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• W2011137269 abstract "GADD45a is a transcription target of the breast tumor suppressor gene BRCA. It was recently shown that mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of Brca1 (Brca1Δ11/Δ11) or a Gadd45A-null mutation (Gadd45a-/-) suffer centrosome amplification. To study genetic interactions between these genes during centrosome duplication, we generated Brca1Δ11/Δ11Gadd45a-/- mice by crossing each mutant. We found that all Brca1Δ11/Δ11Gadd45a-/- embryos at embryonic days 9.5-10.5 were exencephalic and exhibited a high incidence of apoptosis accompanied by altered levels of BAX, BCL-2, and p53. The trigger for these events is likely the genetic instability arising from centrosome amplification that is associated, at least in part, with decreased expression of the NIMA-related kinase NEK2. We demonstrate that small interfering RNA-mediated suppression of Brca1 decreased Nek2 more dramatically in Gadd45a-/- cells than in wild-type cells and, conversely, that overexpression of Brca1 and/or Gadd45a up-regulated transcription of Nek2. Furthermore, we show that overexpression of Nek2 in Brca1-specific small interfering RNA-treated wild-type and Gadd45a-/- cells repressed abnormal centrosome amplification. These observations suggest that NEK2 plays a role in mediating the actions of BRCA1 and GADD45a in regulating centrosome duplication and in maintaining genetic stability. GADD45a is a transcription target of the breast tumor suppressor gene BRCA. It was recently shown that mouse embryonic fibroblast cells carrying a targeted deletion of exon 11 of Brca1 (Brca1Δ11/Δ11) or a Gadd45A-null mutation (Gadd45a-/-) suffer centrosome amplification. To study genetic interactions between these genes during centrosome duplication, we generated Brca1Δ11/Δ11Gadd45a-/- mice by crossing each mutant. We found that all Brca1Δ11/Δ11Gadd45a-/- embryos at embryonic days 9.5-10.5 were exencephalic and exhibited a high incidence of apoptosis accompanied by altered levels of BAX, BCL-2, and p53. The trigger for these events is likely the genetic instability arising from centrosome amplification that is associated, at least in part, with decreased expression of the NIMA-related kinase NEK2. We demonstrate that small interfering RNA-mediated suppression of Brca1 decreased Nek2 more dramatically in Gadd45a-/- cells than in wild-type cells and, conversely, that overexpression of Brca1 and/or Gadd45a up-regulated transcription of Nek2. Furthermore, we show that overexpression of Nek2 in Brca1-specific small interfering RNA-treated wild-type and Gadd45a-/- cells repressed abnormal centrosome amplification. These observations suggest that NEK2 plays a role in mediating the actions of BRCA1 and GADD45a in regulating centrosome duplication and in maintaining genetic stability. Cells normally contain one or two centrosomes depending on their phases in the cell cycle. Centrosome duplication starts at later G1 phase and proceeds through S phase (1Winey M. Curr. Biol. 1996; 6: 962-964Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 2Rieder C.L. Faruki S. Khodjakov A. Trends Cell Biol. 2001; 11: 413-419Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 3Fukuda S. Foster R.G. Porter S.B. Pelus L.M. Blood. 