Mapping DNA Repair and Error-Prone DNA Synthesis in Geriatric Skin

项目摘要/摘要 非黑色素瘤皮肤癌(NMSCs)在我国老年人口中是一个日益严重的问题， 而且目前的治疗方法既昂贵，又可能毁容。影响NMSC发展的主要风险因素 暴露于紫外线B(UVB)波长的阳光和高龄。尽管病毒的诱变作用 中波紫外线诱导基因组DNA中的联嘧啶二聚体是众所周知的，年龄影响中波紫外线的机制。 依赖NMSC在皮肤角质形成细胞中的启动不太清楚。独特的结构和生理特性 老年性皮肤的缺陷可能会影响整个基因组中形成的光斑的数量和分布 紫外线照射后的基底层角质形成细胞以及这些细胞对损伤的反应方式。此外， 众所周知，包括皮肤在内的衰老组织中含有比年轻时更多的衰老真皮成纤维细胞。 皮肤，这可能会改变细胞因子和生长因子的产生，并有助于促肿瘤 微环境。在老年皮肤中表达水平较低的一种这样的因子是胰岛素样生长因子 1(IGF-1)。我们已发表的和初步的数据表明，胰岛素样生长因子-1/胰岛素样生长因子-1受体(IGF-1R)信号的丢失在 表皮角质形成细胞经UVB照射后不能有效去除UVB 来自DNA的光产物或正确激活顶端DNA损伤反应激酶ATR，它的功能 抑制受损DNA模板上容易出错的DNA合成。这项建议的目的是 确定年龄，特别是IGF-1受体的激活如何影响UVB DNA的形成和复制 人皮肤基底层角质形成细胞中的光产物。研究团队的背景，这是 包括DNA修复、复制应激反应信号、基因组学、角质形成细胞生物学以及 临床皮肤科，使这一群体唯一有资格解决这一重要的临床问题。中环 这一建议的假设是，老年人的皮肤容易发生更多的突变形式的 紫外线照射后的DNA合成比年轻个体的皮肤合成更多。这一假说已被推出。 来自体外培养的角质形成细胞和人类皮肤活检的重要初步和已发表的数据 体外和体内。这项拟议研究的基本原理是，它将定义 老年皮肤易发生UVB诱变和致癌作用。我们的假设将通过携带 提出以下三个具体目标：目标1)确定年龄和IGF-1状态如何影响紫外线诱导的DNA 人皮肤表皮角质形成细胞基因组中光产物的形成和修复；目的2)确定 IGF-1信号如何影响UVB照射的人皮肤中易出错的DNA聚合酶的利用 外植体和体外培养的角质形成细胞；目的3)确定受试者的年龄和IGF-1状态如何影响 中波紫外线损伤的DNA在人皮肤表皮中的体内复制我们的方法是创新的，因为它 将提供皮肤表皮中DNA损伤形成和复制的详细、定量的表征 使用以前从未应用于人类皮肤的新技术方法。拟议的研究 对退伍军人有重要意义，因为它将显示老年皮肤微环境如何对紫外线做出贡献 诱变和致癌。最终，我们希望在这里获得的知识将提供 使用旨在预防NMSC的临床皮肤年轻化干预措施的进一步机制基础 特定的患者群体。最后，因为许多其他癌症都与高龄和 上皮组织暴露在环境和饮食DNA损伤剂中，我们的工作可能有 对了解老年退伍军人肺癌、结肠癌和食道癌发病机制的启示。

PROJECT SUMMARY/ABSTRACT Non-melanoma skin cancers (NMSCs) are a growing problem among our nation’s Veteran population, and current treatments are both costly and potentially disfiguring. The primary risk factors for NMSC development are exposure to ultraviolet B (UVB) wavelengths of sunlight and advanced age. Though the mutagenic effects of UVB-induced bipyrimidine dimers in genomic DNA are well known, the mechanisms by which age impacts UVB- dependent NMSC initiation in skin keratinocytes are less clear. The unique structural and physiological properties of geriatric skin likely influence both the number and distribution of photolesions that form across the genome of basal keratinocytes after UV exposure and the ways in which these cells respond to the damage. Moreover, aged tissues including skin are known to contain a higher number of senescent dermal fibroblasts than younger skin, which may alter cytokine and growth factor production and contribute to a pro-tumorigenic microenvironment. One such factor that is expressed at lower levels in geriatric skin is insulin-like growth factor 1 (IGF-1). Our published and preliminary data show that the loss of IGF-1/IGF-1 receptor (IGF-1R) signaling in epidermal keratinocytes results in UVB-irradiated keratinocytes that are unable to efficiently remove UVB photoproducts from DNA or to properly activate the apical DNA damage response kinase ATR, which functions to suppress error-prone DNA synthesis on damaged DNA templates. The objective of this proposal is to determine how age and specifically IGF-1 receptor activation affect the formation and replication of UVB DNA photoproducts in basal epidermal keratinocytes in human skin. The background of the research team, which includes expertise in DNA repair, replication stress response signaling, genomics, keratinocyte biology, and clinical dermatology, makes this group uniquely qualified to address this important clinical issue. The central hypothesis of this proposal is that the skin of geriatric individuals is prone to carry out a more mutagenic form of DNA synthesis following UVB exposure than the skin of younger individuals. This hypothesis has been derived from significant preliminary and published data with both cultured keratinocytes in vitro and human skin biopsies ex vivo and in vivo. The rationale for this proposed research is that it will define how the unique features of geriatric skin predispose it to UVB mutagenesis and carcinogenesis. Our hypothesis will be tested by carrying out the following three specific aims: Aim 1) Define how age and IGF-1 status influence UVB-induced DNA photoproduct formation and repair across the keratinocyte genome in human skin epidermis; Aim 2) Determine how IGF-1 signaling impacts the utilization of error-prone DNA polymerases in UVB-irradiated human skin explants ex vivo and cultured keratinocytes in vitro; and Aim 3) Define how subject age and IGF-1 status affect the replication of UVB-damaged DNA in human skin epidermis in vivo. Our approach is innovative because it will provide a detailed, quantitative characterization of DNA damage formation and replication in skin epidermis using novel technical approaches that have not previously been applied to human skin. The proposed research is significant to the VA because it will show how the geriatric skin microenvironment contributes to UV mutagenesis and carcinogenesis. Ultimately, we expect that the knowledge that will be gained here will provide further mechanistic rationale for the use of clinical skin rejuvenation interventions aimed at preventing NMSC in specific patient populations. Lastly, because many other cancers are associated with both advanced age and the exposure of epithelial tissues to environmental and dietary DNA damaging agents, our work likely has implications for understanding the pathogenesis of lung, colon, and esophageal cancers in older veterans.