Genotoxicity of Cytolethal Distending Toxin (CDT) on Isogenic Human Colorectal Cell Lines: Potential Promoting Effects for Colorectal Carcinogenesis

Abstract

The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichia coli possesses a variety of virulent factors, among them the Cytolethal Distending Toxin (CDT). CDT displays dual DNase and phosphatase activities and induces DNA double strand breaks, cell cycle arrest and apoptosis in a broad range of mammalian cells. As CDT could promote malignant transformation, we investigated the cellular outcomes induced by acute and chronic exposures to E. coli CDT in normal human colon epithelial cells (HCECs). Moreover, we conducted a comparative study between isogenic derivatives cell lines of the normal HCECs in order to mimic the mutation of three major genes found in CRC genetic models: APC, KRAS, and TP53. Our results demonstrate that APC and p53 deficient cells showed impaired DNA damage response after CDT exposure, whereas HCECs expressing oncogenic KRAS (V12) were more resistant to CDT. Compared to normal HCECs, the precancerous derivatives exhibit hallmarks of malignant transformation after a chronic exposure to CDT. HCECs defective in APC and p53 showed enhanced anchorage independent growth and genetic instability, assessed by the micronucleus formation assay. In contrast, the ability to grow independently of anchorage was not impacted by CDT chronic exposure in KRAS(V12) HCECs, but micronucleus formation is dramatically increased. Thus, CDT does not initiate CRC by itself, but may have promoting effects in premalignant HCECs, involving different mechanisms in function of the genetic alterations associated to CRC.

Keywords: APC; DNA double strand breaks; KRAS; colorectal cancer; cytolethal distending toxin; genotoxicity; p53.

Figure 1

EcolCDT exposure affects cell viability of human colonic epithelial cells. (A) 1CT, 1CTA, 1CTR, and 1CTP human colonic epithelial cells (HCECs) were cultured for 3 days in the presence of different doses of EcolCDT (indicated above the graph). Cell viability was assessed using the PrestoBlue Cell Viability Reagent. Results present the mean ± SEM of at least three independent experiments. (B) Cell Index was measured in real-time and representative cell growth curves of the first 76 h of CDT treatment are shown. (C) The Cell Index-values at 76 h post-treatment are represented. Results represent the mean ± SD of three independent experiments; statistical differences were analyzed by a Student's t-test between the indicated conditions (*P < 0.05; **P < 0.01).

Figure 2

EcolCDT exposure induces the 53BP1 recruitment to sites of DNA damage in HCECs. (A) Representative images of 53BP1 immunostaining (red) in HCECs cells treated with the indicated doses of wild-type CDT or a catalytically dead mutant (CDT^H153A) for 24 h. DNA was stained with DAPI (blue). NT represents non-treated cells. Scale bar = 50 μm. (B) Quantification of HCECs positive for 53BP1 presented in (A). Cells were scored positive when containing more than five 53BP1 foci. Results represent the mean ± SEM of at least four independent experiments; statistical differences were analyzed by a Student's t-test (indicated by asterisks) between the indicated conditions, or by one-way ANOVA (indicated by dollars) for multiple comparisons, followed by Tukey's HSD post-hoc test (* or $ P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

EcolCDT exposure induces γH2AX increase in HCECs. (A) Representative images of 53BP1 immunostaining (red) and γH2AX (green) in HCECs cells treated with the indicated doses of wild-type CDT for 24 h, showing the colocalization of 53BP1 and γH2AX foci (MERGE). DNA was stained with DAPI (blue). NT represents non-treated cells. White asterisks marks cells with γH2AX staining unrelated to DSB. Scale bar = 20 μm. (B) Quantification of HCECs positive for γH2AX or 53BP1 presented in (A). Cells were scored positive when containing more than five foci. Results represent the mean ± SEM of at least four independent experiments. (C) In-Cell Western of γH2AX on HCECs. HCECs were treated with the indicated doses of EcolCDT for 24 h, or treated with 10 μM of etoposide and released or not in fresh media. Results represent the mean ± SEM of at least four independent experiments; statistical differences were analyzed by a Student's t-test between a condition and the non-treated cells or between the indicated conditions (**P < 0.01; ***P < 0.001).

Figure 4

Comparative analyses of HCECs chronically exposed or not to EcolCDT. (A,B) HCECs (A) and HCECs^CE (B) were exposed for 5 days to EcolCDT and cell viability was determined by crystal violet staining. NT represents non-treated cells. Results present the mean ± SEM of at least three independent experiments; statistical differences were analyzed by one-way ANOVA followed by Tukey's HSD post-hoc test ($$$P < 0.001) (C) Quantification of HCECs and HCECs^CE positive for 53BP1 after a 24 h treatment with EcolCDT. Cells were scored positive when containing more than five 53BP1 foci. Results represent the mean ± SEM of at least three independent experiments. (D–G) In-Cell Western of γH2AX on chronically exposed (HCECs^CE) or not (HCECs) 1CT (D), 1CTA (E), 1CTR (F), or 1CTP (G). HCECs were treated with 0.25 ng/ml of EcolCDT for 24 h, or treated with 10 μM of etoposide and released or not in fresh media. Results represent the mean ± SEM of at least three independent experiments; statistical differences were analyzed by a Student's t-test between the indicated conditions (*P < 0.05).

Figure 5

Micronucleus frequency in HCECs and HCECs^CE. (A) Nuclei of HCECs and HCECs^CE were stained with DAPI. The frequency of cells with micronuclei (white arrows) was quantified by fluorescence visualization. (B) Quantification of HCECs with micronuclei presented in (A). Results represent the mean ± SEM of at least three independent experiments; statistical differences were analyzed by a Student's t-test (indicated by asterisks) between the indicated conditions, or by one-way ANOVA (indicated by dollars) for multiple comparisons, followed by Tukey's HSD post-hoc test (** or $$ P < 0.01; *** or $$$ P < 0.001).

Figure 6

Chronic exposure to EcolCDT enhances anchorage-independent growth in 1CTA and 1CTP but not in 1CT and 1CTR. (A) HCECs and HCECs^CE were cultured in soft-agar in 6-well plates for 3 weeks in triplicates, and colonies larger than 100 μm in size were scored. (B) Quantification of the soft-agar assay presented in (A). Results represent the mean ± SEM of three independent experiments; statistical differences were analyzed by a Student's t-test (indicated by asterisks) between the indicated conditions, or by one-way ANOVA (indicated by dollars) for multiple comparisons, followed by Tukey's HSD post-hoc test (** or $$ P < 0.01; $$$ P < 0.001).