Human tissue kallikreins: physiologic roles and applications in cancer

Tissue kallikreins are members of the S1 family (clan SA) of trypsin-like serine proteases and are present in at least six mammalian orders. In humans, tissue kallikreins (hK) are encoded by 15 structurally similar, steroid hormone-regulated genes (KLK) that colocalize to chromosome 19q13.4, representing the largest cluster of contiguous protease genes in the entire genome. hKs are widely expressed in diverse tissues and implicated in a range of normal physiologic functions from the regulation of blood pressure and electrolyte balance to tissue remodeling, prohormone processing, neural plasticity, and skin desquamation. Several lines of evidence suggest that hKs may be involved in cascade reactions and that cross-talk may exist with proteases of other catalytic classes. The proteolytic activity of hKs is regulated in several ways including zymogen activation, endogenous inhibitors, such as serpins, and via internal (auto)cleavage leading to inactivation. Dysregulated hK expression is associated with multiple diseases, primarily cancer. As a consequence, many kallikreins, in addition to hK3/PSA, have been identified as promising diagnostic and/or prognostic biomarkers for several cancer types, including ovarian, breast, and prostate. Recent data also suggest that hKs may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion, and, thus, may represent attractive drug targets to consider for therapeutic intervention.     

Human tissue kallikrein 5 is a member of a proteolytic cascade pathway involved in seminal clot liquefaction and potentially in prostate cancer progression

Human tissue kallikreins (hKs) are a family of fifteen serine proteases. Several lines of evidence suggest that hKs participate in proteolytic cascade pathways. Human kallikrein 5 (hK5) has trypsin-like activity, is able to self-activate, and is co-expressed in various tissues with other hKs. In this study, we examined the ability of hK5 to activate other hKs. By using synthetic heptapeptides that encompass the activation site of each kallikrein and recombinant pro-hKs, we demonstrated that hK5 is able to activate pro-hK2 and pro-hK3. We then showed that, following their activation, hK5 can internally cleave and deactivate hK2 and hK3. Given the predominant expression of hK2 and hK3 in the prostate, we examined the pathophysiological role of hK5 in this tissue. We studied the regulation of hK5 activity by cations (Zn2+, Ca2+, Mg2+, Na2+, and K+) and citrate and showed that Zn can efficiently inhibit hK5 activity at levels well below its normal concentration in the prostate. We also show that hK5 can degrade semenogelins I and II, the major components of the seminal clot. Semenogelins can reverse the inhibition of hK5 by Zn2+, providing a novel regulatory mechanism of its serine protease activity. hK5 is also able to internally cleave insulin-like growth factor-binding proteins 1, 2, 3, 4, and 5, but not 6, suggesting that it might be involved in prostate cancer progression through growth factor regulation. Our results uncover a kallikrein proteolytic cascade pathway in the prostate that participates in seminal clot liquefaction and probably in prostate cancer progression.     

Inhibition profiles of human tissue kallikreins by serine protease inhibitors

Accumulated evidence has shown that human tissue kallikreins (hKs), a group of 15 homologous secreted serine proteases, are novel cancer biomarkers. We report here the inhibition profiles of selected hKs, including hK5, hK7, hK8, hK11, hK12, hK13, and hK14, by several common serine protease inhibitors (serpins) found in plasma. The association constants for the binding of serpins to kallikreins were determined and compared. Protein C inhibitor was found to be the fastest-binding serpin for most of these hKs. alpha2-Antiplasmin, alpha1-antichymotrypsin, and alpha1-antitrypsin also showed rapid inhibition of certain hKs. Kallistatin exhibited fast inhibition only with hK7. Our data demonstrate that these hKs are specifically regulated by certain serpins and their distinct inhibition profiles will be valuable aids in various aspects of kallikrein research.     

Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.     

Human kallikrein 13 expression in normal tissues: an immunohistochemical study.

The human tissue kallikrein 13 gene (KLK13), encoding for hK13 protein, was recently cloned and characterized. Here we describe the immunohistochemical (IHC) localization of hK13 in normal human tissues and compare it with the expression of two other kallikreins, hK6 and hK10. We performed the streptavidin-biotin IHC method on 204 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue, using a polyclonal and a monoclonal hK13 antibody. The staining was cytoplasmic and both antibodies yielded similar results. The hK13 protein was revealed in a variety of tissues, mainly in glandular epithelia. Other epithelia that expressed hK13 included the urothelium, the spermatic epithelium, and the epithelium of the choroid plexus. hK13 was intensely immunoexpressed by some endocrine organs, such as the adenohypophysis, the thyroid gland, the parathyroid glands, the adrenal medulla, the Leydig cells of the testis, and the cells of the endocrine pancreas. Immunoreactivity was also observed in the primordial follicles, the corpus luteum, and sparse luteinized cells in the stroma of the ovary, the trophoblastic cells of the placenta, the Hassall's corpuscles of the thymus, and chondrocytes. Nerves and ganglia of the peripheral nervous system, and both neurons and glial cells in the central nervous system, were positive. In short, hK13 was expressed by many glandular epithelia, some endocrine organs, and some specialized epithelia and cells. Comparison of these data with hK6 and hK10 expression suggests that the three kallikreins have a similar IHC pattern in normal human tissues.     

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.     

Kallikrein-mediated cell signalling: targeting proteinase-activated receptors (PARs)

We tested the hypothesis that human tissue kallikreins (hKs) may regulate signal transduction by cleaving and activating proteinase-activated receptors (PARs). We found that hK5, 6 and 14 cleaved PAR N-terminal peptide sequences representing the cleavage/activation motifs of human PAR1 and PAR2 to yield receptor-activating peptides. hK5, 6 and 14 activated calcium signalling in rat PAR2-expressing (but not background) KNRK cells. Calcium signalling in HEK cells co-expressing human PAR1 and PAR2 was also triggered by hK14 (via PAR1 and PAR2) and hK6 (via PAR2). In isolated rat platelets that do not express PAR1, but signal via PAR4, hK14 also activated PAR-dependent calcium signalling responses and triggered aggregation. The aggregation response elicited by hK14 was in contrast to the lack of aggregation triggered by hK5 and 6. hK14 also caused vasorelaxation in a phenylephrine-preconstricted rat aorta ring assay and triggered oedema in an in vivo model of murine paw inflammation. We propose that, like thrombin and trypsin, the kallikreins must now be considered as important 'hormonal' regulators of tissue function, very likely acting in part via PARs.     

Human kallikrein 10 expression in normal tissues by immunohistochemistry.

The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was recently cloned and encodes for a putative secreted serine protease (human kallikrein 10, hK10). Several studies have confirmed that hK10 shares many similarities with the other kallikrein members at the DNA, mRNA, and protein levels. The enzyme was found in biological fluids, tissue extracts, and serum. Here we report the first detailed immunohistochemical (IHC) localization of hK10 in normal human tissues. We used the streptavidin-biotin method with two hK10-specific antibodies, a polyclonal rabbit and a monoclonal mouse antibody, developed in house. We analyzed 184 paraffin blocks from archival, current, and autopsy material, prepared from almost every normal human tissue. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. Previously, we reported the expression of another novel human kallikrein, hK6, by using similar techniques. The IHC expression of hK10 was generally cytoplasmic and not organ-specific. A variety of normal human tissues expressed the protein. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, epididymis, endometrium, fallopian tubes, gastrointestinal tract, bronchus, salivary glands, bile ducts, and gallbladder. The choroid plexus epithelium, the peripheral nerves, and some neuroendocrine organs (including the islets of Langerhans, cells of the adenohypophysis, the adrenal medulla, and Leydig cells) expressed the protein strongly and diffusely. The spermatic epithelium of the testis expressed the protein moderately. A characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells of the thyroid and parathyroid glands, and chondrocytes. Comparing these results with those of hK6, we observed that both kallikreins had a similar IHC expression pattern.     

