Sónia Rocha

HIF (hypoxia-inducible factor) is the main transcription factor activated by low oxygen tensions. HIF-1α (and other α subunits) is tightly controlled mostly at the protein level, through the concerted action of a class of enzymes called PHDs (prolyl hydroxylases) 1, 2 and 3. Most of the knowledge of HIF derives from studies following hypoxic stress; however, HIF-1α stabilization is also found in non-hypoxic conditions through an unknown mechanism. In the present study, we demonstrate that NF-κB (nuclear factor κB) is a direct modulator of HIF-1α expression. The HIF-1α promoter is responsive to selective NF-κB subunits. siRNA (small interfering RNA) studies for individual NF-κB members revealed differential effects on HIF-1α mRNA levels, indicating that NF-κB can regulate basal HIF-1α expression. Finally, when endogenous NF-κB is induced by TNFα (tumour necrosis factor α) treatment, HIF-1α levels also change in an NF-κB-dependent manner. In conclusion, we find that NF-κB can regulate basal TNFα and, in certain circumstances, the hypoxia-induced HIF-1α.

With the emerging role of NF-κB in cancer it is important that its responses to stimuli relevant to tumor progression and therapy are understood. Here, we demonstrate that NF-κB induced by cytotoxic stimuli, such as ultraviolet light (UV-C) and the chemotherapeutic drugs daunorubicin/doxorubicin, is functionally distinct to that seen with the inflammatory cytokine TNF and is an active repressor of antiapoptotic gene expression. Surprisingly, these effects are mediated by the RelA(p65) NF-κB subunit. Furthermore, UV-C and daunorubicin inhibit TNF-induced NF-κB transactivation, indicating that this is a dominant effect. Consistent with this, mechanistic studies reveal that UV-C and daunorubicin induce the association of RelA with histone deacetylases. RelA can therefore be both an activator and repressor of its target genes, dependent upon the manner in which it is induced. This has important implications for the role of NF-κB in tumorigenesis and the use of NF-κB inhibitors in cancer therapy.

Cell proliferation is a metabolically demanding process. It requires active reprogramming of cellular bioenergetic pathways towards glucose metabolism to support anabolic growth. NF-κB/Rel transcription factors coordinate many of the signals that drive proliferation during immunity, inflammation and oncogenesis, but whether NF-κB regulates the metabolic reprogramming required for cell division during these processes is unknown. Here, we report that NF-κB organizes energy metabolism networks by controlling the balance between the utilization of glycolysis and mitochondrial respiration. NF-κB inhibition causes cellular reprogramming to aerobic glycolysis under basal conditions and induces necrosis on glucose starvation. The metabolic reorganization that results from NF-κB inhibition overcomes the requirement for tumour suppressor mutation in oncogenic transformation and impairs metabolic adaptation in cancer in vivo. This NF-κB-dependent metabolic pathway involves stimulation of oxidative phosphorylation through upregulation of mitochondrial synthesis of cytochrome c oxidase 2 (SCO2; ref. ). Our findings identify NF-κB as a physiological regulator of mitochondrial respiration and establish a role for NF-κB in metabolic adaptation in normal cells and cancer.

Oxygen is essential for the life of most multicellular organisms. Cells possess enzymes called molecular dioxygenases that depend on oxygen for activity. A subclass of molecular dioxygenases is the histone demethylase enzymes, which are characterized by the presence of a Jumanji-C (JmjC) domain. Hypoxia can alter chromatin, but whether this is a direct effect on JmjC-histone demethylases or due to other mechanisms is unknown. Here, we report that hypoxia induces a rapid and hypoxia-inducible factor-independent induction of histone methylation in a range of human cultured cells. Genomic locations of histone-3 lysine-4 trimethylation (H3K4me3) and H3K36me3 after a brief exposure of cultured cells to hypoxia predict the cell's transcriptional response several hours later. We show that inactivation of one of the JmjC-containing enzymes, lysine demethylase 5A (KDM5A), mimics hypoxia-induced cellular responses. These results demonstrate that oxygen sensing by chromatin occurs via JmjC-histone demethylase inhibition.

Hypoxia and inflammation have been associated with a number of pathological conditions, in particular inflammatory diseases. While hypoxia is mainly associated with the activation of hypoxia‐inducible factors ( HIF s), inflammation activates the family of transcription factor called nuclear factor‐kappa B ( NF ‐κB). An extensive crosstalk between these two main molecular players involved in hypoxia and inflammation has been demonstrated. This crosstalk includes common activating stimuli, shared regulators and targets. In this review, we discuss the current understanding of the role of NF ‐κB and HIF in the context of the immune response. We review the crosstalk between HIF and NF ‐κB in the control of the immune response in different immune cell types including macrophages, neutrophils and B and T cells. Furthermore the importance of the molecular crosstalk between HIF s and NF ‐κB for a variety of medical conditions will be discussed.

E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for 'proteolysis-targeting chimeras'). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases.Targeting the ubiquitin proteasome system to modulate protein homeostasis using small molecules has promising therapeutic potential. Here the authors describe Homo-PROTACS: small molecules that can induce the homo-dimerization of E3 ubiquitin ligases and cause their proteasome-dependent degradation.

Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

In order to improve the efficacy of conventional radiotherapy, attention has been paid to immune cells, which not only modulate cancer cell response to therapy but are also highly recruited to tumours after irradiation. Particularly, the effect of ionizing radiation on macrophages, using therapeutically relevant doses, is not well understood. To evaluate how radiotherapy affects macrophage behaviour and macrophage-mediated cancer cell activity, human monocyte derived-macrophages were subjected, for a week, to cumulative ionizing radiation doses, as used during cancer treatment (2 Gy/fraction/day). Irradiated macrophages remained viable and metabolically active, despite DNA damage. NF-kappaB transcription activation and increased Bcl-xL expression evidenced the promotion of pro-survival activity. A significant increase of pro-inflammatory macrophage markers CD80, CD86 and HLA-DR, but not CCR7, TNF and IL1B was observed after 10 Gy cumulative doses, while anti-inflammatory markers CD163, MRC1, VCAN and IL-10 expression decreased, suggesting the modulation towards a more pro-inflammatory phenotype. Moreover, ionizing radiation induced macrophage morphological alterations and increased their phagocytic rate, without affecting matrix metalloproteases (MMP)2 and MMP9 activity. Importantly, irradiated macrophages promoted cancer cell-invasion and cancer cell-induced angiogenesis. Our work highlights macrophage ability to sustain cancer cell activities as a major concern that needs to be addressed to improve radiotherapy efficacy.

Cancer is often characterised by the presence of hypoxia and inflammation. Paramount to the mechanisms controlling cellular responses under such stress stimuli, are the transcription factor families of Hypoxia Inducible Factor (HIF) and Nuclear Factor of κ-light-chain-enhancer of activated B cells (NF-κB). Although, a detailed understating of how these transcription factors respond to their cognate stimulus is well established, it is now appreciated that HIF and NF-κB undergo extensive crosstalk, in particular in pathological situations such as cancer. Here, we focus on the current knowledge on how HIF is activated by inflammation and how NF-κB is modulated by hypoxia. We summarise the evidence for the possible mechanism behind this activation and how HIF and NF-κB function impacts cancer, focusing on colorectal, breast and lung cancer. We discuss possible new points of therapeutic intervention aiming to harness the current understanding of the HIF-NF-κB crosstalk.

Lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders individually considered as rare, and few data on its prevalence has been reported in the literature. The overall birth prevalence of the 29 different LSDs studied in the Portuguese population was calculated to be 25/100000 live births, twice the prevalence previously described in Australia and in The Netherlands. The comparison of the prevalence profile of the LSDs presenting a prevalence higher than 0.5/100000 in the Portuguese, Dutch and Australian populations showed, in the Portuguese, the existence of a higher prevalence of GM2 gangliosidoses (B variant), mucolipidoses (II and III), Niemman-Pick type C and metachromatic leukodystrophy (MLD), and a lower prevalence of Pompe and Fabry. The highest prevalence value for a single LSD is the one of GM2 gangliosidoses (B variant), corresponding to 3/100000, a value which is significantly higher than the prevalence of the most frequent LSD in Dutch, Pompe disease (2/100000) and Australians, Gaucher's disease (GD) (1.8/100000). It is worth noting that the highest prevalence of GM2 gangliosidoses found in the Portuguese is mainly due to the existence of a unique subtype, the rare juvenile B1 variant.

