title: Effects of receptor tyrosine kinase inhibitors on VEGF165 a- and VEGF165 b-stimulated gene transcription in HEK-293 cells expressing human VEGFR2.
abstract: OBJECTIVE
  Receptor tyrosine kinase inhibitors (RTKIs) targeted at VEGF receptor 2 (VEGFR2) have proved to be attractive approaches to cancer therapy based on their ability to reduce angiogenesis. Here we have undertaken a quantitative analysis of the interaction of RTKIs and two VEGF splice variants, VEGF(165)a and VEGF(165)b, with VEGFR2 by studying nuclear factor of activated T-cells (NFAT) reporter gene activity in live HEK-293 cells.
  
  
  METHODS
  HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF(165)a- and VEGF(165)b-stimulated luciferase gene expression.
  
  
  RESULTS
  VEGF(165)a produced a concentration-dependent activation of the NFAT-luciferase reporter gene in living cells that was inhibited in a non-competitive fashion by four different RTKIs (cediranib, pazopanib, sorafenib and vandetanib). The potency obtained for each RTKI from this analysis was similar to those obtained in binding studies using purified VEGFR2 kinase domains. VEGF(165)b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF(165)a. Analysis of the concentration-response data using the operational model of agonism indicated that both VEGF(165) isoforms had similar affinity for VEGFR2.
  
  
  CONCLUSIONS
  Quantitative pharmacological analysis of the interaction of VEGF(165) isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF(165)a and VEGF(165)b for activation of the calcineurin- NFAT signalling pathway by this tyrosine kinase receptor.
authors: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.
authors: Joanne J
authors: JJ
authors: Carter
authors: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.
authors: Amanda J
authors: AJ
authors: Wheal
authors: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.
authors: Stephen J
authors: SJ
authors: Hill
authors: Cell Signalling Research Group, School of Life Sciences, University of Nottingham, Nottingham, UK.
authors: Jeanette
authors: J
authors: Woolard
mesh_terms: D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics; D050778:NFATC Transcription Factors / Q000235:genetics*; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D050778:NFATC Transcription Factors; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D049409:Luciferases, Firefly; D040301:Vascular Endothelial Growth Factor Receptor-2