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.

Diallyl sulfide (DAS), an organosulfur compound from garlic, is recognized as a potential chemo‐preventive agent, and has a wide range of pharmacological effects, including anti‐leukemia cells in vitro. Many investigators have already demonstrated that DAS induces apoptosis from human leukemia HL‐60 and K562 leukemic cells, but there are no reports regarding whether or not it affects leukemia cells in vivo. However, the exact effect of DAS on leukemia cells in murine in vivo is unknown. In this study, we focused on the effects of DAS in vivo affecting murine leukemia WEHI‐3 cells. The primary studies tested the effects of DAS on the changes of percentage of viable cells of WEHI‐3 in vitro by MTT assay and the results showed that DAS decreased the % of viable cells and those effects are dose‐and time‐dependent manners. The following experiments examined the effects of DAS on leukemia WEHI‐3 cells in BLAB/c mice in vivo after i.p. was injected with WEHI‐3 cells before being cotreated with or without DAS, and the results indicate that DAS decreased the percentage of Mac‐3 marker, inhibiting the differentiation of precursor of macrophage and T cells. The final experiments measured the weights of liver and spleen and the results indicated that DAS decreased the weights of liver and spleen. It also brought attention to the enlarged spleens in murine after i.p. was injected with WEHI‐3 cells. However the DAS decreased the size of the spleen. The results showed that DAS affects WEHI‐3 cells in vitro and in vivo. Further investigations are needed to find out the molecular mechanism of DAS on the leukemia mice in vivo.

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