Abstract DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. We have used the topoisomerase I inhibitor SN38 to induce DNA damage in three p53 wild-type cell lines: the immortalized MCF10A, and tumorigenic HCT116 and U2OS. Both phospho-Chk1 and p53 were elevated to a similar extent in all three cell lines. However, the HCT116 and U2OS cells exhibited far less accumulation of p21waf1 protein. The p21waf1 mRNA was induced to a similar extent, and the protein half-life was similar (∼ 30 min) in all three cell lines. These results suggest that the two tumor cell lines have a reduced rate of translation of the p21waf1 mRNA. To obtain further insight into the regulation of p21waf1, we incubated the three cell lines with the non-genotoxic drug nutlin-3, which disrupts p53:MDM2 binding thereby inducing a p53 response. Nutlin-3 induced similar levels of p53 in the three cell lines, and these levels were comparable to those seen with SN38. While nutlin-3 had little additional impact on p21waf1 induction in MCF10A cells and the half-life remained at ∼ 30 min, there was a much stronger induction in HCT116 and U2OS cells which was attributed to a much longer protein half-life (∼ 80 to 90 min). Incubation of p53 mutant cells with Chk1 inhibitors such as SCH900776 can overcome SN38-induced cell cycle arrest and drive cells through a lethal mitosis. p53 wild-type cells can be protected from this lethal mitosis through induction of p21waf1. However, the defective p21waf1 induction in HCT116 and U2OS cells is insufficient to protect them from Chk1 inhibition. In contrast, incubation with nutlin-3 induced sufficient p21waf1 that the cells arrested in either G1 or G2. This arrest protected the cells from subsequent incubation with the S phase-specific DNA damaging agents SN38 and hydroxyurea, either alone, or when combined with checkpoint inhibition. This protection was not observed in MCF10A or HCT116 cells deleted for p21waf1, or in MDA-MB-231 cells that have mutant p53. The results demonstrate that some p53 wild-type tumors have defects in p21waf1 expression, and may retain sensitivity to a combination of DNA damage plus checkpoint inhibition. Furthermore, the results suggest nutlin-3 could be used to protect normal cells from these combinations such that therapy can be targeted selectively to p53-defective tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2973. doi:10.1158/1538-7445.AM2011-2973