Assessing homologous recombination and interstrand cross-link repair in embryonal carcinoma testicular germ cell tumor cell lines Journal Article


Authors: Cavallo, F.; Caggiano, C.; Jasin, M.; Barchi, M.
Article Title: Assessing homologous recombination and interstrand cross-link repair in embryonal carcinoma testicular germ cell tumor cell lines
Abstract: Testicular germ cell tumors (TGCTs) are typically exquisitely sensitive to DNA interstrand cross-link (ICLs) agents. ICLs covalently link both strands of the DNA duplex, impeding fundamental cellular processes like DNA replication to cause cell death. A leading drug used for the treatment of TGCTs is cisplatin, which introduces ICLs and leads to formation of double strand breaks (DSBs), a DNA lesion that can be repaired in the S/G2 phases of the cell cycle by homologous recombination (HR, also termed homology-direct repair). Although most TGCTs respond to cisplatin-induced ICLs, a fraction is resistant to treatment. One proposed mechanism of TGCT resistance to cisplatin is an enhanced ability to repair DSBs by HR. Other than HR, repair of the ICL-lesions requires additional DNA repair mechanisms, whose action might also be implemented in therapy-resistant cells. This chapter describes GFP assays to measure (a) HR proficiency following formation of a DSB by the endonuclease I-SceI, and (b) HR repair induced by site-specific ICL formation involving psoralen. These experimental approaches can be used to determine the proficiency of TGCT cell lines in DSB repair by HR in comparison to HR repair of ICLs, providing tools to better characterize their recombination profile. Protocols of these assays have been adapted for use in Embryonal Carcinoma (EC) TGCT cell lines. Assays only require transient introduction of plasmids within cells, affording the advantage of testing multiple cell lines in a relatively short time. © Springer Science+Business Media, LLC, part of Springer Nature 2021.
Keywords: homologous recombination; psoralen; embryonal carcinoma (ec); gfp reporters (dr-gfp, tr-orip-gfp); interstrand cross-link (icl) repair; tgcts
Journal Title: Methods in Molecular Biology
Volume: 2195
ISSN: 1064-3745
Publisher: Humana Press Inc  
Date Published: 2021-01-01
Start Page: 113
End Page: 123
Language: English
DOI: 10.1007/978-1-0716-0860-9_9
PUBMED: 32852761
PROVIDER: scopus
DOI/URL:
Notes: Chapter 9 in "Testicular Germ Cell Tumors: Methods and Protocols" (ISBN: 978-1-0716-0859-3) -- Export Date: 1 October 2020 -- Source: Scopus
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  1. Maria Jasin
    227 Jasin