Introduction: Pro or precursor forms of prostate-specific antigen (PSA) have emerged as potentially important diagnostic serum markers for prostate cancer detection. Immunoassays were developed to measure specific proPSA forms containing propeptides of 2, 4, and 7 amino acids [(-2)proPSA, (-4)proPSA, and (-7)proPSA, respectively]. Methods: Research-use dual monoclonal antibody immunoassays using europium-labeled detection monoclonal antibodies were developed for each form of proPSA. Sera from patients with prostate cancer or benign prostate disease containing 4 -10 g/L PSA were assayed and analyzed by area under the ROC curve (AUC) for specificity and sensitivity. Results: The proPSA forms had quantification limits of 0.015-0.025 g/L in serum, with cross-reactivities <1% with PSA. The sum of the proPSA forms divided by free PSA (percentage proPSA) had a higher AUC than did percentage of (-2)proPSA, free PSA, and complexed PSA with AUC (95% confidence intervals) of 0.69 (0.64 -0.74), 0.64 (0.58 -0.68), 0.63 (0.58 -0.68), and 0.57 (0.51-0.62), respectively. The proPSA comprised a median of 33% of the free PSA in cancer and 25% in noncancer sera (P <0.0001). One-third (33%) of cancer samples had >40% proPSA, whereas only 8% of noncancer samples did (P <0.0001). In men with cancer and >25% free PSA, the (-2)proPSA had an AUC of 0.77 (0.66 -0.86), with 90% sensitivity and 36% specificity at 0.04 g/L.

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Prostate-specific human kallikrein, hK2, is a serine protease found in prostate tissues that has 78 % amino acid sequence identity with prostate-specific antigen (PSA). We have previously reported the affinity purification of hK2 heterologously expressed in a hamster cell line and demonstrated an argininerestricted substrate specificity. Here, we describe the cloning, expression, purification, and enzymatic activity of a mutant form of hK2 containing an alanine to valine substitution at residue 217 ([Va1217] [Va1217]hK2 also showed altered specificity on a synthetic peptide substrate compared to wild-type hK2, which exhibited partial hydrolysis at a PSA chymotrypsin-like cleavage site as well as the trypsin-like site cleaved by hK2. These results indicate that Ala217 is a key residue affecting the catalytic properties of hK2.Keywords : human glandular kallikrein ; prostate cancer ; prostate-specific antigen ; serine protease.Prostate-specific human kallikrein, hK2, is one of three members of the human glandular kallikrein family [I, 21. The other two kallikreins consist of prostate-specific antigen (PSA), which has the nomenclature hK3, and the salivary/pancreatic/ renal kallikrein, designated hK1 131. All three kallikreins are serine proteases, but have different substrate specificities. hK2 has the highest similarity to PSA, a widely used marker for prostate cancer [2, 4, 51. Evidence for the similarity of hK2 with PSA includes the following: (a) a 78% amino acid sequence identity with PSA [6, 71; (b) androgen regulation [ l , 81; (c) prostate localization [9, 101 ; (d) natural expression in LNCaP cells, a human prostate cancer cell line [ I l l . These similarities with PSA make hK2 an important target for investigation since hK2 could potentially improve the diagnostic value of PSA in the detection of prostate cancer. Immunohistochemical staining using hK2-specific monoclonal antibodies has shown hK2 to be highly expressed in prostate adenocarcinoma compared to benign hyperplastic or normal tissues [12, 131. This observation suggests that hK2 could play a significant role in cancer biology and increases the interest in understanding hK2 enzymatic function.In our previous report, we described the recombinant expression of hK2 from AV12 cells [14]. We have demonstrated that hK2 hydrolyzes synthetic peptides exclusively at the C-terminus of selected arginine residues and that hK2 activity can be measured spectrophotometrically with the chromogenic substrate S-The general finding of trypsin-like activity for hK2 had been predicted by molecular modeling studies due to the presence of aspartic acid at residue 189 in the substrate specificity pocket for hK2, compared to a serine at the same position for the chymotrypsin-like PSA [6, 7, 161. Residue 217 has been proposed by molecular modeling studies to be in the substrate-binding pocket for hK2 and several homologous kallikreins [6, 71. It has not been generally recognized as a critical residue, for instance, in the rat kallikrein family [ l , 171. In this stu...

Human kallikrein 2 (hK2) is a secreted, trypsin-like protease that shares 80% amino acid sequence identity with prostate-specific antigen (PSA). hK2 has been shown to be a serum marker for prostate cancer and may also play a role in cancer progression and metastasis. We have previously identified a novel complex between human kallikrein 2 (hK2) and protease inhibitor 6 (PI-6) in prostate cancer tissue. PI-6 is an intracellular serine protease inhibitor with both antitrypsin and antichymotrypsin activity. In the current study we have shown that PI-6 forms a rapid in vitro complex with hK2 but does not complex with PSA. Recombinant mammalian cells expressing both hK2 and PI-6 showed hK2-PI-6 complex in the spent media only after cell death and lysis. Similarly, LNCaP cells expressing endogenous hK2 and PI-6 showed extracellular hK2-PI-6 complex formation concurrently with cell death. Immunostaining of prostate cancer tissues with PI-6 monoclonal antibodies showed a marked preferential staining pattern in cancerous epithelial cells compared with noncancerous tissue. These results indicate that the hK2-PI-6 complex may be a naturally occurring marker of tissue damage and necrosis associated with neoplasia. Both hK2 and PI-6 were shed into the lumen of prostate cancer glands as granular material that appeared to be cellular necrotic debris. The differential staining pattern of PI6 in tissues suggests a complex regulation of PI-6 expression that may play a role in other aspects of neoplastic progression.

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