DIFFERENTIALLY EXPRESSED GENES IN PRIMARY BREAST CANCER

在最初的拨款中，我们的目标是选择候选肿瘤 抑制基因通过其在乳腺癌中的表达缺失 与匹配良好的正常乳腺癌相比， 上皮细胞，使用差异显示。 这一目标现已 办妥了一批多年想 我们已经鉴定并克隆了100多个 在我们的乳腺癌系统中下调基因。 此外我们 已经研究了几个候选的肿瘤抑制基因， maspin是一种蛋白酶抑制剂， 诊断和治疗应用。 这次补助金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， 抑制子通过其与丝氨酸蛋白酶、组织 纤溶酶原激活物。 maspin的结构，从其 序列，不支持关于 蛋白酶抑制活性的蛋白质，直到现在，它的 分子作用模式仍然不清楚。 在 然而，在细胞水平上，我们已经证明maspin抑制了 在细胞培养测定中的侵袭和运动性，并抑制生长和 在裸鼠测定中观察到转移。 通过延时录像 在显微镜下，我们发现运动被阻断了12小时， 用maspin处理肿瘤细胞。 我们现在的目的是 组织型纤溶酶原激活剂如何促进这些生物学效应 方面的影响. 我们克隆并测序了maspin的启动子区， CAT分析确定了maspin的转录调控 在乳腺和前列腺细胞中均有表达。 我们现在提议 以确定转录因子负责差异 在两种组织的正常细胞与肿瘤细胞中的表达。 我们克隆 并对小鼠maspin进行了测序， 与人maspin的同源性百分比， 抑制侵袭和运动。 现在建议寻找 在转基因小鼠与小鼠杂交的肿瘤抑制活性中， 表达自发性乳腺肿瘤的高频率;以及 生产maspin基因敲除小鼠，研究maspin在 发展 第二个目标是利用一个网格系统的基础上， 用北方区组比较100例受试者的基因表达模式。 我们通过DD分离的下调基因。 基因探针 排列在网格上，将与标记为反向的32 P杂交 从癌细胞系转录单链cDNA， 患者的家属。 其目的是确定 乳腺癌的协调表达特征， 选择特别感兴趣的基因用于诊断和治疗 应用程序.

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.