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We report here that the E7 oncoprotein encoded by the oncogenic human papillomavirus (HPV) type 16 binds to the glycolytic enzyme type M 2 pyruvate kinase (M2-PK). M2-PK occurs in a tetrameric form with a high affinity to its substrate phosphoenolpyruvate and a dimeric form with a low affinity to phosphoenolpyruvate, and the transition between both conformations regulates the glycolytic f lux in tumor cells. The glycolytic intermediate fructose 1,6-bisphosphate induces the reassociation of the dimeric to the tetrameric form of M2-PK. The expression of E7 in an experimental cell line shifts the equilibrium to the dimeric state despite a significant increase in the fructose 1,6-bisphosphate levels. Investigations of HPV-16 E7 mutants and the nononcogenic HPV-11 subtype suggest that the interaction of HPV-16 E7 with M2-PK may be linked to the transforming potential of the viral oncoprotein.Unicellular organisms have a variety of sensing mechanisms to adapt the cell proliferation rate to variations in their environmental nutrient supply. Several gene products, like the ras or cdc kinase proteins, which are involved in nutrient sensing in yeast (1, 2), are conserved during the evolution of multicellular organisms, and in mammals, these gene products often are altered in tumors. Despite our knowledge about the protein machinery regulating cell proliferation increasing tremendously over the recent years, we are still at the beginning to understand how nutrients contribute to proliferation control in multicellular organisms. There is, however, quite good evidence that phosphometabolites derived from both glycolysis (for recent review, see ref.3) and the pentose phosphate pathway (ref. 4 and references therein) provide some of the signals linking metabolic conditions to cell proliferation. The glycolytic phosphometabolites, which are necessary for the biosynthesis of nucleic acids, phospholipids, and complex carbohydrates, are up-regulated in the G 1 phase of the cell cycle (for recent review, see

Cellular senescence is considered a major tumour-suppressor mechanism in mammals, and many oncogenic insults, such as the activation of the ras proto-oncogene, trigger initiation of the senescence programme. Although it was shown that activation of the senescence programme involves the up-regulation of cell-cycle regulators such as the inhibitors of cyclin-dependent kinases p16INK4A and p21CIP-1, the mechanisms underlying the senescence response remain to be resolved. In the case of stress-induced premature senescence, reactive oxygen species are considered important intermediates contributing to the phenotype. Moreover, distinct alterations of the cellular carbohydrate metabolism are known to contribute to oncogenic transformation, as is best documented for the phenomenon of aerobic glycolysis. These findings suggest that metabolic alterations are involved in tumourigenesis and tumour suppression; however, little is known about the metabolic pathways that contribute to these processes. Using the human fibroblast model of in vitro senescence, we analysed age-dependent changes in the cellular carbohydrate metabolism. Here we show that senescent fibroblasts enter into a metabolic imbalance, associated with a strong reduction in the levels of ribonucleotide triphosphates, including ATP, which are required for nucleotide biosynthesis and hence proliferation. ATP depletion in senescent fibroblasts is due to dysregulation of glycolytic enzymes, and finally leads to a drastic increase in cellular AMP, which is shown here to induce premature senescence. These results suggest that metabolic regulation plays an important role during cellular senescence and hence tumour suppression.

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In differentiated tissues, such as muscle and brain, increased adenosine monophosphate (AMP) levels stimulate glycolytic flux rates. In the breast cancer cell line MCF-7, which characteristically has a constantly high glycolytic flux rate, AMP induces a strong inhibition of glycolysis. The human breast cancer cell line MDA-MB-453, on the other hand, is characterized by a more differentiated metabolic phenotype. MDA-MB-453 cells have a lower glycolytic flux rate and higher pyruvate consumption than MCF-7 cells. In addition, they have an active glycerol 3-phosphate shuttle. AMP inhibits cell proliferation as well as NAD and NADH synthesis in both MCF-7 and MDA-MB-453 cells. However, in MDA-MB-453 cells glycolysis is slightly activated by AMP. This disparate response of glycolytic flux rate to AMP treatment is presumably caused by the fact that the reduced NAD and NADH levels in AMP-treated MDA-MB-453 cells reduce lactate dehydrogenase but not cytosolic glycerol-3-phosphate dehydrogenase reaction. Due to the different enzymatic complement in MCF-7 cells, proliferation is inhibited under glucose starvation, whereas MDA-MB-453 cells grow under these conditions. The inhibition of cell proliferation correlates with a reduction in glycolytic carbon flow to synthetic processes and a decrease in phosphotyrosine content of several proteins in both cell lines.Both proliferating cells and tumor cells maintain a high glycolytic rate even under aerobic conditions, a process referred to as aerobic glycolysis. Observations on aerobic glycolysis in tumor cells prompted Warburg (1) to postulate an altered respiratory function leading to an increased glycolytic capacity and a high rate of lactate formation from glucose in the presence of oxygen. Data from former reports suggest that there are many factors contributing to the origin of aerobic glycolysis (2). The altered control of glycolysis by expression of certain isoenzymes is one important factor (2-12). Furthermore, the glycerol 3-phosphate shuttle and the malate-aspartate shuttle are altered in such a way that transport of cytosolic hydrogen into the mitochondria is reduced, requiring tumor cells to reoxidize NADH cytosolically by lactate dehydrogenase (13-15). Additionally, oxidation of pyruvate is reduced in favor of glutamine oxidation (16 -25). Due to the expression of the mitochondrial, NAD-dependent malate decarboxylase, malate is converted to pyruvate and lactate (22)(23)(24). The conversion of glutamine to lactate is called, in analogy to glycolysis, glutaminolysis (25). In tumor cells the glycolytic capacity can be so great that all of the cell's energy requirements are derived from glycolysis (2, 26). Therefore, high glycolytic activity ensures the survival and the migration of tumor cells in hypoxic areas (2,26,27). The main role of the glutaminolytic pathway is the generation of energy (2, 25). However, a high glycolytic rate is not always linked to cell proliferation or tumor formation. There are several cell lines that are able to grow in a medium with 5 mM gala...