Glucagon response to oral glucose challenge in type 1 diabetes: lack of impact of euglycemia.

OBJECTIVE Previous studies have demonstrated aberrant glucagon physiology in the setting of type 1 diabetes (T1D) but have not addressed the potential impact of ambient glycemia on this glucagon response. Thus, our objective was to evaluate the impact of euglycemia versus hyperglycemia on the glucagon response to an oral glucose challenge in T1D. RESEARCH DESIGN AND METHODS Ten adults with T1D (mean age 56.6 ± 9.0 years, duration of diabetes 26.4 ± 7.5 years, HbA1c 7.5% ± 0.77, and BMI 24.1 kg/m(2) [22.6-25.4]) underwent 3-h 50-g oral glucose tolerance tests (OGTTs) on two separate days at least 24 h apart in random order under conditions of pretest euglycemia (plasma glucose [PG] between 4 and 6 mmol/L) and hyperglycemia (PG between 9 and 11 mmol/L), respectively. RESULTS Glycemic excursion on the OGTT was similar between the euglycemic and hyperglycemic tests (P = 0.72 for interaction between time postchallenge and glycemic setting). Interestingly, glucagon levels increased in response to the OGTT under both glycemic conditions (P < 0.001) and there were no differences in glucagon response between the euglycemic and hyperglycemic days (P = 0.40 for interaction between time postchallenge and glycemic setting). In addition, the incretin responses to the OGTT (glucose-dependent insulinotropic polypeptide, glucagon-like peptide-1, glucagon-like peptide-2) were also not different between the euglycemic and hyperglycemic settings. CONCLUSIONS In patients with T1D, there is a paradoxical increase in glucagon in response to oral glucose that is not reversed when euglycemia is achieved prior to the test. This abnormal glucagon response likely contributes to the postprandial hyperglycemia in T1D irrespective of ambient glycemia.

Determinants of insulin resistance in infants at age 1 year: impact of gestational diabetes mellitus.

OBJECTIVE: The offspring of women with gestational diabetes mellitus (GDM) display a propensity for the early accrual of cardiometabolic risk factors, including insulin resistance, in childhood and adolescence. Thus, we sought to identify early life determinants of insulin resistance in infants of women with and without GDM. RESEARCH DESIGN AND METHODS: In total, 104 full-term, singleton infants born to women with (n = 36) and without (n = 68) GDM were evaluated at age 1 year, with insulin resistance assessed by homeostasis model (HOMA-IR). RESULTS: HOMA-IR at 1 year did not differ between infants born to mothers with and without GDM (P = 0.74). The sole independent predictor of infant HOMA-IR in the non-GDM group was birth weight (t = 3.33, P = 0.002). In contrast, weight gain in the 1st year was the only independent predictor of HOMA-IR in infants of women with GDM (t = 2.19, P = 0.039). CONCLUSIONS: In the 1st year of life, weight gain in infants born to women with GDM is associated with insulin resistance, unlike in their peers.

Insulins: past, present, and future.

This article highlights selected milestones in insulin discovery and its continued development as a pivotal therapy for diabetes. The last 90 years have witnessed tremendous progress in insulin therapy, from the initial crude, yet life-saving, animal insulin extracts to novel human insulin analogues. Although the complete physiologic replacement of insulin is inherently difficult to achieve with open-loop subcutaneously administered insulin, the continued development of improved injectable insulin formulations with superior pharmacokinetics and pharmacodynamics will enhance glucose control, and represents important clinical advances in the treatment of both type 1 and type 2 diabetes.

Defining the extended substrate specificity of kallikrein 1-related peptidases.

Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1-P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2-P4 specificities, attributable to structural variations in their surface loops. The preferred P4-P1 substrate recognition motifs based on optimal subsite occupancy were as follows: VI-QSAV-QL-R for KLK4; YFWGPV-RK-NSFAM-R for KLK5; VY-R-LFM-R for KLK13; and YW-KRSAM-HNSPA-R for KLK14. Protein database queries using these motifs yielded many extracellular targets, some of which represent plausible KLK substrates. For instance, cathelicidin, urokinase-type plasminogen activator, laminin and transmembrane protease serine 3 were retrieved as novel putative substrates for KLK4, 5, 13 and 14, respectively. Our findings may facilitate studies on the role of KLKs in (patho)physiology and can be used in the development of selective KLK inhibitors.

