PARP1 Knockout Cell Line (A549)

PURPOSE:Proton beam radiation therapy (PBT) offers superior accuracy of dose deposition, reducing the risk of adverse effects to surrounding healthy tissues. However, despite high medical need, molecular and cellular determinants of radiosensitivity to PBT remain underexplored, and prognostic biomarkers and therapeutic targets informing precision medicine strategies for PBT are mostly missing. This study aimed to investigate the role of DNA double-strand break (DSB) repair pathways in shaping tumor response to proton versus photon radiation. METHODS AND MATERIALS:The study employed genetic and pharmacologic methods to impair DSB repair, including CRISPR-Cas9 gene editing to generate DSB repair deficient (ATM, PARP1, and BRCA2 knockout) A549 and HCT116 cell lines, and pharmacologic inhibitors of ATM and PARP using KU55933 or AZD1390 and olaparib, respectively. Cellular responses to photon (x-rays) and proton irradiation were evaluated through clonogenic survival assays, crystal violet proliferation, and annexin V/7AAD apoptosis assays. To investigate DNA repair mechanisms, U2OS reporter systems were employed, complemented by chromosomal aberration analysis, and pulsed-field gel electrophoresis. Finally, the translational relevance of the findings was validated using the chorioallantoic membrane assay closer representing an in vivo situation. RESULTS:PBT triggered a stronger activation of resection-dependent DNA repair pathways, primarily homologous recombination and alternative end-joining (alt-EJ), compared with photon irradiation. This increased activation was further supported by classical cytogenetics results. Tumor cells deficient in BRCA2, ATM, or PARP1 showed significantly increased sensitivity to PBT, highlighting enhanced relative biological effectiveness in both, in vitro and in the chorioallantoic membrane model. Importantly, combining PBT with olaparib, AZD1390 or KU55933 potentiated tumor cell killing, even in repair-proficient models, showing synergy not observed with photons. CONCLUSIONS:The observed genotype-specific or drug-induced increase in radiosensitivity toward PBT highlights the promise of genetic profiling of DSB repair defects for biology-driven patient stratification and the use of PARP inhibitors in guiding personalized proton radiation therapy strategies.

As a group I carcinogen, environmental exposures to formaldehyde (FA) have been associated with various types of malignancies. However, exact mechanisms of FA-triggered carcinogenesis are still not clear. Lactylation is recently identified as a post-translational modification driven by overproduced lactic acid (LA) that regulates protein activities in different cellular processes. Our previous studies clearly demonstrated that environmentally relevant levels of FA could elevate LA in tumor cells. Poly (ADP-ribose) polymerase 1 (PARP1) is a major player in DNA repair and tumor cell survival, which has been shown to be activated by lactylation. In order to examine if PARP1 lactylation is promoted by FA environmental exposure, subcutaneous tumor models were established using BALB/c nude mice, which were exposed to 2.0 mg/m FA for 14 days. FA significantly elevated LA concentrations (p = 0.011) in the tumor tissues, which was confirmed in A549 cells treated with 100 μM FA in vitro. Both activity and lactylation of PARP1 were found to be induced by FA, which also enhanced DNA repair and tumor-promotive functions in vitro. Inhibition of LA production through lactate dehydrogenase A (LDHA) knockout reduced FA-potentiated PARP1 lactylation and activity. Collectively, these results revealed for the first time that FA promoted tumor cell growth through enhanced PARP1 lactylation, which could be the underlying mechanism of FA-related carcinogenesis.