The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug-metabolizing enzymes andmany other genes. Radiolabeled ³²P-DNA-probes had been used in studies of PXR-DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.
- MeSH
- cytochrom P-450 CYP3A genetika metabolismus MeSH
- HeLa buňky MeSH
- lidé MeSH
- molekulární sondy genetika metabolismus MeSH
- responzivní elementy * MeSH
- retardační test metody MeSH
- steroidní receptory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
AIMS: The aim was develop stable human cell line stable over-expressing transcription co-activator peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) with restored hepatospecific functions and increased expression of major xenobiotic metabolizing enzymes. METHODS: Six clones of HepG2-PGC-1α and one control clone HepG2-pcDNA3 were isolated and analyzed for secretion of hepatospecific markers, fibrinogen, albumin and alpha1-antitrypsin. Expression levels of protein and mRNA of hepatocyte nuclear factor (HNF4α), pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) were determined. We measured basal and ligand inducible expression of CYP1A1 and CYP3A4. RESULTS: Stably transfected cell line HepG2-PGC-1α derived from HepG2 cells over-expressing PGC-1α displayed increased secretion of fibrinogen, but not albumin or alpha1-antitrypsin compared to parent HepG2 cells. We found increased levels of HNF4α, PXR and AhR proteins but not their mRNAs in HepG2-PGC1 cells. Basal expression of CYP3A4 protein in HepG2-PGC-1α cells was increased but rifampicin-inducible expression of CYP3A4 protein was lowered in comparison with parent HepG2 cells. Induction of CYP3A4 mRNA varied between 1.3 - 1.9 fold in individual clones. Expression of TCDD-inducible CYP1A1 protein was lower in HepG2-PGC-1α cells than in parent HepG2 cells. Induction of CYP1A1 mRNA by TCDD in HepG2-PGC-1α cells was comparable with that in parent HepG2 cells and ranged between 103 - 198 fold. CONCLUSION: Stable expression of PGC-1α in HepG2 cells restores several hepatospecific functions, such as secretion of fibrinogen, expression of HNF4α1 and xenoreceptors PXR and AhR. However, the expression and induction of key drug-metabolizing enzymes (CYP1A1 and CYP3A4) were not improved.
- MeSH
- buňky Hep G2 MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- cytochrom P-450 CYP3A metabolismus MeSH
- fibrinogen sekrece MeSH
- gentamiciny farmakologie MeSH
- hepatocytární jaderný faktor 4 metabolismus MeSH
- hepatocyty metabolismus MeSH
- lidé MeSH
- nádorové biomarkery metabolismus MeSH
- polychlorované dibenzodioxiny farmakologie MeSH
- PPAR gama metabolismus MeSH
- receptory aromatických uhlovodíků metabolismus MeSH
- steroidní receptory metabolismus MeSH
- teratogeny farmakologie MeSH
- transfekce metody MeSH
- transkripční faktory bHLH metabolismus MeSH
- transkripční faktory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Valproic acid (VPA) is a wide spread anticonvulsant and mood-stabilizing agent, the use of which is associated with hepatotoxicity, bone marrow suppression and osteomalacia. In the current paper we propose a possible mechanism of VPA-induced osteomalacia involving accelerated catabolism of 1α,25(OH)(2)-vitamin D3 (VD3) due to increased expression of CYP24. We demonstrate that VPA strongly potentiates CYP24 mRNA expression by VD3 in human hepatocytes (HH) and in human embryonic kidney cells (HEK293). By the method of gene reporter assay we found that VPA increases basal and VD3-inducible activity of CYP24 promoter (pCYP24-luc) in human liver adenocarcinoma (HepG2) and in HEK293 cells in dose-dependent manner. In order to delineate the role of inhibitory effects of VPA on histone deacetylase 1 (HDAC1), we compared the effects of VPA with trichostatin A (TSA) on basal and inducible levels of CYP24 mRNA and pCYP24-luc transactivation. Transactivation of CYP24 promoter by VD3 was enhanced in the presence of both TSA and VPA. In contrast, VD3-inducible expression of CYP24 mRNA was enhanced by VPA but not by TSA, implying that HDAC1 inhibition is not the major reason for VPA effects on CYP24. We examined the effects of VPA on mitogen-activated protein kinases as the important transcriptional regulators of VDR. VPA activated extracellular signal-regulated kinase (ERK) but not c-Jun-N-terminal kinase (JNK) and p38 MAPKs. In conclusion, VPA enhances transcriptional activity of VDR and increases expression of CYP24 mRNA in the presence of VD3 in physiological concentrations. The mechanism involves activation of ERK and partly the inhibition of HDAC1.
- MeSH
- antikonvulziva toxicita MeSH
- cholekalciferol farmakologie MeSH
- extracelulárním signálem regulované MAP kinasy genetika metabolismus MeSH
- HEK293 buňky MeSH
- hepatocyty účinky léků MeSH
- histondeacetylasa 1 biosyntéza genetika MeSH
- kyselina valproová toxicita MeSH
- lidé MeSH
- luciferasy genetika MeSH
- messenger RNA biosyntéza genetika MeSH
- osteomalacie chemicky indukované patologie MeSH
- plazmidy genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- receptory kalcitriolu účinky léků MeSH
- steroidhydroxylasy biosyntéza genetika MeSH
- steroidní receptory účinky léků MeSH
- transfekce MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cancer cell lines derived from hepatocytes have an altered phenotype and they lack hepatocyte-specific functions. It is at least partly due to the under-expression of transcription factors such as hepatocyte nuclear factor 4α (HNF4α), steroid receptor co-activator 1 (SRC1) etc. Recently, a strategy of transient transfection of human hepatic cells with HNF4α revealed improved hepatospecific functions, including the expression of drug-metabolizing enzymes. In the current study we established a human cell line derived from HepG2 cells stably transfected with human HNF4α, and we examined this line for hepatospecific markers. Of the 9 clones analyzed, we found an increased secretion of fibrinogen (9 clones), albumin (5 clones) and plasminogen (3 clones), while secretion of alpha1-antitrypsin was not changed. The expression of pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) proteins but not mRNAs was slightly increased. TCDD-dependent induction of CYP1A1 mRNA and protein was augmented in 50% of clones, but there was no correlation between the CYP1A1 inducibility and expression levels of AhR and HNF4α. Induction of CYP3A4 mRNA by rifampicin was about 1.5-2.5 fold (clones 2, 4, 6, 7) and it was not significantly different from CYP3A4 mRNA induction in parent HepG2. The basal expression of CYP3A4 protein was increased in all clones, but rifampicin-induced expression of CYP3A4 protein was in all clones lower than in parent HepG2. Overall, the stable over-expression of HNF4α in HepG2 cells restores some of the hepatospecific functions, but it has a minor effect on the expression of xenobiotic-metabolizing enzymes and their regulators.
- MeSH
- albuminy metabolismus MeSH
- alfa-1-antitrypsin metabolismus MeSH
- antiprotozoální látky farmakologie MeSH
- cytochrom P-450 CYP1A1 metabolismus MeSH
- cytochrom P-450 CYP3A metabolismus MeSH
- fibrinogen metabolismus MeSH
- gentamiciny farmakologie MeSH
- glyceraldehyd-3-fosfátdehydrogenasy metabolismus MeSH
- hepatocytární jaderný faktor 4 genetika metabolismus MeSH
- hepatocyty metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie metabolismus MeSH
- plazminogen metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- receptory aromatických uhlovodíků metabolismus MeSH
- steroidní receptory metabolismus MeSH
- transfekce MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Autoři článku se snaží ve zkratce přiblížit čtenářům možnost využití oboru ergoterapie, respektive erudovaného ergoterapeuta, v oblasti akutní i následné gerontopsychiatrie. V oblasti nefarmakologické intervence pacientů s demencí se setkáváme se širokým spektrem profesního zastoupení od sester, psychologů, psychiatrů, činnostních terapeutů, sanitářů a dalších. Zastoupení ergoterapeutů je stále ještě poměrně málo rozšířené, i když je z naší vlastní zkušenosti zřejmé, že práce ergoterapeuta je v oblasti gerontopsychiatrie nezastupitelná.
The authors aim at acquainting the reader briefly with the possible use of ergotherapy, or an experienced ergotherapist, in the field of acute as well as follow-up gerontopsychiatry. In the area of nonpharmacological intervention in dementia patients, a wide range of professionals are encountered including nurses, psychologists, occupational therapists, orderlies and others. The involvement of ergotherapists is still relatively little widespread even though it is apparent from our experience that, in the field of gerontopsychiatry, the work of an ergotherapist is irreplaceable.
- Klíčová slova
- terapeutická jednotka,
- MeSH
- cévní mozková příhoda MeSH
- ergoterapie metody MeSH
- geriatrická psychiatrie metody MeSH
- lidé MeSH
- rehabilitace po cévní mozkové příhodě MeSH
- rehabilitace MeSH
- senioři MeSH
- Check Tag
- lidé MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- přehledy MeSH
