International Journal of Molecular and Cellular Medicine (IJMCM)

The DNA damage response (DDR) safeguards genomic integrity through lesion sensing, checkpoint activation, and repair. While ATM/ATR-centered DDR signaling is well defined, how growth-associated pathways such as MAPK/ERK and PI3K/AKT co-engage with DDR programs across distinct genotoxic stresses remains incompletely resolved. U2OS and HCT116 cells were challenged with ionizing radiation (4 Gy), replication stress (hydroxyurea, 2 mM), or oxidative stress (H₂O₂, 200 µM, 30 min). DDR and growth-pathway phosphorylation kinetics were quantified by immune-blot densitometry (phosphor /total; fold-change vs. 0 h vehicle baseline). Replication fork dynamics were assessed by DNA fiber assays under HU with ATR or PI3K inhibition. DNA damage burden was measured by alkaline comet tail moment and 53BP1 foci at 6 h and 24 h. Long-term proliferative capacity was quantified by clonogenic survival (SF4) after IR. Pharmacologic perturbations included trametinib (MEK), BKM120 (PI3K), VE-821 (ATR), NU7441 (DNA-PK), and olaparib (PARP). Analyses used biological replicate–level inference. Stressors produced expected checkpoint signatures (IR: ATM–CHK2; HU: ATR–CHK1) with temporally overlapping ERK and AKT phosphorylation changes. ATR inhibition markedly increased fork stalling and reduced fork progression under HU. PI3K inhibition was associated with higher early DNA-damage readouts (comet tail moment and 53BP1 foci at 6 h) and produced larger reductions in clonogenic survival after IR than MEK inhibition in both cell lines. Canonical DDR activation and growth-signaling phosphorylation changes co-occur after genotoxic, replication, and oxidative stress and are associated with distinct functional outcomes: ATR predominates in fork stability, whereas PI3K activity is linked to early damage burden and post-IR survival. These findings motivate mechanistic follow-up to define causal nodes for combination targeting in genomically unstable cells.