姜黄素对人淋巴瘤Raji细胞组蛋白H3乙酰化作用的影响

背景与目的:表观遗传改变是肿瘤发生的一个重要原因,具有表观遗传修饰作用的甲基化转移酶抑制剂和去乙酰化酶抑制剂可以抑制肿瘤增殖诱导凋亡。本研究探讨姜黄素对Raji细胞组蛋白H3的乙酰化作用和细胞周期素依赖性激酶抑制剂p21WAF1/CIP1基因表达的影响。方法:用25μmol/L姜黄素作用Raji细胞不同时间,Westernblot分析乙酰化组蛋白H3和p21WAF1/CIP1变化;RT-PCR检测p21WAF1/CIP1基因表达;染色质免疫沉淀分析p21WAF1/CIP1的启动子基因位点组蛋白H3乙酰化水平;流式细胞术检测细胞周期变化。结果:姜黄素提高p21WAF1/CIP1的启动子基因位点组蛋白H3乙酰化水平1.9倍;使p21WAF1/CIP1mRNA合成增加和蛋白表达上调,在24h时分别增加了4.2倍和5.1倍;24h阻滞细胞在G2/M期,36h阻滞在G0/G1期。结论:姜黄素通过表观遗传修饰作用,调节p21WAF1/CIP1启动子基因位点组蛋白H3乙酰化水平,促进p21WAF1/CIP1基因转录,阻滞Raji细胞周期进程。     

NS-398调节HepG2细胞组蛋白乙酰化和p21WAF1/CIP1表达

背景与目的:研究非甾体药物NS-398对人肝癌细胞株HepG2细胞组蛋白H3乙酰化水平的调节作用及对细胞周期素依赖性激酶抑制p21WAF1/CIP1表达的影响.材料与方法:用不同浓度(100、200、300、400μmol/L)的NS-398处理HepG2细胞,以四甲基偶氮唑蓝(MTT)法测定肿瘤细胞增殖抑制率,流式细胞仪(FCM)检测细胞周期的改变及凋亡百分率的变化,应用NS-398分别作用HepG2细胞4、8、12、24、48 h,非药物作用组作为对照,提取细胞的总RNA和总蛋白,采用RT-PCR技术检测p21WAF1/CIP1 mRNA表达情况,并用免疫印迹技术(Western blot)观察组蛋白H3的乙酰化水平变化及p21WAF1/CIP1蛋白的表达水平.结果:NS-398抑制HepG2细胞增殖,且呈剂量依赖性,并诱导其凋亡.且呈浓度依赖性改变细胞周期的分布,一方面增高G0/G1期细胞比例,另一方面降低S期和G2/M期细胞比例,与对照组相比差异具有统计学意义(P＜0.05).NS-398对组蛋白H3乙酰化作用随时间改变而变化,可引起组蛋白H3的乙酰化.NS-398对p21WAF1/CIP1 mRNA和p21WAF1/CIP1蛋白表达的影响呈时间依赖性.结论:NS-398明显上调HepG2细胞组蛋白H3的乙酰化水平,促进细胞周期依赖性激酶抑制剂p21WAF1/CIP1的表达.     

RNA激活上调p21~(WAF1/CIP1)基因表达对肺癌细胞增殖和凋亡影响的研究

目的:探讨应用RNA激活(RNAa)技术上调p21WAF1/CIP1(p21)表达对人肺癌H441细胞增殖和凋亡的影响。方法:设计靶向抑癌基因p21WAF1/CIP1启动子DNA序列互补的双链RNA分子(dsp21),转染H441细胞后应用半定量逆转录聚合酶链反应(RT-PCR)及蛋白质印迹法检测p21WAF1/CIP1基因mRNA及蛋白质的表达变化,噻唑蓝(MTT)法检测H441细胞增殖速度的变化,流式细胞仪检测H441细胞凋亡率的变化。结果:dsp21转染H441细胞72 h后p21WAF1/CIP1表达显著上调。RT-PCR灰度比值结果显示,空白对照组、阴性对照组和实验组p21WAF1/CIP1mRNA相对表达量分别为(38.1±2.5)%、(33.5±3.9)%和(96.5±2.3)%,差异有统计学意义,F=87.0,P<0.01;蛋白质印迹法结果显示,空白对照组、阴性对照组和实验组p21WAF1/CIP1蛋白相对表达量分别为(45.7±2.2)%、(43.2±3.1)%和(93.6±2.5)%,差异有统计学意义,F=79.0,P<0.01;经dsp21作用的H441细胞增殖受到明显抑制,凋亡率较对照...     

人乳腺癌中p21WAF1组蛋白H3、H4乙酰化水平的表达变化

[目的]探讨人乳腺癌中p21WAF1组蛋白H3、H4乙酰化水平的变化及其意义。[方法]应用HE染色鉴定乳腺癌的病理形态变化,RT—PCR检测p21WAF1mRNA的表达,染色质免疫沉淀法检测p21WAF1组蛋白H3、H4乙酰化的状态。[结果]HE染色可见,与癌旁组织及正常乳腺组织相比,乳腺癌组织结构及细胞形态有明显的异型性。RT—PCR检测结果显示,乳腺癌组织中p21WAF1mRNA的表达水平明显低于癌旁组织及正常乳腺组织（P〈0．05）;p21WAF1mRNA在组蛋白H3、H4乙酰化水平降低乳腺癌中的表达明显低于组蛋白H3、H4乙酰化水平非降低乳腺癌中的表达（P〈0．05）;p21WAF1mRNA表达降低与人乳腺癌的临床分期、分化程度和淋巴结转移有关。染色质免疫沉淀法显示,乳腺癌组织中p21WAF1组蛋白H3、H4的乙酰化水平明显低于癌旁组织及正常乳腺组织;p21WAF1乙酰化水平降低与人乳腺癌的分化程度和淋巴结转移有关。[结论]p21WAF1组蛋白H3、H4乙酰化的表达变化与乳腺癌的发生发展密切相关。     

p21WAF1蛋白的表达与非小细胞肺癌增殖和预后的关系

目的检测人非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的p21WAF1/CIP1表达水平,探讨NSCLC中p21WAF1/CIP1的表达与细胞增殖活性和预后的关系.方法采用流式细胞术检测60例NSCLC中p21WAF1/CIP1的表达量及DNA含量;分析p21WAF1/CIP1表达水平与S期细胞比例、预后的关系.结果正常肺组织中未见p21WAF1/CIP1的表达,60例NSCLC组织中p21WAF1/CIP1阳性率为60%(36/60),p21WAF1/CIP1阳性表达者的S期细胞比例(10.87±0.69)%低于阴性表达者(13.30±0.93)%,t=3.232,P= 0.001.p21WAF1/CIP1的表达与患者的术后累积生存月数和5年生存率有关,p21WAF1/CIP1 阳性表达者的术后平均生存月数(40个月)明显高于阴性表达者(25个月),t=2.885,P=0.003;5年生存率(29%)也高于阴性表达者(9%),t=2.712,P=0.004.结论 p21WAF1/CIP1表达是判断NSCLC预后的一个可参考指标.     

去甲二氢愈创木酸抑制宫颈癌SiHa细胞的生长与上调p21基因组蛋白乙酰化相关性的研究

目的:研究去甲二氢愈创木酸(nordihydroguaiaretic acid, NDGA)对宫颈癌SiHa细胞增殖的影响及作用机制.方法:MTT法检测NDGA对SiHa细胞生长的影响,FCM法检测NDGA对SiHa细胞周期及凋亡的影响,RT-PCR检测NDGA对p21基因转录的影响,Western印迹法检测NDGA对组蛋白H3总乙酰化水平的影响,染色质免疫沉淀(chromatin immunoprecipitation,ChIP)-PCR法检测NDGA对p21基因近启动子区域组蛋白H3乙酰化的影响.结果:NDGA能明显抑制SiHa细胞的生长,且呈时间和剂量依赖性;可使SiHa细胞的细胞周期阻滞于G1期,而凋亡率却下降;能明显提高p21基因的转录水平和组蛋白H3的总乙酰化水平,ChIP-PCR检测结果表明NDGA可明显促进p21基因近启动子区域组蛋白H3的乙酰化水平.结论:NDGA促进p21基因近启动子区域及细胞内总体组蛋白H3的乙酰化可能是其抑制宫颈癌SiHa细胞生长的潜在机制.     

丙戊酸钠诱导kasumi-1细胞分化与凋亡机制的探讨

目的:探讨组蛋白去乙酰化酶抑制剂丙戊酸钠(sodium valproate,VPA)体外诱导白血病细胞kasumi-1的分化和凋亡作用.方法:MTT实验和台盼蓝拒染法检测VPA对细胞的增殖抑制,流式细胞仪检测细胞分化和凋亡及进行细胞周期分析,蛋白质印迹方法检测组蛋白H3(lys9)的乙酰化水平变化及p21waf1/Gp1蛋白的表达.结果:VPA可以抑制kasumi-1细胞增殖.VPA作用后,细胞周期阻滞在G0/G1期,该期细胞由(53.07±6.05)%增加到(78.97±5.82)%,P<0.05.在较低浓度(0.5 mmol/L),VPA可以诱导kasumi-1细胞髓系分化,其表面抗原CD13表达从(20.47±0.21)%上升到(68.67±1.15)%,P:0.000.联合G-CSF后作用更为显著[(87.80±1.21)%],P=0.000.在较高浓度(2 mmol/L)下,VPA则诱导kash-mi-1细胞凋亡,流式检测Annexin V结合力上升.VPA能够上调kasumi-1细胞组蛋白H3乙酰化水平,增加p21waf1/Gp1蛋白的表达.结论:VPA能通过抑制HDAC活性,抑制细胞增殖,诱导kasumi-1细胞分化和凋亡.     

曲古霉素A对肺腺癌细胞株NCI-H1299增殖的抑制作用及其机制

背景与目的：曲古霉素A（trichostatin A,TSA）是具有组蛋白去乙酰化酶（histone deacetylases,HDAC@强效非竞争性抑制剂,对血液系统肿瘤和实质性肿瘤均有较强的生长抑制作用。本文观察HDACs抑制剂TSA对体外培养的肺腺癌NCI-H1299细胞株的增殖、凋亡和周期以及相关基因表达的影响,并探讨其可能的作用机制。方法：MTT法检测不同浓度（0.1、0.2、0.4、2.0μmol/L@的TSA对人肺腺癌NCI-H1299细胞株体外增殖的影响,流式细胞术检测药物处理后细胞周期及凋亡率的变化;Western blot法检测细胞内组蛋白H4乙酰化水平的变化;Real-time PCR检测NCI-H1299细胞内p21、CyclinB1、Bcl-2和Bax的基因表达。结果：TSA能明显抑制NCI-H1299细胞的体外生长,其抑制作用呈明显的剂量和时间依赖性。TSA诱导后,流式细胞术检测结果显示细胞阻滞于G2/M期,细胞凋亡增加。TSA可明显提高NCI-H1299细胞内组蛋白H4的乙酰化水平,诱导p21和Bax的mRNA表达增加,同时抑制Bcl-2和CyclinB1表达。结论：TSA可通过诱导细胞凋亡及阻滞细胞周期而发挥体外抗肺腺癌细胞生长的作用,其机制可能与组蛋白乙酰化水平的提高以及调控相关基因p21、Bax、Bcl-2和CyclinB1的表达变化有关。     

胃癌中p21WAF1/CIP1、细胞周期素D1、p53表达的相关性

目的探讨p21WAF1/CIP1、细胞周期素D1(cyclin D1)、p53在胃癌中表达之间的相关性.方法应用原位杂交技术检测p21WAF1/CIP1 mRNA、细胞周期素D1 mRNA及免疫组化技术检测p53蛋白在胃癌中的表达.结果 p21WAF1/CIP1 mRNA在癌组织及癌旁正常粘膜中阳性表达率各为93.15%(68/73)及76.71%(56/73),二者相比具有显著差异(P<0.05).Cyclin D1 mRNA在癌组织及癌旁正常粘膜中阳性表达率各为54.79%(40/73)及30.16%(22/73),二者具有显著差异(P<0.05).p53蛋白在胃癌中的阳性表达率为32.87%(24/73), p53过表达者,其p21WAF1/CIP1 mRNA表达较p53阴性者为低,二者存在显著差异(P<0.05).p21WAF1/CIP1表达与细胞周期素D1表达呈负相关.结论 p21WAF1/CIP1、Cyclin D1、p53的异常表达及它们之间可能存在的相互作用,对于胃癌的发生发展具有重要意义.     

Farnesyltransferase inhibitors: antineoplastic properties, mechanisms of action, and clinical prospects

Farnesyltransferase (FTase) inhibitors are among the current wave of molecularly targeted anti-cancer agents being used to attack malignancy in a rational manner. A large body of preclinical data indicates that FTase inhibitors block cancer cell proliferation through both cytostatic and cytotoxic effects. Interestingly, FTase inhibitors have rather limited effects on normal cell function, suggesting that they may target unique aspects of cancer cell pathophysiology. The development of FTase inhibitors was predicated on the discovery that the Ras oncoproteins must be post-translationally modified to transform cells. However, recent work indicates that the anti-neoplastic effects of FTase inhibitors depend on altering the post-translational modifications of non-Ras proteins as well. In particular, a critical target protein that responds to FTase inhibition by blocking tumor cell growth is RhoB, an endosomal Rho protein that functions in receptor trafficking. In this review, we survey the biological foundations for the clinical development of FTase inhibitors, and consider some of the latest mechanistic studies that reveal how these agents affect cellular physiology.     

