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Donald J Tindall - One of the best experts on this subject based on the ideXlab platform.
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kininogenase activity of prostate derived human glandular kallikrein HK2 purified from seminal fluid
Journal of Andrology, 1999Co-Authors: Cristine M Charlesworth, Charles Y E Young, Virginia M Miller, Donald J TindallAbstract:Prostate-specific human glandular kallikrein (HK2) is an active enzyme in human seminal fluid. It is one of three serine proteases in the human kallikrein gene family, which includes hK1 (tissue kallikrein) and hK3 (prostate-specific antigen [PSA]). In order to examine kininogenase activity (i.e., production of kinin by these enzymes), we tested for bradykinin and/or Lys-bradykinin release upon incubation of HK2 and for other kallikreins with high-molecular weight kininogen (HMWK), which contains the nonapeptide bradykinin. Kinins are important regulatory peptides (especially for vascular permeability), and they may have a role in enhancing sperm motility. High-molecular weight kininogen is the substrate for plasma kallikrein (PK-a potent kinin-generating enzyme circulating in blood, not of the same gene family) and for hK1. Glandular kallikrein and protein-C inhibitor (PCI)-HK2 complex, a serpin protease inhibitor that binds HK2, were purified to homogeneity by affinity and size-exclusion chromatography. About one-half of the HK2 is found in complex with PCI. The kallikrein enzymes were incubated with HMWK, and the resulting cleavage products were analyzed for kinin activity using enzyme immunoassay, high-performance liquid chromatography and mass spectrometry, and in vitro bioassay. Our results show that HK2 cleaves HMWK to produce bradykinin, not Lys-bradykinin (like hK1 ), and the resultant heavy (56-kDa) and light (42-kDa) chains of HMWK show similar electrophoretic mobility to those cleaved by PK. Prostate-specific antigen (hK3) had no kinin-generating activity. We also identified three other intemal cleavage sites for HK2 in HMWK (Arg 427 , Arg 437 , and Arg 457 ) that yielded two peptides, one of which is identical to a PK-cleaved peptide. Glandular kallikrein is about 500-fold less active than is PK or tissue kallikrein, but it may play a physiologically important role in bradykinin release in seminal fluid.
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Kininogenase Activity of Prostate‐Derived Human Glandular Kallikrein (HK2) Purified From Seminal Fluid
Journal of Andrology, 1999Co-Authors: M. Cristine Charlesworth, Charles Y E Young, Virginia M Miller, Donald J TindallAbstract:Prostate-specific human glandular kallikrein (HK2) is an active enzyme in human seminal fluid. It is one of three serine proteases in the human kallikrein gene family, which includes hK1 (tissue kallikrein) and hK3 (prostate-specific antigen [PSA]). In order to examine kininogenase activity (i.e., production of kinin by these enzymes), we tested for bradykinin and/or Lys-bradykinin release upon incubation of HK2 and for other kallikreins with high-molecular weight kininogen (HMWK), which contains the nonapeptide bradykinin. Kinins are important regulatory peptides (especially for vascular permeability), and they may have a role in enhancing sperm motility. High-molecular weight kininogen is the substrate for plasma kallikrein (PK-a potent kinin-generating enzyme circulating in blood, not of the same gene family) and for hK1. Glandular kallikrein and protein-C inhibitor (PCI)-HK2 complex, a serpin protease inhibitor that binds HK2, were purified to homogeneity by affinity and size-exclusion chromatography. About one-half of the HK2 is found in complex with PCI. The kallikrein enzymes were incubated with HMWK, and the resulting cleavage products were analyzed for kinin activity using enzyme immunoassay, high-performance liquid chromatography and mass spectrometry, and in vitro bioassay. Our results show that HK2 cleaves HMWK to produce bradykinin, not Lys-bradykinin (like hK1 ), and the resultant heavy (56-kDa) and light (42-kDa) chains of HMWK show similar electrophoretic mobility to those cleaved by PK. Prostate-specific antigen (hK3) had no kinin-generating activity. We also identified three other intemal cleavage sites for HK2 in HMWK (Arg 427 , Arg 437 , and Arg 457 ) that yielded two peptides, one of which is identical to a PK-cleaved peptide. Glandular kallikrein is about 500-fold less active than is PK or tissue kallikrein, but it may play a physiologically important role in bradykinin release in seminal fluid.
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expression of human prostate specific glandular kallikrein protein HK2 in the breast cancer cell line t47 d
Cancer Research, 1997Co-Authors: Ming Li Hsieh, Cristine M Charlesworth, Donald J Tindall, Thomas Seay, George G Klee, Marcia K Goodmanson, Shaobo Zhang, Charles Y F YoungAbstract:Abstract Human glandular kallikrein (HK2) protein, like prostate-specific antigen (PSA), is produced mainly in prostatic epithelium. It may be useful as a new diagnostic indicator for prostate cancer. Recently, a number of HK2-specific monoclonal antibodies have been developed that enable us to detect HK2 protein in human prostate tissue, seminal fluid, and sera. Whether HK2 can be expressed, like PSA, in nonprostatic cells is not known. In this study, we have characterized the presence of HK2 in an androgen-responsive breast cancer cell line T47-D at both the protein and mRNA levels with an immunoassay, Western blot analysis, Northern blot analysis, and the reverse transcription-PCR. Using a sensitive immunoassay with monoclonal antibodies to HK2, we found that T47-D cells could be induced with androgens, mineralocorticoids, glucocorticoids, and progestins to produce significantly more HK2 than PSA. Estrogens failed to mimic the effect of the other steroids, blocking instead the stimulatory effect of androgens. Androgen induction of HK2 in T47-D cells was dose dependent. More interestingly, we found that the HK2 in androgen-induced T47-D cell spent media appears to be the pro-form of HK2 rather than mature HK2. Our study demonstrates that HK2, a serine protease thought to be found only in prostate-related tissues and fluids, is also produced in a breast cancer cell line T47-D after steroid stimulation. This finding suggests that HK2 may have a potential role in breast cancer as well as prostatic cancer and will be the impetus for further studies of HK2 distribution and function.
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human glandular kallikrein 2 HK2 expression in prostatic intraepithelial neoplasia and adenocarcinoma a novel prostate cancer marker
Urology, 1997Co-Authors: Micheal F Darson, Donald J Tindall, Robert L Wolfert, Harry G Rittenhouse, Patrick C Roche, George G Klee, Charles Y F Young, Anna Pacelli, David G BostwickAbstract:Abstract Objectives. We describe the expression of a potentially new tumor marker, human glandular kallikrein 2 (HK2), that may be useful as an adjunct to prostate-specific antigen (PSA) in the diagnosis and monitoring of prostate cancer. Methods. We evaluated 257 radical prostatectomy specimens removed at the Mayo Clinic with pathologic Stage T2 adenocarcinoma to compare the cytoplasmic expression of HK2, PSA, and prostatic acid phosphatase (PAP) in benign tissue, high-grade prostatic intraepithelial neoplasia (PIN), and adenocarcinoma. Two monoclonal antibodies, HK2-A523 and HK2-G586, specific for HK2 were used, as well as antibodies against PSA (PSM-773) and PAP (polyclonal). Results. Intense epithelial cytoplasmic immunoreactivity was observed in every case for HK2-A523, HK2-G586, PSA, and PAP (100% of cases, respectively). The intensity and extent of HK2 expression for both antibodies were greater in cancer than high-grade PIN; furthermore, high-grade PIN was greater than benign epithelium. Cases of Gleason primary grade 4 and 5 cancer showed HK2 staining in almost every cell, whereas there was greater heterogeneity of staining in lower grades of cancer. In marked contrast to HK2, PSA and PAP immunoreactivity was most intense in benign epithelium and stained to a lesser extent in PIN and carcinoma. The number of immunoreactive cells for HK2 and PSA was not predictive of cancer recurrence. Conclusions. HK2 was expressed in every cancer, and the expression incrementally increased from benign epithelium to high-grade PIN and adenocarcinoma. PSA and PAP displayed inverse immunoreactivity compared with HK2. The expression of HK2 and PSA was not predictive of cancer recurrence in patients with Stage T2 carcinoma. Expression of HK2 indicates that this kallikrein antigen is both prostate localized and tumor associated. Tissue expression of HK2 appears to be regulated independently of PSA and PAP. Further studies are needed to determine whether tissue immunoreactivity of HK2 will prove clinically useful in the diagnosis and monitoring of prostate cancer.
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detection of a prostate specific protein human glandular kallikrein HK2 in sera of patients with elevated prostate specific antigen levels
Urology, 1997Co-Authors: Cristine M Charlesworth, Stephen D Mikolajczyk, Mohammad S Saedi, Judith A Finlay, George G Klee, Charles Y F Young, Donald J TindallAbstract:Abstract Objectives Messenger ribonucleic acid for human glandular kallikrein (HK2), a protein similar to prostatespecific 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 proand 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.
Hans Lilja - One of the best experts on this subject based on the ideXlab platform.
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pharmacokinetics biodistribution and antitumor efficacy of a human glandular kallikrein 2 HK2 activated thapsigargin prodrug
The Prostate, 2006Co-Authors: Samuel Janssen, Hans Lilja, John T Isaacs, Rebecca M. Ricklis, Craig A. Dionne, Marc D Rosen, Soeren Christensen, Samuel R DenmeadeAbstract:BACKGROUND: Prostate cancer cells secrete unique proteases such as prostate-specific antigen (PSA) and human glandular kallikrein 2 (HK2) that represent targets for the activation of prodrugs as systemic treatment of metastatic prostate cancer. Previously, a combinatorial peptide library was screened to identify a highly active peptide substrate for HK2. The peptide was coupled to an analog of the potent cytotoxin thapsigargin, L12ADT, to generate an HK2-activated prodrug that was efficiently hydrolyzed by purified HK2, stable to hydrolysis in human and mouse plasma in vitro and selectively toxic to HK2 producing prostate cancer cells in vitro. METHODS: In the current study, toxicology, pharmacokinetics, prodrug biodistribution, and antitumor efficacy studies were performed to evaluate the HK2-activated prodrug in vivo. RESULTS: The single intravenous maximally tolerated dose of prodrug was 6 mg/kg (i.e., 3.67 micromole/kg) which produced peak serum concentration of approximately 36 microM and had a half-life of approximately 40 min. In addition, over a 24 hr period <0.5% of free L12ADT analog was observed in plasma. The prodrug demonstrated significant antitumor effect in vivo while it was being administered, but prolonged intravenous administration was not possible due to local toxicity to tail veins. Subcutaneous administration of equimolar doses produced lower plasma AUC compared to intravenous dosing but equivalent intratumoral levels of prodrug following multiple doses. CONCLUSIONS: The HK2-activated prodrug was stable in vivo. The prodrug, however, was rapidly cleared and difficult to administer over prolonged dosing interval. Additional studies are underway to assess antitumor efficacy with prolonged administration of higher subcutaneous doses of prodrug. Second-generation HK2-activated thapsigargin prodrugs with increased half-lives and improved formulations are also under development.
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Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (HK2)‐activated thapsigargin prodrug
The Prostate, 2006Co-Authors: Samuel Janssen, Hans Lilja, John T Isaacs, D. Marc Rosen, Rebecca M. Ricklis, Craig A. Dionne, Soeren B. Christensen, Samuel R DenmeadeAbstract:BACKGROUND: Prostate cancer cells secrete unique proteases such as prostate-specific antigen (PSA) and human glandular kallikrein 2 (HK2) that represent targets for the activation of prodrugs as systemic treatment of metastatic prostate cancer. Previously, a combinatorial peptide library was screened to identify a highly active peptide substrate for HK2. The peptide was coupled to an analog of the potent cytotoxin thapsigargin, L12ADT, to generate an HK2-activated prodrug that was efficiently hydrolyzed by purified HK2, stable to hydrolysis in human and mouse plasma in vitro and selectively toxic to HK2 producing prostate cancer cells in vitro. METHODS: In the current study, toxicology, pharmacokinetics, prodrug biodistribution, and antitumor efficacy studies were performed to evaluate the HK2-activated prodrug in vivo. RESULTS: The single intravenous maximally tolerated dose of prodrug was 6 mg/kg (i.e., 3.67 micromole/kg) which produced peak serum concentration of approximately 36 microM and had a half-life of approximately 40 min. In addition, over a 24 hr period
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the role of molecular forms of prostate specific antigen psa or hk3 and of human glandular kallikrein 2 HK2 in the diagnosis and monitoring of prostate cancer and in extra prostatic disease
Critical Reviews in Clinical Laboratory Sciences, 2001Co-Authors: Charlotte Becker, Eleftherios P Diamandis, Joachim Noldus, Hans LiljaAbstract:Prostate-specific antigen (PSA or hK3) is a glandular kallikrein with abundant expression in the prostate that is widely used to detect and monitor prostate cancer (PCa), although the serum level is frequently elevated also in benign and inflammatory prostatic diseases. PSA testing is useful for early detection of localized PCa and for the detection of disease recurrence after treatment. However, PSA has failed to accurately estimate cancer volume and preoperative staging. There is no PSA level in serum that definitively distinguishes men with benign conditions from those with prostate cancer, although PCa is rare in men with PSA levels in serum < 2.0 ng/ml. This prompted searches for enhancing parameters to combine with PSA testing, such as PSA density, PSA velocity, and age-specific reference ranges. Due to the protease structure, PSA occurs in different molecular forms in serum and their concentrations vary according to the type of prostatic disease. Human glandular kallikrein 2 (HK2) is very similar to PSA, but expressed at higher levels in prostate adenocarcinoma than in normal prostate epithelium. Blood testing for HK2 combined with different PSA forms improves discrimination of men with benign prostatic disease from those with prostate cancer. Many data have also been reported on the extra-prostatic expression of both PSA and HK2, and it is now believed that they may both have functions in tissues outside the prostate.
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activation of latent protease function of pro HK2 but not pro psa involves autoprocessing
The Prostate, 2001Co-Authors: Samuel R Denmeade, Hans Lilja, Janita Lovgren, Saeed R Khan, John T IsaacsAbstract:BACKGROUND: Human glandular kallikrein 2 (HK2) and prostate-specific antigen (PSA) are members of an extensive kallikrein family of proteases. Both proteases are secreted as zymogens or proenzymes containing a seven amino acid propeptide that must be proteolytically removed for enzymatic activation. The physiological proteases that activate pro-HK2 and pro-PSA are not known. METHODS: The pro-HK2 peptide sequence is Val-Pro-Leu-Ile-Gln-Ser-Arg (VPLIQSR). For PSA, the amino acid sequence of the propeptide is Ala-Pro-Leu-Ile-Leu-Ser-Arg (APLILSR). Fluorescent substrates were made by coupling these peptide sequences to 7-amino-4-methylcoumarin (AMC). The hydrolysis of the VPLIQSR-AMC and APLILSR-AMC substrates by HK2, PSA, and a panel of purified proteases was determined. RESULTS: HK2 readily cleaved the pro-HK2 peptide substrate VPLIQSR-AMC with a rate of hydrolysis that was approximately 8-fold higher than an equimolar amount of purified trypsin. HK2 also had the highest hydrolysis rate from among a group of other trypsin-like proteases. In contrast, neither HK2 nor PSA was able to appreciably cleave the pro-PSA substrate APLILSR-AMC. The pro-PSA substrate was most readily hydrolyzed by urokinase and trypsin. CONCLUSIONS: HK2 can hydrolyze the pro-HK2 substrate suggesting that maturation of pro-HK2 to enzymatically active HK2 involves autoprocessing. As expected, PSA, a chymotrypsin-like protease, was unable to hydrolyze either of the propeptide substrates. Therefore, it is unlikely that PSA can auto-process its own enzymatic function. HK2 has trypsin-like specificity but was unable to hydrolyze the pro-PSA substrate. These results raise the possibility that an additional processing protease may be required to fully process PSA to an enzymatically active form.
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Activation of latent protease function of pro‐HK2, but not pro‐PSA, involves autoprocessing
The Prostate, 2001Co-Authors: Samuel R Denmeade, Hans Lilja, Janita Lovgren, Saeed R Khan, John T IsaacsAbstract:BACKGROUND: Human glandular kallikrein 2 (HK2) and prostate-specific antigen (PSA) are members of an extensive kallikrein family of proteases. Both proteases are secreted as zymogens or proenzymes containing a seven amino acid propeptide that must be proteolytically removed for enzymatic activation. The physiological proteases that activate pro-HK2 and pro-PSA are not known. METHODS: The pro-HK2 peptide sequence is Val-Pro-Leu-Ile-Gln-Ser-Arg (VPLIQSR). For PSA, the amino acid sequence of the propeptide is Ala-Pro-Leu-Ile-Leu-Ser-Arg (APLILSR). Fluorescent substrates were made by coupling these peptide sequences to 7-amino-4-methylcoumarin (AMC). The hydrolysis of the VPLIQSR-AMC and APLILSR-AMC substrates by HK2, PSA, and a panel of purified proteases was determined. RESULTS: HK2 readily cleaved the pro-HK2 peptide substrate VPLIQSR-AMC with a rate of hydrolysis that was approximately 8-fold higher than an equimolar amount of purified trypsin. HK2 also had the highest hydrolysis rate from among a group of other trypsin-like proteases. In contrast, neither HK2 nor PSA was able to appreciably cleave the pro-PSA substrate APLILSR-AMC. The pro-PSA substrate was most readily hydrolyzed by urokinase and trypsin. CONCLUSIONS: HK2 can hydrolyze the pro-HK2 substrate suggesting that maturation of pro-HK2 to enzymatically active HK2 involves autoprocessing. As expected, PSA, a chymotrypsin-like protease, was unable to hydrolyze either of the propeptide substrates. Therefore, it is unlikely that PSA can auto-process its own enzymatic function. HK2 has trypsin-like specificity but was unable to hydrolyze the pro-PSA substrate. These results raise the possibility that an additional processing protease may be required to fully process PSA to an enzymatically active form. (Less)
Eleftherios P Diamandis - One of the best experts on this subject based on the ideXlab platform.
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Coordinated steroid hormone-dependent and independent expression of multiple kallikreins in breast cancer cell lines
Breast Cancer Research and Treatment, 2007Co-Authors: Miltiadis Paliouras, Eleftherios P DiamandisAbstract:The regulation of gene expression by steroid hormones plays an important role in the normal development and function of many organs, as well in the pathogenesis of endocrine-related cancers. Previous experiments have shown that many kallikrein genes are under steroid hormone regulation in breast cancer cell lines. We here examine the coordinated expression of multiple kallikrein genes in several breast cancer cell lines after steroid hormone stimulation. Breast cancer cell lines were treated with various steroid hormones and kallikrein ( KLK/ hK) expression of hK3 (prostate-specific antigen, PSA), hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 was analyzed at the RNA level via RT-PCR and at the protein level by immunofluorometric ELISA assays. We identified several distinct hK hormone-dependent and hormone-independent expression patterns. Hormone-specific modulation of expression was seen for several kallikreins in BT-474, MCF-7, and T-47D cell lines. hK6 was specifically up-regulated upon estradiol treatment in all three cell lines whereas PSA expression was induced by dihydrotestosterone (DHT) and norgestrel stimulation in BT-474 and T-47D. hK10, hK11, hK13, and hK14 were specifically up-regulated by DHT in T-47D and by estradiol in BT-474 cells. Bioinformatic analysis of upstream proximal promoter sequences for these hKs did not identify any recognizable hormone-response elements (HREs), suggesting that the coordinated activation of these four hKs represents a unique expression “cassette”, utilizing a common hormone-dependent mechanism. We conclude that groups of human hKs are coordinately expressed in a steroid hormone-dependent manner. Our data supports clinical observations linking expression of multiple hKs with breast cancer prognosis.
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Serum human glandular kallikrein 2 (HK2) for distinguishing stage and grade of prostate cancer
International Journal of Urology, 2006Co-Authors: Carsten Stephan, George M Yousef, Klaus Jung, Terukazu Nakamura, Glen Kristiansen, Eleftherios P DiamandisAbstract:Background: Human glandular kallikrein (HK2) has been shown to add important information regarding the early detection and staging of prostate cancer. Preliminary analysis pointed out that HK2 may discriminate between pT2 and pT3 tumors, and that HK2 may predict Gleason grade 4/5 cancer volume, better than prostate-specific antigen (PSA) or percent free PSA (% fPSA). We investigated the role of HK2 serum values for predicting pathological stage, grade and Gleason score. Methods: Prostate-specific antigen, free PSA and HK2 were measured on 222 untreated prostate cancer patients who had received radical prostatectomy at the Charite Hospital, Berlin, Germany. Pathological work up revealed pT2-cancer in 111 patients and pT3-cancer in 111 patients. Grade 2 was found in 118 patients whereas grade 3 tumors were found in 104 patients. Results: For pT2 and pT3 patients, the % fPSA (P = 0.006), the ratios HK2/fPSA (P = 0.08) and HK2 × tPSA/fPSA (P = 0.002) were all significant different whereas HK2 (P = 0.143) and PSA (P = 0.1) did not differ. Between grade 2 and grade 3 tumors, the HK2 alone (P = 0.27), the % fPSA (P = 0.13), the ratios HK2/fPSA (P = 0.94) and HK2 × tPSA/fPSA (P = 0.12) did not separate, whereas PSA (P = 0.039) showed a difference. The same relationships were found between the two groups in Gleason score
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serum human glandular kallikrein 2 HK2 for distinguishing stage and grade of prostate cancer
International Journal of Urology, 2006Co-Authors: Carsten Stephan, Eleftherios P Diamandis, George M Yousef, Klaus Jung, Terukazu Nakamura, Glen KristiansenAbstract:Background: Human glandular kallikrein (HK2) has been shown to add important information regarding the early detection and staging of prostate cancer. Preliminary analysis pointed out that HK2 may discriminate between pT2 and pT3 tumors, and that HK2 may predict Gleason grade 4/5 cancer volume, better than prostate-specific antigen (PSA) or percent free PSA (% fPSA). We investigated the role of HK2 serum values for predicting pathological stage, grade and Gleason score. Methods: Prostate-specific antigen, free PSA and HK2 were measured on 222 untreated prostate cancer patients who had received radical prostatectomy at the Charite Hospital, Berlin, Germany. Pathological work up revealed pT2-cancer in 111 patients and pT3-cancer in 111 patients. Grade 2 was found in 118 patients whereas grade 3 tumors were found in 104 patients. Results: For pT2 and pT3 patients, the % fPSA (P = 0.006), the ratios HK2/fPSA (P = 0.08) and HK2 × tPSA/fPSA (P = 0.002) were all significant different whereas HK2 (P = 0.143) and PSA (P = 0.1) did not differ. Between grade 2 and grade 3 tumors, the HK2 alone (P = 0.27), the % fPSA (P = 0.13), the ratios HK2/fPSA (P = 0.94) and HK2 × tPSA/fPSA (P = 0.12) did not separate, whereas PSA (P = 0.039) showed a difference. The same relationships were found between the two groups in Gleason score <7 and ≥7. Neither the HK2 ratio, nor % fPSA was different. Conclusion: Human glandular kallikrein was not different between pT2 and pT3, nor between G2 versus G3 or Gleason scores <7 and ≥7 prostate cancer. Together with % fPSA, HK2 may only help to distinguish preoperatively between pT2 and pT3 prostate cancer but cannot add further information.
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serum human glandular kallikrein HK2 and insulin like growth factor 1 igf 1 improve the discrimination between prostate cancer and benign prostatic hyperplasia in combination with total and free psa
The Prostate, 2003Co-Authors: Andreas Scorilas, Eleftherios P Diamandis, Mario Plebani, S Mazza, Daniela Basso, Antoninus Soosaipillai, Nikos Katsaros, F PaganoAbstract:BACKGROUND There is growing evidence describing an association of HK2 and IGFs with cancer. The aim of this study is to investigate the differences in serum levels of HK2 and IGFs in a large group of patients with benign prostatic hyperplasia (BPH) or prostatic carcinoma (CaP) and to examine the value of these variables, as well as their various combinations with PSA, for discriminating between these two clinical entities. METHODS Human glandular kallikrein 2 (HK2), insulin-like growth factor-1 (IGF-1), free and total PSA concentrations were measured with non-competitive immunological procedures. Receiver operating characteristic (ROC) analysis as well as univariate and multivariate logistic regression analysis were performed to investigate the potential utility of the various markers and their combinations for discriminating between BPH and CaP. RESULTS HK2 and IGF-1 concentrations were increased in CaP patients, in comparison to BPH patients. HK2/free PSA and free/total PSA ratios (area under the curve, AUC = 0.70) were stronger predictors of prostate cancer than the IGF-1/total PSA ratio (AUC = 0.56) in the group of patients with total PSA <4 μg/L. The HK2/free PSA ratio (AUC = 0.74) was found to have significant discriminatory value in patients with total PSA within the “gray zone” (4–10 μg/L). Multivariate logistic regression models confirmed the observed relationships and identified IGF-1/free PSA and HK2/free PSA as independent predictors of CaP. CONCLUSIONS HK2/free PSA and IGF-1/free PSA ratios may be useful adjuncts in improving patient selection for prostate biopsy. Prostate 54: 220–229, 2003. © 2002 Wiley-Liss, Inc.
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the role of molecular forms of prostate specific antigen psa or hk3 and of human glandular kallikrein 2 HK2 in the diagnosis and monitoring of prostate cancer and in extra prostatic disease
Critical Reviews in Clinical Laboratory Sciences, 2001Co-Authors: Charlotte Becker, Eleftherios P Diamandis, Joachim Noldus, Hans LiljaAbstract:Prostate-specific antigen (PSA or hK3) is a glandular kallikrein with abundant expression in the prostate that is widely used to detect and monitor prostate cancer (PCa), although the serum level is frequently elevated also in benign and inflammatory prostatic diseases. PSA testing is useful for early detection of localized PCa and for the detection of disease recurrence after treatment. However, PSA has failed to accurately estimate cancer volume and preoperative staging. There is no PSA level in serum that definitively distinguishes men with benign conditions from those with prostate cancer, although PCa is rare in men with PSA levels in serum < 2.0 ng/ml. This prompted searches for enhancing parameters to combine with PSA testing, such as PSA density, PSA velocity, and age-specific reference ranges. Due to the protease structure, PSA occurs in different molecular forms in serum and their concentrations vary according to the type of prostatic disease. Human glandular kallikrein 2 (HK2) is very similar to PSA, but expressed at higher levels in prostate adenocarcinoma than in normal prostate epithelium. Blood testing for HK2 combined with different PSA forms improves discrimination of men with benign prostatic disease from those with prostate cancer. Many data have also been reported on the extra-prostatic expression of both PSA and HK2, and it is now believed that they may both have functions in tissues outside the prostate.
