Q77798271 Dotaz Zobrazit nápovědu
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
BACKGROUND: Prostate specific membrane antigen (PSMA) is a type II transmembrane protein overexpressed in prostate cancer as well as in the neovasculature of several non-prostatic solid tumors. In addition to full-length PSMA, several splice variants exist in prostatic tissue. Notably, the N-terminally truncated PSMA variant, termed PSM', is prevalent in healthy prostate, and the ratio of PSMA/PSM' mRNA has been shown to correlate with cancer progression. The widely accepted hypothesis is that the PSM' protein is a translation product arising from the alternatively spliced PSM' mRNA. METHODS: Differential ultracentrifugation, cell surface biotinylation, Western blotting, and enzyme activity measurement were used to study the origin and localization of the PSMA/PSM' variants in prostatic (LNCaP; lymph-node carcinoma of the prostate) and non-prostatic (HEK293) cell lines. These experiments were further complemented by analysis of the N-glycosylation patterns of the PSMA/PSM' proteins and by site-directed mutagenesis. RESULTS: We identified PSM' protein expression in both the LNCaP cell line and a non-cancerous HEK293 human cell line transfected with a plasmid encoding full-length PSMA. Differential centrifugation revealed that PSM' is localized predominantly to the cytosol of both these cell lines and is proteolytically active. Furthermore, the PSM' protein is N-glycosylated by a mixture of high-mannose and complex type oligosaccharides and therefore trafficked beyond the cis-Golgi compartment. CONCLUSIONS: Our data suggest that the PSM' protein is likely not generated by alternative splicing of the PSMA gene but by different mechanism, probably via an endoproteolytic cleavage of the full-length PSMA.
- MeSH
- adenokarcinom metabolismus patologie MeSH
- buněčné linie MeSH
- financování organizované MeSH
- glykosylace MeSH
- ledviny cytologie embryologie metabolismus MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- mikrozomy metabolismus MeSH
- místa sestřihu RNA genetika MeSH
- mitochondrie metabolismus MeSH
- nádorové buněčné linie MeSH
- nádory prostaty metabolismus patologie MeSH
- prostatický specifický antigen genetika chemie metabolismus MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
BACKGROUND: Prostate specific membrane antigen (PSMA), also called glutamate carboxypeptidase II (GCPII), is a target enzyme for diagnosis and treatment of prostate cancer. Moreover, it is upregulated in the vasculature of most solid tumors and is therefore a potential target for the generation of novel antineoplastics. In this context, we analyze the possibility of using rat and pig as animal models for enzymologic and in vivo studies. METHODS: We prepared the recombinant extracellular part of human, rat, and pig GCPII in S2 cell media and characterized the activity and inhibition profiles of the three orthologs by radioenzymatic assay. We performed Western blot analysis of GCPII expression in human, rat, and pig tissues using the monoclonal antibody GCP-04 and confirmed these findings by activity measurements and immunohistochemistry. RESULTS: The three recombinant proteins show similar specific enzymatic activities and inhibition profiles. Tissue expression analysis revealed that most of the pig and human tissues show at least some GCPII-positivity, while the expression pattern in rat is more restricted. Moreover, tissues such as prostate and testes exhibit different GCPII expression levels among the species studied. CONCLUSIONS: The rat and pig orthologs of GCPII seem to be suitable to approximate human GCPII in enzymologic studies. However, the diffuse expression pattern of GCPII in animal and human tissues could be a caveat for the potential utilization of GCPII-targeted anticancer drugs. Furthermore, variations in GCPII tissue distribution among the species studied should be considered when using rat or pig as models for antineoplastic drug discovery.
- MeSH
- druhová specificita MeSH
- financování organizované MeSH
- krysa rodu rattus MeSH
- ledviny enzymologie patologie MeSH
- lidé MeSH
- mícha enzymologie patologie MeSH
- miniaturní prasata MeSH
- modely u zvířat MeSH
- molekulární sekvence - údaje MeSH
- potkani inbrední LEW MeSH
- prasata MeSH
- prostata enzymologie patologie MeSH
- prostatický specifický antigen analýza genetika metabolismus MeSH
- regulace genové exprese enzymů MeSH
- sekvence aminokyselin MeSH
- testis enzymologie patologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
Nejpoužívanějším nádorovým markerem u karcinomu prostaty je od konce 80-tých let PSA (prostatický specifický antigen). Jeho zavedení do klinické praxe zcela změnilo nejen diagnostiku, ale i celou terapii karcinomu prostaty. Hlavním nedostatkem se ukázala orgánová a nikoliv nádorová specifika. Snahou posledních let je nalézt biomarker, který je produkován pouze buňkou nádorovou a nikoli buňkou nebo orgánem jako takovým. V loňském roce byly publikovány první klinické zkušenosti s EPCA-2 (early prostate cancer antigen – 2), jehož specifika i senzitivita jasně předčí dosavadní zkušenosti s PSA. Vývoj ale pokračuje dále a objevují se další medicinsky mimořádně zajímavé molekuly jako např. PSMA (prostatický specifický membránový antigen) respektive GCPII (glutamátkarboxypeptidáza II) v jehož výzkumu byl v posledních deseti letech zaznamenán zásadní posun. V současné době lze konstatovat, že GCPII, respektive PSMA představuje vysoce specifický nádorový marker pro karcinom prostaty s jasnou korelací k agresivitě nádoru a prognostickými možnostmi. Dále je prokázán jeho vztah k angiogenezi většiny solidních tumorů a jeho souvislost s neurologickým postižením při ischemických atakách centrálního nervového systému.
PSA has been the most commonly used prostate cancer marker since the end of the 1980s. Its introduction into clinical practice has changed prostate cancer diagnosis and treatment dramatically. The major disadvantage with PSA is its organ specificity and not cancer specificity. The major research task in recent years has been an attempt to define a biomarker that is produced specifically by the cancer cell only. The first clinical experience with EPCA-2 was published last year. It is clear that its specificity and sensitivity are better than for PSA. Nevertheless, the research has been ongoing and new molecules which are very interesting from a medical point of view have been discovered. One of which is PSMA (GCP II) and major progress towards understanding the function of this enzyme has been made in the past ten years. Currently, it can be stated that GCP II (PSMA) is a highly specific tumour marker for prostate cancer, which correlates with differentiation of the tumour and has prognostic potential. Furthermore, there is evidence supporting correlations between PSMA expression and angiogenesis in most solid tumours and neurological defects after ischemic attacks in the central nervous system.
- MeSH
- diagnostické techniky urologické trendy využití MeSH
- financování organizované MeSH
- hyperplazie prostaty diagnóza krev prevence a kontrola MeSH
- lidé MeSH
- nádorové biomarkery izolace a purifikace krev MeSH
- nádory prostaty komplikace krev prevence a kontrola MeSH
- nemoci prostaty diagnóza MeSH
- prostatický specifický antigen izolace a purifikace krev MeSH
- protilátky nádorové izolace a purifikace krev MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a number of pathological conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approximately 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochemical analysis revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymatically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.
- MeSH
- aktivace enzymů genetika MeSH
- antigeny povrchové analýza imunologie metabolismus MeSH
- astrocyty enzymologie MeSH
- dipeptidy metabolismus MeSH
- financování organizované MeSH
- glutamátkarboxypeptidasa II analýza imunologie metabolismus MeSH
- imunohistochemie metody MeSH
- kyselina glutamová biosyntéza MeSH
- lidé středního věku MeSH
- lidé MeSH
- mapování epitopu metody MeSH
- molekulární modely MeSH
- mozek anatomie a histologie enzymologie MeSH
- protilátky imunologie MeSH
- radioligandová zkouška metody MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- terciární struktura proteinů genetika MeSH
- western blotting MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
