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, 15 (10), 1188-93

Tumorigenesis in Mice Carrying a Truncating Brca1 Mutation

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Tumorigenesis in Mice Carrying a Truncating Brca1 Mutation

T Ludwig et al. Genes Dev.

Abstract

We generated mouse mutants carrying in the Brca1 locus a modification (Brca1(tr)) that eliminates the C-terminal half of the protein product and obtained results indicating that, depending on genetic background, the missing BRCT and/or other domains are dispensable for survival, but essential for tumor suppression. Most of the apparently hypomorphic Brca1(tr/tr) mutants developed various tumors. Lymphomas were detected at all ages, whereas sarcomas and carcinomas, including breast cancer, appeared after a long latency. The mammary tumors showed striking variability in histopathological patterns suggesting stochastic engagement of tumorigenic pathways in their progression, to which the Brca1(tr/tr) mutation was apparently a late participant.

Figures

Figure 1
Figure 1
Generation of Brca1 hypomorphic mouse mutants. (A) Targeting scheme. A partial restriction map (exons 9–13; black rectangles) of the Brca1 locus is shown on top, followed by a diagram of the targeting vector used to insert a tk-neo cassette flanked by loxP sites (open triangles; not to scale) into a unique NheI site of exon 11 by homologous recombination (large X symbols). An open rectangle and a wavy line represent a diptheria toxin gene (DT) and the plasmid vector. The tk-neo cassette was removed by transient expression of cre in targeted embryonic stem (ES) cells (generation of a Brca1tr allele carrying a single loxP site insert). (E) EcoRI; (RV) EcoRV; (N) NheI; (P) PacI. The positions of the probes used for Southern analyses and the sizes of the endogenous and targeted DNA fragments recognized by these probes are shown. (B) Structure of exon 11 in the region of insertion in Brca1tr as determined by DNA sequencing of PCR and RT-PCR products (the NheI site at position 2882 was destroyed, whereas a PacI site was introduced in the mutant allele). The appearance of a stop codon (TAA; asterisk) in the Brca1tr sequence is shown. (C) Southern analysis of ES cell DNA digested with EcoRV, to confirm the initial targeting. Because of introduction of an additional EcoRV site by the inserted cassette, the 11-kb EcoRV wild-type fragment detected by probe A (lane 1) is reduced to 5.9 kb in targeted ES clones (lanes 2,3). (D) Genotyping by Southern analysis with probe B by using tail DNA digested with EcoRI/NheI (left) or EcoRI/PacI (right) from wild-type (lane 1), Brca1tr/+ heterozygous (lanes 24), and Brca1tr/tr homozygous (lane 5) mice. (E) Northern analysis of total RNA (15 μg per lane) from e11.5 wild-type (lanes 1,6), Brca1tr/+ heterozygous (lanes 3,5), and Brca1tr/tr homozygous (lanes 2,4) embryos. The blot was hybridized sequentially (after stripping) with cDNA probes for Brca1, p21Waf1, and Gapd (loading control). (F) Western analysis to assay for the presence of Brca1 in protein extracts from wild-type (lanes 1,2) and Brca1tr/tr homozygous mutant (lanes 3,4) embryos by using monoclonal GH118 either directly (left) or after immunoprecipitation with the polyclonal antibody B28 (right; see Materials and Methods).
Figure 2
Figure 2
Brca1tr/tr mutants develop a variety of tumors. (AI) Examples of histological sections from nonmammary neoplasms are shown. (A) Mediastinal lymphoma (ly) invading soft tissues around pulmonary bronchi (br) and pulmonary vessels (pv). (B) The same lymphoma as in (A) shown at higher magnification. (insets) The immunophenotype of the tumor is B220 and CD3+ (brown staining) and therefore of T-cell origin. (C) Nodal B cell lymphoma (B220+ and CD3; insets). (D) Angiosarcoma with atypical endothelial cells (arrows) lining anastomosing vascular channels. (E) Spindle cell sarcoma (retroperitoneal). (F) Bronchioloalveolar neoplasm (bn). The interface with normal lung tissue (lu) is shown. Positive nuclear immunostaining for Titf1 (thyroid transcription factor 1; inset) confirms that the tumor is of primary lung origin. (G) Nodules of hepatocellular carcinoma (hc) lacking bile ductular epithelium and therefore being negative for cytokeratin (CK) immunostaining (inset), in contrast with adjacent normal liver parenchyma (nl). (H) Colorectal mucinous adenocarcinoma with distended glands (gl) invading the bowel wall. (I) Endometrial carcinoma. Original magnifications: A,G,H, 40×; F,I, 100×; D,E, 200×; B,C 400×.
Figure 3
Figure 3
Mammary tumors in Brca1tr/tr mutants. (A) Examples of tumor carriers. (BL) Examples of histological sections showing the diverse patterns of breast carcinomas developing in Brca1tr/tr mice. (B) Solid tumor (s) with focal squamous differentiation (arrow) forming keratin (k) (case 1; Table 2). (C) Stromal desmoplasia in solid tumor (s). The arrows indicate collagen bands (case 2). (D) Papillary (arrows) and cribriform (cr) architectural pattern (case 3). (E) Adenoacanthoma showing a combination of small glands (arrows) and squamous epithelium (arrowheads) with keratin pearls (k) (case 4). (F) Mammary carcinoma in a male animal (case 5) that is cytokeratin-positive (inset) and shows a characteristic invasion pattern of cells in single files (Indian-file pattern) closely resembling infiltrating lobular human breast carcinoma. (G) Carcinoma composed of numerous papillary fronds with fibrovascular cores (arrows; case 6). (H) Mixed papillary (arrows), glandular (large arrowheads), and acinar (small arrowheads) growth patterns (case 7a). (inset) myc-like cytology (see text). (I) Cystic component (cy) in solid tumor (case 8). (J) Carcinoma with sarcomatous metaplasia (case 10). Poorly formed glands (arrows) retaining cytokeratin expression (CK; inset) are overrun by spindle-like cells expressing vimentin (VM; inset). (K) Atypical epithelial cells of a ductal carcinoma in situ (arrows) are filling and expanding ductules within a hyperplastic alveolar nodule (absence of invasion; case 11). (L) Invasive carcinoma forming tubular structures (t) (case 12). Original magnifications 200× for each panel except H (100×).

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