Inducible loss of NF-kappaB 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-kappaB activity by targeting IkappaBalpha. To understand the role of NF-kappaB activation in EBV-related oncogenesis, we have subcloned mutated IkappaBalpha(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 IkappaBalpha(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible IkappaBalpha(32/36A) peaked at day 2. Inhibition of NF-kappaB activity was closely correlated with levels of inducible IkappaBalpha(32/36A). Levels of 3 well-known NF-kappaB-dependent genes, CD54, p105, and endogenous IkappaBalpha, were decreased when IkappaBalpha(32/36A) was induced, and the growth of IkappaBalpha(32/36A)-induced EBV-infected cells was slightly reduced. Loss of NF-kappaB activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased in IkappaBalpha(32/36A)-overexpressing cells. Together these results show that it is possible to control IkappaBalpha(32/36A) levels, ie, NF-kappaB activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-kappaB can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible IkappaBalpha(32/36A) may be useful in the identification of genuine NF-kappaB target genes in EBV-infected B cells. (Blood. 2000;95:2068-2075)
Feuillard
J
J
Biochimie Cellulaire des Hémopathies Lymphoïdes, Université Paris, Bobigny, France. jean.feuillard@avc.ap-hop-paris.fr
Schuhmacher
M
M
Kohanna
S
S
Asso-Bonnet
M
M
Ledeur
F
F
Joubert-Caron
R
R
Bissières
P
P
Polack
A
A
Bornkamm
G W
GW
Raphaël
M
M
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis; D001402:B-Lymphocytes / Q000473:pathology*; D015153:Blotting, Western; D002461:Cell Line, Transformed; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004318:Doxycycline / Q000494:pharmacology; D005434:Flow Cytometry; D019254:Genes, bcl-2 / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007700:Kinetics; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000502:physiology*; D010957:Plasmids; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D014162:Transfection
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D019253:Proto-Oncogene Proteins c-bcl-2; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D004318:Doxycycline