RASAL1基因启动子甲基化及RAS活性与结肠癌临床病理的关系

目的:检测RASAL1(ras GTPase-activatinglike protein 1)基因甲基化率及RAS活性在人结肠癌组织中的表达,探讨其与临床病理资料间的关系.方法:以40例结肠癌标本为研究对象,相应的正常组织标本为对照.甲基化特异性PCR(methylation-specific PCR,MSP)检测RASAL1启动子CpG岛甲基化状态,免疫共沉淀法检测RAS活性,分析肿瘤组织中RASAL1基因甲基化率及RAS活性与临床病理参数间的关系.结果:40例肿瘤组织中有26例检测出RASAL1基因甲基化(26/40,67.5%),对照组40例中有12例出现甲基化(12/40,30%),肿瘤组织甲基化率明显高于正常组织(P=0.0017).肿瘤组织中RAS活性的中位数为1.07,正常组织中RAS活性的中位数为0.52,P<0.001,差异有统计学意义.结肠癌组织中RASAL1基因的甲基化率、RAS活性与患者肿瘤分化程度、侵袭深度、淋巴结转移、TNM分期有统计学差异(均P<0.05).结论:RASAL1甲基化状态与RAS活性增加有关,可能在结肠癌的发生发展中起重要作用.抑制RASAL1甲基化及RAS活性,可能成为治疗结肠癌的新靶点.     

ras癌基因与膀胱癌

肿瘤的发生、发展及转移均与基因异常有密切关系 ,在以往被肯定的膀胱癌致癌因素中 ,ras癌基因与膀胱癌的关系已被有关的基因学研究证实。现结合文献将二者的关系综述如下。1　 ras癌基因与膀胱癌的关系1982年 ,Weinberg和 Barbacid从人膀胱癌细胞株中克隆到第 1个人类癌基因 ,即 ras基因。 ras基因家族包括 K-ras、H- ras和 N- ras,是人类肿瘤中最常见的癌基因。其编码蛋白质分子量为 2 1KD,称为 P2 1 ,能与细胞膜结合 ,具有 GPT酶的活性。 P2 1 与其它 G蛋白具有同源性 ,可在信号转导中发挥作用。有报道 ,7%～ 16 %的膀胱癌中有 ras…     

胰腺癌ras p21蛋白表达与AgNORs计数及组织学分级的关系

目的研究胰腺癌ras p21蛋白表达与AgNORs计数及组织学分级的关系。方法用免疫组化法检测了50例胰腺癌组织及50例正常胰腺组织中ras p21蛋白的表达；AgNORs染色技术检测胰腺癌细胞的AgNORs含量；观察肿瘤的组织学分级。结果ras p21蛋自在胰腺癌组织和正常胰腺组织中的表达有显著差异；ras p21蛋白阳性胰腺癌的AgNORs计数比阴性的AgNORs计数明显增多（P〈0．001）：ras p21蛋白表达与肿瘤组织学分级呈显著负相关（r5==0．99，P〈0．001）。结论胰腺癌的发生发展过程包含了ras p21基因的突变。ras p21蛋白表达可反映胰腺癌的恶性程度，是一个有效的判断胰腺癌预后的指标。     

