Loss of Brca2 and p53 synergistically promotes genomic instability and deregulation of T-cell apoptosis

BRCA2 is a breast cancer susceptibility gene of which the product is thought to be involved in monitoring genome integrity and cell cycle progression. Brca2-null mice have a defect in embryonic cellular proliferation and die in utero. Here we report the generation of T-cell lineage-specific Brca2-deficient (tBrca2(-/-)) mice using the Cre-loxP system. Mice with a flanked by loxP allele of Brca2 were crossed to transgenic mice bearing Cre recombinase driven by the T cell-specific promoter Lck. Thymic cellularity and distribution of subset populations were normal in tBrca2(-/-) mutants. Thymocytes from tBrca2(-/-) mice underwent normal apoptosis in response to a variety of stimuli, and activated tBrca2(-/-) T cells had normal proliferative capacity. tBrca2(-/-) T cells were more likely than wild-type cells to undergo spontaneous apoptosis, but apoptosed normally in response to restimulation or DNA-damaging stress signals. Examination of metaphase spreads of tBrca2(-/-) T cells revealed that the chromosomes often exhibited aberrations such as breaks and tri-radial structures. The level of chromosomal abnormalities was enhanced in T cells from tBrca2(-/-); p53(-/-) double-mutant mice. However, tBrca2(-/-); p53(-/-) T cells did not show the enhanced level of spontaneous apoptosis demonstrated by tBrca2(-/-) T cells, a difference that likely accounts for an increase in cell number and (3)[H]thymidine incorporation of double-mutant T cells in culture compared with either single mutant. Despite this increased T-cell number, the onset of T-cell lymphomas was only marginally accelerated in tBrca2(-/-); p53(-/-) mice compared with p53(-/-) mice. Our results support a role for Brca2 in repairing spontaneous DNA lesions, and suggest that loss of Brca2 enhances the susceptibility of mouse T-lineage cells to chromosomal aberrations and deregulation of apoptosis in the absence of p53.     

Differential p53-dependent mechanism of radiosensitization in vitro and in vivo by the protein kinase C-specific inhibitor PKC412

The cellular response to ionizing radiation is governed by the DNA-damage recognition process but is also modulated by cytoplasmic signal transduction cascades that are part of the cellular stress response. Growth-promoting protein kinase C activity antagonizes irradiation-induced cell death, and, therefore, protein kinase C inhibitors might be potent radiosensitizers. The antiproliferative and radiosensitizing effect of the novel N-benzoylated staurosporine analogue PKC412 was tested in vitro against genetically defined p53-wild type (+/+) and p53-deficient (-/-) murine fibrosarcoma cells and in vivo against radioresistant p53-/- murine fibrosarcoma and human colon adenocarcinoma tumor xenograft (SW480, p53-mutated). PKC412 sensitized both p53+/+ and p53-/- tumor cells in vitro and in vivo for treatment with ionizing radiation but with a different mechanism of radiosensitization depending on the p53 status. In p53+/+, cells combined treatment with PKC412 and ionizing radiation drastically induced apoptotic cell death, whereas no apoptosis induction could be observed in p53-deficient cells in vitro and in histological tumor sections. Combined treatment resulted in an increased G2 cell cycle distribution in p53-/- cells at PKC412 concentrations that did not alter cell cycle distribution when applied alone. In vivo, a minimal treatment regimen during 4 consecutive days of PKC412 (4 x 100 mg/kg) in combination with ionizing radiation (4 x 3 Gy) exerted a substantial tumor growth delay for both p53-disfunctional tumor xenografts and showed that the clinically relevant protein kinase C inhibitor PKC412 is a promising new radiosensitizer with a potentially broad therapeutic window.     

