INHIBITION OF FARNESYL PROTEIN TRANSFERASE AND P21^(RAS) MEMEBRANE ASSOCIATION BY D-LIMONENE IN HUMAN PANCREAS TUMOR CELLS IN VITRO

The monoterpene d limonene inhibit the plasma membrane associated P21 ras expression and the posttranslational isoprenylation of P21 ras , a mechanism that may contribute to its efficacy in the chemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study,the relative abilities of d limonene to inhibit membrane associated P21 ras expression in pancreas tumor cell(PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FTPase) activity during the Ras protein isoprenylation and cell proliferation were determined.Concomitantly,the effects of d limonene on P21 ras localization by immunohistochemistry and H ras oncogene expression in PaCa tumor cell line by Northern blotting were observed. The results showed that d limonene inhibited FPTase activity, thus to reduce P21H ras isoprenylation. d limonene could decrease P21 ras membrane association and increase cytosolic accumulation of P21 ras . This phenomenon was also noted when d limonene treated PaCa cells were stained immunohistochemically with anti P21 ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21 ras membrane association and the alteration of P21 ras localization. Inhibition of farnesylation of P21 ras altered their intracellular localization and, hence, disrupted their biological activity,but no relationship with H ras oncogene expression was found.

PDRG1 at the interface between intermediary metabolism and oncogenesis

PDRG1 is a small oncogenic protein of 133 residues. In normal human tissues, the p53 and DNA damageregulated gene 1(PDRG1) gene exhibits maximal expression in the testis and minimal levels in the liver. Increased expression has been detected in several tumor cells and in response to genotoxic stress. High-throughput studies identified the PDRG1 protein in a variety of macromolecular complexes involved in processes that are altered in cancer cells. For example, this oncogene has been found as part of the RNA polymerase Ⅱ complex, the splicing machinery and nutrient sensing machinery, although its role in these complexes remains unclear. More recently, the PDRG1 protein was found as an interaction target for the catalytic subunits of methionine adenosyltransferases. These enzymes synthesize S-adenosylmethionine, the methyl donor for, among others, epigenetic methylations that occur on the DNA and histones. In fact, downregulation of S-adenosylmethionine synthesis is the first functional effect directly ascribed to PDRG1. The existence of global DNA hypomethylation, together with increased PDRG1 expression, in many tumor cells highlights the importance of this interaction as one of the putative underlying causes for cell transformation. Here, we will review the accumulated knowledge on this oncogene, emphasizing the numerous aspects that remain to be explored.

P21^(ras)、P16在大肠癌中的表达及临床意义

目的探讨癌基因P21^(ras)及抑癌基因P16与大肠癌的关系。方法应用免疫组化SP法分析、研究了P21^(ras)蛋白、P16蛋白在80例大肠癌、20例大肠腺瘤及20例正常大肠组织中的表达。结果①P21^(ras)在前二者中的阳性表达率分别为70.1％(57/80)和65％(13/20),明显高于在正常组织中的30％(6/20),其比较具有显著性差异(x^2=17.66、P<0.005;x^2=5.23、P<0.05);P16在大肠癌中的阳性表达率为36.3％(29/80)低于在腺瘤及正常组织中的65％(13/20)和75％(15/20),其比较亦具有显著性差异。②P21^(ras)、P16的阳性表达率在大肠癌患者的年龄、性别及肿瘤大小方面无显著性差异,而与肿瘤的临床Dukes分期(x^2=6.20、P<0.025;x^2=6.25、P<0.05)、有无淋巴结转移及术后5a生存率密切相关。③P21^(ras)在不同的大肠癌组织病理类型中,其表达率无显著性差异;P16的阳性表达率随着肿瘤浸润深度的增加有下降趋势,但无统计学意义。④在80例大肠癌患者中,P21^(ras)阳性而P16阴性者比率高于其他三者,而且这类大肠癌患者的术后5a生存率下降。结论大肠癌的发生、发展,是由包括癌基因、抑癌基因在内的多种因素作用的结果;在临床工作中联合检测P21^(ras)及P16蛋白在大肠癌中的表达水平,对我们估计患者的病情、推测其预后有指导性意义。

洛伐他汀对NB4细胞ras基因p21^(Ras)膜结合作用影响的体外试验

目的 :探讨洛伐他汀 (LOV)对人白血病NB4细胞的作用及其机制。方法 :以MTT比色法首先观察LOV对NB4细胞增殖的影响 ;利用逆转录 -聚合酶链反应半定量测定LOV作用于NB4细胞不同时间H -ras、K -ras、N -ras癌基因mRNA表达水平 ;同时采用流式细胞术测定NB4细胞p2 1Ras总蛋白、膜蛋白的表达。结果 :①LOV抑制NB4细胞增殖 ,IC5 0为 12 5 9μmol/L。②NB4细胞H、K、N -ras基因表达均为阳性。③LOV处理不同时间的NB4细胞H、K、N -ras基因转录水平无明显变化 ;p2 1Ras总蛋白水平也无变化 ,而细胞膜表面p2 1Ras蛋白水平随时间进行性下降。结论 :LOV抑制NB4细胞增殖。LOV靶向HMG -CoA还原酶 ,抑制p2 1Ras蛋白异戊二烯化、阻滞p2 1Ras蛋白与细胞膜结合 ;不影响ras癌基因以及p2 1Ras总蛋白的表达。

肺鳞癌细胞p^(53)蛋白及ras p^(21)的免疫组织化学定量研究

应用免疫组织化学方法检测２２例肺鳞癌组织标本的ｐ５３及ｒａｓｐ２１表达，用显微图像分析仪测定阳性细胞百分率Ｐ／Ａ（％）和平均光密度（ＡＯＤ），并采用阳性水平指数（ｐｏｓｉｔｉｖｅｌｅｖｅｌｉｎｄｅｘ，ＰＬＩ）作为免疫组化反应水平指标，对癌基因蛋白进行比较研究。结果显示：ｐ５３表达阳性率为５０％，ｒａｓｐ２１表达阳性率为６３６％。ｐ５３蛋白表达在非角化型鳞癌组高于角化型鳞癌组；ｒａｓｐ２１的表达随组织分化程度的增高而增高，ｒａｓｐ２１与ｐ５３的表达水平无直线相关关系。

胃癌术后硫酸性肠化切缘P^（53）和ras P^（21）蛋白的表达及其意义

目的探讨硫酸性肠化切缘Ｐ５３和ｒａｓＰ２１蛋白的表达，进一步从分子生物学水平证实硫酸性肠化与胃癌术后切缘复发的关系。方法应用流式细胞术和细胞免疫荧光染色技术，对１８例硫酸性和１７例非硫酸性肠化切缘粘膜上皮细胞的Ｐ５３和ｒａｓＰ２１蛋白的表达量进行了定量研究．结果硫酸性肠化切缘上皮的Ｐ５３和ｒａｓｐ２１蛋白的表达量显著高于非硫酸性肠化切缘和正常粘膜上皮，而低于胃癌细胞．结论硫酸性肠化切缘的ｐ５３和ｒａｓＰ２１蛋白有过量表达，可能与胃癌术后复发有关。

蛋白激酶C抑制剂对SACC-83系P_(21)^(ras)、c-myc表达的影响

本实验采用ＰＫＣ抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ作用于ＳＡＣＣ－８３系，观察对Ｐ２１ｒａｓ、ｃ－ｍｙｃ表达的影响．结果表明，Ｓｔａｕｒｏｐｏｒｉｎｅ可明显降低Ｈ－ｒａｓ、ｃ－ｍｙｃ表达程度（ｐ＜０．０１），这提示ＰＫＣ对ｒａｓ、ｃ－ｍｙｃ基因表达具有调控作用，ＰＫＣ抑制剂可能通过调控癌基因的表达来表现抗肿瘤作用的．

ras-P_(21)过表达和 P_(53)蛋白蓄积对肺癌预后的研究

应用Ｈａ－ｒａｓＰ２１和Ｐ５３（Ｄｏ－１）单克隆抗体，采用Ｓ－Ｐ免疫组化法对７７例原发性肺癌进行研究。结果表明：Ｐ２１强阳性（）率在细支气管肺泡癌的表达明显高于腺泡状腺癌和大细胞癌（Ｐ＜０．０５），分化程度越高，染色越强。Ｐ５３蛋白蓄积与肺癌的分型、分化、ＴＮＭ分期无关，但Ｐ５３染色阳性与阴性患者的术后平均存活月数间有显著差异（Ｐ＜０．０５），Ｐ５３染色越强术后存活时间越短，提示Ｐ５３蛋白蓄积是判定肺癌预后的指标之一。

P_(21)^(ras)在胃良、恶性病变中的表达及与幽门螺旋杆菌感染的关系

本文Ｐ２１ｒａｓ免疫组化采用链霉菌抗生物素蛋白——过氧化酶（ＳＰ）法检测了６１例胃粘膜标本。结果表明胃癌（５６．０％）与胃癌前病变（６０．０％）阳性率高于胃良性病变（１４．０％），Ｐ值分别＜０．０５与＜０．００５。并进行了诊断胃癌价值评估，其敏感性、特异性和准确性分别为５６．０％，６６．７．与６２．３％，说明Ｐ２１ｒａｓ诊断胃癌有一定价值。同时检测了幽门螺旋杆菌（ＨＰ），计算Ｐ２１ｒａｓ阳性与ＨＰ感染的相关系数为０．８７９，呈正相关。

子宫内膜腺癌组织中P^(21ras)的表达及意义

应用Ｐ２１ｒａｓ单克隆抗体，用ＡＢＣ免疫组织化学法分析子宫内膜腺癌组织中Ｐ２１ｒａｓ的表达。结果表明：子宫内膜腺癌组织中Ｐ２１ｒａｓ阳性表达率及表达强度明显高于其他组织（Ｐ＜０．０５，Ｐ＜０．００５），且随组织学分级增高而增高。说明：子宫内膜腺癌的发生、发展均与ｒａｓ基因变异有关。提示：Ｐ２１ｒａｓ单抗做为子宫内膜腺癌诊断标志物及临床应用的可能性。

Effects of endotoxin on expression of ras, p53 and bcl-2 oncoprotein in hepatocarcinogenesis induced by thioacetamide in rats

AIM To evaluate the relationship between expression of ras, p53, bcl 2 gene products, and hepatocarcinogenesis since endotoxemia produced from lipopolysaccharide admi nistration and/or the hypophagocytic state of splenectomy significantly accelerated hepatocarcinogenesis induced by thioacetamide. 〖WTH4〗METHODS〓〖WTXFZ〗The hepatocarcinoma model was induced by oral intake of 0 03% thioacetamide for six months. During the induction of hepatocarcinoma model, rats were additionally treated with splenectomy and/or lipopolysaccharide administration. The techniques of flow cytometry, immunohistochemistry and immunoelectronmicroscopy were applied to quantitative analysis of the expression of oncogene proteins. RESULTS In this model system, overexpression of ras p21 protein mainly occurred on precancerous cell population or in early stage of hepatocyte transformation. And the levels of ras p21 declined when nuclear DNA aneuploid increased. Expression of bcl 2 protein slowly and steadily rose with more hepatocytes staying in S+G2M phases as the hepatocarcinoma became more malignant. P53 was moderately expressed during the hepatocarcinogenesis. There was no statistical correlation between endotoxemia levels and the changes of ras, p53 and bcl 2 gene products. CONCLUSION Over expression of oncogene ras p21 was likely to be a precursor of the premalignant hepatocytes and it might be responsible for the initiation of hepatocarcinogenesis. Bcl 2 protein expression is proportional to the severity of the malignancies. P53 may be a key pathway on the transformation and development of hepatocarcinoma. This study confirmed the hypothesis that there are multiple genes and multiple steps involved in hepatocarcinogenesis. Expressions of oncogene proteins reflected the properties of the premalignant and malignant cells, but not directly related to endotoxemia statistically.[JP]

AgNOR and rasp21 expression in gastric mucosal lesions with Helicobacter pylori infection

IM To study the number of AgNOR and rasp21 expression in different gastric mucosal lesions with Helicobacter pylori (Hp) infection to evaluate their biological behaviour and possible mechanism of Hp. METHODS Hp (using CLO test combined with WathinStarry staining), AgNOR (silver colloid technique) and rasp21 (monoclonal antibody and immunohistochemical staining—ABC method) were detected in 278 patients with endoscopically and pathologically confirmed gastric mucosal lesions, including chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), intestinal metaplasia (IM), dysplasia (Dys), and gastric cancer (GC). Among them, 146 cases were Hp positive, and 132 cases Hp negative. RESULTS The mean number of AgNOR in Hp positive group was significantly higher than that in Hp negative group in the gastric mucosal lesions except for CSG (P<005 or P<001). The positive rate of rasp21 expression in Hp positive group was also significantly higher than that in Hp negative group in gastric mucosal lesions except for CSG and CAG (P<005).CONCLUSION Hp+ gastric mucosal lesions have more biological behaviour of tumors. Hp may act as a promoter to activate ras gene and to stimulate cell over proliferation.

食管癌DNA倍体异质性肿瘤的P^(53)和ras P^(21)蛋白的表达

目的 在ＤＮＡ含量研究的基础上，进一步从蛋白质表达水平上探讨ＤＮＡ 倍体异质性肿瘤的生物学特性。方法 应用流式细胞术和细胞免疫荧光技术，对１５ 例异质性肿瘤和非异质性肿瘤细胞的Ｐ５３ 和ｒａｓＰ２１ 蛋白表达量进行了定量和比较。结果 异质性肿瘤细胞的Ｐ５３ 和ｒａｓＰ２１ 蛋白的表达量显著高于非异质性肿瘤和正常组织（ Ｐ＜ ０．０５ ～０．０１）。结论 ＤＮＡ 倍体异质性肿瘤的癌基因激活和抑癌基因失活的程度高于非异质性肿瘤，异质性肿瘤的恶性程度高于非异质性肿瘤。

ras癌基因蛋白P^(21)表达与膀胱移行细胞癌生物学行为的关系

应用流式细胞术和细胞免疫荧光染色技术,对42例膀胱移行细胞癌基因ras P^(21)表达进行了定量分析,并结合患者随访结果进行回顾性研究。结果表明,ras P^(21)表达与肿瘤细胞分化程度及浸润情况密切相关。ras P^(21)表达与肿瘤复发也具有相关性,在复发的膀胱肿瘤中,P^(21)阳性表达(87.5％),显著高于P^(21)阴性表达(12.5％)。

子宫内膜癌细胞rasP^(21)、P^(53)表达、DNA含量和临床病理特征及预后的关系

为探讨ｒａｓ癌基因和Ｐ５３抗癌基因表达产物、ＤＮＡ含量及其它临床病理因素对子宫内膜癌预后的影响，本文采用单因素、Ｋａｐｌａｎ－Ｍｅｉｅｒ生存曲线和ＳＰＳＳ多元相关与逐步回归软件将患者年龄、临床期别、病理类型、临床分型、组织学分级、肌层侵犯深度、局部免疫反应、ＤＮＡ指数（ＤＩ）、Ｓ期细胞比率（ＳＰＦ）、增殖指数（ＰＩ）、Ｐ２１荧光指数（Ｐ２１ＦＩ）、Ｐ５３荧光指数（Ｐ５３ＦＩ）和Ｐ５３免疫组织化学表达强度等１３个因素与生存的关系进行了分析。结果表明：Ｐ５３表达强度和临床期别为最有意义的预后指标，Ｐ５３表达强阳性和临床Ⅲ期与短生存期有关；另外，肌层侵犯超过２／３、乳头状腺癌和年龄大也提示不良预后。

Correlated Flow Cytometric Analysis of H－ras p21 and DNA Ploidy in Acute Myelogenous Leukemia

The flow cytometric immunoassay was used to study the correlation between the H-ras oncogene product p21 and the DNA ploidy in 30 de novo cases of acute myelogenous leukemia (AML). The results showed that 17 cases were negative for p21 expression and 13 positive for p21. The patients with positive p21 had higher percentage of bone marrow and peripheral blasts and lower peripheral leukocyte count. The expression of p21 had no influence on the therapeutic effect. Before treatment,DNA diploidy occurred in 18 cases including 13 p21 negative ones,and DNA aneuploidy was revealed in 12 cases including 8 p21 positive ones. Patients with positive p21 or having aneuploidy in complete remission were at risk for early relapse. Our results suggest that p21 may be involved in the process of leukemogenesis and progression in AML.

42例膀胱癌及其癌旁组织中ras癌基因P^（21）蛋白的表达

应用免疫组织化学技术（ＡＢＣ法），对４２例膀胱癌及癌旁组织中ｒａｓ癌基因Ｐ￣（２１）蛋白进行了检测。结果表明，膀胱癌组织中Ｐ￣（２１）蛋白阳性率为７３．８％，而４２例癌旁组织中Ｐ￣（２１）蛋白阳性率为１４．３％，Ｐ￣（２１）蛋白的阳性率随着病理分级、临床分期的升级增高，值得注意的是癌旁组织中Ｐ￣（２１）蛋白阳性者癌症易于复发，提示预后不良。

Ras基因P_(53)基因在食管癌贲门癌的过度表达及相互关系的探讨

目的 探讨食管癌、贲门癌的发生与癌基因、抑癌基因的作用及相互关系。方法 对 6 0例食管癌、贲门癌进行了病理分析及Ras癌基因蛋白和P53抑癌基因蛋白单克隆抗体免疫组化对比分析。结果 P2 1蛋白总阳性率为 90 0 % (占 36例食管癌 86 1 % ,2 4例贲门癌 95 8% ) ,P53蛋白总阳性率为 93 3% (食管癌 94 4 % ,贲门癌 91 7% ) ,且 2组病例在P2 1蛋白阳性表达和P53蛋白阳性表达方面均无显著性差异 (P >0 0 5 ) ;同时还发现P2 1阳性表达在肿瘤分化程度上不具有显著性差异 ,而P53阳性表达在中、低分化组明显增强 ,说明P2 1阳性表达在肿瘤发生发展过程中起着持续作用 ,而P53阳性表达在肿瘤发展的某一阶段起着重要作用。结论 提示食管癌和贲门癌的发生发展在癌基因激活和抑癌基因失活等方面具有相似之处 ,支持食管癌和贲门癌在肿瘤发生及其它生物学特性方面属同一类肿瘤的观点 ;P53蛋白的过度表达可作为判断该肿瘤生物学行为的一个敏感可靠的指标 ;Ras癌基因的激活。

Epidermal growth factor receptor and K-Ras in non-small cell lung cancer-molecular pathways involved and targeted therapies

Lung cancer is currently the leading cause of cancer death in Western nations.Non-small cell lung cancer(NSCLC)represents 80%of all lung cancers,and adenocarcinoma is the predominant histological type.Despite the intensive research carried out on this field and therapeutic advances,the overall prognosis of these patients remains unsatisfactory,with a 5-year overall survival rate of less than 15%.Nowadays,pharmacogenetics and pharmacogenomics represent the key to successful treatment.Recent studies suggest the existence of two distinct molecular pathways in the carcinogenesis of lung adenocarcinoma:one associated with smoking and activation of the K-Ras oncogene and the other not associated with smoking and activation of the epidermal growth factor receptor(EGFR).The K-ras mutation is mainly responsible for primary resistance to new molecules which inhibit tyrosine kinase EGFR(erlotinib and gefitinib)and most of the EGFR mutations are responsible for increased tumor sensitivity to these drugs.This article aims to conduct a systematic review of the literature regarding the molecular pathways involving the EGFR,K-Ras and EGFR targeted therapies in NSCLC tumor behavior.

P21 and CEA expression and AgNOR counts in DMH induced colon carcinoma in rats

AIM To study the expression of P21 and CEA, and AgNOR counts in various lesions of colonic mucosa and the mechanism of carcinogenesis. METHODS Thirty eight male Wistar rats were injected with DMH once a week, the experiment lasted 25 weeks. The expression of P21 and CEA was detected by the immunohistochemical methods and AgNOR was counted with silver staining in paraffin sections of various colonic lesions. RESULTS The incidence of colonic carcinoma in DMH treated rats was 71 05%(27/38), and lymphnode metastasis occurred in 6 rats. Immunohistochemical study showed that the expression of P21 was mainly in dysplasia and carcinomas, while CEA expressed in carcinoma and metastasis. The AgNOR counts was also increased in dysplasia and carcinomas. There were significant differences in P21 and CEA expression between benign and malignant lesions ( P <0 05). Difference in AgNOR counts was also significant between normal and dysplasia, dysplasia and malignant lesions ( P <0 05). CONCLUSION Dysplasia is a premalignant change of colonic carcinoma. The detection of P21 with immunohistochemistry and AgNOR counting might be important for screening colonic carcinoma and premalignant lesions clinically.