小干扰RNA靶向突变K-Ras基因对人胰腺癌细胞TGFβ1表达的影响

背景与目的研究显示ras基因活化可促使肿瘤细胞分泌多种细胞因子,而ras基因与TGFβ1基因表达之间的关系还未可知。本研究利用小干扰RNA敲除突变k-ras基因,探讨突变k-ras基因对TGFβ1表达及分泌的影响。方法小干扰RNA瞬时转染靶向突变k-ras后,应用real-time PCR和Western blot方法检测胰腺癌细胞株CFPAC-1、Aspc-1中ras mRNA和蛋白的表达;应用real-time PCR和流式细胞仪检测对胰腺癌细胞TGFβ1表达的影响;应用ELISA方法检测转染前后胰腺癌细胞上清液中TGFβ1浓度。结果①以人已知基因无影响的序列阴性对照组为基准计算k-ras mRNA的相对表达量,实验组胰腺癌细胞CFPAC-1和ASPC-1转染后k-ras mRNA表达明显减低,k-ras mRNA表达在转染后48h为最低,抑制率达(75.33±5.50)%。Western blot检测各组质粒转染的细胞ras蛋白表达,结果显示实验组ras蛋白量较转染试剂对照组、已知基因无影响的序列阴性对照组明显降低。②Real-timePCR结果显示实验组CFPAC-1和ASPC-1细胞转染后,TGFβ1mRNA表达量(CFPAC-10.68±0.08;Aspc-10.59±0.09)明显降低(P0.05)。而流式细胞仪结果显示TGFβ1蛋白表达降低(CFPAC-1组0.30±0.08,ASPC-1组0.33±0.10,P0.05)。③ELISA法检测实验组CFPAC-1上清液TGFβ1浓度为(1906.87±50.75)ng/ml,较阴性对照组(2072.79±96.98)ng/ml有所降低(P=0.036);实验组Aspc-1细胞上清液TGFβ1浓度(851.47±41.08)ng/ml,较阴性对照组(950.93±31.34)ng/ml有所降低(P0.05)。结论人胰腺癌细胞CFPAC-1、Aspc-1中TGFβ1mRNA和蛋白的表达与ras基因相关。TGFβ1可能是ras基因活化后驱动胰腺癌发生及演变的重要物质。本研究为k-ras突变的胰腺癌治疗提供有益的信息。     

肿瘤相关基因RASAL1在结肠癌中的表达及临床意义

目的:探讨Ras三磷酸鸟苷酶激活样蛋白(RASAL1)及基因在人结肠癌中的表达及其与临床病理参数间的关系.方法:收集2009-04/2010-05经手术切除的结肠腺癌标本50例作为研究组,相应正常组织为对照组,做成蜡块标本;其中20例另取其癌组织、癌旁组织及正常组织的新鲜标本;用免疫组织化学染色观察50例蜡块标本RASAL1蛋白的表达;用RT-PCR检测20例新鲜标本RASAL1 mRNA的表达,并分析其与临床病理参数的关系.结果:RASAL1蛋白主要在细胞质中表达;RASAL1蛋白在结肠癌中的阳性表达率明显低于癌旁组织及正常组织[46%(23/50)vs85%(17/20),96%(48/50),均P<0.05];RASAL1 mRNA在结肠癌中的阳性表达率较癌旁组织及正常组织明显减低[50%(10/20)vs90%(18/20),95%(19/20),均P<0.05],RASAL1蛋白与mRNA的表达呈明显正相关(r=0.686,P<0.01),与肿瘤的分化程度(P<0.05)、侵袭深度(P<0.01)、淋巴结转移(P<0.05)、TNM分期(P<0.05)呈负相关.结论:RASAL1在结肠腺癌组织中低表...     

抑癌基因Smad4与胃肠道肿瘤

Smad4是一种肿瘤抑制基因,其编码的蛋白是转化生长因子-β(TGF-β)超家族细胞内信号转导和调节的中心分子。Smad4突变失活引起的TGF-β信号转导紊乱,导致肿瘤细胞对TGF-β的生长抑制作用逃逸,与胃肠道肿瘤的发生发展存在一定关系。突变的Smad4蛋白较野生型更易被泛素介导的蛋白酶体降解。     

白血病的ras癌基因点突变

人类白血病发生的病理机制仍不明,癌基因与某些血液系统肿瘤有关,如c-abl癌基因与慢性髓性白血病(CML)、c-myc癌基因与Burkitt’s淋巴瘤。近年来注意到ras癌基因与白血病有一定关系,且具有某些特点和意义,现简述如下。1 急性髓性白血病(AML) ras癌基因突变是AML最常检测到的癌基因异常,因作者采用的检测方法不同,报道的ras突变检出率有差异。1985年前后多采用NIH/3T3细胞转化试验,AML患者存在的活化ras阳性率是58%(11/19例);1987年前后多用寡核苷酸探针杂交     

胃癌组织H-ras基因点突变与预后的关系

目的:胃癌发生发展的分子基础仍不甚明了,为了明确H—ras点突变在胃癌发生发展中的作用,本研究对胃癌组织H—ras点突变进行检测。方法:采用多聚酶链延伸反应—限制性片段长度多态性分析法(PCR—RFLP)对88例福尔马林液固定、石蜡包埋胃癌组织H—ras第12位和61位密码子点突变作了检测,并对点突变与肿瘤生物学行为及预后的关系进行分析。结果:H—ras总突变率为14.8％(13/88),点突变的发生与肿瘤将膜浸润,淋巴结转移、临床分期及术后生存期密切相关。结论:检测胃癌组织H—ras基因点突变有助于判断胃癌患者的预后。     

抑癌基因Smad4与胃肠道肿瘤

Smad4是一种肿瘤抑制基因，其编码的蛋白是转化生长因子-β（TGF-β）超家族细胞内信号转导和调节的中心分子。Smad4突变失活引起的TGF-β信号转导紊乱，导致肿瘤细胞对TGF-β的生长抑制作用逃逸，与胃肠道肿瘤的发生发展存在一定关系。突变的Smad4蛋白较野生型更易被泛素介导的蛋白酶体降解。     

胃肠道间质瘤与原癌基因c-kit的研究进展

胃肠道间质瘤起源于胃肠道间质细胞,原癌基因c-kit突变是其主要发病机制之一。c-kit突变形式多样,其突变类型与胃肠道间质瘤的生物学特性及预后相关。伊马替尼通过阻滞c-kit受体从而抑制肿瘤生长。     

Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers

Ras has achieved notoriety as an oncogene aberrantly activated in multiple human tumors. Approximately 30% of all human tumors express an oncogenic form of this GTPase that is locked in an active conformation as a result of being insensitive to Ras GTPase-activating proteins (GAPs), proteins that normally regulate the inactivation of Ras by enhancing its intrinsic GTPase activity. Besides oncogenic mutations in Ras, signaling by wild-type Ras is also frequently deregulated in tumors through aberrant coupling to activated cell surface receptors. This indicates that alternative mechanisms of aberrant wild-type Ras activation may be involved in tumorigenesis. Here, we describe another mechanism through which aberrant Ras activation is achieved in human cancers. We have established that Ras GTPase-activating-like protein (RASAL), a Ca(2+)-regulated Ras GAP that decodes the frequency of Ca(2+) oscillations, is silenced through CpG methylation in multiple tumors. With the finding that ectopic expression of catalytically active RASAL leads to growth inhibition of these tumor cells by Ras inactivation, we have provided evidence that epigenetically silencing of this Ras GAP represents a mechanism of aberrant Ras activation in certain cancers. Our demonstration that RASAL constitutes a tumor suppressor gene has therefore further emphasized the importance of Ca(2+) in the regulation of Ras signaling and has established that deregulation of this pathway is an important step in Ras-mediated tumorigenesis.     

Farnesyl transferase inhibitors: the next targeted therapies for breast cancer?

The ras family of proto-oncogenes are upstream mediators of several essential cellular signal transduction pathways involved in cell proliferation and survival. Point mutations of ras oncogenes result in constitutively active Ras and have been shown to be oncogenic. However, ras activation can occur in the absence of ras mutations secondary to upstream receptor activation. The first important step in Ras activation is farnesylation by farnesyl transferase, and inhibitors of this enzyme have been demonstrated to inhibit Ras signaling, and have anti-tumor effects. However, it is now clear that farnesyl transferase inhibitors (FTIs) have activity independent of Ras, most likely due to effects on prenylated proteins downstream of Ras, which explains their activity in several malignancies, including breast cancer, where ras mutations are rare. Several FTIs are in clinical development for the treatment of solid tumors. Preclinical evidence suggests that FTIs can inhibit breast cancers in vitro and in vivo, and a phase II trial of the FTI, R115777, in patients with advanced breast cancer produced encouraging results. Based on prior successful outcomes with agents targeting the estrogen and epidermal growth factor receptor pathways in breast cancer, the FTIs, used alone or more likely with other agents, may be the next exciting targeted therapy in breast cancer.     

