POLYAMINES AND EPITHELIAL TUMORIGENESIS

鸟氨酸脱羧酶(ODC)是一种重要的生物活性调节酶。 多胺的生物合成，这是正常细胞生长和 差异化。申请者已经证明，ODC水平的升高 多胺与突变的Ha-ras基因协同促进上皮性肿瘤 形成和入侵。申请人的假设是，高水平的 ODC和多胺“激活”角质形成细胞和真皮基质细胞以产生 一种与受伤皮肤相似的表型，其特征是 增殖和迁移增加，新血管生成，改变 分化，增加蛋白水解酶的表达。推论就是 维持这种多胺诱导的激活的条件(或癌基因) 细胞会导致肿瘤的发展。这个项目的总体目标是 项目是确定在调解过程中发挥关键作用的下游效应器 ODC促进了皮肤肿瘤的发生。目标是复杂的，当肿瘤 作为突变的RAS的结果(如通常在启动的 皮肤)，因为多个效应器通路参与RAS介导 细胞的转化。因此，为了实现这些目标， 申请者还计划剖析多胺的下游效应。 作为与ODC合作的激活的RAS的下游效应器 多胺推动上皮肿瘤的发生。为此，吉尔莫博士建议 以下具体目标：1.确定ODC水平升高和 多胺以相似的方式激活角质形成细胞和真皮基质细胞 与创伤皮肤中的ODC一样，ODC在正常的静止成人中也有表达 表皮(使用可诱导的ODC转基因小鼠模型)，以及在增殖期 活化的表皮(磨损K6/ODC转基因小鼠皮肤)。通过控制 ODC在静息或活化角质形成细胞中的表达 申请者将评估这种未启动的(即没有突变的ras基因)皮肤 诱导：a.伤口愈合表型，b.形成良性 皮肤肿瘤依赖于ODC诱导的细胞持续激活。2.使用 可诱导的ODC转基因小鼠模型，ODC过表达皮肤中的mRNA 将与使用表征差异的正常皮肤进行比较 分析(RDA)和微阵列方法。上调的基因或 由ODC下调的将因其在调解 突变的H-ras表达ODC的致瘤和侵袭作用。3. Gilmour博士将确定激活的Ha-RAS的哪些下游效应器 可以与高水平的ODC和多胺合作，将表皮转化为 细胞分化为恶性侵袭性表型。特别是，她计划 研究ODC和各种部分功能之间可能的合作 激活的Ha-RAS的效应器突变体，它能特异性地诱导 Raf-1/ERK、RalGDS或磷酸肌醇3-OH激酶途径。

Ornithine decarboxylase (ODC) is a key regulatory enzyme in the biosynthesis of polyamines which are essential for normal cell growth and differentiation. The applicants have demonstrated that elevated levels of ODC and polyamines cooperate with a mutated Ha-ras gene to promote epithelial tumor formation and invasion. The applicant's hypothesis is that elevated levels of ODC and polyamines "activate" keratinocytes and dermal stromal cells to produce a phenotype similar to that in wounded skin, which is characterized by increased proliferation and migration, neoangiogenesis, altered differentiation, and increased expression of proteases. A corollary is that conditions (or oncogenes) that sustain this polyamine-induced activation of cells will result in the development of a tumor. The overall goal of this project is to identify downstream effectors that play a key role in mediating ODC-promoted tumorigenesis in skin. The objective is complicated when tumors arise as a consequence of a mutated ras (as is generally found in initiated skin) since multiple effector pathways contribute to Ras-mediated transformation of cells. Therefore, in order to achieve these goals, the applicant plans to dissect out the downstream effectors of polyamines as well as the downstream effectors of an activated ras that cooperate with ODC and polyamines to drive epithelial tumorigenesis. To this end, Dr, Gilmour proposes the following specific aims: 1. To determine if elevated levels of ODC and polyamines activate keratinocytes and dermal stromal cells in a manner similar to that in wounded skin, ODC will be expressed in both normal quiescent adult epidermis (using an inducible ODC transgenic mouse model), and in proliferative activated epidermis (abrading K6/ODC transgenic mouse skin). By controlling expression of ODC in either sedentary or activated keratinocytes, the applicants will evaluate this noninitiated (i.e. no mutated ras gene) skin for the induction of: A. a wound healing phenotype, and B. the formation of benign skin tumors dependent upon sustained ODC-induced activation of cells. 2. Using the inducible ODC transgenic mouse model, mRNA from ODC overexpressing skin will be compared with that from normal skin using representational difference analysis (RDA) and microarray approaches. Genes that are upregulated or downregulated by ODC will be evaluated for their role in mediating the tumorigenic and invasive effects of ODC when expressed with a mutated H-ras. 3. Dr. Gilmour will determine which downstream effectors of an activated Ha-Ras can cooperate with elevated levels of ODC and polyamines to convert epidermal cells to a malignant invasive phenotype. In particular, she plans to investigate possible cooperation between ODC and various partial function effector mutants of an activate Ha-Ras that specifically induce either the raf-1/ERK, RalGDS, or phosphoinostitie 3-OH kinase pathway.