V. Analysis of Cfp1 Function in DNA Damage Sensitivity
1. CXXC1 -/- ES cells exhibit hypersensitivity to DNA damaging agents
deoxyribose or by formation of oxidized bases that are substrates for DNA glycosylases (Mitra 2002). IRcauses DNA DSBs either directly or indirectly by forming clustersof oxidative damage, which can be converted to DNA breaks duringrepair (Courtemanche 2004). These lesions are particularly dangerous for proliferating cells and DNA
damage must be efficiently recognized and removed before DNA replication and chromosome segregation (Costelloe 2006). CXXC1+/+ and CXXC1-/- ES cells were exposed to increasing amounts of IR before being plated for assessment of clonogenic survival. CXXC1-/- ES cells exhibit decreased cell survival compared to CXXC1+/+ at 100 rad and a significant decrease in cell survival at 200 rad and 400 rad treatment (Fig. 44A).
In order to determine if CXXC1+/+ and CXXC1-/- ES cells showed differences in clonogenic survival after treatment with an alkylating DNA damaging agent,
CXXC1+/+ and CXXC1-/- ES cells were treated with increasing amounts of methyl methanesulfonate (MMS) and analyzed for colony forming ability. The majority (50- 80%) of damaged DNA bases caused by therapeutic alkylating agents are N-alkyl purines, which can be converted to abasic sites either by DNA glycosylase-mediated cleavage or by spontaneous hydrolysis of the glycosylic linkage between deoxyribose
of bases. The most abundant lesions produced by MMS are N-methylpurines, which are repaired by BER (Atain 2006). CXXC1-/- ES cells exhibit increased sensitivity to MMS compared to CXXC1+/+ cells at every concentration of MMS treatment, and a significant decrease was observed at 250 nM, 500 nM, and 800 nM treatment (Fig.
44B).
Etoposide is a DNA damaging agent that causes DNA single strand breaks and double strand breaks (DSBs) (Ross 1978). Etoposide causes DNA damage through inhibition of topoisomerase II and activation of oxidation-reduction reactions to produce derivatives that bind directly to DNA (Liu 1989). Topoisomerase II carries out
breakage and reunion reactions of DNA which are necessary for transcription and DNA replication. CXXC1-/- ES cells exhibit an increased sensitivity to etoposide at every concentration analyzed, and a significant decrease in cell survival compared to
CXXC1+/+ ES cells at 100 nM, 500 nM, and 1 àM etoposide concentrations (Fig. 44C).
Cisplatin (cis-diamminedichloroplatinum, cis-DDP) is one of the most effective chemotherapeutic anti-neoplastic agents, whose action is believed to result from its interactions with DNA. The N7 atoms of guanine and adenine are the main binding sites for platinum complexes in DNA (Widlak 2006). The DNA adducts induced by cisplatin include monoadducts, interstrand and intrastrand DNA cross-links and DNA- protein cross-links that interfere with DNA replication and transcription (Widlak 2006).
In addition, ROS are generated following cisplatin treatment (Yang 2005). CXXC1-/- ES cells exhibit a significant decrease in cell survival compared to CXXC1+/+ ES cells at all concentrations tested (0.5 àM, 1 àM, 5 àM, and 10 àM) (Fig. 44D).
FIGURE 44. Sensitivity of CXXC1+/+ and CXXC1-/- ES cells to DNA damaging agents.
Clongogenic survival of CXXC1+/+ and CXXC1-/- ES cells after treatment with ionizing radiation (A), methyl methanesulfonate (B), etoposide (C), cisplatin (D), temozolomide (E), or hydrogen peroxide (F). Each point represents the mean ± standard error from the results of at least 3 experiments, with each experiment representing 3 dishes per treatment group. Asterisks denote a statistically significant
Temozolomide (TMZ) is an alkylating agent that is non-enzymatically
hydrolyzed in solution to an active compound that methylates DNA primarly at the N7 and O6 positions of guanine and the N3 of adenine (Mutter 2006). Hypersensitivity to TMZ was observed in CXXC1-/- ES cells, and a significant decrease was observed at all concentrations tested (25 àM, 50 àM, and 100 àM) (Fig. 44E).
Hydrogen peroxide (H2O2) is an agent that generates ROS that lead to oxidative DNA damage, the most abundant being a base modification to 7,8-dihydro-8-
oxoguanine (8-oxoG) that can cause DNA DSBs (Daroui 2004). Similar to cisplatin and TMZ, hypersensitivity to H2O2 was observed in CXXC1-/- ES cells, and a significant decrease in cell survival was observed at all concentrations (1 àM, 5 àM, and 10 àM) (Fig. 44F).
Taken together, these results indicate that CXXC1-/- ES cells are hypersensitive to DNA damaging agents that cause DNA strand breaks (IR and etoposide), alkylation (MMS, TMZ), DNA cross-links (cisplatin), and oxidative (IR, H2O2, cisplatin) damage compared to CXXC1+/+ ES cells. CXXC1-/- ES cells exhibit an extended population doubling time due to an increase in apoptosis (Carlone 2005). Therefore, longer time points for colony formation were analyzed in case CXXC1-/- ES cells colonies take longer to arise. However, there was no difference in number of colonies formed for either the CXXC1+/+ orCXXC1-/- ES cell lines after an extended incubation time (data not shown).
FIGURE 45. Sensitivity of CXXC1+/+ and CXXC1-/- ES cells to non-genotoxic agents.
Clonogenic survival of CXXC1+/+ and CXXC1-/- ES cells after treatment with methotrexate (A), or paclitaxel (B). Each point represents the mean ± standard error from the results of at least 3 experiments, with each experiment representing 3 dishes per treatment group.