天花粉蛋白对宫颈癌HeLa细胞TSLC1基因甲基化及其表达的影响

目的：检测人宫颈癌HeLa细胞中TSLC1基因甲基化的状况,研究在人宫颈癌HeLa细胞凋亡过程中TSLC1基因甲基化的变化情况,探讨肿瘤细胞凋亡与抑癌基因甲基化的相关性,并进一步证实天花粉蛋白（TCS）去甲基化作用是否存在普遍性,以促进天花粉蛋白的临床应用。方法：应用甲基化特异性PCR（MSP）法检测人宫颈癌HeLa细胞及其凋亡过程中TSLC1基因甲基化的状况;采用实时定量RT-PCR技术检测TCS处理前、后HeLa细胞TSLC1基因表达的变化。结果：肿瘤抑制基因TSLC1在人宫颈癌HeLa细胞中呈高度甲基化状态,经40μg／mL TCS处理48h后,TSLC1基因甲基化程度明显降低;RT-PCR检测结果显示,TCS处理组HeLa细胞中TSLC1 mRNA的表达量高于未处理组,提示TSLC1基因启动子区CpG岛甲基化是导致其低表达的重要机制。结论：肿瘤抑制基因TSLC1启动子甲基化在人宫颈癌癌变过程中可能是一种重要的分子调控机制;人宫颈癌HeLa细胞凋亡与抑癌基因的去甲基化之间可能存在某些密切的相关性;TCS对肿瘤抑制基因TSLC1有一定的去甲基化作用。     

DNA甲基化与肿瘤

DNA甲基化参与基因的表达调控。DNA甲基化异常与肿瘤等疾病有关,抑癌基因启动子区的异常甲基化和癌基因的去甲基化均影响肿瘤的发生发展过程,肿瘤细胞的总体甲基化水平比正常细胞低,但是伴有某些CpG岛甲基化程度增高。     

斜纹夜蛾视黄酸结合蛋白(Slcrabp)基因的克隆、表达及功能

视黄酸结合蛋白(Cellular retinoic acid binding protein,CRABP)属于胞内脂质结合蛋白超基因家族,参与了许多生理活动,如细胞分化、组织重建和信号转导等,但其在昆虫中肠的作用尚不明确。研究从斜纹夜蛾Spodoptera litura中肠基因表达序列标签(EST)文库中克隆获得一个编码Slcrabp的全长c DNA,该c DNA由396个核苷酸组成,编码132个氨基酸。预测CRABP蛋白质的空间结构与脂肪酸结合蛋白非常相似,含一个由10个反平行的β折叠和2个α螺旋形成的配体结合中心结构域。Sl CRABP基因具有4个外显子,与脊椎动物crabp基因类似。RT-PCR检测表明,在转录水平上,Slcrabp在6龄幼虫中肠的各个时期均有较高表达。Western blot分析结果显示,Sl CRABP蛋白分布广泛,在中肠、脂肪体、精巢等组织上大量表达。在中肠,其表达峰值出现在预蛹期。利用荧光标记物质8-苯胺基-1-萘磺酸(1,8-ANS)分析了重组Sl CRABP蛋白与不同底物的亲和力,发现Sl CRABP与不饱和长链脂肪酸有较高结合活性,如花生四烯酸钠、亚麻酸、油酸钠和油酸,但与视黄酸、视黄醇的结合力却很弱或几乎不结合,暗示斜纹夜蛾Sl CRABP功能与同家族脂肪酸结合蛋白(FABP)性质相似。对6龄幼虫进行饥饿处理,结果显示经过经24 h和48 h饥饿处理后,虫体中肠Sl CRABP蛋白质的表达量有显著上升,暗示Sl CRABP可能参与了斜纹夜蛾体内脂类的转运过程。     

DNA甲基化与癌症

DNA甲基化是重要的表观遗传修饰,主要发生在DNA的CpG岛. DNA的甲基化通过DNA甲基转移酶（DNA methyltransferases, DNMTs）完成. DNA甲基化参与了细胞分化、基因组稳定性、X染色体失活、基因印记等多种细胞生物学过程.单基因水平及基因组范围内的DNA甲基化改变在肿瘤发生发展中亦发挥重要作用. 抑癌基因的异常甲基化引起的表达抑制,可导致肿瘤细胞的增殖失控和侵袭转移,并参与肿瘤组织的血管生成过程.在许多肿瘤的研究中都发现了基因组整体DNA低甲基化所导致的染色体不稳定性. 本文从DNA的异常高甲基化和低甲基化两方面论述了DNA甲基化在细胞恶变发生发展过程中的改变及其影响,并阐述了DNA甲基化改变在肿瘤诊断和治疗中的作用.     

DNA 甲基化及其对肿瘤的作用

DNA甲基化是一种重要的表遗传修饰,在肿瘤细胞中表现为基因组整体水平的低甲基化和一些特定区域的高甲基化,发生低甲基化的序列主要是一些高度和中度重复序列,而发生高甲基化序列通常是跨越管家基因和肿瘤抑制基因启动子的CpG岛,它们在肿瘤的发生发展中起重要的作用,本文就这些序列的异常甲基化和其对肿瘤作用一些进展和观点作一介绍。     

基因启动子异常甲基化及其作为肿瘤生物标志物的研究进展

基因启动子异常甲基化与肿瘤的发生关系密切,它可导致抑癌基因或其它肿瘤相关基因的表达失活。研究显示基因启动子异常甲基化是肿瘤形成过程中的一个早期、频发事件,广泛存在于几乎所有种类的人类肿瘤中,因此,基因启动子区异常甲基化是一个高灵敏度的肿瘤生物标志物,检测外周血血清或肿瘤累及器官体液中肿瘤相关基因的甲基化状态将为肿瘤的早期诊断、疗效观察及预后判断提供非常有价值的信息。     

肿瘤诊断新的标记物--甲基化谱

肿瘤的形成与有关基因的异常有一定的关系,但是目前还没有可以用于检测与肿瘤有关的基因异常系统。通过对肿瘤的不同基因甲基化检测,认为甲基化谱可以达到上述目的。对十几种基因在十几种肿瘤中的甲基化情况分析,发现不同基因和肿瘤甲基化的特点。由于启动子甲基化改变在肿瘤初期即可发生,有利于肿瘤的早期诊断。     

非小细胞性肺癌患者Runx3基因启动子区甲基化状态研究

目的:分析非小细胞性肺癌(NSCLC)中Runx3基因启动子区甲基化状态.方法:运用甲基化特异性PCR技术检测62例NSCLC组织和癌旁正常肺组织中Runx3基因启动子甲基化,并分析该基因启动子甲基化Runx3基因mRNA和蛋白表达的影响及其与临床特征之间的关系.结果:NSCLC组织中Runx3基因异常甲基化率(48.4%)显著高于癌旁正常肺组织中Runx3基因的异常甲基化率(17.7%,P＝0.000);发生完全或者不完全甲基化的NSCLC组织或者正常肺组织中Runx3基因mRNA和蛋白表达显著降低.Runx3基因启动子区高甲基化和肿瘤分化程度及临床分期有相关性(P＝0.041和0.009),而与NSCLC患者性别、年龄、有无吸烟史及肿瘤类型等临床特征无关(P=0.400,0.301,0.290和0.965).结论:Runx3基因启动子区异常甲基化是导致Runx3基因在NSCLC中表达下调的重要因素,有望成为NSCLC早期辅助诊断的分子标志物之一.     

DNA甲基化抑制鼻咽癌细胞系膜联蛋白A1基因表达

为了研究不同分化程度和转移潜能鼻咽癌(NPC)细胞系膜联蛋白A1(ANXA1)mRNA和蛋白质表达情况及其与基因甲基化的关系．培养NPC细胞系CNE1、CNE2、5-8F、6-10B和永生化非癌性人鼻咽黏膜上皮细胞NP69细胞用于实验,用甲基化特异性聚合酶链反应(MSP)方法检测ANXA1基因甲基化状态,同时利用逆转录-聚合酶链反应(RT-PCR)方法检测ANXA1基因的mRNA表达水平．然后用不同浓度的5-杂氮-2′-脱氧胞苷(5-aza-2dC)对NPC细胞进行去甲基化处理,MSP和RT-PCR方法检测处理组和对照组细胞ANXA1基因甲基化状况和mRNA表达水平,并用Western-blotting方法检测ANXA1基因蛋白质表达水平．结果发现,NP69细胞ANXA1基因无甲基化,4株NPC细胞系ANXA1基因都存在不同程度的甲基化,甲基化程度与细胞的分化程度和转移潜能相关．NPC细胞ANXA1基因mRNA表达水平降低,低于NP69细胞,其降低的程度与基因的甲基化程度相关．5-aza-2dC能够剂量依赖性地引起ANXA1基因去甲基化,经去甲基化处理后,NPC细胞系ANXA1基因的mRNA和蛋白质的表达水平相应提高．研究证明,NPC细胞系ANXA1基因的mRNA和蛋白质表达水平出现下调,甲基化是导致表达下调的主要原因,5-aza-2dC去甲基化处理能够恢复ANXA1基因的表达水平．     

肺癌肿瘤阻抑基因1:一种新的抑癌基因

新的抑癌基因TSLC1属于免疫球蛋白超家族,位于人染色体11q23.2。TSLC1编码的蛋白质参与细胞间黏附、细胞运动、信号转导及免疫调节。研究显示,TSLC1在多种肿瘤中表达异常,它对肿瘤的影响主要表现为抑制瘤细胞增殖及诱导凋亡、改变瘤细胞的生长特性及基因表达,和抑制上皮间质转化。TSLC1的缺失与启动子甲基化关系密切,但其发挥抑癌作用的分子机制及信号转导途径尚有待研究。     

The molecular cloning and expression of two CRABP cDNAs from human skin

Retinoic acid (RA) is known to have a profound effect on the growth and differentiation of human epidermal cells in vivo and in vitro. One of the proteins thought to be involved in mediating the action of RA is the cellular retinoic acid-binding protein (CRABP). We have used PCR technology to generate cDNAs for two distinct CRABPs from human skin and skin-derived cells. One is highly homologous to the CRABP I cDNAs previously cloned from bovine and murine sources. The second shares extensive deduced amino acid homology with CRABP II, a protein recently described in newborn rat and embryonic chick. Although both mRNAs can be detected in neonatal foreskin, CRABP II mRNA is the predominant one in this tissue, as well as in cultured newborn fibroblasts and keratinocytes. Northern blot analysis showed CRABP II mRNA level was only slightly reduced by addition of 10(-6) or 10(-5) M RA to cultures of neonatal foreskin-derived fibroblasts, as was the CRABP I mRNA level in cultured human gut epithelial cells. In contrast, expression of CRABP II mRNA by cultured neonatal keratinocytes was strongly downregulated by RA. We conclude that CRABP II is the predominant CRABP in human skin, at least in the newborn period, and that it is differentially regulated in fibroblasts versus keratinocytes. Our data are consistent with a role for CRABP in regulating the amount of RA delivered to the nucleus.     

The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.     

Dynamic regulation of retinoic acid-binding proteins in developing, adult and neoplastic skin reveals roles for beta-catenin and Notch signalling

Retinoic acid (RA) signalling is essential for epidermal differentiation; however, the mechanisms by which it acts are largely unexplored. Partitioning of RA between different nuclear receptors is regulated by RA-binding proteins. We show that cellular RA-binding proteins CRABP1 and CRABP2 and the fatty acid-binding protein FABP5 are dynamically expressed during skin development and in adult tissue. CRABP1 is expressed in embryonic dermis and in the stroma of skin tumours, but confined to the hair follicle dermal papilla in normal postnatal skin. CRABP2 and FABP5 are expressed in the differentiating cells of sebaceous gland, interfollicular epidermis and hair follicles, with FABP5 being a prominent marker of sebaceous glands and anagen follicle bulbs. All three proteins are upregulated in response to RA treatment or Notch activation and are negatively regulated by Wnt/β-catenin signalling. Ectopic follicles induced by β-catenin arise from areas of the sebaceous gland that have lost CRABP2 and FABP5; conversely, inhibition of hair follicle formation by N-terminally truncated Lef1 results in upregulation of CRABP2 and FABP5. Our findings demonstrate that there is dynamic regulation of RA signalling in different regions of the skin and provide evidence for interactions between the RA, β-catenin and Notch pathways.     

Cellular retinoic acid-binding protein II gene expression is directly induced by estrogen,but not retinoic acid,in rat uterus

It has been suggested that cellular retinoic acid-binding protein (II) (CRABP(II)) may have a role in the movement of retinoic acid (RA) to its nuclear receptors, thereby enhancing the action of RA in the cells in which it is expressed. RA has also been shown to increase expression of CRABP(II). Previous work from our laboratory has shown that 17 beta-estradiol (E2) administration to prepubertal female rats leads to acquisition of the ability of the lining epithelium to synthesize RA as well as to express CRABP(II). To determine whether this appearance of CRABP(II) was dependent on the production of RA, both E2 and RA were administered to ovariectomized rats. E2 administration induced expression of the CRABP(II) gene in the uterus within 4 h, and this induction was not inhibited by prior administration of puromycin, indicating that the induction was direct. In contrast, RA caused no change in CRABP(II) message level, even at times as late as 48 h after administration. Isolation and analysis of 4.5 kb of the 5'-flanking region of the gene revealed no apparent E2-response element. Using this portion of the gene to drive expression of the luciferase gene in transfected cells allowed identification of a region containing an imperfect estrogen-response element and estrogen-response element half-site, necessary for E2-driven induction. A possible Sp1 binding site in the 5'-flanking region of the CRABP(II) gene was also required for this induction. The ability of E2 to induce expression of CRABP(II) suggests that it can enhance the activity of RA, directly affecting expression of retinoid-responsive genes.     

The relationship among retinoid structure, affinity for retinoic acid-binding protein, and ability to respecify pattern in the regenerating axolotl limb

To further our understanding of the action of retinoids on the respecification of pattern in the regenerating axolotl limb we have studied the relative potencies of a range of synthetic and natural retinoids administered locally to the blastema. Alterations in the polar end group of the retinoic acid (RA) molecule to produce esters, the alcohol, or the aldehyde abolish the ability of the molecule to respecify pattern. On the other hand, alterations of the ring or side chain to produce the synthetic retinoids arotinoid and TTNPB considerably increases the potency of the molecule to respecify pattern--TTNPB is at least 100X more potent than retinoic acid. To examine the role of cellular retinoic acid-binding protein (CRABP) in the respecification process we determined the relative binding affinities of these retinoids for CRABP. These data correlated well with the respecification series: retinoids which showed no affinity for CRABP did not respecify pattern and those which did show affinity for CRABP did respecify pattern. Furthermore the most potent retinoid, TTNPB, has a higher affinity for CRABP than RA itself. This suggests that CRABP may be playing an important role in the action of RA on pattern formation in the regenerating limb.