HIF-Independent Regulation of Thioredoxin Reductase 1 Contributes to the High Levels of Reactive Oxygen Species Induced by Hypoxia

Cellular adaptation to hypoxic conditions mainly involves transcriptional changes in which hypoxia inducible factors (HIFs) play a critical role. Under hypoxic conditions, HIF protein is stabilized due to inhibition of the activity of prolyl hydroxylases (EGLNs). Because the reaction carried out by these enzymes uses oxygen as a co-substrate it is generally accepted that the hypoxic inhibition of EGLNs is due to the reduction in oxygen levels. However, several studies have reported that hypoxic generation of mitochondrial reactive oxygen species (ROS) is required for HIF stabilization. Here, we show that hypoxia downregulates thioredoxin reductase 1 (TR1) mRNA and protein levels. This hypoxic TR1 regulation is HIF independent, as HIF stabilization by EGLNs inhibitors does not affect TR1 expression and HIF deficiency does not block TR1 hypoxic-regulation, and it has an effect on TR1 function, as hypoxic conditions also reduce TR1 activity. We found that, when cultured under hypoxic conditions, TR1 deficient cells showed a larger accumulation of ROS compared to control cells, whereas TR1 over-expression was able to block the hypoxic generation of ROS. Furthermore, the changes in ROS levels observed in TR1 deficient or TR1 over-expressing cells did not affect HIF stabilization or function. These results indicate that hypoxic TR1 down-regulation is important in maintaining high levels of ROS under hypoxic conditions and that HIF stabilization and activity do not require hypoxic generation of ROS

Apoptosis in cancer: from pathogenesis to treatment

Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. It is also one of the most studied topics among cell biologists. An understanding of the underlying mechanism of apoptosis is important as it plays a pivotal role in the pathogenesis of many diseases. In some, the problem is due to too much apoptosis, such as in the case of degenerative diseases while in others, too little apoptosis is the culprit. Cancer is one of the scenarios where too little apoptosis occurs, resulting in malignant cells that will not die. The mechanism of apoptosis is complex and involves many pathways. Defects can occur at any point along these pathways, leading to malignant transformation of the affected cells, tumour metastasis and resistance to anticancer drugs. Despite being the cause of problem, apoptosis plays an important role in the treatment of cancer as it is a popular target of many treatment strategies. The abundance of literature suggests that targeting apoptosis in cancer is feasible. However, many troubling questions arise with the use of new drugs or treatment strategies that are designed to enhance apoptosis and critical tests must be passed before they can be used safely in human subjects

Regulation of hypoxia-inducible factor is preserved in the absence of a functioning mitochondrial respiratory chain.

Hypoxia-inducible factor (HIF) mediates a large number of transcriptional responses to hypoxia and has an important role in processes that include angiogenesis and erythropoiesis. The HIF DNA binding complex consists of 2 basic-helix-loop-helix PAS proteins designated alpha and beta subunits. Regulation occurs principally through the alpha subunits, which are stabilized and activated in hypoxia. Although substantial evidence implicates reactive oxygen species (ROS) in the regulatory process, the precise mechanisms remain unclear. Mitochondria are an important source of ROS, and in one model it has been proposed that hypoxia increases the generation of ROS at complex III in the mitochondrion and that this signal acts through a transduction pathway to stabilize HIF-1alpha and to activate HIF. To test this model the induction of the HIF-1alpha subunit and the HIF target gene, glucose-transporter-1, was examined in a variety of mutant cells that lacked mitochondrial DNA (rho0) or had other genetic defects in mitochondrial respiration. HIF induction by hypoxia was essentially normal in all cells tested. Hydrogen peroxide production was measured by the luminol/peroxidase method and found to be reduced in rho0 versus wild-type cells and reduced by hypoxia in both rho0 and wild-type cells. Furthermore, concentrations of rotenone that maximally inhibited respiration did not affect HIF activation by hypoxia. These data do not support the model outlined above and indicate that a functional respiratory chain is not necessary for the regulation of HIF by oxygen

Does prophylactic anticoagulation reduce the risk of femoral tunneled dialysis catheter-related complications?

Purpose: to determine the incidence and predictors of femoral tunneled dialysis catheter (tdc)-related complications and whether prophylactic anticoagulation is associated with reduced catheter-related deep vein thrombosis (crt) or prolonged patency. Methods: A retrospective review of femoral tdcs inserted for maintenance hemodialysis in patients from two dialysis units that have used two different strategies to reduce thrombotic complications. one center routinely considered all femoral tdcs for prophylactic anticoagulation, whilst the other restricted anticoagulation to tdcs that had required repeated treatment with urokinase locks to maintain patency. Survival analyses were performed to establish complication rates, identify predictors of complications and assess the effect of prophylactic anticoagulation use. Results: Of the 194 femoral TDCs identified, 178 (92%) were associated with at least one complication. Approximately three quarters did not provide adequate small solute clearance; one half were not in use by three months; one quarter had at least one catheter-related infection (2.3 per 1000 catheter days); and one in ten developed a crt (1.1 per 1000 catheter days). Prophylactic anticoagulation was not associated with significant improvements in rates of catheter occlusion, CRT, catheter-related infection or dialysis adequacy. A previous ipsilateral femoral TDC was identified as a statistically significant predictor of a CRT (adjusted hazard ratio 3.7 [95% confdence interval 1.4-9.8]; P=.007). Conclusions: Femoral tdcs are associated with poor patency rates and high complication rates; reusing femoral veins for tdcs should be avoided where possible, and this study provides no evidence to support routine prophylactic anticoagulation in all patients with femoral tdcs. © 2012 Wichtig Editore

Selection of mutant CHO cells with constitutive activation of the HIF system and inactivation of the von Hippel-Lindau tumor suppressor.

Hypoxia-inducible factor (HIF) mediates a widespread transcriptional response to hypoxia through binding to cis-acting DNA sequences termed hypoxia response elements (HREs). Activity of the transcriptional complex is suppressed in the presence of oxygen by processes that include the targeting of HIF-alpha subunits for ubiquitin-mediated proteolysis. To provide further insights into these processes we constructed Chinese hamster ovary (CHO) cells bearing stably integrated plasmids that expressed HRE-linked surface antigens and used these cells in genetic screens for mutants that demonstrated constitutive up-regulation of HRE activity. From mutagenized cultures, clones were isolated that demonstrated up-regulation of HRE activity and increased HIF-1alpha protein levels in normoxic culture. Transfection and cell fusion studies suggested that these cells possess recessive defects that affect one or more pathways involved in HIF-alpha proteolysis. Two lines were demonstrated to harbor truncating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. In these cells, defects in ubiquitylation of exogenous human HIF-1alpha in vitro could be complemented by wild type pVHL, and re-expression of a wild type VHL gene restored a normal pattern of HIF/HRE activity, demonstrating the critical dependence of HIF regulation on pVHL in CHO cells. In contrast, other mutant cells had no demonstrable mutation in the VHL gene, and ubiquitylated exogenous HIF-1alpha normally, suggesting that they contain defects at other points in the oxygen-regulated processing of HIF-alpha subunits

The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.

Hypoxia-inducible factor-1 (HIF-1) has a key role in cellular responses to hypoxia, including the regulation of genes involved in energy metabolism, angiogenesis and apoptosis. The alpha subunits of HIF are rapidly degraded by the proteasome under normal conditions, but are stabilized by hypoxia. Cobaltous ions or iron chelators mimic hypoxia, indicating that the stimuli may interact through effects on a ferroprotein oxygen sensor. Here we demonstrate a critical role for the von Hippel-Lindau (VHL) tumour suppressor gene product pVHL in HIF-1 regulation. In VHL-defective cells, HIF alpha-subunits are constitutively stabilized and HIF-1 is activated. Re-expression of pVHL restored oxygen-dependent instability. pVHL and HIF alpha-subunits co-immunoprecipitate, and pVHL is present in the hypoxic HIF-1 DNA-binding complex. In cells exposed to iron chelation or cobaltous ions, HIF-1 is dissociated from pVHL. These findings indicate that the interaction between HIF-1 and pVHL is iron dependent, and that it is necessary for the oxygen-dependent degradation of HIF alpha-subunits. Thus, constitutive HIF-1 activation may underlie the angiogenic phenotype of VHL-associated tumours. The pVHL/HIF-1 interaction provides a new focus for understanding cellular oxygen sensing