In the present study we show thatN-acetylsphingosine (C2-ceramide),N-hexanoylsphingosine (C6-ceramide), and, to a much lesser extent, C2-dihydroceramide induce cytochromec (cyto c) release from isolated rat liver mitochondria. Ceramide-induced cyto c release is prevented by preincubation of mitochondria with a low concentration (40 nm) of Bcl-2. The release takes place when cytoc is oxidized but not when it is reduced. Upon cytoc loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (ΔΨ), and Ca2+ retention are diminished. Incubation with Bcl-2 prevents, and addition of cytoc reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter ΔΨ, neither when cyto c is oxidized nor when it is reduced, ruling out a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cytoc. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cytoc followed by its release and that the redox state of cytoc influences its detachment by ceramide from the inner mitochondrial membrane. In the present study we show thatN-acetylsphingosine (C2-ceramide),N-hexanoylsphingosine (C6-ceramide), and, to a much lesser extent, C2-dihydroceramide induce cytochromec (cyto c) release from isolated rat liver mitochondria. Ceramide-induced cyto c release is prevented by preincubation of mitochondria with a low concentration (40 nm) of Bcl-2. The release takes place when cytoc is oxidized but not when it is reduced. Upon cytoc loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (ΔΨ), and Ca2+ retention are diminished. Incubation with Bcl-2 prevents, and addition of cytoc reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter ΔΨ, neither when cyto c is oxidized nor when it is reduced, ruling out a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cytoc. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cytoc followed by its release and that the redox state of cytoc influences its detachment by ceramide from the inner mitochondrial membrane. Recently, the importance of ceramide in cell metabolism has been broadly investigated. It is now evident that ceramide is involved as a second messenger in what has become known as the sphingomyelin cycle (1Hannun Y.A. J. Biol. Chem. 1994; 269: 3125-3128Abstract Full Text PDF PubMed Google Scholar), apoptosis, and differentiation in many cell types (2Bose R. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (783) Google Scholar). The mechanisms by which ceramide mediates apoptosis have not yet been fully addressed, however, it is known that mitochondria are targets of ceramide. Thus, direct inhibition of complex III of the mitochondrial respiratory chain by ceramide (3Gudz T.I. Tserng K.Y. Hoppel C.L. J. Biol. Chem. 1997; 272: 24154-24158Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar), ceramide-induced generation of reactive oxygen species in intact mitochondria (4Garcia-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar) and in cells (5Quillet-Mary A. Jaffrézou J.-P. Mansat V. Bordier C. Naval J. Laurent G. J. Biol. Chem. 1997; 272: 21388-21395Crossref PubMed Scopus (445) Google Scholar, 6FranceLanord V. Brugg B. Michel P.P. Agid Y. Ruberg M. J. Neurochem. 1997; 69: 1612-1621Crossref PubMed Scopus (155) Google Scholar), and ceramide-induced cell death via disruption of mitochondrial functions (7Arora A.S. Jones B.J. Patel T.C. Bronk S.F. Gores G.J. Hepatology. 1997; 25: 958-963Crossref PubMed Scopus (149) Google Scholar) are lines of evidence of the strong influence of ceramide on mitochondria. Cytochrome c (cytoc) 1The abbreviations used are: cyto c , cytochrome c ; AA, antimycin A; Asc, ascorbate; TMPD, tetramethyl-1,4-phenylenediamine; C2-ceramide, N-acetylsphingosine; C6-ceramide, N-hexanoylsphingosine; DHC, C2-dihydroceramide; ΔΨ, mitochondrial transmembrane potential; PAGE, polyacrylamide gel electrophoresis.1The abbreviations used are: cyto c , cytochrome c ; AA, antimycin A; Asc, ascorbate; TMPD, tetramethyl-1,4-phenylenediamine; C2-ceramide, N-acetylsphingosine; C6-ceramide, N-hexanoylsphingosine; DHC, C2-dihydroceramide; ΔΨ, mitochondrial transmembrane potential; PAGE, polyacrylamide gel electrophoresis. plays a dual role in cell homeostasis. As a part of the respiratory chain, it is needed for cell life, and as one of the triggers of apoptosis, it is needed for cell death. It is now well accepted that many apoptogenic factors induce cell death via mitochondrial cyto c release (8Kluck R.M. Bossy Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 5303: 1132-1136Crossref Scopus (4266) Google Scholar). The released cytochrome switches on the death machinery, for example, by activation of caspases (9Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4448) Google Scholar, 10Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6199) Google Scholar). The anti-apoptoic protein, Bcl-2, was shown to prevent apoptosis both upstream (8Kluck R.M. Bossy Wetzel E. Green D.R. Newmeyer D.D. Science. 1997; 5303: 1132-1136Crossref Scopus (4266) Google Scholar) and downstream (11Zhivotovsky B. Orrenius S. Brustugun O.T. Doskeland S.O. Nature. 1998; 391: 469Crossref Scopus (271) Google Scholar) of cyto c release. In the present study we show that 1) C2-ceramide (N-acetylsphingosine), C6-ceramide (N-hexanoylsphingosine), and, to a much lesser extent, DHC (C2-dihydroceramide) release cyto c from isolated mitochondria, 2) ceramide-induced cyto c release occurs when cyto c is oxidized but not when it is reduced, 3) this release is prevented by Bcl-2, 4) cyto c release causes a decrease in mitochondrial oxygen consumption, transmembrane potential (ΔΨ), and Ca2+ retention, all of which are prevented by preincubation of mitochondria with Bcl-2 and reversed by addition of cyto c, and 5) ceramide interacts with cytoc and changes its reducibility. C2- and C6-ceramide were obtained from Alexis Biochemicals (Läufelfingen, Switzerland), DHC from Calbiochem (Luzern, Switzerland), horse heart cytochrome cfrom Sigma, mouse monoclonal cyto c antibody from RDI (Flanders, NJ), anti-mouse Ig and horseradish peroxidase from Amersham and His6-human Bcl-2 from Novartis (Basel, Switzerland). Ceramide stock solutions were prepared at a 500 times concentration in ethanol (containing 1% Me2SO) and kept at −20 °C. The vehicle always served as control. Isolation of rat liver mitochondria was performed by differential centrifugation as described (12Lötscher H.R. Winterhalter K.H. Carafoli E. Richter C. J. Biol. Chem. 1980; 255: 9325-9330Abstract Full Text PDF PubMed Google Scholar). The protein content of mitochondria and the mitochondrial supernatants were determined by the Biuret method with bovine serum albumin as standard. Freshly isolated mitochondria (10 mg protein/ml) were incubated at room temperature in 0.1 m HEPES buffer, pH 7.0, containing aprotinin, pepstatin A, and leupeptin (1.5 μg/ml each). To investigate the effect of ceramide when cyto c is oxidized, mitochondrial respiratory chain complex III was blocked by 50 nm antimycin A (AA), and after 1 min, ceramide (20 μm) or the vehicle was added. Mitochondria were incubated for 2 min and then energized with 1 mm ascorbate (Asc) plus 0.4 mmtetramethyl-1,4-phenylenediamine (TMPD) (Asc/TMPD). The effect of ceramide on mitochondria when cyto c is reduced was studied by addition of ceramide 1 min after Asc/TMPD. Bcl-2 (40 nm) was added 5 min before AA. After 10 min of incubation at room temperature, mitochondria were spun at 12,000 × g for 10 min at 4 °C, and the resulting supernatant was spun at 100,000 × g for 15 min at 4 °C. The supernatant of the second centrifugation was used for the detection of cytoc either spectrophotometrically or by gel electrophoresis. Spectrophotometric measurements were done in a Varian Cray spectrophotometer. As the blank sample, 10 mg of mitochondrial protein was diluted in 1 ml of the buffer, mixed gently, and spun immediately in 2 steps as mentioned above, and the supernatant was considered as the blank. The absorption of the supernatants of mitochondria incubated with ceramide or the vehicle was then recorded before and after reduction with sodium dithionite. The concentration of cytoc was calculated using an extinction coefficient of 19.0 mm−1 cm−1 for reduced cytoc at 550–540 nm (13Dutton P.L. Petty K.M. Bonner H.S. Morse S.D. Biochim. Biophys. Acta. 1975; 387: 536-556Crossref PubMed Scopus (212) Google Scholar) and is expressed as the percentage of the control. For gel analysis, 20 μl of the supernatant of mitochondria incubated with ceramide or the vehicle was separated by 15% SDS-PAGE and stained with Coomassie Blue. For Western blotting, 20 μg of protein of supernatants were separated by 15% SDS-PAGE, blotted onto a nitrocellulose membrane, probed by a monoclonal mouse anti-cyto c antibody, and developed by enhanced chemiluminescence. For studying the possible influence of ΔΨ on mitochondrial cytoc release, ΔΨ was fully abrogated by incubation of mitochondria with 5 μm rotenone, 50 nm AA, and 1 μm carbonyl cyanidem-chlorophenylhydrazone. The absence of ΔΨ was verified as explained below. After 10 min, mitochondria were spun as described above, and the supernatant was tested for the presence of cytoc by Western blotting. For studying mitochondrial functions, 1 mg of mitochondrial protein/ml was incubated in 0.1 m HEPES buffer, pH 7.0, with the following compounds present where appropriate: 10–20 μm ceramide, 50 nm AA to block complex III, Asc/TMPD to reduce cytoc, 1 mm KCN to block complex IV, 1 mm ATP as the substrate of ATPase, 1 μmcarbonyl cyanide m-chlorophenylhydrazone to uncouple mitochondria, and 1.7 μg/ml oligomycin to block ATPase. Ceramide was added when cyto c was oxidized, i.e. after AA, or when it was reduced, i.e., after Asc/TMPD. Bcl-2 was added 5 min prior to the above mentioned compounds. Oxygen consumption was measured at room temperature with a Clark-type oxygen electrode (Yellow Spring Instruments, Yellow Spring, OH) under continuous stirring. Mitochondrial transmembrane potential was measured in an Aminco DW-2A spectrophotometer at 511–533 nm in the presence of 10 μm safranin as described (14Schweizer M. Richter C. Biochemistry. 1996; 35: 4524-4528Crossref PubMed Scopus (68) Google Scholar). Mitochondrial Ca2+ uptake and release was measured in 0.1 mHEPES buffer, pH 7.0, containing 10 μm CaCl2(10 nmol of Ca2+/mg of mitochondrial protein) at 685–675 nm in the presence of 50 μm Arsenazo III as described (15Frei B. Winterhalter K.H. Richter C. J. Biol. Chem. 1985; 260: 7394-7401Abstract Full Text PDF PubMed Google Scholar). The optical density of a 10 μm oxidized horse heart cyto c solution in the presence of 10 μm ceramide or the vehicle was recorded between 380 and 600 nm at 1-nm intervals with a Varian Cray spectrophotometer. Oxidized cyto c was then reduced stepwise by the repeated addition of 2 μm ascorbate (taken from a 2 mm freshly prepared stock solution) to the cuvette. After each reduction step, the optical density was again recorded. The recorded optical density of the oxidized cyto c in each wavelength was subtracted from the corresponding one of the reduced cyto c and plotted against the wavelengths. Ceramide-induced mitochondrial cyto c release was determined by SDS-PAGE (Fig.1 A), by Western blotting (Fig.1 B), and spectrophotometrically (Fig. 1 C). In these experiments, ceramide was added when cyto c was mainly oxidized, i.e. after blocking the complex III. Addition of ceramide to mitochondria when cyto c was mainly reduced,i.e., in the presence of Asc/TMPD, did not increase the released cyto c, compared to the control (Fig.1 D). Preincubation of mitochondria with 40 nmBcl-2 (4 pmol of Bcl-2/mg of mitochondrial protein) fully prevented the release of cyto c induced by ceramide (see Fig.1 B). When equal volumes (20 μl) of the supernatants of mitochondria incubated with ceramides were separated by SDS-PAGE, an increase in the total protein amount released into the supernatant was detected (Fig. 1 A). When equal amounts (20 μg) of the released proteins were analyzed by Western blot, a specific increase in the cyto c was found (Fig. 1 B). To investigate the consequences of cyto c release on mitochondrial functions, we measured mitochondrial oxygen consumption, ΔΨ, and Ca2+ homeostasis. Fig.2 A shows that addition of ceramide to mitochondria when cyto c was oxidized decreased the oxygen consumption supported by Asc/TMPD. Conversely, ceramide added to mitochondria when cyto c was reduced did not change the oxygen consumption (Fig. 2 B). The decreased oxygen consumption caused by ceramide was prevented by preincubation of mitochondria with 40 nm Bcl-2 (Fig. 2 C) and was reversed by the addition of 200 nm exogenous cytoc (200 pmol of cyto c/mg of mitochondrial protein) (Fig. 2 D). A decrease in ΔΨ is considered important when cells commit suicide (16Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (705) Google Scholar). Fig. 3 A shows that addition of ceramide to mitochondria when cyto c was oxidized caused a decrease in ΔΨ. This figure also shows that addition of 200 nm exogenous cyto c resulted in a full gain of ΔΨ. Incubation of mitochondria with 40 nm Bcl-2 prevented the loss of ΔΨ caused by ceramide (not shown). Fig. 3 B shows that ceramide did not alter ΔΨ when cyto c was mainly reduced. When ΔΨ was built up as a consequence of ATP hydrolysis instead of respiration, ceramide did not change ΔΨ, neither when cyto c was oxidized nor when it was reduced (not shown). Mitochondria are important calcium buffers in eukaryotic cells, and mitochondrial calcium release is involved in apoptosis (17Kruman I. Guo Q. Mattson M.P. J. Neurosci. Res. 1998; 51: 293-308Crossref PubMed Scopus (343) Google Scholar). Fig.4 shows that C6-ceramide, added to mitochondria when cyto c was mainly oxidized, caused a decrease in Ca2+ retention by mitochondria, in a Bcl-2 sensitive manner. The same results were obtained with C2-ceramide and, to a minor extent, with DHC (not shown). Addition of ceramide to mitochondria when cyto c was reduced did not change the mitochondrial Ca2+ homeostasis (not shown). Reduction of cyto c can be followed photometrically. Fig.5 shows that in the presence of C2-ceramide, the reduction of cyto c by Asc was hampered, as evidenced by the smaller increase in the optical densities of the γ- and α-regions. Again, C2-ceramide was most effective, followed by C6-ceramide and DHC (not shown). The present study shows that ceramide induces cyto crelease from isolated mitochondria, an event strongly influenced by the redox state of cyto c, and that incubation of mitochondria with Bcl-2 prevents the cyto c release. Release of cytoc decreases mitochondrial oxygen consumption, ΔΨ, and the Ca2+ buffering capacity of mitochondria, all of which are reversed by addition of exogenous cyto c. For all parameters measured, the observed rank order of potency is C2 > C6 > DHC. This study also provides evidence for a possible direct interaction of ceramide and cytoc. There is growing evidence that mitochondria are involved in apoptosis (16Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (705) Google Scholar). The release of cyto c from mitochondria triggers apoptosis (18Rossé T. Olivier R. Monney L. Rager M. Conus S. Fellay I. Jansen B. Borner C. Nature. 1998; 391: 496-499Crossref PubMed Scopus (793) Google Scholar), and ΔΨ decreases during apoptosis (16Mignotte B. Vayssiere J.L. Eur. J. Biochem. 1998; 252: 1-15Crossref PubMed Scopus (705) Google Scholar, 19Shidoji Y. Nakamura N. Moriwaki H. Muto Y. Biochem. Biophys. Res. Commun. 1997; 230: 58-63Crossref PubMed Scopus (56) Google Scholar, 20Inai Y. Yabuki M. Kanno T. Akiyama J. Yasuda T. Utsumi K. Cell Struct. Func. 1997; 22: 555-563Crossref PubMed Scopus (78) Google Scholar, 21Wadia J.S. ChalmersRedman R.M.E. Ju W.J.H. Carlile G.W. Phillips J.L. Fraser A.D. Tatton W.G. J. Neurosci. 1998; 18: 932-947Crossref PubMed Google Scholar). Accordingly, prevention of cyto c release (22Macho A. Hirsch T. Marzo I. Marchetti P. Dallaporta B. Susin S.A. Zamzami N. Kroemer G. J. Immunol. 1998; 158: 4612-4619Google Scholar) as well as stabilization of ΔΨ (23Hennet T. Bertoni G. Richter C. Peterhans E. Cancer Res. 1993; 53: 1456-1460PubMed Google Scholar) prevent apoptosis. Furthermore, mitochondria carry the pro- and antiapoptotic proteins, cytoc and Bcl-2. The presence of procaspase-3 in mitochondria was also shown recently (24Mancine M. Nicholson D.W. Roy S. Thornberry N.A. Peterson E.P. Casciola-Rosen L.A. Rosen A. J. Cell Biol. 1998; 140: 1485-1495Crossref PubMed Scopus (372) Google Scholar). Several mediators, pathways, and factors are involved in apoptosis (25Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6179) Google Scholar,26Hermann S. Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2425) Google Scholar). Among them, ceramide has been shown to directly target mitochondria. Zhang et al. (27Zhang P. Liu B. Kang S.W. Seo M.S. Rhee S.G. Obeid L.M. J. Biol. Chem. 1997; 272: 30615-30618Crossref PubMed Scopus (336) Google Scholar) showed that in Molt-4 leukemic cells, 6 h of incubation with C6-ceramide increased the cytosolic concentration of cyto c, which was preventable by overexpression of Bcl-2. In a study by Amarante-Mendeset al. (28Amarante-Mendes G.P. Naekyung Kim C. Liu L. Huang Y. Perkins C.L. Green D.R. Bhalla K. Blood. 1998; 91: 1700-1705Crossref PubMed Google Scholar) 6 h of incubation with C2-ceramide caused the cytosolic accumulation of cytoc in control but not in Bcr-Abl-overexpressing HL-60 cells. In the present study, we show that ceramide directly causes the release of cyto c from isolated mitochondria and accordingly hypothesize that cyto c is the prime mitochondrial target of ceramide. The fact that incubation of mitochondria with a low concentration of Bcl-2, used in this study, prevents ceramide-induced cyto c release and its consequences suggests a specific function of the oncoprotein in ceramide-mediated apoptotic signals. To investigate a possible direct interaction of ceramide with cytoc, we primed Sepharose columns with the biologically actived-C2-ceramide or biologically inactivel-C2-ceramide. 2P. Ghafourifar, C. Richter, and J. Brunner, unpublished observation.We observed that d-C2-ceramide but notl-C2-ceramide columns selectively retained cytoc and that the retained protein could be eluted withd-C2-ceramide. We also observed that reduced cyto c had a much lower affinity tod-C2-ceramide column, compared to the oxidized cyto c. Because of that observation together with the fact that ceramide affects the reducibility of cyto c (Fig. 5), we hypothesize that ceramide may directly interact with cytoc, with a higher affinity for the oxidized protein, and that this interaction changes the physicochemical properties of cytoc, leading to its rejection from mitochondria. The fact that cyto c has multiple lipid binding sites and that the lipid-bound cyto c shows a lower affinity for attachment to the artificial membranes (29Subramanian M. Jutila A. Kinnunen P.K.J. Biochemistry. 1998; 37: 1394-1402Crossref PubMed Scopus (43) Google Scholar) strengthens this hypothesis. Under our experimental conditions, prevention of mitochondrial cytoc reduction is paralleled by the disappearance of ΔΨ. To distinguish which of these two is decisive for ceramide-induced cytoc release, we investigated by Western blot analysis whether cyto c is released because of ΔΨ collapse. We found that the absence of ΔΨ, achieved by blocking respiration and uncoupling of mitochondria, does not result in cyto c release, indicating that binding of cyto c to the outer side of the inner mitochondrial membrane does not require ΔΨ. These results also indicate that the release of cyto c by ceramide is not a consequence of a nonspecific solubilization of mitochondrial membranes due to the lipophilicity of ceramide, but rather is a specific event. The release of other mitochondrial proteins apart from cytoc is also increased upon treatment with ceramide (Fig.1 A). We argue that this apparently nonspecific protein release by ceramide is due to a general weakening of mitochondria upon cyto c release. Fig. 1 D supports this argument by showing that when ceramide does not cause cyto c loss, other mitochondrial proteins are also not released. This notion, together with the fact that addition of exogenous cyto c reverses all the altered mitochondrial functions, leads us to conclude that mitochondrial cyto c is a prime target for ceramide. This conclusion does not rule out other possibilities, for example, the modification of Bcl-2 by ceramide. In contrast to the finding that binding of cyto c to the inner mitochondrial membrane is not a function of ΔΨ, the formation and the maintenance of ΔΨ is dependent on the presence of a functional cyto c (cf. Fig. 3 A). Disruption of ΔΨ was shown to be involved in ceramide-induced apoptosis (30De-Maria R. Lenti L. Malisan F. d'Agostino F. Tomassini B. Zeuner A. Rippo M.R. Testi R. Science. 1997; 277: 1652-1655Crossref PubMed Scopus (374) Google Scholar). According to our study, cyto c has a critical role in ΔΨ formation, in that stabilization of cytoc stabilizes ΔΨ. Bcl-2 is located at the outer mitochondrial membrane (31Lithgow T. van Driel R. Bertram J.F. Strasser A. Cell Growth Differ. 1994; 4: 411-417Google Scholar) and has been shown to stabilize ΔΨ (23Hennet T. Bertoni G. Richter C. Peterhans E. Cancer Res. 1993; 53: 1456-1460PubMed Google Scholar). It is known that Bcl-xL, another oncoprotein with mitochondrial location, binds cyto c (32Kharbanda S. Pandey P. Schofield L. Israels S. Roncinske R. Yoshida K. Bharti A. Yuan Z. Saxena S. Weichselbaum R. Nalin C. Kufe D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942Crossref PubMed Scopus (368) Google Scholar). Based on the present results and the close proximity of Bcl-2 to mitochondrial cyto c, it may be speculated that Bcl-2 stabilizes ΔΨ by preventing cytoc loss. Nonspecific solute transport across the inner mitochondrial membrane, operated via a Bcl-2-sensitive megachannel (the “permeability transition pore”) is considered to be the reason for the collapse of ΔΨ and many other features of apoptosis (reviewed in Ref. 33Hirsch T. Marzo I. Kroemer G. Biosci. Rep. 1997; 17: 67-76Crossref PubMed Scopus (191) Google Scholar). It was also reported that operation of such a pore causes cytoc release, and thus it was concluded that cyto crelease occurs downstream to pore formation and ΔΨ collapse (34Yang J.C. Cortopassi G.A. Free Radical Biol. Med. 1998; 24: 624-631Crossref PubMed Scopus (198) Google Scholar). Because addition of cyto c reverses the ceramide-induced decrease in mitochondrial oxygen consumption, ΔΨ, and Ca2+-maintaining capacity (see Figs. Figure 2, Figure 3, Figure 4), we argue that ceramide-induced apoptosis is not mediated via opening a nonspecific megachannel or pore. The fact that ceramide does not affect ΔΨ when it is supported by ATP gives further support to this argument. It was reported by Gudz et al. (3Gudz T.I. Tserng K.Y. Hoppel C.L. J. Biol. Chem. 1997; 272: 24154-24158Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) that ceramide decreases the activity of complex III of the mitochondrial respiratory chain, as deduced from the measurement of mitochondrial oxygen consumption supported by Asc/TMPD. Cyto c shuttles electrons between complex III and IV, and oxygen is consumed at the level of complex IV. Figs. 2 D and 3 A show that the addition of cytoc to mitochondria recovered the decreased respiration and the reduced ΔΨ. Therefore, we conclude that parts of the ceramide-induced reduction of complex III activity is due to cytoc loss. Garcia-Ruiz et al. (4Garcia-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar) showed that ceramide induces superoxide radical formation in isolated rat liver mitochondria. From the elegant recent study by Cai and Jones (35Cai J. Jones D.P. J. Biol. Chem. 1998; 273: 11401-11404Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar), it is evident that superoxide formation by mitochondria is a consequence, and not the cause, of cyto c loss. Release of cyto c from mitochondria produces a gap in the hierarchically arranged mitochondrial respiratory complexes III and IV, and therefore a site for electron leakage. Present finding explains the ceramide-induced superoxide formation, reported by Garcia-Ruiz et al. (4Garcia-Ruiz C. Colell A. Mari M. Morales A. Fernandez-Checa J. J. Biol. Chem. 1997; 272: 11369-11377Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar), and supports the hypothesis that cyto c is the prime mitochondrial site of action of ceramide. We reported (23Hennet T. Bertoni G. Richter C. Peterhans E. Cancer Res. 1993; 53: 1456-1460PubMed Google Scholar) that TNF-α added to cells induces a drop in ΔΨ that is accompanied by increased reactive oxygen species formation in mitochondria and that both events are prevented in Bcl-2-overexpressing cells. It is known that de novo ceramide synthesis is part of the transmembrane signaling transduction mechanism of TNF-α receptor (36Kuroki J. Hirokawa M. Kitabayashi A. Lee M. Horiuchi T. Kawabata Y. Miura A.B. Leukemia. 1996; 10: 1950-1958PubMed Google Scholar, 37Dbaibo G.S. Perry D.K. Gamard C.J. Platt R. Poirier G.G. Obeid L.M. Hannun Y.A. J. Exp. Med. 1997; 185: 481-490Crossref PubMed Scopus (208) Google Scholar, 38Higuchi M. Singh S. Jaffrezou J.P. Aggarwal B.B. J. Immunol. 1996; 157: 297-304PubMed Google Scholar, 39Kitajima I. Soejima Y. Takasaki I. Beppu H. Tokioka T. Maruyama I. Bone. 1996; 19: 263-270Crossref PubMed Scopus (116) Google Scholar, 40Bezombes C. Maestre N. Laurent G. Levade T. Bettaieb A. Jaffrezou J.P. FASEB J. 1998; 12: 101-109PubMed Google Scholar). It was also reported that stimulation of TNF-α receptors causes apoptotic cell death by releasing mitochondrial cytoc (41Tang D.G. Li L. Zhu Z.Y. Joshi B. Biochem. Biophys. Res. Commun. 1998; 242: 380-384Crossref PubMed Scopus (116) Google Scholar). The present study makes the link: after de novo ceramide synthesis upon TNF-α receptor stimulation, mitochondrial cyto c is released, ΔΨ is decreased, and reactive oxygen species formation is favored. Accordingly, overexpression of Bcl-2 prevents the apoptogenic properties of TNF-α (37Dbaibo G.S. Perry D.K. Gamard C.J. Platt R. Poirier G.G. Obeid L.M. Hannun Y.A. J. Exp. Med. 1997; 185: 481-490Crossref PubMed Scopus (208) Google Scholar) by prevention of ceramide-induced cyto c release. It was reported by Hampton et al. (42Hampton M.B. Zhivotovsky B. Slater A.F.G. Burgess D.H. Orrenius S. Biochem. J. 1998; 329: 95-99Crossref PubMed Scopus (114) Google Scholar) that cytoc does not necessarily need to be reduced to activate caspases. The investigators acknowledged, however, that they were unable to keep cyto c oxidized in the presence of cytosolic extracts. In the present study, we show that oxidized cyto cis released by ceramide (Fig. 1, A–C) and that ceramide decreases the reducibility of oxidized cyto c (Fig. 5). Regarding the fact that both ceramide and cyto c are strong mediators of caspase-induced apoptosis (43Tepper A.D. Boesen-de-Cock J.G.R. de Vries E. Borst J. van Blitterswijk W.J. J. Biol. Chem. 1997; 272: 24308-24312Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), the possible interaction of ceramide and cyto c may thus amplify the progression of the apoptotic cascade. We thank Dr. Wolfgang Blodig for very useful discussions, Dr. Theresa Visarius for critically reading the manuscript, Novartis Pharma AG for providing Bcl-2, and Dr. Kaspar H. Winterhalter for his interest and support. d- andl-ceramide Sepharose affinity chromatography columns were a generous gift of Dr. Joseph Brunner, Federal Institute of Technology (Zurich, Switzerland).

Hypoxia induces profound changes in the cellular gene expression profile. The discovery of a major transcription factor family activated by hypoxia, HIF (hypoxia-inducible factor), and the factors that contribute to HIF regulation have greatly enhanced our knowledge of the molecular aspects of the hypoxic response. However, in addition to HIF, other transcription factors and cellular pathways are activated by exposure to reduced oxygen. In the present review, we summarize the current knowledge of how additional hypoxia-responsive transcription factors integrate with HIF and how other cellular pathways such as chromatin remodelling, translation regulation and microRNA induction, contribute to the co-ordinated cellular response observed following hypoxic stress.

The p53 and NF-κB transcription factor families are important, multifunctional regulators of the cellular response to stress. Here we have investigated the regulatory mechanisms controlling p53-dependent cell cycle arrest and cross talk with NF-κB. Upon induction of p53 in H1299 or U-2 OS cells, we observed specific repression of cyclin D1 promoter activity, correlating with a decrease in cyclin D1 protein and mRNA levels. This repression was dependent on the proximal NF-κB binding site of the cyclin D1 promoter, which has been shown to bind the p52 NF-κB subunit. p53 inhibited the expression of Bcl-3 protein, a member of the IκB family that functions as a transcriptional coactivator for p52 NF-κB and also reduced p52/Bcl-3 complex levels. Concomitant with this, p53 induced a significant increase in the association of p52 and histone deacetylase 1 (HDAC1). Importantly, p53-mediated suppression of the cyclin D1 promoter was reversed by coexpression of Bcl-3 and inhibition of p52 or deacetylase activity. p53 therefore induces a transcriptional switch in which p52/Bcl-3 activator complexes are replaced by p52/HDAC1 repressor complexes, resulting in active repression of cyclin D1 transcription. These results reveal a unique mechanism by which p53 regulates NF-κB function and cell cycle progression.

Oxygen is both an environmental and developmental signal that governs important cellular pathways. Therefore, hypoxia (or low oxygen tensions) is part of both physiological and pathological processes. To deal with hypoxic conditions, cells and organisms have evolved exquisite mechanisms for adaptation and survival. The cellular responses are reliant on controlled transcriptional and post-transcriptional events, where certain genes are positively regulated and others either remain inactive or are actively repressed. It has been known for some time that, during hypoxia, transcription is mainly regulated by the hypoxia inducible factor (HIF). However, recently it has been demonstrated that additional transcription factors are also activated and that non-HIF-dependent processes are involved in the hypoxic stress response. Therefore, gene expression following hypoxia is the result of combined effects on transcription, translation and adjustment mechanisms such as the induction of microRNAs and changes in chromatin.

NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease.

<h2>Abstract</h2> One mechanism by which a cell affords protection from the transforming effects of oncogenes is via the action of the tumor suppressor, ARF, which activates p53 by inactivating Mdm2. Many oncogenes have also been shown to activate the transcription factor NF-κB, which can contribute toward the malignant phenotype in many ways, including an ability to antagonize p53. Here we find that ARF inhibits NF-κB function and its antiapoptotic activity independent of Mdm2 and p53. ARF represses the transcriptional activation domain of the NF-κB family member RelA by inducing its association with the histone deacetylase, HDAC1. Further, we show that the response of NF-κB to the oncogene Bcr-Abl is determined by the ARF status of the cell. These results reveal an important function of ARF that can regulate the NF-κB response to oncogene activation.

The ARF tumour suppressor is a central component of the cellular defence against oncogene activation. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF-kappaB subunit. Here we demonstrate that ARF induces the ATR- and Chk1-dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF-dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF-induced sensitivity to tumour necrosis factor alpha-induced cell death. Significantly, ATR activity is also required for ARF-induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF-kappaB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor.

Mammals have developed evolutionarily conserved programs of transcriptional response to hypoxia and inflammation. These stimuli commonly occur together in vivo and there is significant crosstalk between the transcription factors that are classically understood to respond to either hypoxia or inflammation. This crosstalk can be used to modulate the overall response to environmental stress. Several common disease processes are characterised by aberrant transcriptional programs in response to environmental stress. In this review, we discuss the current understanding of the role of the hypoxia-responsive (hypoxia-inducible factor) and inflammatory (nuclear factor-κB) transcription factor families and their crosstalk in rheumatoid arthritis, inflammatory bowel disease and colorectal cancer, with relevance for future therapies for the management of these conditions.

Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1β mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1β result in modulation of HIF-2α protein. HIF-1β overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1β (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.

As Nuclear Factor-κB (NF-κB) is a major transcription factor responding to cellular stress, it is perhaps not surprising that is activated by hypoxia, or decreased oxygen availability. However, how NF-κB becomes activated in hypoxia is still not completely understood. Several mechanisms have been proposed and this review will focus on the main findings highlighting the molecules that have been identified in the process of hypoxia induced NF-κB. In addition, we will discuss the role of NF-κB in the control of the cellular response to hypoxia.

Hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs) are α-ketoglutarate (αKG)-dependent dioxygenases that function as cellular oxygen sensors. However, PHD activity also depends on factors other than oxygen, especially αKG, a key metabolic compound closely linked to amino-acid metabolism. We examined the connection between amino-acid availability and PHD activity. We found that amino-acid starvation leads to αKG depletion and to PHD inactivation but not to HIF stabilization. Furthermore, pharmacologic or genetic inhibition of PHDs induced autophagy and prevented mammalian target of rapamycin complex 1 (mTORC1) activation by amino acids in a HIF-independent manner. Therefore, PHDs sense not only oxygen but also respond to amino acids, constituting a broad intracellular nutrient-sensing network.

Changes in the availability or demand for oxygen induce dramatic changes at the cellular level. Primarily, activation of a family of oxygen labile transcription factors, Hypoxia Inducible Factor (HIF), initiates a variety of cellular processes required to re-instate oxygen homeostasis. Oxygen is sensed by molecular dioxygenases in cells, such as the prolyl-hydroxylases (PHDs), enzymes which are responsible for the oxygen-dependent regulation of HIF. As HIF is a transcription factor it must bind DNA sequences of its target genes possibly in the context of a complex chromatin structure. How chromatin structure changes in response to hypoxia is currently unknown. However, the identification of a novel class of histone demethylases as true dioxygenases suggests that chromatin can act as an oxygen sensor and plays an active role in the coordination of the cellular response to hypoxia. This review will discuss the current knowledge on how hypoxia engages with different proteins involved in chromatin organisation and dynamics.

The von Hippel-Lindau tumor suppressor protein is the substrate binding subunit of the VHL E3 ubiquitin ligase, which targets hydroxylated α subunit of hypoxia inducible factors (HIFs) for ubiquitination and subsequent proteasomal degradation. VHL is a potential target for treating anemia and ischemic diseases, motivating the development of inhibitors of the VHL:HIF-α protein-protein interaction. Additionally, bifunctional proteolysis targeting chimeras (PROTACs) containing a VHL ligand can hijack the E3 ligase activity to induce degradation of target proteins. We report the structure-guided design and group-based optimization of a series of VHL inhibitors with low nanomolar potencies and improved cellular permeability. Structure-activity relationships led to the discovery of potent inhibitors 10 and chemical probe VH298, with dissociation constants <100 nM, which induced marked HIF-1α intracellular stabilization. Our study provides new chemical tools to probe the VHL-HIF pathways and new VHL ligands for next-generation PROTACs.

Within the genomes of metazoans, nucleosomes are highly organised adjacent to the binding sites for a subset of transcription factors. Here we have sought to investigate which chromatin remodelling enzymes are responsible for this. We find that the ATP-dependent chromatin remodelling enzyme SNF2H plays a major role organising arrays of nucleosomes adjacent to the binding sites for the architectural transcription factor CTCF sites and acts to promote CTCF binding. At many other factor binding sites SNF2H and the related enzyme SNF2L contribute to nucleosome organisation. The action of SNF2H at CTCF sites is functionally important as depletion of CTCF or SNF2H affects transcription of a common group of genes. This suggests that chromatin remodelling ATPase’s most closely related to the Drosophila ISWI protein contribute to the function of many human gene regulatory elements.

Hypoxia' or decreases in oxygen availability' results in the activation of a number of different responses at both the whole organism and the cellular level. These responses include drastic changes in gene expression, which allow the organism (or cell) to cope efficiently with the stresses associated with the hypoxic insult. A major breakthrough in the understanding of the cellular response to hypoxia was the discovery of a hypoxia sensitive family of transcription factors known as the hypoxia inducible factors (HIFs). The hypoxia response mounted by the HIFs promotes cell survival and energy conservation. As such, this response has to deal with important cellular process such as cell division. In this review, the integration of oxygen sensing with the cell cycle will be discussed. HIFs, as well as other components of the hypoxia pathway, can influence cell cycle progression. The role of HIF and the cell molecular oxygen sensors in the control of the cell cycle will be reviewed.

Abstract Hypoxia and inflammation are intimately linked. It is known that NF-κB regulates the HIF system but little is known about how HIF regulates NF-κB. Here, we show that HIF-1α represses NF-κB dependent gene expression. HIF-1α depletion results in increased NF-κB transcriptional activity both in mammalian cells and in the model organism Drosophila melanogaster. HIF-1α depletion enhanced the NF-κB response and this required not only the TAK-IKK complex, but also CDK6. Loss of HIF-1α results in an increased angiogenic response in mammalian cancer cells and increased mortality in Drosophila following infection. These results indicate that HIF-1α is required to restrain the NF-κB response, and thus prevents excessive and damaging pro-inflammatory responses.

Responding appropriately to changes in oxygen availability is essential for multicellular organism survival. Molecularly, cells have evolved intricate gene expression programmes to handle this stressful condition. Although it is appreciated that gene expression is co-ordinated by changes in transcription and translation in hypoxia, much less is known about how chromatin changes allow for transcription to take place. The missing link between co-ordinating chromatin structure and the hypoxia-induced transcriptional programme could be in the form of a class of dioxygenases called JmjC (Jumonji C) enzymes, the majority of which are histone demethylases. In the present review, we will focus on the function of JmjC histone demethylases, and how these could act as oxygen sensors for chromatin in hypoxia. The current knowledge concerning the role of JmjC histone demethylases in the process of organism development and human disease will also be reviewed.

The importance of oxygen for the survival of multicellular and aerobic organisms is well established and documented. Over the years, increased knowledge of its use for bioenergetics has placed oxygen at the centre of research on mitochondria and ATP‐generating processes. Understanding the molecular mechanisms governing cellular oxygen sensing and response has allowed for the discovery of novel pathways oxygen is involved in, culminating with the award of the Nobel Prize for Medicine and Physiology in 2019 to the pioneers of this field, Greg Semenza, Peter Ratcliffe and William Kaelin. However, it is now beginning to be appreciated that oxygen can be a signalling molecule involved in a vast array of molecular processes, most of which impinge on gene expression control. This review will focus on the knowns and unknowns of oxygen as a signalling molecule, highlighting the role of 2‐oxoglutarate‐dependent dioxygenases as central players in the cellular response to deviations in oxygen tension.

The p52/p100 nuclear factor kappa B (NF-kappaB) subunit (NF-kappaB2) is aberrantly expressed in many tumour types and has been implicated as a regulator of cell proliferation. Here, we demonstrate that endogenous p52 is a direct regulator of Cyclin D1 expression. However, stimulation of Cyclin D1 expression alone cannot account for all the cell cycle effects of p52/p100 and we also find that p52 represses expression of the Cyclin-dependent kinase inhibitor p21(WAF/CIP1). Significantly, this latter effect is dependent upon basal levels of the tumour suppressor p53. By contrast, p52 cooperates with p53 to regulate other known p53 target genes such as PUMA, DR5, Gadd45alpha and Chk1. p52 associates directly with these p53-regulated promoters where it regulates coactivator and corepressor binding. Moreover, recruitment of p52 is p53 dependent and does not require p52-DNA-binding activity. These results reveal a complex role for p52 as regulator of cell proliferation and p53 transcriptional activity. Furthermore, they imply that in some cell types, p52 can regulate p53 function and influence p53-regulated decision-making following DNA damage and oncogene activation.

Hypoxia induces a variety of cellular responses such as cell cycle arrest, apoptosis, and autophagy. Most of these responses are mediated by the hypoxia-inducible factor-1alpha. To induce target genes, hypoxia-inducible factor-1alpha requires a chromatin environment conducive to allow binding to specific sequences. Here, we have studied the role of the chromatin-remodeling complex SWI/SNF in the cellular response to hypoxia. We find that SWI/SNF is required for several of the cellular responses induced by hypoxia. Surprisingly, hypoxia-inducible factor-1alpha is a direct target of the SWI/SNF chromatin-remodeling complex. SWI/SNF components are found associated with the hypoxia-inducible factor-1alpha promoter and modulation of SWI/SNF levels results in pronounced changes in hypoxia-inducible factor-1alpha expression and its ability to transactivate target genes. Furthermore, impairment of SWI/SNF function renders cells resistant to hypoxia-induced cell cycle arrest. These results reveal a previously uncharacterized dependence of hypoxia signaling on the SWI/SNF complex and demonstrate a new level of control over the hypoxia-inducible factor-1alpha system.

Fbw7 is a ubiquitin-ligase that targets several oncoproteins for proteolysis, but the full range of Fbw7 substrates is not known. Here we show that by performing quantitative proteomics combined with degron motif searches, we effectively screened for a more complete set of Fbw7 targets. We identify 89 putative Fbw7 substrates, including several disease-associated proteins. The transcription factor NF-κB2 (p100/p52) is one of the candidate Fbw7 substrates. We show that Fbw7 interacts with p100 via a conserved degron and that it promotes degradation of p100 in a GSK3β phosphorylation-dependent manner. Fbw7 inactivation increases p100 levels, which in the presence of NF-κB pathway stimuli, leads to increased p52 levels and activity. Accordingly, the apoptotic threshold can be increased by loss of Fbw7 in a p100-dependent manner. In conclusion, Fbw7-mediated destruction of p100 is a regulatory component restricting the response to NF-κB2 pathway stimulation.

Abstract The transcription factor HIF‐1α is essential for cells to rapidly adapt to low oxygen levels (hypoxia). HIF‐1α is frequently deregulated in cancer and correlates with poor patient prognosis. Here, we demonstrate that the deubiquitinase Cezanne regulates HIF‐1α homeostasis. Loss of Cezanne decreases HIF‐1α target gene expression due to a reduction in HIF‐1α protein levels. Surprisingly, although the Cezanne‐regulated degradation of HIF‐1α depends on the tumour suppressor pVHL , hydroxylase and proteasome activity are dispensable. Our data suggest that Cezanne is essential for HIF‐1α protein stability and that loss of Cezanne stimulates HIF‐1α degradation via proteasome‐independent routes, possibly through chaperone‐mediated autophagy.

PHD1 belongs to the family of prolyl-4-hydroxylases (PHDs) that is responsible for posttranslational modification of prolines on specific target proteins. Because PHD activity is sensitive to oxygen levels and certain byproducts of the tricarboxylic acid cycle, PHDs act as sensors of the cell's metabolic state. Here, we identify PHD1 as a critical molecular link between oxygen sensing and cell-cycle control. We show that PHD1 function is required for centrosome duplication and maturation through modification of the critical centrosome component Cep192. Importantly, PHD1 is also required for primary cilia formation. Cep192 is hydroxylated by PHD1 on proline residue 1717. This hydroxylation is required for binding of the E3 ubiquitin ligase SCF(Skp2), which ubiquitinates Cep192, targeting it for proteasomal degradation. By modulating Cep192 levels, PHD1 thereby affects the processes of centriole duplication and centrosome maturation and contributes to the regulation of cell-cycle progression.

Reduced oxygen availability (hypoxia) can act as a signalling cue in physiological processes such as development, but also in pathological conditions such as cancer or ischaemic disease. As such, understanding how cells and organisms respond to hypoxia is of great importance. The family of transcription factors called Hypoxia Inducible Factors (HIFs) co-ordinate a transcriptional programme required for survival and adaptation to hypoxia. However, the effects of HIF on chromatin accessibility are currently unclear. Here, using genome wide mapping of chromatin accessibility via ATAC-seq, we find hypoxia induces loci specific changes in chromatin accessibility are enriched at a subset hypoxia transcriptionally responsive genes, agreeing with previous data using other models. We show for the first time that hypoxia inducible changes in chromatin accessibility across the genome are predominantly HIF dependent, rapidly reversible upon reoxygenation and partially mimicked by HIF-α stabilisation independent of molecular dioxygenase inhibition. This work demonstrates that HIF is central to chromatin accessibility alterations in hypoxia, and has implications for our understanding of gene expression regulation by hypoxia and HIF.

Abstract The RelA (p65) nuclear factor-κB (NF-κB) subunit can contribute towards tumor cell survival through inducing the expression of a variety of antiapoptotic genes. However, the NF-κB response can show great diversity and is not always antiapoptotic. Here, we find that cisplatin, a DNA cross-linking agent and commonly used anticancer compound, does not affect RelA nuclear translocation but modulates its transcriptional activity. Similar to other genotoxic agents, such as daunorubicin and UV light, cisplatin treatment in the U-2 OS osteosarcoma cell line represses RelA activity and inhibits expression of the NF-κB antiapoptotic target gene Bcl-xL. The mechanism through which cisplatin achieves these effects is different to daunorubicin and UV light but shows great similarity to the RelA regulatory pathway induced by the ARF tumor suppressor: cisplatin regulation of RelA requires ATR/Chk1 activity, represses Bcl-xL but not XIAP expression, and results in phosphorylation of RelA at Thr505. In contrast to these results, another chemotherapeutic drug etoposide activates NF-κB and induces expression of these target genes. Thus, within a single tumor cell line, there is great heterogeneity in the NF-κB response to different, commonly used chemotherapeutic drugs. These observations suggest that it might be possible to minimize the ability of RelA to inhibit cancer therapy by diagnostically predicting the type of chemotherapeutic drug most compatible with NF-κB functionality in a tumor cell type. Moreover, our data indicate that at least with respect to RelA, cisplatin functions as an ARF mimic. Other drugs capable of mimicking this aspect of ARF function might therefore have therapeutic potential. (Cancer Res 2006; 66(2): 929-35)

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3'-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling.

Hypoxia or reduced oxygen availability has been studied extensively for its ability to activate specific genes. Hypoxia-induced gene expression is mediated by the HIF transcription factors, but not exclusively so. Despite the extensive knowledge about how hypoxia activates genes, much less is known about how hypoxia promotes gene repression. In this review, we discuss the potential mechanisms underlying hypoxia-induced transcriptional repression responses. We highlight HIF-dependent and independent mechanisms as well as the potential roles of dioxygenases with functions at the nucleosome and DNA level. Lastly, we discuss recent evidence regarding the involvement of transcriptional repressor complexes in hypoxia.

The cell cycle is an important cellular process whereby the cell attempts to replicate its genome in an error-free manner. As such, mechanisms must exist for the cell cycle to respond to stress signals such as those elicited by hypoxia or reduced oxygen availability. This review focuses on the role of transcriptional and post-transcriptional mechanisms initiated in hypoxia that interface with cell cycle control. In addition, we discuss how the cell cycle can alter the hypoxia response. Overall, the cellular response to hypoxia and the cell cycle are linked through a variety of mechanisms, allowing cells to respond to hypoxia in a manner that ensures survival and minimal errors throughout cell division.

The oxygen-dependent regulation of hypoxia-inducible factors is more complex than previously appreciated.

Hypoxia-reduction in oxygen availability-plays key roles in both physiological and pathological processes. Given the importance of oxygen for cell and organism viability, mechanisms to sense and respond to hypoxia are in place. A variety of enzymes utilise molecular oxygen, but of particular importance to oxygen sensing are the 2-oxoglutarate (2-OG) dependent dioxygenases (2-OGDs). Of these, Prolyl-hydroxylases have long been recognised to control the levels and function of Hypoxia Inducible Factor (HIF), a master transcriptional regulator in hypoxia, via their hydroxylase activity. However, recent studies are revealing that dioxygenases are involved in almost all aspects of gene regulation, including chromatin organisation, transcription and translation. We highlight the relevance of HIF and 2-OGDs in the control of gene expression in response to hypoxia and their relevance to human biology and health.

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

The IKK [inhibitor of NF-κB (nuclear factor κB) kinase] complex has an essential role in the activation of the family of NF-κB transcription factors in response to a variety of stimuli. To identify novel IKK-interacting proteins, we performed an unbiased proteomics screen where we identified TfR1 (transferrin receptor 1). TfR1 is required for transferrin binding and internalization and ultimately for iron homoeostasis. TfR1 depletion does not lead to changes in IKK subunit protein levels; however, it does reduce the formation of the IKK complex, and inhibits TNFα (tumour necrosis factor α)-induced NF-κB-dependent transcription. We find that, in the absence of TfR1, NF-κB does not translocate to the nucleus efficiently, and there is a reduction in the binding to target gene promoters and consequentially less target gene activation. Significantly, depletion of TfR1 results in an increase in apoptosis in response to TNFα treatment, which is rescued by elevating the levels of RelA/NF-κB. Taken together, these results indicate a new function for TfR1 in the control of IKK and NF-κB. Our data indicate that IKK-NF-κB responds to changes in iron within the cell.

