Abstract 1596: Effects of folylpolyglutamyl synthase and γ-glutamyl hydrolase modulation on DNA methylation

Abstract

Abstract Background: Folate mediates the transfer of one-carbon units for the provision of S-adenosylmethionine, the primary methyl group donor for most biological methylation reactions including DNA methylation, which is catalyzed by DNA methyltransferase (DNMT). Both genomic DNA hypomethylation and gene-specific promoter CpG island hypermethylation are important epigenetic mechanisms of carcinogenesis. DNA methylation and DNMT also are potential therapeutic targets and may modify the effect of specific chemotherapeutic agents. Intracellular folate homeostasis is maintained by folylpolyglutamate synthase (FPGS) that facilitate intracellular retention of folate by polyglutamylation and by γ -glutamyl hydrolase (GGH) that catalyzes the hydrolysis of polyglutamylated folate into monoglutamates, thereby facilitating export of folate out of the cell. We investigated whether GGH and FPGS modulation would affect genomic DNA methylation and DNMT activity. Methods: We generated an in vitro model of GGH overexpression and inhibition in human HCT116 colon and MDA-MB-435 breast cancer cells by stably transfecting the cells with the sense GGH cDNA or GGH-targeted siRNA, respectively. An in vitro model of FPGS overexpression and inhibition in HCT116 cells was generated by stably transfecting the cells with the sense or antisense FPGS cDNA, respectively. Similarly, MDA-MB-435 cells were transfected with the sense FPGS cDNA or FPGS-targeted siRNA, respectively, to generate an in vitro model of FPGS overexpression and inhibition. Genomic DNA methylation and DNMT activity were determined. Results: Functionally significant GGH/FPGS overexpression and inhibition were confirmed by GGH and FPGS protein expression, GGH and FPGS activity, and total and long-chain polyglutamylated intracellular folate concentrations. In both cell lines, GGH overexpression was associated with significantly lower genomic DNA methylation and DNMT activity than controls, while GGH inhibition was associated with significantly higher genomic DNA methylation and DNMT activity than controls (P<0.05). FPGS overexpression was associated with lower genomic DNA methylation and DNMT activity than controls in HCT116 cells while it was associated with higher genomic DNA methylation and DNMT activity than controls in MDA-MB-435 cells (P<0.05). FPGS inhibition was associated with higher genomic DNA methylation and lower DNMT activity compared with controls in HCT116 cells (P<0.05) whereas it did not affect genomic DNA methylation in MDA-MB-435 cells. Conclusions: Our data indicate that GGH and FPGS modulation can affect genomic DNA methylation and DNMT activity of colon and breast cancer cells. Studies are underway to determine whether these altered DNA methylation and DNMT activity influence chemosensitivity of these cancer cells to 5-fluorouracil and antifolates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1596.