Analysis of glucosinolates from broccoli and other cruciferous vegetables by hydrophilic interaction liquid chromatography.

While methods for the identification and quantification of total plant glucosinolate content typically utilize desulfation of glucosinolates followed by reversed-phase chromatography, the analysis of intact glucosinolates has been problematic. Hydrophilic interaction chromatography offers a novel method for analyzing intact glucosinolates and when performed along with ion-pair reversed-phase chromatography offers a powerful and complementary method for glucosinolate analysis.

Location of prostate-specific membrane antigen in the LNCaP prostate carcinoma cell line.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a novel prostate biomarker overexpressed in poorly differentiated and metastatic prostate carcinomas and apparently upregulated following hormone-ablation therapy. PSMA appears to be a satisfactory target for antibody-directed imaging of prostate carcinomas despite the recent finding that the antigenic epitope recognized by monoclonal antibody (MAb) 7E11-C5 is found in the cytoplasmic domain of this transmembrane glycoprotein [Troyer et al.: Urol Oncol 1:29-37, 1995]. This finding prompted the present investigation to precisely define the cellular location of PSMA in the LNCaP prostate carcinoma cell line, the line used to generate MAb 7E11-C5. METHODS: Subcellular fractionation, immunofluorescence and immunoperoxidase staining of live and fixed cells, and immunoelectron microscopy were used to determine the localization of PSMA in LNCaP cells. RESULTS: PSMA was found to be localized at the inner face of the plasma membrane as well as being associated with mitochondria. Staining of LNCaP cells, treated by serum starvation followed by serum stimulation, showed no changes in the typical cytoplasmic staining pattern. CONCLUSIONS: The data suggest that the PSMA target epitope for antibody-directed imaging with MAb 7E11-C5 only becomes accessible upon apoptosis or necrosis. This further suggests that antibodies directed at the extracellular domain may enhance the sensitivity of antibody-directed imaging and therapy of prostate carcinomas by recognizing surface epitopes of PSMA on living cancer cells.

Detection and characterization of the prostate-specific membrane antigen (PSMA) in tissue extracts and body fluids.

The prostate-specific membrane antigen (PSMA) glycoprotein is recognized by the monoclonal antibody (MAb) 7E11-C5.3 as a predominant 100 kDa and minor 180 kDa component in LNCaP cell line extracts and its expression has been shown by immunohistochemistry to be highly restricted to prostate epithelium. The aim of the present study was to utilize Western blot analysis to determine if PSMA could be detected in human tissue extracts and body fluids and if so, which molecular forms were present. PSMA was detected as 120 and 200 kDa bands in normal, benign and malignant prostate tissues and seminal plasma. Further analysis demonstrated that the larger molecular form of PSMA may be a dimer of the lower m.w. species. The PSMA glycoprotein was not detected in the majority of non-prostate tissue extracts examined except for a low yet significant amount in normal salivary gland, brain and small intestine, suggesting that PSMA may not be as prostate-specific as originally thought. Since the prostate-specific antigen (PSA) has been shown to be maximally shed into the serum in high-grade and metastatic prostate carcinomas, it was surprising that PSMA could not be detected in serum by Western blot analysis even in patients with actively progressive metastatic disease. Second generation antibodies generated against different epitopes may be required to determine if PSMA is shed into serum. Our results support the hypothesis that PSMA is a novel prostate biomarker.

Biochemical characterization and mapping of the 7E11-C5.3 epitope of the prostate-specific membrane antigen.

The expression of the prostate-specific membrane antigen (PSMA) glycoprotein recognized by the murine monoclonal antibody (MAb) 7E11-C5.3 has been shown to be highly restricted to prostate epithelium. Although the conjugated form of this MAb (CYT-356) may soon be used clinically for in vivo imaging of extraprostatic disease, few details regarding the nature of the antigenic epitope of PSMA have been reported. This study was carried out to analyze the MAb 7E11-C5.3 epitope on PSMA using standard biochemical techniques, and the antigenic epitope was mapped with synthetic peptides. The MAb 7E11-C5.3 epitope was susceptible to both periodic acid oxidation and proteolytic digestion, which indicated that the antigen consisted of a glycoprotein. However, additional biochemical assays such as sodium borohydride, tunicamycin treatment, and digestion with glycosidases failed to abrogate MAb 7E11-C5.3 binding. Epitope mapping with synthetic peptides demonstrated the epitope to be localized to the intracellular domain at the N-terminus of the PSMA molecule with a minimal reactive peptide consisting of six amino acids (MWNLLH). The synthetic peptides were treated with periodic acid, which resulted in inhibition of antibody binding, suggesting that treatment of the PSMA antigen resulted in damage to the peptide chain. These data suggest that the MAb 7E11-C5.3 does not recognize a glycopeptide as was initially thought, but recognizes an intracellular epitope consisting of only the primary polypeptide chain. Further studies are needed to determine how CYT-356 is able to image tumors in vivo when the antigenic epitope is intracellular.