Prolyl-4-hydroxylase 2 enhances hypoxia-induced glioblastoma cell death by regulating the gene expression of hypoxia-inducible factor-α.

Oxygen deprivation (hypoxia) is a common feature of solid tumors in advanced stages. The primary cellular transcriptional responses to hypoxia are mainly mediated by the transcription factor hypoxia-inducible factor (HIF). HIF consists of an oxygen-labile α-subunit (HIF-1α, -2α) and a stable ß-subunit (ARNT). Prolyl-4-hydroxylase 2 (PHD2) is known as an important mediator of the oxygen-dependent degradation of HIF-α subunits. As HIF-α subunits are not confirmed to be the only substrates of PHD2, it is unknown whether PHD2 regulates HIF-1α and HIF-2α by interacting with other intracellular molecules. In this study, we found that in the glioblastoma cells, PHD2 maintains the gene expression of HIF-1α in dependence of nuclear factor κB and suppresses the gene expression of HIF-2α through HIF-1α. The PHD2-mediated degradation of HIF-1α and HIF-2α seems less important. Furthermore, PHD2 enhances hypoxia-induced glioblastoma cell death by modulating the expression of the HIF target genes glucose transporter 1, vascular endothelial growth factor-A and Bcl-2 binding protein 3. Our findings show that PHD2 inhibits the adaptation of glioblastoma cells to hypoxia by regulating the HIF-α subunits in a non-canonical way. Modulation of PHD2 activity might be considered as a new way to inhibit glioblastoma progression.

Interleukin-1ß promotes hypoxia-induced apoptosis of glioblastoma cells by inhibiting hypoxia-inducible factor-1 mediated adrenomedullin production.

Glioblastoma is the most common brain tumor in adults. Advanced glioblastomas normally contain hypoxic areas. The primary cellular responses to hypoxia are generally mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1). Interleukin-1ß (IL-1ß) is a cytokine that is often present in the glioblastoma microenvironment and is known to be a modulator of glioblastoma progression. However, the role of IL-1ß in regulating glioblastoma progression is still controversial. In this study, we found that in the human glioblastoma cell lines U87MG and U138MG, IL-1ß inhibits the transactivation activity of HIF-1 by promoting the ubiquitin-independent proteasomal degradation of the oxygen-labile α-subunit of HIF-1 and downregulates the expression of the HIF-1 target gene adrenomedullin (AM). Apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays showed that AM protects glioblastoma cells against hypoxia-induced apoptosis in a dose-dependent manner. Thus, in the presence of IL-1ß more glioblastoma cells undergo hypoxia-induced cell death. Our findings suggest that when estimating the influence of IL-1ß on the prognosis of glioblastoma patients, factors such as the degree of hypoxia, the expression levels of HIF-1 and AM should be taken into consideration. For the AM-producing glioblastoma cells, IL-1ß represents a potent apoptosis inducer.

Identification and complete genome analysis of a virus variant or putative new foveavirus associated with apple green crinkle disease.

A virus identified as "apple green crinkle associated virus" (AGCaV) was isolated from Aurora Golden Gala apple showing severe symptoms of green crinkle disease. Evidence was obtained of a potential causal relationship to the disease. The viral genome consists of 9266 nucleotides, excluding the poly(A) tail at the 3'-terminus. It has a genome organization similar to that of members of the species Apple stem pitting virus (ASPV), the type species of the genus Foveavirus, family Betaflexiviridae. ORF1 of AGCaV encodes a replicase-complex polyprotein with a molecular mass of 247 kDa; the proteins of ORFs 2, 3, and 4 (TGB proteins) are estimated to be 25.1 kDa, 12.8 kDa, and 7.4 kDa, respectively; and ORF5 encodes the CP, with an estimated molecular mass of 43.3 kDa. Interestingly, AGCaV utilizes different stop codons for ORF1, ORF3, and ORF5 compared to the ASPV type isolate PA66, and between the two viruses, six distinct indel events were observed within ORF5. AGCaV has four non-coding regions (NCRs), including a 5'-NCR (60 nt), a 3'-NCR (134 nt), and two intergenic (IG) NCRs: IG-NCR1 (69 nt) and IG-NCR2 (91 nt). A conserved stable hairpin structure was identified in the variable 5'-NCR of members of the genus Foveavirus. AGCaV may be a variant or strain of ASPV with unique biological properties, but there is evidence that it may be a distinct putative foveavirus.

