Role of ubiquitination in degradation of p21^CIP1/WAF1 after Fe chelation. (A-C) Iron chelators decrease p21^CIP1/WAF1 polyubiquitination when proteasomal activity is inhibited. (D) A simplified schematic illustration showing the multiple molecular effectors involved in G₁/S arrest of the cell cycle, inhibition of proliferation, metastasis suppression, and induction of apoptosis. MCF-7 cells were incubated with control media (Con) or media containing 311 (25 μM) in the presence or absence of MG132 (20 μM) for 8 or 24 hours. Immunoprecipitation was performed using anti-p21^CIP1/WAF1 antibody. Complexes were then separated by SDS-PAGE and probed with anti-p21^CIP1/WAF1 or antiubiquitin antibody using Western analysis. (B) Iron chelators induce ubiquitin-independent degradation of p21^CIP1/WAF1 in A31N-ts20 cells. A31N-ts20 cells were used, as they have a temperature-sensitive E1 ubiquitin–activating enzyme that is active at 32°C, but which inactivates at 39°C, preventing ubiquitin-dependent proteasomal degradation.^40,62  Cells were grown at 32°C or 39°C for 24 hours before the experiment. The media was then removed, and the cells were incubated for 24 hours at either temperature with control media, or media containing 311 (25 μM), and Western analysis was performed. (C) Iron chelators induce ubiquitin-independent degradation of p21^CIP1/WAF1 in p21-K6R–transfected cells. Mouse fibroblast NIH3T3 cells were transiently transfected with pCS2⁺p21WT or pCS2⁺p21-K6R plasmids, which contain wild-type (p21-WT) or mutant forms for human p21^CIP1/WAF1, respectively. At 24 hours after transfection, cells were incubated for 24 hours with either control medium, 311 (25 μM), MG132 (20 μM), or the combination of 311 (25 μM) and MG132 (20 μM). Western blotting was performed using antihuman p21^CIP1/WAF1 antibody, which only detects the transfected wild-type and mutant human p21^CIP1/WAF1. Results are a representative experiment from 3 performed. (D) Multiple molecular effectors involved in G₁/S arrest, inhibition of proliferation, apoptosis, and metastasis suppression are modulated in response to Fe deprivation. These responses include down-regulation of p21^CIP1/WAF1 protein,^18,21  down-regulation of cyclin D1 protein,^20,21,23  decreased activity of ribonucleotide reductase,^63,64  increased p53 protein expression,^17,25  increased expression of the metastasis suppressor protein Ndrg-1,¹⁹  and the up-regulation of the apoptosis-inducing protein NIP3.⁶⁵  Both NIP3 and Ndrg-1 are targets of the hypoxia inducible factor-1α (HIF-1α) transcription factor.^19,65  Other HIF-1α targets are probably also involved in the Fe depletion–induced inhibition of proliferation and induction of apoptosis.³⁰