ACTIVATION OF COLLAGENASE IN ORAL TUMORS

基质金属蛋白酶(MMPs)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，为细胞迁移创造了一条途径。 胶原酶对纤维性胶原的定点切割是必要的 以开始降解过程，并为进一步的 蛋白质分解。 我们在这个项目中的重点是活体事件 胶原酶从正常潜伏期到酶活性 形式。虽然我们的实验室和其他人已经研究了体外 胶原酶和其他基质金属蛋白酶的激活，对此知之甚少 在肿瘤组织中的过程。需要检验的假设是， 口腔肿瘤细胞分泌的胶原酶比口腔癌细胞更容易被激活 正常的口腔组织。激活过程中的这种改变可能 由肿瘤细胞分泌胶原酶激活剂所致 例如，蛋白分解激活剂，例如另一种类似基质分解蛋白-1的基质金属蛋白酶 或明胶酶)或通过破坏胶原酶和其 天然抑制物，如金属蛋白酶的组织抑制物； TIMPS)。在过去的八年里，我们积累了两个cdna探针。 和独特的抗体试剂来研究这一过程，我们建议 将这些材料应用于胶原酶体内激活的研究 口腔肿瘤。 我们提出了三个具体目标来解决为此而提出的假设 研究： 1.肿瘤中是否含有活化的胶原酶和其他基质金属蛋白酶？我们会 结合以前的原位杂交和免疫化学方法来 利用一个新的新斑点检测口腔肿瘤材料中基质金属蛋白酶的表达 降解测定法测定胶原膜的溶解性能 口腔肿瘤长出的原代细胞斑点。初步结果 用SCC-25细胞(来源于口腔鳞状细胞癌)获得 已经显示出可检测到的激活胶原酶水平，我们建议 确定大多数口腔肿瘤是否含有活化的胶原酶。 2.口腔肿瘤细胞中胶原酶是如何被激活的？我们将净化 激活过程中的中间体。如果中间体是 蛋白水解性处理，我们将确定切割位置和 确定激活剂(胶原酶本身、其他MMPs、其他 蛋白酶)。 3.口腔肿瘤中是否有其他基质金属蛋白酶参与胶原酶的激活？ 我们将使用手头的抑制性抗体来阻断特定的 MMPs或基质金属蛋白酶抑制剂可能存在于细胞培养上清液中 我们的斑点胶原蛋白降解测试。人工合成的基质金属蛋白酶抑制剂也将 作为一种验证性的方法。

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.