title: G-protein-coupled receptor-mediated MAPK and PI3-kinase signaling is maintained in Chinese hamster ovary cells after γ-irradiation.
abstract: To expedite G-protein-coupled receptor (GPCR) drug screening studies, cell lines amenable to transfection (e.g. CHO cells) have been widely used as cellular models. These cells can be frozen in a ready-to-use format, allowing screening of a single batch of cells and validation of the cellular material prior to the screening run. A common method used to deliver frozen cells to screening programs is to γ-irradiate the cells, abrogating cell division after thawing and ensuring consistency in the number of cells analyzed per well. With the recognition that signaling proteins such as ERK and Akt are important markers of GPCR activation, along with the availability of suitable assays for their measurement, these outputs have become important for GPCR screening programs. Here we show that several γ-irradiated and frozen CHO-K1 cell lines expressing transfected GPCRs, initially optimized for performing cAMP or AequoScreen calcium flux assays, can be used for the measurement of GPCR-mediated ERK and Akt phosphorylation. Furthermore, CHO-K1 cells transfected with NOP or GAL(1) receptors show pharmacology for a number of agonists and antagonists that is consistent with non-irradiated cultured lines. These data indicate that γ-irradiated CHO-K1 cells can be reliably used for the measurement of GPCR-mediated kinase signaling outputs.
authors.affiliation: TGR Biosciences, Thebarton, SA, Australia. rosmond@tgrbio.com
authors.forename: Ronald I W
authors.initials: RI
authors.lastname: Osmond
authors.forename: M Henry
authors.initials: MH
authors.lastname: Martin-Harris
authors.forename: Michael F
authors.initials: MF
authors.lastname: Crouch
authors.forename: Janet
authors.initials: J
authors.lastname: Park
authors.forename: Eric
authors.initials: E
authors.lastname: Morreale
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Dupriez
mesh_terms: D000818:Animals; D016466:CHO Cells / Q000528:radiation effects; D002455:Cell Division / Q000528:radiation effects; D006224:Cricetinae; D004353:Drug Evaluation, Preclinical; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D043562:Receptors, G-Protein-Coupled; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases