Aberrant DNA methylation is a critical epigenetic process involved in gene 
expression of tumor cells. Diverse DNA methyltransferase inhibitors are being 
studied as potential anticancer drugs, and there is interest in developing novel 
and more effective DNMTIs. We evaluated zebularine, a stable and low-toxic 
cytidine analog, effects on human promyelocytic leukemia cell lines, NB4 and 
KG1. Zebularine caused a dose- and time-dependent NB4 and KG1 cell growth 
inhibition, did not induce myeloid differentiation but triggered 
concentration-dependent apoptosis as manifested by procaspase-3 and PAR-1 
cleavage and the occurrence of early apoptosis detected by Annexin-V-propidium 
iodide. Zebularine co-treatment with all-trans retinoic acid (RA) at 
pharmacological dose (1 μM for NB4 cells) and higher (3 μM for KG1 cells) 
increased granulocytic differentiation in both cell lines. Pretreatment for 24 
or 48 h with zebularine before the treatment with different doses of RA alone or 
RA with histone deacetylase inhibitors, phenyl butyrate, and BML-210, resulted 
in significant acceleration and enhancement of differentiation and cell cycle 
arrest at G0/1. Zebularine alone or in sequential combination with RA decreased 
expression of DNMT1, caused fast and time-dependent expression of pan-cadherin 
and partial demethylation of E-cadherin but not tumor suppressor p15. When used 
in combination with RA, zebularine increased expression of both genes transcript 
and protein. Zebularine induced regional chromatin remodeling by local histone 
H4 acetylation and histone H3-K4 methylation in promoter sites of methylated 
E-cadherin and also in the promoter of unmethylated p21 as evidenced by 
chromatin immunoprecipitation assay. Our results extend the spectrum of 
zebularine effects and the evaluation its utility in acute myeloid leukemia 
therapy based on epigenetics.