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Carcinogenesis Advance Access originally published online on January 7, 2006
Carcinogenesis 2006 27(4):848-855; doi:10.1093/carcin/bgi302
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Atm-haploinsufficiency enhances susceptibility to carcinogen-induced mammary tumors

Shu Lu 1, {dagger}, Kate Shen 1, {dagger}, Yaolin Wang 4, Steven J. Santner 1, Jie Chen 5, S.C. Brooks 1, 2 and Y.Alan Wang 1, 3, *

1 Karmanos Cancer Institute, 2 Department of Biochemistry and Molecular Biology, 3 Department of Pathology, Wayne State University, School of Medicine, 110 E. Warren Avenue, Detroit, MI 48201, USA, 4 Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA and 5 Department of Mathematics, University of Massachusetts, Boston, MA 02215, USA

* To whom correspondence should be addressed. Tel: +1 313 833 0715; Fax: +1 313 831 7518; E-mail: wangya{at}kci.wayne.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ataxia-telangiectasia (A-T), which is due to mutations in the ATM gene, is a rare autosomal recessive genomic instability syndrome characterized by radiosensitivity and predisposition to cancer. Epidemiological studies have suggested that relatives of A-T patients (A-T carriers) have increased risks of developing breast cancer. We propose that increased breast cancer risks in A-T carriers may be due to exposure to various environmental carcinogens and/or dietary consumption. To test this hypothesis, we treated a congenic strain of Atm+/– mice with DMBA (7,12-dimethylbenz({alpha})anthracene), a mammary carcinogen, and observed mammary tumor incidence. It was found that Atm+/– mice have a 2-fold increase, as well as early onset, in mammary tumor incidence relative to wild-type mice (P < 0.005). The increased mammary tumor development is correlated with a 3-fold increase in the development of mammary dysplasia in Atm+/– compared with wild-type mice (P < 0.05). We also found that Ras signaling pathway was not activated in DMBA-induced mammary tumors irrespective of the Atm status. At the cellular level, Atm-haploinsufficiency confers increased cellular stress manifested by an increased p53 expression and a slightly enhanced survival of mammary epithelial cells in response to radiation. Our results demonstrate that Atm heterozygotes are predisposed to mammary tumor development and support the hypothesis that exposure to environmental carcinogens contributes to the increased rate of breast cancer development in A-T carriers. Given that 1% of the general population are ATM heterozygotes (A-T carriers), this study has great implications in breast cancer development in this population.

Abbreviations: A-T, ataxia-telangiectasia; DMBA, 7,12-dimethylbenz({alpha})anthracene; LOH, loss of heterozygosity; MAPK, mitogen-activated protein kinase.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
ATM, which encodes a serine/threonine protein kinase of the PIKK family, is known to ensure genomic integrity by controlling cell cycle checkpoint in response to DNA damage signals. ATM is required for the phosphorylation and activation of downstream genes such as p53, hChk2/hCds1, MDM2 and BRCA1. Functional mutations or amplifications of these genes have frequently been associated with the development of breast cancer (1–3). The importance of ATM in suppressing breast cancer development is supported by recent studies demonstrating that ATM is epigenetically silenced in advanced breast cancer (4) and that ATM mutations contribute to the development of familial breast and ovarian cancer (5).

Consistent with these observations, a number of epidemiological studies have suggested that blood relatives of ataxia-telangiectasia (A-T) patients have increased risks of developing breast cancer (6–12). Indeed, haploinsufficiency of Atm has been shown to result in increased sensitivity to radiation-induced aging phenotypes, such as hair graying and cataracts, in mouse models (13,14). About 80% of the mutations in A-T patients are due to truncating mutations that lead to the loss of ATM protein (15,16). Atm truncating/knockout mouse models have faithfully recapitulated most of the A-T-associated phenotypes (17–20). However, none of the truncated Atm knockout mouse models develop mammary tumors when housed in a controlled environment. On the other hand, ~10% of the mothers of A-T patients (obligate A-T carriers) are more likely to develop breast cancers at an early age (8,10–12). These observations suggest that additional environmental factors may influence the breast cancer development in A-T carriers.

In light of the varied environmental exposures encountered by the A-T carriers, it is suggested that A-T carriers are more sensitive to environmental carcinogens, and hence have higher incidence of breast cancer than the general population. In this study, we tested this hypothesis and analyzed the incidence of carcinogen-induced mammary tumors in an A-T carrier mouse model system.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Mice
Male Atm mice in a mixed genetic background [129SvEv x Black Swiss, (20)] were backcrossed with female FVB/N mice to generate FVB.129S6-Atm F1 mice. The F1 mice were then backcrossed again with female FVB/N mice for a total of 13 generations to obtain FVB.129S6(BLKSW)-Atmtm1Led mice. PCR assay was used to identify Atm heterozygotes with the following primers: M1:5'-tgtagataggtcagcattggat-3'; M10: 5'-gtcaaaattcgatctgctgct-3'; Neo1: 5'-gctctttactgaaggctctttac-3'. PCR amplification was performed in 1x GPCR buffer [16.6 mM (NH4)2SO4, 67 mM Tris–HCl (pH 8.3), 6.7 mM MgCl2, 5 mM ßME, 1% DMSO, 80 µg/ml BSA, 1 mM dNTP, 150 nM PCR primers and 2 U Taq polymerase]. PCR amplification was initially denatured for 2 min at 94°C, followed by 30 cycles of 30 s at 94°C, 45 s at 56°C and 60 s at 72°C. PCR product was resolved onto 2% agarose gel and wild-type and mutant genotypes were identified as 125 and 400 bp bands, respectively, on agarose gel in the presence of ethidium bromide. Mice were fed ad libitum and housed in a barrier facility with 12-h light cycle.

DMBA mammary carcinogenesis
Wild-type (n = 8) and Atm+/– mice (n = 17) were used for 7,12-dimethylbenz({alpha})anthracene (DMBA) treatment in FVB congenic background as described (21). In brief, DMBA (Sigma) was dissolved in olive oil at a concentration of 5 mg/ml and administrated to mice ~6 weeks of age by oral gavage at a dose of 1 mg once a week for five consecutive weeks. Mammary tumor development was followed by weekly palpation of DMBA-treated mice.

Whole mount and histological analysis of mammary gland
Whole mount analysis was performed as described (22). For histological analysis of the whole mount mammary gland, the tissue was removed from the slide and placed in xylene for 30 min. The whole mount was then paraffin-embedded and sectioned with a Zeiss microtome. Sections were rehydrated in a series of descending concentrations of alcohol followed by counterstaining in fast green (Sigma). Histological analysis of mammary tumors was performed as described (23).

Protein expression analysis
Frozen mammary glands or mammary tumors were thawed on ice followed by homogenization in NP 40 buffer (150 mM NaCl, 50 mM Tris–HCl, pH 8.0 and 1% IGEPAL CA630) containing a cocktail of proteinase inhibitors (Roche). Cleared lysate were resolved on SDS–PAGE gel and transferred to PVDF membrane. Protein expression was detected by enhanced chemiluminescence following incubations of various antibodies. The following antibodies were used: anti-ATM (MAT3), 1:1000 (kindly provided by Y.Shiloh); anti-Mcm2 (Santa Cruz, N-19, sc-9839), 1:200; anti-Smc1 (Novus, NB-100-204), 1:5000; AhR (Santa Cruz, H-211, sc-5579), 1:500; anti-H-Ras (Santa Cruz, F235, sc-29), 1:500; anti-Raf (Pharmingen, Cat#13981A), 1 µg/ml; anti-MAPKpT202/Y204 (Cell Signaling, #9101), 1:1000; anti-MAPK (ERK2, Santa Cruz, C-14, sc-154), 1:400; anti-p53-S15 (Cell Signaling, #9284), 1:1000. For quantitative analysis, the scanned gel images were analyzed by ImagePro 5.0 software, and relative levels were normalized against the expression levels of ß-actin.

Tumor genomic DNA isolation and Ras mutation assay
Following NP40 extraction of tumor sample as described above, insoluble pellet was briefly rinsed in PBS, and 200 µl of lysis buffer (100 mM NaCl, 10 mM EDTA, 20 mM Tris–HCl, pH 7.5 and 0.5% SDS) containing 100 µg/ml proteinase K (Roche) was added followed by overnight incubation at 50°C. Saturated NaCl (100 µl) was then added to the samples and this was followed by vigorous shaking for 5 min. Tumor samples were spun at 6000 g for 10 min after sitting on ice for 10 min. Supernatant was transferred to a new tube and tumor genomic DNA was precipitated by addition of 100% ethanol. Tumor DNA was dissolved in TE and was used for PCR assay directly without further treatment.

