Clinical Utility of Mutant Antibody-Based Assays for Determination of Internally Cleaved and Intact Forms of Free Prostate-Specific Antigen.

BACKGROUND: Subforms of prostate-specific antigen (PSA) have been a subject of intensive research, and use of multikallikrein immunoassays can add clinical value to the early detection of prostate cancer, overcoming known limitations of PSA. In this study, we evaluated mutant 4D4 (L3-2) antibody-assisted assay constructs against reference wild-type (wt)-4D4-based assays for determination of intact PSA (iPSA) and nicked PSA (nPSA) in plasma samples. METHODS: Perioperative plasma samples obtained from 105 men who underwent biopsy (73 cancer, 32 noncancer) were analyzed with sandwich immunoassays for total PSA (tPSA), free PSA (fPSA), iPSA (3 constructs), and measured nPSA (2 constructs). Calculated nPSA (CN) was obtained from total fPSA - iPSA. RESULTS: Mutant-assisted iPSA assays measured lower concentrations than the reference in both patient groups. CN separated the 2 groups with the iPSA using the mutant for capture (I-MC) performing the best (P = 0.008). In prostate volume group > median, only measured nPSA provided significant discrimination [area under the curve (AUC), 0.71; P = 0.016] but equally using mutant and wt antibodies. In the whole cohort, all ratios to tPSA performed well (AUC, 0.819-0.870; P ≤ 0.0001) with CN based on I-MC scoring highest (AUC, 0.870). Importantly, in the ≤ median volume group, the I-MC/F and CN(I-MC)/T ratios stand out as the best performing parameters (AUC, 0.825 and 0.861; P = 0.001 and P = 0.0003, respectively). CONCLUSIONS: The new assay construct using the mutant 4D4 (L3-2) as a capture provides clear improvement in separating cancer from noncancer in all subgroups analyzed but especially in patients with prostate volume ≤ median.Clinical Trial Registration: ClinicalTrials.gov Identifier NCT01864135.

Indirect Back-Titration ELISA: A New Format for Estimation of Human Tissue Kallikreins.

Enzyme-linked immunosorbent assay (ELISA) is either based on sandwich, competitive, or inhibition type of format. However, these formats need 2 or 3 monoclonal antibodies (moAB) to estimate 1 antigen. To get a cost-effective, high throughput, ELISA for estimation of human tissue kallikreins we have now developed an indirect, back-titration style, Time Resolved ImmunoFluorometric (TRIF) ELISA that uses only 1 antigen-specific moAB and a general polyclonal antibody. Polystyrene microtiter plate wells coated with a capture antibody, a mouse moAB prepared against a specific human tissue kallikrein are allowed to interact either with the corresponding pure antigen, as the calibrator, or with the corresponding antigen present in a biological fluid or tissue extract. The detection antibody, anti-mouse IgG conjugated with alkaline phosphatase, is added to find the antigen-free immobilized capture moAB. Conjugated enzyme is allowed to hydrolyze diflunisal phosphate to produce a highly fluorescent complex. The fluorescence measured in TRIF mode corresponds to the antigen-free immobilized capture moAB and is used to quantify antigen-bound capture moAB. The detection antibody binds with the antigen-free capture moAB and strength of the signal correlates inversely with the amount of antigen bound to the capture moAB. With a minimum detection level of 20 ng/L the assay has no cross-reactivity with several test molecules. The method is sensitive, specific, applicable to a variety of biological samples, and cost-effective as it uses only 1 moAB and a polyclonal antibody. Using this assay, a single epitope can be estimated without purification.

A Highly Sensitive Porous Silicon (P-Si)-Based Human Kallikrein 2 (hK2) Immunoassay Platform toward Accurate Diagnosis of Prostate Cancer.

Levels of total human kallikrein 2 (hK2), a protein involved the pathology of prostate cancer (PCa), could be used as a biomarker to aid in the diagnosis of this disease. In this study, we report on a porous silicon antibody immunoassay platform for the detection of serum levels of total hK2. The surface of porous silicon has a 3-dimensional macro- and nanoporous structure, which offers a large binding capacity for capturing probe molecules. The tailored pore size of the porous silicon also allows efficient immobilization of antibodies by surface adsorption, and does not require chemical immobilization. Monoclonal hK2 capture antibody (6B7) was dispensed onto P-Si chip using a piezoelectric dispenser. In total 13 × 13 arrays (169 spots) were spotted on the chip with its single spot volume of 300 pL. For an optimization of capture antibody condition, we firstly performed an immunoassay of the P-Si microarray under a titration series of hK2 in pure buffer (PBS) at three different antibody densities (75, 100 and 145 µg/mL). The best performance of the microarray platform was seen at 100 µg/mL of the capture antibody concentration (LOD was 100 fg/mL). The platform then was subsequently evaluated for a titration series of serum-spiked hK2 samples. The developed platform utilizes only 15 µL of serum per test and the total assay time is about 3 h, including immobilization of the capture antibody. The detection limit of the hK2 assay was 100 fg/mL in PBS buffer and 1 pg/mL in serum with a dynamic range of 106 (10(-4) to 10(2) ng/mL).

Genetic variation in KLK2 and KLK3 is associated with concentrations of hK2 and PSA in serum and seminal plasma in young men.

BACKGROUND: Genetic variants in KLK2 and KLK3 have been associated with increased serum concentrations of their encoded proteins, human kallikrein-related peptidase 2 (hK2) and prostate-specific antigen (PSA), and with prostate cancer in older men. Low PSA concentrations in seminal plasma (SP) have been associated with low sperm motility. To evaluate whether KLK2 and KLK3 genetic variants affect physiological prostatic secretion, we studied the association of SNPs with hK2 and PSA concentrations in SP and serum of young, healthy men. METHODS: Leukocyte DNA was extracted from 303 male military conscripts (median age 18.1 years). Nine SNPs across KLK2-KLK3 were genotyped. We measured PSA and hK2 in SP and serum using immunofluorometric assays. The association of genotype frequencies with hK2 and PSA concentrations was tested with the Kruskal-Wallis test. RESULTS: Four KLK2 SNPs (rs198972, rs198977, rs198978, and rs80050017) were strongly associated with hK2 concentrations in SP and serum, with individuals homozygous for the major alleles having 3- to 7-fold higher concentrations than the intermediate concentrations found in other homozygotes and heterozygotes (all P < 0.001). Three of these SNPs were significantly associated with percentage of free PSA (%fPSA) in serum (all P < 0.007). Three KLK3 SNPs showed associations with PSA in SP, and the rs1058205 SNP was associated with total PSA in serum (P = 0.001) and %fPSA (P = 0.015). CONCLUSIONS: Associations observed in young, healthy men between the SP and serum concentrations of hK2 and PSA and several genetic variants in KLK2 and KLK3 could be useful to refine models of PSA cutoff values in prostate cancer testing.

Glandular kallikrein in the innate immune system of Atlantic salmon (Salmo salar).

Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon (Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish.

Polymorphisms at the Microseminoprotein-beta locus associated with physiologic variation in beta-microseminoprotein and prostate-specific antigen levels.

BACKGROUND: rs10993994, a single nucleotide polymorphism (SNP) at the genetic locus encoding beta-microseminoprotein (beta-MSP), is associated with both prostate cancer risk and levels of blood prostate-specific antigen (PSA), a biomarker used in prostate cancer screening. Therefore, we wished to determine the association between SNPs at MSMB, the gene encoding beta-MSP, and the levels of prostate-produced biomarkers beta-MSP, PSA, and human kallikrein 2 (hK2) in blood and semen. METHODS: Blood and semen from 304 healthy young Swedish men (ages 18-21) were assayed for beta-MSP, PSA, and hK2. SNPs around MSMB were genotyped from matched DNA and analyzed for quantitative association with biomarker levels. Empirical P values were multiple test-corrected and the independence of each SNP's effect was determined. RESULTS: rs10993994 was significantly associated with the blood and semen levels of beta-MSP (both P < 1.0 x 10(-7)) and PSA (P = 0.00014 and P = 0.0019), and semen levels of hK2 (P = 0.00027). Additional copies of the prostate cancer risk allele resulted in lower beta-MSP but higher PSA levels, and singly explained 23% and 5% of the variation seen in semen beta-MSP and PSA, respectively. Additional SNPs at MSMB are associated with beta-MSP and PSA independently of rs10993994. CONCLUSIONS: SNPs at MSMB correlate with physiologic variation in beta-MSP and PSA levels in the blood and semen of healthy young Swedish men. In particular, rs10993994 has a strong effect on beta-MSP levels. IMPACT: Our results suggest a mechanism by which rs10993994 might predispose to prostate cancer and raise the possibility that genetic variation might need to be considered in interpreting the levels of these biomarkers.

Determination of kininogens levels and kallikrein/kininase II activities in patients with thromboangiitis obliterans.

Some components of the kinin system such as plasma kallikrein levels, the activities of tissue kallikrein (including saliva) and kininase II and the concentrations of kininogen fractions (low-molecular weight/LKg and high-molecular weight/HKg) were evaluated in the plasma of patients with thromboangiitis obliterans (TAO) presenting clinical symptoms of the condition. Twenty TAO were diagnosed by means of the traditional Shionoya and Olin criteria and later classified into non-smokers (n = 11) and active smokers (n = 9). Fifty-three normal, non-smoking/smoking individuals (control) were also studied. Kininogen levels were determined by ELISA; the activities of kallikreins and kininase II were determined using selective substrates. The levels of enzymes (kallikreins and kininase II) and protein (kininogens) were significantly higher in patients with TAO who were active smokers compared to the control groups (no matter whether control individuals were active smokers or non-smokers, P < 0.001 for all comparisons). Interestingly, regardless of the time of disease onset, a significant increase in the levels of these components of the kinin system was also observed in patients when TAO active smokers were compared with TAO ex-smokers (P < 0.01 for all analysed parameters). Activation of the kinin system in patients with TAO may indicate the involvement of vasodilatation in an attempt to control vascular changes, thereby favouring the deposition of immune complexes at the vascular level because of nicotine stimulation. Moreover, our results corroborate the idea that TAO can be an autoimmune disorder with specific mechanisms.

Angiogenesis in cervical cancer is mediated by HeLa metabolites through endothelial cell tissue kallikrein.

High vascularity correlates with poor clinical outcome in cancer of the uterine cervix. We investigated whether human cervical cancer cell (HeLa) metabolites influenced endothelial cell proliferation through the serine protease, tissue kallikrein. The angiogenic potential of tissue kallikrein is proposed due to its proteolytic, mitogenic and invasive properties. Under pre-defined conditions, we examined the regulation of tissue kallikrein simultaneously in both endothelial and HeLa cells using immunochemistry, ELISA, cell proliferation assays and in situ RT-PCR. In an endothelial-cervical carcinoma conditioned-medium model, HeLa metabolites caused a dramatic decrease in endothelial cellular tissue kallikrein and a concomitant proliferation of endothelial cells. ELISA on the conditioned media showed a dose-dependent increase of tissue kallikrein, while in situ RT-PCR demonstrated no change in tissue kallikrein mRNA in both endothelial and HeLa cells when challenged with each other's metabolites. This demonstration of the ability of cervical cancer to simultaneously manipulate both tissue kallikrein processing within endothelial cells and angiogenesis is novel. Should this occur in vivo, the tissue kallikrein released from the endothelial cells into the microenvironment may simultaneously degrade the matrix and elicit a mitogenic effect by promoting angiogenesis. Pre-treatment with TK inhibitors and/or anti-angiogenic therapies may prove to benefit future cervical cancer patients.

Immunocytochemical study of granular duct cells with a hormonally enhanced granular cell phenotype in the mouse parotid gland.