Brca1 heterozygous mice have shortened life span and are prone to ovarian tumorigenesis with haploinsufficiency upon ionizing irradiation

BRCA1 mutation carriers have an 85% lifetime risk of breast cancer and 60% for ovarian cancer. BRCA1 facilitates DNA double-strand break repair, and dysfunction of BRCA1 leads to hypersensitivity to DNA damaging agents and consequently genomic instability of cells. In this communication, we have examined the tumor incidence and survival of Brca1 heterozygous female mice. Brca1 heterozygotes appear to have a shortened life span with 70% tumor incidence. Lymphoma, but not ovarian and mammary gland tumors, occurs commonly in these mice. After a whole-body exposure to ionizing radiation, Brca1 heterozygous mice have a 3-5-fold higher incidence specific to ovarian tumors, but not lymphoma, when compared with the Brca1+/+ mice. All the tumors from heterozygous mice examined retain the wild-type allele and the cancer cells express Brca1 protein, precluding the chromosomal mechanism for loss of heterozygosity of Brca1 locus. Although the manifestation of BRCA1 haploinsufficiency may be different between human and mouse, this study suggests that women carrying Brca1 mutations may be more prone to ovarian tumor formation after IR exposure than nonmutation carriers.     

Role of cell cycle in mediating sensitivity to radiotherapy

Multiple pathways are involved in maintaining the genetic integrity of a cell after its exposure to ionizing radiation. Although repair mechanisms such as homologous recombination and nonhomologous end-joining are important mammalian responses to double-strand DNA damage, cell cycle regulation is perhaps the most important determinant of ionizing radiation sensitivity. A common cellular response to DNA-damaging agents is the activation of cell cycle checkpoints. The DNA damage induced by ionizing radiation initiates signals that can ultimately activate either temporary checkpoints that permit time for genetic repair or irreversible growth arrest that results in cell death (necrosis or apoptosis). Such checkpoint activation constitutes an integrated response that involves sensor (RAD, BRCA, NBS1), transducer (ATM, CHK), and effector (p53, p21, CDK) genes. One of the key proteins in the checkpoint pathways is the tumor suppressor gene p53, which coordinates DNA repair with cell cycle progression and apoptosis. Specifically, in addition to other mediators of the checkpoint response (CHK kinases, p21), p53 mediates the two major DNA damage-dependent cellular checkpoints, one at the G(1)-S transition and the other at the G(2)-M transition, although the influence on the former process is more direct and significant. The cell cycle phase also determines a cell's relative radiosensitivity, with cells being most radiosensitive in the G(2)-M phase, less sensitive in the G(1) phase, and least sensitive during the latter part of the S phase. This understanding has, therefore, led to the realization that one way in which chemotherapy and fractionated radiotherapy may work better is by partial synchronization of cells in the most radiosensitive phase of the cell cycle. We describe how cell cycle and DNA damage checkpoint control relates to exposure to ionizing radiation.     

Effects of p53 mutations on cellular sensitivity to ionizing radiation

Mutations in the p53 tumor suppressor gene have been found in more than 50% of human tumors including those in breast, colon, lung, and oral cavity. However, the significance of p53 mutation in radiation sensitivity and its underlying mechanisms still remains unclear. In this study, we have measured the effects of p53 mutation on cell cycle delay, apoptosis, and radiation sensitivity using mouse cells transfected with different forms of p53 mutations. Wild-type p53 and p53-Null mouse embryo fibroblast cells were used as positive and negative controls, respectively. Exponentially growing cells were irradiated with 0- to 9-Gy gamma rays and then assayed for cell survival, p53 expression, cell cycle checkpoint, and apoptosis. Cell survivals determined by clonogenic assay show that p53 mutant cells are generally more sensitive to ionizing radiation than cells with wild-type p53. Western blot analysis indicates that exposure to 6-Gy gamma rays increases the p53 expression levels by two- to threefold in wild-type p53 cells. However, the p53 level remains unchanged in cells with mutant p53 during the same postirradiation period. Irradiation with 6-Gy gamma rays produces G2/M arrest in all cell lines, indicating that p53 is probably not involved in the G2/M checkpoint. However, all mutant cells fail to show any significant G1/S arrest after irradiation, suggesting that G1/S arrest may be implicated in radiation sensitivity. Finally, there is very little apoptosis (<3% by Tat-mediated dUTP nick-end labeling [TUNNEL] and morphologic assays) detected in wild-type and p53 mutant cell lines after 6-Gy gamma rays. Our results suggest that mutant forms of p53 represent a phenotype that affects the radiation sensitivity and is not dependent on the apoptotic pathway.     

