RESUMEN
The human kallikrein gene 10 (KLK10) is a member of the kallikrein gene family on chromosome 19q13.4. This gene was identified by its downregulation in breast cancer, and preliminary evidence suggests that it may act as a tumor suppressor. A computer-based analysis was performed on EST and SAGE clones from the Cancer Genome Anatomy Project and other databases. Experimental verification of differential expression of KLK10 in cancer was performed by PCR using gene-specific primers. The mRNA and EST analysis allowed the construction of the longest transcript of the gene and characterization of a 5' extension of the reported mRNA. In addition, seven new splice variants of KLK10 were identified. One of these variants, named KLK10 splice variant 3 (KLK10-SV3) which starts with a novel first exon, was experimentally verified. This variant is predicted to encode for the same protein as the 'classical' KLK10 mRNA, since the first exon is untranslated. One variant mRNA partially matches with the sequence of KLK10, while the rest of the mRNA matches with a portion of the polycystic kidney disease gene, found on chromosome 15. This variant could not be experimentally verified in either normal or cancerous tissues. There are 39 reported single nucleotide polymorphisms (SNPs) for the gene, in which three result in amino acid substitutions. SAGE analysis shows a clear upregulation of KLK10 in ovarian, pancreatic, colon, and gastric cancers. The gene is, however, downregulated in breast and prostate cancers. A three-fold decrease in expression levels was noted in actinic keratosis, compared to normal skin from the same patient. The differential regulation of KLK10 in ovarian and prostate cancers was experimentally verified by RT-PCR analysis. In addition, a significant number of clones were isolated from carcinomas of the head and neck. Fewer clones were found in carcinomas of the skin, brain and prostate. Orthologues were identified in three other species, with the highest degree of homology observed with the mouse and rat orthologues (42% in each). In conclusion new splice variants of the KLK10 gene were identified. These in silico analyses show a differential expression of the gene in various malignancies and provide the basis for directing experimental efforts to investigate the possible role of the gene as a cancer biomarker.
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Perfilación de la Expresión Génica , Variación Genética , Calicreínas/genética , Neoplasias/genética , Bases de Datos Genéticas , Regulación hacia Abajo , Femenino , Humanos , Masculino , Sitios de Empalme de ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salvia officinalisAsunto(s)
Biomarcadores de Tumor/análisis , Calicreínas de Tejido/fisiología , Animales , Perfilación de la Expresión Génica , Hormonas/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Enfermedades de la Piel/genética , Enfermedades de la Piel/metabolismo , Enfermedades de la Piel/patología , Especificidad de la Especie , Calicreínas de Tejido/química , Calicreínas de Tejido/genéticaRESUMEN
Alternative splicing is prevalent within the human tissue kallikrein gene locus. Aside from being the most important source of protein diversity in eukaryotes, this process plays a significant role in development, physiology and disease. A better understanding of alternative splicing could lead to the use of gene variants as drug targets, therapeutic agents or diagnostic markers. With the rapidly rising number of alternative kallikrein transcripts, classifying new transcripts and piecing together the significance of existing data are becoming increasingly challenging. In this review, we present a systematic analysis of all currently known kallikrein alternative transcripts. By defining a reference form for each of the 15 kallikrein genes (KLK1 to KLK15), we were able to classify alternative splicing patterns. We identified 82 different kallikrein gene transcript forms, including reference forms. Alternative splicing may lead to the synthesis of 56 different protein forms for KLK1-15. In the kallikrein locus, the majority of alternative splicing events occur within the protein-coding region, and to a lesser extent in the 5' untranslated regions (UTRs). The most common alternative splicing event is exon skipping (35%) and the least common events are cryptic exons (3%) and internal exon deletion (3%). Seventy-six percent of kallikrein splice variants that are predicted to encode truncated proteins are the result of frameshifts. Eighty-nine percent of putative proteins encoded by splice variants are predicted to be secreted. Although several reports describe the identification of kallikrein splice variants and their potential clinical utility, this is the first extensive review on this subject. Accumulating evidence suggests that alternative kallikrein forms could be involved in many pathologic conditions or could have practical applications as biomarkers. The organization and analysis of the kallikrein transcripts will facilitate future work in this area and may lead to novel clinical and diagnostic applications.
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Empalme Alternativo , Calicreínas de Tejido/genética , Codón , Humanos , Isoformas de Proteínas/clasificación , ARN Mensajero , Terminología como Asunto , Calicreínas de Tejido/clasificaciónRESUMEN
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.
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Empalme Alternativo , Calicreínas/genética , Secuencia de Bases , Clonación Molecular , Humanos , Intrones , Isoenzimas/biosíntesis , Isoenzimas/genética , Calicreínas/biosíntesis , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Próstata/metabolismo , Neoplasias de la Próstata/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Calicreínas de Tejido/biosíntesis , Calicreínas de Tejido/genéticaRESUMEN
The presence of more than one mRNA form is common among kallikrein genes. We identified an mRNA transcript of the human kallikrein gene 5 (KLK5), denoted KLK5 splice variant 1 (KLK5-SV1). This variant has a different 5'-splice site, but encodes the same protein as the classical KLK5 transcript. RT-PCR analysis of this variant transcript expression in 29 human tissues indicated highest expression in the cervix, salivary gland, kidney, mammary gland, and skin. Comparative analysis of the expression levels of KLK5-SV1, another splice variant named KLK5 splice variant 2 (KLK5-SV2), and the classical KLK5 form showed that out of all three mRNA transcripts, the classical form is predominantly expressed (found in more tissues and at higher expression levels) followed by KLK5-SV1. KLK5-SV1 is expressed at high levels in ovarian, pancreatic, breast and prostate cancer cell lines. KLK5-SV1 was also found to be expressed in 9/10 ovarian cancer tissues, but it was not found in one normal ovarian tissue tested. Hormonal regulation experiments suggest that KLK5-SV1 is regulated by steroid hormones in the BT-474 breast cancer cell line. Furthermore, this variant had significantly higher expression in normal prostate tissues compared to their matched cancer tissue counterparts. KLK5-SV1 may have clinical utility in various malignancies and should be further explored as a potential new biomarker for prostate and ovarian cancer.
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Biomarcadores de Tumor/análisis , Perfilación de la Expresión Génica , Calicreínas/biosíntesis , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Secuencia de Bases , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Sitios de Empalme de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Células Tumorales CultivadasRESUMEN
Human kallikrein 6 (protease M/zyme/neurosin) was originally identified based on its aberrant expression in tumor cells and is considered a biomarker for ovarian cancer. Here, we describe the identification, cloning, and tissue expression of three novel transcript variants of the KLK6 gene that encode for wild-type kallikrein 6. Contrary to the classical form, transcript variants contain one untranslated exon, exploit intronic sequences, and are likely products of alternative promoters. In addition, we cloned splice variants 2 and 3 produced by splicing out exons 3 and 4, respectively. Given the potential diagnostic applications of kallikrein 6 at both the mRNA and protein levels, we developed a duplex RT-PCR, in order to differentially detect and quantitate mRNA species corresponding to splice variants. We show that in normal mammary epithelial cells and mammary tumor cell lines that overexpress the KLK6 gene, splice variants account for approximately 10-20% of all mRNA species.
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Neoplasias de la Mama/metabolismo , Clonación Molecular/métodos , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica/métodos , Calicreínas/genética , Calicreínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Secuencia de Aminoácidos , Mama/metabolismo , Neoplasias de la Mama/genética , Línea Celular , Activación Enzimática , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Calicreínas/química , Datos de Secuencia Molecular , Especificidad de Órganos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribución TisularRESUMEN
The presence of more than one mRNA form for the same gene is common among kallikreins, and many of the kallikrein splice variants may hold significant clinical value. The human kallikrein gene 5 (KLK5) is a member of the human kallikrein gene family of serine proteases on chromosome 19q13.4. KLK5 has been shown to be differentially expressed in a variety of endocrine tumors including ovarian, breast and prostate cancer. Utilizing Expressed Sequence Tag database analysis and reverse transcriptase polymerase chain reaction, we identified a new alternatively spliced form of KLK5(KLK5-splice variant 2, KLK5-SV2). This variant mRNA is 1,438 bp in length; formed of 195 bp of 5' untranslated region, 882 bp of protein coding sequence and a 3' untranslated region of 326 nucleotides. KLK5-SV2 has 7 exons, the first 2 of which are untranslated, and 6 intervening introns. KLK5-SV2 is different from the classic form of the KLK5 mRNA in its 5' untranslated region, where the first 5' untranslated exon of the classic form is split into 2 exons with an intervening intron of 135 nucleotides. KLK5-SV2 is expressed in a variety of tissues, with higher expression levels in the mammary gland, cervix, salivary gland and trachea. The steroid hormone receptor-positive breast cancer cell line BT-474 was used to examine the effect of different steroids on the expression levels of KLK5-SV2. Expression levels were significantly higher after stimulation with androgens, but not estrogens, progestins, aldosterone or corticosteroids. While relatively high levels of expression were found in all 10 normal breast tissues examined, no expression was detected in 16 breast cancer tissues, and expression was significantly lower than normal in the remaining 4 cancers. Expression levels comparable to normal were found in only 1 breast cancer cell line. Weak to no expression was detected in 3 other breast cancer cell lines. KLK5-SV2 was not detectable in any of the 10 normal ovarian tissues examined. It was, however, expressed at relatively high levels in 10 out of 20 ovarian cancer tissues, and lower levels were found in 4 other cancers. No expression was detected in the remaining 6 cancers. High expression levels were also detected in the CAOV-3 ovarian cancer cell line. KLK5-SV2 is a potential biomarker for breast and ovarian cancers.