title: Quantifying nuclear p65 as a parameter for NF-κB activation: Correlation between ImageStream cytometry, microscopy, and Western blot.
abstract: The nuclear factor kappa B (NF-κB) pathway, which regulates many cellular processes including proliferation, apoptosis, and survival, has emerged as an important therapeutic target in cancer. Activation of the NF-κB transcription factor is associated with nuclear translocation of the p65 component of the complex. Conventional methods employed to determine nuclear translocation of NF-κB either lack statistical robustness (microscopy) or the ability to discern heterogeneity within the sampled populations (Western blotting and Gel Shift assays). The ImageStream platform combines the high image content information of microscopy with the high throughput and multiparameter analysis of flow cytometry which overcomes the aforementioned limitations of conventional assays. It is demonstrated that ImageStream assessment of receptor-mediated (TNFα) and drug (Daunorubicin, DNR)-induced NF-κB translocation in leukemic cell lines correlates well with microscopy analysis and Western blot analysis. It is further demonstrated that ImageStream cytometry enables quantitative assessment of p65 translocation in immunophenotypically defined subpopulations; and that this assessment is highly reproducible. It is also demonstrated that, quantitatively, the DNR-induced nuclear translocation of NF-κB correlates well with a biological response (apoptosis). We conclude that the ImageStream has the potential to be a powerful tool to evaluate NF-κB /p65 activity as a determinant of response to therapies designed to target aberrant NF-κB signaling activities.
authors.affiliation: Department of Flow and Image Cytometry, Roswell Park Cancer Institute, Buffalo, New York, USA. orla.maguire@roswellpark.org
authors.forename: Orla
authors.initials: O
authors.lastname: Maguire
authors.forename: Christine
authors.initials: C
authors.lastname: Collins
authors.forename: Kieran
authors.initials: K
authors.lastname: O'Loughlin
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Miecznikowski
authors.forename: Hans
authors.initials: H
authors.lastname: Minderman
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003630:Daunorubicin / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D051996:Transcription Factor RelA / Q000819:agonists / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D003630:Daunorubicin