睾丸生殖细胞肿瘤(TGCT)通常对DNA链间交联(ICL)试剂非常敏感。ICL共价连接DNA双链体的两条链,阻碍DNA复制等基本细胞过程导致细胞死亡。用于治疗TGCT的主要药物是顺铂,引入ICL并导致双链断裂(DSB)的形成,可以通过同源重组在细胞周期的S/G2期修复的DNA损伤(HR,也称为同源性-直接修复)。虽然大多数TGCT对顺铂诱导的ICL有反应,一小部分对治疗有抵抗力。一种提出的TGCT对顺铂的抗性机制是通过HR修复DSB的能力增强。除了HR,ICL损伤的修复需要额外的DNA修复机制,其作用也可能在治疗抗性细胞中实现。本章描述了GFP测定以测量(a)通过核酸内切酶I-SceI形成DSB后的HR能力,和(b)由涉及补骨脂素的位点特异性ICL形成诱导的HR修复。与ICL的HR修复相比,这些实验方法可用于确定TGCT细胞系在通过HR进行DSB修复中的熟练程度。提供工具来更好地表征它们的重组概况。这些测定的方案已经适用于胚胎癌(EC)TGCT细胞系。检测只需要在细胞内瞬时引入质粒,提供了在相对短的时间内测试多个细胞系的优点。
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.