Zebularine inhibits human acute myeloid leukemia cell growth in vitro in association with p15INK4B demethylation and reexpression.
OBJECTIVE
The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine, can reverse p15INK4B promoter methylation, but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies.


METHODS
We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples.


RESULTS
Zebularine treatment of AML193, which has a densely methylated p15INK4B promoter, results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation, arrests cells at G(2)/M, and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis.


CONCLUSIONS
Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies.
Scott
Stuart A
SA
Department of Human Genetics, Mount Sinai School of Medicine, New York, NY., USA.
Lakshimikuttysamma
Ashakumary
A
Sheridan
David P
DP
Sanche
Stephen E
SE
Geyer
C Ronald
CR
DeCoteau
John F
JF
D000107:Acetylation; D000208:Acute Disease; D017209:Apoptosis / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019175:DNA Methylation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D016195:G2 Phase / Q000187:drug effects; D020869:Gene Expression Profiling; D018922:HL-60 Cells; D006657:Histones / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D050762:Cyclin-Dependent Kinase Inhibitor p15; D006657:Histones; D006877:Hydroxamic Acids; C012589:trichostatin A; D003562:Cytidine; C009131:pyrimidin-2-one beta-ribofuranoside