Development of an immunofluorometric assay for human kallikrein 15 (KLK15) and identification of KLK15 in tissues and biological fluids.

BACKGROUND: Human kallikrein 15 (KLK15) may have some utility as a prostate, ovarian, and breast cancer biomarker, based on previous studies, which examined mRNA levels of KLK15. The aim of this study was to develop analytical technology for human kallikrein 15, including recombinant protein, specific antibodies, and a sensitive and specific ELISA immunoassay. The assay was then used to examine levels of KLK15 in tissues and biological fluids. METHODS: We produced human, recombinant pro-KLK15 in HEK 293 cells. Recombinant KLK15 was purified with various chromatographic steps and used to immunize rabbits and mice for production of KLK15 polyclonal antibodies. We used these antibodies to develop a highly sensitive and specific KLK15 immunoassay and to study KLK15 expression in various tissues and biological fluids. RESULTS: Large amounts of pure, recombinant KLK15 have been produced and characterized. KLK15 mouse and rabbit polyclonal antibodies have been employed for development of a KLK15 immunoassay. This assay has a lower detection limit of 0.05 microg/L, and no cross-reactivity with any of the other fourteen kallikreins. Using this assay, KLK15 was detected in prostate, colon, and thyroid tissues, as well as in breast milk and seminal plasma. CONCLUSIONS: The KLK15 reagents developed here will allow for analysis of KLK15 protein expression levels in tissues and biological fluids, both normal and cancerous. This will expand upon previously characterized tissue KLK15 mRNA expression studies which suggested that KLK15 might be useful as a biomarker for breast, ovarian, and prostate cancer. KLK15 is another serine protease that is produced in prostate and other tissues and is secreted in seminal plasma and other fluids. Its physiological function needs to be further elucidated.

Human kallikrein 14: a new potential biomarker for ovarian and breast cancer.

Human kallikrein gene 14 (KLK14) is a recently discovered member of the tissue kallikrein family of secreted serine proteases, which includes hK3/prostate-specific antigen, the best cancer biomarker to date. Given that KLK14 is hormonally regulated, differentially expressed in endocrine-related cancers, and a prognostic marker for breast and ovarian cancer at the mRNA level, we hypothesize that its encoded protein, hK14, like hK3/prostate-specific antigen, may constitute a new biomarker for endocrine-related malignancies. The objective of this study was to generate immunological reagents for hK14, to develop an ELISA and immunohistochemical techniques to study its expression in normal and cancerous tissues and biological fluids. Recombinant hK14 was produced in Pichia pastoris, purified by affinity chromatography, and injected into mice and rabbits for polyclonal antibody generation. Using the mouse and rabbit antisera, a sandwich-type immunofluorometric ELISA and immunohistochemical methodologies were developed for hK14. The ELISA was sensitive (detection limit of 0.1 micro g/liter), specific for hK14, linear from 0 to 20 micro g/liter with between-run and within-run coefficients of variation of <10%. hK14 was quantified in human tissue extracts and biological fluids. Highest levels were observed in the breast, skin, prostate, seminal plasma, and amniotic fluid, with almost undetectable levels in normal serum. hK14 concentration was higher in 40% of ovarian cancer tissues compared with normal ovarian tissues. Serum hK14 levels were elevated in a proportion of patients with ovarian (65%) and breast (40%) cancers. Immunohistochemical analyses indicated strong cytoplasmic staining of hK14 by the epithelial cells of normal and malignant skin, ovary, breast, and testis. In conclusion, we report the first ELISA and immunohistochemical assays for hK14 and describe its distribution in tissues and biological fluids. Our preliminary data indicate that hK14 is a potential biomarker for breast and ovarian cancers.

Human kallikrein 8, a novel biomarker for ovarian carcinoma.

