6-Thioguanine and zebularine down-regulate DNMT1 and globally demethylate canine malignant lymphoid cells

Abstract

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.

Figure 1

Western blot of DNMT1 expression. This image is a western blot of DNMT1 protein expression in CLGL 90 and CLL 17–7 cells treated with 1.5 μM or 6 μM 6-TG or 50 μM or 200 μM Zeb concentrations when compared to control. DNMT bands are evident at approximately 180,000 molecular weight. The treated groups had a reduction in intensity of DNMT1 expression as evident by bands in the 180 kDA region. Reduction in the expression of DNMT1 treated CLGL 90 cells (6-TG low, high and Zeb low, high groups) was 0.81, 0.76, 0.89, and 0.67 when compared to untreated control, respectively. Reduction in the expression of DNMT1 treated CLL 17–7 cells (6-TG and Zeb high dose) is 0.27 and 0.40, respectively. Loading control is Beta-actin.

Figure 2

DNA gel electrophoresis showing endonuclease activity in restriction landmark genomic scanning. Lanes: Lad = ladder, 1 = Control, 2 = Control + Hpa, 3 = Control + Msp, 4 = 6TG, 5 = 6TG + Hpa, 6 = Hpa + Msp, 7 = Zeb, 8 = Zeb + Hpa, 9 = Zeb + Msp. 1 kb pair DNA ladders were loaded into lane 1, followed by control, HpaII, and MspI incubated extracted DNA from the lymphoma cell line for low and high doses of 6-TG and Zeb respectively. The MSV equation $\left(\mathrm{M}\mathrm{S}\mathrm{V}=\frac{\mathrm{Densit}{\mathrm{y}}_{\mathrm{HpaII}}‐\mathrm{Densit}{\mathrm{y}}_{\mathrm{Background}}}{\mathrm{Densit}{\mathrm{y}}_{\mathrm{MspI}}‐\mathrm{Densit}{\mathrm{y}}_{\mathrm{Background}}}\right)$ was used for each treatment subset. Densities were calculated at the 2.0-2.3 kb pair region. Boxes shown in the first three lanes demonstrate the region of analysis.

Figure 3

A and B. Relative global DNA methylation with and without treatment by 6-TG or zebularine. RLGS global methylation results after treatment with 6-TG and Zeb. 6-TG and Zeb had a statistically significant (P < 0.001) reduction in relative methylation when compared to control for each cell line. All results were normalized to control values of 1 for purposes of comparison. CLGL-90 is presented in panel A and CLL 17–7 in panel B. The central bar represents the mean of the data and error bars represent the standard deviation.

Figure 4

CLGL-90 cell survival at 24 and 48-hour following treatment high dose 6-TG and zebularine compared to untreated controls. 6-TG and Zeb were applied at 6 uM and 200 uM respectively. The number of alive and dead cells was significantly different for treated groups zebularine (p = 0.026) compared to controls, but not for 6-TG (p = 0.092). The error bars represent the standard deviation.

Figure 5

Proportion of cells surviving following 48 hours of exposure to 6-G or Zeb normalized to controls. Proportion of viable cells decreased significantly compared to control at 48 hours (P < 0.001). When comparisons were made inside groups, low vs. high treated cells showed a dose-dependent response to 6-TG (P = 0.013). The error bars represent the standard deviation.

Figure 6

Fluorescent intensity of activated Caspase 3/7 assay following treatment with 6-TG or Zeb. Shown is the absolute intensity of fluorescence of 15,000 cells evaluated following no treatment (control) or low or high dose of 6-TG or Zeb. All treated groups are significantly different from control (P = 0.0011), but there is no difference between concentrations. The error bars represent the standard deviation.