Mohammad S. Saedi

A precursor form of PSA (pPSA) is a component of the free PSA in prostate cancer serum

OBJECTIVES Prostate-specific antigen (PSA) is a widely used serum marker for human prostate cancer (PCa). The majority of PSA in serum is present as a complex with alpha-1-antichymotrypsin (ACT). In recent years, the ratio of free (uncomplexed) to total PSA has shown improved discrimination of PCa from benign prostatic hyperplasia. This study examines the nature of the free PSA from detected in PCa serum and shows that some of the uncomplexed PSA is an inactive precursor of PSA (pPSA). METHODS Western blot analysis was used to detect clipped, fragment forms of PSA in sera and seminal fluid. Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) was used to identify forms of PSA present in the free PSA population. Pooled sera was passed over a PSA immunoaffinity column, and the eluted PSA components were further resolved by HIC-HPLC. RESULTS Western blot analysis of whole sera showed complexed PSA and the intact, approximately 34 kilodalton free PSA. Only negligible levels of clipped or degraded forms of PSA, as found in seminal fluid, were detected. Column fractions measured for uncomplexed PSA using the Tandem-MP free PSA assay showed that about 25% of the free PSA eluted as pPSA beginning at the [-4]amino acid. Studies with purified recombinant [-4]pPSA showed that this proenzyme form is inactive and does not complex with ACT. CONCLUSIONS These results suggest that the uncomplexed PSA in PCa serum is primarily unclipped PSA that contains a significant fraction of pPSA.

Detection of a prostate-specific protein, human glandular kallikrein (hK2), in sera of patients with elevated prostate-specific antigen levels

OBJECTIVES Messenger ribonucleic acid for human glandular kallikrein (hK2), a protein similar to prostate-specific antigen (PSA), is expressed in the prostate. Quantitative tests for the relative amounts of PSA in serum have become important in the diagnosis and management of patients with prostate cancer. Measurement of hK2 in serum may also serve as a diagnostic indicator of disease. The object of this study was to determine if hK2 is present in the serum of patients with high serum concentrations of PSA. METHODS Recombinant prohK2 with an alanine to valine mutation at aa217 (phK2v217) was expressed in a hamster tumor cell line, AV12. The propeptide was treated with trypsin to yield the mature form of hK2 (hK2v217). Using a monoclonal antibody, HK1G586.1, which recognized wild type and mutant forms of pro- and mature hK2, an hK2-specific radioimmunoassay was developed. RESULTS PSA cross-reactivity in the radioimmunoassay (RIA) was 0.23%. hK2 was detected in the sera of 51 of 76 patients with PSA levels above 100 ng/mL. The dose-response curve of hK2-positive samples was linear, and recovery of phK2v217-spiked serum samples was close to 100%. The correlation between PSA and hK2 values in the patient sera was low (r = 0.168). CONCLUSIONS Given the importance of the role of PSA as a serologic indicator of prostate cancer, the demonstration that hK2 is also circulating in the blood of patients in different relative proportions to PSA suggests that it may be a significant novel marker for prostate cancer.

Overexpression of a human prostate-specific glandular kallikrein, hK2, in E. coli and generation of antibodies

The genomic and the cDNA clones of human glandular kallikrein (hK2), a member of the kallikrein family, have been isolated; however, the hK2 protein has not yet been identified and characterized. The deduced sequence of hK2 is highly homologous to prostate specific antigen (PSA), a widely accepted prognostic indicator of prostate carcinoma. Also, hK2 mRNA, like PSA mRNA, is exclusively expressed in prostatic epithelia. These two properties make hK2 a potentially useful marker for studying prostate cancer. In this paper, we describe for the first time the overexpression of the entire hK2 protein (pre-pro hK2:pphK2) in the E. coli system. Our system yields high levels of authentic pphK2 (as determined by partial amino acid sequence analysis) comprising about 40% of total cellular protein. pphK2 was purified to near homogeneity by preparative SDS/PAGE and used to generate anti-pphK2 antibodies in rabbits. The antibodies recognize the recombinant hK2 protein and a major band of approximately 34 kDa in seminal fluid.

Human kallikrein 2 (hK2), but not prostate‐specific antigen (PSA), rapidly complexes with protease inhibitor 6 (PI‐6) released from prostate carcinoma cells

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.