Characterization Of Follicular Stem Cells In Tg.AC Mice

皮肤是一种不断更新的组织，由大量增殖潜力有限的传递扩增(TA)细胞和少量具有高增殖潜力和克隆性的角质形成干细胞(KSCs)组成。KSCs更新干细胞群体并产生TA细胞，TA细胞移位到超基底层，并因终末分化而丢失。在皮肤中，干细胞群体驻留在毛囊隆起中，毛囊隆起位于毛囊的永久部分，在毛囊从静止(休眠)到生长活跃(生长期)的周期中，它受到保护，免受身体损伤和毛囊经历的变化。在众所周知的两阶段小鼠表皮癌变模型中，人们普遍认为KSCs是主要的致癌靶细胞(即潜伏的肿瘤细胞)。支持这一观点的一个关键因素是，DMBA启动的小鼠在接触肿瘤促进剂如TPA时，无论是在启动后一周还是一年后使用，都会患上皮肤肿瘤。鉴于小鼠的角质形成细胞群体每6天自我更新一次，启动的细胞持续存在的事实表明，这些细胞肯定是处于受保护的微环境中的缓慢循环的细胞。我们目前的重点是鉴定和表征导致小鼠皮肤肿瘤的细胞。作为这一目标的一部分，我们研究了皮肤中的造血干和祖细胞标记物CD34，并将该标记物与α-6整合素和荧光激活细胞分类(FACS)相结合，表明CD34特异性地标记毛囊隆起角质形成细胞，并有助于分离活的毛囊隆起角质形成细胞，这些角质形成细胞代表静止的α-6整合素亮细胞亚群(即主要处于G1/G0期)。这项工作代表了首次使用隆起特异性细胞表面标记来物理浓缩活的角质形成细胞、干细胞和祖细胞。在骨髓和包括皮肤在内的其他组织中使用活性染料Hoechst 33342，已经确定了一小部分细胞(称为侧群，或SP细胞)，这些细胞可能是非常早期的原始干细胞/祖细胞。我们已经在我们的实验室开发了这种检测方法，目前正在表征和研究这一群体作为祖细胞和致癌靶细胞的增殖潜力。 从TPA处理和未处理的Tg.AC小鼠获取的CD34角质形成细胞之间进行了比较，以研究肿瘤促进后的差异基因表达模式。用Clontech公司的尼龙基因芯片与从TPA处理或未处理的皮肤分离的CD34细胞制备的基于PCR的SMART扩增cDNA进行探针，鉴定出11个基因的表达随TPA处理而发生显著变化。在与异质性肢体发育障碍相关的分手/分足1(Dss 1)基因中，特别感兴趣的基因被删除。RT-PCR和Northern分析检测到Dss1和NDPK-B在TPA处理的皮肤(非肿瘤；与未处理的皮肤低水平相比)以及皮肤肿瘤(包括乳头状瘤、鳞状细胞癌和梭形细胞肿瘤)中过表达。功能研究表明，组成表达Dss-1的癌前表皮细胞的病灶形成活性和增殖能力增加。有趣的是，Dss1诱导的稳定表达的JB6表皮细胞的转化可通过添加蛋白激酶C(PKC)特异性抑制剂而被取消，这意味着PKC可能在Dss1的表达中发挥调节作用。综上所述，这些结果表明，Dss1是一种TPA诱导的基因，可能在皮肤癌的早期阶段发挥重要作用。此外，最近的研究表明，NDBK-B可能参与了化学诱导的皮肤癌发生的早期肿瘤发展。 为了进一步评估毛囊在皮肤肿瘤发展中的作用，我们利用了表皮磨损技术，即物理地去除毛囊间表皮。由此产生的表皮再生来自于角质形成细胞从下面的毛囊中迁移出来。任何在DMBA引发的野生型或基因引发的Tg.AC中发展的肿瘤都必须来自毛囊，这为研究肿瘤的毛囊来源提供了一种直接的方法。接受一次擦伤的Tg.AC小鼠发生良性乳头状瘤的潜伏期和多发性与TPA治疗的对照组相似。我们已经开始研究，在磨损后的第3天、第5天、第9天和第18天，将磨损的Tg.AC与类似磨损的年龄匹配的FVB/N(亲本株)小鼠进行比较，以使用高密度微阵列分析来开发基因表达谱。这些研究的主要目标是深入了解受ras转基因差异调控并有助于肿瘤发展的基因。

The skin is a continually renewing tissue consisting of a large population of transit amplifying (TA) cells with a limited proliferative potential, and a smaller population of keratinocyte stem cells (KSCs) that have a high proliferative potential and are clonogenic. KSCs renew the stem cell population and give rise to TA cells, which are displaced to the suprabasal layers and are lost by terminal differentiation. In the skin, the stem cell population resides in the hair follicle bulge, which is located in the permanent portion of the hair follicle and is protected from both physical damage and the changes the hair follicle undergoes as it cycles from resting (telogen) to active growth (anagen). In the well-known two- stage murine epidermal carcinogenesis model, it is a widely held belief that KSCs are the primary carcinogen target cells (i.e., latent neoplastic cells). A key factor supporting this belief is the fact that DMBA-initiated mice will develop skin tumors upon exposure to a tumor promoter such as TPA whether it is applied a week or a year after initiation. Given that the keratinocyte population renews itself every 6 days in the mouse, the fact that initiated cells persist suggests that these must be slowly cycling cells located in a protected microenvironment. Our current focus is on identification and characterization of the cells that give rise to cutaneous neoplasms in the mouse. As part of this objective, we have investigated the hematopoietic stem and progenitor cell marker, CD34, in the skin and using this marker in combination with alpha-6 integrin and fluorescence activated cell sorting (FACS), have shown that CD34 specifically marks hair follicle bulge keratinocytes, and facilitates isolation of live follicular bulge keratinocytes that represent a subset of alpha-6 integrin bright cells that are quiescent (i.e., predominantly in G1/G0). This work represents the first use of a bulge-specific cell surface marker for physical enrichment of live keratinocyte stem and progenitor cells. The use of the vital dye Hoechst 33342 in the bone marrow and other tissues, including the skin, has identified a small population of cells (called Side Population, or SP cells) that are potentially very early, primitive stem/progenitor cells. We have developed this assay in our lab and are currently characterizing and investigating the proliferative potential of this population, both as progenitor and carcinogen target cells. A comparison was made between CD34+ keratinocytes harvested from either TPA-treated or untreated Tg.AC mice to investigate differential gene expression patterns following tumor promotion. Using nylon cDNA arrays from Clontech probed with PCR-based SMART-amplified cDNA prepared from CD34+ cells isolated from TPA-treated or untreated skin, eleven genes were identified whose expression changed significantly in response to treatment with TPA. Of particular interest was Deleted in Split Hand/Split Foot 1 (Dss1), which is associated with a heterogeneous limb developmental disorder. Overexpression of Dss1 and NDPK-B was detected by RT-PCR and Northern analysis in TPA treated skin (non-tumor bearing; compared to low levels in untreated skin), as well as in cutaneous tumors, including papillomas, squamous cell carcinomas, and spindle cell tumors. Functional studies revealed an increase in foci-forming activity and proliferation of preneoplastic epidermal cells constitutively expressing Dss1. Interestingly, Dss1 induced transformation of stably transfected JB6 epidermal cells was abrogated by addition of a protein kinase C (PKC) specific inhibitor, implicating a possible PKC regulatory role in Dss1 expression. Taken, together, these results suggest that Dss1 is a TPA-inducible gene that may play an important role in the early stages of skin carcinogenesis. In addition, recent studies have implicated a potential involvement of NDBK-B in early-stage neoplastic development in chemically-induced skin carcinogenesis. To assess further the role of hair follicle in cutaneous tumor development, we have utilized the technique of epidermal abrasion, in which the interfollicular epidermis is physically removed. The resulting epidermal regeneration is derived from keratinocytes migrating out from the underlying hair follicles. Any tumors that develop in DMBA-initated wild type or genetically initiated Tg.AC must come from the hair follicle, providing a direct method of investigating the follicular origin of tumors. Tg.AC mice subjected to a single abrasion develop benign papillomas at a similar latency and multiplicity as TPA-treated controls. We have initiated studies in which abraded Tg.AC were compared to similarly abraded, age-matched, FVB/N (parental strain) mice at days 3, 5, 9, and 18 post-abrasion to develop gene expression profiles using high density microarray analysis. The primary goal of these studies is to gain insight into genes that are differentially regulated by the ras transgene and contribute to tumor development.