STAT3 regulated ARF expression suppresses prostate cancer metastasis

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.

Id1 regulation of cellular senescence through transcriptional repression of p16/Ink4a

The Id family of helix–loop–helix (HLH) transcriptional regulatory proteins does not possess a basic DNA-binding domain and functions as a negative regulator of basic HLH transcription factors. Id proteins coordinate cell growth and differentiation pathways within mammalian cells and have been shown to regulate G1-S cell-cycle transitions. Although much recent data has implicated Id1 in playing a critical role in modulating cellular senescence, no direct genetic evidence has been reported to substantiate such work. Here we show that Id1-null primary mouse embryo fibroblasts undergo premature senescence despite normal growth profiles at early passage. These cells possess increased expression of the tumor-suppressor protein p16/Ink4a but not p19/ARF, and have decreased cyclin-dependent kinase (cdk) 2 and cdk4 kinase activity. We also show that Id1 is able to directly inhibit p16/Ink4a but not p19/ARF promoter activity via its HLH domain, and that Id1inhibits transcriptional activation at E-boxes within the p16/Ink4a promoter. Our data provide, to our knowledge, the first genetic evidence for a role for Id1 as an inhibitor of cellular senescence and suggest that Id1 functions to delay cellular senescence through repression of p16/Ink4a. Because epigenetic and genetic abrogation of p16/Ink4a function has been implicated in the evolution of several human malignancies, we propose that transcriptional regulation of p16/Ink4a may also provide a mechanism for the dysregulation of normal cellular growth controls during the evolution of human malignancies.

Spontaneous and UV radiation-induced multiple metastatic melanomas in Cdk4(R24C/R24C)/TPras mice

Human melanoma susceptibility is often characterized by germ-line inactivating CDKN2A (INK4A/ARF) mutations, or mutations that activate CDK4 by preventing its binding to and inhibition by INK4A. We have previously shown that a single neonatal UV radiation (UVR) dose delivered to mice that carry melanocyte-specific activation of Hras (TPras) increases melanoma penetrance from 0% to 57%. Here, we report that activated Cdk4 cooperates with activated Hras to enhance susceptibility to melanoma in mice. Whereas UVR treatment failed to induce melanomas in Cdk4(R24C/R24C) mice, it greatly increased the penetrance and decreased the age of onset of melanoma development in Cdk4(R24C/R24C)/TPras animals compared with TPras alone. This increased penetrance was dependent on the threshold of Cdk4 activation as Cdk4(R24C/+)/TPras animals did not show an increase in UVR-induced melanoma penetrance compared with TPras alone. In addition, Cdk4(R24C/R24C)/TPras mice invariably developed multiple lesions, which occurred rarely in TPras mice. These results indicate that germ-line defects abrogating the pRb pathway may enhance UVR-induced melanoma. TPras and Cdk4(R24C/R24C)/TPras tumors were comparable histopathologically but the latter were larger and more aggressive and cultured cells derived from such melanomas were also larger and had higher levels of nuclear atypia. Moreover, the melanomas in Cdk4(R24C/R24C)/TPras mice, but not in TPras mice, readily metastasized to regional lymph nodes. Thus, it seems that in the mouse, Hras activation initiates UVR-induced melanoma development whereas the cell cycle defect introduced by mutant Cdk4 contributes to tumor progression, producing more aggressive, metastatic tumors.

Differential roles of the pRb and Arf/p53 pathways in murine naevus and melanoma genesis

We report on a systematic analysis of genotype-specific melanocyte (MC) UVR responses in transgenic mouse melanoma models along with tumour penetrance and comparative histopathology. pRb or p53 pathway mutations cooperated with NrasQ61K to transform MCs. We previously reported that MCs migrate from the follicular outer root sheath into the epidermis after neonatal UVR. Here, we found that Arf or p53 loss markedly diminished this response. Despite this, mice carrying these mutations developed melanoma with very early age of onset after neonatal UVR. Cdk4R24C did not affect the MC migration. Instead, independent of UVR exposure, interfollicular dermal MCs were more prevalent in Cdk4R24C mice. Subsequently, in adulthood, these mutants developed dermal MC proliferations reminiscent of superficial congenital naevi. Two types of melanoma were observed in this model. The location and growth pattern of the first was consistent with derivation from the naevi, while the second appeared to be of deep dermal origin. In animals carrying the Arf or p53 defects, no naevi were detected, with all tumours ostensibly skipping the benign precursor stage in progression.

Aza-induced cardiomyocyte differentiation of P19 EC-cells by epigenetic co-regulation and ERK signaling

Stem cells in cell based therapy for cardiac injury is being potentially considered. However, genetic regulatory networks involved in cardiac differentiation are not clearly understood. Among stem cell differentiation models, mouse P19 embryonic carcinoma (EC) cells, are employed for studying (epi)genetic regulation of cardiomyocyte differentiation. Here, we comprehensively assessed cardiogenic differentiation potential of 5-azacytidine (Aza) on P19 EC-cells, associated gene expression profiles and the changes in DNA methylation, histone acetylation and activated-ERK signaling status during differentiation. Initial exposure of Aza to cultured EC-cells leads to an efficient (55%) differentiation to cardiomyocyte-rich embryoid bodies with a threefold (16.8%) increase in the cTnI(+) cardiomyocytes. Expression levels of cardiac-specific gene markers i.e., Isl-1, BMP-2, GATA-4, and alpha-MHC were up-regulated following Aza induction, accompanied by differential changes in their methylation status particularly that of BMP-2 and alpha-MHC. Additionally, increases in the levels of acetylated-H3 and pERK were observed during Aza-induced cardiac differentiation. These studies demonstrate that Aza is a potent cardiac inducer when treated during the initial phase of differentiation of mouse P19 EC-cells and its effect is brought about epigenetically and co-ordinatedly by hypo-methylation and histone acetylation-mediated hyper-expression of cardiogenesis-associated genes and involving activation of ERK signaling.

Structural study of CUG-repeating small RNAs complexed with silencing suppressor P19

The study is focused on structural aspects of interaction between silencing suppressor p19 and CUG-repeating small RNAs. The work involves crystal structure determination of a protein-unbound RNA form and RNA fragments of various lengths (19, 20, 21 nucleotides) complexed with p19-suppressor. Results prove the ability of silencing suppressor p19 to bind CUG-repeating small RNAs, as well as reveal features of U•U mismatches flanked by Watson-Crick C•G base pairs in p19-bound and p19-unbound states. In addition, structural data reveal a p19 specific site for anchoring extra nucleotides in small RNAs. In general, the study extends our knowledge about the mechanism of small RNA recognition by silencing suppressor p19.

Impact of polarized illumination on high NA imaging in ArF immersion lithography at 45 nm node

Immersion lithography has been considered as the mainstream technology to extend the feasibility of optical lithography to further technology nodes. Using proper polarized illumination in an immersion lithographic tool is a powerful means to enhance the image quality and process capability for high numerical aperture (NA) imaging. In this paper, the impact of polarized illumination on high NA imaging in ArF immersion lithography for 45 nm dense lines and semi-dense lines is studied by PROLITH simulation. The normalized image log slope (NILS) and exposure defocus (ED) window are simulated under various polarized illumination modes, and the impact of polarized illumination on image quality and process latitude is analyzed. (C) 2007 Elsevier GmbH. All rights reserved.

