CATHEPSIN B-LIKE CYSTEINE PROTEINASES AND TUMOR INVASION

蛋白水解酶参与了 转移级联反应 我们的长期目标是确定 半胱氨酸蛋白酶组织蛋白酶B和内源性 半胱氨酸蛋白酶抑制剂（CPI）在肿瘤细胞转移中的作用。 我们 已经纯化了两种形式的组织蛋白酶B（35和25 kDa）， 人类肿瘤，并显示其免疫相关性， 人肝组织蛋白酶B（25 kDa）蛋白质印迹法。 我们有 显示肿瘤组织蛋白酶B具有增强的抗 细胞外基质底物层粘连蛋白， 相关pH值（6.5）。 组织蛋白酶B参与肿瘤细胞的假说 转移级联过程中的外渗将在 在一系列包括侵入性过程的体外测定中： 1)肿瘤细胞粘附基底膜，2）肿瘤细胞 外渗过程中的趋化性，3） 基底膜或分离的组分和/或4）肿瘤细胞 通过羊膜基底膜侵入。 两类 将在以下试验中检测组织蛋白酶B抑制剂： 内源性低Mr蛋白CPI和一系列合成抑制剂 筛选对纯化的肿瘤组织蛋白酶B的选择性。 CPI将从人肝脏和肿瘤中纯化至均一。 迄今为止的研究表明，肿瘤细胞的趋化性，粘附性和 CPI和特定合成物都能阻断入侵 抑制剂的 在小鼠肿瘤（黑色素瘤和肝癌）和人类乳腺中 肿瘤组织蛋白酶的亚细胞分布 组织蛋白酶B是双峰的，其中多达37%的组织蛋白酶B 活性与质膜组分共沉淀， 高转移性B16无黑色素性黑色素瘤（B16 a），而不是， 与正常细胞中的溶浆部分一样。 35 kDa肿瘤 组织蛋白酶B很可能是成熟溶酶体组织蛋白酶前体 B作为一个35 kDa的前体预测从最近分离的 cDNA克隆。 一个将被检验的假设是， 肿瘤中组织蛋白酶B的加工可能是有缺陷的。 拟议的研究应能加深我们对 组织蛋白酶B在蛋白水解级联反应中的作用导致转移。 它 也许可以通过设计一个 抑制组织蛋白酶B和/或纠正 所提出的处理中的缺陷，即，直接肿瘤组织蛋白酶B 溶菌

Proteolytic enzymes have been implicated in invasive processes of the metastatic cascade. Our long term objective is to determine the role(s) of the cysteine proteinase cathepsin B and endogenous cysteine proteinase inhibitors (CPI) in tumor cell metastasis. We have purified two forms of cathepsin B (35 and 25 kDa) from human tumors and shown their immunological relatedness to human liver cathepsin B (25 kDa) by Western blots. We have shown that tumor cathepsin B's have enhanced activity against the extracellular matrix substrate laminin at a physiologically relevant pH (6.5). The hypothesis that cathepsin B participates in tumor cell extravasation during the metastatic cascade will be tested in vitro in a series of assays which encompass the invasive process: 1) tumor cell adhesion the basement membrane, 2) tumor cell chemotaxis during extravasation, 3) tumor cell degradation of the basement membrane or isolated components and/or 4) tumor cell invasion through the amnion basement membrane. Two classes of inhibitors of cathepsin B will be tested in these assays: endogenous low Mr protein CPI and a series of synthetic inhibitors screened for selectivity against the purified tumor cathepsin B's. CPI will be purified to homogeneity from human liver and tumors. Studies to date indicate that tumor cell chemotaxis, adhesion and invasion are all blocked by CPI as well as by specific synthetic inhibitors. In murine tumors (melanoma and hepatoma) and in human breast tumors, the subcellular distribution of cathepsin distribution of cathepsin B is bimodal with as much as 37% of cathepsin B activity cosedimenting with plasma membrane fractions in the highly metastatic B16 amelanotic melanoma (B16a) rather that with the lysosmal fraction as in normal cells. The 35 kDa tumor cathepsin B may well be a precursor of mature lysosmal cathepsin B as a 35 kDa precursor predicted from the recently isolated cDNA clones. One hypothesis which will be tested is that the processing of cathepsin B in tumors may be defective. The proposed studies should further our understanding of the role of cathepsin B in a proteolytic cascade resulting in metastasis. It might be possible to interrupt this cascade by designing therapeutic interventions to inhibit cathepsin B and/or to correct the proposed defect in processing, i.e., direct tumor cathepsin B to lysosmes.