ROLE OF COLLAGENOLYTIC METALLOPROTEINASES IN METASTASES

为了探讨IV型胶原酶在肿瘤侵袭中的作用 和转移，我们专注于多层次的调控， 酵素 我们已经研究了72 kDa的转录调控， 胶原酶IV酶在正常和人肿瘤细胞系中也是如此 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 IV型 胶原酶mRNA水平在对TGF β 1的反应中增加， 不受促进肿瘤的佛波醇酯的影响， 与邻近正常组织相比， 粘膜组织 转染具有表达的人肿瘤细胞 含有72 kDa IV型胶原酶基因的构建体导致 表型变化，如细胞生长速率减慢和细胞标记 空泡化 这些发现表明，未复合的72 kDa IV型 这些转化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型胶原酶相关且特异，和 其可以通过用佛波醇酯或伴刀豆球蛋白预处理来诱导 A. 这种细胞激活机制不影响其他成员， 胶原酶基因家族。 进一步表征和纯化 这一启动机制的组成部分正在进行中。 我们还 检测了72 kDa IV型胶原酶的自身蛋白水解分解 然后从复合物中除去TIMP-2。 酶具有 自身蛋白水解消化的特征模式产生特异性 42 kDa和37.5 kDa明胶结合片段，其中一个（37.5 kDa） 保持蛋白水解活性。 最后，我们制备了抗体， 胶原酶家族其他成员的cDNA探针， 一个筛查项目，检查人类肺肿瘤组织， 72 kDa IV型胶原酶的mRNA水平和蛋白表达， 溶基质素-1、溶基质素-2、溶基质素-3、溶基质素-1和92 kDa型 IV胶原酶。

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.