CATHEPSIN B-LIKE CYSTEINE PROTEINASES AND TUMOR INVASION

最近的证据已经确定了组织蛋白酶B样半胱氨酸蛋白酶（CB） 这可能促进体液侵袭和转移的过程。 CB 已经显示，从肝脏中提取的胶原蛋白可以降解胶原蛋白和蛋白聚糖， 激活潜伏的胶原酶。 因此，CB可能通过以下方式在肿瘤转移中起作用： 增强肿瘤细胞侵入周围组织的能力， 原发部位和转移部位以及内渗和外渗。 我们将从小鼠B16无黑色素瘤的皮下肿瘤中纯化CB， 黑色素瘤（B16a）和来自B16a-条件培养基。 CB发布 从肿瘤中分离出来的蛋白质显然是一种酶原， 中性和碱性pH。这些酶将表征其 物理和动力学性质。 proCB的蛋白水解激活将是 使用活性CB、纤溶酶、纤溶酶原激活剂、激肽释放酶等进行评估。 将开发针对活性CB和proCB的单克隆抗体， 它们的交叉反应性相互测试以及对CB 从其他鼠和人肿瘤、其他组织、木瓜蛋白酶和 组织蛋白酶H和L。 内源性半胱氨酸蛋白酶抑制剂将是 纯化并与合成抑制剂一起沿着测试其能力， CSB和proCB。 使用纯化的CB和proCB，我们将确定它们在体外降解的能力， 存在和不存在单克隆抗体和半胱氨酸蛋白酶 抑制剂，细胞外基质的组分[胶原蛋白（I-V型）， 层粘连蛋白、纤连蛋白]。 CB和proCB激活 还将测定肿瘤衍生的潜伏性IV型胶原酶。 体外 研究肿瘤细胞通过内皮单层侵袭的测定 并通过细胞膜形成基底膜。 入侵 将在存在下跟踪125I-Udr标记的肿瘤细胞， 不存在CB和proCB的单克隆抗体以及半胱氨酸蛋白酶 抑制剂的 这些研究应使我们能够评估CB在以下方面的假定作用： 肿瘤细胞转移，并评估CB是否可能是一种蛋白水解 转移性蛋白水解级联中的酶。 例如，纤溶酶衍生的 从纤溶酶原激活剂（PA）对纤溶酶原的作用可以激活 proCB和CB又可以激活潜伏的IV型胶原酶。 它可能是 可以通过引导治疗来中断这种蛋白水解级联反应 为防止proCB释放或为预防 （因为proCB似乎不会从正常细胞中释放）。

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).