Role of CtIP in BRCA1-Mediated Tumor Suppression

摘要

CtIP is reported to function as a tumor suppressor; however, it is not known whether this function reflects its activity in the BRCA1 pathway and, as such, would be relevant to human breast and ovarian cancer. Indeed, since Ctip-null mice die at an earlier stage of embryogenesis than Brca1-null animals, CtIP is likely to execute a least some functions independently of BRCA1. Therefore, to study specifically the BRCA1-dependent functions of CtIP, I will examine tumor suppression and genomic stability in cells/mice that expresses a CtIP polypeptide that fails to interact with BRCA1, the CtIP-S326A mutant. To do this, I prepared knock-in targeting constructs that were designed to generate conditional-null (CtipCo) and S326A-mutant (CtipS326A) alleles of the mouse Ctip gene. These constructs were independently electroporated into 129Sv ES cells. Following selection, resistant colonies were examined by Southern analysis to identify correctly targeted clones. Several independent CtipCo/+ clones or CtipS326A/+ clones were injected into blastocysts to derive germline chimeras. Control (CtipCo/+, WapCre/+) and experimental (CtipS326A/Co, WapCre/+) cohorts are currently being generated to determine whether the BRCA1- CtIP interaction is required for mammary-epithelial cell specific tumor suppression. Furthermore, we obtained isogenic ES cells that are either Ctip+/- or CtipS326A/- by targeting the wildtype allele of Ctip+/+ or CtipS326A/+ ES cells with a Ctip-null allele. We are currently assessing the genomic stability function of the BRCA1-CtIP interaction by comparing the CtipS326A-mutant's ability to repair DNA double strand breaks, resist genotoxic stresses, and suppress spontaneous and damage-induced chromosomal defects to the matched wildtype cell line (Ctip+/-).