Sinha A A, Jamuar M P, Wilson M J, Rozhin J, Sloane B F
Department of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, USA.
Prostate. 2001 Nov 1;49(3):172-84. doi: 10.1002/pros.1132.
Cathepsin B (CB), a cysteine protease, is usually found in perinuclear lysosomes of epithelial cells of normal organs and non-malignant tumors, but is associated with the plasma membranes of many solid organ malignant tumors. Plasma membrane localized CB facilitates degradation of extracellular matrix proteins and progression of tumor cells from one biological compartment to another. The activities of CB and its subcellular distribution have not been investigated in malignant prostate. Our objective was to examine the subcellular distribution of CB by determining the activities of CB in lysosome and plasma membrane/endosome subcellular fractions and its subcellular localization by immunogold electron microscopy.
Prostate tissue pieces obtained immediately after prostatectomy were homogenized and fractionated into subcellular components for determining biochemical activities of CB and cysteine protease inhibitors (CPIs). Distribution of CB was compared with that of prostate specific antigen (PSA, a serine protease), which is abundant in secretory vesicles and granules of normal prostate, benign prostatic hyperplasia (BPH) and malignant prostate cells. Localization of CB was investigated in resin embedded lysosomes and plasma membrane/endosome subcellular fractions and in prostate tissue sections by immunogold electron microscopy.
We have demonstrated the specificity of CB activity in human prostate homogenates by using a variety of inhibitors in our assay. We did not find any difference in the specific activity of CB based on protein or DNA content in homogenates of malignant prostate (Gleason histologic scores 5-7) and BPH (no histological evidence of cancer) whether it was measured by chromogenic or fluorogenic peptide substrate assay techniques. We found significantly higher activities of CB in the plasma membrane/endosome fractions of malignant prostate than in BPH. In contrast, CPI activity was increased relative to CB activity in plasma membrane/endosome fraction of BPH versus prostate cancer. Our data indicated a shift in the balance of enzyme to inhibitor that would favor increased activities of CB in prostate cancer. The immunogold microscopic study showed specific localization of CB in plasma membrane. They also showed localization of CB in lysosomes that were often adjacent to luminal and/or basal surfaces of malignant cells in contrast to the usual perinuclear distribution of lysosomes in hyperplastic prostate glands. PSA was localized in secretory granules and vesicles, including the plasma membranes and secretory blebs in malignant prostate cells. Occasional PSA positive secretory vesicles or membrane profiles were seen in the plasma membrane/endosomal and lysosomal fractions.
The increased activity of CB in plasma membrane/endosomal fractions is associated with malignant prostate and not with BPH or normal prostate. Morphologic distribution CB is associated with the plasma membranes or lysosomes adjacent to apical and basal cell surfaces. This distribution is characteristic feature prostate cancer cells, but not in BPH or normal prostate cells. Subcellular distribution of PSA occurs in secretory vesicles and granules of the cytoplasm, but not in lysosomes. Our biochemical and morphological data could be used to distinguish malignant prostates from non-malignant tumors.
组织蛋白酶B（CB）是一种半胱氨酸蛋白酶，通常存在于正常器官和非恶性肿瘤上皮细胞的核周溶酶体中，但与许多实体器官恶性肿瘤的质膜相关。定位在质膜上的CB促进细胞外基质蛋白的降解以及肿瘤细胞从一个生物隔室转移到另一个隔室。尚未在恶性前列腺组织中研究CB的活性及其亚细胞分布。我们的目的是通过测定溶酶体和质膜/内体亚细胞组分中CB的活性来检查CB的亚细胞分布，并通过免疫金电子显微镜确定其亚细胞定位。
前列腺切除术后立即获取的前列腺组织块被匀浆并分离成亚细胞组分，以测定CB和半胱氨酸蛋白酶抑制剂（CPI）的生化活性。将CB的分布与前列腺特异性抗原（PSA，一种丝氨酸蛋白酶）的分布进行比较，PSA在正常前列腺、良性前列腺增生（BPH）和恶性前列腺细胞的分泌小泡和颗粒中含量丰富。通过免疫金电子显微镜在树脂包埋的溶酶体和质膜/内体亚细胞组分以及前列腺组织切片中研究CB的定位。
我们通过在检测中使用多种抑制剂证明了人前列腺匀浆中CB活性的特异性。基于恶性前列腺（Gleason组织学评分5 - 7）和BPH（无癌症组织学证据）匀浆中的蛋白质或DNA含量，无论通过显色或荧光肽底物检测技术测量，我们都未发现CB的比活性有任何差异。我们发现恶性前列腺质膜/内体组分中CB的活性明显高于BPH。相比之下，BPH与前列腺癌质膜/内体组分中CPI活性相对于CB活性增加。我们的数据表明酶与抑制剂的平衡发生了变化，这有利于前列腺癌中CB活性的增加。免疫金显微镜研究显示CB在质膜中有特异性定位。他们还显示CB在溶酶体中的定位，这些溶酶体通常与恶性细胞的管腔和/或基底表面相邻，这与增生性前列腺腺体中溶酶体通常的核周分布形成对比。PSA定位于分泌颗粒和小泡中，包括恶性前列腺细胞质膜和分泌泡。在质膜/内体和溶酶体组分中偶尔可见PSA阳性分泌小泡或膜结构。
质膜/内体组分中CB活性的增加与恶性前列腺相关，而与BPH或正常前列腺无关。CB的形态学分布与顶端和基底细胞表面相邻的质膜或溶酶体相关。这种分布是前列腺癌细胞的特征，但在BPH或正常前列腺细胞中不存在。PSA的亚细胞分布发生在细胞质的分泌小泡和颗粒中，而不是溶酶体中。我们的生化和形态学数据可用于区分恶性前列腺与非恶性肿瘤。
