Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.10 (IKK)
 4,900 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:A cell-based assay for IkappaBalpha stabilization using a two-color dual luciferase-based sensor. 1735 2

Emerging studies indicate that DNA damage in cancer cells triggers antitumor immunity, but its intrinsic regulatory mechanism in breast cancer cells remains poorly understood. Here, we show that ZMYND8 is upregulated and inhibits micronucleus formation and DNA damage in breast cancer cells. Loss of ZMYND8 triggered activation of the DNA sensor cyclic guanosine monophosphate-adenosine monophosphate synthase in micronuclei, leading to further activation of the downstream signaling effectors stimulator of interferon genes and NF-kappaB, but not TANK-binding kinase 1 and interferon regulatory factor 3, thereby inducing the expression of interferon-beta and interferon-stimulated genes (ISGs) in breast cancer cells in vitro and tumors in vivo. ZMYND8 knockout (KO) in breast cancer cells promoted infiltration of CD4+ and CD8+ T cells leading to tumor inhibition in syngeneic mouse models, which was significantly attenuated by treatment of anti-CD4/CD8 depleting antibodies or anti-IFNAR1 antibody and in immunodeficient Rag1 KO mice. In human breast tumors, ZMYND8 was negatively correlated with ISGs, CD4, CD8A, CD8B, and the tumor-lymphocyte infiltration phenotype. Collectively, these findings demonstrate that maintenance of genome stability by ZMYND8 causes breast cancer cells to evade cytotoxic T-lymphocyte surveillance which leads to tumor growth.
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PMID:ZMYND8 expression in breast cancer cells blocks T-lymphocyte surveillance to promote tumor growth. 3314 60