scholarly journals Low replication stress leads to specific replication timing advances associated to chromatin remodelling in cancer cells

2020 ◽  
Author(s):  
Lilas Courtot ◽  
Elodie Bournique ◽  
Chrystelle Maric ◽  
Laure Guitton-Sert ◽  
Miguel Madrid-Mencía ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Replication Timing ◽  
Replication Stress ◽  
Chromatin Accessibility ◽  
Genome Replication ◽  
Origin Activation ◽  
Early Replication ◽  
Dna Damage Signalling ◽  
The Impact

ABSTRACTDNA replication is well orchestrated in mammalian cells through a tight regulation of the temporal order of replication origin activation, named the replication timing, a robust and conserved process in each cell type. Upon low replication stress, the slowing of replication forks induces delayed replication of fragile regions leading to genetic instability. The impact of low replication stress on the replication timing in different cellular backgrounds has not been explored yet. Here we analysed the whole genome replication timing in a panel of 6 human cell lines under low replication stress. We first demonstrated that cancer cells were more impacted than non-tumour cells. Strikingly, we unveiled an enrichment of specific replication domains undergoing a switch from late to early replication in some cancer cells. We found that advances in replication timing correlate with heterochromatin regions poorly sensitive to DNA damage signalling while being subject to an increase of chromatin accessibility. Finally, our data indicate that, following release from replication stress conditions, replication timing advances can be inherited by the next cellular generation, suggesting a new mechanism by which cancer cells would adapt to cellular or environmental stress.

scholarly journals Low Replicative Stress Triggers Cell-Type Specific Inheritable Advanced Replication Timing

2021 ◽  
Vol 22 (9) ◽  
pp. 4959
Author(s):  
Lilas Courtot ◽  
Elodie Bournique ◽  
Chrystelle Maric ◽  
Laure Guitton-Sert ◽  
Miguel Madrid-Mencía ◽  
...  
Keyword(s):  
Replication Timing ◽  
Replication Stress ◽  
Chromatin Accessibility ◽  
Cell Type ◽  
Replicative Stress ◽  
Daughter Cells ◽  
Origin Activation ◽  
Cell Type Specific ◽  
Cellular Identity ◽  
Dna Replication Timing

DNA replication timing (RT), reflecting the temporal order of origin activation, is known as a robust and conserved cell-type specific process. Upon low replication stress, the slowing of replication forks induces well-documented RT delays associated to genetic instability, but it can also generate RT advances that are still uncharacterized. In order to characterize these advanced initiation events, we monitored the whole genome RT from six independent human cell lines treated with low doses of aphidicolin. We report that RT advances are cell-type-specific and involve large heterochromatin domains. Importantly, we found that some major late to early RT advances can be inherited by the unstressed next-cellular generation, which is a unique process that correlates with enhanced chromatin accessibility, as well as modified replication origin landscape and gene expression in daughter cells. Collectively, this work highlights how low replication stress may impact cellular identity by RT advances events at a subset of chromosomal domains.

scholarly journals Camptothecin-Induced Replication Stress Affects DNA Replication Profiling by E/L Repli-Seq

2021 ◽  
pp. 1-8
Author(s):  
Takuya Hayakawa ◽  
Rino Suzuki ◽  
Kazuhiro Kagotani ◽  
Katsuzumi Okumura ◽  
Shin-ichiro Takebayashi
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Replication Timing ◽  
Replication Stress ◽  
Replication Initiation ◽  
Domain Structures ◽  
Early Replication

E/L Repli-seq is a powerful tool for detecting cell type-specific replication landscapes in mammalian cells, but its potential to monitor DNA replication under replication stress awaits better understanding. Here, we used E/L Repli-seq to examine the temporal order of DNA replication in human retinal pigment epithelium cells treated with the topoisomerase I inhibitor camptothecin. We found that the replication profiles by E/L Repli-seq exhibit characteristic patterns after replication-stress induction, including the loss of specific initiation zones within individual early replication timing domains. We also observed global disappearance of the replication timing domain structures in the profiles, which can be explained by checkpoint-dependent suppression of replication initiation. Thus, our results demonstrate the effectiveness of E/L Repli-seq at identifying cells with replication-stress-induced altered DNA replication programs.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

scholarly journals Differential Effects of Endoplasmic Reticulum Stress-induced Autophagy on Cell Survival

2006 ◽  
Vol 282 (7) ◽  
pp. 4702-4710 ◽  
Author(s):  
Wen-Xing Ding ◽  
Hong-Min Ni ◽  
Wentao Gao ◽  
Yi-Feng Hou ◽  
Melissa A. Melan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Endoplasmic Reticulum Stress ◽  
Cell Survival ◽  
Cancer Cells ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Brefeldin A ◽  
Human Colon ◽  
The Impact

Autophagy is a cellular response to adverse environment and stress, but its significance in cell survival is not always clear. Here we show that autophagy could be induced in the mammalian cells by chemicals, such as A23187, tunicamycin, thapsigargin, and brefeldin A, that cause endoplasmic reticulum stress. Endoplasmic reticulum stress-induced autophagy is important for clearing polyubiquitinated protein aggregates and for reducing cellular vacuolization in HCT116 colon cancer cells and DU145 prostate cancer cells, thus mitigating endoplasmic reticulum stress and protecting against cell death. In contrast, autophagy induced by the same chemicals does not confer protection in a normal human colon cell line and in the non-transformed murine embryonic fibroblasts but rather contributes to cell death. Thus the impact of autophagy on cell survival during endoplasmic reticulum stress is likely contingent on the status of cells, which could be explored for tumor-specific therapy.

scholarly journals The impact of transcription-mediated replication stress on genome instability and human disease

2020 ◽  
Vol 1 (5) ◽  
pp. 207-234
Author(s):  
Stefano Gnan ◽  
Yaqun Liu ◽  
Manuela Spagnuolo ◽  
Chun-Long Chen
Keyword(s):  
Dna Replication ◽  
Gene Transcription ◽  
Human Disease ◽  
Genome Stability ◽  
Genome Instability ◽  
Current Knowledge ◽  
Replication Stress ◽  
Genome Replication ◽  
Living Organisms ◽  
The Impact

Abstract DNA replication is a vital process in all living organisms. At each cell division, > 30,000 replication origins are activated in a coordinated manner to ensure the duplication of > 6 billion base pairs of the human genome. During differentiation and development, this program must adapt to changes in chromatin organization and gene transcription: its deregulation can challenge genome stability, which is a leading cause of many diseases including cancers and neurological disorders. Over the past decade, great progress has been made to better understand the mechanisms of DNA replication regulation and how its deregulation challenges genome integrity and leads to human disease. Growing evidence shows that gene transcription has an essential role in shaping the landscape of genome replication, while it is also a major source of endogenous replication stress inducing genome instability. In this review, we discuss the current knowledge on the various mechanisms by which gene transcription can impact on DNA replication, leading to genome instability and human disease.

scholarly journals Dynamics of replication origin over-activation

2020 ◽  
Author(s):  
Haiqing Fu ◽  
Christophe E. Redon ◽  
Koichi Utani ◽  
Bhushan L. Thakur ◽  
Sangmin Jang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Molecular Interactions ◽  
Replication Origin ◽  
Replication Timing ◽  
Replication Stress ◽  
Single Fiber ◽  
Replication Origins ◽  
Initiation Frequency

AbstractWe determined replication patterns in cancer cells in which the controls that normally prevent excess replication were disrupted (“re-replicating cells”). Single-fiber analyses suggested that replication origins were activated at a higher frequency in re-replicating cells. However, nascent strand sequencing demonstrated that re-replicating cells utilized the same pool of potential replication origins as normally replicating cells. Surprisingly, re-replicating cells exhibited a skewed initiation frequency correlating with replication timing. These patterns differed from the replication profiles observed in non-re-replicating cells exposed to replication stress, which activated a novel group of dormant origins not typically activated during normal mitotic growth. Hence, disruption of the molecular interactions that regulates origin initiation can activate two distinct pools of potential replication origins: re-replicating cells over-activate flexible origins while replication stress in normal mitotic growth activates dormant origins.

scholarly journals Simultaneous quantification of protein-DNA contacts and transcriptomes in single cells

2019 ◽  
Author(s):  
Koos Rooijers ◽  
Corina M. Markodimitraki ◽  
Franka J. Rang ◽  
Sandra S. de Vries ◽  
Alex Chialastri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Single Cells ◽  
Critical Role ◽  
Nuclear Lamina ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Expression Variability ◽  
The Impact

