INDUCTION OF MURINE ERYTHROLEUKEMIA DIFFERENTIATION BY ACTINOMYCIN-D

Murine erythroleukemia cells are induced to differentiate by 0.5-5 ng of actinomycin D per ml. Murine erythroleukemia cells cultured with actinomycin D prolong cell doubling time but achieve the same density after 5 days as cells without inducer. Actinomycin D causes over 95% of the cells to become benzidine-reactive. [3H]Actinomycin D uptake into DNA can be detected within 2 hr and reaches a maximum (approximately 0.1 pmol/106 cells) by 10-12 hr. It is estimated that about one out of 105dG.dC pairs is bound to actinomycin D. Commitment to differentiation, assayed by transfer of cells to culture without inducer, was detected as early as 5 hr. Unlike Me2SO, which causes a transient prolongation in G1 at about 15-20 hr, cells cultured with actinomycin D show a more sustained increase in the proportion of the cells in G1. Globin mRNA accumulation was detectable by 19 hr in culture. Alteration in DNA stability in alkaline sucrose gradients was detected by 19 hr. Actinomycin D induces synthesis of Hb(maj) and Hb(min) in approximately equal amounts. A decrease in rates of synthesis of RNA, DNA, and total protein occurs in cells cultured with actinomycin D, as well as in cells cultured with Me2SO. No evidence for an early action of actinomycin D at the plasma membrane was obtained by measurement of changes in cell volume or 86RbCl uptake. Taken together, the present results indicate that actinomycin D is a potent inducer of differentiation of murine erythroleukemia cells and suggest that the target of its effect may be at the level of DNA.