Base Excision Repair Glycosylase Activity Is Impaired in a Subgroup of Acute Myeloid Leukemia Resulting in Increased Levels of Oxidative Base Lesions

Abstract

Base excision repair (BER) is the primary DNA repair mechanism dealing with oxidative base lesions. Oxidative DNA base lesions are the predominant type of DNA damage in mammalian cells. Deficiencies in glycosylases, the BER initiating enzymes, have been associated with increased genomic instability and increased frequencies of cancer. Here we investigated the role of oxidative BER in acute myeloid leukemia (AML).We determined oxidative BER activity in 99 primary AML blast cell samples, 34 CD34+ umbilical cord blood cell samples and 27 AML cell lines using the alkaline comet assay. Oxidative base lesion levels were determined in 10 AML cell lines using a modified version of the Comet assay with the bacterial enzymes Fpg and Endo III as well as using liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Using nuclear protein extracts in an oligonucleotide incision assay we tested the enzymatic activity of oxidative glycosylases. Finally, mutational analysis, gene expression analysis and protein expression of oxidative glycosylases was used using Sanger sequencing, real time PCR and western blot of nuclear extracts, respectively.We found DNA strand incision of oxidatively damaged bases significantly impaired in primary AML cells as compared to UCB cells (p= 0.003) suggesting a deficiency in BER glycosylases. In addition, 5/27 AML cell lines showed impaired DNA strand incision activity. We hypothesized that BER deficient cells harbor an increased number of oxidative base lesions compared to BER proficient cells. Using a modified comet assay and LC-MS/MS we were able to show that increased numbers of unrepaired oxidative base lesions were indeed present in glycosylase deficient AML cells (comet assay: p= 0.0001; mass spec: p= 0.03). We then evaluated the activity of the predominant oxidative DNA glycosylase, OGG1, and found significantly decreased DNA strand incision activity in BER deficient cells as compared to proficient cells (p= 0.002) further supporting the fact that glycosylases are impaired in BER deficient cells. Determining causes of BER deficiency preliminary experiments showed significantly decreased expression of nuclear OGG1 protein in BER deficient cells but did not reveal novel non-synonymous mutations or a difference in gene expression.Taken together we found impaired BER glycosylases in a substantial number of primary AML samples and AML cell lines resulting in increased levels of potentially mutagenic oxidative DNA base lesions DisclosuresNo relevant conflicts of interest to declare.