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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The discrepancy between the values measured by equimolar and skewed assays depends on the ratio of free to total PSA in the sample.

High concentrations of the macrophage migration inhibitory factor in human seminal plasma and prostatic tissues.

During purification procedures to isolate kallikrein hK2 from human seminal plasma, kallikrein hK2 was found to be associated with another protein after several chromatographic steps. This study was conducted to identify the hK2 companion protein and characterize its properties and distribution. The protein was identified as macrophage migration inhibitory factor (MIF) by its NH2-terminal amino acid sequence. It had an enzymatic activity identical to that of recombinant MIF. Its concentration varied between 1 and 10 micrograms/mL in various seminal plasma. By immunohistochemical analysis, MIF was found to be localized mainly in the epithelial cells of normal and cancerous prostates. Since MIF is a well-known proinflammatory mediator, these results suggest that it may have important functions in both human reproduction and prostatic physiology.

Simple purification procedure for human prostatic kallikrein hK2 in its active form.

Kallikrein hK2 is a new potential marker of prostate cancer. It is the last member of the human kallikrein gene family to be isolated. We propose a simple purification procedure permitting us to obtain the active form of hK2 starting from human seminal plasma and using commonly available chromatography matrices. In contrast to recently published papers, this procedure is carried out without any immunoaffinity chromatography step and without the need for any antibody to follow the purification. Furthermore, it does not require any recombinant DNA technology nor sophisticated instruments.

Assessment of the trypsin-like human prostatic kallikrein, also known as hK2, in the seminal plasma of infertile men: respective contributions of an ELISA procedure and of Western blotting.

Human seminal plasma (SP) is a unique source of kallikreins. Prostate-specific antigen (hK3), which is a chymotrypsin-like human prostatic kallikrein (CHPK), and its cousin protein (hK2), which is recognized as a trypsin-like human prostatic kallikrein (THPK), have been assessed in infertility disorders to test the hypothesis that oligoasthenoteratozoospermia (OAT) is associated with an abnormal prostatic function. Monoclonal antibodies specific for THPK (hK2) were produced by Immunova, Canada, and used to develop a new enzyme-linked immunosorbent assay procedure and to perform Western blot analyses in SP. The immunoradiometric assay from Hybritech Inc., San Diego, Calif., was selected for CHPK (hK3) measurements in SP. Determinations of the THPK and of CHPK contents in SP from four groups of subjects were performed after validation of the assays. The concentration of both kallikreins was similar in three groups of infertile men, and no statistical difference from the control group was recorded. Western blot analysis confirmed the existence of different molecular forms of both kallikreins in SP. Generally, these molecular forms were not affected by infertility disorders except when obstructive azoospermia led to the exclusion of seminal vesicles, which are the sources of protein C inhibitor (PCI). No THPK-PCI complex was observed because THPK, unlike CHPK, is bound mainly to PCI within a few minutes after ejaculation. These data suggest that measurements of kallikreins in the SP of infertile men are much less useful than evaluation of their different molecular forms. Specifically, the absence of THPK-PCI appears to be a reliable feature of obstructive azoospermia, and this test should be routinely practiced in andrology laboratories.

Abundant cysteine-rich protein-1 is localized in the stromal compartment of the human prostate.

The cysteine-rich protein-1 (CRP1) is one of the major proteins of the human prostate. Because of the suspected importance of that protein in cell proliferation and differentiation, its expression was investigated in the prostate, prostatic cancer cells, and other organs of the body. At the mRNA level, the highest concentrations of CRP1 were found in the prostate and the colon followed by the brain and the testis. It was virtually absent from the spleen, liver, heart, and kidney. Prostatic cancer cells PC-3, DU-145, and LNCaP also expressed CRP1 mRNA but virtually no protein. CRP1 protein localization in tissues was determined by immunohistochemical analysis using polyclonal antibodies developed against recombinant CRP1 protein. Strong positive cytoplasmic immunoreactions were observed only in the stromal compartment of the prostate and of other smooth muscle-rich tissues without significant staining in any secretory epithelium. These results, along with previously reported data of colocalization of CRP1 with stress fibers and adhesion plaques, suggest that the main function of CRP1 may be structural.

Contamination of purified prostate-specific antigen preparations by kallikrein hK2.

This paper ascertained the contamination by hK2 of two types of PSA preparation, that of Sensabaugh and Blake (J. Urol., 144: 1523, 1990) and that of Deperthes et al (J. Androl., 17: 659, 1996). In the first procedure, the free forms of hK2 co-migrated with PSA during the CM-Sephadex and the Sephacryl S-200 steps. By contrast, in the second procedure a very high proportion of hK2 was separated from PSA. In two different Sensabaugh and Blake procedures, the hK2 contamination per microg. of PSA was found to be respectively 0.3 and 1.0 ng. We conclude that hK2 is a quantitatively minor contaminant of some PSA preparations. That contamination is probably of little consequence for PSA standardization but it could lead to erroneous conclusions in enzymatic studies of PSA.

Serpin-derived peptide substrates for investigating the substrate specificity of human tissue kallikreins hK1 and hK2.

The third human tissue kallikrein to be identified, hK2, could be an alternate or complementary marker to kallikrein hK3 (prostate-specific antigen) for prostate diseases. Most of the hK2 in seminal plasma forms an inactive complex with protein C inhibitor (PCI), a serpin secreted by seminal vesicles. As serpin inhibitors behave as suicide substrates that are cleaved early in the interaction with their target enzyme, and kallikreins have different sensitivities to serpin inhibitors, we prepared a series of substrates with intramolecularly quenched fluorescence based on the sequences of the serpin reactive loops. They were used to compare the substrate specificities of hK1 and hK2, which both have trypsin-like specificity, and thus differ from chymotrypsin-like hK3. The serpin-derived peptides behaved as kallikrein substrates whose sensitivities reflected the specificity of the parent inhibitory proteins. Substrates derived from PCI were the most sensitive for both hK1 and hK2 with specificity constants of about 10(7) M-1. s-1. Those derived from antithrombin III and alpha2-antiplasmin were more specific for hK2 while a kallistatin-derived substrate was specifically cleaved by hK1. hK1 and hK2 substrates of greater specificity were obtained using chimeric peptides based on the sequence of serpin reactive loops. The main difference between specificities of hK1 and hK2 arise because hK2 can accommodate positively charged as well as small residues at P2 and requires an arginyl residue at P1. Thus, unlike hK1, hK2 does not cleave kininogen-derived substrates overlapping the region of N-terminal insertion of bradykinin in human kininogens.

Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator.

Our work was undertaken to compare the relative efficiency of 2 purified prostatic kallikreins, namely, hK2 and prostate-specific antigen (PSA or hK3), in the activation of single-chain urokinase (scuPA). We found that hK2 converts scuPA into an active enzyme with an efficiency equal to approximately 1/50 that of plasmin. During the activation of scuPA by hK2, two fragments of 33 and 22 kDa were generated. The NH2-terminal amino acid sequence of the 33 kDa fragment showed that hK2 cleaved scuPA between Lys158 and Ile159. In contrast to a previous report by another group, our purified hK3 preparation containing no trypsin-like contaminants was totally unable to activate scuPA. Our results show that kallikrein hK2 has plasmin-like activity and suggest that it could be the initiator of a proteolytic cascade leading to prostatic cancer invasion.

Purification of enzymatically active kallikrein hK2 from human seminal plasma.

Kallikrein hK2 is a member of the human glandular kallikrein family which includes prostate-specific antigen (PSA) and pancreatic-renal kallikrein. The purpose of this work was to isolate and characterize for the first time the enzymatically active form of the hK2 protein starting from the PCI-hK2 complex isolated from human seminal plasma (Deperthes, D., Chapdelaine, P., Tremblay, R.R., Brunet, C., Berton, J., Hébert, J., Lazure, C. and Dubé, J.Y. (1995) Biochim. Biophys. Acta 1245, 311-316). That complex was dissociated by an incubation at alkaline pH and final purification was achieved by C-18 reverse phase HPLC. The purified material contained a 27 kDa band by SDS gel electrophoresis and had the expected NH2-terminal amino acid sequence of hK2. It hydrolyzed synthetic chromogenic substrates containing esters of lysine and arginine but not of phenylalanine. Furthermore, hK2 formed molecular complexes with alpha 2 -antiplasmin, alpha 1-antichymotrypsin, antithrombin III and alpha 2-macroglobulin but not with alpha 1-antitrypsin. In conlusion, the new findings of the present paper are that the PCI-hK2 complex can be dissociated by mild procedures, that the free hK2 protein can be purified thereafter by standard HPLC procedures, that the recovered free hK2 is a trypsin-like enzyme and that it can form molecular complexes with many of the major serum proteinase inhibitors.

Immunohistochemical study suggesting a complementary role of kallikreins hK2 and hK3 (prostate-specific antigen) in the functional analysis of human prostate tumors.

The development of monoclonal antibodies directed against prostatic kallikrein hK2 prompted us to evaluate its content, along with that of hK3 (prostate-specific antigen), in human prostate carcinoma. Seventy tumors categorized according to the M.D. Anderson Hospital classification (grade I to IV) were analyzed by immunohistochemistry. The staining intensity or the kallikrein content of benign prostatic hyperplasia glandular tissue (used as control) and of grade I tumors appeared similar. In grade II to IV tumors, histochemical data revealed highly variable hK2 or hK3 content in approximately 25% of tumors. Such patterns are consistent with a current observation related to heterogeneity of prostate tumors. In addition, a few tumors did not express hK3 (n = 3), hK2 (n = 3), or both (n = 3), indicating that some growth patterns of prostatic neoplasia are associated with a lack of secretion or storage of hK3 or hK2 for immunodetection. This statement also appears relevant to metastases. It was interesting to note that 4% of hK3-negative tumors had detectable hK2. Because of the importance of hK3 as a serum marker of prostate disorder, this study addresses for the first time the question of the relative importance of both hK3 and hK2 in the immunohistochemical diagnosis of prostatic tumors. We conclude that hK2 may add new information to prostate cancer diagnosis and characterization.

Human kallikrein hK2 has low kininogenase activity while prostate-specific antigen (hK3) has none.

In the present paper, we determined the kinin-releasing activity of human prostatic kallikrein hK2 and compared it to one of the kallikreins hK1 and prostate specific antigen (hK3). Kinin-like substances active on the rabbit jugular vein were progressively produced when nanomolar concentrations of hK2 were incubated with heated plasma. However in these experiments, hK1 appeared much more potent than hK2 while hK3 was totally inactive. When hK2 was incubated with purified high molecular weight kininogen, several peptides were generated as shown by the analysis on C18 reverse-phase HPLC. Kinin activity was localized exclusively in a small peak having an elution time identical to that of bradykinin while the only important peak obtained with hK1 corresponded to Lys-bradykinin. Finally, the rate of kinin production of hK2 was found to be more than a thousandfold lower than that of hK1. These experiments show that kallikreins hK2 has only a low kininogenase activity. However, it is not excluded that some of the peptides produced by hK2 action could have other types of biological activity.

Potential involvement of kallikrein hK2 in the hydrolysis of the human seminal vesicle proteins after ejaculation.

We have recently demonstrated in liquefied human seminal plasma the presence of the novel kallikrein hK2 in association with protein C inhibitor (PCI) as a 75-kDa complex. In the present study, we showed that hK2, immediately after ejaculation, was recovered only in its free form but complex formation with PCI occurred rapidly thereafter and was completed within 10 minutes. That reaction required an enzymatically active kallikrein. In order to determine the patterns of hydrolysis of major seminal vesicle proteins, semenogelins and fibronectin were exposed to hK2 and to hK3 (prostate-specific antigen or PSA) and cleavage sequences were identified by N-terminal sequencing. Free hK2 was able to hydrolyze semenogelins and fibronectin in vitro. Most of cleavage sites were at the carboxyl-side of arginyl residues. Semenogelins were hydrolyzed to a similar extent by catalytic (and similar) concentration of either hK2 or PSA though no common cleavage sites was identified for both proteinases. Unlike semenogelins, fibronectin was hydrolyzed much more efficiently by hK2 than by PSA. These results show that hK2 is enzymatically active during a short period of time after ejaculation, that major seminal vesicle proteins can be the target of this proteolytic activity, and that hK2 and PSA have different substrate specificities.

Kallikreins expression in a mature cystic ovarian teratoma.

We report an example of benign cystic ovarian teratoma that was incidentally discovered in a nulliparous 24-year-old woman taking contraceptive pills. Histological examination of the cyst revealed the presence of prostatelike tissue in association with a wide variety of other tissues. The use of highly specific monoclonal antibodies developed against the two prostate-specific kallikreins (hK2 and hK3) in humans allowed the demonstration that the multiple islets of epithelial cells were prostatic tissue in nature and that only part of these cells had conserved their intrinsic property of producing kallikreins.

Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma.

To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.

Search for androgen response elements in the proximal promoter of the canine prostate arginine esterase gene.

We have demonstrated the binding of the recombinant DNA binding domain of the rat androgen receptor to a DNA sequence of the canine prostate arginine esterase gene and have determined the functional significance of this sequence in transient transfection experiments. One of the binding sites was localized to a region (-172 to -148 bp) containing the sequence AGGACAACAGGTGTT that has 73% homology with the prostate-specific antigen (PSA) androgen response element (ARE) found at a similar position in the PSA promoter. Competition experiments showed that the androgen receptor had an approximately 100-fold more affinity for the PSA ARE than for the arginine sequence at -172 to -148. Transient cotransfection of 5'-deletion mutants of the arginine esterase promoter and 5'-flanking sequences driving the activity of the reporter gene along with the rat androgen receptor expression vector yielded only negligible inductions of chloramphenicol acetyl transferase (CAT) activity when dihydrotestosterone (DHT) was added to the culture medium. The introduction of one to four repeats of the -172 to -148 sequence of the arginine esterase gene upstream of the basal promoter of the mouse p12 gene in p12.108 also resulted in a minimal induction of CAT activity compared with a 10-fold induction of PSA AREs under similar conditions. These results suggest that the regulation of the canine arginine esterase gene by androgens is most probably achieved by mechanisms that differ from the ones prevailing with the human PSA and kallikrein-2 (hKLK2) genes.

Identification of glandular kallikrein in dog pancreas and determination of its tissue distribution.

In order to establish a formal link between previously purified canine urinary kallikrein and dog pancreatic kallikrein whose cDNA sequence has recently been published, we have isolated the pancreatic kallikrein from that animal species. Pancreatic cytosol proteins were sequentially subjected to chromatography on DEAE-Sepharose CL-6B and Concanavalin A-Sepharose, to an autolysis step and finally to two-dimensional gel electrophoresis. Kallikrein immunoreactive spots were identified with an antibody directed against canine urinary kallikrein. These proteins were isolated after electroblotting and the amino acid sequence of their NH2-terminal portion was determined by microsequencing. The sequence was found to be identical to the one deduced from pancreatic kallikrein cDNA. Using the same antibody and immunohistochemical procedures, kallikrein was found to be present in the pancreas, the salivary glands, the kidney, the colon, the lungs and the testis. These results thus confirm the molecular nature of a glandular kallikrein in the canine species.

Characterization of canine pancreas kallikrein cDNA.

Using a combination of primer extension and RT-PCR, the cDNA encoding a canine tissue kallikrein expressed in the pancreas was cloned and sequenced. The cloned 0.85 kbp cDNA contained a complete open reading frame encoding a polypeptide of 261 amino acids. The calculated molecular mass of the processed, unglycosylated, 237 amino acid protein was 26,428 Da. Its mRNA was expressed at high levels in the pancreas, kidney and submaxillary gland. The sequence of the encoded protein was highly homologous with canine prostatic arginine esterase (66%) and human renal/pancreatic kallikrein (74%). Therefore, the cloned cDNA encoded a previously uncharacterized canine kallikrein enzyme which was named dog renal/pancreatic kallikrein or dK2 according to the new nomenclature for kallikrein gene family members. Because of its specific pattern of tissue expression and the presence of all the amino acid residues necessary for kininogenase activity, we suggest that dK2 is the canine true tissue kallikrein.

