Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21

Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c(-/-), mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence.

Coordinate Hyperactivation of Notch1 and Ras/MAPK Pathways Correlates with Poor Patient Survival: Novel Therapeutic Strategy for Aggressive Breast Cancers

Aberrant activation of Notch and Ras pathways has been detected in breast cancers. A synergy between these two pathways has also been shown in breast cell transformation in culture. Yet, the clinical relevance of Notch-Ras cooperation in breast cancer progression remains unexplored. In this study, we show that coordinate hyperactivation of Notch1 and Ras/MAPK pathways in breast cancer patient specimens, as assessed by IHC for cleaved Notch1 and pErk1/2, respectively, correlated with early relapse to vital organs and poor overall survival. Interestingly, majority of such Notch1 (high)Erk(high) cases encompassed the highly aggressive triple-negative breast cancers (TNBC), and were enriched in stem cell markers. We further show that combinatorial inhibition of Notch1 and Ras/MAPK pathways, using a novel mAb against Notch1 and a MEK inhibitor, respectively, led to a significant reduction in proliferation and survival of breast cancer cells compared with individual inhibition. Combined inhibition also abrogated sphere-forming potential, and depleted the putative cancer stem-like cell subpopulation. Most importantly, combinatorial inhibition of Notch1 and Ras/MAPK pathways completely blocked tumor growth in a panel of breast cancer xenografts, including the TNBCs. Thus, our study identifies coordinate hyperactivation of Notch1 and Ras/MAPK pathways as novel biomarkers for poor breast cancer outcome. Furthermore, based on our preclinical data, we propose combinatorial targeting of these two pathways as a treatment strategy for highly aggressive breast cancers, particularly the TNBCs that currently lack any targeted therapeutic module. (C) 2014 AACR.

Ras Behari’s guide to buying fish seed

Document contains 4 pages.

Regulación de las GTPasas de la familia Ras y la viabilidad de neuronas dopaminérgicas en respuesta a la señalización purinérgica

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Estudo de polimorfismos nos genes TP53 e p21(WAF1) e do perfil imunohistoquímico das proteínas p53, p21(WAF1), p16(INK4a) e ciclina D1 pela técnica de Tissue Microarray (TMA) e sua importância para o desenvolvimento e/ou severidade das neoplasias cervicais

O câncer de colo do útero é o terceiro tipo de câncer mais frequente em mulheres no mundo, e a infecção persistente pelo papilomavirus humano (HPV) oncogênico é condição necessária, mas não suficiente para seu desenvolvimento. As oncoproteínas virais E6 e E7 interferem direta ou indiretamente na ação de várias proteínas celulares. Entretanto, as variantes proteicas, resultantes de polimorfismos genéticos, podem apresentar comportamento distinto mediante a infecção pelo HPV. O objetivo deste estudo foi avaliar possíveis associações entre polimorfismos nos genes TP53 (p53 PIN3, p53 72C>G) e p21 (p21 31C>A) e o desenvolvimento de neoplasias cervicais, considerando os níveis de expressão das proteínas p53, p21, p16 e ciclina D1, e fatores de risco clássicos para o câncer cervical. Foram selecionadas 466 mulheres residentes no Rio de Janeiro, 281 com diagnóstico histopatológico de neoplasia cervical de baixo (LSIL) e alto grau (HSIL) e câncer (grupo de casos) e 185 sem história atual ou pregressa de alteração citológica do colo uterino (grupo controle). A técnica de PCR-RFLP (reação em cadeia da polimerase - polimorfismo de comprimento de fragmento de restrição), foi empregada na análise dos polimorfismos p53 72C>G e p21 31C>A, usando as enzimas de restrição BstUI e BsmaI, respectivamente. A avaliação do polimorfismo p53 PIN3 (duplicação de 16 pb) foi feita por meio da análise eletroforética direta dos produtos de PCR. A expressão das proteínas p53, p21, p16, ciclina D1 e Ki-67 e a pesquisa de anticorpos anti-HPV 16 e HPV pool foram avaliadas por imunohistoquímica (Tissue Microarray - TMA) em 196 biópsias do grupo de casos. O grupo controle se mostrou em equilíbrio de Hardy-Weinberg em relação aos três polimorfismos avaliados. As distribuições genotípicas e alélicas relativas a p53 PIN3 e p53 72C>G nos grupos controles e de casos não apresentaram diferenças significativas, embora o genótipo p53 72CC tenha aumentado o risco atribuído ao uso de contraceptivos das pacientes apresentarem lesões mais severas (OR=4,33; IC 95%=1,19-15,83). O genótipo p21 31CA(Ser/Arg) conferiu proteção ao desenvolvimento de HSIL ou câncer (OR=0,61, IC 95%=0,39-0,97), e modificou o efeito de fatores de risco associados à severidade das lesões. A interação multiplicativa de alelos mostrou que a combinação p53 PIN3A1, p53 72C(Pro) e p21 31C(Ser), representou risco (OR=1,67, IC95%=1,03-2,72) e a combinação p53 PIN3A1, p53 72C(Pro) e p21 31A(Arg) conferiu efeito protetor (OR=0,26, IC95%=0,08-0,78) para o desenvolvimento de HSIL e câncer cervical. Observou-se correlação positiva da expressão de p16 e p21 e negativa da ciclina D1 com o grau da lesão. A distribuição epitelial de p16, Ki-67, p21 e p53 se mostrou associada à severidade da lesão. Os polimorfismos analisados não apresentaram associação com a expressão dos biomarcadores ou positividade para HPV. Nossos resultados sugerem a importância do polimorfismo p21 31C>A para o desenvolvimento das neoplasias cervicais e ausência de correlação dos polimorfismos p53 PIN3 e p53 72C>G com a carcinogênese cervical, embora alguns genótipos tenham se comportado como modificadores de risco. Nossos resultados de TMA corroboram o potencial de uso de biomarcadores do ciclo celular para diferenciar as lesões precursoras do câncer cervical.

