Keratin immunoreactivity in the benign and neoplastic human prostate

Keratin immunoreactivity in the benign and neoplastic human prostate was examined immunohistochemically using two monoclonal antibodies with differing specificities. One of these antibodies stained only the basal cells of the normal and hyperplastic prostatic epithelium, with no reactivity in tumor cells of prostatic adenocarcinoma. The other monoclonal antibody recognized a keratin protein present in all normal and hyperplastic columnar (secretory) epithelial cells, as well as in all cancer cells regardless of degree of tumor differentiation. In addition, the second antibody stained acinar and ductal epithelial cells exhibiting premalignant changes. Our findings indicate that keratin immunoreactivity differs among the epithelial cell populations of the human prostate, probably reflecting expression of different keratin proteins. The distinctive patterns of staining obtained with these two antibodies may assist in distinguishing hyperplastic from neoplastic prostatic epithelium, as well as in the recognition of basal cell hyperplasia, transitional cell metaplasia, and premalignant changes.     

Chromatin-associated protein kinases in human normal and benign hyperplastic prostate

Nuclear phosphoproteins and protein kinases of human normal and benign hyperplastic prostate (BPH) were studied in an effort to delineate their properties and to identify any underlying differences therein. Chromatin-associated protein kinases active towards phosvitin, lysine-rich histone, and endogenous nonhistone proteins were characterized in human prostatic nuclei. The general properties of the human chromatin-associated prostatic protein kinases were similar to those of rat ventral prostate chromatin. Polyamines stimulated the phosphorylation of endogenous nonhistone proteins and phosvitin. Protein kinases active towards phosvitin and lysine-rich histones were unaltered in chromatin from BPH tissue as compared with the normal prostate. However, phosphorylation of chromatin-associated nonhistone proteins was markedly enhanced (average, 123%) in BPH tissue as compared with the normal tissue. The results indicate a change in the protein kinase reaction specifically involving chromatin-associated nonhistone proteins of BPH tissue as compared with normal human prostate.     

Plasminogen activator content and secretion in explants of neoplastic and benign human prostate tissues

The plasminogen activators of surgically excised prostate cancers (43 specimens) and benign prostatic hyperplasias (33 specimens) were extracted with an acetate:arginine:detergent buffer, and the activities were quantitated with azocaseinolytic tests. Immunoinhibition with anti-urokinase antibody served to distinguish between activator types. The mean activator activities (total; urokinase type; and nonurokinase type) of the neoplastic group were about 2 times higher (p less than 0.05) than that of the benign prostatic hyperplasia tissues. Each group had more non-urokinase-type activator activity relative to urokinase type. Studies with autopsy tissues (13 specimens) revealed that different anatomical compartments of the prostate contain about the same mean activator activity, indicating that the site of origin of the disease did not influence the results. Immunoperoxidase staining for urokinase revealed its presence in the tumor cells and, to a lesser extent, in the epithelial elements of some benign ducts and glands but not in the connective tissue. The secretion and synthesis of activator activities were monitored in short-term (approximately 120 hr) organ cultures (serum-free media) of 21 neoplastic tissues. On the average, about 12 times more activity (approximately 80% as urokinase type) was recovered in the media and postculture tissue extracts than was present in preculture tissues. Similar results were observed with 10 benign prostatic hyperplasia specimens. Wide individual variations were present in both groups (1.5 to 322 times more activity than the initial values). Except for one case, urokinase-type activity was secreted continuously, while nonurokinase was secreted only initially in quantities similar to that present in preculture tissue extracts. After culture, tissue explants contained higher quantities of both activator activities than were present initially. Dexamethasone (10 or 100 microM) decreased secretion and/or synthesis of activators by about 70%. This human organ culture model appears to be a reproducible system for individual tissues and may prove to be a valuable tool for further studies.     

Plasminogen activator content of neoplastic and benign human prostate tissues; fibrin augmentation of an activator activity

The plasminogen activator content of the extracts of excised prostate cancers (25 specimens) was determined with an azocasein assay and found to be on the average 1.7 times higher than that of extracts of excised prostate benign hyperplasias (29 specimens). Both groups contained the same average percentage of human urokinase type activator (～45%) as determined by the inhibition of activity when anti-human urokinase antibody was included in the assay system. The two types of activators were partially purified and found to have distinctly different properties. The most striking difference was the large augmentation of activity of the non-urokinase enzyme in fibrinolysis. The implications of an enhanced fibrinolysis relative to azocaseinolysis (or other) is discussed, particularly with respect to its importance in the quantitation and characterization of activators by different investigators. Highly purified urokinase-like activator was found to be similar to commercial urokinase preparation with respect to molecular weight, isoelectric point, inhibition by the antibody, and inhibition by placenta inhibitor.     

Cellular retinol-binding protein in normal and neoplastic human mammary gland

The concentration of cellular retinol-binding protein (CRBP) was determined in samples of normal and neoplastic mammary gland, using a specific and sensitive radioimmunoassay. The CRBP concentration was significantly higher in neoplastic tissue, but detectable levels were also present in all samples of normal gland. Tubulo-ductal cancers had significantly lower CRBP levels than other cancer types. The CRBP concentration of the neoplastic tissue showed no correlation with the concentration of progesterone or estrogen receptor.     

Differential expression of p62c-yes in normal, hyperplastic and neoplastic human epidermis

The protein product of the c-yes proto-oncogene, p62c-yes, is highly expressed in a variety of mammalian cell types, including neurons, spermatozoa, platelets, and epithelial cells. In order to understand the function of p62c-yes in epithelial cells, the expression and localization of p62c-yes was studied in cultured human epidermal keratinocytes and in normal, hyperplastic, and neoplastic human epidermis. Human keratinocytes in culture produce a single 4kb c-yes mRNA and a 62kd protein product, p62c-yes, which is active as a protein tyrosine kinase. Using affinity-purified antibodies generated to the amino-terminus of the human c-yes protein, the expression of p62c-yes was localized to keratinocytes in the basal epidermal layer of normal neonatal and adult epidermis. There was a marked reduction in expression of p62c-yes by suprabasal keratinocytes undergoing progressive differentiation. By immunofluorescence microscopy, p62c-yes was localized to the plasma membrane and to a perinuclear cytoplasmic area in cultured keratinocytes. The apparent association of p62c-yes with plasma membranes was particularly evident in suprabasal keratinocytes from hyperplastic epidermis. Neoplastic keratinocytes in basal cell carcinomas showed a marked reduction in p62c-yes expression compared to normal basal keratinocytes in epidermis or to proliferating cultured keratinocytes. Thus the expression of p62c-yes by one epithelial cell type, the keratinocyte, is altered by cellular differentiation and neoplastic transformation. Keratinocytes provide a normal epithelial cell model in which the biochemical function of p62c-yes can be studied.     

Proteomics-based signature for human benign prostate hyperplasia and prostate adenocarcinoma

Prostate adenocarcinoma often presents at a late stage, due to a lack of early clinical symptoms and lack of accurate objective markers. This study aimed to identify and validate proteomics-based biomarkers useful for prostate cancer diagnosis and to establish a marker-panel for prostate cancer and benign prostate hyperplasia (BPH). Global protein expression patterns in fresh tissue specimens from 8 patients with prostate carcinoma and 16 with BPH were analyzed by two-dimensional gel electrophoresis. Differentially expressed proteins were identified by MALDI-TOF mass spectrometry. We compared our results with those of published studies and defined a set of common biomarkers. We identified 22 differentially expressed proteins between BPH and prostate carcinomas. The up-regulated proteins in cancer compared to BPH included protein disulfide-isomerase, 14-3-3-protein, Enoyl CoA-hydrase, prohibitin and B-tubulin β-2. Keratin-II, desmin, HSP71, ATP-synthase-β-chain and creatine kinase-β-chain were down-regulated. Survey of the literature showed that 15 of our 22 identified proteins have been previously reported to differ in their expression levels between BPH and prostate cancer by other laboratories. The expression patterns of these biomarkers could successfully cluster BPH and adenocarcinomas as well as prostate cancer of low and high Gleason scores. This study validates protein-biomarkers that can be useful for accurate diagnosis and prognostic monitoring of prostate adenocarcinoma. Despite varied prevalence of the disease between different ethnic populations (i.e., high in Sweden, low in Saudi Arabia); the biomarkers indicate that BPH and prostate cancers are biologically 'homogeneous' in their protein expression patterns across wide geographical regions.     

Basement membrane components in normal hyperplastic and neoplastic endometrium

The major basement membrane (BM) components, laminin and type IV collagen, were studied by immunochemistry in normal, hyperplastic, and neoplastic endometrium. By immunoperoxidase technique, proliferative and secretive endometrium showed capillary and epithelial cell basement membranes with linear staining with antibodies to both laminin and type IV collagen. Immunostaining of laminin and type IV collagen showed that capillaries were surrounded by a continuous perivascular sheath of these matrices in specimens of adenomatous hyperplasia and in nearly all specimens of endometrial adenocarcinoma. Laminin and type IV collagen were found to accumulate around glandular epithelial cells of adenomatous hyperplastic endometrium, but in several specimens these linear surrounding formations were defective and discontinuous. In several areas of well-differentiated endometrial adenocarcinomas BM-like structures were found around glandular epithelial cells as shadows without staining for laminin and type IV collagen. These basement membrane components accumulate around stromal cells to encircle each cell with a gradual, progressive, and cyclic process depending on the phase of the menstrual cycle. Laminin and type IV collagen were clearly detected around stromal cells at days 20 to 22 of the menstrual cycle and more thickly at days 26 to 28. The accumulation of these matrices around stromal cells is a progesterone/progestin-related process. In the well-differentiated adenocarcinoma a mid-term treatment with progestin (Danatrol Maggioni-Winthrop, SPA, Milan, Italy) was found to be effective on laminin and type IV collagen accumulation around stromal cells.     

Angiogenic activity of hyperplastic and neoplastic lymph nodes

Angiogenic capacity was tested in 14 non-Hodgkin's lymphomas, 7 Hodgkin's lymphomas and 15 cervical lymph nodes nonneoplastic but draining a territory with a laryngeal carcinoma. The objective was to find out whether different groups of lymphomas showed differences in their angiogenic capacity and to compare the ability to induce neovascularization of neoplastic lymphocytes. Frequency and intensity of the angiogenic response were similar for classes of lymphomas different for morphologic and immunologic characteristics. The presence of a carcinoma was sufficient to induce in tributary, nonmetastatic lymph nodes an angiogenic activity comparable to that known to characterize antigenically stimulated lymphocytes.     

Cadmium-induced neoplastic transformation of human prostate epithelial cells

Cadmium is a ubiquitous environmental human carcinogen. Epidemiological and animal studies have suggested its carcinogenic potential on the prostate. In the present study, non-tumorigenic human prostate epithelial cells (pRNS-1-1) immortalized by simian papovavirus (SV40) were transformed after repeated exposures to cadmium. Such transformants showed morphological alterations, anchorage-independent growth in soft agar, and formed tumors when transplanted into SCID mice. The tumors were characterized histologically as poorly-differentiated adenocarcinomas, expressing prostate-specific antigen (PSA), androgen receptor (AR), prostate stem cell antigen (PSCA), NKX3.1 and cytokeratin 8 (CK8). These findings provide evidence of malignant transformation of human prostate epithelial cells exposed to this environmentally important chemical.     

Tyrosine protein kinase activity of human hyperplastic prostate and carcinoma cell lines PC3 and DU145

Using the substrate poly[Glu80Na,Tyr20] [poly(GT)] and the autoradiographic detection of alkali-resistant phosphoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, tyrosine protein kinase (TPK) has been evidenced in human hyperplastic prostates (BPH) and the prostatic carcinoma cell lines PC3 and DU145. The enzyme was mainly found in the soluble fractions from hyperplastic tissues and in Triton extracts from the cell lines. However, its specific activity in tissues was 1.5- to 4.5-fold times higher in particulate than in soluble fractions and it was of the same order of magnitude as that of neoplastic cells. Under these conditions, no activity was detected in human seminal plasma and in sera from normal adult males or patients with BPH and/or prostatic carcinoma. On the other hand, some TPK activity was associated with human spermatozoa, with a specific activity 4- to 6-fold lower than in BPH tissue fractions and a total activity, per 10(6) cells, 5- to 20-fold lower than that in prostatic carcinoma cells. The activity of prostatic TPK was dependent upon the presence of the divalent cations Mn2+ or Mg2+ and it was completely abolished by heat denaturation. Angiotensin II, casein, and histone H2B were poor substrates compared to poly(GT). The TPK activities towards poly(GT) as well as endogenous proteins were not stimulated by epidermal growth factor and insulin or by dihydrotestosterone and estradiol. The autoradiography of alkali-resistant phosphoproteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed several bands in both BPH tissues and neoplastic cells (molecular weight ranging from 17,000 to 200,000). Preliminary characterization of TPK by gel filtration on Sephacryl S-300 showed that the soluble enzyme from BPH tissues had a molecular weight of 50,000, while the particulate-associated TPK, when assayed on poly(GT), eluted with proteins of Mr 210,000. When these peak fractions were used for endogenous phosphorylation, several major alkali-resistant phosphoproteins in the range of Mr 40,000-60,000 were evidenced, together with a Mr 110,000 band phosphorylated by the particulate TPK of Mr 210,000. In similar conditions, the TPK solubilized from rat liver membranes and partially purified by gel filtration was associated with a Mr 170,000 alkali-resistant phosphoprotein. Thus, TPKs are expressed in BPH tissues and carcinoma cell lines. In BPH tissues, two forms of TPK are expressed and the predominant enzyme is soluble and of low molecular weight (Mr 40,000-60,000).