Volume 44, Issue 4, Pages 415-422 (October 2003) Expression and Immunolocalisation of Neutral Endopeptidase in Prostate Cancer  Martin Albrecht, Anja Mittler, Beate Wilhelm, Åke Lundwall, Hans Lilja, Gerhard Aumüller, Anders Bjartell  European Urology  Volume 44, Issue 4, Pages 415-422 (October 2003) DOI: 10.1016/S0302-2838(03)00322-1

Fig. 1 Immunohistochemistry showing the distribution of NEP in the normal prostate (A), BPH (B), PC Gleason grade 2 (C), 3 (D), 4 (E) and 5 (F). In the normal prostate and tissue from BPH, NEP is confined to the apical plasma membrane of the epithelial cells (A and B). In specimens derived from low grade PC of Gleason grade 2 some acini lack NEP, whereas others show a strong NEP staining inside the lumen and in the apical plasma membrane (C). In PC of Gleason grade 3 an additional intracytoplasmic localisation of NEP can be detected (D). In high grade prostate tumours (Gleason 4 and 5) NEP is distributed heterogeneously throughout the tissue with an extracellular, intracytoplasmic and partly plasma membrane bound localisation (E and F). Magnification: A, C, D, E ×200; B ×50; F ×300. European Urology 2003 44, 415-422DOI: (10.1016/S0302-2838(03)00322-1)

Fig. 2 Confocal laser scanning microscopy showing the localisation of NEP (green) and beta-actin (red) in the BPH prostate (A) and in high grade PC (B). An apical localisation of NEP and an apical, basal and lateral distribution of actin is found in glandular cells of BPH tissue (A). Combination of the green (NEP) and red (actin) fluorescence signals reveals a distinct colocalisation of actin and NEP in BPH tissue (A, merge). This distribution pattern is lost in PC of Gleason grade 5 (B). While some of the actin is still confined to the terminal web and the region beneath the lateral and basal plasma membrane, NEP shows a granular distribution throughout the cytoplasm. Merging the red and the green channel, approves the loss of actin/NEP colocalisation (B, merge). Magnification: A, B ×200; B lower panel ×800. European Urology 2003 44, 415-422DOI: (10.1016/S0302-2838(03)00322-1)

Fig. 3 Dual immunohistochemical staining of PC tissue for the proliferation associated antigen Ki-67 (brown cells) and NEP (blue cells). Ki-67 is mainly located in regions with a low or no expression of NEP (A). Vice versa, PC areas with strong NEP expression (B) display low Ki-67 reactivity and hence a diminished cell proliferation. Magnification: ×200; A, B ×400. European Urology 2003 44, 415-422DOI: (10.1016/S0302-2838(03)00322-1)

Fig. 4 NEP gene expression in BPH (A) and PC (B) tissue. Using in situ hybridisation studies a strong NEP mRNA expression can be detected in glandular epithelial cells of BPH (A) and high grade PC tissue (B). Only weak signals are confined to the stromal cells surrounding the acini in BPH and PC (A and B). Magnification: ×200. European Urology 2003 44, 415-422DOI: (10.1016/S0302-2838(03)00322-1)

Fig. 5 Expression of NEP protein in BPH tissue and PC tissue derived from 8 different patients undergoing androgen ablation therapy. Western blotting experiments were performed with 30μg protein extracts per lane. Tissue homogenates of BPH specimens contain large amounts of NEP, whereas a low varying expression of the protein is detected in tissue derived from different PC patients. To prove that approximately same amounts of protein were separated on each lane Western blotting experiment using anti-actin antibodies were performed in parallel (lower panel). Due to the missing intracytoplasmic domain [30], the NEP positive control appears somewhat lower on the gel. European Urology 2003 44, 415-422DOI: (10.1016/S0302-2838(03)00322-1)