Post-transcriptional Modulation of Iron Homeostasis during p53-dependent Growth Arrest

Abstract

Iron plays an essential role in cell proliferation and is a required cofactor for a number of critical cellular enzymes. In this report we investigate changes in proteins of iron metabolism during p53-mediated replicative arrest. Following the induction of p53 in H1299 lung cancer cells containing a doxycycline-inducible p53, an increase in both H and L subunits of ferritin protein was observed. To determine the mechanism of this effect, we investigated the ability of p53 to regulate ferritin. Real time reverse transcription-PCR demonstrated no difference in levels of ferritin H mRNA in the presence and absence of p53. Because these results suggested that transcriptional mechanisms were not responsible for the p53-dependent increase in ferritin, we tested whether a post-transcriptional mechanism was involved. RNA bandshift assays revealed that induction of p53 decreased iron regulatory protein binding. Consistent with this observation, Western blot analysis revealed a decline in transferrin receptor 1 protein levels following induction of p53. Collectively, these results suggest that p53 may induce cell cycle arrest not only by well described mechanisms involving the induction of cyclin-dependent kinase inhibitors but also by the recruitment of pathways that reduce the availability of intracellular iron.The p53 tumor suppressor protein is a critical mediator of cell cycle arrest and apoptosis. p53 is a nuclear transcription factor that is activated by cellular stress, including DNA damage, hypoxia, ribosomal stress, and loss of adhesion (reviewed in Ref. 1). Following its induction, p53 transcriptionally induces a set of genes that can promote growth arrest, cell death, or senescence, including p21 waf1 , bax, fas, and KILLER/DR5 (2). p53 can also function as a transcriptional repressor (3). Whether a cell undergoes cell cycle arrest or apoptosis in response to p53 depends on several factors, including the presence of extracellular survival factors, the presence of other oncogenic alterations, and the availability of additional transcription factors or cofactors, as well as the character and magnitude of p53 induction (4 -6). In addition to its role as a transcription factor, p53 exhibits transcription-independent functions in the cytosol that augment its pro-apoptotic activity (1). Although an increase in reactive oxygen species may contribute to p53-mediated apoptosis (7), p53 can also play a role as an antioxidant (8) by inducing genes that reduce reactive oxygen species, such as TIGAR (9), sestrins (10), and ALDH4 (11).Iron is critically important for cell growth and proliferation. Iron is required for the activity of ribonucleotide reductase, the rate-limiting enzyme for de novo DNA synthesis (12). It is also an important cofactor of the G 1 phase cyclins E/cdk2, D/cdk5 (13), D1 (14), and the S phase cyclin A/cdk2 (15), as well as numerous proteins involved in respiration and energy metabolism. Accordingly, iron deprivation inhibits proliferation. For example, small molecule iron chelators that sequester ir...

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