Role of ornithine decarboxylase in Ras transformation

鸟氨酸脱羧酶在 Ras 转化中的作用

基本信息

批准号：
7081242
负责人：
LISA M SHANTZ
金额：
$ 20.58万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-07-01 至 2008-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The goal of this work is to understand the mechanism by which ornithine decarboxylase (ODC) activity is induced during carcinogenesis by oncogenic ras, andto define the role of ODC in this process. Results in the Progress Report show for the first time that induction of ODC activity in Ras-transformed cells requires the coordinate activation of the Raf/MEK/ERK and PI 3-kinase pathways. ODC RNA and protein are increased, with the primary regulation being post-transcriptional. We will use constitutively active forms of Ras, Raf and Akt to activate the effector pathways of interest in RIE-1 epithelial cells. These cells are appropriate since the Raf/ME/ERK and PI 3-kinase pathways cooperate in their transformation. We will use this model to study translational regulation of ODC in response to these signaling cascades, and to assess the role of sustained ODC activation in transformation, cell cycle control, and survival. We also show that inhibiting ODC with the irreversible inactivator alpha-difluoromethylornithine (DFMO) delays the onset of spontaneous tumors in mice overexpressing an active MEK mutant in the skin (K14-MEK mice), and causes regression of established tumors. To test the hypothesis that suppression of ODC blocks the promotion of target cells initiated by the Raf/MEK/ERK pathway, we will cross K14-MEK mice with two transgenic lines overexpressing antizyme (AZ), which binds to ODC and targets it for degradation. We have shown using K5-AZ and K6-AZ mice that AZ suppresses tumor growth in the two-stage model of skin carcinogenesis. The K6 promoter requires hyperproliferation for maximal expression. Since MEK overexpression causes hyperplasia, MEK/K6-AZ mice represent a model to test if ODC inhibition is effective in preventing tumor formation after establishment of a carcinogenic environment. The K5 promoter is constitutive, and MEK/K5-AZ mice have a constant expression of AZ from birth. These mice represent a chemoprevention model, in which ODC is inhibited at the time of the initiating stimulus. We will examine tumors from MEK/AZ mice to determine effects on cell cycle proteins, apoptosis, and proliferation. To specifically address the role of ODC in cell cycle progression during tumorigenesis, we will use transgenic mice that overexpress AZ and cyclin D1 in the skin. Keratinocytes from D1/AZ mice will also be used to analyze direct interactions of AZ with cyclin D1, to test whether other properties of AZ are important for deregulation of cell proliferation in tumor development.

描述（由申请人提供）：这项工作的目的是了解在致癌性Ras期间诱导鸟氨酸脱羧酶（ODC）活性的机制，并定义ODC在此过程中的作用。进度报告的结果首次表明，RAS转换细胞中ODC活性的诱导需要RAF/MEK/ERK和PI 3-激酶途径的坐标激活。 ODC RNA和蛋白质增加，主要调节后转录后。我们将使用RAS，RAF和AKT组成型活性形式来激活RIE-1上皮细胞感兴趣的效应途径。这些细胞是合适的，因为RAF/ME/ERK和PI 3-激酶途径在其转化中进行了配合。我们将使用该模型来研究ODC对这些信号级联反应的翻译调节，并评估持续的ODC激活在转化，细胞周期控制和生存中的作用。我们还表明，抑制不可逆的灭活剂α-二氟甲基氨酸氨基氨酸（DFMO）延迟了过表达皮肤中活性MEK突变体（K14-MEK小鼠）的自发性肿瘤的发作，并导致既定肿瘤的消退。为了检验ODC抑制阻止RAF/MEK/ERK途径促进的靶细胞的假设，我们将使用两个过表达抗卵的转基因线（AZ）跨K14-MEK小鼠，该抗酶（AZ）与ODC结合并靶向其降级。我们已经表明，使用K5-AZ和K6-AZ小鼠，AZ在皮肤致癌的两阶段模型中抑制了肿瘤的生长。 K6启动子需要过度增殖才能最大表达。由于MEK过表达会引起增生，因此MEK/K6-AZ小鼠代表了一个模型，用于测试ODC抑制是否有效预防致癌环境后的肿瘤形成。 K5启动子是本构的，MEK/K5-AZ小鼠从出生时就会持续表达AZ。这些小鼠代表了化学预防模型，其中在启动刺激时抑制ODC。我们将检查MEK/AZ小鼠的肿瘤，以确定对细胞周期蛋白，凋亡和增殖的影响。为了特别解决ODC在肿瘤发生过程中细胞周期进程中的作用，我们将使用过表达AZ和细胞周期蛋白D1在皮肤中的转基因小鼠。来自D1/AZ小鼠的角质形成细胞也将用于分析AZ与Cyclin D1的直接相互作用，以测试AZ的其他特性是否对于消除肿瘤发育中细胞增殖的管制是否重要。