INDUCTION OF DIFFERENTIATION IN HUMAN PROMYELOCYTIC HL-60 LEUKEMIA-CELLS ACTIVATES P21, WAF1 CIP1, EXPRESSION IN THE ABSENCE OF P53/

The melanoma differentiation associated gene, mda-6, which is identica l to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin depen dent kinases, mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to t erminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound mezerein (Jiang and Fisher, 1993). In t he present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediat e early response gene induced during differentiation of the promyelocy tic HL-60 leukemia cell line along the granulocytic or macrophage/mono cyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway e voked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxy vitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic a cid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses usi ng an anti-p21 antibody indicate a temporal induction of p21 protein f ollowing treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA- induced growth arrest and differentiation. A similar delay in mda 6 ge ne expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiatio n. Since HL-60 cells have a null-p53 phenotype, these results demonstr ate that p21 induction occurs during initiation of terminal differenti ation in a p53-independent manner. In this context, p21 may play a mor e global role in growth control and differentiation than originally en visioned.