title: Tumor necrosis factor alpha inhibits cyclin A expression and retinoblastoma hyperphosphorylation triggered by insulin-like growth factor-I induction of new E2F-1 synthesis.
abstract: Cyclin A is required for cell cycle S phase entry, and its overexpression contributes to tumorigenesis. Release of pre-existing E2Fs from inactive complexes of E2F and hypophosphorylated retinoblastoma (RB) is the prevailing dogma for E2F transcriptional activation of target genes such as cyclin A. Here we explored the hypothesis that new synthesis of E2F-1 is required for insulin-like growth factor-I (IGF-I) to induce cyclin A accumulation and RB hyperphosphorylation, events that are targeted by tumor necrosis factor alpha (TNFalpha) to arrest cell cycle progression. We first established that IGF-I increases expression of cyclin A, causes hyperphosphorylation of RB, and augments the mass of E2F-1 in a time-dependent manner. As expected, E2F-1 small interfering RNA blocks the ability of IGF-I to increase synthesis of E2F-1. Most important, this E2F-1 small interfering RNA also blocks the ability of IGF-I to increase cyclin A accumulation and to hyperphosphorylate RB. We next established that TNFalpha dose-dependently inhibits IGF-I-induced phosphorylation of both RB and histone H1 by cyclin A-dependent cyclin-dependent kinases. Cyclin-dependent kinase 2 (Cdk2) mediates this suppression because co-immunoprecipitation experiments revealed that TNFalpha reduces the amount of IGF-I-induced cyclin A that binds Cdk2, leading to a reduction in Cdk2 enzymatic activity. TNFalpha antagonizes the ability of IGF-I to increase mass of both E2F-1 and cyclin A but not cyclin E or D1. The cytostatic property of TNFalpha is also shown by its ability to block IGF-I-stimulated luciferase activity of a cyclin A promoter reporter. Deletion of an E2F recognition site from this reporter eliminates the regulatory effects of both IGF-I and TNFalpha on cyclin A transcription, indicating the essential role of E2F-1 in mediating their cross-talk. Collectively, these results establish that TNFalpha targets IGF-I-induced E2F-1 synthesis, leading to inhibition of the subsequent accumulation in cyclin A, formation of cyclin A-Cdk2 complexes, hyperphosphorylation of RB, and cell cycle arrest.
authors.affiliation: Laboratory of Immunophysiology, Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801, USA.
authors.forename: Wen Hong
authors.initials: WH
authors.lastname: Shen
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Yin
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Broussard
authors.forename: Robert H
authors.initials: RH
authors.lastname: McCusker
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Freund
authors.forename: Robert
authors.initials: R
authors.lastname: Dantzer
authors.forename: Keith W
authors.initials: KW
authors.lastname: Kelley
mesh_terms: D000230:Adenocarcinoma; D001943:Breast Neoplasms; D042846:CDC2-CDC28 Kinases / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D019925:Cyclin A / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D051357:Cyclin-Dependent Kinase 2; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D016160:Retinoblastoma Protein / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D016160:Retinoblastoma Protein; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D010768:Phosphoserine; D007334:Insulin-Like Growth Factor I; D008156:Luciferases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2