Abstract While DNA damage response pathways are well characterized in cancer cells, much less is known about their status in normal cells. These pathways protect tumor cells from DNA damage and replication stress and consequently present potential therapeutic targets. However, a better understanding of the response of normal cells to DNA damage is needed before a role in cancer treatment can be pursued. Here we characterize the response of hTERT-immortalized normal human urothelial (NHU) cells to agents that disrupt DNA replication. In contrast to malignant cells, the ATR-CHK1 signaling cascade is activated late after treatment with a replication inhibitor in hTERT-NHU cells and these cells do not depend upon the ATR-CHK1 signaling cascade for protection from apoptosis during DNA replication stress. Instead ATM signaling is more rapidly activated under these conditions. Intriguingly, an ATM inhibitor not only suppressed checkpoint activation after exposure to replication inhibitors but also stopped entry of cells into S phase suggesting activation of a G1 checkpoint. Consistent with this, cells treated with the ATM inhibitor and thymidine showed increased levels of cyclin-dependent kinase inhibitor p19INK4D and reduced phosphorylation of the retinoblastoma protein. Nevertheless ATM inhibitor-treated hTERT-NHU cells re-entered S-phase upon release into inhibitor-free medium. In contrast, a bladder cancer cell line treated with the ATM inhibitor progressed more slowly through S-phase and showed a dramatic increase in apoptosis following co-treatment with ATM and replication inhibitors. Taken together, our findings suggest that ATM and CHK1 signaling cascades play different roles in tumor and normal epithelial cells, confirming these as promising therapeutic targets. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B61. Citation Format: Wei-Ting Wang, James Catto, Mark Meuth. Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B61.