Cell resistance to the Cytolethal Distending Toxin involves an association of DNA repair mechanisms

The Cytolethal Distending Toxin (CDT), produced by many bacteria, has been associated with various diseases including cancer. CDT induces DNA double-strand breaks (DSBs), leading to cell death or mutagenesis if misrepaired. At low doses of CDT, other DNA lesions precede replication-dependent DSB formation, implying that non-DSB repair mechanisms may contribute to CDT cell resistance. To address this question, we developed a proliferation assay using human cell lines specifically depleted in each of the main DNA repair pathways. Here, we validate the involvement of the two major DSB repair mechanisms, Homologous Recombination and Non Homologous End Joining, in the management of CDT-induced lesions. We show that impairment of single-strand break repair (SSBR), but not nucleotide excision repair, sensitizes cells to CDT, and we explore the interplay of SSBR with the DSB repair mechanisms. Finally, we document the role of the replicative stress response and demonstrate the involvement of the Fanconi Anemia repair pathway in response to CDT. In conclusion, our work indicates that cellular survival to CDT-induced DNA damage involves different repair pathways, in particular SSBR. This reinforces a model where CDT-related genotoxicity primarily involves SSBs rather than DSBs, underlining the importance of cell proliferation during CDT intoxication and pathogenicity.


PrestoBlue Cell Viability Assay
PrestoBlue Cell Viability Assay (Invitrogen) has been performed according to the manufacturer's instructions. Briefly, HCT116 cells were grown on 96 well plates (10 000 cells/well) during 24 h. Then, cells were incubated in duplicate at three concentrations of CDT (2.5, 0.25, 0.025 µg/ml). After 72 h of treatment, cells were incubated with the PrestoBlue Cell Viability Reagent (1X) for 2 h at 37°C and absorbance (570 nm measurement wavelength, 600 nm reference wavelength) was read using an Infinite 200 PRO reader (TECAN). The percentage of cytotoxicity was determined by comparing results with non-treated cells.
Immunoprecipitation 10 7 HCT116 cells exposed for 24 h to 250 pg/ml of CDT wt before to be collected and lyzed in IP250 buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 0.05% NP40), and soluble extract was incubated with antibodies against FANCD2 from GeneTex (GTX116037) or from Novus Biologicals (NB100-182) overnight at 4°C under constant agitation. Then, 20 µl of equilibrated Protein A Magnetic Beads (Thermo Scientific) were added for 1 h before to be washed for 5 minutes three times in IP250 buffer before to proceed to immunoblots. Figure   Fig. S1 Effects on cell viability and cell cycle arrest caused by p53 deficiency on HCT116 cells exposed to CDT. B. Cell cycle analysis by flow-cytometry of HCT116 p53 +/+ and HCT116 p53 -/cells non-treated (NT) or exposed for 48 h to etoposide (5 µM) or CDT wt (2.5 ng/ml). Graphs show the cell cycle profiles obtained for one representative experiment.  represent the mean ± SD of at least three independent experiments. Statistics were calculated by unpaired Student's t-test (* P < 0.05).

Fig. S4
Quantification of alkaline Comet assay on HCT116 cells after a 4 h pulse treatment of CDT wt followed by different release times. Tail DNA percentage. Data are the mean ± SD of at least 3 independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001).

Fig. S5 FANCD2 depletion sensitizes HeLa cells to CDT.
A. FANCD2 protein level analyzed by Western blot in soluble cell extracts of HeLa cells treated with control or FANCD2 siRNA for 72 h. β-actin is shown as a loading control.
B. HeLa cells treated with control or FANCD2 siRNA were exposed for 5 days to CDT wt or CDT H153A and cell viability was analyzed by crystal violet staining (* P < 0.05; ** P < 0.01; *** P < 0.001).

Fig. S6
Western blot with mono-and polyubiquitinylated conjugates (FK2) and FANCD2 antibodies after FANCD2 immunoprecipitation with two different FANCD2 antibodies. Immunoprecipitations on soluble extracts from HCT116 exposed to 250 pg/ml of CDT wt for 24 h were conducted with beads alone or two different polyclonal antibodies against FANCD2 and blotted against anti mono-and polyubiquitinylated conjugates (FK2) or FANCD2, as indicated.

Fig. S7
Effect of p53 inhibition on cell survival of MEFs and PD20 cells exposed to CDT.
A. Clonogenic survival of XRCC4 +/+ and XRCC4 -/-MEFs treated or not with 10 µM of Pifithrin-α (PFT) and exposed to CDT wt or CDT H153A . Results present the mean ± SD of at least 3 independent experiments.
C. Clonogenic survival of PD20 D2 and PD20 cells treated or not with 10 µM of Pifithrin-α (PFT) and exposed to CDT wt or CDT H153A . Results are the mean ± SD of at least 3 independent experiments.