6-Thioguanine and zebularine globally demethylate a canine lyphoma cell line

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Epigenetic modifications of chromatin, including methylation, histone modifications, and protein chaperoning, play a significant role in the biology of cancer. In concert with mutations, epigenetic modifications alter gene expression and protein translation/activity to confer a neoplastic phenotype. Hypermethylation of tumor suppressor genes or hypomethylation of proto-oncogenes or oncogenes can abrogate cell-cycle controls. These processes are reversible, resulting in restoration of normal gene expression. Relationships between methylation aberrations in human NHL have already been made; work is yet to be done for canine lymphoma. Recent treatment alterations are now being directed at specific epigenetic changes. Many of the common epigenetic modifiers have previously been used in both humans and dogs. This lends drugs already proven to be safe available for research opportunities in vivo. The purpose of this research project is to determine if epigenetically-active drugs could result in phenotypic reversion in a canine lymphoma cell line. To test this, we evaluated a cells in vitro, exposing them to demethylating agents. Our end analysis was generation of cell proliferation and viability assays, quantitation of global methylation levels, and confirmation of demethylating drug targets. We hypothesized that canine lymphoma cells will undergo global demethylation in vitro after treatment with demethylating agents including 6-thioguanine (6-TG) and zebularine (Zeb). As in humans, 6-TG will downregulate DNMT- 1. The demethylating agents will have a dose dependent effect on global methylation and cell kill. The canine lymphoma cell line, CLGL-90, was grown in vitro with custom RPMI media using sterile techniques. The cells were treated with control (buffer solution), 6-TG, or Zeb in varying concentrations. Initial cell counts were collected prior to the drugs being added, and at 24 and 48-hour time points. Half of the originally plated cells were harvested for methylation analysis and cell survival analysis at 24 hours and replaced with appropriate media. The remaining cells were then re-treated and removed for analysis at 48 hours. Methylation analysis was HpaI/MspII (selective and non-selective methylation restriction enzymes) ratio. Western blot was used to determine DNMT1 protein levels before and after treatment. Cell survival curves were calculated using hemocytometer and trypan blue staining. The CLGL-90 cells were globally demethylated with increasing concentrations of 6-TG and Zeb. DNMT1 was reduced in cells treated with 6-TG and Zeb compared to controls, in a dose-dependent manner. Increasing concentrations of the demethylating agents also resulted in decreased cell survival, with 6-TG more effective than Zeb. 6-TG and Zeb globally demethylate the CLGL-90 canine lymphoma cell line and result in decreased amounts of DNMT-1 protein present. Increasing concentrations of the drugs also results in increasing cell kill. Future directions include in vitro work using these drugs in concert with cytotoxic chemotherapy drugs against resistant lymphoma cells. We also plan to begin using these agents in vivo, targeting resistant canine lymphoma patients in the clinic.