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.

BACKGROUND. Kallikreins are a subgroup of serine proteases with diverse physiological functions, Many kallikrein genes are differentially expressed in various malignancies and prostate specific antigen (PSA; encoded by the KLK3 gene) is the best tumor marker for prostate cancer. Human glandular kallikrein (hK2; encoded by the KLK2 gene) is an emerging tumor marker for prostate cancer. KLK5 is a newly discovered human kallikrein gene which shares a high degree of homology and is located adjacent to KLK29 and KLK3 genes on chromosome 19q13.4. Like KLK2 and KLK3, the KLK5 gene is regulated by steroid hormones in the BT-474 breast cancer cell line. We have previously shown that KLK5 is differentially expressed in ovarian and breast cancer.METHODS. We compared the expression of KLK5 in 29 pairs of histologically confirmed normal and prostate cancer tissues by quantitative RT-PCR using the LightCycler technology.RESULTS. KLK5 expression was significantly lower in cancer tissues compared to their normal counterparts. Lowest levels of expression were found in T3 stage tumors compared with T1 and T2. Also, a significant negative correlation was found between Gleason score and KLK5 xpression.CONCLUSIONS. KLK5 should be further studied as a potential new prognostic marker in prostate cancer, whose expression is negatively correlated with cancer aggressiveness. (C) 2002 Wiley-Liss, Inc.

Kallikreins are a group of serine proteases with diverse physiological functions. KLK13 (previously known as KLK-L4) is a novel kallikrein gene located on chromosome 19q13.4 and shares a high degree of homology with other kallikrein family members. Many kallikrein genes were found to be differentially expressed in various malignancies, and their regulation is controlled by steroid hormones in prostate and breast cancer cell lines. We studied the expression of KLK13 by quantitative reverse transcriptase–polymerase chain reaction in 173 patients with epithelial breast carcinoma. An optimal cutoff point equal to the 40th percentile was defined, based on the ability of KLK13 to predict disease-free survival. KLK13 values were then associated with other established prognostic factors and with disease-free survival and overall survival. Higher positivity for KLK13 expression was found in older, oestrogen receptor positive patients. In univariate analysis, KLK13 expression is a significant predictor of improved disease-free survival and overall survival ( P <0.001 and P =0.009, respectively). Cox multivariate analysis indicated that KLK13 was an independent prognostic variable in the subgroups of patients with Grade I–II tumours and in patients who were oestrogen receptor and progesterone receptor positive, and node positive. Hazard ratios derived from Cox analysis, related to disease-free survival and overall survival were 0.22 ( P =0.001) and 0.24 ( P =0.008), respectively, for the Grade I–II group; 0.36 ( P =0.008) and 0.44 ( P =0.038), respectively, for the node positive group and 0.36 ( P =0.008) and 0.18 ( P =0.008), respectively, for the oestrogen receptor positive group. The adjusted hazard ratio for progesterone receptor positive patients for disease-free survival was 0.25 ( P =0.012). For patients in the node positive and oestrogen receptor positive subgroup ( n =51) the adjusted hazard ratio was 0.25 ( P =0.006) and for the node positive and progesterone receptor positive subgroup ( n =46) the hazard ratio was 0.24 ( P =0.008). Taken together, these data suggest that higher KLK13 expression in these subgroups of breast cancer patients is associated with an approximately 55 to 80% reduction in the risk of relapse or death. We conclude that KLK13 expression, as assessed by quantitative reverse transcriptase–polymerase chain reaction, is an independent favourable prognostic marker for breast carcinoma.

