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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jostes S, Nettersheim D, Fellermeyer M et al (2017) The bromodomain inhibitor JQ1 triggers growth arrest and apoptosis in testicular germ cell tumours in vitro and in vivo. J Cell Mol Med 21(7):1300–1314
Oosterhuis JW, Looijenga LH (2005) Testicular germ-cell tumours in a broader perspective. Nat Rev Cancer 5(3):210–222
Bagrodia A, Lee BH, Lee W et al (2016) Genetic determinants of cisplatin resistance in patients with advanced germ cell tumors. J Clin Oncol 34(33):4000–4007
Pont J, Holtl W, Kosak D et al (1990) Risk-adapted treatment choice in stage I nonseminomatous testicular germ cell cancer by regarding vascular invasion in the primary tumor: a prospective trial. J Clin Oncol 8(1):16–20
Moul JW, McCarthy WF, Fernandez EB et al (1994) Percentage of embryonal carcinoma and of vascular invasion predicts pathological stage in clinical stage I nonseminomatous testicular cancer. Cancer Res 54(2):362–364
Heidenreich A, Sesterhenn IA, Mostofi FK et al (1998) Prognostic risk factors that identify patients with clinical stage I nonseminomatous germ cell tumors at low risk and high risk for metastasis. Cancer 83(5):1002–1011
Einhorn LH, Donohue J (1977) Cis-diamminedichloroplatinum, vinblastine, and bleomycin combination chemotherapy in disseminated testicular cancer. Ann Intern Med 87(3):293–298
Ceccaldi R, Sarangi P, D’Andrea AD (2016) The Fanconi anaemia pathway: new players and new functions. Nat Rev Mol Cell Biol 17(6):337–349
Pierce AJ, Hu P, Han M et al (2001) Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev 15(24):3237–3242
Cavallo F, Graziani G, Antinozzi C et al (2012) Reduced proficiency in homologous recombination underlies the high sensitivity of embryonal carcinoma testicular germ cell tumors to Cisplatin and poly (adp-ribose) polymerase inhibition. PLoS One 7(12):e51563
Moynahan ME, Jasin M (2010) Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis. Nat Rev Mol Cell Biol 11(3):196–207
Nakanishi K, Cavallo F, Perrouault L et al (2011) Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication. Nat Struct Mol Biol 18(4):500–503
Nakanishi K, Cavallo F, Brunet E et al (2011) Homologous recombination assay for interstrand cross-link repair. Methods Mol Biol 745:283–291
Richardson C, Moynahan ME, Jasin M (1998) Double-strand break repair by interchromosomal recombination: suppression of chromosomal translocations. Genes Dev 12(24):3831–3842
Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108(2):193–199
Pierce AJ, Jasin M (2005) Measuring recombination proficiency in mouse embryonic stem cells. Methods Mol Biol 291:373–384
Weinstock DM, Nakanishi K, Helgadottir HR et al (2006) Assaying double-strand break repair pathway choice in mammalian cells using a targeted endonuclease or the RAG recombinase. Methods Enzymol 409:524–540
Acknowledgments
This work was supported by My First AIRC Grant (MFAG) n. 4765 (M.B.), Cooperlink grant n. CII11RLETZ (M.B.), PRIN 2010/2010M4NEFY_004 (M.B.) and R01CA185660 (M.J.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Cavallo, F., Caggiano, C., Jasin, M., Barchi, M. (2021). Assessing Homologous Recombination and Interstrand Cross-Link Repair in Embryonal Carcinoma Testicular Germ Cell Tumor Cell Lines. In: Bagrodia, A., Amatruda, J.F. (eds) Testicular Germ Cell Tumors. Methods in Molecular Biology, vol 2195. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0860-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0860-9_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0859-3
Online ISBN: 978-1-0716-0860-9
eBook Packages: Springer Protocols