Identification of metastatic cell nucleus in human prostate cancer by electron microscopy.

AIM: Metastatic prostate cancer is responsible for a large proportion of deaths worldwide. The aim of this study was to identify metastatic cells and determine if stromal invasion by cancer cells differs from those during metastasis. METHODS & RESULTS: Tissue biopsy/prostatectomy samples, visualized by transmission electron microscopy, identified that metastatic cells are a lineage of stem cells, which have dedifferentiated into cancerous columnar/cuboidal cells. These cells demonstrate nuclear plasticity; the loss of nuclear membranes and boundary between nucleus and cytoplasm; and the presence of electron dense molecules, which can readily pass through basement membranes and enter the capillary, ready for dissemination to metastatic sites. CONCLUSION: This is the first study to demonstrate differences between invasive and metastatic cell types.

Electron Microscopic Analysis of Stem Cells in Human Prostate Cancer, Including Inverted Capsule Embedding Methods for Archival Sections and Falcon Films for Prostate Cancer Cell Lines.

BACKGROUND/AIM: Identification of prostatic stem cells in primary prostate tissue sections, organ cultures of prostate and cell lines requires a range of techniques that allows characterization of stem cells for their potential use in the treatment of patients. Isolated cells usually round-up and develop changes in shape, size and cellular characteristics. The aim of this study was to provide a range of methods for identifying prostatic stem cells and characterizing them with regard to ultrastructure, nuclear morphology, cytoplasmic organelles, and/or expression stem cell marker CD133. MATERIALS AND METHODS: Prostate biopsy and prostatectomy specimens were used for studying prostatic stem cells and their known marker CD133 in tissue sections by light and/or electron microscopy. Inverted capsule embedding was used to study archival metastatic prostate in pelvic nodes and Du145 cell line in a monolayer culture. RESULTS: Staining for CD133 positively identified stem cells that were found in benign prostatic hyperplasia, benign prostate, and prostate cancer cells. Paraffin embedded sections showed a single type of stem cells, whereas methylene blue-stained Epon sections showed both light and dark stem cells. Electron microscopy showed that both basal and stem cells were closely associated with the basement membrane (basal lamina). Stem cells had smooth plasma and nuclear membranes, a prominent nucleolus, small mitochondria, and few ribosomes. Du145 cells were separated by intercellular spaces in monolayer culture. CONCLUSION: The inverted capsule embedding method allowed the study of metastasized prostate cancer in pelvic lymph nodes. Our approach enabled the assessment of stem cells in tissue sections by light and electron microscopy.

Identification of Two Types of Stem Cells in Methylene Blue-stained Sections of Untreated and Diethylstilbestrol-treated Human Prostate Cancer and Their Characterization by Immunogold Localization of CD133.

BACKGROUND: The basal compartment of the prostatic acinus harbors stem and basal cells, whereas the luminal compartment contains cuboidal and columnar cells. Mutation in the genes of stem cells is required for benign (normal) prostate to develop into prostatic adenocarcinoma. Stem/basal cells survive androgen deprivation therapy in humans and castrated mice to repopulate glandular cells by proliferation when stimulated by androgen. We hypothesized that using different embedding and staining methods, it would be possible to identify two types of stem cells in human prostate by localization of CD133. MATERIALS AND METHODS: Prostate biopsy or prostatectomy pieces from 13 untreated and eight diethylstilbestrol-treated men with prostate cancer were sectioned, stained by methylene blue and CD133 was localized by immunogold technique. RESULTS: Methylene blue stained basic proteins in dark basal cells, but not in light cells. Light basal cells expressed androgen receptors and dark cells estrogen receptors. Light and dark cells expressed CD133, indicating them to be stem cells. Light stem cells produced the lineage of columnar/cuboidal cells. Estrogen-dependent dark cells produced a lineage of columnar/cuboidal cells, that also expressed estrogen receptors. CONCLUSION: Our analysis indicates that stem/basal cells are privileged cells in the basal compartment. Stem cells are not under the regulation of steroid hormones, whereas their lineage of cuboidal/columnar cells are. The lineage of androgen-dependent cells are columnar/cuboidal cells and the lineage of estrogen-dependent cells are also columnar/cuboidal cells. Epon-embedding and methylene blue staining showed two types of CD133-positive stem cells in prostate. Paraffin sections did not show two types of stem cells in prostate and bone marrow leukemia cells. Our study indicates the continuity of embryonic stem cells into adult prostate as organ-specific stem cells. To our knowledge, this is the first study to identify two types of stem cells in human prostate.

Matrix metalloproteinase and heparin-stimulated serine proteinase activities in post-prostate massage urine of men with prostate cancer.

Proteinases secreted by the prostate gland have a reproductive function in cleaving proteins in the ejaculate and in the female reproductive tract, but some may have a fundamental role in disease and pathological processes including cancer. The purpose of this study was to determine if there were differences in proteinase activities in urine samples collected following prostate massage of men positive (CaP) or negative (no evidence of malignancy, NEM) for biopsy determined prostate cancer. Matrix metalloproteinase (MMP) and serine proteinase activities were detected using protein substrate zymography. There were no differences in activities of MMP-2, proMMP-9, and MMP-9/NGAL (neutrophil gelatinase associated lipocalin) complex (gelatin substrate) in men with detected prostate cancer, although the latter two were somewhat diminished. A caseinolytic activity of about 75kDa inhibited by calcium did not differ between the NEM and CaP groups. Heparin stimulated calcium sensitive gelatinolytic activities of approximately 22, 42, and 60kDa, but did not affect activities of MMP-2, MMP-9, or the 75kDa caseinolytic activity. The 22, 42, and 60kDa activities appear to be serine proteinases since they were inhibited by benzamidine. There was a significant decrease in the 22kDa heparin-stimulated serine proteinase activity in urines of men with cancer. Proteinase expression and activities, perhaps in combination with other potential markers, may prove useful in urine for detection and evaluation of prostate cancer.

Concurrent Androgen and Estrogen Ablation and Inhibition of Steroid Biosynthetic Enzyme Treatment for Castration-resistant Prostate Cancer.

BACKGROUND/AIM: About 80 to 90% of prostate cancer (PCa) is androgen-dependent at diagnosis, but patients ultimately develop castration-resistant prostate cancer (CRPC), which is usually not amenable to androgen deprivation (ablation) therapy (ADT). Patients with CRPC usually succumb to death in less than 5 years and there is no cure. Here, we investigated reasons for ADT failure. MATERIALS AND METHODS: Biopsy specimens from untreated and diethylstilbestrol (DES)-treated patients were assessed for localization of antibody IgGs against androgen (AR) and estrogen (ER) receptors. RESULTS: In untreated and DES-treated sections, methylene blue stained basic proteins in dark basal (undifferentiated) PCa cells, whereas light basal cells were not stained. AR localized to light basal cells which showed widespread degeneration in sections from DES-treated patients, indicating their dependence on androgen. In contrast, dark basal cells did not show widespread degeneration in DES-treated patients; ER was usually localized in dark cells. The number of dark cells progressively increased in DES-treated patients indicating their androgen-independence. The localization of AR and ER in some light and dark basal cells indicated that the supply of androgen/estrogen was not inhibited during ADT. Dark basal cells had emerged prior to treatment and proliferated during DES treatment, that also indicated their androgen-independence. CONCLUSION: PCa has at least two populations of cells: androgen-dependent light basal and estrogen-dependent dark basal cells. ADT did not destroy estrogen-dependent cells which may have given rise to CRPC tumors. Therefore, ADT is an incomplete treatment. For a more complete treatment of PCa, we recommend concurrent androgen and estrogen ablation, together with the inhibition of selected steroid biosynthetic enzymes.

Induction of a heparin-stimulated serine proteinase in sex accessory gland tumors of the Lobund-Wistar rat.

Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10-14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5-3.0µg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26-30kDa Ca(2+)-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not leak into or is inactivated in the blood. The role of this proteinase remains to be determined, but its possible interaction with extracellular glycosaminoglycans could focus its proteolytic activity in the tumor microenvironment and affect tumor growth.

Prediction of immune surveillance responsive metastatic prostate cancer in pelvic lymph node and emergence of surveillance unresponsive/resistant metastatic cells.

BACKGROUND: Immune cells (lymphocytes and macrophages) provide the microenvironment for immune surveillance of metastatic prostate cancer (PCa) cells in pelvic lymph nodes. We have hypothesized that degeneration and/or apoptosis of metastatic PCa cells in pelvic lymph nodes can distinguish between aggressive and non-aggressive metastatic disease in patients. Our objective was to define the relationship between metastatic cell lysis and the presence of immune cells. MATERIALS AND METHODS: We studied archival primary PCa (n=38) and cancer-positive regional pelvic nodes (n=32) from the same patients undergoing radical retropubic prostatectomy at the Minneapolis Veterans Affairs Medical Center. RESULTS: Using morphological and immunohistochemical features of immune and metastatic cancer cells, we have identified progression of metastasis in the nodal compartments. Nodal parenchyma contained small, intermediate and large metastatic nodules/tumors. Immune surveillance occurred primarily in small tumors and surveillance was either absent or greatly reduced in intermediate and large tumors in nodes. Metastatic nodules/cells were lysed or became apoptotic when under immune-surveillance, as indicated by pyknotic nuclei and cytoplasm, the latter still had remnants of prostate specific antigen (PSA) staining. Metastatic cells without surveillance did not exhibit morphological features of cell degeneration (lysis) or apoptosis. Metastatic cells under immune-surveillance had an inverse relationship with those without immune-surveillance. This relationship differed from node to node and patient to patient. CONCLUSION: We have shown that at least two populations of metastatic cells were present in the nodes; the first group of cells was under immune surveillance, as indicated by limited to wide-spread cell lysis/apoptosis, and the second group did not exhibit morphological evidence of cell lysis indicating emergence of surveillance-unresponsive (resistant) metastatic cells. These criteria can be used to distinguish metastatic cancer that is expected to be responsive to immunotherapy from that which would show little or no benefit from such treatment. Enhancement of immune surveillance and other treatments can be used to treat surveillance-unresponsive (resistant) disease to improve survival of patients.

Characterization of prostate cancer in needle biopsy by cathepsin B, cell proliferation and DNA ploidy.

BACKGROUND: Our objective was to determine localization patterns of three distinct groups of biomarkers (cathepsin B, MIB-1 and DNA ploidy) in prostate needle biopsy sections to establish localization similarities (or differences) in biopsy and retropubic prostatectomy specimens (RPs). MATERIALS AND METHODS: Prostate needle biopsy specimens and matched RPs from 47 patients with cancer were evaluated. Biopsy and RP sections were stained with anti-cathepsin B (CB) and anti-stefin (cystatin) A (SA) and for cell proliferation and DNA ploidy. The ratio of CB to SA in stained cells was calculated for each biopsy cancer and matched benign prostatic hyperplasia (BPH) sample. RESULTS: The geometric mean of CB to SA was 1.45 in BPH and 2.99 in cancer specimens (p=0.0001). The percentage of S-phase cells and DNA ploidy status in needle biopsy was associated with cancer volume in RP cases (p=0.03). CONCLUSION: Our study has indicated that the ratio of CB to SA is significantly higher in prostate cancer biopsy specimens than in BPH. The percentage of S-phase cells and DNA ploidy in needle biopsies predicts cancer volume of RPs. We have shown that localization of three distinct biomarkers in biopsies reliably assesses the nature of prostate cancer in biopsy sections.

Patterns, art, and context: Donald Floyd Gleason and the development of the Gleason grading system.

