Abstract 4351: Effects of epigenetic agents on methylation of DNA in vitro and in vivo, as measured with stable isotopically labeled methionine
Authors: Anderson Lawrence , Majerova Eva , Hill Kimberly D. , Carter John , Stottlemyer Jesse , Stotler Howard , Hollingshead Melinda G. , Collins Jerry M. 
Published: July 2017

Abstract
Background: Modulation of cytidine methylation in DNA continues to be actively investigated as a target for chemotherapy. The primary source of methyl groups is methionine, via S-adenosyl-methionine (SAM) as the direct methyl donor for DNA methyl transferases, e.g., DNMT1. Methods were developed using stable isotopes and mass spec detection to examine the flux of the DNMT reaction and modification to this flux by cytidine analogs such as decitabine, an established hypomethylation agent, and 5-aza-4’-thio-2’-deoxy-cytidine (AzaTdC), currently under development at NCI.
Methods: For in vitro studies; A549, H23, HCT116, H522, and OVCAR3 cell lines grown in RPMI1640 media supplemented with 10% FBS were exposed to D3-methionine(20uM) and either decitabine or AzaTdC (NSC77586) at 0 to 5uM in the media for 0 to 48hr. For in vivo studies; mice were maintained under standard ACUC protocols and practices. Tumors were grown SC in the flank area. Stable labeled methionine, decitabine and AzaTdC were administered as an oral bolus. At set times, mice were sacrificed and tumors, tissues and plasma samples collected. DNA was extracted and purified from cells and tissues using PureLink Genomic DNA kits (Invitrogen) according to manufacturer’s instruction. The purified DNA was digested to nucleosides for analysis. The isotopic enrichment in 5-methyl-deoxy-cytidine (mdC) was determined by analysis using HPLC coupled to a QE mass spectrometer (Thermo Scientific).
Results: D3-methionine was found to be an efficient precursor via SAM for the methylation of cytidine in DNA. Fortification of basal media (100uM methionine) with 20uM D3-methionine yielded D3-mdC enrichment of 5-15% in 24 hr, depending on cell line. All cell lines were found to have some enrichment. Treatment of cells with either decitabine or AzaTdC caused a concentration dependent reduction of the enrichment for mdC in DNA. In mice, after an oral dose of D3-methionine, enrichment of methionine and SAM in plasma peaked within 1hr and fell to 5% of peak levels within 5hr. Methionine and SAM enrichment in tissues followed similar time curves. At 50mg/kg of D3-methionine, no perturbation was observed for endogenous levels of either methionine or SAM. Enrichment of mdC in DNA was detectable in 1hr, maximal in 5hr, and remained constant for up to 48hr. When mice were treated with 1-2 mg/kg of either decitabine or AzaTdC along with D3-methionine, the level of enrichment of mdC in DNA was reduced by 20-70% in tumors, bone marrow and intestine.
Conclusions: Stable labeled methionine can be efficiently used to monitor the flux of methyl groups into mdC in DNA, both in vitro and in vivo. Changes in the flux of the methylation reaction can be used to assess the extent and time course for inhibition caused by cytidine analogs. These techniques may have relevance for improvement of doses and schedules to produce epigenetic modulation. Funded by NCI Contract No. HHSN261200800001E
Citation Format: Lawrence Anderson, Eva Majerova, Kimberly D. Hill, John Carter, Jesse Stottlemyer, Howard Stotler, Melinda G. Hollingshead, Jerry M. Collins. Effects of epigenetic agents on methylation of DNA in vitro and in vivo, as measured with stable isotopically labeled methionine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4351. doi:10.1158/1538-7445.AM2017-4351