Somatic Apc mutations are selected upon their capacity to inactivate the beta-catenin downregulating activity

The APC gene, originally identified as the gene for familial adenomatous polyposis (FAP), is now considered as the true "gatekeeper" of colonic epithelial proliferation. Its main tumor suppressing activity seems to reside in the capacity to properly regulate intracellular beta-catenin signaling. Most somatic APC mutations are detected between codons 1286 and 1513, the mutation cluster region (MCR). This clustering can be explained either by the presence of mutation-prone sequences within the MCR, or by the selective advantage provided by the resulting truncated polypeptides. Here, a Msh2-deficient mouse model (Msh2(delta 7N) ) was generated and bred with Apc(1638N) and Apc(Min) that allowed the comparison of the somatic mutation spectra along the Apc gene in the different allelic combinations. Mutations identified in Msh2(delta 7N/delta 7N) tumors are predominantly dinucleotide deletions at simple sequence repeats leading to truncated Apc polypeptides that partially retain the 20 a.a. beta-catenin downregulating motifs. In contrast, the somatic mutations identified in the wild type Apc allele of Msh2(delta 7N/delta 7N) /Apc(+/1638N) and Msh2(delta 7N/delta 7N) /Apc(+/Min) tumors are clustered more to the 5' end, thereby completely inactivating the beta-catenin downregulating activity of APC. These results indicate that somatic Apc mutations are selected during intestinal tumorigenesis and that inactivation of the beta-catenin downregulating function of APC is likely to represent the main selective factor.     

The C-terminus of Apc does not influence intestinal adenoma development or progression

Adenomatous polyposis coli (APC ) mutations are found in most colorectal tumours. These mutations are almost always protein-truncating, deleting both central domains that regulate Wnt signalling and C-terminal domains that interact with the cytoskeleton. The importance of Wnt dysregulation for colorectal tumourigenesis is well characterized. It is, however, unclear whether loss of C-terminal functions contributes to tumourigenesis, although this protein region has been implicated in cellular processes--including polarity, migration, mitosis, and chromosomal instability (CIN)—that have been postulated as critical for the development and progression of intestinal tumours. Since almost all APC mutations in human patients disrupt both central and C-terminal regions, we created a mouse model to test the role of the C-terminus of APC in intestinal tumourigenesis. This mouse (Apc(ΔSAMP)) carries an internal deletion within Apc that dysregulates Wnt by removing the beta-catenin-binding and SAMP repeats, but leaves the C-terminus intact. We compared Apc(ΔSAMP) mice with Apc(1322T) animals. The latter allele represented the most commonly found human APC mutation and was identical to Apc(ΔSAMP) except for absence of the entire C-terminus. Apc(ΔSAMP) mice developed numerous intestinal adenomas indistinguishable in number, location, and dysplasia from those seen in Apc(1322T) mice. No carcinomas were found in Apc(ΔSAMP) or Apc(1322T) animals. While similar disruption of the Wnt signalling pathway was observed in tumours from both mice, no evidence of differential C-terminus functions (such as cell migration, CIN, or localization of APC and EB1) was seen. We conclude that the C-terminus of APC does not influence intestinal adenoma development or progression.     

The C-terminal domain of the adenomatous polyposis coli (Apc) protein is involved in thyroid morphogenesis and function

Adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we have investigated Apc ( 1638T/1638T ) mice, which express a truncated Apc that lacks the C-terminal domain. Apc ( 1638T/1638T ) mice are tumor free and exhibit growth retardation. In the present study, we analyzed the morphology and functions of the thyroid gland in Apc ( 1638T/1638T ) mice. There was no significant difference in the basal concentration of serum thyroid hormones between Apc ( 1638T/1638T ) and Apc (+/+) mice. Thyroid follicle size was significantly larger in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. The extent of serum T4 elevation following exogenous thyroid-stimulating hormone (TSH) injection was lower in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. TSH also induced a greater reduction in thyroid follicle size in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. Analyses using immunohistochemistry and electron microscopy indicated that follicular epithelial cells in Apc ( 1638T/1638T ) mice had an enlarged rough endoplasmic reticulum of irregular shape. These results suggest that the C-terminal domain of Apc is involved in thyroid morphology and function.     

Adenomatous polyposis coli (<Emphasis Type="Italic">Apc</Emphasis>) tumor suppressor gene as a multifunctional gene

The adenomatous polyposis coli (Apc) gene is mutated in familial adenomatous polyposis and in sporadic colorectal tumors. The Apc gene product (APC), basically a cytoplasmic protein, blocks cell cycle progression and plays crucial roles in development. The APC binds to beta-catenin, axin and glycogen synthase kinase 3beta to form a large protein complex, in which beta-catenin is phosphorylated and broken down, resulting in negative regulation of the Wnt signaling pathway. Most of the mutated Apc genes in colorectal tumors lack beta-catenin-binding regions and fail to inhibit Wnt signaling, leading to overproliferation of tumor cells. The APC, having some nuclear localizing signals in its molecule, can also be localized in the nucleus. The nuclear APC exports excess beta-catenin to the cytoplasm. Through its C-terminus, APC binds to post-synaptic density discs large zonula occludens domain-containing proteins, such as discs large (DLG) and post-synaptic density (PSD)-95, and may play important roles in epithelial morphogenesis, brain development and neuronal functions. In addition, APC is involved in cell motility through its association with microtubules and APC-stimulated guanine nucleotide exchange factor. Colocalization of APC and DLG is dependent on microtubules. The Apc gene is highly expressed in the embryonic and postnatal developing brain. Recently, we found that APC is required for the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by facilitating the clustering of PSD-95 and these receptors at the postsynapse. In addition, APC is present in astrocytes, although its role in astrocytes is, as yet, unknown.     

Aneuploidy arises at early stages of Apc-driven intestinal tumorigenesis and pinpoints conserved chromosomal loci of allelic imbalance between mouse and human

Although chromosomal instability characterizes the majority of human colorectal cancers, the contribution of genes such as adenomatous polyposis coli (APC), KRAS, and p53 to this form of genetic instability is still under debate. Here, we have assessed chromosomal imbalances in tumors from mouse models of intestinal cancer, namely Apc(+/1638N), Apc(+/1638N)/KRAS(V12G), and Apc(+/1638N)/Tp53-/-, by array comparative genomic hybridization. All intestinal adenomas from Apc(+/1638N) mice displayed chromosomal alterations, thus confirming the presence of a chromosomal instability defect at early stages of the adenoma-carcinoma sequence. Moreover, loss of the Tp53 tumor suppressor gene, but not KRAS oncogenic activation, results in an increase of gains and losses of whole chromosomes in the Apc-mutant genetic background. Comparative analysis of the overall genomic alterations found in mouse intestinal tumors allowed us to identify a subset of loci syntenic with human chromosomal regions (eg, 1p34-p36, 12q24, 9q34, and 22q) frequently gained or lost in familial adenomas and sporadic colorectal cancers. The latter indicate that, during intestinal tumor development, the genetic mechanisms and the underlying functional defects are conserved across species. Hence, our array comparative genomic hybridization analysis of Apc-mutant intestinal tumors allows the definition of minimal aneuploidy regions conserved between mouse and human and likely to encompass rate-limiting genes for intestinal tumor initiation and progression.     

The genetic background modifies the spontaneous and X-ray-induced tumor spectrum in the Apc1638N mouse model.

The effect of the genetic background on the tumor spectrum of Apc1638N, a mouse model for attenuated familial adenomatous polyposis (FAP), has been investigated in X-irradiated and untreated F1 hybrids between C57BL/6JIco-Apc1638N (B6) and A/JCrIBR (A/J), BALB/cByJIco (C) or C3H/HeOuJIco (C3). Similar to the ApcMin model, the Apc1638N intestinal tumor multiplicity seems to be modulated by Mom1. Moreover, several additional (X-ray-responsive) modifier loci appear also to affect the Apc1638N intestinal tumor number. The genetic background did not significantly influence the number of spontaneous desmoids and cutaneous cysts in Apc1638N. In general, X-irradiation increased the desmoid multiplicity in Apc1638N females but had no effect in males. The opposite was noted for the cyst multiplicity after X-rays. Surprisingly, X-irradiated CB6F1-Apc1638N females were highly susceptible to the development of ovarian tumors, which displayed clear loss of the wild-type Apc allele.     