Many members of the human kallikrein gene family were found to be differentially expressed in various malignancies and some are useful cancer diagnostic/prognostic markers. KLK9 is a newly discovered human kallikrein gene that is expressed in several tissues including thymus, testis, spinal cord, salivary gland, ovary, and skin. Like other kallikreins, the KLK9 gene was found to be regulated by steroid hormones in cancer cell lines. Our purpose is to examine whether quantitative analysis of KLK9 expression has prognostic value in ovarian cancer. We studied the expression of KLK9 by quantitative reverse transcription-PCR in 168 consecutive ovarian tumors of different stages, grades, and histological types, and correlated the expression with clinicopathological parameters, response to chemotherapy, and patients' survival. We found that KLK9 expression was significantly higher in patients with early disease stages (I or II; P = 0.044) and in patients with optimal debulking (P = 0.019). Kaplan-Meier survival curves demonstrated that patients with KLK9-positive tumors have substantially longer progression-free and overall survival (P < 0.001 and P = 0.016, respectively). When the Cox proportional hazard regression analysis was applied to subgroups of patients, KLK9 expression was found to be a significant predictor of progression-free survival in the subgroup of patients with low-grade tumors [hazard ratio (HR), 0.13; P = 0.0015], early stage (HR, 0.099; P = 0.031); and those with optimal debulking (HR, 0.26; P = 0.012). After adjusting for other known prognostic variables, KLK9 retained its independent prognostic value in all of these subgroups of patients. A negative correlation was found between the expression levels of CA125 and KLK9 (rs, 0.350; P = 0.002). Our results indicate that KLK9 is under steroid hormone regulation in ovarian and breast cancer cell lines. Immmunohistochemically, human kallikrein protein (hK9) was localized in the cytoplasm, but not in the nuclei, of the epithelial cells of ovarian cancer tissues. We conclude that KLK9 is a potential new independent favorable prognostic marker for early stage, low-grade, optimally debulked ovarian cancer patients.

The kallikrein gene family, is comprised of genes that have either established or potential applications in prostate and breast cancer diagnostics. New members of the human kallikrein gene family, have been recently identified. By, using the positional candidate gene approach, we were able to clone a novel human serine protease gene that maps to chromosome 19q13.3-q13.4, the location of the kallikrein gene family. We named this gene KLK-L4 (now also known as KLK13). Here, we describe the identification of five new KLK-L4 splice variants which are not expressed in any other tissue except the human testis. We have further established that these splice variants can be detected in normal testis but not in the adjacent matched testicular tumors. In addition, differential expression of the KLK-L4 gene was found in various histological types of testicular cancer. Our results suggest that the KLK-L4 gene is expressed in normal and cancerous testicular tissue; however, its five variants are all expressed in normal tissue but not in testicular tumors. The physiological relevance of these variants and the implications of their differential expression between cancerous and normal tissues are currently unknown.

Kallikreins (KLKs) belong to the serine protease family of proteolytic enzymes. Human pancreatic/renal KLK (KLK1) encodes for an enzyme that is involved in posttranslational processing of polypeptide precursors. The function of the other members of this gene family is currently unknown, but growing evidence suggests that many KLKs are implicated in carcinogenesis. By using the positional candidate approach, we were able to identify a new human KLK-like gene, KLK14 (also known as KLK-L6). This new gene maps to chromosome 19q13.3-q13.4 and is formed of seven exons (two untranslated and five coding exons) and six intervening introns. KLK14 was defined as a KLK gene based on structural and mapping criteria, in relation to other known KLK genes. KLK14 is expressed in a variety of tissues, but the highest levels of KLK14 are found in the central nervous system, including brain, cerebellum, and spinal cord. Our preliminary results show that KLK14 is down-regulated, at the mRNA level, in breast, testicular, prostatic, and ovarian cancer.

