The HIF-1 transcription factor drives hypoxic gene expression changes that are thought to be adaptive for cells exposed to a reduced-oxygen environment. For example, HIF-1 induces the expression of glycolytic genes. It is presumed that increased glycolysis is necessary to produce energy when low oxygen will not support oxidative phosphorylation at the mitochondria. However, we find that while HIF-1 stimulates glycolysis, it also actively represses mitochondrial function and oxygen consumption by inducing pyruvate dehydrogenase kinase 1 (PDK1). PDK1 phosphorylates and inhibits pyruvate dehydrogenase from using pyruvate to fuel the mitochondrial TCA cycle. This causes a drop in mitochondrial oxygen consumption and results in a relative increase in intracellular oxygen tension. We show by genetic means that HIF-1-dependent block to oxygen utilization results in increased oxygen availability, decreased cell death when total oxygen is limiting, and reduced cell death in response to the hypoxic cytotoxin tirapazamine.

Clinical evidence shows that tumor hypoxia is an independent prognostic indicator of poor patient outcome. Hypoxic tumors have altered physiologic processes, including increased regions of angiogenesis, increased local invasion, increased distant metastasis and altered apoptotic programs. Since hypoxia is a potent controller of gene expression, identifying hypoxia-regulated genes is a means to investigate the molecular response to hypoxic stress. Traditional experimental approaches have identified physiologic changes in hypoxic cells. Recent studies have identified hypoxia-responsive genes that may define the mechanism(s) underlying these physiologic changes. For example, the regulation of glycolytic genes by hypoxia can explain some characteristics of the Warburg effect. The converse of this logic is also true. By identifying new classes of hypoxia-regulated gene(s), we can infer the physiologic pressures that require the induction of these genes and their protein products. Furthermore, these physiologically driven hypoxic gene expression changes give us insight as to the poor outcome of patients with hypoxic tumors. Approximately 1-1.5% of the genome is transcriptionally responsive to hypoxia. However, there is significant heterogeneity in the transcriptional response to hypoxia between different cell types. Moreover, the coordinated change in the expression of families of genes supports the model of physiologic pressure leading to expression changes. Understanding the evolutionary pressure to develop a 'hypoxic response' provides a framework to investigate the biology of the hypoxic tumor microenvironment.

The hypoxia-inducible factors 1␣ (HIF-1␣) and 2␣ (HIF-2␣) have extensive structural homology and have been identified as key transcription factors responsible for gene expression in response to hypoxia. They play critical roles not only in normal development, but also in tumor progression. Here we report on the differential regulation of protein expression and transcriptional activity of HIF-1␣ and -2␣ by hypoxia in immortalized mouse embryo fibroblasts (MEFs). We show that oxygen-dependent protein degradation is restricted to HIF-1␣, as HIF-2␣ protein is detected in MEFs regardless of oxygenation and is localized primarily to the cytoplasm. Endogenous HIF-2␣ remained transcriptionally inactive under hypoxic conditions; however, ectopically overexpressed HIF-2␣ translocated into the nucleus and could stimulate expression of hypoxia-inducible genes. We show that the factor inhibiting HIF-1 can selectively inhibit the transcriptional activity of HIF-1␣ but has no effect on HIF-2␣-mediated transcription in MEFs. We propose that HIF-2␣ is not a redundant transcription factor of HIF-1␣ for hypoxia-induced gene expression and show evidence that there is a cell type-specific modulator(s) that enables selective activation of HIF-1␣ but not HIF-2␣ in response to low-oxygen stress.

Summary HERG K + channels, besides contributing to regulate cardiac and neuronal excitability, are preferentially expressed in tumour cell lines of different histogenesis, where their role in the development and maintenance of the neoplastic phenotype is under study. We show here that both herg gene and HERG protein are expressed with high frequency in primary human endometrial cancers, as compared to normal and hyperplastic endometrium. RT-PCR and immunohistochemistry, using specific anti-HERG antibodies developed in our laboratory, were applied to tissue specimens obtained from 18 endometrial cancers and 11 non-cancerous endometrial tissues. herg RNA and HERG protein are expressed in 67% and 82%, respectively, of cancerous, while in only 18% of non-cancerous tissues. In particular, no expression was found in endometrial hyperplasia. Moreover, electrophysiological experiments confirmed the presence of functioning HERG channels on the plasma membrane of tumour cells. On the whole, these data are the first demonstration of the presence of HERG channels in primary human neoplasias, and could candidate HERG as a potential tool capable of marking cancerous versus hyperplastic endometrial growth.

Quail neural crest cells were cultured in a differentiative medium to study the inward K+ channel profile in neuronal precursors at various stages of maturation. Between 12 and 24 h of culture, neural crest-derived neurons displayed, in addition to the previously described outward depolarization-activated K+ currents, an inward current enhanced in high K+ medium. A biophysical and pharmacological analysis led us to conclude that this inward K+ current is identical to that previously demonstrated in mouse and human neuroblastoma cell lines (I[IR]). This current (quail I[IR] or ql[IR]), which is active at membrane potentials positive to -35 mV, was blocked by Cs+ and by class III antiarrhythmic drugs, thus resembling the K+ current encoded by the human ether-a-gò-gò-related gene (HERG). At later stages of incubation (>48 h), neural crest-derived neurons underwent morphological and biochemical differentiation and expressed fast Na+ currents. At this stage the cells lost qI[IR], displaying instead a classical inward rectifier K+ (IRK) current (quail I[IRK] = qI[IRK]). This substitution was reflected in the resting potential (VREST), which became hyperpolarized by >20 mV compared with the 24 h cells. Neurons were also harvested from peripheral ganglia and other derivatives originating from the migration of neural crest cells, viz. ciliary ganglia, dorsal root ganglia, adrenal medulla and sympathetic chain ganglia. After brief culture following harvesting from young embryos, ganglionic neurons always expressed qI(IR). On the other hand, when ganglia were explanted from older embryos (7-12 days), briefly cultured neurons displayed the IRK-like current. Again, in all the above derivatives the qI(IR) substitution by qI(IRK) was accompanied by a 20 mV hyperpolarization of VREST. Together, these data indicate that the VREST of normal neuronal precursors is sequentially regulated by HERG- and IRK-like currents, suggesting that HERG-like channels mark an immature and transient stage of neuronal differentiation, probably the same stage frozen in neuroblastomas by neoplastic transformation.