Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species.

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.

Differences in DNA-binding efficiency of Sp1 to aldolase and pyruvate kinase promoter correlate with altered redox states in resting and proliferating rat thymocytes.

Thymocytes induce their glycolytic enzymes as they undergo transition from the resting to the proliferating state. Corresponding increases in mRNA levels point to a transcriptional regulation. Electrophoretic mobility shift assays revealed that the DNA-binding efficiency of Sp1 is increased when nuclear extracts from proliferating compared to resting rat thymocytes were used. Here we demonstrate that hydrogen peroxide, added to nuclear extract from proliferating cells, decreases the Sp1 DNA-binding activity, whereas in nuclear extracts from resting cells dithioerythritol fully restores DNA-binding efficiency. Moreover we show that in contrast to resting thymocytes, production of reactive peroxide anions upon priming with phorbol 12-myristate 13-acetate is nearly abolished in the proliferating cells. From these results we propose that reactive oxygen intermediates affect the interaction of the Sp1 transcription factor with its consensus sequence and subsequently regulate glycolytic gene expression.

The aldolase A promoter in proliferating rat thymocytes is regulated by a cluster of SP1 sites and a distal modulator.

In mitogen-stimulated rat thymocytes the activities and mRNA levels of aldolase A increase remarkably during proliferation pointing to a transcriptional regulation of this enzyme. By DNAse I footprinting and mobility shift competition assays five binding sites for the activating transcription factor Sp1 and one site for an AP-1 like nuclear factor could be identified in the core activating region of the proximal aldolase AH1 promoter downstream of -400. Transfection data and differences found in nuclear protein binding of resting and proliferating cells to DNA sites suggest that Sp1 is an integral part of the mechanism by which the AH1 promoter achieves high level transcription during proliferation. Moreover we demonstrate that an element between positions -1066/-731 significantly attenuates the AH1 promoter driven transcription as well as transcription regulated by the heterologous SV40 promoter. From this effect a functional linkage between the distal muscle-restricted M1 promoter and the active AH1 promoter can be suggested.

Expression of glycolytic isozymes in rat thymocytes during cell cycle progression.

The time courses of activities of aldolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase and pyruvate kinase were determined in stimulated rat thymocytes at 24 h intervals during a period of 72 h of culture. In parallel the mRNA levels of these enzymes were analysed by Northern blotting with specific probes. Both the enzyme activities and the corresponding mRNA levels reached their maxima 48 h after stimulation coinciding with the S-phase of the cell cycle. The isozyme types of aldolase and hexokinase in resting and in mitogen-stimulated rat thymocytes were identified by Northern blot hybridisation using isozyme-specific probes. In these cells the aldolase A is expressed, whereas type B and C could not be detected. The transcription of the aldolase A gene can be regulated by two different promoters. Depending on the alternative usage of the promoters the aldolase A-specific mRNA either contains the non-translated exons M1 or AH1. In rat thymocytes the promoter proximal to the exon AH1 is used while the expression of mRNA I, the type characteristic for muscle tissue, was not observed. In contrast to aldolase two isozyme types of hexokinase were detected. Hexokinase I as well as hexokinase II were present in thymocytes whereas hexokinase III was not detectable. A shift in the isozyme pattern was not observed during the cell cycle progression.