Repair of Clustered DNA Damages

修复DNA簇损伤

• 批准号: 7728506
• 负责人: YOKE W KOW
• 金额: $ 23.89万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-11 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7728506
• 关键词: Accounting; Americas; Appearance; Area; Aspartic Acid; Attention; Biological; Biological Assay; Biological Process; Bypass; Cell Line; Cells; Codon Nucleotides; Complex; Contracts; Cutaneous Melanoma; Cyclobutanes; DNA; DNA Damage; DNA Repair Enzymes; DNA Repair Gene; DNA Sequence; Databases; Development; Environmental Risk Factor; Exons; Frequencies; GAG Gene; Generations; Genes; Genetic; Genomic Instability; Human; In Vitro; Individual; Ionizing radiation; Laboratories; Lead; Lesion; Measures; Melanocytic nevus; Molecular Genetics; Mus; Mutation; Mutation Spectra; NRAS gene; Nature; Nevi and Melanomas; Occupational; Oxidative Stress; Play; Process; Pyrimidine; Pyrimidines; Reactive Oxygen Species; Research; Risk Factors; Role; Single base substitution; Site; Skin Cancer; Small Interfering RNA; Sunlight; TP53 gene; Tissues; UV Radiation Exposure; Valine; Woman; base; cancer diagnosis; cancer type; environmental agent; immortalized cell; in vivo; insight; interest; keratinocyte; melanocyte; melanoma; men; mutant; novel; oxidative DNA damage; plasmid DNA; repaired; transversion mutation

Melanoma is the fifth and seventh most commonly diagnosed cancer in America men and women. The molecular and genetic basis for the formation of melanoma is still largely unclear. The majority of melanoma (90%) is of sporadic origin, and only about 10% appears to have familial clustering. Familiar melanoma has been observed to be associated with CDKN2a/ARF and CDK mutations. In contract, a high proportion of the sporadic melanoma shows mutation in the N-RAS and B-RAF gene. B-RAF mutations, in particular mutations in exon 15 were found to be associated with greater than 65% of melanoma. It is of interest to note that more than 90% of B-RAF mutations at exon 15 occur at V599. The predominant mutation signature at V599 is T to A transversion mutation (GTG to GAG), changing valine to aspartic acid. Tandem mutation at GTG(V599) site can account for up to 30 % of the B-RAF mutations observed in primary invasive melanoma. B-RAF mutations also occur at relatively high frequency (70-80%) in melanocytic nevi, but not the surrounding tissue. It is clear that UV exposure is a major etiological risk factor for cutaneous melanoma(CM); however, UV is not sufficient for the generation of the unique B-RAF mutation that is present in high frequencies in melanocytic nevi and melanoma. Genetic and environmental (occupational?) factors are potentially contributing to the development of sporadic melanoma and the appearance of the unique B-RAF mutation. We will focus on identifying the potential environmental and genetic factors that might cooperate with UV that lead to the generation of the unique V599 mutation. Two cell lines will be used for the proposed research, the immortalized normal keratinocytes, HaCat cells and immortalized normal melanocytes, PIG1 cells. These two cell lines will be exposed to various genotoxicants, singly or in combination with UV exposure. The mutation frequency and spectrum at the V599 codon will be determined for the mutants generated in both cell lines. Comparison of the mutation frequency and spectrum at V599 codon generated in these two cell lines is expected to help to delineate the potential agents that will lead to the generation of expected double mutations generated only in melanocytes and not in keratinocytes. In order to determine the potential genetic factors that might be involved, we will use siRNA to knockdown various DNA repair genes in both HaCat and PIG1 cells and determine which DNA repair enzyme plays a role in the generation of the unique V599 mutation. The mutation frequency will be determined by the random mutation assay developed by Loeb’s. The mutation spectrum at the V599 codon will be obtained by DNA sequencing of PCR fragments generated from the mutant cells that harbor the V599 mutation. Completion of the proposed research is expected to yield crucial information on the nature of the potential environmental agents that play an essential role in the formation of melanoma. In addition, it will provide significant insight as to the potential mechanism involved in melanoma progression.

黑色素瘤是美国男性和女性第五和第七常见的癌症。黑色素瘤形成的分子和遗传基础仍然在很大程度上不清楚。大多数黑色素瘤（90％）具有弹性，只有大约10％的家庭聚类。已经观察到熟悉的黑色素瘤与CDKN2A/ARF和CDK突变有关。在合同中，很大一部分的弹性黑色素瘤显示出N-RAS和B-RAF基因的突变。 B-RAF突变，特别是外显子15突变为 与大于65％的黑色素瘤有关。值得注意的是，在V599时出现外显子15的B-RAF突变中有90％以上。 V599处的主要突变特征是t转向突变（GTG至GAG），将缬氨酸更改为天冬酸。 GTG（V599）部位的串联突变可以占原发性浸润性黑色素瘤中观察到的B-RAF突变的30％。 B-RAF突变也发生在相对较高的频率（70-80％）中 黑素细胞nevi，但没有周围的组织。 显然，紫外线暴露是皮肤黑色素瘤（CM）的主要病因危险因素。然而，紫外线不足以产生在黑色素细胞NEVI和黑色素瘤中高频出现的独特B-RAF突变。遗传和环境（职业？）因素可能有助于零星黑色素瘤的发展和独特的B-RAF突变的出现。 我们将专注于确定可能与紫外线协调的潜在环境和遗传因素，从而导致独特的V599突变产生。拟议的研究将使用两种细胞系，即永生的正常角质形成细胞，HACAT细胞和永生的正常黑色素细胞PIG1细胞。这两种细胞系将单独或与紫外线暴露结合使用各种遗传毒性。 V599密码子的突变频率和光谱将针对两种细胞系中产生的突变体确定。 预计在这两种细胞系中生成的V599密码子上的突变频率和光谱的比较有助于描绘出可能导致仅在黑素细胞而不是在角质形成细胞中产生的预期双突变的潜在药物。为了确定可能涉及的潜在遗传因素，我们将使用siRNA敲低HACAT和PIG1细胞中的各种DNA修复基因，并确定哪种DNA修复 酶在独特的V599突变的产生中起作用。突变频率将由勒布开发的随机突变测定法确定。 V599密码子的突变光谱将通过从含有V599突变的突变细胞产生的PCR片段来获得。预计拟议研究的完成将产生有关潜在环境代理的性质的重要信息 黑色素瘤形成中的重要作用。此外，它将就黑色素瘤进展的潜在机制提供重要的见解。