Abstract A67: Mechanisms involved in regulating the expression of the cyclin-dependent kinase (cdk) inhibitor, p21, by intracellular iron levels.

Iron is known to be important for cell cycle progression and intracellular iron (Fe) chelation has been shown to down-regulate the expression of the cyclin-dependent kinase inhibitor, p21 CIP1/WAF1 (p21) in MCF-7 breast cancer cells [1]. p21 has been demonstrated to have both positive and negative effects on G 1 /S cell cycle progression and regulate cancer cell apoptosis and senescence [2, 3]. Our preliminary studies have suggested that Fe-depletion affects p21 expression differentially depending upon the cell type. Therefore, understanding the ability of Fe chelators to regulate the expression of p21 differently in cancer cells and the biological consequences thereof may provide an insight into the mechanisms involved in the anti-tumor efficacy of these agents. The current study examined the mechanisms involved in regulating p21 expression by Fe in a number of cancer cell-types with different p53 status. RT-PCR demonstrated an increase in p21 mRNA levels in all cells in response to Fe chelation. Western blotting demonstrated that the expression of p21 after incubation with chelators differed between the cell-types. Down-regulation in p21 protein expression was observed in the MCF-7 breast cancer and SK-N-MC neuroepithelioma cells, while up-regulation of p21 protein was evident in the SK-MEL-28 melanoma, CFPAC-1 pancreatic and LNCaP prostate cancer cell-types. However, the effect of the Fe chelator on p21 expression was not correlated to the p53 status of these cells. Furthermore, we showed that nuclear expression levels of p21 in cancer cells were much higher than cytoplasmic expression after Fe chelation. It has been shown that the variety of physiological roles that p21 plays, including cell cycle arrest, rely on its nuclear localisation [4]. These results were confirmed using cytoplasmic and nuclear fractionation, western analysis and immunofluorescence studies. The roles of MDM2 and Np63 in regulating p21 in response to Fe-depletion were also assessed, as these latter molecules can modulate p21 expression in a p53-independent manner [5, 6]. Results demonstrated that after incubation with Fe chelators, the expression pattern of the p75 isoform of MDM2 (p75 MDM2 ) closely resembled that of p21 in 4 of the 5 cell lines investigated. This indicated that MDM2 may be one pathway through which these drugs regulate p21 expression. There was no significant change in Np63 expression after incubating these cells with Fe chelators, suggesting that the alterations in p21 expression observed were independent of Np63. Further studies are focusing on the possible transcriptional regulation of p21 by Fe-depletion. This is being carried out by using p21 promoter constructs to decipher the region of the p21 promoter that is involved in the Fe-regulated expression of p21. Overall, these studies will provide significant insight into the mechanism of action of Fe chelators at the molecular level. References : 1. Fu, D. and D.R. Richardson, Blood, 2007. 110 (2): p. 752–61. 2. Ogryzko, V.V., et. al Mol Cell Biol, 1997. 17 (8): p. 4877–82. 3. Gartel, A.L. and S.K. Radhakrishnan, Cancer Res, 2005. 65 (10): p. 3980–5. 4. Child, E.S. and D.J. Mann, Cell Cycle, 2006. 5 (12): p. 1313–9. 5. Zhang, Z., et al., J Biol Chem, 2004. 279 (16): p. 16000–6. 6. Yang, A., et al., Mol Cell, 1998. 2 (3): p. 305–16. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A67.