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Cancer Susceptibility of Mice with a Homozygous Deletion in the COOH-Terminal Domain of the Brca2 Gene¹

Laboratory of Women’s Health [K. A. M., C. D. H., T. W., J. M., N. K. C., B. J. D., R. W. W.], Biostatistics Branch [J. H.], and Laboratory of Reproductive Development and Toxicology [E. H. G., E. M. E.], National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709; Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551 [L. M. B.]; Integrated Laboratory Systems, Inc., Durham, North Carolina 27713 [C. C.]; and Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599 [D. B.]

	ABSTRACT

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Inherited mutations of the human BRCA2 gene confer increased risks for developing breast, ovarian, and several other cancers. Unlike previously described Brca2 knockout mice that display predominantly embryonic lethal phenotypes, we developed mice with a homozygous germ-line deletion of Brca2 exon 27 that exhibit a moderate decrease in perinatal viability and are fertile. We deleted this Brca2 COOH-terminal domain because it interacts directly with the Rad51 protein, contains a nuclear localization signal, and is required to maintain genomic stability in response to various types of DNA damage. These homozygous Brca2-mutant mice have a significantly increased overall tumor incidence and decreased survival compared with their heterozygous littermates. Virgin female mice homozygous for this Brca2 mutation also display an inhibition of ductal side branching in the mammary gland at 6 months of age. Given their substantial viability and cancer predisposition, these mutant mice will be useful to further define the role of the COOH-terminal Brca2 domain in tumorigenesis both in vivo and in vitro.

	Introduction

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Women who inherit germ-line defects of the BRCA2 breast cancer susceptibility gene have up to an 85% risk of breast cancer development by 70 years of age (1 , 2) . BRCA2 mutation carriers also have increased risks for a variety of cancers including ovarian, pancreatic, colon, prostate, stomach, laryngeal, thyroid, male breast cancer, and ocular melanoma (3) . Substantial evidence from studies of human and mouse cells indicates that the BRCA2 protein is an important component in DNA damage response pathways, and loss of this function is considered a major factor in cancer predisposition (4 , 5) . Brca2-mutant cells and embryos are hypersensitive to ionizing radiation and other DNA-damaging agents and develop numerous spontaneous chromosomal abnormalities (6, 7, 8, 9, 10) . In addition, several domains of the BRCA2 protein appear to interact directly with RAD51, a protein having distinct roles in normal meiotic and mitotic recombination, DNA damage repair, and chromosome segregation. These BRCA2 domains include the BRC repeats in exon 11 and a highly conserved RAD51 binding domain in exon 27 (6 , 11, 12, 13) . The interaction of Brca2 with Rad51 is pivotal for the role Brca2 plays in the activation of double-strand break repair and/or homologous recombination, and disruption of these processes may predispose individuals to cancer development.

Functional studies of Brca2 in adult tissues have been hindered by the embryonic lethal phenotypes of Brca2 knockout mice reported to date (14) . We and others previously described mutant mice with disruptions in the 5' half of Brca2 that result in embryonic lethality during early to mid-gestation (6 , 15, 16, 17) . The few homozygous mutant Brca2 mice that have been reported to survive to adulthood display gross developmental abnormalities, are infertile, and develop thymic lymphomas by 5 months of age (7 , 8) . Here, we describe the creation and initial phenotypic characterization of mutant mice that carry a homozygous germ-line deletion of exon 27 of the Brca2 gene. We chose to target exon 27 because a Rad51 binding domain has been identified between Brca2 amino acids 3196–3232 in yeast two-hybrid studies (6 , 11) . In addition, this COOH-terminal domain of the Brca2 protein contains nuclear localization signals that have been highly conserved among species (18 , 19) . Finally, cells lacking this COOH-terminal domain are hypersensitive to -radiation (10) , and they have recently been shown to be deficient in error-free, homology-directed DNA repair (20 , 21) . The Brca2-mutant mice described in this report exhibit a low penetrance of perinatal lethality and an increased susceptibility to spontaneous tumorigenesis in a variety of tissues.