Proteolytic processing of human growth hormone by multiple tissue kallikreins and regulation by the serine protease inhibitor Kazal-Type5 (SPINK5) protein.

BACKGROUND: Human growth hormone (hGH) is naturally present in numerous isoforms, some of which arise from proteolytic processing in both the pituitary and periphery. The nature of the enzymes that proteolytically cleave hGH and the regulation of this process are not fully understood. Our objective is to examine if members of a newly discovered human tissue kallikrein family (KLKs) are expressed in the pituitary and if these enzymes can cleave hGH in-vitro. METHODS: Expression of 12 of the KLKs (KLKs 4-15) and serine protease inhibitor Kazal-type 5 (SPINK5) genes and their proteins in the pituitary was examined by RT-PCR and immunohistochemistry. Recombinant hGH was digested by various recombinant KLKs and fragments were characterized by N-terminal sequencing. SPINK5 recombinant fragments were used for inhibition of KLK activities. RESULTS: We here describe for the first time expression of numerous KLKs (KLKs 5-8, 10-14) and SPINK5 in the pituitary. KLK6 and SPINK5 appeared to be localized to hGH-producing cells. KLKs 4-6, 8, 13 and 14 were able to cleave hGH, yielding various isoforms, in vitro. Inhibitor SPINK5 fragments were able to suppress activity of KLKs 4, 5 and 14 in vitro. Based on these data, we propose a model for the proteolytic processing of hGH in the pituitary and the regulation of this system by SPINK5 inhibitory domains. We speculate that loss of SPINK5 inhibitory domains, as in the case of Netherton syndrome, may lead to proteolytic over-processing of hGH and to growth retardation. CONCLUSION: We conclude that many KLKs and SPINK5 are expressed in the pituitary. This serine protease-inhibitor system is likely to participate in the regulated proteolytic processing of hGH in the pituitary, leading to generation of hGH fragments. Our data suggest that KLKs 5, 6 and 14 might be involved in this process.

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.

A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation.

Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1-6 (rLEKTI(1-6)), domains 6-8 and partial domain 9 (rLEKTI(6-9')), domains 9-12 (rLEKTI(9-12)), and domains 12-15 (rLEKTI(12-15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1-6), rLEKTI(6-9'), and rLEKTI(9-12) with Ki values in the range of 2.3-28.4 nm, 6.1-221 nm, and 2.7-416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12-15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated.

Mutant recombinant serpins as highly specific inhibitors of human kallikrein 14.

The reactive center loop (RCL) of serpins plays an essential role in the inhibition mechanism acting as a substrate for their target proteases. Changes within the RCL sequence modulate the specificity and reactivity of the serpin molecule. Recently, we reported the construction of alpha1-antichymotrypsin (ACT) variants with high specificity towards human kallikrein 2 (hK2) [Cloutier SM, Kündig C, Felber LM, Fattah OM, Chagas JR, Gygi CM, Jichlinski P, Leisinger HJ & Deperthes D (2004) Eur J Biochem271, 607-613] by changing amino acids surrounding the scissile bond of the RCL and obtained specific inhibitors towards hK2. Based on this approach, we developed highly specific recombinant inhibitors of human kallikrein 14 (hK14), a protease correlated with increased aggressiveness of prostate and breast cancers. In addition to the RCL permutation with hK14 phage display-selected substrates E8 (LQRAI) and G9 (TVDYA) [Felber LM, Borgoño CA, Cloutier SM, Kündig C, Kishi T, Chagas JR, Jichlinski P, Gygi CM, Leisinger HJ, Diamandis EP & Deperthes D (2005) Biol Chem386, 291-298], we studied the importance of the scaffold, serpins alpha1-antitrypsin (AAT) or ACT, to confer inhibitory specificity. All four resulting serpin variants ACT(E8), ACT(G9), AAT(E8) and AAT(G9) showed hK14 inhibitory activity and were able to form covalent complex with hK14. ACT inhibitors formed more stable complexes with hK14 than AAT variants. Whereas E8-based inhibitors demonstrated a rather relaxed specificity reacting with various proteases with trypsin-like activity including several human kallikreins, the two serpins variants containing the G9 sequence showed a very high selectivity for hK14. Such specific inhibitors might prove useful to elucidate the biological role of hK14 and/or its implication in cancer.

