Gi-protein inhibitor, guanosine 5'-O-(2-thiodiphosphate), induces senescence-associated beta-galactosidase positive cell formation through CREB phosphorylation.
OBJECTIVE
We evaluated Gi-protein inhibitor, guanosine 5'-O-(2-thiodiphosphate)(GOT)-induced senescence-associated(SA)-beta-galactosidase(Gal) positive cell formation to determine if it occurred through phosphorylation of cyclic AMP-dependent response element binding protein (CREB).


METHODS
IMR-90 human lung fibroblast cells were used. SA-beta-Gal positive cells and senescence-associated heterochromatic foci (SAHF) were determined by assessing blue color formation of substrate, X-gal inside cells and DAPI staining, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis. CREB phosphorylation and molecular changes were analyzed by western blot.


RESULTS
GOT treatment led to SA-beta-Gal positive cell formation and SAHF. CREB phosphorylation increased in response to GOT treatment but then decreased over 24h. SA-beta-Gal positive cell formation increased in response to transient transfection of pS6-RSV-CREB and no changes were detected following CREB knockdown with CREB-siRNA. In addition, CREB phosphorylation was delayed by treatment with the anti-cellular senescence agents, clitocybins which also reduced the number of SA-beta-Gal positive cells. Collectively, our data showed that GOT-induced CREB phosphorylation initiated SA-beta-Gal positive cell formation after which decreased in SA-beta-Gal positive cells.


CONCLUSIONS
These findings suggest for the first time that CREB phosphorylation by GOT could induce cellular senescence as judged by SA-beta-Gal positive cell formation.
Moon
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EY
Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Republic of Korea.
Kim
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HS
Im
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D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D016922:Cellular Senescence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004171:Diploidy; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D013873:Thionucleotides / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D013873:Thionucleotides; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D001616:beta-Galactosidase; D019206:GTP-Binding Protein alpha Subunits, Gi-Go