DNA Damage as Women Age: Implications for Breast Cancer

描述（由申请人提供）：拟议研究的目的是描述健康和肿瘤性乳腺上皮、肌上皮和间质中DNA结构的年龄相关变化。这些细胞级分之间的相互作用已被证明在乳腺和其他激素反应组织中的肿瘤发展中是关键的，并且可能至少部分地由活性氧产生的DNA结构的改变引起（例如，自由基）和微环境中的其他因素。我们的具体目标是： I.从20-85岁女性的非癌性乳腺（NN）正常组织和癌性乳腺（NC）组织学正常组织中分离纯上皮、肌上皮和富含成纤维细胞的基质的核DNA。我们将获得进行目标二和目标三所述分析所需数量的DNA。 二.确定8-羟基腺嘌呤的基底病变浓度（8-OH-Ade），8-羟基鸟嘌呤（8-OH-Gua），4，6-二氨基-5-甲酰胺基嘧啶（Fapyadenine [FapyAde]），和2，6-二氨基-4-羟基-5-甲酰胺基嘧啶（Fapyguanine [FapyGua]）和致突变性8-羟基（8-OH）与非-使用气相色谱-质谱法（GC-MS）测定来自每个分离的细胞级分的DNA中与女性年龄相关的致突变性损伤。我们假设：（a）诱变和其他基础损伤谱将反映相对较高程度的基础氧化，其将作为女性年龄的函数而变化，以及（B）对于三种细胞组分中的每一种，年龄-基础损伤关系将是不同的。 三.为了确定分离的DNA的傅里叶变换红外（FT-IR）光谱曲线，反映碱基和磷酸二酯-脱氧核糖骨架结构的振动，从而获得对官能团（例如，NH 2，C-O）和构象结构作为与女性年龄相关的每个细胞组分中DNA变化的生物标志物。我们假设（a）FT-IR光谱曲线将作为女性年龄的函数而变化，以及（B）年龄-光谱曲线关系对于三种细胞组分中的每一种都是不同的。 四.使用已建立的统计方案，将年龄数据与从两种组织类型（癌性和非癌性乳腺的组织学正常组织）分离的三种细胞组分（上皮、肌上皮和基质）的GC-MS和FT-IR生物标志物数据整合。我们假设，这种综合方法将导致对细胞组分中与年龄相关的DNA变化的初步，第一层次的理解，其相互作用已被广泛证明是乳腺癌发展的关键。

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.