Introduction Fast-growing tumour cells show enhanced protein synthesis and therefore depend on efficient folding for nascent export proteins in the endoplasmic reticulum (ER). Herein, the two most prominent ER resident thiol oxidoreductases ERp57 and PDI play important parts in formation of disulfide bonds in client proteins. This and the finding that both proteins fulfil various roles also in other compartments (i.e. cytoplasm, nucleus, cell membrane) encouraged us to investigate the impact of their depletion on colorectal cancer cells. Material and methods Using an inducible knockdown (KD) system we tested ERp57 and PDI deficiency in long term survival assays in normoxia and hypoxia combined with irradiation. Results and discussions KD of ERp57 or PDI triggered a severe attenuation of proliferation, but only ERp57 deficiency led to activation of the PERK-dependent UPR and apoptosis. When combined with an ERp57 KD, irradiation displayed the most dramatic growth reduction even under 1% oxygen. The absence of ERp57 reduced expression of cellular proliferation factors like c-Myc, PLK-1, AKT, PDPK1, ERK1,2 and others. Further, we demonstrated for the first time that PDI is an essential activator of the ER stress sensor PERK that enforces cancer cell survival under global ER stress in hypoxia. In the absense of ER stress, ERp57 functions as a reductase for PDI that keeps PERK in an inactive state. Conclusion Our data identified ERp57 and PDI as promising new targets for a mono- and combination anti-cancer therapy due to multiple cellular points of attack.

Introduction Poor prognosis of many solid tumours is often associated with hypoxic regions and an increased level of hypoxia-inducible-factor-1α (HIF-1α). Previous findings indicate that HIF-1α expression is relevant for radiation resistance. It was shown that HIF-1α is connected to molecules involved in DNA damage repair and checkpoint control. HIF-1α has an enhanced activity in tumours after radiation treatment (IR) and also mediates low radiation sensitivity. Material and methods In vitro cultured Lewis Lung Carcinoma (LLC) cells with doxycycline (Dox)-inducible knockdown (KD) of HIF-1α were used. Cells were incubated under hypoxic conditions (Hx, 1%) and irradiated with 5 Gy. Cell cycle distribution analysis was performed by PI staining and FACS. Colony formation assay, caspase-3 assay, PARP-1 cleavage and Annexin/PI staining were used to quantify apoptosis and long term survival. DNA double strand break (DSB) repair was investigated by pulse field gel electrophoresis, Western blot and immunofluorescent staining with γH2AX, 53 BP1, RAD51, BRCA1 antibodies. In vivo the induction of HIF-1α KD was performed by tamoxifen administration to HIF-1αfl/flCre-ERki/+ C57BL/6 mice. The LLC cells with constitutive HIF-1α targeting (shHIF) or scrambled (shscr) shRNA-expressing vectors were inoculated into the flank of the mice. IR was delivered once at day 10 with a dose of 15 Gy using an X-ray source. Results and discussions We demonstrated an inhibition of decrement of phosphorylated H2AX after Hx and IR in HIF-1α deficient cells. Fast repair kinetics of the cells remained unchanged, whereas the long term survival of the cells with reduced HIF-1α was decreased. Moreover, these cells displayed an increased rate of apoptosis after IR. We also observed persisted RAD51 foci after 24 hour in HIF-1α deficient cells which indicates an alteration in homologous recombination repair (HRR) of the cells. Furthermore, in vivo experiments with a HIF-1α deficient stromal cells in the mice and injected HIF-1α deficient tumours cells indicate a decrease of tumour growth. Conclusion These results imply that inactivation of HIF-1α disrupts DSB repair, in particular HRR. Furthermore, they highlight the importance of the interaction of tumour and microenvironment with respect to radiation sensitivity. It was shown that inhibition of HIF-1α enhances radiation sensitivity of tumour cells which is potentially helpful for the development of novel tumour therapies.

Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels ( n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47 phox and p67 phox . In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells ( n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O 2 -sensitive dye. We conclude that attenuated p47 phox subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.

Purpose: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1 α . The effects of PHD3 in tumour growth are largely unknown. Experimental design: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model. Results: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo , PHD3 inhibited tumour growth by abrogation of tumour angiogenesis. Conclusion: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

In this issue of Blood, van Rooijen and colleagues demonstrate that zebrafish carrying a mutation in the von Hippel Lindau gene (VHL) develop Chuvash polycythemia, a hereditary human disease characterized by the dysregulation of hypoxia-inducible proteins including erythropoietin.

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.

Anämie ist ein wichtiger und unabhängiger Prognosefaktor bei Patienten mit Plattenepithelkarzinomen im Kopf-Hals-Bereich, insbesondere bei definitiver Strahlentherapie. Dies beruht wahrscheinlich auf verschiedenen Ursachen, von denen die Verstärkung einer Tumorhypoxie durch Anämie ein wichtiger Faktor ist. Im Tiermodell kann man durch Anämiekorrektur mittels Erythropoetinen die Strahlenempfindlichkeit verbessern. Der Einsatz von Erythropoetin bei Kopf-Hals-Tumoren ist deshalb logisch und konsequent. Allerdings zeigen die bisherigen klinischen Ergebnisse keine Vorteile hinsichtlich des Überlebens.

Anemia is a major independent risk factor in patients with squamous cell cancer of the head and neck, and particularly in those undergoing radiotherapy with curative intent. There are probably various reasons for this, the most important of which seems to be the induction of tumor hypoxia via anemia. Tumor hypoxia is associated with radiation resistance. In animal models, it has proved possible to improve radiosensitivity by correcting anemia. It is therefore logical to use erythropoietins in head and neck cancer patients. However, the data currently available from randomized studies have not demonstrated any survival benefit.

The major pathological processes of systemic scleroderma (SSc) comprise inflammation and microvascular damage in the early or acute progressive stage as well as tissue fibrosis and hypoxia in the chronic end stage. Fibrosis seems to be a general phenomenon characterized by an increase of hydroxylysine aldehyde derived collagen cross-links which has been shown in vitro for systemic scleroderma fibroblasts. In the present study, we analyzed the cross-link pattern and the gene expression of lysyl hydroxylase 2 (LH2) in the skin of SSc. Furthermore, we determined the modulatory impact of inflammatory cytokines (interleukin 4, TNF- alphaand interleukin 1alpha/beta) and prolonged hypoxia on the cross-link profile and the gene expression of LH2, respectively. The concentration of hydroxylysine aldehyde derived cross-links was significantly increased in SSc, while the level of lysine aldehyde derived cross-links was not changed. Accordingly, a marked increase of the transcriptional level of LH2 was found. In long term dermal fibroblast cultures, only interleukin 4 induced an increase of hydroxylysine aldehyde derived cross-links accompanied by a higher gene expression of LH2. Furthermore, prolonged hypoxia induced a marked increase of the mRNA level of LH2 in relation to collagen I. The skin of SSc is characterized by an increase of the transcriptional activity of LH2 leading to an altered cross-link pattern. The changes in the quality of the collagenous matrix can also be obtained in cell culture by the exposure of fibroblasts to interleukin 4 or prolonged hypoxia emphasizing the role of this mediator in the acute and the low oxygen tension in the chronic phase of the disease.

The vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. Mediated by the hypoxia-inducible transcription factor HIF-I~fl , a reduction in 02 tension (pO2) leads to increased VEGF gene expression in nonmalignant tissues. In tumor cells VEGF mRNA levels are often constitutively elevated. We examined pO2-dependent VEGF mRNA expression and VEGF protein formation in the human breast cancer cell line MX-1 in vitro and in vivo. For in vitro study MX-1 cultures were grown on dishes with a gas-permeable bottom to expose the cells to defined 02 concentrations (from 95% to 0%) for 4h. Northern blot analysis showed significant VEGF mRNA in MX-1 cultures under normoxic conditions which was further increased by hypoxia. The amount of secreted VEGF was also elevated in hypoxic cultures. Western blot analysis revealed a correlation between the severity of hypoxia and HIF-lo¢ protein amounts in the nucleus. Furthermore, DNA-binding activity of HIF-1 could be demonstrated by gel-shift assays. For in vivo study immunodeficient nude mice bearing MX-1 tumor transplants were exposed to inspiratory hypoxia (10% 02). Northern blot and immunohistochemical analyses of MX-1 tumor transplants showed that VEGF mRNA and VEGF protein levels were increased in mice 17 h after the induction of inspiratory hypoxia. Thus, pO2-dependence of VEGF gene expression can be maintained in cancer cells, even in vivo, which may be relevant in regard to therapeutic attempts to inhibit tumor angiogenesis by increasing tumor oxygenation.