Homologs of eukaryotic Ras superfamily proteins in prokaryotes and their novel phylogenetic correlation with their eukaryotic analogs

Ras superfamily proteins are key regulators in a wide variety of cellular processes. Previously, they were considered to be specific to eukaryotes, and MglA, a group of obviously different prokaryotic proteins, were recognized as their only prokaryotic an

Ras 超家族蛋白的起源演化研究

Ras 超家族蛋白是真核生物中普遍存在的一类小分子GTP 结合蛋白。它们 具有高度保守的GTP 结合结构域，根据序列结构和细胞功能被分为七个家族： Sar1、Arf、SRβ、Ran、Rab、Rho 和Ras。这些蛋白分别行使着真核生物特有的 细胞功能，诸如运输小泡的形成和转运（Sar1、Arf、Rab），胞质骨架的建成（Rho）， 细胞核-胞质运输及核膜重建（Ran）等，其起源演化和真核细胞的起源密切相关。 本文利用生物信息学手段和分子生物学实验调查研究了原核生物和原生生物中 Ras 超家族蛋白同源物的存在情况，并进行了分子系统分析，对Ras 超家族蛋白 的起源演化问题进行了较为深入、系统的探讨。获得了以下结果和结论： 1）通过原核生物基因组的搜索和序列结构分析，在一些真细菌中首次鉴定 出了高度相似于真核生物Ras 超家族蛋白的原核生物同源物，且实验证明它们的 基因具有表达活性；在原细菌中的产甲烷菌和热原体中也发现有序列分歧较大的 同源物。并在更多的真细菌种类中鉴定出了更多的前人已报道的另一种小分子 GTP 结合蛋白—MglA。序列比对分析表明MglA 蛋白具有自己独特的序列特征， 与真核生物的Ras 超家族蛋白序列差异较大。进一步的分子系统分析显示：真核 生物Ras 超家族蛋白的七个家族中，Ran、Rab、Rho 和Ras 等四个家族聚在一 起，上述我们所鉴定的真细菌的Ras 超家族蛋白同源物则紧聚在其外围；真核生 物的另三个家族（Sar1、Arf、SRβ）聚成另一枝，并接着与产甲烷原细菌的的同 源物及真细菌的MglA 蛋白聚在一起。这些结果表明：Ras 超家族蛋白不是前人 所认为的为真核生物所特有，实际上在一些原核生物中就已产生；真核生物Ras 超家族蛋白的祖先也不太可能是前人所认为的为真细菌的MglA；真核生物Ras 超家族蛋白的七个家族可能有两种不同的起源：Ran、Rab、Rho 和Ras 等可能 来源于蓝细菌或蛋白菌，或二者的共同祖先，而Sar1、Arf 和SRβ 可能来源于产 甲烷原细菌，这也可能反映了真核细胞“融合起源”的历史。 2）通过搜索一些较为低等的单细胞真核生物——原生生物基因组中Ras 超 家族蛋白，并结合一系列其他处在不同进化地位真核生物的Ras 超家族蛋白进行 分析，发现Sar1、Arf、Rab 和Ran 家族的蛋白在真核生物中普遍存在，而SRβ、 Rho 和Ras 家族蛋白在有些真核生物中未找到。根据各家族蛋白在真核生物中的分布情况推测在真核生物的最近共同祖先中存在的Ras 超家族蛋白可能有下列 两种情况：（1）最近的共同祖先已经具有了所有七个家族的蛋白，并且至少有 11 个成员：1 个Sar1、1 个SRβ、3 个Arf（Arf1、Arl1、Arl2）、3 个Rab（Rab1、 Rab6、Rab11）、1 个Ran、1 个Rho（Rac）和1 个Ras（RheB）。因而，部分真 核生物中缺少SRβ、Rho 和Ras 家族蛋白很可能是因基因丢失所致。植物中Ras 家族蛋白的缺少应该是由于在进化早期，其祖先绿藻丢失了单个Ras 家族蛋白基 因所致；（2）根据Cavalier-Smith 的真核生物划分为单鞭毛（变形虫类、真菌和 后生动物）和双鞭毛（藻类、植物和除变形虫外的原生动物）两大类的分类观点， 真核生物最近的共同祖先可能只具有除Ras 家族而外的六个家族的成员，而Ras 家族蛋白则是在此两大类群分化以后在单鞭毛类生物中才产生的，多数双鞭毛类 生物如原生动物、绿藻和植物中没有Ras 的情况应该是一种祖征，而个别双鞭毛 类生物如红藻具有的Ras 家族蛋白则很可能是从单鞭毛类生物那里水平基因转 移而来的。至于SRβ 和Rho 家族蛋白在部分物种中的缺少，则还是可能因为基 因丢失所致。此外，变形虫类生物中大量的Ras 超家族蛋白提示基因组的大小或 进化地位的高低并不是Ras 超家族蛋白成员多少的决定性因素，而细胞相应生理 活动的需求才是家族成员增多的关键。

碳离子辐射对人肝癌细胞SMMC-7721细胞周期和P53、MDM2及P21表达的影响

研究12C6+离子辐照对体外培养人肝癌细胞SMMC-7721细胞周期和P53、MDM2及P21表达的影响。采用0、1.0、2.0、4.0、6.0Gy12C6+离子束辐照细胞,用克隆形成法观察细胞存活情况;同时在辐照24h后用流式细胞仪检测细胞周期的变化,Western-blot检测细胞中P53、MDM2及P21蛋白表达情况。结果发现,重离子辐照后细胞存活率显著下降;1.0Gy、4.0Gy和6.0Gy照射组发生G0/G1期阻滞,而2.0Gy照射组出现G2/M期阻滞;Western-blot结果显示细胞辐照后MDM2的57kD蛋白表达水平无明显变化,而76kD蛋白表达水平随辐照剂量逐渐上升;P53和P21蛋白表达水平随辐照剂量增高。以上结果提示不同剂量的12C6+离子束照射可激活SMMC-7721细胞不同的细胞周期检测点,其中G0/G1期阻滞与P53和P21蛋白以及MDM2截短体76kD蛋白的表达水平升高有关。

Potentiality of phosphorylation of BRCA1 at Ser 1524 to activate p21 in response to X-ray irradiation

The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The percentages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activatep21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis.

Detection of K-ras exon 1 mutations by constant denaturant capillary electrophoresis

Among various mutation detection methods, constant denaturant capillary electrophoresis (CDCE) is one of the most common techniques for rapid identification of known or unknown mutations. In this report, a CDCE analysis method with homemade linear polyacrylamide (LPA) kit was developed on ABI 310 genetic analyzer, the effect and relationship of various denaturing factors in CDCE analysis were investigated and K-ras gene mutations of 31 coloerctal cancer patients were detected. Results indicate that, with the increase of chemical danaturant concentration, the optimum temperature was lowered, and when the concentration of urea (formamide) was higher than 7 M (40%), the homoduplex and heteroduplex of mutant samples were separated with difficulty. Detection results of K-ras gene in colorectal samples indicated that mutations were present in eight (26%) of 31 patients; most mutations were localized in codon 12, which is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. Copyright (C) 2004 John Wiley Sons, Ltd.

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. (C) 2003 Elsevier B.V. All rights reserved.

Biophysical characterization of the interaction of p21 with calmodulin: A mechanistic study

p21 is a protein with important roles in cell proliferation, cell cycle regulation and apoptosis. Several studies have demonstrated that its intracellular localization plays an important role in the functional regulation and binding of calmodulin favors its nuclear translocation. However, the detail mechanism of the interaction with p21 and calmodulin is not well understood. In this report, peptides derived from the C-terminal of p21 that cover the binding domain of calmodulin were used to investigate the association of p21 with calmodulin.