Abstract
BackgroundThe 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.ResultsFollowing 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.ConclusionsThese 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.
Highlights
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
Cell culture Two cell lines, a canine large T cell leukemia line CLGL90 and a canine B cell line CLL 17–7 [25] were grown in custom RPMI media containing 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 0.4 mg/mL gentamicin, supplemented with 10% fetal bovine serum, and maintained at 37°C in a 5% CO2 air atmosphere
Cells were centrifuged and the cell pellet was collected for DNMT1 and methylation analysis
Summary
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. 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. 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. 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. Despite an over 90% remission rate, almost all dogs relapse and eventually become resistant to cytotoxic chemotherapy drugs. Rescue protocols have been suboptimal, with median remission durations ranging between 40 and 60 days after initiation [3,4,5,6,7]. Recent work in human non-Hodgkin lymphoma (nHL), the group of diseases most similar to canine
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