ACTIVATION OF COLLAGENASE IN ORAL TUMORS

口腔肿瘤中胶原酶的激活

基本信息

批准号：
6501048
负责人：
JEFFREY ALLEN ENGLER
金额：
$ 26.84万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-08-01 至 2002-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6501048
关键词：
biomarker collagenase enzyme activity human tissue immunochemistry immunologic assay /test in situ hybridization laboratory rabbit metalloendopeptidases neoplastic growth neoplastic transformation oral pharyngeal neoplasm protein purification squamous cell carcinoma tissue /cell culture

项目摘要

Matrix metalloproteinases (MMPs) have long been implicated in the degradation of the extracellular matrix (ECM) during tumor invasion and metastasis. One of the early steps to the invasion process is the localized dissolution of the ECM, to create a pathway for cell migration. A site-specific cleavage of fibrillar collagen by collagenase is necessary to begin the degradation process and to prepare the collagen for further proteolysis. Our focus in this project is on the in vivo events that activate collagenase from its normally latent form to its enzymatically active form. Although our laboratory and others have studied the in vitro activation of collagenase and other MMPs, little is known about this process in tumor tissue. The hypothesis to be tested is that the collagenase secreted by oral tumor cells is more easily activated than in normal oral tissue. This alteration in the activation process could result from secretion by tumor cells of an activator of collagenase (for example, a proteolytic activator, such as another MMP like stromelysin-1 or gelatinase) or by disruption of the balance between collagenase and its natural inhibitors such as the tissue inhibitors of metalloproteinases; TIMPs). Over the last eight years, we have accumulated both cDNA probes and unique antibody reagents for studying this process, and we propose to apply these materials to this study of collagenase activation in vivo in oral tumors. We propose three specific aims to address the hypothesis proposed for this study: 1. Do tumors contain activated collagenase and other MMPs? We will combine previous in situ hybridization and immunochemistry approaches to detect MMP expression in oral tumor material with a novel new spot degradation assay to measure dissolution of collagen films underneath spots of primary cells grown from oral tumors. Preliminary results obtained with SCC-25 cells (derived from an oral squamous cell carcinoma) have shown detectable levels of activated collagenase and we propose to determine whether most oral tumors contain activated collagenase. 2. How is collagenase activated in oral tumor cells? We will purify intermediates in the activation process. If the intermediates are proteolytically processed, we will determine the sites of cleavage and identify the activating agent (collagenase itself, other MMPs, other proteases). 3. Do other MMPs play a role in collagenase activation in oral tumors? We will use inhibitory antibodies that we have on hand to block specific MMPs or MMP inhibitors that might be found in the cell culture medium of our spot collagen degradation assay. Synthetic MMP inhibitors will also be used as a confirmatory approach.

长期以来，基质金属蛋白酶（MMP）与肿瘤入侵期间细胞外基质（ECM）的降解和转移。入侵过程的早期步骤之一是 ECM的局部溶解，以创建细胞迁移的途径。有必要通过胶原酶对纤维状胶原蛋白的特定位点特异性切割开始降解过程并准备胶原蛋白以进一步蛋白水解。我们在这个项目上的重点是激活的体内事件胶原酶从正常的潜在形式到酶促活性形式。尽管我们的实验室和其他实验室研究了体外激活胶原酶和其他MMP，对此知之甚少肿瘤组织中的过程。要检验的假设是口腔肿瘤细胞分泌的胶原酶比在正常的口腔组织。激活过程中的这种改变可能由肿瘤细胞分泌的胶原酶激活剂的分泌（对于例如，蛋白水解激活剂，例如另一个MMP，例如Stromelysin-1 或明胶酶）或通过破坏胶原酶及其之间的平衡天然抑制剂，例如金属蛋白酶的组织抑制剂； Timps）。在过去的八年中，我们积累了两个cDNA探针以及用于研究此过程的独特抗体试剂，我们建议将这些材料应用于本体内胶原酶激活的研究口腔肿瘤。我们提出了三个特定的目的，以解决为此提出的假设学习： 1。肿瘤是否包含活化的胶原酶和其他MMP？我们将结合以前的原位杂交和免疫化学方法用新的新位置检测口腔肿瘤材料中的MMP表达降解测定以测量下面胶原蛋白膜的溶解由口腔肿瘤生长的原代细胞的斑点。初步结果用SCC-25细胞获得（源自口服鳞状细胞癌）已经显示出可检测的活化胶原酶水平，我们建议确定大多数口腔肿瘤是否含有活化的胶原酶。 2。如何在口腔肿瘤细胞中激活胶原酶？我们将净化中间在激活过程中。如果中间体是通过蛋白水解处理，我们将确定乳沟的位置识别激活剂（胶原酶本身，其他MMP，其他蛋白酶）。 3。其他MMP是否在口腔肿瘤中的胶原酶激活中起作用？我们将使用抑制性抗体来阻止特定 MMP或MMP抑制剂可能在细胞培养基中发现我们的胶原蛋白降解测定法。合成的MMP抑制剂也将用作验证方法。