Palmitoyl ascorbic acid glucoside enhanced cell survival with post irradiation treatment.

Radiation exposure poses a significant threat to cellular integrity by inducing DNA damage through the generation of free radicals and reactive oxygen species. Ascorbic acid, particularly its derivative Palmitoyl Ascorbic Acid 2-Glucoside (PA2G), has demonstrated remarkable radioprotective properties. While previous research focused on its pre-irradiation application, this study explores the post-irradiation radiomitigation potential of PA2G. Our findings reveal that post-irradiation treatment with PA2G enhances cell survival and accelerates DNA repair processes, particularly the non-homologous end-joining (NHEJ) repair pathway. Notably, PA2G treatment reduces the frequency of lethal chromosomal aberrations and micronuclei formation, indicating its ability to enhance the repair of complex DNA lesions. Furthermore, PA2G is shown to play a role in potentially lethal damage repair (PLDR). These radioprotective effects are specific to NHEJ and ATM pathways, as cells deficient in these mechanisms do not benefit from PA2G treatment. This study highlights PA2G as a versatile radioprotector, both pre- and post-irradiation, with significant potential for applications in radiation therapy and protection, offering new insights into its mechanism of action. Further research is required to elucidate the precise molecular mechanisms underlying PA2G's radiomitigation effects and its potential clinical applications.

ATM dysfunction in Chinese hamster XRCC8 mutants.

XRCC8 is a member of the X-ray cross-complementing (XRCC) family, whose responsible gene has not been identified. Previous studies suggested ATM and other genes were potential candidates for XRCC8, but this was not confirmed. In this study, we characterized three V79-derived XRCC8 mutant cells: V-C4, V-E5, and V-G8. Western blot analysis showed reduced expression of the ATM protein in three XRCC8 mutants, and radiation-induced phosphorylated ATM foci were not detected by fluorescence immunocytochemistry. Both ATM knockout cells and XRCC8 mutants exhibited hypersensitivity to camptothecin. Through a cell fusion-based complementation test, we found that XRCC8 mutants were complemented by ATM-proficient cells, but not by ATM knockout cells, in terms of camptothecin sensitivity. Comprehensive sequencing of the ATM genome in XRCC8 mutants revealed unique mutations in each mutant. These results suggest that XRCC8 mutants carry ATM mutations, and their ATM is not properly functional, despite protein expression being detected. This is similar to missense mutations in some Ataxia Telangiectasia patients.

Simultaneous inhibition of ATM, ATR, and DNA-PK causes synergistic lethality.

Here we report that simultaneous inhibition of the three primary DNA damage recognition PI3 kinase-like kinases (PIKKs) -ATM, ATR, and DNA-PK- induces severe combinatorial synthetic lethality in mammalian cells. Utilizing Chinese hamster cell lines CHO and V79 and their respective PIKK mutants, we evaluated effects of inhibiting these three kinases on cell viability, DNA damage response, and chromosomal integrity. Our results demonstrate that while single or dual kinase inhibition increased cytotoxicity, inhibition of all three PIKKs results in significantly higher synergistic lethality, chromosomal aberrations, and DNA double-strand break (DSB) induction as calculated by their synergy scores. These findings suggest that the overlapping redundancy of ATM, ATR, and DNA-PK functions is critical for cell survival, and their combined inhibition greatly disrupts DNA damage signaling and repair processes, leading to cell death. This study provides insights into the potential of multi-targeted DDR kinase inhibition as an effective anticancer strategy, necessitating further research to elucidate underlying mechanisms and therapeutic applications.

ATM and ATR gene editing mediated by CRISPR/Cas9 in Chinese Hamster cells.

Chinese hamster-derived cell lines including Chinese hamster lung fibroblasts (V79) have been used as model somatic cell lines in radiation biology and toxicology research for decades and have been instrumental in advancing our understanding of DNA damage response (DDR) mechanisms. Whereas many mutant lines deficient in DDR genes have been generated more than over decades, several key DDR genes such as ATM and ATR have not been established in the Chinese hamster system. Here, we transfected CRISPR/Cas9 vectors targeting Chinese hamster ATM or ATR into V79 cells and investigated whether the isolated clones had the characteristics reported in human and mouse studies. We obtained two clones of ATM knockout cells containing an insertion or deletions in the targeted locus. The ATM knockouts with no detectable ATM protein expression exhibited increased sensitivity to radiation and DNA double strand break inducing agents, cell cycle checkpoint defects and defective chromatid break repair. These are all characteristics of defective ATM function. Among the obtained ATR cells, which contained mutations in both ATR alleles while maintaining normal levels of ATR protein expression, one clone exhibited hypersensitivity to UV and replication stress agents. In the present study, we successfully established CRISPR-Cas9 derived ATM knockout cells. We couldn't knock out the ATR gene but obtained ATR mutant cells. Our results showed that Chinese hamster origin ATM knockout cells and ATR mutant cells could be useful tools for further research to reveal oncogenic functions and effects of developing anti-cancer therapeutics.