RESUMEN
Burkitt Lymphoma (BL) cells are highly sensitive to suboptimal growth conditions and undergo apoptosis when seeded at low cell density or reduced serum concentration. Irradiated fibroblasts or a mix of pruvate, alpha-thioglycerol, and bathocuproine disulfonate can protect BL cells from apoptosis induced by lowering cell density or serum concentration by promoting cystine uptake in the cells. The availability of cystine is the limiting factor for glutathione biosynthesis in BL cells and thus for the ability of the cells to cope with oxidative stress. We have set up an expression cloning strategy to clone genes that protect BL cells from apoptosis induced by low cell density and/or serum. Using this approach we have cloned among others the cDNA for Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx).
Asunto(s)
Apoptosis/fisiología , Linfoma de Burkitt/patología , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Secuencia de Bases , División Celular , Línea Celular , Clonación Molecular , Citosol/enzimología , ADN Complementario/química , Escherichia coli , Biblioteca de Genes , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Plásmidos , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , TransfecciónAsunto(s)
Núcleo Celular/enzimología , Glutatión Peroxidasa/metabolismo , Compuestos de Organoselenio/metabolismo , Maduración del Esperma/fisiología , Espermatozoides/fisiología , Animales , Secuencia de Bases , Núcleo Celular/fisiología , Cromatina/genética , Cromatina/metabolismo , Electroforesis en Gel de Poliacrilamida , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Humanos , Masculino , Datos de Secuencia Molecular , Compuestos de Organoselenio/química , Protaminas/química , Protaminas/metabolismo , Ratas , Selenio/metabolismo , Alineación de Secuencia , Espermatozoides/citología , Compuestos de Sulfhidrilo/químicaRESUMEN
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)