MN antagonizes biologic effects of ATRA in vitro. (A) In vitro cytotoxicity assay. ND13 + MN1, ND13 + CTL, or MN1 cells were incubated at a cell density of 4 × 104 cells/mL in the presence of either DMSO (1/1000th of total volume, equal to control) or increasing concentrations of ATRA. After 72 hours, viable cells were counted. Proliferation inhibition is expressed as percentage growth of control (mean ± SD of 3 independent experiments; P = .007 for ND13 + MN1 vs ND13 + CTL). (B) In vitro differentiation assay with ND13 + MN1 or ND13 + CTL cells using either DMSO (1/1000th of total volume, equal ot control) or ATRA (1 μM). Cell cultures were propagated every 3 days, and DMSO or ATRA were re-added. Morphology was analyzed after 10 days (magnification, × 1000; see “Materials and methods” for more image acquisition information). (C-E) In vitro differentiation assay with ND13 + MN1 or ND13 + CTL cells. Percentage change of cells expressing Gr-1 (C), Mac-1 (D), or c-kit (E) in ATRA-treated (0.1, 1, or 2 μM) compared with DMSO-treated (1/1000th of total volume, equal to control) cells after incubation for 48 hours (mean ± SD of 3 independent experiments; *P < .05 for each comparison). (F) Relative gene expression of p21, p27, PU.1, and C/ebpa in ND13 + MN1 cells at baseline compared with relative gene expression of these genes in ND13 + CTL cells at baseline (mean ± SD of 3 independent experiments). (G) Change of relative gene expression of p27 over time during in vitro differentiation of ND13 + MN1 or ND13 + CTL cells comparing ATRA-treated (1 μM) to DMSO-treated cells (mean ± SD of 3 independent experiments; *P < .01 for each time point).
