Effects of 5-aza-2'-deoxycytidine on differentiation and oncogene expression in the human monoblastic leukemia cell line U-937.

The DNA hypomethylating agent 5-aza-2'-deoxycytidine was able to induce irreversible terminal differentiation of the human monoblastic leukemia cell line U-937, when administered at a concentration of 0.1 microM every 12 hours for six times (72 hours). Differentiation occurred after removal of the drug, as shown by the gradual appearance of morphological, cytochemical, phenotypical, and functional cell maturation, along with the loss of the proliferative potential. Adherence to the plastic surface, a further marker of monocytic differentiation, was observed in long-term cultures of treated cells. Molecular events induced by 5-aza-2'-deoxycytidine included a decrease in DNA methylation, along with a dramatic, permanent reduction in the levels of c-myc transcripts; both these events were detectable early (24 to 48 hours) after the start of drug administration. A stable increase in c-fos and c-fms mRNAs, regarded as molecular markers of monocytic differentiation, was observed only after the end of treatment, in concomitance with the appearance of differentiation markers. The latency between early and late effects elicited by 5-aza-2'-deoxycytidine in U-937 cells suggests that the drug, presumably through DNA hypomethylation, is able to promote 'competence' to differentiate, via the activation of the regulatory program(s) needed for a monoblast to maturate, rather than directly inducing the expression of differentiation-specific genes. The temporal order of events described renders the present model suitable for the study of the human monocytic developmental program and of the molecular regulatory steps entailing differentiation by 5-aza-2'-deoxycytidine.