Activation of NF-κB following detachment delays apoptosis in intestinal epithelial cells

We reported earlier that IL-1 beta, an NF-kappa B-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-kappa B- regulated products and therefore aimed to determine whether NF-kappa B signalling plays any role in regulating apoptosis. Using Western blotting, we detected that I kappa B alpha becomes phosphorylated immediately following detachment and that levels of phospho-I kappa B alpha peaked within 20 min. Phosphorylation of I kappa B alpha was followed by Rel A (p65) nuclear translocation. Increased NF-kappa B activity following detachment was confirmed using the detection of NF-kappa B-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor I kappa B alpha protein and pharmacological inhibitors of NF-kappa B resulted in the failure to phosphorylate I kappa B alpha, a more rapid activation of caspases and earlier apoptosis. We also detected that I kappa B kinase alpha (IKK alpha) and not IKK beta became phosphorylated following detachment. Since IKK alpha is activated by NF-kappa B-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-I kappa B alpha and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24 h. We conclude that detachment directly activates NF-kappa B, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells.