MODULATION OF DNA REPAIR BY P53 IN HUMAN UROEPITHELIUM

野生型p53（wt-p53）调节DNA修复的假说 由人类膀胱致癌物4-氨基联苯（ABP）引起的损伤， 将在其实际的体内靶细胞类型中进行测试，即人 尿上皮细胞（HUC）。 使用一组独特的同基因细胞系， 不同的wt-p53功能状态（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。 导致什么类型的DNA损伤（共价加合物和链断裂） ABP在HUC中的N-羟基代谢产物？ 功能的丧失 wt-p53的表达导致DNA修复速率的降低， 在整个基因组DNA水平上或在单个基因水平上， 次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）基因;和 如果是，wt-p53的作用是否依赖于转录， 身份？ 为了回答上述问题，DNA链断裂和共价加合物的形成 将通过碱性洗脱、蔗糖梯度沉降和 32 P后标记法。 wt-p53功能丧失的影响 对DNA损伤的影响将通过比较DNA损伤的动力学来确定。 在wt-p53不同的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测量：1）监测 ABP-DNA加合物的消失由32 P-后标记; 2）由 通过用E.大肠杆菌UvrABC 核酸酶;和3）通过估计DNA修复补丁合成。 的 wt-p53对转录依赖性与非依赖性的影响 修复将通过测量修复合成的动力学来确定 使用对转录的和非转录的具有选择性的核糖核酸探针 HGPRT链。 我们的HUC系统的一个罕见的功能是，它允许我们测试 环境致癌物ABP的影响，直接影响其在体内 靶细胞，从而克服严重的局限性， 成纤维细胞或啮齿动物培养系统。 这些研究的结果 对人类重要病因学、机制和治疗意义 癌的

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.