Results represent the mean SEM of three independent experiments; statistical differences were analyzed by a Student’s test (** or $$ < 0.01; $$$ < 0.001). Discussion In the last decade, there is mounting evidence directly implicating the human microbiome in carcinogenesis in various organs (Bultman, 2014). models, contamination with CDT-positive is usually associated with gastritis and gastric dysplasia (Fox et al., 2004), and contamination with CDT-positive is usually accompanied with hepatitis and hepatic dysplastic nodules (Ge et al., 2007). These studies could be interpreted to suggest that CDT participates in the acquisition of a tumorigenic phenotype, probably through the induction of DNA damage. Actually, chronic exposure of mammalian cells with sublethal doses of CDT promotes the acquisition of cancer cells Sulpiride characteristics, namely genetic instability, enhanced anchorage-independent growth and defective DNA damage responses (Guidi et al., 2013). Thus, according to the growing evidence of bacterial contamination associated with increased risk of cancer (Bultman, 2014), deciphering the possible role of CDT in the induction or promotion of carcinogenesis in different niches is usually of particular concern. Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide in both men and women. Sporadic cancers represent the majority of CRC cases, and only 5C10% are attributable to inherited mutations of familial cancer syndromes (Pancione et al., 2012). Genetic models Sulpiride of CRC identified key tumor suppressors and oncogenes whose mutations drive multiple Sulpiride pathways for CRC progression from healthy tissue to dysplastic adenoma and finally carcinoma (Fearon and Vogelstein, 1990). Truncating mutations in the ((encodes a small GTPase and plays a key role in transduction of extracellular mitogenic signals to control cell proliferation. Finally, the tumor suppressor TP53 (p53), a multi-functional transcription factor mutated in up to 70% of CRC, regulates genes involved in cell cycle control, apoptosis, senescence and DNA repair in response to DNA damage and other cellular stresses (Toledo and Wahl, 2006). Several parameters influence CRC, including Sulpiride bacterial pathogens from the gut microbiota that represent important risk factors (Allen-Vercoe and Jobin, 2014; Yu and Fang, 2015). Various bacteria have been associated with CRC including possesses numerous virulence factors important for host tissue colonization, some of which potentially may be implicated in CRC initiation or progression. Indeed, colibactin, the product of the (have been associated with human CRC, including CDT (Buc et al., 2013; Bonnet et al., 2014). As colibactin and CDT are the two only known DNA damaging toxins produced by (EcolCDT) exposure on a colonic cell culture model. As we aimed to observe the possible acquisition of some hallmarks of cancer, we worked on non-transformed human colonic epithelial cells (HCEC) derived from healthy patient biopsies. These cells have been immortalized with the non-oncogenic proteins cyclin-dependent kinase 4 (Cdk4) and the catalytic component of the human ribonucleoprotein enzyme telomerase (hTERT) (Roig et al., 2010), a strategy used to immortalize various epithelial cell types without conferring tumorigenic properties. The cellular defects induced by CDT have been compared between isogenic derivative cell lines mimicking the mutation of three major genes found in CRC genetic models: loss of and (Smith et al., 2002). In the present studies these isogenically experimentally derived cells have been chronically exposed to sublethal doses of EcolCDT and analyzed for cancer hallmark acquisition. This study will allow for a better understanding of the carcinogenic potential of CDT from in normal or preneoplasic colonic tissues. Materials and methods Chemicals and supplements for cell-culture media The cytolethal distending toxin from (CDT-I) was produced and purified in the lab at 25 mg/ml (Fedor et al., 2013) and preserved in 10% glycerol PBS (Sigma-Aldrich). Fetal Bovine Serum (FBS), puromycin, hydromycin, and zeocin were BMPR2 provided by Fisher Scientific. Epidermal growth factor (EGF), hydrocortisone, insulin, transferrin, sodium selenite (5 nM), and Gentamycin sulfate (50 g/ml) were provided by Sigma-Aldrich. Antibodies Anti 53BP1 (Novus Biological) from rabbit is usually diluted 1/3000 in PBS made up Sulpiride of 3% bovine serum albumin (BSA), and anti H2AX (Merck/Millipore) from mouse is usually diluted 1/3000. For.