H3K9me3 facilitates hypoxia-induced p53-dependent apoptosis through repression of APAK.

Regions of hypoxia occur in most solid tumors, and they are associated with a poor prognostic outcome. Despite the absence of detectable DNA damage, severe hypoxia (<0.1% O2) induces a DNA damage response, including the activation of p53 and subsequent induction of p53-dependent apoptosis. Factors affecting hypoxia-induced p53-dependent apoptosis are unclear. Here we asked whether H3K9me3, through mediating gene repression, could regulate hypoxia-induced p53-dependent apoptosis. Under hypoxic conditions, increases in H3K9me3 occur in an oxygen-dependent but HIF-1-independent manner. We demonstrate that under hypoxic conditions, which induce p53 activity, the negative regulator of p53, APAK, is repressed by increases in H3K9me3 along the APAK loci. APAK repression in hypoxia is mediated by the methyltransferase SETDB1 but not Suv39h1 or G9a. Interestingly, increasing hypoxia-induced H3K9me3 through pharmacological inhibition of JMJD2 family members leads to an increase in apoptosis and decreased clonogenic survival and again correlates with APAK expression. The relevance of understanding the mechanisms of APAK expression regulation to human disease was suggested by analysis of patients with colorectal cancer, which demonstrates that high APAK expression correlates with poor prognosis. Together, these data demonstrate the functional importance of H3K9me3 in hypoxia, and they provide a novel mechanistic link between H3K9me3, p53 and apoptosis in physiologically relevant conditions of hypoxia.

Targeting chromatin to improve radiation response.

Chromatin, the structure formed by the wrapping of approximately 146 base pairs of DNA around an octamer of histones, has a profound impact on numerous DNA-based processes. Chromatin modifications and chromatin remodellers have recently been implicated in important aspects of the DNA damage response including facilitating the initial sensing of the damage as well as subsequent recruitment of repair factors. Radiation is an effective cancer therapy for a large number of tumours, and there is considerable interest in finding approaches that might further increase the efficacy of radiotherapy. The use of radiation leads to the generation of DNA damage and, therefore, agents that can affect the sensing and repair of DNA damage may have an impact on overall radiation efficacy. The chromatin modifications as well as chromatin modifiers that have been associated with the DNA damage response will be summarized in this review. An emphasis will be placed on those processes that can be pharmacologically manipulated with currently available inhibitors. The rationale for the use of these inhibitors in combination with radiation will also be described.

Aberrant IDH3α expression promotes malignant tumor growth by inducing HIF-1-mediated metabolic reprogramming and angiogenesis.

Cancer cells gain a growth advantage through the so-called Warburg effect by shifting glucose metabolism from oxidative phosphorylation to aerobic glycolysis. Hypoxia-inducible factor 1 (HIF-1) has been suggested to function in metabolic reprogramming; however, the underlying mechanism has not been fully elucidated. We found that the aberrant expression of wild-type isocitrate dehydrogenase 3α (IDH3α), a subunit of the IDH3 heterotetramer, decreased α-ketoglutarate levels and increased the stability and transactivation activity of HIF-1α in cancer cells. The silencing of IDH3α significantly delayed tumor growth by suppressing the HIF-1-mediated Warburg effect and angiogenesis. IDH3α expression was associated with the poor postoperative overall survival of lung and breast cancer patients. These results justify the exploitation of IDH3 as a novel target for the diagnosis and treatment of cancers.

The meaning, measurement and modification of hypoxia in the laboratory and the clinic.

Hypoxia was identified as a microenvironmental component of solid tumours over 60 years ago and was immediately recognised as a potential barrier to therapy through the reliance of radiotherapy on oxygen to elicit maximal cytotoxicity. Over the last two decades both clinical and experimental studies have markedly enhanced our understanding of how hypoxia influences cellular behaviour and therapy response. Furthermore, they have confirmed early assumptions that low oxygenation status in tumours is an exploitable target in cancer therapy. Generally such approaches will be more beneficial to patients with hypoxic tumours, necessitating the use of biomarkers that reflect oxygenation status. Tissue biomarkers have shown utility in many studies. Further significant advances have been made in the non-invasive measurement of tumour hypoxia with positron emission tomography, magnetic resonance imaging and other imaging modalities. Here, we describe the complexities of defining and measuring tumour hypoxia and highlight the therapeutic approaches to combat it.

Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pancreatic tumors to radiation.

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.

Targeting radiation-resistant hypoxic tumour cells through ATR inhibition.

BACKGROUND: Most solid tumours contain regions of sub-optimal oxygen concentration (hypoxia). Hypoxic cancer cells are more resistant to radiotherapy and represent the most aggressive fraction of a tumour. It is therefore essential that strategies continue to be developed to target hypoxic cancer cells. Inhibition of the DNA damage response (DDR) might be an effective way of sensitising hypoxic tumour cells to radiotherapy. METHODS: Here, we describe the cellular effects of pharmacological inhibition of the apical DDR kinase ATR (Ataxia Telangiectasia and Rad 3 related) with a highly selective inhibitor, VE-821, in hypoxic conditions and its potential as a radiosensitiser. RESULTS: VE-821 was shown to inhibit ATR-mediated signalling in response to replication arrest induced by severe hypoxia. In these same conditions, VE-821 induced DNA damage and consequently increased Ataxia Telangiectasia Mutated-mediated phosphorylation of H2AX and KAP1. Consistently, ATR inhibition sensitised tumour cell lines to a range of oxygen tensions. Most importantly, VE-821 increased radiation-induced loss of viability in hypoxic conditions. Using this inhibitor we have also demonstrated for the first time a link between ATR and the key regulator of the hypoxic response, HIF-1. HIF-1 stabilisation and transcriptional activity were both decreased in response to ATR inhibition. CONCLUSION: These findings suggest that ATR inhibition represents a novel strategy to target tumour cells in conditions relevant to pathophysiology and enhance the efficacy of radiotherapy.

Hypoxia-driven cell motility reflects the interplay between JMY and HIF-1α.

Junction-mediating and regulatory protein (JMY) is a novel p53 cofactor that regulates p53 activity during stress. JMY interacts with p300/CBP, which are ubiquitous transcriptional co-activators that interact with a variety of sequence-specific transcription factors, including hypoxia-inducible factor-1α (HIF-1α). In addition, JMY is an actin-nucleating protein, which, through its WH2 domains, stimulates cell motility. In this study, we show that JMY is upregulated during hypoxia in a HIF-1α-dependent manner. The JMY gene contains HIF-responsive elements in its promoter region and HIF-1α is recruited to its promoter during hypoxia. HIF-1α drives transcription of JMY, which accounts for its induction under hypoxia. Moreover, the enhanced cell motility and invasion that occurs during hypoxia requires JMY, as depleting JMY under hypoxic conditions causes decreased cell motility. Our results establish the interplay between JMY and HIF-1α as a new mechanism that controls cell motility under hypoxic stress.

Induction of activating transcription factor 3 by anoxia is independent of p53 and the hypoxic HIF signalling pathway.

Solid tumors often have an inadequate blood supply, which results in large regions that are subjected to hypoxic or anoxic stress. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates much of the transcriptional response of cells to hypoxia. Activating transcription factor 3 (ATF3) is another transcription factor that responds to a variety of stresses and is often upregulated in cancer. We investigated the regulation of ATF3 by oxygen deprivation. ATF3 induction occurred most robustly under anoxia, is common, and it is not dependent on presence of HIF-1 or p53, but is sensitive to the inhibition of c-Jun NH2-terminal kinase activation and the antioxidant N-acetylcystein. ATF3 could also be induced by desferrioxamine but not by the mitochondrial poison cyanide or the nonspecific 2-oxoglutarate dioxygenase inhibitor dimethyloxalylglycine. We also show that anoxic ATF3 mRNA is more stable than normoxic mRNA providing a mechanism for this induction. Thus, this study demonstrates that the regulation of ATF3 under anoxia is independent of 2-oxoglutarate dioxygenase, HIF-1 and p53, presumably involving multiple regulatory pathways.

Specific cleavage of gamma catenin by caspases during apoptosis.

Caspase-mediated proteolysis of cytoskeletal proteins during apoptosis appears to be commonplace. Enlarging on previous studies we have shown here that gamma catenin, like beta catenin, was degraded during cisplatin-induced apoptosis, initially giving a major product of 75 kDa. This truncated protein could be co-immunoprecipitated with alpha catenin. Addition of caspase inhibitors to cells in the presence of cisplatin appreciably reduced the proteolysis of gamma catenin as well as the level of apoptosis. Only limited degradation of alpha catenin was observed even at very late times when over 90% of cells in the culture were apoptotic. Immunohistochemical staining showed that during apoptosis there was a relocation of alpha, beta, and gamma catenin from the periphery of the cell to the cytoplasm, at the same time as other morphological changes commonly associated with apoptosis occurred. Interestingly, the changes in localisation of the catenins preceded proteolysis by several hours. In the presence of cisplatin and caspase inhibitor no change in distribution of catenins was observed, suggesting that re-localisation requires caspase activity but not necessarily directed against beta and gamma catenins.

Homology between a human apoptosis specific protein and the product of APG5, a gene involved in autophagy in yeast.

Apoptosis specific proteins (ASP) are expressed in the cytoplasm of cultured mammalian cells of various lineages following induction of apoptosis. The cDNA encoding ASP has been cloned from a human expression library and has significant homology to the Saccharomyces cerevisiae APG5 gene which is essential for yeast autophagy. The ASP gene, known as hAPG5, can be transcribed to give mRNAs of 3.3 kbp, 2.5 kbp and 1.8 kbp which are present at comparable levels in viable and apoptotic cells, demonstrating that protein expression must be regulated at the translational level. These data indicate a possible relationship between apoptosis and autophagy and suggest evolutionary conservation in mammalian apoptosis of a degradative process present in yeast.

ATP depletion causes a reversible decrease in Na+ pump density in cultured ventricular myocytes.

To examine factors contributing to impaired K+ homeostasis induced by prolonged but sublethal ATP depletion, we subjected cultured chick ventricular myocytes to metabolic inhibition with 20 mM 2-deoxy-D-glucose plus 1 mM NaCN for 2 h and then allowed myocytes to recover for 5 days in medium containing 6% fetal calf serum (FCS) or in hormone-supplemented serum-free medium. We measured spontaneous contractions (with a video motion detector), K+ content, K+ uptake, membrane potential, and Na+ pump density ([3H]ouabain binding). Exposure to metabolic inhibition for 2 h caused an acute decrease in Na+ pump site density [8.2 +/- 1.1 to 3.8 +/- 0.8 (SE) pmol/mg protein; n = 9, P < 0.02]. Compared with control cells (no metabolic inhibition, cultured for 5 days in serum-free medium), Na+ pump density remained depressed in cells recovered from metabolic inhibition in serum-free medium (3.0 +/- 0.7 pmol/mg), and this was associated with persistently depressed K+ uptake (54% of control), K+ content (67% of control), and membrane depolarization (-19 +/- 2 mV), a significant decrease in cell number (79% of control), and failure to resume spontaneous contractions. Exposure of cells inhibited for 2 h to culture medium containing 6% FCS resulted in a return of Na+ pump site density toward normal levels by 5 days, associated with recovery of K+ uptake and K+ content, preservation of cell number, and resumption of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Amplification of nitric oxide signaling by interstitial cells isolated from canine colon.

The effects of nitric oxide (NO) on intracellular Ca2+ concentration ([Ca2+]i) were studied in enzymatically dispersed interstitial cells (ICs) and smooth muscle cells (SMCs) isolated from canine colon. [Ca2+]i was monitored by using fluo-3 and video fluorescence imaging techniques. Exogenous NO caused an increase in [Ca2+]i in ICs and a decrease in [Ca2+]i in SMCs. Effects of NO on ICs were not blocked by removal of extracellular Ca2+ but were blocked by ryanodine, suggesting that NO caused release of Ca2+ from intracellular stores. When [Ca2+]i was elevated in an IC by micropressure ejection of Bay K 8644, [Ca2+]i decreased in nearby SMCs, suggesting release of a diffusible substance. The diffusible substance may be NO or an NO-related substance based on blockade of transmission by NG-nitro-L-arginine methyl ester, NG-monomethyl-L-arginine, or oxyhemoglobin. The elevation of [Ca2+]i in ICs by NO, which, in turn, might cause further release of NO and elevation of [Ca2+]i, suggests a positive feedback and amplification mechanism in these cells. Elevation of [Ca2+]i in SMCs had no effect on adjacent SMCs. Our data suggest that ICs may play a central role in amplification of NO signaling and propagation of inhibitory wave fronts.

Neuroblastoma--when are urinary catecholamines and their metabolites 'normal'?

The overproduction of catecholamines and their metabolites is a well recognised feature of neuroblastoma. Published data are scarce for their urinary excretion in children with neuroblastoma and in ill children in whom this diagnosis may be considered. We have determined a graphical upper reference limit for total catecholamines, total metadrenalines and HMMA in urine, expressed as a ratio to the creatinine concentration, for a group of 174 children with neuroblastoma and 704 hospitalised children with other disorders. This graph has been determined by examining the overlap region between the results for the two groups of children and avoids the irregularities caused by statistical outliers. The sensitivity and specificity of the individual tests indicate that total catecholamines is marginally the best single test to perform when trying to diagnose neuroblastoma, with the best clinical sensitivity being achieved by examining both total catecholamines and HMMA. Only two of the 174 children with neuroblastoma would not have been detected using these two tests. Total metadrenalines did not appear to add any further information and could be dropped from the repertoire in favour of the other two measurements.

Urinary catecholamines and their metabolites in management of neuroblastoma.

Measurements of urinary total catecholamines expressed as dopamine (TC) and their metabolites, total metadrenalines (TM) and 3-methoxy-4-hydroxy mandelic acid (HMMA) were made on 24-h urine collections from 56 previously untreated children with neuroblastoma. All results were expressed as a ratio to the creatinine excretion and were compared with "smoothed" age-related reference ranges derived from results in 704 children with other illnesses. Urinary catecholamines/metabolites excretion was elevated in 55 of the 56 patients, the exception being a baby with opsomyoclonus. TC was raised in 47, HMMA in 41, and TM in 37. Eleven patients had elevations of one parameter (TC in 10, HMMA in 1), 15 had 2 elevated levels, and 29 had elevations of all three. The TM level was the least contributory, as in no patient was it the only parameter elevated. Serial measurements were performed in 40 children, and the results correlated closely with the clinical progress of the disease and in some children permitted early detection of recurrence.

Serum dopamine beta hydroxylase in children with neuroblastoma.

Serum dopamine-beta-hydroxylase (DBH) activity has been reported to be raised in some patients with neuoblastoma but this has been challenged. We have studied serum DBH levels on 26 children with neuroblastoma and 58 age-matched controls. Only in 2 patients were the levels higher than in the controls, and then only transiently. In both, the rise in DBH levels could be accounted for by the transfusion of adult blood. Serum DBH levels in children with neuroblastoma were unrelated to the response of this neoplasm to treatment or to urinary catecholamine output and thus are unlikely to have any value in diagnosis or as a marker of tumour activity.