Utilization of a fos-lacZ plasmid to investigate the activation of c-fos during cellular senescence and okadaic acid-induced apoptosis.
C-fos is an immediate-early gene that is induced by external stimuli and is possibly involved in initiation of DNA synthesis by such stimuli. In these studies, we used the murine c-fos promoter coupled to a lacZ reporter gene to study fos induction in senescent and quiescent cells. In transfected, quiescent, immortal Syrian hamster embryo (SHE) cells (10W), serum stimulation induced the expression of the fos construct to the same extent that DNA synthesis was stimulated. In contrast, in transfected normal cells that have a finite life span, we observed that the cells failed to display upregulation of fos-lacZ in response to serum in individual cells as they senesced. High doses of the phosphatase inhibitor okadaic acid (160-1000 nM) also induced fos-lacZ expression in quiescent immortal cells; however, induction of DNA synthesis and expression of fos-lacZ were not coordinately induced as a function of okadaic acid concentration. Low concentrations of okadaic acid (0.16 nM) induced DNA synthesis but not fos-lacZ expression, indicating that induction of DNA synthesis by phosphatase inhibitors may bypass, at least quantitatively, the requirement for c-fos induction. At the levels of okadaic acid that induced fos-lacZ expression, cell death, rather than DNA synthesis, was observed. The cells died by apoptosis, thereby implicating a signaling pathway that includes c-fos induction in this process.
Afshari
C A
CA
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina.
Bivins
H M
HM
Barrett
J C
JC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics*; D001769:Blood; D015152:Blotting, Northern; D002273:Carcinogens / Q000494:pharmacology*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects / Q000235:genetics; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004622:Embryo, Mammalian; D004988:Ethers, Cyclic / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D016762:Genes, fos / Q000187:drug effects / Q000235:genetics*; D008647:Mesocricetus; D008854:Microscopy, Electron; D019319:Okadaic Acid; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000187:drug effects / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects; D001616:beta-Galactosidase / Q000235:genetics
D002273:Carcinogens; D004988:Ethers, Cyclic; D012333:RNA, Messenger; D019319:Okadaic Acid; D012313:RNA; D004247:DNA; D001616:beta-Galactosidase