The ARF tumor suppressor is a central component of the cellular defense against oncogene activation in mammals. p14ARF activates p53 by binding and inhibiting HDM2, resulting, inter alia, in increased transcription and expression of the cyclin-dependent kinase inhibitor p21 and consequent cell-cycle arrest. We analyzed the effect of p14ARF induction on nucleolar protein dynamics using SILAC mass spectrometry and have identified the human Formin-2 (FMN2) protein as a component of the p14ARF tumor suppressor pathway. We show that FMN2 is increased upon p14ARF induction at both the mRNA and the protein level via a NF-κB-dependent mechanism that is independent of p53. FMN2 enhances expression of the cell-cycle inhibitor p21 by preventing its degradation. FMN2 is also induced by activation of other oncogenes, hypoxia, and DNA damage. These results identify FMN2 as a crucial component in the regulation of p21 and consequent oncogene/stress-induced cell-cycle arrest in human cells.

The SIN3A-HDAC complex deacetylates histones thereby repressing gene transcription. Here we describe family with sequence similarity 60A (FAM60A), a cell cycle-regulated protein that binds to the SIN3-HDAC complex. FAM60A expression peaks during G(1) and S phases of the cell cycle in U2OS cells, in a manner similar to the G(1) regulator cyclin D1, which is a known target of SIN3-HDAC. In this light we found that FAM60A binds to SIN3-HDAC-regulated promoters such as cyclin D1 in G(1) and S phases. Cells depleted of FAM60A show increased histone acetylation at the cyclin D1 promoter and elevated levels of cyclin D1 mRNA and protein. Furthermore, depletion of FAM60A altered the periodic association of HDAC1 with the cyclin D1 promoter, increased cyclin D1 expression at all cell cycle phases, and caused premature S phase entry. The data in this study introduce FAM60A as a novel regulator of SIN3-HDAC function and gene expression.

Hypoxia is not only a developmental cue but also a stress and pathological stimulus in many human diseases. The response to hypoxia at the cellular level relies on the activity of the transcription factor family, hypoxia inducible factor (HIF). HIF-1 is responsible for the acute response and transactivates a variety of genes involved in cellular metabolism, cell death, and cell growth. Here, we show that hypoxia results in increased mRNA levels for human lysine (K)-specific demethylase 2 (KDM2) family members, KDM2A and KDM2B, and also for Drosophila melanogaster KDM2, a histone and protein demethylase. In human cells, KDM2 family member's mRNA levels are regulated by HIF-1 but not HIF-2 in hypoxia. Interestingly, only KDM2A protein levels are significantly induced in a HIF-1-dependent manner, while KDM2B protein changes in a cell type-dependent manner. Importantly, we demonstrate that in human cells, KDM2A regulation by hypoxia and HIF-1 occurs at the level of promoter, with HIF-1 binding to the KDM2A promoter being required for RNA polymerase II recruitment. Taken together, these results demonstrate that KDM2 is a novel HIF target that can help coordinate the cellular response to hypoxia. In addition, these results might explain why KDM2 levels are often deregulated in human cancers.

COPD is a heterogeneous disease with multiple clinical phenotypes. COPD endotypes can be determined by different expressions of hypoxia-inducible factors (HIFs), which, in combination with individual susceptibility and environmental factors, may cause predominant airway or vascular changes in the lung. The pulmonary vascular phenotype is relatively rare among COPD patients and characterised by out-of-proportion pulmonary hypertension (PH) and low diffusing capacity of the lung for carbon monoxide, but only mild-to-moderate airway obstruction. Its histologic feature, severe remodelling of the small pulmonary arteries, can be mediated by HIF-2 overexpression in experimental PH models. HIF-2 is not only involved in the vascular remodelling but also in the parenchyma destruction. Endothelial cells from human emphysema lungs express reduced HIF-2α levels, and the deletion of pulmonary endothelial Hif-2α leads to emphysema in mice. This means that both upregulation and downregulation of HIF-2 have adverse effects and that HIF-2 may represent a molecular "switch" between the development of the vascular and airway phenotypes in COPD. The mechanisms of HIF-2 dysregulation in the lung are only partly understood. HIF-2 levels may be controlled by NAD(P)H oxidases via iron- and redox-dependent mechanisms. A better understanding of these mechanisms may lead to the development of new therapeutic targets.

Eukaryotic genomes are organised in a structure called chromatin, comprising of DNA and histone proteins. Chromatin is thus a fundamental regulator of gene expression, as it offers storage and protection but also controls accessibility to DNA. Sensing and responding to reductions in oxygen availability (hypoxia) have recognised importance in both physiological and pathological processes in multicellular organisms. One of the main mechanisms controlling these responses is control of gene expression. Recent findings in the field of hypoxia have highlighted how oxygen and chromatin are intricately linked. This review will focus on mechanisms controlling chromatin in hypoxia, including chromatin regulators such as histone modifications and chromatin remodellers. It will also highlight how these are integrated with hypoxia inducible factors and the knowledge gaps that persist.

Mistletoe extracts are used as alternative cancer treatment in addition to standard chemotherapy and radiation treatment and have an immunostimulatory and pain-relieving effect. A direct antitumour effect of mistletoe extracts against tumour cells of lymphoid origin has been linked to the D-galactoside-specific mistletoe lectin I. In this study, we investigated the cellular effect of bacterially expressed, recombinant mistletoe lectin alone or in combination with ionising radiation in a genetically defined p53-wild-type and p53-deficient E1A/ras-transformed murine tumour cells system. Downregulation of the proliferative activity and cell killing by recombinant mistletoe lectin occurred in a clear dose response (0.1-1 ng ml(-1)). Induction of apoptosis was p53-independent, but apoptosis-associated factor-1-dependent. Cellular treatment with lectin in combination with ionising radiation resulted in both p53-wild-type and p53-deficient tumour cells in an at least additive, antiproliferative effect and enhanced activation of caspase-3. Combined treatment with ionising radiation and lectin revealed a similar cytotoxic effect in human, p53-mutated adenocarcinoma cells. Thus, recombinant mistletoe lectin alone and in combination with ionising radiation bypasses often prevalent apoptotic deficiencies in treatment-resistant tumour cells.

The tumor suppressor adenomatous polyposis coli (APC) is mutated in the majority of colorectal cancers and is best known for its role as a scaffold in a Wnt-regulated protein complex that determines the availability of β-catenin. Another common feature of solid tumors is the presence of hypoxia as indicated by the up-regulation of hypoxia-inducible factors (HIFs) such as HIF-1α. Here, we demonstrate a novel link between APC and hypoxia and show that APC and HIF-1α antagonize each other. Hypoxia results in reduced levels of APC mRNA and protein via a HIF-1α-dependent mechanism. HIF-1α represses the APC gene via a functional hypoxia-responsive element on the APC promoter. In contrast, APC-mediated repression of HIF-1α requires wild-type APC, low levels of β-catenin, and nuclear factor-κB activity. These results reveal down-regulation of APC as a new mechanism that contributes to the survival advantage induced by hypoxia and also show that loss of APC mutations produces a survival advantage by mimicking hypoxic conditions.

The NF-κB family of transcription factors is a well-established regulator of the immune and inflammatory responses and also plays a key role in other cellular processes, including cell death, proliferation, and migration. Conserved residues in the trans-activation domain of RelA, which can be posttranslationally modified, regulate divergent NF-κB functions in response to different cellular stimuli. Using rela(-/-) mouse embryonic fibroblasts reconstituted with RelA, we find that mutation of the threonine 505 (T505) phospho site to alanine has wide-ranging effects on NF-κB function. These include previously described effects on chemotherapeutic drug-induced apoptosis, as well as new roles for this modification in autophagy, cell proliferation, and migration. This last effect was associated with alterations in the actin cytoskeleton and expression of cellular migration-associated genes such as WAVE3 and α-actinin 4. We also define a new component of cisplatin-induced, RelA T505-dependent apoptosis, involving induction of NOXA gene expression, an effect explained at least in part through induction of the p53 homologue, p73. Therefore, in contrast to other RelA phosphorylation events, which positively regulate NF-κB function, we identified RelA T505 phosphorylation as a negative regulator of its ability to induce diverse cellular processes such as apoptosis, autophagy, proliferation, and migration.

The hypoxia-inducible factor (HIF) is a master regulator of the cellular response to hypoxia. Its levels and activity are controlled by dioxygenases called prolyl-hydroxylases and factor inhibiting HIF (FIH). To activate genes, HIF has to access sequences in DNA that are integrated in chromatin. It is known that the chromatin-remodeling complex switch/sucrose nonfermentable (SWI/SNF) is essential for HIF activity. However, no additional information exists about the role of other chromatin-remodeling enzymes in hypoxia. Here we describe the role of imitation switch (ISWI) in the cellular response to hypoxia. We find that unlike SWI/SNF, ISWI depletion enhances HIF activity without altering its levels. Furthermore, ISWI knockdown only alters a subset of HIF target genes. Mechanistically, we find that ISWI is required for full expression of FIH mRNA and protein levels by changing RNA polymerase II loading to the FIH promoter. Of interest, exogenous FIH can rescue the ISWI-mediated upregulation of CA9 but not BNIP3, suggesting that FIH-independent mechanisms are also involved. Of importance, ISWI depletion alters the cellular response to hypoxia by reducing autophagy and increasing apoptosis. These results demonstrate a novel role for ISWI as a survival factor during the cellular response to hypoxia.

Hypoxia, or low oxygen availability, is an important physiological and pathological stimulus for multicellular organisms. Molecularly, hypoxia activates a transcriptional programme directed at restoration of oxygen homoeostasis and cellular survival. In mammalian cells, hypoxia not only activates the HIF (hypoxia-inducible factor) family, but also additional transcription factors such as NF-κB (nuclear factor κB). Here we show that hypoxia activates the IKK-NF-κB [IκB (inhibitor of nuclear factor κB)-NF-κB] pathway and the immune response in Drosophila melanogaster. We show that NF-κB activation is required for organism survival in hypoxia. Finally, we identify a role for the tumour suppressor Cyld, as a negative regulator of NF-κB in response to hypoxia in Drosophila. The results indicate that hypoxia activation of the IKK-NF-κB pathway and the immune response is an important and evolutionary conserved response.

Oxygen sensing is an essential feature of metazoan biology and reductions in oxygen availability (hypoxia) have both physiological and pathophysiological implications. Co-ordinated mechanisms have evolved for sensing and responding to hypoxia, which involve diverse biological outputs, with the main aim of restoring oxygen homeostasis. This includes a dynamic gene transcriptional response, the central drivers of which are the hypoxia-inducible factor (HIF) family of transcription factors. HIFs are regulated in an oxygen-dependent manner and while their role in hypoxia is well established, it is apparent that other key players are required for gene expression control in hypoxia. In this review, we highlight the current understanding of the known and potential molecular mechanisms underpinning gene transcriptional responses to hypoxia in mammals, with a focus on oxygen-dependent effects on chromatin structure.

Von Hippel-Lindau (VHL) disease is characterized by frequent mutation of VHL protein, a tumor suppressor that functions as the substrate recognition subunit of a Cullin2 RING E3 ligase complex (CRL2VHL). CRL2VHL plays important roles in oxygen sensing by targeting hypoxia-inducible factor-alpha (HIF-α) subunits for ubiquitination and degradation. VHL is also commonly hijacked by bifunctional molecules such as proteolysis-targeting chimeras to induce degradation of target molecules. We previously reported the design and characterization of VHL inhibitors VH032 and VH298 that block the VHL:HIF-α interaction, activate the HIF transcription factor, and induce a hypoxic response, which can be beneficial to treat anemia and mitochondrial diseases. How these compounds affect the global cellular proteome remains unknown. Here, we use unbiased quantitative MS to identify the proteomic changes elicited by the VHL inhibitor compared with hypoxia or the broad-spectrum prolyl-hydroxylase domain enzyme inhibitor IOX2. Our results demonstrate that VHL inhibitors selectively activate the HIF response similar to the changes induced in hypoxia and IOX2 treatment. Interestingly, VHL inhibitors were found to specifically upregulate VHL itself. Our analysis revealed that this occurs via protein stabilization of VHL isoforms and not via changes in transcript levels. Increased VHL levels upon VH298 treatment resulted in turn in reduced levels of HIF-1α protein. This work demonstrates the specificity of VHL inhibitors and reveals different antagonistic effects upon their acute versus prolonged treatment in cells. These findings suggest that therapeutic use of VHL inhibitors may not produce overt side effects from HIF stabilization as previously thought.

Abstract Oxygen is essential for viability in mammalian organisms. However, cells are often exposed to changes in oxygen availability, due to either increased demand or reduced oxygen supply, herein called hypoxia. To be able to survive and/or adapt to hypoxia, cells activate a variety of signalling cascades resulting in changes to chromatin, gene expression, metabolism and viability. Cellular signalling is often mediated via post‐translational modifications (PTMs), and this is no different in response to hypoxia. Many enzymes require oxygen for their activity and oxygen can directly influence several PTMS. Here, we review the direct impact of changes in oxygen availability on PTMs such as proline, asparagine, histidine and lysine hydroxylation, lysine and arginine methylation and cysteine dioxygenation, with a focus on mammalian systems. In addition, indirect hypoxia‐dependent effects on phosphorylation, ubiquitination and sumoylation will also be discussed. Direct and indirect oxygen‐regulated changes to PTMs are coordinated to achieve the cell's ultimate response to hypoxia. However, specific oxygen sensitivity and the functional relevance of some of the identified PTMs still require significant research.

Pifithrin-alpha is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-alpha in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-alpha provides a useful tool in the laboratory to investigate the function of p53 in model systems.While investigating the effects of pifithrin-alpha on the transcriptional activity of NF-kappaB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-alpha was observed both in vivo and in vitro. Pifithrin-alpha did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-alpha to active extracts inhibited this activity. Furthermore, pifithrin-alpha also inhibited recombinant firefly luciferase protein activity.Among its other biological activities, pifithrin-alpha is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.

The disclosure of information to patients about diagnosis and prognosis and other aspects of care is still a matter of debate. We have conducted a study in advanced cancer patients about their experiences and preferences concerning this issue. A questionnaire was developed and completed during the first appointment in the outpatient clinic of an oncology centre's palliative care unit, before the patient had any contact with the staff of the unit. The study was conducted on a convenience sample of 47 patients. We found that 34 (72%) of those patients thought they had been informed of their diagnosis, most of them by the hospital doctor. However, not all stated the diagnosis in a manner clearly showing that they were aware of the nature of their disease. Most patients were with a family member when the diagnosis was disclosed, which is what the majority had preferred. Of the 13 uninformed patients, only one preferred to remain uninformed. Most patients (89%) participated in decisions concerning treatment, although only 68% thought they should have participated. Some 39 patients (83%) were informed that they were being referred to the palliative care unit, but surprisingly only eight had received an explanation of the unit's function. We concluded that, although most patients had been informed of their disease, there remains many problems, the most important of which, in our view, is the difference between the information provided and the patients' needs.