Jean Dube - One of the best experts on this subject based on the ideXlab platform.
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CONTAMINATION OF PURIFIED PROSTATE-SPECIFIC ANTIGEN PREPARATIONS BY KALLIKREIN HK2
The Journal of Urology, 1998Co-Authors: Gilles Frenette, Roland R Tremblay, Yannick Gervais, Jean DubeAbstract:AbstractThis 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 micro g. 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.
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human kallikrein HK2 has low kininogenase activity while prostate specific antigen hk3 has none
Biochimica et Biophysica Acta, 1997Co-Authors: David Deperthes, Francois Marceau, Gilles Frenette, Claude Lazure, Roland R Tremblay, Jean DubeAbstract:Abstract 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.
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prostatic kallikrein HK2 but not prostate specific antigen hk3 activates single chain urokinase type plasminogen activator
International Journal of Cancer, 1997Co-Authors: Gilles Frenette, Claude Lazure, Roland R Tremblay, Jean DubeAbstract: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. Int. J.Cancer 71: 897-899, 1997. © 1997 Wiley-Liss Inc.
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purification of enzymatically active kallikrein HK2 from human seminal plasma
Biochimica et Biophysica Acta, 1997Co-Authors: Gilles Frenette, David Deperthes, Claude Lazure, Roland R Tremblay, Jean DubeAbstract:Abstract 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., Hebert, J., Lazure, C. and Dube, 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 α2-antiplasmin, α1-antichymotrypsin, antithrombin III and α2-macroglobulin but not with α1-antitrypsin. In conclusion, 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.
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immunohistochemical study suggesting a complementary role of kallikreins HK2 and hk3 prostate specific antigen in the functional analysis of human prostate tumors
American Journal of Pathology, 1997Co-Authors: Roland R Tremblay, David Deperthes, B Tetu, Jean DubeAbstract: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.
Charles Y F Young - One of the best experts on this subject based on the ideXlab platform.
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an androgen response element mediates lncap cell dependent androgen induction of the HK2 gene
Molecular and Cellular Endocrinology, 2000Co-Authors: Susan H Mitchell, Shaobo Zhang, Patricia E Murtha, Charles Y F YoungAbstract:Abstract Human glandular kallikrein (HK2) is an androgen regulated protein primarily expressed in the prostate and recently identified as a novel prostate cancer marker. A 5 kb 5′ flanking region of the HK2 gene was isolated and sequenced to characterize the regulatory mechanisms for the expression of HK2 in the androgen responsive prostate cell line, LNCaP. Using gene transfer, gel shift, and mutagenesis assays we have identified an ARE in the 5′ far upstream promoter region of the HK2 gene that is crucial for its regulation in LNCaP cells. This study further demonstrated that the HK2 upstream ARE plays a predominant role in androgenic response. More interestingly, previously identified AREs in the prostate specific antigen promoter and the HK2 proximal promoter exert little activity in LNCaP cells. This study for the first time identifies a unique ARE that alone mediates the function of the androgen receptor in LNCaP cells in a cell dependent manner. This study also examines the activity of this ARE with 1α, 25 dihydroxy-vitamin D3 on the expression of the HK2 gene in LNCaP cells.
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expression of human prostate specific glandular kallikrein protein HK2 in the breast cancer cell line t47 d
Cancer Research, 1997Co-Authors: Ming Li Hsieh, Cristine M Charlesworth, Donald J Tindall, Thomas Seay, George G Klee, Marcia K Goodmanson, Shaobo Zhang, Charles Y F YoungAbstract:Abstract Human glandular kallikrein (HK2) protein, like prostate-specific antigen (PSA), is produced mainly in prostatic epithelium. It may be useful as a new diagnostic indicator for prostate cancer. Recently, a number of HK2-specific monoclonal antibodies have been developed that enable us to detect HK2 protein in human prostate tissue, seminal fluid, and sera. Whether HK2 can be expressed, like PSA, in nonprostatic cells is not known. In this study, we have characterized the presence of HK2 in an androgen-responsive breast cancer cell line T47-D at both the protein and mRNA levels with an immunoassay, Western blot analysis, Northern blot analysis, and the reverse transcription-PCR. Using a sensitive immunoassay with monoclonal antibodies to HK2, we found that T47-D cells could be induced with androgens, mineralocorticoids, glucocorticoids, and progestins to produce significantly more HK2 than PSA. Estrogens failed to mimic the effect of the other steroids, blocking instead the stimulatory effect of androgens. Androgen induction of HK2 in T47-D cells was dose dependent. More interestingly, we found that the HK2 in androgen-induced T47-D cell spent media appears to be the pro-form of HK2 rather than mature HK2. Our study demonstrates that HK2, a serine protease thought to be found only in prostate-related tissues and fluids, is also produced in a breast cancer cell line T47-D after steroid stimulation. This finding suggests that HK2 may have a potential role in breast cancer as well as prostatic cancer and will be the impetus for further studies of HK2 distribution and function.
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human glandular kallikrein 2 HK2 expression in prostatic intraepithelial neoplasia and adenocarcinoma a novel prostate cancer marker
Urology, 1997Co-Authors: Micheal F Darson, Donald J Tindall, Robert L Wolfert, Harry G Rittenhouse, Patrick C Roche, George G Klee, Charles Y F Young, Anna Pacelli, David G BostwickAbstract:Abstract Objectives. We describe the expression of a potentially new tumor marker, human glandular kallikrein 2 (HK2), that may be useful as an adjunct to prostate-specific antigen (PSA) in the diagnosis and monitoring of prostate cancer. Methods. We evaluated 257 radical prostatectomy specimens removed at the Mayo Clinic with pathologic Stage T2 adenocarcinoma to compare the cytoplasmic expression of HK2, PSA, and prostatic acid phosphatase (PAP) in benign tissue, high-grade prostatic intraepithelial neoplasia (PIN), and adenocarcinoma. Two monoclonal antibodies, HK2-A523 and HK2-G586, specific for HK2 were used, as well as antibodies against PSA (PSM-773) and PAP (polyclonal). Results. Intense epithelial cytoplasmic immunoreactivity was observed in every case for HK2-A523, HK2-G586, PSA, and PAP (100% of cases, respectively). The intensity and extent of HK2 expression for both antibodies were greater in cancer than high-grade PIN; furthermore, high-grade PIN was greater than benign epithelium. Cases of Gleason primary grade 4 and 5 cancer showed HK2 staining in almost every cell, whereas there was greater heterogeneity of staining in lower grades of cancer. In marked contrast to HK2, PSA and PAP immunoreactivity was most intense in benign epithelium and stained to a lesser extent in PIN and carcinoma. The number of immunoreactive cells for HK2 and PSA was not predictive of cancer recurrence. Conclusions. HK2 was expressed in every cancer, and the expression incrementally increased from benign epithelium to high-grade PIN and adenocarcinoma. PSA and PAP displayed inverse immunoreactivity compared with HK2. The expression of HK2 and PSA was not predictive of cancer recurrence in patients with Stage T2 carcinoma. Expression of HK2 indicates that this kallikrein antigen is both prostate localized and tumor associated. Tissue expression of HK2 appears to be regulated independently of PSA and PAP. Further studies are needed to determine whether tissue immunoreactivity of HK2 will prove clinically useful in the diagnosis and monitoring of prostate cancer.
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detection of a prostate specific protein human glandular kallikrein HK2 in sera of patients with elevated prostate specific antigen levels
Urology, 1997Co-Authors: Cristine M Charlesworth, Stephen D Mikolajczyk, Mohammad S Saedi, Judith A Finlay, George G Klee, Charles Y F Young, Donald J TindallAbstract:Abstract Objectives Messenger ribonucleic acid for human glandular kallikrein (HK2), a protein similar to prostatespecific 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 proand 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.
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identification of human glandular kallikrein HK2 from lncap cells
Journal of Andrology, 1996Co-Authors: Lana S Grauer, Kristine Kuusreichel, Mohammad S Saedi, M C Charlesworth, J A Finlay, Charles Y F Young, Donald J TindallAbstract:: Based on studies indicating that human glandular kallikrein (HK2) mRNA is present in the prostate, we prepared a monoclonal antibody to a synthetic peptide corresponding to the 41–56 region of HK2 to try to identify the HK2 protein. Although prostate-specific antigen (PSA) and HK2 share 80% homology, the 41–56 amino acid sequence of HK2 is only 50% homologous with PSA. A monoclonal antibody, HK1A523, was identified that demonstrates high specificity for HK2. In western blot analysis, the antibody has a 1,000-fold greater sensitivity for the detection of HK2 than for PSA. The antibody was used to probe spent media from the prostate carcinoma cell line, LNCaP. An immunoreactive species was N-terminally sequenced and identified as mature HK2. HK1A523 was also utilized to probe prostate tumor cytosols and seminal fluid where putative forms of HK2 were also identified. The HK2 protein therefore is expressed and secreted from prostate carcinoma cells.