2002; 100: 2463-2471Crossref PubMed Scopus (126) Google Scholar, 4Lange B.M. Curr. Opin. Cell Biol. 2002; 14: 35-43Crossref PubMed Scopus (88) Google Scholar). Prior to mitosis, the duplicated centrosomes separate and move to the future poles of the spindle to initiate the bipolar spindle, which is required for equal segregation of chromosomes. Dysregulation of this process can cause centrosome malformation, chromosome unequal segregation, and, consequently, malignant transformation (5Lingle W.L. Barrett S.L. Negron V.C. D'Assoro A.B. Boeneman K. Liu W. Whitehead C.M. Reynolds C. Salisbury J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1978-1983Crossref PubMed Scopus (475) Google Scholar, 6Pihan G.A. Purohit A. Wallace J. Knecht H. Woda B. Quesenberry P. Doxsey S.J. Cancer Res. 1998; 58: 3974-3985PubMed Google Scholar). It has been shown that alterations of many factors, including Brca1 (the mouse homolog of human BRCA1), Gadd45a (growth arrest and DNA damage-inducible gene) and Nek2, affect centrosome duplication (7Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar, 8Xu X. Weaver Z. Linke S.P. Li C. Gotay J. Wang X.W. Harris C.C. Ried T. Deng C.-X. Mol. Cell. 1999; 3: 389-395Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 9Fry A.M. Meraldi P. Nigg E.A. EMBO J. 1998; 17: 470-481Crossref PubMed Scopus (343) Google Scholar, 10Hsu L.C. Doan T.P. White R.L. Cancer Res. 2001; 61: 7713-7718PubMed Google Scholar). However, the underlying mechanisms remain illusive. The functions of BRCA1 have been the subject of extensive research since its cloning in 1994 (11Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Crossref PubMed Scopus (5302) Google Scholar). Mounting evidence reveals that BRCA1 plays essential roles in many biological processes, including transcription activation and repression, cell cycle regulation, chromatin remodeling, DNA damage repair, and centrosome duplication (reviewed in Refs. 12Zheng L. Li S. Boyer T.G. Lee W.H. Oncogene. 2000; 19: 6159-6175Crossref PubMed Scopus (135) Google Scholar, 13Deng C.-X. Wang R.-H. Hum. Mol. Genet. 2003; 12: R113-R123Crossref PubMed Google Scholar, 14Venkitaraman A.R. Cell. 2002; 108: 171-182Abstract Full Text Full Text PDF PubMed Scopus (1401) Google Scholar). It has been shown that mouse embryos carrying Brca1-null mutations die early in gestation, displaying proliferation defects (15Gowen L.C. Johnson B.L. Latour A.M. Sulik K.K. Koller B.H. Nat. Genet. 1996; 12: 191-194Crossref PubMed Scopus (396) Google Scholar, 16Hakem R. de la Pompa J.L. Sirard C. Mo R. Woo M. Hakem A. Wakeham A. Potter J. Reitmair A. Billia F. Firpo E. Hui C.C. Roberts J. Rossant J. Mak T.W. Cell. 1996; 85: 1009-1023Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar, 17Liu C.Y. Flesken-Nikitin A. Li S. Zeng Y. Lee W.H. Genes Dev. 1996; 10: 1835-1843Crossref PubMed Scopus (274) Google Scholar, 18Ludwig T. Chapman D.L. Papaioannou V.E. Efstratiadis A. Genes Dev. 1997; 11: 1226-1241Crossref PubMed Scopus (462) Google Scholar, 19Shen S.X. Weaver Z. Xu X. Li C. Weinstein M. Chen L. Guan X.Y. Ried T. Deng C.-X. Oncogene. 1998; 17: 3115-3124Crossref PubMed Scopus (294) Google Scholar). Embryos carrying a homozygous deletion of Brca1 exon 11 (Brca1Δ11/Δ11), which encodes 60% of the amino acids of the protein, die at later stages of gestation because of widespread apoptosis (20Xu X. Qiao W. Linke S.P. Cao L. Li W.M. Furth P.A. Harris C.C. Deng C.-X. Nat. Genet. 2001; 28: 266-271Crossref PubMed Scopus (300) Google Scholar). We have shown that haploinsufficiency of p53 can suppress apoptosis in Brca1Δ11/Δ11 embryos and allow them to develop to adulthood; however, the survivors (Brca1Δ11/Δ11p53+/-) exhibit apoptosis in testes and thymus and eventually die of premature aging and tumorigenesis (20Xu X. Qiao W. Linke S.P. Cao L. Li W.M. Furth P.A. Harris C.C. Deng C.