Related Experiment Video
Updated: Aug 29, 2026
Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos
Published on: August 18, 2022
Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells
Jonathan C Cheng1, Daniel J Weisenberger, Felicidad A Gonzales
1Department of Urology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California 90089-9181, USA.
Abstract:
During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Recently, we reported that zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one] acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. Here we show that continuous application of zebularine to T24 cells induces and maintains p16 gene expression and sustains demethylation of the 5' region for over 40 days, preventing remethylation. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. The drug caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1) and partial depletion of DNMT3a and DNMT3b3. Last, sequential treatment with 5-aza-2'-deoxycytidine followed by zebularine hindered the remethylation of the p16 5' region and gene resilencing, suggesting the possible combination use of both drugs as a potential anticancer regimen.
Insights
Zebularine continuously applied to cancer cells maintains gene expression by preventing DNA methylation. This drug also depletes DNA methyltransferases, offering a potential new cancer treatment strategy.
Area of Science:
- Epigenetics
- Cancer Biology
- Pharmacology
Background:
- Aberrant DNA methylation silences tumor suppressor genes during cancer development.
- Zebularine is a stable, low-cytotoxicity inhibitor of DNA methylation.
Purpose of the Study:
- To investigate the long-term effects of zebularine on gene expression and DNA methylation in cancer cells.
- To assess zebularine's impact on DNA methyltransferases and its potential as an anticancer agent.
Main Methods:
- Continuous zebularine treatment of T24 cells.
- Analysis of p16 gene expression and promoter methylation.
- Global DNA methylation profiling.
- Assessment of DNA methyltransferase (DNMT) levels.
- Sequential treatment with 5-aza-2'-deoxycytidine and zebularine.
Main Results:
- Continuous zebularine treatment induced sustained p16 gene expression and demethylation for over 40 days.
- Zebularine caused global demethylation, particularly in CpG-poor regions.
- The drug depleted DNA methyltransferase 1 (DNMT1) and partially depleted DNMT3a/3b.
- Sequential treatment prevented p16 gene resilencing.
Conclusions:
- Zebularine effectively induces and maintains gene expression by preventing DNA methylation.
- Zebularine's ability to deplete DNMTs and prevent remethylation suggests its potential as a novel anticancer therapeutic.
- Combination therapy with 5-aza-2'-deoxycytidine may enhance zebularine's efficacy.
