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Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors
Published on: August 5, 2022
6-Thioguanine and zebularine down-regulate DNMT1 and globally demethylate canine malignant lymphoid cells
Brian K Flesner1,2, Senthil R Kumar3, Jeffrey N Bryan4
1Department of Veterinary Medicine and Surgery, Comparative Oncology and Epigenetics Laboratory, College of Veterinary Medicine, University of Missouri-Columbia, 900 E. Campus Drive, Columbia, MO, 65211, USA. flesnerb@lsu.edu.
Background:
The antimetabolite 6-thioguanine (6-TG) has been used to treat both human and canine lymphoid malignancies. 6-TG has been shown to be epigenetically active as a demethylating agent in a human lymphoma cell line, causing downregulation of DNA methyltransferase 1 (DNMT1) through ubiquitin-targeted degradation. Zebularine (Zeb), a similar cytidine analog, also has demethylating activity as well as oral bioavailability. The hypothesis of the present study was that 6-TG and Zeb would cause downregulation of DNMT1 and globally demethylate the genomic DNA of canine lymphoma cells. The secondary hypothesis was that these agents would cause a dose-dependent decrease in cell proliferation in canine lymphoma cells. Canine CLGL-90 malignant T cells and CLL 17-7 cells were incubated in modified RPMI media. They were treated with 6-TG, Zeb, or control media at biologically relevant concentrations.
Results:
Following treatment with each agent, DNMT1 protein and global DNA methylation were significantly decreased. A dose-dependent decrease in cell survival was also observed, with apoptosis being the primary mode of cell death in the CLGL-90 cell line.
Conclusions:
These results confirm the demethylating action of 6-TG and Zeb in canine cells which is similar to that shown in human cell lines. Confirmation of this mechanism supports the clinical application of these compounds as demethylating drugs in veterinary patients.
Insights
The antimetabolite 6-thioguanine (6-TG) and Zebularine (Zeb) demethylate canine lymphoma cells by downregulating DNA methyltransferase 1 (DNMT1). These epigenetic drugs reduce cell proliferation and support their use in veterinary medicine.
Area of Science:
- Oncology
- Epigenetics
- Pharmacology
Background:
- 6-thioguanine (6-TG) and Zebularine (Zeb) are antimetabolites used in lymphoid malignancies.
- 6-TG exhibits epigenetic activity, downregulating DNA methyltransferase 1 (DNMT1) in human lymphoma cells.
- Zebularine (Zeb) is a cytidine analog with demethylating activity and oral bioavailability.
Purpose of the Study:
- To investigate the demethylating effects of 6-TG and Zeb on canine lymphoma cells.
- To determine if these agents downregulate DNMT1 and globally demethylate genomic DNA.
- To assess the impact of 6-TG and Zeb on canine lymphoma cell proliferation.
Main Methods:
- Canine malignant T cells (CLGL-90 and CLL 17-7) were cultured in modified RPMI media.
- Cells were treated with biologically relevant concentrations of 6-TG, Zeb, or control media.
- DNMT1 protein levels, global DNA methylation, and cell survival were analyzed.
Main Results:
- Treatment with 6-TG and Zeb significantly decreased DNMT1 protein and global DNA methylation.
- A dose-dependent reduction in cell survival was observed.
- Apoptosis was identified as the primary mechanism of cell death in CLGL-90 cells.
Conclusions:
- 6-TG and Zeb demonstrate demethylating activity in canine lymphoma cells, mirroring effects in human cell lines.
- The downregulation of DNMT1 and global DNA demethylation were confirmed.
- These findings support the potential clinical use of 6-TG and Zeb as demethylating agents in veterinary oncology.
