Hypoxia-simulating agents and selective stimulation of arsenic trioxide-induced growth arrest and cell differentiation in acute promyelocytic leukemic cells

Background and Objectives. We recently reported that hypoxia-mimetic agents cobalt chloride (CoCl2) and desferrioxamine (DFO) could induce differentiation of acute myeloid leukemic (AML) cells. Here, we investigate whether these two agents influence the in vitro differentiation-inducing effect of arsenic trioxide (As2O3) on AML cells, an effective drug for the treatment of acute promyelocytic leukemia (APL) that is a unique subtype of AML with a specific fusion protein, PML-RAR alpha. Design and Methods. The APL cell line NB4 and non-APL promonocytic leukemic cell line U937 were treated with As2O3 (0.5 mu M) combined with CoCl2 (50 mu M) or DFO (10 mu M). The U937/PR9 subclone, whose expression of PML-RAR alpha protein can be induced by Zn2+, was also investigated. Cellular differentiation was evaluated by morphological criteria and myeloid differentiation-related antigens and marker gene expression. The hypoxia-inducible factor-1 alpha (HIF-1 alpha) mRNA and protein were detected, respectively, by semi-quantitative/real-time quantitative reverse transcription polymerase chain reaction and immunoblots. PML-RAR alpha protein was also analyzed. Results. CoCl2 and DFO potentiated the growth-inhibiting and differentiation-inducing effects of low-dose As2O3, the latter enhancing CoCl2 and DFO-induced accumulation of HIF-1 alpha protein in NB4 cells but not in U937 cells. These two hypoxia-mimetic agents also accelerated As2O3-induced modulation and degradation of PML-RAR alpha protein in NB4 cells. Furthermore, inducible expression of the fusion gene restored the co-operative effects of As2O3 and CoCl2/DFO on U937/PR9 cells in terms of growth arrest, differentiation induction and HIF-1 alpha protein accumulation. Interpretation and Conclusions. Mimicked hypoxia enhanced As2O3-induced differentiation, in which HIF-1 alpha and PML/RARa proteins played an important role. These data provide new insights into the understanding of the mechanisms of the action of As2O3 in the treatment of patients with APL.