INTRODUCTION: Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand. METHODS: To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-α) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain. RESULTS: Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-α, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement. DISCUSSION: The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.
- MeSH
- časové faktory MeSH
- epidermální růstový faktor chemie metabolismus MeSH
- erbB receptory antagonisté a inhibitory chemie metabolismus MeSH
- humanizované monoklonální protilátky chemie farmakologie MeSH
- kompetitivní vazba účinky léků MeSH
- lidé MeSH
- ligandy MeSH
- monoklonální protilátky chemie farmakologie MeSH
- nádorové buňky kultivované MeSH
- substrátová specifita MeSH
- transformující růstový faktor alfa chemie metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus-oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2, TNFAIP6, and HAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression of CYP11A1 in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.
- MeSH
- buněčná diferenciace účinky léků genetika MeSH
- embryonální vývoj účinky léků genetika MeSH
- epidermální růstový faktor chemie farmakologie MeSH
- folikuly stimulující hormon farmakologie MeSH
- gonadotropiny farmakologie MeSH
- kultivace embrya MeSH
- kultivované buňky MeSH
- kumulární buňky účinky léků metabolismus fyziologie MeSH
- oocyty účinky léků metabolismus fyziologie MeSH
- oogeneze účinky léků genetika MeSH
- partenogeneze účinky léků genetika fyziologie MeSH
- peptidové fragmenty chemie farmakologie MeSH
- prasata genetika metabolismus fyziologie MeSH
- proliferace buněk účinky léků MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- MeSH
- epidermální růstový faktor chemie metabolismus MeSH
- erythropoetin metabolismus MeSH
- insulinu podobný růstový faktor I metabolismus MeSH
- růstové látky MeSH
- transformující růstový faktor alfa chemie metabolismus MeSH
- transformující růstový faktor beta chemie metabolismus MeSH
- Publikační typ
- přehledy MeSH
