Re-evaluation of the Mutagenic Response to Phosphorothioate Nucleotides in Human Lymphoblastoid TK6 Cells

Oligonucleotide (OND)-based therapeutics and antisense ONDs in particular, are widely used for modulating gene expression inside cells with promising clinical potential (Graham et al., 2013; Thomas et al., 2013). Various chemical modifications to the native ONDs structures including the backbone, sugar, and/or base have been developed to improve their therapeutic potential (Deleavey and Damha, 2012). Chemically modified ONDs, in which one of the nonbridging oxygen atoms of the phosphodiester backbone is replaced with a sulphur atom, are commonly utilized in antisense applications with several candidates currently explored in clinical trials. This phosphorothioate (PS) substitution increases the nuclease resistance of PS-ONDs while maintaining target mRNA binding and degradation by intracellular ribonuclease H (RNase H) (Eckstein, 2000). Following systemic administration, PS-ONDs are rapidly cleared from the blood and distributed into all tissues with the kidney and the liver showing the highest accumulation (Geary et al., 1997). Clearance from tissues results mainly from the exonuclease metabolism of PS-ONDs into shortened ONDs and the release of mononucleotide analogs. These mononucleotides could potentially enter and disrupt the endogenous nucleotide pools, which are known to have genotoxic consequences, including mutations (Anderson et al., 1981; Mattano et al., 1990), DNA strand breaks (Brox et al., 1984), and chromosomal abnormalities (Ryan et al., 1965). However, perhaps of more concern, the liberated nucleotide analogs could be a substrate for various kinases and become incorporated into the genomic DNA during synthesis. If base pairing with the nucleotide analog occurred with reduced fidelity, a mutagenic event may arise http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500003149.pdf; Fersht, 1979). Recently, it has been reported that a PS nucleotide analog, deoxyadenosine monophosphorothioate (dAMPαS), had a potent mutagenic response at the endogenous thymidine kinase (TK) gene in human lymphoblastoid TK6 cells (Reshat et al., 2012). In a different study, Liber et al. (1985) likewise have reported that the nucleoside analog, 5-bromodeoxyuridine (BrdUrd), induces mutations at the TK locus, whereas no mutation was observed at the phosphoribosyl-transferase (HPRT) locus. However, because isolated BrdUrd-induced colonies were sensitive to the selective agent, trifluorothymidine (TFT), the authors concluded that BrdUrd produced a transient TFT-resistant phenotype that did not result from a mutagenic mechanism. It should be noted that BrdUrd, as a thymidine analog, has been utilized as a selective agent for TK mutants in L5178Y mouse lymphoma cells (Clive et al., 1972), and thus may exert selective toxicity to TK-competent cells resulting in the outgrowth of pre-existing TK mutants. In addition, two phenotypic classes of TK mutants are known to occur in the TK6 assay and are usually characterized as normal growing (NG), which are normally scored within 14 days, or slow growing (SG) mutants which require extended incubation time (Liber et al., 1989). However, the late appearing colonies isolated from BrdUrd-induced cultures were TFT-sensitive and thus are not actually mutants. This prompted us to explore further if dAMPαS could induce a similar unstable TFT-resistant phenotype and, if that was the case, to provide insight as to the mechanism of such effect. In this study, we characterized the TFT-resistant colonies induced by dAMPαS by assessing their ability to survive following TFT-rechallenge, and investigated the influence of dAMPαS on the cell cycle. We found that dAMPαS-induced colonies were SG and lost the TFT-resistant phenotype when re-challenged with TFT. Our studies indicate that cell cycle arrest and loss of TFT efficacy lead to the growth of non-mutant colonies.