CATHEPSIN B-LIKE CYSTEINE PROTEINASES AND TUMOR INVASION

蛋白水解酶被认为与侵袭过程有关 转移级联反应。我们的长期目标是确定 组织蛋白酶B与内源性半胱氨酸蛋白酶的作用(S) 半胱氨酸蛋白酶抑制物(CPI)与肿瘤细胞转移我们 提纯了两种形式的组织蛋白酶B(35和25 kDa) 人类肿瘤，并显示其免疫学与 人肝组织蛋白酶B(25 KDa)的免疫印迹分析。我们有 表明肿瘤组织蛋白酶B具有增强抗肿瘤活性的作用 生理状态下细胞外基质底物层粘连蛋白 相应pH值(6.5)。 组织蛋白酶B参与肿瘤细胞的假说 在转移性级联过程中的外渗将在 在一系列围绕侵入性过程的测试中进行体外实验： 1)肿瘤细胞黏附于基底膜，2)肿瘤细胞 外渗过程中的趋化作用，3)肿瘤细胞的降解 基底膜或分离成分和/或肿瘤细胞 通过羊膜基底膜侵入。两个班级 组织蛋白酶B的抑制剂将在以下检测中进行测试： 内源性低MR蛋白CPI和一系列合成抑制剂 对纯化的肿瘤组织蛋白酶B的选择性进行筛选。 CPI将从人的肝脏和肿瘤中纯化成均一种。 到目前为止的研究表明，肿瘤细胞的趋化、黏附和 侵袭都被CPI以及特定的人工合成物质所阻断 抑制剂。 在小鼠肿瘤(黑色素瘤和肝癌)和人类乳腺中 肿瘤的亚细胞分布中组织蛋白酶的分布 组织蛋白酶B是双峰的，高达37%的组织蛋白酶B 与质膜组分共降解的活性 高度转移性B16无色素性黑色素瘤(B16a) 与正常细胞中的溶酶体部分一样。35 kDa的肿瘤 组织蛋白酶B很可能是成熟溶酶组织蛋白酶的前体 B作为35 kDa的前体从最近分离出来的 CDNA克隆。一个将被检验的假设是， 组织蛋白酶B在肿瘤中的处理可能是有缺陷的。 拟议的研究应进一步加深我们对这一角色的理解 组织蛋白酶B在蛋白水解级联反应中的作用导致转移。它 有可能通过设计来中断这种级联 抑制组织蛋白酶B和/或纠正组织蛋白酶B的治疗干预 加工过程中的拟议缺陷，即直接肿瘤组织蛋白酶B 到溶酶。

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.