George DD Jones

Aim: In response to DNA damage the serine/threonine-specific protein kinase checkpoint kinase 1 (CHK1) is activated allowing cells to enter S phase (S) and G2 phase (G2) cell-cycle arrest. CHK1 inhibitors are expected to prevent cells from entering such arrest, thereby enhancing DNA damage-induced cytotoxicity. In contrast, normal cells with intact ataxia–telangiectasia mutated (ATM), CHK2 and tumour suppressor protein 53 (P53) signalling are still able to enter cell-cycle arrest using the functioning G1/S checkpoint, thereby being rescued from enhanced cytotoxicity. The main objective of this work is to investigate the in vitro effects of the novel CHK1 inhibitor SRA737 on pairs of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines, all with genetic aberrations rendering them susceptible to replication stress but of differing tumour protein 53 (TP53) gene status, focusing on DNA damage induction and the subsequent effects on cell proliferation and viability. Methods: NSCLC cell lines H23 [TP53 mutant (MUT)] and A549 [TP53 wild-type (WT)] and CRC cell lines HT29 (TP53 MUT) and HCT116 (TP53 WT) were incubated with differing micromolar concentrations of SRA737 for 24 h and then analysed using alkaline comet and phosphorylated H2A.X variant histone (γH2AX)-foci assays to assess mostly DNA single strand break and double strand break damage, respectively. Cell-counting/trypan blue staining was also performed to assess cell proliferation/viability. Results: Clear concentration-dependent increases in comet formation and γH2AX-foci/cell were noted for the TP53 MUT cells with no or lower increases being noted in the corresponding TP53 WT cells. Also, greater anti-proliferative and cell killing effects were noted in the TP53 MUT cells than in the TP53 WT cells. Conclusions: This study’s data suggests that P53 status/functioning is a key factor in determining the sensitivity of NSCLC and CRC cancer cells towards CHK1 inhibition, even in circumstances conducive to high replicative stress.

Numerous studies have demonstrated the normal tissue-sparing effects of ultra-high dose rate ‘FLASH’ irradiation in vivo, with an associated reduction in damage burden being reported in vitro. Towards this, two key radiochemical mechanisms have been proposed: radical–radical recombination (RRR) and transient oxygen depletion (TOD), with both being proposed to lead to reduced levels of induced damage. Previously, we reported that FLASH induces lower levels of DNA strand break damage in whole-blood peripheral blood lymphocytes (WB-PBL) ex vivo, but our study failed to distinguish the mechanism(s) involved. A potential outcome of RRR is the formation of crosslink damage (particularly, if any organic radicals recombine), whilst a possible outcome of TOD is a more anoxic profile of induced damage resulting from FLASH. Therefore, the aim of the current study was to profile FLASH-induced damage via the Comet assay, assessing any DNA crosslink formation as a putative marker of RRR and/or anoxic DNA damage formation as an indicative marker of TOD, to determine the extent to which either mechanism contributes to the “FLASH effect”. Following FLASH irradiation, we see no evidence of any crosslink formation; however, FLASH irradiation induces a more anoxic profile of induced damage, supporting the TOD mechanism. Furthermore, treatment of WB-PBLs pre-irradiation with BSO abrogates the reduced strand break damage burden mediated by FLASH exposures. In summary, we do not see any experimental evidence to support the RRR mechanism contributing to the reduced damage burden induced by FLASH. However, the observation of a greater anoxic profile of damage following FLASH irradiation, together with the BSO abrogation of the reduced strand break damage burden mediated by FLASH, lends further support to TOD being a driver of the reduced damage burden plus a change in the damage profile mediated by FLASH.