1. Volume 140, Issue 4, Pages 1303-1313.e3 (April 2011)
Inactivation of Brca2 Promotes Trp53-Associated but Inhibits KrasG12D-Dependent Pancreatic Cancer Development in Mice 
Matthew Rowley, Akihiro Ohashi, Gourish Mondal, Lisa Mills, Lin Yang, Lizhi Zhang, Rhianna Sundsbak, Virginia Shapiro, Michael H. Muders, Thomas Smyrk, Fergus J. Couch 
Gastroenterology 
Volume 140, Issue 4, Pages e3 (April 2011)
DOI: /j.gastro
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2. Figure 1
Brca2 inactivation promotes pancreatic cancer when combined with Trp53 inactivation. (A) Schematic representation of the Brca2F11 allele and the Trp53F2-10 allele before and after pdx-1-cre–dependent recombination. (B) PCR analysis showing rearrangement of the Brca2 and Trp53 alleles in response to pdx-1-cre expression using DNA from mouse tail and pancreas. (C) Kaplan–Meier plots showing pancreatic cancer–free survival of aged CPB2Δ11/Δ11 (n = 47), CPB2wt/Δ11 (n = 41), and CPB2wt/wt (n = 34) mice. P values were determined by a log-rank test. (D) Frequency of histologic subtypes of tumors detected in CPB2Δ11/Δ11, CPB2wt/Δ11, and CPB2wt/wt mice. (E) Representative images from tumors derived from CPB2Δ11/Δ11 mice stained with H&E (i, iv, vii, and x), cytokeratin 19 (ii, v, viii, and xi), or amylase (iii, vi, ix, and xii). (F) >PanIN lesions (i and ii) and multinucleated cells (iii) from a CPB2Δ11/Δ11 pancreas.
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3. Figure 2
Inactivation of Brca2 in pancreatic cancer cell lines induces sensitivity to DNA damage and promotes chromosomal instability. (A–C) Apoptosis in cell lines in response to ABT-888, gemcitabine, and cisplatin. (D) Quantification of radial structures in tumor cell lines treated with 100 nmol/L mitomycin-c. (E) Multinucleation and centrosome amplification in murine pancreatic tumor cell lines. (F) Aneuploidy and polyploidy in tumor cell lines with and without BRCA2.
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4. Figure 3
Inactivation of Brca2 in pancreatic cancer cell lines disrupts localization of midbody proteins during cytokinesis. (A) Immunofluorescence images of intercellular bridges and midbody structures in tumor cell lines stained with antibodies against α-tubulin, BRCA2, CEP55, and CHMP1B. (B) Expression of BRCA2 in a reconstituted CPB2Δ11/Δ11 cell line. (C) Immunofluorescence images showing the presence of endobrevin (red) and BRCA2 (green) at the midbody of a CPB2Δ11/Δ11 cell line (top) reconstituted with green fluorescent protein (GFP)-BRCA2 (bottom). (D) Multinucleation and centrosome amplification measured by immunofluorescence in a CPB2Δ11/Δ11 cell line reconstituted with empty vector or wild-type BRCA2. Error bars represent standard error of the mean.
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5. Figure 4
Brca2 mutant alleles prevent development of premalignant lesions and pancreatic tumors in LSL-KrasG12D mice. (A) Schematic representation of the LSL-KrasG12D allele before and after pdx-1-cre–dependent rearrangement. (B) PCR analysis of the Brca2F11 and KrasG12D alleles in response to pdx-1-cre expression in the mouse tail and pancreas. (C) H&E (i), CK19 (ii), amylase (iii), and insulin (iv) staining of a normal pancreas from a CKB2Δ11/Δ11 mouse, H&E (v), CK19 (vi), and amylase (vii) staining of a metaplastic lesion from a CKB2wt/wt mouse. H&E staining of PanIN lesions from an 8-month-old KrasG12D mouse (viii and xii). H&E (ix), CK19 (x), and amylase (xi) staining of a ductal tumor from a CKB2Δ11/Δ11 mouse. (D and E) Quantitation of (D) PanINs and (E) metaplastic lesions from the pancreata of CKB2wt/wt, CKB2wt/Δ11, and CKB2Δ11/Δ11 mice. Error bars represent the standard error of the mean. (F) Tumor incidence in CKB2Δ11/Δ11 mice (n = 28), CKB2wt/Δ11 mice (n = 35), and CKB2wt/wt mice (n = 30). P values were determined by a log-rank test.
