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

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 induces NF-kappa B activity by targeting I kappa B alpha. To understand the role of NF-kappa B activation in EBV-related oncogenesis, we have subcloned mutated (I kappa B alpha(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 I kappa B alpha(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible I kappa B alpha(32/36A) peaked at day 2, inhibition of NF-KB activity was closely correlated with levels of inducible I kappa B alpha(32/36A) Levels of 3 well-known NF-kappa B-dependent genes, CD54,p105,and endogenous I kappa B alpha, were decreased when (I kappa B alpha(32/36A) was induced, and the growth of I kappa B alpha(32/36A)-induced EBV-infected cells was slightly reduced. Loss of NF-kappa B activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased In I kappa B alpha(32/36A)-overexpressing cells. Together these results show that it is possible to control I kappa B alpha(32/36A) levels is, NF-kappa B activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-kappa B can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible I kappa B alpha 32/36A may be useful in the identification of genuine NF-kappa B target genes in EBV-infected B cells. (C) 2000 by The American Society of Hematology.