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

To investigate the role of iron in hematopoiesis, we studied effects of iro n deprivation on PMA-induced monocyte/macrophage differentiation in HL-60 c ells. Iron deprivation induced by desferrioxamine (DF) blocked PMA-induced differentiation and induced S-phase arrest and apoptosis in up to 60% of ce lls. Apoptosis was not related to a decrease of bcl-2 or to c-myc overexpre ssion. In the presence of DF, PMA-induced upregulation of the cyclin depend ent kinase inhibitor (CDKI), p21(WAF1/CIP1), was blocked and its expression could be restored in the presence of DF by supplementation with ferric cit rate. Furthermore, ferrioxamine (iron saturated DF) did not block induction of p21(WAF1/CIP1) indicating that the changes were not due to a nonspecifi c toxic effect of DF. Similarly, hydroxyurea, an inhibitor of ribonucleotid e reductase, did not block p21 expression. p21(WAF1/CIP1) antisense oligonu cleotides caused cell cycle alterations similar to DF and p21 overexpressio n overcame effects of iron deprivation on both cell cycling and differentia tion. Therefore, p21 is a key target for the effects of iron deprivation on HL-60 cell cycling and differentiation. Nuclear run-on transcription assay s and p21 mRNA half-life studies indicated that iron was required to suppor t transcriptional activation of p21(WAF1/CIP1) after a PMA stimulus. By con trast, iron deprivation did not inhibit expression of a second CDKI, p27(KI P1), These data demonstrate a new role for iron during monocyte/ macrophage differentiation. A key role of iron is to allow induction of p21(WAF1/ CIP 1) in response to a differentiation stimulus subsequently blocking cells at the G(1)/S cell cycle interface and preventing premature apoptosis. This e ffect 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 requireme nt for iron to support p21 expression represents an important mechanism by which iron may modulate hematopoietic cell growth and differentiation. Publ ished 2001 Wiley-Liss, Inc.(dagger).