Normal lymphocyte development and thymic lymphoma formation in Brca1 exon-11-deficient mice

Breast-cancer-associated gene 1 (BRCA1) is highly expressed in thymus and spleen. In this paper, we have studied lymphocyte development and tumorigenesis in mice carrying mutations in Brca1 and p53. We show that the deletion of Brca1 exon 11 (Brca1-delta11), which disrupts the full-length isoform, but not the short isoform of Brca1, does not interfere with lymphocyte development. This is true irrespective of p53 status, that is, whether it is wild type, heterozygous or homozygous for a null mutation. These data suggest that the expression of Brca1 short isoform alone is enough to maintain normal development of lymphocytes. However, it cannot suppress tumorigenesis as about 30% of Brca1(delta11/delta11)p53(+/-) mice develop thymic lymphoma between 3 and 7 months of age. We demonstrate that p53 plays an essential role in Brca1-associated lymphoma, as all the tumors from Brca1(delta11/delta11)p53(+/-) mice exhibit LOH of p53 and Brca1(delta11/delta11)p53(-/-) mice exhibited accelerated tumorigenesis. We further demonstrate that the Brca1-delta11 deficiency does not affect thymocyte proliferation; however, it increases genetic instability and triggers gamma-irradiation-induced apoptosis. The loss of p53 attenuates apoptosis and allows accumulation of further mutations in Brca1-delta11 thymocytes, eventually leading to thymic lymphoma formation.     

A delayed chemically induced tumorigenesis in Brca2 mutant mice

BRCA2 is a breast cancer susceptibility gene. Germline mutations of BRCA2 account for about 10-30% of familial breast cancer cases. Consistent with its tumor-suppressor activity, BRCA2 plays an important role in DNA repair. To assess the susceptibility of carriers of mutant BRCA2 to tumorigenesis induced by DNA-damaging carcinogens, we generated a Brca2 knockout mouse strain and studied its susceptibility to chemically induced tumorigenesis. Similar to previously reported Brca2 knockout mice, our Brca2-/- embryos die at E8.5-9.5, while the Brca2+/- mice are tumor-free and fertile. Unexpectedly, Brca2+/- mice developed tumors slower than did their wild-type littermates when treated with a potent carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). In vitro experiments showed that Brca2+/- mouse cells and Capan-1 cells, a human pancreatic cancer cell line deficient of BRCA2, were more sensitive to DMBA-induced apoptosis, than were Brca2+/+ mouse cells and a derivative of Capan-1 cells that expressed exogenous wild-type BRCA2, respectively. Our results suggest that enhanced sensitivity of Brca2 mutant cells to DMBA-induced apoptosis at the dose of DMBA we used contributes to the delayed tumorigenesis of Brca2+/- animals. This suggestion may also provide a rational explanation for a previous unexpected finding that cigarette smoking appears to reduce the breast cancer risk of BRCA2 mutation carriers.     

A member of the Pyrin family, IFI16, is a novel BRCA1-associated protein involved in the p53-mediated apoptosis pathway

We identified IFI16 as a BRCA1-associated protein involved in p53-mediated apoptosis. IFI16 contains the Pyrin/PAAD/DAPIN domain, commonly found in cell death-associated proteins. BRCA1 (aa 502-802) interacted with the IFI16 Pyrin domain (aa 1-130). We found that IFI16 was localized in the nucleoplasm and nucleoli. Clear nucleolar IFI16 localization was not observed in HCC1937 BRCA1 mutant cells, but reintroduction of wild-type BRCA1 restored IFI16 nuclear relocalization following IR (ionizing radiation). Coexpression of IFI16 and BRCA1 enhanced DNA damage-induced apoptosis in mouse embryonic fibroblasts from BRCA1 mutant mice expressing wild-type p53, although mutant IFI16 deficient in binding to BRCA1 did not induce apoptosis. Furthermore, tetracycline-induced IFI16 collaborated in inducing apoptosis when adenovirus p53 was expressed in DNA-damaged p53-deficient EJ cells. These results indicate a BRCA1-IFI16 role in p53-mediated transmission of DNA damage signals and apoptosis.     

The effect of loss of Brca1 on the sensitivity to anticancer agents in p53-deficient cells

BRCA1 is implicated in cellular responses to DNA damage, thereby substantially contributing to maintenance of the genome integrity. Mutations in the BRCA1 gene occur in breast and ovarian cancer and mutations that disable p53 are frequently found in human cancers, often accompanied by mutations in additional genes, contributing to tumor progression or high-grade malignancy. Therefore, the role of BRCA1 in the sensitivity to anticancer agents in p53-deficient cells was investigated using p53-deficient mouse knockout cell lines either deficient or proficient in Brca1 function. We report that Brca1-deficiency in p53-null cells was associated with increased sensitivity to the topoisomerase I poisons camptothecin and topotecan, the topoisomerase II poisons doxorubicin, mitoxantrone and etoposide, and to the platinum compounds carboplatin and oxaliplatin, but not to the antimetabolites 5-fluorouracil and gemcitabine and the taxanes docetaxel and paclitaxel. The increased growth inhibition to doxorubicin after loss of Brca1 correlated with increased cell killing caused by increased apoptosis. The data presented here indicate that Brca1 modulates p53-independent DNA damage response pathways and they support the case of a role of Brca1 to protect cells from apoptosis-mediated cell death in p53-deficient cells. These results suggest a higher chemotherapy susceptibility of cells disabled in both functions and they foster the concept that functional inhibition of BRCA1 may be a valuable adjunct to anticancer agents to increase the efficacy of chemotherapy in the treatment of p53-mutated cancers.     

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest

Hypersensitivity of Brca1-deficient cells to interstrand crosslinking (ICL) agents such as cisplatin and mitomycin C (MMC) implicates an important role for Brca1 in cellular response to the ICL DNA damage repair. However, the detailed mechanism of how Brca1 is involved in the ICL response remains unclear. In this study, we analysed the cellular response to MMC treatment using isogenic mouse embryonic fibroblasts (MEFs) including wild type, p53-/- and p53-/-Brca1-/-. Marked hypersensitivity of p53-/- Brca1-/- MEFs to MMC was found, and the reconstitution of Brca1 expression in these cells restored resistance to MMC. Upon MMC treatment, wild-type MEF was temporarily arrested at G2/M phase but subsequently resumed a normal cell cycle progression. In contrast, Brca1-deficient MEF exhibited a marked time-dependent accumulation of cells arrested at S phase and a prolonged increase in the G2/M population, followed by extensive cell deaths. Importantly, DNA damage-induced Rad51 foci were not formed in these cells, suggesting a defect in homologous recombination. Such defects are fully rescued by reconstitution of Brca1 expression in Brca1-deficient MEF, suggesting that Brca1 directly plays an essential role in ICL repair, which depends on homologous recombination during S phase.     

Multiple genetic changes are associated with mammary tumorigenesis in Brca1 conditional knockout mice.

