Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.

Identification of a BRCA1-mRNA Splicing Complex Required for Efficient DNA Repair and Maintenance of Genomic Stability

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.

Increased susceptibility to delayed genetic effects of low dose X-irradiation in DNA repair deficient cells

Purpose: To examine whether the levels of micronuclei induction, as a marker for genomic instability in the progeny of X-irradiated cells, correlates with DNA repair function.

Materials and methods: Two repair deficient cell lines (X-ray repair cross-complementing 1 [XRCC1] deficient cell line [EM9] and X-ray repair cross complementing 5 [XRCC5; Ku80] deficient X-ray sensitive Chinese hamster ovary [CHO] cell line [xrs5]) were used in addition to wild-type CHO cells. These cells were irradiated with low doses of X-rays (up to 1 Gy). Seven days after irradiation, micronuclei formed in binucleated cells were counted. To assess the contribution of the bystander effect micronuclei induction was measured in progeny of non-irradiated cells co-cultured with cells that had been irradiated with 1Gy.

Results: The delayed induction of micronuclei in 1 Gy-irradiated cells was observed in normal CHO and EM9 but not in xrs5. In the clone analysis, progenies of xrs5 under bystander conditions showed significantly higher levels of micronuclei, while CHO and EM9 did not.

Conclusion: Genomic instability induced by X-irradiation is associated with DSB (double-strand break) repair, even at low doses. It is also suggested that bystander signals, which lead to genomic instability, may be enhanced when DSB repair is compromised.

Marine organisms for bone repair and regeneration

Bioprospecting has led to increased interest in potential applications for marine organisms and their by-products. As a rich source of mineralising porous organisms, our seas and oceans could provide new directions for bone tissue engineering particularly in the supply of biomimetic templates that may enhance in vivo and ex vivo bone formation. In this chapter we examine the history of marine organism use in this field; exploring how these organisms could be utilised, given the problems of sustainability, and reviewing the current evidence to support their use for bone repair and regeneration.

A randomized clinical trial of ascorbic acid in open abdominal aortic aneurysm repair

BACKGROUND: Open AAA repair is associated with ischaemia-reperfusion injury where systemic inflammation and endothelial dysfunction can lead to multiple organ injury including acute lung injury. Oxidative stress plays a role that may be inhibited by ascorbic acid.

METHODS: A double blind, allocation concealed, randomized placebo-controlled trial was performed to test the hypothesis that a single bolus dose (2g) of intra-operative parenteral ascorbic acid would attenuate biomarkers of ischaemia-reperfusion injury in patients undergoing elective open AAA repair.

RESULTS: Thirty one patients completed the study; 18 received placebo and 13 ascorbic acid. Groups were comparable demographically. Open AAA repair caused an increase in urinary Albumin:Creatinine Ratio (ACR) as well as plasma IL-6 and IL-8. There was a decrease in exhaled breath pH and oxygenation. Lipid hydroperoxides were significantly higher in the ascorbic acid group following open AAA repair. There were no other differences between the ascorbic acid or placebo groups up to 4 hours after removal of the aortic clamping.

CONCLUSIONS: Open AAA repair caused an increase in markers of systemic endothelial damage and systemic inflammation. Administration of 2g parenteral ascorbic acid did not attenuate this response and with higher levels of lipid hydroperoxides post-operatively a pro-oxidant effect could not be excluded.

TRIAL REGISTRATION: ISRCTN27369400.