CAMP-INDUCED NF-KAPPA-B (P50 RELB) BINDING TO A C-MYB INTRONIC ENHANCER CORRELATES WITH C-MYB UP-REGULATION AND INHIBITION OF ERYTHROLEUKEMIA CELL-DIFFERENTIATION/

During hexamethylene bisactamide (HMBA)-induced differentiation of mur ine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are do wn-regulated. Differentiation is inhibited by cAMP analogues and the a denyl cyclase-stimulating agent forskolin. We found that these drugs p revented the late down-regulation of c-myb which is known to be critic al for MEL cell differentiation. Since the increase in c-myb mRNA leve ls was due to increased mRNA transcription, we examined the transcript ion factors NF-kappa B and AP-1 which have been implicated in the regu lation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappa B recognition sequence in c-myb intron 1 was strongly induced by 8-Br-cAMP or forskolin; induction was delayed and correlated with the up-regulation of c-myb. The cAMP-induced NF-kappa B complex contai ned p50 and RelB; in co-transfection assays, p50 and RelB transactivat ed a reporter construct containing the c-myb intronic NF-kappa B site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increased the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells Which could cooperate with NF-kappa B. We conclude that inhibition of MEL cell differentiation by pharmacological doses o f cAMP can be explained by the up-regulation of c-myb which is mediate d, at least in part, by NF-kappa B p50/RelB heterodimers.