A new molecular role for iron in regulation of cell cycling and differentiation of HL-60 human leukemia cells: Iron is required for transcription of p21(WAF1/CIP1) in cells induced by phorbol myristate acetate

Abstract

To investigate the role of iron in hematopoiesis, we studied effects of iron deprivation on PMA-induced monocyte/macrophage differentiation in HL-60 cells. Iron deprivation induced by desferrioxamine (DF) blocked PMA-induced differentiation and induced S-phase arrest and apoptosis in up to 60% of cells. Apoptosis was not related to a decrease of bcl-2 or to c-myc overexpression. In the presence of DF, PMA-induced upregulation of the cyclin dependent kinase inhibitor (CDKI), p21(WAF1/CIP1), was blocked and its expression could be restored in the presence of DF by supplementation with ferric citrate. Furthermore, ferrioxamine (iron saturated DF) did not block induction of p21(WAF1/CIP1) indicating that the changes were not due to a nonspecific toxic effect of DF. Similarly, hydroxyurea, an inhibitor of ribonucleotide reductase, did not block p21 expression. p21(WAF1/CIP1) antisense oligonucleotides caused cell cycle alterations similar to DF and p21 overexpression overcame effects of iron deprivation on both cell cycling and differentiation. Therefore, p21 is a key target for the effects of iron deprivation on HL-60 cell cycling and differentiation. Nuclear run-on transcription assays and p21 mRNA half-life studies indicated that iron was required to support transcriptional activation of p21(WAF1/CIP1) after a PMA stimulus. By contrast, iron deprivation did not inhibit expression of a second CDKI, p27(KIP1). These data demonstrate a new role for iron during monocyte/macrophage differentiation. A key role of iron is to allow induction of p21(WAF1/CIP1) in response to a differentiation stimulus subsequently blocking cells at the G(1)/S cell cycle interface and preventing premature apoptosis. This effect of iron is independent of its requirement in supporting the activity of the enzyme, ribonucleotide reductase. Because of the central role of p21(WAF1/CIP1) as regulator of the G(1)/S cell cycle checkpoint this requirement for iron to support p21 expression represents an important mechanism by which iron may modulate hematopoietic cell growth and differentiation. Published 2001 Wiley-Liss, Inc.

Tuning Cell Cycle Regulation with an Iron Key

“…151 Therefore, the down-regulation of cyclin D1 and p21 CIP1/WAF1 following Fe-depletion may facilitate degradation of each of these molecules via this pathway. 151 Under very different experimental conditions, Gazitt and colleagues 152 identified that HL60 leukemia cells need to be Fe-replete to transcribe p21 CIP1/WAF1 when induced by the phorbol ester, phorbol myristate acetate (PMA). In these studies, Fe-deprivation induced by DFO blocked PMA-induced differentiation and induced S-phase arrest and apoptosis.…”

“…In these studies, Fe-deprivation induced by DFO blocked PMA-induced differentiation and induced S-phase arrest and apoptosis. 152 Clearly, the difference on whether there is up-or down-regulation of p21 CIP1/WAF1 mRNA after Fe-depletion can be ascribed to the experimental conditions used Figure 3. Iron-depletion induces Ub-independent degradation of cyclin D1 and p21 CIP1/WAF1 .…”

“…Furthermore, the effect of PMA on p21 CIP1/WAF1 has only been found in HL60 cells. 152 In contrast, the effects of Fe-depletion on up-regulating p21 CIP1/WAF1 mRNA have been observed in a variety of cell types 72,92,101,107,148,153 and are relevant to the pharmacological effects of chelators as anti-tumor agents. 75,84,93 Examination of the Fe chelator-mediated downregulation of p21 CIP1/WAF1 is important, as apart from being a cdk inhibitor and positive regulator of the cell cycle, this protein also has anti-apoptotic activity.…”

Cobalt protoporphyrin induces differentiation of monocytic THP-1 cells through regulation of cytoplasmic Ref-1-related NADPH oxidase activity

“…Our data indicate that iron is an essential factor in CoPP-induced differentiation of THP-1 cells, suggesting a positive role for iron in monocyte differentiation. Similarly, other reports have demonstrated that iron is required for cell growth, viability, and differentiation of hematopoietic cells (26,27). In contrast, it has also been reported that DFO induces THP-1 cell differentiation by modulating intracellular GSH levels (28).…”

Ethanol Extract of Tripterygium wilfordii Hook. F. Induces G0/G1 Phase Arrest and Apoptosis in Human Leukemia HL-60 Cells Through c-myc and Mitochondria-Dependent Caspase Signaling Pathways

“…Previous studies have demonstrated that HL-60 cells exhibited an overexpression of Bcl-2 and c-Myc proto-oncogene and that alteration of cellular oncogenes occur during the differentiation of HL-60 cells (Kumakura et al, 1996). Within myeloid lineage, Bcl-2 is over-expressed in early myeloid precursors but under-expressed or absent in matured myeloid cells and neutrophils (Gazitt et al, 2001;Blagosklonny et al, 1996).…”