RESUMO
p53 tumor suppressor is a transcription factor that functions, in part, through many of its downstream target genes. We have identified a p53-inducible gene by performing mRNA differential display on IW32 murine erythroleukemia cells containing a temperature-sensitive p53 mutant allele, tsp53(Val-135). Sequence analysis of the full-length cDNA revealed its identity as the mouse homologue of the human thiamine transporter 1 (THTR-1). Induction of the mouse THTR-1 (mTHTR-1) mRNA was detectable as early as 1 h at 32.5 degrees C; upon shifting back to 38.5 degrees C, mTHTR-1 transcript was rapidly degraded with a half-life of less than 2 h. Elevation of mTHTR-1 expression was found in DNA damage-induced normal mouse embryonic fibroblast cells, but not in p53(-/-) mouse embryonic fibroblast cells, suggesting that mTHTR-1 induction was p53-dependent. A region within the first intron of the mTHTR-1 gene bound to p53 and conferred the p53-mediated transactivation. Furthermore, increased thiamine transporter activities were found in cells overexpressing mTHTR-1 and under conditions of DNA damage or p53 activation. Our findings indicate that p53 may be involved in maintaining thiamine homeostasis through transactivation of THTR-1.
Assuntos
Proteínas de Membrana Transportadoras/genética , Tiamina/metabolismo , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , DNA Complementar/análise , Humanos , Camundongos , Dados de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/fisiologia , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
The biological activity of p53 in IW32 erythroleukemia cells was investigated. IW32 cells had no detectable levels of p53 mRNA and protein expression. By transfecting a temperature-sensitive mutant p53 cDNA, tsp53val135, into the cells, we have established several clones stably expressing the mutant p53 allele. At permissive temperature, these p53 transfectants were arrested in G1 phase and underwent apoptosis. Moreover, differentiation along the erythroid pathway was observed as evidenced by increased benzidine staining and mRNA expression of beta-globin and the erythroid-specific delta-aminolevulinic acid synthase (ALAS-E). Treatment of cells with protein tyrosine phosphatase inhibitor vanadate blocked the p53-induced differentiation, but not that of cell death or growth arrest. Increased protein tyrosine phosphatase activity as well as mRNA levels of PTPbeta2 and PTPepsilon could be observed by wildtype p53 overexpression. These results indicate that p53 induced multiple phenotypic consequences through separate signal pathways in IW32 erythroleukemia cells, and protein tyrosine phosphatase is required for the induced differentiation.
Assuntos
Genes p53 , Leucemia Eritroblástica Aguda/patologia , Proteínas de Neoplasias/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Fosfatases/fisiologia , Proteína Supressora de Tumor p53/fisiologia , 5-Aminolevulinato Sintetase/biossíntese , 5-Aminolevulinato Sintetase/genética , Animais , Apoptose , Diferenciação Celular , DNA Complementar/genética , Indução Enzimática , Fase G1 , Regulação Leucêmica da Expressão Gênica , Globinas/biossíntese , Globinas/genética , Leucemia Eritroblástica Aguda/enzimologia , Leucemia Eritroblástica Aguda/genética , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fenótipo , Fosforilação , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
Changes in phosphate energy metabolism with time in a rat flap model were followed noninvasively with in vivo 31P-NMR. The influence of age on high-energy phosphate metabolites in perfused and ischemic ends of 3 x 10 cm dorsal flaps was noted from 30 minutes to 7 days after closure in 6-, 12-, and 24-month-old (n = 4, 7, and 8, respectively) male Fischer 344 rats. Phosphocreatine to inorganic phosphate ratios showed younger animals exhibiting significant returns to preinjury energy status in 2- and 3-mm ischemic layers. This behavior, 24 to 72 hours after closure, coincides with neovascularization of the flap tissue. By contrast, 12- and 24-month-old animals experienced statistically significantly lesser high-energy rebound, developing greater necrosis in the ischemic regions. Early intracellular pH lowering, indicative of lactate production, was somewhat greater in the flaps of younger animals. The in vivo 31P-NMR methods thus provide metabolic insights into flap behavior correlating with physiologic influences of aging.