Mitochondrial DNA Mutations in Keratinocyte Hyperplasia

描述(由申请人提供)：表皮的异常增殖与累积的无保护的紫外线照射有关，导致体细胞突变的积累。最近的研究表明，线粒体不仅在提供细胞能量方面发挥着重要作用，而且可能在细胞凋亡和肿瘤发生中起着中心作用。现在有几个关于线粒体基因的报道，这些基因以前被认为只在能量产生中起作用，但在常见的肿瘤综合征中没有被证明发生突变，以及关于人类肿瘤中获得性同质线粒体DNA(MtDNA)突变的报道。这项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，紫外线将被用来诱导无毛小鼠的光损伤和肿瘤产生。将研究光损伤皮肤以及良性和恶性表皮肿瘤的mtDNA，以确定光损伤小鼠皮肤的体细胞mtDNA突变类型。接下来，mtDNA突变将从过度增殖的角质形成细胞中恢复，方法是分离线粒体并融合到缺乏自身mtDNA的成纤维细胞中，以创建细胞质杂交(Cybrid)文库。将对单个环状克隆进行mtDNA突变筛查，并分析这些DNA变化的影响。这些细胞将被研究氧化磷酸化生物化学和ATP产生的变化，以及细胞表型的变化，包括生长特征、基质金属蛋白酶表达、逃脱细胞凋亡的能力和形成肿瘤的能力。突变的mtDNA对野生型的显性能力也将在融合分析中进行遗传研究。最后，恢复的mtDNA突变将通过产生传输线粒体转基因小鼠而返回到活的表皮，以研究这些突变改变完整小鼠表皮生长和肿瘤发展的能力。

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.