Human kallikrein 8 protein is a favorable prognostic marker in ovarian cancer.

Human kallikrein 8 (hK8/neuropsin/ovasin; encoded by KLK8) is a steroid hormone-regulated secreted serine protease differentially expressed in ovarian carcinoma. KLK8 mRNA levels are associated with a favorable patient prognosis and hK8 protein levels are elevated in the sera of 62% ovarian cancer patients, suggesting that KLK8/hK8 is a prospective biomarker. Given the above, the aim of the present study was to determine if tissue hK8 bears any prognostic significance in ovarian cancer. Using a newly developed ELISA, hK8 was quantified in 136 ovarian tumor extracts and correlated with clinicopathologic variables and outcome [progression-free survival (PFS); overall survival (OS)] over a median follow-up period of 42 months. hK8 levels in ovarian tumor cytosols ranged from 0 to 478 ng/mg total protein, with a median of 30 ng/mg. An optimal cutoff value of 25.8 ng/mg total protein (74th percentile) was selected based on the ability of hK8 values to predict the PFS of the study population and to categorize tumors as hK8 positive or negative. Women with hK8-positive tumors most often had lower-grade tumors (G1), no residual tumor after surgery, and optimal debulking success (P < 0.05). Univariate and multivariate analyses revealed that patients with hK8-positive tumors had a significantly longer PFS and OS than hK8-negative patients (P < 0.05). Kaplan-Meier survival curves further confirmed a reduced risk of relapse and death in women with hK8-positive tumors (P = 0.001 and P = 0.014, respectively). These results indicate that hK8 is an independent marker of favorable prognosis in ovarian cancer.

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology.

The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.

The emerging roles of human tissue kallikreins in cancer.

Human tissue kallikreins (hKs), which are encoded by the largest contiguous cluster of protease genes in the human genome, are secreted serine proteases with diverse expression patterns and physiological roles. Although primarily known for their clinical applicability as cancer biomarkers, recent evidence implicates hKs in many cancer-related processes, including cell-growth regulation, angiogenesis, invasion and metastasis. They have been shown to promote or inhibit neoplastic progression, acting individually and/or in cascades with other hKs and proteases, and might represent attractive targets for therapeutic intervention.

Cloning of a kallikrein pseudogene.

OBJECTIVES: Kallikreins are a group of serine proteases clustered together on a small region of chromosome 19q13.4. Recent reports suggest that kallikreins are differentially expressed in malignancy and have potential as cancer biomarkers. The human kallikrein gene locus has now been fully characterized and 15 functional kallikreins were identified. Although many kallikrein pseudogenes have already been characterized in rodents, none have been identified in humans. METHODS AND RESULTS: In the current study, we identified the first human kallikrein pseudogene named PsiKLK1 and mapped it between the KLK2 and KLK4 genes. This pseudogene shares a moderate degree of similarity with the adjacent functional kallikreins. It has a conserved histidine residue of the catalytic triad of serine proteases and its surrounding motif, but lacks the aspartate and serine residues. Positions of some cysteine residues are also conserved in the pseudogene. This pseudogene lacks intronic sequences and should thus be classified as a processed pseudogene. EST and PCR analyses indicate that this pseudogene may be transcriptionally active, because mRNA was detected in many tissues including the prostate, testis, pituitary, and adrenal glands, as well as in tissues of the female genital organs. DISCUSSION: The mRNA sequence of the gene is, however, defective and is not predicted to code for a protein. Highly conserved sequences were found in the flanking region of the pseudogene, thus supporting the view that it evolved by retrotransposition. We also identified another serine protease fragment that has only the conserved histidine residue. The functional significance of the pseudogene and the other fragment is yet to be identified.

