{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T04:59:30Z","timestamp":1785473970077,"version":"3.56.0"},"reference-count":40,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T00:00:00Z","timestamp":1785283200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003141","name":"Secretar\u00eda de Ciencia, Humanidades, Tecnolog\u00eda e Innovaci\u00f3n","doi-asserted-by":"crossref","award":["SEP-243102"],"award-info":[{"award-number":["SEP-243102"]}],"id":[{"id":"10.13039\/501100003141","id-type":"DOI","asserted-by":"crossref"}]},{"award":["SEP-243102"],"award-info":[{"award-number":["SEP-243102"]}],"id":[{"id":"https:\/\/ror.org\/059ex5q34","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Instituto Nacional de Pediatr\u00eda","award":["032\/2015"],"award-info":[{"award-number":["032\/2015"]}]},{"award":["032\/2015"],"award-info":[{"award-number":["032\/2015"]}],"id":[{"id":"https:\/\/ror.org\/05adj5455","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Direcci\u00f3n General de Asuntos del Personal Acad\u00e9mico (DGAPA), Universidad Nacional Aut\u00f3noma de M\u00e9xico","award":["IN205120"],"award-info":[{"award-number":["IN205120"]}]},{"award":["IN205120"],"award-info":[{"award-number":["IN205120"]}],"id":[{"id":"https:\/\/ror.org\/01tmp8f25","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003141","name":"Secretar\u00eda de Ciencia, Humanidades, Tecnolog\u00eda e Innovaci\u00f3n","doi-asserted-by":"crossref","award":["237968"],"award-info":[{"award-number":["237968"]}],"id":[{"id":"10.13039\/501100003141","id-type":"DOI","asserted-by":"crossref"}]},{"award":["237968"],"award-info":[{"award-number":["237968"]}],"id":[{"id":"https:\/\/ror.org\/059ex5q34","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Int. J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Fanconi anemia (FA) is a rare disease with a deficient homologous recombination DNA repair pathway and high sensitivity to mitomycin C (MMC). In FA cells, hydroxyurea (HU), when applied in the G2 phase of the cell cycle, exacerbates the chromosomal aberrations (CAs) induced by MMC. Here, we study how exposure to HU in the G2 phase results in an increased CAs frequency in FA cells with or without prior treatment with MMC. We performed chromosome aberration analysis, flow cytometry, and mRNA and protein expression in lymphoblastoid cell lines. We found that HU alone induces post-replicative DNA damage in the form of DNA double-strand breaks (DSBs) in both wild-type and FA cells and a similar increase in the activation of DSBs marker \u0263H2AX. HU potentiates the amount of CAs only in MMC-treated FA cells by multiple mechanisms, including (1) hindering the \u0263H2AX signal, (2) impairing the translocation of ribonucleotide reductase (RNR) into the cell nucleus, and (3) promoting the activity of WIP1 phosphatase. Concomitantly, increased levels of cell death were observed in FA cell cultures. HU induces DNA damage and potentiates pre-existent damage in FA cells by preventing the translocation of the RNR component p53R2 into the nucleus and activating checkpoint recovery, thus allowing the survival of a proportion of FA cells that have adapted to DNA damage.<\/jats:p>","DOI":"10.3390\/ijms27156782","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T08:16:52Z","timestamp":1785313012000},"page":"6782","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Mechanisms of Hydroxyurea-Mediated DNA Damage Potentiation in Fanconi Anemia Cells"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-7396-6068","authenticated-orcid":false,"given":"Benjamin","family":"Bustamante","sequence":"first","affiliation":[{"name":"Laboratorio de Citogen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Insurgentes Sur 3700 C, Col. Insurgentes Cuicuilco, Mexico City 04530, Mexico"},{"name":"Doctorado en Biolog\u00eda Experimental, Universidad Aut\u00f3noma Metropolitana-Iztapalapa, Mexico City 09310, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9583-0925","authenticated-orcid":false,"given":"David","family":"Sosa","sequence":"additional","affiliation":[{"name":"Laboratorio de Citogen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Insurgentes Sur 3700 C, Col. Insurgentes Cuicuilco, Mexico City 04530, Mexico"},{"name":"Laboratorio de An\u00e1lisis Gen\u00e9ticos Especializados M\u00e9xico (LAGEM), Mexico City 04730, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2209-1607","authenticated-orcid":false,"given":"Jorge","family":"Melendez-Zajgla","sequence":"additional","affiliation":[{"name":"Functional Cancer Genomics Laboratory, Instituto Nacional de Medicina Gen\u00f3mica (INMEGEN), Mexico City 14610, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0470-1069","authenticated-orcid":false,"given":"Gabriel","family":"L\u00f3pez-Vel\u00e1zquez","sequence":"additional","affiliation":[{"name":"Laboratory of Biomolecules and Infant Health, Instituto Nacional de Pediatr\u00eda, Mexico City 04530, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia","family":"Ostrosky-Wegman","sequence":"additional","affiliation":[{"name":"Departamento de Medicina Gen\u00f3mica y Toxicolog\u00eda Ambiental, Instituto de Investigaciones Biom\u00e9dicas, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Mexico City 04510, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfredo","family":"Rodriguez","sequence":"additional","affiliation":[{"name":"Departamento de Medicina Gen\u00f3mica y Toxicolog\u00eda Ambiental, Instituto de Investigaciones Biom\u00e9dicas, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Mexico City 04510, Mexico"},{"name":"Laboratorio de Falla Medular y Carcinog\u00e9nesis, Instituto Nacional de Pediatr\u00eda, Mexico City 04530, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leda","family":"Torres","sequence":"additional","affiliation":[{"name":"Laboratorio de Citogen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Insurgentes Sur 3700 C, Col. Insurgentes Cuicuilco, Mexico City 04530, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ulises","family":"Ju\u00e1rez-Figueroa","sequence":"additional","affiliation":[{"name":"Laboratorio de Citogen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Insurgentes Sur 3700 C, Col. Insurgentes Cuicuilco, Mexico City 04530, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberto","family":"S\u00e1nchez-Olea","sequence":"additional","affiliation":[{"name":"Instituto de F\u00edsica, Universidad Aut\u00f3noma de San Luis Potos\u00ed, San Luis Potos\u00ed 78295, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elsa","family":"Cervantes-R\u00edos","sequence":"additional","affiliation":[{"name":"Laboratorio de Biolog\u00eda Celular y Citometr\u00eda de Flujo, Departamento de Ciencias de la Salud, Universidad Aut\u00f3noma Metropolitana-Iztapalapa, Mexico City 09310, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2501-2916","authenticated-orcid":false,"given":"Roc\u00edo","family":"Ortiz-Mu\u00f1iz","sequence":"additional","affiliation":[{"name":"Laboratorio de Biolog\u00eda Celular y Citometr\u00eda de Flujo, Departamento de Ciencias de la Salud, Universidad Aut\u00f3noma Metropolitana-Iztapalapa, Mexico City 09310, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bertha","family":"Molina","sequence":"additional","affiliation":[{"name":"Laboratorio de Citogen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Insurgentes Sur 3700 C, Col. Insurgentes Cuicuilco, Mexico City 04530, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3097-6368","authenticated-orcid":false,"given":"Sara","family":"Frias","sequence":"additional","affiliation":[{"name":"Laboratorio de Citogen\u00e9tica, Instituto Nacional de Pediatr\u00eda, Insurgentes Sur 3700 C, Col. Insurgentes Cuicuilco, Mexico City 04530, Mexico"},{"name":"Departamento de Medicina Gen\u00f3mica y Toxicolog\u00eda Ambiental, Instituto de Investigaciones Biom\u00e9dicas, Universidad Nacional Aut\u00f3noma de M\u00e9xico, Mexico City 04510, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1136\/jmg-2022-108714","article-title":"Comprehensive laboratory diagnosis of Fanconi anaemia: Comparison of cellular and molecular analysis","volume":"60","author":"Joshi","year":"2023","journal-title":"J. Med. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/S0921-8777(99)00041-5","article-title":"Drug sensitivity spectra in Fanconi anemia lymphoblastoid cell lines of defined complementation groups","volume":"435","author":"Carreau","year":"1999","journal-title":"Mutat. Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"102","DOI":"10.18233\/APM43No2pp102-1282393","article-title":"Anemia de Fanconi, Parte 1. Diagn\u00f3stico citogen\u00e9tico","volume":"43","author":"Molina","year":"2022","journal-title":"Acta Pedi\u00e1tr. M\u00e9x."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e185126","DOI":"10.1172\/JCI185126","article-title":"Deficiency of the Fanconi anemia core complex protein FAAP100 results in severe Fanconi anemia","volume":"135","author":"Harrison","year":"2025","journal-title":"J. Clin. Investig."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3013","DOI":"10.1172\/JCI92069","article-title":"Biallelic mutations in the ubiquitin ligase RFWD3 cause Fanconi anemia","volume":"127","author":"Knies","year":"2017","journal-title":"J. Clin. Investig."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"111600","DOI":"10.1016\/j.celrep.2022.111600","article-title":"NEIL3 contributes to the Fanconi anemia\/BRCA pathway by promoting the downstream double-strand break repair step","volume":"41","author":"Li","year":"2022","journal-title":"Cell Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"112428","DOI":"10.1016\/j.celrep.2023.112428","article-title":"Fanconi anemia-associated chromosomal radial formation is dependent on POLtheta-mediated alternative end joining","volume":"42","author":"Rogers","year":"2023","journal-title":"Cell Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e53639","DOI":"10.15252\/embr.202153639","article-title":"SCAI promotes error-free repair of DNA interstrand crosslinks via the Fanconi anemia pathway","volume":"23","author":"Schubert","year":"2022","journal-title":"EMBO Rep."},{"key":"ref_9","first-page":"115","article-title":"Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients","volume":"357","author":"Frias","year":"1996","journal-title":"Mutat. Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1038\/onc.2015.68","article-title":"A novel role for non-ubiquitinated FANCD2 in response to hydroxyurea-induced DNA damage","volume":"35","author":"Chen","year":"2016","journal-title":"Oncogene"},{"key":"ref_11","first-page":"1559","article-title":"Inhibition of ribonucleoside diphosphate reductase by hydroxyurea","volume":"28","author":"Krakoff","year":"1968","journal-title":"Cancer Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"40647","DOI":"10.1074\/jbc.M106088200","article-title":"Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells","volume":"276","author":"Guittet","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1038\/35003506","article-title":"A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage","volume":"404","author":"Tanaka","year":"2000","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1101\/gr.180497.114","article-title":"Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription","volume":"25","author":"Hoffman","year":"2015","journal-title":"Genome Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"29361","DOI":"10.1038\/srep29361","article-title":"DNA replication inhibitor hydroxyurea alters Fe-S centers by producing reactive oxygen species in vivo","volume":"6","author":"Huang","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Singh, A., and Xu, Y.J. (2016). The Cell Killing Mechanisms of Hydroxyurea. Genes, 7.","DOI":"10.3390\/genes7110099"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1791","DOI":"10.1038\/s41467-023-37341-y","article-title":"Excessive reactive oxygen species induce transcription-dependent replication stress","volume":"14","author":"Andrs","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1021\/jm00338a026","article-title":"Hydroxyurea. A New Type of Potential Antitumor Agent","volume":"6","author":"Stearns","year":"1963","journal-title":"J. Med. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e2404470121","DOI":"10.1073\/pnas.2404470121","article-title":"Revised mechanism of hydroxyurea-induced cell cycle arrest and an improved alternative","volume":"121","author":"Shaw","year":"2024","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1002\/em.21938","article-title":"Hydroxyurea induces chromosomal damage in G2 and enhances the clastogenic effect of mitomycin C in Fanconi anemia cells","volume":"56","author":"Molina","year":"2015","journal-title":"Environ. Mol. Mutagen."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"350","DOI":"10.1016\/j.yexcr.2015.03.011","article-title":"DNA damage checkpoint recovery and cancer development","volume":"334","author":"Wang","year":"2015","journal-title":"Exp. Cell Res."},{"key":"ref_22","first-page":"1829","article-title":"A high susceptibility of Fanconi\u2019s anemia to chromosome breakage by DNA cross-linking agents","volume":"33","author":"Sasaki","year":"1973","journal-title":"Cancer Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"110701","DOI":"10.1016\/j.celrep.2022.110701","article-title":"DNA replication is highly resilient and persistent under the challenge of mild replication stress","volume":"39","author":"Mocanu","year":"2022","journal-title":"Cell Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1186\/s12976-015-0011-4","article-title":"Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process","volume":"12","author":"Rodriguez","year":"2015","journal-title":"Theor. Biol. Med. Model."