Characterization of intracellular prostate-specific antigen from laser capture microdissected benign and malignant prostatic epithelium

The proportion of unbound serum prostate-specific antigen (PSA; percent-fre e PSA) is reported to be lower in men with prostate cancer compared to men with benign prostates (U. H. Stenman et al,, Cancer Res., 51: 222-226, 1991 ; H, Lilja et at, Clin, Chem,, 37: 1618-1625, 1991; D. L. Woodrum ed at, J. Urol., 159: 5-12, 1998; W. J. Catalona et al., J. Am. Med. Assoc., 279: 15 42-1547, 1998), The majority of immunoreactive PSA in serum is complexed to alpha-1-antichymotrypsin (ACT), Two major mechanistic questions have previ ously been unknown: (a) Does PSA in human prostate cancer cells in tissue e xist in a free or bound form? and (b) Is PSA produced by malignant cells in the free form because it has lost the ability to form a complex with ACT? Laser capture microdissection (LCM) enables the acquisition of pure populat ions of defined cell types from tissue (M. R. Emmert-Buck et al., Science, 274: 998-1001, 1996; R. F. Bonner et al,, Science, 278: 1481-1483, 1997), T his technology provides a unique opportunity to study intracellular protein composition and structure from human cells, In this study, we used LCM to assess the bound versus free form of intracellular PSA in both benign and m alignant epithelium procured from prostate tissue, One-dimensional and two-dimensional PAGE were performed on cellular lysates from LCM-procured benign and malignant prostate epithelium from frozen tis sue specimens. Western blotting analysis of one-dimensional PAGE gels revea led a strong band at nl, 30,000 (expected molecular weight of unbound PSA) in ail cases demonstrating that the vast majority of intracellular tumor an d normal PSA exists within cells in the "free" form. Binding studies showed that PSA recovered from LCM-procured cells retained the full ability to bi nd ACT, and two-dimensional PAGE Western analysis demonstrated that the PSA /ACT complex was stable under strong reducing conditions. We conclude that intracellular PSA exists in the "free" form and that binding to ACT occurs exclusively outside of the cell.