The Gleason grading system is a standard method of assessing prostate cancer. Very little is known, however, about the person behind the system, Donald Floyd Gleason, MD, PhD. Our objective was to construct a biography of Gleason and show how his work advanced prostate cancer diagnosis. We reviewed Gleason's notes, letters, and publications with the Veteran's Administration Cooperative Urology Research group (VACURG) between 1960 and 1980. Gleason described seeing five recurring histologic "pictures" in his review of 280 initial cases of prostate cancer from 1960 to 1964. By 1966, NIH statisticians combined Gleason's "pictures" with VACURG clinical data into a scoring system. By 1974, over 4,000 cases had been analyzed, and by 1978 the Gleason scale had reached widespread use. Donald F. Gleason recognized trends in histologic patterns which had eluded earlier, more sophisticated pathologic approaches and which lie as the basis for the sophisticated neural networks and nomograms in use today.

Increased aggressiveness of human prostate PC-3 tumor cells expressing cell surface localized membrane type-1 matrix metalloproteinase (MT1-MMP).

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a multidomain transmembrane endopeptidase with a major role in physiological and pathological processes through proteolysis of extracellular matrix and other pericellular proteins. We examined cell surface function of MT1-MMP in PC-3 human prostate tumor cells selected for metastasis in nude mice (PC-3-LN4), or transfected with the full-length wild-type (WT) MT1-MMP or with the mutant form lacking the cytoplasmic tail (Delta C-MT1-MMP). Enhanced cell surface MT1-MMP was determined by fluorescence-activated cell sorting analysis and evidenced mechanistically by increased activation of proMMP-2 and invasion into type-I collagen gels. PC-3 cells overexpressing MT1-MMP grew faster than mock-transfected control cells subcutaneously in nude mice. MT1-MMP localized in caveolae, as judged by immunofluorescence microscopy and sucrose-gradient, detergent-resistant cell fractionation. Delta C-MT1-MMP was strongly associated with caveolae, whereas the WT form was present in both caveolae and noncaveolae fractions. The role of plasma membrane MT1-MMP was supported by localization of MT1-MMP by immunofluorescence microscopy at the cell surface of tumor cells in primary prostate cancers. Increased plasma membrane localization of MT1-MMP, either in caveolae or in other lipid raft structures, is a mechanism to localize this proteinase in contact with extracellular matrix and other pericellular proteins, the cleavage of which can facilitate prostate cancer cell invasion and metastasis.

Cathepsin B expression in prostate cancer of native Japanese and Japanese-American patients: an immunohistochemical study.

BACKGROUND: Japanese-American (J-A) men who have immigrated to the U.S.A. and acquired the Western lifestyle usually have more invasive prostate cancer (PCa) than native Japanese (NJ) living in Japan. The specific reasons for these differences remain unknown. The objective of this study was to examine immunostainings of cathepsin B (CB) and its endogenous inhibitor stefin A (SA) in tissue microarray (TMA) and radical prostatectomy (RP) tissue sections in the hope of obtaining insights into the invasiveness of PCa in Japanese patients. PATIENTS AND METHODS: TMA and RP sections were evaluated in 50 men (25 NJ and 25 J-A) for CB and SA reaction products. The CB and SA immunostainings were imaged directly from microscope slides to a computer using a high performance charge coupled device (CCD) digital camera, quantified using Metamorph software, analyzed using the two-sample t-test, and confirmed by multiple regression analysis. RESULTS: The CB and SA proteins were localized in the carcinomatous glands and isolated cancer cells in the TMA and RP sections. The Gleason scores and pre-surgery serum total prostate-specific antigen (PSA) levels did not differ significantly in the NJ and J-A patients (p = 0.14, p = 0.16, respectively). The Chi-square analysis of clinical stage versus place of birth showed that the NJ patients had significantly more T2a and T2b clinical stages than the J-A patients who had more advanced T2c and T3a stages (p = 0.003). The CB and SA immunostainings and their ratios in Gleason score 6 tumors did not show any difference, but the CB:SA ratios in score > or = 7 tumors approached significance levels. CONCLUSION: The overall matching of specimens according to the Gleason grade/score, pre-RP serum total PSA levels, clinical stage and age prior to evaluation of immunostainings greatly minimizes subjectivity associated with the evaluation of markers in this ethnic sub-population of PCa patients. CB and SA immunostaining is similar in Japanese patients who have organ-confined and moderately-differentiated PCa. Analysis of the reaction product data provides indirect evidence that invasiveness of PCa is similar in the two Japanese patient populations.

The effect of dietary supplementation with limonene or myo-inositol on the induction of neoplasia and matrix metalloproteinase and plasminogen activator activities in accessory sex organs of male Lobund-Wistar rats.

Prostate cancer, the most prevalent non-cutaneous cancer in men, is associated with increased age. This suggests that dietary chemopreventive measures could be effective in delaying the onset or decreasing the severity of the disease. We utilized the Lobund-Wistar rat nitrosomethylurea induced, testosterone promoted (NMU-T) model of male sex accessory gland cancer to test the potential chemopreventive effects of myo-inositol and limonene on tumor incidence and associated protease activities. Tumors were found to arise in the seminal vesicles and dorsal and anterior prostate lobes. There were also some tumors that appeared to arise in both the seminal vesicles and anterior prostate, and in some cases the tissue of origin was not clear. The distribution of tumors as to site of origin in limonene or myo-inositol treated animals did not vary from that of the starch fed control animals, and the number of animals presenting with metastases did not vary significantly between treatment groups. There was a statistically significant delay in onset of tumors in myo-inositol, but not limonene fed rats, at 10 months post-induction of carcinogenesis; however, at 12 and 15 months this was not significant. The ventral prostate and seminal vesicles expressed pro-MMP-2 and plasminogen activator (PA) activities. Based on sensitivity to amiloride, the PA activities were predominately urokinase (uPA) in the ventral prostate and a mixture of tissue-type activator (tPA) and uPA in the seminal vesicles of non-treated rats. Sex accessory gland tumors, and metastases, expressed increased levels PA and pro- and active forms of MMP-2 and -9. The PA activities of the tumors were a mixture of uPA and tPA. There was no difference in the levels of these protease activities based on the tissue of tumor origin, nor in tumor vs metastasis. These studies indicate that MMP and PA activities play a role in sex accessory gland tumor biology and that dietary supplementation with myo-inositol can delay but not ultimately prevent the development of such tumors.

Cathepsin B expression is similar in African-American and Caucasian prostate cancer patients.

BACKGROUND: Increased incidence and mortality of prostate cancer (PCa) suggest that U.S. African-American men have more invasive cancer than Caucasian men. Invasive PCa requires several proteases, including the cysteine protease cathepsin B (CB), for degradation of basement membrane and extracellular matrix proteins prior to cancer cell migration across biological compartments. Our objective was to determine whether CB immunostaining patterns, in relation to clinical data, could distinguish invasive PCa in African-American and Caucasian patients. PATIENTS AND METHODS: Fifty Gleason score 6/7 PCa cases were selected for similar clinical data with benign prostatic hyperplasia (BPH) samples as controls. Immunostainings were imaged directly from microscope slides to a computer using a digital camera. Data were quantified using Metamorph software, analyzed using the two-sample t-test and confirmed by multiple regression. RESULTS: Ratios of CB to its endogenous inhibitor stefin A (SA) immunostainings were greater in PCa than BPH, but were not significantly different in PCa of either race. The African-American patients did not show increased CB immunostaining, indicating that the contribution of this protease to invasiveness was similar in both races. CONCLUSION: When veterans received equal medical care at the Minneapolis Veterans Affairs Medical Center, African-American patients did not show increased PCa invasiveness. Our conclusion is supported by analysis of post-surgery serum total PSA levels and cancer cell invasion to margins/capsules, seminal vesicles and/or lymph nodes. Invasiveness of PCa does not appear to be race-dependent. The previous conclusion of race-based differences in PCa requires re-evaluation with respect to the role of proteases (such as CB, matrix metalloproteinase) in invasion and metastasis of cancer cells.

Heterogeneity of cathepsin B and stefin A expression in Gleason pattern 3+3 (score 6) prostate cancer needle biopsies.

