The Kinetics of Keratinocyte Stem Cell Division

描述（由申请人提供）： 现在，众所周知，在各种上皮组织中的干细胞，例如表皮的基底层、毛囊的隆起区域和小肠的隐窝，可以在发育的关键阶段连续施用氚化胸苷（[3 H]TdR）后标记。最初，在这样的标记程序之后，所有增殖细胞都掺入标记。然而，在对每个组织特定的追踪期之后，特定位置的极少数细胞保留标记。这些标记保留细胞已被确定为缓慢循环标记保留细胞（LRC）在光显微镜放射自显影。这些观察结果与以下假设一致，即表皮增殖单位（EPU）的中心位置、毛囊的隆起区域和肠隐窝的细胞位置4中的这些细胞是干细胞。在这些组织中存在LRC的原因通常是LRC比其他增殖细胞分裂得更慢，后者通过随机DNA链分离快速稀释DNA标记。这种解释的问题在于，其他证据，包括来自肠或表皮内稳态中细胞更新的数学模型的证据，强烈表明LRC的周期时间大约是过境扩增细胞的两倍，因此应该在四次分裂内将其标记减少到背景水平。因此，必须找到干细胞标记保留的另一种解释。我们的目标是确定表皮LRC标记保留的机制。我们假设，表皮LRC有一个p53依赖的机制，选择性地分离其模板DNA链，以及防止DNA复制诱导的错误的机制。我们有两个具体目标：1）确定p53+/+、p53+/-和p53-/-小鼠表皮中的干细胞是否保留永生DNA链，和2）确定p53+/+、p53+/-和p53-/-表皮干细胞是否具有损伤诱导的细胞凋亡的机制。该建议中详细介绍的方法可能回答上皮干细胞生物学中一个令人信服的基本问题：处于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。本文提出的研究不仅对表皮的结构和功能具有重要意义，而且对慢性皮肤病和癌症中角质形成干细胞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.