RESUMO
Multigenic programs controlling susceptibility to apoptosis in response to ionizing radiation have not yet been defined. Here, using DNA microarrays, we show gene expression patterns in an apoptosis-sensitive and apoptosis-resistant murine B cell lymphoma model system both before and after irradiation. From the 11,000 genes interrogated by the arrays, two major patterns emerged. First, before radiation exposure the radioresistant LYar cells expressed significantly greater levels of message for several genes involved in regulating intracellular redox potential. Compared with LYas cells, LYar cells express 20- to 50-fold more mRNA for the tetraspanin CD53 and for fructose-1,6-bisphosphatase. Expression of both of these genes can lead to the increase of total cellular glutathione, which is the principle intracellular antioxidant and has been shown to inhibit many forms of apoptosis. A second pattern emerged after radiation, when the apoptosis-sensitive LYas cells induced rapid expression of a unique cluster of genes characterized by their involvement in mitochondrial electron transport. Some of these genes have been previously recognized as proapoptotic; however others, such as uncoupling protein 2, were not previously known to be apoptotic regulatory proteins. From these observations we propose that a multigenic program for sensitivity to apoptosis involves induction of transcripts for genes participating in mitochondrial uncoupling and loss of membrane potential. This program triggers mitochondrial release of apoptogenic factors and induces the "caspase cascade." Conversely, cells resistant to apoptosis down-regulate these biochemical pathways, while activating pathways for establishment and maintenance of high intracellular redox potential by means of elevated glutathione.
Assuntos
Apoptose/genética , Proteínas de Membrana Transportadoras , Mitocôndrias/genética , Proteínas Mitocondriais , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Animais , Anexinas/metabolismo , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Proteínas de Transporte/genética , Análise por Conglomerados , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Frutose-Bifosfatase/genética , Regulação Neoplásica da Expressão Gênica , Canais Iônicos , Cinética , Camundongos , Modelos Biológicos , Proteína P2 de Mielina/genética , Porinas/genética , Proteínas/genética , Espectrometria de Fluorescência , Tetraspanina 25 , Fatores de Tempo , Células Tumorais Cultivadas , Proteína Desacopladora 2 , Regulação para Cima , Canais de Ânion Dependentes de VoltagemAssuntos
Doenças Autoimunes/terapia , Epitopos/imunologia , Imunoterapia , Sequência de Aminoácidos , Animais , Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/terapia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/terapia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Dados de Sequência Molecular , Proteína Básica da Mielina/uso terapêutico , Peptídeos/uso terapêuticoRESUMO
We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation.