,BRD7蛋白包含多个磷酸化位点和一个保守bromodomain功能域,Blast显示BRD7蛋白与人的Celtix-1及鼠的bromodomain蛋白BP75具有高度的同源性．利用转基因技术已证实,在鼻咽癌细胞系HNEl中过表达BRD7基因可以抑制其细胞生长和细胞周期G1-S的进程,并部分逆转鼻咽痛细胞HNE1的恶性表型．为了全面地揭示BRD7基因的细胞内生物学功能,深入了解BRD7基因的细胞内整体信息流向,中南大学肿瘤研究所细胞遗传室已从上、中、下游三个不同层面对BRD7基因的功能研究展开了初步的探索．     

候选抑瘤基因 BRD7 及家族蛋白的功能研究进展

溴区结构(bromodomain)是近年来发现的广泛分布于多种生物中的一种高度保守的结构域,溴区结构蛋白通过参与信号依赖性的基因转录调控而广泛参与细胞内重要的生命活动.BRD7基因是1999年克隆的一个新的bromodomain基因,GenBank登录号为AF152604或AF152605.eMotif分析表明,BRD7蛋白包含多个磷酸化位点和一个保守bromodomain功能域,Blast显示BRD7蛋白与人的Celtix-1及鼠的bromodomain蛋白BP75具有高度的同源性.利用转基因技术已证实,在鼻咽癌细胞系HNE1中过表达BRD7基因可以抑制其细胞生长和细胞周期G1-S的进程,并部分逆转鼻咽癌细胞HNE1的恶性表型.为了全面地揭示BRD7基因的细胞内生物学功能,深入了解BRD7基因的细胞内整体信息流向,中南大学肿瘤研究所细胞遗传室已从上、中、下游三个不同层面对BRD7基因的功能研究展开了初步的探索.     

Invited review: the circle of life: cell cycle regulation in airway smooth muscle.

Severe asthma is characterized by increased airway smooth muscle (ASM) mass, due predominantly to ASM hyperplasia. Diverse stimuli, which include growth factors, plasma- or inflammatory cell-derived mediators, contractile agonists, cytokines, and extracellular matrix proteins, induce ASM proliferation. Mitogens act via receptor tyrosine kinase, G protein-coupled receptors, or cytokine receptors, to activate p21ras and stimulate two parallel signaling pathways in ASM cells, namely, the extracellular signal-regulated kinase (ERK) or the phosphatidylinositol 3-kinase (PI3K) pathways. ERK and PI3K regulate cell cycle protein expression and thus modulate cell cycle traversal. ERK activation and downstream effectors of PI3K, such as Rac1 and Cdc42, stimulate expression of cyclin D1, a key regulator of G(1) progression in the mammalian cell cycle. In addition, PI3K activates 70-kDa ribosomal S6 kinase, an enzyme that also regulates the translation of many cell cycle proteins, including the elongation factor E2F. The present review examines the mitogens and critical signal transduction pathways that stimulate ASM cell proliferation. Further study in this area may reveal new therapeutic targets to abrogate ASM hyperplasia in diseases such as asthma and chronic obstructive pulmonary disease.     

An intrinsic cell cycle checkpoint pathway mediated by MEK and ERK in Drosophila

Cell cycle checkpoints are surveillance mechanisms that safeguard genome integrity. While the extrinsic pathways that halt the cell cycle in response to DNA damages have been well documented, the intrinsic pathways that ensure orderly progression of cell cycle events are not well understood. We demonstrate that Drosophila MEK and ERK constitute an essential intrinsic checkpoint pathway that restrains cell cycle progression in the absence of DNA damage and also responds to ionizing radiation to arrest the cell cycle. Embryos lacking MEK exhibit faster and extra division cycles and fail to undergo timely midblastula transition (MBT) or arrest following ionizing radiation. Conversely, constitutively activated MEK causes cell cycle arrest. Further, MEK activation in the early embryo is cell cycle-dependent and Raf independent and increases in response to ionizing radiation or in the absence of Chk1. Thus, MEK/ERK activation is required for multiple checkpoints and is essential for orderly cell cycle progression.