Nuclear factor kappa B dominant negative genetic constructs inhibit X-ray induction of cell adhesion molecules in the vascular endothelium

X-ray-induced expression of inflammatory mediators has been proposed to con tribute to radiation injury in normal tissues. Radiation-inducible inflamma tory mediators include the cell adhesion molecule (CAM) E-selectin and the intercellular adhesion molecule (ICAM)-1, Nuclear factor (NF)KB is activate d by X-rays and may participate in the transcriptional regulation of each o f these inflammatory mediators. To determine whether NF kappa B inhibition abrogates X-ray induction of inflammatory mediators, we used two experiment al approaches including NF kappa B inhibitory drugs and a dominant negative genetic construct. Human umbilical vein endothelial cells (HUVEC) and huma n microvascular endothelial cells were treated with the NF kappa B inhibito rs ALLN, PDTC, NAG, and MG132, After irradiation, E-selectin or ICAM-1 was measured by fluorescence-activated cell-sorting analysis. E-selectin and IC AM-1 expression was measured by use of immunofluorescence and fluorescence- activated cell-sorting analysis. E-selectin expression increased 7-fold, an d ICAM-1 expression increased 4-fold after irradiation, All of the inhibito rs attenuated E-selectin expression after irradiation, ALLN and MG132 atten uated radiation-induced ICAM expression, However, PDTC and NAC induced incr eased expression of ICAM-1 in HUVECs, Inhibition of X-ray induction of ICAM by these agents could not he demonstrated. In separate experiments, the NF kappa B dominant negative genetic construct was cotransfected with the pro moter-reporter constructs by means of Lipofectin reagent, The ICAM promoter -reporter construct consists of the 1.2-kb segment of the human ICAM promot er upstream of the transcriptional start site linked to the luciferase repo rter gene (pGL.FL-Luc). The E-selectin promoter-reporter construct consists of 525 bp upstream of the transcriptional start site of the human E-select in promoter linked to the human growth hormone reporter gene (pE525-GH). En dothelial cells transfected with the ICAM-1 promoter-reporter construct sho wed a 3-fold induction after irradiation. Likewise, cells transfected with pE525-GH showed a 7-fold induction after irradiation. When cotransfected wi th the CAM reporter-promoter constructs, the NF kappa B dominant negative g enetic construct abolished X-ray-induced transcriptional activation of the E-selectin and ICAM-1 promoters. NF kappa B inhibition is, therefore, a mea ns of abrogating radiation-induced expression of CAMs.