-X. Nat. Genet. 2001; 28: 266-271Crossref PubMed Scopus (300) Google Scholar, 21Xu X. Aprelikova O. Moens P. Deng C.-X. Furth P.A. Development (Camb.). 2003; 130: 2001-2012Crossref PubMed Scopus (129) Google Scholar, 22Bachelier R. Xu X. Wang X. Li W. Naramura M. Gu H. Deng C.-X. Oncogene. 2003; 22: 528-537Crossref PubMed Scopus (28) Google Scholar, 23Cao L. Li W. Kim S. Brodie B.G. Deng C.-X. Genes Dev. 2003; 17: 201-213Crossref PubMed Scopus (239) Google Scholar). In mutant mice in which the Brca1 exon 11 was specifically disrupted in mammary epithelium using a Cre/loxP approach, mammary tumors develop at low frequency after long latency (24Xu X. Wagner K.U. Larson D. Weaver Z. Li C. Ried T. Hennighausen L. Wynshaw-Boris A. Deng C.-X. Nat. Genet. 1999; 22: 37-43Crossref PubMed Scopus (625) Google Scholar). Further analysis revealed the involvement of multiple factors in the Brca1-associated tumorigenesis, including overexpression of the erbB2,c-myc, p27, and cyclin D1 genes as well as down-regulation or loss of p53 and p16 in the majority of tumors (25Brodie S.G. Xu X. Qiao W. Li W.M. Cao L. Deng C.-X. Oncogene. 2001; 20: 7514-7523Crossref PubMed Scopus (161) Google Scholar). As Brca1 is a transcription activator that regulates expression of a number of important genes, including p21, 14-3-3, Chk-1, and Gadd45a (26Harkin D.P. Bean J.M. Miklos D. Song Y.H. Truong V.B. Englert C. Christians F.C. Ellisen L.W. Maheswaran S. Oliner J.D. Haber D.A. Cell. 1999; 97: 575-586Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 27Aprelikova O. Pace A.J. Fang B. Koller B.H. Liu E.T. J. Biol. Chem. 2001; 276: 25647-25650Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 28Yarden R.I. Pardo-Reoyo S. Sgagias M. Cowan K.H. Brody L.C. Nat. Genet. 2002; 30: 285-289Crossref PubMed Scopus (401) Google Scholar, 29Somasundaram K. Zhang H. Zeng Y.X. Houvras Y. Peng Y. Wu G.S. Licht J.D. Weber B.L. El-Deiry W.S. Nature. 1997; 389: 187-190Crossref PubMed Scopus (471) Google Scholar), part of the Brca1 mutant phenotypes could be due to changes in its downstream transcription targets. GADD45a is one of several growth arrest- and DNA damage-inducible genes (30Sheikh M.S. Hollander M.C. Fornance Jr., A.J. Biochem. Pharmacol. 2000; 59: 43-45Crossref PubMed Scopus (166) Google Scholar). Its expression can be regulated by a variety of genotoxic and non-genotoxic stresses, including UV radiation, methy1 methanesulfonate, and ionizing radiation. GADD45a interacts with proliferating the cell nuclear antigen, p21, Cdc2, core histone, and MTK/MEKK4 genes, indicating that GADD45a may be involved in multiple important cellular events (31Carrier F. Georgel P.T. Pourquier P. Blake M. Kontny H.U. Antinore M.J. Gariboldi M. Myers T.G. Weinstein J.N. Pommier Y. Fornace Jr., A.J. Mol. Cell. Biol. 1999; 19: 1673-1685Crossref PubMed Scopus (248) Google Scholar, 32Hildesheim J. Bulavin D.V. Anver M.R. Alvord W.G. Hollander M.C. Vardanian L. Fornace Jr., A.J. Cancer Res. 2002; 62: 7305-7315PubMed Google Scholar). GADD45a may also serve as a tumor suppressor, as its expression in multiple tumor lines suppresses their growth (33Wang X.W. Zhan Q. Coursen J.D. Khan M.A. Kontny H.U. Yu L. Hollander M.C. O'Connor P.M. Fornace Jr., A.J. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3706-3711Crossref PubMed Scopus (533) Google Scholar). It was shown that expression of BRCA1 induces expression of GADD45a, leading to JNK 1The abbreviations used are: JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; MEF, mouse embryonic fibroblast; E, embryonic day; BrdUrd, bromodeoxyuridine; HA, hemagglutinin; siRNA, small interfering RNA; RT, reverse transcription; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; MAPK, mitogen-activated protein kinase; NTD, neural tube closure defect./SAPK-dependent apoptosis (26Harkin D.P. Bean J.M. Miklos D. Song Y.H. Truong V.B. Englert C. Christians F.C. Ellisen L.W. Maheswaran S. Oliner J.D. Haber D.A. Cell. 1999; 97: 575-586Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). Conversely, both the tumor-derived BRCA1 mutants and truncated BRCA1 mutants, which lack transactivation activity, are unable to activate the GADD45a promoter, indicating that BRCA1-mediated activation of the GADD45a promoter requires normal transcriptional properties of BRCA1 (34Jin S. Zhao H. Fan F. Blanck P. Fan W. Colchagie A.B. Fornace Jr., A.J. Zhan Q. Oncogene. 2000; 19: 4050-4057Crossref PubMed Scopus (85) Google Scholar). Mouse Gadd45a-/- embryos exhibit a low frequency of exencephaly, but otherwise are developmentally normal. Mutant mice survive to adulthood and exhibit increased γ-irradiation- or UV light-induced carcinogenesis (7Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar). Similar to Brca1Δ11/Δ11 mouse embryonic fibroblast (MEF) cells, Gadd45a-/- cells display genomic instability as exemplified by aneuploidy, chromosome aberration, defective global genomic repair, and centrosome amplification (7Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar, 35Smith M.L. Ford J.M. Hollander M.C. Bortnick R.A. Amundson S.A. Seo Y.R. Deng C.-X. Hanawalt P.C. Fornace Jr., A.J. Mol. Cell. Biol. 2000; 20: 3705-3714Crossref PubMed Scopus (396) Google Scholar). The phenotypic similarities between Brca1Δ11/Δ11 and Gadd45a-/- cells prompted us to study the possible genetic interaction between Brca1 and Gadd45a in maintaining genome integrity. Using the existing Brca1Δ11/Δ11 (20Xu X. Qiao W. Linke S.P. Cao L. Li W.M. Furth P.A. Harris C.C. Deng C.-X. Nat. Genet. 2001; 28: 266-271Crossref PubMed Scopus (300) Google Scholar) and Gadd45a-/- (7Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar) mice, we generated mice that are homozygous for mutations of both genes (Brca1Δ11/Δ11Gadd45a-/-). We found that virtually all Brca1Δ11/Δ11Gadd45a-/- embryos exhibited exencephaly, showing increased apoptosis in their neuroepithelia due to p53 activation, as haploid or complete loss of p53 repressed apoptosis and rescued embryonic lethality. Our further analysis uncovered a synergistic role of Brca1 and Gadd45a in regulating centrosome duplication and in maintaining genome integrity. Mating and Genotyping of Mice—Genotyping of Brca1+/Δ11 and Gadd45a+/- mutant mice was as described (7Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar, 20Xu X. Qiao W. Linke S.P. Cao L. Li W.M. Furth P.A. Harris C.C. Deng C.-X. Nat. Genet. 2001; 28: 266-271Crossref PubMed Scopus (300) Google Scholar). Brca1+/Δ11 and Gadd45a+/- mice were crossed to generate Brca1 and Gadd45a double heterozygous (Brca1+/Δ11Gadd45a+/-) mice, which were further crossed to generate Brca1Δ11/Δ11Gadd45a-/- mice. The genetic background is a mixture of 129, B6, and Black Swiss. Apoptosis and Proliferation Analysis—Sections from E9.5 to E13.5 were analyzed for apoptosis using the ApoTag kit (Intergen Co., Purchase, NY) as recommended by the manufacturer. To evaluate cell proliferation rates, bromodeoxyuridine (BrdUrd) incorporation was measured using a cell proliferation kit (Amersham Biosciences) following the manufacturer's directions. Cell Cultures and Treatments—Primary MEFs were obtained from E14.5 embryos using a standard procedure. UBR-60 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and glutamine in the presence or absence of 1 μg/ml tetracycline for controlling BRCA1 expression as described (26Harkin D.P. Bean J.M. Miklos D. Song Y.H. Truong V.B. Englert C. Christians F.C. Ellisen L.W. Maheswaran S. Oliner J.D. Haber D.A. Cell. 1999; 97: 575-586Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar). Plasmids bearing hemagglutinin (HA)-tagged GADD45a (36Bulavin D.V. Kovalsky O. Hollander M.C. Fornace Jr., A.J. Mol. Cell. Biol. 2003; 23: 3859-3871Crossref PubMed Scopus (136) Google Scholar, 37Kovalsky O. Lung F.D. Roller P.P. Fornace Jr., A.J. J. Biol. Chem. 2001; 276: 39330-39339Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) or pEGFP-NEK2A (38Hames R.S. Fry A.M. Biochem. J. 2002; 361: 77-85Crossref PubMed Scopus (68) Google Scholar) were transfected into cultured cells using LipofectAMINE™ 2000 (Invitrogen). For small interfering RNA (siRNA) transfection, we plated 2.5 × 105 cells onto a 60-mm dish 1 day before transfection. siRNA specific for Brca1 (made by Dharmacon Research) and control siRNA (made by George Ploy) at a concentration of 0.36 μm were transfected into MEF cells at passage 2 using OligofectAMINE as described (39Elbashir S.M. Harborth J. Lendeckel W. Yalcin A. Weber K. Tuschl T. Nature. 2001; 411: 494-498Crossref PubMed Scopus (8160) Google Scholar). After transfection, cells were harvested at 24, 48, 72, and 96 h and processed for immunofluorescence, Western blotting, and/or reverse transcription (RT)-PCR. The siRNA sequence for murine Brca1 was GAGACAGUAACUAAGCCAG. The control siRNA was derived from human BRCA1 and differs from the corresponding region of mouse Brca1 by 2 bases. The siRNA sequences were also tagged with a fluorescence group at the 3′-end. The transfection efficiencies for siRNA were ∼70% as directly reviewed under a fluorescence microscope 48 h after transfection. RT-PCR Analysis—Total RNAs were extracted from E10 embryos or MEFs. RT reactions were carried out using a first strand cDNA synthesis kit (Roche Applied Science). The cDNA samples were stored at -20 °C. One μg of RNA from each sample was used as template for each reaction, and 1 μl of cDNA from each sample was used for PCR. The optimal number of cycles for amplification was chosen according to the cycle number that yielded the strongest band while staying within the liner range. The ranges of cycles varied from 25 to 28 according to the specific target of RNA and primer set. The samples were heated to 94 °C for 2 min and then run through 25-28 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 10 min and then 4 °C. The primers used in this study were as follows: Nek2-1, 5′-GAA GAT TCG GAG GAA GAG CG-3′; Nek2-2, 5′-GAG GCA TTA GTG CAC ACA GC-3′; Gadd45a-1, 5′-GCA CTT GCA ATA TGA CTT TGG-3′; Gadd45a-2, 5′-GTT CCG GGA GAT TAA TCA CG-3′; Brca1-1, 5′-CT CAA GAA GCT GGA GAT GAA GG-3′; Brca1-2, 5′-CAA TAA ACT GCT GGT CTC AGG-3′; glyceraldehyde-3-phosphate dehydrogenase-1, 5′-ACA GCC GCA TCT TCT TGT GC-3′; and glyceraldehyde-3-phosphate dehydrogenase-2, 5′-TTT GAT GTT AGA GGG GTC TGC-3′. Centrosome Staining and Analysis—Cells grown on chamber slides (Falcon) were fixed in 2.5% paraformaldehyde, 25 mm MgCl2, and phosphate-buffered saline (PBS) for 10 min at room temperature. The slides were then washed with 0.3 m glycine and PBS, permeabilized in 0.2% Triton X-100 and PBS, and incubated overnight with anti-γ-tubulin polyclonal antibody (Sigma) diluted 1:1000 in 5% goat serum and PBS. The antibody complexes were detected with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Roche Applied Science) and stained with 4′,6-diamidino-2-phenylindole. For dual detection of centrosomes and microtubules, cells were fixed in ice-cold methanol for 10 min, and the permeabilization step was eliminated. Immunostaining was performed in four layers: anti-α-tubulin antibody (Sigma) followed by Texas Red-conjugated goat anti-mouse IgG (Vector Laboratories) and then anti-γ-tubulin antibody diluted 1:500 in 5% goat serum and PBS followed by fluorescein isothiocyanate-conjugated sheep anti-rabbit IgG (Roche Applied Science). Gray level images were acquired using a CCD camera (CH250, Photometrics Ltd., Tucson, AZ) mounted on a Leica DMRBE epifluorescence microscope and pseudocolored using registration software. The centrosome/nucleus ratio is determined by counting centrosome numbers/cell, which contains only one nucleus. Western Blot Analysis—Western blot analysis was accomplished according to standard procedures by ECL detection (Amersham Biosciences). The following primary antibodies were used: anti-p53 (Ab-7, Oncogene); anti-JNK1/JNK2 (BD Biosciences); anti-JNK3 (Upstate Biotechnology, Inc.); and anti-Bax, anti-Bcl-2, anti-HA, and anti-NEK2 (Santa Cruz Biotechnology). Peroxidase-labeled goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) antibodies (Kirkegaard & Perry Laboratories) were used as secondary antibodies. Chromosome Preparation from E9.5 Embryos—E9 embryos were dissected and incubated at 37 °C for 2 h in medium containing 0.1 μg/ml Colcemid. The embryos were placed into 0.56% potassium chloride for 5 min and then fixed in a freshly prepared 3:1 mixture of methanol and glacial acetic acid at 4 °C overnight, followed by disaggregation in a 5-fold excess of aqueous 60% glacial acetic acid at room temperature for 5 min. The suspension was dropped across the surface of a slide, and the cells were spread and allowed to dry on the surface. The slides were stained with Giemsa. Absence of Gadd45a Accelerates Embryonic Lethality of Brca1Δ11/Δ11 Embryos—A previous investigation showed that the majority of Brca1Δ11/Δ11 embryos die before birth, whereas Gadd45a-/- mice survive to adulthood (7Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar). The animals double heterozygous for Brca1 and Gadd45a mutations were indistinguishable from wild-type controls (data not shown). However, no Brca1Δ11/Δ11Gadd45a-/- mice were found among 1246 pups generated from interbreeding with mice with several combinations of genotypes (Table I, Crosses 1-3), suggesting that the Brca1Δ11/Δ11Gadd45a-/- mutation is recessive lethal.Table IGenotypes of animals derived from crosses between Brca1 and Gadd45a mutant miceCrossesTotalNo. of each genotypeBr+/+ G−/−Br+/Δ G−/−BrΔ/Δ G−/−Br+/+ G+/−Br+/Δ G+/−BrΔ/Δ G+/−Br+/+ G+/+Br+/Δ G+/+BrΔ/Δ G+/+Cross 1 (Br+/Δ G+/− × Br+/Δ G+/−)At weaning14511002654114381E12.5-15.526111476231Br+/+ G−/−Br+/Δ G−/−BrΔ/Δ G−/−Br+/+ G+/−Br+/Δ G+/−BrΔ/Δ G+/−Cross 2 (Br+/Δ G+/− × Br+/Δ G−/−)At weaning4251004230E12.5-15.5916144134113Br+/+ G−/−Br+/Δ G−/−BrΔ/Δ G−/−Resorbed embryosCross 3 (Br+/Δ G−/− × Br+/Δ G−/−At weaning3411212200E13.5-15.521249111 (2)52 (41)23E11.5-12.519060111 (4)29 (14)13E9.5-10.519948 (4)107 (13)44 (44)Br+/+Br+/ΔBrΔ/ΔResorbed embryosCross 4 (Br+/Δ × Br+/Δ)At weaning182751034E13.5-14.52305712152 (3)6E11.5-12.585174226 (1)8E9.5-10.5176398057 (2)1 Open table in a new tab Next, we dissected pregnant females to study possible phenotypes of Brca1Δ11/Δ11Gadd45a-/- embryos during gestation. We found that Brca1Δ11/Δ11Gadd45a-/- embryos were presented at the expected ratios at E9.5-10.5 (Table I, Cross 3). However, all of the mutant embryos, especially those of older stages (from E11.5 to E15.5) were significantly smaller than the controls, and many of them were dying or dead (Fig. 1) (data not shown). We also analyzed Brca1Δ11/Δ11 embryos that were in the same genetic background. We found that, although Brca1Δ11/Δ11 embryos were distinguishably smaller than the wild-type controls at these stages of development, their development was relative normally and that they did not die until after E14.5 (Table I, Cross 4) (data not shown). Moreover, ∼1-2% of the Brca1Δ11/Δ11 mice survived to adulthood (Table I, Cross 1, 1/145; and Cross 4, 4/182), whereas no Brca1Δ11/Δ11Gadd45a-/- mice were found in 1246 offspring generated from our crosses (Table I, Crosses 1-3). These observations indicate that the absence of Gadd45a accelerates embryonic lethality caused by the Brca1Δ11/Δ11 mutation. Brca1Δ11/Δ11Gadd45a-/- Embryos Display Exencephaly—One prominent feature of Brca1Δ11/Δ11Gadd45a-/- embryos was that the majority of them exhibited exencephaly because of failed closure of the anterior neural tube (Table I). In a normal embryo, the neural tube begins to close at E8.5 from multiple sites in the middle portion of the embryo and extends both anteriorly and posteriorly in a zipper-like fashion. By E9.5, most parts of the neural tube have already closed, and only small openings, called the neural pores, are left in both the anterior and posterior ends of the embryo. Our examination of E9.5-10.5 embryos indicated that all Brca1Δ11/Δ11Gadd45a-/- embryos failed to close their anterior neural tubes with varying severity (Fig. 1, A and B; and Table I, Cross 3). Whole-mount in situ hybridization using Otx2, which marks the neuroepithelium of the forebrain and midbrain, revealed that the unclosed region extended from Otx2-positive portions of the brain throughout the Otx2-negative hindbrain (Fig. 1E, arrow). We also analyzed Brca1Δ11/Δ11Gadd45a-/- embryos at E11.5-15.5 and found that ∼80% of them exhibited exencephaly because of failure to close the anterior neural tube (Fig. 1, F and G; and Table I, Cross 3). Exencephaly also occurred in Brca1Δ11/Δ11 and Gadd45a-/- embryos at much lower frequencies (Table I, Crosses 3 and 4). These observations indicate that the combined effect of the Brca1 exon 11 deletion and the Gadd45a-null mutation blocks anterior neural tube closure, leading to exencephaly. Next, we performed histological analysis on the double mutant embryos. Fig. 2A shows that the mutant neural plate was elevated in E9.5 embryos, but its lateral edges did not bend toward each other. Although the thickness of the neuroepithelium was comparable with that of wild-type embryos, the lumina of the brain vesicles were not formed (Fig. 2, A-D). Examination of older embryos (E11.5-13.5) revealed that the mutant brains failed to develop further (Fig. 2E). Consequently, they contained only one layer of the neuroepithelium without any structures that were observed in normal brains. We also performed histological analysis on other parts of embryos and found that the mutant embryos contained all major internal organs despite being significantly smaller than the wild-type embryos (data not shown). This observation indicates that the combined mutations of Brca1 and Gadd45a do not affect organogenesis. Brca1Δ11/Δ11Gadd45a-/- Embryos Exhibit Increased Apoptosis—Previous investigations revealed that altered cell death and proliferation are responsible for failure to close neural tubes in some mutant embryos (40Sabapathy K. Jochum W. Hochedlinger K. Chang L. Karin M. Wagner E.F. Mech. Dev. 1999; 89: 115-124Crossref PubMed Scopus (301) Google Scholar, 41Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar). To determine whether this is the case, we first analyzed E9.5 embryos for apoptosis. Our analysis revealed a significantly higher rate of apoptosis in the neuroepithelia of Brca1Δ11/Δ11Gadd45a-/- embryos compared with control embryos (Fig. 3, A-C and enlargements). In the midbrain and hindbrain, ∼40% of the Brca1Δ11/Δ11-Gadd45a-/- cells, 20% of the Brca1Δ11/Δ11 cells, and ∼5% of the wild-type cells were TUNEL-positive. In the forebrain, the apoptotic rates for all embryos were similar, except for Brca1Δ11/Δ11 embryos, which contained more apoptotic cells in the tip of the forebrain (Fig. 3B, arrow). Our examination of older embryos at E10.5-13.5 revealed that Brca1Δ11/Δ11-Gadd45a-/- embryos continued to maintain higher apoptotic rates compared with Brca1Δ11/Δ11 embryos and other controls. These observations indicate that mutations of both Brca1 and Gadd45a synergistically increase cell death. Next, we analyzed cell proliferation using BrdUrd incorporation in E9.5-13.5 embryos. Our analysis revealed no significant differences in proliferation in E9.5 embryos of all different genotypes (Fig. 3, D and E), although proliferation of Brca1Δ11/Δ11Gadd45a-/- and Brca1Δ11/Δ11 embryos gradually decreased after E10.5 compared with Gadd45a-/- and wild-type embryos (data not shown). A careful examination of E9.5 Brca1Δ11/Δ11Gadd45a-/- embryos revealed that many nuclei, including BrdUrd-positive ones, broke into pieces (Fig. 3F, arrowheads). This observation suggests that the primary effect of the doubly deficiency of Brca1 and Gadd45a at E9.5 is apoptosis, which occurs at different phases in the cell cycle, rather than limitation of cell proliferation. As the high rate of apoptosis at E9.5 correlated with exencephaly exhibited by all Brca1Δ11/Δ11Gadd45a-/- embryos, and the decreased proliferation occurred 1 or 2 days later, we believe that the alteration in cell death is responsible for the failure to close neural tubes. Brca1 and Gadd45a Deficiency Synergistically Induces Centrosome Amplification—We have shown previously that Brca1Δ11/Δ11 and Gadd45a-/- MEF cells exhibit centrosome amplification (7Hollander M.C. Sheikh M.S. Bulavin D.V. Lundgren K. Augeri-Henmueller L. Shehee R. Molinaro T.A. Kim K.E. Tolosa E. Ashwell J.D. Rosenberg M.P. Zhan Q. Fernandez-Salguero P.M. Morgan W.F. Deng C.-X. Fornace Jr., A.J. Nat. Genet. 1999; 23: 176-184Crossref PubMed Scopus (442) Google Scholar, 8Xu X. Weaver Z. Linke S.P." @default.
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• W2011137269 title "Genetic Interactions between Brca1 and Gadd45a in Centrosome Duplication, Genetic Stability, and Neural Tube Closure" @default.
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