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

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 IκBα(32/36A) 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 IκBα(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible IκBα(32/36A) peaked at day 2. Inhibition of NF-κB activity was closely correlated with levels of inducible IκBα(32/36A). Levels of 3 well-known NF-κB-dependent genes, CD54, p105, and endogenous IκBα, were decreased when IκBα(32/36A) was induced, and the growth of IκBα(32/36A)-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 IκBα(32/36A)-overexpressing cells. Together these results show that it is possible to control IκBα(32/36A) 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 IκBα(32/36A) may be useful in the identification of genuine NF-κB target genes in EBV- infected B cells. (C) 2000 by The American Society of Hematology.