Atm-haploinsufficiency enhances susceptibility to carcinogen-induced
mammary tumors
Shu Lu
1,†, Kate Shen
1,†, Yaolin Wang
4, Steven J.Santner
1,
Jie Chen
5, S.C.Brooks
1,2and Y.Alan Wang
1,3,1Karmanos Cancer Institute,2Department of Biochemistry and Molecular
Biology,3Department of Pathology, Wayne State University, School of Medicine, 110 E. Warren Avenue, Detroit, MI 48201, USA,4Department of
Tumor Biology, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA and5Department 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@kci.wayne.edu
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
pro-pose 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(
a)anthracene), a mammary
carcino-gen, 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
+/com-pared 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.
Introduction
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
activa-tion 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
epidemio-logical 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
MiceMale 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)-Atmtm1Ledmice. PCR assay was used to identify Atm heterozygotes with the following primers: M1:50-tgtagataggtcagcattggat-30; M10: 50-gtcaaaattcgatctgctgct-30; Neo1: 50-gctctttactgaaggctctttac-30. PCR amplification was performed in 1· GPCR
buffer [16.6 mM (NH4)2SO4, 67 mM Tris–HCl (pH 8.3), 6.7 mM MgCl2, 5 mM
bME, 1% DMSO, 80 mg/ml BSA, 1 mM dNTP, 150 nM PCR primers and 2 U Taq polymerase]. PCR amplification was initially denatured for 2 min at 94C, followed by 30 cycles of 30 s at 94C, 45 s at 56C and 60 s at 72C. 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 (n¼ 8) and Atm+/mice (n¼ 17) were used for 7,12-dimethyl-benz(a)anthracene (DMBA) treatment in FVB congenic background as described (21). In brief, DMBA (Sigma) was dissolved in olive oil at a Abbreviations:A-T, ataxia-telangiectasia; DMBA,
7,12-dimethylbenz(a)anthracene; LOH, loss of heterozygosity; MAPK, mitogen-activated protein kinase.
†These authors contributed equally to this work.
concentration of 5 mg/ml and administrated to mice6 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 mg/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 b-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 200ml of lysis buffer (100 mM NaCl, 10 mM EDTA, 20 mM Tris–HCl, pH 7.5 and 0.5% SDS) containing 100mg/ml proteinase K (Roche) was added followed by overnight incubation at 50C.
Saturated NaCl (100ml) 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 37C. Digested samples were
resolved onto 5% polyacylamide gel in 1· TBE buffer to distinguish the parental DNA (207 bp) and the mutated HRas gene (116 and 91 bp) along with thefX174DNA/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 50 -gaacatcgtgtgatctcactgtcaggagag-30; SODKOEX5 50
-caagcggctcccagcatttc-cagtctttgt-30. 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 37C, 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), 10mg/ml insulin (Sigma), 20 ng/ml EGF (Upstate Biotechnology), 0.5mg/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 mg/ml insulin, 5 ng/ml EGF and 1· penicillin/streptomycin in DMEM/F-12 media).
Clonogenic survival assay
Clonogenic survival assay was done essentially as described (26), with modi-fication. 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 a137Cs 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
In an effort to better understand the function of Atm in
mam-mary tumor development, we generated a congenic strain of
Atm knockout mice by backcrossing 129SvEv x Black Swiss
Atm
tm1Ledmice (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)-Atm
tm1Led(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
popula-tion, we performed the following experiments with mice
and cells derived from FVB.129S6(BLKSW)-Atm
tm1Led(Atm
+/hereafter).
Wild-type and Atm
+/nulliparous female mice were treated
with DMBA at 6 weeks of age, and mammary tumor
devel-opment 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
¼ 6) and Atm
+/(n
¼ 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).
Since tissue dysplasia is an early event during
tumorigen-esis, the extent of DMBA-induced dysplasia was determined in
the mammary glands of the treated mice. In these experiments,
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
¼ 0.169, Chi-Square test). Over 56% (n ¼ 53) of the
Atm
+/mammary glands exhibited severe dysplasia, compared
with 21% (n
¼ 28) of the wild-type mammary glands that
showed only mild dysplasia (Figure 1B and Figure 2, P
¼
0.0248 by Poisson regression analysis). The dysplastic
mam-mary 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).
Histopatho-logical 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.
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
ovar-ian 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.
Fur-thermore, 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
Table I. DMBA-induced tumors in wild-type and Atm+/miceAge (days) Tumor pathology Mice, Atm+
1 112 Lymphoma
2 142 Mammary adenocarcinoma 3 168 Mammary adenocarcinoma 4 169 Mammary adenosquamous tumor,
uterine adenocarcinoma 5 171 Mammary adenosquamous tumor 6 183 Mammary adenoma
7 185 Mammary papillary tumor 8 202 Mammary cribriform tumor
9 204 Mammary solid tumor, uterine adenocarcinoma 10 205 Mammary adenocarcinoma
11 206 Mammary adenocarcinoma 12 208 Uterine squamous cell carcinoma 13 212 Ovarine granulosa cell tumor 14 215 Ovarine granulosa cell tumor
15 253 Lung adenocarcinoma, gastric adenocarcinoma 16 254 Mammary squamous carcinoma and
mammary solid tumor
17 263 Uterine adenosquamous carcinoma, lung adenocarcinoma
Mice, WT
1 218 Mammary adenocarcinoma 2 232 Mammary adenosquamous tumor 3 237 Mammary solid undifferentiated tumor
and ovary granulosa cell tumor 4 272 Ovarine granulosa cell tumor 5 272 Ovarine granulosa cell tumor
6 272 No tumor
7 272 No tumor
8 272 No tumor
Six wild-type tumors and 22 Atm+/tumors were analyzed. Three of the eight wild-type mice developed mammary tumors compared with 11 of the 17 Atm+/mice.
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 mice6 weeks of age by oral gavages at a dose of 1 mg once a week for five consecutive weeks. Wild-type mice, (n¼ 8); Atm+/mice, (n¼ 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, n¼ 28; Atm+/mice, n¼ 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.
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.
It was shown previously that DMBA-induced skin
carcino-genesis 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
dis-tinguish 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,
phosphoryla-tion 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
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, M¼ 7.5·; B, F, J, N ¼ 20·; C, G, K, O ¼ 100·; D, H, L, P ¼ 200·.Fig. 3. AtmLOH 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.
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
mam-mary tumor development in an Atm
+/background, we
gen-erated 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
phosphoryla-tion at serine 18 (equivalent to human Ser15) was detected
in AHME1 (2.07 ± 0.58, n
¼ 3) and AHME2 (3.1-fold) Atm
+/mammary cell lines without DNA damage (Figure 5),
suggest-ing 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
con-firmed 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
mam-mary epithelial cells in response to irradiation, while a slightly
increased expression of Atm was seen in Atm
+/mammary
epi-thelial cells after normalization to the expression levels of
b-actin
(Figure 6A).
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
per-formed 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
Fig. 4.Ras signaling in DMBA-induced mammary tumors. All tumorsample 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).
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.b-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.
confers enhanced survival in response to radiation in
mam-mary epithelial cells.
Discussion
It was estimated that 1% of the general population are A-T
carriers who are at an increased risk of breast cancer
devel-opment (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
remain-ing 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
predis-pose 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
environ-mental 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
muta-tions 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
differ-ence, 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-DSRI) 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
heterozy-gotes of ATM-DSRI mice developed tumors, a 3-fold increase
over that of the wild-type mice (43). However, only 2% of the
ATM-DSRI 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-DSRI 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
with-out 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
Fig. 6. Atmheterozygotes 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 againstb-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.
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
gen-eral (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
path-ways 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
interpreta-tion, 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
con-fers 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
tumori-genesis in response to chemical carcinogens. In summary, our
results suggest that gene–environmental interactions may
dic-tate the breast cancer development in ATM heterozygotes.
Acknowledgements
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.
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Received July 11, 2005; revised October 26, 2005; accepted December 6, 2005