Detection of H-Ras mutation at codon 61 was done essentially as described, with modification (24). In brief, following amplification of exon 2 of HRas, DNA sample was digested with XbaI for 2 h at 37°C. Digested samples were resolved onto 5% polyacylamide gel in 1x TBE buffer to distinguish the parental DNA (207 bp) and the mutated HRas gene (116 and 91 bp) along with the {phi}X174DNA/HaeIII molecular weight marker (Promega).

Atm LOH analysis of mammary tumor DNA was identical to that described for mice genotyping. LOH in the Atm heterozygotes tumor samples would be revealed by the disappearance of 125 bp wild-type PCR product. Sod1 locus was also examined as PCR control with the following primers: SODKOEX4 5'-gaacatcgtgtgatctcactgtcaggagag-3'; SODKOEX5 5'-caagcggctcccagcatttccagtctttgt-3'. Sod1 primers were kindly provided by Dr. Y.Ho.

Isolation of mammary epithelial cell lines
Isolation of epithelial cells from mouse mammary gland was similar to that described previously (25). Briefly, the mammary tissues from wild-type or Atm+/– mice were chopped fine and the pieces were suspended in DMEM/F12 (Invitrogen) containing 100 U/ml hyaluronidase (Sigma) and 150 U/ml collagenase (Sigma). After overnight incubation at 37°C, the organoids were transferred to flasks in attachment medium (DMEM/F12 with 10 mM HEPES, 20% calf serum, pen/strep and Fungizone). After attachment of a significant number of organoids, the flasks were refed with high-calcium growth medium [DMEM/F12 with 10 mM HEPES, 1.05 mM CaCl2, 5% horse serum (GIBCO), 10 µg/ml insulin (Sigma), 20 ng/ml EGF (Upstate Biotechnology), 0.5 µg/ml hydrocortisone (Sigma) and 100 ng/ml cholera toxin (Calbiochem)] to allow cells to spread over the plastic. After the attached pieces had started to grow out (about 10 days), the cells were stripped of fibroblasts by a short trypsinization and refed in low calcium growth medium (containing 0.04 mM CaCl2) to start cells growing as floaters. Within a month, growth in this medium and production of floaters became extremely rapid. After 8 weeks some cells were transferred back into high-calcium medium to determine whether they had spontaneously immortalized. Wild-type (WTME1, WTME2) and Atm+/– (AHME1, AHME2) mammary epithelial cell lines were maintained in mammary media (2.5% fetal bovine serum, 5% horse serum, 10 µg/ml insulin, 5 ng/ml EGF and 1x penicillin/streptomycin in DMEM/F-12 media).

Clonogenic survival assay
Clonogenic survival assay was done essentially as described (26), with modification. In brief, exponentially growing cell lines were seeded in triplicate at 300 cells/per 6-well dish and irradiated at the indicated dose the day after plating. Radiation was performed using a 137Cs source (Mark 1 Irradiator, J.L.Shepherd & Sons, San Bernardino, CA) with a dose rate of 1.1 Gy/min. The surviving clones were fixed in cold methanol and stained with 0.1% crystal violet 2 weeks post-radiation. The survival fractions were expressed as a percentage of number of irradiated survival clones divided by the number of non-irradiated surviving clones.

Statistical analysis
Mammary tumor-free survival was performed with Kaplan–Meier analysis. Log-rank test was performed to determine statistical significance. A P-value of <0.05 was considered to be statistically significant. The significance of mammary dysplasia in Atm+/– mice compared with wild-type was performed by Poisson regression analysis.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
In an effort to better understand the function of Atm in mammary tumor development, we generated a congenic strain of Atm knockout mice by backcrossing 129SvEv x Black Swiss Atmtm1Led mice (20) into FVB/N congenic background for 13 generations. Reciprocal mammary gland transplantation can be performed with this new strain, which we referred to as FVB.129S6(BLKSW)-Atmtm1Led (Lu and Wang, unpublished data). To test the hypothesis that A-T heterozygotes are predisposed to mammary tumorigenesis and to identify the environmental factors that may influence the development of breast cancer of A-T heterozygotes in the general population, we performed the following experiments with mice and cells derived from FVB.129S6(BLKSW)-Atmtm1Led (Atm+/– hereafter).

Wild-type and Atm+/– nulliparous female mice were treated with DMBA at 6 weeks of age, and mammary tumor development was monitored weekly by palpation. Once tumor mass was identified, the mice were killed and the tumors dissected for histopathological analysis (Table I). Nearly twice as many Atm heterozygotes developed mammary tumors (64.7%) as the wild-type mice (37.5%). The relative risk factor for DMBA-induced mammary tumors is 1.7 for Atm heterozygotes. Histopathological analysis of tumor samples indicates a wide variety of mammary tumor histological types in both wild-type and Atm heterozygotes (Table I and data not shown). Most importantly, Atm heterozygotes developed mammary tumors earlier with an average onset of 189 days compared with 229 days for wild-type mice (Figure 1A, P < 0.005 by log-rank test). In contrast, control untreated wild-type (n = 6) and Atm+/– (n = 11) mice did not develop mammary tumors over a 1-year period (data not shown). Interestingly, following DMBA treatment, ovarian tumors were observed in both wild-type and Atm+/– mice, whereas uterine tumors were observed in some of the Atm+/– mice but not in wild-type mice (Table I).


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  		Table I. DMBA-induced tumors in wild-type and Atm+/– mice

 

Figure 1
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  		Fig. 1. Tumor incidence and mammary dysplasia in wild-type and Atm+/– mice. (A) Kaplane–Meier survival analysis of mammary tumor-free mice. DMBA (Sigma) was dissolved in olive oil at a concentration of 5 mg/ml and administrated to mice ~6 weeks of age by oral gavages at a dose of 1 mg once a week for five consecutive weeks. Wild-type mice, (n = 8); Atm+/– mice, (n = 17). P < 0.005 by log-rank test. (B) Increased mammary dysplasia in Atm+/– mice compared with wild-type mice. Whole mount analysis of mammary glands from wild-type or Atm+/– mice treated with DMBA. Number of mammary glands analyzed; wild-type mice, n = 28; Atm+/– mice, n = 53. On the basis of the Poisson regression model, the estimated coefficient is 0.9988, which is significant with P-value of 0.0248. It is indicated that Atm+/– mice had higher rate of dysplasia.

 
Since tissue dysplasia is an early event during tumorigenesis, the extent of DMBA-induced dysplasia was determined in the mammary glands of the treated mice. In these experiments, tumor-free mammary glands were harvested from tumor-bearing mice and processed for whole mount analysis. Poisson regression analysis was performed to determine whether mammary dysplasia might be affected by mammary tumor development. Our analysis suggests that mammary tumor development had no effect on mammary dysplasia of adjacent glands (P = 0.169, Chi-Square test). Over 56% (n = 53) of the Atm+/– mammary glands exhibited severe dysplasia, compared with 21% (n = 28) of the wild-type mammary glands that showed only mild dysplasia (Figure 1B and Figure 2, P = 0.0248 by Poisson regression analysis). The dysplastic mammary structures in the wild-type mice were more localized by whole mount analysis and exhibited a single epithelial layer by histological analysis (Figure 2A–D). For example, after DMBA treatment, 58% of the Atm heterozygotes with dysplastic mammary glands appeared to be diffusive, whereas only 11% of the wild-type with dysplastic mammary glands had such feature (Figure 2E–L, data not shown). Histopathological analysis revealed the presence of various stages of mammary tumorigenesis including hyperplastic alveolar nodule (HAN) (Figure 2M–P) and mammary intraductal neoplasia (MIN) (Figure 2G–H) in Atm+/– mammary glands.


Figure 2
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  		Fig. 2. Whole mount and histopathological analysis of mammary dysplasia. (A–D) Wild-type mammary gland. (E–P) Atm+/– mammary gland. (A, B, E, F, I, J, M and N) Whole mount analysis of mammary gland. (C, D, G, H, K, L, O and P) Histopathological analysis of the corresponding whole mount mammary gland. (G and H) Representative images showing MIN. (K and L) Representative images of ductal epitheliums hyperplasia were presented. (M–P) Representative images indicating the presence of HAN. Magnifications, A, E, I, M = 7.5x; B, F, J, N = 20x; C, G, K, O = 100x; D, H, L, P = 200x.

 
Although increased mammary dysplasia in Atm+/– mice could contribute to enhanced mammary tumorigenesis in those mice, it is not clear whether mammary tumors from Atm+/– mice exhibit loss of heterozygosity (LOH) at the Atm locus. To further characterize the enhanced carcinogen-induced mammary tumor development in Atm+/– mice, genomic DNA from three wild-type and five Atm+/– mammary tumors was harvested from fresh frozen tissues. PCR analysis was performed to determine whether LOH occurred in tumors from Atm+/– mice. Our results demonstrated that the wild-type Atm allele was retained in all five mammary tumors analyzed (Figure 3A). It is possible that Atm LOH might have occurred in the tumors and that wild-type PCR products detected may be derived from normal cells intermingling with tumor cells. Therefore, we compared PCR products derived from wild-type and Atm+/– mammary tissues with that derived from tumors and found that the intensity of the wild-type bands were comparable in tissues or tumors derived from Atm+/– mice (Figure 3B). Moreover, Atm expression was clearly detectable in all five mammary tumor samples and three ovarian tumor samples derived from Atm+/– mice (Figure 3C). Therefore, increased mammary tumor development in Atm+/– mice is not due to loss of Atm expression from the wild-type allele. Collectively, these results suggest that Atm-haploinsufficiency confers increased sensitivity to chemical carcinogenesis. Interestingly, Mini-Chromosome Maintenance protein 2 (Mcm2), which was recently described as a target of ATM/ATR (27,28), was strongly expressed in three of the five Atm+/– mammary tumors but none in wild-type tumors. Furthermore, Mcm2 expression was only weakly detected in one of the three Atm+/– ovarian tumors (Figure 3B). In addition, the expression of Structural Maintenance Chromosome 1 (Smc1), another downstream target of Atm (29,30), appears to be positively correlated with the levels of Atm protein except in one wild-type tumor (#2) expressing high amount of Atm. The levels of Atm protein in these tumors were not due to differential protein loading since Aryl hydrocarbon Receptor (AhR) and Raf were all expressed similarly in these tumors (Figures 3B and 4B). It has been demonstrated recently that ATM is epigenetically silenced in advanced breast cancers (4). It is possible that variable expression of Atm in the mammary tumors may also be under epigenetic regulation.


Figure 3
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  		Fig. 3. Atm LOH analysis in DMBA-induced tumor samples. (A) PCR genotyping of Atm locus in three wild-type mammary tumors and five Atm+/– mammary tumors. Three ovarian tumors from Atm+/– mice were also examined for Atm LOH. Mut, Atm mutant genotype; WT, wild-type genotype. All tumors retain wild-type Atm, demonstrating no LOH at Atm locus. Control PCR analysis of Sod1 was also performed. (B) PCR genotyping of Atm locus in genomic DNA derived from mouse tails, mammary gland and mammary tumors. (C) Nuclear protein expression in mammary and ovarian tumors by immunoblot analysis. Mcm2 expression can only be detected in some of the mammary tumors derived from Atm+/– mice but none in the tumors from wild-type mice. The expression of Smc1 was strongly elevated in three of the mammary tumors from Atm+/– mice but only weakly in one of the three wild-type tumors. The Atm monoclonal antibody used here is not expected to recognize any truncated Atm protein in this mouse strain.

 

Figure 4
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  		Fig. 4. Ras signaling in DMBA-induced mammary tumors. All tumor sample numberings were identical to Figure 3. (A) DMBA-induced mammary and ovarian tumors do not contain activating H-Ras mutation at codon 61. PCR-amplified DNA was digested by XbaI DNA restriction enzyme to distinguish mutated H-Ras from normal H-Ras. WT indicates a 216 bp PCR product of the normal H-Ras on a 5% polyacrylamide gel stained with ethidium bromide. (B) Ras signaling pathway was not altered in mammary tumors derived from either wild-type or Atm+/– mice. The expression of Ras, Raf, activated MAPKpT202/Y204 and MAPKp44/42 was determined by immunoblot analysis. (C) Comparison of Ras signaling in mammary gland (MG) and mammary tumor (MT) derived from wild-type and Atm+/– mice. Immunoblot analysis was performed as in (B).

 
It was shown previously that DMBA-induced skin carcinogenesis was due to mutation of the Hras gene at codon 61, which could be detected in over 90% of the DMBA-induced skin tumor samples (31,24). In addition, mammary tumors induced by DMBA appear to be correlated with ras mutations (32–34). To determine whether mammary tumor induced by DMBA harbours Hras mutation at codon 61 in our experimental model, an allele-specific Hras mutation assay was performed (24). This assay takes advantage of the fact that DMBA-induced Hras mutation creates a novel XbaI restriction site, which could be conveniently employed to distinguish the wild-type allele from mutated Hras allele after PCR amplification of sequence surrounding codon 61. Our data clearly indicate that Hras mutation at codon 61 was not involved in either DMBA-induced mammary or ovarian tumor development since all tumors from wild-type and Atm+/– mice display only wild-type Hras allele following XbaI digestion of the PCR products (Figure 4A). Although we were unable to detect Hras mutation at codon 61, the presence of other activating mutations in Ras gene cannot be excluded. Since Ras mutations would activate mitogen-activated protein kinase (MAPK) signaling pathway, we therefore determined the expression of activated MAPK in these mammary tumors. As shown in Figure 4B, phosphorylation of MAPK at Thr202 and Tyr204 was variably observed in mammary and ovarian tumors derived from both wild-type and Atm+/– mice. In a limited comparison of Ras signaling between mammary gland and mammary tumors from the same wild-type or Atm+/– mice, we also could not find correlative evidence of Ras activation and mammary tumor development since phosphorylated MAPK was expressed at a lower level in the Atm+/– mammary tumors than that in the mammary glands (Figure 4C). Although our data did not provide evidence for a maintenance role of Ras activation in DMBA-induced mammary tumors, we cannot exclude the possibility that Ras activation may be required for the initiation of DMBA-induced mammary tumor development.

It was shown previously that ionizing radiation induces mammary dysplasia in about 10% of the Atm+/– mice although the mechanism of dysplasia induction was not determined (35). To further characterize the molecular mechanism of mammary tumor development in an Atm+/– background, we generated mammary epithelial cell lines from Atm+/– (AHME1 and AHME2) and wild-type control (WTME1 and WTME2) littermates. Both wild-type and Atm+/– cell lines display epithelial morphology and express pan-cytokeratin marker, confirming the nature of mammary epithelial cells (data not shown). To determine whether Atm-haploinsufficiency affects DNA damage signaling, we examined the induction of p53 in both wild-type and Atm+/– mammary cells in response to DNA damage. Interestingly, ~2-fold increased p53 phosphorylation at serine 18 (equivalent to human Ser15) was detected in AHME1 (2.07 ± 0.58, n = 3) and AHME2 (3.1-fold) Atm+/– mammary cell lines without DNA damage (Figure 5), suggesting that Atm-haploinsufficiency elevated endogenous DNA damage in unstressed cells. However, upon DNA damage, Ser18 phosphorylation was detected to a similar extent in both wild-type and Atm+/– mammary epithelial cells in a range of doses and time points examined (Figure 5). The expression of Atm was verified in these cells and it was confirmed that Atm+/– cells expressed half the amount of Atm protein found in wild-type cells (Figure 6A). Interestingly, a slightly decreased expression of Atm was observed in wild-type mammary epithelial cells in response to irradiation, while a slightly increased expression of Atm was seen in Atm+/– mammary epithelial cells after normalization to the expression levels of ß-actin (Figure 6A).


Figure 5
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  		Fig. 5. The induction of p53 in response to radiation in Atm+/– mammary epithelial cell lines. Wild-type (WTME1, WTME2) and Atm+/– (AHME1, AHME2) mammary epithelial cell lines were established from respective mouse mammary gland. Active proliferating cells were irradiated at given doses, and protein extracts were prepared at the time indicated. Phosphorylated p53 at serine 18 was examined in these cells. ß-actin was used as loading control. (A–C) WTME1 and AHME1 cells were irradiated at 1 Gy (A) or 8 Gy (B) of various time points, or (C) irradiated at various doses for 30 min. (D) WTME2 and AHME2 cells were irradiated at given doses and the expression of phosphorylated p53 was determined at indicated time points.

 

Figure 6
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  		Fig. 6. Atm heterozygotes displayed enhanced survival to radiation. (A) Atm expression in wild-type (WTME2) or Atm+/– (AHME1) mammary epithelial cell line in response to different doses of irradiation. The expression of Atm was determined 1 h post-radiation. The relative expression levels were quantitated using ImagePro 5.0 software after normalization against ß-actin, and fold differences compared with the level of Atm in non-irradiated wild-type cells were shown at bottom of the gel. (B) Mammary epithelial cell lines (WTME1, WTME2, AHME1 and AHME2) were irradiated at given doses and survival clones were scored after 2 weeks in culture. Survival fractions were expressed as percentage of irradiated survival clones normalized to non-irradiated survival clones. The experiment was performed in triplicate and results were presented as mean ± SEM.

 
One of the major features of A-T patients is increased sensitivity to radiation (3). At the cellular level, the increased radiation sensitivity can be determined by clonogenic survival assay. To determine whether Atm-haploinsufficiency could confer radiosensitivity in mammary epithelial cells, we performed clonogenic survival assay in response to various doses of ionizing radiation in both wild-type and Atm+/– mammary cell lines. Surprisingly, we found that both Atm+/– mammary epithelial cell lines (AHME1 and AHME2) were less sensitive than wild-type mammary epithelial cell lines (WTME1 and WTME2) to radiation in this clonogenic survival assay (Figure 6B). Hence, Atm-haploinsufficiency confers enhanced survival in response to radiation in mammary epithelial cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
It was estimated that 1% of the general population are A-T carriers who are at an increased risk of breast cancer development (6). This hypothesis is supported by the observations that obligate A-T carriers are more likely to develop breast cancer at an early age (8,10–12). The majority of the mutations in A-T patients are truncating mutations, whereas the remaining are missense mutations (1,15). However, the relative contribution of the truncating and missense mutations of ATM to breast cancer development in A-T family is not clear. It is inferred that blood relatives of A-T patients would most probably have truncating mutations that predispose this population to breast cancer. However, since only a small percentage of these A-T carriers develop breast cancer at an early age, it is also probable that missense ATM mutations preferentially contributed to breast cancer in relatives of A-T patients. Gatti et al. (15) had proposed a model suggesting that missense mutations of ATM predispose to cancer in the heterozygotic or homozygotic state in the general population. It was shown previously that 7271T>G and IVS10-6T>G missense mutations have been associated with multiple cases of breast cancers in the A-T family and in the non-A-T Australia family (36). However, the association of these two mutations with breast cancer was not supported by large-scale family-history-based or population-based screening for these two mutations in breast cancer patients (37,38). In addition, ATM mutations are more frequently associated with early onset familial breast cancer patients (5,39) while these studies have not been replicated by others (40,41). To reconcile these differences, we propose that the expression of ATM mutations is modified by environmental factors, such as local environmental carcinogen exposure or a difference in dietary intake.

Animal models for both truncating mutations and missense mutations have been developed (17–20,42). Using these models, it was shown that Atm+/– mice with truncating mutations are more sensitive to radiation-induced aging problems, such as cataracts and hair graying (13,14). Mammary epithelial cells from Atm heterozygotes with truncating mutations appear to be sensitive to radiation-induced mammary dysplasia in 10% of the transplanted tissues (35). Interestingly, Atm+/– mice with truncating mutations have not been shown to develop mammary tumors. The difference in the occurrence of mammary tumors between mouse and human in the Atm+/– background is not clear. However, one important difference is that all animals studied are housed in similar controlled environment and fed similar diets. To account for this difference, we propose that A-T carriers who developed breast cancers might have encountered additional mutagenic events from exposure to environmental toxic compounds through dietary consumption.

A mouse model with missense mutation (ATM-{Delta}SRI) in which three amino acids of ATM at positions 2556–2558 have been deleted has been developed recently (42). This model is representative of missense mutations observed in some A-T patients where full-length ATM protein can be detected. However, ATM protein kinase activity is lost in this mutant protein (42). Interestingly, ~9% of the heterozygotes of ATM-{Delta}SRI mice developed tumors, a 3-fold increase over that of the wild-type mice (43). However, only 2% of the ATM-{Delta}SRI heterozygotes developed mammary tumors (43), suggesting that additional factors are required for mammary tumor development in this model. It will be interesting to determine whether ATM-{Delta}SRI mice are also sensitive to chemical carcinogen-induced mammary tumorigenesis.

In Atm+/– mice treated with ethylnitrosourea (ENU, a direct alkylating agent that is thought to produce DNA lesions without DNA double-strand breaks), no increased mammary tumors were observed within 100 days of treatment (44). The inability to detect increased tumors induced by ENU in Atm+/– mice may be due to a short incubation time or that Atm+/– mammary epithelial cells are not sensitive to ENU treatment. Umesaka et al. (45) have recently reported that Atm-haploinsufficiency promotes mammary tumor development in a p53 heterozygotic background and that the Atm-haploinsufficiency effect was further enhanced by ionizing radiation. However, it is not clear whether mammary dysplasia occurred in this cohort. The importance of ATM in suppressing breast cancer development is also supported by a recent study demonstrating that ATM is epigenetically silenced in advanced breast cancer (4).

In this study, we found that truncating Atm+/– mice had a 2-fold increased mammary tumor incidence in the DMBA-induced mammary tumorigenesis model. In addition, we found that there was about a 3-fold increase in mammary dysplasia induced by DMBA treatment in Atm+/– mice compared with wild-type mice. Importantly, there was more severe mammary dysplasia in Atm heterozygotes than in wild-type mice (see Figure 2E–L), which is consistent with the notion that DMBA may interfere with the hormone response pathway (46,47). Mammary dysplasia appears to be specifically associated in mice treated with either DMBA (this study) or ionizing radiation (35) since untreated Atm+/– mice do not develop this phenotype (data not shown, also see ref. 35). The severe mammary dysplasia observed in Atm heterozygotes implies that Atm+/– mammary epithelial cells are more sensitive to hormone-induced proliferation than wild-type cells. Therefore, increased tumor incidence and dysplasia in Atm heterozygotes may be partly explained by the combined actions of increased mutation accumulations (deficient DNA repair system) and increased sensitivity to hormone-induced proliferation of mammary epithelial cells. DMBA causes bulky DNA–protein adducts formation, which could be repaired by base/nucleotide excision repair. Activation of Ras pathway has been implicated in DMBA-induced carcinogenesis in general (32–34,48,49). The possibility of DMBA-induced Ras mutation in the initiation of mammary tumorigenesis was evaluated in this study. Our results suggest that Ras mutation at codon 61 was not involved in DMBA-induced mammary tumors irrespective of the status of Atm. However, it is likely that DMBA may also induce other Ras mutations. If true, then Ras mutations would activate downstream signaling, and such events should be detected. Surprisingly, examination of Raf and MAPK levels did not provide such evidence. Therefore, our results suggest that DMBA induces distinct signaling pathways in tumor progression dependent on cell types. However, we could not exclude the possibility that activation of Ras signaling pathway is involved in the initiation of mammary carcinogenesis but is not required for the maintenance once mammary tumor mass formed. Alternatively, DMBA-induced DNA-adducts may interfere with replication fork movement and therefore may cause replication fork collapse. Restart of the replication fork is thought to invoke a DNA recombination step induced by double-strand breaks. It is possible that in Atm heterozygotes the restart of the replication fork progression is defective, thereby leading to the accumulation of mutations and chromosome instability. Consistent with this interpretation, DMBA and other bulky-adduct-forming agents, such as PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine), have been shown to induce aneuploidy in Chinese hamster cells and colon cancer cells, respectively (50,51).

Mammary epithelial cells from Atm heterozygotes might be deficient in the correction of environmental mutagenic insults and hence predisposed to breast cancer. Consistent with this hypothesis, we found that Atm+/– mammary epithelial cells exhibit elevated basal levels of p53 phosphorylation compared with that in wild-type cells. Constitutive induction of p53 phosphorylation in Atm+/– mammary cells indicates that these cells are constantly under low levels of stresses compared with wild-type cells. Therefore, Atm-haploinsufficiency confers elevated cellular stresses in mammary epithelial cells.

Atm-deficient cells exhibit increased radiation sensitivity. The cause of radiation sensitivity in Atm-deficient cells is not well understood although both cell cycle checkpoints and DNA repair defects could contribute to such effect (26,52). It was shown previously that ATM heterozygotic lymphocytes showed intermediate radiation sensitivity using a short-term survival assay (43,53), whereas radiation sensitivity was not observed in mouse Atm+/– ES cells using a clonogenic survival assay (18). In this study, we were therefore surprised to found that Atm-haploinsufficiency confers slightly enhanced survival in mammary epithelial cells in response to DNA damage. The variations in radiation sensitivity in these different cell types are not clear at present but could be dependent on cell context and/or due to the history of cellular stresses. Atm-haploinsufficiency may predispose mammary epithelial cells to genome instability and therefore leads to enhanced tumorigenesis in response to chemical carcinogens. In summary, our results suggest that gene–environmental interactions may dictate the breast cancer development in ATM heterozygotes.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   Acknowledgments
 
We thank Dr Y.Shiloh for Atm monoclonal antibody, Drs B.Reizis and A.Elson for Atm PCR primer sequences, and Dr Y.Ho for Sod1 PCR primers. This research was partly supported by Grants from Department of Defense (DAMD17-02-1-0619) and NIH (RO1 CA89526) and (P30 ES06639).

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 11, 2005; revised October 26, 2005; accepted December 6, 2005.


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