Immunofluorometric assay of human kallikrein 10 and its identification in biological fluids and tissues

BACKGROUND: The human kallikrein 10 gene [KLK10, also known as normal epithelial cell-specific 1 gene (NES1)] is a member of the human kallikrein gene family. The KLK10 gene encodes for a secreted serine protease (hK10). We hypothesize that hK10 is secreted into various biological fluids and that its concentration changes in some disease states. The aim of this study was to develop a sensitive and specific immunoassay for hK10. METHODS: Recombinant hK10 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-hK10 antisera. A sandwich-type immunofluorometric assay was then developed using these antibodies. RESULTS: The hK10 immunoassay has a detection limit of 0.05 microg/L. The assay is specific for hK10 and has no detectable cross-reactivity with other homologous kallikrein proteins, such as prostate-specific antigen (hK3), human glandular kallikrein 2 (hK2), and human kallikrein 6 (hK6). The assay was linear from 0 to 20 microg/L with within- and between-run CVs <10%. hK10 is expressed in many tissues, including the salivary glands, skin, and colon and is also detectable in biological fluids, including breast milk, seminal plasma, cerebrospinal fluid, amniotic fluid, and serum. CONCLUSIONS: We report development of the first immunofluorometric assay for hK10 and describe the distribution of hK10 in biological fluids and tissue extracts. This assay can be used to examine the value of hK10 as a disease biomarker.     

Human kallikrein 13: production and purification of recombinant protein and monoclonal and polyclonal antibodies,and development of a sensitive and specific immunofluorometric assay

BACKGROUND: The aims of this study were to develop immunologic reagents and a sensitive and specific immunoassay for human kallikrein 13 (hK13) and to examine the presence of hK13 in human tissues and biological fluids. METHODS: Recombinant hK13 protein was produced and purified with use of a Pichia pastoris yeast expression system. The protein was used as an immunogen to generate mouse monoclonal and rabbit polyclonal anti-hK13 antibodies. A sandwich-type immunoassay was developed with these antibodies. The assay was used to measure hK13 in various biological fluids and tissue extracts. Immunohistochemical analysis was also performed on nondiseased and cancerous prostatic sections. RESULTS: The hK13 immunoassay had a detection limit of 0.05 micro g/L and showed no cross-reactivity with homologous kallikreins. The assay was linear at 0-20 micro g/L, and within-and between-run CVs were <10% (n = 12). hK13 was detected in tissues, including esophagus, tonsil, trachea, lung, cervix, and prostate. hK13 was also found in seminal plasma, amniotic fluid, follicular fluid, ascites of ovarian cancer patients, breast milk, and cytosolic extracts of ovarian cancer tissues. hK13 was immunohistochemically localized in epithelial cells of both nondiseased and cancerous prostate. hK13 appears to be overexpressed in 50% of ovarian cancer tissues compared with healthy ovarian tissues. Recovery of active enzyme added to milk or amniotic fluid was 70-98%, but was <20% when added to serum, suggesting rapid sequestration by protease inhibitors. In fluids and tissue extracts, hK13 was found in its free (approximately 30 kDa) form. CONCLUSIONS: This immunofluorometric assay for hK13 may be used to examine the value of hK13 as a disease biomarker and to further explore the physiologic and pathobiologic role of this enzyme in human disease.     

Expression profile of human tissue kallikrein 15 provides preliminary insights into its roles in the prostate and testis

BackgroundHuman tissue kallikrein 15 (KLK15) is the latest member of the kallikrein-related peptidase family. Little is known about the pathophysiological roles of KLK15. Previous studies implied a role of KLK15 in prostate cancer.MethodsIn the present study, we examined KLK15 protein expression using a new immunoassay (ELISA) and immunohistochemistry (IHC).ResultsHighest KLK15 levels were detected in the testis and seminal fluid, whereas lower levels were observed in prostate and other tissues. Immunohistochemical analysis of testis suggests that KLK15 is strongly expressed in mature spermatids, but not in immature germ cells. KLK15 displayed predominantly nuclear localization in the basal cell layer of the prostatic epithelium. We also measured KLK15 in supernatants of various cell lines. Highest KLK15 levels were primarily detected in prostate cancer cell lines and KLK15 expression was hormone-independent, in contrast to KLK3.ConclusionsCollectively, our data provide insights into the localization and possible role of KLK15 in human physiology.     

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.     

Prognostic value of quantitatively assessed KLK7 expression in ovarian cancer

BACKGROUND: Among females, ovarian cancer is the sixth most common malignancy. Women with ovarian cancer have poor overall survival rates, largely because the disease is often diagnosed at an advanced, less curable stage. Several lines of evidence suggest that members of the kallikrein family are involved in various malignancies such as prostate (PSA, KLK2, KLK15), ovarian (KLK4, KLK5, KLK6, KLK8, KLK10), and breast cancer (KLK10, KLK13, KLK14). Recent evidence has indicated that expression of KLK7 appears to be increased in ovarian cancer. We hypothesized that overexpression of the KLK7 gene in ovarian cancer may serve as a prognostic marker of the disease. METHODS: Using the LightCycler technology we quantified the level of KLK7 mRNA expression in 125 ovarian tumors. Different disease stages and tumor grades were analyzed. Univariate and multivariate analyses were performed to establish the associations between clinicopathological parameters and KLK7 expression. RESULTS: We here report that patients with KLK7-negative tumors have a significantly higher disease-free survival than patients with KLK7-positive tumors. KLK7 expression levels were significantly higher in patients with grade 3 than in patients with grade 1 to 2 tumors (p = 0.030). KLK7 status also correlated with size of residual tumor postsurgery. KLK7 expression is an independent predictor of both disease-free and overall survival for patients with low grade tumors. In this subgroup of patients the hazard ratios for disease-free and overall survival were 3.28 and 3.09, respectively. Similarly, patients who had undergone optimal debulking but harbored KLK7-positive tumors had a high hazard ratio (HR) for relapse (HR = 8.2) and death (HR = 4.6). CONCLUSIONS: We conclude that higher KLK7 expression in ovarian cancer tissue is associated with poorer prognosis of ovarian cancer patients, especially those with lower grade disease and those who have been optimally debulked.     

