Growth, Differentiation, and Death of Retinoic Acid-Treated Human Acute Promyelocytic Leukemia NB4 Cells

Published reports vary markedly on some characteristics of retinoic acid (RA) effects on cell growth and differentiation of the human acute promyelocytic leukemia cell line NB4. We explored possible reasons for this variability and found that the initial cell density of the experimental culture and the stage of growth of the cells used to inoculate experimental cultures were critical parameters for obtaining reproducible growth and differentiation responses of NB4 cells. Thus, the time to reach 50% differentiation in the presence of 1 μMRA and various initial concentrations of cells was 1.9 days with 2 × 106cells/ml, 3.5 days with 2 × 105cells/ml, and 4.7 days with 2 × 104cells/ml. With an initial concentration of 2 × 105cells/ml we saw time- and dose-dependent differentiation with RA concentrations >250 nM.However, in the presence of 25–250 nMRA, differentiation reached a maximum of about 20% on either Day 1 or Day 2 and then declined with time. The catabolism of RA appeared to be responsible for the decline in differentiation. In addition, large decreases in viability occurred after NB4 cultures, growing without or with RA, reached a density of only 1 × 106cells/ml. The decreases in viability were greater at intermediate concentrations of RA with a nadir at about 100 nM.Loss of viability was associated with DNA fragmentation and changes in morphology typical of apoptosis and necrosis. The loss of viability occurring in control cultures necessitates caution when these cultures are used to seed experimental cultures.