Plasma membrane association of cathepsin B in human prostate cancer: biochemical and immunogold electron microscopic analysis.

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.

Freeze-fracture, ultrastructural and autoradiographic analysis of the ventral prostate glands in castrated mice.

Ultrastructure of the ventral prostate glands was studied in mice castrated for 1 through 60 days and for 11 and 17 months and in age-matched normals. We have described freeze-fracture and ultrastructural characteristics of acinar epithelial cells in addition to the patterns of thymidine incorporation in the cells of castrates and normal animals. Our study has shown a biphasic pattern of prostatic involution in the long-term castrated mice. In castrates the initial atrophy of prostate glands occurred by sloughing of the apical portions of columnar cells, autophagia of the cytoplasmic organelles as well as by occasional sloughing of the individual cells into the acinar lumen. Concurrent with the initial atrophy, the glands and stroma were infiltrated by neutrophils and lymphocytes. The cell loss by sloughing and leucocyte infiltration of glands became infrequent in 7- to 21-day castrates. However, the cell loss by sloughing increased secondarily in mice castrated for 21 to 37 days along with the increased leucocyte infiltration of the glands. The cell loss became minimal in castrates of 60 days and beyond. Our evidence suggests that the cell loss by sloughing was an active process in the involution of prostate glands which also showed differential sensitivity to castration stimuli in mice.

The fate of blood granulocytes.

The present study was undertaken to determine whether neutrophilic granulocytes leave the vascular bed and enter body tissues, particularly the lung, in pathogen-free mice under normal steady state conditions and also under inflammatory stress. Tissues from normal mice and from mice with granulocytosis-producing tumor were processed for electron microscopic studies. Neutrophils have not been seen outside the capillary or in the pulmonary epithelium in alveoli under normal steady state or in mice with leukocyte counts of 300,000/mm3 in which capillaries were replete with neutrophils. Emigration of neutrophils outside the capillaries was observed under inflammatory conditions. Granulocytes were also observed to be concentrated for destruction in the spleen, perhaps the major site for removal of granulocytes. In the pathogen-free mice granulocytes did not appear to leave blood vessels and enter tissues.

Studies of spermiogenesis in a nematode, Nippostrongylus brasiliensis.

The fine structure of the developing spermatids and the mature sperm of Nippostrongylus brasiliensis was investigated. Immature spermatids are found at one end of the tubelike testis, and the mature sperm at the other. The spermatid has a prominent nucleus, with the chromatin clumped at the margin. It also contains a pair of centrioles, located near the nucleus. The cytoplasm is filled with ribosomal clusters, but it lacks an organized Golgi area or endoplasmic reticulum. Besides the normal mitochondria, the spermatid has specialized mitochondrionlike inclusions with dense matrix, few broad cristae, and a crystalloid structure always facing the nucleus. As spermiogenesis proceeds, the nucleus elongates, comes to lie at one end, and later evaginates to form a separate head structure, leaving the mitochondria and other cytoplasmic organelles in a broad cytoplasmic region. The nuclear material becomes filamentous and spiral, and the centrioles come to lie at one end near the junction of the head and the cytoplasmic portion of the sperm. Microtubules are found in the cytoplasmic region extending from the tubelike nucleus. The specialized mitochondria are about eighteen in number, and are arranged in rows in staggered groups of three around the microtubules in the cytoplasmic region. The mature sperm is aflagellate and lacks an acrosome. No movement of the sperm was ever observed.