Human kallikrein 8 (hK8; neuropsin) is a serine protease and new member of the hK family. The aim of this study was to examine if hK8 may serve as a novel cancer biomarker. An hk8-ELISA, developed in-house, was used to study the distribution of hK8 in various biological fluids and tissue extracts from healthy individuals and ovarian cancer patients of different stages of the disease (International Federation of Obstetrics and Gynecology II-IV). For ovarian cancer patients, very high levels in ascites fluid were observed (

Human kallikrein 11: a new biomarker of prostate and ovarian carcinoma.

Human kallikrein 11 (hK11) is a putative serine protease of the human kallikrein gene family. Currently, no methods are available for measuring hK11 in biological fluids and tissues. Our aim was to develop immunological reagents and assays for measuring hK11 and examine if the concentration of this kallikrein is altered in disease states. We produced recombinant hK11 protein in a baculovirus system and used it to develop monoclonal and polyclonal antibodies against hK11. We then developed an immunofluorometric procedure for measuring hK11 in biological fluids and tissue extracts with high sensitivity and specificity. We further quantified hK11 in various biological fluids and in serum of patients with various cancers. The hK11 immunofluorometric assay is highly sensitive (detection limit, 0.1 microg/l) and specific (no detectable cross-reactivity for other homologous kallikreins). We established the tissue expression pattern of hK11 at the protein level and found the highest levels in the prostate, followed by stomach, trachea, skin, and colon. We have immunohistochemically localized hK11 in epithelial cells of various organs. We further detected hK11 in amniotic fluid, milk of lactating women, cerebrospinal fluid, follicular fluid, and breast cancer cytosols. However, highest levels were seen in prostatic tissue extracts and seminal plasma. hK11 in seminal plasma and prostatic extracts is present at approximately 300-fold lower levels than prostate-specific antigen and at approximately the same levels as hK2. hK11 expression in breast cancer cell lines is up-regulated by estradiol. Elevated serum levels of hK11 were found in 70% of women with ovarian cancer and in 60% of men with prostate cancer. This is the first reported immunological assay for hK11. Analysis of this biomarker in serum may aid in the diagnosis and monitoring of ovarian and prostatic carcinoma.

Human kallikrein 5: a potential novel serum biomarker for breast and ovarian cancer.

The kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4. Human kallikrein (hK) gene 5 (KLK5) is a member of this family and encodes for a secreted serine protease (hK5). KLK5 was shown to be differentially expressed at the mRNA level in breast and ovarian cancer. Until now, detection of hK5 protein in either biological fluids or tissues has not been described due to lack of suitable reagents and methods. The aim of this study was to develop immunological reagents and a sensitive and specific fluorometric immunoassay (ELISA) for hK5, to examine the presence of hK5 in human tissues and biological fluids, and to study the possible clinical utility of hK5 as a biomarker for endocrine-related malignancies. Recombinant hK5 protein was produced and purified using a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse and rabbit polyclonal anti-hK5 antibodies. A sandwich-type microplate immunoassay (ELISA) was developed using these antibodies, coupled with a time-resolved fluorometric detection technique. The ELISA assay was then used to measure hK5 in various biological fluids, tissue extracts, and serum samples from normal individuals and patients with various malignancies. The hK5 ELISA immunoassay has a lower detection limit of 0.1 micro g/liter, is specific for hK5, and has no cross-reactivity with other homologous kallikreins. The dynamic range is 0.1-25 micro g/liter, and within-run and between-run coefficients of variation within this range are <10%. hK5 is found in many tissues, with the highest expression levels seen in the skin, breast, salivary gland, and esophagus. hK5 is present at relatively high levels in milk of lactating women. Whereas the levels of hK5 are almost undetectable in serum of normal individuals (male and female) and patients with diverse malignancies, higher concentrations were found in a proportion of patients with ovarian (69%) and breast (49%) cancer. High levels were also detected in ascites fluid from metastatic ovarian cancer patients and in ovarian cancer tissue extracts. In conclusion, we report development of the first immunofluorometric assay for hK5 and describe the distribution of hK5 in biological fluids and tissue extracts. Our preliminary data indicate that hK5 is a potential biomarker in patients with ovarian and breast cancer.

