Localization of IFN-gamma-activated Stat1 and IFN regulatory factors 1 and 2 in breast cancer cells.
The aim of the present work was to evaluate the induction and localization of Stat1, interferon (IFN) regulatory factor-1 (IRF-1), and IRF-2 after IFN-gamma exposure of human breast cancer cell lines, SKBR3, MDA468, MCF7, and BT20. Results from growth assays, Western staining, electrophoretic mobility shift assay (EMSA), and immunohistochemical staining were collated to test our hypothesis that immunohistochemical analysis of Stat1, IRF-1, and IRF-2 would provide additional information about the functionality of the IFN-gamma signaling pathway in human tumor lines. EMSA results showed that in each of four cell lines, Stat1 expression was increased and demonstrated functional activity after IFN-gamma stimulation. Western and EMSA analysis showed upregulation of IRF-1 but not IRF-2 in each cell line. Confocal microscopy of cells stained for Stat1, IRF-1, and IRF-2 confirmed the results and also provided novel information about the intracellular localization of proteins and intercellular variations in responses. The proportion of cells with IRF-1 stimulation and translocation was positively correlated with the IFN-gamma growth suppression in vitro. In conclusion, using four independent assays, we have demonstrated that heterogeneity in IFN-gamma-mediated upregulation of signal transduction proteins can be detected in vitro and that these differences can explain distinct cellular growth effects.
Connett
Judith M
JM
Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA.
Hunt
Steven R
SR
Hickerson
Suzanne M
SM
Wu
Susan J
SJ
Doherty
Gerard M
GM
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D005786:Gene Expression Regulation; D006801:Humans; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D007371:Interferon-gamma / Q000494:pharmacology*; D018613:Microscopy, Confocal; D010750:Phosphoproteins / Q000378:metabolism*; D011994:Recombinant Proteins; D012097:Repressor Proteins; D050794:STAT1 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; C494229:IRF2 protein, human; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D010750:Phosphoproteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D007371:Interferon-gamma