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Clinical Chemistry 51: 506-515, 2005. First published January 13, 2005; 10.1373/clinchem.2004.042341
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(Clinical Chemistry. 2005;51:506-515.)
© 2005 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Intron Retention: A Common Splicing Event within the Human Kallikrein Gene Family

Iacovos P. Michael1,2, Lisa Kurlender1,2, Nader Memari1,2, George M. Yousef3, Daisy Du1, Linda Grass1, Carsten Stephan4, Klaus Jung4 and Eleftherios P. Diamandis1,2,a

1 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada.
2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.
3 Discipline of Pathology, Memorial University, St. John’s, NL, Canada.
4 Urology, University Hospital Charité, Humboldt University, Berlin, Germany.

aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, ON, Canada M5G 1X5. Fax 416-586-8628; e-mail ediamandis{at}mtsinai.on.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: All human kallikrein (KLK) genes have at least one splice variant, some of which possess clinical utility in cancer diagnostics/prognostics. Given that introns <100 bp in length are retained in 95% of human genes and that splice variants of KLK3 and KLK4 retain intron III, we hypothesized that other proteins in this family, with a small intron III, may also retain it.

Methods: Variant-specific reverse transcription-PCRs (RT-PCRs) for KLK1, KLK2, KLK5, and KLK15 were used to identify and clone the full coding sequence of intron III-containing splice variants. In addition, variant-specific RT-PCRs for the cloned KLK3 and KLK4 variants as well as for the "classical" forms of the six genes were used to determine their expression profiles in healthy tissues, their regulation by steroids, and their differential expression in prostate cancer.

Results: KLK1, KLK2, KLK3, KLK4, KLK5, and KLK15 showed a common type of splice variant in which intron III is retained. Expression profiling of these splice variants revealed expression profiles similar to those of the classical mRNA forms, although the pattern of hormonal regulation was different. The KLK15 splice variant was up-regulated in 8 of 12 cancerous prostate tissues. All encoded variant proteins were predicted to be truncated and catalytically inactive because of a lack of the serine residue of the catalytic triad.

Conclusions: The first six centromeric members of the KLK gene family have splice variants that retain intron III. Some variants show tissue-specific expression. The KLK15 splice variant appears to be a candidate biomarker for prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The mechanism of alternative splicing was first proposed by Gilbert (1), who postulated that different combinations of exons could be spliced together to produce different mRNA isoforms of a gene, and was a major contribution after the discovery of exons and introns in the adenovirus hexon gene in 1977 (2). Alternative splicing is now considered one of the main mechanisms responsible for increasing the diversity and complexity of the human genome (3)(4). Genome-wide analyses indicate that 40–60% of human genes have alternative splicing forms (5). Furthermore, recent reports indicate that 10–30% of alternatively spliced human genes possess tissue-specific splice forms (6) and that at least 300 genes possess cancer-specific mRNA variants (7). Among the hallmarks of cancer are genomic mutations. It has been estimated that at least 15% of mutations that cause genetic diseases affect pre-mRNA splicing (8). Indeed, numerous studies have indicated that alternative splicing occurs frequently in cancer cells, and a plethora of cancer-specific splice variants have been reported. Some of them are considered to be candidate cancer biomarkers, such as CD44 (9) and the Wilms tumor (WT1) (10) genes.

The human kallikrein (KLK) 1 genes are a family of 15 serine protease genes that map to chromosome 19q13.4 (11)(12)(13). Kallikreins represent the largest cluster of protease genes of any kind within the human genome. Although the function of many of these proteases is currently unknown, their clinical utilities have been successfully exploited. For example, human kallikrein 3 [prostate-specific antigen (PSA)] is the best biomarker for prostate cancer screening, diagnosis, staging, and monitoring (14). Other members of this family are promising biomarkers for prostate, breast, and ovarian cancer (15)(16)(17)(18). The association of this gene family with other disorders, such as Alzheimer disease (19), and physiologic processes, such as skin desquamation (20), has also been suggested.

Alternative pre-mRNA splicing is a common event among members of the KLK gene family. To date, ~70 KLK splice variants have been reported, and each KLK gene possesses at least one variant (13). Splicing events such as exon skipping, extension, truncation, cryptic exons, and intron retention have been observed in both the coding and noncoding regions of the genes. Furthermore, alternative transcriptional start sites and polyadenylation sites have also been reported. Most of these variant transcripts are predicted to encode for truncated proteins lacking one or more residues of the catalytic triad because of frameshifts. An exception is one KLK4 isoform, which retains all of the residues of the catalytic triad but lacks the signal peptide and is thought to act intracellularly (21). Despite the fact that alternative splicing substantially increases the diversity of this locus, most of these putative protein isoforms have not been isolated, with the exception of a few proteins encoded by KLK2 and KLK3 variants (22). The association of some of these splice variants with cancer has been examined (23)(24)(25)(26)(27)(28)(29). Some of them are tissue, developmental stage, stimulus, or disease specific (our unpublished data).

Common patterns of alternative splicing have been observed within several gene families. For example, a study examining the effects of alternative splicing on transcripts encoding membrane proteins revealed that a common splice form leads to the removal of the transmembrane domain of single-pass transmembrane proteins, producing a soluble protein isoform (30). Another study has shown that 50 protein domain types were selectively removed by alternative splicing (31).

In the present study, we examined the frequency of a common type of splicing (intron retention) within the KLK gene family. Intron retention has been reported for several other genes. Recently, a study examining intron retention in a set of 21 106 known human genes revealed that 14.8% retained at least one intron (32). The probability of intron retention increases as intron length decreases because introns <100 bp in length are retained in 95% of cases. Among human kallikrein splice variants reported to date, two of them, Psa-rp2 for the KLK3 gene (GenBank accession no. AJ310938) and KLK4 variant 1 for the KLK4 gene (GenBank accession no. AF148532), retain intron III, which is relatively short (143 and 83 bp, respectively). Because other kallikrein genes, such as KLK1, -2, -5, and -15, also possess a short intron III, we speculated that retention of intron III might be a common splicing event among members of the KLK family.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
Total RNA isolated from human adrenal gland, adult brain, colon, fetal brain, fetal liver, heart, kidney, liver, lung, mammary gland, pancreas, placenta, prostate, salivary gland, skeletal muscle, spinal cord, spleen, small intestine, stomach, testis, thymus, thyroid, trachea, and uterus was obtained from Clontech. RNA from adipose, cervical, and vaginal tissue was from Ambion, whereas RNA from skin was from Invitrogen. We used the breast cancer cell lines T-47D, ZR-75-1, BT-474, MCF-7; the prostate cancer cell lines DU 145 and PC-3; the neuroblastoma cancer cell lines SK-N-BE and SW 1088; and the ovarian cancer cell lines PA-1, ES-2, TOV-21G, and BG-1. All cell lines were obtained from ATCC except for BG-1, which was a gift from Dr. H. Rocheford (INSERM, Montpellier, France).

matched noncancerous/cancerous tissues from prostate cancer patients
We obtained 12 pairs of matched tissue samples (noncancerous/cancerous) from patients with a median age of 63 years who underwent radical prostatectomy for prostatic adenocarcinoma at the University Hospital Charité, Berlin, Germany. Fresh prostate tissue samples were obtained from the cancerous and noncancerous parts of prostatectomy specimens. Small pieces of tissue were gross-dissected by a pathologist immediately after prostate removal, snap-frozen, and stored in liquid nitrogen until analysis. To ensure that the tissue was either malignant or benign, histologic analysis was performed by the same pathologist, as described previously (33). Only samples that were fully surrounded by malignant tissue were used. Tissue characterized as noncancerous was usually taken from the inner zone of the contralateral lobe.

The samples were collected with informed consent, and the study was approved by the Ethics Committee of the Charité Hospital.

hormonal regulation experiments with cancer cell lines
Cells were cultured to near confluency in RPMI medium (Life Technologies, Inc.) supplemented with glutamine (200 mmol/L), bovine insulin (10 mg/L), fetal bovine serum (100 mL/L), antibiotics, and antimycotics. The cells were then aliquoted into 24-well tissue culture plates and cultured to ~50% confluency. Twenty-four hours before the experiments, the culture media were replaced with phenol red-free medium containing 100 mL/L charcoal-stripped fetal bovine serum. For stimulation experiments, the steroid hormones estradiol (estrogen), dihydrotestosterone (androgen), norgestrel (synthetic progestin), aldosterone (mineralocorticoid), and dexamethasone (synthetic glucocorticoid) dissolved in absolute ethanol were added to the culture medium at a final concentration of 10–8 mol/L. Cells stimulated with ethanol were included as controls. In all cases, the final ethanol concentration was 1 mL/L. The cells were cultured for 24 h and then harvested for mRNA extraction. All experiments were performed in triplicate.

rna extraction
Prostate tissues and other healthy human tissues (esophagus, fallopian tube, hippocampus, ovary, pituitary, and tonsil) were pulverized with a hammer under liquid nitrogen. Total RNA from these tissues, as well as from cell line pellets, was extracted with TRIzol reagent (Life Technologies) and treated with DNase I (Invitrogen) according to the manufacturers’ instructions. The RNA concentration and purity were determined spectrophotometrically.

reverse transcription
We reverse-transcribed 2 µg of total RNA into first-strand cDNA, using the SuperScriptTM First-Strand Synthesis System for reverse-transcription-PCR (RT-PCR; Invitrogen). The final volume was 20 µL. We diluted 1 µL of the cDNA 100-fold and performed a PCR reaction for the housekeeping gene ß-actin, as described below, to check the quality of the first-strand cDNA synthesis.

pcr
Three PCR reactions were performed for each splice variant. In the first reaction, we used the primers F1 and R1 to simultaneously amplify both the "classical" as well as the splice variant form of interest (Fig. 1 ). Using primers sets F2/R2 and F3/R3, we amplified and characterized the whole coding region of each splice variant (Fig. 1 ). Furthermore, using the primers identified by asterisks in Table 1 of the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol51/issue3/, we were able to achieve specific amplification of each splice variant alone (one of the two primers binds within intron III). To examine the tissue expression of each splice variant, we used both splice-variant specific primers (F2/R2 or F3/R3) and the F1/R1 pair of primers in different reactions. For the steroid hormone regulation experiments, we used the F1/R1 pair of primers, whereas for the prostate cancer/noncancer pairs, we used the splice variant-specific primers.



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  		Figure 1. Primers sets (arrows) used in this study.

The set F1/R1 was used to amplify both the classical form and the corresponding splice variant, whereas the sets F2/R2 and F3/R3 were used for specific amplification of the splice variants. Only coding exons ({blacksquare}) are shown. {cjs2113}, intron III.

Each PCR reaction was carried out in a reaction mixture containing 1 µL of cDNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphates, 100 ng of primers, and 2.5 U of Hot Star Taq DNA polymerase (Qiagen Inc.) on a Eppendorf thermocycler. The cycling conditions were 95 °C for 15 min to activate the Taq polymerase followed by 35 or 40 cycles (Table 1 in the online Data Supplement) of 94 °C for 30 s, the annealing temperature (Ta, in °C; see Table 1 in the online Data Supplement) for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min. Equal amounts of PCR products were electrophoresed on 1.5% agarose gels and visualized by ethidium bromide staining.

To verify the identity of the PCR products, we purified them, using gel extraction reagents (Qiagen), and cloned them into the TOPO TA cloning vector (Invitrogen) according to the manufacturer’s instructions. The inserts were sequenced from both directions by use of vector-specific primers with an automated DNA sequencer.

in silico analysis
Sequence homology searching was performed with the basic local alignment research tool (BLAST), available from the National Center of Biotechnology Information, against the human expressed sequence tag (EST) database (dbEST). Sequences with >95% homology were considered as putative ESTs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cloning of four novel splice variants
Given that two splice variants with intron retention were already identified, i.e., Psa-rp2 for KLK3 (GenBank accession no. AJ310938) and KLK4 variant 1 (AF148532), we designed primers that bound in coding exons 3 and 5 (F1 and R1 primers, respectively; Fig. 1Up ; also see Table 1 in the online Data Supplement) to examine whether additional kallikrein genes have splice variants with a retained intron III. When we performed PCR on 36 healthy human tissue samples, we observed a second band, higher than the one expected for the classical form for the genes KLK1, KLK2, KLK5, and KLK15. After cloning and sequencing these bands, we verified that they represent splice variants with retained intron III (Fig. 2 ). To further characterize these splice variants, we designed two splice variant-specific RT-PCRs, with one primer of each pair binding within intron III (F3 and R2 primers; Fig. 1Up ; also see Table 1 in the online Data Supplement). Thus, we were able to clone the complete coding sequence of each splice variant. The splice variants for the KLK3 and KLK4 genes, described previously, were verified by cloning and sequencing. The GenBank accession numbers, names, and mRNA lengths for the newly identified splice variants are shown in Table 1 . The new variants will be referred to as KLK1-IRIII, KLK2-IRIII, KLK5-IRIII, and KLK15-IRIII, and for those that have been published previously as KLK3-IRIII and KLK4-IRIII.



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  		Figure 2. Genomic organization of the classical and the corresponding splice variant of the genes KLK1 through -5 and KLK15.

{blacksquare}, coding exons; {square}, noncoding exons; —–, introns. Numbers in the boxes and above the lines represent the lengths of the exons and the introns, respectively, in base pairs. *, start codon; {blacktriangledown}, stop codon. The amino acids of the catalytic triad are indicated: H, histidine; D, aspartic acid, S, serine.


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  		Table 1. Splice variants of the KLK1 through -5 and KLK15 genes with retained intron III.

genomic organization
Because all of these splice variants retain intron III, they have four instead of five coding exons (Fig. 2Up ). A new exon is created by merging coding exons 3 and 4 of the classical form and the intron between them.

We defined the 5' and 3' splice sites [branch point, polypyrimidine (pY) tract, and acceptor site] of the classical form of the corresponding splice variant (Fig. 3 ) to explore the reasons and possible mechanism that gives rise to this splicing pattern. Knowing that the optimal donor and acceptor sites are denoted by the sequences AG/gtRagt and (Y)ncag/GT, respectively, we observed that none (except KLK5) of the classical forms had optimal splice sites in either the donor or acceptor sites (Fig. 3 ). KLK5 was the only gene that had an optimal donor site. Regarding the possible branch point (YNY YRA Y), again the KLK5 gene was the only one with an optimal branch point. Finally, all of the genes have a pY tract located –18 to –40 bp before the acceptor site (Fig. 3 ).



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  		Figure 3. The 5' and 3' splice sites (branch point, pY tract, and acceptor site) of the classical forms of six kallikrein genes.

The uppercase letters represent the exon sequences, and the lowercase letters represent intron III sequences. The branch point is indicated with a solid underline and the pY tract with a dotted underline. The nonconserved base pairs are shaded (see text for more details).

Lander et al. (3) showed that shorter introns have a higher GC content in humans. On average, the GC content (60.8%) of intron III of kallikrein genes is higher than all other introns and is the only intron with a higher GC content than its flanking exons (data not shown). Furthermore, the GC content for sequences upstream and downstream from the premature termination codon is 66.8% and 58.2%, respectively (data not shown).

predicted protein sequence
Using the open reading frame finder program (ORF Finder) from the National Center of Biotechnology Information (http://www.ncbi.nlm.nih.gov/gorf/gorf.html), we identified the predicted protein sequence for each splice variant (Table 1Up ; also see Fig. 1 in the online Data Supplement). Comparison with KLK3-IRIII and KLK4-IRIII revealed the following features: (a) they retain the signal peptide, suggesting that they will be secreted proteins; (b) they have a premature termination codon (PTC), and thus encode truncated proteins (Fig. 2Up ); (c) they lack the "Ser" residue of the catalytic triad, and thus will not function as serine proteases; and (d) they contain a unique C-terminal sequence encoded from the intronic sequence (Fig. 4 in the online Data Supplement).

in silico analysis
Using BLAST, we identified ESTs that correspond to our experimentally defined splice variants (Table 2 ). We found 2 ESTs for KLK1-IRIII, 10 for KLK2-IRIII, and 1 for KLK15-IRIII. No EST was found for KLK5-IRIII. For the previously identified splice variants, we found five ESTs for KLK3-IRIII and one EST for KLK4-IRIII.


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  		Table 2. EST clones with >95% homology with the exons of the splice variants.

tissue expression
The tissue expression profile of the six splice variants was elucidated by RT-PCR using total RNA from 36 healthy human tissues. By performing two PCR reactions for each splice variant (as described in the Materials and Methods), we were able to compare the expression of each splice variant with its corresponding classical form. Generally, the concentrations of the splice variant mRNAs were similar to the concentrations of the corresponding classical forms (Table 3 ). Thus, KLK1-IRIII was highly expressed in kidney, pancreas, salivary gland, and thyroid. The splice variants KLK2-IRIII, KLK3-IRIII, and KLK4-IRIII were highly expressed in the prostate. The expression of these variants appears to be more prostate specific (particularly for KLK3-IRIII) compared with that of the corresponding classical forms. KLK15-IRIII was more highly expressed in prostate, salivary gland, testis, and thyroid. Finally, KLK5-IRIII appears to have a broader expression pattern than that of the classical form (Fig. 2 in the online Data Supplement). High expression was seen in the fallopian tube, esophagus, and pituitary. We observed no expression of the classical form in these tissues. On the other hand, in the cervix, salivary gland, spinal cord, stomach, and thyroid, in which the classical form was expressed in high concentrations, we observed no expression of the splice variant.


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  		Table 3. Tissue expression of the classical kallikrein genes and the corresponding splice variants with retained intron III.

hormonal regulation
We examined the hormonal regulation patterns of the splice variants and classical forms of the six kallikrein genes by RT-PCR. Splice variants KLK2-IRIII, KLK3-IRIII, KLK4-IRIII, and KLK15-IRIII followed the same pattern as the corresponding classical forms; i.e., they were regulated mainly by the androgen dihydrotestosterone (DHT) and the androgenic progestin norgestrel. The splice variants KLK4-IRIII, KLK5-IRIII, and KLK15-IRIII were down-regulated by dexamethasone (Fig. 4 ).



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  		Figure 4. Hormonal regulation of the classical forms and the corresponding splice variants of the genes KLK1 through -5 and KLK15 in cell lines.

Shown are four breast cell lines (T-47D, ZR75-1, BT-474, and MCF-7), four ovarian cell lines (PA-1, ES-2, BG-1, and TOV 21G), two prostate cell lines (DU145 and PC-3), and two neuroblastoma cell lines (SK-N-BE and HTB12). Steroid hormones (estradiol, DHT, norgestrel, aldosterone, and dexamethasone) dissolved in absolute ethanol were added to the culture media at a final concentration of 10–8 mol/L and an ethanol concentration 1 mL/L. The lower band for each cell line represents the classical form, and the upper band the splice variant form. There was no DNA template used in the PCR negative control. The primer set used was F1/R1 (Fig. 1Up ). For interpretations, see the text. Actin was used as a positive and nonhormonally regulated gene (not shown).

Of interest is the pattern of regulation observed for KLK5-IRIII. Previous studies indicated that the classical form, KLK5, is up-regulated by estradiol and norgestrel in the cell line BT474 (34). In the present study, we observed that in the cell line BT474, KLK5-IRIII was down-regulated by estradiol, whereas KLK5 was up-regulated; in the cell lines PA-1 and HTB12, estradiol up-regulated KLK5-IRIII. In the cell line ES-2, only KLK5-IRIII was expressed and was down-regulated by norgestrel. Down-regulation by norgestrel was observed in the BG-1 cell line and by dexamethasone in the cell line HTB12. On the other hand, DHT up-regulated KLK5-IRIII in the cell line BG-1 (Fig. 4Up ).

differential expression in cancerous vs noncancerous matched prostate samples
We examined the expression of the six splice variants in 12 pairs of matched noncancerous and cancerous prostate tissues. We observed no differential expression for the splice variants KLK2-IRIII, KLK3-IRIII, and KLK4-IRIII. The KLK1-IRIII splice variant was down-regulated in cancer in four pairs, whereas it was up-regulated in two (data not shown). KLK15-IRIII was up-regulated in 8 of 12 pairs (pairs 1–5 and 9–11), whereas we observed no difference for the remaining pairs (Fig. 5 ).



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  		Figure 5. KLK15 splice variant expression in paired cancerous/healthy prostate samples, as assessed by RT-PCR.

The primer set used was F3/R3 (see Fig. 1Up ). There was no DNA template used in the PCR negative control. Actin was used as a positive control. Note the up-regulation of KLK15-IRIII in 8 of 12 cancerous (C) tissues compared with the matched healthy tissues (N). For more discussion, see the text.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Alternative splicing is a major mechanism for increasing the diversity of the proteome. As many as 70 splice variants of kallikrein genes have been published to date. In this study, we examined a specific splicing event (intron retention) within the human kallikrein gene family. We found that the first six centromeric members of this family, KLK1 through -5 and KLK15, have a common splice variant derived from retention of intron III.

Intron retention is a relatively common event. For example, in Drosophila P elements, an intron may be either spliced or retained, depending on the cell type in which it is expressed (35). In mammals, intron retention has been observed in many genes, including human tumor necrosis factor-ß (36), human growth hormone (37), bovine growth hormone (38), and rat {gamma}-fibrinogen (39). Dirksen et al. (40) have shown that both suboptimal 5' and 3' splice sites are required for intron retention of bovine growth hormone. In the present study, examination of 5' and 3' splice sites of the third intron of KLK1 through -5 and KLK15 indicated that all of them, except for KLK5, have suboptimal sites. Furthermore, mutational analysis of the pY tract of introns has shown that a minimum of five uninterrupted thymidines are required for strong binding of the heterogeneous ribonucleoprotein C (hnRNP C) and for correct splicing to take place (41). Only KLK1, -2, and -3 have an uninterrupted sequence of five thymidines, whereas KLK5 and -15 seem to have a very weak pY tract. The branch point was also shown to be important for effective splicing (41). Of the genes examined here, only KLK5 has a fully conserved branch point. In conclusion, it seems that a suboptimal 5' splice site (donor site) and 3' splice site (branch point, pY tract, and acceptor site) play a major role in intron III retention of these genes.

The higher GC content of intron III compared with the rest of the introns is in agreement with the results reported by Goodall and Filipowics (42), who demonstrated in plants that a higher GC content might have a lower excision rate, and with the results of a global analysis of the human transcriptome for intron retention (32). High GC content is a characteristic feature of active euchromatin and transcriptional activity. This feature of intron III might indicate that this area of the gene is more susceptible to transcription factor binding and higher transcriptional activity.

Nonsense-mediated mRNA decay (NMD) is a surveillance posttranslational mechanism that controls the quality of the mRNA function by degrading all abnormal mRNA transcripts that contain a PTC (43). The retention of intron III changes the reading frame and creates a PTC. It seems the splice variant transcripts reported here are immune to NMD because high concentrations of cytoplasmic mRNAs were detected. NMD-resistant mRNAs for other genes, such as ß-globin (44), von Willebrand factor (45), cystic fibrosis transmembrane conductance regulator (46), LDL receptor (47), and apolipoprotein B (48), have been reported. It is possible that some genes contain cis-acting sequences that confer resistance to NMD. Such sequences have been found in yeast (49)(50).

Many proteins encoded by intron-retaining splice variants have been reported. These include isoforms of cofactor proteins CD44 and CD46 (51)(52), human growth hormone (53), human gonadotropin-related hormone gene (54), effector cell protease receptor-1 (55), and murine vitamin D receptor (VDR0) (56). Interestingly, in the latter case, the vitamin D receptor isoform (VDR1) was shown to act as a dominant-negative receptor against VDR0 transactivation. The predicted proteins of the kallikrein splice variants described here will be truncated and lack the serine residue of the catalytic triad. These proteins may act in a dominant-negative manner, regulating the function of the corresponding classical forms, or may display an as yet unknown nonprotease function.

Tissue-specific splicing has been reported for 10–30% of the human genes (6). We have shown here that the splice variant of the KLK3 gene seems to be expressed exclusively in the prostate. Very high expression in the prostate was also observed for the KLK2 gene splice variant. These data agree with our in silico analysis, according to which the KLK2 and KLK3 splice variants are expressed predominantly in the prostate. The finding of ESTs for these variants in sciatic nerve libraries is interesting because the classical KLK2 and KLK3 genes are known to be expressed almost exclusively in the prostate. The KLK5 splice variant seems to have a pattern of expression different from that of the classical form because in some tissues, either the splice variant or the classical form is expressed. The tissue-specific expression of the KLK2, KLK3, and KLK5 splice variants might be valuable if these genes find application as diagnostic and/or prognostic biomarkers in cancer. The differential tissue expression and hormonal regulation between the splice variants and the corresponding classical forms suggests that different cis- and/or trans-acting elements might regulate their transcription.

The connection between human kallikreins and cancer has been reported in many studies (11). Numerous kallikreins are established or emerging biomarkers for the diagnosis, prognosis, and monitoring of cancer at the mRNA and/or protein level. Splice variants of these genes display cancer-specific expression, or they are differentially expressed in cancer (22)(23)(24)(25)(57). In this study, we found that the splice variant of the KLK15 gene is up-regulated in 8 of 12 prostate cancer tissues compared with the corresponding healthy tissue samples. The classical form of the KLK15 gene has also been shown to be up-regulated at the mRNA level in prostate cancer and was associated with more aggressive forms (58).

A relationship between splice variants that retain introns and human diseases, including malignancies, has been revealed by global analysis of the human transcriptome (32). Among 88 genes that generate putative truncated proteins, there are genes associated with Williams–Beuren syndrome and Batten–Spielmayer–Vogt disease. Furthermore, several genes are related to the tumorigenic process, including the p19A, tumor necrosis factor receptor, BCL2-like 11, and CDC2-like 10 genes.

Our unpublished data indicate that 3' extension of coding exon 3 in the rest of the genes may produce similar isoforms. For example, we found that KLK6 and KLK13 have a splice variant with 3'-coding exon 3 extension by ~100 bp (close to the length of the retained intron III in the genes examined here). The extension also leads to a frameshift and creation of a PTC. The predicted encoded proteins will be truncated and lack the catalytic serine residue. Bioinformatic analysis of ESTs revealed that 3'-coding exon 3 extension or complete retention is universal among all kallikrein genes.

In this study, we showed a common type of alternative splicing, characterized by complete retention of intron III, in 6 (KLK1 through -5 and KLK15) of the 15 human kallikrein genes. Some of these variants may have diagnostic and/or prognostic value because they show tissue-specific expression or differential expression in comparison with the classical form. The up-regulation of the KLK15 splice variant in prostate cancer warrants examination of the encoded protein as a biomarker of prostatic cancer.


   Acknowledgments
 
This work was supported by a grant to E.P. Diamandis from Natural Sciences and Engineering Research Council of Canada (NSERC) and IBEX Technologies Inc., under a University/Industry program, and by a grant from Deutsche Forschungsgemeinschaft (JU 365/6-1) to K. Jung and C. Stephan.


   Footnotes
 
1 Nonstandard abbreviations: KLK, kallikrein gene; RT-PCR, reverse transcription-PCR; EST, expressed sequence tag; pY, polypyrimidine; PTC, premature termination codon; DHT, dihydrotestosterone; and NMD, nonsense-mediated mRNA decay.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Gilbert W. Why genes in pieces?. Nature 1978;271:501.[CrossRef][Medline] [Order article via Infotrieve]
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