Growth factor-induced transcription via the serum response element is inhibited by cyclic adenosine 3',5'-monophosphate in MCF-7 breast cancer cells.
The effect of increased intracellular cAMP on MCF-7 breast cancer cell growth was examined by treating cells with either forskolin, an activator of adenylate cyclase, or 8-[4-chlorophenylthio]-cAMP (8-CPT-cAMP), a cAMP analog. Compared to cells maintained in control medium, treatment with either 1 or 10 microM forskolin decreased cell growth by 17% and 68%, respectively, whereas treatment with 250 microM 8-CPT-cAMP decreased cell growth by 29%. To determine whether this effect of cAMP on cell growth was mediated by inhibition of the activity of extracellular signal-regulated kinases 1 and 2 (ERK1 and -2), two mitogen-activated protein kinases, the effect of cAMP on growth factor-induced ERK activity in MCF-7 cells was examined. Treatment with either insulin-like growth factor I (IGF-I) or epidermal growth factor (EGF) for 10 min stimulated a 4- to 8-fold increase in ERK1 and -2 activity. This effect of IGF-I and EGF was not inhibited by increased intracellular cAMP generated by pretreatment of the cells with 10 microM forskolin. Similarly, 10 microM forskolin had no effect on IGF-I- or EGF-induced ERK activity in cells treated with growth factor for 30 min. To determine whether cAMP inhibits other growth factor-mediated effects, its effect on the activity of the serum response element (SRE), a DNA promoter element whose activity is regulated by a variety of growth-promoting events, was examined. For these assays, MCF-7 cells were transiently transfected with pTK81-SRE-Luc, a luciferase fusion gene that contains the SRE cloned 5' to a minimal thymidine kinase promoter and the luciferase gene. Treatment with either IGF-I or EGF increased pTK81-SRE-Luc activity in a dose-dependent fashion. Pretreatment of cells with 10 microM forskolin decreased IGF-I- and EGF-stimulated luciferase activity by approximately 75%. An intermediate effect was observed using 1 microM forskolin. When intracellular cAMP levels were increased using 8-CPT-cAMP, similar results were obtained. SRE activity is dependent upon the activation by phosphorylation of a ternary complex factor; included among the ternary complex factors is Elk-1. When MCF-7 cells were cotransfected with a vector that expresses a Gal4/Elk-1 fusion protein and UAS-TK-Luc, a plasmid that contains two Gal4 DNA recognition sites cloned 5' to a thymidine kinase promoter and the luciferase gene, treatment with forskolin partially inhibited the activation of Elk-1 by IGF-I and EGF. These data demonstrate that in MCF-7 breast cancer cells, cAMP has no effect on IGF-I- or EGF-induced ERK activity, but it inhibits growth factor-induced transcription. Taken together with the effects of cAMP on IGF-I- and EGF-induced Elk-1 activation, these data suggest that the effect of cAMP on SRE activity occurs distal to ERK activation, possibly via inhibition of an ERK-independent pathway. Finally, these data indicate that the effect of increased intracellular cAMP on breast cancer growth may be mediated through inhibition of specific growth factor-induced effects, including gene transcription.
Lowe
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Department of Medicine, Veterans Administration Chicago Healthcare System and Northwestern University Medical School, Chicago, Illinois 60611, USA. wlowe@nwu.edu
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D001943:Breast Neoplasms; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000502:physiology; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D026362:Serum Response Factor; D013873:Thionucleotides / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D026362:Serum Response Factor; D013873:Thionucleotides; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP; D008156:Luciferases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases