Concentrations of As2O3 that do not cause apoptosis activate key late-differentiation genes (CEBPE, MXD1) that repress MYC. (A) Concentrations of As2O3 that do not cause early apoptosis in NB4 cells were identified. Apoptosis measured by annexin-staining and flow cytometry. (B) As2O3 0.25μM activated the myeloid late-differentiation genes CEBPE and MXD1, and repressed MYC. mRNA levels measured by QRT-PCR. *P < .05, **P < .01 compared with baseline (t test). (C) Time-course changes in protein expression reiterated changes in mRNA expression. Protein expression measured by Western blot after As2O3 0.25μM or ATRA 1μM. Antibodies for MXD1 did not work. (D) As2O3 was more anti-proliferative than ATRA. Cumulative cell counts by automated counter. Error bars = SD, 3 experiments. (E) Both As2O3 and ATRA induced morphologic differentiation, although this was much more extensive with ATRA. Giemsa-stained cytospins 96 hours after addition of As2O3 0.5μm or ATRA 1μm. Black arrows indicate cells with maturation changes.