Germline mutations in the tumor suppressor gene BRCA1 predispose women to breast cancer, however somatic mutations in the gene are rarely detected in sporadic cancers. To understand this phenomenon, we examined mouse models carrying conditional disruption of Brca1 in mammary epithelium in either p53 wild type (wt) or heterozygous backgrounds. Although a p53(+/-) mutation significantly accelerated tumorigenesis, both strains developed mammary tumors in a stochastic fashion, suggesting that multiple factors, in addition to p53 mutations, may be involved in Brca1 related tumorigenesis. A unique feature of Brca1 mammary tumors is their highly diverse histopathology accompanied by severe chromosome abnormalities. The tumors also display extensive genetic/molecular alterations, including overexpression of ErbB2, c-Myc, p27 and Cyclin D1 in the majority of tumors, while they were virtually ERalpha and p16 negative. Translocations involving p53 were also identified which lead to abnormal RNA and protein products. In addition, we generated cell lines from mammary tumors and found that the cells retained many of the genetic changes found in the primary tumors, suggesting that these genes may be players in Brca1-associated tumorigenesis. Despite their distinct morphology, all cultured tumor cells were Tamoxifen resistant but highly sensitive to Doxorubicin or gamma-irradiation, suggesting that these methods would be effective in treatment of this disease.     

Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM

The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio- and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3'-kinase-like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC(50) of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3'-kinase-like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, gammaH2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.     

Isogenic normal basal and luminal mammary epithelial isolated by a novel method show a differential response to ionizing radiation

Epithelial cells within the normal breast duct seem to be the primary target for neoplastic transformation events that eventually produce breast cancer. Normal epithelial cells are easily isolated and propagated using standard techniques. However, these techniques almost invariably result in populations of cells that are largely basal in character. Because only approximately 20% of human breast cancers exhibit a basal phenotype, our understanding of the disease may be skewed by using these cells as the primary comparator to cancer. Further, because germ line mutations in BRCA1 yield breast cancers that are most often of the basal type, a comparison of normal basal and luminal cells could yield insight into the tissue and cell type specificity of this hereditary cancer susceptibility gene. In this report, we describe a simplified and efficient method for isolating basal and luminal cells from normal human breast tissue. These isogenic cells can be independently propagated and maintain phenotypic markers consistent with their respective lineages. Using these cultured cells, we show that basal and luminal cells exhibit distinct responses to ionizing radiation. Basal cells undergo a rapid but labile cell cycle arrest, whereas luminal cells show a much more durable arrest, primarily at the G(2)-M boundary. Molecular markers, including p53 protein accumulation, p53-activated genes, and BRCA1 nuclear focus formation all correlate with the respective cell cycle responses. Further, we show that short-term cultures of human breast tissue fragments treated with ionizing radiation show a similar phenomenon as indicated by the biphasic accumulation of p53 protein in the basal versus luminal layer. Together, these results indicate that normal basal cells have a transitory cell cycle arrest after DNA damage that may underlie their increased susceptibility to transformation after the loss of functional BRCA1.     

A targeted mouse Brca1 mutation removing the last BRCT repeat results in apoptosis and embryonic lethality at the headfold stage.

A mouse model with a targeted mutation in the 3' end of the endogenous Brca1 gene, Brca1(1700T), was generated to compare the phenotypic consequences of truncated Brca1 proteins with other mutant Brca1 models reported in the literature to date. Mice heterozygous for the Brca1(1700T) mutation do not show any predisposition to tumorigenesis. Treatment of these mice with ionizing radiation or breeding with Apc, Msh-2 or Tp53 mutant mouse models did not show any change in the tumor phenotype. Like other Brca1 mouse models, the Brca1(1700T) mutation is embryonic lethal in homozygous state. However, homozygous Brca1(1700T) embryos reach the headfold stage but are delayed in their development and fail to turn. Thus, in contrast to Brca1(null) models, the mutant embryos do not undergo growth arrest leading to a developmental block at 6.5 dpc, but continue to proliferate and differentiate until 9.5 dpc. Homozygous embryos die between 9.5-10.5 dpc due to massive apoptosis throughout the embryo. These results indicate that a C-terminal truncating Brca1 mutation removing the last BRCT repeat has a different effect on normal cell function than does the complete absence of Brca1.     

