Mitochondrial DNA Mutations in Keratinocyte Hyperplasia

描述（由申请方提供）：表皮的异常增殖与紫外线辐射的累积无保护暴露相关，导致体细胞突变的累积。最近的研究表明，线粒体不仅在提供细胞能量方面发挥重要作用，而且可能在细胞凋亡和肿瘤形成中发挥核心作用。现在有几个报告的线粒体基因，以前被认为只在能量生产中发挥作用，尚未显示在熟悉的肿瘤综合征中发生突变，以及人类肿瘤中获得性同质线粒体DNA（mtDNA）突变的报告。该建议旨在测试线粒体DNA变化在光老化和异常角质形成细胞增生过程中的作用。首先，紫外线辐射将用于诱导无毛小鼠的光损伤和肿瘤产生。将研究光损伤皮肤和良性和恶性表皮肿瘤的mtDNA，以确定光损伤小鼠皮肤中体细胞mtDNA突变的类型。接下来，将通过分离线粒体并融合到缺乏其自身mtDNA的成纤维细胞中以创建细胞质杂交体（cybrid）文库来从过度增殖的角质形成细胞中回收mtDNA突变。将筛选单个胞质杂种克隆的mtDNA突变，并分析这些DNA变化的影响。将研究胞质杂交体的氧化磷酸化生物化学和ATP产生的改变，以及细胞表型的改变，包括生长特征、基质金属蛋白酶表达、逃避细胞凋亡的能力和形成肿瘤的能力。突变型mtDNA相对于野生型显性的能力也将在融合试验中进行遗传学研究。最后，恢复的mtDNA突变将返回到活的表皮，通过transmitochondrial转基因小鼠的一代，以研究这些突变的能力，改变生长和肿瘤的发展，在完整的小鼠表皮。

DESCRIPTION (provided by applicant): Abnormal proliferation of the epidermis is associated with cumulative unprotected exposure to ultraviolet radiation leading to the accumulation of somatic mutations. Recent research has shown that mitochondria not only play an important role in providing cellular energy, but also may have a central role in apoptosis and neoplasia. There are now several reports of mitochondrial genes that were previously thought to function only in energy production that have not been shown to be mutated in familiar tumor syndromes, and reports of acquired homoplasmic mitochondrial DNA (mtDNA) mutations in human tumors. This proposal seeks to test the role of mtDNA changes in the process of photoaging and aberrant keratinocyte hyperplasia. First, ultraviolet radiation will be used to induce photodamage and tumor production in the hairless mouse. The mtDNA will be studied from photodamaged skin and from benign and malignant epidermal neoplasms to determine the types of somatic mtDNA mutations in photodamaged mouse skin. Next, mtDNA mutations will be recovered from hyperproliferating keratinocytes by isolation of mitochondria and fusion into fibroblast cells that lack their own mtDNA to create cytoplasmic hybrid (cybrid) libraries. Individual cybrid clones will be screened for mtDNA mutations and the effect of these DNA changes will be analyzed. The cybrids will be studied for alterations in oxidative phosphorylation biochemistry and ATP production, as well as for alterations in cellular phenotype including growth characteristics, matrix metalloproteinases expression, ability to escape apoptosis, and ability to form tumors. The ability of mutant mtDNAs to become dominant over wild type will also be studied genetically in fusion assays. Finally, recovered mtDNA mutations will be returned to the live epidermis by the generation of transmitochondrial transgenic mice to study the ability of these mutations to alter growth and tumor development in the intact mouse epidermis.