To prevent rereplication of genomic segments, the eukaryotic cell cycle is divided into two nonoverlapping phases. During late mitosis and G1 replication origins are "licensed" by loading MCM2-7 double hexamers and during S phase licensed replication origins activate to initiate bidirectional replication forks. Replication forks can stall irreversibly, and if two converging forks stall with no intervening licensed origin-a "double fork stall" (DFS)-replication cannot be completed by conventional means. We previously showed how the distribution of replication origins in yeasts promotes complete genome replication even in the presence of irreversible fork stalling. This analysis predicts that DFSs are rare in yeasts but highly likely in large mammalian genomes. Here we show that complementary strand synthesis in early mitosis, ultrafine anaphase bridges, and G1-specific p53-binding protein 1 (53BP1) nuclear bodies provide a mechanism for resolving unreplicated DNA at DFSs in human cells. When origin number was experimentally altered, the number of these structures closely agreed with theoretical predictions of DFSs. The 53BP1 is preferentially bound to larger replicons, where the probability of DFSs is higher. Loss of 53BP1 caused hypersensitivity to licensing inhibition when replication origins were removed. These results provide a striking convergence of experimental and theoretical evidence that unreplicated DNA can pass through mitosis for resolution in the following cell cycle.uring the eukaryotic cell cycle, the genome must be precisely duplicated with no sections left unreplicated and no sections replicated more than once. To prevent rereplication, the process is divided into two nonoverlapping phases: during late mitosis and G1 replication origins are "licensed" for subsequent use by loading MCM2-7 double hexamers, and during S phase DNAbound MCM2-7 is activated to form processive CMG (CDC45-MCM-GINS) helicases that drive replication fork progression. The prohibition of origin licensing during S phase and G2 ensures that rereplication of DNA cannot occur. However, the inability to license new origins after the onset of S phase provides a challenge for the cell to fully replicate the genome using its finite supply of licensed origins. Replication forks can irreversibly stall when they encounter unusual structures on the DNA, such as DNA damage or tightly bound protein-DNA complexes.When replication initiation occurs at a licensed replication origin the MCM2-7 double hexamer forms a pair of bidirectionally orientated CMG helicases (1-3). If one fork irreversibly stalls, the converging fork from a neighboring origin can compensate by replicating all of the DNA up to the stalled fork. However, if two converging forks both stall and there is no licensed origin between them-a "double fork stall" (DFS)-new replicative machinery cannot be recruited to replicate the intervening DNA (4). To compensate for this potential for underreplication, origins are licensed redundantly, with most (typically >70%) remaining dorm...
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.
An amendment to this paper has been published and can be accessed via the original article.
The maintenance of biomolecules functionality is essential for the assurance of cellular homeostasis. At the proteome level this is achieved by the action of a modular, yet integrated subcellular compartment-specific system which ensures proteome quality control and it is called the proteostasis network (PN). PN is orchestrated by a plethora of different mechanisms and complex protein machines aiming to respond to stressors, recognize and either rescue (via the action of chaperones) or degrade unfolded, misfolded or damaged polypeptides at the two main cellular proteolytic systems, namely the proteasome and the lysosome. We have found that aging is accompanied by increased proteotoxic stress and a reduction of PN modules functionality. Interestingly, proteotoxic stress is also an emerging hallmark of age-related diseases, including tumorigenesis, and we recently proposed that age-related disruption of proteostasis contributes to tumor formation by (among others) increasing genomic instability due to reduced fidelity in processes like DNA replication or repair. We also hypothesized that the carcinogenesis-related increasing proteome instability eventually triggers the reactivation of the PN modules at advanced and/or metastatic stages in order for the tumor to survive and enable its various malignant phenotypes. We are testing these hypotheses by examining the differential regulation of PN components in precancerous and cancerous lesions of tumor biopsies, as well as, in cellular models of step-wise carcinogenesis. Specifically, precancerous and cancerous lesions of the larynx along with normal epithelium were examined by immunohistochemical staining for the expression levels of Apolipoprotein J/Clusterin (CLU); a molecular chaperone that has been implicated in tumor formation, metastasis and tumor cells resistance to chemotherapeutic drugs. Additionally, human bronchial epithelial cells into which various oncogenes were sequentially introduced, as well as, a mouse skin carcinogenesis model have been used to: a) investigate the status of PN components as a response to oncogenic hits and, b) to test the differential sensitivity of tumor vs. normal cells to stressors and to PN modules inhibitors. We found that CLU expression is induced by de novo synthesis in larynx dysplasia. In support, at both human and mouse cellular models of carcinogenesis we noted higher levels and activities of PN modules in advanced tumorigenesis; these phenotypes were accompanied with increased oxidative damage and proteome instability. Interestingly, advanced and/or metastatic mouse tumor cells were more sensitive to disruption of proteostasis (e.g. proteasome inhibition) or increased oxidative load compared to normal cells, while combined proteasome and lysosomal inhibition further sensitized metastatic tumor cells. These observations indicate that while tumor evolves over a cellular landscape of increased proteome instability, tumor cells eventually become “addicted” to higher activities of the PN modules in order to survive. Thus, inhibition of PN components provides a strategy for the development of novel tumor specific therapies. We are currently investigating this option by screening for natural compounds functioning as potent inhibitors of PN modules. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B73. Citation Format: Eirini-Stavroula Komseli, Fabiola Sesti, Konstantinos Evangelou, Christina Cheimonidou, Athanassios Kotsinas, Vassilis Gorgoulis, Ioannis P. Trougakos. Proteostasis network modules as molecular targets for cancer therapeutics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B73.
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