Kallikrein hK2 Research Articles

Prostate kallikrein markers in diagnosis, risk stratification and prognosis

The kallikrein, prostate-specific antigen (PSA), is one of the world's most frequently used disease biomarkers. After almost two decades of research and clinical experience, the diagnostic and monitoring limitations of PSA are beginning to be understood. Most physicians are aware of PSA's low specificity for cancer among older men with benign prostatic conditions; fewer are aware of recent data, which show that a prior negative biopsy or a prior PSA value below the threshold for biopsy might compromise the predictive accuracy of PSA even further. Furthermore, a subtle increase in serum PSA level during early middle age is strongly correlated with clinically important prostate cancer. We review current and past reports on the prostate kallikreins PSA and hK2 in relation to pathology and epidemiology.

Predicting Prostate Cancer Biochemical Recurrence Using a Panel of Serum Proteomic Biomarkers

The pathological state of the prostate may be reflected by serum proteome in a man. We hypothesized that biomarkers are present in preoperative serum, which may be used to predict the probability of biochemical recurrence following radical prostatectomy. Mass spectrometry analysis was used to compare 52 men who experienced biochemical recurrence with 52 who remained biochemical recurrence-free for approximately 5 years after radical retropubic prostatectomy. A total of 30 matched pairs of recurrent and nonrecurrent serum samples were randomly selected as a training set for biomarker discovery and model development. Selected mass spectrometry peaks were combined with pre-radical retropubic prostatectomy prostate specific antigen in a multivariate algorithm to predict recurrence. The algorithm was evaluated using the remaining 22 recurrent and 22 nonrecurrent subjects as test samples. Protein identities of the selected mass spectrometry peaks were investigated. Two serum biomarkers for recurrence, P1 and P2, were combined with preoperative prostate specific antigen to predict biochemical recurrence. The ROC AUC for prostate specific antigen and the predicted outcome was 0.606 and 0.691 in the testing data, respectively. Using a single cutoff the samples were divided into 2 groups that were predictive of biochemical recurrence (p = 0.026). In contrast, preoperative prostate specific antigen did not differ between recurrent and nonrecurrent cases (Wilcoxon matched pairs test p = 0.07). The protein identity of P1 was determined to be a truncated form of C4a (C4a des-Arg). Preliminary data indicated that P2 was an N-terminal fragment of protein C inhibitor. In the current study population, which was matched on Gleason score and TNM staging, pre-radical retropubic prostatectomy prostate specific antigen retained no independent power to predict recurrence. However, by adding 2 proteomic biomarkers to preoperative prostate specific antigen the combined model demonstrated statistically significant value for predicting prostate cancer recurrence in men who underwent radical retropubic prostatectomy.

Prostate-Specific Kallikreins-2 and -4 Enhance the Proliferation of DU-145 Prostate Cancer Cells through Protease-Activated Receptors-1 and -2

A major characteristic of prostate cancer is the elevation of serum levels of prostate-specific antigen (hK3) and hK2, which are tumor markers that correlate with advancing stages of disease. Including hK4, these three kallikrein serine proteases are almost exclusively produced by the prostate. Prostate cancer cells have been recently shown to overexpress protease-activated receptors (PAR), which can be potentially activated by kallikreins and can regulate tumor growth. Here, we show that recombinant hK2 and hK4 activate ERK1/2 signaling of DU-145, PC-3, and LNCaP prostate cancer cells, which express both PAR1 and PAR2. These kallikreins also stimulate the proliferation of DU-145 cells. Pretreatment of hK2 and hK4 with the serine protease inhibitor, aprotinin, blocks the responses in DU-145 cells, and small interfering RNA against PAR1 and PAR2 also inhibits ERK1/2 signaling. To determine which PAR is activated by hK2 and hK4, a cell line that expresses a single PAR, a PAR1 knockout mouse lung fibroblast cell line transfected with PAR1 (KOLF-PAR1) or PAR2 (KOLF-PAR2) was used. hK4 activates both PAR1 and PAR2, whereas hK2 activates PAR2. hK4 generates more phosphorylated ERK1/2 than hK2. These data indicate that prostatic kallikreins (hK2 and hK4) directly stimulate prostate cancer cell proliferation through PAR1 and/or PAR2 and may be potentially important targets for future drug therapy for prostate cancer.