Molecular cloning of a new gene which is differentially expressed in breast and prostate cancers.

OBJECTIVE: The chromosomal region 19q13 is non-randomly rearranged in many solid tumors. METHODS: Using the positional candidate gene approach, we cloned a new gene, tentatively named cancer-associated gene (CAG), which is differentially expressed in breast and prostate cancers. RESULTS: The gene is formed of 3 exons and 2 intervening introns. Its coding region is 1,047 bp in length and is predicted to encode a 348-amino-acid polypeptide. The new gene maps to chromosome 19q13.4 and is located 14 kb telomeric to the kallikrein gene locus (KLK14 gene) and 17 kb centromeric from the Siglec family of genes (Siglec-9). The gene is expressed in a wide variety of tissues including the brain, colon, kidney and pancreas. The CAG protein shows a high degree of conservation among species and phylogenetically is most closely related to its mouse ortholog. In silico analysis indicates that this gene is differentially expressed in a variety of tumors including brain, colon, ovarian and prostate cancers. CONCLUSIONS: Our preliminary experimental data show that CAG is upregulated in prostate cancer tissues compared to normal prostatic tissues. CAG also appears to be downregulated in breast cancer tissues. The physiological function of the CAG protein is currently unknown.

Human kallikrein 11: an indicator of favorable prognosis in ovarian cancer patients.

OBJECTIVES: Human kallikrein 11 (hK11) is a secreted serine protease, highly expressed in hormonally regulated tissues, including the prostate and the ovary. Our preliminary studies indicate that hK11 may represent a diagnostic and prognostic biomarker for ovarian cancer. The aim of the present study was to examine the prognostic value of hK11 expression in ovarian tumors. METHODS: Using our established immunofluorometric assay, hK11 levels were quantified (ng per mg of total protein) in 134 ovarian tumor extracts and correlated with various clinicopathological variables and outcome [progression-free survival (PFS), overall survival (OS)], over a median follow-up period of 42 months. RESULTS: hK11 concentration in ovarian tumor cytosols ranged from 0 to 155 ng/mg of total protein, with a median of 1.45 ng/mg. An optimal cutoff value of 6.3 ng/mg was selected to categorize tumors as hK11-positive or negative. hK11-positive tumors were most often of early stage (Stage I/II) and grade (G1/G2) (P < 0.05). Univariate analysis revealed that patients with hK11-positive tumors had a significantly longer PFS (HR of 0.39, P = 0.005) and OS (HR of 0.44, P = 0.033). Cox multivariate analysis indicated that hK11 was an independent prognostic indicator of PFS (HR of 0.47, P = 0.042). Kaplan-Meier survival curves further confirmed that women with hK11-positive tumors have longer PFS and OS (P = 0.003 and P = 0.028, respectively). Also, a weak positive correlation was found between the expression levels of tissue hK11 and tissue CA125 (rs = 0.508; P < 0.001). CONCLUSIONS: These results further validate our initial findings that hK11 is an independent marker of favorable prognosis in ovarian cancer patients.

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.

In-silico analysis of kallikrein gene expression in pancreatic and colon cancers.

Human kallikreins are a cluster of 15 serine protease genes located in the chromosomal band 19q13.4, a non-randomly rearranged region in many solid tumors, including pancreatic cancer. We utilized the SAGE and EST databases of the Cancer Genome Anatomy Project to perform in-silico analysis of kallikrein gene expression in normal and cancerous pancreatic and colon tissues and cell lines using virtual Northern blotting (VNB), digital differential display (DDD) and X-profiler. At least two kallikreins, KLK6 and KLK10, are significantly up-regulated in pancreatic cancer. We probed 2 normal and 6 pancreatic cancer SAGE libraries with gene-specific tags for each of these kallikreins. KLK6 was found to be expressed in 5/6 cancer libraries and showed the most marked (5-fold) increase in average expression levels in cancer vs. normal. These data were verified by screening the EST databases, where all mRNA clones isolated were from cancerous libraries, with no clones detected in normal pancreatic tissues or cell lines. X-profiler comparison of two pools of normal and cancerous pancreatic libraries further verified the significant increase of KLK6 expression levels in pancreatic cancer. DDD data showed a 13-fold increase in KLK10 expression in pancreatic cancer. Three kallikrein genes, KLK6, 8 and 10 are overexpressed in colon cancer compared to normal colon, while one kallikrein, KLK1, is down-regulated. While no expression of KLK6 was detected in normal colon, KLK6-specific tags were detectable in 2 cancer libraries. Similar results were obtained by EST screening; no KLK6 clones were detected in any of the 28 normal libraries examined, while 10 KLK6 EST clones were found in colon adenocarcinoma. KLK10 was not detectable in normal colon. Gene-specific tags were, however, detectable with high density in colon cancer and 7 EST clones were found to be expressed in colon Adenocarcinoma.

Human kallikrein 13 protein in ovarian cancer cytosols: a new favorable prognostic marker.

PURPOSE: Human kallikrein 13 (hK13; encoded by the KLK13 gene) is a secreted serine protease expressed in endocrine tissues, including the prostate, testis, breast, and ovary. We have previously reported steroid hormone regulation of the KLK13 gene and its clinical value as a marker of favorable prognosis in breast cancer at the mRNA level. We hypothesized that hK13 may represent a potential biomarker for ovarian carcinomas. PATIENTS AND METHODS: Using a newly developed enzyme-linked immunosorbent assay (ELISA), hK13 levels were quantified in 131 ovarian tumor extracts and correlated with various clinicopathological variables and outcome (progression-free survival [PFS], overall survival [OS]), over a median follow-up period of 42 months. RESULTS: hK13 concentration in ovarian tumor cytosols ranged from 0 to 18.4 ng/mg of total protein. An optimal cutoff value of 0.13 ng/mg (67(th) percentile) was selected, based on the ability of hK13 values to predict the PFS of the study population, to categorize tumors as hK13-positive or negative. Women with hK13-positive tumors most often had early stage (stage I/II) disease, no residual tumor after surgery and optimal debulking success (P <.05). Univariate and multivariate Cox regression analyses revealed that patients with hK13-positive tumors had a significantly longer PFS and OS than hK13-negative patients (P <.05). Kaplan-Meier survival curves further confirmed a reduced risk of relapse and death in women with hK13-positive tumors (P =.007 and P =.002, respectively). CONCLUSION: These results indicate that hK13 is an independent marker of favorable prognosis in ovarian cancer.

In silico analysis of the human kallikrein gene 6.

Kallikreins are a family of 15 serine proteases clustered together on the long arm of chromosome 19. Recent reports have linked kallikreins to malignancy. The human kallikrein gene 6 (KLK6) is a newly characterized member of the human kallikrein gene family. Recent work has focused on the possible role of this gene and its protein product as a tumor marker and its involvement in diseases of the central nervous system. In this study, we performed extensive in silico analyses of KLK6 expression from different databases using various bioinformatic tools. These data enabled us to construct and verify the longest transcript for this kallikrein, to identify several polymorphisms among published sequences and to summarize the 21 single-nucleotide polymorphisms of the gene. Our expressed sequence tag (EST) analyses suggest the existence of seven new splice variants of the gene, in addition to the already reported ones. Most of these variants were identified in libraries from cancerous tissues. KLK6 orthologues were identified from three other species with approximately 86% overall homology with rat and mouse orthologues. We also utilized several databases to compare KLK6 gene expression in normal and cancerous tissues. The serial analysis of gene expression and EST expression profiles showed upregulation of the gene in female genital (ovarian and uterine) and gastrointestinal (gastric, colon, esophageal and pancreatic) cancers. Significant downregulation was observed in breast cancers and brain tumors, in relation to their normal counterparts.