Effects of acute versus chronic hypoxia on DNA damage responses and genomic instability.

Questions exist concerning the effects of acute versus chronic hypoxic conditions on DNA replication and genomic stability that may influence tumorigenesis. Severe hypoxia causes replication arrest independent of S-phase checkpoint, DNA damage response, or transformation status. Arrests occur during both the initiation and elongation phases of DNA replication, correlated with a rapid decrease in available deoxynucleotide triphosphates. With fluctuating oxygen tensions in tumors, arrested hypoxic cells may undergo rapid reperfusion and reoxygenation that leads to reoxygenation-induced DNA damage. In cells subjected to chronic hypoxia, we found that replicative restart was inhibited along with numerous replication factors, including MCM6 and RPA, the latter of which limits the hypoxia-induced DNA damage response. In contrast, in cells where replicative restart occurred, it was accompanied by extensive reoxygenation-induced DNA damage and compromised DNA repair. We found that cells reoxygenated after acute hypoxia underwent rapid p53-dependent apoptosis. Our findings suggest that cells lacking functional p53 are more susceptible to genomic instability and potentially tumorigenesis if they experience reoxygenation after acute exposure to hypoxia

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same regio

The role of nitric oxide as a hypoxic cell radiosensitizer

Many tumours contain regions of hypoxia which are difficult to treat by conventional radiotherapy. There is much interest in the ability of nitric oxide (•NO) to radiosensitize hypoxic mammalian cells as a possible adjunct to radiotherapy but mechanisms for its action are unclear. It has been proposed that •NO may radiosensitize cells by ‘fixing’ radiation-induced DNA free radicals, and elevated radiation response by •NO in cells has been partly attributed to increased formation of DNA double strand breaks. In the work carried out for this thesis it is shown that reaction of •NO with radiation-induced nucleobase radicals produces some novel products. New pathways for the reactions of radiation-induced hydroxyl radicals with purine radicals are proposed. In addition, the effects of •NO on the yields of radiation-induced single strand breaks in anoxic plasmid DNA, and on anoxic mammalian cell radiosensitivity are investigated. Kinetics of formation and repair of radiation-induced double strand breaks indicate different effects of •NO on radiation-induced clustered and non-clustered DNA damage involving replication-induced DNA breaks. As •NO is an inhibitor of ribonucleotide reductase, some of the radiosensitizing properties of •NO may be due to reduction in the availability of 2-deoxyribonucleotides. Through studying reactions of •NO with tyrosine radicals, essential components of ribonucleotide reductase, this work has enhanced understanding into how •NO may inhibit the enzyme, which may offer new insights into the development of •NO-releasing anti-cancer agents. The potential for delivery of •NO to hypoxic tissue for radiotherapy has also been investigated in this work, through the development of bioreductively-activated pro-drugs. These novel agents are stable until reduced by one-electron reductants, when a •NO-releasing pro-drug is rapidly evolved, only in those regions which are sufficiently hypoxic. By increasing our understanding into the mechanisms involved in the ability of •NO to radiosensitize hypoxic cells, especially the reactivity of •NO with DNA radicals, knowledge has been gained into the identification, development and repair of radiation-induced DNA damage in cells, including clustered damage, in the presence of •NO. These studies contribute to further development of novel anti-cancer therapies based upon the release of •NO in hypoxic cells.</p

Radiolysis Studies of Oxidation and Nitration of Tyrosine and Some Other Biological Targets by Peroxynitrite-Derived Radicals

The widespread interest in free radicals in biology extends far beyond the effects of ionizing radiation, with recent attention largely focusing on reactions of free radicals derived from peroxynitrite (i.e., hydroxyl, nitrogen dioxide, and carbonate radicals). These radicals can easily be generated individually by reactions of radiolytically-produced radicals in aqueous solutions and their reactions can be monitored either in real time or by analysis of products. This review first describes the general principles of selective radical generation by radiolysis, the yields of individual species, the advantages and limitations of either pulsed or continuous radiolysis, and the quantitation of oxidizing power of radicals by electrode potentials. Some key reactions of peroxynitrite-derived radicals with potential biological targets are then discussed, including the characterization of reactions of tyrosine with a model alkoxyl radical, reactions of tyrosyl radicals with nitric oxide, and routes to nitrotyrosine formation. This is followed by a brief outline of studies involving the reactions of peroxynitrite-derived radicals with lipoic acid/dihydrolipoic acid, hydrogen sulphide, and the metal chelator desferrioxamine. For biological diagnostic probes such as &lsquo;spin traps&rsquo; to be used with confidence, their reactivities with radical species have to be characterized, and the application of radiolysis methods in this context is also illustrated

The role of nitric oxide as a hypoxic cell radiosensitizer

Many tumours contain regions of hypoxia which are difficult to treat by conventional radiotherapy. There is much interest in the ability of nitric oxide (•NO) to radiosensitize hypoxic mammalian cells as a possible adjunct to radiotherapy but mechanisms for its action are unclear. It has been proposed that •NO may radiosensitize cells by ‘fixing’ radiation-induced DNA free radicals, and elevated radiation response by •NO in cells has been partly attributed to increased formation of DNA double strand breaks. In the work carried out for this thesis it is shown that reaction of •NO with radiation-induced nucleobase radicals produces some novel products. New pathways for the reactions of radiation-induced hydroxyl radicals with purine radicals are proposed. In addition, the effects of •NO on the yields of radiation-induced single strand breaks in anoxic plasmid DNA, and on anoxic mammalian cell radiosensitivity are investigated. Kinetics of formation and repair of radiation-induced double strand breaks indicate different effects of •NO on radiation-induced clustered and non-clustered DNA damage involving replication-induced DNA breaks. As •NO is an inhibitor of ribonucleotide reductase, some of the radiosensitizing properties of •NO may be due to reduction in the availability of 2-deoxyribonucleotides. Through studying reactions of •NO with tyrosine radicals, essential components of ribonucleotide reductase, this work has enhanced understanding into how •NO may inhibit the enzyme, which may offer new insights into the development of •NO-releasing anti-cancer agents. The potential for delivery of •NO to hypoxic tissue for radiotherapy has also been investigated in this work, through the development of bioreductively-activated pro-drugs. These novel agents are stable until reduced by one-electron reductants, when a •NO-releasing pro-drug is rapidly evolved, only in those regions which are sufficiently hypoxic. By increasing our understanding into the mechanisms involved in the ability of •NO to radiosensitize hypoxic cells, especially the reactivity of •NO with DNA radicals, knowledge has been gained into the identification, development and repair of radiation-induced DNA damage in cells, including clustered damage, in the presence of •NO. These studies contribute to further development of novel anti-cancer therapies based upon the release of •NO in hypoxic cells.This thesis is not currently available in OR