Comparison of hk2, pro-hk2 Failure With Conventional PSA Failure in Permanent Brachytherapy and External Beam Prostate Cancer Patients in a Prospective Biomarker Protocol

A recent study from our institution demonstrated correlation of overall survival in patients (pts) with hormone refractory prostate cancer (PCa) with human kallikrein (hk2) and pro-hk2 levels at presentation. The purpose of this study is to compare outcome using conventional biochemical failure definitions with hk2 and pro-hk2 progression in pts treated with radiotherapy.

Serum and Urine Tissue Kallikrein Concentrations in Male-to-Female Transsexuals Treated with Antiandrogens and Estrogens

The expression of human tissue kallikrein genes is regulated by steroid hormones, but most studies have been conducted with cancer cell lines. Our purpose was to examine serum and urinary tissue kallikrein concentration changes in male-to-female transsexuals before and after treatment with antiandrogens and estrogens. Thirty-five male-to-female transsexuals receiving cyproterone acetate and estrogens (orally or transdermally) were included in this study. Serum and urine samples were collected before initiation of therapy and 4 and 12 months post therapy. ELISAs were used to measure multiple kallikreins in serum and urine. After antiandrogen and estrogen therapy, serum testosterone concentrations decreased dramatically, as did serum and urinary concentrations of human glandular kallikrein (hK2) and prostate-specific antigen (PSA; hK3). Statistically significant but relatively small changes in serum and urinary concentrations of many other kallikreins were also seen. Kallikreins in serum and urine were correlated before and after treatment. The concentrations of hK2 and hK3, but not of any other kallikreins, decrease dramatically after combined antiandrogen and estrogen treatment in male-to-female transsexuals. The smaller responses of the other kallikreins presumably reflect their expression in multiple tissues.

Serum human glandular kallikrein 2 (hK2) for distinguishing stage and grade of prostate cancer

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. Prostate-specific antigen, free PSA and hK2 were measured on 222 untreated prostate cancer patients who had received radical prostatectomy at the Charité 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. For pT2 and pT3 patients, the %fPSA (P=0.006), the ratios hK2/fPSA (P=0.08) and hK2xtPSA/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 hK2xtPSA/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 >or=7. Neither the hK2 ratio, nor % fPSA was different. Human glandular kallikrein was not different between pT2 and pT3, nor between G2 versus G3 or Gleason scores<7 and >or=7 prostate cancer. Together with %fPSA, hK2 may only help to distinguish preoperatively between pT2 and pT3 prostate cancer but cannot add further information.

A sensitive proximity ligation assay for active PSA

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 microg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.

Spotlight

Steuber et al., pp. 1234–1240 Radical prostatectomy is a very effective treatment for localized prostate cancer. Biochemical recurrence (BCR) of prostate cancer—as indicated by serum prostate-specific antigen (PSA)—is the most likely sign of cancer recurrence after surgical treatment. In about 30% of patients with BCR, the cancer will recur within 10 years of surgery and thus the need to identify this subgroup of patients is crucial. Clinical stage, prostate biopsy histology (the so-called Gleason grade) and pretreatment total serum PSA levels can be used in a model to predict BCR after radical prostatectomy. However, when pretreatment PSA levels are <10 ng/ml, the predictive value of PSA is questionable. This caveat is not trivial as a high proportion of patients present with PSA levels in this range at diagnosis. In an effort to overcome this limitation, the authors investigated whether preoperative levels of human glandular kallikrein (hK2)—a serine protease expressed predominantly in the prostate epithelium—and of free PSA could enhance the prediction model. Increased levels of both proteins have been associated with advanced prostate pathology. By testing serum samples in 461 patients, the authors established a “base” predictive model using the standard factors of total serum PSA, clinical stage and Gleason grade and compared this with a model with hK2 and fPSA as additional predictors. The predictive accuracy of the new model was, in general, not superior. However, it was enhanced significantly (p=0.005) in patients with PSA pretreatment levels <10 ng/ml. Clinically these finding are very relevant as a high proportion of men present with serum PSA <10 ng/ml. Thus, testing hK2 and fPSA levels in preoperative patients to enhance the accuracy of the base predictive model shows promise for this cohort of patients. Porter et al., pp. 1241–1247 ADAMTS is a family of 19 metallopeptidases in which all members are predicted to be catalytically active. Previously, the authors examined ADAMTS gene expression in malignant and normal breast tissue and showed that 11 ADAMTS genes are abnormally expressed. They also demonstrated that one family member—ADAMTS15—is an independent predictor of relapse-free survival in breast cancer patients. In this study, the authors broadened their investigation by determining ADAMTS8 expression, in addition to ADAMTS15, in tumor tissue from 229 breast cancer patients. The median follow-up period for the cohort was 7 years. Using real-time PCR, 23.6% of patients were defined as having high levels of ADAMTS8. This ADAMTS8 high expressing group had a shorter overall survival (OS) time: a 2-fold higher chance of death (hazard ratio (HR) = 2.2, p = 0.004) when compared to the ADAMTS8 low expressing group. On the other hand, ADAMTS15 expression in tumor tissue did not impact OS in patients but, as previously demonstrated, high levels were significantly associated with a prolonged relapse-free survival. Using multivariate Cox analysis regression to estimate the prognostic value of ADAMTS8 and ADAMTS15 expression, the authors demonstrated that the patient subgroup with high ADAMTS8 and low ADAMTS15 had the worst prognosis. This group had a 5-fold higher risk of death (HR=5.4, p < 0.001) when compared to the most favorable group with low ADAMTS8 and high ADAMTS15 levels. These findings strongly suggest that expression levels of ADAMTS8 and ADAMTS15 in breast cancer tissue could be an important prognostic tool. Lee et al., pp. 1285–1291 Previous studies have shown a link between the use of menopausal hormone therapy (HT) and breast cancer risk. This risk is most pronounced with the use of combined estrogen-progestin therapy (EPT) although estrogen therapy (ET) also carries a risk. To date, most data on HT and breast cancer risk have been generated by examining white populations. Using data from an ongoing multi-ethnic cohort -- where the population was enrolled in the USA in California and Hawaii—the authors could address the question of whether breast cancer risk is present in the following ethnic groups: African-American, Native Hawaiian, Japanese-American, Latina, in addition to a White population. Analysis of over 55,000 postmenopausal women showed that current EPT use was associated with an increased risk of breast cancer in all ethnic groups and even for durations of use as short as 0-5 years. The breast cancer risk associated with using EPT was estimated as 29% per 5 years of use (RR5) (RR5=1.29, 95% CI =1.23-1.35). Current ET therapy was associated with a 10% increased risk; past EPT was also associated with an increased risk but this was not statistically significant. Although current use of EPT per 5 years showed an increased risk in all ethnic groups examined, it was most pronounced in Japanese-American (33%) Latina (36%), and White (26%). Current ET use was associated with a risk in all ethnic groups except African-American. This study reaffirms the breast cancer risk associated with HT, in particular with current EPT use, and importantly highlights that this phenomenon occurs across various ethnic groups.

Perillyl alcohol inhibits the expression and function of the androgen receptor in human prostate cancer cells

Perillyl alcohol is a hydroxylated monocyclic monoterpene. In animal study, monoterpene has shown to have an anti-tumor effect. The aim of this study is to evaluate whether POH plays an important role in the development and progression of prostate cancer (pCa). We treated LNCaP cells with different concentrations of perillyl alcohol (POH). First of all, we performed cell proliferation assay and prostate-specific antigen (PSA) and human glandular kallikrein (hK2) quantification assays. LNCaP cells were treated with or without POH for Western blot analysis of androgen receptor (AR) and c-Jun. Finally, we performed transient transfection assay by transfecting LNCaP cells—which were treated with or without POH—with pGL-3 luciferase vector containing PSA promoter and AR promoter. We observed inhibition of the expression and function of the AR by POH, through inhibition of androgen-induced cell growth and androgen-stimulated secretion of prostate-specific antigen and hK2, in human pCa cell line LNCaP. In addition, we demonstrated, for the first time, that POH inhibits the transcription activities of the AR gene promoter by over-expression of c-Jun protein. These novel properties of POH strongly suggest that POH could be highly useful for intervention of pCa.

Epithelial and prostatic marker expression in short-term primary cultures of human prostate tissue samples

Despite the high incidence and mortality of prostate cancer (PCa), molecular and genetic events involved in its progression remain poorly understood due to difficulty in establishing premalignant lesions and primary tumors in vitro. The most used cancer cell lines, which have been established primarily from metastatic lesions, do not accurately recapitulate the biological behaviour of primary tumors as compared to primary cultures generated from clinical PCa specimens. However, prostate primary cultures contain a mixture of different cell types which must be characterized completely to obtain reproducible information for studying the biology of single tumors and for evaluating the effectiveness of therapeutical approaches. In this report we show the differential expression of epithelial and prostatic markers in 30 PCa-, 6 high grade prostate intraepithelial neoplasia (PIN)- and 6 BPH-derived primary cell cultures. After organoids attached, outgrowths appeared with cells maintaining close cell-to-cell associations: cell colonies express either cyto-keratin 14 [K14 (20-60%)], or cytokeratin 18 [K18 (10-70%)] with moderately high levels of androgen receptor (AR), prostate-specific antigen (PSA) and kallikrein (hK2). The differences observed for K14 immunostaining was not statistically different between PIN- and BPH-derived cultures, whereas the difference of expression of the same marker resulted highly significant (p<0.001) in the comparison between PIN- and PCa-derived cultures and between BPH- and PCa-derived cultures. In addition, the percentage of positivity for lumenal K18 was statistically lower for BPH cultures respect to the positivity observed for both PIN and PCa-derived cultures (p<0.05 and p<0.001, respectively). A reduced expression of K18+ cells, without modification in K14 expression, was evident in high grade PCa in which we observed also an increment in K5 expression representing an intermediate basal/differentiating epithelial cell marker. The primary cultures derived from prostatic tissues can be an extremely important method to study genetic and molecular changes involved in PCa progression.

Trypsin stimulates the phosphorylation of p42,44 mitogen-activated protein kinases via the proteinase-activated receptor-2 and protein kinase C epsilon in human cultured prostate stromal cells

The pathogenesis of benign prostatic hyperplasia (BPH) is not well understood. It involves the proliferation of prostate stromal cells. The proteinase-activated receptor subtype 2 (PAR-2) receptor is expressed by human prostate tissue and can be stimulated by serine proteases. Prostate epithelial cells secrete serine proteases such as trypsin, prostate specific antigen (PSA), and human glandular kallikrein (hK2). The p42,44 mitogen activated protein kinase (MAP kinase) pathway regulates cell proliferation. Trypsin can stimulate this pathway via the PAR-2 receptor and protein kinase C (PKC) in other tissues. Serine proteases secreted by prostate epithelial cells may interact with PAR-2 receptors expressed by prostate stromal cells causing them to proliferate. The aim of the present study was to establish whether functional PAR-2 receptors are expressed by human prostate stromal cells (HPSCs) and to determine whether PAR-2 stimulation can activate p42,44 MAP kinase via a pathway involving PKC. HPSCs were cultured from patients undergoing trans urethral resection of the prostate (TURP). HPSCs were stimulated with PAR agonists. Immunoblotting of HPSC lysate with anti-p42,44 MAP kinase and -PKC isoforms. Data were analyzed with densitometry. Trypsin and the PAR-2 synthetic peptide SLIGKV caused significant increases in MAP kinase phosphorylation and calcium mobilization in HPSCs. The MAP kinase response was attenuated by pertussis toxin (PTX), phorbol 12,13 dibutyrate, Go6983, and Ro 318220. The PKC isoforms alpha, delta, epsilon, and zeta were detected in HPSCs. Trypsin caused the translocation of PKC(epsilon) from the cytosol to the membrane in HPSCs and was able to stimulate cellular proliferation. The PAR-2 selective serine protease trypsin activates p42,44 MAP kinase phosphorylation via PKC(epsilon). This may be an important mechanism of BPH pathophysiology.

Development of recombinant inhibitors specific to human kallikrein 2 using phage-display selected substrates.

The reactive site loop of serpins undoubtedly defines in part their ability to inhibit a particular enzyme. Exchanges in the reactive loop of serpins might reassign the targets and modify the serpin-protease interaction kinetics. Based on this concept, we have developed a procedure to change the specificity of known serpins. First, reactive loops are very good substrates for the target enzymes. Therefore, we have used the phage-display technology to select from a pentapeptide phage library the best substrates for the human prostate kallikrein hK2 [Cloutier, S.M., Chagas, J.R., Mach, J.P., Gygi, C.M., Leisinger, H.J. & Deperthes, D. (2002) Eur. J. Biochem. 269, 2747-2754]. Selected substrates were then transplanted into the reactive site loop of alpha1-antichymotrypsin to generate new variants of this serpin, able to inhibit the serine protease. Thus, we have developed some highly specific alpha1-antichymotrypsin variants toward human kallikrein 2 which also show high reactivity. These inhibitors might be useful to help elucidate the importance of hK2 in prostate cancer progression.

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.

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. 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. 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 microg/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 microg/L). Multivariate logistic regression models confirmed the observed relationships and identified IGF-1/free PSA and hK2/free PSA as independent predictors of CaP. hK2/free PSA and IGF-1/free PSA ratios may be useful adjuncts in improving patient selection for prostate biopsy.

Human Tissue Kallikreins: A Family of New Cancer Biomarkers

Kallikreins are a subgroup of the serine protease enzyme family. Until recently, it was thought that the human kallikrein gene family contained only three members. In the past 3 years, the entire human kallikrein gene locus was discovered and found to contain 15 kallikrein genes. Kallikreins are expressed in many tissues, including steroid hormone-producing or hormone-dependent tissues such as the prostate, breast, ovary, and testis. Most, if not all, kallikreins are regulated by steroid hormones in cancer cell lines. There is strong but circumstantial evidence linking kallikreins and cancer. Prostate-specific antigen (PSA; hK3) and, more recently, human glandular kallikrein (hK2) are widely used tumor markers for prostate cancer. Three other kallikreins, hK6, hK10, and hK11, are emerging new serum biomarkers for ovarian and prostate cancer diagnosis and prognosis. Several other kallikreins are differentially expressed at both the mRNA and protein levels in various endocrine-related malignancies, and they have prognostic value. The coexpression of many kallikreins in the same tissues (healthy and malignant) points to the possible involvement of kallikreins in cascade enzymatic pathways. In addition to their diagnostic/prognostic potential, kallikreins may also emerge as attractive targets for therapeutics.

Silymarin inhibits function of the androgen receptor by reducing nuclear localization of the receptor in the human prostate cancer cell line LNCaP.

A number of reports have shown that the polyphenolic flavonoid silymarin (SM) is an effective anticancer agent. Agents with novel mechanisms of blocking androgen receptor (AR) function may be useful for prostate cancer prevention and therapy. Previous studies showed that silibinin (SB), the major active component of SM, could inhibit cell proliferation of a human prostate cancer cell line, LNCaP, by arresting the cell cycle at the G(1) phase without causing cell death. This study further delineates the potential molecular mechanism by which SM and SB exhibit antiproliferative effects on androgen-responsive prostate cancer cells by inhibiting function of the AR. We observed that SM and SB inhibited androgen-stimulated cell proliferation as well as androgen-stimulated secretion of both prostate-specific antigen (PSA) and human glandular kallikrein (hK2). Additionally, for the first time, we show that an immunophilin, FKBP51, is androgen regulated and that this up-regulation is suppressed by SM and SB. We further demonstrate that transactivation activity of the AR was diminished by SM and SB using gene transfer of PSA promoter and hK2 androgen-responsive element constructs. However, expression and steroid-binding ability of total AR were not affected by SM in western blotting and ligand-binding assays. Intriguingly, we found that nuclear AR levels are significantly reduced by SM and SB in the presence of androgens using western blotting assay and immunocytochemical staining. This study provides a new insight into how SM and SB negatively modulate androgen action in prostate cancer cells.

Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3.

Insulin-like growth factors (IGFs) are important growth regulators of both normal and malignant prostate cells. Their action is regulated by six insulin-like growth factor binding proteins (IGFBPs). The proteolytic cleavage of IGFBPs by various proteases decreases dramatically their affinity for their ligands and therefore enhances the bioavailability of IGFs. To elucidate the putative biological role of prostatic kallikreins hK2 and hK3 (prostate-specific antigen) in tumour progression, we analyzed the degradation of IGFBP-2, -3, -4 and -5 by these two tissue kallikreins. We found that hK3, already characterized as an IGFBP-3 degrading protease, cleaved IGFBP-4 but not IGFBP-2 and -5, whereas hK2 cleaved all of the IGFBPs much more effectively, and at concentrations far lower than those reported for other IGFBP-degrading proteases. The proteolytic patterns after cleavage of IGFBPs by hK2 and hK3 were similar and were not modified in the presence of IGF-I. Heparin, but not other glycosaminoglycans, enhanced dramatically the ability of hK3 but not hK2 to degrade IGFBP-3 and IGFBP-4. More importantly, the IGFBP fragments generated by hK2 and hK3 had no IGF-binding capacity, as assessed by Western ligand blotting. Our results suggest that the prostatic kallikreins hK2 and hK3 may influence specifically the tumoral growth of prostate cells through the degradation of IGFBPs, to increase IGF bioavailability.

An androgen response element mediates LNCaP cell dependent androgen induction of the hK2 gene

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.

The preparation and catalytic properties of recombinant human prostate-specific antigen (rPSA)

The serine proteinase prostate-specific antigen (PSA), and its complex with the serine proteinase inhibitor α 1-antichymotrypsin (ACT), have been used as markers for the diagnosis of prostate cancer. PSA prepared from seminal fluid is typically contaminated with the trypsin-like glandular kallikrein (hK2). Here we describe a convenient and reproducible preparation of catalytically active recombinant PSA (rPSA) and demonstrate an overall similarity in the properties of cloned and refolded rPSA to PSA purified from seminal fluid. We also present results that are relevant for increasing the sensitivity of assays of PSA activity in biological fluids, for the putative role of PSA activity in physiologically important processes, including prostate cancer metastasis, and for the design of PSA inhibitors. Specifically, we find that added salts, in particular NaCl, give rise to dramatic increases in rPSA catalytic activity, as does added glycerol. On the other hand, Zn 2+, spermine, and spermidine, each a major component of seminal and prostatic fluid, strongly inhibit rPSA activity, with Zn 2+ being a non-competitive inhibitor while spermine is a competitive inhibitor. Citrate, also a major component of seminal and prostatic fluid, spermine, and spermidine each protect rPSA from Zn 2+ inhibition, presumably via Zn 2+ sequestration. Finally, rPSA efficiently proteolyzes several protein substrates.

Serum and Urinary Prostate-specific Antigen and Urinary Human Glandular Kallikrein Concentrations Are Significantly Increased after Testosterone Administration in Female-to-Male Transsexuals

The genes that encode prostate-specific antigen (PSA) and human glandular kallikrein (hK2) are up-regulated by androgens and progestins in cultured cells, but no published studies have described the effect of androgen administration in women on serum and urinary PSA or hK2. We measured serum and urinary PSA and hK2 before, and 4 and 12 months post testosterone treatment by immunofluorometric methods in 32 female-to-male transsexuals. Mean serum PSA increased from 1.1 ng/L to 11.1 ng/L and then to 22 ng/L by 4 and 12 months post treatment, respectively; the corresponding mean values in urine were 17, 1420, and 18 130 ng/L, respectively. Serum hK2, another kallikrein closely related to PSA, remained undetectable at the three time points. However, urinary hK2 concentration rose from below the detection limit (<6 ng/L) before treatment to 18 and 179 ng/L by the 4th and the 12th month of treatment, respectively. All changes were statistically significant (P <0.001) at 4 months. Testosterone administration increases serum and urinary PSA and urinary hK2 in women. These measurements may be useful as indicators of androgenic stimulation in women.

Human glandular kallikrein as a tool to improve discrimination of poorly differentiated and non-organ-confined prostate cancer compared with prostate-specific antigen

Objectives. Human glandular kallikrein (hK2) possesses 80% structure identity with prostate-specific antigen (PSA) and is secreted by identical prostate epithelial cells. Although increasing with pathologic stage, PSA is not clinically sufficient to predict histologic grade and pathologic stage of prostate cancer (PCa) in individual cases. To address this issue, serum hK2 in various PCa grades was investigated. Methods. Sera from 122 consecutive patients with PCa, graded as well-differentiated (G1, n = 35); moderately differentiated (G2, n = 61), and poorly differentiated (G3, n = 26) PCa, was studied. In patients who underwent radical prostatectomy (n = 42), 24 had organ-confined (pT2a-b) and 18 extracapsular (pT3a or greater) disease. hK2 was measured by an indirect immunofluorometric assay with a functional sensitivity of 0.03 ng/mL. Total PSA (tPSA), free PSA (fPSA), and PSA bound to alpha 1-antichymotrypsin (PSA-ACT) were also measured. Multivariate logistic regression analysis was used for evaluation of the best combinations of tumor markers. Results. Median hK2 and tPSA increased twofold from G1 to G2 tumors (hK2 0.07 versus 0.14 ng/mL, P <0.002; tPSA 6.1 versus 12.1 ng/mL, P <0.0002). Between G2 and G3 tumors, hK2 increased threefold (0.14 versus 0.43 ng/mL, P <0.02), and tPSA showed no significant increase (12.1 versus 26.5 ng/mL, P <0.18). The f/t PSA ratio decreased between G1 and G2 cancers (0.15 vs. 010, P <0.001); no difference was found between G2 and G3 tumors (0.10 versus 0.11, P = 0.93). However, the hK2/fPSA ratio distinguished between G1 and G3 tumors and G2 and G3 tumors (0.085 [G1] and 0.11 [G2] versus 0.22 [G3], P <0.0002 and P <0.002, respectively). Using multivariate regression analysis, the fPSA/(tPSA × hK2) ratio differentiated G2 and G3 tumors ( P <0.01). In the tPSA range of 3 to 15 ng/mL, hK2, the hK2/fPSA ratio, and the fPSA/(tPSA × hK2) ratio differentiated between the G1/G2 and G3 tumors, and tPSA, the f/t PSA ratio, and PSA-ACT did not. In radical prostatectomy cases, hK2 (0.06 versus 0.156, P <0.005) and the fPSA/(tPSA × hK2) ratio (2.104 versus 0.828, P <0.005) discriminated between pT2a-b and pT3a or greater PCa. Conclusions. hK2 significantly improved the identification of poorly differentiated (G3) tumors compared with PSA. By multivariate logistic regression analysis, the hK2/fPSA and fPSA/(tPSA × hK2) ratios further improved the detection of PCa grade. This improvement was also seen with the intermediate range of tPSA. hK2 was also helpful in the prediction of organ-confined disease. Thus, hK2 may be a useful tool for more accurate prediction of tumor grade or stage and allow better clinical decision-making.