MODULATION OF DNA REPAIR BY P53 IN HUMAN UROEPITHELIUM

P53 对人尿上皮 DNA 修复的调节

• 批准号: 2870290
• 负责人: SANTHANAM SWAMINATHAN
• 金额: $ 20.39万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-17 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2870290
• 关键词: DNA damage; DNA repair; biphenylamines; bladder neoplasm; carcinogen testing; chemical carcinogen; chemical carcinogenesis; enzyme activity; human genetic material tag; human tissue; hypoxanthine phosphoribosyltransferase; mutagen testing; mutagens; neoplasm /cancer genetics; p53 gene /protein; tissue /cell culture; transitional cell carcinoma; urinary bladder epithelium

The hypothesis that wild-type p53 (wt-p53) modulates repair of DNA damage caused by the human bladder carcinogen, 4-aminobiphenyl (ABP), will be tested in its actual in vivo target cell types, namely the human uroepithelial cells (HUC). Using a unique set of isogeneic cell lines, differing in wt-p53 functional status (due to the expression of HPV-E6 oncoprotein or a transdominant mutant of p53), the following questions will be addressed. What types of DNA damage (covalent adducts and strand breaks) are caused by the N-hydroxy metabolites of ABP in HUC? Does the loss of function of wt-p53 cause a reduction in the rate of DNA repair either at the level of overall genomic DNA or at the level of an individual gene, such as hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene?; and if so, does the effect of wt-p53 dependent upon the transcription status? To answer the above, DNA strand breaks and covalent adduct formation will be analyzed by alkaline elution, sucrose gradient sedimentation and by 32P-postlabeling methods. The effect of loss of function of wt-p53 on DNA lesion will be determined by comparison of the kinetics of DNA repair between the set of isogeneic cell lines, differing in wt-p53 activity. The kinetics of DNA repair will be measured by: 1) monitoring the disappearance of ABP-DNA adducts by 32P-postlabeling; 2) by quantifying the unrepaired DNA adducts by digestion with E. Coli UvrABC nuclease; and 3) by estimating the DNA repair patch synthesis. The effect of wt-p53 on the transcription dependent versus -independent repair will be determined by measuring the kinetics of repair synthesis using riboprobes selective for the transcribed versus nontranscribed strand of HGPRT. A rare feature of our HUC system is that it permits us to test the effect of the environmental carcinogen ABP, directly on its in vivo target cells, and thus surmount the serious limitations of the fibroblast or rodent culture system. The results of these studies have important etiologic, mechanistic and therapeutic implications in human cancers.

野生型p53(wt-p53)调控DNA修复的假说 人类膀胱癌致癌物质4-氨基联苯(ABP)造成的损害， 将在其实际的体内靶细胞类型，即人类 尿上皮细胞(Huc)。使用一组独特的同源细胞系， Wt-p53功能状态不同(由于HPV-E6的表达 癌蛋白或P53的反显性突变体)，以下问题 将会得到解决。 造成哪些类型的DNA损伤(共价加合物和链断裂) ABP的N-羟基代谢物？功能的丧失 Wt-p53的缺失会导致DNA修复率的降低 整体基因组DNA水平或单个基因水平，如 作为次黄嘌呤鸟嘌呤磷酸核糖基转移酶基因？；以及 如果是这样，wt-p53的作用是否依赖于转录 情况如何？ 为了回答上述问题，DNA链断裂和共价加合物的形成 将通过碱洗脱法、蔗糖梯度沉淀法和 ~(32)P-后标记法。Wt-p53功能丧失的影响 将通过比较DNA的动力学来确定对DNA的损伤 在wt-p53不同的一组同基因细胞系之间的修复 活动。DNA修复的动力学将通过以下方式进行测量：1)监测 ~(32)P-后标记ABP-DNA加合物的消失； 用E.Coli UvrABC消化定量未修复的DNA加合物 核酸酶；以及3)通过估计DNA修复斑块的合成。这个 Wt-p53对转录依赖与非依赖转录的影响 修复将通过测量修复合成的动力学来确定 使用核探针选择性地检测转录的和非转录的 HGPRT链。 我们的Huc系统的一个罕见功能是它允许我们测试 环境致癌物ABP对其体内的直接作用 目标细胞，从而克服了 成纤维细胞或啮齿动物培养系统。这些研究的结果是 人类的重要病因学、机制和治疗意义 癌症。