The major dog pancreas protein recognized by an antiserum to dog prostate kallikrein is the anionic trypsin.

1. On the basis of its immunoreactivity with a polyclonal antiserum to dog prostate kallikrein in Western blot experiments, a 30 kDa protein was purified from the pancreas of the dog using ion-exchange and gel filtration chromatography. 2. That protein was identified as the anionic trypsin by its NH2-terminal amino acid sequence. 3. The immunoreaction occurred despite an overall amino acid homology which was limited to 39% between the prostate kallikrein and anionic trypsin. 4. Otherwise, the anti-prostatic kallikrein antiserum was rather specific since it did not react with dog cationic trypsin, dog renal kallikrein and human prostate specific antigen.

Characterization of rhesus monkey prostate specific antigen cDNA.

Using a RT-PCR approach, we obtained two overlapping cDNA clones containing the entire 1.5 kb sequence of rhesus monkey prostate specific antigen (rmPSA). The sequence obtained revealed an open reading frame of 261 amino acids. One potential N-glycosylation site was identified at Asn-78. The calculated molecular mass for the unglycosylated mature protein was 26,147 Da. Extensive amino acid homology (89%) was observed between rmPSA and its human counterpart. These results demonstrate that rhesus monkey and man prostate share a major biochemical component, and suggest that this animal species might be useful to answer specific questions related to human prostatic function and pathology.

Effect of combination treatment with zanoterone (WIN 49596), a steroidal androgen receptor antagonist, and finasteride (MK-906), a steroidal 5 alpha-reductase inhibitor, on the prostate and testes of beagle dogs.

The effects of the steroidal androgen receptor antagonist zanoterone (WIN 49596) and the steroidal 5 alpha-reductase inhibitor finasteride (MK-906) either alone or in combination on prostatic size, histomorphology, and biochemistry were determined in the intact male dog. Additionally, the effects of treatment with zanoterone and/or finasteride on testicular size, serum testosterone and LH levels, and spermatogenesis were determined in the same dogs. Daily oral treatment for 16 weeks with either zanoterone alone at 10 mg/kg.day or finasteride alone at 1.0 mg/kg.day reduced (P < 0.05) the size of the prostate, resulted in mild to moderate diffuse glandular atrophy of the prostate, and decreased prostatic DNA and prostatic arginine esterase (the primary canine prostatic protein) levels compared to those in intact controls. These changes occurred with no effect on testicular weight, testicular histomorphology, daily sperm production, or serum LH levels. Serum testosterone concentrations were increased (P < 0.05) approximately 3-fold in the 10 mg/kg.day zanoterone treatment group compared to those in intact controls. Combination treatment of male dogs for 16 weeks with zanoterone (10 mg/kg.day) plus finasteride (1.0 mg/kg.day) orally also reduced (P < 0.05) prostate size, resulted in moderate to marked diffuse prostatic glandular atrophy, and decreased prostatic DNA and arginine esterase levels more than either drug alone, without affecting testicular size, testicular histomorphology, serum LH concentrations, or serum testosterone concentrations compared to those in intact controls. The effects of combination treatment with zanoterone and finasteride on prostatic size; histomorphology; and DNA, arginine esterase protein, and arginine esterase mRNA levels were similar to those observed in castrate controls. In addition, in situ estimates of prostatic size using transrectal ultrasonography indicated that the median time to 70% prostatic regression in dogs administered combination zanoterone plus finasteride was similar to that in castrate controls (9.6 and 9.3 weeks, respectively), indicating that the combination was more effective in causing prostatic regression than either drug alone. Finally, at the dosages used, no adverse effects of combination treatment with zanoterone plus finasteride on testicular or other major body organ weights were observed. Based on these results, combination therapy using zanoterone and finasteride for the treatment of human androgen-dependent disorders such as benign prostatic hyperplasia and prostate cancer has potential utility.