KI RAS ACTIVATION AND ADDUCT FORMATION BY CYCLOPENTA PAH

这项研究的长期目标是深入了解 化学诱导的DNA损伤被加工成突变， 可能最终导致肿瘤发生。 该项目旨在 将环戊二烯基PAH形成的DNA加合物与Ki- ras原癌基因在A/J系小鼠肺肿瘤中的表达。环戊多环芳烃是 生物活性非交替PAH，在不完全燃烧中形成 过程，似乎是由不同的代谢途径激活 从海湾地区特征的交替PAH推断。 的 迄今为止测试的环戊二烯基PAH在A/J小鼠肺中都是有效的致瘤剂， 许多由此产生的肿瘤携带一种不寻常的GGT到CGT的颠换 Ki-ras基因第12位密码子缺失与PAH交替发生无关。的可用性 通过环戊二烯基PAH共价修饰的核苷使我们能够开始 将特定的加合物与突变相关联，并使 产生位点特异性修饰的DNA序列， 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：评估四种药物的相对致癌效力 结构相关环戊二烯并[cd]芘，苯并[j] A/J小鼠中的醋蒽烯、苯并[l]醋蒽烯和醋蒽烯 肺;验证肿瘤组织中激活的Ki-ras的存在;检查 产生的突变谱，特别是相对于丰度 Ki-ras基因第12位密码子GGT突变为CGT。 二.环戊二烯基PAH的代谢和活化：鉴别和定量 在A/J小鼠肺中发现的最终活性代谢物，并验证 与形成的DNA加合物一致。 确定身份， 形成的加合物的立体化学，以及它们与 给药剂量和观察到的致瘤效力。 三.加合物化学：合成和表征推定的主要加合物 环戊二烯基多环芳烃与脱氧腺苷和脱氧鸟苷的加合物的制备 用作色谱标准品的3 '-磷酸核苷酸，和 用于合成的亚磷酰胺-5 '-DMT加合物衍生物， 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复制导致的突变谱 模板修饰的cyclopentaPAH加合物。 描述亲属的特征 环戊二烯基PAH诱导的主要突变的转化效率 Ki-ras基因第12位密码子突变，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.