Hypoxia inducible factor (HIF) is the major transcription factor involved in the regulation of the cellular response to hypoxia, or low oxygen tensions. Even though HIF-1 function is mostly studied following hypoxic stress, well oxygenated areas of several diseased tissues have detectable levels of this transcription factor. Therefore, it is surprising how little is known about the function of HIF in normoxia. This study seeks to fill this gap. Using transient HIF-1α knockdown, as well as, stable cell lines generated using short hairpin RNAs (shRNA), we have further characterized the role of HIF-1α in normoxia. Our data reveals that knockdown of HIF-1α results in a significant increase in cells in the G1 phase of the cell cycle. We find that HIF-1α depletion increases the protein and mRNA of both p21 and p27. p21 is induced via, at least in part, p53-independent but SP1-dependent mechanisms. Interestingly, HIF-1α knockdown also alters the cellular response to chemotherapeutic agents. These data have important implications in not only for the further understanding of HIF-1α, a major transcription factor, but also for the use of HIF-targeted and combination therapies in cancer treatment.

The cellular response to hypoxia relies on the activation of a specific transcriptional program. Although, most of the attention is focused on the transcription factor HIF, other transcription factors are also activated in hypoxia. We have recently described the mechanism for hypoxia induced NFκB. We have demonstrated the crucial dependency on the IKK complex as well as in the upstream IKK kinase TAK1. TAK1 and IKK activation is dependent upon the calcium calmodulin kinase, CaMK2 and requires Ubc13 as the E2 ubiquitin conjugation enzyme. We report a role for XIAP as the possible E3-ubiquitin ligase for this system. Interestingly, hypoxia induced IKK mediated phosphorylation of IκBα, does not lead to degradation. Hypoxia prevents IκBα de-sumoylation of Sumo-2/3 chains on critical lysine residues, normally required for K-48 linked polyubiquitination. Our results define a novel pathway regulating NFκB activation.

Hypoxia is very common in most solid tumours and is a driving force for malignant progression as well as radiotherapy and chemotherapy resistance. Incidences of head and neck squamous cell carcinoma (HNSCC) have increased in the last decade and radiotherapy is a major therapeutic technique utilised in the treatment of the tumours. However, effectiveness of radiotherapy is hindered by resistance mechanisms and most notably by hypoxia, leading to poor patient prognosis of HNSCC patients. The phenomenon of hypoxia-induced radioresistance was identified nearly half a century ago, yet despite this, little progress has been made in overcoming the physical lack of oxygen. Therefore, a more detailed understanding of the molecular mechanisms of hypoxia and the underpinning radiobiological response of tumours to this phenotype is much needed. In this review, we will provide an up-to-date overview of how hypoxia alters molecular and cellular processes contributing to radioresistance, particularly in the context of HNSCC, and what strategies have and could be explored to overcome hypoxia-induced radioresistance.

Abstract Acquired platinum resistance poses a significant therapeutic impediment to ovarian cancer patient care, accounting for more than 200,000 deaths annually worldwide. We previously identified that overexpression of the antioxidant superoxide dismutase 1 ( SOD1 ) in ovarian cancer is associated with a platinum-resistant phenotype via conferring oxidative stress resistance against platinum compounds. We further demonstrated that enzymatic inhibition using small-molecule inhibitors or silencing of SOD1 via RNA interference (RNAi) increased cisplatin sensitivity and potency in vitro. We launched this study to explore the potential therapeutic applications of SOD1 silencing in vivo in order to reverse cisplatin resistance using a graphene-based siRNA delivery platform. PEGylated graphene oxide (GO) polyethyleneimine (GO PEI -mPEG) nanoparticle was complexed with SOD1 siRNA. GO PEI -mPEG-siSOD1 exhibited high biocompatibility, siRNA loading capacity, and serum stability, and showed potent downregulation of SOD1 mRNA and protein levels. We further observed that cisplatin and PEI elicited mitochondrial dysfunction and transcriptionally activated the mitochondrial unfolded protein response (UPR mt ) used as a reporter for their respective cytotoxicities. SOD1 silencing was found to augment cisplatin-induced cytotoxicity resulting in considerable tumour growth inhibition in cisplatin-sensitive A2780 and cisplatin-resistant A2780 DDP subcutaneous mouse xenografts. Our study highlights the potential therapeutic applicability of RNAi-mediated targeting of SOD1 as a chemosensitizer for platinum-resistant ovarian cancers.

In response to replication stress, Claspin mediates the phosphorylation and activation of Chk1 by ATR. Claspin is not only necessary for the propagation of the DNA-damage signal, but its destruction by the ubiquitin-proteosome pathway is required to allow the cell to continue the cell cycle allowing checkpoint recovery. Here, we demonstrate that both the NF-kappaB family of transcription factors and their upstream kinase IKK can regulate Claspin levels by controlling its mRNA expression. Furthermore, we show that c-Rel directly controls Claspin gene transcription. Disruption of IKK and specific NF-kappaB members impairs ATR-mediated checkpoint function following DNA damage. Importantly, hyperactivation of IKK results in a failure to inactivate Chk1 and impairs the recovery from the DNA checkpoint. These results uncover a novel function for IKK and NF-kappaB modulating the DNA-damage checkpoint response, allowing the cell to integrate different signalling pathways with the DNA-damage response.

Cell cycle progression is an energy demanding process and requires fine-tuned metabolic regulation. Cells must overcome an energy restriction checkpoint before becoming committed to progress through the cell cycle. Aerobic organisms need oxygen for the metabolic conversion of nutrients into energy. As such, environmental oxygen is a critical signalling molecule regulating cell fate. The Hypoxia Inducible Factors (HIFs) are a family of transcription factors that respond to changes in environmental oxygen and cell energy and coordinate a transcriptional program which forms an important part of the cellular response to a hostile environment. A significant proportion of HIF-dependent transcriptional target genes, code for proteins that are involved in energy homeostasis. In this review we discuss the role of the HIF system in the regulation of energy homeostasis in response to changes in environmental oxygen and the impact on cell cycle control, and address the implications of the deregulation of this effect in cancer.

Both cancer and tumour-associated host cells are exposed to ionizing radiation when a tumour is subjected to radiotherapy. Macrophages frequently constitute the most abundant tumour-associated immune population, playing a role in tumour progression and response to therapy. The present work aimed to evaluate the importance of macrophage-cancer cell communication in the cellular response to radiation. To address this question, we established monocultures and indirect co-cultures of human monocyte-derived macrophages with RKO or SW1463 colorectal cancer cells, which exhibit higher and lower radiation sensitivity, respectively. Mono- and co-cultures were then irradiated with 5 cumulative doses, in a similar fractionated scheme to that used during cancer patients' treatment (2 Gy/fraction/day). Our results demonstrated that macrophages sensitize RKO to radiation-induced apoptosis, while protecting SW1463 cells. Additionally, the co-culture with macrophages increased the mRNA expression of metabolism- and survival-related genes more in SW1463 than in RKO. The presence of macrophages also upregulated glucose transporter 1 expression in irradiated SW1463, but not in RKO cells. In addition, the influence of cancer cells on the expression of pro- and anti-inflammatory macrophage markers, upon radiation exposure, was also evaluated. In the presence of RKO or SW1463, irradiated macrophages exhibit higher levels of pro-inflammatory TNF, IL6, CCL2 and CCR7, and of anti-inflammatory CCL18. However, RKO cells induce an increase of macrophage pro-inflammatory IL1B, while SW1463 cells promote higher pro-inflammatory CXCL8 and CD80, and also anti-inflammatory VCAN and IL10 levels. Thus, our data demonstrated that macrophages and cancer cells mutually influence their response to radiation. Notably, conditioned medium from irradiated co-cultures increased non-irradiated RKO cell migration and invasion and did not impact on angiogenesis in a chicken embryo chorioallantoic membrane assay. Overall, the establishment of primary human macrophage-cancer cell co-cultures revealed an intricate cell communication in response to ionizing radiation, which should be considered when developing therapies adjuvant to radiotherapy.

In many human hematologic and solid malignancies, intrinsic or acquired treatment resistance remains a major obstacle for successful cancer therapy. The molecular understanding of how tumor cells respond to chemotherapy and ionizing radiation is rapidly evolving. Induction of programmed cell death, apoptosis, is one important strategy for successful cancer therapy. This has been shown convincingly for oncogene-transformed normal cells as well as tumor cells of lymphoid origin. However, the relevance of apoptosis in solid human malignancies is less clear. Loss of apoptosis might be linked to specific mutations in the often tissue-specific apoptotic pathways due to aberrations in the stress-related signal transduction cascades. Restoration of a dysfunctional apoptotic program in cancer tissue where apoptosis has been identified as an important mechanism for tissue homeostasis is one rational approach for innovative cancer therapy. In this review, we focus on the relevance of the tumor suppressor p53 for apoptosis-induction and successful cancer therapy outlining the importance of an intact caspase machinery for apoptosis execution. Strategies are discussed to overcome treatment resistance and a high apoptotic threshold in human malignancies where apoptosis is the dominant mode of cell death and the status of p53 is an important determinant for apoptosis induction.

Oxygen sensing is important in physiology but also in disease. We find that hypoxia (oxygen deficiency) triggers rapid and hypoxia-inducible factor (HIF)-independent histone methylation changes which are reversible upon reoxygenation. Hypoxia-induced histone methylation genomic distribution precedes transcriptional changes and is mimicked by specific Jumonji-C (JmjC) histone demethylase depletion. Oxygen sensing by JmjC histone demethylases is required for the cellular response to hypoxia.

NF-κB signalling is crucial for cellular responses to inflammation but is also associated with the hypoxia response. NF-κB and hypoxia inducible factor (HIF) transcription factors possess an intense molecular crosstalk. Although it is known that HIF-1α modulates NF-κB transcriptional response, very little is understood regarding how HIF-1β contributes to NF-κB signalling. Here, we demonstrate that HIF-1β is required for full NF-κB activation in cells following canonical and non-canonical stimuli. We found that HIF-1β specifically controls TRAF6 expression in human cells but also in Drosophila melanogaster. HIF-1β binds to the TRAF6 gene and controls its expression independently of HIF-1α. Furthermore, exogenous TRAF6 expression is able to rescue all of the cellular phenotypes observed in the absence of HIF-1β. These results indicate that HIF-1β is an important regulator of NF-κB with consequences for homeostasis and human disease.

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here, we investigated how PBRM1 controls HIF-1α activity. We found that PBRM1 is required for HIF-1α transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1α mRNA translation, as absence of PBRM1 results in reduced actively translating HIF-1α mRNA. Interestingly, we found that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1α mRNA is m6A-modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1α mRNA and reduction of YTHDF2 results in reduced HIF-1α protein expression in cells. Our results identify a SWI/SNF-independent function for PBRM1, interacting with HIF-1α mRNA and the epitranscriptome machinery. Furthermore, our results suggest that the epitranscriptome-associated proteins play a role in the control of hypoxia signalling pathways.

Activation of the phosphatidylinositol 3'-kinase (PI3K)/Akt survival pathway protects against apoptotic stress stimuli. Therefore, compounds that down-regulate this pathway are of clinical interest for single and combined anticancer treatment modalities. Here we demonstrate that the cytotoxic effect of the protein kinase C (PKC)-inhibitor N-benzoylated staurosporine (PKC412) is mediated via the PI3K/Akt pathway. Dose-dependent down-regulation of the proliferative activity, activation of the apoptotic machinery, and cell killing by PKC412 (0-1 microM) in Rat1a-fibroblasts and H-ras-oncogene-transformed fibroblasts correlated with a decrease of Akt phosphorylation and a reduced phosphorylation of the endogenous Akt-substrate GSK3-alpha. Expression of the dominant-active myristoylated form of Akt abrogated this cytotoxic effect of PKC412. Experiments with Apaf-1-deficient cells revealed that PKC412-induced cytotoxicity depends on an intact apoptosome but that the decrease of Akt phosphorylation is not attributable to apoptosis execution. Comparative experiments indicate that PKC412 and the parent-compound staurosporine down-regulate this survival pathway upstream or at the level of Akt but by a different mechanism than the PI3K-inhibitor LY294002. Furthermore, inhibition of this pathway by PKC412 is relevant for sensitization to ionizing radiation. These results demonstrate the specific role of this signaling pathway for the PKC412-mediated down-regulation of an apoptotic threshold and its cytotoxicity.

The ARF tumor suppressor initiates the cellular response to aberrant oncogene activation through binding to and inhibiting the activity of Hdm2/Mdm2, the inhibitor of p53. However, many pathways also active in the cell will oppose p53 function if left unchecked. An example of this, is the RelA (p65) NF-kappaB subunit. Frequently activated by oncogenes, RelA is a potent inducer of anti-apoptotic gene expression, which has the potential to inhibit the pro-apoptotic functions of p53. We have recently discovered that by inducing the activity of the checkpoint kinases ATR and Chk1, ARF neutralises this opposing pathway. ARF-induced Chk1 phosphorylates RelA on threonine 505, a residue in its transactivation domain, thus inhibiting NF-kappaB's ability to stimulate anti-apoptotic gene expression. Furthermore, ARF-induced ATR is required for efficient induction and activation of p53. We propose that this pathway will target other proteins with pro-proliferative or anti-apoptotic functions. Therefore, through this mechanism, ARF can integrate the cellular response to an oncogene, thus maximising the effectiveness of the p53 tumor suppressor pathway.

Introduction Hypoxia-inducible factor is a key transcriptional factor involved in the cellular response to low levels of oxygen, hypoxia. Moreover, hypoxiainducible factor has been recently associated with a role in inflammation and immunity. Importantly, hypoxiainducible factor is regulated by the major inflammatory responsive transcription factor, nuclear factor-κB. These two major pathways have been intimately linked. On one hand, they share a number of common target genes; on the other hand, physical interactions between hypoxiainducible factor subunits and nuclear factor-κB have been observed. Even though the role of nuclear factor-κB over hypoxia-inducible factor is fairly well-known, the involvement of hypoxia-inducible factor over the nuclear factor-κB pathway is not. Given the overlap between these pathways, it would not be surprising to find a functional involvement of hypoxiainducible factor in processes where nuclear factor-κB is involved. In this review, we will describe the communalities between hypoxia-inducible factor and nuclear factor-κB pathways, highlighting the crosstalk that occurs in a variety of conditions. Conclusion Taken together all the communalities between hypoxia-inducible factor and nuclear factor-κB pathways, there is no doubt that a crosstalk occurs, which can potentially bring new insights for therapeutic intervention in situations of disease such as cancer, stroke or rheumatoid arthritis. Introduction Oxygen is essential for multicellular organisms. As such, being able to respond to variations in oxygen availability is a requirement for the survival and homoeostasis of the organism. Sensing and responding appropriately to oxygen changes is important for a variety of important physiological processes, which include high altitude living, intense exercise and embryo development. However, lowering of the oxygen concentration or availability (hypoxia) is part and/or contributes to a number of human pathologies, such as cancer, stroke/infarction, diabetes and ageing1–2. Understanding the molecular mechanisms controlling the cellular response to hypoxia is, therefore, of great importance. One master regulator of the cellular response to oxygen changes is the family of transcription factors, hypoxia-inducible factor (HIF). However, HIF activity has been associated with additional stimuli that do not involve changes in oxygen, such as infection and inflammation3. These findings led to the discovery that HIF is controlled by a transcription factor, mostly involved in immune responses, nuclear factorκB (NF-κB). In this review, we will highlight the shared features of these transcription factors, from activating stimulus to common targets. Discussion The authors have referenced some of their own studies in this review. The protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. HIF pathway At the molecular level, the cellular response to hypoxia relies on HIF. HIF was first identified in 1995 together with hypoxia response element (HRE, 5ʹ-RCGTG-3ʹ) of the erythropoietin gene (EPO). Further studies revealed that HIF is actually a heterodimeric complex comprising an αand a β-subunit, which exist as a series of isoforms: -1α, -2α, and 3α. HIF-1α is constitutively expressed, while HIF-2α and HIF-3α expression is restricted to a subset of tissues. Even though HIF-1β expression and protein are not dependent on oxygen changes, HIF-α subunits are extremely labile at normal oxygen levels. This occurs mostly at the protein level, with HIF-α half-life being very short (~ 5 min), while transcription changes in response to oxygen have not been widely reported thus far. The activity of the complex HIF-1α– HIF-1β is determined by the stabilisation of the α subunit during hypoxia. In the presence of oxygen (normoxia), HIF-α is regulated by a class of dioxygenases called prolyl hydroxylases (PHDs), of which four isoforms have been identified so far (PHD1, PHD2, PHD3 and PHD4). These proteins use iron, 2-oxoglutarate, ascorbate and molecular oxygen as co-factors to catalyse the hydroxylation of HIF-α. The hydroxylation of specific prolyl residues promotes the interaction of HIF with von Hippel-Lindauprotein (VHL) containing E3 ligase complex, which mediates proteasomalmediated * Corresponding author Email: s.rocha@dundee.ac.uk Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow street, DD1 5EH, United Kingdom

Most colorectal cancers have mutations in the tumor suppressor APC. The best-understood function of APC is its participation in a protein complex that regulates the availability of β-catenin. Solid tumors are characterized by the presence of hypoxia as well as inflammation, which leads to the upregulation of Hypoxia Inducible Factors like HIF-1α. We recently demonstrated a novel antagonistic link between APC and HIF-1α. We found that hypoxia results in reduced levels of APC mRNA and protein via a direct HIF-1α-dependent mechanism. Similarly, APC mediates the repression of HIF-1α. However, this requires wild-type APC, low levels of β-catenin and NFκB activity. These results reveal the downregulation of APC as a novel mechanism that contributes to the survival advantage induced by hypoxia and cytokines such as TNFα. Our data indicate that loss-of-function mutations in APC result in the engagement of the hypoxia response. Importantly, this suggests that other stimuli that induce HIF, such as inflammatory cytokines and oncogenes, alter APC function.

Non-canonical NF-κB signalling plays important roles in the development and function of the immune system but it also is deregulated in a number of inflammatory diseases. Although, NF-κB and HIF crosstalk has been documented, this has only been described following canonical NF-κB stimulation, involving RelA/p50 and the HIF-1 dimer. Here, we report that the non-canonical inducer TNFSF14/LIGHT leads to HIF induction and activation in cancer cells. We demonstrate that only HIF-2α is induced at the transcriptional level following non-canonical NF-κB activation, via a mechanism that is dependent on the p52 subunit. Furthermore, we demonstrate that p52 can bind to the HIF-2α promoter in cells. These results indicate that non-canonical NF-κB can lead to HIF signalling implicating HIF-2α as one of the downstream effectors of this pathway in cells.

Hypoxia is an important developmental cue for multicellular organisms but it is also a contributing factor for several human pathologies, such as stroke, cardiovascular diseases and cancer. In cells, hypoxia activates a major transcriptional program coordinated by the Hypoxia Inducible Factor (HIF) family. HIF can activate more than one hundred targets but not all of them are activated at the same time, and there is considerable cell type variability. In this report we identified the paired-like homeodomain pituitary transcription factor (PITX1), as a transcription factor that helps promote specificity in HIF-1α dependent target gene activation. Mechanistically, PITX1 associates with HIF-1β and it is important for the induction of certain HIF-1 dependent genes but not all. In particular, PITX1 controls the HIF-1α-dependent expression of the histone demethylases; JMJD2B, JMJD2A, JMJD2C and JMJD1B. Functionally, PITX1 is required for the survival and proliferation responses in hypoxia, as PITX1 depleted cells have higher levels of apoptotic markers and reduced proliferation. Overall, our study identified PITX1 as a key specificity factor in HIF-1α dependent responses, suggesting PITX1 as a protein to target in hypoxic cancers.

Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insight into how cells detect, and respond to low oxygen is crucial to understanding the role of hypoxia in disease. Central to the hypoxic response is rapid changes in the expression of genes essential to carry out a wide range of functions to adapt the cell/tissue to decreased oxygen availability. These changes in gene expression are co-ordinated by specialised transcription factors, changes to chromatin architecture and intricate balances between protein synthesis and destruction that together establish changes to the cellular proteome. In this article, we will discuss the advances of our understanding of the cellular oxygen sensing machinery achieved through the application of 'omics-based experimental approaches.

<h2>Abstract</h2> Radiotherapy is a widely used treatment modality against cancer, and although survival rates are increasing, radioresistant properties of tumours remain a significant barrier for curative treatment. Tumour hypoxia is one of the main contributors to radioresistance and is common in most solid tumours. Hypoxia is responsible for many molecular changes within the cell which helps tumours to survive under such challenging conditions. These hypoxia-induced molecular changes are predominantly coordinated by the hypoxia inducible factor (HIF) and have been linked with the ability to confer resistance to radiation-induced cell death. To overcome this obstacle research has been directed towards autophagy, a cellular process involved in self degradation and recycling of macromolecules, as HIF plays a large role in its coordination under hypoxic conditions. The role that autophagy has following radiotherapy treatment is conflicted with evidence of both cytoprotective and cytotoxic effects. This literature review aims to explore the intricate relationship between radiotherapy, hypoxia, and autophagy in the context of cancer treatment. It provides valuable insights into the potential of targeting autophagy as a therapeutic strategy to improve the response of hypoxic tumours to radiotherapy.

Neoadjuvant radiotherapy is successfully used in rectal cancer to improve overall survival. However, treatment response is both unpredictable and variable. There is strong evidence to show that the phenomenon of tumour hypoxia is associated with radioresistance, however the mechanism(s) behind this are poorly understood. Consequently, there have only been a small number of studies evaluating methods targeting hypoxia-induced radioresistance. The purpose of this systematic review is to evaluate the potential effectiveness of targeting hypoxia-induced radioresistance in rectal cancer and provide recommendations for future research in this area.A comprehensive literature search was performed following the PRISMA guidelines. This study was registered on the Prospero database (CRD42023441983).Eight articles met the inclusion criteria. All studies identified were in vitro or in vivo studies, there were no clinical trials. Of the 8 studies identified, 5 assessed the efficacy of drugs which directly or indirectly targeted hypoxia and three that identified potential targets. There was conflicting in vivo evidence for the use of metformin to overcome hypoxia induced radioresistance. Vorinostat, atovaquone, and evofosfamide showed promising preclinical evidence that they can overcome hypoxia-induced radioresistance.The importance of investigating hypoxia-induced radioresistance in rectal cancer is crucial. However, to date, only a small number of preclinical studies exist evaluating this phenomenon. This systematic review highlights the importance of further research to fully understand the mechanism behind this radioresistance. There are promising targets identified in this systematic review however, substantially more pre-clinical and clinical research as a priority for future research is needed.

Three new aurantiactinomyxon types are described from the oligochaete Ilyodrilus templetoni (Southern, 1909) (Naididae) collected from a northern Portuguese estuary, based on light microscopy and sequencing of the 18S rDNA. The addition of I. templetoni to the group of freshwater annelids known to be permissive for aurantiactinomyxon development reinforces the crucial role of naidids in the evolution and settlement of myxozoans in estuarine environments. Maximum likelihood and Bayesian inference analyses of a comprehensive 18S rDNA dataset placed the novel types within the Paramyxidium clade. This positioning suggests them as probable life cycle counterparts to Paramyxidium spp. that most likely infect the European eel Anguilla anguilla, as the sole representative of Elopomorpha in Portuguese rivers. Although distance estimation revealed a genetic difference of only 0.4 % between Aurantiactinomyxon types 1 and 3, this value was determined to be representative of interspecific variability based on the consistent matching of both genotypes with distinct actinospore morphologies, and potential richness of closely related species of Paramyxidium infecting the European eel in Portuguese waters. The clustering of aurantiactinomyxon types within distinct myxosporean lineages, representative of the suborders Variisporina and Platysporina, demonstrates that the aurantiactinomyxon morphotype is highly functional in promoting myxozoan infections in estuarine environments.

Abstract Prostate cancer (PCa) is one of the leading causes of cancer morbidity and mortality in men. Metastasis is the main cause of PCa‐associated death. Recent evidence indicated a significant reduction in PCa mortality associated with higher ω‐3 polyunsaturated fatty acids (PUFAs) consumption. However, the underlying mechanisms remained elusive. In this study, we applied global acetylome profiling to study the effect of fatty acids treatment. Results indicated that oleic acid (OA, monounsaturated fatty acid, MUFA, 100 µM) elevates while EPA (eicosapentaenoic acid, 100 µM) reduces the acetyl‐CoA level, which alters the global acetylome. After treatment, two crucial cell motility regulators, PFN1 and FLNA, were found with altered acetylation levels. OA increased the acetylation of PFN1 and FLNA, whereas EPA decreased PFN1 acetylation level. Furthermore, OA promotes while EPA inhibits PCa migration and invasion. Immunofluorescence assay indicated that EPA impedes the formation of lamellipodia or filopodia through reduced localization of PFN1 and FLNA to the leading edge of cells. Therefore, perturbed acetylome may be one critical step in fatty acid‐affected cancer cell motility. This study provides some new insights into the response of ω‐3 PUFAs treatment and a better understanding of cancer cell migration and invasion modulation.

Investigating how cells and organisms sense and respond to O2 levels is essential to our understanding of physiology and pathology. This field has advanced considerably since the discovery of the major transcription factor family, hypoxia-inducible factor (HIF), and the enzymes that control its levels: prolyl hydroxylases (PHDs). However, with its expansion, new complexities have emerged. Herein we highlight three main areas where, in our opinion, the research community could direct some of their attention. These include non-transcriptional roles of HIFs, specificity and O2 sensitivity of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), and new tools and methods to detect O2 concentrations in cells and organs. A greater understanding of these areas would answer big questions and help drive our knowledge of cellular responses to hypoxia forward.

The cellular response to ionizing radiation is governed by the DNA-damage recognition process but is also modulated by cytoplasmic signal transduction cascades that are part of the cellular stress response. Growth-promoting protein kinase C activity antagonizes irradiation-induced cell death, and, therefore, protein kinase C inhibitors might be potent radiosensitizers. The antiproliferative and radiosensitizing effect of the novel N-benzoylated staurosporine analogue PKC412 was tested in vitro against genetically defined p53-wild type (+/+) and p53-deficient (-/-) murine fibrosarcoma cells and in vivo against radioresistant p53-/- murine fibrosarcoma and human colon adenocarcinoma tumor xenograft (SW480, p53-mutated). PKC412 sensitized both p53+/+ and p53-/- tumor cells in vitro and in vivo for treatment with ionizing radiation but with a different mechanism of radiosensitization depending on the p53 status. In p53+/+, cells combined treatment with PKC412 and ionizing radiation drastically induced apoptotic cell death, whereas no apoptosis induction could be observed in p53-deficient cells in vitro and in histological tumor sections. Combined treatment resulted in an increased G2 cell cycle distribution in p53-/- cells at PKC412 concentrations that did not alter cell cycle distribution when applied alone. In vivo, a minimal treatment regimen during 4 consecutive days of PKC412 (4 x 100 mg/kg) in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumor growth delay for both p53-disfunctional tumor xenografts and showed that the clinically relevant protein kinase C inhibitor PKC412 is a promising new radiosensitizer with a potentially broad therapeutic window.

PHD1 (also known as EGLN2) belongs to a family of prolyl hydroxylases (PHDs) that are involved in the control of the cellular response to hypoxia. PHD1 is also able to regulate mitotic progression through the regulation of the crucial centrosomal protein Cep192, establishing a link between the oxygen-sensing and the cell cycle machinery. Here, we demonstrate that PHD1 is phosphorylated by CDK2, CDK4 and CDK6 at S130. This phosphorylation fluctuates with the cell cycle and can be induced through oncogenic activation. Functionally, PHD1 phosphorylation leads to increased induction of hypoxia-inducible factor (HIF) protein levels and activity during hypoxia. PHD1 phosphorylation does not alter its intrinsic enzymatic activity, but instead decreases the interaction between PHD1 and HIF1α. Interestingly, although phosphorylation of PHD1 at S130 lowers its activity towards HIF1α, this modification increases the activity of PHD1 towards Cep192. These results establish a mechanism by which cell cycle mediators, such as CDKs, temporally control the activity of PHD1, directly altering the regulation of HIF1α and Cep192.

