Activation of nuclear factor kappa B (NF-kappaB) assayed by laser scanning cytometry (LSC).
Nuclear factor kappa B (NF-kappaB)/rel is the family of ubiquitous transcriptional activators involved in regulation of diverse immune and inflammatory responses. It also plays a role in control of cell growth and apoptosis. In its inactive form NF-kappaB remains in the cytoplasm sequestered through interaction with IkappaB protein. Rapid translocation of NF-kappaB from cytoplasm to nucleus that occurs in response to extracellular signals is considered to be a hallmark feature of its activation. The translocation of NF-kappaB in HL-60, U-937 and Jurkat leukemic cells as well as in human fibroblasts induced by tumor necrosis factor alpha (TNF-alpha) or phorbol myristate acetate (PMA) was presently measured by laser scanning cytometry (LSC). NF-kappaB was detected immunocytochemically with FITC-tagged antibody and its presence in the nucleus vis-a-vis cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide (PI), and over the cytoplasm, respectively. Activation of NF-kappaB led to a rapid increase in NF-kappaB-associated fluorescence measured over the nucleus (FN) concomitant with a decrease in fluorescence over the cytoplasm (F(C)), which was reflected by an increase in F(N)/F(C) ratio. This rapid assay of NF-kappaB activation can be combined with morphological identification of the activated cells or with their immunophenotype. Bivariate analysis of NF-kappaB expression versus cellular DNA content makes it possible to correlate its activation with the cell cycle position. The described method has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators such as p53 tumor suppressor protein or signal transduction molecules.
Deptala
A
A
Brander Cancer Research Institute, New York Medical College, Valhalla, USA.
Bedner
E
E
Gorczyca
W
W
Darzynkiewicz
Z
Z
D002453:Cell Cycle; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D004247:DNA / Q000032:analysis; D018922:HL-60 Cells; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007150:Immunohistochemistry; D019169:Jurkat Cells; D016328:NF-kappa B / Q000378:metabolism*; D010641:Phenotype; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020298:U937 Cells
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D013755:Tetradecanoylphorbol Acetate