Abstract
Abstract Methylation-induced silencing of gene promoters is widely documented in numerous cancer types, and the anti-tumor activity of agents that target this phenomenon has established DNA methyltransferase (DNMT1) as a valid drug target. We have investigated the combination of 5-fluorodeoxycytidine with Tetrahydrouridine (FdCyd + THU) in vitro and in vivo using continuous exposures up to 12 weeks. Supplementation of the medium with Deoxythymidine (10 μM) was required to prevent FdCyd cytotoxicity and allow sufficient time for demethylation to occur. Decitabine was used as the positive control throughout the evaluation. The bladder cancer line EJ6 was sensitive to FdCyd + THU, as evidenced by an increase in cell doubling time, a decrease in LINE1 methylation, and an initial decrease in total DNMT1 concentrations, determined with a newly-developed, quantitative two-site immunoassay. The triple negative breast cancer line MDA-MB-231 was resistant to the regimen, as assessed by all three criteria. The mechanism of this combination regimen in tumor cells isolated from paracentesis specimens from patients enrolled on an NCI-supported Phase 1 clinical trial of FdCyd + THU; and was confirmed by finding changes in both DNMT1 levels and LINE1 methylation. Upregulation of expression of p16 (INK4a), but not GSTP1 or RASSF1, was observed in EJ6 cells following FdCyd + THU by Western blotting. An immunofluorescence assay for p16 expression in circulating tumor cells (CTCs) has been developed and implemented in the Phase 1 trial. The ongoing Phase 2 trial of this regimen will include measurements of DNMT1 and LINE1 methylation in tumor biopsies and p16 in CTCs to directly assess drug effect on the DNMT1 target. This Research has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E and NCI RAID Project #266 Grant. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A106.
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