水稻小G蛋白Arf GTPase激活蛋白OsAGAP功能研究

　　锌指蛋白在植物生长发育中具有重要功能，它们可以识别并结合特定的DNA序列进行转录调控，还能够参与蛋白之间相互作用的调节。我们根据锌指蛋白等转录因子特征结构域的序列特点，从来自10 K水稻芯片的EST数据库中筛选出编码58个EST序列。通过对器官表达特异性的比较分析，从中选出七个只在单一器官表达的基因，并对这七个基因的功能进行研究。对其转基因水稻的表型分析发现，C1基因调节水稻的株高和穗的发育;LIM 家族的F9影响小花的形态，主要体现在雌蕊与雄蕊的发育;锌指蛋白S34调控叶倾角的变化;F14基因编码一个核定位的TFIIIA类锌指蛋白，具体功能尚不清楚;锌指蛋白F35转基因水稻主根缩短，侧根数目显著减少。它编码一个推测的ArfGAP (Arf GTPase activating protein)，据此我们将其命名为OsAGAP，并对其进行深入研究。 　　OsAGAP的cDNA全长为1328bp，编码的蛋白由320个氨基酸组成，含有两个保守结构域：锌指结构域和C2 结构域。其中锌指结构域属于CX2CX16CX2C类，即ArfGAP domain的特征结构。GTP酶活性测定试验表明，OsAGAP蛋白能够激活水稻Arf的GTP酶活性，另外，OsAGAP还能够恢复酵母ArfGAP缺失突变体的表型。说明OsAGAP编码的蛋白是水稻中的一个ArfGAP。 　　OsAGAP在水稻各器官中均有表达，但强弱有所不同。RNA原位杂交结果显示，它在茎尖分生组织与侧生原基及侧根部位表达强烈;它在根尖主要分布于中央维管组织、分生区、皮层细胞，最有趣的是恰好与生长素在根尖极性运输路径相吻合。在亚细胞水平，OsAGAP广泛分布于细胞膜、细胞质、细胞核。 　　OsAGAP超表达水稻主根、不定根长度缩短，侧根数目显著减少表现出类似于生长素极性运输突变体的表型。其主根伸长对TIBA的抑制作用不敏感，这暗示OsAGAP超表达水稻的生长素极性运输被破坏;另外，其对各种生长素的作用敏感性也发生变化，对IAA、2,4-D的不敏感，而对NAA的反应与野生型一致，根据各类生长素进出细胞机制不同，可以推测超表达水稻的输入能力存在缺陷。极性运输实验结果表明，超表达水稻极性运输能力被破坏;对生长素输入能力的测定进一步表明，超表达水稻根载体的介导的生长素输入能力显著下降。另外，NAA处理能够恢复超表达水稻中侧根发育受抑的表型缺陷。由此可见，OsAGAP在水稻中超表达破坏了生长素极性运输的输入能力。 　　FM1-43是一类特异标记囊泡运输的荧光染料。经其染色标记后，OsAGAP超表达水稻细胞内囊泡成片聚集，形成“BFA区间”，表现出囊泡运输被破坏的典型特征。透射电镜观察发现，超表达水稻细胞内有大量的小液泡，其中积累了电子密度很高的颗粒物质。由此推测，可能由于细胞的囊泡运输被破坏，导致胞内的代谢物质不能被正常运送或分泌，而在液泡中暂时贮存以维持细胞环境的稳定。 　　在酵母和动物细胞中的研究表明， ArfGAP是调控囊泡运输的一个重要因子，然而目前还没有关于ArfGAP在植物细胞中生理作用的报道。我们的结果说明，OsAGAP作为的一个ArfGAP，它通过调控水稻中的囊泡运输，而影响了生长素的极性运输，具体表现在对生长素输入能力的调控。由此，我们推测ArfGAP可能在生长素的极性运输中也起着重要的调控作用。 　　但OsAGAP在拟南芥中却通过调控植株生长素的水平，而影响了转基因拟南芥根的发育。每种生物都有多个ArfGAP，它们之间的分工存在联系，但各不相同。OsAGAP是拟南芥的外源基因，它在拟南芥中可能以不同于水稻的机制起作用。

D-UHV/CVD 外延生长SiGe和ArF准分子激光退火制备量子点的研究

Submitted by zhangdi (zhangdi@red.semi.ac.cn) on 2009-04-13T11:45:31Z

Smaller Ge Quantum Dots Obtained by ArF Excimer Laser Annealing

Ge self-assembled quantum dots (SAQDs) are grown with a self-assembled UHV/CVD epitaxy system. Then,the as-grown Ge quantum dots are annealed by ArF excimer laser. In the ultra-shot laser pulse duration,～20ns, bulk diffusion is forbidden, and only surface diffusion occurs, resulting in a laser induced quantum dot (LIQD). The diameter of the LIQD is 20～25nm which is much smaller than the as-grown dot and the LIQD has a higher density of about 6 × 10~(10)cm~(-2). The surface morphology evolution is investigated by AFM.

P14~(ARF)相关信号通路；P14~(ARF) Signaling Pathway

p14ARF是新近发现的一种具有细胞周期调节功能的抑癌基因,P14ARF主要定位于核仁,但也有少部分位于核质。p14ARF在部分人类肿瘤中频发失活,其表达异常导致肿瘤的发生机制及以P14ARF为靶点进行的肿瘤治疗越来越受到重视。现对于Pl4ARF相关信号通路的研究进展进行了综述。