Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
Received on May 13, 1999; revised on July 26, 1999; accepted on July 27, 1999.
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Abstract |
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Key words: carbohydrate/metastasis/oligosaccharides/prostate specific antigen/tumor marker
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Introduction |
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Prostate specific antigen (PSA), a glycoprotein secreted by the prostatic epithelium has been demonstrated to be clinically important for the detection and monitoring of prostate cancer (Hudson et al., 1989). It is believed that PSA has the highest validity of any circulating cancer screening marker discovered thus far. Elevated serum concentration has become a common tool for detecting early stage prostate cancer and monitoring therapy of this disease (Staney et al., 1987
; Catalona et al., 1991
). However, the use of this screening method remains controversial.
Since oncogenesis is often correlated with a change in carbohydrate structure (Fukuda, 1985; Dennis and Lafarté, 1989
), a number of carbohydrate markers have been used for clinical characterisation of human carcinomas (Hakomori, 1991a
,b). In the current study, we sought to determine if the carbohydrate moeity on normal PSA is distinguishable from the carbohydrate moiety of PSA derived from cancerous prostatic epithelium. As an initial effort we investigated the PSA derived from a cell line (LnCaP) which was established from the lymph node of a patient with metastatic prostate carcinoma (Horoszewicz et al., 1980
). We report here that the carbohydrate derived from the metastatic cell line (LnCaP) is different from that derived from normal PSA. Further investigations will reveal whether these differences are useful for clinical diagnosis.
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Results |
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Discussion |
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Our data using the ANTS labeled (GLYKO FACE) analysis of the carbohydrate moiety of the normal PSA (Figures 2, 3) is in agreement with the previously determined structure of the carbohydrate moiety of PSA. Upon treatment with neuraminidase, a majority of the carbohydrate appears to be fucosylated biantennary oligosaccharide. In comparison, carbohydrates derived from the PSA of a metastatic prostate carcinoma cell line is a mixture of biantennary, triantennary, and possibly tetraantennary oligosaccharides. It is possible that oncogenic transformation of the prostate epithelium may differentially affect the N-linked glycan processing of the prostate specific antigen.
Other studies have shown that oncogenic transformation of cells can profoundly affect the processing of some glycosyalation sitesyielding higher levels of tri- and tetraantennary oligosaccharides (Feizi, 1985; Fernandés et al., 1991
; Matsumoto et al., 1992
). However, the regulation of this process of oligosaccharide modification is not completely understood. It has been noted that oncogenic transformation may not only selectively affect N-linked glycan processing of different glycoproteins but may actually result in selective glycosylation of different sites on a single polypeptide. In one particular study, it was shown that transformation associated increase in complex N-linked oligosaccharide branching synthesis in hamster fibroblasts transformed by hamster sarcoma virus was shown to selectively affect processing at one of the two VSV glycosylation sites, Asn 336, but not at Asn 179 (Hubbard, 1987
). This study as well as other studies have shown that the activity of ß1,6-N-acetylglucosaminyl transferase (GnT-V) which catalyzes the synthesis of elongated ß1-6-N-linked oligosaccharide branches correlates in many instances with the metastatic potential of cells (Dennis et al., 1987
, 1989). Thus, artificial introduction and expression of the GnT-III gene into B16 mouse melanoma cells decreased production of ß1-6 branches by experimental models of lung metastatic potential in experimental models of lung metastasis (Yoshimura et al., 1995
).
Development of accurate markers are needed not only in early detection, but also in predicting response to chemoprevention and chemotherapy, or other forms of therapy. Further investigations will be required to determine whether the carbohydrate profile of PSA can be correlated with tumor grade, metastatic potential, and whether it can be used for prognosis of human prostate cancer, and finally for evaluation of preventive or therapeutic strategies.
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Materials and methods |
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Purification of PSA by immunoprecipitation
PSA was purified by use of anti-PSA antibody linked gel. A polyclonal rabbit anti human PSA antibody (AXL 685, Accurate Chemical & Scientific Corp.) was cross linked to Protein G Sepharose using an Immunopure crosslinking kit (Pierce, Rockford, IL). Before crosslinking, protein G Sepharose was equilibrated with Immunopure binding buffer and then mixed with anti PSA IgG at a concentration of 34 mg IgG/ml of gel. The solution was mixed by gentle inversion at room temperature. After 3060 min, gel was washed with wash buffer and the antibody was bound using a solution of DMP (dimethyl-pimelimidate·2HCl) for 12 h at room temperature; the remaining active sites were blocked using immunopure blocking buffer. Unbound IgG was eluted with glycine-HCl (pH 2.5), gel was washed and then stored in PBS containing 0.02% NaN3. For immunoprecipitation, approximately 1520 ml of medium containing PSA derived from hormone stimulated LnCap cells was incubated with 0.30.4 ml of washed anti-PSA bound gel. After incubation at room temperature for 3060 min, unbound fraction was withdrawn and the gel was washed 34x with PBS. Bound PSA was eluted in a batchwise procedure using an equal volume of 100 mM acetic acid. Resulting fractions (3 or 4) were collected and concentrated using a Speed Vac. Concentrated fractions were then resolved by SDS-PAGE for carbohydrate analysis. In some cases, the fractions eluted were placed in tubes containing 50 µl of Tris-HCl (pH 8.5). These fractions were used for estimating concentrations of recovered PSA.
SDS polyacrylamide gel electrophoresis
Samples were subjected to electrophoresis through 10% separating, 5% stacking precast gels (Bio-Rad, Richmond, CA) under reducing conditions according to the method of Laemmli (Laemmli, 1970). Gels were stained for protein by a silver stain using a Bio-Rad kit. Transfer to PVDF (Immobilon membranes, Bio-Rad) was as described previously (Towbin et al., 1979
). Transfers were done for 1.52 h at a constant voltage of 8590 V at 4°C. The PVDF membranes were stained with Ponceau S (Bio-Rad; 0.1% v/v in 1% acetic acid) destained with 1% acetic acid and then washed with water. The appropriate bands were excised and either stored at 70°C or used immediately for releasing oligosaccharides.
Preparation and analysis of oligosaccharides from PSA
PSA bands were excised from the stained membranes and placed in tubes containing 35 µl of sodium phosphate buffer (50 mM, pH 7.7) and 0.5 µl of SDS (5%). Samples were denatured for 5 min at 100°C and cooled on ice; 2.7 µl of 7.5% NP40 was added to each tube, followed by 4 µl of PNGase F (New England Biolabs, Beverly, MA). Samples were incubated at 37°C for 2 h; control samples were treated as above without the addition of PNGase F. For treatment with NaNases III (Glyko), samples were acidified by addition of 12 µl of acetic acid; ~2 µl of the enzyme (Glyko) was added subsequently. The samples were incubated for a duration of 2 h at 37°C, and the digests were dried in a Speed-Vac. The released oligosaccharides were labeled for 18 h at 37°C using reagents from Glyko with fluorophore, 8 aminonapthalene1,3,6 trisulfonate (ANTS), by reductive deamination (Friedman and Higgins, 1995; Starr et al., 1996
). Once the oligosaccharide were labeled with ANTS they were separated by polyacrylamide gel electrophoresis using precast polyacrylamide gels (Glyko). The ANTS labeled dextran and other oligosaccharide standards as well as running and loading buffers for oligosaccharide PAGE were obtained from Glyko.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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