Formation of the early-region-2 transcription-factor-1-retinoblastoma-protein (E2F-1-RB) transrepressor and release of the retinoblastoma protein from nuclear complexes containing cyclin A is induced by interferon alpha in U937V cells but not in interferon-alpha-resistant U937VR cells.
We have analysed the different regulation of cell-cycle-relevant proteins by interferon alpha (IFN alpha) in IFN alpha-sensitive and resistant U937 leukemic cell lines. In contrast to the INF alpha-sensitive U937 variant cell line U937V, the IFN alpha-resistant derivative (U937VR) is insensitive to the antiproliferative activity of IFN alpha. As we found no differences between these cell lines concerning the induction by IFN alpha of the pathway involving tyrosine-protein kinases and the signal transducer and activator of transcription (Jak-Stat), we examined whether cell-cycle-regulating proteins are differently affected by IFN alpha in U937VR and U937VR cells. In U937V cells IFN alpha induced the formation of the complex between early-region-2 transcription factor 1 (E2F-1) and retinoblastoma protein (RB) which is known to repress transcription of E2F-1-inducible genes, necessary for cell cycle progression. Formation of this complex was not inducible by IFN alpha in U937VR cells, although the suitable binding partners (E2F-1 and under-phosphorylated RB) were present. Interestingly, treatment of nuclear extracts from logarithmically growing U937V and U937VR cells with an antiserum against cyclin A that disrupts cyclin-A-containing complexes, led to the formation of the E2F-1-RB complex, suggesting the presence of under-phosphorylated (active) RB, trapped in nuclear complexes that contain cyclin A. This suggestion was supported by combined immunoprecipitation/western blot experiments that revealed a physical interaction between phosphorylated as well as under-phosphorylated forms of RB and cyclin A complex(es) in U937V and U937VR cells. RB, especially the under-phosphorylated form, was released by treatment with IFN alpha from this complex(es) in the case of U937V cells but not U937VR cells. We conclude that the missing induction of the E2F-1-RB transrepressor by IFN alpha and the failure to release RB from cyclin-A-containing complexes might contribute to the resistance of U937VR cells to the antiproliferative effects of IFN alpha.
Kirch
H C
HC
Department of Molecular Biology, West German Cancer Centre, University of Essen Medical School, Germany. h-c.kirch@uni-essen.de
Putzer
B
B
Brockmann
D
D
Esche
H
H
Kloke
O
O
D000906:Antibodies / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D016193:G1 Phase; D005786:Gene Expression Regulation; D006801:Humans; D016898:Interferon-alpha / Q000502:physiology*; D046911:Macromolecular Substances; D008954:Models, Biological; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D056465:Retinoblastoma-Binding Protein 1; D050794:STAT1 Transcription Factor; D050795:STAT2 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D016898:Interferon-alpha; D046911:Macromolecular Substances; D012097:Repressor Proteins; D016160:Retinoblastoma Protein; D056465:Retinoblastoma-Binding Protein 1; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050795:STAT2 Transcription Factor; D015534:Trans-Activators; D050680:Transcription Factor DP1; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases