KI RAS ACTIVATION AND ADDUCT FORMATION BY CYCLOPENTA PAH

这项研究的长期目标是深入了解机制 化学诱导的 DNA 损伤被加工成突变， 最终可能导致肿瘤发生。 这个具体项目旨在 将环戊基多环芳烃 (cyclopentaPAH) 形成 DNA 加合物与 Ki- 的激活联系起来 A/J 品系小鼠肺部肿瘤中的 ras 原癌基因。环五PAH是 具有生物活性的非交替多环芳烃，在不完全燃烧中形成 过程，似乎是由不同的代谢途径激活的 从那些推断具有湾区特征的交替多环芳烃。 的 迄今为止测试的环戊多环芳烃都是 A/J 小鼠肺中的强致肿瘤原， 许多由此产生的肿瘤携带不寻常的 GGT 到 CGT 转换 Ki-ras 的密码子 l2 中未发现交替 PAH 的情况。的可用性 由环戊基多环芳烃共价修饰的核苷使我们能够开始 将特定加合物与突变相关联，并使 生成位点特异性修饰的 DNA 序列，用作 用于研究化学诱导的复制和加工的模板 病变，进行如下： I. 肿瘤生物学：评估四种物质的相对致癌能力 结构相关的环五PAH、环五[cd] 芘、苯[j) 苊，苯并 [l) 苊和苊，在 A/J 小鼠中 肺；验证肿瘤组织中是否存在激活的 Ki-ras；检查 由此产生的突变谱，特别是丰度方面 Ki-ras 密码子 12 中独特的 GGT 到 CGT 突变。 二.环戊多环芳烃的代谢和激活：识别和定量 在 A/J 小鼠肺中发现最终活性代谢物，并验证 与形成的DNA加合物的一致性。 确定身份并 形成的加合物的立体化学及其定量关系 给药剂量和观察到的致瘤效力。 三．加合物化学：合成和表征假定的专业 环戊多环芳烃与脱氧腺苷和脱氧鸟苷的加合物，制备 3'-磷酸核苷酸用作色谱标准品，以及 3'- 亚磷酰胺-5'-DMT加合物衍生物的合成和 修饰的 Ki-ras 寡核苷酸序列的表征。 四．位点特异性修饰的 Ki-ras 序列的分子生物学： 研究 Ki-ras 上复制产生的突变谱 由环戊基多环芳烃加合物修饰的模板。 描述亲属的特征 环戊多环芳烃 (cyclopentaPAH) 诱导的主要突变的转化效率 Ki-ras 密码子 12，并与突变谱相关。

The long-term goals of this research are to gain insight into mechanisms by which chemically-induced DNA lesions are processed into mutations that may ultimately lead to tumorigenesis. This specific project aims to relate formation of DNA adducts by cyclopentaPAH to activation of the Ki- ras protooncogene in strain A/J mouse lung tumors. The cyclopentaPAH are biologically active non-alternant PAH, formed in incomplete combustion processes, that appear to be activated by metabolic pathways differing from those inferred for alternant PAH with bay-region features. The cyclopentaPAH tested to date are all potent tumorigens in A/J mouse lung, and many of the resulting tumors carry an unusual GGT to CGT transversion in codon l2 of Ki-ras not seen with alternant PAH. The availability of nucleosides covalently modified by cyclopentaPAH enables us to begin to correlate specific adducts with mutations, and renders feasible the generation of site-specifically modified DNA sequences for use as templates to investigate replication and processing of chemically-induced lesions, proceeding as follows: I. Tumor Biology: Evaluate the relative carcinogenic potencies of four structurally-related cyclopentaPAH, cyclopenta [cd) pyrene, benz [j) aceanthrylene, benz [l) aceanthrylene and aceanthrylene, in A/J mouse lung; verify the presence of activated Ki-ras in tumor tissue; examine the resulting mutational spectra, particularly with respect to the abundance of the unique GGT to CGT mutation in codon 12 of Ki-ras. II. Metabolism and Activation of CyclopentaPAH: Identify and quantitate ultimate active metabolites found in A/J mouse lung, and verify consistency with the DNA adducts formed. Determine the identity and stereochemistry of adducts formed, and their quantitative relationship to administered dose and observed tumorigenic potency. III. Chemistry of Adducts: Synthesize and characterize the putative major adducts of cyclopentaPAH with deoxyadenosine and deoxyguanosine, prepare 3'-phosphate nucleotides to use as chromatography standards, and 3'- phosphoramidite-5'-DMT adduct derivatives for synthesis and characterization of modified Ki-ras oligonucleotide sequences. IV. Molecular Biology of Site-Specifically Modified Ki-ras sequences: Investigate mutational spectra resulting from replication on Ki-ras templates modified by cyclopentaPAH adducts. Characterize the relative transforming efficiencies of the major cyclopentaPAH-induced mutations of Ki-ras codon 12, and correlate with mutational spectra.