title: Plasma membrane association of cathepsin B in human prostate cancer: biochemical and immunogold electron microscopic analysis.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, USA. sinha001@tc.umn.edu
authors.forename: A A
authors.initials: AA
authors.lastname: Sinha
authors.forename: M P
authors.initials: MP
authors.lastname: Jamuar
authors.forename: M J
authors.initials: MJ
authors.lastname: Wilson
authors.forename: J
authors.initials: J
authors.lastname: Rozhin
authors.forename: B F
authors.initials: BF
authors.lastname: Sloane
mesh_terms: D002401:Cathepsin B / Q000096:biosynthesis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D016253:Microscopy, Immunoelectron; D017430:Prostate-Specific Antigen / Q000032:analysis / Q000096:biosynthesis; D011470:Prostatic Hyperplasia / Q000378:metabolism / Q000473:pathology; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D015853:Cysteine Proteinase Inhibitors; D017430:Prostate-Specific Antigen; D002401:Cathepsin B