The 'just-right' signaling model: APC somatic mutations are selected based on a specific level of activation of the beta-catenin signaling cascade

According to the classical interpretation of Knudson's 'two-hit' hypothesis for tumorigenesis, the two 'hits' are independent mutation events, the end result of which is loss of a tumor suppressing function. Recently, it has been shown that the APC (adenomatous polyposis coli) gene does not entirely follow this model. Both the position and type of the second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germline mutation. This non-random distribution of somatic hits has been interpreted as the result of selection for more advantageous mutations during tumor formation. However, the APC gene encodes for a multifunctional protein, and the exact cellular function upon which this selection is based is yet unknown. In this study, we have analyzed somatic APC point mutations and loss of heterozygosity (LOH) in 133 colorectal adenomas from six FAP patients. We observed that when germline mutations result in truncated proteins without any of the seven beta-catenin downregulating 20-amino-acid repeats distributed in the central domain of APC, the majority of the corresponding somatic point mutations retain one or, less frequently, two of the same 20-amino-acid repeats. Conversely, when the germline mutation results in a truncated protein retaining one 20-amino-acid repeat, most second hits remove all 20-amino-acid repeats. The latter is frequently accomplished by allelic loss. Notably, and in contrast to previous observations, in a patient where the germline APC mutation retains two such repeats, the majority of the somatic hits are point mutations (and not LOH) located upstream and removing all of the 20-amino-acid repeats. These results indicate selection for APC genotypes that are likely to retain some activity in downregulating beta-catenin signaling. We propose that this selection process is aimed at a specific degree of beta-catenin signaling optimal for tumor formation, rather than at its constitutive activation by deletion of all of the beta-catenin downregulating motifs in APC.     

Some fine-structural findings on the thyroid gland in Apc 1638T/1638T mice that express a C-terminus lacking truncated Apc

Adenomatous polyposis coli (Apc) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we examined Apc ^1638T/1638T mice that express a truncated Apc lacking the C-terminal domain. The Apc ^1638T/1638T mice were tumor free and exhibited growth retardation. We recently reported abnormalities in thyroid morphology and functions of Apc ^1638T/1638T mice, although the mechanisms underlying these abnormalities are not known. In the present study, we further compared thyroid gland morphology in Apc ^1638T/1638T and Apc ^+/+ mice. The diameters of thyroid follicles in the left and right lobes of the same thyroid gland of Apc ^1638T/1638T mice were significantly different whereas the Apc ^+/+ mice showed no significant differences in thyroid follicle diameter between these lobes. To assess the secretory activities of thyroid follicular cells, we performed double-immunostaining of thyroglobulin, a major secretory protein of these cells, and the rough endoplasmic reticulum (rER) marker calreticulin. In the Apc ^1638T/1638T follicular epithelial cells, thyroglobulin was mostly colocalized with calreticulin whereas in the Apc ^+/+ follicular epithelial cells, a significant amount of the cytoplasmic thyroglobulin did not colocalize with calreticulin. In addition, in thyroid-stimulating hormone (TSH)-treated Apc ^1638T/1638T mice, electron microscopic analysis indicated less frequent pseudopod formation at the apical surface of the thyroid follicular cells than in Apc ^+/+ mice, indicating that reuptake of colloid droplets containing iodized thyroglobulin is less active. These results imply defects in intracellular thyroglobulin transport and in pseudopod formation in the follicular epithelial cells of Apc ^1638T/1638T mice and suggest suppressed secretory activities of these cells.     

