Antiproton induced DNA damage : proton like in flight, carbon-ion light near rest

Biological validation of new radiotherapy modalities is essential to understand their therapeutic potential. Antiprotons have been proposed for cancer therapy due to enhanced dose deposition provided by antiproton-nucleon annihilation. We assessed cellular DNA damage and relative biological effectiveness (RBE) of a clinically relevant antiproton beam. Despite a modest LET (~19 keV/μm), antiproton spread out Bragg peak (SOBP) irradiation caused significant residual γ-H2AX foci compared to X-ray, proton and antiproton plateau irradiation. RBE of ~1.48 in the SOBP and ~1 in the plateau were measured and used for a qualitative effective dose curve comparison with proton and carbon-ions. Foci in the antiproton SOBP were larger and more structured compared to X-rays, protons and carbon-ions. This is likely due to overlapping particle tracks near the annihilation vertex, creating spatially correlated DNA lesions. No biological effects were observed at 28–42 mm away from the primary beam suggesting minimal risk from long-range secondary particles.

BRCA1 deficiency exacerbates estrogen-induced DNA damage and genomic instability

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell cycle arrest and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here we report that estrogen and estrogen metabolites can cause DNA double strand breaks (DSB) in estrogen receptor-α negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolising enzymes, such as CYP1A1, in breast cells. Lastly, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumours in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types.

Exercise-induced DNA damage: Is there a relationship with inflammatory responses?

Both a systemic inflammatory response as well as DNA damage has been observed following exhaustive endurance exercise. Hypothetically, exercise-induced DNA damage might either be a consequence of inflammatory processes or causally involved in inflammation and immunological alterations after strenuous prolonged exercise (e.g. by inducing lymphocyte apoptosis and lymphocytopenia). Nevertheless, up to now only few studies have addressed this issue and there is hardly any evidence regarding a direct relationship between DNA or chromosomal damage and inflammatory responses in the context of exercise. The most conclusive picture that emerges from available data is that reactive oxygen and nitrogen species (RONS) appear to be the key effectors which link inflammation with DNA damage. Considering the time-courses of inflammatory and oxidative stress responses on the one hand and DNA effects on the other the lack of correlations between these responses might also be explained by too short observation periods. This review summarizes and discusses the recent findings on this topic. Furthermore, data from our own study are presented that aimed to verify potential associations between several endpoints of genome stability and inflammatory, immune-endocrine and muscle damage parameters in competitors of an Ironman triathlon until 19 days into recovery. The current results indicate that DNA effects in lymphocytes are not responsible for exercise-induced inflammatory responses. Furthermore, this investigation shows that inflammatory processes, vice versa, do not promote DNA damage, neither directly nor via an increased formation of RONS derived from inflammatory cells. Oxidative DNA damage might have been counteracted by training- and exercise-induced antioxidant responses. However, further studies are needed that combine advanced -omics based techniques (transcriptomics, proteomics) with state-of-the-art biochemical biomarkers to gain more insights into the underlying mechanisms.

Effect of phytoestrogen and antioxidant supplementation on oxidative DNA damage assessed using the comet assay

Antioxidant species may act in vivo to decrease oxidative damage to DNA, protein and lipids thus reducing the risk of coronary heart disease and cancer. Phytoestrogens are plant compounds which are a major component of traditional Asian diets and which may be protective against certain hormone-dependent cancers (breast and prostate) and against coronary heart disease. They may also be able to function as antioxidants, scavenging potentially harmful free radicals. In this study, the effects of the isoflavonoids (a class of phytoestrogen) genistein and equol on hydrogen peroxide-mediated DNA damage in human lymphocytes were determined using alkaline single-cell gel electrophoresis (the comet assay). Treatment with hydrogen peroxide significantly increased the levels of DNA strand breaks. Pre-treatment of the cells with both genistein and equol offered protection against this damage at concentrations within the physiological range. This protection was greater than that offered by addition of the known antioxidant vitamins ascorbic acid and alpha -tocopherol, or the compounds 17 beta -oestradiol and Tamoxifen which have similar structures to isoflavonoids and are known to have weak antioxidant properties. These findings are consistent with the hypothesis that phytoestrogens can, under certain conditions, function as antioxidants and protect against oxidatively-induced DNA damage. (C) 2001 Elsevier Science B.V. All rights reserved.

In vitro isoflavone supplementation reduces hydrogen peroxide-induced DNA damage in sperm

Isoflavones are plant compounds, proposed to have health benefits in a variety of human diseases, including coronary heart disease and endocrine-responsive cancers. Their physiological effects include possible antioxidant activity, therefore suggesting a role for isoflavones in the prevention of male infertility. The aim of this study was to test the antioxidant effects of the isoflavones genistein and equol on sperm DNA integrity, assessed in vitro after hydrogen peroxide-mediated damage, using the cornet assay. Pre-treatment with genistein or equol at doses of 0.01-100 mumol/l significantly protected sperm DNA against oxidative damage. Both ascorbic acid (10-600 mumol/l) and alpha-tocopherol (1-100 mumol/l) also protected. Compared with ascorbic acid and alpha-tocopherol, added at physiological concentrations, genistein was the most potent antioxidant, followed by equol, ascorbic acid, and alpha-tocopherol. Genistein and equol added in combination were more protective than when added singly. Based on these preliminary data, which are similar to those observed previously in lymphocytes, these compounds may have a role to play in antioxidant protection against male infertility.

BRCA1-BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage

BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to become sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ATR (ataxia-telangiectasia-mutated/ATM and Rad3-related)-mediated phosphorylation of p53(Ser-15) following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53(Ser-15) efficiently. Phosphorylation of p53(Ser-15) is necessary for an IR-induced G(1)/S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, repressing p53(Ser-15) phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G(1)/S checkpoint arrest in response to IR but not UV radia-tion. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G(1)/S cell cycle progression after DNA damage.

Antiproton induced DNA damage: proton like in flight, carbon-ion like near rest

Biological validation of new radiotherapy modalities is essential to understand their therapeutic potential. Antiprotons have been proposed for cancer therapy due to enhanced dose deposition provided by antiproton-nucleon annihilation. We assessed cellular DNA damage and relative biological effectiveness (RBE) of a clinically relevant antiproton beam. Despite a modest LET (,19 keV/mm), antiproton spread out Bragg peak (SOBP) irradiation caused significant residual c-H2AX foci compared to X-ray, proton and antiproton plateau irradiation. RBE of ,1.48 in the SOBP and ,1 in the plateau were measured and used for a qualitative effective dose curve comparison with proton and carbon-ions. Foci in the antiproton SOBP were larger and more structured compared to X-rays, protons and carbon-ions. This is likely due to overlapping particle tracks near the annihilation vertex, creating spatially correlated DNA lesions. No biological effects were observed at 28–42 mm away from the primary beam suggesting minimal risk from long-range secondary particles.

BRCA1 Deficiency Exacerbates Estrogen-Induced DNA Damage and Genomic Instability

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptora- negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability.We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types. © 2014 American Association for Cancer Research.

BRCA1, FANCD2 and Chk1 are potential molecular targets for the modulation of a radiation-induced DNA damage response in bystander cells

Radiotherapy is an important treatment option for many human cancers. Current research is investigating the use of molecular targeted drugs in order to improve responses to radiotherapy in various cancers. The cellular response to irradiation is driven by both direct DNA damage in the targeted cell and intercellular signalling leading to a broad range of bystander effects. This study aims to elucidate radiation-induced DNA damage response signalling in bystander cells and to identify potential molecular targets to modulate the radiation induced bystander response in a therapeutic setting. Stalled replication forks in T98G bystander cells were visualised via bromodeoxyuridine (BrdU) nuclear foci detection at sites of single stranded DNA. γH2AX co-localised with these BrdU foci. BRCA1 and FANCD2 foci formed in T98G bystander cells. Using ATR mutant F02-98 hTERT and ATM deficient GM05849 fibroblasts it could be shown that ATR but not ATM was required for the recruitment of FANCD2 to sites of replication associated DNA damage in bystander cells whereas BRCA1 bystander foci were ATM-dependent. Phospho-Chk1 foci formation was observed in T98G bystander cells. Clonogenic survival assays showed moderate radiosensitisation of directly irradiated cells by the Chk1 inhibitor UCN-01 but increased radioresistance of bystander cells. This study identifies BRCA1, FANCD2 and Chk1 as potential targets for the modulation of radiation response in bystander cells. It adds to our understanding of the key molecular events propagating out-of-field effects of radiation and provides a rationale for the development of novel molecular targeted drugs for radiotherapy optimisation.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.

Antigenotoxic effect of kefir and ayran supernatants on fecal water-induced DNA damage in human colon cells

Fermented dairy products and their component bacteria have been shown to possess health-promoting functions in consumers and recently have been suggested to reduce the risk of colorectal cancer. Kefir and ayran are two popular fermented milk drinks that have their origins in the Caucasus region of Russia. The present study aimed to evaluate their potential anticancer properties in colon cells in vitro. The comet assay and transepithelial resistance assay were used to assess the effect of kefir and ayran supernatants on genotoxicity of fecal water samples and on intestinal tight junction integrity. Their antioxidant capacity was measured by trolox equivalent antioxidant capacity assay and compared with that of unfermented milk. The results showed that DNA damage induced by 2 of 4 fecal water samples was significantly decreased by kefir and ayran supernatants and with ayran the effect was dose-dependent. However no effect on intestinal tight junctions was observed. The supernatants of kefir and ayran contained high amounts of acetic and lactic acid but only a very small quantity of caproic and butyric acid, and they showed significantly greater antioxidant capacity than milk. These findings suggest kefir and ayran can reduce DNA damage, which might be due to their antioxidant capacities.

Genistein protects prostate cells against hydrogen peroxide-induced DNA damage and induces expression of genes involved in the defence against oxidative stress

Prostate cancer is one of the most frequent cancer types in Western societies and predominately occurs in the elderly male. The strong age-related increase of prostate cancer is associated with a progressive accumulation of oxidative DNA damage which is presumably supported by a decline of the cellular antioxidative defence during ageing. Risk of developing prostate cancer is much lower in many Asian countries where soy food is an integral part of diet. Therefore, isoflavones from soy were suggested to have chemopreventive activities in prostate cells. Here, we have investigated the hypothesis that the soy-isoflavone genistein could protect DNA of LAPC-4 prostate cells from oxidative stress-related damage by enhancing the expression of antioxidative genes and proteins. A 24 h preincubation with genistein (1-30 microM) protected cells from hydrogen peroxide-induced DNA damage, as determined by the comet assay. Analysis of two cDNA macroarrays, each containing 96 genes of biotransformation and stress response, revealed a modulated expression of 3 genes at 1 microM and of 19 genes at 10 microM genistein. Real-time PCR confirmed the induction of three genes encoding products with antioxidant activities, namely glutathione reductase (2.7-fold), microsomal glutathione S-transferase 1 (1.9-fold) and metallothionein 1X (6.3-fold), at 1-30 microM genistein. 17Beta-estradiol, in contrast, decreased the expression of metallothionein 1X at 0.3 microM (2.0-fold), possibly pointing to an estrogen receptor-mediated regulation of this gene. Immunocytochemical staining revealed an induction of metallothionein proteins at 30 microM genistein, while their intracellular localization was unaffected. Metallothioneins were previously found to protect cells from hydrogen peroxide-induced DNA damage. Hence, our findings indicate that genistein protects prostate cells from oxidative stress-related DNA damage presumably by inducing the expression of antioxidative products, such as metallothioneins. Genistein, therefore, might counteract the age-related decline of important antioxidative defence systems which in turn maintain DNA integrity.