DNA Damage as Women Age: Implications for Breast Cancer

描述(由申请人提供)：拟议研究的目标是描绘健康和肿瘤乳腺的上皮、肌上皮和间质中DNA结构随年龄的变化。这些细胞组分之间的相互作用已被证明在乳腺和其他激素反应组织的肿瘤发展中起关键作用，至少部分原因可能是由微环境中的活性氧物种(例如，自由基)和其他因素产生的DNA结构的改变。我们的具体目标是： 1.从20-85岁女性乳腺癌(NC)正常组织和非癌乳腺(NN)正常组织中分离纯上皮、肌上皮和富含成纤维细胞的间质的核DNA。我们将获得所需数量的DNA，以进行AIMS II和AIMS III中概述的分析。 2.用气相色谱-质谱法(GC-MS)测定8-羟基腺嘌呤(8-OH-Ade)、8-羟基鸟嘌呤(8-OH-Gua)、4，6-二氨基-5-甲酰胺(Fapyadine[FapyAde])和2，6-二氨基-4-羟基-5-甲酰胺(Fapygua[Fapygua])在DNA中的碱基损伤浓度以及致突变的8-羟基(8-OH)与非致突变的Fapy损伤的比例。我们假设(A)突变和其他碱基病变特征将反映相对较高的碱基氧化程度，这将随着女性年龄的变化而变化，(B)三个细胞组分中的每一个与年龄的基础病变关系都将不同。 3.确定反映碱基和磷酸二酯-脱氧核糖骨架结构振动的分离DNA的傅立叶变换红外(FT-IR)谱，从而广泛了解官能团(如NH2、C-O)和构象结构的变化，作为DNA随女性年龄变化的生物标记物。我们假设(A)FT-IR光谱曲线将随着女性年龄的变化而变化，以及(B)年龄-光谱曲线关系对于三个细胞组分中的每一个都将是不同的。利用已建立的统计方案，将年龄数据与GC-MS和FT-IR生物标志物数据进行整合，这些数据是从两种组织类型(乳腺癌和非癌乳腺的组织学正常组织)中分离出来的三种细胞组分(上皮、肌上皮和间质)的数据。我们假设，这种综合的方法将导致对细胞部分中与年龄相关的DNA变化的初步、第一水平的了解，这些细胞部分的相互作用已被广泛表明在乳腺癌的发展中起着关键作用。

DESCRIPTION (provided by applicant): The objectives of the proposed research are to delineate age-related changes in DNA structure in the epithelium, myoepithelium, and stroma of the healthy and neoplastic breast. Reciprocal interactions between these cellular fractions have been shown to be pivotal in tumor development in the breast and other hormone response tissues and may at least partially result from alterations in DNA structure produced by reactive oxygen species (e.g., free radicals) and other factors in the microenvironment. Our specific aims are: I. To isolate nuclear DNA of pure epithelium, myoepithelium, and fibroblast-enriched stroma from normal tissue from the non-cancerous breast (NN) and from histologically normal tissue from the cancerous breast (NC) of women aged 20-85 years. We will obtain the required quantities of DNA to perform the analyses outlined in aims II and III. II. To determine base lesion concentrations of 8-hydroxyadenine (8-OH-Ade), 8-hydroxyguanine (8-OH-Gua), 4,6- diamino-5-formamidopyrimide (Fapyadenine [FapyAde]), and 2,6-diamino-4-hydroxy-5-formamidopyrimide (Fapyguanine [FapyGua]) and the ratio of mutagenic 8-hydroxy (8-OH) to non-mutagenic Fapy lesions in DNA from each of the isolated cellular fractions in relation to a woman's age using gas chromatography-mass spectrometry (GC-MS). We hypothesize (a) that the mutagenic and other base lesion profiles will reflect a relatively high degree of base oxidation which will change as a function of a woman's age, and (b) that age-base lesion relationships will be different for each of the three cellular fractions. III. To determine Fourier transform-infrared (FT-IR) spectral profiles for the isolated DNA reflecting vibrations in base and phosphodiester-deoxyribose backbone structures, thus obtaining a broad understanding of alterations in functional group (e.g., NH2, C-O) and conformational structure as biomarkers for DNA changes in each cellular fraction in relation to a woman's age. We hypothesize (a) that the FT-IR spectral profiles will change as a function of a woman's age, and (b) that age-spectral profile relationships will be different for each of the three cellular fractions. IV. To integrate, using established statistical protocols, the age data with the GC-MS and FT-IR biomarker data for the three cellular fractions (epithelium, myoepithelium, and stroma) isolated from the two tissue types (histologically normal tissues from cancerous and non-cancerous breasts). We hypothesize that this integrated approach will lead to an initial, first level of understanding of age-related DNA changes in cellular fractions whose reciprocal interactions have been widely shown to be pivotal in breast cancer development.