BackgroundSenescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now.MethodsIn the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs.ResultsThis experimental setting has three advantages that are presented and discussed: i) it covers a “gap” in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics.ConclusionsConsequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4375-1) contains supplementary material, which is available to authorized users.
Highlights d Typhoid toxin causes DNA fragmentation in vivo not associated with inflammation d Infection with a genotoxin-producing bacterium induces senescence in vivo d The presence of senescent cells is associated with an antiinflammatory response d The anti-inflammatory effect is lost in mice with acute colitis
An amendment to this paper has been published and can be accessed via the original article.
30Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA 31 damage response (DDR) in vitro. These toxins are produced by Gram negative bacteria, en-32 riched in the microbiota of Inflammatory Bowel Disease (IBD) and colorectal cancer (CRC) 33 patients. However, their role in infection remains poorly characterized. We have addressed the 34 role of the typhoid toxin in the modulation of the host-microbial interaction in health and dis-35 ease. 36Infection with a genotoxigenic Salmonella protected mice from intestinal inflammation. The 37 toxin-induced DNA damage caused senescence in vivo, which was uncoupled from the inflam-38 matory response, and associated with the maintenance of an anti-inflammatory environment. 39This effect was lost when infection occurred in mice suffering from inflammatory conditions 40 that mimic Ulcerative Colitis, a form of IBD. 41These data highlight a complex context-dependent crosstalk between bacterial genotoxins-in-42 duced DDR and the host immune response, underlining an unexpected role for bacterial geno-43 toxins. 44 45 cumulates in the cytoplasm of senescent cells (Evangelou et al., 2017). As shown in Figures 2C 130 and S3A, we detected a significant increase in GL13 epithelial positive cells in the colon and 131 small intestine of mice infected with the MC1 TT and MC71 TT strains compared to the levels 132 detected in mice infected with the control Salmonella, with the most prominent effect observed 133 in the C57BL/6 mouse strain (cp Figure 2C and Figure S3A). In our model, induction of senes-134 cence was uncoupled from the activation of a pro-inflammatory response (Figures 1, 3 and S2). 135 136The typhoid toxin maintains an anti-inflammatory microenvironment 137 Supplementary Information
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