GGTase-I或FTase基因敲除在抑制K-ras突变诱导肺癌发生中的分子机制研究
|
摘要
第一部分体内实验
背景
肺癌是目前发病率和病死率均很高的恶性肿瘤,严重影响着人类健康。肺癌是一种基因病,癌基因激活和抑癌基因失活是其重要的分子事件。因此,阐明肺癌发生的分子机制,从分子水平阻断其发生的相关链接具有极为重要的临床意义。K-ras是目前研究最多的癌基因之一,在许多肿瘤中均存在K-ras基因的扩增或过度表达,其中在肺腺癌中大约有50%左右的患者存在K-ras突变,说明其在肺癌发生、发展中发挥重要作用。深入研究K-ras基因在肺癌发生中的作用机制,尽早阻断其信号系统已成为近年来肺癌研究领域中一个新的热点。
大约有100多种细胞内蛋白具有CAAX基序,K-ras基因编码蛋白属于其中的一种,这些蛋白可以进行多种转录后修饰,其中半胱氨酸残基(C末端CAAX基序)可直接进行异戊二烯化修饰。一部分CAAX蛋白,其末端可以在牛龙酰基转移酶Ⅰ(GGTase-I)的作用下进行牛龙基化修饰,还有一部分CAAX蛋白,例如K-Ras,在法呢酰基转移酶(FTase)的作用下进行法尼基化修饰,其中的“X”残基表明这种CAAX蛋白是被法尼基化还是牛龙基化修饰:一般情况下,如果“X”是亮氨酸,这种蛋白就会被牛龙基化修饰,否则就会被法尼基化修饰。CAAX蛋白末端进行异戊二烯化修饰后可以增强其蛋白羧基端的疏水性,进而增强膜的亲和力及与配体的结合能力,从而增强其生物学活性。FTase和GGTase-I有一个相同的α亚单位,但是分别具有各自独特的表明其底物特异性的β亚单位。例如,GGTase-I是由Fnta编码的一个α亚单位和Pggtlb编码的独特的β亚单位组成。
GGTase-I是许多真核细胞的必需酶,在果蝇和酵母菌种GGTase-I酶的β亚单位的无效突变通常是致死性的。GGTase-I缺乏导致生物死亡的主要原因是Rho1p和Cdc42p蛋白无法进行牛龙基化,而这种牛龙基化在特定条件下可通过FTase进行法尼基化弥补。RAS蛋白的法尼基化已引起学者的足够重视,研究表明,Ras蛋白的法尼基化对其靶向定位于细胞膜上的结合位点和保持正常的转化能力是非常必须的。在动物模型中,法呢酰基转移酶抑制剂(FTIs)具有高效的抗肿瘤活性且毒性低,而在临床试验中,FTIs的疗效并不理想,其可能原因是FTase缺乏时RAS蛋白进行了牛龙基化。许多研究已证实GGTIs能抑制多种K-ras突变肿瘤细胞株的增殖,同时研究还表明GGTIs能改善多发性硬化症动物模型的表型以及抑制肝癌中肝炎病毒的复制等。
鉴于GGTase-I和FTase在K-ras突变诱导肿瘤发生中的作用以及前期研究结果,本研究旨在应用条件性敲基因技术建立动物模型,并运用组织病理学、分子生物学、免疫学等研究手段,充分阐明条件性敲除GGTase-I的β亚单位(Pggtlb)或FTase的β亚单位(Fntb)后,阻断CAAX蛋白异戊二烯化修饰,从而抑制K-ras基因突变诱导的肺癌发生的作用,为肺癌的基因治疗提供科学依据。
目的
(1)利用条件性基因敲除技术建立符合人类肺癌病变特征的动物模型;(2)应用组织病理学等方法观察肺癌组织病理形态,探讨Pggtlb或Fntb基因敲除对K-ras基因突变诱导肺癌形成的抑制作用;(3)探讨Pggtlb或Fntb基因敲除抑制K-ras基因突变诱导肺癌发生的分子生物学机制。
方法
1.条件性敲基因动物模型的建立及其基因型验证
利用Cre/lox P系统条件性敲入K-ras基因,敲除GGTase-I或FTase基因,分别建立K-ras(K~(LSL))、GGTase-I(Pggtlb~(fl/fl))和FTase(Fntb~(fl/fl))条件性敲基因鼠模型。然后以K~(LSL)鼠和Pggtlb~(fl/fl)鼠或Fntb~(fl/fl)鼠杂交产生K~(LSL)Pggtlb~(fl/fl)鼠和K~(LSL)Fntb~(fl/fl)鼠,经基因分型后筛选出符合要求的小鼠。应用PCR技术进行基因型验证。
2.实验动物分组
按基因型分为三组:即K~(LSL)(A组)、K~(LSL)Pggtlb~(fl/fl)(B组)和K~(LSL)Fntb~(fl/fl)(C组),各组根据感染病毒种类的不同又分为两个亚组即:cre腺病毒载体感染组(实验组:A1组、B1组和C1组)和β-gal腺病毒载体感染组(对照组:A2组、B2组和C2组)。
3.干预方法
第14天龄时应用雾化吸入法进行腺病毒载体干预,分别敲除或激活K~(LSL)、K~(LSL)Pggtlb~(fl/fl)和K~(LSL)Fntb~(fl/fl)基因。
4.称量体重
病毒感染前及以后每周末称量所有实验鼠体重。
5.各组动物生存率测定
自基因干预起,观察各组动物的生存时间。
6.病理学检查
不同时间点处死动物,留取肺脏、肝脏、脾脏等组织器官并称量其重量,一部分组织进行苏木素-伊红(HE)染色,观察肿瘤细胞形态;另一部分留取标本进行分子生物学检测。
7.免疫组织化学染色
进行CD4、CC10、CD8、SP-C、非异戊烯化Rap1A及Ki-67的免疫组织化学染色。
8.Real-time PCR
检测肺脏、肝脏和脾脏等组织器官中Pggtlb、Fntb和K~(LSL)等基因的mRNA表达水平。
9.Western blot
检测肺脏肿瘤组织中非异戊烯化RAP1A、总RAP1A、p-ERK1/2、p-AKT、总AKT、总ERK1/2等的蛋白表达水平。
10.统计学分析
连续性数据用(?)±SD表示,离散数据用例数和百分数表示。三组间生存率的比较应用Kaplan-Meier方法,其余指标应用单因素方差分析。应用SPSS统计软件进行统计学处理(version 11.0;SPSS Inc),P＜0.05有统计学差异。
结果
1.实验动物基本情况
腺病毒载体干预动物的共60只。最终完成基因干预的共58只,其中A1组10只,A2组9只,B1组10只,B2组9只,C1组10只,C2组10只。本文对完成实验的58只实验鼠的资料进行分析。
2.各组动物的基因分型
共有6个亚组,分别是A1组(K~(G12D)组);B1组(K~(G12D)Pggtlb~(△/△)组);C1组(K~(G12D)Fntb~(△/△)组)和A2组(K~(LSL)组);B2组(K~(LSL)Pggtlb~(fl/fl)组):C2组(K~(LSL)Fntb~(fl/fl)组)。
3.基因敲除率测定
为验证Cre介导的基因敲除效果,应用定量PCR分析了基因组DNA和cDNA。与脾脏比较,肺脏各基因的敲除率均显著增高(P均＜0.05)。
4.各组实验动物体重的变化
各组实验鼠的基础体重无显著性差异。A1组、B1组和C1组治疗后体重增加,但增加程度均显著低于对照组(P＜0.01)。在A1组体重增加缓慢,由基础状态的(9.16±0.42)增至3周末的(10.10±0.36),B1组和C1组体重增加趋势相似,组间未见显著性差异(P＞0.05)。
5.各组动物肺脏与体重比值(LW/BW)变化
与A1组比较,B1组、C1组及对照组均显著降低(P均＜0.01),在Cre感染后3周时,A1组肺体重显著增加,这可能与其肿瘤形成有关。
6.各组实验动物脾脏与体重(SW/BW)比值变化
3周末时,与对照组比较,A1组、B1组和C1组显著增高(P均＜0.01);而A1组、B1组和C1组间无统计学意义。5周末时,与A1组比较,B1组、C1组及对照组均显著降低(P均＜0.01)。
7.各组实验动物生存率分析
对照组生存率显著高于实验组(P均＜0.01);与A1组比较,B1和C1组生存率显著延长(P＜0.01),而B1组和C1组间无统计学意义。A1组在Cre感染后10天内未见有肺癌相关的死亡,但其后生存状态迅速恶化,绝大部分在3周内死于呼吸困难。
8.病理学检查
3周时均可见非典型腺瘤性增生(AAH)、支气管上皮细胞增生(EH)和腺瘤三种病理改变,但A1组病变更为明显且出现弥漫性腺癌阻塞肺泡腔。5周时,可见弥漫性腺癌细胞堵塞了大部分肺泡腔。肿瘤细胞具有深染的、不规则的细胞核,核浆比例明显失调,癌细胞散在或融合成片,肿瘤细胞团直径显著增大,B1组和C1组病变程度较A1组明显减轻。正常的肺脏未见肿瘤组织,说明相关基因未被激活。
9.免疫组织化学染色
免疫组化染色显示在肺肿瘤组织中单核细胞/巨噬细胞标志CD11b、CD4/CD8染色均为阴性,细支气管细胞标志CC10染色部分为阴性,部分阳性;肺泡Ⅱ型细胞标记SP-C染色为阳性。与A1组比较,B1组和C1组SP-C和Ki-67染色明显减弱。
10.Western blot检测
B1组和C1组中可见非异戊烯化RAP1蛋白表达明显增高,对照组和A1组无表达;在B1组和C1组均可见P-AKT蛋白表达水平显著升高,A1组、B2组和C2组无明显变化;在实验组P-ERK1/2蛋白表达均降低,其中B1组降低最为显著。各组间总AKT和总ERK1/2蛋白表达水平无显著差异。
结论
(1)利用条件性敲基因技术定时、定点的敲入癌基因K-ras能建立符合人类肺癌病变特征的动物模型;(2)条件性敲除Pggtlb基因或Fntb基因能抑制K-ras突变诱导肺癌的发生和发展;(3)Pggtlb基因或Fntb基因敲除后抑制K-ras突变诱导肺癌发生和发展的分子机制是抑制Ras蛋白的转录后修饰,从而下调Ras蛋白依赖性激活的ERK的蛋白表达、抑制肿瘤细胞的增殖能力。
第二部分体外部分
背景
基因突变是肿瘤发生、发展的重要原因,在多种肿瘤中均存在癌基因的突变。非小细胞肺癌(Non-small cell lung cancer,NSCLC)约占肺癌的70%,它是目前世界上发病率和死亡率最高的恶性肿瘤之一,严重影响着人类健康。在NSCLC中,K-ras是最常见的癌基因之一,它可促进肺癌的发生、发展。因此,K-ras基因在诱发肺癌发生、发展中的作用日益引起学者的重视。
研究表明,RAS蛋白属于具有CAAX基序的细胞内蛋白的一种,这类蛋白末端的半胱氨酸羧基可在牛龙酰基转移酶Ⅰ(GGTase-I)催化下进行牛龙基化修饰,亦可在法呢酰基转移酶(FTase)催化下进行法尼基化修饰,这两种转录后修饰被称之为异戊二烯化。GGTase-I和FTase具有共同的α亚单位,而其β亚单位的差异决定了其底物的特异性。在动物实验中,利用条件性基因敲除技术敲除GGTase-I或FTase基因后可抑制K-ras诱导肺癌的发生、发展,但敲除GGTase-I或FTase基因对鼠胚胎成纤维细胞(mouse embryo fibroblast,MEF)活力影响的文献尚未见报道。
因此,为进一步探讨GGTase-I或FTase基因MEF的影响,本研究旨在应用条件性敲基因技术建立K-ras、GGTase-I和FTase基因的条件性敲除细胞模型,并运用分子生物学、免疫学等研究手段,阐明GGTase-I或FTase基因对K-ras突变细胞株的影响及其分子机制,为诊治K-ras基因诱导的肺癌提供科学依据。
目的
(1)利用条件性敲基因技术建立建立K-ras和GGTase-I或FTase基因的条件性敲基因MEF模型;(2)观察K-ras基因突变对各组细胞株生物特性的影响;(3)研究条件性敲除GGTase-I或FTase基因后对K-ras突变生物特性的影响;(4)探讨条件性敲除GGTase-I或FTase基因后抑制K-ras突变MEF生物特性的分子机制。
方法
1.MEF细胞株的获取
以胎龄为12.5-17.5天的K~(LSL)、K~(LSL)Pggtlb~(fl/fl)和K~(LSL)Fntb~(fl/fl)鼠为对象,取其胚胎,获取K~(LSL)MEF、K~(LSL)Pggtlb~(fl/fl)和K~(LSL)Fntb~(fl/fl)MEF。
2.实验细胞分组:按基因型分为三组:即K~(LSL)(A组)、K~(LSL)Pggtlb~(fl/fl)(B组)和K~(LSL)Fntb~(fl/fl)(C组),各组根据感染病毒种类的不同又分为两个亚组即:Cre腺病毒载体感染组(实验组,A1组、B1组和C1组)和β-gal腺病毒载体感染组(对照组,A2组、B2组和C2组)。
3.实验细胞腺病毒感染:应用cre和β-gal腺病毒载体分别感染各组细胞株。
4.基因分型:利用PCR技术对各组细胞株进行基因分型。
5.Real-time PCR:检测各组细胞株GGTase-I、FTase和K~(LSL)的mRNA的表达水平。
6.各组MEF生物特性检测:分别检测各组细胞株的生长情况、克隆形成能力、迁移能力和凋亡情况。
7.统计学分析:连续性数据用(?)±SD表示,离散数据用例数和百分数表示。组间生长曲线比较应用单因素方差分析,其余指标应用T检验。应用SPSS统计软件进行统计学处理(version 11.0;SPSS Inc),P＜0.05有统计学差异。
结果
1.MEF基因敲除率检测:实验组中均可见K~(LSL)的活化及GGTase-I和FTase的敲除且其敲除率显著高于体内实验,而对照组中未见K~(LSL)的活化及GGTase-I或FTase的敲除。
2.各组MEF基因干预后基因型检测
各组基因分型与体内部分相同。
3.各组MEF生长能力测定
在实验组中,与B1组、C1组比较,A1组生长显著增快,而B1组和C1组间无显著性差异。对照组中各亚组间无显著性差异。A1组和A2组、B1组和B2组、C1组和C2组间比较均有显著性差异。
4.各组MEF迁移能力测定
12h和24h时A1组与A2组、B1组与B2组和C1组与C2组之间均有显著性差异;A1组显著高于B1组和C1组,而对照组中各亚组间无显著性差异。
5.各组MEF克隆形成检测
只有A1组形成克隆,而B1组和C1组细胞未见克隆形成。对照组中亦未见克隆形成。
6.各组MEF凋亡检测
A1组MEF的凋亡率为3.31±1.34%,B1组为13.91%±1.88%,C1组为9.57%±2.45%。与A1组比较,B1组和C1组的细胞凋亡率显著增高,且B1组显著高于C1组,而对照组中各亚组间无显著性差异。
7.Western blot
与A2组比较,A1组Caspase-3的蛋白表达水平显著降低,在B组和C组中,与各自亚组比较,B1组和C1组Caspase-3的蛋白表达水平显著增高。磷酸化-P70蛋白表达水平在各组间无显著性差异。在B组和C组中,与各自亚组比较,B1组和C1组PARP的蛋白表达水平显著增高,而A1组与A2组间无显著性差异。
结论
(1)应用条件性敲基因技术可建立同时伴有K-ras活化和GGTase-I或FTase基因敲除的细胞株;(2)K-ras基因突变显著增强细胞的增殖能力、迁移能力和克隆形成能力并抑制其凋亡能力;(3)条件性敲除GGTase-I或FTase基因后可显著抑制细胞的增殖能力、迁移能力和克隆形成能力并增强其凋亡能力;(4)GGTase-I或FTase可作为治疗K-ras基因活化诱发的肺癌的靶基因;(5)敲除GGTase-I或FTase基因作用于K-ras突变MEF的主要机制是:抑制K-ras蛋白转录后修饰,增强凋亡基因表达,促进细胞凋亡。
PartⅠstudy in vivo
Background
Recent studies have demonstrated that lung cancer is the leading morbility and fatality rate among malignance tumor in the worldwide.Lung cancer is a gene disease; its genesis is a multistep process involving the cooperation of oncogenic mutations and antioncogenic inactivation.Therefore,elucidating the molecular mechanism and blockage the correlated link of lung cancer is very significance in clinical study.K-ras and P53 gene are the most common oncogenes and anti-oncogene.For example,K-ras mutations occur with high frequency in human colon,pancreas and lung cancer,and the presence of this strongly correlates with disease progression to malignancy. Moreover,there is recent evidence that activation of Ras to promote cancer progression in mouse models for lung.Recently,the study of the role of K-ras gene in the genesis of lung cancer has been the hot study.
More than 100 intracellular proteins contain a CAAX motif that directs isoprenylation at a carboxyterminal cysteine(the "C" of the CAAX motif).Some CAAX proteins,such as RHOA,cell division cycle 42(CDC42),and RAP1,are geranylgeranylated by protein geranylgeranyltransferase typeⅠ(GGTase-I).Others, such as K-RAS and N-RAS,are farnesylated by protein farnesyltransferase(FTase). If the "X" of the CAAX motif is a leucine,the protein is generally geranylgeranylated; otherwise,it is farnesylated.Isoprenylation renders the carboxyl terminus of the CAAX proteins more hydrophobic,enhancing their ability to bind to membranes within cells,and also regulates protein-protein interactions.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.Theirβsubunits are called Pggtlb or Fntbs,respectively.
In some eukaryotic cells,GGTase-I is an essential enzyme.Null mutations in theβsubunit of GGTase-I are lethal in both Drosophila melanogaster and Saccharomyces cerevisiae.The lethality of GGTase-I deficiency in eukaryotic cells was likely due to the failure to geranylgeranylate Rho1p and Cdc42p,as the lethality could be overcome by expressing mutant Rho1p and Cdc42p proteins engineered to undergo farnesylation by FTase.The realization that the RAS proteins are farnesylated has fueled interest in protein isoprenylation.Farnesylation is important for the proper membrane targeting of RAS proteins and for their transforming ability. In mouse models,farnesyltransferase inhibitors(FTIs) have significant antitumor activity and minimal toxicity.In human clinical trials,however,FTIs have been disappointing,at least for the treatment of solid tumors,likely because K-ras and N-ras -the Ras isoforms most often implicated in human cancer-can be geranylgeranylated in the presence of an FTI.At the same time,inhibition of GGTase-I ameliorated disease phenotypes in a mouse model of multiple sclerosis, inhibited hepatitis C viral replication in hepatoma cell.
According to the inhibitory role of GGTase-I or FTase gene in K-ras mutation induced lung cancer and the results of fundament study.The present study was designed to test the hypothesis that the role of inactivation of Pggtlb or Fntb gene in K-ras induced lung cancer due to its anti-proliferation in a mouse model of lung cancer created mice with a conditional knockout allele.The result of this study will provide science evidence for gene therapy.
Objective
(1) To establish an animal model of lung cancer that are mimic to human pathological changes and convenient for study;(2) To investigate the inhibitory role of knockout GGTase-I or FTase gene on K-ras induced lung cancer transformation;(3) To investigate the molecular mechanisms of GGTase-I or FTase gene on the genesis of K-ras induced lung cancer in vivo.
Methods
1.Animal model:we created mice with a conditional knockout or knockin allele for GGTase-I(Pggtlb~(fl/fl))、FTase(Fntb~(fl/fl)) and K-ras(K~(LSL)).And then K~(LSL) mice were bred with Pggtlb~(fl/fl) mice or Fntb~(fl/fl)mice,generating K~(LSL)Pggtlb~(fl/fl) mice and K~(LSL) Fntb~(fl/fl)mice.
2.According the genotype of mice,they were divided into three groups:K~(LSL)(A group)、K~(LSL)Pggtlb~(fl/fl)(B group) and K~(LSL)Fntb~(fl/fl)(C group),then they were divided into two subgroups according to different adenovirus:cre-adenovirus groups(Study groups:A1 group、B1 group and C1 group) and gal-adenovirus groups(Control groups:A2 group、B2 group and C2 group).
3.Adenovirus infection of animals:On the day of 14,all animals were infected with Cre-adenovirus orβ-gal-adenovirus by aerosol rebreathing method.
4.Genotype:The K~(LSL),Fntb~(fl) and Pggtlb~(fl) allele was genotyped by PCR amplification of genomic DNA from tail biopsies.
5.Body weight:after infection with adenovirus,the weight of all mousse was measured every week.
6.Survival for all animals:the life span of all animals was detected from the treatment.
7.Histopathological analysis:The lung,spleen and liver were processed and examined by hematoxylin and eosin staining.
8.Immunohistochemical staining was performed and the expressions of CD11b、SP-C、CC10 and Ki-67 were detected.
9.Real-time PCR:The mRNA expressions of Pggtlb~(fl/fl)、Fntb~(fl/fl) and K~(LSL) in the lung,liver,cell lines and spleen tissue were analyzed using real-time PCR technique.
10.Western blot:The protein expressions of nonprenylated RAP1、total RAP1、RHOA、phosphorylated-ERK1/2、phosphorylated-AKT、total AKT and total ERK1/2 in lung,liver,cell lines and spleen tissue were analyzed using western blot technique.
11.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival rates of animal were compared with Kaplan-Meier curve test and other indexes were compared with One-Way ANOVA comparison test.P＜0.05 was considered statistically significant.
Results
1.General state of the experimental animals:60 mice were infected with adenovirus in all groups.19 mice in group A,19 mouse in group B and 20 mouse in group C.
2.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I or FTased was silencing in the tumor tissue.
3.Body weight:body weights in the control groups were essentially equal.Since two weeks after infection,body weight in group B1 and C1 were significantly higher than that in group A1(P all＜0.01).Compared with the control group,body weight in the group A1,group B1,and group C1 were significantly decreased(P all＜0.01).
4.The rate of lung or spleen weight on body weight
Compared A1 group,the lung weight relative to total body weight(LW/BW) of group B1 and group C1 were significantly decreased(P all＜0.01),while the spleen weight relative to total body(SW/BW) of group B1 and group C1 were significantly decreased(P all＜0.01).Compared the group B1,the LW/BW of group C1 is significantly increased(P＜0.01),and the SW/BW of group C1 is significantly increased(P＜0.01).
5.Pathologic staining:Most mouse in the trial groups developed tumor cells with large and trachychromatic nuclear,diffuse hyperplasia and leukocyte infiltration were seen in the lung tumor tissue.Compared with group A1,there are little tumor cells and leukocyte infiltration in the group B1 and C1.In the group A1,the diffuse adenocarcinoma has obliterated the majority of alveolar spaces in lung.
6.Immunohistochemical staining:The lung tumors of K~(LSL)、K~(LSL)Pggtlb~(fl/fl) and K~(LSL)Fntb~(fl/fl) mice were negative for the monocyte/macrophage marker,the Clara cell marker,and the T cell markers but were positive for the typeⅡpneumocyte marker SP-C as judged by immunohistochemistry.Compared with group A1,the expression of SP-C in lung tumor tissue was significantly decreased in group B1 and C1,while Ki-67 was significantly enhanced.
7.Real-time PCR:The mRNA expressions of Pggtlb~(fl/fl),Fntb~(fl/fl) and K~(LSL)mRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2(P＜0.01 or 0.05).Comparison the group A2,the group A1 had higher K~(LSL) mRNA expressions(P＜0.01).
8.Western blot:The protein expression of P-AKT in the tissues of trial group were increased,and P-ERK1/2 were decreased.The nonprenylated RAP1 was only detected in the group B1 and C1.No difference was found about the total AKT and total ERK1/2.
9.Survival rate:Kaplan-Meier curve showing the survival of mouse in the group B1 and group C1 was significantly improved than that in group A1,while there is no significant difference between the group B1 and group C1.Compared with the trial groups,the survival the control group was significantly improved(P＜0.001).
Conclusions
(1) The method of conditional knockout allele technique is an efficient and time-saving way to establish lung cancer model induced by K-ras;(2) The special gene can be expressed in lung by inhale adenovirus.(3) Inactivation of Pggtlb or Fntb by conditional knockout technique can reduce the tumor formation and improves survival in mice with K-ras-induced lung cancer;(4) GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras;(5) The molecular mechanisms of Pggtlb or Fntb inactivation to treat the lung cancer induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein.
PartⅡin vitro study
Background
Gene mutation is the main cause of the genesis of cancer and there is gene mutation in many solid tumor.Non-small cell lung cancer(NSCLC) may be occupy more than 70%of all the pulmonary carcinoma.It is the leading morbility and fatality rate among malignance tumor in the worldwide and shorten the life span.K-ras gene is the most common oncogene in NSCLC.Therefore,the study of the role of K-ras in the genesis of lung cancer has been the hot study.
Recent studies have demonstrated that RAS protein belong to the intracellular proteins contain a CAAX motif.Parts of these proteins can be geranylgeranylated by protein geranylgeranyltransferase typeⅠ(GGTase-I) and the others can farnesylated by protein farnesyltransferase(FTase) alternatively.This is the prenylation process by post-transcriptional modification.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.In mouse study,We have proved that inactivation of GGTase-I or FTase can inhibit the development of lung cancer induced by K-ras.But we do not know the effects of inactivation of GGTase-I or FTase on the mouse embryo fibroblast(MEF) with K-ras mutation.
To investigate the effects of the inactivation of GGTase-I or FTase on K-ras mutation MEF,The present study was designed to test the role of the inactivation GGTase-I or FTase on K-ras induced MEF through a conditional knockout technique.
Objective
(1) To establish the cell model with the K-ras and GGTase-I or FTase knockout; (2) To observe the effects of K-ras on the bionomics in vitro,such as curve growth, clone formation in soft agar and migration;(3) To investigate the inhibitory role of the inactivation of GGTase-I or FTase on K-ras induced MEF;(4) To investigate the molecular mechanisms of the inactivation of GGTase-I or FTase on the K-ras induced MEF.
Methods
1.Isolation of embryonic fibroblasts:The mouse embryonic fibroblasts(MEF) were isolated from K~(LSL)、K~(LSL)Pggtlb~(fl/fl) and K~(LSL)Fntb~(fl/fl) embryos on day 15.5 post coitum.
2.cell lines groups:According the genetype of cell lines,they were divided into three groups:K~(LSL)(A group)、K~(LSL)Pggtlb~(fl/fl)(B group) and K~(LSL)Fntb~(fl/fl)(C group), then they were divided into two subgroups according to different adenovirus:creadenovirus groups(Trial groups,A1 group、B1 group and C1 group) andβ-gal-adenovirus groups(Control groups,A2 group、B2 group and C2 group).
3.Adenovirus infection of cell lines:To inactivate Pggtlb,Fntb~(fl) and activate K-RAS~(G12D) expression,all cells were seeded and infected with Cre-adenovirus orβ-gal-adenovirus for two times.
4.Genotype:The K~(LSL),Fntb~(fl) and Pggtlb~(fl) allele was genotyped by PCR amplification of genomic DNA from tail biopsies.
5.Real-time PCR:The mRNA expressions of GGTase-I、FTase and K~(LSL) in the cell lines were analyzed using real-time PCR technique.
6.Bionomics of cell lines:The bionomics of cell lines was detected,such as cell growth curve,clone formation in soft agar,migration and apoptosis.
7.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival curve of cell lines were compared with One-Way ANOVA comparison test and other indexes were compared with T test.P＜0.05 was considered statistically significant.
Results
1.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I and FTased was inactived.
2.Real-time PCR:The mRNA expressions of GGTase-I,FTase and K~(LSL) mRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2(P＜0.01 or 0.05).Comparison of the group A1 and group A2 revealed that group A1 had higher K~(LSL) mRNA expressions than group A2(P＜0.01).
2.Genetype of the MEF lines
The genetype of the cell lines are the same as that in the animal.
3.The growth of MEF lines
In the trial group,compared with group B1 and group C1,the group A1 grows significantly quickly.No significant difference was found between the group B1 and group C1.No significant difference was found between the trial group and the control group.There is no significant difference in the control group.
4.Migration of the MEF lines
Compared with the control group,the ability of migration in the trial group was increased significantly.In the trial group,the number of migration cells in the group A1 is much more than that in the group B1 and C1.There is no significant difference in the control group.
5.Clone formation of MEF lines
The clone formation was only seen in the group A1,and no clone in the group B1 and C1.There is also no clone in the control group.
6.Apoptosis of MEF lines
The rate of apoptosis in the group A1 is 3.31±1.34%,the group B1 is 13. 91%±1.88%,and the group C1 is 9.57%±2.45%.Compared with the group A1,the rates of apoptosis in the group B1 and C1 are increased significantly,and the group B1 is significant higher than that in the group C1.There is no significant difference in the control group.
7.Western blot
Compared with the group A2,the expression of Caspase-3 in the group A1 was significantly decreased.The expression of Caspase-3 in the group B1 and C1 was significantly lower than that in the group B2 and C2.The expression of phos-P70 in all the cell lines was no significant difference.The expression of PARP in the group B1 and C1 was significantly higher than that in the group B2 and C2,and no significant difference in the group A1 and A2.
Conclusions
(1) The method of conditional knockout allele technique is an efficient and time-saving way to establish MEF model induced by K-ras;(2) Activation of K-ras can promote the ability of cancer cell growth,migration,clone formation,and inhibit the apoptosis;(3) Inactivation of GGTase-I or FTase by conditional knockout technique can reduce the K~(LSL) cell viability,proliferation and enhanced the apoptosis;; (4) GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras; (5) The molecular mechanisms of the inactivation of GGTase-I or FTase to affect the MEF induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein,enhance the expression of apoptosis gene,promote the apoptosis,and suppressor cell division.
背景
肺癌是目前发病率和病死率均很高的恶性肿瘤,严重影响着人类健康。肺癌是一种基因病,癌基因激活和抑癌基因失活是其重要的分子事件。因此,阐明肺癌发生的分子机制,从分子水平阻断其发生的相关链接具有极为重要的临床意义。K-ras是目前研究最多的癌基因之一,在许多肿瘤中均存在K-ras基因的扩增或过度表达,其中在肺腺癌中大约有50%左右的患者存在K-ras突变,说明其在肺癌发生、发展中发挥重要作用。深入研究K-ras基因在肺癌发生中的作用机制,尽早阻断其信号系统已成为近年来肺癌研究领域中一个新的热点。
大约有100多种细胞内蛋白具有CAAX基序,K-ras基因编码蛋白属于其中的一种,这些蛋白可以进行多种转录后修饰,其中半胱氨酸残基(C末端CAAX基序)可直接进行异戊二烯化修饰。一部分CAAX蛋白,其末端可以在牛龙酰基转移酶Ⅰ(GGTase-I)的作用下进行牛龙基化修饰,还有一部分CAAX蛋白,例如K-Ras,在法呢酰基转移酶(FTase)的作用下进行法尼基化修饰,其中的“X”残基表明这种CAAX蛋白是被法尼基化还是牛龙基化修饰:一般情况下,如果“X”是亮氨酸,这种蛋白就会被牛龙基化修饰,否则就会被法尼基化修饰。CAAX蛋白末端进行异戊二烯化修饰后可以增强其蛋白羧基端的疏水性,进而增强膜的亲和力及与配体的结合能力,从而增强其生物学活性。FTase和GGTase-I有一个相同的α亚单位,但是分别具有各自独特的表明其底物特异性的β亚单位。例如,GGTase-I是由Fnta编码的一个α亚单位和Pggtlb编码的独特的β亚单位组成。
GGTase-I是许多真核细胞的必需酶,在果蝇和酵母菌种GGTase-I酶的β亚单位的无效突变通常是致死性的。GGTase-I缺乏导致生物死亡的主要原因是Rho1p和Cdc42p蛋白无法进行牛龙基化,而这种牛龙基化在特定条件下可通过FTase进行法尼基化弥补。RAS蛋白的法尼基化已引起学者的足够重视,研究表明,Ras蛋白的法尼基化对其靶向定位于细胞膜上的结合位点和保持正常的转化能力是非常必须的。在动物模型中,法呢酰基转移酶抑制剂(FTIs)具有高效的抗肿瘤活性且毒性低,而在临床试验中,FTIs的疗效并不理想,其可能原因是FTase缺乏时RAS蛋白进行了牛龙基化。许多研究已证实GGTIs能抑制多种K-ras突变肿瘤细胞株的增殖,同时研究还表明GGTIs能改善多发性硬化症动物模型的表型以及抑制肝癌中肝炎病毒的复制等。
鉴于GGTase-I和FTase在K-ras突变诱导肿瘤发生中的作用以及前期研究结果,本研究旨在应用条件性敲基因技术建立动物模型,并运用组织病理学、分子生物学、免疫学等研究手段,充分阐明条件性敲除GGTase-I的β亚单位(Pggtlb)或FTase的β亚单位(Fntb)后,阻断CAAX蛋白异戊二烯化修饰,从而抑制K-ras基因突变诱导的肺癌发生的作用,为肺癌的基因治疗提供科学依据。
目的
(1)利用条件性基因敲除技术建立符合人类肺癌病变特征的动物模型;(2)应用组织病理学等方法观察肺癌组织病理形态,探讨Pggtlb或Fntb基因敲除对K-ras基因突变诱导肺癌形成的抑制作用;(3)探讨Pggtlb或Fntb基因敲除抑制K-ras基因突变诱导肺癌发生的分子生物学机制。
方法
1.条件性敲基因动物模型的建立及其基因型验证
利用Cre/lox P系统条件性敲入K-ras基因,敲除GGTase-I或FTase基因,分别建立K-ras(K~(LSL))、GGTase-I(Pggtlb~(fl/fl))和FTase(Fntb~(fl/fl))条件性敲基因鼠模型。然后以K~(LSL)鼠和Pggtlb~(fl/fl)鼠或Fntb~(fl/fl)鼠杂交产生K~(LSL)Pggtlb~(fl/fl)鼠和K~(LSL)Fntb~(fl/fl)鼠,经基因分型后筛选出符合要求的小鼠。应用PCR技术进行基因型验证。
2.实验动物分组
按基因型分为三组:即K~(LSL)(A组)、K~(LSL)Pggtlb~(fl/fl)(B组)和K~(LSL)Fntb~(fl/fl)(C组),各组根据感染病毒种类的不同又分为两个亚组即:cre腺病毒载体感染组(实验组:A1组、B1组和C1组)和β-gal腺病毒载体感染组(对照组:A2组、B2组和C2组)。
3.干预方法
第14天龄时应用雾化吸入法进行腺病毒载体干预,分别敲除或激活K~(LSL)、K~(LSL)Pggtlb~(fl/fl)和K~(LSL)Fntb~(fl/fl)基因。
4.称量体重
病毒感染前及以后每周末称量所有实验鼠体重。
5.各组动物生存率测定
自基因干预起,观察各组动物的生存时间。
6.病理学检查
不同时间点处死动物,留取肺脏、肝脏、脾脏等组织器官并称量其重量,一部分组织进行苏木素-伊红(HE)染色,观察肿瘤细胞形态;另一部分留取标本进行分子生物学检测。
7.免疫组织化学染色
进行CD4、CC10、CD8、SP-C、非异戊烯化Rap1A及Ki-67的免疫组织化学染色。
8.Real-time PCR
检测肺脏、肝脏和脾脏等组织器官中Pggtlb、Fntb和K~(LSL)等基因的mRNA表达水平。
9.Western blot
检测肺脏肿瘤组织中非异戊烯化RAP1A、总RAP1A、p-ERK1/2、p-AKT、总AKT、总ERK1/2等的蛋白表达水平。
10.统计学分析
连续性数据用(?)±SD表示,离散数据用例数和百分数表示。三组间生存率的比较应用Kaplan-Meier方法,其余指标应用单因素方差分析。应用SPSS统计软件进行统计学处理(version 11.0;SPSS Inc),P＜0.05有统计学差异。
结果
1.实验动物基本情况
腺病毒载体干预动物的共60只。最终完成基因干预的共58只,其中A1组10只,A2组9只,B1组10只,B2组9只,C1组10只,C2组10只。本文对完成实验的58只实验鼠的资料进行分析。
2.各组动物的基因分型
共有6个亚组,分别是A1组(K~(G12D)组);B1组(K~(G12D)Pggtlb~(△/△)组);C1组(K~(G12D)Fntb~(△/△)组)和A2组(K~(LSL)组);B2组(K~(LSL)Pggtlb~(fl/fl)组):C2组(K~(LSL)Fntb~(fl/fl)组)。
3.基因敲除率测定
为验证Cre介导的基因敲除效果,应用定量PCR分析了基因组DNA和cDNA。与脾脏比较,肺脏各基因的敲除率均显著增高(P均＜0.05)。
4.各组实验动物体重的变化
各组实验鼠的基础体重无显著性差异。A1组、B1组和C1组治疗后体重增加,但增加程度均显著低于对照组(P＜0.01)。在A1组体重增加缓慢,由基础状态的(9.16±0.42)增至3周末的(10.10±0.36),B1组和C1组体重增加趋势相似,组间未见显著性差异(P＞0.05)。
5.各组动物肺脏与体重比值(LW/BW)变化
与A1组比较,B1组、C1组及对照组均显著降低(P均＜0.01),在Cre感染后3周时,A1组肺体重显著增加,这可能与其肿瘤形成有关。
6.各组实验动物脾脏与体重(SW/BW)比值变化
3周末时,与对照组比较,A1组、B1组和C1组显著增高(P均＜0.01);而A1组、B1组和C1组间无统计学意义。5周末时,与A1组比较,B1组、C1组及对照组均显著降低(P均＜0.01)。
7.各组实验动物生存率分析
对照组生存率显著高于实验组(P均＜0.01);与A1组比较,B1和C1组生存率显著延长(P＜0.01),而B1组和C1组间无统计学意义。A1组在Cre感染后10天内未见有肺癌相关的死亡,但其后生存状态迅速恶化,绝大部分在3周内死于呼吸困难。
8.病理学检查
3周时均可见非典型腺瘤性增生(AAH)、支气管上皮细胞增生(EH)和腺瘤三种病理改变,但A1组病变更为明显且出现弥漫性腺癌阻塞肺泡腔。5周时,可见弥漫性腺癌细胞堵塞了大部分肺泡腔。肿瘤细胞具有深染的、不规则的细胞核,核浆比例明显失调,癌细胞散在或融合成片,肿瘤细胞团直径显著增大,B1组和C1组病变程度较A1组明显减轻。正常的肺脏未见肿瘤组织,说明相关基因未被激活。
9.免疫组织化学染色
免疫组化染色显示在肺肿瘤组织中单核细胞/巨噬细胞标志CD11b、CD4/CD8染色均为阴性,细支气管细胞标志CC10染色部分为阴性,部分阳性;肺泡Ⅱ型细胞标记SP-C染色为阳性。与A1组比较,B1组和C1组SP-C和Ki-67染色明显减弱。
10.Western blot检测
B1组和C1组中可见非异戊烯化RAP1蛋白表达明显增高,对照组和A1组无表达;在B1组和C1组均可见P-AKT蛋白表达水平显著升高,A1组、B2组和C2组无明显变化;在实验组P-ERK1/2蛋白表达均降低,其中B1组降低最为显著。各组间总AKT和总ERK1/2蛋白表达水平无显著差异。
结论
(1)利用条件性敲基因技术定时、定点的敲入癌基因K-ras能建立符合人类肺癌病变特征的动物模型;(2)条件性敲除Pggtlb基因或Fntb基因能抑制K-ras突变诱导肺癌的发生和发展;(3)Pggtlb基因或Fntb基因敲除后抑制K-ras突变诱导肺癌发生和发展的分子机制是抑制Ras蛋白的转录后修饰,从而下调Ras蛋白依赖性激活的ERK的蛋白表达、抑制肿瘤细胞的增殖能力。
第二部分体外部分
背景
基因突变是肿瘤发生、发展的重要原因,在多种肿瘤中均存在癌基因的突变。非小细胞肺癌(Non-small cell lung cancer,NSCLC)约占肺癌的70%,它是目前世界上发病率和死亡率最高的恶性肿瘤之一,严重影响着人类健康。在NSCLC中,K-ras是最常见的癌基因之一,它可促进肺癌的发生、发展。因此,K-ras基因在诱发肺癌发生、发展中的作用日益引起学者的重视。
研究表明,RAS蛋白属于具有CAAX基序的细胞内蛋白的一种,这类蛋白末端的半胱氨酸羧基可在牛龙酰基转移酶Ⅰ(GGTase-I)催化下进行牛龙基化修饰,亦可在法呢酰基转移酶(FTase)催化下进行法尼基化修饰,这两种转录后修饰被称之为异戊二烯化。GGTase-I和FTase具有共同的α亚单位,而其β亚单位的差异决定了其底物的特异性。在动物实验中,利用条件性基因敲除技术敲除GGTase-I或FTase基因后可抑制K-ras诱导肺癌的发生、发展,但敲除GGTase-I或FTase基因对鼠胚胎成纤维细胞(mouse embryo fibroblast,MEF)活力影响的文献尚未见报道。
因此,为进一步探讨GGTase-I或FTase基因MEF的影响,本研究旨在应用条件性敲基因技术建立K-ras、GGTase-I和FTase基因的条件性敲除细胞模型,并运用分子生物学、免疫学等研究手段,阐明GGTase-I或FTase基因对K-ras突变细胞株的影响及其分子机制,为诊治K-ras基因诱导的肺癌提供科学依据。
目的
(1)利用条件性敲基因技术建立建立K-ras和GGTase-I或FTase基因的条件性敲基因MEF模型;(2)观察K-ras基因突变对各组细胞株生物特性的影响;(3)研究条件性敲除GGTase-I或FTase基因后对K-ras突变生物特性的影响;(4)探讨条件性敲除GGTase-I或FTase基因后抑制K-ras突变MEF生物特性的分子机制。
方法
1.MEF细胞株的获取
以胎龄为12.5-17.5天的K~(LSL)、K~(LSL)Pggtlb~(fl/fl)和K~(LSL)Fntb~(fl/fl)鼠为对象,取其胚胎,获取K~(LSL)MEF、K~(LSL)Pggtlb~(fl/fl)和K~(LSL)Fntb~(fl/fl)MEF。
2.实验细胞分组:按基因型分为三组:即K~(LSL)(A组)、K~(LSL)Pggtlb~(fl/fl)(B组)和K~(LSL)Fntb~(fl/fl)(C组),各组根据感染病毒种类的不同又分为两个亚组即:Cre腺病毒载体感染组(实验组,A1组、B1组和C1组)和β-gal腺病毒载体感染组(对照组,A2组、B2组和C2组)。
3.实验细胞腺病毒感染:应用cre和β-gal腺病毒载体分别感染各组细胞株。
4.基因分型:利用PCR技术对各组细胞株进行基因分型。
5.Real-time PCR:检测各组细胞株GGTase-I、FTase和K~(LSL)的mRNA的表达水平。
6.各组MEF生物特性检测:分别检测各组细胞株的生长情况、克隆形成能力、迁移能力和凋亡情况。
7.统计学分析:连续性数据用(?)±SD表示,离散数据用例数和百分数表示。组间生长曲线比较应用单因素方差分析,其余指标应用T检验。应用SPSS统计软件进行统计学处理(version 11.0;SPSS Inc),P＜0.05有统计学差异。
结果
1.MEF基因敲除率检测:实验组中均可见K~(LSL)的活化及GGTase-I和FTase的敲除且其敲除率显著高于体内实验,而对照组中未见K~(LSL)的活化及GGTase-I或FTase的敲除。
2.各组MEF基因干预后基因型检测
各组基因分型与体内部分相同。
3.各组MEF生长能力测定
在实验组中,与B1组、C1组比较,A1组生长显著增快,而B1组和C1组间无显著性差异。对照组中各亚组间无显著性差异。A1组和A2组、B1组和B2组、C1组和C2组间比较均有显著性差异。
4.各组MEF迁移能力测定
12h和24h时A1组与A2组、B1组与B2组和C1组与C2组之间均有显著性差异;A1组显著高于B1组和C1组,而对照组中各亚组间无显著性差异。
5.各组MEF克隆形成检测
只有A1组形成克隆,而B1组和C1组细胞未见克隆形成。对照组中亦未见克隆形成。
6.各组MEF凋亡检测
A1组MEF的凋亡率为3.31±1.34%,B1组为13.91%±1.88%,C1组为9.57%±2.45%。与A1组比较,B1组和C1组的细胞凋亡率显著增高,且B1组显著高于C1组,而对照组中各亚组间无显著性差异。
7.Western blot
与A2组比较,A1组Caspase-3的蛋白表达水平显著降低,在B组和C组中,与各自亚组比较,B1组和C1组Caspase-3的蛋白表达水平显著增高。磷酸化-P70蛋白表达水平在各组间无显著性差异。在B组和C组中,与各自亚组比较,B1组和C1组PARP的蛋白表达水平显著增高,而A1组与A2组间无显著性差异。
结论
(1)应用条件性敲基因技术可建立同时伴有K-ras活化和GGTase-I或FTase基因敲除的细胞株;(2)K-ras基因突变显著增强细胞的增殖能力、迁移能力和克隆形成能力并抑制其凋亡能力;(3)条件性敲除GGTase-I或FTase基因后可显著抑制细胞的增殖能力、迁移能力和克隆形成能力并增强其凋亡能力;(4)GGTase-I或FTase可作为治疗K-ras基因活化诱发的肺癌的靶基因;(5)敲除GGTase-I或FTase基因作用于K-ras突变MEF的主要机制是:抑制K-ras蛋白转录后修饰,增强凋亡基因表达,促进细胞凋亡。
PartⅠstudy in vivo
Background
Recent studies have demonstrated that lung cancer is the leading morbility and fatality rate among malignance tumor in the worldwide.Lung cancer is a gene disease; its genesis is a multistep process involving the cooperation of oncogenic mutations and antioncogenic inactivation.Therefore,elucidating the molecular mechanism and blockage the correlated link of lung cancer is very significance in clinical study.K-ras and P53 gene are the most common oncogenes and anti-oncogene.For example,K-ras mutations occur with high frequency in human colon,pancreas and lung cancer,and the presence of this strongly correlates with disease progression to malignancy. Moreover,there is recent evidence that activation of Ras to promote cancer progression in mouse models for lung.Recently,the study of the role of K-ras gene in the genesis of lung cancer has been the hot study.
More than 100 intracellular proteins contain a CAAX motif that directs isoprenylation at a carboxyterminal cysteine(the "C" of the CAAX motif).Some CAAX proteins,such as RHOA,cell division cycle 42(CDC42),and RAP1,are geranylgeranylated by protein geranylgeranyltransferase typeⅠ(GGTase-I).Others, such as K-RAS and N-RAS,are farnesylated by protein farnesyltransferase(FTase). If the "X" of the CAAX motif is a leucine,the protein is generally geranylgeranylated; otherwise,it is farnesylated.Isoprenylation renders the carboxyl terminus of the CAAX proteins more hydrophobic,enhancing their ability to bind to membranes within cells,and also regulates protein-protein interactions.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.Theirβsubunits are called Pggtlb or Fntbs,respectively.
In some eukaryotic cells,GGTase-I is an essential enzyme.Null mutations in theβsubunit of GGTase-I are lethal in both Drosophila melanogaster and Saccharomyces cerevisiae.The lethality of GGTase-I deficiency in eukaryotic cells was likely due to the failure to geranylgeranylate Rho1p and Cdc42p,as the lethality could be overcome by expressing mutant Rho1p and Cdc42p proteins engineered to undergo farnesylation by FTase.The realization that the RAS proteins are farnesylated has fueled interest in protein isoprenylation.Farnesylation is important for the proper membrane targeting of RAS proteins and for their transforming ability. In mouse models,farnesyltransferase inhibitors(FTIs) have significant antitumor activity and minimal toxicity.In human clinical trials,however,FTIs have been disappointing,at least for the treatment of solid tumors,likely because K-ras and N-ras -the Ras isoforms most often implicated in human cancer-can be geranylgeranylated in the presence of an FTI.At the same time,inhibition of GGTase-I ameliorated disease phenotypes in a mouse model of multiple sclerosis, inhibited hepatitis C viral replication in hepatoma cell.
According to the inhibitory role of GGTase-I or FTase gene in K-ras mutation induced lung cancer and the results of fundament study.The present study was designed to test the hypothesis that the role of inactivation of Pggtlb or Fntb gene in K-ras induced lung cancer due to its anti-proliferation in a mouse model of lung cancer created mice with a conditional knockout allele.The result of this study will provide science evidence for gene therapy.
Objective
(1) To establish an animal model of lung cancer that are mimic to human pathological changes and convenient for study;(2) To investigate the inhibitory role of knockout GGTase-I or FTase gene on K-ras induced lung cancer transformation;(3) To investigate the molecular mechanisms of GGTase-I or FTase gene on the genesis of K-ras induced lung cancer in vivo.
Methods
1.Animal model:we created mice with a conditional knockout or knockin allele for GGTase-I(Pggtlb~(fl/fl))、FTase(Fntb~(fl/fl)) and K-ras(K~(LSL)).And then K~(LSL) mice were bred with Pggtlb~(fl/fl) mice or Fntb~(fl/fl)mice,generating K~(LSL)Pggtlb~(fl/fl) mice and K~(LSL) Fntb~(fl/fl)mice.
2.According the genotype of mice,they were divided into three groups:K~(LSL)(A group)、K~(LSL)Pggtlb~(fl/fl)(B group) and K~(LSL)Fntb~(fl/fl)(C group),then they were divided into two subgroups according to different adenovirus:cre-adenovirus groups(Study groups:A1 group、B1 group and C1 group) and gal-adenovirus groups(Control groups:A2 group、B2 group and C2 group).
3.Adenovirus infection of animals:On the day of 14,all animals were infected with Cre-adenovirus orβ-gal-adenovirus by aerosol rebreathing method.
4.Genotype:The K~(LSL),Fntb~(fl) and Pggtlb~(fl) allele was genotyped by PCR amplification of genomic DNA from tail biopsies.
5.Body weight:after infection with adenovirus,the weight of all mousse was measured every week.
6.Survival for all animals:the life span of all animals was detected from the treatment.
7.Histopathological analysis:The lung,spleen and liver were processed and examined by hematoxylin and eosin staining.
8.Immunohistochemical staining was performed and the expressions of CD11b、SP-C、CC10 and Ki-67 were detected.
9.Real-time PCR:The mRNA expressions of Pggtlb~(fl/fl)、Fntb~(fl/fl) and K~(LSL) in the lung,liver,cell lines and spleen tissue were analyzed using real-time PCR technique.
10.Western blot:The protein expressions of nonprenylated RAP1、total RAP1、RHOA、phosphorylated-ERK1/2、phosphorylated-AKT、total AKT and total ERK1/2 in lung,liver,cell lines and spleen tissue were analyzed using western blot technique.
11.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival rates of animal were compared with Kaplan-Meier curve test and other indexes were compared with One-Way ANOVA comparison test.P＜0.05 was considered statistically significant.
Results
1.General state of the experimental animals:60 mice were infected with adenovirus in all groups.19 mice in group A,19 mouse in group B and 20 mouse in group C.
2.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I or FTased was silencing in the tumor tissue.
3.Body weight:body weights in the control groups were essentially equal.Since two weeks after infection,body weight in group B1 and C1 were significantly higher than that in group A1(P all＜0.01).Compared with the control group,body weight in the group A1,group B1,and group C1 were significantly decreased(P all＜0.01).
4.The rate of lung or spleen weight on body weight
Compared A1 group,the lung weight relative to total body weight(LW/BW) of group B1 and group C1 were significantly decreased(P all＜0.01),while the spleen weight relative to total body(SW/BW) of group B1 and group C1 were significantly decreased(P all＜0.01).Compared the group B1,the LW/BW of group C1 is significantly increased(P＜0.01),and the SW/BW of group C1 is significantly increased(P＜0.01).
5.Pathologic staining:Most mouse in the trial groups developed tumor cells with large and trachychromatic nuclear,diffuse hyperplasia and leukocyte infiltration were seen in the lung tumor tissue.Compared with group A1,there are little tumor cells and leukocyte infiltration in the group B1 and C1.In the group A1,the diffuse adenocarcinoma has obliterated the majority of alveolar spaces in lung.
6.Immunohistochemical staining:The lung tumors of K~(LSL)、K~(LSL)Pggtlb~(fl/fl) and K~(LSL)Fntb~(fl/fl) mice were negative for the monocyte/macrophage marker,the Clara cell marker,and the T cell markers but were positive for the typeⅡpneumocyte marker SP-C as judged by immunohistochemistry.Compared with group A1,the expression of SP-C in lung tumor tissue was significantly decreased in group B1 and C1,while Ki-67 was significantly enhanced.
7.Real-time PCR:The mRNA expressions of Pggtlb~(fl/fl),Fntb~(fl/fl) and K~(LSL)mRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2(P＜0.01 or 0.05).Comparison the group A2,the group A1 had higher K~(LSL) mRNA expressions(P＜0.01).
8.Western blot:The protein expression of P-AKT in the tissues of trial group were increased,and P-ERK1/2 were decreased.The nonprenylated RAP1 was only detected in the group B1 and C1.No difference was found about the total AKT and total ERK1/2.
9.Survival rate:Kaplan-Meier curve showing the survival of mouse in the group B1 and group C1 was significantly improved than that in group A1,while there is no significant difference between the group B1 and group C1.Compared with the trial groups,the survival the control group was significantly improved(P＜0.001).
Conclusions
(1) The method of conditional knockout allele technique is an efficient and time-saving way to establish lung cancer model induced by K-ras;(2) The special gene can be expressed in lung by inhale adenovirus.(3) Inactivation of Pggtlb or Fntb by conditional knockout technique can reduce the tumor formation and improves survival in mice with K-ras-induced lung cancer;(4) GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras;(5) The molecular mechanisms of Pggtlb or Fntb inactivation to treat the lung cancer induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein.
PartⅡin vitro study
Background
Gene mutation is the main cause of the genesis of cancer and there is gene mutation in many solid tumor.Non-small cell lung cancer(NSCLC) may be occupy more than 70%of all the pulmonary carcinoma.It is the leading morbility and fatality rate among malignance tumor in the worldwide and shorten the life span.K-ras gene is the most common oncogene in NSCLC.Therefore,the study of the role of K-ras in the genesis of lung cancer has been the hot study.
Recent studies have demonstrated that RAS protein belong to the intracellular proteins contain a CAAX motif.Parts of these proteins can be geranylgeranylated by protein geranylgeranyltransferase typeⅠ(GGTase-I) and the others can farnesylated by protein farnesyltransferase(FTase) alternatively.This is the prenylation process by post-transcriptional modification.GGTase-I and FTase share a commonαsubunit but have uniqueβsubunits that dictate their substrate specificities.In mouse study,We have proved that inactivation of GGTase-I or FTase can inhibit the development of lung cancer induced by K-ras.But we do not know the effects of inactivation of GGTase-I or FTase on the mouse embryo fibroblast(MEF) with K-ras mutation.
To investigate the effects of the inactivation of GGTase-I or FTase on K-ras mutation MEF,The present study was designed to test the role of the inactivation GGTase-I or FTase on K-ras induced MEF through a conditional knockout technique.
Objective
(1) To establish the cell model with the K-ras and GGTase-I or FTase knockout; (2) To observe the effects of K-ras on the bionomics in vitro,such as curve growth, clone formation in soft agar and migration;(3) To investigate the inhibitory role of the inactivation of GGTase-I or FTase on K-ras induced MEF;(4) To investigate the molecular mechanisms of the inactivation of GGTase-I or FTase on the K-ras induced MEF.
Methods
1.Isolation of embryonic fibroblasts:The mouse embryonic fibroblasts(MEF) were isolated from K~(LSL)、K~(LSL)Pggtlb~(fl/fl) and K~(LSL)Fntb~(fl/fl) embryos on day 15.5 post coitum.
2.cell lines groups:According the genetype of cell lines,they were divided into three groups:K~(LSL)(A group)、K~(LSL)Pggtlb~(fl/fl)(B group) and K~(LSL)Fntb~(fl/fl)(C group), then they were divided into two subgroups according to different adenovirus:creadenovirus groups(Trial groups,A1 group、B1 group and C1 group) andβ-gal-adenovirus groups(Control groups,A2 group、B2 group and C2 group).
3.Adenovirus infection of cell lines:To inactivate Pggtlb,Fntb~(fl) and activate K-RAS~(G12D) expression,all cells were seeded and infected with Cre-adenovirus orβ-gal-adenovirus for two times.
4.Genotype:The K~(LSL),Fntb~(fl) and Pggtlb~(fl) allele was genotyped by PCR amplification of genomic DNA from tail biopsies.
5.Real-time PCR:The mRNA expressions of GGTase-I、FTase and K~(LSL) in the cell lines were analyzed using real-time PCR technique.
6.Bionomics of cell lines:The bionomics of cell lines was detected,such as cell growth curve,clone formation in soft agar,migration and apoptosis.
7.Statistical analysis:Data are expressed as means for continuous variables and by frequency count and percentage for qualitative variables.Survival curve of cell lines were compared with One-Way ANOVA comparison test and other indexes were compared with T test.P＜0.05 was considered statistically significant.
Results
1.Cre-adenovirus infection rate:After infected with Cre-adenovirus,K-ras gene was activation and GGTase-I and FTased was inactived.
2.Real-time PCR:The mRNA expressions of GGTase-I,FTase and K~(LSL) mRNA in the tumor tissue of the lung,liver and spleen in group B1 and group C1 were significantly lower than those in group B2 and C2(P＜0.01 or 0.05).Comparison of the group A1 and group A2 revealed that group A1 had higher K~(LSL) mRNA expressions than group A2(P＜0.01).
2.Genetype of the MEF lines
The genetype of the cell lines are the same as that in the animal.
3.The growth of MEF lines
In the trial group,compared with group B1 and group C1,the group A1 grows significantly quickly.No significant difference was found between the group B1 and group C1.No significant difference was found between the trial group and the control group.There is no significant difference in the control group.
4.Migration of the MEF lines
Compared with the control group,the ability of migration in the trial group was increased significantly.In the trial group,the number of migration cells in the group A1 is much more than that in the group B1 and C1.There is no significant difference in the control group.
5.Clone formation of MEF lines
The clone formation was only seen in the group A1,and no clone in the group B1 and C1.There is also no clone in the control group.
6.Apoptosis of MEF lines
The rate of apoptosis in the group A1 is 3.31±1.34%,the group B1 is 13. 91%±1.88%,and the group C1 is 9.57%±2.45%.Compared with the group A1,the rates of apoptosis in the group B1 and C1 are increased significantly,and the group B1 is significant higher than that in the group C1.There is no significant difference in the control group.
7.Western blot
Compared with the group A2,the expression of Caspase-3 in the group A1 was significantly decreased.The expression of Caspase-3 in the group B1 and C1 was significantly lower than that in the group B2 and C2.The expression of phos-P70 in all the cell lines was no significant difference.The expression of PARP in the group B1 and C1 was significantly higher than that in the group B2 and C2,and no significant difference in the group A1 and A2.
Conclusions
(1) The method of conditional knockout allele technique is an efficient and time-saving way to establish MEF model induced by K-ras;(2) Activation of K-ras can promote the ability of cancer cell growth,migration,clone formation,and inhibit the apoptosis;(3) Inactivation of GGTase-I or FTase by conditional knockout technique can reduce the K~(LSL) cell viability,proliferation and enhanced the apoptosis;; (4) GGTase-I or FTase can be as the target to treated the lung cancer induced by K-ras; (5) The molecular mechanisms of the inactivation of GGTase-I or FTase to affect the MEF induced by K-ras is to inhibit the post-transcriptional modification of K-ras protein,enhance the expression of apoptosis gene,promote the apoptosis,and suppressor cell division.
引文
1. Alvarado Y, Giles FJ. Ras as a therapeutic target in hematologic malignancies. Expert Opin Emerg Drugs. 2007;12(2):271-284.
2. Wasylyk G, Zheng H, Castell C,et al. Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules. Cancer Res. 2008;68(5):1275
3. Caulin C, Nguyen T, Lang GA, et al. An inducible mouse model for skin cancer reveals distinct roles for gain- and loss-of-function p53 mutations. J Clin Invest 2007;117(7):1893-1901
4. Ventura JJ, Tenbaum S, Perdiguero E, et al. p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation. Nat Genet 2007; 39(6): 750-758
5. Mallakin A, Sugiyama T, Taneja P, et al. Mutually exclusive inactivation of DMP1 and ARF/p53 in lung cancer. Cancer Cell 2007;12(4):381-394.
6. Sjogren AK, Andersson KM, Liu M, et al. GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer. J Clin Invest 2007;117(5): 1294-1304.
7. Basso AD, Kirschmeier P and Bishop WR. Lipid posttranslational modifications. Farnesyltransferase inhibitors. J Lipid Res. 2006;47:15-31.
8.Reid TS, Terry KL, Casey PJ, et al. Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity. J Mol Biol. 2004;343:417-433
9.Annika M. Wahlstrom, Briony A, et al. Rce1 deficiency accelerates the development of K-RAS-induced myeloproliferative disease.Blood.2007; 109(2): 763-768
10. Therrien M, et al. KSR, a novel protein kinase required for RAS signal transduction. Cell 1995;83:879-888.
11.Ohya Y, Qadota H, Anraku Y, et al. Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Mol Biol Cell. 1993;4:1017-1025.
12. Running MP, et al. Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases. Proc Natl Acad Sci USA.2004;101: 7815-7820
13. Casey PJ, Solski PA, Der CJ,et al. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci. USA. 1989;86:8323-8327
14. Kato K, Cox AD, Hisaka MM, et al. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci USA.1992;89:6403-6407.
15. Kohl NE, Omer CA, Conner MW, et al. Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med. 1995; 1:792-797
16. Le Gouill, Pellat-Deceunynck C, Harousseau JL, et al.Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells.Leukemia. 2002; 16(9): 1664-7
17.James G, Goldstein JL and Brown MS. Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells. Proc Natl Acad Sci.USA. 1996;93:4454-4458.
18. Peterson YK, Kelly P, Weinbaum CA, et al A novel protein geranylgeranyltransferase- I inhibitor with high potency, selectivity, and cellular activity. J Biol Chem. 2006; 281:12445-12450.
19. Gelb MH, Brunsveld L, Hrycyna CA, et al. 2006. Therapeutic intervention based on protein prenylation and associated modifications. Nat Chem Biol. 2006; 2(10):518-528.
20. Ye J, Wang C, Sumpter R Jr, et al. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci. USA. 2003;100(26):15865-15870.
21. O'Meara SJ, Kinsella BT. O'Meara SJ,et al. Evaluation of farnesyl: protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models. Cancer Res. 2001; 61:8758-8768
22. Dan HC, Jiang K, Coppola D,et al. Phosphatidylinositol-3- OH kinase/AKT and survivin pathways as critical targets for geranylgeranyltransferase I inhibitorinduced apoptosis. Oncogene. 2004;23(3): 706-715.
23. Sun J, Ohkanda J, Coppola D, et al. Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice. Cancer Res.2003;63(24):8922-8929.
24. Campbell SL, Khosravi-Far R, Rossman KL, et al. Increasing complexity of Ras signaling. Oncogene 1998;17:1395-1413.
25. Malkinson AM. Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp Lung Res. 1998;24: 541-555.
26. Johnson L, Mercer K, Greenbaum D, et al. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 2001 ;410:1111-1116.
27. Jackson EL, Willis N, Mercer K, et al.Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes development 2001; 15:3243-3248
28. Sartori G, Cavazza A, Bertolini F, et al. subset of lung adenocarcinomas and atypical adenomatous hyperplasia-associated foci are genotypically related: an EGFR, HER2, and K-ras mutational analysis. Am J Clin Pathol. 2008; 129(2): 202-10.
29. Mori M Chiba R and Takahashi T. Atypical adenomatous hyperplasia of the lung and its differentiation from adenocarcinoma Characterization of atypical cells by morphometry and multivariate cluster analysis. Cancer 1993;72:2331-2340
30. Westra WH, Baas IO, Hruban RH, et al. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res. 1996;56:2224-2228.
31. Dermer GB. Origin of bronchioloalveolar carcinoma and peripheral bronchial adenocarcinoma. Cancer 1982;49(5):881-887.
32. Wikenheiser KA, Clark JC, Linnoila RI, et al. Simian virus 40 large T antigen directed by transcriptional elements of the human surfactant protein C gene produces pulmonary adenocarcinomas in transgenic mice. Cancer Res. 1992;52: 5342-5352.
33. Magdaleno SM, Wang G, Mireles VL, et al. Cyclin-dependent kinase inhibitor expression in pulmonary Clara cells transformed with SV40 large T antigen in transgenic mice. Cell Growth Differ. 1997;8:145-155.
34. Annika MW, Briony AC, Christin K, et al. Reel deficiency accelerates the development of K-RAS-induced myeloproliferative disease. Blood 2007; 109(2): 763-768.
35. Chan IT, Kutok JL, Williams IR, et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest. 2004;113:528-538.
36.Sarrabayrouse G, Synaeve C, Leveque K, et al.Statins stimulate in vitro membrane FasL expression and lymphocyte apoptosis through RhoA/ROCK pathway in murine melanoma cells.Neoplasia. 2007;9(12): 1078-90
37.Niemeyer CM, Kratz CP. Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia: molecular classification and treatment options. Br J Haematol. 2008; 140(6):610-24.
38. Leung KF, Baron R, Ali BR, et al. Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation. J Biol Chem. 2007;282(2): 1487-97.
39. Takahashi K, Nakagawa M, Young SG, et al. Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway. J Biol Chem. 2005;280(38):32768-74.
40.Wright LP and Philips MR. Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras. J Lipid Res. 2006;47(5):883-91.
41. Sun J, Blaskovich MA, Knowles D,et al. Antitumor efficacy of a novel class of non-thiol-containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcitabine. Cancer Res. 1999;59(19):4919-26.
42. Raponi M, Lancet JE, Fan H, et al. A 2-gene classifier for predicting response to the farnesyltransferase inhibitor tipifarnib in acute myeloid leukemia.Blood. 2008; 111(5):2589-96.
43.Karp JE and Lancet JE. Development of farnesyltransferase inhibitors for clinical cancer therapy: focus on hematologic malignancies. Cancer Invest. 2007;25(6): 484-94
44. Omer CA, Chen Z, Diehl RE, et al. Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor. Cancer Res. 2000;60(10):2680-8
45. Sebti SM. Protein farnesylation: implications for normal physiology, malignant transformation, and cancer therapy.Cancer Cell. 2005;7(4):297-300
46. Lancet JE and Karp JE.Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy. Blood. 2003;102(12):3880-9
47. Falsetti SC, Wang DA, Peng H, et al.Geranylgeranyltransferase I inhibitors target RalB to inhibit anchorage-dependent growth and induce apoptosis and RalA to inhibit anchorage-independent growth.Mol Cell Biol.2007;27(22):8003-14.
48.Lobell RB, Omer CA, Abrams MT, et al.Evaluation of farnesykprotein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.Cancer Res. 2001;61(24):8758-68
49. Sebti SM and Hamilton AD.Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside translational studies.Oncogene. 2000;19(56):6584-93
50. Blum R, Jacob-Hirsch J, Rechavi G, et al. Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.Mol Cancer Ther 2006;5(9):2337-2347
51.Cobb MH and Goldsmith EJ. How MAP kinases are regulated. J Biol Chem, 1995;70:14843-14846
52. Simons K and Ikonen E. Functional rafts in cell membranes. Nature (Lond.), 1997;387:569-572
53. Hay N. The Akt-mTOR tango and its relevance to cancer. Cancer Cell 2005; 8:179-183.
54. Aoki M, Batista O, Bellacosa A, et al.The akt kinase: Molecular determinants of oncogenicity. Proc Natl Acad Sci. USA 1998;95:14950-14955.
55. Majumder PK, Yeh JJ, George DJ, et al. Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: The MPAKT model. Proc Natl Acad Sci USA 2003; 100:7841-7846.
56. Skeen JE, Bhaskar PT, Chen CC, et al. Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner. Cancer cell.2006; 10: 269-280,
57. Balkwill F, Charles KA, and Mantovani A.Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 2005;7:211-217.
58. Chen ML, Xu PZ, Peng X, et al. The deficiency of Akt1 is sufficient to suppress tumor development in Pten+/2mice. Genes Dev. 2006;20:1569-1574.
59. Tamanoi F, Kato-Stankiewicz J, Jiang C, et al. Farnesylated proteins and cell cycle progression. J Cell Biochem 2001;37:64-70.
60. O'Meara SJ and Kinsella BT. Investigation of the effect of the farnesyl protein transferase inhibitor R115777 on isoprenylation and intracellular signalling by the prostacyclin receptor. Br J Pharmacol 2004; 143:318-330.
61. Takada Y, Khuri FR and Aggarwal BB.Protein farnesyltransferase inhibitor (SCH 66336) abolishes NF-kappaB activation induced by various carcinogens and inflammatory stimuli leading to suppression of NF-kappaB-regulated gene expression and up-regulation of apoptosis. J Biol Chem 2004;279:26287-26299.
62. Na HJ, Lee SJ, Kang YC, et al. Inhibition of farnesyltransferase prevents collagen-induced arthritis by down-regulation of inflammatory gene expression through suppression of p21(ras)-dependent NF-kappaB activation. J Immunol 2004;173:1276-1283.
63. Yu HM, Liu YF, Cheng YF, et al. Effects of rhubarb extract on radiation induced lung toxicity via decreasing transforming growth factor-beta-1 and interleukin-6 in lung cancer patients treated with radiotherapy. Lung Cancer 2008;59:219-226.
1. Beaupre DM and Kurzrock R. RAS and leukemia: from basic mechanisms to gene-directed therapy. J Clin Oncol. 1999; 17:1071-9.
2. Mali Gana-Weisz, Julius Halaschek-Wiener, Burkhard Jansen, et al. The Ras Inhibitor S-trans,trans-Farnesylthiosalicylic Acid Chemosensitizes Human Tumor Cells Without Causing Resistance. Clin Cancer Res 2002;8:555-65
3. Campbell PM, Groehler AL, Lee KM, et al. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling. Cancer Res 2007;67:2098-106
4. Ventura JJ, Tenbaum S, Perdiguero E, et al. p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation. Nat Genet 2007;39:750-8
6.Gelb MH, Brunsveld L, Hrycyna CA, et al. Therapeutic intervention based on protein prenylation and associated modifications. Nat Chem Biol. 2006;2:518-28
7.Dan HC, Jiang K, Coppola D,et al. Phosphatidylinositol-3- OH kinase/AKT and survivin pathways as critical targets for geranylgeranyltransferase I inhibitorinduced apoptosis.Oncogene. 2004;23:706-15
8. Serrano M, Lin AW, McCurrach ME, et al. Oncogenic ras provokes premature cell senescence associated with accu-mulation of p53 and p16INK4a. Cell 1997; 88:593-602.
9. Tuveson DA, Shaw AT, Nicholas A, et al. Endogenous oncogenic K-rasG12D stimulates proliferation and widespread neoplastic and developmental defects. Cancer cell.2004;5:375-87
10. Sharpless NE, Bardeesy N, Lee KH, et al.Loss of pl6Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 2001;413:86-91
11. Malumbres M and Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003;3:459-65.
12. Lamphier MS, Aizawa S, Mak TW, et al. Cellular transforming genes to transform primary cells in culture. Nature 1994;304:602-6
13. Bergo MO, Ambroziak P, Gregory C, et al. Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation. Mol Cell Biol.2002;22:171-81.
14. Sun J, et al. Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice. Cancer Res. 2003; 63:8922-9
15. Anna KMS, Karin ME,Liu M,et al. GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer. J Clin Invest. 117: 1294-304
16. Marciano D, Ben Baruch G, Marom M, et al. Farnesyl derivatives of rigid carboxylic acids-inhibitors of ras-dependent cell growth. J Med Chem, 1995;38: 1267-72
17.Whyte DB. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem.1997;272: 14459-64.
18. Mijimolle N. Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development. Cancer Cell. 2005;7:313-24.
19. Reid TS, Terry KL, Casey PJ, et al. Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity. J Mol Biol. 2004;343:417-33.
20. Silvius JR and l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry. 1994;33: 3014-22.
21. Zhang FL and PJ Casey. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 1996;65:241-69
22.0hya Y, Qadota H, Anraku Y, et al. Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Mol Biol Cell. 1993;4: 1017-25
23. Samuel CF, De-an W, Hairuo P, et al.Geranylgeranyltransferase I Inhibitors Target RalB To Inhibit Anchorage-Dependent Growth and Induce Apoptosis and RalA To Inhibit Anchorage-Independent Growth. Mol Biol Cell.2007;27: 8003-14
24.Blum R, Jacob-Hirsch J, Amariglio N, et al. Ras inhibition in glioblastoma down-regulates hypoxia-inducible factor-1a, causing glycolysis shutdown and cell death. Cancer Res 2005;65: 999-1006
25. Prendergast GC. Actin up: RhoB in cancer and apoptosis. Nat Rev Cancer 2001;1:162-8
26.Joyce PL and AD Cox. Rac1 and Rac3 are targets for geranylgeranyltransferase I inhibitor-mediated inhibition of signaling, transformation, and membrane ruffling. Cancer Res.2003;63: 7959-67
27. Alnemri ES , Livingston DJ , Nicholson DW, et al. Human ICE/ CED23 protease nomenclature. Cell 1996;87:171-9
28.van Gurp M, Festjens N,van LooG,et al. Mitochondrial intennembrane proteins in cell death. Biochem Biophys Res Commun 2003;304: 487-97
29.Blum R, Jacob-Hirsch J, Rechavi G, et al. Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.Mol Cancer Ther 2006;5:2337-47.
30.KoomagiR,VolmM.Relationship between the expression of caspase3 and the clinical outcome of patients with non small cell lung cancer. Anticnacer Res, 2000;20:493-8
31. Pieper AA, Blackshaw S, Clements EE, et al.Poly(ADP-ribosyl)ation basally activated by DNA strand breaks reflects glutamate-nitric oxide neurotransmission. Proc Natl Acad Sci USA. 2000;97:1845-50
32. CoosonMR, IncePG, UsherPA, etal. Poly(ADP2ribose polymerase is found in both thenucleus and cytoplasm of human CNS neurons. BrainRes, 1999; 834: 182-5.
33. Oliver FJ, de la Rubia G, Rolli V, Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant.J Biol Chem, 1998;273:33533-9
34. Lekmine F, Uddin S, Sassano S, et al. Activation of the P70 S6 kinase and phosphorylation of the 4E2BP1 repressor ofmRNA translation by type I interferons. J Biol Chem 2003; 278:27772-77
35.Thomas G. The S6 kinase signaling pathway in the control of development and growth. Biol Res 2002; 35:305-13
1. Movsas B, Raffin TA, Epstein AH, Link CJ. Pulmonary radiation injury. Chest 1997;111: 1061-76.
2. Ohe Y, Yamamoto S, Suzuki K, Hojo F, Kakinuma R, Matsumoto T, et al. Risk factors of treatment-related death in chemotherapy and thoracic radiotherapy for lung cancer. Eur J Cancer 2001;37:54- 63.
3. Ziai WC, Ardelt AA, Llinas RH. Brainstem stroke following uncomplicated cervical epidural steroid injection. Arch Neurol 2006;63:1643-6.
4. Zhu W, Wang XM, Zhang L, Li XY, Wang BX. Pharmacokinetic of rhein in healthy male volunteers following oral and retention enema administration of rhubarb extract: a single dose study. Am J Chin Med 2005;33:839-50.
5. Chang CH, Lin CC, Yang JJ, Namba T, Hattori M. Anti-inflammatory effects of emodin from ventilago leiocarpa. Am J Chin Med 1996;24:139-42.
6. Kuo YC, Meng HC, Tsai WJ. Regulation of cell proliferation, inflammatory cytokine production and calcium mobilization in primary human T lymphocytes by emodin from Polygonum hypoleucum Ohwi. Inflamm Res 2001;50:73-82.
7. Zhao YQ, Liu XH, Ito T, Qian JM. Protective effects of rhubarb on experimental severe acute pancreatitis. World J Gastroenterol 2004; 10:1005-9.
8. Zhan Y, Li D, Wei H, Wang Z, Huang X, Xu Q, et al. Emodin on hepatic fibrosis in rats. Chin Med J (Engl) 2000;113:599-601.
9. ICRU-50: Prescribing, Recording, Reporting, Photon Beam Therapy. Washington, DC, International Commission on Radiation Units and Measurements, 1994.
10. LENT SOMA tables. Radiother Oncol 1995;35:17-60.
11. Adamson IY, Prieditis H, Bowden DH. Instillation of chemotactic factor to silica-injected lungs lowers interstitial particle content and reduces pulmonary fibrosis. Am J Pathol 1992;141:319-26.
12. Tsoutsou PG, Koukourakis MI. Radiation pneumonitis and fibrosis: mechanisms underlying its pathogenesis and implications for future research. Int J Radiat Oncol Biol Phys 2006;66:1281-93.
13. Chen Y, Williams J, Ding I, Hernady E, Liu W, Smudzin T, et al. Radiation pneumonitis and early circulatory cytokine markers. Semin Radiat Oncol 2002; 12 (Suppl 1):26-33.
14. Evans ES, Kocak Z, Zhou SM, Kahn DA, Huang H, Hollis DR, et al. Does transforming growth factor-β1 predict for radiation-induced pneumonitis in patients treated for lung cancer? cytokine 2006;35:186-92.
15. Evans ES, Kocak Z, Zhou SM, Kahn DA, Huang H, Hollis DR, et al.Predicting the risk of symptomatic radiation-induced lung injury using both the physical and biologic parameters V(30) and transforming growth factor beta. Int J Radiat Oncol Biol Phys 2001; 50: 899-908.
16. Anscher MS, Marks LB, Shafman TD, Clough R,Huang H, Tisch A, et al.Risk of long-term complications after TGF-β1-guided very-high-dose thoracic radiotherapy. Int J Radiat Oncol Biol Phys 2003; 56:988-95.
17. Novakova-Jiresova A, Van Gameren MM, Coppes RP, Kampinga HH, Groen HJ. Transforming growth factor-β plasma dynamics and post-irradiation lung injury in lung cancer patients. Radiother Oncol 2004;71:183-9.
18. Anscher MS, Thrasher B, Rabbani Z, Teicher B, Vujaskovic Z. Antitransforming growth factor-β antibody 1D11 ameliorates normal tissue damage caused by high-dose radiation. Int J Radiat Oncol Biol Phys 2006;65:876-81.
19. Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: A regulator of the transition from neutrophil to monocyte recruitment during inflammation.Trends Immunol 2003;24:25-9.
20. Barthelemy-Brichant N, Bosquee L, Cataldo D, Corhay JL, Gustin M, Seidel L, et al: Increased IL-6 and TGF-beta 1 concentrations in bronchoalveolar lavage fluid associated with thoracic radiotherapy. Int J Radiat Oncol Biol Phys 2004;58:758-67.
21. Arpin D, Perol D, Blay JY, Falchero L, Claude L, Vuillermoz-Blas S, et al. Early Variations of Circulating Interleukin-6 and Interleukin-10 Levels During Thoracic Radiotherapy Are Predictive for Radiation Pneumonitis. J Clin Oncol. 2005;23:8748-56.
22. Chen Y, Hyrien O, Williams J, Okunieff P, Smudzin T, Rubin P. Interleukin (IL)-1A and IL-6: applications to the predictive diagnostic testing of radiation pneumonitis. Int J Radiat Oncol Biol Phys 2005;62:260-6.
23. Moon MK, Kang DG, Lee JK, Kim JS, Lee HS. Vasodilatory and anti-inflammatory effects of the aqueous extract of rhubarb via a NO-cGMP pathway. Life Sci 2006;78:1550- 7.
24. Zhang J, Liu ZH, Chen ZH, Li YJ, Li LS. Effect of rhein on glucose transport-1 expression and its function in glomerular mesangial cells.Chin Med J (Engl) 1999;112:1070-9.
25. Guo XH, Liu ZH, Dai CS, Li H, Liu D, Li LS.Rhein inhibits renal tubular epithelial cell hypertrophy and extracellular matrix accumulation induced by transforming growth factor beta1. Acta Pharmacol Sin 2001;22:934-8.
26. Zhu J, Liu Z, Huang H, Chen Z, Li L. Rhein inhibits transforming growth factor betal induced plasminogen activator inhibitor-1 in endothelial cells. Chin Med J (Engl) 2003;116:354-9.
27. Li L.Rheum officinale: a new lead in preventing progression of chronic renal failure.Chin Med J(Engl) 1996;109:35-7.
28.Spencer CM,Wilde MI.Diacerein.Drugs 1997;53:98-106.
29.Lee TY,Chang HH,Chen JH,Hsueh ML,Kuo JJ.Herb medicine Yin-Chen-Hao-Tang ameliorates hepatic fibrosis in bile duct ligation rats.J Ethnopharmacol.2007,109:318-24.
30.Zhang XP,Li ZF,Liu XG,Wu YT,Wang JX,Wang KM,et al.Effects of emodin and baicalein on rats with severe acute pancreatitis.World J Gastroenterol 2005;11:2095-100.
1. Suit H and du Bois W. The importance of optimal treatment planning in radiation therapy. Int J Radiat Oncol Biol Phys 1991 ;21:1471-1478
2. International Commission on Radiation Units and Measurements.ICRU report 62: Prescribing, Recording and Reporting Photon Beam Therapy. (Suppl. To ICRU report 50) Bethesda, MD: International Commission on Radiation Units and Measurements; 19991
3. Chan R, He Y, Haque A and Zwischenberger J. Computed tomographic- pathologic correlation of gross tumour volume and clinical target volume in non-small cell lung cancer: a pilot experience. Arch Pathol Lab Med 2001; 125:1469-1472
4. Grills IS, Yan D, Black QC, et al. Clinical implications of defining the gross tumor volume with combination of CT and (18)FDG-positron emission tomography in non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 2006; 67:709-719
5. Brianzoni E, Rossi G, Ancidei S, et al. Radiotherapy planning: PET/CT scanner performances in the definition of gross tumour volume and clinical target volume. Eur J Nucl Med Mol Imaging 2005;32:1392-1399
6. Pfannenberg AC, Aschoff P, Brechtel K, et al. Low dose non-enhanced CT versus standard dose contrast-enhanced CT in combined PET/CT protocols for staging and therapy planning in non-small cell lung cancer.Eur J Nucl Med Mol Imaging 2007;34:36-44
7. Takiguchi Y, Uruma R, Asaka-Amano Y, et al. Phase I study of cisplatin and irinotecan combined with concurrent hyperfractionated accelerated thoracic radiotherapy for locally advanced non-small cell lung carcinoma. Int J Clin Oncol 2005;10:418-424
8. Beyer T, Townsend DW, Brun T, et al. A combined PET/CT scanner for clinical oncology. J Nucl Med 2000;41:1369-1379
9. Erdi YE, Mawlawi 0, Larson SM, et al. Segmentation of lung lesion volume by adaptive positron emission tomography image thresholding. Cancer 1997;80:2505-2509
10. Giraud P, Antoine M, Larrouy A, et al. Evaluation of microscopic tumour extension in non-small-cell lung cancer for three-dimensional conformal radiotherapy planning. Int J Radiat Oncol Biol Phys 2000;48:1015-1024
11. Al-Sugair A and Coleman RE. Applications of PET in lung cancer. Semin Nucl Med 1998;28:303-319
12. Hoekstra CJ, Stroobants SG, Smit EF, et al. Prognostic relevance of response evaluation using [18F]-2-fluoro- 2-deoxy-D-glucose positron emission tomography in patients with locally advanced non-small-cell lung cancer. J Clin Oncol 2005; 23:8362-8370
13. Steenbakkers RJ, Duppen JC, Fitton I, et al. Redution of observer variation using matched CT-PET for lung cancer delineation: a three-dimensional analysis. Int J Radiat Oncol Biol Phys 2006;64:435-448
14. Greco C, Rosenzweig K, Cascini GL and Tamburrini O. Current status of PET/CT for tumour volume definition in radiotherapy treatment planning for non-small cell lung cancer (NSCLC). Lung cancer 2007; 57:125-134
15. Xia SA, Wu GH, Lin Q and Chen Y. Influence of Window Wide and Window Level on Outlining the Target Volume in CT Simulation. Academic Journal of Shanghai Second Medical University 2005;25:741-743
16. Grills IS, Fitch DL, Goldstein NS, et al. Clinicopathologic analysis of microscopic extension in lung adenocarcinoma: defining clinical target volume for radiotherapy. Int J Radiat Oncol Biol Phys. 2007;69:334-341
17. Sakao Y, Nakazono T, Tomimitsu S, et al. Lung adenocarcinoma can be subtyped according to tumor dimension by computed tomography mediastinal-window setting. Additional size criteria for clinical T1 adenocarcinoma. Eur J Cardiothorac Surg2004;26:1211-1215
18. Harris KM, Adams H, Lloyd DC, Harvey DJ. The effect on apparent size of simulated pulmonary nodules of using three standard CT window settings. Clin Radiol 1993;47:241-214
19. Xing LG, Sun XR, Yu JM, Lu J, Liu J, Yin Y. Impact of PET/CT image coregistration accuracy on radiation treatment planning for non-small cell lung cancer. Chin J Radiat Oncol 2005;14:314-318
20. Nehmeh SA, Erdi YE, Rosenzweig KE, et al. Reduction of respiratory motion artifacts in PET imaging of lung cancer by respiratory correlated dynamic PET: methodology and comparison with respiratory gated PET. J Nucl Med 2003; 44: 1644-1648
2. Wasylyk G, Zheng H, Castell C,et al. Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules. Cancer Res. 2008;68(5):1275
3. Caulin C, Nguyen T, Lang GA, et al. An inducible mouse model for skin cancer reveals distinct roles for gain- and loss-of-function p53 mutations. J Clin Invest 2007;117(7):1893-1901
4. Ventura JJ, Tenbaum S, Perdiguero E, et al. p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation. Nat Genet 2007; 39(6): 750-758
5. Mallakin A, Sugiyama T, Taneja P, et al. Mutually exclusive inactivation of DMP1 and ARF/p53 in lung cancer. Cancer Cell 2007;12(4):381-394.
6. Sjogren AK, Andersson KM, Liu M, et al. GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer. J Clin Invest 2007;117(5): 1294-1304.
7. Basso AD, Kirschmeier P and Bishop WR. Lipid posttranslational modifications. Farnesyltransferase inhibitors. J Lipid Res. 2006;47:15-31.
8.Reid TS, Terry KL, Casey PJ, et al. Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity. J Mol Biol. 2004;343:417-433
9.Annika M. Wahlstrom, Briony A, et al. Rce1 deficiency accelerates the development of K-RAS-induced myeloproliferative disease.Blood.2007; 109(2): 763-768
10. Therrien M, et al. KSR, a novel protein kinase required for RAS signal transduction. Cell 1995;83:879-888.
11.Ohya Y, Qadota H, Anraku Y, et al. Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Mol Biol Cell. 1993;4:1017-1025.
12. Running MP, et al. Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases. Proc Natl Acad Sci USA.2004;101: 7815-7820
13. Casey PJ, Solski PA, Der CJ,et al. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci. USA. 1989;86:8323-8327
14. Kato K, Cox AD, Hisaka MM, et al. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci USA.1992;89:6403-6407.
15. Kohl NE, Omer CA, Conner MW, et al. Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice. Nat Med. 1995; 1:792-797
16. Le Gouill, Pellat-Deceunynck C, Harousseau JL, et al.Farnesyl transferase inhibitor R115777 induces apoptosis of human myeloma cells.Leukemia. 2002; 16(9): 1664-7
17.James G, Goldstein JL and Brown MS. Resistance of K-RasBV12 proteins to farnesyltransferase inhibitors in Rat1 cells. Proc Natl Acad Sci.USA. 1996;93:4454-4458.
18. Peterson YK, Kelly P, Weinbaum CA, et al A novel protein geranylgeranyltransferase- I inhibitor with high potency, selectivity, and cellular activity. J Biol Chem. 2006; 281:12445-12450.
19. Gelb MH, Brunsveld L, Hrycyna CA, et al. 2006. Therapeutic intervention based on protein prenylation and associated modifications. Nat Chem Biol. 2006; 2(10):518-528.
20. Ye J, Wang C, Sumpter R Jr, et al. Disruption of hepatitis C virus RNA replication through inhibition of host protein geranylgeranylation. Proc Natl Acad Sci. USA. 2003;100(26):15865-15870.
21. O'Meara SJ, Kinsella BT. O'Meara SJ,et al. Evaluation of farnesyl: protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models. Cancer Res. 2001; 61:8758-8768
22. Dan HC, Jiang K, Coppola D,et al. Phosphatidylinositol-3- OH kinase/AKT and survivin pathways as critical targets for geranylgeranyltransferase I inhibitorinduced apoptosis. Oncogene. 2004;23(3): 706-715.
23. Sun J, Ohkanda J, Coppola D, et al. Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice. Cancer Res.2003;63(24):8922-8929.
24. Campbell SL, Khosravi-Far R, Rossman KL, et al. Increasing complexity of Ras signaling. Oncogene 1998;17:1395-1413.
25. Malkinson AM. Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp Lung Res. 1998;24: 541-555.
26. Johnson L, Mercer K, Greenbaum D, et al. Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature 2001 ;410:1111-1116.
27. Jackson EL, Willis N, Mercer K, et al.Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes development 2001; 15:3243-3248
28. Sartori G, Cavazza A, Bertolini F, et al. subset of lung adenocarcinomas and atypical adenomatous hyperplasia-associated foci are genotypically related: an EGFR, HER2, and K-ras mutational analysis. Am J Clin Pathol. 2008; 129(2): 202-10.
29. Mori M Chiba R and Takahashi T. Atypical adenomatous hyperplasia of the lung and its differentiation from adenocarcinoma Characterization of atypical cells by morphometry and multivariate cluster analysis. Cancer 1993;72:2331-2340
30. Westra WH, Baas IO, Hruban RH, et al. K-ras oncogene activation in atypical alveolar hyperplasias of the human lung. Cancer Res. 1996;56:2224-2228.
31. Dermer GB. Origin of bronchioloalveolar carcinoma and peripheral bronchial adenocarcinoma. Cancer 1982;49(5):881-887.
32. Wikenheiser KA, Clark JC, Linnoila RI, et al. Simian virus 40 large T antigen directed by transcriptional elements of the human surfactant protein C gene produces pulmonary adenocarcinomas in transgenic mice. Cancer Res. 1992;52: 5342-5352.
33. Magdaleno SM, Wang G, Mireles VL, et al. Cyclin-dependent kinase inhibitor expression in pulmonary Clara cells transformed with SV40 large T antigen in transgenic mice. Cell Growth Differ. 1997;8:145-155.
34. Annika MW, Briony AC, Christin K, et al. Reel deficiency accelerates the development of K-RAS-induced myeloproliferative disease. Blood 2007; 109(2): 763-768.
35. Chan IT, Kutok JL, Williams IR, et al. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest. 2004;113:528-538.
36.Sarrabayrouse G, Synaeve C, Leveque K, et al.Statins stimulate in vitro membrane FasL expression and lymphocyte apoptosis through RhoA/ROCK pathway in murine melanoma cells.Neoplasia. 2007;9(12): 1078-90
37.Niemeyer CM, Kratz CP. Paediatric myelodysplastic syndromes and juvenile myelomonocytic leukaemia: molecular classification and treatment options. Br J Haematol. 2008; 140(6):610-24.
38. Leung KF, Baron R, Ali BR, et al. Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation. J Biol Chem. 2007;282(2): 1487-97.
39. Takahashi K, Nakagawa M, Young SG, et al. Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway. J Biol Chem. 2005;280(38):32768-74.
40.Wright LP and Philips MR. Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras. J Lipid Res. 2006;47(5):883-91.
41. Sun J, Blaskovich MA, Knowles D,et al. Antitumor efficacy of a novel class of non-thiol-containing peptidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with the cytotoxic agents cisplatin, Taxol, and gemcitabine. Cancer Res. 1999;59(19):4919-26.
42. Raponi M, Lancet JE, Fan H, et al. A 2-gene classifier for predicting response to the farnesyltransferase inhibitor tipifarnib in acute myeloid leukemia.Blood. 2008; 111(5):2589-96.
43.Karp JE and Lancet JE. Development of farnesyltransferase inhibitors for clinical cancer therapy: focus on hematologic malignancies. Cancer Invest. 2007;25(6): 484-94
44. Omer CA, Chen Z, Diehl RE, et al. Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor. Cancer Res. 2000;60(10):2680-8
45. Sebti SM. Protein farnesylation: implications for normal physiology, malignant transformation, and cancer therapy.Cancer Cell. 2005;7(4):297-300
46. Lancet JE and Karp JE.Farnesyltransferase inhibitors in hematologic malignancies: new horizons in therapy. Blood. 2003;102(12):3880-9
47. Falsetti SC, Wang DA, Peng H, et al.Geranylgeranyltransferase I inhibitors target RalB to inhibit anchorage-dependent growth and induce apoptosis and RalA to inhibit anchorage-independent growth.Mol Cell Biol.2007;27(22):8003-14.
48.Lobell RB, Omer CA, Abrams MT, et al.Evaluation of farnesykprotein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.Cancer Res. 2001;61(24):8758-68
49. Sebti SM and Hamilton AD.Farnesyltransferase and geranylgeranyltransferase I inhibitors and cancer therapy: lessons from mechanism and bench-to-bedside translational studies.Oncogene. 2000;19(56):6584-93
50. Blum R, Jacob-Hirsch J, Rechavi G, et al. Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.Mol Cancer Ther 2006;5(9):2337-2347
51.Cobb MH and Goldsmith EJ. How MAP kinases are regulated. J Biol Chem, 1995;70:14843-14846
52. Simons K and Ikonen E. Functional rafts in cell membranes. Nature (Lond.), 1997;387:569-572
53. Hay N. The Akt-mTOR tango and its relevance to cancer. Cancer Cell 2005; 8:179-183.
54. Aoki M, Batista O, Bellacosa A, et al.The akt kinase: Molecular determinants of oncogenicity. Proc Natl Acad Sci. USA 1998;95:14950-14955.
55. Majumder PK, Yeh JJ, George DJ, et al. Prostate intraepithelial neoplasia induced by prostate restricted Akt activation: The MPAKT model. Proc Natl Acad Sci USA 2003; 100:7841-7846.
56. Skeen JE, Bhaskar PT, Chen CC, et al. Akt deficiency impairs normal cell proliferation and suppresses oncogenesis in a p53-independent and mTORC1-dependent manner. Cancer cell.2006; 10: 269-280,
57. Balkwill F, Charles KA, and Mantovani A.Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell 2005;7:211-217.
58. Chen ML, Xu PZ, Peng X, et al. The deficiency of Akt1 is sufficient to suppress tumor development in Pten+/2mice. Genes Dev. 2006;20:1569-1574.
59. Tamanoi F, Kato-Stankiewicz J, Jiang C, et al. Farnesylated proteins and cell cycle progression. J Cell Biochem 2001;37:64-70.
60. O'Meara SJ and Kinsella BT. Investigation of the effect of the farnesyl protein transferase inhibitor R115777 on isoprenylation and intracellular signalling by the prostacyclin receptor. Br J Pharmacol 2004; 143:318-330.
61. Takada Y, Khuri FR and Aggarwal BB.Protein farnesyltransferase inhibitor (SCH 66336) abolishes NF-kappaB activation induced by various carcinogens and inflammatory stimuli leading to suppression of NF-kappaB-regulated gene expression and up-regulation of apoptosis. J Biol Chem 2004;279:26287-26299.
62. Na HJ, Lee SJ, Kang YC, et al. Inhibition of farnesyltransferase prevents collagen-induced arthritis by down-regulation of inflammatory gene expression through suppression of p21(ras)-dependent NF-kappaB activation. J Immunol 2004;173:1276-1283.
63. Yu HM, Liu YF, Cheng YF, et al. Effects of rhubarb extract on radiation induced lung toxicity via decreasing transforming growth factor-beta-1 and interleukin-6 in lung cancer patients treated with radiotherapy. Lung Cancer 2008;59:219-226.
1. Beaupre DM and Kurzrock R. RAS and leukemia: from basic mechanisms to gene-directed therapy. J Clin Oncol. 1999; 17:1071-9.
2. Mali Gana-Weisz, Julius Halaschek-Wiener, Burkhard Jansen, et al. The Ras Inhibitor S-trans,trans-Farnesylthiosalicylic Acid Chemosensitizes Human Tumor Cells Without Causing Resistance. Clin Cancer Res 2002;8:555-65
3. Campbell PM, Groehler AL, Lee KM, et al. K-Ras promotes growth transformation and invasion of immortalized human pancreatic cells by Raf and phosphatidylinositol 3-kinase signaling. Cancer Res 2007;67:2098-106
4. Ventura JJ, Tenbaum S, Perdiguero E, et al. p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation. Nat Genet 2007;39:750-8
6.Gelb MH, Brunsveld L, Hrycyna CA, et al. Therapeutic intervention based on protein prenylation and associated modifications. Nat Chem Biol. 2006;2:518-28
7.Dan HC, Jiang K, Coppola D,et al. Phosphatidylinositol-3- OH kinase/AKT and survivin pathways as critical targets for geranylgeranyltransferase I inhibitorinduced apoptosis.Oncogene. 2004;23:706-15
8. Serrano M, Lin AW, McCurrach ME, et al. Oncogenic ras provokes premature cell senescence associated with accu-mulation of p53 and p16INK4a. Cell 1997; 88:593-602.
9. Tuveson DA, Shaw AT, Nicholas A, et al. Endogenous oncogenic K-rasG12D stimulates proliferation and widespread neoplastic and developmental defects. Cancer cell.2004;5:375-87
10. Sharpless NE, Bardeesy N, Lee KH, et al.Loss of pl6Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 2001;413:86-91
11. Malumbres M and Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003;3:459-65.
12. Lamphier MS, Aizawa S, Mak TW, et al. Cellular transforming genes to transform primary cells in culture. Nature 1994;304:602-6
13. Bergo MO, Ambroziak P, Gregory C, et al. Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation. Mol Cell Biol.2002;22:171-81.
14. Sun J, et al. Geranylgeranyltransferase I inhibitor GGTI-2154 induces breast carcinoma apoptosis and tumor regression in H-Ras transgenic mice. Cancer Res. 2003; 63:8922-9
15. Anna KMS, Karin ME,Liu M,et al. GGTase-I deficiency reduces tumor formation and improves survival in mice with K-RAS-induced lung cancer. J Clin Invest. 117: 1294-304
16. Marciano D, Ben Baruch G, Marom M, et al. Farnesyl derivatives of rigid carboxylic acids-inhibitors of ras-dependent cell growth. J Med Chem, 1995;38: 1267-72
17.Whyte DB. K- and N-Ras are geranylgeranylated in cells treated with farnesyl protein transferase inhibitors. J Biol Chem.1997;272: 14459-64.
18. Mijimolle N. Protein farnesyltransferase in embryogenesis, adult homeostasis, and tumor development. Cancer Cell. 2005;7:313-24.
19. Reid TS, Terry KL, Casey PJ, et al. Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity. J Mol Biol. 2004;343:417-33.
20. Silvius JR and l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry. 1994;33: 3014-22.
21. Zhang FL and PJ Casey. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 1996;65:241-69
22.0hya Y, Qadota H, Anraku Y, et al. Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p. Mol Biol Cell. 1993;4: 1017-25
23. Samuel CF, De-an W, Hairuo P, et al.Geranylgeranyltransferase I Inhibitors Target RalB To Inhibit Anchorage-Dependent Growth and Induce Apoptosis and RalA To Inhibit Anchorage-Independent Growth. Mol Biol Cell.2007;27: 8003-14
24.Blum R, Jacob-Hirsch J, Amariglio N, et al. Ras inhibition in glioblastoma down-regulates hypoxia-inducible factor-1a, causing glycolysis shutdown and cell death. Cancer Res 2005;65: 999-1006
25. Prendergast GC. Actin up: RhoB in cancer and apoptosis. Nat Rev Cancer 2001;1:162-8
26.Joyce PL and AD Cox. Rac1 and Rac3 are targets for geranylgeranyltransferase I inhibitor-mediated inhibition of signaling, transformation, and membrane ruffling. Cancer Res.2003;63: 7959-67
27. Alnemri ES , Livingston DJ , Nicholson DW, et al. Human ICE/ CED23 protease nomenclature. Cell 1996;87:171-9
28.van Gurp M, Festjens N,van LooG,et al. Mitochondrial intennembrane proteins in cell death. Biochem Biophys Res Commun 2003;304: 487-97
29.Blum R, Jacob-Hirsch J, Rechavi G, et al. Suppression of survivin expression in glioblastoma cells by the Ras inhibitor farnesylthiosalicylic acid promotes caspase-dependent apoptosis.Mol Cancer Ther 2006;5:2337-47.
30.KoomagiR,VolmM.Relationship between the expression of caspase3 and the clinical outcome of patients with non small cell lung cancer. Anticnacer Res, 2000;20:493-8
31. Pieper AA, Blackshaw S, Clements EE, et al.Poly(ADP-ribosyl)ation basally activated by DNA strand breaks reflects glutamate-nitric oxide neurotransmission. Proc Natl Acad Sci USA. 2000;97:1845-50
32. CoosonMR, IncePG, UsherPA, etal. Poly(ADP2ribose polymerase is found in both thenucleus and cytoplasm of human CNS neurons. BrainRes, 1999; 834: 182-5.
33. Oliver FJ, de la Rubia G, Rolli V, Importance of poly(ADP-ribose) polymerase and its cleavage in apoptosis. Lesson from an uncleavable mutant.J Biol Chem, 1998;273:33533-9
34. Lekmine F, Uddin S, Sassano S, et al. Activation of the P70 S6 kinase and phosphorylation of the 4E2BP1 repressor ofmRNA translation by type I interferons. J Biol Chem 2003; 278:27772-77
35.Thomas G. The S6 kinase signaling pathway in the control of development and growth. Biol Res 2002; 35:305-13
1. Movsas B, Raffin TA, Epstein AH, Link CJ. Pulmonary radiation injury. Chest 1997;111: 1061-76.
2. Ohe Y, Yamamoto S, Suzuki K, Hojo F, Kakinuma R, Matsumoto T, et al. Risk factors of treatment-related death in chemotherapy and thoracic radiotherapy for lung cancer. Eur J Cancer 2001;37:54- 63.
3. Ziai WC, Ardelt AA, Llinas RH. Brainstem stroke following uncomplicated cervical epidural steroid injection. Arch Neurol 2006;63:1643-6.
4. Zhu W, Wang XM, Zhang L, Li XY, Wang BX. Pharmacokinetic of rhein in healthy male volunteers following oral and retention enema administration of rhubarb extract: a single dose study. Am J Chin Med 2005;33:839-50.
5. Chang CH, Lin CC, Yang JJ, Namba T, Hattori M. Anti-inflammatory effects of emodin from ventilago leiocarpa. Am J Chin Med 1996;24:139-42.
6. Kuo YC, Meng HC, Tsai WJ. Regulation of cell proliferation, inflammatory cytokine production and calcium mobilization in primary human T lymphocytes by emodin from Polygonum hypoleucum Ohwi. Inflamm Res 2001;50:73-82.
7. Zhao YQ, Liu XH, Ito T, Qian JM. Protective effects of rhubarb on experimental severe acute pancreatitis. World J Gastroenterol 2004; 10:1005-9.
8. Zhan Y, Li D, Wei H, Wang Z, Huang X, Xu Q, et al. Emodin on hepatic fibrosis in rats. Chin Med J (Engl) 2000;113:599-601.
9. ICRU-50: Prescribing, Recording, Reporting, Photon Beam Therapy. Washington, DC, International Commission on Radiation Units and Measurements, 1994.
10. LENT SOMA tables. Radiother Oncol 1995;35:17-60.
11. Adamson IY, Prieditis H, Bowden DH. Instillation of chemotactic factor to silica-injected lungs lowers interstitial particle content and reduces pulmonary fibrosis. Am J Pathol 1992;141:319-26.
12. Tsoutsou PG, Koukourakis MI. Radiation pneumonitis and fibrosis: mechanisms underlying its pathogenesis and implications for future research. Int J Radiat Oncol Biol Phys 2006;66:1281-93.
13. Chen Y, Williams J, Ding I, Hernady E, Liu W, Smudzin T, et al. Radiation pneumonitis and early circulatory cytokine markers. Semin Radiat Oncol 2002; 12 (Suppl 1):26-33.
14. Evans ES, Kocak Z, Zhou SM, Kahn DA, Huang H, Hollis DR, et al. Does transforming growth factor-β1 predict for radiation-induced pneumonitis in patients treated for lung cancer? cytokine 2006;35:186-92.
15. Evans ES, Kocak Z, Zhou SM, Kahn DA, Huang H, Hollis DR, et al.Predicting the risk of symptomatic radiation-induced lung injury using both the physical and biologic parameters V(30) and transforming growth factor beta. Int J Radiat Oncol Biol Phys 2001; 50: 899-908.
16. Anscher MS, Marks LB, Shafman TD, Clough R,Huang H, Tisch A, et al.Risk of long-term complications after TGF-β1-guided very-high-dose thoracic radiotherapy. Int J Radiat Oncol Biol Phys 2003; 56:988-95.
17. Novakova-Jiresova A, Van Gameren MM, Coppes RP, Kampinga HH, Groen HJ. Transforming growth factor-β plasma dynamics and post-irradiation lung injury in lung cancer patients. Radiother Oncol 2004;71:183-9.
18. Anscher MS, Thrasher B, Rabbani Z, Teicher B, Vujaskovic Z. Antitransforming growth factor-β antibody 1D11 ameliorates normal tissue damage caused by high-dose radiation. Int J Radiat Oncol Biol Phys 2006;65:876-81.
19. Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: A regulator of the transition from neutrophil to monocyte recruitment during inflammation.Trends Immunol 2003;24:25-9.
20. Barthelemy-Brichant N, Bosquee L, Cataldo D, Corhay JL, Gustin M, Seidel L, et al: Increased IL-6 and TGF-beta 1 concentrations in bronchoalveolar lavage fluid associated with thoracic radiotherapy. Int J Radiat Oncol Biol Phys 2004;58:758-67.
21. Arpin D, Perol D, Blay JY, Falchero L, Claude L, Vuillermoz-Blas S, et al. Early Variations of Circulating Interleukin-6 and Interleukin-10 Levels During Thoracic Radiotherapy Are Predictive for Radiation Pneumonitis. J Clin Oncol. 2005;23:8748-56.
22. Chen Y, Hyrien O, Williams J, Okunieff P, Smudzin T, Rubin P. Interleukin (IL)-1A and IL-6: applications to the predictive diagnostic testing of radiation pneumonitis. Int J Radiat Oncol Biol Phys 2005;62:260-6.
23. Moon MK, Kang DG, Lee JK, Kim JS, Lee HS. Vasodilatory and anti-inflammatory effects of the aqueous extract of rhubarb via a NO-cGMP pathway. Life Sci 2006;78:1550- 7.
24. Zhang J, Liu ZH, Chen ZH, Li YJ, Li LS. Effect of rhein on glucose transport-1 expression and its function in glomerular mesangial cells.Chin Med J (Engl) 1999;112:1070-9.
25. Guo XH, Liu ZH, Dai CS, Li H, Liu D, Li LS.Rhein inhibits renal tubular epithelial cell hypertrophy and extracellular matrix accumulation induced by transforming growth factor beta1. Acta Pharmacol Sin 2001;22:934-8.
26. Zhu J, Liu Z, Huang H, Chen Z, Li L. Rhein inhibits transforming growth factor betal induced plasminogen activator inhibitor-1 in endothelial cells. Chin Med J (Engl) 2003;116:354-9.
27. Li L.Rheum officinale: a new lead in preventing progression of chronic renal failure.Chin Med J(Engl) 1996;109:35-7.
28.Spencer CM,Wilde MI.Diacerein.Drugs 1997;53:98-106.
29.Lee TY,Chang HH,Chen JH,Hsueh ML,Kuo JJ.Herb medicine Yin-Chen-Hao-Tang ameliorates hepatic fibrosis in bile duct ligation rats.J Ethnopharmacol.2007,109:318-24.
30.Zhang XP,Li ZF,Liu XG,Wu YT,Wang JX,Wang KM,et al.Effects of emodin and baicalein on rats with severe acute pancreatitis.World J Gastroenterol 2005;11:2095-100.
1. Suit H and du Bois W. The importance of optimal treatment planning in radiation therapy. Int J Radiat Oncol Biol Phys 1991 ;21:1471-1478
2. International Commission on Radiation Units and Measurements.ICRU report 62: Prescribing, Recording and Reporting Photon Beam Therapy. (Suppl. To ICRU report 50) Bethesda, MD: International Commission on Radiation Units and Measurements; 19991
3. Chan R, He Y, Haque A and Zwischenberger J. Computed tomographic- pathologic correlation of gross tumour volume and clinical target volume in non-small cell lung cancer: a pilot experience. Arch Pathol Lab Med 2001; 125:1469-1472
4. Grills IS, Yan D, Black QC, et al. Clinical implications of defining the gross tumor volume with combination of CT and (18)FDG-positron emission tomography in non-small-cell lung cancer. Int J Radiat Oncol Biol Phys 2006; 67:709-719
5. Brianzoni E, Rossi G, Ancidei S, et al. Radiotherapy planning: PET/CT scanner performances in the definition of gross tumour volume and clinical target volume. Eur J Nucl Med Mol Imaging 2005;32:1392-1399
6. Pfannenberg AC, Aschoff P, Brechtel K, et al. Low dose non-enhanced CT versus standard dose contrast-enhanced CT in combined PET/CT protocols for staging and therapy planning in non-small cell lung cancer.Eur J Nucl Med Mol Imaging 2007;34:36-44
7. Takiguchi Y, Uruma R, Asaka-Amano Y, et al. Phase I study of cisplatin and irinotecan combined with concurrent hyperfractionated accelerated thoracic radiotherapy for locally advanced non-small cell lung carcinoma. Int J Clin Oncol 2005;10:418-424
8. Beyer T, Townsend DW, Brun T, et al. A combined PET/CT scanner for clinical oncology. J Nucl Med 2000;41:1369-1379
9. Erdi YE, Mawlawi 0, Larson SM, et al. Segmentation of lung lesion volume by adaptive positron emission tomography image thresholding. Cancer 1997;80:2505-2509
10. Giraud P, Antoine M, Larrouy A, et al. Evaluation of microscopic tumour extension in non-small-cell lung cancer for three-dimensional conformal radiotherapy planning. Int J Radiat Oncol Biol Phys 2000;48:1015-1024
11. Al-Sugair A and Coleman RE. Applications of PET in lung cancer. Semin Nucl Med 1998;28:303-319
12. Hoekstra CJ, Stroobants SG, Smit EF, et al. Prognostic relevance of response evaluation using [18F]-2-fluoro- 2-deoxy-D-glucose positron emission tomography in patients with locally advanced non-small-cell lung cancer. J Clin Oncol 2005; 23:8362-8370
13. Steenbakkers RJ, Duppen JC, Fitton I, et al. Redution of observer variation using matched CT-PET for lung cancer delineation: a three-dimensional analysis. Int J Radiat Oncol Biol Phys 2006;64:435-448
14. Greco C, Rosenzweig K, Cascini GL and Tamburrini O. Current status of PET/CT for tumour volume definition in radiotherapy treatment planning for non-small cell lung cancer (NSCLC). Lung cancer 2007; 57:125-134
15. Xia SA, Wu GH, Lin Q and Chen Y. Influence of Window Wide and Window Level on Outlining the Target Volume in CT Simulation. Academic Journal of Shanghai Second Medical University 2005;25:741-743
16. Grills IS, Fitch DL, Goldstein NS, et al. Clinicopathologic analysis of microscopic extension in lung adenocarcinoma: defining clinical target volume for radiotherapy. Int J Radiat Oncol Biol Phys. 2007;69:334-341
17. Sakao Y, Nakazono T, Tomimitsu S, et al. Lung adenocarcinoma can be subtyped according to tumor dimension by computed tomography mediastinal-window setting. Additional size criteria for clinical T1 adenocarcinoma. Eur J Cardiothorac Surg2004;26:1211-1215
18. Harris KM, Adams H, Lloyd DC, Harvey DJ. The effect on apparent size of simulated pulmonary nodules of using three standard CT window settings. Clin Radiol 1993;47:241-214
19. Xing LG, Sun XR, Yu JM, Lu J, Liu J, Yin Y. Impact of PET/CT image coregistration accuracy on radiation treatment planning for non-small cell lung cancer. Chin J Radiat Oncol 2005;14:314-318
20. Nehmeh SA, Erdi YE, Rosenzweig KE, et al. Reduction of respiratory motion artifacts in PET imaging of lung cancer by respiratory correlated dynamic PET: methodology and comparison with respiratory gated PET. J Nucl Med 2003; 44: 1644-1648
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |