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1.
Nat Commun ; 14(1): 6140, 2023 10 02.
Article in English | MEDLINE | ID: mdl-37783689

ABSTRACT

DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that basal levels of the IFN-stimulated gene 15, ISG15, and its conjugation (ISGylation) are essential to protect nascent DNA from degradation. Moreover, IFNß treatment restores replication fork stability in BRCA1/2-deficient cells, which strictly depends on topoisomerase-1, and rescues lethality of BRCA2-deficient mouse embryonic stem cells. Although IFNß activates hundreds of genes, these effects are specifically mediated by ISG15 and ISGylation, as their inactivation suppresses the impact of IFNß on DNA replication. ISG15 depletion significantly reduces cell proliferation rates in human BRCA1-mutated triple-negative, whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, which we propose as an in-built mechanism of drug resistance linked to BRCAness.


Subject(s)
BRCA1 Protein , Interferons , Animals , Humans , Mice , BRCA1 Protein/genetics , Cell Survival , BRCA2 Protein/metabolism , Ubiquitins/genetics , Ubiquitins/metabolism , Cytokines/metabolism
3.
Sci Adv ; 7(19)2021 05.
Article in English | MEDLINE | ID: mdl-33952518

ABSTRACT

The stalled fork protection pathway mediated by breast cancer 1/2 (BRCA1/2) proteins is critical for replication fork stability. However, it is unclear whether additional mechanisms are required to maintain replication fork stability. We describe a hitherto unknown mechanism, by which the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily-A containing DEAD/H box-1 (SMARCAD1) stabilizes active replication forks, that is essential to maintaining resistance towards replication poisons. We find that SMARCAD1 prevents accumulation of 53BP1-associated nucleosomes to preclude toxic enrichment of 53BP1 at the forks. In the absence of SMARCAD1, 53BP1 mediates untimely dissociation of PCNA via the PCNA-unloader ATAD5, causing frequent fork stalling, inefficient fork restart, and accumulation of single-stranded DNA. Although loss of 53BP1 in SMARCAD1 mutants rescues these defects and restores genome stability, this rescued stabilization also requires BRCA1-mediated fork protection. Notably, fork protection-challenged BRCA1-deficient naïve- or chemoresistant tumors require SMARCAD1-mediated active fork stabilization to maintain unperturbed fork progression and cellular proliferation.

4.
Cancer Res ; 78(2): 528-541, 2018 01 15.
Article in English | MEDLINE | ID: mdl-29141899

ABSTRACT

The clinical use of multiple classes of cancer chemotherapeutics is limited by irreversible, dose-dependent, and sometimes life-threatening cardiotoxicity. Though distinct in their mechanisms of action, doxorubicin, paclitaxel, and 5-FU all induce rapid and robust upregulation of atypical G protein Gß5 in the myocardium correlating with oxidative stress, myocyte apoptosis, and the accumulation of proinflammatory and profibrotic cytokines. In ventricular cardiac myocytes (VCM), Gß5 deficiency provided substantial protection against the cytotoxic actions of chemotherapeutics, including reductions in oxidative stress and simultaneous attenuation of ROS-dependent activation of the ATM and CaMKII proapoptotic signaling cascades. In addition, Gß5 loss allowed for maintenance of Δψm, basal mitochondrial calcium uniporter expression, and mitochondrial Ca2+ levels, effects likely to preserve functional myocyte excitation-contraction coupling. The deleterious effects of Gß5 are not restricted to VCM, however, as Gß5 knockdown also reduces chemotherapy-induced release of proinflammatory cytokines (e.g., TNFα), hypertrophic factors (e.g., ANP), and profibrotic factors (e.g., TGFß1) from both VCM and ventricular cardiac fibroblasts, with the most dramatic reduction occurring in cocultured cells. Our experiments suggest that Gß5 facilitates the myofibroblast transition, the persistence of which contributes to pathologic remodeling and heart failure. The convergence of Gß5-mediated, ROS-dependent signaling pathways in both cell types represents a critical etiological factor in the pathogenesis of chemotherapy-induced cardiotoxicity. Indeed, intracardiac injection of Gß5-targeted shRNA allowed for heart-specific protection against the damaging impact of chronic chemotherapy. Together, our results suggest that inhibition of Gß5 might represent a novel means to circumvent cardiotoxicity in cancer patients whose treatment regimens include anthracyclines, taxanes, or fluoropyrimidines.Significance: These findings suggest that inhibiting an atypical G-protein might provide a strategy to limit the cardiotoxicity in cancer patients treated with anthracyclines, taxanes, or fluoropyrimidines. Cancer Res; 78(2); 528-41. ©2017 AACR.


Subject(s)
Antineoplastic Agents/toxicity , Apoptosis/drug effects , Fibrosis/pathology , GTP-Binding Protein beta Subunits/metabolism , Myocytes, Cardiac/pathology , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolism , Animals , Cell Proliferation/drug effects , Cells, Cultured , Fibrosis/chemically induced , Fibrosis/metabolism , Male , Mice , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Signal Transduction/drug effects
6.
Nat Struct Mol Biol ; 23(8): 699-701, 2016 08 03.
Article in English | MEDLINE | ID: mdl-27487392
7.
Methods Mol Biol ; 1094: 177-208, 2014.
Article in English | MEDLINE | ID: mdl-24162989

ABSTRACT

The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by various forms of replication stress. The replication structures are stabilized in vivo (by psoralen cross-linking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of genomic DNA, extracted from budding yeast, Xenopus egg extracts, or cultured mammalian cells.


Subject(s)
DNA Replication , Eukaryotic Cells/cytology , Eukaryotic Cells/ultrastructure , Microscopy, Electron/methods , Animals , Cell Extracts , Chromatin/metabolism , Chromosomes, Artificial, Bacterial/metabolism , Cross-Linking Reagents/pharmacology , DNA/metabolism , DNA Replication/drug effects , DNA, Cruciform/metabolism , Ficusin/pharmacology , Genome, Fungal , Male , Mammals , Nucleic Acid Denaturation/drug effects , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spermatozoa/cytology , Spermatozoa/drug effects , Xenopus
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