Erythropoietin and the vascular wall: the controversy continues.

BACKGROUND AND AIMS: Erythropoietin (EPO) stimulates erythropoiesis through its specific receptor (EPO-R). Preclinical work has assigned a role for the EPO/EPO-R system in the heart and blood vessels. The potential use of erythropoiesis-stimulating agents (ESAs) for nonhematopoietic indications is a focus of current research. This article considers proven actions of EPO in the cardiovascular system, with emphasis on the human responses. DATA SYNTHESIS: By use of specific anti-EPO-R antibody no EPO-R protein was detected by Western blotting in normal non-erythroid tissues. Clinical trials failed to demonstrate clear beneficial effects of high-dosed ESAs in patients with coronary syndrome or myocardial infarct. While ESA therapy may lead to an elevation in arterial blood pressure in previously anemic patients, several studies have reported no effects on vessels/blood pressure with ESAs. EPO has been reported to stimulate angiogenesis. EPO-R mRNA is detectable in human vascular endothelium. However, in most vitro studies very high concentrations of EPO were applied and well-designed studies have failed to show direct effects of ESAs on endothelial cells. Whether EPO promotes the mobilization of myeloid progenitor cells into the blood stream still needs to be studied in more detail, as this effect may prove useful for augmenting the neovascularization of ischemic tissues. With respect to the administration of ESAs to tumor patients, a deeper insight into the role of EPO for tumor angiogenesis is desirable. CONCLUSIONS: The enthusiastic reports of the nonhematopoietic cytoprotective potential of EPO and its derivatives in the cardiovascular system have not yet been confirmed in placebo-controlled clinical trials.

Effects of erythropoietin receptors and erythropoiesis-stimulating agents on disease progression in cancer.

Erythropoiesis-stimulating agents (ESAs) increase red blood cell (RBC) production in bone marrow by activating the erythropoietin receptor (EpoR) on erythrocytic-progenitor cells. Erythropoiesis-stimulating agents are approved in the United States and Europe for treating anaemia in cancer patients receiving chemotherapy based on randomised, placebo-controlled trials showing that ESAs reduce RBC transfusions. Erythropoiesis-stimulating agent-safety issues include thromboembolic events and concerns regarding whether ESAs increase disease progression and/or mortality in cancer patients. Several trials have reported an association between ESA use and increased disease progression and/or mortality, whereas other trials in the same tumour types have not provided similar findings. This review thoroughly examines available evidence regarding whether ESAs affect disease progression. Both clinical-trial data on ESAs and disease progression, and preclinical data on how ESAs could affect tumour growth are summarised. Preclinical topics include (i) whether tumour cells express EpoR and could be directly stimulated to grow by ESA exposure and (ii) whether endothelial cells express EpoR and could be stimulated by ESA exposure to undergo angiogenesis and indirectly promote tumour growth. Although assessment and definition of disease progression vary across studies, the current clinical data suggest that ESAs may have little effect on disease progression in chemotherapy patients, and preclinical data indicate a direct or indirect effect of ESAs on tumour growth is not strongly supported.

Complete genome sequences of three Erwinia amylovora phages isolated in north america and a bacteriophage induced from an Erwinia tasmaniensis strain.

Fire blight, a plant disease of economic importance caused by Erwinia amylovora, may be controlled by the application of bacteriophages. Here, we provide the complete genome sequences and the annotation of three E. amylovora-specific phages isolated in North America and genomic information about a bacteriophage induced by mitomycin C treatment of an Erwinia tasmaniensis strain that is antagonistic for E. amylovora. The American phages resemble two already-described viral genomes, whereas the E. tasmaniensis phage displays a singular genomic sequence in BLAST searches.

Erythropoiesis stimulating agents and techniques: a challenge for doping analysts.

Recombinant human erythropoietin (rHuEPO) engineered in Chinese hamster ovary (CHO) cell cultures (Epoetin alfa and Epoetin beta) and its hyperglycosylated analogue Darbepoetin alfa are known to be misused by athletes. The drugs can be detected by isoelectric focusing (IEF) and immunoblotting of urine samples, because "EPO" is in reality a mixture of isoforms and the N-glycans of the recombinant products differ from those of the endogenous hormone. However, there is a plethora of novel erythropoiesis stimulating agents (ESAs). Since the originator Epoetins alfa and beta are no longer protected by patent in the European Union, rHuEPO biosimilars have entered the market. In addition, several companies in Asia, Africa and Latin America produce copied rHuEPOs for clinical purposes. While the amino acid sequence of all Epoetins is identical, the structure of their glycans differs depending on the mode of production. Some products contain more acidic and others more basic EPO isoforms. Epoetin delta is special, as it was engineered by homologous recombination in human fibrosarcoma cells (HT-1080), thus lacking N-glycolylneuraminic acid like native human EPO. ESAs under development include EPO fusion proteins, synthetic erythropoiesis stimulating protein (SEP) and peptidic (Hematide(), CNTO 528) as well as non-peptidic EPO mimetics. Furthermore, preclinical respectively clinical trials have been performed with small orally active drugs that stimulate endogenous EPO production by activating the EPO promoter ("GATA-inhibitors": diazepane derivatives) or enhancer ("HIF-stabilizers": 2-oxoglutarate analogues). The prohibited direct EPO gene transfer may become a problem in sports only in the future.

Dexamethasone impairs hypoxia-inducible factor-1 function.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of alpha- and beta-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1alpha levels in the cytosol of HepG2 cells, while nuclear HIF-1alpha levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients.

Hydroxylation of hypoxia-inducible transcription factors and chemical compounds targeting the HIF-alpha hydroxylases.

The hypoxia-inducible transcription factors (HIFs) are central components in the cellular responses to a lack of O(2), i.e. hypoxia. Homologs of the HIF system (HIF-1, -2 and -3) are detectable in all nucleated cells of multicellular organisms. Active HIFs are heterodimers (HIF-alpha/ beta). In hypoxia the O(2)-labile alpha-subunit is translocated to the nucleus where it binds HIF-beta. Over 100 HIF target genes have already been identified. The translational products of these genes increase O(2) delivery to hypoxic tissues, such as erythropoietin which stimulates the production of red blood cells, and they adapt cellular metabolism to hypoxia, such as glycolytic enzymes. HIFs are inactive in normoxia because of O(2)-dependent enzymatic hydroxylation and subsequent degradation of their alpha-subunit. Three HIF-alpha prolyl hydroxylases (PHD1, 2 and 3) initiate proteasomal degradation while an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1) inhibits the function of the C-terminal transactivation domain of HIF-alpha. In addition to O(2) and 2-oxoglutarate, the HIF-alpha hydroxylases require Fe(2+) and ascorbate as co-factors. Products of glycolysis can act as endogenous inhibitors of HIF hydroxylases which may lead to sustained activation of HIFs in cancer cells. The cofactor requirements define the routes to inhibition of the enzymes when HIF activation is desirable. In particular, 2-oxoglutarate analogues have emerged as promising tools for stimulation of erythropoiesis and angiogenesis ("HIF-stabilizers"). However, as the HIF system promotes the transcription of many genes, and other 2-oxoglutarate dependent dioxygenases are likely to be inhibited by the same analogues, careful evaluation of the inhibitors seems mandatory prior to their clinical use.

Interleukin-1beta inhibits the hypoxic inducibility of the erythropoietin enhancer by suppressing hepatocyte nuclear factor-4alpha.

The suppression of hypoxia-induced erythropoietin (EPO) expression by inflammatory cytokines like interleukin-1 (IL-1) contributes to the development of the anemia of chronic disease (ACD). However, the precise mechanism of this suppression is unclear. The 3'-EPO enhancer mediates the transcriptional response to hypoxia by binding several transcription factors, including hypoxia-inducible factor, hepatocyte nuclear factor-4alpha (HNF-4alpha) and chicken ovalbumin upstream promoter transcription factor. We investigated whether IL-1beta inhibits the activity of the 3'-EPO enhancer via HNF-4alpha. IL-1beta inhibited HNF-4alpha mRNA expression and caused proteasome-dependent degradation of HNF-4alpha protein, which resulted in a strongly reduced DNA-binding activity of HNF-4alpha. Reporter gene assays revealed that IL-1beta caused a complete suppression of the hypoxic inducibility of the 3' enhancer via inhibition of HNF-4alpha. We conclude that IL-1beta, at least partially, reduces hypoxia-induced EPO expression by down-regulation of HNF-4alpha.

High plasma VEGF relates to low carbohydrate intake in patients with type 2 diabetes.

OBJECTIVE: Vascular endothelial growth factor (VEGF) has been suggested to enhance glucose transport across the blood-brain barrier, thereby increasing brain glucose supply. Increased brain glucose concentration is known to suppress food intake and to decrease body mass via action on hypothalamic regulation centers. Based on the crucial role of VEGF on brain glucose supply, we hypothesized that higher VEGF concentrations are associated with lower food intake and body mass in humans. METHODS: Intending to investigate subjects with high variance of blood glucose, we examined patients with type 2 diabetes mellitus. Our hypothesis was tested in a population-based cohort of 190 subjects with type 2 diabetes. Plasma VEGF levels in conjunction with other parameters known to modulate food intake were measured and subsequently correlated with food intake patterns at a breakfast buffet as well as with body mass. RESULTS: We found that subjects with higher concentrations of plasma VEGF had 17% less carbohydrate intake (P=0.003) and 4.8% lower body mass (P=0.017) than those with lower VEGF concentrations. Intake of protein and fat did not correlate with VEGF concentrations. These associations of plasma VEGF were confirmed in multiple linear regression analyses controlling for several parameters interacting with food intake. CONCLUSION: We conclude that high plasma VEGF concentrations are associated with less carbohydrate intake and lower body mass in type 2 diabetes. The role VEGF plays in facilitating glucose access to the brain represents a new aspect of food intake regulation and energy homeostasis, with relevance for diseases with body mass disturbances.

Effects of erythropoietin on brain function.

This article is a selective extension of a review on recombinant human erythropoietin (rHu-EPO) as an anti-anaemic drug, published in this journal in 2000. It summarises the recent advances in understanding the molecular mechanisms by which the hypoxia-inducible transcription factor 1 (HIF-1) regulates O(2)-dependent genes, including the EPO gene in brain. With respect to brain integrity, EPO exerts positive effects in two different ways. First, rHu-EPO raises the blood haemoglobin concentration and, hence, the O(2) capacity of the blood in anaemic patients. The restored O(2) supply ameliorates attention difficulties and psychomotor slowing, improves memory capacities and normalises neuroendocrine functions. Second, EPO can act as a neurotrophic and neuroprotective factor directly in brain. EPO and its receptor are expressed in the cerebral cortex, cerebellum, hippocampus, pituitary gland and spinal cord. In vitro EPO protects against glutamate-induced cell death in a dose-dependent way. In animal models it reduces volumes of brain ischaemia, protects the cortex from hypoxic damage and leads to survival of neurons and synapses. One can expect that in the near future rHu-EPO will be used therapeutically in cerebral ischaemia, brain trauma, inflammatory diseases, and neural degenerative disorders. A first clinical trial has shown the neuroprotective effectiveness of the drug in cerebral ischaemia.

Little cherry virus-2: sequence and genomic organization of an unusual member of the Closteroviridae.

The complete genomic sequence of variant USA6b of Little cherry virus-2 (LChV-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (ORFs). The sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of LChV-2 (variant USA6a). Both LChV-2/USA6a and LChV-2/USA6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. LChV-2/USA6b is more closely related to the partially determined genomic sequence of a Canadian isolate of LChV-2, strain LC5 (92.9% identity). LChV-2/USA6b has an unusual genomic organization compared to other members of the Closteroviridae. The LChV-2/USA6b genome is potentially ambi-sense, with a negative sense ORF0 at the 5' terminus, from which an 18.1 kDa protein of unknown function can be expressed in vitro. The N-terminal region of the LChV-2/USA6 ORF1a translation product does not code for a papain-like protease motif. ORF1 codes for a novel motif, of unknown function, also present in isolates of the Grapevine leafroll associated virus-3, (genus Ampelovirus) as well as viruses of the family Flexiviridae. ORF3 lacks an AUG start codon, but could potentially be expressed via read-through of the ORF2 stop codon. At the 3' end, there is a re-organization of encoded genes compared with other members of the Closteroviridae including separation of the coat protein and coat protein duplicate genes by 4 other genes as found for LChV-2/LC5.

Strain diversity and conserved genome elements in Strawberry mild yellow edge virus.

The complete nucleotide sequence of Strawberry mild yellow edge virus isolate D74, and the sequences of a 878 nt region of the coat protein and flanking regions of twenty three isolates of SMYEV were obtained and analysed. The full sequence of the aphid transmissible strain D74 was deduced and found to have an 86% sequence identity to the non-transmissible Agrobacterium infectious MY18 strain. In contrast to isolate MY18 the 5' terminal nucleotides (GAAAAC) of D74 are typical of those from other potexviruses. However, both MY18 and D74 have a non-AUG initiation codon for ORF2 encoding the triple gene block protein 1 (TGB1), and an overlapping TGB3 and coat protein (CP), features unique in the Potexvirus genus. Other conserved features of the genome, including stem-loop structures in the untranslated regions, and motifs common to related viruses are described. The previously postulated ORF6 of MY18 is absent in twenty of the isolates sequenced, including D74. Phylogenetic analysis places all isolates in one of three distinct groups/strains named here I (type-D74), II (type-9Redland), and III (type-MY18), with the majority of isolates, including all European isolates tested, belonging to strain I (type-D74).

Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control.

The principal aphid-borne viruses infecting Strawberry (Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry (Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers-which produced a PCR fragment of the expected size in 25 of 27 plant species tested-combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.

Erythropoietin.

This article summarizes recent advances in understanding the production and action of the hormone erythropoietin (Epo) with respect to high altitude physiology and sports medicine. Hypoxia is the main stimulus for Epo gene expression. An O2-labile protein (hypoxia-inducible factor 1, HIF-1) has been identified that is hydroxylated and degraded under normoxic conditions but active in hypoxia, where it enhances Epo gene transcription resulting in elevated hemoglobin levels and O2 capacity of the blood. The stimulation of Epo production at lowered arterial O2 tension can be maladaptive, if erythrocytosis develops such as seen in high altitude habitants. Within physiological limits the aerobic power increases in parallel with blood O2 capacity. Therefore, some elite athletes have misused recombinant human Epo (rhEpo), which is a beneficial anti-anemic drug in clinical practice. Indirect and direct methods to detect rhEpo doping have been recently developed.

The Detection and Variation of Strawberry mottle virus.

Strawberry mottle virus (SMoV) is probably the most important virus to infect strawberry (Fragaria spp.). All species of strawberry are susceptible to SMoV, resulting in severe losses both in fruit and runner yield. However, due to the absence of definitive symptoms in commercial varieties, the only effective means of detecting SMoV is by transmission to susceptible indicator plants. In this study, we describe a reverse transcription-polymerase chain reaction (RT-PCR) method for the detection of SMoV in Fragaria spp. with the use of primers specific for the noncoding regions (NCR) of both RNA1 and RNA2 of the virus. Using this method, all of 16 isolates from various geographical origins were positive for SMoV. Partial sequences of a 327-nt long coding region were obtained for the putative large coat protein of all isolates by RT-PCR using degenerate primers. Nucleotide identities between isolates ranged from 72.8 to 99.7%. A 546-nt sequence of the putative polymerase gene of nine isolates was obtained by RT-PCR and compared. Nucleotide identities ranged from 73.4 to 100%. There was a clear tendency for isolates to group according to their geographical origin. Sequence data obtained of the NCR show four completely conserved regions of 20 or more bases.

Characterization and complete nucleotide sequence of Strawberry mottle virus: a tentative member of a new family of bipartite plant picorna-like viruses.

An isolate of Strawberry mottle virus (SMoV) was transferred from Fragaria vesca to Nicotiana occidentalis and Chenopodium quinoa by mechanical inoculation. Electron micrographs of infected tissues showed the presence of isometric particles of approximately 28 nm in diameter. SMoV-associated tubular structures were also conspicuous, particularly in the plasmodesmata of C. quinoa. DsRNA extraction of SMoV-infected N. occidentalis yielded two bands of 6.3 and 7.8 kbp which were cloned and sequenced. Gaps in the sequence, including the 5' and 3' ends, were filled using RT-PCR and RACE. The genome of SMoV was found to consist of RNA1 and RNA2 of 7036 and 5619 nt, respectively, excluding a poly(A) tail. Each RNA encodes one polyprotein and has a 3' non-coding region of approximately 1150 nt. The polyprotein of RNA1 contains regions with identities to helicase, viral genome-linked protein, protease and polymerase (RdRp), and shares its closest similarity with RNA1 of the tentative nepovirus Satsuma dwarf virus (SDV). The polyprotein of RNA2 displayed some similarity to the large coat protein domain of SDV and related viruses. Phylogenetic analysis of the RdRp region showed that SMoV falls into a separate group containing SDV, Apple latent spherical virus, Naval orange infectious mottling virus and Rice tungro spherical virus. Given the size of RNA2 and the presence of a long 3' non-coding region, SMoV is more typical of a nepovirus, although atypically for a nepovirus it is aphid transmissible. We propose that SMoV is a tentative member of an SDV-like lineage of picorna-like viruses.