Human kallikrein 6 (hK6): a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma.

PURPOSE: The discovery of new ovarian cancer biomarkers that are suitable for early disease diagnosis and prognosis may ultimately lead to improved patient management and outcomes. PATIENTS AND METHODS: We measured, by immunoassay, human kallikrein 6 (hK6) concentration in serum of 97 apparently healthy women, 141 women with benign abdominal diseases, and 146 women with histologically proven primary ovarian carcinoma. We then calculated the diagnostic sensitivity and specificity of this test and examined the association of serum hK6 concentration with various clinicopathologic variables and patient survival. RESULTS: Serum hK6 concentration between normal and benign disease patients was not different (mean, 2.9 and 3.1 micro g/L, respectively). However, hK6 in presurgical serum of ovarian cancer patients was highly elevated (mean, 6.8 micro g/L; P <.001). Serum hK6 decreased after surgery (to a mean of 3.9 micro g/L) in 68% of patients. The diagnostic sensitivity of serum hK6 at 90% and 95% specificity is 52% and 47%, respectively, in the whole patient population. For early stage disease (stage I or II), sensitivity is approximately 21% to 26%. When combined with CA-125, at 90% specificity, sensitivity increases to 72% (for all patients) and to 42% in stage I or II disease. Serum hK6 concentration correlates moderately with CA-125 and is higher in patients with late-stage, higher-grade disease and in patients with serous histotype. Preoperative serum hK6 concentration is a powerful predictor of disease-free and overall survival in both univariate and multivariate analyses. CONCLUSIONS: Serum hK6 concentration seems to be a new biomarker for ovarian carcinoma and may have value for disease diagnosis and prognosis.

Decreased concentration of human kallikrein 6 in brain extracts of Alzheimer's disease patients.

BACKGROUND: The human kallikrein 6 gene (KLK6) encodes for a secreted serine protease, hK6, which is highly expressed in brain. Previous reports have associated hK6 with the pathogenesis of Alzheimer's disease. Our objective was to develop a highly sensitive immunoassay for hK6 and use it to examine the levels of hK6 in brain tissue extracts from Alzheimer's disease patients and control subjects. METHODS: We developed antibodies against hK6 and constructed a 'sandwich' type immunoassay. We then assessed levels of hK6 in brain extracts from normal individuals and patients with Alzheimer's disease. RESULTS: The hK6 assay was developed using a combination of two antibodies (a mouse monoclonal and a rabbit polyclonal). Purified recombinant hK6 was used as a calibrator. The detection limit of the assay was 0.05 microg/L. The intra and inter-assay coefficient of variation was less than 6.5%. We found no detectable cross-reactivity by the homologous proteins hK2, hK3, hK8, hK10, hK11, hK13 and hK14. The hK6 concentration in human brain tissue extracts from healthy (n = 24) and Alzheimer's patients (n = 55) were 10.1 +/- 1.0 and 3.39 +/- 0.26 mcirog/g of total protein (mean +/- SE), respectively (p < 0.001). Similar differences were seen when the tissues were stratified by brain region (occipital, parietal, frontal and temporal cortex). CONCLUSIONS: We conclude that the newly developed hK6 immunoassay is suitable for quantification of hK6 protein in biologic fluids and tissue extracts. The brain of Alzheimer's disease patients contains significantly less hK6 than the brain of nonaffected individuals. The possible connection of hK6 with the pathogenesis of Alzheimer's disease merits further investigation.

A comparison of the anticarcinogenic properties of four red wine polyphenols.

BACKGROUND: There has been growing interest in the analysis of certain polyphenols in wine, especially flavonoids, trihydroxystilbenes and phenolic acids, stimulated by intense research into their potential benefits to human health. One of their main properties in this regard is their antioxidant activity, which enables them to attenuate the development of atherosclerosis, inflammatory diseases, and cancer. METHODS: A two stage CD-1 mouse skin cancer model using 9,10-dimethyl-1,2-benzanthracene (DMBA) as initiator and phorbol 12-myristate 13-acetate (TPA) as promoter was employed to compare the antitumorigenic activities of one polyphenol from each of four different classes: flavanols [(+)-catechin], stilbenes (trans-resveratrol), flavonols (quercetin) and hydroxybenzoic acids (gallic acid). Animals were treated with specific polyphenols at doses ranging from 0 to 25 micromoles (dissolved in 200 microL acetone), twice a week for eighteen weeks. The solution was applied topically to the shaved dorsal region of each animal. The relative potencies of the polyphenols were compared by evaluating the percentage inhibition of tumor formation in individual mice and the number of mice developing one or more tumors with the different dose schedules. RESULTS: Probit analysis revealed that quercetin was the most (ED(50)<1 micromole) and gallic acid the least effective (ED(50) 5-10 micromoles). (+)-Catechin and trans-resveratrol were intermediate, with ED(50) values of 5 and 6 micromoles, respectively. CONCLUSION: We have shown recently that trans-resveratrol is absorbed much more efficiently than (+)-catechin and quercetin in humans after oral consumption. Taking this and the relative concentrations in red wine into account, together with the present results, we conclude that trans-resveratrol may be the most effective anticancer polyphenol present in red wine as consumed po by healthy human subjects.

Steroid hormone regulation of the human kallikrein 10 (KLK10) gene in cancer cell lines and functional characterization of the KLK10 gene promoter.

BACKGROUND: The human kallikrein 10 (KLK10) gene is a new member of the human tissue kallikrein gene family. It encodes for a secreted serine protease (hK10) with predicted trypsin-like enzymatic activity. KLK10 is highly expressed in the sex organs and its expression level changes in malignancy. METHODS: To determine the role of steroid hormones in KLK10 gene expression, we investigated its modulation by 17beta-estradiol, 5alpha-dihydrotestosterone, norgestrel, dexamethasone and aldosterone, at both the transcription and translation level, in a panel of cancer cell lines. After steroid hormone stimulation, the change of KLK10 mRNA was monitored with reverse transcriptase polymerase chain reaction and hK10 protein levels in the culture supernatant were quantified with an hK10-specific immunoassay. The presence of hormone response elements in the KLK10 gene promoter was examined with the chloramphenicol acetyltransferase reporter gene system. RESULTS: The KLK10 expression was mainly up-regulated by estrogens, androgens and progestins, and to a lesser extent by dexamethasone and aldosterone in the breast cancer cell lines BT-474, MCF-7 and T-47D, both at the mRNA and protein levels. The effect of stimulation of these steroids on KLK10 expression varied among the cell lines. Estrogens, androgens and progestins were most potent in the BT-474, T-47D and MCF-7 cells, respectively. The up-regulation effect of estrogens, androgens, and progestins on KLK10 expression can be blocked by their antagonists ICI-182, 780, RU-56,187, and mifepristone, respectively. Time course studies showed that hK10 protein started to increase 1 day after steroid hormone stimulation and this increase persisted for 7 days. These data suggest that steroid hormones up-regulate KLK10 gene expression through direct interaction between hormone-receptor complexes and their cognate hormone response elements. To search for hormone response elements, we functionally characterized the KLK10 promoter by placing it upstream of the chloramphenicol acetyltransferase reporter gene. We found that KLK10 promoter activity did not rely on the presence of functional estrogen and androgen receptors. Also, the presence of functional estrogen and androgen receptors did not increase its constitutive activity. We suggest that the hormone response elements that mediate the transcriptional regulation of KLK10 are unlikely to locate in the KLK10 promoter. CONCLUSIONS: Estrogens, androgens and progestins modulate KLK10 expression through their own receptors but this regulation is not mediated by steroid hormone response elements in the promoter of the KLK10 gene.

Expression and functional characterization of the cancer-related serine protease, human tissue kallikrein 14.

Human tissue kallikrein 14 (KLK14) is a novel extracellular serine protease. Clinical data link KLK14 expression to several diseases, primarily cancer; however, little is known of its (patho)-physiological role. To functionally characterize KLK14, we expressed and purified recombinant KLK14 in mature and proenzyme forms and determined its expression pattern, specificity, regulation, and in vitro substrates. By using our novel immunoassay, the normal and/or diseased skin, breast, prostate, and ovary contained the highest concentration of KLK14. Serum KLK14 levels were significantly elevated in prostate cancer patients compared with healthy males. KLK14 displayed trypsin-like specificity with high selectivity for P1-Arg over Lys. KLK14 activity could be regulated as follows: 1) by autolytic cleavage leading to enzymatic inactivation; 2) by the inhibitory serpins alpha1-antitrypsin, alpha2-antiplasmin, antithrombin III, and alpha1-antichymotrypsin with second order rate constants (k(+2)/Ki) of 49.8, 23.8, 1.48, and 0.224 microM(-1) min(-1), respectively, as well as plasminogen activator inhibitor-1; and 3) by citrate and zinc ions, which exerted stimulatory and inhibitory effects on KLK14 activity, respectively. We also expanded the in vitro target repertoire of KLK14 to include collagens I-IV, fibronectin, laminin, kininogen, fibrinogen, plasminogen, vitronectin, and insulin-like growth factor-binding proteins 2 and 3. Our results indicate that KLK14 may be implicated in several facets of tumor progression, including growth, invasion, and angiogenesis, as well as in arthritic disease via deterioration of cartilage. These findings may have clinical implications for the management of cancer and other disorders in which KLK14 activity is elevated.

Characterization of human kallikreins 6 and 10 in ascites fluid from ovarian cancer patients.

OBJECTIVES: Human kallikreins 6 (hK6) and 10 (hK10) are secreted serine proteases. We previously found that hK6 and hK10 are highly overexpressed in epithelial ovarian tumors and demonstrated that serum levels of hK6 and hK10 are valuable biomarkers for ovarian cancer diagnosis and prognosis. Our aim is to purify and characterize these two kallikreins from ascites fluid of ovarian cancer patients. METHODS: Protein concentrations of hK6 and hK10 in ovarian cancer ascites fluids were measured with ELISA-type immunoassays. hK6 and hK10 were purified from the ascites fluids with immunoaffinity columns, followed by reverse-phase high performance liquid chromatography. Purified hK6 and hK10 were then subjected to N-terminal sequencing. Enzymatic analyses were performed with synthetic fluorogenic peptides. RESULTS: hK6 and hK10 were present in ovarian cancer ascites fluid at concentrations ranging from 0.2-571 and 0.7-220 microg/l, respectively. The majority of hK6 and hK10 in the ascites fluids were present in the free (uncomplexed) form. Both hK6 and hK10 purified from the ascites fluid were zymogens with a molecular mass of 30 kDa. Purified hK6 exhibited trypsin-like enzymatic activity, whereas no enzymatic activity was observed for purified hK10. The enzymatic activity of hK6 could be suppressed by a neutralizing monoclonal antibody. CONCLUSIONS: The majority of hK6 secreted by the ovarian tumor cells into the ascites fluid are present in the uncomplexed, zymogen form, possessing weak trypsin-like enzymatic activity. All hK10 present in ovarian cancer ascites fluids are in the uncomplexed, zymogen form and have no detectable enzymatic activity.

Human tissue kallikrein 9: production of recombinant proteins and specific antibodies.

