Kinetics of Keratinocyte Stem Cell Division

描述(由申请人提供)： 目前人们普遍认为，在发育的关键阶段用氚胸腺嘧啶核苷([~3H]TdR)持续给药，可以标记各种上皮组织中的干细胞，如表皮的基底层、毛囊的隆起区和小肠的隐窝。最初，在这样的标记过程之后，所有的增殖细胞都加入了标记。然而，在每个组织特定的追踪期之后，特定位置的极少数细胞保留了标记。在光镜放射自显影中，这些标记保留细胞被鉴定为缓慢循环的标记保留细胞(LRC)。这些观察结果与位于表皮增殖单位(EPU)中央位置、毛囊隆起区和肠腺第4细胞位置的这些细胞是干细胞的假设是一致的。这些组织中存在LRCs的原因通常是LRCs比其他增殖细胞分裂得更慢，后者通过随机的DNA链分离迅速稀释DNA标记。这种解释的问题是，其他证据，包括来自肠道或表皮内稳态细胞更新的数学模型的证据强烈表明，LRC的周期时间大约是运输放大细胞的两倍，因此应该将它们的标记降低到四个分裂内的背景水平。因此，必须找到干细胞标记保留的另一种解释。我们的目的是确定表皮LRCs保留标记的机制。我们假设，表皮LRC具有选择性地分离其模板DNA链的p53依赖的机制，以及保护DNA复制诱导的错误的机制。我们有两个特定的目标：1)确定P53+/+、P53+/-和P53-/-小鼠的表皮干细胞是否保留了一条不朽的DNA链；2)确定P53+/+、P53+/-和P53-/-的表皮干细胞是否具有损伤诱导细胞凋亡的机制。这项建议中详细介绍的方法使解决上皮干细胞生物学中一个引人注目的基本问题成为可能：处于内稳态的角质形成细胞干细胞是否分离其模板DNA链，以及这是否与受损干细胞中的利他性细胞自杀(凋亡)有关。这里提出的研究不仅对表皮的结构和功能具有重要意义，而且对角质形成细胞干细胞在慢性皮肤病和癌症中的行为也具有重要意义。

DESCRIPTION (provided by applicant): It is now common knowledge that stem cells in various epithelial tissues such as the basal layer of the epidermis, the bulge region of the hair follicle, and the crypt of the small intestine can be labeled following continuous administration with tritiated thymidine ([3H]TdR) at a critical stage of development. Initially, following such labeling procedures, all of the proliferating cells incorporate the label. However, following a chase period specific for each tissue, a very few cells in specific locations retain the label. These label-retaining cells have been identified as slowly cycling label-retaining cells (LRCs) in light microscopic autoradiographs. These observations are consistent with the hypothesis that these cells in the central position of the epidermal proliferative units (EPUs), in the bulge region of the hair follicles, and in cell position 4 of the intestinal crypts are stem cells. The reason usually given for the presence of LRCs in these tissues is that the LRCs divide more slowly than the other proliferating cells that rapidly dilute the DNA label by random DNA strand segregation. The problem with this explanation is that other evidence including that from mathematical models of cell renewal in intestinal or epidermal homeostasis strongly suggests that the LRCs have a cycle time approximately twice as long as the transit amplifying cells, and so should therefore reduce their label to background levels within four divisions. Hence, another explanation for label-retention by the stem cells must be found. Our objective is to determine the mechanism of label-retention by epidermal LRCs. We hypothesize that epidermal LRCs have a p53-dependent mechanism for selectively segregating their template DNA strands as well as a mechanism for protecting against DNA-replication-induced errors. We have two Specific Aims: 1) to determine whether stem cells in epidermis of p53+/+, p53+/-, and p53-/- mice retain an immortal DNA strand, and 2) to determine whether epidermal stem cells of p53+/+, p53+/-, and p53-/- have a mechanism for damage-induced apoptosis. The approach detailed in this proposal makes possible an answer to a compelling and fundamental problem in the biology of epithelial stem cells: whether keratinocyte stem cells in homeostasis segregate their template DNA strands and whether this is linked to altruistic cell suicide (apoptosis) in damaged stem cells. The research proposed here has critical implications not only for the structure and function of the epidermis, but also for the behavior of keratinocyte stem cells in chronic skin disease and cancer.