ROLE OF POLYAMINES IN ESTROGEN FUNCTION IN BREAST CANCER

本提案的目的是了解自然发生的 雌激素在乳腺癌中的作用 的组合 组织培养、物理化学和重组DNA技术将是 用于实现这一目标。 两种细胞应答细胞系（MCF-7和 T-47 D）和两种激素无反应细胞系（BT-20和MDA-MB-231），全部 来源于人乳腺癌，将用于本研究。 雌激素 多胺生物合成酶--鸟氨酸脱羧酶刺激 (ODC)将通过测量ODC和多胺的水平进行研究， 雌激素给药的作用。 的抗增殖作用， ODC抑制剂α-二氟甲基鸟氨酸（DFMO）将在 在激素应答细胞系上与抗雌激素的组合以及与 顺铂、阿霉素和它们的类似物对凋亡无应答细胞的作用。 多胺在雌激素诱导的细胞生长中的作用将通过以下方法进行研究： 将ODC的合成与DNA、RNA和特异性DNA、RNA的合成相关联， 雌激素诱导蛋白，如24和52 K蛋白。 为了了解多胺作用的分子机理 对雌激素功能的影响， 受体（ER）将通过密度梯度分析，DNA-纤维素 竞争测定和配体解离速率， 多胺及其衍生物。 ER与右手B-DNA的结合 左手的Z-DNA将被检测以深入了解 ER-DNA上多胺诱导的DNA构象转变 交互. 这些研究将通过绘制ER图来补充 在多胺存在下DNA上的结合位点。 在分子 水平上，雌激素对ODC基因的调控在人乳腺癌中的作用， 使用人ODC cDNA进行研究。 核转录测定，斑点印迹法 将进行北方转移和S1核酸酶分析， 理解ODC在细胞应答中的转录调控， 免疫无反应细胞系。 这些研究的结果 将推进我们对基因表达的激素调节的知识， 乳腺癌 这些研究还将提供重要信息， 涉及多胺的新型治疗方法的发展 抑制剂与抗雌激素/抗肿瘤药物组合。

The goal of this proposal is to understand the role of naturally occurring polyamines in the function of estrogens in breast cancer. A combination of tissue culture, physico-chemical and recombinant DNA techniques will be used to achieve this goal. Two hormone-responsive cell lines (MCF-7 and T-47D) and two hormone-unresponsive cell lines (BT-20 and MDA-MB-231), all of human breast cancer origin, will be used for this study. The estrogenic stimulation of a polyamine biosynthetic enzyme, ornithine decarboxylase (ODC) will be studied by measuring the levels of ODC and polyamines as a function of estrogen administration. The antiproliferative effect of the ODC inhibitor, Alpha-difluromethylornithine (DFMO) will be examined in combination with antiestrogens on hormone responsive cell lines and with cisplatin, adriamycin, and their analogs on hormone-unresponsive cells. The role of polyamines on estrogen induced cell growth will be studied by correlating the synthesis of ODC with that of DNA, RNA and specific estrogen-induced proteins, such as 24 and 52K proteins. In order to understand the molecular mechanism of the action of polyamines on estrogen function, the stabilization and activation of estrogen receptors (ER) will be studied by density gradient analysis, DNA-cellulose competition assay and ligand dissociation rate in the presence of polyamines, and their derivatives. ER binding to the right-handed B-DNA and the left-handed Z-DNA will be examined to gain insight into the effect of polyamine-induced conformational transitions of DNA on ER-DNA interactions. These studies will be complemented by a mapping of the ER binding sites on DNA in the presence of polyamines. At the molecular level, the regulation of ODC gene by estrogen in human breast cancer will be studied using human ODC cDNA. Nuclear transcription assays, dot-blot analysis, Northern transfer and S1 nuclease analysis will be carried out to understand the transcriptional regulation of ODC in hormone-responsive and hormone-unresponsive cell lines. The results generated from these studies will advance our knowledge of the hormonal regulation of gene expression in breast cancer. These studies will also provide significant information for the development of novel therapeutic approaches involving polyamine inhibitors in combination with antiestrogens/antitumor drugs.