It is widely accepted that the p53 tumor suppressor restricts abnormal cells by induction of growth arrest, or by triggering apoptosis. Here we show that in addition p53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause for DNA damage and genetic instability. In the absence of severe stresses relatively low levels of p53 are sufficient for up-regulation of several antioxidant genes, which is associated with a decrease in intracellular ROS. Down-regulation of p53 results in excessive oxidation of DNA, increased mutation rate, and karyotype instability, which are prevented by incubation with antioxidant N-acetylcysteine (NAC). Dietary supplementation with NAC prevents frequent lymphomas characteristic to p53 knockout mice, and slows down growth of xenografts from A549 cells with p53 inhibited by siRNA. Our results provide novel paradigm for a non-restrictive tumor suppressor function of p53 and highlight potential importance of antioxidants in prophylactics and treatment of cancer.The major function for the p53 tumor suppressor is to restrict abnormal or stress-exposed cells before damage to DNA is converted to inherited mutation 1 . However, even without extended stress the DNA is exposed to endogenous damaging reactive oxygen species (ROS), which are by-products of normal respiration, and important signaling molecules 2,3 . Indeed, endogenous ROS is the major source of DNA damage 4 , and a substantial factor contributing to chromosome instability and accumulation of mutations and deletions leading to cancer 4,5 . As the endogenous ROS modify approximately 20,000 bases of DNA per day in a single cell 6 it is unlikely that the restriction of proliferation of cells with oxidized DNA would be efficient in preventing mutations. Previously it was found that among transcriptional targets of p53 there are several potential ROS-generating genes whose action presumably contributes to p53-mediated cell death 7,8 . However, other p53-upregulated genes, such as glutathione peroxidase (GPX1) 9,10 , Mn-superoxide dismutase (Mn-SOD) 10 and aldehyde dehydrogenase 4 (ALDH4) 11 , would presumably act as antioxidants. In addition, the function of two p53-regulated sestrins (HI95 and PA26) is essential for regeneration of over-oxidized peroxiredoxins 12 , the enzymes involved in the decomposition of hydrogen peroxide 3 . These findings suggest that p53 might play opposite roles in ROS regulation. In this study we Results Down-regulation of p53 elevates intracellular ROSTo reveal effects of p53 on ROS levels in non-stressed cells we inhibited p53 by lentiviralmediated expression of siRNA in a set of human normal and carcinoma cell lines, which have functional p53 ( Supplementary Fig. 1a,b online). Analysis of dichlorodihydrofluorescein (DCF) staining revealed approximately 2-fold increases in ROS 48 h after inhibition of p53 (Fig. 1a). The increase in ROS induced by siRNA to p53 was similar in magnitude to that observed after treatment of RKO cells with hydrogen peroxide and was completely re...