RESUMEN
Glycolytic glucose degradation to lactate is the only way of energy production in mammalian cells that does not use oxygen, and is thus a means of reducing oxidative stress. The aim of this work was to study the effect on proliferation of progressive glucose depletion during the cell cycle of stimulated cultured rat thymocytes. It has been shown that under conditions of limited glucose supply, these cells increase the generation of reactive oxygen species. Because of the sensitivity of the stimulating transcription factor Sp1 to redox changes with respect to its DNA binding, gene expression of glycolytic enzymes is markedly suppressed. As a consequence, cell division does not occur in glucose depleted thymocytes.
Asunto(s)
Glucosa/metabolismo , Especies Reactivas de Oxígeno , Timo/citología , Animales , División Celular , Núcleo Celular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Femenino , Fructosa-Bifosfato Aldolasa/genética , Fructosa-Bifosfato Aldolasa/metabolismo , Glucosa/farmacología , Glucosa/fisiología , Immunoblotting , Cinética , L-Lactato Deshidrogenasa/metabolismo , Mediciones Luminiscentes , Regiones Promotoras Genéticas , Unión Proteica , Piruvato Quinasa/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Espectrometría de Fluorescencia , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
During the cell cycle of mitogen stimulated rat thymocytes, an 8-10-fold induction of glycolytic enzymes and a corresponding increase in the mRNA levels has been observed. This prompted us to study the transcriptional regulation of the rat aldolase A and pyruvate kinase M genes. cis-Regulatory elements of both promoters were evaluated by site-directed mutagenesis of promoter/luciferase constructs and transient transfections of rat hepatoma FTO2B cells. Furthermore, the binding proteins were identified by mobility shift assays in the presence of specific antibodies. In the aldolase AH1 promoter, five binding sites for Sp1 and Sp3 and a TPA responsive element were identified as essential for transcriptional regulation. Most of the promoter activity can be attributed to these regulatory elements. In the pyruvate kinase M promoter three out of five binding sites of Sp1 and Sp3 (B box and GC boxes 1 and 3) turned out to be functional in the transfection assays whereas the disruption of GC box 2 had no effect, and the disruption of the GC box 4 had only a minor effect on the promoter activity. Both promoters are stimulated by Sp1 as well as Sp3, as judged by cotransfection experiments of Drosophila SL2 cells. Therefore, the Sp1- and Sp3-directed transcription provides a means for common regulatory mechanism of the aldolase A and the pyruvate kinase M genes.
Asunto(s)
Fructosa-Bifosfato Aldolasa/genética , Piruvato Quinasa/genética , Factor de Transcripción Sp1/fisiología , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Drosophila , Regulación de la Expresión Génica , Mutagénesis Sitio-Dirigida , Regiones Promotoras Genéticas , Ratas , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Our laboratory has reported that glucose is essential for glycolytic enzyme induction and proliferation of mitogen-activated rat thymocytes (41). Here we show that: 1) Resting thymocytes meet their ATP demand mainly by oxidative glucose breakdown (88%), whereas proliferating thymocytes produce 86% by glycolytic degradation of glucose to lactate and only 14% by oxidation to CO2 and water. 2) In contrast to nonstimulated resting thymocytes, production of PMA primed reactive oxygen species (ROS) in the proliferating cells is nearly abolished. 3) Consistent with this finding, no ROS formation is observed in proliferating human promyelocytic HL-60 cells, whereas differentiated, nonproliferating HL-60 cells exert a marked response upon priming with PMA. 4) The observed reduction of ROS formation by resting thymocytes incubated with pyruvate suggests a function of pyruvate as an H(2)O(2) scavenger. 5) The respiratory chain is a potential origin for ROS because inhibitors of the mitochondrial electron transport strongly reduce H(2)O(2) production by resting thymocytes. The results are discussed in the context of aerobic glycolysis by proliferating cells being a means to minimize oxidative stress during the phases of the cell cycle where maximally enhanced biosynthesis and cell division do occur.