Abstract 1282: Sequence-specific repair of etheno-adenine at mutation hotspots in p53.

Abstract

Abstract Vinyl chloride monomer (VCM) is a colorless gas used in the plastic industry for manufacturing polyvinyl chloride. VCM is mutagenic and carcinogenic, being associated with the development of liver angiosarcoma, hepatocellular carcinoma, and cholangiocarcinoma in humans. The metabolites of VCM chloroethylene oxide and chloroacetaldehyde react with all four bases in DNA by alkylation to form exocyclic etheno adducts. An adenine adduct, 1, N6-etheno adenine (εA), is specifically recognized and excised by N-methylpurine DNA glycosylase (MPG) leaving an abasic (AP) site that is subsequently recognized and cleaved by apurinic/apyrimidinic endonuclease (APE1), forming a nick in the DNA during Base Excision Repair (BER). Unrepaired εA results in A to T transversions, and these mutations are specifically found at hotspot codons in the p53 gene in liver angiosarcoma patients: codons 179, 249, and 255. We hypothesized that εA at these codons is excised less efficiently than at non-mutation hotspot codons due to differences in sequence context. We modified an existing real time PCR-based method to monitor BER in vivo, which utilizes a phagemid construct (M13mp18) containing a single εA placed in the p53 sequence. The phagemid is transfected into mammalian cells and retrieved at various time points post-transfection. The retrieved phagemid is treated with MPG and APE1 to convert any unrepaired εA to nicked DNA. The products are then analyzed by real time PCR utilizing primers that flank the damage site. Nicked DNA will be less amplifiable than intact DNA, which will indicate the amount of repair that occurred in the cell. We transfected HepG2 cells with phagemid constructs containing a single εA at the mutation hotspot codons 179, 249, and 255, as well as the non-mutagenic codon 246. We observed that 50% of eA placed at a non-mutagenic codon in the p53 gene (codon 246) is repaired in less than 5 hours post-transfection, while 50% of εA placed at the hotspot codons (codons 179, 249, or 255) is repaired in more than 16 hours. The in vivo results were confirmed in vitro utilizing purified MPG and oligonucleotide substrates containing εA in the sequence context of the non-hotspot and hotspot codons. We observed a 2.5 fold decrease in product formation by MPG when εA is placed at codon 249 compared to codon 246. We observed a 1.5 fold decrease in product formation by MPG when εA is placed at codon 179 or 255 compared to codon 246. These results indicate that sequence context affects the excision activity of MPG which suggests a potential mechanism for the presence of mutation hotspots in p53 (supported by RO1 CA92306 grant from NCI/NIH). Citation Format: Jordan Woodrick, Suhani Gupta, Sanchita Sarangi, Sanjay Adhikari, Rabindra Roy. Sequence-specific repair of etheno-adenine at mutation hotspots in p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1282. doi:10.1158/1538-7445.AM2013-1282