Visualizing Clonal Expansion in Living Mice

描述(由申请人提供)：推动癌前细胞克隆性扩张的力量，以及通常阻止它的屏障，人们知之甚少。这一探索性资助项目为研究这一问题开发了关键资源。对于皮肤癌，已知的最早的病变是携带p53突变的角质形成细胞克隆。我们发现，这些突变细胞的克隆扩增需要持续的UVB，而不是额外的突变。相邻的干细胞隔间通常起到克隆扩增的屏障作用，但UVB允许p53突变的角质形成细胞突破这一屏障。通过创造一个携带荧光P53-mRFP1融合蛋白的敲入小鼠，我们将第一次能够实时动态观察P53突变克隆的起源、扩展和退化。第二种方法可能适用于人类皮肤，通过局部应用与p53紧密结合的荧光肽来可视化克隆。这些方法将在第二年用于直接观察突变细胞从一个干细胞室到另一个干细胞室的量化跳跃，调查克隆与癌前病变的前体-产物关系，并确定是否可以使用局部RNAi在基因上修改克隆扩张。未来，我们会问：克隆扩增会创造新的干细胞吗？克隆人会通过光化性角化病进展为癌症吗？UVA能通过减少细胞间通讯来驱动克隆性扩张吗？克隆人会通过挤压而倒退吗？抗退化克隆是由干细胞产生的吗？可见的皮损消退后，罕见的突变细胞会持续很长时间吗？皮肤筛选RNAi文库能揭示影响克隆扩张的基因吗？因此，这些技术进步应该会打开克隆扩张的领域。

DESCRIPTION (provided by applicant): The forces that drive clonal expansion of a precancerous cell, and the barriers that normally prevent it, are little understood. This Exploratory Grant project develops key resources for studying this problem. For skin cancer, the earliest known lesions are keratinocyte clones carrying p53 mutations. We find that clonal expansion of these mutant cells requires sustained UVB, but not additional mutations. Neighboring stem cell compartments normally act as a barrier to clonal expansion, but UVB allows p53-mutant keratinocytes to breach this barrier. By creating a knock-in mouse carrying a fluorescent P53-mRFP1 fusion protein, we will be able, for the first time, to dynamically observe the origin, expansion, and regression of p53-mutated clones in real time. A second approach, potentially applicable to human skin, visualizes clones by topically applying fluorescent peptides that tightly bind P53. These methods will be used in the second year to directly observe quantized jumping of mutant cells from one stem cell compartment to another, investigate the precursor-product relation of clones to precancers, and determine whether clonal expansion can be modified genetically using topical RNAi. In the future, we will ask: Does clonal expansion create new stem cells? Does a clone progress through actinic keratosis to carcinoma? Can UVA drive clonal expansion by reducing intercellular communication? Do clones regress by squaming? Do the regression-resistant clones arise from stem cells? Do rare mutant cells persist long after visible lesions have regressed? Can skin screening of RNAi libraries reveal genes affecting clone expansion? Thus, these technical advances should open up the field of clonal expansion.