Bars indicate the standard error of the mean. Student’s t-test was performed. ns = not significant. Three independent experiments were performed. The expression of p21 (also known as Cip1 and WAF1) in response to genotoxic stress is tightly regulated by p53 (reviewed in ), and we therefore measured it as an additional indicator of p53 activity. The fraction of p21-positive cells was approximately doubled by selenite treatment (Figure 2F–J). Although these changes are statistically significant, the positive fraction Trametinib was very small even after selenite treatment. As a positive control, epithelioid cells were treated
with 2 μM doxorubicin and showed a 22% positive fraction (not shown). Cells of either phenotype treated with the p53 inhibitor Pifithrin did not show a decreased apoptosis frequency as judged by Annexin-PI (Figure 1), nor a smaller loss of δΦm (Table 2). This is particularly interesting since p53 inhibition decreased the baseline apoptosis in untreated cells (Figure
1, Additional file 1). Consequently, p53 was active in the control cells but was inactivated by selenite. Apoptosis was still induced by selenite, implicating p53-independent pathways in this process. To find the mechanism of inhibition, we considered the complex regulation of p53 activity. The central DNA-binding Selleck BIBW2992 core domain of p53 contains one zinc atom. Zinc chelators have been shown to cause accumulation of wild-type p53 in a structurally aberrant form with inhibited DNA-binding activity . Selenium is a known chelator of zinc and when applied in vivo as selenite or Benzatropine its reduced form selenide, it forms nanocrystals
of zinc-selenium with free or loosely bound zinc . Another possibility is that selenite as an oxidizing agent may act directly upon regulatory cysteines on the p53 molecule, leading to an accumulation of oxidized p53 incapable of DNA-binding . Also, secondary mediated redox regulation needs to be considered. The multifunctional protein Redox Effector Factor 1 (Ref-1) is involved in the redox regulation of stress inducible transcription factors such as Activating Protein-1, Nuclear Factor-κB, Hypoxia Inducible Factor-1 and p53, and may play an important role in this system. Ref-1 depends on thioredoxin (Trx) to maintain its active reduced state [49–51]. In a yeast experimental system, it has been shown that deletion of thioredoxin reductase (TrxR) downregulates p53 activity by keeping it in its oxidized form [52, 53]. Trx overexpression on the other hand has been shown to increase p53 transactivation of reporter genes in human cell lines . Protein levels of Trx were reduced by selenite treatment in sarcomatoid cells, from 175 ng/mg to 100 ng/mg. The epithelioid cells had a baseline expression of 57 ng/mg, decreasing slightly to 52 ng/mg after selenite treatment (Figure 3).