MODULATION OF DNA REPAIR BY P53 IN HUMAN UROEPITHELIUM

野生型 p53 (wt-p53) 调节 DNA 修复的假设 人类膀胱致癌物 4-氨基联苯 (ABP) 造成的损害， 将在其实际体内靶细胞类型（即人类）中进行测试 尿上皮细胞（HUC）。 使用一组独特的同基因细胞系， wt-p53 功能状态不同（由于 HPV-E6 的表达） 癌蛋白或 p53 的转显性突变体），以下问题 将得到解决。 引起什么类型的 DNA 损伤（共价加合物和链断裂） HUC 中 ABP 的 N-羟基代谢物？ 是否丧失功能 wt-p53 会导致 DNA 修复率降低 整体基因组 DNA 水平或单个基因水平，例如 作为次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）基因？；和 如果是这样，wt-p53 的作用是否依赖于转录 状态？ 为了回答上述问题，DNA链断裂和共价加合物形成 将通过碱性洗脱、蔗糖梯度沉降和 通过32P后标记方法。 wt-p53 功能丧失的影响 通过比较 DNA 的动力学来确定 DNA 损伤的情况 wt-p53 不同的同基因细胞系组之间的修复 活动。 DNA 修复动力学将通过以下方式测量：1) 监测 32P-后标记导致 ABP-DNA 加合物消失； 2）通过 通过大肠杆菌 UvrABC 消化来定量未修复的 DNA 加合物 核酸酶； 3) 通过估计 DNA 修复补丁的合成。 的 wt-p53 对转录依赖性与非依赖性转录的影响 修复将通过测量修复合成的动力学来确定 使用对转录与非转录有选择性的核糖探针 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的影响，直接对其体内 靶细胞，从而克服了现有技术的严重局限性 成纤维细胞或啮齿动物培养系统。 这些研究的结果 对人类具有重要的病因学、机制和治疗意义 癌症。

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.