Simultaneous induction of dispersed and clustered DNA lesions compromises DNA damage response in human peripheral blood lymphocytes

Abstract

Due to its ability to induce DNA damage in a space and time controlled manner, ionising radiation is a unique tool for studying the mechanisms of DNA repair. The biological effectiveness of ionising radiation is related to the ionisation density which is defined by the linear energy transfer (LET). Alpha particles are characterised by high LET, while X-rays by low LET values. An interesting question is how cells react when exposed to a mixed beam of high and low LET radiation. In an earlier study carried out with human peripheral blood lymphocytes (PBL) we could demonstrate that alpha radiation X-rays interact in producing more chromosomal aberrations than expected based on additivity. The aim of the present investigation was to look at the mechanism of the interaction, especially with respect to the question if it is due to an augmented level of initial damage or impaired DNA repair. PBL were exposed to various doses of alpha particles, X-rays and mixed beams. DNA damage and the kinetics of damage repair was quantified by the alkaline comet assay. The levels of phosphorylated, key DNA damage response (DDR) proteins ATM, p53 and DNA-PK were measured by Western blotting and mRNA levels of 6 damage-responsive genes were measured by qPCR. Alpha particles and X-rays interact in inducing DNA damage above the level predicted by assuming additivity and that the repair of damage occurs with a delay. The activation levels of DDR proteins and mRNA levels of the studied genes were highest in cells exposed to mixed beams. The results substantiate the idea that exposure to mixed beams presents a challenge for the cellular DDR system.

Impact of ATM and DNA-PK Inhibition on Gene Expression and Individual Response of Human Lymphocytes to Mixed Beams of Alpha Particles and X-Rays

“…Overall, the first set of experiments displayed clear synergy for most donors, also when the sum of the four donors was analysed. This result is in agreement with our earlier results on synergy in human PBL using micronuclei [22], chromosomal aberrations [23], comet assay and radiation-induced gene expression [18] as endpoints. Synergy was also detected at the level of 53BP1 foci and the activation of DNA repair proteins using human U2OS osteosarcoma cells [20] and as the hindered mobility of foci in mixed-beam-exposed cells as demonstrated by live cell imaging [21].…”

“…Blood collection and irradiation methods were modified from earlier work [18]. Fresh peripheral blood was collected by venepuncture in heparin blood collection tubes (VACUTEST Kima, Arzergrande, Italy) from four healthy male non-smokers, just before experiments (we have an ethical permit with the number 2010/27-31/1 from the Ethical Committee at the Karolinska University Hospital).…”

“…The 2 −∆∆Ct method was used for calculation of relative mRNA expression, and melting curve analysis was used for testing primer specificity. The same primers were used as previously [18]: BBC3 for: TACGAGCGGCGGAGACAAGA, BBC3 rev: GCAGGAGTCCCATGATGAGATTGTAC; FDXR for: TGGATGTGCCAGGCCTCTAC, FDXR rev: TGAGGAAGCTGTCAGTCATGGTT; GADD45A for: ACTGCGTGCTGGTGACGAAT, GADD45A rev: GTTGACTTAAGGCAGGATCCTTCCA; XPC for: GCTTGGAGAAGTACCCTACAAGATGGT, XPC rev: GGCTTTCCGAGCACGGTTAGA; MDM2 for: TATCAGGCAGGGGAGAGTGATACA, MDM2 rev: CCAACATCTGTTGCAATGTGATGGAA; CDKN1A for: CCTGGAGACTCTCAGGGTCGAAA, CDKN1A rev: GCGTTTGGAGTGGTAGAAATCTGTCA. For 18S, sequences are given in [40].…”

“…IR-induced clustered damage two of the donors. The study is a continuation of our analyses of cellular effects of simultaneous exposure to alpha radiation and X-rays [18,[20][21][22][23]].…”

Hadrontherapy Interactions in Molecular and Cellular Biology

“…At the cellular level, the response to radiation modalities appears to differ due to the quality and quantity of the resulting DNA damage, for example-not to the particle itself. Among protein biomarkers, such as pP53, pATM, MDM2, and XPC, only p-DNA PKC is upregulated after alpha versus gamma radiation [110]. Perhaps by examining the complexity of DNA damage using, for example, gamma-H2AX foci in combination p-DNA PKcs, one could estimate the quality of the radiation that targets the cell.…”