DIFFERENTIALLY EXPRESSED GENES IN PRIMARY BREAST CANCER

原发性乳腺癌中差异表达的基因

• 批准号: 2402746
• 负责人: ARTHUR B PARDEE
• 金额: $ 25.19万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2402746
• 关键词: animal genetic material tag; athymic mouse; breast neoplasms; cell motility; clinical research; extracellular matrix proteins; gene induction /repression; genetically modified animals; human genetic material tag; human subject; laboratory mouse; neoplasm /cancer genetics; neoplasm /cancer invasiveness; nucleic acid probes; oncogenes; oncoproteins; protease inhibitor; serine proteinases; transcription factor; tumor suppressor genes; tumor suppressor proteins

In the initial grant, our aim was to select candidate tumor suppressor genes by their loss of expression in mammary carcinomas compared with well matched normal mammary epithelial cells using differential display. This objective has now been achieved. We have identified and cloned more than 100 down-regulated genes in our breast cancer system. In addition we have investigated several candidate tumor suppressor genes of which maspin, which is a protease inhibitior, shows promise in both diagnostic and therapeutic applications. The first specific aim of this grant renewal is to test the hypothesis that maspin, a serpin (serine protease inhibitor) acts as a tumor suppressor through its interaction with the serine protease, tissue plasminogen activator. The structure of maspin, deduced from its sequence, does not support a strong prediction concerning the protease inhibitory activity of the protein, and until now its molecular mode of action has remained underfined. At the cellular level, however, we have shown that maspin inhibits invasion and motility in cell culture assays, and inhibits growth and metastasis in the nude mouse assay. By time-lapse video microscopy, we showed that motility is blocked for 12 hours when tumor cells are treated with maspin. Our purpose now is to define how tissue plasminogen activator contributes to these biological effects. We cloned and sequenced the promoter region of maspin, and by CAT analysis established the transcriptional regulation of maspin expression in both mammary andprostate cells. We propose now to identify the transcription factors responsible for differential expression in normal vs. Tumor cells of both tissues. We cloned and sequenced the mouse maspin and showed that it has 87 percent homology with human maspin and similar activity in inhibiting invasion and motility. It is proposed now to look for tumor suppressor activity in transgenic mice crossed with mice that express high frequencies of spontaneous mammary tumors; and to produce maspin knockout mice to study effects of maspin in development. The second aim is to utilize a grid system based on reverse Northern bloct to compare patterns of gene expression in the 100 down-regulated genes we have isolated by DD. Gene probes arrayed on grids will be hybridized with 32P labeled reverse transcribed single strand cDNAs from carcinoma cell lines and from patient speciments. The purpose is to identify patterns of coordinate expression characteristic of breast cancer and thereby to select genes of special interest for diagnostic and therapeutic application.

在最初的拨款中，我们的目标是选择候选肿瘤 抑癌基因在乳腺中的表达缺失 乳腺癌与配对良好的正常乳腺的比较 上皮细胞差异显示。这一目标现在已经实现 已经实现了。我们已经鉴定和克隆了100多个 在我们的乳腺癌系统中下调了基因。此外，我们还 已经研究了几个候选的抑癌基因 Maspin是一种蛋白酶抑制剂，它在这两种情况下都显示出希望 诊断和治疗应用。 这次拨款更新的第一个具体目标是检验假设 Maspin，一种丝氨酸蛋白酶抑制剂，起到了肿瘤的作用。 抑制物通过其与丝氨酸蛋白酶的相互作用，组织 纤溶酶原激活剂。Maspin的结构，从它的 序列，并不支持关于 蛋白的蛋白酶抑制活性，到目前为止它的 分子作用模式仍然不够完善。在 然而，我们已经证明，在细胞水平上，maspin可以抑制 在细胞培养中检测侵袭性和运动性，并抑制生长和 裸鼠转移实验。通过延时视频 显微镜下，我们发现运动被阻断了12小时 肿瘤细胞用maspin治疗。我们现在的目标是定义 组织型纤溶酶原激活剂如何对这些生物 效果。 我们克隆并测序了maspin的启动子区域，并通过 CAT分析建立了maspin的转录调控机制 在乳腺和前列腺细胞中均有表达。我们现在求婚 确定与差异有关的转录因子 在两种组织的正常细胞和肿瘤细胞中均有表达。我们克隆了 并对小鼠的maspin进行了测序，结果显示它有87 与人maspin的同源性百分比以及在 抑制侵袭和运动。现在有人提议去寻找 转基因小鼠与小鼠杂交后的肿瘤抑制活性 表达自发性乳腺肿瘤的高频率；以及 建立maspin基因敲除小鼠研究maspin基因敲除对小鼠的影响 发展。 第二个目标是利用基于逆向的网格系统 Northern区块比较100个基因的表达模式 我们通过DD分离到了下调的基因。基因探针 在栅格上排列的将与标记为反向的32P杂交 从癌细胞系转录的单链cDNA和 从病人的样本中提取。其目的是确定 乳腺癌的协同表达特征，从而 选择诊断和治疗中特别感兴趣的基因 申请。