5b4e6e2122140c003c7a2af4f9e455d8 pmc_347717416.pdf 7d2c7dc77b3a59f65e8ca22032ff23026eb7dfa4 pmc_347717416.pdf 234d10687a26b6004e291ed468a098db1de9ffe2bea63c7dc86a5be290b4d8e2 pmc_347717416.pdf Title: Gossypol Treatment Restores Insufficient Apoptotic Function of DFF40/CAD in Human Glioblastoma Cells Subject: Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible. Keywords: apoptosis; caspase-activated DNase (DFF40/CAD); glioblastoma (GBM); gossypol; nuclear fragmentation/disassembly Author: Laura Martínez-Escardó, Montse Alemany, María Sánchez-Osuna, Alejandro Sánchez-Chardi, Meritxell Roig-Martínez, Salvio Suárez-García, Daniel Ruiz-Molina, Noemi Vidal, Gerard Plans, Carles Majós, Judit Ribas, María Antonia Baltrons, Jose R. Bayascas, Carlos Barcia, Jordi Bruna and Victor J. 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yes yes 612 0 JIGDHB+MicrosoftSansSerif CID TrueType Identity-H yes yes yes 618 0 JIGDJB+Calibri,BoldItalic CID TrueType Identity-H yes yes yes 624 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-20 02:31:27 CEST RepresentationInformation: pmc_347717416.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-05-19 10:15:07 CEST Size: 11265079 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 876 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Reagents and Cell Lines Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Patient-Derived Non-Commercial GBM Cell Cultures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: DEVD-Directed Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Immunofluorescence Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Lysosomes Labeling Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Protein Extractions and Western Blotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Transmission and Field Emission Scanning Electron Microscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Transmission Electron Microscope (TEM) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Field Emission-Scanning Electron Microscopy (FE-SEM) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Nuclear Morphology Analysis by Chromatin Staining Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: DFF40/CAD Overexpression and Silencing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: TUNEL Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Statistical Analysis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Children: Item: Title: Human Glioblastoma Cells Show Nuclear Alterations Compatible with Apoptotic Nuclear Morphologies When Challenged with Gossypol Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Caspases and DFF40/CAD Are Required for Gossypol-Induced Apoptotic Nuclear Morphologies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: The Activation of Caspases and DFF40/CAD Are Necessary but Not Sufficient to Trigger Apoptotic Nuclear Disassembling after Gossypol Treatment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Gossypol Induces Both Caspase-Dependent and Caspase-Independent DNA Damage Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Gossypol Facilitates Nuclear DFF40/CAD Assembly into High-Order Structures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Info: Title: Gossypol Treatment Restores Insufficient Apoptotic Function of DFF40/CAD in Human Glioblastoma Cells Author: Laura Martínez-Escardó, Montse Alemany, María Sánchez-Osuna, Alejandro Sánchez-Chardi, Meritxell Roig-Martínez, Salvio Suárez-García, Daniel Ruiz-Molina, Noemi Vidal, Gerard Plans, Carles Majós, Judit Ribas, María Antonia Baltrons, Jose R. Bayascas, Carlos Barcia, Jordi Bruna and Victor J. Yuste Subject: Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible. 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