Immunohistochemical localization of human kallikreins 6 and 10 in pancreatic islets

Tissue kallikreins are thought to be present in the pancreatic islets of Langerhans and to aid in the conversion of proinsulin to insulin. In recent immunohistochemical studies, we observed strong staining of the newly identified human kallikreins 6 and 10 (hK6 and hK10) in the islets of Langerhans. Here, we examine hK6 and hK10 immunoexpression in different types of islet cells of the endocrine pancreas, in order to obtain clues for hK6 and hK10 function in these cells. Ten cases of normal pancreatic tissue, two cases of nesidioblastosis, five insulin-producing tumours and one case of multiple endocrine neoplasia 1 syndrome, containing an insulin-, a somatostatin- and several glucagon-producing tumours, as well as tiny foci of endocrine dysplasia with different predominance of the secreted hormones (mainly glucagon and pancreatic polypeptide) were included in the study. A streptavidin–biotin–peroxidase and an alkaline phosphatase protocol, as well as a sequential immunoenzymatic double staining method were performed, using specific antibodies against hK6, hK10, insulin, glucagon, somatostatin, pancreatic polypeptide, and serotonin. hK6 and hK10 immunoexpression was observed in the islets of Langerhans, including the pancreatic polypeptide-rich islets, in the normal pancreas. Scattered hK6 and hK10 positive cells were localized in relationship with pancreatic acinar cells. In the exocrine pancreas, a cytoplasmic and/or brush border hK6 and hK10 immunoexpression was observed in the median and small sized pancreatic ducts, while the acinar cells were negative. Foci of nesidioblastosis and endocrine dysplasia expressed both kallikreins. hK6 and hK10 were also strongly and diffusely expressed throughout all insulin-, glucagon- and somatostatin-producing tumours. The double staining method revealed co-localization of each hormone and hK6/hK10 respectively, in the same cellular population, in the normal as well as in the diseased pancreas. Our results support the view that hK6 and hK10 may be involved in insulin and other pancreatic hormone processing and/or secretion, as well as in physiological functions related to the endocrine pancreas.     

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.     

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.     

Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells

The human tissue kallikrein family of serine proteases (hK1-hK15 encoded by the genes KLK1-KLK15) is involved in several cancer-related processes. Accumulating evidence suggests that certain tissue kallikreins are part of an enzymatic cascade pathway that is activated in ovarian cancer and other malignant diseases. In the present study, OV-MZ-6 ovarian cancer cells were stably co-transfected with plasmids expressing hK4, hK5, hK6, and hK7. These cells displayed similar proliferative capacity as the vector-transfected control cells (which do not express any of the four tissue kallikreins), but showed significantly increased invasive behavior in an in vitro Matrigel invasion assay (p<0.01; Mann-Whitney U-test). For in vivo analysis, the cancer cells were inoculated into the peritoneum of nude mice. Simultaneous expression of hK4, hK5, hK6, and hK7 resulted in a remarkable 92% mean increase in tumor burden compared to the vector-control cell line. Five out of 14 mice in the 'tissue kallikrein overexpressing' group displayed a tumor/situs ratio greater than 0.198, while this weight limit was not exceeded at all in the vector control group consisting of 13 mice (p=0.017; chi2 test). Our results strongly support the view that tumor-associated overexpression of tissue kallikreins contributes to ovarian cancer progression.     

Effects of long-term androgen administration on breast tissue of female-to-male transsexuals.

Our aim was to examine the effects of androgen administration on breast tissue histology of female-to-male transsexuals and to study the immunohistochemical expression of three human tissue kallikreins, hK3 (PSA), hK6, and hK10. We studied 23 female-to-male transsexuals who were treated with injectable testosterone for 18-24 months. We also used 10 control female breast tissues. All tissues were fixed in buffered formalin, embedded in paraffin, and examined by hematoxylin-eosin staining and immunohistochemical staining for PSA, hK6, and hK10. Females treated with androgens exhibited similar involutionary changes as those seen in breast of menopausal women, such as marked reduction of glandular tissue, involution of the lobuloalveolar structures, and prominence of fibrous connective tissue, but presence of only small amounts of fat tissue. Fibrocystic lesions were generally not observed. In immunohistochemistry, in control breast tissues, we found moderate to strong cytoplasmic immunoexpression of hK6 and hK10 in the epithelial ductal and lobuloalveolar structures, but myoepithelial cells were negative. Luminal secretions were also positive. In menopausal breast, the immunoexpression of hK6 and hK10 was weaker and focal. No control case showed immunoexpression for PSA. In female-to-male transsexuals, one case showed focal PSA cytoplasmic immunoexpression in the epithelium of moderately involuting lobules. Long-term administration of androgens in female-to-male transsexuals causes marked reduction of glandular tissue and prominence of fibrous connective tissue. These changes are similar to those observed at the end-stage of menopausal mammary involution.     