Targeting tumor vasculature with homing peptides from phage display

Tumor vasculature expresses a number of molecular markers at much lower levels than those seen in the blood vessels of normal tissues, and in some cases, such markers are undetectable. The presence of these markers relates to angiogenesis; the same markers are shared by all blood vessels undergoing angiogenesis. The endothelial cells, pericytes and smooth muscle cells, and the vascular extracellular matrix in angiogenic vessels can each express such markers. Molecularly, they represent vascular growth factor receptors, cell adhesion proteins and their receptors. Screening of phage display libraries for peptides that home to tumor vasculature when injected into mice has recently provided a new tool for analyzing the distinguishing features of tumor vasculature. Tumor-homing peptides isolated in this manner, as well as an antibody against a form of fibronectin expressed in tumor blood vessels, have been found to serve as targeting devices to concentrate drugs and other therapeutic materials to tumors in in vivo models. Such a targeting strategy can therefore potentially improve the efficacy of drugs and reduce their side effects.     

Identification of EBV transforming genes by recombinant EBV technology

Epstein-Barr virus (EBV) is able to infect primary B-lymphocytes but usually does not proceed to replicate more virions. Instead, EBV persists as an incomplete virus and expresses 12 gene products that transform the growth of these cells into continuously proliferating lymphoblastoid cell lines. Because EBV is associated with several human malignancies, there is intense interest in delineating the molecular functions of these EBV gene products in transformation. This review focuses on the recombinant EBV technologies that have been developed to introduce specific mutations into EBV and test the functions of these EBV genes in primary B-lymphocyte growth transformation.     

Matrix metalloproteinases in tumor invasion and metastasis

Extensive work on the mechanisms of tumor invasion and metastasis has identified matrix metalloproteinases (MMPs) as key players in the events that underlie tumor dissemination. Studies using natural and synthetic MMP inhibitors, as well as tumor cells transfected with cDNAs encoding the MMPs characterized thus far have provided compelling evidence that MMP activity can induce or enhance tumor survival, invasion and metastasis. Because of the ability of MMPs to degrade extracellular matrix (ECM) proteins, the principal mechanism whereby MMPs promote tumor development has been thought to be the proteolytic breakdown of tissue barriers to invasion and the associated facilitation of circulating tumor cell extravasation. However, recent evidence stemming from the use of novel experimental approaches indicates that MMPs do not play a major role in the process of extravasation itself. Rather, they appear to promote intravasation (the process of penetrating the circulation following invasion of blood vessels) and regulate the relationship between tumor cells and host tissue stroma subsequent to extravasation. In addition, the discoveries that a growing number of proteolytically active MMPs may localize to the cell surface in association with adhesion receptors, and that MMP substrates include latent cytokines and growth factors, provide a new conceptual framework for the mechanisms whereby MMPs influence tumor behavior.     

New aspects of integrin signaling in cancer

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behavior, including motility and invasiveness, cell growth, and cell survival. Engagement of integrins with extracellular matrix (ECM) proteins can activate members of the Rho-family of small GTPases; conversely, Rho- and Ras-family proteins can influence the ability of integrins to bind their ligands. These events impinge on the control of cell motility, and ultimately on invasive and metastatic behavior. Integrin engagement with ECM also has important effects on cell survival, particularly for cells of epithelial origin. In some cases, specific integrins have selective effects on the efficiency of signal transduction in cell survival pathways.     

Role of LMP1 in immune control of EBV infection

The Epstein-Barr virus (EBV) encoded latent membrane protein (LMP1) plays a crucial role in the long-term persistence of this virus within the cells of the immune system. Not only is this protein critical for the transformation of resting B cells by EBV, it also displays pleiotropic effects on various cellular proteins expressed in the host cell. These include up-regulation of expression of B cell activation antigens, adhesion molecules and various components of the antigen processing pathway. Here we discuss how LMP1 acts like an expression 'switch' which, depending on the stage of EBV infection, manoeuvres various pathways that either modulate the immune system towards or against its survival.     

腹部压块对膈肌运动影响的研究

目的 :研究腹部压块对膈肌运动的影响。方法 :选择拟行立体适形放疗患有肺癌或肝脏肿瘤的患者 2 0例。按治疗体位仰卧于体部立体放疗定位负压袋内 ,待患者呼吸平稳后 ,将灯光野的中心点置于膈顶运动的最低点 ,在膈肌运动至最高位时拍摄照片 ,测量膈肌运动的最大幅度 ;然后 ,将心形腹部压块放置于患者剑突下 ,并用定位框架的腹带交叉固定 ,按压程度以不引起患者呼吸困难或其他不适为标准 ,5min后按上述方法再次测量膈肌运动的最大幅度。结果 :2 0例患者未加腹部压块的运动幅度为0 6 2～ 2 6 7cm ,平均 (1 4± 0 6 4)cm ,加腹部压块后的膈肌运动幅度为 0 2 8～ 2 0 8cm ,平均 (1 0±0 5 5 )cm ,加腹部压块后膈肌运动幅度平均减小 (0 4± 0 34)cm ,P =0 0 0 0。加腹部压块后 90 % (18/2 0 )的患者膈肌运动幅度受到不同程度的限制 ,但有 10 % (2 /2 0 )的患者膈肌运动幅度增加。结论 :腹部压块可使大部分患者膈肌运动的幅度减小 ,但少部分患者例外 ,即腹部压块并不能使所有膈肌周围肿瘤的照射容积减少。建议在制定放射治疗计划前应预先进行测量和评价     

ABCG2在肺癌中表达的定量研究

目的 观察ABCG2在肺癌和癌周肺组织的表达,从量化角度阐明其在肺癌组织中表达的病理学意义.方法 常规石蜡包埋、HE切片确诊,用免疫组化SP法检测ABCG2在肺癌和癌周肺组织的定位和表达,用LeicaQ500MC图像分析系统对其表达强度进行定量分析,并用表达的阳性单位(positive unit PU)反映其表达强度.结果 ABCG2蛋白在肺癌和癌周正常肺组织中的表达主要定位在细胞质和细胞膜.在癌周正常肺组织的支气管和细支气管上皮呈弥漫表达,腺上皮呈灶性表达;肺鳞癌和肺腺癌弥漫或大片表达,肺鳞癌表达的PU值高于肺腺癌(P＜0.001),肺大细胞癌和肺小细胞癌不表达,PU值接近于零.癌周肺组织表达的PU值高于各型肺癌(P＜0.05).ABCG2蛋白表达的PU值在肺癌原发灶和转移灶之间无差别(P＞0.05),且与肺癌患者的性别、年龄、转移和TNM分期未见明显相关性(P＞0.05),与肺癌分化程度有关(P＜0.001).分化程度越高,PU值越高,但高分化肺癌和癌周肺组织的表达PU值差异无显著性(P＞0.05).结论 ABCG2蛋白表达程度与肺癌类型及分化程度具有相关性,可能成为判断其指标之一.     

Telomerase and human tumorigenesis

Human cancer cells, unlike their normal counterparts, have shed the molecular restraints to limited cell growth and are immortal. Exactly how cancer cells manage this at the molecular level is beginning to be understood. Human cells must overcome two barriers to cellular proliferation. The first barrier, referred to as senescence, minimally involves the p53 and Rb tumor-suppressor pathways. Inactivation of these pathways results in some extension of lifespan. However, inactivation of these pathways is insufficient for immortalization. As normal cells undergo repeated rounds of DNA replication, their telomeres shorten due to the inability of traditional DNA polymerases to completely replicate the end of the chromosomal DNA. This shortening continues until the cells reach a second proliferative block referred to as crisis, which is characterized by chromosomal instability, end-to-end fusions, and cell death. Stabilization of the telomeric DNA through either telomerase activation or the activation of the alternative mechanism of telomere maintenance (ALT) is essential if the cells are to survive and proliferate indefinitely. Conversely, loss of telomere stabilization by an already-immortalized cell results in loss of immortality and cell death. Together this indicates that telomere maintenance is a critical component of immortality. In this review we attempt to describe our current understanding of the role of telomere maintenance in senescence, crisis, and tumorigenesis.     

Post-translational regulation of COX2 activity by FYN in prostate cancer cells

While increased COX2 expression and prostaglandin levels are elevated in human cancers, the mechanisms of COX2 regulation at the post-translational level are unknown. Initial observation that COX2 forms adduct with non-receptor tyrosine kinase FYN, prompted us to study FYN-mediated post-translational regulation of COX2. We found that FYN increased COX2 activity in prostate cancer cells DU145, independent of changes in COX2 or COX1 protein expression levels. We report that FYN phosphorylates human COX2 on Tyr 446, and while corresponding phospho-mimetic COX2 mutation promotes COX2 activity, the phosphorylation blocking mutation prevents FYN-mediated increase in COX2 activity.