Human tissue kallikreins (genes, KLKs; proteins, hKs) are a subgroup of hormonally regulated serine proteases. Two tissue kallikreins, namely hK2 and hK3 (prostate-specific antigen, PSA), are currently used as serological biomarkers of prostate cancer. Human tissue kallikrein 9 (KLK9) is a newly identified member of the tissue kallikrein gene family. Recent reports have indicated that KLK9 mRNA is differentially expressed in ovarian and breast cancer and has prognostic value. Here, we report the production of recombinant hK9 (classic form) using prokaryotic and mammalian cells and the generation of polyclonal antibodies. Total testis tissue mRNA was reverse-transcribed to cDNA, amplified, cloned into a pET/200 TOPO plasmid vector, and transformed into E. coli cells. hK9 was purified and used as an immunogen to generate polyclonal antibodies. Full-length KLK9 cDNA was also cloned in the vector pcDNA3.1 and was expressed in CHO cells. The identity of hK9 was confirmed by mass spectrometry. hK9 rabbit antiserum displayed no cross-reactivity with other tissue kallikreins and could specifically recognize E. coli- and CHO-derived hK9 on Western blots. hK9 was mainly detected in testis and seminal vesicles by Western blotting. The reagents generated here will help to define the physiological role of this tissue kallikrein and its involvement in human disease.

Kallikreins as markers of disseminated tumour cells in ovarian cancer-- a pilot study.

BACKGROUND: Kallikreins are a family of secreted serine proteases, encoded by 15 genes which all localize in tandem on chromosome 19q13.4. Several members of this family have been previously associated with ovarian cancer. Kallikreins 6 (KLK6) and 10 (KLK10) are elevated in tumour cells, serum and ascites fluid of ovarian cancer patients and correlate with disease prognosis. Other kallikreins that have been related to ovarian cancer include KLK4, 5, 7, 8, 9, 11, 13, 14 and 15. We hypothesized that KLK6 and KLK10 can be utilized to monitor dissemination of ovarian cancer cells in blood and ascites fluid of ovarian cancer patients. METHODS: RNA was isolated by immunomagnetic separation of cancer cells and was amplified by RT-PCR. RESULTS: Screening for disseminated cancer cells in blood from 24 ovarian cancer patients, with RT-PCR for KLK6 mRNA, resulted in 75% positivity; however, this was not different from the positivity of normal controls. By utilizing KLK10 as a marker, the positivity of patients was 40% versus 20% of controls. Screening of ascites fluid of ovarian cancer patients revealed 90% positivity for KLK6 and KLK10 mRNA compared with 33% for other cancer types. Significant correlations were identified among mRNA of KLK4, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 15 in cancer cells isolated from ascites fluid. CONCLUSION: Kallikrein expression by ovarian cancer cells is not specific enough for detecting disseminated disease. Kallikrein expression may have some value for differentiating ovarian cancer from other types of cancer or from non-malignant diseases that lead to ascites accumulation.

Disease processes may be reflected by correlations among tissue kallikrein proteases but not with proteolytic factors uPA and PAI-1 in primary ovarian carcinoma.

In epithelial ovarian cancer, the high mortality rate is usually ascribed to late diagnosis, since these tumors commonly lack early-warning symptoms, but tumor-associated biomarkers useful for prognosis or therapy response prediction are in short supply. However, members of the tissue kallikrein serine protease family, the serine protease uPA and its inhibitor PAI-1, are associated with tumor progression of ovarian cancer. Therefore, we used ELISA to determine uPA, PAI-1, and tissue kallikreins hK5-8, 10, 11, and 13 in extracts of 142 primary tumor tissue specimens from ovarian cancer patients and studied the strength of association between protein expression levels of these tumor tissue-associated factors. uPA, PAI-1, hk5, and hk8 were related to FIGO stage; hK5 expression was higher in FIGO III/IV than in FIGO I/II patient tissues. PAI-1 and hk5 differed significantly according to nuclear grading; expression of hK5 was higher in G3 than in G1/2 tumors. Associations between uPA, PAI-1, and the tissue kallikreins were weak. There were strong pairwise correlations within the cluster of tissue kallikreins hK5, 6, 7, 8, 10, and 11, but their bivariate distributions depended on nuclear grading. These results support the notion that several tissue kallikreins are co-expressed in ovarian cancer patients, substantiating the existence of a steroid hormone-driven tissue kallikrein cascade in this disease.

Intron retention: a common splicing event within the human kallikrein gene family.

BACKGROUND: All human kallikrein (KLK) genes have at least one splice variant, some of which possess clinical utility in cancer diagnostics/prognostics. Given that introns <100 bp in length are retained in 95% of human genes and that splice variants of KLK3 and KLK4 retain intron III, we hypothesized that other proteins in this family, with a small intron III, may also retain it. METHODS: Variant-specific reverse transcription-PCRs (RT-PCRs) for KLK1, KLK2, KLK5, and KLK15 were used to identify and clone the full coding sequence of intron III-containing splice variants. In addition, variant-specific RT-PCRs for the cloned KLK3 and KLK4 variants as well as for the "classical" forms of the six genes were used to determine their expression profiles in healthy tissues, their regulation by steroids, and their differential expression in prostate cancer. RESULTS: KLK1, KLK2, KLK3, KLK4, KLK5, and KLK15 showed a common type of splice variant in which intron III is retained. Expression profiling of these splice variants revealed expression profiles similar to those of the classical mRNA forms, although the pattern of hormonal regulation was different. The KLK15 splice variant was up-regulated in 8 of 12 cancerous prostate tissues. All encoded variant proteins were predicted to be truncated and catalytically inactive because of a lack of the serine residue of the catalytic triad. CONCLUSIONS: The first six centromeric members of the KLK gene family have splice variants that retain intron III. Some variants show tissue-specific expression. The KLK15 splice variant appears to be a candidate biomarker for prostate cancer.

Human kallikrein 4: quantitative study in tissues and evidence for its secretion into biological fluids.

BACKGROUND: Human kallikrein 4 (hK4) is a proteolytic enzyme belonging to the tissue kallikrein family of serine proteases. Previous tissue expression studies have demonstrated highest KLK4 mRNA expression in prostatic tissue, but there has been only limited evidence for the presence of hK4 protein in prostate and other tissues and in corresponding biological secretions. METHODS: To investigate the concentrations of hK4 in tissues and biological fluids, we developed a new hK4-specific sandwich-type immunoassay using a monoclonal antibody as the capture reagent. RESULTS: The assay has a detection limit of 0.02 microg/L and <0.1% cross-reactivity toward any of the other 14 human kallikreins. Twelve of 40 tissue extracts prepared from various human tissues contained detectable hK4 concentrations (0.68-7143 ng/g of total protein), with healthy prostate tissue containing the highest amount of hK4. Examination of 16 malignant and 18 benign prostate tissues revealed no significant differences in hK4 protein content, and the tissues contained a wide range of values (benign, <0.02 to 801 ng/g; malignant, <0.02 to 824 ng/g). Among the biological fluids tested, seminal plasma and urine contained widely varying amounts of hK4; concentrations in 54 urine samples were <0.02 to 2.6 microg/L, whereas concentrations in 58 seminal plasma samples were 0.2-202 microg/L. Affinity purification of hK4 from seminal plasma and subsequent mass spectrometry demonstrated the secreted nature of hK4 in seminal plasma. CONCLUSIONS: hK4 is found primarily in prostate tissue and is secreted in seminal plasma. Its value as a novel prostatic biomarker needs to be defined further.

Human kallikrein 8: immunoassay development and identification in tissue extracts and biological fluids.

