KI RAS ACTIVATION AND ADDUCT FORMATION BY CYCLOPENTA PAH

环戊基 PAH 激活 KI RAS 并形成加合物

• 批准号: 2458998
• 负责人: Avram Gold
• 金额: $ 13.64万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2458998
• 关键词: DNA damage; adduct; carbopolycyclic compound; chemical carcinogen; chemical carcinogenesis; cyclopentane; deoxyadenosines; deoxyguanosine; detoxification; gene mutation; high performance liquid chromatography; laboratory mouse; lung; molecular biology; nucleic acid chemical synthesis; oligonucleotides; oncogenes; polymerase chain reaction; stereochemistry; synthetic nucleic acid; toxin metabolism; transfection /expression vector

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.

这项研究的长期目标是深入了解机制 通过它化学诱导的DNA损伤被处理成突变 可能最终导致肿瘤的发生。这一具体项目旨在 环戊烷多环芳烃DNA加合物的形成与KI-激活的关系 RAS原癌基因在A/J系小鼠肺肿瘤中的表达环戊烷多环芳烃是 生物活性非交替多环芳烃，在不完全燃烧中形成 过程，似乎被不同的代谢途径激活 与海湾地区特征的交替多环芳烃的推断结果不同。这个 到目前为止，环五环芳烃在A/J小鼠肺中都是有效的致癌物质， 许多由此产生的肿瘤携带着一种不寻常的GGT到CGT的转换 在Ki-ras密码子L2中，未见交替PAH。是否可以使用 由环戊烷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 位点特异性修饰DNA序列的生成 用于研究化学诱导的复制和加工的模板 损害，按如下步骤进行： I.肿瘤生物学：评估四种药物的相对致癌能力 结构相关的环戊烷多环芳烃、环戊二烯、苯并[j] 苯并[L]、苯并[L]和苯并[L]对A/J小鼠的影响 肺；验证肿瘤组织中是否存在激活的Ki-ras；检查 由此产生的突变光谱，特别是关于丰度 Ki-ras基因第12位密码子GGT到CGT的独特突变。 II.环戊烷多环芳烃的代谢和活化：鉴定和定量 在A/J小鼠肺中发现最终活性代谢物，并验证 与DNA加合物形成的一致性。确定身份和 形成的加合物的立体化学及其与 给药剂量和观察致瘤能力。 加合物化学：合成和表征假定的主语 环戊多环芳烃与脱氧腺苷和脱氧鸟苷的加合物，制备 用作层析标准的3‘-磷酸核苷酸和3’- 亚磷酰胺-5‘-DMT加合物的合成和应用 修饰的Ki-ras寡核苷酸序列的特征。 四、定点修饰KI-ras序列的分子生物学： 研究Ki-ras基因复制引起的突变谱 由环戊烷多环芳烃加合物修饰的模板。描述亲属的特征 环五环戊烷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。