A cell-based assay for IkappaBalpha stabilization using a two-color dual luciferase-based sensor.
A cell-sensor assay for stabilization of IkappaBalpha was developed in the activated B cell-like diffuse large B-cell lymphoma cell line OCI-Ly3. This cell line expresses known nuclear factor kappaB (NFkappaB) target genes due to high constitutive activity of IkappaB kinase (IKK), which phosphorylates the protein IkappaBalpha leading to proteasomal degradation of IkappaBalpha and activation of NFkappaB. The cell-sensor assay uses green and red light-emitting beetle luciferases, with the green luciferase fused to IkappaBalpha (IkappaBalpha-CBG68) and the red luciferase (CBR) present in its native state. The IkappaBalpha-CBG68 reporter functions as a sensor of IKK and proteasome activity, while CBR serves to normalize for cell number and nonspecific effects. Both reporter constructs were stably integrated and placed under the control of an inducible promoter system, which increased fold responsiveness to inhibitors when assay incubations were performed simultaneous to reporter induction by doxycycline. The assay was miniaturized to a 1,536-well plate format and showed a Z' of 0.6; it was then used to panel 2,677 bioactive compounds by a concentration-response-based screening strategy. The concentration-effect curves for the IkappaBalpha-CBG68 and CBR signals were then used to identify specific stabilizers of IkappaBalpha, such as IKK inhibitors or proteasome inhibitors, which increased the doxycycline-induced rise in IkappaBalpha-CBG68 without affecting the rise in CBR. Known and unexpected inhibitors of NFkappaB signaling were identified from the bioactive collection. We describe here the development and performance of this assay, and discuss the merits of its specific features.
Davis
R Eric
RE
Metabolism Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892-3370, USA.
Zhang
Ya-Qin
YQ
Southall
Noel
N
Staudt
Louis M
LM
Austin
Christopher P
CP
Inglese
James
J
Auld
Douglas S
DS
D001681:Biological Assay / Q000379:methods*; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D020672:I-kappa B Proteins / Q000032:analysis* / Q000378:metabolism*; D008156:Luciferases; D016393:Lymphoma, B-Cell / Q000378:metabolism*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D000072000:NF-KappaB Inhibitor alpha; D009363:Neoplasm Proteins / Q000378:metabolism*
D014408:Biomarkers, Tumor; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D009363:Neoplasm Proteins; D000072000:NF-KappaB Inhibitor alpha; D008156:Luciferases