Glandular Kallikrein Research Articles

Expression of a kallikrein-like protein in prostatic intraepithelial neoplasia in ventral prostate of the noble rat

BACKGROUND In an effort to identify biomarker(s) for prostatic cancer (PCa), we analyzed the changes of secretory proteins in the ventral prostate (VP) of Noble rats at early stages of carcinogenesis. METHODS Ventral prostates were removed from both control (n = 36) and experimental (n = 88) rats implanted with a known ratio of testosterone (T) and 17β-estradiol (E2). Tissue sections were stained by hematoxylin and eosin (H&E) for pathological screening, and secretions were collected for SDS-PAGE analysis followed by N-terminal microsequencing, antiserum production, Western blot, and immunohistochemical study. RESULTS Pathologically, low-grade prostatic intraepithelial neoplasia (LGPIN) and high-grade PIN (HGPIN) were observed in ducts or alveoli after 3 and 5 months of T + E2 treatment, respectively. The results of SDS-PAGE showed an elevated expression of 18-kDa protein (p18) in secretions of VP with HGPIN or cancerous lesions. Analysis of p18 by N-terminal sequencing showed a high score of homology to rat glandular kallikrein. To characterize the expression pattern of the protein in tissue samples, an antiserum was raised against the N-terminus of the p18. The monospecificity of the antiserum against p18 was confirmed by Western blot analysis. Immunohistochemical study showed that in ducts or alveoli of normal and LGPIN samples, a mild positive staining for p18 was observed in secretions. However, the reactivity was intense not only in luminal secretions but also in some luminal secretory cells in HGPIN and cancer cells as well. CONCLUSIONS The high expression of p18 in connection with neoplastic transformation of cells strongly suggests that the potential application of this protein as a marker for early detection of PCa should be further investigated. Prostate 42:8–17, 2000. © 2000 Wiley-Liss, Inc.

Time-resolved fluorescence imaging for quantitative histochemistry using lanthanide chelates in nanoparticles and conjugated to monoclonal antibodies.

Tissue and cell examinations have a potential to produce extremely valuable information about antigen quantities in samples. Using currently available methods, a truly quantitative analysis is nearly impossible. We have previously shown that immunohistochemical (IHC) detection of prostate-specific antigen and human glandular kallikrein from prostatic tissue, together with time-resolved fluorescence imaging (TRFI), is a suitable method for obtaining quantitative data from biological samples and that the signal response is linear. In this paper we show that Eu-chelate containing particles in the nanometer range are suitable labels for quantitative IHC. Even single nanoparticle molecules can be detected by TRFI and the signals measured can be readily quantitated. The signal intensity correlates very well with the amount of bound label, and the use of nanoparticles could markedly improve the sensitivity of quantitative IHC methods. TRFI provides a powerful tool for providing quantitative data about antigens or transcripts in tissue sections or cultured cells. It is also of major importance in standardization and optimization of protocols for fixation and tissue preparation, including antigen retrieval methods.

Plasminogen-independent initiation of the pro-urokinase activation cascade in vivo. Activation of pro-urokinase by glandular kallikrein (mGK-6) in plasminogen-deficient mice.

The plasminogen activation (PA) system is involved in the degradation of fibrin and various extracellular matrix proteins, taking part in a number of physiological and pathological tissue remodeling processes including cancer invasion. This system is organized as a classical proteolytic cascade, and as for other cascade systems, understanding the physiological initiation mechanism is of central importance. The attempts to identify initiation routes for activation of the proform of the key enzyme urokinase-type plasminogen activator (pro-uPA) in vivo have been hampered by the strong activator potency of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes Dev. 9, 794-807], we have now demonstrated and identified a component capable of initiating the cascade by activating pro-uPA. The urine from Plg -/- mice contained active two-chain uPA as well as a proteinase capable of activating exogenously added pro-uPA. The active component was purified and identified by mass spectrometry-based peptide mapping as mouse glandular kallikrein mGK-6 (true tissue kallikrein). The pro-uPA converting activity of the mGK-6 enzyme, as well as its ability to cleave a synthetic substrate for glandular kallikrein, was inhibited by the serine proteinase inhibitor leupeptin but not by other serine proteinase inhibitors such as aprotinin, antithrombin III, or alpha(1)-antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other locations.

Production and activation of recombinant hK2 with propeptide mutations resulting in high expression levels.

Human glandular kallikrein 2 (hK2) is a serine protease expressed mainly by the prostate gland with 80% identity in primary structure to prostate specific antigen (PSA). hK2 has proven to be a useful marker of prostate cancer which can be used in combination with PSA to better discriminate between prostate cancer and benign prostate hyperplasia. The studies on hK2 have been hampered by its very low phyciological levels (6 microgram.mL-1), its close similarity to PSA, and the low expression levels obtained using recombinant procedures to produce hK2 (0.7 mg.L-1). We have now generated propeptide mutations of hK2 which can be used to isolate stable, inactive prohK2 mutants. Compared with wild-type hK2, expression of the propeptide hK2 mutants increases the expression levels up to 15-40-fold giving 10-30 mg hK2.L-1. These results indicate that the low expression levels of wild-type hK2 are related to the activation or autoactivation of the wild-type enzyme and the instability of the active protease in cell culture and possibly also in tissue. The purified mutant hK2 may be activated by either enterokinase or factor Xa to generate an enzyme for use in functional studies with the characteristics of the original wild-type protein. Further, the stable inactive mutant hK2 protein may be used for immunizations to generate novel monoclonal antibodies, used as standard material for clinical assays or in crystallization studies where large quantities of protein are required.

Cleavage of porcine high molecular weight kininogen by the action of porcine pancreatic kallikrein

Pancreatic kallikrein (KK), converts the single chain high molecular weight kininogen (HK) into a two-chain molecule accompanied by kinin release. Further degradation was not observed. Molecular weights of the heavy (H)-chain and light (L)-chain were estimated to be 61 kDa and 56 kDa, respectively, by SDS-PAGE. The amino (N)-terminal sequences of intact HK, reduced and carboxymethylated (RCM)-H-chain, and RCM-L-chain were determined. These results indicate that porcine pancreatic KK initially cleaved the Arg–Ser bond of HK, to form a two chain molecule. Then pancreatic KK cleaved the Met–Lys bond yielding kallidin and a kinin-free protein, which was apparently equal in size to the nicked kininogen.

SIGNIFICANCE AND METABOLISM OF COMPLEXED AND NONCOMPLEXED PROSTATE SPECIFIC ANTIGEN FORMS, AND HUMAN GLANDULAR KALLIKREIN 2 IN CLINICALLY LOCALIZED PROSTATE CANCER BEFORE AND AFTER RADICAL PROSTATECTOMY

We studied plasma concentrations and elimination rates of prostate specific antigen (PSA) complexed to alpha1-antichymotrypsin and alpha2-macroglobulin, free PSA, total PSA (free PSA plus PSA alpha1-antichymotrypsin) and human glandular kallikrein 2 before, during and after radical retropubic prostatectomy for clinically localized prostate cancer. Plasma was collected and frozen within 10 minutes after sampling from 18 patients undergoing radical retropubic prostatectomy for prostate cancer. One sample was drawn preoperatively. Subsequent sampling intervals were 5 to 20 minutes perioperatively, 2 to 4 hours during the first 12 postoperative hours and 24 to 48 hours until postoperative day 14. Free PSA, PSA alpha1-antichymotrypsin, total PSA, PSA alpha2-macroglobulin and human glandular kallikrein 2 were measured with time resolved immunofluorometric assays. Preoperatively PSA alpha2-macroglobulin was undetectable (less than 2 ng./ml.) in 17 of 18 patients. Human glandular kallikrein 2, free PSA and total PSA but not PSA alpha1-antichymotrypsin were significantly higher in patients with extraprostatic cancer (pT3a-pT4a, pN1) compared to those with organ confined cancer (pT2a/b). Surgical manipulation of the prostate caused no detectable elevation of human glandular kallikrein 2, PSA alpha1-antichymotrypsin or PSA alpha2-macroglobulin. In contrast, a mean 9.6-fold increase (range 3.4 to 22) in free PSA was noted 5 minutes after prostatectomy. Free PSA was eliminated from plasma in a biphasic exponential pattern with an early plasma half-life of 55 minutes and a late plasma half-life of 18 hours. PSA alpha1-antichymotrypsin decreased slowly, whereas human glandular kallikrein 2 was detectable only 12 hours after prostatectomy. PSA alpha2-macroglobulin remained at insignificant, nondetectable concentrations during the entire perioperative and postoperative period. Release of free PSA contributes to the elevation of plasma total PSA after prostatectomy. Free PSA is enzymatically inactive as the release does not result in subsequent elevation of PSA alpha1-antichymotrypsin or PSA alpha2-macroglobulin. Biphasic exponential elimination of free PSA may be explained by rapid extracellular redistribution (early half-life) and glomerular filtration in the kidneys (late half-life). Our data suggest rapid metabolism of human glandular kallikrein 2 but do not support suggestions of the significance in vivo of complex formations with alpha2-macroglobulin as a major means to eliminate PSA from plasma in patients with clinically localized prostate cancer.

Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds

Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with Ki values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (Ki=80 nM). The comparison between BuXI and BvTI reactive site structure indicates differences at Met59, Thr66 and Met67 residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (Km=0.68 μM, kcat/Km=1.3×106 M−1 s−1), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (Km=0.66 μM, kcat/Km=2.2×103 M−1 s−1). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. The determined Km and kcat values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes.

Characterization of a tissue kallikrein inhibitor isolated from Bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates

Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (Ki 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI–NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (Ki 2.0 nM) and Abz-FRSSRQ-EDDnp (Ki 2.5 nM).

The Combination of Human Glandular Kallikrein and Free Prostate-specific Antigen (PSA) Enhances Discrimination Between Prostate Cancer and Benign Prostatic Hyperplasia in Patients with Moderately Increased Total PSA

Prostate-specific antigen (PSA) is the most reliable tumor marker available and is widely used for the diagnosis and management of prostate cancer. Unfortunately, PSA cannot distinguish efficiently between benign and malignant disease of the prostate, especially within the range of 4-10 microg/L. Among the refinements developed to enhance PSA specificity is the free/total PSA ratio, which is useful in discriminating between the two diseases within the diagnostic "gray zone". Recent data indicate that human glandular kallikrein (hK2), a protein with high homology to PSA, may be an additional serum marker for the diagnosis and monitoring of prostate cancer. We analyzed 206 serum samples (all before treatment was initiated) from men with histologically confirmed benign prostatic hyperplasia (n = 100) or prostatic carcinoma (n = 106) with total PSA in the range of 2.5-10 microg/L. Total and free PSA and hK2 were measured with noncompetitive immunological procedures. Statistical analysis was performed to investigate the potential utility of the various markers or their combinations in discriminating between benign prostatic hyperplasia and prostatic carcinoma. hK2 concentrations were not statistically different between the two groups of patients. There was a strong positive correlation between hK2 and free PSA in the whole patient population. hK2/free PSA ratio (area under the curve = 0.69) was stronger predictor of prostate cancer than the free/total PSA ratio (area under the curve = 0.64). At 95% specificity, the hK2/free PSA ratio identified 30% of patients with total PSA between 2.5-10 microg/L who had cancer. At 95% specificity, the hK2/free PSA ratio identified 25% of patients with total PSA between 2.5 and 4.5 microg/L who had cancer. Our data suggest that hK2 in combination with free and total PSA can enhance the biochemical detection of prostate cancer in patients with moderately increased total PSA concentrations. More specifically, the hK2/free PSA ratio appears to be valuable in identifying a subset of patients with total PSA between 2.5 and 4.5 microg/L who have high probability of cancer and who should be considered for biopsy.

Identification of Three New Alternate Human Kallikrein 2 Transcripts: Evidence of Long Transcript and Alternative Splicing

In a search for prostate-specific genes in the human expressed sequence tag (EST) database, we identified a seemingly unique EST cluster C81. Experimental data linked C81 to the human hKLK2 gene that encodes a prostate specific serine protease-human glandular kallikrein (hK2). We uncovered a full-length hKLK2 cDNA corresponding to a 3.0 kb hKLK2 mRNA by PCR and sequence analysis. The 3.0 kb transcript accounts for about 25% of the hKLK2 transcripts as compared to the previously known 1.5 kb transcript. We also identified a third spliced form of the hKLK2 gene produced by alternative splicing between intron III and exon 4. This spliced form was detected in normal prostate, prostate cancer and the prostate adenocarcinoma cell line LNCaP. The identification of long hKLK2 transcript and an alternative spliced form of the hKLK2 gene indicates that regulation of the gene is complex.

Use of human glandular kallikrein 2 for the detection of prostate cancer: preliminary analysis

Objectives. Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of a multigene family of serine proteases that share approximately 80% sequence homology. Both are expressed in the prostate epithelium, are under androgen regulation, are present in serum and seminal fluid, and can form complexes with endogenous protease inhibitors (eg, alpha 2-macroglobulin and alpha 1-antichymotrypsin). Differences in immunohistochemistry and substrate specificity suggest hK2 may provide unique information for early detection and characterization of prostate cancer. Methods. Nine hundred thirty-seven archived serum samples from men treated at two academic institutions were studied. All men underwent biopsy, had a histologically confirmed diagnosis of cancer or noncancer, and a total PSA level greater than 2 ng/mL. Samples were tested in Hybritech’s Tandem-R PSA and Tandem-R free PSA (fPSA) assays and a research prototype assay for total hK2 (thK2). Results. The thK2/fPSA ratio provided additional specificity for cancer detection over PSA and the percentage of fPSA (%fPSA). A model for cancer detection using %fPSA and the thK2/fPSA ratio when PSA is 2 to 4 ng/mL is proposed that would identify as many as 40% of the cancers and would require biopsy in only 16.5% of the men in this PSA range. Conclusions. In this study, %fPSA and thK2/fPSA provided unique information for prostate cancer detection and increased the specificity of cancer detection.

Human Glandular Kallikrein in Breast Milk, Amniotic Fluid, and Breast Cyst Fluid

Human glandular kallikrein (hK2) belongs to the serine protease family of enzymes and has high sequence homology with prostate-specific antigen (PSA). The physiological role of hK2 has not as yet been determined, but there is evidence that it can regulate the proteolytic activity of PSA through processing and activating pro-PSA, an inactive precursor. Thus, it is conceivable that these two secreted proteins may coexist in biological fluids. Currently, hK2 is considered an androgen-regulated and prostate-specific protein. Recently, it has been demonstrated that hK2 is expressed in the breast cancer cell line T-47D after stimulation by steroid hormones, and we reported that hK2 can be detected in a subset of breast tumor extracts. These data suggest that hK2 may be expressed in tissues other than the prostate, such as those in which PSA has already been detected. Because hK2 is a secreted protein, it may be present in various biological fluids. We analyzed milk samples from lactating women, amniotic fluid from pregnant women, and breast cyst fluid from patients with gross breast cystic disease, using a highly sensitive and specific immunoassay for hK2. hK2 was present in all three biological fluids. We suggest that the female breast may produce hK2 and provide evidence that hK2 may have value as an additional marker for the discrimination between type I and type II breast cysts. The female breast produces hK2 in addition to PSA. More studies are necessary to establish the role of this kallikrein in nondiseased breast, gross breast cystic disease, and breast cancer.

Highly Sensitive Automated Chemiluminometric Assay for Measuring Free Human Glandular Kallikrein-2

Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to alpha1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results. We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS(R) analyzer. The detection limit of the assay was 1.5 ng/L, the "functional sensitivity" (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations. The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.

Enzymatic action of human glandular kallikrein 2 (hK2). Substrate specificity and regulation by Zn2+ and extracellular protease inhibitors.

