Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

Abstract

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/gammaH2AX(+) foci), enhanced senescence-associated beta-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the 'distended' morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.

Fig 1

Persistent DNA damage foci in HdCDT-intoxicated cells. (A) BJ fibroblasts were fixed and stained with anti-53BP1 (red) or anti-γH2AX (green) antibody and DAPI (blue) at different times after HdCDT intoxication. (B) Control IMR-90 cells and IMR-90 cells exposed to HdCDT for 24 hrs were fixed and stained with anti-53BP1 (purple) and DAPI. Depending on an amount of incorporated DAPI dye into the DNA, the cells were separated into the G₁- and G₂-phase population. (C) Six days after the intoxication with HdCDT, IMR-90 fibroblasts were fixed and stained with anti-P-Chk2 (Thr68) (red) and DAPI (blue). Untreated cells were used as negative control (CTR).

Fig 2

Increased fragmentation of nuclei in HdCDT-intoxicated fibroblasts. (A) Control or HdCDT-treated IMR-90 cells were fixed at different time-points upon intoxication and stained with DAPI. Numbers of micronuclei versus nuclei were calculated using the OSIS Scan^R software. Approximately 1000 nuclei were calculated for each sample. Error bars represent the S.D. of three separate experiments. (B) Eight days after the HdCDT-intoxication IMR-90 fibroblasts were fixed and stained with anti-53BP1 (red) and anti-γH2AX (green) antibodies and DAPI (blue).

Fig 3

Activation of cell cycle inhibitors after HdCTD intoxication. (A) U2-OS p53DD cells were treated with HdCDT in the presence (p53DD OFF) or the absence (p53DD ON) of tetracycline (Tet off system) and lysed at different time-points after the intoxication. Cell extracts were separated by SDS-PAGE and analysed by Western blotting with anti-p53, anti-P-p53 (Ser15) and anti-p21 antibodies. GAPDH expression was used as a loading control. (B) Immunoblot analysis of cell lysates prepared from HdCDT-intoxicated IMR-90 fibroblasts after different time-points. Untreated cells were used as negative control (CTR). Blots were probed with anti-p21, anti-p16 and anti-Rb as indicated; GAPDH was used as a loading control.

Fig 4

HdCDT induces SA-β-gal and increases number of PML bodies in normal and transformed cells. (A) Beta-galactosidase staining on IMR-90 and HeLa cells untreated or exposed to HdCDT for 6 to 8 days. (B) BJ fibroblasts, left untreated or exposed to HdCDT for the indicated periods of time, were fixed and stained with anti-PML (green) antibody and DAPI (blue).

Fig 5

HdCDT evokes expression of pro-inflammatory cytokines IL-6, IL-8 and IL-24. BJ fibroblasts or U2-OS cells were intoxicated with HdCDT and after the indicated time-points the expression levels of IL-6, IL-8 and IL-24 transcripts were determined by RT-PCR. Levels are represented relative to those found in control cycling cells. Error bars represent the S.D. of three separate experiments.