Objective: FLASH irradiation reportedly produces less normal tissue toxicity, while maintaining tumour response. To investigate oxygen’s role in the ‘FLASH effect’, we assessed DNA damage levels following irradiation at different oxygen tensions, doses and dose rates. Methods: Samples of whole blood were irradiated (20 Gy) at various oxygen tensions (0.25–21%) with 6 MeV electrons at dose rates of either 2 kGy/s (FLASH) or 0.1 Gy/s (CONV), and subsequently with various doses (0–40 Gy) and intermediate dose rates (0.3–1000 Gy/s). DNA damage of peripheral blood lymphocytes (PBL) were assessed by the alkaline comet assay. Results: Following 20 Gy irradiation, lower levels of DNA damage were induced for FLASH, the difference being significant at 0.25% (p < 0.05) and 0.5% O2 (p < 0.01). The differential in DNA damage at 0.5% O2 was found to increase with total dose and dose rate, becoming significant for doses ≥20 Gy and dose rates ≥30 Gy/s. Conclusion: This study shows, using the alkaline comet assay, that lower levels of DNA damage are induced following FLASH irradiation, an effect that is modulated by the oxygen tension, and increases with the total dose and dose rate of irradiation, indicating that an oxygen related mechanism, e.g. transient radiation-induced oxygen depletion, may contribute to the tissue sparing effect of FLASH irradiation. Advances in knowledge: This paper is first to directly show that FLASH-induced DNA damage is modulated by oxygen tension, total dose and dose rate, with FLASH inducing significantly lower levels of DNA damage for doses ≥20 Gy and dose rates ≥30 Gy/s, at 0.5% O2.

Radiotherapy is still a treatment of choice for many malignancies, often in combination with other strategies. However, its efficacy is limited by the dose that can be safely administered without eliciting serious side effects, as well as the fact that recurrence is common, particularly in large tumours. Combining radiotherapy with drugs that could sensitize cells to radiation and/or reduce the factors that promote the recovery of the surviving cancer cells is a promising approach. Ionizing radiation has been shown to induce senescence, and the accumulation of senescent cells creates a microenvironment that facilitates neoplastic growth. This provides a rationale to test the addition of anti-senescent drugs, some of them already available in the clinic, to radiotherapy protocols. Here, we discuss the relevance of radiotherapy-induced senescent cell accumulation and the potential interventions to minimize its negative effects.

Clinical Oncology j o ur n a l h o m e p a ge : w w w. c l i n i c a l on c o l o gy o n l i ne. n e t

We have measured by gel electrophoresis the yields of single- and double-strand breaks (SSBs and DSBs) induced in aqueous solutions of SV40 DNA and the SV40 minichromosome by 137Cs gamma rays (mean LET 0.3 keV micron-1) and 4He ions (mean LETs 85, 102, and 152 keV microns-1). DNA SSBs are caused mainly by the hydroxyl radicals under these conditions and are reduced in yield as either the hydroxyl radical scavenger concentration or the LET is increased (over the range studied). The G(SSB) for 4He ion irradiation is less by a factor of up to 10 than the G(SSB) for gamma irradiation, depending upon the scavenger concentration. The difference in the yields of SSBs agrees well with the difference in the yields of hydroxyl radicals for the radiations in question. In contrast, the yields of DSBs are similar for gamma and 4He ion irradiation over much of the range of scavenging capacity studied. However, at the highest scavenger concentrations the yields of DSBs are greater for 4He ion irradiation. In addition, the yields of DSBs remain almost constant with increasing LET (over the range studied). Therefore the relative yield of DSBs per SSB increases with increasing LET, supporting the hypothesis that increasing LET leads to an increased clustering of damage in DNA.

Quantitation in plasma-based proteomics necessitates the reproducible removal of highly abundant proteins to enable the less abundant proteins to be visible to the mass spectrometer. We have evaluated immunodepletion (proteoprep20) and enrichment (Bio-Rad beads), as the current predominant approaches. Label-free analysis offers an opportunity to estimate the effectiveness of this approach without incorporating chemical labels. Human plasma samples were used to quantitatively assess the reproducibility of these two methods using nano-LC-data-independent acquisition MS. We have selected 18 candidate proteins and a comparison of both methodologies showed that both of the methods were reproducible and fell below 20% residual SD. With the same candidate proteins, individual inter-day variability for the samples was also processed, allowing us to monitor instrument reproducibility. Overall, a total of 131 proteins were identified by both methods with 272 proteins identified by enrichment and 200 identified by immunodepletion. Reproducibility of measurements of the amount of protein in the processed sample for individual proteins is within analytically acceptable standards for both methodologies. This enables both methods to be used for biomarker studies. However, when sample is limited, enrichment is not suitable as larger volumes (>1.0 mL) are required. In experiments where sample is not limited then a greater number of proteins can be reliably identified using enrichment.