Human glandular kallikrein 2 (hK2) is a serine protease expressed by the prostate gland with 80% identity in primary structure to prostate-specific antigen (PSA). Recently, hK2 was shown to activate the zymogen form of PSA (proPSA) in vitro and is likely to be the physiological activator of PSA in the prostate. hK2 is also able to activate urokinase and effectively cleave fibronectin. We studied the substrate specificity of hK2 and regulation of its activity by zinc and extracellular protease inhibitors present in the prostate and seminal plasma. The enzymatic activity and substrate specificity was studied by determining hK2 cleavage sites in the major gel proteins in semen, semenogelin I and II, and by measuring hydrolysis of various tripeptide aminomethylcoumarin substrates. HK2 cleaves substrates C-terminal of single or double arginines. Basic amino acids were also occasionally found at several other positions N-terminal of the cleavage site. Therefore, the substrate specificity of hK2 fits in well with that of a processor of protein precursors. Possible regulation mechanisms were studied by testing the ability of Zn2+ and different protease inhibitors to inhibit hK2 by kinetic measurements. Inhibitory constants were determined for the most effective inhibitors PCI and Zn2+. The high affinity of PCI for hK2 (kass = 2.0 x 10(5) M-1 x s-1) and the high concentrations of PCI (4 microM) and hK2 (0.2 microM) in seminal plasma make hK2 a very likely physiological target protease for PCI. hK2 is inhibited by Zn2+ at micromolar concentrations well below the 9 mM zinc concentration found in the prostate. The enzymatic activity of hK2 is likely to be reversibly regulated by Zn2+ in prostatic fluid. This regulation may be impaired in CAP and advanced metastatic cancer resulting in lack of control of the hK2 activity and a need for other means of control.

Development of an Ultrasensitive Immunoassay for Human Glandular Kallikrein with No Cross-Reactivity from Prostate-specific Antigen

Studies demonstrating that human glandular kallikrein (hK2) is increased in prostate cancer patients have prompted speculation that this marker may of use in addition to prostate-specific antigen (PSA). An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients. Our assay has an excellent detection limit (6 ng/L) and precision (>90%). Recovery studies indicated that hK2 binds to serum protease inhibitors. All sera from healthy males had measurable hK2 concentrations (median, 402 ng/L). Almost all female sera had undetectable hK2. Serum hK2 and PSA in males correlated positively (r = 0.44), but hK2 was present at concentrations approximately 2. 5-fold lower than PSA. The PSA/hK2 ratio in male sera was 0.1-34, with a median of 2.6. In seminal plasma, this ratio was 100-500. More than 94% of immunoreactive hK2 in serum was in the free form ( approximately 30 kDa); traces of hK2 complexed to alpha1-antichymotrypsin were present. The limit of detection of the method for hK2 measurement described here ( approximately 20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.

New Ultrasensitive Assays Facilitate Studies on the Role of Human Glandular Kallikrein (hK2) as a Marker for Prostatic Disease

Prostate cancer is the most common non-skin cancer in men, and it is an important cause of morbidity and mortality. The growth rate of prostate cancer is unusually slow. It has been estimated that the tumor starts developing at 20–30 years of age and reaches a detectable stage 30–40 years later (1). This estimate is based on the doubling time of the serum concentrations of prostate-specific antigen (PSA), ∼2 years (2), and the prevalence of high-grade prostatic intraepithelial neoplasia and microscopic cancers in prostates removed at autopsy (3)(4) in various age groups. Studies on archival serum samples from men who later developed prostate cancer have shown that the serum concentrations of PSA begin increasing 5–10 years before clinical presentation(2)(5). When serum PSA exceeds the commonly used cutoff (4 μg/L), the tumor is mostly organ-confined and potentially curable. Therefore, PSA-based screening and case finding is now increasingly used for early detection of this disease (6). However, the value of this screening is debated because the treatment causes morbidity (7) and, although recent data suggest that screening reduces prostate cancer mortality (8), the long-term effect on mortality and quality of life remains to be demonstrated. When prostate cancer is treated without curative intent, the median disease-specific survival is 17 years (5). Thus, it will not be possible to evaluate the long-term effects of screening for another 5–10 years, but it is hardly possible to reverse the trend to screen for prostate cancer until then. However, as laboratorians, we can help to reduce the negative effects of screening by developing better screening tools. Currently, the main screening tool is serum PSA, which suffers from low specificity (6). Approximately two-thirds of the increased …

Measurement of prostate-specific antigen and human glandular kallikrein 2 in different body fluids.