RASAL1基因在胃癌组织中的表达及其临床意义

目的:探讨Ras蛋白激活物类似物-1(RASAL1)基因在胃癌组织中的表达情况及临床意义.方法:应用免疫组织化学SP法分别检测50例正常组织、50例低级别上皮内瘤变组织、50例高级别上皮内瘤变组织+早期胃癌组织及50例进展期胃癌组织中RASAL1基因的表达,并结合其临床病理资料进行综合分析.结果:RASAL1基因在正常...     

Ras as a target in cancer therapy

Ras proteins are guanine nucleotide-binding proteins that are central to the control of normal and transformed cell growth and that are mutated in approximately 30% of human cancers. Binding of ligands to various growth factor receptors activates Ras and subsequently a plethora of downstream effectors including the Raf-1/mitogen-activated protein kinase pathway. For effective ras functioning and for transformation, Ras proteins must undergo post-translational modifications that facilitate their attachment to the plasma membrane. Farnesylation, catalysed by farnesyl protein transferase (FPT), is the first and the most important of these modifications; inhibition of which ablates ras activity, resulting in significant anti-proliferative effect in vitro and in human cancer xenograft models. FPT inhibitors are being assessed in a range of phase I and phase II trials, which incorporate both pharmacokinetic and dynamic end-points. In addition, ras mutations can also generate neo-epitopes for cytotoxic and helper T-cell recognition, rendering ras-mutated tumours a potential target for immunotherapy. Though their clinical evaluation is still in infancy, these two modes of ras targeting represent rational therapeutic strategies that can undergo mechanistic evaluation in the clinic.     

Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.

Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors.     

Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements.     

Detection of ras gene mutations of pancreatic tumors by polymerase chain reaction and direct sequencing method

Ras genes (H-,K-,N-ras) are converted to active oncogenes by point mutations occurring in either codon 12, 13 or 61. We analyzed 19 pancreatic tumors (formalin fixed paraffin embedded tissue) of these codons by a method to directly sequence nucleotides around codons 12/13 and 61 of the three ras genes, using polymerase chain reaction and direct sequencing method. Of 19 pancreatic tumors, all 17 duct cell carcinomas involving 2 mucous producing pancreatic cancers had point mutations of the K-ras codon 12, but 2 islet cell tumors had ano point mutation around codons 12, 13, 61 of the three ras genes. Extremely high incidence of ras gene mutation may be relevant to certain pathogenesis of pancreatic cancers.     

Effect of DNA Demethylation in Experimental Encapsulating Peritoneal Sclerosis

Encapsulating peritoneal sclerosis (EPS) involves excessive peritoneal fibrosis in patients on peritoneal dialysis, eventually leading to visceral constriction and bowel obstruction. Few studies have investigated epigenetic mechanisms relating to EPS. Here we evaluated the therapeutic effects of DNA demethylation in experimental EPS. Experimental EPS was induced by intraperitoneal injection of 0.1% chlorhexidine gluconate (CG) and 15% ethanol in non‐uremic male Sprague–Dawley (SD) rats. Rats were divided into three groups: group C (N = 5) with saline injection only, group CG (N = 7) with EPS induction for 4 weeks, and chlorhexidine gluconate and azacytidine (CGA) treated group (N = 7) with EPS induction for 4 weeks and 5'‐azacytidine injection for the last 2 weeks. Morphometric analysis of peritoneum and immunohistochemical staining for type 1 collagen and α‐smooth muscle actin (α‐SMA) were performed. Expressions of transforming growth factor‐β (TGF‐β), fibroblast‐specific protein 1 (FSP1), and DNA methyltransferase 1 (DNMT1) were analyzed by Western blot. Methylation‐specific polymerase chain reaction (PCR) for Ras GTPase activating‐like protein 1 (RASAL1) was performed with measurement of RASAL1 protein expression. Parietal peritoneal thickness and the number of vessels in omental tissue were significantly decreased in group CGA compared to group CG, as were the expressions of type 1 collagen, α‐SMA, TGF‐β, and FSP1. DNMT1 was significantly increased in group CG, and reduced in group CGA. RASAL1 hypermethylation was associated with decreased RASAL1 protein expression in group CG, which was reversed in group CGA. DNA demethylation by 5'‐azacytidine treatment improved pathologic changes of the peritoneum in experimental EPS, and was associated with reversal of increased DNMT1 expression and RASAL1 hypermethylation.