The up-regulation of human caspase-8 by interferon-gamma in breast tumor cells requires the induction and action of the transcription factor interferon regulatory factor-1.
Treatment of human breast tumor cells with interferon-gamma (IFN-gamma) elevates caspase-8 expression and sensitizes these cells to death receptor-mediated apoptosis through the increased processing and activation of apical procaspase-8. We have characterized the human caspase-8 gene promoter and studied the transcriptional regulation of caspase-8 gene expression in MCF-7 breast tumor cells treated with IFN-gamma. Our findings show that IFN-gamma induces the up-regulation of caspase-8 mRNA expression through a protein synthesis-dependent mechanism involving the action of the IFN-gamma-inducible transcription factor interferon regulatory factor-1 (IRF-1) and without altering mRNA stability. The human caspase-8 gene promoter lacks recognizable TATA and CAAT boxes but contains a consensus Sp1 binding site. We have identified two major IFN-gamma-inducible transcriptional start sites in these cells by S1 nuclease mapping, confirmed by primer extension analysis. Deletion analysis of the promoter defined an 82-bp minimal region responsible for IFN-gamma-inducible promoter activity. In this region, we have identified an IFN-stimulated response element that is important for both the basal and IFN-gamma-enhanced transcriptional activities. Electrophoretic mobility shift assay analysis demonstrated that IFN-gamma induces a complex between an oligonucleotide probe containing the ISRE motif and IRF-1 over a similar time scale to the induction of caspase-8 mRNA. Exogenously expressed IRF-1 in MCF-7 cells up-regulated the activity of a luciferase reporter plasmid containing an 82-bp region of the caspase-8 promoter. These data define a new pathway through which IFN-gamma might control the sensitivity of tumor cell to death receptor-mediated apoptosis.
Instituto de Parasitología y Biomedicina, CSIC, Calle Ventanilla 11, 18001 Granada, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Madrid E-28049, Spain.
Carmen
C
Ruiz-Ruiz
Carmen
C
Ruiz de Almodóvar
Antonio
A
Rodríguez
Gustavo
G
Ortiz-Ferrón
Juan Miguel
JM
Redondo
Abelardo
A
López-Rivas
D020816:Amino Acid Motifs; D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D001943:Breast Neoplasms / Q000378:metabolism*; D053181:Caspase 8; D020169:Caspases / Q000096:biosynthesis* / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000502:physiology*; D017353:Gene Deletion; D006801:Humans; D015151:Immunoblotting; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D010750:Phosphoproteins; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; D007371:Interferon-gamma; D008156:Luciferases; D015719:Single-Strand Specific DNA and RNA Endonucleases; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases