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Keywords:

  • prostate;
  • NEP;
  • in situ RT-PCR;
  • gene expression;
  • immunofluorescence

Abstract

BACKGROUND

Neutral endopeptidase (NEP/CD10) is a cell surface zinc metalloproteinase that functions as part of a regulatory loop controlling local concentrations of peptide substrates and associated peptide-mediated signal transduction processes. In contrast to the encouraging data dealing with NEP activity and regulation in prostate epithelial cells, only a few studies are available on the cellular expression and localization of neutral endopeptidase in the prostatic stromal and cancer cells. Here, we describe the cellular localization of NEP in human prostatic tissue and cells using in situ RT-PCR as a novel molecular biological approach.

METHODS

Immunofluorescence and Western blot experiments were performed to control the expression and distribution of the NEP in normal and malignant human prostatic tissues and cell lines. NEP gene expression was monitored by RT-PCR, NEP mRNA was detected in paraffin tissue sections and cultured cells of human prostate by the highly sensitive method of one step-in situ reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

NEP mRNA was detected in human prostatic tissue and in cultured cells by means of in situ RT-PCR. Prostatic tissue showed strong signals in the glandular epithelium and weak signals in the stroma, cultured cells displayed strong signals in prostate cancer cells (LNCaP) and weak signals in stromal cells (hPCPs). Western blot experiments were performed using whole cell extracts to proof the presence of NEP protein in LNCaP and hPCPs. The experiments confirm the expression of NEP by both cell types, however, the experiment with hPCPs cells showed two bands. NEP-immunofluorescence was strong in normal prostatic epithelium and confined to the apical plasma membrane. In dedifferentiated prostate cancer specimens, immunofluorescence of apical plasma membranes was lost, and both the cytoplasm and portions of the plasma membrane were immunoreactive for NEP. Prostate cancer cells (LNCaP) showed a strong immunoreaction of the plasma membrane and the cytoplasm. In comparison with LNCaP cells, only a weak cytoplasmic immunofluorescence was found in some stromal cells (hPCPs).

CONCLUSIONS

In normal prostatic tissue and specimens derived from human prostate cancer, NEP mRNA and protein are expressed mainly by the epithelial cells and to a minor extent by the stromal cells of human prostate glands. In situ RT-PCR is a powerful and straightforward approach for the routine and rapid detection of cellular specific expression of low copy genes. © 2003 Wiley-Liss, Inc.