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1016\/j.cell.2018.07.011","article-title":"Dual Roles of Poly(dA:dT) Tracts in Replication Initiation and Fork Collapse","volume":"174","author":"Tubbs","year":"2018","journal-title":"Cell"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2715","DOI":"10.1101\/gad.340905","article-title":"Homologous recombination and nonhomologous end-joining repair pathways regulate fragile site stability","volume":"19","author":"Schwartz","year":"2005","journal-title":"Genes Dev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1117","DOI":"10.1016\/j.molcel.2016.10.037","article-title":"RAD52 Facilitates Mitotic DNA Synthesis Following Replication Stress","volume":"64","author":"Bhowmick","year":"2016","journal-title":"Mol. Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1038\/nature25154","article-title":"Alcohol and endogenous aldehydes damage chromosomes and mutate stem cells","volume":"553","author":"Garaycoechea","year":"2018","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"R158","DOI":"10.1093\/hmg\/ddaa087","article-title":"Emerging functions of Fanconi anemia genes in replication fork protection pathways","volume":"29","author":"Kolinjivadi","year":"2020","journal-title":"Hum. Mol. Genet."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"22602","DOI":"10.1074\/jbc.M115.663666","article-title":"FANCD2-associated nuclease 1, but not exonuclease 1 or flap endonuclease 1, is able to unhook DNA interstrand cross-links in vitro","volume":"290","author":"Pizzolato","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_31","first-page":"81","article-title":"Microhomology-mediated end joining: Good, bad and ugly","volume":"809","author":"Seol","year":"2018","journal-title":"Mutat. Res."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Garcia-de-Teresa, B., Rodriguez, A., and Frias, S. (2020). Chromosome Instability in Fanconi Anemia: From Breaks to Phenotypic Consequences. Genes, 11.","DOI":"10.20944\/preprints202011.0563.v2"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1101\/gad.1863810","article-title":"Essential role of Tip60-dependent recruitment of ribonucleotide reductase at DNA damage sites in DNA repair during G1 phase","volume":"24","author":"Niida","year":"2010","journal-title":"Genes Dev."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Zavileyskiy, L., and Bunik, V. (2022). Regulation of p53 Function by Formation of Non-Nuclear Heterologous Protein Complexes. Biomolecules, 12.","DOI":"10.3390\/biom12020327"},{"key":"ref_35","first-page":"980","article-title":"Wild-type p53 regulates human ribonucleotide reductase by protein-protein interaction with p53R2 as well as hRRM2 subunits","volume":"63","author":"Xue","year":"2003","journal-title":"Cancer Res."},{"key":"ref_36","first-page":"83","article-title":"Beyond interstrand crosslinks repair: Contribution of FANCD2 and other Fanconi Anemia proteins to the replication of DNA","volume":"808","author":"Federico","year":"2018","journal-title":"Mutat. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"3196","DOI":"10.1038\/emboj.2009.246","article-title":"Wip1 confers G2 checkpoint recovery competence by counteracting p53-dependent transcriptional repression","volume":"28","author":"Lindqvist","year":"2009","journal-title":"EMBO J."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e45","DOI":"10.1093\/nar\/29.9.e45","article-title":"A new mathematical model for relative quantification in real-time RT-PCR","volume":"29","author":"Pfaffl","year":"2001","journal-title":"Nucleic Acids Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1101\/pdb.prot083733","article-title":"Rapid Isolation of Nuclei from Cells In Vitro","volume":"2015","author":"Nabbi","year":"2015","journal-title":"Cold Spring Harb. Protoc."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"373","DOI":"10.3109\/10520298709108028","article-title":"The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans","volume":"62","author":"Gray","year":"1987","journal-title":"Stain. Technol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/15\/6782\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T04:26:41Z","timestamp":1785472001000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/15\/6782"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,29]]},"references-count":40,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["ijms27156782"],"URL":"https:\/\/doi.org\/10.3390\/ijms27156782","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,29]]}}}