AbstractThe epigenome plays a critical role in regulating gene expression in mammalian cells. However, understanding how cell-to-cell heterogeneity in the epigenome influences gene expression variability remains a major challenge. Here we report a novel method for simultaneous single-cell quantification of protein-DNA contacts with DamID and transcriptomics (scDamID&T). This method enables quantifying the impact of protein-DNA contacts on gene expression from the same cell. By profiling lamina-associated domains (LADs) in human cells, we reveal different dependencies between genome-nuclear lamina (NL) association and gene expression in single cells. In addition, we introduce the E. coli methyltransferase, Dam, as an in vivo marker of chromatin accessibility in single cells and show that scDamID&T can be utilized as a general technology to identify cell types in silico while simultaneously determining the underlying gene-regulatory landscape. With this strategy the effect of chromatin states, transcription factor binding, and genome organization on the acquisition of cell-type specific transcriptional programs can be quantified.

The Impact of Antioxidants from the Diet on Breast Cancer Cells Monitored by Raman Microspectroscopy

2018 ◽  
Vol 16 (2) ◽  
pp. 127-137
Author(s):  
Paula Sofia Coutinho Medeiros ◽  
Ana Lúcia Marques Batista de Carvalho ◽  
Cristina Ruano ◽  
Juan Carlos Otero ◽  
Maria Paula Matos Marques
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Breast Adenocarcinoma ◽  
Coumaric Acid ◽  
Human Breast ◽  
The Impact

Background: The impact of the ubiquitous dietary phenolic compound p-coumaric acid on human breast cancer cells was assessed, through a multidisciplinary approach: Combined biological assays for cytotoxicity evaluation and biochemical profiling by Raman microspectroscopic analysis in cells. </P><P> Methods: Para-coumaric acid was shown to exert in vitro chemoprotective and antitumor activities, depending on the concentration and cell line probed: a significant anti-invasive ability was detected for the triple-negative MDA-MB-231 cells, while a high pro-oxidant effect was found for the estrogen- dependent MCF-7 cells. A striking cell selectivity was obtained, with a more noticeable outcome on the triple-negative MDA-MB-231 cell line. Results: The main impact on the cellular biochemical profile was verified to be on proteins and lipids, thus justifying the compound´s anti-invasive effect and chemoprotective ability. Conclusion: p-Coumaric acid was thus shown to be a promising chemoprotective/chemotherapeutic agent, particularly against the low prognosis triple-negative human breast adenocarcinoma.

scholarly journals Oxidative Stress in the Tumor Microenvironment and Its Relevance to Cancer Immunotherapy

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 986
Author(s):  
Nada S. Aboelella ◽  
Caitlin Brandle ◽  
Timothy Kim ◽  
Zhi-Chun Ding ◽  
Gang Zhou
Keyword(s):  
Oxidative Stress ◽  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Redox Homeostasis ◽  
Tumor Rejection ◽  
Oxygen Species ◽  
Antitumor Effects ◽  
Key Drivers ◽  
Cancer Immunotherapies ◽  
The Impact

It has been well-established that cancer cells are under constant oxidative stress, as reflected by elevated basal level of reactive oxygen species (ROS), due to increased metabolism driven by aberrant cell growth. Cancer cells can adapt to maintain redox homeostasis through a variety of mechanisms. The prevalent perception about ROS is that they are one of the key drivers promoting tumor initiation, progression, metastasis, and drug resistance. Based on this notion, numerous antioxidants that aim to mitigate tumor oxidative stress have been tested for cancer prevention or treatment, although the effectiveness of this strategy has yet to be established. In recent years, it has been increasingly appreciated that ROS have a complex, multifaceted role in the tumor microenvironment (TME), and that tumor redox can be targeted to amplify oxidative stress inside the tumor to cause tumor destruction. Accumulating evidence indicates that cancer immunotherapies can alter tumor redox to intensify tumor oxidative stress, resulting in ROS-dependent tumor rejection. Herein we review the recent progresses regarding the impact of ROS on cancer cells and various immune cells in the TME, and discuss the emerging ROS-modulating strategies that can be used in combination with cancer immunotherapies to achieve enhanced antitumor effects.

scholarly journals High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sarah E. Pierce ◽  
Jeffrey M. Granja ◽  
William J. Greenleaf
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cancer Cells ◽  
High Throughput ◽  
Regulatory Networks ◽  
Temporal Dynamics ◽  
Chromatin Accessibility ◽  
Regulatory Regions ◽  
Factor Binding ◽  
Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

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