Genome-wide single nucleotide resolution of oxaliplatin-DNA adduct repair in drug sensitive and resistant colorectal cancer cell lines

Platinum-based chemotherapies, including oxaliplatin, are a mainstay in solid tumor treatment and induce cell death by forming intrastrand dinucleotide DNA adducts. Despite their common use, they are highly toxic and approximately half of patients have tumors that are either intrinsically resistant, or that develop resistance. Studies suggest that this resistance is mediated by variations in DNA repair levels or net drug influx. We aimed to better define the roles of nucleotide excision repair and DNA damage formation in platinum resistance by profiling DNA damage levels and repair efficiency in a panel of seven oxaliplatin-sensitive and three oxaliplatin-resistant colorectal cancer cell lines. We assayed repair indirectly as toxicity, and directly measured bulky adduct formation and removal from the genome by slot blot, repair capacity by excision assay, and mapped repair events genome-wide at single nucleotide resolution using XR-Seq. Using this comprehensive approach of combining novel methods and in some cases using proxies for oxaliplatin-DNA damage, we found no significant difference in repair efficiency that could explain the relative sensitivity and resistance of our cell lines. In contrast, the overall levels of oxaliplatin-DNA damage were significantly lower in resistant cells, indicating that decreased formation of damage, rather than increased repair of that damage, is a major determinant of oxaliplatin resistance. Analysis of gene expression using our XR-seq data showed upregulation of membrane transport pathways in resistant cells, and these pathways may contribute to resistance. Additional research is needed to characterize factors mitigating cellular DNA damage formation by platinum compounds. Overall design: We applied XR-seq on 10 colorectal cancer cell lines obtained from ATCC (Rko, Ls1034, Dld1, Ls180, Colo205) and from the UNC Tissue Culture Facility (Colo320, Hct116, Ht29, Sw480, Ls174t). For oxaliplatin treatments, cells were plated into R-150 plates and grown to about 90 percent confluence. 200µM oxaliplatin was added to each plate and cells were incubated at 32 degrees celcius for 2 hours. Plates were then placed on ice and cooled cells were harvested by scraping and rinsing with ice-cold PBS. Cells were pelleted, transferred to Eppendorf tubes, and resuspended in 2mL of lysis buffer. Samples were immunoprecipitated with anti-XPG antibody, and then ligated to adapters and processed for sequencing (XR-seq).