Mice with the CHEK2*1100Delc SNP Are Predisposed to Cancer with a Strong Gender Bias

Mice with the CHEK2*1100delC SNP are predisposed to cancer with a strong gender bias

El Mustapha Bahassia, Susan B. Robbinsa, Moying Yina, Gregory P. Boivinb, Raoul Kuiperc, Harry van Steegc, and Peter J. Stambrooka,1

aDepartments of Molecular Genetics and Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, OH 45267; bLaboratory Animal Resources, Wright State University, Dayton, OH 45435; and cLaboratory for Health Protection Research, National Institute of Public Health and the Environment, PO Box 1, 3720BA Bilthoven, The Netherlands

Communicated by Elwood V. Jensen, University of Cincinnati College of Medicine, Cincinnati, OH, August 14, 2009 (received for review March 1, 2009) The CHEK2 kinase (Chk2 in mouse) is a member of a DNA damage sponds to a 37% cumulative risk of breast cancer at 70 years of response pathway that regulates cell cycle arrest at cell cycle age in heterozygotes, which compares with similar estimates of checkpoints and facilitates the repair of dsDNA breaks by a recom- 57% for BRCA1 heterozygotes and 49% for BRCA2 heterozy- bination-mediated mechanism. There are numerous variants of the gotes (13). CHEK2 gene, at least one of which, CHEK2*1100delC (SNP), asso- The CHEK2 protein resides predominantly in the nucleus. In ciates with breast cancer. A mouse model in which the wild-type response to DNA double-strand breaks, it participates in cell Chk2 has been replaced by a Chk2*1100delC allele was tested for cycle arrest and in initiating DNA repair (14, 15). Among its elevated risk of spontaneous cancer and increased sensitivity to many targets, CHEK2 phosphorylates serine 20 on p53 (16–18) challenge by a carcinogenic compound. Mice homozygous for and serine 123 on Cdc25A (19, 20). Phosphorylation of Cdc25A Chk2*1100delC produced more tumors than wild-type mice, by CHEK2 contributes to its ubiquitinylation and consequent whereas heterozygous mice were not statistically different. When proteasome-mediated degradation (21). Cdc25A is a bifunc- fractionated by gender, however, homozygous and heterozygous tional phosphatase whose targets include threonine 14 and mice developed spontaneous tumors more rapidly and to a far tyrosine 15 on Cdk2, dephosphorylation of which is necessary for

greater extent than wild-type mice, indicative of a marked gender cells to transit from the G1 to S phase (21). In the absence of GENETICS bias in mice harboring the variant allele. Consistent with our CHEK2 kinase activity, Cdc25A should be stabilized, and the G1 previous data showing elevated genomic instability in mouse checkpoint should be impaired, leading to a degree of genomic embryonic fibroblasts (MEFs) derived from mice homozygous for instability. Chk2*1100delC, the level of Cdc25A was elevated in heterozygous The single cytosine deletion at nucleotide position 1100 of and homozygous MEFs and tumors. When challenged with the CHEK2 produces a frameshift mutation. The CHEK2*1100delC- carcinogen 7,12-dimethylbenz[a]anthracene, all mice, regardless of encoded protein is truncated with a nonsense amino-terminal genotype, had a reduced lifespan. Latency for mammary tumori- tail and lacks kinase activity (3). Because CHEK2*1100delC genesis was reduced significantly in mice homozygous for mRNA is degraded by nonsense-mediated mRNA decay (NMD) Chk2*1100delC but unexpectedly increased for the development (22), the truncated protein is not detectable by Western blots of lymphomas. An implication from this study is that individuals (23). However, in the Chk2*1100delC mouse model, NMD who harbor the variant CHEK2*1100delC allele not only are at an appears variable between tissues (22). Thus, impaired CHEK2 elevated risk for the development of cancer but also that this risk function may lead to genomic instability and cancer by compro- can be further increased as a result of environmental exposure. mising the regulation of Cdc25A stability. Women who are heterozygous for CHEK2*1100delC develop cancer predisposition ͉ Cdc25A ͉ polymorphism breast tumors that are positive for estrogen receptor (ER) and progesterone receptor (PR) with very high frequencies (91% and amilial early-onset breast cancer is attributable in large part 81%, respectively) compared with noncarriers whose breast Fto variant germ-line alleles of BRCA1 and BRCA2 (1). A tumors are 69% ER-positive and 53% PR-positive (24). Despite substantial fraction of breast cancers with a familial component tumors being ER-positive, patients with the CHEK2*1100delC that affect young women with wild-type BRCA1 and BRCA2 may variant have a less favorable prognosis for disease-free survival be explained by genes with low- or moderate-penetrance alleles and the occurrence of a contralateral tumor (24). Males who are (2), prompting a continued search for allelic variants that are constitutionally heterozygous for CHEK2*1100delC are not only associated with an elevated risk for breast cancer (3, 4). A at a 10-fold higher risk for developing breast cancer than their specific variant of CHEK2 (cell cycle checkpoint kinase 2), noncarrier counterparts (12) but are also at an elevated risk of developing familial prostate cancer (25, 26). designated CHEK2*1100delC, in whichaCisdeleted at nucle- We have described previously the generation of a knockin otide position 1100 (i.e., 1100delC), was identified first in a mouse in which the wild-type CHEK2(Chk2 in mouse) allele has Li-Fraumeni family with wild-type p53 (5). Although an asso- been replaced with the Chk2*1100delC variant (22). Mouse ciation of CHEK2*1100delC with Li-Fraumeni syndrome was embryonic fibroblasts (MEFs) derived from mice homozygous suggested initially (5), such an association has been questioned for Chk2*1100delC have an altered cell cycle profile and show subsequently (6). Heterozygosity at CHEK2*1100delC, however, signs of genomic instability, as indicated by constitutive activa- does contribute to breast cancer susceptibility in European and tion of ataxia telangiectasia mutated (ATM)-mediated signaling North American populations (7–13) and produces a 3-fold pathways, suggestive of persistent DNA damage. Consistent with increase in the risk of breast cancer in women within the general constitutively active ATM-mediated pathways, unperturbed cells population (7). A meta-analysis of 26,000 cases and 27,000 controls further confirmed that the CHEK2*1100delC allele confers a 3- to 5-fold increase in risk for breast cancer (13). With Author contributions: E.M.B., R.K., H.v.S., and P.J.S. designed research; E.M.B., S.B.R., M.Y., fixed-effect models for CHEK2*1100delC heterozygotes versus G.P.B., and H.v.S. performed research; E.M.B., G.P.B., R.K., H.v.S., and P.J.S. analyzed data; noncarriers, the aggregated odds ratios were 2.7 for unselected and E.M.B. and P.J.S. wrote the paper. breast cancer, 2.6 for early onset breast cancer, and 4.8 for The authors declare no conflict of interest. familial breast cancer. In the last case, this odds ratio corre- 1To whom correspondence should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909237106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 Fig. 1. Development of tumors in mice with wild-type and variant Chk2.(A) Wild-type mice and mice heterozygous and homozygous for Chk2*1100delC were maintained for 24 months and monitored for palpable tumors every other day. Mice were killed when tumors reached 0.5 cm in diameter or when mice appeared to be in distress. In all cases, complete pathology was performed, and tumors and tumor types were noted. (B) Spontaneous tumorigenesis in only female mice of each genotype as a function of age. (C) Spontaneous tumorigenesis as a function of age in male and female mice homozygous for Chk2*1100delC. (D) Comparison by gender of mice of each genotype that had developed spontaneous tumors by 24 months of age.

manifest an increase in frequency of DNA double-strand breaks Results compared with control MEFs with wild-type Chk2. Spontaneous Tumors Are More Prevalent in Mice Harboring the Mice in which Chk2 has been knocked out are reported not to Chk2*1100delC Allele Than in Mice with Wild-Type Chk2. MEFs develop spontaneous tumors (27, 28). Topical application of 7,12- derived from mice that are homozygous for Chk2*1100delC dimethylbenz[a]anthracene (DMBA), however, accelerates the for- display characteristics of genomic instability (22). The cells mation of skin tumors (29). In contrast, mice transgenic for a spontaneously accumulate in the G2/M and S phases, indicating kinase-dead, dominant-negative Chk2 under control of a mouse that one or more DNA damage checkpoints are constitutively mammary tumor virus (MMTV) promoter developed mammary active. Consistent with this observation, these MEFs display tumors but only after a latency period of Ϸ20 months (30). nuclear ␥-H2AX foci, suggesting the presence of persistent We now show that Chk2*1100delC mice develop spontaneous dsDNA breaks. The ␥-H2AX foci in these cells are as numerous mammary tumors and other tumor types with a shorter latency and as intensely stained as those in nuclei of irradiated wild-type and a higher frequency than wild-type mice. Furthermore, there cells. These data suggest that cells derived from mice homozy- is a gender bias in cancer predisposition, because the overwhelm- gous for Chk2*1100delC are inherently genetically unstable and ing majority of mice with cancer are female. We also show that perhaps predispose the mice to cancer. the time of tumor onset is much reduced after oral administra- To test whether Chk2*1100delC mice develop spontaneous tion of DMBA. Because CHEK2*1100delC is an allele that mammary tumors or other tumor types, mice heterozygous and enhances susceptibility to breast cancer and probably other homozygous for Chk2*1100delC and their wild-type counter- tumor types in some human populations, this mouse model will parts were maintained for up to 2 years and checked every other help resolve whether individuals with such susceptibility alleles day for tumors. Around the 11th month (46 weeks) of age, both in the DNA damage response pathway are more susceptible to homozygous mutant and heterozygous mice started developing cancer and other diseases than the general population as a tumors (Fig. 1A) that affected several different tissues (Table 1). consequence of environmental exposure. When analyzed together, fewer wild-type mice developed spon-

Table 1. Distribution of tumor types in mice of all three Chk2 genotypes either untreated or treated with DMBA Other Other Genotype Gender N Treatment Mammary Hematopoietic Lung Granulosa epithelial mesenchymal Neuroendrocrine

ϩ/ϩ Both 42 None 1 (14.3) 2 (28.6) 1 (14.3) 0 1 (14.3) 2 (28.6) 0 ϩ/SNP Both 51 None 2 (7.4) 6 (22.2) 6 (22.2) 1 (3.7) 6 (22.2) 6 (22.2) 0 SNP/SNP Both 48 None 2 (7.7) 8 (30.8) 6 (23.1) 0 2 (7.7) 8 (30.8) 0 SNP/SNP Female 22 DMBA 12 (30.0) 7 (17.5) 8 (20.0) 3 (17.5) 3 (17.5) 6 (15.0) 1 (2.5)

The numbers in parentheses indicate the percentage of tumors of a given tumor type based on the total number of tumors in that genotype. N represents the total number of animals in each group.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909237106 Bahassi et al. Downloaded by guest on September 25, 2021 taneous tumors than mice homozygous or heterozygous for Chk2*1100delC, suggesting that the presence of this allele elevated the risk for cancer. The difference in tumor formation as a function of age between homozygous mutant and wild-type mice is statistically significant (P ϭ 0.025). The difference between heterozygous and wild-type mice is not statistically significant (P ϭ 0.127) but shows a developing trend by 24 months. On the basis of pathology, the primary tumors were of multiple types and widely distributed (Table 1). Many of the tissues that produced spontaneous tumors are hormonally responsive and express ER (ER␣ or ER␤). Prominent among these is lung, which produces a greater proportion of spontaneous tumors in mice with Chk2*1100delC than wild-type mice. Some of the tumors were metastatic and invaded other organs, dem- onstrating their potentially aggressive nature.

Gender Bias in CHEK2*1100delC Predisposition to Cancer. Although differences in tumor burden as a function of age between homozygous and wild-type mice are statistically significant but less so between heterozygotes and wild-type mice, there were marked differences when the mice were fractionated by sex. In females, the differences in tumorigenesis between each of the genotypes were striking (Fig. 1 B and D). Furthermore, a sizable majority of mice that carried the Chk2*1100delC allele and that developed tumors were female (Fig. 1C). These gender biases were particularly apparent in comparisons between homozygous ϭ females and males (P 0.001) but less obvious in comparisons GENETICS between heterozygous females and males (P ϭ 0.11) and absent in wild-type mice (P ϭ 0.67). When compared exclusively at the 24-month end point, a larger number of heterozygous female mice had more tumors than wild-type female mice, and homozygous females had even a greater number (Fig. 1D). In contrast, the male mice homozygous for Chk2*1100delC had significantly fewer tumors than wild-type males. That females carrying the Chk2*1100delC allele are more prone to developing tumors suggests that spontaneous tumorigenesis in these mice is partly hormone dependent, an observation that is consistent with human epidemiological data. Approximately 90% of CHEK2*1100delC-related cancers are ER-positive (24), suggesting that hormones may contribute to CHEK2*1100delC-related human cancers.

Administration of DMBA to Chk2*1100delC Mutant Mice Reduces Latency for Mammary Tumorigenesis but Not Lymphoma Develop- ment Compared with Treated Wild-Type Mice. Although epidemi- ology has shown that the CHEK2*1100delC allele predisposes to breast cancer (13), whether individuals harboring this allele are also more susceptible to cancer after exposure to a carcinogen is not known. The possibility of elevated cancer risk after Fig. 2. Tumor response in female mice homozygous for Chk2*1100delC after exposure was tested in the Chk2*1100delC mouse model by administration of DMBA. (A) Percentage of tumor-free mice as a function of administering DMBA orally, by gavage, to female mice of each time after exposure. (B) Percentage of mice free of mammary tumors as a genotype. DMBA is a known carcinogen that induces predom- function of time after exposure. (C) Percentage of mice free of lymphoma as a function of time after exposure. inantly mammary tumors in rodents and other tumor types (31). To assess whether or not the presence of Chk2*1100delC elevates risk for tumor formation after carcinogenic challenge, female mice for the Chk2*1100delC allele (P ϭ 0.5 and 0.2, respectively), which of all three genotypes were treated with DMBA and monitored for probably accounts, in part, for the absence of a difference in the the development of tumors. Exposure to DMBA resulted in sig- overall survival of the three genotypes after exposure to DMBA nificantly decreased tumor latency and accelerated death in each of the three groups of mice compared with mice that were not exposed (Fig. 2A). The distribution of tumor types is different in female (compare time frames in Figs. 1 and 2A). By 40 weeks of age, wild-type mice and female mice that are heterozygous and ho- 80–90% of the treated mice of all three genotypes were dead (Fig. mozygous for Chk2*1100delC. The distribution is yet again differ- 2A). When fractionated by cause of death (Fig. 2B), mice that were ent for those mice challenged with DMBA (Table 1). Again, the homozygous but not heterozygous for Chk2*1100delC had a sig- tumors were metastatic and invaded other organs. Histopathology nificantly reduced latency for mammary tumor onset (P ϭ 0.02 and of some representative examples is shown in Fig. 3. 0.81, respectively), and increased frequency of mammary tumors compared with treated wild-type mice. Surprisingly, mice homozy- Loss of Chk2 Function in Chk2*1100delC Mice Correlates with an gous for wild-type Chk2 had a significantly higher frequency of Increase in Cdc25A Protein. The mechanism by which the lymphomas (Fig. 2C) than either mice heterozygous or homozygous Chk2*1100delC confers an elevated cancer risk is not known.

Bahassi et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 Fig. 3. Histopathology of tumors from DMBA-treated mice depicting some representative examples.

One of the targets of Chk2 kinase activity is the Cdc25A mutant BRCA1 or BRCA2. Among these are ATM (36), CHEK2 bifunctional phosphatase that plays a critical role in malignant (12, 15), and Cdc25A (33). Some pedigrees in which multiple human cancers and is overexpressed in a majority of solid members present with breast cancer indicate that tumors, including breast tumors (19). Furthermore, MMTV- CHEK2*1100delC is a variant allele that associates with an targeted transgenic expression of Cdc25A results in alveolar elevated frequency of breast cancer in such families. Further- hyperplasia in mammary glands, suggesting that increased ex- more, CHEK2*1100delC confers an increased risk of breast pression of Cdc25A accelerates developmental proliferation of cancer even in women unselected for family history (13). A mammary epithelial cells (32). After DNA damage in normal second CHEK2 variant that appears to be overrepresented in cells, the cell cycle checkpoint machinery is activated, and women with breast cancer is CHEK2 I157T (5), but the risk CHEK1 and CHEK2 kinases target Cdc25A for degradation associated with other CHEK2 variants is unclear. (Fig. 4A), thereby decelerating the cell cycle. In the absence of The nucleotide deletion and consequent frameshift in CHEK2 kinase activity, as in CHEK2*1100delC cells, Cdc25A is CHEK2*1100delC result in the loss of much of the CHEK2 degraded ineffectively, resulting in its accumulation (19). Accu- kinase domain. Consequently, some cellular substrates that are mulated Cdc25A allows continued activation of cyclin/Cdk com- targets for CHEK2 kinase activity may have functions that are plexes and disrupts cell cycle regulation by compromising cell compromised. In some cases, kinase redundancy may compen- cycle checkpoints and leading to genomic instability and subse- sate, as in the phosphorylation of serine 20 of p53. This residue quent cancer. The available data relating to Cdc25A over- is phosphorylated by CHEK2 by polo-like kinase 3 (Plk3) (37). expression in cancer show an unequivocal relationship be- Serine 123 of Cdc25A also is phosphorylated by CHEK2, and this tween Cdc25A levels and specific clinical and pathological phosphorylation contributes to Cdc25A proteasome-mediated features, such as higher-grade tumors and poor disease-free degradation after DNA damage in the form of DNA double- survival (33–35). strand breaks. This same residue also is phosphorylated by To assess the status of Cdc25A in cells of CHEK2*1100delC CHEK1, particularly after UV-induced DNA damage (38). mice, the levels of Cdc25A expression were assessed in MEFs There are multiple sites on Cdc25A that can be phosphorylated from each of the three genotypes by Western blot. As illustrated by different kinases under various conditions and that may be in Fig. 4B, the levels of Cdc25A protein in the heterozygous cells partly responsible for its stability or instability (Fig. 4A). Our are elevated compared with those in the wild-type cells and are data with MEFs from mice with different Chk2 genotypes, even higher in the homozygous mutant cells. A similar qualita- however, suggest that even though Cdc25A may be phosphory- tive observation was made when Cdc25A levels were assessed by lated by other proteins, phosphorylation by Chk2 is in large part immunohistochemistry in mammary tumors and normal mam- responsible for Cdc25A degradation (Fig. 4B). There is an mary tissue (Fig. 4C). accumulation of Cdc25A in cells heterozygous for Chk2 and an even higher level in homozygous deficient cells. Immunostaining Discussion of mammary tumor tissues for Cdc25A showed a similar eleva- Familial breast cancer accounts for 15–35% of all breast malig- tion of Cdc25A protein (Fig. 4C). The MEFs homozygous for nancies (1). Variant alleles of several genes are known now to Chk2 kinase deficiency have a compromised cell cycle and significantly elevate risk for breast cancer, the most extensively exhibit genomic instability (22), which is consistent with elevated studied of which include BRCA1 and BRCA2. The majority of Cdc25A levels and with the observation that Cdc25A is overex- familial breast cancers, however, are not directly attributable to pressed in Ͼ50% of solid tumors (33–35).

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0909237106 Bahassi et al. Downloaded by guest on September 25, 2021 A Unknown Chk2 Chk2 Chk2 Kinase defective CHEK2 Chk1 kinase Chk1 Chk1 Chk1 Chk1 P P P P P P P

S76 S82 S88 S124 S178 S279 S293 DNA damage Cdc25A Regulatory domain Catalytic domain 1336524

B ATM SNP/SNP +/SNP +/+ CHEK2*1100delC P-Cdc25A ER Cdc25A

α-tubulin C-Myc Cdc25A C Normal WT

CyclinE/Cdk2

S phase entry and genomic instability

WT/SNP SNP/SNP Fig. 5. Model showing the regulation of Cdc25A levels by ER and CHEK2 in response to DNA damage. A defect in CHEK2 kinase activity leads to elevated levels of Cdc25A. Induction of c-Myc by ER should further elevate levels of Cdc25A and lead to further dysregulation of cell cycle control and ultimately to genomic instability and cancer. GENETICS

threshold at which Cdc25A promotes genomic instability and tumorigenesis. Elevated Cdc25A, which is common to almost Fig. 4. Cdc25A is elevated in cells and tumors from Chk2*1100delC mice. (A) half of all solid tumors, promotes dephosphorylation of Cyclin Schematic of residues that have putative regulatory function for Cdc25A protein levels by phosphorylation and degradation in response to DNA dam- E/CDK 2 and 4 complexes, which facilitates deregulated passage age. (B) Detection of Cdc25A by Western blot in MEFs derived from mice that of cells from the G1 to S phase and consequent genomic are wild type, heterozygous, and homozygous for Chk2*1100delC. ␣-Tubulin instability (31, 41). Consistent with this model, the level of was used as a loading control. (C) Immunohistochemical staining for Cdc25A Cdc25A protein in the CHEK2*1100delC cells and tissues was in normal mammary tissue from mice with wild-type Chk2 and mammary considerably higher compared with that in wild-type cells (Fig. tumors from each of the three genotypes. 4 B and C). The model is depicted schematically in Fig. 5. An alternative scheme might involve physical interaction between ER and one of the proteins that Chk2 regulates directly or To better understand the role of CHEK2*1100delC in cancer indirectly. The androgen receptor (AR) physically binds and predisposition and malignant transformation, we have main- immunoprecipitates with BRCA2. The BRCA2 protein is phos- tained mice that are homozygous and heterozygous for phorylated by CHEK2 kinase to regulate its association with Chk2*1100delC and their wild-type counterparts for up to 24 Rad51 (42). A direct interaction between ER and BRCA2 has months and assessed the effect of this variant allele on cancer not been reported, but given the association between AR and initiation and development. Consistent with cancer predisposi- BRCA2, such an interaction is not unlikely. If truncation of tion in humans, mice harboring this Chk2 variant have a statis- CHEK2 or loss of its kinase activity affects its putative interac- tically significant increase in the rate of tumor formation and in tion with ER and also its ability to phosphorylate BRCA2, then Ϸ the number of tumors after 1 year of age, confirming that this genomic stability may be disrupted. allele can elevate cancer risk. The distribution of tumor types appeared to vary between Of particular note is that almost all of the mice that developed female mice with different genotypes (Table 1). Most of the tumors were female, suggesting hormonal involvement in some tissues affected, however, are hormonally responsive. Lung Ϸ Chk2-associated tumors. In humans, 91% of CHEK2*1100delC- appeared to be most affected and mammary tumors less so. That dependent cancers are ER-positive compared with 69% in the lung tumors were among the most common tumor types in mice general population. The potential involvement of ER in with the Chk2*1100delC allele is consistent with the observation CHEK2*1100delC-dependent cancers suggests a model to pro- that local estrogen levels and ER status may be important in vide a basis for understanding how CHEK2*1100delC and ER controlling the behavior of some lung tumors and that the ER might cooperate to elevate cancer risk. The bifunctional phos- may be useful as a therapeutic target (43–45) or as a predictor phatase Cdc25A is a target of CHEK2 kinase (19), which of disease outcome (46). After treatment with DMBA, mam- phosphorylates Cdc25A on serine 15 and contributes to its mary tumors and lung tumors were the most increased tumor degradation after the introduction of DNA damage as dsDNA types. Mammary tumors showed a clear increase in numbers and breaks (19). Because CHEK2*1100delC lacks kinase activity, it rate of onset after challenge. In contrast, the number of lym- cannot phosphorylate Cdc25A in response to DNA damage, phomas that developed after challenge appeared to be sup- thereby impairing its proteasome-mediated degradation and pressed. Although suppression of lymphomas may be a real increasing its steady-state level. Similarly, 2-estradiol/ER is phenomenon, the mice more likely die of other tumor types or known to regulate the expression and stabilization of c-Myc (39), for different reasons before the lymphomas manifest. which in turn induces transcription of Cdc25A (40). Thus, these The data indicate that mouse Chk2*1100delC, like its human two pathways may act in concert to increase Cdc25A levels to a counterpart, is a low-penetrance allele that recapitulates the

Bahassi et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 25, 2021 human cancer phenotype. The data also suggest that this allele cessed for histopathology, and preserved in 10% neutral buffered formalin may confer an added cancer risk after environmental exposure. (catalog no. 9400–5; Richard-Allan Scientific). The tissues were embedded in The very strong gender bias observed with the Chk2*1100delC paraffin wax, sectioned at 5 ␮m, and stained with hematoxylin and eosin for mouse model predicts an association with the estrogen/ER axis histopathologic evaluation. and, if correct, affords a rationale for therapeutic targets. Tissue Collection and Analyses. Mice were killed, and tissues were dissected, Materials and Methods mounted on glass slides, and fixed and stained in carmine alum solution as described for whole-mount analysis (47). For immunohistochemical analysis, Derivation of MEFs and Western Blot. The Chk2*1100delC mice and MEFs were tissues were fixed as above, processed, and embedded in paraffin. Cdc25A generated as described (22). Western blots for Cdc25A were performed by using staining was done with the same antibody as above. a commercially available polyclonal antibody (sc-97; Santa Cruz Biotechnology). Statistical Analysis. Comparisons of survival, time of tumor onset, and fre- DMBA Treatment. The Chk2*1100delC mice were backcrossed with FVB mice quencies of tumors between cohorts of mice for the three genotypes were for at least five generations. Four-week-old Chk2*1100delC female mice, performed by using the Cox–Mantel log-rank test to evaluate levels of signif- either homozygous or heterozygous for the allele, together with their wild- icance at a 95% confidence interval. Differences were determined to be ␮ type control littermates, were treated by gavage with 100 g of DMBA (Sigma) statistically significant when P Ͻ 0.05, unless otherwise noted. dissolved in 0.1 mL of sunflower oil three times per week for 6 subsequent weeks. Control female animals only received the solvent during the treatment ACKNOWLEDGMENTS. We thank Dr. Linda Levin and Yingying Xu for help period. All mice were weighed weekly and checked for the development of with statistical analyses and Rita Angel for help with mice. This work was tumors until the age of 43 weeks (39 weeks after start of the treatment). supported in part by National Institutes of Health Grants R03 ES015307 (to Moribund animals or those with visible tumors were killed as were surviving E.M.B.) and R01 ES012695, R01 ES016625, and UO1 ES011038 (to P.J.S.) and mice at the end of the experiment. Tumors and tissues were collected, pro- Center for Environmental Genetics Grant P30-ES006096.

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