Dss1, A Novel Tpa-inducible Gene Product Involved In Mur

Dss1，一种参与 Mur 的新型 Tpa 诱导基因产物

• 批准号: 6681917
• 负责人: Raymond W Tennant
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6681917
• 关键词: disease /disorder model; gene environment interaction; gene induction /repression; genetic regulation; genetically modified animals; keratinocyte; laboratory mouse; molecular pathology; neoplasm /cancer genetics; neoplastic process; plasminogen activator; protein structure function; skin neoplasms; stem cells

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. 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 lesions 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. In addition, using the genetically initiated v-Ha-ras transgenic Tg.AC mouse, we have shown that the ras transgene is expressed in CD34+ cells in non-tumor bearing skin following TPA treatment, evidence that the follicular KSC and progenitor cells are among the earliest to show transgene induction following chemical exposure. 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 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 that 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. 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 foll icles. 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 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. Initial analysis of the data has revealed that many of the changes in gene expression are similar between the two strains, particularly with regard to genes associated with terminal differentiation, cell adhesion, cell migration and inflammation. Further data analysis is being undertaken to develop insight into the gene expression differences that occur as a result of ras transgene expression.

皮肤是一种不断更新的组织，由大量增殖潜力有限的转运扩增（TA）细胞和少量增殖潜力高且具有克隆形成能力的角质形成细胞干细胞（KSC）组成。KSC更新干细胞群并产生TA细胞，TA细胞被移位到基底上层并因终末分化而丢失。在皮肤中，干细胞群驻留在毛囊隆起中，其位于毛囊的永久部分中，并且被保护免受物理损伤和毛囊从静止（休止期）到活跃生长（生长期）循环时所经历的变化。在众所周知的两阶段鼠表皮癌发生模型中，广泛认为KSC是主要致癌物靶细胞。支持这一观点的一个关键因素是，DMBA启动的小鼠在暴露于肿瘤促进剂（如TPA）后会发生皮肤肿瘤，无论是在启动后一周还是一年。鉴于小鼠中角质形成细胞群体每6天更新一次，启动细胞持续存在的事实表明这些细胞必须是位于受保护微环境中的缓慢循环细胞。我们目前的重点是识别和表征的细胞，引起皮肤病变的小鼠。作为该目的的一部分，我们研究了皮肤中的造血干细胞和祖细胞标志物CD 34，并将该标志物与α-6整联蛋白和荧光激活细胞分选（FACS）结合使用，已经表明CD 34特异性地标记毛囊隆起角质形成细胞，并有助于分离代表静止的α-6整联蛋白亮细胞亚群的活毛囊隆突角化细胞（即，主要在G1/G 0）。这项工作代表了第一次使用肿胀特异性细胞表面标志物的物理富集活角质形成细胞干细胞和祖细胞。此外，使用遗传启动的v-Ha-ras转基因Tg.AC小鼠，我们已经表明，在TPA处理后，ras转基因在非荷瘤皮肤中的CD 34+细胞中表达，这证明滤泡KSC和祖细胞是在化学暴露后最早显示转基因诱导的细胞之一。 比较从TPA处理或未处理的Tg.AC小鼠收获的CD 34+角质形成细胞，以研究肿瘤促进后的差异基因表达模式。使用来自Clontech的尼龙cDNA阵列，用从TPA处理或未处理的皮肤分离的CD 34+细胞制备的基于PCR的SMART扩增的cDNA探测，鉴定了11个基因，其表达响应于TPA处理而显著改变。特别令人感兴趣的是裂手/裂足1（Dss 1），这是与异质性肢体发育障碍。通过RT-PCR和北方分析在TPA处理的皮肤（非肿瘤承载;与未处理的皮肤中的低水平相比）以及皮肤肿瘤（包括乳头状瘤、鳞状细胞癌和梭形细胞肿瘤）中检测到Dss 1的过表达。功能研究表明，增加病灶形成活性和增殖的癌前表皮细胞组成性表达Dss 1。有趣的是，Dss 1诱导的转化稳定转染的JB 6表皮细胞被废除，通过添加蛋白激酶C（PKC）特异性抑制剂，暗示可能的PKC调节Dss 1表达的作用。两者合计，这些结果表明，Dss 1是TPA诱导的基因，可能在皮肤癌发生的早期阶段发挥重要作用。 为了进一步评估毛囊在皮肤肿瘤发展中的作用，我们采用了表皮磨损技术，其中毛囊间表皮被物理去除。由此产生的表皮再生源自角质形成细胞从下面的毛囊中迁移出来。在DMBA引发的野生型或遗传引发的Tg.AC中发展的任何肿瘤必须来自毛囊，这提供了研究肿瘤的毛囊起源的直接方法。Tg.AC小鼠进行一个单一的磨损发展良性乳头状瘤在类似的潜伏期和多重性TPA治疗的对照。我们已经启动了研究，其中磨损Tg.AC进行了比较，以类似的磨损，年龄匹配，FVB/N（亲本品系）小鼠在磨损后5天，9，和18，以开发基因表达谱使用高密度微阵列分析。这些研究的主要目标是深入了解ras转基因差异调节的基因，并有助于肿瘤的发展。对数据的初步分析表明，两种菌株之间的许多基因表达变化是相似的，特别是与终末分化、细胞粘附、细胞迁移和炎症相关的基因。正在进行进一步的数据分析，以深入了解ras转基因表达导致的基因表达差异。