Inducible loss of NF-κB activity is associated with apoptosis and Bcl-2 down-regulation in Epstein-Barr virus-transformed B lymphocytes

Abstract

Abstract The Epstein–Barr virus (EBV)-encoded latent membrane protein-1 induces NF-κB activity by targeting IκBα. To understand the role of NF-κB activation in EBV-related oncogenesis, we have subcloned mutated IκBα32/36A cDNA into a pHEBo vector containing doxycycline regulatory sequences and stably transfected this construct into a lymphoblastoid cell line. Two tightly regulated clones were obtained in which IκBα32/36A was inducible in a doxycycline dose-dependent manner. Levels of inducible IκBα32/36A peaked at day 2. Inhibition of NF-κB activity was closely correlated with levels of inducible IκBα32/36A. Levels of 3 well-known NF-κB-dependent genes, CD54, p105, and endogenous IκBα, were decreased when IκBα32/36A was induced, and the growth of IκBα32/36A-induced EBV-infected cells was slightly reduced. Loss of NF-κB activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased in IκBα32/36A-overexpressing cells. Together these results show that it is possible to control IκBα32/36A levels, ie, NF-κB activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-κB can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible IκBα32/36A may be useful in the identification of genuine NF-κB target genes in EBV-infected B cells.