BACKGROUND: There is a significant positive association of increased ratios of cathepsin B to its endogenous inhibitor stefin (cystatin) A in prostatectomy tumors with pelvic lymph node metastases. Needle biopsy diagnosis of prostate cancer is critical in initial treatment selection. The objective was to characterize cathepsin B and stefin A immunostaining patterns in needle biopsies of histologically similar Gleason pattern 3+3 (score 6) foci in relation to pretreatment clinical data. MATERIALS AND METHODS: Immunostaining of cathepsin B and stefin A of 65 biopsy sections were imaged, quantified and analyzed with Student's t-test (p < 0.05). RESULTS: Patients had T1c to T3b clinical stages and pre-surgery total prostate-specific antigen serum levels from 1.25 to 20.0 ng/ml. Cathepsin B and stefin A reaction products were found in the cytoplasm of basal and columnar/cuboidal cells of benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and neoplastic cells. Ratios of cathepsin B to stefin A were significantly higher in prostate cancer when compared to that in BPH and PIN glands. CONCLUSION: Small foci of Gleason pattern 3+3 tumors in needle biopsies have heterogeneous cathepsin B and stefin A immunostaining. Stratification of these tumors in relation to clinical stage by cathepsin B and stefin A may assist in treatment selection.

Elevation of dipeptidylpeptidase iv activities in the prostate peripheral zone and prostatic secretions of men with prostate cancer: possible prostate cancer disease marker.

PURPOSE: Dipeptidylpeptidase IV (DPIV) is a multifunctional type II plasma membrane glycoprotein with serine-type exopeptidase activity that is secreted by the prostate and increased in prostate cancer. We determined whether changes in DPIV activities in prostatic tissue zones and expressed secretions were associated with the presence of cancer. MATERIALS AND METHODS: Expressed prostatic secretion (EPS), and biopsy of the transition (TZ) and peripheral (PZ) zones were collected from men undergoing ultrasound guided prostate biopsy. DPIV activities were measured by glypro-p-nitroanalide hydrolysis. RESULTS: DPIV activities were significantly higher in TZ than in PZ tissues in men with no evidence of malignancy. However, activities in EPS were negatively associated with TZ volume and positively associated with PZ volume. Mean and median DPIV activities in EPS from men with biopsy determined cancer were significantly higher than in men with no evidence of malignancy. DPIV activities in TZ and PZ biopsies were higher in men with cancer but most markedly in the PZ. CONCLUSIONS: These data indicate that secreted DPIV originates from the TZ and PZ. Increased DPIV activities in cancer are strongly associated with the PZ, which is the zone most commonly involved with cancer. Measuring DPIV levels in expressed EPS or post-digital rectal prostate examination urine may be useful for evaluating men for prostate cancer.

Microvessel density as a molecular marker for identifying high-grade prostatic intraepithelial neoplasia precursors to prostate cancer.

BACKGROUND: Existing clinical data have shown that high-grade prostatic intraepithelial neoplasia (HGPIN) is the most likely precursor to prostate cancer (CaP). Criteria to distinguish HGPIN that progress to CaP from those that do not remain poorly defined. Our objective was to evaluate microvessel density as a molecular marker for distinguishing HGPINs that have the potential of progressing to cancer. MATERIALS AND METHODS: Human prostatic tissue samples were collected randomly from 50 prostatectomy and cystoprostatectomy patients. Formalin-fixed and paraffin-embedded sections were used for immunohistochemical localization of rabbit anti-human von Willebrand factor VIII (vWF) IgG, mouse anti-high molecular weight cytokeratin 34BE-12 in basal cells, and mouse anti-heparan sulphate proteoglycan (HSPG) IgGs in basement membranes associated with benign prostatic hyperplasia (BPH), PIN associated with some BPH (isolated PIN), and PIN associated with CaP. RESULTS: Analysis of immunostaining data showed that PINs could be categorized according to their distributions within and outside 2 standard deviations (SD) of the mean for microvessel density. The average number of microvessels was significantly higher (P < 0.0001) in PINs associated with Gleason score 7 tumors than those associated with Gleason scores 4-6 (P < 0.1328) or 8 and 9 tumors (P < 0.1708). Morphologically, PINs within 2 SD were composed of low- and high-grade type, whereas those outside 2 SD of microvessel density were predominantly of high-grade type. Cytokeratin and HSPG localization patterns also showed differences in PINs found within and outside 2 SD of microvessel density. We found localization of cytokeratin 34BE-12 in basal cells of specimens with BPH alone, isolated PIN, and PIN associated with CaP within 2 SD, whereas many PINs outside 2 SD showed disruptions in cytokeratin localization. The basement membranes of PINs within 2 SD of microvessel density were relatively intact, whereas those outside 2 SD were fragmented. CONCLUSIONS: Our immunostaining data indicates that once HGPIN is found in the initial prostatic biopsy, it should be evaluated for microvessel density by localization of vWF. Our data indicate that characteristics of HGPIN can be augmented by evaluations of cytokeratin and HSPG molecular markers to assess the potential of HGPIN progression to malignancy. When biopsy samples show HGPIN with increased microvessel density and disrupted cytokeratin and HSPG markers, the patient may be a candidate for repeat biopsy. Since our study is limited to 50 prostate tissue samples, we emphasize that our conclusion is tentative and ought to be confirmed in a study with a larger sample size. This is the first report to show that microvessel density may distinguish HGPIN that is a precursor to prostate cancer.

Matrix metalloproteinases in the pathogenesis of estradiol-induced nonbacterial prostatitis in the lateral prostate lobe of the Wistar rat.

Chronic nonbacterial prostatitis develops spontaneously with age in the lateral lobe of the prostate in some strains of rat. Our objective was to examine the role of matrix metalloproteinases (MMP) in the pathogenesis of chronic nonbacterial prostatitis using a chronic estrogen treatment, Wistar rat model (Prostate 12 (1988) 271). Male Wistar rats, 90 days of age (8 rats/group), were castrated and groups were implanted 8 days later with 1 cm silastic tubings containing estradiol 17 beta (E2). Some animals received 5-cm silastic tubings of dihydrotestosterone (DHT) or testosterone (T) on day 22 and all untreated control and experimental animals were sacrificed on day 36 of the protocol. MMP activities were determined by SDS-gelatin-, casein-, and carboxymethyl transferrin-polyacrylamide gel zymography. A light/mild interstitial monocytic infiltration was found in the ventral lobes, but not other lobes, of half of the untreated control rats. This ventral lobe interstitial inflammation was not affected by E2 treatment. A prominent to heavy inflammation, including both intraluminal neutrophil and interstitial monocytic infiltrates, was produced by E2 treatment at a 100% incidence in the lateral lobes. Prominent MMP activities were detected in the lateral lobes of E2-treated rats, including both the active (55 and 81 kDa) and proenzyme (72 and 92 kDa) forms of MMP-2 and MMP-9, respectively. These activities were strongly attenuated by treatment of E2-implanted animals with T, which also reduced inflammation; but they were only weakly affected by DHT given with E2, which did not reduce inflammation. Similarly, DHT treatment of E2-implanted castrated rats restored the wet weight of the lateral lobe, but it did not fully restore secretion volume production, whereas T treatment of estrogenized rats increased lateral lobe wet weight and secretion volume above that of untreated controls. E2 treatment also induced an activity in casein gels of about 27 kDa with properties of MMP-7; that is, molecular mass, inhibition by EDTA, stimulation by heparin sulfate in casein and carboxymethylated transferrin gels. A high molecular weight nonmetalloproteinase activity (>160 kDa) was detected in gelatin gels in the lateral prostate lobe of both treated and untreated control animals. In comparison to the lateral lobe, E2 treatment produced only minimal effects on MMP activities in the ventral and dorsal prostatic lobes. Thus, elevated MMP-2, MMP-7, and MMP-9 activities in lateral lobe prostatitis correlate with leukocyte infiltration in the inflammatory response. These proteinases may help mediate the accompanying epithelial atrophy and tissue damage in this organ.

Limited processing of pro-matrix metalloprotease-2 (gelatinase A) overexpressed by transfection in PC-3 human prostate tumor cells: association with restricted cell surface localization of membrane-type matrix metalloproteinase-1.

The expression and activation of matrix metalloproteinases (MMPs) by tumor cells is correlated with progression to invasive and metastatic status. The purpose of this study was to examine the role of increased MMP-2 (gelatinase A) expression in prostate cancer progression utilizing human prostate PC-3 cancer cells that overexpress MMP-2 using gene transfection. PC-3 cells were transfected with pCR-3 vector only and pCR-3 MMP-2 plasmids employing the LipofectAMINE method, and stable transfectants were selected with G418. The expression of MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane-type MMP 1 (MT1-MMP) in PC-3 parental and transfected cells under serum-free conditions was determined by zymography, immunoblotting, immunofluorescent microscopy, Northern blotting, and/or reverse transcriptase-polymerase chain reaction (RT-PCR). MMP-2 transfected cells produced primarily the proenzyme form of MMP-2; the parental and vector control transfected PC-3 cells did not express any MMP-2 that was detectable by the methods we employed. Treatment of PC-3 MMP-2 transfected cells with Concanavalin A (Con A), in contrast to HT-1080 cells, processed only a small amount of the secreted 72-kd proenzyme to a 62-kd intermediate and a cell-associated 59-kd active form. The low level of secreted pro-MMP-2 processing induced by Con A was inhibited by serine protease inhibitors and was unaffected by cyclic adenosine monophosphate (cAMP). Immunoblotting showed that these cells produced abundant TIMP-2 and lower amounts of MT1-MMP in comparison with Con A-responding HT-1080 cells. HT-1080 cells respond to Con A by translocating MT1-MMP from intracellular localization sites to the plasma membrane, an effect not observed in PC-3 cells. The molecular basis for the low level of processing of pro-MMP-2 by PC-3 cells may be due to an overabundance of TIMP-2 and/or a low level of cell surface active MT1-MMP.

Effect of vitamin E deficiency on the growth and secretory function of the rat prostatic complex.

Increased intake of vitamin E has been suggested to be protective against prostate cancer in men, but the effects of vitamin E on prostate growth and function remain poorly defined. The purpose of this study was to determine the effects of vitamin E deficiency on pubertal growth and maturation of the prostate in the rat. Animals were placed on a vitamin E deficient diet at 28 days of age and were followed for 15 and 26 weeks. Vitamin E deficient rats had a circulating vitamin E level of less than 1% of control animals and experienced a decrease in body and testis weight. The deficiency did not alter the weights of the ventral and dorsal lobes of the prostate. However, there was an increase in weight, DNA, and protein contents of the lateral lobe in control and vitamin E deficient rats from 15 to 26 weeks of treatment, but these increases were significantly lower in vitamin E deficient 26-week treated rats. The volume of secretion per milligram tissue was greater in the ventral than lateral or dorsal lobes. The volume of secretion and activity of the secretory 26 kDa protease in the ventral prostate was lower in vitamin E deficient rats at 15 weeks, but not at 26 weeks of treatment. In contrast, the relative protein content of lateral lobe secretion increased in both control and vitamin E deficient rats from 15 to 26 weeks of treatment. The lateral, but not ventral or dorsal, lobes of both control and vitamin E deficient rats were affected by chronic prostatitis as evidenced by infiltration of inflammatory cells. The lateral lobes also showed markedly elevated activities of the matrix metalloproteinases gelatinase A (MMP-2) and gelatinase B (MMP-9). These data indicate that vitamin E deficiency does not alter the growth of the prostatic lobes, nor the onset and extent of lateral lobe specific prostatitis, but it may delay some differentiated functions such as secretion of specific proteins in the ventral lobe. Thus, the effects of vitamin E in the prostate of the rat appear to be selective.