Inhibition of DNA methylation and reactivation of silenced genes by zebularine.
BACKGROUND
Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog.


METHODS
We tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided.


RESULTS
In N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001).


CONCLUSIONS
Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.
Cheng
Jonathan C
JC
University of Southern California/Norris Comprehensive Cancer Center and Hospital, Department of Biochemistry and Molecular Biology, USC Keck School of Medicine, Los Angeles 90089, USA.
Matsen
Cindy B
CB
Gonzales
Felicidad A
FA
Ye
Wei
W
Greer
Sheldon
S
Marquez
Victor E
VE
Jones
Peter A
PA
Selker
Eric U
EU
D000284:Administration, Oral; D000818:Animals; D015139:Blotting, Southern; D002934:Cinnamates; D003562:Cytidine / Q000031:analogs & derivatives; D019175:DNA Methylation / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004334:Drug Administration Schedule; D004622:Embryo, Mammalian; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000378:metabolism; D007263:Infusions, Parenteral; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009492:Neurospora crassa / Q000187:drug effects / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000187:drug effects / Q000378:metabolism*; D011741:Pyrimidine Nucleosides / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*
D002934:Cinnamates; D004273:DNA, Neoplasm; D011741:Pyrimidine Nucleosides; D006921:Hygromycin B; C026273:hygromycin A; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase