ROLE OF COLLAGENOLYTIC METALLOPROTEINASES IN METASTASES

胶原蛋白金属蛋白酶在转移中的作用

• 批准号: 3808569
• 负责人: W G STETLER-STEVENSON
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3808569
• 关键词: antibody; binding proteins; collagen; collagenase; colorectal neoplasms; complementary DNA; enzyme induction /repression; enzyme mechanism; fibronectins; gelatin; genetic regulation; genetic transcription; human genetic material tag; human tissue; lung neoplasms; metalloenzyme; metalloproteins; metastasis; molecular cloning; molecular oncology; neoplasm /cancer invasiveness; nucleic acid probes; protein purification; protein structure function; proteolysis; tissue /cell culture; transfection

In order to investigate the role of type IV collagenase in tumor invasion and metastases, we have focused on the multilevel regulation of this enzyme. We have studied the transcriptional regulation of the 72 kDa collagenase IV enzyme in both normal and human tumor cell lines as well as human tumor tissues. These studies have shown that in contrast with other members of the collagenase enzyme family, the 72 kDa type IV collagenase mRNA levels are increased in response to TGFbeta1, are unaffected by the tumor promoting phorbol esters, and show elevated levels in colorectal tumor tissues when compared with adjacent normal mucosa tissues. Transfection of human tumor cells with expression constructs containing the 72 kDa type IV collagenase gene resulted in phenotypic changes, such as slowed cell growth rates and marked cell vacuolization. These findings suggest that uncomplexed 72 kDa type IV collagenase is not readily secreted by these transformed cells. We have identified a cellular activation mechanism which is cell surface associated and specific for the 72 kDa type IV collagenase enzyme, and which can be induced by pretreatment with phorbol esters or concanavalin A. This cellular activation mechanism does not affect other members of the collagenase gene family. Further characterization and purification of components of this activation mechanism are ongoing. We have also examined the autoproteolytic breakdown of the 72 kDa type IV collagenase following removal of TIMP-2 from the complex. The enzyme has a characteristic pattern of autoproteolytic digestion generating specific 42 kDa and 37.5 kDa gelatin binding fragments, one (37.5 kDa) of which retains proteolytic activity. Finally, we have prepared antibodies and cDNA probes to other members of the collagenase family and have initiated a screening project to examine human lung tumor tissues for the relative mRNA levels and protein expression of the 72 kDa type IV collagenase, stromelysin-1, stromelysin-2, stromelysin-3, PUMP-1 and the 92 kDa type IV collagenase.

为探讨IV型胶原酶在肿瘤侵袭中的作用 和转移，我们专注于对这一过程的多层次调控 酵素。我们已经研究了72 kDa的转录调控 胶原酶IV在正常和人类肿瘤细胞系中的表达 作为人类肿瘤组织。这些研究表明，与 胶原酶家族的其他成员，72 kDa的IV型 胶原酶mRNA水平在TGFbeta1的反应中增加，是 不受肿瘤影响促进佛波酯，并显示升高 结直肠肿瘤组织与邻近正常组织的水平比较 粘膜组织。转染人肿瘤细胞的研究 含有72 kDa IV型胶原酶基因的构建导致 表型变化，如细胞生长速度减慢和细胞标记 空泡化。这些发现表明，未复杂的72 kDa IV型 这些转化的细胞不容易分泌胶原酶。我们有 确定了一种细胞激活机制，即细胞表面 与72 kDa IV型胶原酶相关和特异，以及 可通过佛波酯或刀豆蛋白的预处理来诱导。 答：这种细胞激活机制不会影响 胶原酶基因家族。进一步表征和提纯 这一激活机制的组成部分正在进行中。我们还有 检测72 kDa IV型胶原酶的自身蛋白分解 在从复合体中去除TIMP-2之后。这种酶有一种 自身蛋白水解酶产生特异性的特征模式 42 kDa和37.5 kDa明胶结合片段，其中一个(37.5 kDa) 保留蛋白质分解活性。最后，我们准备了抗体和 与胶原酶家族其他成员的cdna探针，并已启动 一种检测人肺癌组织中相关基因的筛查项目 72 kDa IV型胶原酶的mRNA水平和蛋白表达， 基质分解素-1、基质分解素-2、基质分解素-3、Pump-1和92 kDa类型 静脉注射胶原酶。