Zebularine is a novel potent inhibitor of both cytidine deaminase and DNA 
methylation. We examined the effect of zebularine on mammary tumor growth in 
genetically engineered MMTV-PyMT transgenic mice that develop mammary tumors at 
60 days of age with 100% penetrance. The MMTV-PyMT transgenic mice were 
randomized at 46 days of age into control (n = 25) and zebularine (n = 25) 
treatment groups and monitored for parameters of tumor growth. Zebularine was 
administered at 5 mg/mL in drinking water. We observed a significant delay in 
the growth of mammary tumors in zebularine-treated mice with a statistically 
significant reduction (P = 0.0135) in total tumor burden at 94 days of age when 
the mice were sacrificed. After 48 days of zebularine treatment, the tumors were 
predominantly necrotic compared with untreated animals. In addition, a high 
apoptotic index by terminal deoxynucleotidyl transferase-mediated dUTP nick end 
labeling assay was observed as early as 13 days following treatment. Immunoblot 
analysis showed depletion of DNMT1 and partial depletion of DNMT3b after 
zebularine treatment. Microarray analyses of global gene expression identified 
upregulation of twelve methylation-regulated genes as well as a set of candidate 
cancer genes that participate in cell growth and apoptosis. In summary, 
zebularine inhibits the growth of spontaneous mammary tumors and causes early 
onset of tumor cell necrosis and apoptosis in a genetically engineered mouse 
model of breast cancer. Defining the parameters of zebularine-mediated tumor 
inhibition may advance the future development of DNA methyltransferase 
inhibitors as an effective cancer treatment.