The ability to respond to differential levels of oxygen is important to all respiring cells. The response to oxygen deficiency, or hypoxia, takes many forms and ranges from systemic adaptations to those that are cell autonomous. Perhaps the most ancient of the cell-autonomous adaptations to hypoxia is a metabolic one: the Pasteur effect, which includes decreased oxidative phosphorylation and an increase in anaerobic fermentation. Because anaerobic fermentation produces far less ATP than oxidative phosphorylation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell. Here, we present genetic and biochemical evidence that, in mammalian cells, this metabolic switch is regulated by the transcription factor HIF-1. As a result, cells lacking HIF-1␣ exhibit decreased growth rates during hypoxia, as well as decreased levels of lactic acid production and decreased acidosis. We show that this decrease in glycolytic capacity results in dramatically lowered free ATP levels in HIF-1␣-deficient hypoxic cells. Thus, HIF-1 activation is an essential control element of the metabolic state during hypoxia; this requirement has important implications for the regulation of cell growth during development, angiogenesis, and vascular injury.Decreased environmental oxygen forces cells and tissues to adapt in multiple ways. In response to hypoxia, a significant number of changes in gene expression occur, resulting in elevated transcription of angiogenic factors, hematopoietic factors, and some metabolic enzymes (21). The switch between the two forms of respiration utilized by animal cells, aerobic versus anaerobic, was first noted by Pasteur in the late 19th century (12,22). As the oxygen level decreases, the generation of ATP shifts from the oxidative phosphorylation pathway in the mitochondria to the oxygen-independent pathway of glycolysis in the cytoplasm. Although glycolysis is less efficient than oxidative phosphorylation in the generation of ATP, in the presence of sufficient glucose glycolysis can sustain ATP production due to increases in the activity of the glycolytic enzymes (12,22). Perhaps nowhere has this forced adaptation been the focus of so much study as in transformed cells; this is because in solid tumors it is clear that a large percentage of the cell population is at least transiently hypoxic (1).Earlier in the 20th century, Otto Warburg demonstrated that tumors differed from normal tissues in their utilization of the glycolytic pathway (26). For a given amount of glucose, tumor fragments ex vivo produced far more lactate than sections of nontransformed tissues under normoxic conditions. In vivo the situation is likely to be more complex. Within individual tumors, there are some areas that may respond to hypoxia by exhibiting the normal physiological switch to glycolysis similar to that employed by all nontransformed cells in response to lowered oxygen levels. Concurrently, many other areas of transformed cells in solid tumors may adapt to h...

Low oxygen gradients (hypoxia and anoxia) are important determinants of pathological conditions under which the tissue blood supply is deficient or defective, such as in solid tumors. We have been investigating the relationship between the activation of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the mammalian response to hypoxia, and 5-AMP-activated protein kinase (AMPK), another regulatory system important for controlling cellular energy metabolism. In the present study, we used mouse embryo fibroblasts nullizygous for HIF-1␣ or AMPK expression to show that AMPK is rapidly activated in vitro by both physiological and pathophysiological low-oxygen conditions, independently of HIF-1 activity. These findings imply that HIF-1 and AMPK are components of a concerted cellular response to maintain energy homeostasis in low-oxygen or ischemic-tissue microenvironments. Finally, we used transformed derivatives of wild-type and HIF-1␣-or AMPK␣-null mouse embryo fibroblasts to determine whether AMPK is activated in vivo. We obtained evidence that AMPK is activated in authentic hypoxic tumor microenvironments and that this activity overlaps with regions of hypoxia detected by a chemical probe. We also showed that AMPK is important for the growth of this tumor model.We have been studying the relationship between the activity of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the response of mammalian cells to oxygen deprivation (e.g., see references 21, 43, and 50) and the regulation of c-Jun/AP-1 transcription factors (31, 32). We determined that c-Jun N-terminal phosphorylation is induced by low-oxygen conditions (hypoxia or anoxia; called hypoxia hereafter) in an HIF-1-dependent manner (31) and showed that this HIF-1-dependent c-Jun phosphorylation absolutely requires extracellular glucose utilization (32). Together, these findings suggest that enhanced glucose absorption and/or glycolytic activity mediated by HIF-1 in response to hypoxia activates c-Jun/AP-1, as well as other targets of c-Jun N-terminal kinases. To further investigate this potential mechanism, we focused on determining the contribution of bioenergetics-ATP depletion-to hypoxia-inducible c-Jun phosphorylation in wild-type (WT) and HIF-1-null mouse embryo fibroblasts (MEFs). While exploring cellular mechanisms of ATP regulation, we observed that 5Ј-AMP-activated protein kinase (AMPK) activity was induced in both cell types, particularly under conditions of hypoxia and glucose deprivation. This observation suggested the hypothesis that AMPK is important for the adaptive responses of energetically stressed cells in the hypoxic and glucose-deprived microenvironments present in solid tumors (e.g., reviewed in references 35 and 59).AMPK activity is defined by a class of evolutionarily conserved serine/threonine kinases that are sensitive to various environmental stresses, especially those that perturb cellular energy status (reviewed in references 9, 19, and 47). Different members of the AMPK catalyt...

The genomic sequencing of hundreds of organisms including homo sapiens, and the exponential growth in gene expression and proteomic data for many species has revolutionized research in biology. However, the computational analysis of these burgeoning datasets has been hampered by the sparse successes in combinations of data sources, representations, and algorithms. Here we propose the application of symbolic toolsets from the formal methods community to problems of biological interest, particularly signaling pathways, and more specifically mammalian mitogenic and stress responsive pathways. The results of formal symbolic analysis with extremely efficient representations of biological networks provide insights with potential biological impact. In particular, novel hypotheses may be generated which could lead to wet lab validation of new signaling possibilities. We demonstrate the graphic representation of the results of formal analysis of pathways, including navigational abilities, and describe the logical underpinnings of the approach. In summary, we propose and provide an initial description of an algebra and logic of signaling pathways and biologically plausible abstractions that provide the foundation for the application of highpowered tools such as model checkers to problems of biological interest.

Background: Cancer is a heterogeneous disease resulting from the accumulation of genetic defects that negatively impact control of cell division, motility, adhesion and apoptosis. Deregulation in signaling along the EgfR-MAPK pathway is common in breast cancer, though the manner in which deregulation occurs varies between both individuals and cancer subtypes.

No abstract