<ns4:p><ns4:bold>Background:</ns4:bold>Hypoxia-inducible factor (HIF) transcription factors are well known to control the transcriptional response to hypoxia. Given the importance of cellular response to hypoxia, a number of pharmacological agents to interfere with this pathway have been developed and entered pre-clinical or clinical trial phases. However, how similar or divergent the transcriptional response elicited by different points of interference in cells is currently unknown.</ns4:p><ns4:p><ns4:bold>Methods:</ns4:bold>We performed RNA-sequencing to analyse the similarities and differences of transcriptional response in HeLa cells treated with hypoxia or chemical agents that stabilise HIF by inhibiting components of the hypoxia signalling pathway – prolyl hydroxylase (PHD) inhibitor or von Hippel–Lindau (VHL) inhibitor.</ns4:p><ns4:p><ns4:bold>Results:</ns4:bold>This analysis revealed that hypoxia produces the highest changes in gene transcription, with activation and repression of genes being in large numbers. Treatment with the PHD inhibitor IOX2 or the VHL inhibitor VH032 led mostly to gene activation, majorly via a HIF-dependent manner. These results were also confirmed by qRT-PCR using more specific and/or efficient inhibitors, FG-4592 (PHDs) and VH298 (VHL).</ns4:p><ns4:p><ns4:bold>Conclusion:</ns4:bold>PHD inhibition and VHL inhibition mimic gene activation promoted by hypoxia via a HIF-dependent manner. However, gene repression is mostly associated with the hypoxia response and not common to the response elicited by inhibitors of the pathway.</ns4:p>

The forkhead associated (FHA) domain-containing protein Smad nuclear interacting protein 1 (SNIP1) has multiple cellular functions, including the ability to interact with DNA-binding transcription factors and transcriptional coactivators. Moreover, we have demonstrated previously that SNIP1 regulates cyclin D1 expression and promoter activity. Here, we identify a new function for SNIP1 as a regulator of ATR checkpoint kinase-dependent pathways in human U-2 OS osteosarcoma cells: SNIP1 is required for p53 induction in response to ultraviolet light treatment and selectively regulates the phosphorylation of known ATR target proteins, including p53, Chk1 and the histone variant H2AX. These activities are independent of its ability to regulate cyclin D1 expression. Significantly, SNIP1 is also required for ATR-dependent functions of the human p14(ARF) tumour suppressor, including its ability to modulate the activity of the RelA(p65) NF-kappaB subunit. This, together with its other described functions, suggests that SNIP1 could have an important role during tumorigenesis and cancer therapy.

We have identified the human FMN2 gene as a novel target regulated by induction of p14ARF and by multiple other stress responses, including DNA damage and hypoxia, which have in common activation of cell cycle arrest. We showed that increased expression of the FMN2 gene following p14ARF induction is caused, at the transcriptional level, by relief of repression by RelA and E2F1, which, under non-induced conditions, bind the FMN2 promoter. Increased FMN2 protein levels promote cell cycle arrest by inhibiting the degradation of p21, and our data show that control of p21 stability is a key part of the mechanism that regulates p21 induction. Consistent with this model, we have shown that transient expression of exogenous FMN2 protein alone is sufficient to increase p21 protein levels in cells, without altering p21 mRNA levels. Here, we provide additional evidence for the role of the N terminus of FMN2 as being the important domain required for p21 stability. In addition, we also investigate the role of RelA's threonine 505 residue in the control of FMN2. Our results identify FMN2 as a crucial protein involved in the control of p21.

The SIN3A–HDAC (histone deacetylase) complex is a master transcriptional repressor, required for development but often deregulated in disease. Here, we report that the recently identified new component of this complex, SINHCAF (SIN3A and HDAC-associated factor)/FAM60A (family of homology 60A), links the SIN3A–HDAC co-repressor complex function to the hypoxia response. We show that SINHCAF specifically represses HIF-2α mRNA and protein expression, via its interaction with the transcription factor SP1 (specificity protein 1) and recruitment of HDAC1 to the HIF-2α promoter. SINHCAF control over HIF-2α results in functional cellular changes in in vitro angiogenesis and viability. Our analysis reveals an unexpected link between SINHCAF and the regulation of the hypoxia response.

O loxoscelismo cutâneo é um diagnóstico excecional em Portugal, apesar da presença reconhecida da espécie Loxosceles rufescens. Apresentamos uma doente de 33 anos que desenvolveu placa eritematosa quente, dolorosa, com progressão para necrose cutânea e ulceração tórpida na face interna da coxa direita, em relação com mordedura visualizada de aranha. Em virtude da ausência de cicatrização adequada após tratamento conservador, recorreu-se ao desbridamento cirúrgico da úlcera, tendo-se realizado plastia em O-Z para a sua reconstrução, com bom resultado funcional. O diagnóstico de loxoscelismo cutâneo é difícil e obriga idealmente à observação da mordedura, verificação de sinais clínicos compatíveis e captura da aranha para identificação por taxonomista. Estecaso clínico reúne critérios que relacionam uma mordedura de aranha com a necrose cutânea resultante, sendo este, no nossoconhecimento, o primeiro caso relatado em Portugal.Palavras-chave: Doenças da Pele; Mordeduras e Picadas; Venenos de Aranha; Portugal.

Cancer is often characterised by the presence of hypoxia and inflammation. Paramount to the mechanisms controlling cellular responses under such stress stimuli, are the transcription factor families of Hypoxia Inducible Factor (HIF) and Nuclear Factor of kappa-light-chain-enhancer of activated B cells (NF-кB). Although, a detailed understating of how these transcription factors respond to their cognate stimulus is well established, it is now appreciated that HIF and NF-кB undergo extensive crosstalk, in particular in pathological situations such as cancer. Here, we focus on the current knowledge on how HIF is activated by inflammation and how NF-кB is modulated by hypoxia. We summarise the evidence for the possible mechanism behind this activation and how HIF and NF-кB function impacts cancer, focusing on colorectal, breast and lung cancer. We discuss possible new points of therapeutic intervention aiming to harness the current understanding of the HIF-NF-кB crosstalk.

Abstract To improve understanding of the role of site-specific proline hydroxylation in controlling protein function, we have developed a robust workflow for the identification of proline hydroxylation sites in proteins using a combination of hydrophilic interaction chromatography (HILIC) enrichment and high-resolution nano-Liquid Chromatography-Mass Spectrometry (LC-MS). Using this approach, together with refining and filtering parameters during data analysis, by combining the results from cell lines being treated with either the prolyl hydroxylase inhibitor Roxadustat (FG-4592, FG) or the proteasome inhibitor MG-132 (MG), or DMSO, a total of 4,993 and 3,247 proline hydroxylation sites were identified in HEK293 and RCC4 samples, respectively. A subset of 1,954 and 1,253 high confident proline hydroxylation sites (non-collagen) from HEK293 and RCC4 samples were inhibited by FG-4592 treatment. A set of features characteristic of proline hydroxylated peptides were identified in both datasets, which differ from either unmodified peptides, or oxidised peptides. Peptides containing hydroxyproline were enriched in the more hydrophilic HILIC fractions and showed characteristic differences in charge and mass distribution, as compared with unmodified or oxidised peptides. Furthermore, we discovered that the intensity of the diagnostic hydroxyproline immonium ion was greatly influenced by parameters including the MS collision energy setting, parent peptide concentration and the sequence of adjacent amino acids neighbouring the modified proline. We show using synthetic peptides that a combination of retention time in LC and optimised MS parameter settings allows reliable identification of proline hydroxylation sites in peptides, even when multiple prolines residues are present. By matching all the proline hydroxylated, non-collagen proteins to the Pfam database, the most common protein family domains identified in both HEK293 and RCC4 datasets were RNA recognition motif (RRM_1), WD40 repeat (WD40), and protein kinase domain (Pkinase). Sequence analysis of the hydroxylated peptides showed enrichment for the motif GxPGxx, including the Gxx repeats found in collagen proteins, as well as the protein kinase domain GTP motif. Glycine (G), serine (S) and glutamic acid (E) residues were found frequently in the sequence window from the hydroxylated peptides. Reactome pathway analysis for the proteins of these newly identified proline hydroxylation sites (FG inhibited), showed enrichment for proteins involved in metabolism of RNA, mRNA splicing and cell cycle regulation, potentially mediated by prolyl hydroxylase enzymes (PHDs).

Prostate cancer (PCa) poses a significant health threat to males, and research has shown that fish oil (FO) can impede PCa progression by activating multiple mitochondria-related pathways. Our research is focused on investigating the impact of FO on succinylation, a posttranslational modification that is closely associated with mitochondria in PCa cells. This study employed a mass spectrometry-based approach to investigate succinylation in PCa cells. Bioinformatics analysis of these succinylated proteins identified glutamic-oxaloacetic transaminase 2 (GOT2) protein as a key player in PCa cell proliferation. Immunoprecipitation and RNA interference technologies validated the functional data. Further analyses revealed the significance of GOT2 protein in regulating nucleotide synthesis by providing aspartate, which is critical for the survival and proliferation of PCa cells. Our findings suggest that FO-dependent GOT2 succinylation status has the potential to inhibit building block generation. This study lays a solid foundation for future research into the role of succinylation in various biological processes. This study highlights the potential use of FO as a nutrition supplement for managing and slowing down PCa progression.

Introduction: Fabry disease is an X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene, leading to decreased/absent α-galactosidase activity. In clinical practice, enzyme activity and substrate/byproduct accumulation play a role in diagnosis and disease-monitoring biomarkers. However, interpreting biomarker levels is not straightforward and can change according to the underlying GLA protein abnormality. Objective: Our goals were to understand how disrupting specific protein regions changes biomarker behaviour and to establish specific patterns for individual variants. Methodology: We analysed data from the Biochemical Genetics Laboratory regarding GLA variants, GLA enzyme activity (in dried blood spots, plasma or white blood cells), plasma LysoGb3 accumulation, and urinary Gb3 excretion. We assessed correlations, trends, and potential predictor models of biomarker behaviour. Results: We assessed 169 hemizygous male and 255 heterozygous female patients. For both groups, substrate accumulation correlates inversely with GLA activity. Variants affecting residues buried within the protein core or the active site were associated with more severe biomarker changes, while those affecting residues that establish disulfide bonds or are glycosylated were similar to other variants. For each non-truncating variant, we also established specific profiles of biomarker behaviour. Finally, we also designed predictor models of biomarker behaviour based on structural variant information. This study provides the groundwork for the impact of GLA protein variation on GLA activity and substrate accumulation. Conclusion: This knowledge is of extreme relevance for diagnostic labs and clinicians, as some genetic variants are challenging to interpret regarding pathogenicity. Assessing whether biomarker changes are in the expected range for a specific variant may help diagnostic evaluation. This study also contributes to recognising non-disease-causing variants, considering their overall biochemical impact, and providing a comparative reference for biomarker discovery studies. In the future, the correlation of these findings with disease severity may be of great relevance for diagnosis and monitoring progression.

Caspases are a family of cysteine proteases that constitute the apoptotic cell death machinery. We report the importance of the cytochrome c-mediated caspase-9 death pathway for radiosensitization by the protein kinase C (PKC) inhibitors staurosporine (STP) and PKC-412. In our genetically defined tumor cells, treatment with low doses of STP or the conventional PKC-specific inhibitor PKC-412 in combination with irradiation (5 Gy) potently reduced viability, enhanced mitochondrial cytochrome c release into the cytosol, and specifically stimulated the initiator caspase-9. Whereas treatment with each agent alone had a minimal effect, combined treatment resulted in enhanced caspase-3 activation. This was prevented by broad-range and specific caspase-9 inhibitors and absent in caspase-9-deficient cells. The tumor suppressor p53 was required for apoptosis induction by combined treatment but was dispensable for dose-dependent STP-induced caspase activation. These results demonstrate the requirement for an intact caspase-9 pathway for apoptosis-based radiosensitization by PKC inhibitors and show that STP induces apoptosis independent of p53.

Allopurinol, an inhibitor of xanthine oxidase, has been used in clinical trials of patients with cardiovascular and chronic kidney disease. These are two pathologies with extensive links to hypoxia and activation of the transcription factor hypoxia inducible factor (HIF) family. Here we analysed the effects of allopurinol treatment in two different cellular models, and their response to hypoxia. We explored the dose-dependent effect of allopurinol on Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) under hypoxia and normoxia. Under normoxia and hypoxia, high dose allopurinol reduced the accumulation of HIF-1α protein in HFF and HUVEC cells. Allopurinol had only marginal effects on HIF-1α mRNA level in both cellular systems. Interestingly, allopurinol effects over the HIF system were independent of prolyl-hydroxylase activity. Finally, allopurinol treatment reduced angiogenesis traits in HUVEC cells in an in vitro model. Taken together these results indicate that high doses of allopurinol inhibits the HIF system and pro-angiogenic traits in cells.

Hypoxia, or reduced oxygen availability, has been studied extensively for its ability to activate specific genes. Hypoxia induced gene expression is mediated by the HIF transcription factors, although not exclusively so. Despite the great knowledge on the mechanisms by which hypoxia activates genes, much less is known about how hypoxia promotes gene repression. In this review, we discuss the potential mechanisms underlying hypoxia-induced transcriptional repression responses. We highlight HIF-dependent and independent mechanisms, but also the potential roles of dioxygenases with functions at the nucleosome and DNA level. Finally, we discuss recent evidence regarding the involvement of transcriptional repressor complexes in hypoxia.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription. To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA "ubiquitome" library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question. Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter. An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested. Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Overexpression of Bcl-2 enhances sensitivity of L929 cells to a lipophilic cationic photosensitiser

Direct-acting antiviral drugs (DAAs) have recently changed the paradigm of hepatitis C therapy, significantly improving treatment response rates, patient life expectancy and quality of life. In Portugal, sofosbuvir (SOF) and SOF/ledipasvir (SOF/LDV) were fully reimbursed by the National Health System since early 2015 and generalized use of interferon-free DAA based regimens became current practice. During 2016, the remaining DAAs were sequentially added and covered by the same health access policy. The Portuguese Study Group of Hepatitis and HIV Co-infection (GEPCOI) collected data from 15 clinical centres in Portugal, pertaining to the HCV treatment experience with DAA regimens. A cohort of 2133 patients was analysed, representing one of the largest DAA treated HCV/HIV co-infected individuals. The global sustained virologic response (SVR) achieved was 95% in this real-life cohort setting. Linear regression analysis showed significant differences in treatment response rates when using SOF plus ribavirin (RBV) combination in genotype 2 or 3 infected individuals (P < .002) and in those with liver cirrhosis (P < .002). These findings corroborate that early treatment is mandatory in HIV/HCV co-infected patients, as response rates may be negatively influenced by higher fibrosis stages and suboptimal DAA regimens. The current national Portuguese health policy should continue to promote wider treatment access and individualized therapy strategies, aiming at the elimination of HCV infection in this high-risk co-infected population.

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP-HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy.