Proliferating cells and tumour cells maintain a high glycolytic rate even under aerobic conditions. FT02B cells, a rat hepatoma cell line, show high activities of glycolytic enzymes. Within a culture period of 48 h the cell number increases 5-fold. Replacement of glucose by pyruvate in the culture medium lowers glycolytic enzyme activity and prevents proliferation. Transfection assays revealed that glucose deprivation dramatically decreases the transcriptional activities of the Spl-dependent aldolase and pyruvate kinase promoters leading to reduced reporter gene expression. Spl binding activity is also inhibited by ocadaic acid, an inhibitor of protein phosphatase 1. Western blot analyses with nuclear extracts from FT02B cells cultured in the presence or absence of glucose revealed differences in the phosphorylation state of Spl. From these results we conclude that glucose increases the amount of the dephosphorylated form of Spl which has a higher DNA binding activity. As a consequence gene expression of the glycolytic enzymes is increased which is a prerequisite for cell proliferation.

Thymocytes induce their glycolytie 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 Spl 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 Spl 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 Spl transcription factor with its consensus sequence and subsequently regulate glycolytic gene expression.

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