	Materials and Methods

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Targeting Construct Design.
A targeting construct was generated that contained exon 27 of the Brca2 gene flanked by loxP sites (Fig. 1) . The 5' targeting fragment consisted of a 4-kb EcoRI fragment containing exons 25, 26, and 27, and this was subcloned from a genomic mouse bacterial artificial chromosome clone (18) . Likewise, a 3-kb NsiI fragment distal to the 3' untranslated region of Brca2 was also subcloned. Double-stranded loxP oligonucleotides flanked by appropriate restriction sites were inserted into a MunI site in intron 26 of the 5' targeting fragment and a BamHI site of the 3-kb Nsi targeting fragment. Both fragments were then inserted into a previously described pgkNeoTK targeting vector (17) . After linearization with SalI, this targeting vector was introduced into 129/Ola-derived BK-4 ES³ cells by electroporation as described previously (17) . Electroporated cells were subjected to positive and negative selection with geneticin (250 µg/ml; Life Technologies, Inc., Rockville, MD) and gancyclovir (2 µM; Roche, Hertfordshire, United Kingdom). A properly targeted ES cell clone with the "floxed" Brca2 allele (Brca2^Flox27) was identified by Southern analyses with unique probes outside the Brca2 targeting construct as well as PCR analyses using loxP-specific primers.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Generation of Brca2Flox27 and Brca227 ES cells. Homologous recombination between the endogenous Brca2 locus (Brca2Wildtype) and a targeting vector carrying a pair of LoxP sites flanking Brca2 exon 27 and a neomycin resistance cassette yielded a "floxed" Brca2 locus (Brca2Flox27). ES cells containing the properly targeted Brca2Flox27 allele were isolated by positive/negative selection and confirmed by Southern blot and PCR analyses. Conversion of the Brca2Flox27 allele to the Brca227 allele was accomplished in vitro by transient electroporation of Brca2Flox27 ES cells with a Cre expression plasmid. The Cre recombinase induces site-specific recombination between the 5' and 3' loxP sites, resulting in the deletion of exon 27 and the neomycin gene. Germ-line transmission of the desired Brca227 allele was obtained by injection into blastocysts, followed by standard breeding techniques.

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Mice and Tissues.
Mice carrying the Brca2²⁷ mutation were backcrossed from a targeted 129-derived chimeric founder onto the C57BL/6N background for one to three generations, followed by intercrosses to generate mice homozygous for the Brca2-exon 27 deletion. Both virgin and multiparious females as well as males carrying this Brca2²⁷ mutation were group housed in plastic cages on pressed wood-chip bedding. Animals had access to an NIH-31 diet (18% protein, 4% fat, and 5% fiber; Zeigler Bros., Gardeners, PA) and water ad libitum. Mice were monitored for 17 months and were sacrificed when palpable tumors developed or when signs of morbidity became apparent. Terminal sacrifices were performed on all surviving animals between 17 and 19 months of age. All tissue and tumor samples were removed postmortem and fixed in 10% neutral buffered formalin. Specimens were processed for routine histology, embedded in paraffin, sectioned, and stained with H&E. Histological examination was performed by veterinary pathologists (C. H. and B. D.). In all female mice, the right #4 and #5 mammary glands were fixed with neutral buffered formalin on the pelts for 24 h, dissected, fixed, and then stained with carmine as described previously for whole-mount analysis (22) . Routine H&E staining was performed on the left #4 and #5 mammary gland of each animal, followed by histopathological analysis.

Statistical Analysis.
A ² goodness-of-fit test was performed for segregation analysis of the three genotypic classes from heterozygous intercrosses. The overall difference in survival between Brca2^27//27 and Brca2^27/+ virgin females was compared with a life table test. Both a Peto analysis (23) and a Fisher’s exact test were used to test for a difference in tumor rates between genotypic classes. Both procedures produced similar results.

	Results

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Generation of Mice with a Homozygous Germ-Line Deletion of Brca2 Exon 27.
ES cells carrying a targeted Brca2 allele with loxP sites flanking exon 27 were generated by homologous recombination (Fig. 1) . After transient Cre expression in vitro, ES clones with a Brca2²⁷ allele were identified. PCR products generated with the B2F1 and B2R2 primers that flank the 5' and 3' loxP sites (Fig. 1) were sequenced to confirm the expected exon 27 deletion. Germ-line transmission of the Brca2²⁷ allele was identified by standard breeding techniques.

As expected, reverse transcriptase-PCR analysis with primers specific for exon 27 does not yield detectable messages in testes RNA from Brca2^27/27 mice, although this reverse transcription-PCR product is easily detectable for Brca2^27/+ mice. Assuming a mutant transcript is expressed, it could give rise to a truncated protein product of 3142 amino acids compared with the wild-type murine Brca2 protein that contains 3329 amino acids. Unfortunately, we are unable to confirm the presence or relative levels of this putative mutant Brca2 protein because of the unavailability of specific antibodies directed against the murine gene product.

Viability of Brca2^27/27 Mice.
Although the Brca2^27/27 animals are viable compared with previous Brca2 germ-line mutants, survival analysis indicates an overall decrease in viability of homozygous mutants compared with heterozygous and wild-type littermates:

First, cumulative genotyping at weaning from Brca2^27/+ intercrosses revealed a significant (P < 0.008) deficit of Brca2^27/gD27 mice from the expected 1:2:1 Mendelian ratio. When both sexes are combined, only 47 of 278 animals analyzed at weaning were of the homozygous mutant genotype. Thus, there are 33% fewer Brca2^27/27 mice than expected, and both male and female offspring were under represented. To further characterize the decreased overall viability of Brca2^27/27 mice during development, we examined embryonic fibroblasts from Brca2^27/+ intercrosses at 13.5 days of gestation. Analysis of 39 such embryos shows that the genotypic distribution does not deviate from the expected Mendelian ratio. Thus, a subset of Brca2^27/27 offspring appear to either die during late gestation or shortly after birth, suggesting that loss of Brca2 function impacts overall viability.

Secondly, the overall survival of Brca2^27/27 compared with Brca2^27/+ virgin female mice is significantly decreased (P < 0.05) by a life table test, providing additional support that lifetime survival is affected by this Brca2 mutation. Interestingly, test matings of both adult Brca2^27/27 males and females with wild-type mice do not reveal apparent infertility or difficulty in raising litters.

Inhibition of Mammary Side Branching in Brca2^27/27 Mice.
Alterations in normal growth and differentiation of mammary tissue from Brca2-mutant virgin females were determined using whole-mount analysis. At 6 months of age, mammary tissue from three Brca2^27/27 animals exhibited a dramatic lack of side branching and a much lower density of ductules compared with that observed for three heterozygous and wild-type littermates, which were indistinguishable (Fig. 2) . Although the ducts reached the limits of the mammary fat pad in the homozygous mutant animals, a substantial lack of side branching was observed in these Brca2^27/27 females. These observations were confirmed by examining histological sections from these mice. This general trend of inhibited side branching observed in Brca2^27/27 females was maintained in the nine animals of all three genotypic classes that were examined subsequently at 9 months of age.

View larger version (122K): [in this window] [in a new window]   Fig. 2. Altered mammary ductal morphology in Brca227/27 mice. Representative mammary gland whole mounts stained with carmine are illustrated from 6-month-old virgin female mice. A, Brca227/+ mammary gland. B, Brca227/27 mammary gland. Ductal side branching is clearly reduced in Brca227/27 mammary tissue compared with Brca227/+ littermates.

Predisposition of Brca2^27/27 Mice to Spontaneous Tumor Development.

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Brca2^+/+ littermates (Tables 1 and 2 ). We observed untreated animals on a mixed 129 x C57BL/6N genetic background for tumor development or signs of morbidity. Prior to 17 months, 9 spontaneous tumors were detected in Brca2^27/27 mice, whereas only a single tumor was observed in the Brca2^27/+ animals, and no tumors were observed in Brca2^+/+

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The tumor spectrum is diverse and includes carcinomas, adenomas, lymphomas, and sarcomas (Table 2) . Of particular interest is the exclusive appearance of various types of carcinomas in the Brca2^27/27 mice. These include one mammary adenosquamous carcinoma (Fig. 3) , five squamous cell carcinomas (three of gastric origin), two gastric adenocarcinomas, one endometrial carcinoma, and two A/B lung carcinomas. Fig. 3 illustrates representative histological sections for the two types of gastric tumors that were observed. Interestingly, a single mammary adenomyoepithelioma was detected in one Brca2^27/+ mouse. The sarcoma incidence also appears to be increased in the Brca2^27/27 mice compared with their littermates. Three histiocytic sarcomas, along with a hemangiosarcoma and a leiomyosarcoma, arose in Brca2^27/27 mice, whereas only two histiocytic sarcomas were identified in their Brca2^27/+ and Brca2^+/+ littermates. We found a wide variety of tumor types in the Brca2^27/27 mice that were sacrificed before 17 months of age because of morbidity (Table 2) . Therefore, no obvious differences in latency between distinct tumor types were observed.

View larger version (201K): [in this window] [in a new window]   Fig. 3. Photomicrographs of carcinomas from Brca227/27 mice. A, mammary gland adenosquamous carcinoma characterized by irregular islands of neoplastic squamous cells admixed with small and large cysts filled with keratin debris (arrows) and inflammatory cells. x40. H&E stain. B, x400 disorganized island of neoplastic squamous cells in mammary adenosquamous carcinoma pictured in A. C, gastric squamous cell carcinoma demonstrating islands of keratin-forming neoplastic cells invading into the submucosa and muscle wall (arrows). x40. H&E stain. D, gastric adenocarcinoma demonstrating various-sized acini and tubules separated by fine fibrovascular stroma and invading into the submucosa and wall of the stomach (arrows). x40. H&E stain.

	Discussion

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This report supports a strong correlation between the Brca2 mutation position and the resulting Brca2 mouse knockout phenotype. We and other laboratories have shown previously that mice homozygous for targeted Brca2 mutations 5' of the BRC repeats in exon 11 exhibit early embryonic lethal phenotypes (6, 7, 8 , 15, 16, 17) . These BRC repeats interact with Rad51 (12 , 13) and have been highly conserved in evolution (18 , 26) , which suggests that they are important functional domains of the Brca2 protein. A few viable Brca2-mutant mice have been generated that retain at least some of these BRC repeats, and these animals display multiple severe developmental abnormalities, infertility, and early thymic lymphoma development (7 , 8) . The Brca2^27/27 mice lack only the COOH-terminal domain and could produce a truncated Brca2 gene product that preserves all eight BRC repeats. In contrast to all other Brca2-null mice reported to date, these Brca2^27/27 mice display a modest loss of viability and have no gross developmental abnormalities or obvious infertility. Thus, full retention of the BRC repeats and other functional domains may direct a genotype-phenotype correlation.

This study extends previous findings by several laboratories that have demonstrated the functional significance of the COOH-terminal domain of both human and rodent BRCA2. Morimatsu et al. (10) demonstrated that mouse ES cells and embryonic fibroblasts lacking exon 27 are hypersensitive to -radiation and undergo premature senescence. Recent reports indicate that the COOH-terminus of Brca2 is required for error-free, homology-directed repair of DNA double strand breaks and the ability to facilitate the induction of Rad51 nuclear foci after ionizing radiation (20 , 21) . Thus, disruption of the exon 27 Brca2-Rad51 interaction appears to lead to defective repair of DNA damage and generalized chromosomal instability with a subsequent increased risk of neoplastic progression in Brca2-deficient cells (4 , 5) . The human COOH-terminal region of BRCA2 also appears to be critical because a truncating mutation (9808delCC) at position 3195, which occurs just upstream of the predicted Rad51-interacting domain in human BRCA2 exon 27, is associated with an elevated breast cancer risk (27) .

Despite the increased susceptibility of Brca2^27/27 mice to tumorigenesis, these animals are not highly susceptible to mammary tumorigenesis. Several factors may account for this observation, but the most likely reason is the fact that the Brca2²⁷ mutation was examined on a mixed C57BL/6N and 129 genetic background. C57BL/6N and 129/SvEvS6 mice are both extremely resistant to spontaneous as well as radiation-induced mammary carcinogenesis.⁴ Thus, we are currently using microsatellite marker-assisted breeding techniques to transfer this Brca2²⁷ mutation onto inbred strains such as BALB/cJ that are susceptible to mammary carcinogenesis.

The reduced ductal branching phenotype seen in Brca2^27/27 virgin female mice may be associated with increased mammary tumor risk in combination with other environmental or genetic interactions. We cannot exclude the possibility that the subtle mammary gland phenotype we have observed may be attributable to hormonal differences in the Brca2^27/27 mice compared with their littermates rather than a more direct Brca2 effect. However, Deng and Brodie (14) and Xu et al. (28) described a blunted ductal branching phenotype in the mammary glands of mice with a mammary-specific targeted Brca1 mutation that was associated with subsequent tumor development after a long latency. In addition, a high incidence of mammary adenocarcinomas was reported recently in mice carrying a mammary tissue-specific mutation that completely disrupts the Brca2 gene (29) . Given their substantial viability and a cancer predisposition, Brca2^27/27 mice and cells derived from them will be useful to further define the role of Brca2 in tumorigenesis.

	ACKNOWLEDGMENTS

 
We thank Drs. Cynthia Afshari and Richard DiAugustine for critical review of the manuscript. The expert technical assistance of William Gersch, Brian Register, Sarah Hagevik, Christine Furmick, Laura Wharey, and David Godley is greatly appreciated. We thank Dr. John Seely for his pathology support. We also thank the staff and managers of Integrated Laboratory Systems, Inc. for their substantial animal husbandry, necropsy, and histology support.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This research was funded in part by Department of Defense Breast Cancer Research Grant DAMD17-98-1-8131 (to K. M.).

² To whom requests for reprints should be addressed, at Laboratory of Women’s Health, National Institute of Environmental Health Sciences, NIH, MD C4-06, 111 T. W. Alexander Drive, Research Triangle Park, NC 27709. Phone: (919) 541-3229; Fax: (919) 541-3720; E-mail: mcallis2{at}niehs.nih.gov

³ The abbreviation used is: ES, embryonic stem.

⁴ L. Michelle Bennett, Jason Malphurs, Toni Ward, John C. Seely, Barbara J. Davis, and Roger W. Wiseman. Radiation-induced mammary carcinogenesis and ductal morphology in inbred mouse strains, manuscript in preparation.

Received 10/25/01. Accepted 1/ 2/02.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 

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