Characterization and processing of prostate specific antigen (hk3) and human glandular kallikrein (hK2) secreted by LNCaP cells

Prostate specific antigen (PSA, hK3) in serum is predominantly complexed to alpha-1-antichymotrypsin (ACT), but a minor fraction remains in a free for m despite the very large excess of serine protease inhibitors and alpha-2-m acroglobulin. The fraction of free to total PSA is significantly lower in p rostate cancer (CaP) compared to benign prostatic hyperplasia (BPH) which p rovides improved discrimination of these conditions. The molecular nature o f free PSA in the circulation and the reason for its varying concentration in malignant and benign conditions is currently not known. The objective of the present investigation was to study the secretion of PSA and human glan dular kallikrein 2 (hK2) by the LNCaP prostate cancer cell line, and to pur ify and characterize both proteins. LNCaP PSA was thoroughly characterized by immunological characterization, S DS-PAGE, isoelectric focusing, gel filtration, aminoterminal sequencing, re verse-phase chromatography, mass spectrometry and enzymatic activity measur ements. LNCAP cells produced approximately equal amounts of zymogen (proPSA) and th e one-chain mature form of PSA, whereas there was no evidence for the secre tion of any internally cleaved forms. LNCaP cells secreted hK2 into the gro wth medium at about 3-5% of the amount of PSA. One-chain, mature PSA and hK 2 obtained when LNCaP cells were grown in the presence of fetal bovine seru m, had no enzymatic activity, but were active when the cells were grown! in the absence of serum. Using enzymatically active recombinant hK2, it was p ossible to activate proPSA secreted by LNCaP cells. ProPSA formed two bands with high isoelectric points (8.2 and 8.4), which disappeared when proPSA was converted to mature PSA with hK2. Cancerous cells produce the zymogen forms of PSA, which by their isoelectri c pi points seem to be found in serum of prostate cancer patients, but not BPH patients. Mature, one-chain PSA is inactive in the presence of serum. T hese findings may be highly relevant for the understanding of the generatio n of free and complexed PSA in the circulation.