Weiss R E, Liu B C, Ahlering T, Dubeau L, Droller M J
Department of Urology, Mount Sinai School of Medicine, New York, NY 10029.
J Urol. 1990 Sep;144(3):798-804. doi: 10.1016/s0022-5347(17)39595-2.
To study the biochemical mechanisms of bladder tumor invasion, we analyzed specimens of invasive transitional cell carcinoma cell line EJ and non-invasive transitional cell carcinoma cell line RT4 which had been implanted into the bladders of nude mice. Using immunoprobes specific to basement membrane laminin, we observed that superficial but not invasive tumors were surrounded by intact laminin. With immunoprobes specific to cathepsin B, a cysteine proteinase which has the ability to degrade laminin, we demonstrated that cathepsin B is localized in discrete cytoplasmic granules in the non-invasive tumors, and in a more diffuse cytoplasmic pattern in the invasive tumors. Subcellular fractionation followed by immunoblot analysis and enzymatic analysis confirmed that the invasive EJ cells had active cathepsin B localized to its plasma membrane, while non-invasive RT4 cells had cathepsin B confined to lysosomes. Furthermore, immunoblot analysis revealed that invasive EJ cells had the mature form of cathepsin B with a molecular weight of 25 kD, while non-invasive RT4 cells had predominantly precursor forms with molecular weights between 30 and 35 kD. In vitro degradation assays with plasma membrane fractions isolated from invasive EJ cells and non-invasive RT4 cells demonstrated that the plasma membrane of EJ cells but not that of the RT4 cells had the ability to degrade purified laminin, and that the degradative products were similar to those obtained with purified cathepsin B. We conclude that invasive tumor cells have enhanced cathepsin B in their plasma membranes which may be used to degrade basement membrane components such as laminin and thereby facilitate tumor invasion.
为研究膀胱肿瘤侵袭的生化机制，我们分析了已植入裸鼠膀胱的侵袭性移行细胞癌细胞系EJ和非侵袭性移行细胞癌细胞系RT4的标本。使用针对基底膜层粘连蛋白的免疫探针，我们观察到浅表性而非侵袭性肿瘤被完整的层粘连蛋白所包围。使用针对组织蛋白酶B的免疫探针，组织蛋白酶B是一种能够降解层粘连蛋白的半胱氨酸蛋白酶，我们证明组织蛋白酶B定位于非侵袭性肿瘤中离散的细胞质颗粒中，而在侵袭性肿瘤中则呈更弥散的细胞质模式。进行亚细胞分级分离，随后进行免疫印迹分析和酶分析，证实侵袭性EJ细胞的活性组织蛋白酶B定位于其质膜，而非侵袭性RT4细胞的组织蛋白酶B局限于溶酶体。此外，免疫印迹分析显示，侵袭性EJ细胞具有分子量为25 kD的成熟形式的组织蛋白酶B，而非侵袭性RT4细胞主要具有分子量在30至35 kD之间的前体形式。用从侵袭性EJ细胞和非侵袭性RT4细胞分离的质膜部分进行的体外降解试验表明，EJ细胞的质膜而非RT4细胞的质膜具有降解纯化层粘连蛋白的能力，并且降解产物与用纯化的组织蛋白酶B获得的产物相似。我们得出结论，侵袭性肿瘤细胞在其质膜中具有增强的组织蛋白酶B，这可能用于降解基底膜成分如层粘连蛋白，从而促进肿瘤侵袭。