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.     

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.     

Immunofluorometric assay of human kallikrein 6 (zyme/protease M/neurosin) and preliminary clinical applications

BACKGROUND: The human kallikrein gene family has contributed the best prostatic biomarkers currently available, including prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2). Recently, new members of the human kallikrein gene family have been identified. One new member is the KLK6 gene, encoding for human kallikrein 6 (hK6), which is also known as zyme/protease M/neurosin. In this paper, we describe development of antibodies and a sensitive immunofluorometric procedure for hK6 protein. METHODS: Recombinant hK6 protein was used as immunogen to develop polyclonal antibodies in rabbits and mice. These antibodies were used to develop a sandwich-type time-resolved immunofluorometric procedure for hK6. RESULTS: The newly developed hK6 immunofluorometric assay has a detection limit of 0.5 microg/L and upper concentration range of 200 microg/L. The assay is highly specific (no detectable cross-reactivity from PSA and hK2) and was used to quantify hK6 protein in various biologic fluids. Highest concentrations of hK6 were found in milk of lactating women, cerebral spinal fluid, nipple aspirate fluid, and breast cyst fluid. hK6 was also detected in male and female serum, in the majority of seminal plasmas and in a small fraction of amniotic fluids and breast tumor cytosols. hK6 was not detectable in urine. Chromatographic studies indicated that hK6 is present in these biologic fluids in its free, 30-kDa form. CONCLUSIONS: This is the first reported sensitive immunofluorometric procedure for quantifying hK6 protein. hK6 is a secreted proteolytic enzyme that is found at high levels in cerebrospinal fluid and all breast secretions. This assay will facilitate further studies to examine the possible application of hK6 in diagnostics, including cancer and neurodegenerative disorders.     

乳腺癌组织KLK7 mRNA的表达及其与临床病理特征的关系

目的建立人KLK7mRNA荧光定量检测方法,对正常乳腺组织和癌组织KLK7 mRNA进行定量,分析其表达与部分临床病理特征的关系,探讨其临床意义。方法应用实时荧光定量PCR检测48例乳腺癌切除的组织中KLK7基因mRNA的表达量,并分析KLK7 mRNA的表达量与年龄、TNM分期、组织学类型、淋巴结转移、雌激素受体（ER）、孕激素受体（PR）等临床病理特征的关系。结果KLK7 mRNA在乳腺正常组织相对表达水平为0．091±0．139,在乳腺癌组织中的相对表达水平为0．107±0．122,无统计学意义（P〉0．05）。但KLK7 mRNA在有淋巴结转移的患者乳腺癌组织中的表达水平高于未转移组（P〈0．05）;在Ⅲ-Ⅳ期患者乳腺癌组织中高于Ⅰ．Ⅱ期患者（P〈0．05）;ER（＋）阳性乳腺癌组织明显低于ER（-）乳腺癌组织（P〈0．05）。KLK7 mRNA表达水平与患者年龄、组织病理分类以及孕激素受体状态无相关性（P〉0．05）。结论乳腺癌组织KLK7 mRNA表达与TNM分期、ER状态及肿瘤转移情况有关。     

The tissue kallikrein family of serine proteases: functional roles in human disease and potential as clinical biomarkers

Prostate specific antigen (PSA) or human kallikrein 3 (hK3) has long been an effective biomarker for prostate cancer. Now, other members of the tissue kallikrein (KLK) gene family are fast becoming of clinical interest due to their potential as prognostic biomarkers. particularly for hormone dependent cancers. The tissue kallikreins are serine proteases that are encoded by highly conserved multi-gene family clusters in rodents and humans. The rat and mouse loci contain 10 and 25 functional genes, respectively, while the human locus at 19q 13.4 contains 15 genes. The structural organization and size of these genes are similar across species; all genes have 5 coding exons that encode a prepro-enzyme. Although the physiological activators of these zymogens have not been described, in vitro biochemical studies show that some kallikreins can auto-activate and others can activate each other, suggesting that the kallikreins may participate in an enzymatic cascade similar to that of the coagulation cascade. These genes are expressed, to varying degrees, in a wide range of tissues suggesting a functional involvement in a diverse range of physiological and pathophysiological processes. These include roles in normal skin desquamation and psoriatic lesions, tooth development, neural plasticity, and Alzheimer's disease (AD). Of particular interest is the expression of many kallikreins in prostate, ovarian, and breast cancers where they are emerging as useful prognostic indicators of disease progression.     

Human glandular kallikrein in breast milk, amniotic fluid, and breast cyst fluid.

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