Prostate androgen regulated transcript 1 (PART-1), is a gene predominantly expressed in the prostate gland and is regulated by androgens in human prostate cancer cell lines. Here, we report additional characteristics of PART-1 tissue expression and hormonal regulation and study its expression profile in human normal and matched prostate cancer tissues. Since PART-1 shows similarity to prostate-specific antigen (PSA) in prostate specificity and regulation, we hypothesized that it may be implicated in prostate carcinogenesis or may be a potential new biomarker. We used reverse transcriptase polymerase chain reaction (RT-PCR) to further characterize PART-1 tissue expression and hormonal regulation in the LNCaP prostate cancer cell line. RT-PCR analysis revealed that PART-1 is expressed not only in the prostate and salivary gland, but also in other tissues, including the thymus and placenta. In addition to androgen stimulation, PART-1 is also up-regulated by progestins, oestrogens and glucocorticoids. We further studied the expression of PART-1 in 27 paired (from the same patient) cancerous and non-cancerous prostatic tissues, with qualitative and quantitative RT-PCR (LightCycler® technology), in order to examine whether PART-1 is overexpressed or underexpressed in cancer. Our results indicated that PART-1 is more frequently overexpressed in the cancerous prostatic tissue. We conclude that this gene is overexpressed in prostate cancer and may represent a novel prostate cancer tumour marker. © 2001 Cancer Research Campaign

m g t t l l h t ( k s 1 i j a c l ( s m r Kallikreins are a subgroup of serine proteases with iverse physiological functions. Recently, growing evdence indicates that many kallikrein genes are inolved in malignancy. In rodents, kallikreins are enoded by a large multigene family, but in humans only hree kallikreins were thought to exist. Based on the omology between the human and rodent kallikrein oci, we studied a 300 kb region of genomic sequences round the putative KLK1 gene locus on chromosome 9q13.3–q13.4. By using linear sequence information, estriction analysis, end sequencing, PCR and blotting echniques, as well as bioinformatic approaches, we ere able to construct the first detailed map of the uman kallikrein gene family. Comparative analysis f genes located in this area, provides strong evidence hat the human kallikrein gene family locus on chroosome 19 is considerably larger than previously hought, containing at least fifteen genes. We have stablished, for the first time, the common structural eatures that apply to all members of the expanded allikrein multigene family. Our map specifies the disance between genes to one base pair accuracy, the elative location, and the direction of transcription of ll 15 genes. Determination of the true size of the kalikrein family in humans is important for our under-

Kallikreins are a subgroup of serine proteases and these proteolytic enzymes have diverse physiological functions in many tissues. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins constitute a large multigene family, but in humans, only three genes were identified. By using the positional candidate gene approach, we were able to identify a new kallikrein-like gene, tentatively named KLK-L4 (for kallikrein-like gene 4). This new gene maps to chromosome 19q13. 3-q13.4, is formed of five coding exons and four introns, and shows structural similarity to other kallikreins and kallikrein-like genes. KLK-L4 is expressed in a variety of tissues including prostate, salivary gland, breast, and testis. Our preliminary results show that KLK-L4 is down-regulated, at the mRNA level, in breast cancer tissues and breast cancer cell lines. Its expression is regulated by steroid hormones in the breast cancer cell line BT-474. This gene may be involved in the pathogenesis and/or progression of breast cancer and may find applicability as a novel cancer biomarker.

Kallikreins are a subgroup of serine proteases with diverse physiological functions. Recently, growing evidence indicates that many kallikrein genes are involved in malignancy. In rodents, kallikreins are encoded by a large multigene family, but in humans only three kallikreins were thought to exist. Based on the homology between the human and rodent kallikrein loci, we studied a 300 kb region of genomic sequences around the putative KLK1 gene locus on chromosome 19q13.3-q13.4. By using linear sequence information, restriction analysis, end sequencing, PCR and blotting techniques, as well as bioinformatic approaches, we were able to construct the first detailed map of the human kallikrein gene family. Comparative analysis of genes located in this area, provides strong evidence that the human kallikrein gene family locus on chromosome 19 is considerably larger than previously thought, containing at least fifteen genes. We have established, for the first time, the common structural features that apply to all members of the expanded kallikrein multigene family. Our map specifies the distance between genes to one base pair accuracy, the relative location, and the direction of transcription of all 15 genes. Determination of the true size of the kallikrein family in humans is important for our understanding of the contribution of the kallikreins to human biology and pathophysiology.