Abstract Head and neck squamous cell carcinoma (HNSCC) is the fifth leading cause of cancer worldwide and often requires complex, multimodality therapy, frequently including radiation therapy. The majority of deaths from HNSCC are due to loco-regional failure, which is largely dependent on the response of a tumor to external beam radiation therapy (EBRT). Currently, there are no biomarkers predictive of radio-sensitivity in HNSCC. One potential biomarker is TP53, the most commonly altered gene in HNSCC, whose alterations have shown to be prognostic of poor survival. Since TP53 plays a key role in response to DNA damage, a mechanism of EBRT, we hypothesized that HNSCC cell lines having mutant p53 (mutp53) or lacking wild-type p53 (wtp53) expression will be more resistant to EBRT. To test our hypothesis, we used isogenic paired cell lines with endogenous wtp53 and mutp53 genes and also, generated isogenic cell lines harboring either wtp53 or 12 different p53 mutants in a p53 null HNSCC cell line background. Clonogenic assays showed that the 4Gy surviving fraction (SF4) in cells with mutp53 was significantly higher compared to HNSCC cells with wtp53. This radio-resistance was associated with a lack of radiation-induced senescence. Staining with senescence associated β-galactosidase (SA-β-gal), showed that after treatment with EBRT, wtp53 cells had an average of 50% positive staining compared to <5% SA-β-gal staining in mutp53 cells. In addition, radiation treatment of wtp53 cells led to induction of CDKN1A (p21Waf1/Cip1), and knockdown of endogenous p21 in wtp53 cells inhibited EBRT-induced senescence of HNSCC cells, indicating the critical role of p21 in the therapeutic response to radiation. In addition to causing double stranded DNA breaks, thus activating the p53 cascade, EBRT also generates reactive oxygen species (ROS) which have been implicated in causing senescence. The use of DCFDA, a fluorescent intracellular marker of ROS, showed a significantly higher initial burst of ROS levels after ERBT in wtp53 cells compared to mutp53 cells. In addition, the senescence seen after ERBT could be abrogated by use of the antioxidant, N-acetyl cysteine. Furthermore, the treatment of wtp53 HNSCC cells with hydrogen peroxide caused a significant level of SA-β-gal staining and increased p21 protein levels. Taken together, these findings suggest a model of EBRT induced ROS leading to p21 induction and subsequent senescence that is disrupted by the loss of wtp53 expression. Therefore, treatments which could induce p21 expression in HNSCC cells deficient of wtp53 may potentially sensitize these radiation-resistant cells to EBRT. In preliminary studies, we have found that the HDAC inhibitor, Vorinostat, can upregulate p21 transcription independent of p53, and sensitize mutp53 cells to ERBT. Citation Format: Alison Fitzgerald, Abdullah Osman, David Neskey, Ameeta Patel, Heath Skinner, Timothy McDonnell, Jeffrey Myers. Reactive oxygen species and p21Waf1/Cip1 are essential for radiation-induced accelerated senescence of head and neck cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 432. doi:10.1158/1538-7445.AM2013-432