Function of REIC/Dkk-3, a putative Wntregulator in normal skin and cancer

1.筛选携带ReIC/DKK-3的重组腺病毒(Ad-ReIC)感染正常人角质形成细胞后表达上调的基因，结果表明该细胞因子具有较强的诱导性。通过体外和体内实验，我们得出结论：Ad-reic治疗通过体内的细胞因子激活了对癌细胞的免疫反应。当使用Ad-REIC过表达时，REIC/DKK-3可诱导癌细胞凋亡，但不能诱导正常细胞。阿德瑞克对人前列腺癌(PC3)、睾丸癌(NCCIT)和小鼠肾癌(RencA)均有效，但并不是所有肿瘤都对阿德瑞克敏感。为了了解腺病毒敏感性的重要机制，我们比较了腺病毒敏感的肾癌细胞(RECA)和正常成纤维细胞(N1H3T3)。我们发现Hsp70/72的表达水平影响Ad-REIC对肿瘤细胞的特异性诱导凋亡。我们从一株前列腺癌细胞系(PC3)中建立了Adreic耐药亚系。一项概述这些基因表达谱的微阵列分析正在进行中。通过比较Adreic处理后的内质网应激反应，我们发现了一个与Adreic敏感性有关的关键分子。我们还建立了一种从人成纤维细胞中纯化REIC/DKK-3蛋白的方法。我们正在比较添加这种类型的纯化蛋白和通过Ad-REIC5强制表达系统。新一代REIC/DKK-3基因敲除小鼠正在研究中。我们已经得到了敲除的ES细胞，我们正在等待嵌合体的分生。

1. The screening of genes which expression level were elevated after the infection of an adenovirus canying REIC/Dkk-3 (Ad-REIC) into normal human keratinocytes showed that a specific cytokine was strongly induced. After in vitro and in vivo experiments, we conclude that Ad-REIC treatment activates immune reaction against cancer cells through the cytokine in vivo.2. When over expressed using Ad-REIC, REIC/Dkk-3 induced apoptosisin cancer cells but not in normal cells. Ad-REIC was effective on human prostate cancer (PC3), testicular cancer (NCCIT) and mouse renal carcinoma (RENCA), but not all cancers are sensitive to Ad-REIC. To know the crucial mechanism of Ad-REIC sensitivity, we compared Ad-REIC sensitive renal cancer cells (RENCA) with normal fibroblast (N1H3T3). We found that the expression level of Hsp70/72 affected on tumor-cell specific induction of apoptosis by Ad-REIC.3. We established Ad-REIC-resistant sub-lines from a prostate cancer cell line (PC3). A micro array analysis to overview the expression profile of these lines is on the way. After the comparison of ER stress response after Ad-REIC treatment, we found a key molecule to be responsible for the Ad-REIC sensitivity.4. We also established a method for the purification of REIC/Dkk-3 protein from human fibroblasts. We are comparing addition of this type of purified protein with forced expression system by Ad-REIC.5. Generation of REIC/Dkk-3 knockout mouse is under way. We already got knockout ES cells and we are waiting chimera littermates.