title: Production of milligram concentrations of free prostate specific antigen (fPSA) from LNCaP cell culture: difference between fPSA from LNCaP cell and seminal plasma.
abstract: We have established a procedure for the production of milligrams of free PSA (fPSA) from LNCaP cells derived from a human carcinoma of the prostate. By growing LNCaP cells in a serum-free medium in the presence of a synthetic androgen (R1881) and taking advantage of the special design of the Micro-mouse Hollow Fiber Bioreactor, relatively pure fPSA could be obtained. We found that columns containing either Sephacryl S-100 or S-200 could be used to remove the small amount of bovine serum albumin (BSA) and PSA-alpha 1-antichymotrypsin complex (PSA-ACT) from the preparation. More than 90% of the PSA from LNCaP cell cultures are fPSA. Like fPSA from seminal plasma, two fractions of fPSA differing in protease activity can be separated by DEAE-Sepharose chromatography. Based on the band pattern exhibited on the Western blot following sodium dodecyl sulfate-polyacrylamide electrophoresis separation, fPSA from LNCaP contains more inactive PSA isoforms. This was confirmed by chromatofocusing: the isoelectric point (pl) of the major PSA isoforms from the LNCaP cell culture were higher (6.8 and 6.6) than that (6.4 and 6.1) of fPSA from seminal fluid. We conclude that the LNCaP cell culture is a reliable source for obtaining large quantities of pure fPSA both for the preparation of assay calibrators and controls and for studying the difference in fPSA between benign prostate disease and prostate cancer.
authors.affiliation: Department of Pathology, University of Utah Health Science Complex, Salt Lake City, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Wu
authors.forename: B W
authors.initials: BW
authors.lastname: Lyons
authors.forename: G H
authors.initials: GH
authors.lastname: Liu
authors.forename: L L
authors.initials: LL
authors.lastname: Wu
mesh_terms: D000818:Animals; D019149:Bioreactors; D015153:Blotting, Western; D002847:Chromatography, Agarose; D003470:Culture Media; D006801:Humans; D008297:Male; D051379:Mice; D017430:Prostate-Specific Antigen / Q000096:biosynthesis* / Q000302:isolation & purification; D011471:Prostatic Neoplasms / Q000378:metabolism*; D012661:Semen / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D000514:alpha 1-Antichymotrypsin / Q000378:metabolism
chemical_list: D003470:Culture Media; D000514:alpha 1-Antichymotrypsin; D017430:Prostate-Specific Antigen