Psma expression in prostate benign prostate hyperplasia and prostate cancer

Prostate-specific membrane antigen has been detected in both benign tissue and prostate cancer and is clinically designated as prostate-specific (2). In the past, various methods have been employed to distinguish BPH from among various grades of prostate cancer. By employing in situ hybridization, Kawakami et al. (51) increased expression of PSMA-specific transcripts in poorly differentiated adenocarcinoma in 15 tumors (from a range of Gleason scores). Similarly, by employing immunohistochemistry analysis with PSMA-specific antibody, Burger et al. (52) confirmed that PSMA expression reflects the Gleason score of the tumor with PSMA expression localized in secretary epithelial cells. Bostwick et al. (53) has shown intense cytoplasmic immune reactivity for PSMA in every prostate tissue examined. The number of immune-reactive cells increased incrementally from benign epithelium to high-grade prostatic intraepithelial neoplasia (PIN) and prostatic adenocarci-

Prostate Cancer Number Scale

noma. The most extensive and intense staining for PSMA was observed in high-grade carcinoma with immune reactivity in virtually every cell in specimen with Gleason primary pattern 4 or 5 (with greater heterogeneity of staining in primary pattern 3 adenocarcinoma). It has been shown by immunohistochemistry analysis by various other groups that prostate epithelial cells in a large majority of BPH tissues did not express PSMA at a detectable level (53). PSMA enzyme activity (NAALADase) was found to be increased 10-fold in prostate cancer samples as compared to normal prostate and BPH (11). Until now, all reports were on PSMA protein expression in tissue sections. The first report of PSMA as the second most highly expressed gene in prostate cancer (by employing more powerful and advance techniques such as gene-array technology) came from the study of Stamey et al. (54). Molecular profiling of Gleason grade 4/5 cancer (which is the primary cause of failure to cure prostate cancer) was done by taking frozen tissues from nine men with Gleason grade 4/5 cancer and eight men with BPH. Labeled complementary RNA from each of the 17 tissues was applied to HuGene FL probe arrays representing approx 6800 genes. After removing all genes undetectable in BPH and grade 4/5 cancers and transforming the data into a parametric distribution, only those upregulated and downregulated genes were chosen with a p difference in fluorescence between grades 4/5 cancer and BPH of p < 0.0005. Further elimination of genes (not expressed in all 8 BPH and 9 grade 4/5

Fig. 3. (Opposite page) PSMA has a MXXXL motif at its N-terminal cytoplasmic tail, and the first methionine and fifth leucine residues are important for its internalization; the fourth leucine is redundant. PSMA has a dileucine motif at its N-terminal cytoplasmic tail. Dileucine motifs serve as lysosomal targeting signal and usually reside within the cytoplasmic tail of the protein (either N-terminal or C-terminal). (A) The peptide sequence N-terminal cytoplasmic sequence is shown here with putative internalization motif (shown in bold). The actual internalization signal of PSMA is the first five amino acids MXXXL. (B) The helical wheel projection of the C-terminal tail region (N-terminal 19 amino acid). By using the predict protein program (http://cubic.bioc.columbia.edu/pre-dict-protein), it was found that the N-terminal region contains an a-helical region. Using this sequence, we could make a helical wheel projection by using the site http://www.site.uottawa.ca/~turcotte/ resources/Helixwheel and found that one face of the helix contains the hydrophobic residues and the other face of the helix contains the hydrophilic residue, indicating that this region is important for protein-protein interaction. (C) Internalization assay of PSMA and its single-leucine and dileucine repeat mutant (L5A and L4AL5A) in PC3 cells. Cover-slip cultures of the PC3 cells expressing PSMA or L5A or L4AL5A have been incubated at 4°C or 37°C with or without J591 for 60 min. The cells were washed and processed for immunofluorescence. The internalized J591 was detected by incubating the cover slips with goat-anti-mouse biotin, followed by streptavidin-FITC; for Lampl detection, the cover slips were incubated with polyclonal antibody against Lampl, followed by detection with goat-anti-rabbit secondary antibody tagged with Texas red. The J591-induced internalization of PSMA and its recycling through endocytic compartment and localization with lysosome has been studied here. Panel A: PC3-PSMA, internalization of J591 at 37°C for 5 min in green; panel B: staining for the lysosomal marker Lamp1 is shown in red; panel C: overlay of A and B showing the colocal-ization of internalized J591 and Lamp1in yellow; panel D: J591-induced internalization of PC3-PSMA at 37°C for 60 min shown in green; panel E: staining for Lamp1 shown in red; panel F: overlay of D and E shown in yellow; panel G: PC3-L4A, internalization assay with J591 at 37°C for 60 min shown in green; panel H: staining for the lysosome marker Lamp1 is shown in red; panel I: overlay of G and H; panel J: PC3-L5A, internalization assay with J591 at 37°C for 60 min shown in green; panel K: staining for Lamp1 is shown in red; panel L: the overlay ofJ and K; panel M: PC3-L4AL5A internalization assay with J591 at 37°C for 60 min shown in green; panel N: staining for Lamp1 is shown in red; panel O: overlay of M and N; panel P: PC3-Ndel19 internalization assay with J591 at 37°C for 60 min shown in green; panel Q: staining for lysosome marker Lamp1 is shown in red; panel R: overlay of P and Q. (See Color Plate 12 following p. 302.)

Table 2

Summary of Markers Identified by Array Analysis

Fold Increase

Table 2

Summary of Markers Identified by Array Analysis

Fold Increase

Genbank

in Expression

Marker

Reference no.

Gene Function

Detected by Arrays*

5-Catenin

U96136

Cell-cell adhesion molecule

9.5

PSMA

M99487

Prostate-specific membrane antigen

4-6

NEK3

Z29067

Serine/threonine protein kinase

5.5

CCK4

U33635

Receptor tyrosine kinase

4.5

TPSP

X87852

Transmembrane sex protein receptor

4

PCTK3

X66362

Serine/threonine protein kinase

4

EGR1

X52541

Early growth response protein

3.5

1-transcription factor

PLA2

M22430

Phospholipase A2 group IIA-inflammatory

Data from ref. 52.

response regulator

Data from ref. 52.

tissues) produced a final set of 86 genes, of which 22 were upregulated and 64 were down-regulated. The most upregulated gene found to be Hepsin (a trypsin-like serine protease) and the second most upregulated gene was found to be PSMA (54). Later, Burger et al. (52) have shown (by using techniques such as cDNA microarray profiling and real-time reverse transcription-polymerase chain [RT-PCR]) that PSMA is one of the very few markers that could discriminate prostate cancer from BPH, in which cancer cells showed a fourfold to sixfold increase in expression from BPH as detected by arrays (Table 2). There was no overlap between the 17 prostate cancers (of different stages and grades) and 11 BPH samples studied. However in both groups, there were considerable variations among individual samples. Similarly, Zhou et al. (55) have shown that 5-catenin (a cell-cell adhesion molecule) was found to be significantly overexpressed in tumors compared to BPH (9.5-fold increase in expression detected by arrays).

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