ACTIVATION OF COLLAGENASE IN ORAL TUMORS

口腔肿瘤中胶原酶的激活

• 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
• 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

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.

基质金属蛋白酶（MMP）长期以来一直被认为与 肿瘤侵袭过程中细胞外基质（ECM）的降解 转移。 入侵过程的早期步骤之一是 ECM 的局部溶解，为细胞迁移创造一条途径。 胶原酶对原纤维胶原进行位点特异性切割是必要的 开始降解过程并为进一步的胶原蛋白做准备 蛋白水解。 我们在这个项目中的重点是激活体内事件 胶原酶从正常潜伏状态转变为酶活性状态 形式。 虽然我们实验室和其他人已经研究了体外 胶原酶和其他 MMP 的激活，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口腔肿瘤细胞更容易被激活 正常口腔组织。 激活过程中的这种改变可以 肿瘤细胞分泌胶原酶激活剂的结果（对于 例如，蛋白水解激活剂，例如另一种 MMP，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； TIMP）。 在过去的八年里，我们积累了两种 cDNA 探针 和独特的抗体试剂来研究这个过程，我们建议 将这些材料应用于体内胶原酶活化的研究 口腔肿瘤。 我们提出三个具体目标来解决为此提出的假设 学习： 1. 肿瘤中是否含有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 用一个新的新点检测口腔肿瘤材料中 MMP 的表达 降解测定法测量下方胶原蛋白膜的溶解情况 从口腔肿瘤中生长出来的原代细胞斑点。 初步结果 使用 SCC-25 细胞（源自口腔鳞状细胞癌）获得 已显示出可检测水平的活化胶原酶，我们建议 确定大多数口腔肿瘤是否含有活化的胶原酶。 2.口腔肿瘤细胞中的胶原酶是如何被激活的？ 我们会净化 活化过程中的中间体。 如果中间体是 经过蛋白水解处理后，我们将确定切割位点和 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发挥作用？ 我们将使用我们现有的抑制性抗体来阻止特定的 细胞培养基中可能存在的 MMP 或 MMP 抑制剂 我们的现货胶原蛋白降解测定。 合成的 MMP 抑制剂也将 用作确认方法。