HORMONAL REGULATION OF C-HA-RAS AND ROLE IN METASTASIS

可以影响肿瘤进展的癌基因和抑制基因可能 控制基因调控中的关键点，最终是 负责在高转移细胞中看到的改变。因此， 对基因表达调控机制的理解 能够影响肿瘤的发展和进展是至关重要的 提供更有效的预后或治疗的见解。这个 该项目的长期目标是识别和表征 Re参与了乳腺肿瘤的发生和发展。高架 据报道，未突变的c-Ha-ras蛋白水平在 在乳腺组织中的肿瘤发生中起关键作用。我们已经展示了一个 内源性c-Ha-ras蛋白水平与肿瘤的关系 小鼠乳腺肿瘤亚群的转移行为 发现一种新的激素反应转录调控元件 在小鼠Ha-ras基因的内含子-1中。的增强剂活性 内含子-1元件中的内含子-1元件被强烈地诱导 糖皮质激素(伊达米松，Dx)和孕酮(PG) 非转移性亚系。在转移性乳腺亚系中， 由Dx、PG和E2诱导。形成了两个特定的络合物A1和A2 当核提取液S形成转移细胞和非转移细胞时 与内含子-1元件一起孵化。只有在以下情况下才形成复合体B 内含子-1元件与转移细胞的核蛋白发生反应。 我们显示出c-Has-ras蛋白水平的显著差异，复合体 与内含子-1元件的形成和ras的激素反应性 转移性与非转移性乳腺亚系的内含子-1元件 在我们的研究中使用。需要检验的假设是，在小鼠身上 乳腺系统肿瘤进展与转移行为的表达 与调节/解除调节Ha-ras基因的机制有关 表情。 我们的具体目标是：1)确定ras的激素反应性 表达与乳腺肿瘤表型有关；即，DO细胞与 低水平的p21ras表现出更大的激素映射，无论是否有其他 小鼠Ha-ras基因内含子-1的调控位点及其与 内含子-1的调控位点5-与小鼠的内含子-1相互作用 HA-ras基因及其调控自相似基序包含 由于外显子-1，ERE-和GRE-半位点存在于内含子-1元件中 小鼠、大鼠和人类的c-Ha-ras基因。3)定义和描述 与ras内含子-1元件相互作用的调节因子 转移细胞和非转移细胞。4)确定内含子-1是否 在体外转录中，元素可以赋予激素反应性 系统。5)确定E3和糖皮质激素是否调节c-Has-Has- RAS在小鼠乳腺亚系中的水平主要是转录 也可以通过改变蛋白质的稳定性来实现。 在明确定义的小鼠转移模型中完成这些研究 系统将提供有关调解机制的有价值的信息 Ha-ras基因在乳腺肿瘤细胞中的转录调控 转移性乳腺特异的反式作用因子(S) 细胞将用于功能分析，以更完整地定义 Ras在肿瘤转移中的作用。

Oncogenes and suppressor genes that can affect tumor progression may control important points in the regulation of genes that are ultimately responsible for alteration seen in highly metastatic cells. Hence, understanding of the mechanisms regulating expression of the genes capable of influencing tumor development and progression is crucial for providing insights leading to more effective prognosis or therapy. The long term goal of this project is to identify and characterize genes that re involved in mammary tumor development and tumor progression. Elevated levels of nonmutated c-Ha-ras proteins have been reported to play a crucial role in tumorigenesis in the mammary tissue. We have shown a direct correlation between endogenous c-Ha-ras protein levels and metastatic behavior of mouse mammary tumor subpopulations, and have identified an novel hormone responsive transcriptional regulatory element in the intron-1 of the mouse Ha-ras gene. The enhancer activity of intron-1 element in the intron-1 element is induced strongly by glucocorticoids (edamethasone, Dx), and progesterone (Pg) in nonmetastatic sublines. IN metastatic mammary sublines it is moderately induce by Dx, Pg, and E2. Two specific complexes , A1 and A2, are formed when nuclear extract s form metastatic and nonmetastatic cells are incubated with the intron-1 element. Complex B is formed only when the intron-1 element is reacted with nuclear proteins of metastatic cells. We show significant differences in c-Has-ras protein levels, complex formation with intron-1 element, and hormonal responsiveness of the ras intron-1 element between metastatic and nonmetastatic mammary sublines used in our study. The hypothesis to be tested is that in the mouse mammary system tumor progression and expression of metastatic behavior is linked to the mechanisms regulating/deregulating Ha-ras gene expression. Our specific aims are: 1) To determine if hormonal responsiveness of ras expression is related to a mammary tumor phenotype; i.e., do cells with low p21ras levels exhibit greater hormone mapping whether there are other regulatory sites in intron-1 of the mouse Ha-ras gene and how they and regulatory sites 5-of intron-1 interact with the intron-1 of the mouse Ha-ras gene and how they and regulatory since similar motifs containing the ERE-and GRE-half sites are present in intron-1 element, since exon-1 of mouse, rat and human c-Ha-ras genes. 3) To define and characterize the transacting factors interacting with the ras intron-1 element in metastatic and nonmetastatic cells. 4) To determine whether the intron-1 element can confer hormonal responsiveness in in vitro transcription systems. 5) To determine whether E3 and glucocorticoid regulates c-Has- ras levels in the mouse mammary sublines primarily a the transcriptional levels or also by altering the stability of the proteins. Completion of these studies in a well-defined mouse metastasis a model system will provide valuable information on the mechanisms mediating transcriptions regulation of the Ha-ras gene in mammary tumor cells. Once characterized, trans-acting factor(s) specific to metastatic mammary cells will be of use in functional assays to more completely define the role of ras in metastasis.