The role of human tissue kallikreins 7 and 8 in intracranial malignancies

Recent evidence suggests that many tissue kallikreins are implicated in carcinogenesis. Kallikrein 8 (KLK8) plays a role in the physiology of the central nervous system. Kallikrein 7 (KLK7) takes part in skin desquamation. Both show altered expression in ovarian and breast cancer. In this study, we examined the level of mRNA expression of the KLK7 and KLK8 genes in 73 intracranial tumors using qualitative RT-PCR. The results were correlated with clinical and histomorphological variables and patient outcome. The expression of both genes was also examined in the brain cancer cell lines U-251 MG, D54 and SH-SY5Y and the invasive capacity of glioblastoma cells U-251 MG overexpressing hK7 or hK8 was also investigated in an in vitro Matrigel assay. Follow-up analysis revealed that expression of KLK7 mRNA was associated with shorter overall survival (OS) compared to patients with no KLK7 expression, as determined by Cox proportional hazard regression analysis. Overexpression of hK7 protein by cultivated brain tumor cells significantly enhanced the invasive potential in the Matrigel invasion assay, in contrast to cells overexpressing hK8 protein. Our data suggest that hK7 protein overexpression is associated with a more aggressive phenotype in brain cancer cells.     

Proteinase-activated receptors, targets for kallikrein signaling

Serine proteinases like thrombin can signal to cells by the cleavage/activation of proteinase-activated receptors (PARs). Although thrombin is a recognized physiological activator of PAR(1) and PAR(4), the endogenous enzymes responsible for activating PAR(2) in settings other than the gastrointestinal system, where trypsin can activate PAR(2), are unknown. We tested the hypothesis that the human tissue kallikrein (hK) family of proteinases regulates PAR signaling by using the following: 1) a high pressure liquid chromatography (HPLC)-mass spectral analysis of the cleavage products yielded upon incubation of hK5, -6, and -14 with synthetic PAR N-terminal peptide sequences representing the cleavage/activation motifs of PAR(1), PAR(2), and PAR(4); 2) PAR-dependent calcium signaling responses in cells expressing PAR(1), PAR(2), and PAR(4) and in human platelets; 3) a vascular ring vasorelaxation assay; and 4) a PAR(4)-dependent rat and human platelet aggregation assay. We found that hK5, -6, and -14 all yielded PAR peptide cleavage sequences consistent with either receptor activation or inactivation/disarming. Furthermore, hK14 was able to activate PAR(1), PAR(2), and PAR(4) and to disarm/inhibit PAR(1). Although hK5 and -6 were also able to activate PAR(2), they failed to cause PAR(4)-dependent aggregation of rat and human platelets, although hK14 did. Furthermore, the relative potencies and maximum effects of hK14 and -6 to activate PAR(2)-mediated calcium signaling differed. Our data indicate that in physiological settings, hKs may represent important endogenous regulators of the PARs and that different hKs can have differential actions on PAR(1), PAR(2), and PAR(4).     

The spectrum of human kallikrein 6 (zyme/protease M/neurosin) expression in human tissues as assessed by immunohistochemistry.

The KLK6 gene is a new member of the human kallikrein gene family and encodes for a secreted protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin). No study has as yet reported detailed immunohistochemical localization of hK6 in human tissues. Our purpose was to examine the expression of hK6 in human tissues by immunohistochemistry. We have analyzed 199 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue. We employed an hK6-specific polyclonal rabbit antibody and avidin-biotin to localize hK6 by IHC. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. The IHC expression of zyme was generally cytoplasmic. Various normal human tissues expressed the protein abundantly. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, endometrium, colon, appendix, salivary glands, bile ducts, and gallbladder. The small intestine, stomach, endocervix, Fallopian tube, epididymis, bronchus, and upper respiratory tract showed a focal expression as well. Choroid plexus epithelium, peripheral nerves, and some neuroendocrine cells (including the islets of Langerhans, cells in the anterior pituitary gland, and adrenal medulla) expressed the protein strongly and diffusely. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells of the thyroid and parathyroid glands, the primordial follicles of the ovary, dendritic cells mainly in the spleen, and in various cells of the placenta.     

