【作者】 李春笋；

【导师】 刘芝华；

【作者基本信息】 中国协和医科大学 ， 细胞生物学， 2008， 博士

【摘要】 肿瘤抑制因子p53在细胞癌变进程中扮演重要角色,主要通过对细胞发挥细胞周期阻滞或者诱导细胞凋亡的功能来影响细胞生长,这些功能通常通过p53激活其下游靶基因来实现。我室前期工作中从特异性cDNA微阵列筛选出92个食管癌差异表达基因,聚类分析发现一组分化相关基因在食管癌中表达明显下调,进一步分析表明这些分化相关基因多定位于人类染色体1q21,表达产物属钙依赖或钙调节蛋白,参与形成表皮分化复合物。我们对其中一个下调非常明显的S100A9基因进行了研究。S100蛋白家族是一个钙结合蛋白家族,它凭借与钙离子的相互作用在体内发挥重要生物学活性,且多种S100蛋白与肿瘤的发生发展关系密切。该家族部分成员(如S100B、S100A2、S100A4)的启动子区存在肿瘤抑制蛋白p53的结合位点,它们的表达受p53蛋白的转录调控。将食管鳞癌中表达明显下调的S100A9基因的启动子区进行生物信息学分析发现,S100A9的启动子区存在p53蛋白的潜在结合位点。在食管癌标本中验证发现,在p53发生突变的标本中S100A9的表达也明显下调。通过构建一系列包含S100A9启动子区不同长度片段的双荧光素报告基因载体进行双荧光素报告实验显示S100A9的转录活性与p53蛋白成剂量依赖关系,并明确了S100A9启动子区p53蛋白的两个结合位点。染色质免疫共沉淀实验(ChIP)体内证实了p53蛋白和S100A9启动子区的潜在结合位点存在结合,电泳迁移率变动分析实验(EMSA)也在体外确证了这种结合。细胞免疫荧光检测显示p53转染阳性的细胞内S100A9的表达水平也增高。腺病毒Ad-p53外源感染HaCaT细胞以及喜树碱处理内源升高HCT116细胞内p53蛋白水平时,利用Northern Blot和Western Blot分别检测发现S100A9的mRNA水平和蛋白水平都随之增高。S100A9能与S100A8形成异源二聚体在胞外诱导细胞凋亡,我们在胞内过表达S100A9发现同样能明显诱导细胞发生凋亡,S100A9的稳定克隆的生长也受到抑制。进一步在p53野生和缺失的HCT116细胞系中研究发现,S100A9诱导的细胞凋亡部分依赖于p53。我们的研究明确表明,肿瘤抑制蛋白p53能结合于3100A9基因的启动子区且发挥转录活性,并诱导S100A9的表达。S100A9是一个新的p53靶基因,同时S100A9诱导的细胞凋亡部分依赖于p53。这些研究将增加人们对S100家族与肿瘤抑制因子p53之间相互关系的认识,为阐明这些S100蛋白在肿瘤中扮演的确切角色提供了新的理论基础。更多还原

【Abstract】 Tumor suppressor p53 plays an important role in the progression of carcinogenesis. In response to inappropriate growth signals and various kinds of cellular stress, the most important tumor suppressor p53 functions by inducing either cell growth arrest or apoptosis, which are mainly mediated through the activation of its downstream target genes.We have previously found by cDNA microarray that a series of differentiation -associated genes were significantly down-regulated in esophageal squamous cell carcinoma compared with normal esophageal epithelium, most of them located on human chromosome 1q21. These differentiation-associated genes encode calcium-dependent or calcium regulated proteins, and involved in the formation of epidermis differentiation complex. We further studied one of these down-regulated genes, S100A9.S100A9 is a member of S100 protein family which exerts many biological activities in vivo through the interaction with calcium. Many S100 proteins are correlated with tumor development or progression. Some S100 proteins (S100B, S100A2, S100A4) carry p53 binding sites in their promoters, and their expression is transcriptionally regulated by p53. By the bioinformatics analysis, we found that p53 binding sites did exist on S100A9 promoter region. Further immunohistochemistry analysis for p53 and S100A9 on paired esophageal normal and cancer tissue microarray revealed that the tissue with high p53 expression, which indicated the mutant p53 status, express S100A9 at a low degree. We constructed a series of dual-luciferase reporter plasmids including different S100A9 promoter fragments, and found that S100A9 transactivity was p53 dose-dependent and affirmed two p53 binding sites. Chromatin immunoprecipitation (ChEP) and electrophoretic mobility shift assays (EMSA) showed that p53 was capable of binding to these putative p53 bindind sites in vivo and in vitro, respectively. Further immunofluorescence analysis showed that cells transiently transfected with p53 expressed more S100A9 protein consistently. Both Ad-p53 infection and camptothecin treatment could increase cellular p53 level, and S100A9 mRNA and protein expression could be positively regulated in a p53-dependent manner.Extracellular heterodimer composed of S100A8 and S100A9 can induce apoptosis. In this study, we showed that overexpression of S100A9 alone could also induce a significant degree of apoptosis and a slower growth rate compared with the control cells. Analysis of apoptosis in wild type p53 and knock out p53 HCT 116 cell lines showed that apoptosis induced by S100A9 was partly p53-dependent.Taken together, we conclude that a down-regulated gene in esophageal squamous cell carcinoma, S100A9 is a novel p53 transcriptional target gene and induce cellular apoptosis in a partly p53-dependent manner. This work would therefore help us know more about the relationship between S100 family and tumor suppressor p53, and shed light on the role of S100 proteins in carcinogenesis.更多还原