Results Intense and prolonged ER stress induces p53 protein expression Many studies have indicated that p53 tumor suppressor is one of the central players in the response of cells to various forms of stress. MCF-7 human breast adenocarcinoma cells were treated with tunicamycin or brefeldin A, and no changes in p53 protein level in the control cells were observed (Fig. 1A, 1B, upper panel). In contrast, when MCF-7 cells were incubated with tunicamycin or brefeldin A, an increase in the level of p53 was observed in a dose and time-dependent manner. In a dose- dependent manner, p53 protein started to increase in 1 mg/ml of tunicamycin and 0.025 mg/ml of brefeldin A (Fig. 1A, 1B, upper panel). In a time-dependent manner, the level of p53 protein was induced at 12–48 h after treatment with tunicamycin (Fig. 1A lower panel) and the p53 level at 24 hr was approximately 3-fold higher than that expressed at 0 hr. Similarly, using another ER stress inducer, brefeldin A, p53 protein was significantly induced with time up to 48 hr. For example, the p53 level at 48 hr was enhanced up to about 4-fold compared with 0 hr (Fig. 1B Lower panel). Expression of GRP78 protein was used to check that treatments in our system indeed resulted in ER stress. Notably, we observed that under the tunicamycin or brefeldin A treatment, p53 protein was first downregulated at the early time course point of 3 and 6 hr before its expression was increased. This finding is consistent with previous studies showing that mild ER stress increased cytoplasmic localization and degradation of endogenous p53 in human primary WI-38 cells and HT1080 cells [30] and led to downregulation of p53 protein in human lung adenocarcinoma A549 cells [26]. Furthermore, we demon- strated that upregulation of p53 expression by ER stress is not restricted to a particular cell line. In the human cervical cancer Hela cells, treatment of tunicamycin or brefeldin A also induced p53 expression to the extent as seen in the MCF-7 cells (Fig. 1C). All these results suggested that increase of p53 protein expression level by ER stress is a general phenomenon that can occur in a variety of human cells regardless of stimulus types that initiate this process. Induction of p53 is transcription-dependent To establish the mechanism of p53 expression by ER stress, protein biosynthesis and transcription inhibitors were used in brefeldin A-induced p53 expression in MFC-7 cells. A treatment of a low dose of cycloheximide (1.25 mg/ml) was sufficient to reduce ER stress-activated p53 protein expression, almost 100% to its basal level (Fig. 2A). In addition, we also used transcriptional inhibitor, actinomycin D, to examine whether the event of induction of p53 is transcription-dependent. MFC-7 cells were co- treatment of actinomycin D with brefeldin A and, we used reverse transcription-PCR to analyze the expression levels of p53 mRNA. The p53 mRNA levels were dramatically decreased by actinomy- cin D in response to ER stress (Fig. 2B). Furthermore, by the analyses of conventional and real-time RT-PCR, we observed that p53 expression was already increased at an mRNA level when MCF-7 cells were treated with tunicamycin or brefeldin A (Fig. 2C, 2D). These data together suggested that the elevated expression of p53 protein during ER stress, at least partially, is contributed by increasing the expression of p53 transcripts through a regulation on p53 promoter activity. Regulation of p53 Expression by NF-kB in ER Stress Figure 1. p53 expression is elevated in response to endoplas- mic reticulum stress. (A) and (B) present the dose- and time- dependent effect of ER stress inducer, tunicamycin and brefeldin A, on p53 expression. MCF-7 Cells were exposed to tunicamycin and brefeldin A in 10% FBS–supplemented DMEM as time and dose indicated. The cell lysates were analyzed by western blotting with antibodies for p53, GRP78, and a-tubulin. The p53 protein expression level was quantified densitometrically. GRP78 served as an ER stress marker. (C) Induction of p53 expression by ER stress in three different cell lines. Hela cells were treated with 5 mg/ml tunicamycin in 10% FBS–supplemented DMEM as time indicated. The cell lysates were analyzed by western blotting with specific antibodies for p53 and b-actin. doi:10.1371/journal.pone.0039120.g001 ER stress enhances nuclear localization and phosphorylation of p53, as well as p53-mediated transcriptional activation The main function of p53 protein is work as a transcription factor that regulates its target genes to mediate cellular activities. Nuclear localization and phosphorylation of p53 on its N-terminal transactivation domain are both essential for p53 function [31,32]. Therefore, we next investigated the distribution of p53 in MCF-7 cells following the treatment of ER stress inducers. As indicated in Fig. 3A, very little p53 was detected in the untreated cells; however, p53 signal was significantly enhanced and presented exclusively in nuclei in the tunicamycin or brefeldin A-treated cells. To validate this microscopically visualized p53 localization, total cell lysates collected from MCF-7 cells were fractionated to separate cytoplasmic and nuclear components, and a western blotting analysis was performed to measure p53 protein level in each subcellular compartment. The majority of p53 protein was found to reside in nuclei. A sharp accumulation of p53 in the nuclear fractions was detected when cells were treated with brefeldin A for 12 and 24 hours (Fig. 3B). This result agreed with the immunostaining data shown in Fig. 3A, exhibiting a clear nuclear localization of p53 induced by ER stress. Subsequently, we examined phosphorylation status of p53 to see whether any serine residue on the N-terminus of p53 is phosphorylated when ER stress was induced. To this end, MCF-7 cells were treated with brefeldin A, and phosphorylation of p53 was analyzed by western blotting using p53 phospho-specific antibodies. Based on the fact that UV light can induce p53 phosphorylation at multiple sites PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e39120