Inactivation of Brca2 cooperates with Trp53(R172H) to induce invasive pancreatic ductal adenocarcinomas in mice: a mouse model of familial pancreatic cancer

An inactivating germline mutation in BRCA2 is the most common known genetic basis for familial pancreatic cancer (FPC), accounting for 5-10% of inherited cases. A genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) arising on the backdrop of Brca2 deficiency is likely to elucidate valuable diagnostic and therapeutic insights for FPC. Both Brca2 alleles were conditionally deleted during development within the pancreatic epithelium by generating Pdx1-Cre; Brca2(f/f) (CB) mice; in addition, triple transgenic Pdx1-Cre; Brca2(f/f); LSL-Trp53(R172H) (CBP) mice were generated, in order to determine the impact of p53 deregulation on Brca2-deficient carcinogenesis. Both CB and CBP mice developed non-invasive ductal precursor lesions (murine pancreatic intraepithelial neoplasia or mPanIN), although these were observed at an earlier time point (5 versus 8 months) and with higher prevalence in CBP mice. A minority of CB mice (15%) developed invasive and metastatic PDAC at a latency of 15 months or greater; in contrast, CBP mice of comparable age uniformly developed PDAC with variable histological features. Mortality in the absence of neoplasia in CB and CBP mice was associated with profound loss of pancreatic parenchyma, consistent with progressive elimination of Brca2-deficient cells. Widespread DNA damage, as evidenced by overexpression of the phosphorylated histone H(2)AX(Ser139), was observed in the non-neoplastic exocrine pancreas, as well as in the mPanIN and PDAC lesions of Brca2-deficient mice, independent of p53 status. Loss of Brca2 function predisposes the exocrine pancreas to profound DNA damage, and the frequency of invasive neoplasia is accentuated by the concomitant deregulation of p53.     

Repression of mRNA for the PLK cell cycle gene after DNA damage requires BRCA1

DNA damage activates the G2 cell cycle checkpoint to allow time for DNA repair before mitotic entry. The mechanism involves inhibition of the enzymatic activity for polo-like kinase 1 (Plk1), rendering Cdc25C with a basal phosphatase activity that is insufficient for converting Cdc2 to the fully active G2/M transition kinase. We found that cell cycle arrest at the G2/M boundary after ionizing radiation (IR) of breast carcinoma cells may involve repression of the gene for Plk1, PLK, mediated by the tumor-suppressor protein BRCA1. The p53-defective MT-1 cell line had an apparent accumulation of G2/M phase cells 12 h after irradiation. This response was preceded by a transient downregulation of PLK mRNA expression with a barely detectable level 6 h after exposure to IR but recovered after 12 h. A significantly lower fraction of irradiated BRCA1(-/-) HCC1937 cells arrested in the G2/M phase after 12 h, and the transient response of PLK mRNA was also considerably impaired. After reconstitution of wild-type BRCA1 in the HCC1937 cells however, downregulation of PLK mRNA as well as Plk1 protein expression after IR was restored. Moreover, the suppression of PLK mRNA expression 6 h after irradiation was completely abolished by the specific CHEK1 kinase inhibitor UCN-01, further indicating that the effector mechanism of DNA damage on PLK signals through BRCA1 and its downstream CHEK1. Our observations provide new information about the diversity of regulatory mechanisms governed by BRCA1 in DNA damage checkpoint control.     

Brca1 regulates in vitro differentiation of mammary epithelial cells

Murine Brca1 is widely expressed during development in different tissues. Why alterations of BRCA1 lead specifically to breast and ovarian cancer is currently not clarified. Here we show that Brca1 protein expression is upregulated during mammary epithelial differentiation of HC11 cells, during differentiation of C2C12 myoblasts into myotubes and during neuronal differentiation of N1E-115 cells. Ectopic overexpression of BRCA1 and downregulation of endogenous Brca1 expression specifically affect the regulation of mammary epithelial cell differentiation. Accelerated mammary epithelial cell differentiation upon high ectopic BRCA1 expression is not a consequence of the anti-proliferative capacity of this tumor suppressor and independent of functional p53. Overexpression of the BRCA1 variant lacking the large central exon 11 has no effects on mammary epithelial cell differentiation. These data provide new insights into the cellular role of Brca1.