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. 2004 Nov;24(21):9498-507.
doi: 10.1128/MCB.24.21.9498-9507.2004.

Uterus hyperplasia and increased carcinogen-induced tumorigenesis in mice carrying a _targeted mutation of the Chk2 phosphorylation site in Brca1

Sang Soo Kim  1 , Liu Cao, Cuiling Li, Xiaoling Xu, L Julie Huber, Lewis A Chodosh, Chu-Xia Deng
Affiliations

Uterus hyperplasia and increased carcinogen-induced tumorigenesis in mice carrying a _targeted mutation of the Chk2 phosphorylation site in Brca1

Sang Soo Kim et al. Mol Cell Biol. 2004 Nov.

Abstract

The tumor suppressor BRCA1 contains multiple functional domains that interact with many proteins. After DNA damage, BRCA1 is phosphorylated by CHK2 at serine 988, followed by a change in its intracellular location. To study the functions of CHK2-dependent phosphorylation of BRCA1, we generated a mouse model carrying the mutation S971A (S971 in mouse Brca1 corresponds to S988 in human BRCA1) by gene _targeting. Brca1(S971A/S971A) mice were born at the expected ratio without a developmental defect, unlike previously reported Brca1 mutant mice. However, Brca1(S971A/S971A) mice suffered a moderately increased risk of spontaneous tumor formation, with a majority of females developing uterus hyperplasia and ovarian abnormalities by 2 years of age. After treatment with DNA-damaging agents, Brca1(S971A/S971A) mice exhibited several abnormalities, including increased body weight, abnormal hair growth pattern, lymphoma, mammary tumors, and endometrial tumors. In addition, the onset of tumor formation became accelerated, and 80% of the mutant mice had developed tumors by 1 year of age. We demonstrated that the Brca1(S971A/S971A) cells displayed reduced ability to activate the G(2)/M cell cycle checkpoint upon gamma-irradiation and to stabilize p53 following N-methyl-N'-nitro-N-nitrosoguanidine treatment. These observations suggest that Chk2 phosphorylation of S971 is involved in Brca1 function in modulating the DNA damage response and repressing tumor formation.

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Figures

FIG. 1.
FIG. 1.
Introduction of the S971A mutation into mouse Brca1 locus. (A) _targeting vector and final structure of the mutant allele. The point mutation S971A in exon 11 was cotransferred with ploxPneo through homologous recombination in ES cells. Once germ line transmission was established, mice heterozygous for the mutation (+/S971A-neo) were crossed with EIIa-Cre transgenic mice to delete ploxPneo. H, HindIII; Ev, EcoRV; X, XhoI Xb, XbaI TK, thymidine kinase; open triangles, loxp sites. The positions of the probes used for Southern blot analyses and the sizes of the endogenous and _targeted DNA fragments recognized by these probes are shown. (B) Southern analysis of ES cell DNA digested with HindIII to identify the _targeting event that is characterized by a fragment shift from 10 to 12 kb due to the presence of neo. Same samples digested with HindIII and EcoRV were subjected to Southern analysis with an internal BamHI probe (data not shown). (C) Reverse transcription-PCR analysis to check the proper expression of Brca1 was performed in wild-type and Brca1S971A/S971A mutant MEFs by using primers against exon 11 of Brca1 with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) control. (D) DNA fragments containing the S971A mutation were amplified by PCR, followed by digestion with SfaNI. The PCR product (500 bp) from S971A was cleaved by SfaNI (the cleaved fragment [250 bp] is indicated by an arrow). The presence of the mutation was also confirmed by sequencing of the PCR product (data not shown). WT, wild type. (E) Southern blot analysis of XbaI-digested tail DNA isolated from wild-type, heterozygous (+/S971A), and homozygous (S971A/S971A) mice after neo excision. The presence of a 7-kb fragment by Southern blotting represented the removal of neo.
FIG. 2.
FIG. 2.
Phenotypes in Brca1S971A/S971A mice. Whole-mount staining of mammary glands from Brca1S971A/S971A (A) and age-matched wild-type (B) mice. The boxed areas are amplified. (C) Uteri and ovaries from wild-type (WT) and Brca1S971A/S971A mice. Examples of histological sections of uterus (D to G) and ovary (H to K) from wild-type (D, F, H, and J) and Brca1S971A/S971A (E, G, I, and K) mice. J and K represent the magnification of boxed areas of H and I, respectively.
FIG. 3.
FIG. 3.
Phenotypes in Brca1S971A/S971A mouse response upon γ-irradiation. (A) Wild-type (WT) and Brca1S971A/S971A mice 10 months of age after irradiation. (B) Body weights of wild-type (n = 14 at 10 and 11 months [M]) and Brca1S971A/S971A mice (n = 5 at 10 months; n = 3 at 11 months). (C) Percentages of abnormal awl-type hairs determined under the microscope. The error bars indicate standard deviations. (D) Microscopic image of normal and defective hairs from wild-type and Brca1S971A/S971A mice, respectively. (E and F) Whole-mount staining of mammary glands from wild-type (E) and Brca1S971A/S971A (F) mice 13 months after irradiation. (G to J) More highly magnified whole-mount images of wild type (G) and Brca1S971A/S971A (H to J).
FIG. 4.
FIG. 4.
Reduced survival with high tumor incidence in Brca1S971A/S971A mice treated with IR or MNU. (A) Kaplan-Meier survival curve of wild-type (open circles; n = 19) and Brca1 mutant (filled circles; Brca1KO/S971A, n = 5; Brca1S971A/S971A, n = 19) mice after irradiation. The data are combined from two separate experiments. (B) Kaplan-Meier survival curve of wild-type (n = 15) and Brca1S971A/S971A (n = 15) mice after injection of MNU (50 mg/kg) at 6 weeks of age. Red and blue circles in the curves represent the mammary tumors and colon-uterus tumors, respectively. (C) Tumor spectrum of mice after treatment. Parentheses represent the number of mice that died without detectable tumors. *, three mice had tumors in more than one organ. WT, wild type. (D to I) Brca1S971A/S971A mutants develop a variety of tumors after DNA damage. (D) A Brca1S971A/S971A mouse bearing a mammary tumor (arrow). Histological sections from mammary (E to G), colon (H), and uterus (I) tumors.
FIG. 5.
FIG. 5.
Characterization of tumors from Brca1S971A/S971A mice. (A) Soft-agar colony formation assay of three different clones (94A, 94B, and 94C) of V94 mammary tumor. (B) Tumorigenicity assay of mammary tumor cells in nude mice. Tumors (arrows) were observed 3 weeks after injection of 94A, 94B, and 94C. (C) Hematoxylin- and eosin-stained sections of original mammary tumor from V94 and three different tumors from injection of clones into nude mice. (D) Chromosome spreads from V94 and V296 tumors. (E) Microsatellite analysis of tumors of Brca1S971A/S971A mice. T and N represent the DNAs from tumor and normal tissue, respectively.
FIG. 6.
FIG. 6.
Response to γ-irradiation and MNNG in Brca1S971A/S971A MEFs and tumor cells. (A to D) Partially defective G2/M checkpoint activation in Brca1S971A/S971A MEFs. (A) Mitotic index of irradiated or unirradiated wild-type (WT) and Brca1S971A/S971A MEFs was determined at the indicated times after 10 Gy of IR. The mitotic index of irradiated (B) or unirradiated (C) wild-type and Brca1S971A/S971A MEFs was determined in the presence of nocodazole (100 ng/ml). Means ± standard errors of the mean from at least three different littermate experiments are given. Significant differences are indicated by asterisks. (D) Representative fluorescence-activated cell sorter plots of wild-type and Brca1S971A/S971A MEFs 4 h after 0 or 10 Gy of irradiation. For each analysis, 10,000 cells of each genotype were analyzed. (E) DNA synthesis was measured 1 h after the indicated dose of irradiation in wild-type and Brca1S971A/S971A MEFs. (F) Expression patterns of p53 and p21 after treatment with γ-irradiation (10 Gy). C and IR, unirradiated and irradiated MEFs, respectively. (G) p53 expression levels in wild-type (W) and Brca1S971A/S971A (M) cells in the absence (CTL) and presence (MNNG) of MNNG for 12 h were measured and compared as described in Materials and Methods. (H) p53 levels in wild-type and Brca1S971A/S971A MEFs from 0 to 12 h after MNNG (250 μM) treatment. (I) Levels of p53 after treatment with MNNG in wild-type and Chk2−/− MEFs generated independently from two different littermates.
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