Potent iron chelators increase the mRNA levels of the universal cyclin-dependent kinase inhibitor p21CIP1/WAF1, but paradoxically inhibit its translation: A potential mechanism of cell cycle dysregulation

Abstract

Iron (Fe) chelators are potential antitumor agents. Cellular Fe depletion results in a G1/S arrest but the precise molecular mechanisms involved remain unclear. Recent studies have shown that this process is complex with multiple cell cycle molecules being involved. We previously showed that Fe chelators such as 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) were far more potent antitumor agents than the clinically used ligand, desferrioxamine (DFO). To further characterize the effects of chelators on cell cycle arrest, we compared their activity with the DNAd-amaging agents actinomycin D (Act D) and cisplatin (CP). These latter two compounds increase the expression of p53 and its target genes such as the universal cyclin-dependent kinase inhibitor, p21CIP1/WAF1. Incubation of normal and neoplastic cells with all agents resulted in increased nuclear p53, the effect being pronounced for Act D and CP. As expected, both Act D and CP also markedly increased nuclear P21CIP1/WAF1 protein levels, while DFO and 311 caused a significant (P < 0.0004) decrease. This latter effect was surprising, as these chelators markedly increased mRNA levels of this molecule. Immunofluorescence studies showed that Act D and CP caused nuclear localization of p21CIP1/WAF1. In contrast, the chelators prevented translation of p21CIP1/WAF1. This did not appear to be due to a general effect of the chelators on preventing translation, as transferrin receptor 1 was markedly upregulated 15- to 21-fold by DFO and 311. Combination of 311 with Act D or CP prevented translation of p21CIP1/WAF1 and its nuclear localization observed with these DNA-damaging agents. Significantly, the effect of chelation on reducing nuclear p21CIP1/WAF1 was reversed by the Fe donor ferric ammonium citrate, indicating that p21CIP1/WAF1 translation was dependent on intracellular Fe levels. This study demonstrates that while Fe chelators markedly up-regulate the mRNA levels of p21CIP1/WAF1 they paradoxically inhibit translation.