Autocrine lymphotoxin production in Epstein-Barr virus-immortalized B cells: induction via NF-kappaB activation mediated by EBV-derived latent membrane protein 1.
Epstein-Barr virus (EBV)-immortalized lymphoblastoid cells express high levels of lymphotoxin and use this molecule as an autocrine growth factor. We hypothesized that the EBV-derived latent membrane protein 1 (LMP1) mediates lymphotoxin production by inducing NF-kappaB binding to the lymphotoxin promoter. We assessed lymphotoxin production, LMP1 expression, and NF-kappaB activation in Z-43 (EBV-positive lymphoblastoid cells), Daudi (EBV-positive Burkitt's cells), and 3A4 (EBV-negative Burkitt's cells containing a stably transfected tetracycline-inducible LMP1 construct). Z-43 cells expressed high levels of LMP1 (immunoblot) and lymphotoxin (ELISA); the EBV-positive Burkitt's lymphoma line Daudi expressed neither LMP1 nor lymphotoxin. Similarly, induction of LMP1 in the 3A4 cells (exposed to tetracycline) was accompanied by a 13-fold increase in lymphotoxin levels (ELISA) as compared to uninduced (LMP1-negative) cells. EMSAs demonstrated high levels of NF-kappaB activation in Z-43 and tetracycline-induced 3A4 cells, but much lower levels in the uninduced 3A4 cells. Exposure of these cells to Bay 11-7082 (an inhibitor of IkappaB phosphorylation and, therefore, NF-kappaB activation) abrogated NF-kappaB binding and lymphotoxin production in a dose-dependent manner in both Z-43 and 3A4 cells. Therefore, in our model system, autocrine lymphotoxin production is largely driven by NF-kappaB activation, which is in turn mediated by EBV-derived LMP1 signaling.
Thompson
M P
MP
Department of Bioimmunotherapy, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Aggarwal
B B
BB
Shishodia
S
S
Estrov
Z
Z
Kurzrock
R
R
D000818:Animals; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000276:immunology*; D002455:Cell Division; D002470:Cell Survival; D002472:Cell Transformation, Viral; D003124:Colorimetry / Q000379:methods; D003513:Cycloheximide / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D008233:Lymphotoxin-alpha / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000276:immunology*; D014176:Protein Biosynthesis / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D008233:Lymphotoxin-alpha; D016328:NF-kappa B; D009856:Oncogene Proteins, Viral; D014763:Viral Matrix Proteins; D003513:Cycloheximide