Search

Cancer cells, when exposed to the hypoxic tumour microenvironment, respond by activating hypoxia‐inducible factors (HIFs). HIF‐1 mediates extensive metabolic re‐programming, and expression of HIF‐1α, its oxygen‐regulated subunit, is associated with poor prognosis in cancer. Here we analyse the role of pyruvate dehydrogenase phosphatase 1 (PDP1) in the regulation of HIF‐1 activity. PDP1 is a key hormone‐regulated metabolic enzyme that dephosphorylates and activates pyruvate dehydrogenase (PDH), thereby stimulating the conversion of pyruvate into acetyl‐CoA. Silencing of PDP1 down‐regulated HIF transcriptional activity and the expression of HIF‐dependent genes, including that of PDK1, the kinase that phosphorylates and inactivates PDH, opposing the effects of PDP1. Inversely, PDP1 stimulation enhanced HIF activity under hypoxia. Alteration of PDP1 levels or activity did not have an effect on HIF‐1α protein levels, nuclear accumulation or interaction with its partners ARNT and NPM1. However, depletion of PDP‐1 decreased histone H3 acetylation of HIF‐1 target gene promoters and inhibited binding of HIF‐1 to the respective hypoxia‐response elements (HREs) under hypoxia. Furthermore, the decrease of HIF transcriptional activity upon PDP1 depletion could be reversed by treating the cells with acetate, as an exogenous source of acetyl‐CoA, or the histone deacetylase (HDAC) inhibitor trichostatin A. These data suggest that the PDP1/PDH/HIF‐1/PDK1 axis is part of a homeostatic loop which, under hypoxia, preserves cellular acetyl‐CoA production to a level sufficient to sustain chromatin acetylation and transcription of hypoxia‐inducible genes. [ABSTRACT FROM AUTHOR]

Airway smooth muscle (ASM) cells are characterized by phenotypic plasticity and can switch between differentiated and proliferative phenotypes. In rabbit tracheal ASM cells that had been differentiated in vitro by serum starvation, readdition of FBS caused initiation of proliferation and induction of nuclear and transcriptionally active hypoxia-inducible factor (HIF)-1[alpha]. In addition, FBS stimulated the induction of HIF-1[alpha] by the hypoxia mimetic cobalt. Treatment with actinomycin D, cycloheximide, the phosphatidylinositol 3-kinase inhibitors LY-294002 and wortmannin or the reactive oxygen species scavenger diphenyleneiodonium inhibited the FBS-dependent induction of HIF-1[alpha]. These data indicate that, in differentiated ASM cells, FBS upregulates HIF-1[alpha] by a transcription-, translation-, phosphatidylinositol 3-kinase-, and reactive oxygen species-dependent mechanism. Interestingly, addition of FBS and cobalt also induced HIF-l[alpha] in organ cultures of rabbit trachea strips and synergistically increased their contractile response to ACh, suggesting that HIF-1[alpha] might be implicated in airway hypercontractility. cobalt; fetal bovine serum

The Drosophila melanogaster Ketel gene was identified via the [Ketel.sup.D] dominant female sterile mutations and their [ketel.sup.r] revertant alleles that are recessive zygotic lethals. The maternally acting [Ketel.sup.D] mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1.2-3 in four [ketel.sup.r] alleles. The [Ketel.sup.+] transgenes rescue [ketel.sup.r]-associated zygotic lethality and slightly reduce [Ketel.sup.D]-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-[Beta], the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm from stage 11 of oogenesis. In cleavage embryos the Ketel protein is cytoplasmic. The Ketel gene appears to be ubiquitously expressed in embryonic cells. Western blot analysis revealed that the Ketel gene is not expressed in several larval cell types of late third instar larvae.

Airway smooth muscle (ASM) cells are characterized by phenotypic plasticity and can switch between differentiated and proliferative phenotypes. In rabbit tracheal ASM cells that had been differentiated in vitro by serum starvation, readdition of FBS caused initiation of proliferation and induction of nuclear and transcriptionally active hypoxia-inducible factor (HIF)- lα In addition, FBS stimulated the induction of HIF-lα by the hypoxia mimetic cobalt. Treatment with actinomycin D, cycloheximide, the phosphatidylinositol 3-kinase inhibitors LY-294002 and wortmannin or the reactive oxygen species scavenger diphenylenei- odonium inhibited the FBS-dependent induction of HIF-lα. These data indicate that, in differentiated ASM cells, FBS upregulates HIF-lα by a transcription-, translation-, phosphatidylinositol 3-kinase-, and reactive oxygen species-dependent mechanism. Interestingly, addition of FBS and cobalt also induced HIF-lα in organ cultures of rabbit trachea strips and synergistically increased their contractile response to ACh, suggesting that HIF-lα might be implicated in airway hypercontractility. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most common cancer worldwide and despite the abundance of molecular pathways and markers continually being reported, the mortality rates remain high. Hypoxia inducible factor 1alpha (HIF-1α) plays a major role in the response of tumors to hypoxia, and contributes to tumor aggressiveness, invasiveness and resistance to radiotherapy and chemotherapy. Targeting HIF-1α is an attractive strategy, with the potential for disrupting multiple pathways crucial for tumor growth. In the current study, HIF-1α immunohistochemical expression in CRC is reviewed along with the relation to clinical outcome and prognosis. In addition, the significant correlation of HIF-1α to vascular endothelial growth factor (VEGF) expression is reported, as well as the possible role of HIF-1α in predicting the therapeutic response to anti-EGFR therapies. Herein, an overview of the HIF-1α expression in CRC is presented. This review delineates the crucial role that HIF-1α plays in carcinogenesis, tumor angiogenesis and cancer progression. The evaluation of HIF-1α in patient biopsies could be useful as a prognostic and/or predictive biomarker in personalized cancer treatment.

The immunoreactivity of hypoxia inducible factor 1alpha (HIF-1alpha) has been considered a reliable indicator of the HIF-1 pathway activation in tissue hypoxia. However, HIF-1alpha immunoreactivity has been evaluated with different antibodies and heterogeneous protocols. The need to interpret contradictory findings requires, among other things, a comparison of the antibodies. This could be accomplished by using identical, well characterized antigenic targets and by decreasing the influence of other variables. We applied most of the commercially available antibodies, and an antibody developed in our laboratories, to the human cervical cancer HeLa cell line and tissue sections from a renal cell carcinoma systematically, and to other tumors selectively. The expression of HIF-1alpha in HeLa cells was induced by the hypoxia-mimetic DFO. Non-induced HeLa cells were used as 'genuine' negative controls in addition to routine ones. HeLa cells (both induced and not induced) were also examined by immunofluorescence and Western blotting. We found that the antibodies showed immunostaining patterns with remarkable qualitative and quantitative differences, an observation not emphasized in previous literature. Certain antibodies require careful application to avoid specificity issues, and others to avoid low sensitivity problems. Pairing certain antibodies can optimize evaluation of HIF-1alpha expression. Most previous immunohistochemical studies of HIF-1alpha have attempted to map hypoxic neoplastic tissues or to demonstrate hypoxia in studies of neoangiogenesis, rather than 'measuring' HIF-1alpha expression or activation, because this requires a validated immunoassay. Our study thus allows for the development of a controlled and comparative HIF-1alpha immunoassay, which could be valuable if HIF-1alpha becomes a therapeutic target.