Brca2 deficiency in the murine small intestine sensitizes to p53-dependent apoptosis and leads to the spontaneous deletion of stem cells

Abstract

The gene encoding the human BRCA2 tumour suppressor is mutated in a number of different tumour types, most notably inherited breast cancers. The primary role of BRCA2 is thought to lie in the maintenance of genomic stability via its role in the homologous recombination pathway. We generated mice in which Brca2 was deleted from virtually all cells within the adult small intestine, using a CYP1A1-driven Cre-Lox approach. We noted a significant p53-dependent increase in the levels of spontaneous apoptosis which persisted for several months after removal of the gene and ultimately we observed the spontaneous deletion of Brca2-deficient stem cells. Brca2 deficiency did not lead to gross changes in intestinal physiology but did enhance sensitivity to a variety of DNA crosslinking agents. Keywords: Brca2; p53; small intestine; apoptosis; DNA damageIn humans, inheritance of a single mutated copy of either of the tumour suppressor genes BRCA1 or BRCA2 predisposes to breast cancer, usually after loss of heterozygosity at the second allele (Scully, 2000). Rarely, inherited mutation of BRCA2 predisposes to cancer in other tissues, most commonly the ovary (The Breast Cancer Linkage Consortium, 1999). The products of the BRCA1 and BRCA2 genes are large, unrelated proteins, both believed to be independently involved in homologous recombination (HR), a process which allows for the error-free repair of DNA doublestrand breaks (DSBs) (Liu and West, 2002). BRCA2 is known to participate in this process through its direct interaction with RAD51, the mammalian equivalent of the bacterial RecA protein (Pellegrini et al., 2002). It is the breakdown in the HR pathway which is believed to be responsible for tumorigenesis in the absence of BRCA2, due to resulting genomic instability (Davies et al., 2001). Owing to embryonic lethality in mice homozygous for the Brca2-null allele (Ludwig et al. , 2004). Brca2 has also been deleted from cells of the developing thymus using an Lck-driven Cre-Lox system, resulting in a p53-dependent increase in the rate of spontaneous apoptosis in activated T cells (Cheung et al., 2002). These cells showed no significant difference in their apoptotic response to a variety of stimuli, including ionising radiation (IR) (Cheung et al., 2002). However, several studies have shown that cells lacking fully functional BRCA2, in both humans or mice, do exhibit increased sensitivity to DNA-damaging agents (Sharan et al., 1997;Abbott et al., 1998;Chen et al., 1998;Morimatsu et al., 1998;Patel et al., 1998;D'Andrea and Grompe, 2002;Kraakman-van der Zwet et al., 2002;Donoho et al., 2003).The purpose of our study was to analyse the effects of Brca2 deficiency in the murine adult small intestine, a tissue characterized by rapid cell turnover. We utilized a CYP1A1-specific inducible Cre-lox system to efficiently delete the Brca2 gene from crypt cells, including stem cells Sansom et al., 2004). Our results indicate that Brca2 plays an important role in the response to DNA damage in this tissue, that loss of Brca2 sensitizes c...

Efficient Deletion of Normal Brca2-Deficient Intestinal Epithelium by Poly(ADP-Ribose) Polymerase Inhibition Models Potential Prophylactic Therapy

“…To test whether treatment with the PARP inhibitor increased the rate of repopulation in Brca2-deficient tissue, we used mice which carried the Rosa26R reporter allele (20) as well as the Ah-Cre and floxed Brca2 genes. Mice were subjected to twice-weekly injections of 15 mg/kg KU0058948 for 3 weeks and then scored for recombined crypts using a simple whole-mount stain for the reporter allele (11). Figure 3A shows representative areas of small intestine from mice, either heterozygous or homozygous for Brca2, treated with the PARP inhibitor or saline, and clearly shows a reduction in recombined stem cells in Brca2-deficient intestine in which PARP inhibition has taken place.…”

“…We have previously shown that despite the increased levels of apoptosis after deletion of Brca2, the small intestine repopulates very slowly, with f50% of recombined stem cells replaced by those which had failed to recombine 6 months after Brca2 deletion (11). To test whether treatment with the PARP inhibitor increased the rate of repopulation in Brca2-deficient tissue, we used mice which carried the Rosa26R reporter allele (20) as well as the Ah-Cre and floxed Brca2 genes.…”

“…Indeed, studies have already shown the effectiveness of PARP inhibition in combination with either radiotherapy or chemotherapy in a range of human tumor mouse xenograft models (16,17). We have previously shown that conditional deletion of the Brca2 gene, using a wellcharacterized CYP1A1-driven cre-loxP approach (18,19), sensitizes the mouse small intestine to DNA damage and eventually leads to repopulation by stem cells in which recombination of the floxed Brca2 allele has not taken place (11). In this study, we have used our model Brca2-deficient system to analyze whether PARP inhibition further sensitizes the intestine to DNA damage in vivo.…”

“…Cells lacking either protein are prone to genomic instability due to the alternative employment of the error-prone nonhomologous endjoining pathway of DNA repair (2). Cells lacking Brca2 have been shown to be sensitive to a variety of DNA damaging agents, either in vitro or in vivo, particularly following treatment with DNA crosslinking agents such as mitomycin C (3)(4)(5)(6)(7)(8)(9)(10)(11).…”

Mouse models of BRCA1 and BRCA2 deficiency: past lessons, current understanding and future prospects

“…In both studies, treatment with PARP1 inhibitors induced regression of Brca2-deficient tumors in transplantation models. To further validate this approach to targeting BRCAdeficient cells, a small intestine-specific inducible Cre line (Ah-Cre) was crossed to mice carrying conditional Brca2 F9-10 alleles as well as a reporter for Cre-mediated recombination, ROSA26R (Soriano, 1999;Hay et al, 2005b). Subsequent treatment of mice with PARP1 inhibitors resulted in depletion of reporter-positive cells specifically in Ah-Cre;Brca2 F9-10/F9-10 ;Rosa26R intestines but not in Ah-Cre;Brca2 F9-10/ þ ;Rosa26R intestines, suggesting specific depletion of Brca2-deficient cells (Hay et al, 2005a).…”