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.     

Human kallikrein 13 expression in salivary gland tumors

The human kallikrein 13 protein (hK13) is expressed in many normal tissues. Petraki et al have previously described presence of hK13 in salivary gland tissue, localized to duct epithelia and some acinar cells. The aim of this study was to determine whether hK13 is expressed in salivary gland tissues and salivary gland tumors (both benign and malignant), in order to compare normal with tumor tissues. Pleomorphic adenomas (PA), adenoid cystic carcinomas (ACC), polymorphous low grade adenocarcinomas (PLGA), acinic cell carcinomas (ACI), mucoepidermoid carcinomas (MEC) and adenocarcinomas not otherwise specified (ANOS) of both minor and major salivary glands were examined. The results of this study indicate that most salivary gland tumors show high levels of expression of hK13. Overall, staining in PA was significantly less than that seen in normal salivary gland tissue. PLGA, ACC and ANOS each stained significantly more than normal salivary gland tissue while MEC and ACI did not. Ductal cells and cells lining duct-like structures showed a higher intensity of staining than non-ductal cells in most tumors. Tumors which exhibited only non-ductal cells also exhibited cytoplasmic staining. In conclusion, we demonstrate the high expression of hK13 in several common salivary gland tumors.     

Novel expression of kallikreins, kallikrein-related peptidases and kinin receptors in human pleural mesothelioma

Malignant mesothelioma is an aggressive cancer of the pleura that is causally related to exposure to asbestos fibres. The kallikrein serine proteases [tissue (hK1) and plasma (hKB1) kallikreins, and kallikrein-related peptidases (KRP/hK2-15)] and the mitogenic kinin peptides may have a role in tumourigenesis. However, it is not known whether hK1, hKB1, KRP/hK proteins or kinin receptors are expressed in pleural mesotheliomas. The expression of hK1, hKB1, KRP/hK2, 5, 6, 7, 8 and 9, and kinin B(1) and B(2) receptors was assessed in archived selected normal tissue and mesothelioma tumour sections by immunoperoxidase and immunofluorescence labelling. hK1, hKB1 and kinin B(1) and B(2) receptors were expressed in malignant cells of the epithelioid and sarcomatoid components of biphasic mesothelioma tumour cells. The percentage of cells with cytoplasmic and nuclear labelling and the intensity of labelling were similar for hK1, hKB1 and the kinin receptors. KRP/hK2, 6, 8 and 9 were also expressed in the cytoplasm and nuclei of mesothelioma cells, whereas KRP/hK5 and hK7 showed predominantly cytoplasmic localisation. This is a first report, but further studies are required to determine whether these proteins have a functional role in the pathogenesis of mesothelioma and/or may be potential biomarkers for pleural mesothelioma.     

Expression of tissue and plasma kallikreins and kinin B1 and B2 receptors in lung cancer

Abstract Tissue kallikrein (hK1) and plasma kallikrein (PK, hKB1) are serine proteases that produce biologically active kinin peptides from endogenous kininogen substrates. There is evidence linking the kallikreins and the mitogenic kinin peptides to carcinogenesis. The aim of this study was to investigate the expression of tissue prokallikrein (pro-hK1), plasma prekallikrein (PPK, pre-hKB1) and kinin B(1) and B(2) receptor proteins in different subtypes of lung cancer. Immunohistochemistry, using specific antibodies, was performed on archived normal lung sections and sections from adenocarcinomas, squamous cell carcinomas, large cell carcinomas, small cell carcinomas and carcinoid tumours of the lung. Immunoperoxidase labelling was visualised by brightfield microscopy and immunofluorescence labelling by confocal microscopy. Extensive cytoplasmic expression of pro-hK1 and PPK was observed, which was similar in small cell and non-small cell tumours. However, nuclear labelling for the kallikreins was absent or limited. The kinin B(1) and B(2) receptors were highly expressed in the cytoplasm of all tumour types and in the nuclei of non-small cell tumours. Further studies are required to assess the functional significance of the expression of hK1, PK and kinin receptors in lung tumours, and whether any of these proteins may be potential biomarkers for specific subtypes of lung carcinoma.