BACKGROUND: The serine protease human kallikrein 8 (hK8; neuropsin), a new member of the human kallikrein family, was predicted to be secreted; thus, it is expected to be present in biological fluids. The aim of this study was to develop a sensitive and specific immunoassay for hK8 (hK8-ELISA) and establish the distribution of hK8 in tissue extracts and biological fluids. METHODS: Recombinant hK8 was produced in a baculovirus expression system and purified with a three-step chromatographic procedure. Purified hK8 was injected into mice and rabbits for antibody generation. A highly specific and sensitive sandwich-type immunoassay (ELISA) was developed using the rabbit and mouse antisera to hK8. The hK8-ELISA was then used to study the distribution of hK8 in various biological fluids and tissue extracts. RESULTS: The dynamic range of the hK8-ELISA was 0.2 (detection limit) to 20 micro g/L, and imprecision (CV) was <10% within this range. This hK8-ELISA was specific for hK8 and had no detectable cross-reactivity with other members of the human kallikrein family. With this assay, hK8 was detected in tissue extracts of esophagus (highest concentrations), skin, testis, tonsil, kidney, breast, and salivary gland and in the biological fluids breast milk (highest concentrations), amniotic fluid, seminal plasma, and serum. Furthermore, in some cancer cell lines, the concentration of hK8 was regulated by steroid hormones. CONCLUSIONS: We report for the first time production of recombinant hK8 protein, generation of antibodies, and development of a highly sensitive and specific immunoassay for quantification of hK8 in tissue extracts and biological fluids. This assay can be used to explore the potential of hK8 as a marker of cancer or other conditions.

Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells.

Human kallikrein 6 (hK6), a trypsin-like serine protease, is a newly identified member of the kallikrein gene family. Its involvement in inflammatory CNS lesions and in demyelination has been reported. Recent work has suggested that expression of this enzyme is significantly elevated in patients with ovarian cancer. We have identified many tumour cell lines that secrete hK6, but its physiological role is unknown. Here, we try to unveil the role of this kallikrein in the metastasis and invasion of tumour cells. We demonstrate that purified human recombinant hK6 can cleave gelatin in zymography and can efficiently degrade high-molecular-weight extracellular matrix proteins such as fibronectin, laminin, vitronectin and collagen. In Boyden chamber assays, we found that tumour cells treated with a neutralizing hK6 antibody migrate less than control cells. We conclude that hK6 might play a role in the invasion and metastasis of tumour cells and may be a candidate therapeutic target.

Human kallikrein 13 involvement in extracellular matrix degradation.

The human kallikrein family is a group of 15 serine protease genes clustered on chromosome 19q13.4 and shares a high degree of homology. These proteolytic enzymes have diverse physiological functions in many different tissues. Growing evidence suggests that many kallikreins are differentially expressed in cancer and may play a role in metastasis. Human kallikrein gene 13 (KLK13) is a member of this family and codes for a trypsin-like, secreted serine protease (hK13) that is overexpressed in ovarian cancer patients. The aim of this study was to determine if hK13 can degrade extracellular matrix components. Recombinant hK13 was produced in yeast and purified using cation exchange and reverse-phase chromatography. The protein was used as an immunogen to generate mouse monoclonal antibodies. Enzymatic activity of hK13 was verified by using synthetic tri-peptide fluorogenic substrates and gelatin zymography. Active hK13 was incubated with biotinylated extracellular matrix (ECM) proteins and degradation was evaluated by Western blot analysis. hK13-secreting cancer cell lines were treated in a chemotaxis invasion chamber that was coated with various ECM proteins, to determine if hK13 plays a role in tumor cell migration and invasion. Assay with the synthetic substrates and zymography have shown that recombinant hK13 was enzymatically active. The Western blot results showed that hK13 was able to cleave the major components of the extracellular matrix. In the chemotaxis invasion chamber experiment, it was found that ovarian cancer cell lines that secreted hK13 and were treated with an hK13 neutralizing antibody migrated less than untreated cells. Human kallikrein13 may play a role in tissue remodeling and/or tumor invasion and metastasis. Targeting hK13 activity with neutralizing antibodies may have therapeutic applications.

Development of an immunofluorometric assay and quantification of human kallikrein 7 in tissue extracts and biological fluids.

BACKGROUND: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids. METHODS: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies. RESULTS: The assay had imprecision (CV) <10% through the dynamic range of 0.2-20 microg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine. CONCLUSIONS: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.