It has been demonstrated that prostate-specific antigen (PSA), in spite of its name, can be detected in body fluids and tumors from a variety of organs. Investigations have shown that human glandular kallikrein 2 (hK2), a related prostate-secreted protease, can activate the zymogen form of PSA, suggesting that the two enzymes might work as a functional unit, with hK2 as the activator molecule and PSA as the effector molecule. If this is true, then hK2 should be found together with PSA in body fluids other than seminal plasma, as well. Recently, a sensitive and specific assay was devised for hK2, enabling its measurement in picogram quantities. With this assay, the concentration of hK2 was determined in samples of seminal plasma, amniotic fluid, breast milk, and saliva. Simultaneously, the samples were assayed for molecular forms of PSA. In seminal plasma, the mean PSA concentration was 0.82 mg/ml, while the hK2 level was around two orders of magnitude lower: mean value, 6.4 microg/ml. Approximately the same ratio of PSA to hK2 as in seminal plasma was found in amniotic fluid and breast milk, but in most samples, the hK2 values were too low for direct measurements and had to be concentrated prior to analysis. Measurable levels of PSA, all in the free form, were detected in amniotic fluid at the thirteenth week of gestation and then gradually increased to levels around and over 1 microg/L from the twentieth week. Significant levels of PSA were detected in amniotic fluid collected at delivery, also. Measurable levels of mammary PSA were primarily detected in colostrum, with a range from less than 0.03 microg/L to 2.1 mg/L. Around half of the molecules were in complex with protease inhibitor. Most surprisingly, determinations on saliva samples showed that none of them had detectable PSA levels but had measurable concentrations of hK2 with a mean value, 0.09 microg/L. The presence in saliva suggests that hK2 can be the human equivalent to one of the mouse salivary kallikreins with important biological function, like the epidermal growth factor-binding protein or the gamma subunit of nerve growth factor. However, this was ruled out, as a phylogenetic analysis showed that the human and mouse glandular kallikreins evolved independently from a common precursor after the separation of the primate and rodent lineages. In conclusion, the measurements show that in addition to the previously known secretion in seminal plasma, hK2 is secreted in amniotic fluid, breast milk, and saliva. Furthermore, the concerted expression of PSA and hK2 in seminal plasma, amniotic fluid, and breast milk suggests that the two proteases might form a functional unit but not always as demonstrated by the sole presence of hK2 in saliva.

Human glandular kallikrein 2 expression in prostate adenocarcinoma and lymph node metastases

Objectives. To describe the expression of a potential new tumor marker, human glandular kallikrein 2 (hK2), in primary adenocarcinoma and lymph node metastases that may be useful as an adjunct to prostate-specific antigen (PSA) in the diagnosis and monitoring of prostate cancer.Methods. We evaluated 151 radical prostatectomy specimens removed at Mayo Clinic with node-positive adenocarcinoma to compare cytoplasmic expression of hK2, pro-hK2, and PSA in benign tissue, prostate adenocarcinoma, and lymph node metastases. Monoclonal antibodies for mature hK2 (hK2-G586), pro-hK2 (pro-hK2-G464), and PSA (PSA-773) were used. A polyclonal antibody for PSA was also used. Immunoreactivity in each case was tested to determine whether cancer recurrence could be predicted.Results. Intense epithelial cytoplasmic immunoreactivity was observed in every case for hK2-G586, pro-hK2-G464, PSA-773, and polyclonal PSA (100% of cases, respectively). The intensity and extent of hK2 expression was greater in lymph node metastases than in primary cancer; furthermore, the expression in primary cancer was greater than in benign epithelium. Pro-hK2 was expressed in a greater percentage of cells in primary cancer than in benign tissue; furthermore, pro-hK2 was expressed to a greater extent in primary cancer than in lymph node metastases. In marked contrast to mature hK2, monoclonal PSA immunoreactivity was expressed to a higher extent in primary cancer than in lymph node metastases. Polyclonal PSA showed an incremental increase in expression from benign tissue to primary cancer and a further increase in expression in lymph node metastases.Conclusions. hK2 was expressed in every cancer, and the expression incrementally increased from benign epithelium to primary cancer and lymph node metastases. Pro-hK2 was expressed to the greatest extent in primary cancer. Monoclonal PSA displayed inverse immunoreactivity compared with hK2. Polyclonal PSA showed incremental increases, suggesting that both hK2 and PSA were being detected. Tissue expression of hK2 appears to be regulated independently of PSA in benign epithelium, adenocarcinoma, and lymph node metastases.

