CATHEPSIN B-LIKE CYSTEINE PROTEINASES AND TUMOR INVASION

最近的证据发现了一种类似组织蛋白酶B的半胱氨酸蛋白酶(CB) 这可能促进体液侵袭和转移的过程。CB 从肝脏中提取的蛋白质可以降解胶原蛋白和蛋白多糖 激活潜伏胶原酶。因此CB可能通过以下途径在肿瘤转移中起作用 增强肿瘤细胞侵袭周围组织的能力 原发灶和转移灶，以及渗出和渗出。 我们将从小鼠B16无色素性皮下肿瘤中提纯CB 黑色素瘤(B16a)和B16a条件培养液。CB获释 从肿瘤中分离出来的是一种酶原，它在 中性和碱性pH。这些酶将被表征为它们的 物理和动力学性质。原CB的蛋白水解性激活将是 用活性CB、纤溶酶、纤溶酶原激活物、激肽释放酶等进行评估。 将开发抗活性CB和抗ProCB的单抗，并 它们的交叉反应测试彼此之间以及与CB的交叉反应 从其他小鼠和人类肿瘤、其他组织、木瓜酶和 组织蛋白酶H和L内源性半胱氨酸蛋白酶抑制剂 与合成的抑制剂一起提纯和测试它们的能力 灭活CB和ProCB。 使用纯化的CB和proCB，我们将确定它们的降解能力， 单抗和半胱氨酸蛋白酶的存在与缺失 抑制物，细胞外基质的成分[胶原(I-V型)， 层粘连蛋白、纤维连接蛋白]。CB和ProCB的激活能力 肿瘤来源的潜伏型IV型胶原酶也将被检测。离体 研究肿瘤细胞通过内皮单分子层侵袭的方法 并通过羊膜衍生基底膜建立。入侵 通过125I-UdR标记的肿瘤细胞将在存在和 缺少抗CB、ProCB和半胱氨酸蛋白酶的单抗 抑制剂。 这些研究应该使我们能够评估CB在 肿瘤细胞转移和评估CB是否可能是一种蛋白水解物 转移性蛋白水解级联反应中的酶。例如，纤溶酶衍生 通过纤溶酶原激活物(PA)对纤溶酶原的作用而激活 ProCB和CB依次激活潜伏型IV型胶原酶。它可能是 有可能通过指导治疗来中断这种蛋白分解级联反应 对防止释放前CB的干预或对预防的干预 ProCB(因为ProCB似乎不能从正常细胞中释放)。

Recent evidence has identified a cathepsin B-like cysteine proteinase (CB) which may facilitate the processes of humor invasion and metastasis. CB from liver has been shown to degrade collagen and proteoglycans and to activate latent collagenase. Thus CB may act in tumor metastasis by enhancing the ability of tumor cells to invade surrounding tissue at primary and metastatic sites and to intravasate and extravasate. We will purify CB from subcutaneous tumors of the murine B16 amelanotic melanoma (B16a) and from B16a - conditioned culture media. CB released from tumors is apparently a proenzyme which has increased stability at neutral and alkaline pH. These enzymes will be characterized for their physical and kinetic properties. Proteolytic activation of proCB will be assessed using active CB, plasmin, plasminogen activator, kallikrein etc. Monoclonal antibodies to active CB, and to proCB will be developed and their cross-reactivities tested against one another as well as against CB purified from other murine and human tumors, other tissues, papain, and cathepsins H and L. Endogenous cysteine proteinases inhibitors will be purified and tested along with synthetic inhibitors for their ability to inactivate CB and proCB. Using purified CB and proCB we will determine their ability to degrade, in the presence and absence of monoclonal antibodies and cysteine proteinase inhibitors, components of the extracellular matrix [collagen (Types I-V), laminin, fibronectin]. The ability of CB and proCB to activate tumor-derived latent type IV collagenase will also be determined. In vitro assays to study invasion of tumor cells through an endothelial monolayer and through amnion-derived basement membrane will be established. Invasion by 125I-Udr labeled tumor cells will be followed in the presence and absence of monoclonal antibodies to CB and proCB and of cysteine proteinase inhibitors. These studies should enable us to assess the presumptive role of CB in tumor cell metastasis and to assess whether CB may be one proteolytic enzyme in a metastatic proteolytic cascade. For example, plasmin derived from the action of plasminogen activator (PA) on plasminogen may activate proCB and CB in turn may activate latent type IV collagenase. It might be possible to interrupt such a proteolytic cascade by directing therapeutic intervention toward preventing release of proCB or towards the pro frament of proCB (since proCB does not seem to be released from normal cells).