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6. Figure 5
Tumors from CKB2wt/wt, CPB2wt/wt, and CPB2Δ11/Δ11 mice display differences in biomarker expression. Representative images of pancreatic tumors from CPB2Δ11/Δ11, CPB2wt/wt, and CKB2wt/wt stained by immunohistochemistry for Hes1 (i–iii), Sonic hedgehog (Shh) (iv–vi), Ki-67 (vii–ix), phospho-Erk1/2 (x–xii), and Alcian blue dye (xiii–xv).
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7. Figure 6
Brca2 mutant alleles promote chromosomal instability and apoptosis in mouse pancreatic tissue and MEFs. (A) Fluorescence in situ hybridization analysis of pancreatic tissue from mice showing the percentage of cells with aneuploidy. (B) Metaphase spreads of MEFs scored for the presence of aneuploidy. (C) Percentage of MEFs with multinucleation measured by immunofluorescence microscopy after staining with α-tubulin antibody. (D and E) Quantitation of cleaved caspase-3 expression measured by immunohistochemistry in the pancreas of (D) 4-month-old CPB2 and CB2 mice and (E) CKB2 mice. Error bars represent the standard error of the mean.
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8. Figure 7
A model of BRCA2-deficient tumorigenesis in the pancreas. The model reflects germline inheritance of a BRCA2 mutation. Inactivation of Tp53 signaling precedes inactivation or loss of the second BRCA2 allele and facilitates cancer development. Early loss of the second BRCA2 allele before disruption of Tp53 is inconsistent with cell growth/cell survival and tumor formation. Activation of Kras or other oncogenes before disruption of the second BRCA2 allele is insufficient to maintain tumor formation if wild-type Tp53 signaling remains intact. Activation of oncogenes after inactivation of the second BRCA2 allele and Tp53 signaling may promote tumor development.
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9. Supplementary Figure 1
Aged CB2Δ11/Δ11 mice show normal histology in the pancreas. H&E, CK19, amylase, and insulin staining of a normal pancreas from a CB2Δ11/Δ11 mouse.
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10. Supplementary Figure 2
Cell lines generated from CPB2Δ11/Δ11 mice have a suppressed DNA damage repair response. (A) PCR analysis showing rearrangement of Brca2 and Trp53 alleles in cell lines generated from tumors isolated from CPB2Δ11/Δ11, CPB2wt/Δ11, and CPB2wt/wt mice. (B) Metaphase spreads from a CPB2Δ11/Δ11 pancreatic tumor cell line either untreated or treated with 100 nmol/L mitomycin-c (MMC). Insets show examples of a ring chromosome (1), chromosome break (2), gaps (3), and radial structures (4). (C) Images of Rad51 and γH2AX foci in CPB2Δ11/Δ11, CPB2wt/Δ11, and CPB2wt/wt tumor cell lines stained by immunofluorescence with Rad51 or γH2AX antibodies 3 hours after 10 Gy γ-irradiation.
Gastroenterology  , e3DOI: ( /j.gastro )
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11. Supplementary Figure 3
Sequencing to identify mutations in Kras and Trp53 alleles. (A) Sequence of the Kras gene in DNA isolated from pancreatic tumor tissue derived from CPB2Δ11/Δ11 mice. The underlined sequence represents the antisense sequence of codon 12 and the arrow points to the mutation. (B) Sequence of the Trp53 gene in DNA isolated from pancreatic tumor tissue derived from CKB2Δ11/Δ11 mice. The underlined sequence represents codon 239 and the arrow points to the mutation.
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12. Supplementary Figure 4
Disruption of BRCA2 promotes genomic instability and apoptosis. (A) Fluorescence in situ hybridization analysis of normal mouse pancreatic tissues stained with centromeric probes for chromosome 9 (red) or 12 (green). (B) PCR analysis of MEF cell lines with or without adenoviral-cre showing rearrangement of the Brca2 and Kras loci. Top panel shows B2F11 or B2wt alleles, middle panel shows the Brca2Δ11 allele, and the bottom panel shows the KrasG12D allele (upper band), Kraswt allele (middle band), and LSL-KrasG12D allele (lower band). (C) Cleaved caspase-3 staining in pancreatic tissue from 4-month-old CKB2Δ11/Δ11 and CKB2wt/wt mice. Arrows show cells positive for cleaved caspase-3.
Gastroenterology  , e3DOI: ( /j.gastro )
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