Inactivation of p21WAF1/cip1 enhances intestinal tumor formation in Muc2-/- mice

In the Apc1638(+/-) mouse model of intestinal tumorigenesis, targeted inactivation of the cyclin-dependent kinase inhibitor p21(WAF1/cip1) is highly effective in enhancing Apc-initiated tumor formation in the intestine. Because p21(WAF1/cip1) plays a critical role in regulating intestinal cell proliferation, maturation, and tumorigenesis, we examined whether its inactivation would enhance tumor formation in a different mouse model of colon cancer. Therefore, we mated p21(-/-) mice with mice carrying a genetic deficiency of the Muc2 gene, which encodes the major gastrointestinal mucin. Muc2(-/-) mice develop tumors in the small and large intestine and the rectum, but in contrast to tumors in Apc1638(+/-) mice, this does not involve increased expression or nuclear localization of beta-catenin. We found that inactivation of p21(WAF1/cip1) significantly increased the frequency and size of intestinal tumors in Muc2 knockout mice and also led to development of more invasive adenocarcinomas. This enhanced tumorigenesis significantly decreased mouse life span. Further, inactivation of p21(WAF1/cip1) increased cell proliferation, decreased apoptosis, and decreased intestinal trefoil factor expression in the mucosa of both the small and large intestine. Surprisingly, reduced expression of p27(kip1) was also observed in the Muc2(-/-), p21(+/-), and p21(-/-) mice. In contrast, the expression of c-myc was significantly elevated. Thus, p21 modulates the formation of tumors whose initiation does (Apc) or does not (Muc2) involve altered beta-catenin-Tcf4 signaling, but which may converge on common elements downstream of this signaling pathway.     

Adenomatous polyposis coli (APC) plays multiple roles in the intestinal and colorectal epithelia

The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in most sporadic colorectal tumors. During both embryonic and postnatal periods, APC is widely expressed in a variety of tissues, including the brain and gastrointestinal tract. The APC gene product (APC) is a large multidomain protein consisting of 2843 amino acids. APC downregulates the Wnt signaling pathway through its binding to β-catenin and Axin. Most mutated APC proteins in colorectal tumors lack the β-catenin-binding regions and fail to inhibit Wnt signaling, leading to the overproliferation of tumor cells. Several mouse models (APC ^580D , APC ^Δ716 , APC ¹³⁰⁹ , APC ^Min , APC ^1638T ) have been established to investigate carcinogenesis caused by APC mutations. APC also binds to APC-stimulated guanine nucleotide exchange factor, the kinesin superfamily-associated protein 3, IQGAP1, microtubules, EB1, and discs large (DLG). APC has both nuclear localization signals and nuclear export signals in its molecule, suggesting its occasional nuclear localization and export of β-catenin from the nucleus. APC is highly expressed in the intestinal and colorectal epithelia and may be involved in homeostasis of the enterocyte renewal phenomena, in which proliferation, migration, differentiation, and apoptosis are highly regulated both temporally and spatially. Through the many binding proteins mentioned, APC can exert multiple functions involved in epithelial homeostasis.     

Efficacy of chemopreventive agents for growth inhibition of Apc [+/-] 1638NCOL colonic epithelial cells

Germline mutations of the Apc tumor suppressor gene result in increased risk for gastrointestinal carcinogenesis. The Apc1638N [+/-] mouse exhibits accelerated gastrointestinal carcinogenesis that is modifiable by select pharmacological and dietary agents. Experiments in the present study were conducted on a subculturable epithelial 1638NCOL cell line established from histologically normal colon of Apc1638N [+/-] mouse to examine the effects of selected chemopreventive agents that differ in their mechanism of action. Extent of growth arrest, number of cell population doublings, cell cycle progression and aneuploid G0/G1: S + G2/M ratio represented the quantitative endpoints for the susceptibility and efficacy of chemopreventive agents. Treatment of exponentially growing 1638NCOL cells with maximum cytostatic dose of 9cisRA, DFMO or SUL (100 microM) produced a 60-70% growth arrest, that with TAM and AMF (10 microM) produced a 20-40% growth arrest, while that with OLT (100 microM) produced a 25% growth arrest. This response was associated with corresponding decrease in the number of cell population doubling. 9cisRA, SUL or AMF increased the aneuploid G0/G1: S + G2/M ratio by inducing G1 checkpoint arrest, while DFMO, TAM and OLT decreased the ratio by inducing G2 checkpoint arrest. Thus, cell cycle phase-dependent susceptibility of the Apc [+/-] 1638NCOL cell line to mechanistically distinct chemopreventive agents validates a novel colon epithelial cell culture model for mechanistic, preventive or therapeutic studies on Apc regulated colon carcinogenesis.     

Genetic inactivation of the APC gene contributes to the malignant progression of sporadic hepatocellular carcinoma: a case report

Although activated Wnt/beta-catenin pathway is considered to be one of the main driving forces of hepatocarcinogenesis, no somatic mutations of the adenomatous polyposis coli (APC) gene have been found in sporadic hepatocellular carcinoma (HCC) to date. Here we present a case of a sporadic nodule-in-nodule-type HCC that provides the first evidence that biallelic genetic inactivation of the APC gene contributed to the development of the tumor. High-density array-based comparative genomic hybridization (aCGH) was performed to clarify genome-wide chromosomal structural alteration profiles of both early and advanced components of this nodule-in-nodule HCC. aCGH analysis revealed a chromosomal loss of the APC gene locus only in the inner advanced component of this tumor. Direct sequencing of the remaining allele revealed a nonsense mutation at codon 682 in the Armadillo repeats, resulting in a truncated protein that lacked all of the beta-catenin-binding motifs. Nonsense mutations at this location are rare among other types of cancer. In conclusion, combined with an immunohistochemical analysis of the beta-catenin protein, this case provides the first evidence that genetic inactivation of the APC gene can play a significant role in the progression of sporadic HCC, probably by activating the Wnt/beta-catenin pathway.     

Adenomatous polyposis coli (APC) protein regulates epithelial cell migration and morphogenesis via PDZ domain-based interactions with plasma membranes

The tumor suppressor adenomatous polyposis coli (APC) protein is localized at the plus ends of microtubules (MTs) at the migrating edges of cells. Here, we established Xenopus A6 epithelial cell transfectants expressing GFP-fused full-length APC (GFP-fAPC) or truncated APC lacking the COOH-terminal PDZ-binding motif TSV (GFP-APC(DeltaTSV)). Although both APC proteins were similarly accumulated at the MT ends, GFP-fAPC, but not GFP-APC(DeltaTSV), was associated with the basal and lateral plasma membranes and co-localized with a PDZ protein, DLG1. Stable over-expression of GFP-fAPC enforced cell-substrate attachment and thereby enhanced cell spreading on the substratum and induced polarized extension of lamellipodia and MTs during scratch-induced migration. Truncation of the PDZ-binding motif was sufficient to abolish these effects of GFP-fAPC. Furthermore, expression of GFP-APC(DeltaTSV) disturbed the establishment of a continuous epithelial monolayer. These results suggest that APC links MTs to plasma membranes through interactions with PDZ proteins, such that the migration and morphogenesis of epithelial cells can be properly regulated.     

Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers

Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc(+/-) mice, such as the well-characterized Apc(Min/+) model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc(Delta14/+) mice to that of the classical Apc(Min/+). The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Delta14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated beta-catenin, overexpressed the beta-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a beta-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc(Delta14/+) model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.     

Functional definition of the mutation cluster region of adenomatous polyposis coli in colorectal tumours

The mutation cluster region (MCR) of adenomatous polyposis coli (APC) is located within the central part of the open reading frame, overlapping with the region encoding the 20 amino acid repeats (20R) that are beta-catenin-binding sites. Each mutation in the MCR leads to the synthesis of a truncated APC product expressed in a colorectal tumour. The MCR extends from the 3' border of the first 20R coding region to approximately the middle of the third 20R coding region, reflecting both positive and negative selections of the N- and C-terminal halves of the APC protein in colon cancer cells, respectively. In contrast, the second 20R escapes selection and can be either included or excluded from the truncated APC products found in colon cancer cells. To specify the functional outcome of the selection of the mutations, we investigated the beta-catenin binding capacity of the first three 20R in N-terminal APC fragments. We found in co-immunoprecipitation and intracellular co-localization experiments that the second 20R is lacking any beta-catenin binding activity. Similarly, we also show that the tumour-associated truncations abolish the interaction of beta-catenin with the third 20R. Thus, our data provide a functional definition of the MCR: the APC fragments typical of colon cancer are selected for the presence of a single functional 20R, the first one, and are therefore equivalent relative to beta-catenin binding.     

Crystal structure of a beta-catenin/axin complex suggests a mechanism for the beta-catenin destruction complex

The "beta-catenin destruction complex" is central to canonical Wnt/beta-catenin signaling. The scaffolding protein Axin and the tumor suppressor adenomatous polyposis coli protein (APC) are critical components of this complex, required for rapid beta-catenin turnover. We determined the crystal structure of a complex between beta-catenin and the beta-catenin-binding domain of Axin (Axin-CBD). The Axin-CBD forms a helix that occupies the groove formed by the third and fourth armadillo repeats of beta-catenin and thus precludes the simultaneous binding of other beta-catenin partners in this region. Our biochemical studies demonstrate that, when phosphorylated, the 20-amino acid repeat region of APC competes with Axin for binding to beta-catenin. We propose that a key function of APC in the beta-catenin destruction complex is to remove phosphorylated beta-catenin product from the active site.     

Spontaneous aberrant crypt foci in Apc1638N mice with a mutant Apc allele

The Apc1638N/+ mouse has a chain-terminating mutation in one allele of the adenomatous polyposis coli (Apc) gene that is similar to most mutations observed in the human familial adenomatous polyposis syndrome. Aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon, are morphologically abnormal structures that are identifiedmicroscopically in the grossly normal colonic mucosas of rodents treated with colon carcinogens and of human patients. The colons and cecums of 62 Apc1638N/+ mice were evaluated for the spontaneous occurrence of ACF and tumors. Both male and female mice were killed at different times between 5 and 28 weeks of age. Wild-type littermates, ie, Apc(+/+) mice, at 22 to 26 weeks of age served as controls. ACF were identified in 97% of the Apc1638N/+ mice starting at 5 weeks of age and not in any wild-type littermates. Although the number of ACF increased with age (P < 0.0001), the average number of crypts per focus of the ACF did not increase significantly. In addition, wild-type Apc protein was detected by immunohistochemistry in all 22 ACF evaluated. Together these data suggest that heterozygous loss of Apc may be sufficient to initiate ACF in these mice and that these mice may be suitable models to study the interaction of environmental factors with an inherited mutation of the Apc gene that is associated with colon cancer.     

Mammary tumor induction and premature ovarian failure in ApcMin mice are not enhanced by Brca2 deficiency

Inherited BRCA2 mutations predispose individuals to breast cancer and increase risk at other sites. Recent studies have suggested a role for the APC I1307K allele as a low-penetrance breast cancer susceptibility gene that enhances the phenotypic effects of BRCA1 and BRCA2 mutations. To model the consequences of inheriting mutant alleles of the BRCA2 and APC tumor suppressor genes, we examined tumor outcome in C57BL/6 mice with mutations in the Brca2 and Apc genes. We hypothesized that if the Brca2 and Apc genes were interacting to influence mammary tumor susceptibility, then mammary tumor incidence and/or multiplicity would be altered in mice that had inherited mutations in both genes. Female and male offspring treated with a single IP injection of 50 mg/kg N-ethyl-N-nitrosourea (ENU) at 35 days of age developed mammary adenoacanthomas by 100 days of age. The female Apc-mutant and Brca2/Apc double-mutant progeny had mean mammary tumor multiplicities of 6.7+/-2.8 and 7.2+/-2.7, respectively, compared to wild-type and Brca2-mutant females, which had mean mammary tumor multiplicities of 0.1+/-0.4 and 0.3+/-0.5, respectively. Female ENU-treated Apc-mutant and Brca2/Apc double heterozygotes were also susceptible to premature ovarian failure. Thus, the inheritance of an Apc mutation predisposes ENU-treated female and male mice to mammary tumors and, in the case of female mice, to ovarian failure. These results indicate that mammary tumor development in Apc-mutant mice can progress independently of ovarian hormones. The Apc mutation-driven phenotypes were not modified by mutation of Brca2, perhaps because Brca2 acts in a hormonally dependent pathway of mammary carcinogenesis.