Abstract 3638: DNA strand breaks induced by 2′-C-cyano-2′-deoxy-nucleoside analogs are repaired by homologous recombination
Authors: Liu Xiaojun , Matsuda Akira , Plunkett William 
Published: February 2011

Abstract
CNDAC [2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosyl-cytosine], which is undergoing clinical trials, and its congener CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl) guanine] likely share the unique action mechanism of inducing single strand breaks (SSBs) through β-elimination which generates the chain-terminating analog CNddC(G) following incorporation into DNA. We postulate that subsequent processing or DNA replication across the unrepaired nicks would generate double strand breaks (DSBs). Cellular responses to these 2′-C-cyano-2′-deoxy-nucleoside analogs are concentration dependent. Cells were arrested in G2 or S- phase when exposed to high concentrations of CNDAC(G), while at lesser concentrations cells could progress into a second S-phase. This study used cytogenetic approaches to define the mechanisms of DSB formation and damage repair in the above biological context. First, metaphase chromosomal aberrations (including breaks and gaps) were measured in Chinese hamster ovary line AA8 and its mutant UV41, which is sensitized to CNDAC(G) due to lack of XPF, a key endonuclease in the nucleotide excision repair (NER) pathway. The mean values of chromosomal aberrations increased 6(7)-fold and &gt; 6(3)-fold in AA8 and UV41 cells, respectively, after exposure to CNDAC(G) for two population doubling times, compared to those treated for only one population doubling time (n&gt;50). These data clearly demonstrate that SSBs induced by CNDAC(G) in the first S-phase can be converted into one-ended DSBs during the second S-phase when replication forks encountering unrepaired SSBs. Second, the frequencies of sister chromatid exchanges (SCEs) in AA8 cells incubated with CNDAC(G) for two cell cycles (mean 14.2-17.6 per metaphase) were 2-fold of those exposed for one cell cycle (mean 7.4-8.4 per metaphase, n&gt;20, p&lt;0.001), the latter greater than control (mean 6 per metaphase, p&lt;0.05). Since homologous recombination (HR) is considered the mechanism for SCE, these results indicate that CNDAC(G)-caused DNA damage is repaired by HR. Third, spontaneous chromosomal alterations in UV41 cells were 7-fold greater than those in parental AA8 cells, suggesting genetic instability conferred by XPF deficiency. The increase in chromosomal aberrations after CNDAC(G) exposure, regardless of length of time, was greater in UV41 cells compared to the wild-type. This suggests that XPF is likely needed to remove CNddC(G) preceding HR in the first cell cycle. Thus, NER and HR may work in concert to repair CNDAC(G)-induced damage. In addition, UV41 cells presented greater incidence of SCE after treatment with CNDAC for one population doubling time (n=30, p&lt;0.01), suggesting nuclease(s) other than XPF may function in removal of CNddC. Together these results demonstrate that HR is responsible for repair of CNDAC(G)-induced DSBs generated during the second replication across initial SSBs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3638.