In the parotid glands (PGs) of intact male mice (12 weeks of age, ICR strain), immunofluorescence labels for a true tissue kallikrein, mK1, and for nerve growth factor (NGF) were recognized through the subluminal edges of the striated duct (SD) segments and interlobular duct segments. Because of their small size, secretory granules were not detectable by light microscopy in any of the duct cells. Full-fledged granular cells, containing large secretory granules that were visible by light microscopy, were induced in the SD segments of male mice after the injection of 5alpha-dehydrotestosterone (DHT) and triiodothyronine (T(3)), given either alone or in combination every other day for 2 weeks. A stronger effect was detected in the mice that were concomitantly injected with DHT and T(3), and more abundant, fully developed granular cells appeared in the SD segments of these mice. These full-fledged granular cells were immunoreactive for mK1, NGF, and epidermal growth factor, but not for renin. The present results indicate that some of the SD cells with small granules in the mouse PG can develop a granular cell phenotype, producing more kinds of growth factors, as a result of the actions of androgen and thyroid hormone.

Biomarkers associated with breast cancer are associated with obesity.

BACKGROUND: Obesity is linked to the development of postmenopausal breast cancer, and some studies indicate obesity predicts a worse prognosis for premenopausal women who develop the disease. It was our hypothesis that proteins associated with breast cancer would be associated with body mass index (BMI). METHODS: We searched our database of women enrolled in breast health translational research trials for information on BMI and markers predictive of breast cancer (basic fibroblast growth factor (bFGF), prostate-specific antigen (PSA), human kallikrein (hK)2, and urinary plasminogen activator (uPA). Information on BMI and one or more nipple aspirate fluid (NAF) or serum biomarkers was available from 382 women. RESULTS: In this data set, NAF and serum levels of PSA (nPSA and sPSA), and NAF levels hK2, bFGF and uPA were each associated with pre- and/or postmenopausal breast cancer. sPSA was inversely associated with BMI in both pre- (r=-.56, p=.001) and postmenopausal women (r=-.62, p=.0035) without breast cancer. This association was lost when controlling for plasma volume. In women without breast cancer, NAF bFGF (p=.07, premenopausal subjects) and NAF hK2 (p=.09, postmenopausal subjects) were borderline associated with BMI. In women with breast cancer, nPSA was inversely (r=-.53, p=.049) associated with BMI in premenopausal women and directly associated with BMI in postmenopausal women (r=.37, p=.017). nPSA trended higher in hormone sensitive cancers, especially those that expressed progesterone receptor (p=.059). CONCLUSIONS: sPSA was inversely associated with BMI in all pre- and postmenopausal women and specifically in pre- and postmenopausal women without breast cancer. NAF PSA was associated with BMI in pre- and postmenopausal women with breast cancer. Evaluating the change in PSA with changes in weight may provide clues regarding a subject's breast cancer risk.

Biologic markers of breast cancer in nipple aspirate fluid and nipple discharge are associated with clinical findings.

BACKGROUND: The aim of this prospective study was to assess predictive markers in nipple aspirate fluid (NAF) and pathologic nipple discharge (PND) collected prior to excisional breast biopsy, as well as clinical factors available prior to biopsy, with histopathologic results in women with a radiographically suspicious and/or palpable breast lesion. METHODS: 208 NAF samples from 191 women were evaluated for the following candidate predictive proteins and cellular markers: prostate-specific antigen (PSA), human glandular kallikrein 2 (hK2), basic fibroblast growth factor (bFGF), S phase fraction (SPF), DNA index, and cytology. Clinical factors included whether or not the lesion was palpable, menopausal status, history of pregnancy, history of birth control or hormone replacement use, and PND. RESULTS: Considering all women, bFGF (p=0.005) and SPF (0.031) were associated, and abnormal cytology approached an association (p=0.056) with the presence of breast cancer. Women with PND were less likely to have breast cancer (4 vs. 37%, p<0.001) or palpable lesions (10 vs.43%, p<0.001), were younger, had lower PSA levels (p=0.046), and were more likely to have atypical NAF cytology (p=0.002). Excluding PND, increased age, postmenopause (both p<0.01), high bFGF (p=0.004) and low PSA (p=0.05) were associated with cancer. The best breast cancer predictive model included cytology, bFGF, and age (88% sensitive and 57% specific). When the data were divided by menopausal status, the optimal models to predict breast cancer, which included NAF hK2 or PSA and age, were 100% sensitive and 41% specific in pre- vs. 93% sensitive and 12% specific in postmenopausal women. CONCLUSION: NAF and clinical biomarkers are sensitive predictors of whether a breast contains cancer, and may ultimately help guide treatment. Future studies to determine the optimal combination of predictive markers are warranted.

Tissue kallikrein protects against pressure overload-induced cardiac hypertrophy through kinin B2 receptor and glycogen synthase kinase-3beta activation.

OBJECTIVE: We assessed the role of glycogen synthase kinase-3beta (GSK-3beta) and kinin B2 receptor in mediating tissue kallikrein's protective effects against cardiac hypertrophy. METHODS: We investigated the effect and mechanisms of tissue kallikrein using hypertrophic animal models of rats as well as mice deficient in kinin B1 or B2 receptor after aortic constriction (AC). RESULTS: Intramyocardial delivery of adenovirus containing the human tissue kallikrein gene resulted in expression of recombinant kallikrein in rat myocardium. Kallikrein gene delivery improved cardiac function and reduced heart weight/body weight ratio and cardiomyocyte size without affecting mean arterial pressure 28 days after AC. Icatibant and adenovirus carrying a catalytically inactive GSK-3beta mutant (Ad.GSK-3beta-KM) abolished kallikrein's effects. Kallikrein treatment increased cardiac nitric oxide (NO) levels and reduced NAD(P)H oxidase activity and superoxide production. Furthermore, kallikrein reduced the phosphorylation of apoptosis signal-regulating kinase1, mitogen-activated protein kinases (MAPKs), Akt, GSK-3beta, and cAMP-response element binding (CREB) protein, and decreased nuclear factor-kappaB (NF-kappaB) activation in the myocardium. Ad.GSK-3beta-KM abrogated kallikrein's actions on GSK-3beta and CREB phosphorylation and NF-kappaB activation, whereas icatibant blocked all kallikrein's effects. The protective role of kinin B2 receptor in cardiac hypertrophy was further confirmed in kinin receptor knockout mice as heart weight/body weight ratio and cardiomyocyte size increased significantly in kinin B2 receptor knockout mice after AC compared to wild type and B1 receptor knockout mice. CONCLUSIONS: These findings indicate that tissue kallikrein, through kinin B2 receptor and GSK-3beta signaling, protects against pressure overload-induced cardiomyocyte hypertrophy by increased NO formation and oxidative stress-induced Akt-GSK-3beta-mediated signaling events, MAPK and NF-kappaB activation.

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.

Free and total human glandular kallikrein 2 in patients with prostate cancer.

OBJECTIVES: The use of prostate-specific antigen (PSA, hK3) results in the overdiagnosis and overtreatment of prostate cancer. Markers are needed that could identify aggressive, fast-growing tumors and help decide which patients would benefit most from aggressive treatment. Human glandular kallikrein 2 (hK2) could be such a marker. The aim of this study was to test how PSA and hK2 could predict the pathologic stage and grade in a set of patients with clinically organ-confined disease. METHODS: Heparin plasma was collected from 188 patients who had undergone radical prostatectomy at the Turku University Central Hospital. Total and free PSA, as well as total and free hK2, were measured and the results compared with the pathologic TNM stage, tumor World Health Organization grade, and Gleason score. RESULTS: Free and total hK2 performed similarly to PSA in their ability to separate groups of patients with different stages or grades. Concentrations of both kallikreins were significantly different in patients with World Health Organization grade 1 cancer compared with grade 2. Neither marker could separate patients with different Gleason scores. Although PSA concentrations increased most between patients with Stage pT2b and those with pT3a, the increase in hK2 was most pronounced between those with Stage pT3a and those with pT3b. CONCLUSIONS: Although hK2 could not predict the cancer stage or grade better than PSA, changes in the hK2 and PSA concentrations occurred at different points in cancer progression. hK2 may have a role in the prognosis of prostate cancer, but additional studies with longer follow-up are required to determine whether hK2 can help when selecting treatment options.

[The search for better markers for prostate cancer than prostate-specific antigen]. / De zoektocht naar betere markers voor prostaatkanker dan prostaatspecifiek antigeen.

Prostate-specific antigen (PSA) is currently the most important biochemical marker for the diagnosis of prostate cancer. Because of the limited specificity of PSA, clinically irrelevant tumours and benign abnormalities are also detected that potentially lead to over-treatment and the accompanying physical and emotional burden for the patient. In addition, PSA is used as an indicator of progression or clinical response after treatment for prostate cancer, but the prognostic value of this marker is limited. Current studies are evaluating a number of alternative markers, such as PSA-related parameters, human kallikrein 2, osteoprotegerin and the gene DD3(PCA3), that may improve the specificity of current PSA-based diagnostics and the prognostic value of PSA.

The kallikrein world: an update on the human tissue kallikreins.

Human tissue kallikreins (hKs) are attracting increased attention owing to their association with various forms of cancer and other diseases. Human tissue kallikrein genes represent the largest contiguous group of proteases within the human genome. There are many areas of kallikrein research that need to be further explored, including their tissue expression patterns, their regulation, identification of specific substrates, their participation in proteolytic cascades, and their clinical applicability as cancer biomarkers and therapeutic targets. In this review, we briefly describe the current status of kallikrein research and identify future avenues that will enhance our understanding of their function and involvement in human diseases.

High-speed biomarker identification utilizing porous silicon nanovial arrays and MALDI-TOF mass spectrometry.

Speed and accuracy are crucial prerequisites in the application of proteomic methods to clinical medicine. We describe a microfluidic-based nanovial array for rapid proteolytic processing linked to MALDI-TOF MS. This microscale format consumes only minute amounts of sample, and it is compatible with rapid bioanalytical protocols and high-sensitivity readouts. Arrays of vials (300 microm in diameter and 25 microm deep), isotropically etched in silicon wafers were electrochemically porosified. Automated picoliter microdispensing was employed for precise fluid handling in the microarray format. Vials were prefilled with trypsin solution, which was allowed to dry. Porosified and nonporosified nanovials were compared for trypsin digestion and subsequent MS identification of three model proteins: lysozyme, alcohol dehydrogenase, and serum albumin at levels of 100 and 20 fmol. In an effort to assess the rapid digestion platform in a context of putative clinical applications, two prostate cancer biomarkers, prostate-specific antigen (PSA) and human glandular kallikrein 2 (hK2), were digested at levels of 100 fmol (PSA), 20 fmol (PSA) and 8 fmol (hK2). All biomarker digestions were completed in less than 30 s, with successful MS identification in the porous nanovial setting.

KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that is expressed at lower levels in prostate cancer cells than in normal prostate cells.

BACKGROUND: Fifteen human tissue kallikrein (KLK) genes have been identified as a cluster on chromosome 19. KLK expression is associated with various human diseases including cancers. Noncoding RNAs such as PCA3/DD3 and PCGEM1 have been identified in prostate cancer cells. METHODS: Using massively parallel signature sequencing (MPSS) technology, RT-PCR, and 5' rapid amplification of cDNA ends (RACE), we identified and cloned a novel gene that maps to the KLK locus. RESULTS: We have characterized this gene, named as KLK31P by the HUGO Gene Nomenclature Committee, as an unprocessed KLK pseudogene. It contains five exons, two of which are KLK-derived while the rest are "exonized" interspersed repeats. KLK31P is expressed abundantly in prostate tissues and is androgen regulated. KLK31P is expressed at lower levels in localized and metastatic prostate cancer cells than in normal prostate cells. CONCLUSIONS: KLK31P is a novel androgen regulated and transcribed pseudogene of kallikreins that may play a role in prostate carcinogenesis or maintenance.