Epitope Analysis of a Prostate-specific Antigen (PSA) C-Terminal-specific Monoclonal Antibody and New Aspects for the Discrepancy between Equimolar and Skewed PSA Assays

Immunoassays to measure prostate-specific antigen (PSA) often give different values for the same patient samples, and the calibrators among commercial immunoassays are not interchangeable. We developed three novel assays to quantify the free and complexed forms of PSA in serum. We synthesized 46 peptides, which encompassed the entire PSA molecule, and determined the interactions between selected monoclonal antibodies (MAbs) and those peptides or the intact PSA molecule. MAb PA313 did not cross-react with human glandular kallikrein (hK2), which has 78% amino acid homology to PSA. This MAb bound with KD = 40 nmol/L to the C-terminal peptide of PSA and distinguished between a synthetic peptide derived from PSA (PSA46A: NH2-C-R226KWIKDTIVANP237-COOH) that differed from one derived from hK2 (PSA46B: NH2-C-R226KWIKDTAANP237-COOH) by a single amino acid. Only the MAb combination of PA313/PA121 showed equimolar reactivity with PSA and with PSA complexed with alpha1-antichymotrypsin (PSA-ACT). The free form of PSA (F-PSA) was determined by MAbs PA313/FPA503, and the amount of complexed PSA (C-PSA) in PSA-ACT was determined by alphaACT/PA313. The total PSA (T-PSA) measured by either of the equimolar assays (PA313/PA121 or Tandem-R) was consistent with the sum of F-PSA and C-PSA. In contrast, T-PSA by a skewed assay (IMx) was higher than F-PSA + C-PSA when the ratio of F-PSA to T-PSA (F/T) was >0.15. T-PSA measured by IMx was nearly equal to F-PSA/0.55 + C-PSA. The coefficient 0.55 reflected different reactivities of the IMx assay with PSA-ACT and PSA. The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.

Interactive effects of triiodothyronine and androgens on prostate cell growth and gene expression.

T3 plays an important role in the regulation of cell growth and differentiation. In this study, we show the interactive effects of T3 and androgens on the growth response and expression of the prostate-specific genes, PSA (prostate-specific antigen) and hK2 (human glandular kallikrein), in the human prostate cancer cell line, LNCaP. T3 alone showed pronounced growth enhancement in a dose-dependent fashion. However, in the presence of androgens, higher concentrations of T3 were required to produce additional proliferative effects. T3, androgens, or a combination of the two up-regulated PSA protein production in a dose-dependent fashion, but T3 had little stimulatory effect on hK2 protein expression, regardless of the presence or absence of androgens. Using gene transfer assays, T3 alone showed no effect on transcriptional activation of a reporter gene mediated by the PSA or hK2 enhancer/promoters. T3 potentiated the androgen-mediated transcription of the PSA gene but not that of the hK2 gene. A previous study suggested that the T3 effect on PSA protein expression was caused by an up-regulation of the androgen receptor (AR) protein by T3. Our results contradict these. Although AR expression was increased by T3 alone, Western blot analysis showed that the total cellular AR level was not further increased by T3 in the presence of androgens, in comparison with cells stimulated by androgens alone. Both Western blot analysis and a gel DNA band shift assay revealed that nuclear AR was not increased by T3. This study suggests that transcription factor(s) other than the AR may mediate T3 enhancement of androgenic induction of PSA expression.