No abstract

Cellular and systemic O 2 concentrations are tightly regulated to maintain delicate oxygen homeostasis. Although the roles of hypoxia in solid tumors have been widely studied, few studies were reported regarding the possible effects of hypoxia on leukemic cells. Here, we showed for the first time that low concentrations of cobalt chloride (CoCl 2 ), a hypoxia-mimicking agent, and 2-3% O 2 triggered differentiation of various subtypes of human acute myeloid leukemic (AML) cell lines, including NB4, U937 and Kasumi-1 cells, respectively, from M3, M5 and M2b-type AML, but CoCl 2 did not modulate AML subtype-specific fusion proteins promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa) and AML1-ETO. Treatment with CoCl 2 also induced primary leukemic cells from some AML patients to undergo differentiation. Similar to what occurs in solid tumor cells, CoCl 2 -mimicked hypoxia also increased the level of hypoxia-inducible factor (HIF)-1a protein and its DNAbinding activity in leukemic cells. The CoCl 2 induction of HIF-1a protein and its DNA-binding activity were inhibited by 3-morpholinosydnonimine, which also blocked CoCl 2 -induced cell differentiation in leukemic cells. These results provide an insight into a possible link of hypoxia or HIF-1a and leukemic cell differentiation, and are possibly of significance to explore clinical potentials of hypoxia or hypoxia-mimicking agents and novel target-based drugs for differentiation therapy of leukemia

The second messenger cyclic adenosine monophosphate (cAMP) plays an important role in cell proliferation, differentiation and apoptosis. In the present work, we evaluated the cAMP signaling in acute promyelocytic leukemia (APL) cells in the context of differentiation induced by all-trans retinoic acid (ATRA). There was a marked increase in the intracellular cAMP level within a few minutes after treatment with ATRA in APL cell line NB4 and fresh APL cells, whereas no such phenomenon was observed in NB4-R1 cells that are resistant to ATRAinduced maturation. In addition, the basal level of intracellular cAMP was lower in NB4-R1 than in NB4 cells. Mechanistic study showed that this induction of cAMP was mediated through the activation of adenylate cyclase. Moreover, we found that cAMPdependent protein kinase (PKA) activity was quickly upregulated in parallel in ATRA-treated NB4 cells, and the phosphorylation of RARa by PKA could increase its transactivation effect. Use of H-89, an inhibitor of PKA, could partially suppress the transcriptional expression of ATRA target genes and ATRAinduced differentiation of APL cells. Taken together, we suggested a crosstalk between ATRA-induced cytosolic pathway and nuclear pathway in APL cell differentiation.

We reported recently that cobalt chloride-simulated hypoxia and mild hypoxia modified the differentiation of human acute myeloid leukemic (AML) cells, probably acting via a hypoxiainducible factor-1 alpha (HIF-1a)-dependent mechanism. In this study, we investigated the effect of desferrioxamine (DFO), an iron chelator with 'hypoxia-mimetic' activity, on the differentiation of AML cells. The results showed that DFO at nontoxic concentrations induced the differentiation of AML cell lines NB4 and U937, as assessed by morphological criteria and differentiation-associated antigens. DFO-induced differentiation parallel to the rapid accumulation of HIF-1a protein in these two cell lines. Of importance, the transient transfection of HIF-1a cDNA induced U937 cells to develop the differentiationrelated alterations such as growth arrest and increased CD11b expression. Furthermore, the inducible expression of chromosome translocation t(8;21)-generated leukemogenic AML1-ETO fusion gene attenuated DFO-induced differentiation of U937 cells with the decrease of CCAAT/enhancer-binding protein alpha (C/EBPa), a critical factor for granulocytic differentiation. Using immunoprecipitation and luciferase reporter assay, HIF1a was also shown to interact physically with and to increase the transcriptional activity of C/EBPa. Taken together, these results provided novel evidence for a role of HIF-1a in AML cell differentiation, and suggested that C/EBPa might be a downstream effector for HIF-1a-mediated differentiation.