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  1. Article ; Online: Eukaryotic stress-induced mutagenesis is limited by a local control of translesion synthesis.

    Masłowska, Katarzyna H / Villafañez, Florencia / Laureti, Luisa / Iwai, Shigenori / Pagès, Vincent

    Nucleic acids research

    2022  Volume 50, Issue 4, Page(s) 2074–2080

    Abstract: The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this ... ...

    Abstract The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this work, we raise the question whether TLS is controlled locally or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that (i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, (ii) a single lesion is enough to induce PCNA ubiquitination and (iii) PCNA ubiquitination is imperative for TLS to occur. More importantly, we show that the activation of the DDR that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.
    MeSH term(s) DNA Damage ; DNA Repair/genetics ; DNA Replication/genetics ; Mutagenesis ; Proliferating Cell Nuclear Antigen/genetics ; Proliferating Cell Nuclear Antigen/metabolism ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Ubiquitination
    Chemical Substances Proliferating Cell Nuclear Antigen
    Language English
    Publishing date 2022-01-29
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkac044
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1067: Show issues Location:
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  2. Article ; Online: Development and Optimization of a Miniaturized Western Blot-Based Screening Platform to Identify Regulators of Post-Translational Modifications.

    Villafañez, Florencia / Gottifredi, Vanesa / Soria, Gastón

    High-throughput

    2019  Volume 8, Issue 2

    Abstract: Post-translational modifications (PTMs) are fundamental traits of protein functionality and their study has been addressed using several approaches over the past years. However, screening methods developed to detect regulators of PTMs imply many ... ...

    Abstract Post-translational modifications (PTMs) are fundamental traits of protein functionality and their study has been addressed using several approaches over the past years. However, screening methods developed to detect regulators of PTMs imply many challenges and are usually based on expensive techniques. Herein, we described the development and optimization of a western blot-based platform for identification of regulators of a specific PTM-mono-ubiquitylation of proliferating cell nuclear antigen (PCNA). This cell-based method does not require specific equipment, apart from the basic western blot (WB) devices and minor accessories, which are accessible for most research labs. The modifications introduced to the classical WB protocol allow the performance of PTM analysis from a single well of a 96-well plate with minimal sample manipulation and low intra- and inter-plate variability, making this method ideal to screen arrayed compound libraries in a 96-well format. As such, our experimental pipeline provides the proof of concept to design small screenings of PTM regulators by improving the quantitative accuracy and throughput capacity of classical western blots.
    Language English
    Publishing date 2019-06-03
    Publishing country Switzerland
    Document type Journal Article
    ISSN 2571-5135
    ISSN (online) 2571-5135
    DOI 10.3390/ht8020015
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: GSK-3 is an RNA polymerase II phospho-CTD kinase.

    Nieto Moreno, Nicolás / Villafañez, Florencia / Giono, Luciana E / Cuenca, Carmen / Soria, Gastón / Muñoz, Manuel J / Kornblihtt, Alberto R

    Nucleic acids research

    2020  Volume 48, Issue 11, Page(s) 6068–6080

    Abstract: We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling ... ...

    Abstract We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.
    MeSH term(s) Alternative Splicing/genetics ; Alternative Splicing/radiation effects ; Apoptosis/radiation effects ; DNA Damage/radiation effects ; Fluorescence ; Genes, Dominant ; Genes, Reporter ; Glycogen Synthase Kinase 3/antagonists & inhibitors ; Glycogen Synthase Kinase 3/genetics ; Glycogen Synthase Kinase 3/metabolism ; HEK293 Cells ; HeLa Cells ; Histones/metabolism ; Humans ; Mutant Proteins/genetics ; Mutant Proteins/metabolism ; Phosphorylation/radiation effects ; Protein Kinases/genetics ; Protein Kinases/metabolism ; RNA Polymerase II/chemistry ; RNA Polymerase II/metabolism ; Transcription, Genetic/radiation effects ; Ultraviolet Rays
    Chemical Substances Histones ; Mutant Proteins ; Protein Kinases (EC 2.7.-) ; carboxy-terminal domain kinase (EC 2.7.1.-) ; Glycogen Synthase Kinase 3 (EC 2.7.11.26) ; RNA Polymerase II (EC 2.7.7.-)
    Language English
    Publishing date 2020-05-06
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkaa322
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1067: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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  4. Article ; Online: AKT inhibition impairs PCNA ubiquitylation and triggers synthetic lethality in homologous recombination-deficient cells submitted to replication stress.

    Villafañez, Florencia / García, Iris Alejandra / Carbajosa, Sofia / Pansa, María Florencia / Mansilla, Sabrina / Llorens, María Candelaria / Angiolini, Virginia / Guantay, Laura / Jacobs, Heinz / Madauss, Kevin P / Gloger, Israel / Gottifredi, Vanesa / Bocco, Jose Luis / Soria, Gaston

    Oncogene

    2019  Volume 38, Issue 22, Page(s) 4310–4324

    Abstract: Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS ... ...

    Abstract Translesion DNA synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to safeguard replication forks integrity. Thus, the inhibition of TLS becomes a promising point of therapeutic intervention in HR-deficient cancers, where TLS impairment might trigger synthetic lethality (SL). The main limitation to test this hypothesis is the current lack of selective pharmacological inhibitors of TLS. Herein, we developed a miniaturized screening assay to identify inhibitors of PCNA ubiquitylation, a key post-translational modification required for efficient TLS activation. After screening a library of 627 kinase inhibitors, we found that targeting the pro-survival kinase AKT leads to strong impairment of PCNA ubiquitylation. Mechanistically, we found that AKT-mediated modulation of Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation after UV requires the upstream activity of DNA PKcs, without affecting PCNA ubiquitylation levels in unperturbed cells. Moreover, we confirmed that persistent AKT inhibition blocks the recruitment of TLS polymerases to sites of DNA damage and impairs DNA replication forks processivity after UV irradiation, leading to increased DNA replication stress and cell death. Remarkably, when we compared the differential survival of HR-proficient vs HR-deficient cells, we found that the combination of UV irradiation and AKT inhibition leads to robust SL induction in HR-deficient cells. We link this phenotype to AKT ability to inhibit PCNA ubiquitylation, since the targeted knockdown of PCNA E3-ligase (RAD18) and a non-ubiquitylable (PCNA K164R) knock-in model recapitulate the observed SL induction. Collectively, this work identifies AKT as a novel regulator of PCNA ubiquitylation and provides the proof-of-concept of inhibiting TLS as a therapeutic approach to selectively kill HR-deficient cells submitted to replication stress.
    MeSH term(s) Cell Death/genetics ; Cell Line ; Cell Line, Tumor ; DNA/genetics ; DNA Damage/genetics ; DNA Replication/genetics ; DNA-Directed DNA Polymerase/genetics ; HCT116 Cells ; HEK293 Cells ; Homologous Recombination/genetics ; Humans ; Proliferating Cell Nuclear Antigen/genetics ; Proto-Oncogene Proteins c-akt/genetics ; Ubiquitin-Protein Ligases/genetics ; Ubiquitination/genetics
    Chemical Substances PCNA protein, human ; Proliferating Cell Nuclear Antigen ; DNA (9007-49-2) ; Ubiquitin-Protein Ligases (EC 2.3.2.27) ; Proto-Oncogene Proteins c-akt (EC 2.7.11.1) ; DNA-Directed DNA Polymerase (EC 2.7.7.7)
    Language English
    Publishing date 2019-01-31
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 639046-8
    ISSN 1476-5594 ; 0950-9232
    ISSN (online) 1476-5594
    ISSN 0950-9232
    DOI 10.1038/s41388-019-0724-7
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 2218: Show issues Location:
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  5. Article ; Online: P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach.

    Joray, Mariana Belén / Villafañez, Florencia / González, María Laura / Crespo, María Inés / Laiolo, Jerónimo / Palacios, Sara María / Bocco, José Luis / Soria, Gastón / Carpinella, María Cecilia

    Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association

    2017  Volume 109, Issue Pt 2, Page(s) 888–897

    Abstract: This work examines the antitumor activity of an isomeric mixture (1), composed of the limonoids meliartenin and its interchangeable isomer 12-hydroxyamoorastatin. The results obtained showed that 1 displayed outstanding cytotoxic activity against CCRF- ... ...

    Abstract This work examines the antitumor activity of an isomeric mixture (1), composed of the limonoids meliartenin and its interchangeable isomer 12-hydroxyamoorastatin. The results obtained showed that 1 displayed outstanding cytotoxic activity against CCRF-CEM, K562, A549 and HCT116 cells, with a highly selective effect on the latter, with an IC
    Language English
    Publishing date 2017-11
    Publishing country England
    Document type Journal Article
    ZDB-ID 782617-5
    ISSN 1873-6351 ; 0278-6915
    ISSN (online) 1873-6351
    ISSN 0278-6915
    DOI 10.1016/j.fct.2017.04.039
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    Zs.A 529: Show issues Location:
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  6. Article ; Online: Polo-like Kinase 1 Inhibition as a Therapeutic Approach to Selectively Target BRCA1-Deficient Cancer Cells by Synthetic Lethality Induction.

    Carbajosa, Sofía / Pansa, María Florencia / Paviolo, Natalia S / Castellaro, Andrés M / Andino, Diego L / Nigra, Ayelén D / García, Iris Alejandra / Racca, Ana C / Rodriguez-Berdini, Lucía / Angiolini, Virginia / Guantay, Laura / Villafañez, Florencia / Federico, María Belén / Rodríguez-Baili, María Celeste / Caputto, Beatriz L / Drewes, Gerard / Madauss, Kevin P / Gloger, Israel / Fernandez, Elmer /
    Gil, Germán A / Bocco, José Luis / Gottifredi, Vanesa / Soria, Gastón

    Clinical cancer research : an official journal of the American Association for Cancer Research

    2019  Volume 25, Issue 13, Page(s) 4049–4062

    Abstract: Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic ... ...

    Abstract Purpose: BRCA1 and BRCA2 deficiencies are widespread drivers of human cancers that await the development of targeted therapies. We aimed to identify novel synthetic lethal relationships with therapeutic potential using BRCA-deficient isogenic backgrounds.
    Experimental design: We developed a phenotypic screening technology to simultaneously search for synthetic lethal (SL) interactions in BRCA1- and BRCA2-deficient contexts. For validation, we developed chimeric spheroids and a dual-tumor xenograft model that allowed the confirmation of SL induction with the concomitant evaluation of undesired cytotoxicity on BRCA-proficient cells. To extend our results using clinical data, we performed retrospective analysis on The Cancer Genome Atlas (TCGA) breast cancer database.
    Results: The screening of a kinase inhibitors library revealed that Polo-like kinase 1 (PLK1) inhibition triggers strong SL induction in BRCA1-deficient cells. Mechanistically, we found no connection between the SL induced by PLK1 inhibition and PARP inhibitors. Instead, we uncovered that BRCA1 downregulation and PLK1 inhibition lead to aberrant mitotic phenotypes with altered centrosomal duplication and cytokinesis, which severely reduced the clonogenic potential of these cells. The penetrance of PLK1/BRCA1 SL interaction was validated using several isogenic and nonisogenic cellular models, chimeric spheroids, and mice xenografts. Moreover, bioinformatic analysis revealed high-PLK1 expression in BRCA1-deficient tumors, a phenotype that was consistently recapitulated by inducing BRCA1 deficiency in multiple cell lines as well as in BRCA1-mutant cells.
    Conclusions: We uncovered an unforeseen addiction of BRCA1-deficient cancer cells to PLK1 expression, which provides a new means to exploit the therapeutic potential of PLK1 inhibitors in clinical trials, by generating stratification schemes that consider this molecular trait in patient cohorts.
    MeSH term(s) Animals ; Apoptosis/drug effects ; Apoptosis/genetics ; BRCA1 Protein/deficiency ; BRCA2 Protein/deficiency ; BRCA2 Protein/genetics ; Cell Cycle/drug effects ; Cell Cycle/genetics ; Cell Cycle Proteins/antagonists & inhibitors ; Cell Line, Tumor ; Cells, Cultured ; Chromosome Aberrations ; DNA Damage ; Disease Models, Animal ; Gene Expression ; Gene Knockdown Techniques ; Humans ; Mice ; Protein Kinase Inhibitors/pharmacology ; Protein Serine-Threonine Kinases/antagonists & inhibitors ; Proto-Oncogene Proteins/antagonists & inhibitors ; Synthetic Lethal Mutations/drug effects ; Xenograft Model Antitumor Assays ; Polo-Like Kinase 1
    Chemical Substances BRCA1 Protein ; BRCA1 protein, human ; BRCA2 Protein ; BRCA2 protein, human ; Cell Cycle Proteins ; Protein Kinase Inhibitors ; Proto-Oncogene Proteins ; Protein Serine-Threonine Kinases (EC 2.7.11.1)
    Language English
    Publishing date 2019-03-19
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 1225457-5
    ISSN 1557-3265 ; 1078-0432
    ISSN (online) 1557-3265
    ISSN 1078-0432
    DOI 10.1158/1078-0432.CCR-18-3516
    Database MEDical Literature Analysis and Retrieval System OnLINE

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