Exposure to DNA damaging agents and mutagens often occurs as combinations of agents, or as complex mixtures of chemicals. We found that plasmid DNA adducted with benzo[a]pyrene diol epoxide (BPDE) was more susceptible to UV-induced single strand breaks than was control DNA. To determine whether the increase in DNA damage also applied to mutagenic lesions, the supF gene forward mutation assay was used to compare mutations induced by BPDE alone, UVB, UVC, BPDE followed by UVB and BPDE followed by UVC. It was found that the mutation frequency for BPDE + UVB (1167 in 10(4) transformants) was higher than BPDE alone (12 in 10(4) transformants) or UVB alone (446 in 10(4) transformants), and the mutation frequency for BPDE + UVC (197 in 10(4) transformants) was higher than BPDE alone or UVC alone (26 in 10(4) transformants). For BPDE + UVB and BPDE + UVC there was a significant increase in plasmids with multiple mutations. Whilst these indicate error prone repair due to the single strand breaks, the different mutation frequencies in plasmids treated to give similar levels of strand breaks suggest other mechanisms for the mutations in plasmids with single mutation events. The spectrum of non-multiple mutations in the two combined treatments included both UV signature mutations (GC-->AT as the most common mutation) and BPDE signature mutations (GC-->TA and GC-->CG as the most common mutations). However, the increase in absolute mutation frequency of BPDE signature mutations between BPDE treatment and BPDE + UV treatment was greater than the increase in absolute mutation frequency of UV signature mutations, even though the level of BPDE adducts was identical in each case. These results suggest two possibilities: (i) the BPDE adducts are photoactivated to a more mutagenic lesion, or (ii) the presence of UV lesions lead to the BPDE adducts becoming more mutagenic.

CD40L/interleukin-4 (IL-4) stimulation occurs in vivo in the tumor microenvironment and induces global translation to varying degrees in individuals with chronic lymphocytic leukemia (CLL) in vitro. However, the implications of CD40L/IL-4 for the translation of specific genes is not known. To determine the most highly translationally regulated genes in response to CD40L/IL-4, we carried out ribosome profiling, a next-generation sequencing method. Significant differences in the translational efficiency of DNA damage response genes, specifically ataxia-telangiectasia-mutated kinase (ATM) and the MRE11/RAD50/NBN (MRN) complex, were observed between patients, suggesting different patterns of translational regulation. We confirmed associations between CD40L/IL-4 response and baseline ATM levels, induction of ATM, and phosphorylation of the ATM targets, p53 and H2AX. X-irradiation was used to demonstrate that CD40L/IL-4 stimulation tended to improve DNA damage repair. Baseline ATM levels, independent of the presence of 11q deletion, correlated with overall survival (OS). Overall, we suggest that there are individual differences in translation of specific genes, including ATM, in response to CD40L/IL-4 and that these interpatient differences might be clinically important.

The distribution of DNA damage and repair is considered to occur heterogeneously across the genome. However, commonly available techniques, such as the alkaline comet assay or HPLC-MS/MS, measure global genome levels of DNA damage, and do not reflect potentially significant events occurring at the gene/sequence-specific level, in the nuclear or mitochondrial genomes. We developed a method, which comprises a combination of Damaged DNA Immunoprecipitation and next generation sequencing (DDIP-seq), to assess the induction and repair of DNA damage induced by 0.1 J/cm2 solar-simulated radiation at the sequence-specific level, across both the entire nuclear and mitochondrial genomes. DDIP-seq generated a genome-wide, high-resolution map of cyclobutane thymine dimer (T<>T) location and intensity. In addition to being a straightforward approach, our results demonstrated a clear differential distribution of T<>T induction and loss, across both the nuclear and mitochondrial genomes. For nuclear DNA, this differential distribution existed at both the sequence and chromosome level. Levels of T<>T were much higher in the mitochondrial DNA, compared to nuclear DNA, and decreased with time, confirmed by qPCR, despite no reported mechanisms for their repair in this organelle. These data indicate the existence of regions of sensitivity and resistance to damage formation, together with regions that are fully repaired, and those for which > 90% of damage remains, after 24 h. This approach offers a simple, yet more detailed approach to studying cellular DNA damage and repair, which will aid our understanding of the link between DNA damage and disease.