2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) retards mammary gland involution in lactating SpragueDawley rats
Meenakshi Venugopal,
Andrew Callaway and
Elizabeth G. Snyderwine1
Chemical Carcinogenesis Section, Laboratory of Experimental Carcinogenesis, Division of Basic Sciences, National Cancer Institute, Bethesda, MD 20892-4255, USA
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Abstract
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2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a compound from cooked meat, is an established mammary gland carcinogen in female rats. Four doses of PhIP (150 mg/kg, p.o., once per day) were given to lactating SpragueDawley rats separated from their 10-day-old pups to initiate involution of the gland. Twenty-four hours after the last dose, apoptotic index in the mammary gland, as measured by the TUNEL assay, was significantly higher in the gland from control rats than in the PhIP-treated rats (4.757 ± 1.066 versus 1.905 ± 0.248%; P < 0.05). In comparison with controls, alveoli in the mammary gland of PhIP-treated rats were also visibly larger and contained more secretory epithelial cells. The expression of Bax, a stimulator of apoptosis, and Bcl-2, an inhibitor of apoptosis, were quantitated by western blotting. Accordingly, Bax expression was 2.7-fold higher in control rats, whereas Bcl-2 expression was 3.1-fold higher in PhIP-treated rats, both changes being statistically different (Student's t-test, P < 0.05). Immunohistochemistry further confirmed a lower expression of Bax and higher expression of Bcl-2 in secretory alveolar epithelial cells of the PhIP-treated mammary gland. The findings are consistent with the notion that exposure to PhIP retarded involution via partial inhibition of programmed cell death. To investigate possible mechanisms for the inhibitory effects of PhIP on mammary gland involution, serum levels of prolactin, an important hormone for the maintenance of lactation, were measured in virgin rats with regular estrous cycles given PhIP (150 mg/kg, p.o.) on the morning of diestrous. After one estrous cycle, on proestrous morning, serum prolactin levels were 1.3-fold higher after PhIP than after control vehicle (one-way ANOVA, Fisher LSD multiple comparison test, P < 0.05). PhIP exposure during involution was associated with the induction of benign mammary tumors. Seven out of 12 rats developed fibroadenomas, and one developed a tubulopapillary carcinoma within 1 year of receiving PhIP administration during involution (150 mg/kg, p.o., once per day for 5 days), and a high-fat diet (23.5% corn oil). An increase in serum prolactin level and the effects on mammary gland apoptosis seen with PhIP may have implications for the mechanisms of carcinogenic targeting of PhIP to the mammary gland.
Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; HCA, heterocyclic amine; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
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Introduction
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It is generally recognized that diet influences the risk of human breast cancer (1). Whether specific dietary carcinogens/mutagens play a role in human breast cancer etiology is not yet known. Well-done meat contains potent mutagens and rodent carcinogens belonging to the heterocyclic amine (HCA) family of compounds (2,3). One HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), has been shown to be a mammary gland carcinogen in rats. PhIP is formed when meat is cooked by ordinary household methods such as broiling, barbecuing, frying and baking (3,4). Several studies have shown that PhIP induces mammary gland tumors in female rats (58). In addition, a high-fat diet has been shown to increase the incidence of carcinomas induced by PhIP (7,9).
The mechanisms involved in the mammary gland carcinogenicity by PhIP are not entirely understood. Previous studies have shown that carcinogenesis is associated with the formation of PhIPDNA adducts and alterations in the proliferation and development of the mammary gland of rats (10). It has been known for many years that multiple hormonal factors are critical in mammary gland carcinogenesis. In rats, mammary tumors are dependent on hormones for growth and development (11). Two of the principal hormones involved in mammary gland carcinogenesis are estrogen, produced largely by the ovaries, and prolactin, synthesized in the lactotrophic cells of the anterior pituitary (12). Both prolactin and estrogens are potent stimulators of mammary carcinogenesis in rats (13,14). Indeed, studies have shown that ovarectomy, pituitary ablation or lesions in the median eminence, can completely inhibit the induction of mammary gland tumors by the potent experimental mammary gland carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA) (13,15).
Both estrogen and prolactin are also recognized as critical hormones for the normal development and maturation of the mammary gland. Prolactin has been shown to play a role in the proliferation and functional differentiation of the mammary gland during pregnancy and to be an important hormone in the maintenance of lactation (12). During maturation of the gland, ductal epithelial cells give rise to alveolar buds and to lobules. During pregnancy and lactations, lobules proliferate to fill the mammary fat pad (16). The epithelium of the lactating mammary gland is at its most differentiated state and consists of multiple lobular structures of secretory epithelial cells (12). During involution of the rat mammary gland, the removal of the suckling stimulus results in a decline in prolactin levels (17). Mammary gland involution is a well-characterized process by which the secretory epithelial cells die by apoptosis (18).
In the current study, we address whether PhIP affects the normal process of involution in the rat mammary gland. The purpose of this query was twofold: (i) to assess whether PhIP exposure might potentially alter apoptosis in the mammary gland, and (ii) to gain insight into whether PhIP exposure alters the levels of certain mammotrophic hormones, especially prolactin. The effects on mammary gland apoptosis and serum prolactin levels were also studied because of their possible relevance to the mechanisms of mammary gland carcinogenicity of PhIP.
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Materials and methods
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Materials
PhIP was purchased from Toronto Research Chemicals (North York, ON). DMBA and human prolactin were obtained from Sigma (St Louis, MO). Anti-Bax polyclonal (N-20) and anti-Bcl-2 polyclonal (N-19) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Bcl-x monoclonal antibody (B22620) was obtained from Transduction Laboratories (Lexington, KY). Anti-actin monoclonal antibody (Ab-1) was purchased from Oncogene Research products (Cambridge, MA). Enhanced chemiluminescence western blotting detection system was purchased from Amersham (Little Chalfont, UK).
Animals
For studies on the effects of carcinogens on mammary gland involution and for carcinogenicity testing, lactating SpragueDawley rats, age 7090 days, with first litters were purchased from Taconic (Germantown, NY). The litter size was normalized to 10 when the pups were 1 day old. For the prolactin studies, 40-day-old virgin female SpragueDawley rats were also purchased from Taconic. All the animals were provided food (NIH Lab Chow) and water ad libitum and were maintained on a 12 h lightdark cycle.
Mammary gland involution studies
The pups were separated from the mothers at 10 days of age, and the dams were then given a total of four doses of PhIP (150 mg/kg, p.o., in 7.5 ml corn oil/kg, once per day for 4 days). Accordingly, control animals were given four doses of corn oil only. The rats were killed 24 h after the final dose, and the abdominal mammary glands (nos 4 and 5) were removed and processed for immunohistochemistry and mammary epithelial cell isolation.
Detection of apoptotic cells
In the regressing mammary gland, the apoptotic cells were immunohistochemically detected by the in situ cell death detection (TUNEL) method (Boehringer Mannheim, Mannheim, Germany) as described in detail previously (9). The number of stained cells per 1000 cells was counted under a light microscope and the apoptotic index was calculated as the percentage of stained cells.
Immunohistochemistry
Immunohistochemical detection of Bcl-2, Bax and Bcl-x in the regressing mammary gland was performed according to the method of Krajewska et al. (19).
Western blotting
Mammary epithelial cells were isolated from the regressing mammary glands using the method of Ghoshal et al. (20). Mammary epithelial cells were sonicated in lysis buffer and the protein content of the cell extract determined by the Bradford's assay (21). Then, 40 µg of protein was used to detect the expression of Bax, Bcl-2 and Bcl-x by western blotting. HeLa cells, MCF7 cells and mouse macrophage were used as positive controls for the detection of Bax, Bcl-2 and Bcl-x, respectively. Expression of Bax, Bcl-2 and Bcl-x was normalized to actin. Proteins were visualized with the enhanced chemiluminescence detection system (22) and quantitated with ImageQuant software (v.3.3).
Carcinogenicity testing of PhIP in rats during involution
Pups were separated from the mothers at 10 days of age, and the dams were given five daily doses of PhIP (150 mg/kg, p.o., in 7.5 ml corn oil/kg). Control mothers received corn oil vehicle only. One day after the final dose, both the control (n = 10) and the PhIP-treated (n = 12) rats were placed on a defined high-fat diet (23.5% corn oil). The composition of the diet has been described in detail previously (7). The rats were palpated at 2 week intervals for the development of mammary tumors. Control and PhIP-treated rats were maintained up to 1 year after dosing. Histological classification of tumors was based on the criteria of Russo et al. (23,24).
Treatment of animals for studies on prolactin levels
Since serum prolactin levels are known to vary during the estrous cycle, as well as during lactation and weaning (17,25,26), virgin rats were used to study the effect of PhIP and DMBA on serum prolactin levels. The estrous cycle was determined by daily vaginal smears. Following the initial screening, three rats were placed in each cage according to the stage of the estrous cycle. The rats were monitored over three estrous cycles and only rats that experienced regular 4 day estrous cycles were used for the experiment. The rats received a single dose of either PhIP (150 mg/kg, p.o., in 7.5 ml corn oil/kg), DMBA (25 mg/kg in 7.5 ml corn oil/kg) or vehicle only (7.5 ml/kg), on the morning of diestrous. After dosing, the rats were maintained through one estrous cycle. On the morning of proestrous during the second cycle, the rats were killed by decapitation, and serum collected for the analysis of prolactin and estradiol-17ß (26).
Determination of serum prolactin by the nb2 assay
Prolactin levels were determined by the method of Tanaka et al. (27). Nb2 rat lymphoma cells were grown in Fisher's medium supplemented with fetal bovine serum, horse serum, penicillin, streptomycin and ß-mercaptoethanol. Each well of a 6-well culture dish was seeded with 4x105 cells suspended in media. Then, 10 µl of rat serum was added to each well, and the cells incubated at 37°C in the presence of CO2 for 3 days. After incubation, the cells were counted using a coulter counter and compared with a standard curve generated using 0500 pg/ml human prolactin.
Determination of serum estradiol-17ß level by radioimmunoassay
Estradiol-17ß levels in rat serum were measured at Covance (Vienna, VA) using the method of Abraham (28). Briefly, steroids were isolated from the serum samples by solvent extraction, and the estradiol was further purified from the extract by Celite chromatography. The level of estradiol-17ß was then quantitated by radioimmunoassay.
Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) or Student's t-test. All statistical analyses were performed by using SigmaStat software program (v.2.0; Jandel Scientific Software, San Rafael, CA).
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Results
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Effects of PhIP on mammary gland apoptosis during involution
Histological examination of the abdominal mammary glands from rats 4 days after removal of their pups showed that the glands from both control rats and PhIP-treated rats exhibited lobuloalveolar structures with some surrounding stromal tissue typical of the involuting mammary gland (Figure 1A and B
). Apoptotic cells were clearly detectable by TUNEL staining in glands from control and PhIP-treated rats (Figure 1A and B
). These cells contained darkly stained nuclei with a large cytoplasmic area. In comparison with the control, fewer apoptotic cells were visible in the glands from PhIP-treated rats and it appeared that the lobules were larger in glands from the treated rats (compare Figure 1A and B
). More stromal tissue was seen surrounding the lobules of the control gland than the gland from the PhIP-treated rats. These observations suggest that the extent of involution was more pronounced in the controls than in PhIP-treated rats. Quantitation of the percentage of apoptotic cells showed that the apoptotic index in the mammary gland was 2.5-fold higher in controls than in PhIP-treated rats, a difference that was statistically significant (Figure 2
; n = 6, one-way ANOVA, P < 0.05). Dams that received DMBA after weaning also showed a statistically lower percentage of apoptosing cells in the mammary gland in comparison with control rats. The apoptotic index in glands from PhIP- and DMBA-treated rats, however, was not statistically different. By the tenth day after weaning, the apoptotic index remained statistically higher in control animals than in PhIP-treated animals (0.13 ± 0.04 versus 0.026 ± 0.01%, n = 4, Student's t-test, P < 0.05).

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Fig. 1. Appearance of the involuting mammary gland of control (A) and PhIP-treated (B) rats 5 days after weaning. Sections of the abdominal glands (nos 4 and 5) were stained by the TUNEL method. Several apoptotic cells are shown by arrow heads. [Magnification 20x (A) and 10x (B)].
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Fig. 2. The apoptotic index in mammary glands from rats exposed to PhIP, DMBA or control vehicle during mammary gland involution. Data are the mean percentages ± SEM, n = 6, rats per group. Bars with different letters are statistically different (P < 0.05, one-way ANOVA with all pairwise multiple comparison procedures by Tukey test).
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To further examine the effect of PhIP on apoptosis occurring with involution, the expression of Bax, a pro-apoptotic protein, and Bcl-2, an anti-apoptotic protein, was examined in the regressing mammary glands of rats. By immunohistochemistry, both Bax and Bcl-2 were located in the cytoplasm of the mammary epithelial cells. The degree of staining suggested that the expression of Bax was higher in the glands of control rats than in the glands of PhIP-treated rats (compare Figure 3A and B
). In contrast, the expression of Bcl-2 appeared to be higher in the glands of PhIP-treated rats than in the controls (compare Figure 3C and D
). The expression of both Bax and Bcl-2 was quantitated by standard western blotting. Although there was some apparent variation in the expression of Bax and Bcl-2 among the mammary glands from different rats, there were clear differences in the means between the PhIP treatment group and control group (Figure 4
; Table I
). Bax expression was 2.7-fold higher in glands from control animals than in PhIP-treated animals. Concomitantly, the expression of Bcl-2 was 3.1-fold greater in PhIP-treated mammary glands than in the control mammary glands. In some rat samples, a lower molecular weight band was detected with the Bcl-2 antibody suggesting that there was cross-reactivity of the antibody with an unidentified rat protein; however, this band appeared to be unrelated to PhIP treatment. Bcl-x could not be detected by western blotting even when high concentrations of the primary antibody was used (3 µg/ml Bcl-x).


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Fig. 3. Immunohistochemical detection of Bax (A and B) and Bcl-2 (C and D) in the regressing mammary gland. Abdominal mammary glands (nos 4 and 5) from representative rats in the control group (A and C) and PhIP-treated group (B and D) are shown. (Magnification 20x.)
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Fig. 4. Bcl-2 and Bax expression in the involuting mammary gland from three control (C13) and three PhIP-treated rats (P13) detected by western blot analysis. Significant differences in the mean expression of Bcl-2 and Bax was observed between control and PhIP-treated rats (Table I ).
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Table I. Effect of PhIP exposure on apoptosis and the expression of Bax and Bcl-2 in the involuting mammary gland
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Mammary gland tumorigenicity of PhIP after administration during involution.
To assess the effects of PhIP exposure during mammary gland involution on mammary gland carcinogenesis, rats were given PhIP after removal of pups and subsequently placed on a high-fat diet. After 1 year, seven out of 12 (58.3%) PhIP-treated animals developed mammary tumors, whereas none of the 10 control animals developed mammary tumors. Histological examination of the mammary tumors revealed that six of the tumors were fibroadenomas and one was a non-invasive tubulopapillary carcinoma (Table II
). Six of the seven rats presented with a single fibroadenoma. One rat had harbored three fibroadenomas. The average latency from the time of dosing to the detection of palpable tumors was 9 months.
Serum prolactin and estradiol levels after PhIP administration
Since the levels of serum prolactin and estradiol are known to differ throughout the estrous cycle of rats (17,25,26), virgin rats with regular 4 day estrous cycles, rather than the lactating rats undergoing mammary gland regression, were used to determine the effect of PhIP on prolactin and estradiol-17ß levels. DMBA, which has been shown to have an effect on prolactin and estradiol levels in virgin rats (26,29,30), was used as a positive control. The level of serum prolactin in control animals on the morning of proestrus (one complete estrous cycle since dosing with carcinogen or vehicle), was significantly higher in either PhIP- or DMBA-treated rats than in control rats (Figure 5
) (n = 6, one-way ANOVA, P < 0.05). The level of serum estradiol-17ß, as detected by radioimmunoassay, was not altered by dosing with PhIP (n = 6). After DMBA administration, several rats showed high blood levels of estradiol-17ß (Figure 6
); however, in light of the variation among the animals, the mean level of estradiol-17ß in the DMBA rats was not statistically different from control.

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Fig. 5. Effect of PhIP and DMBA on serum prolactin level. Virgin rats received a single dose of PhIP (150 mg/kg, p.o.) or DMBA (25 mg/kg, p.o.) as described in the Materials and methods. Bars with different letters are statistically different (n = 6, P < 0.05, one-way ANOVA, Fisher LSD multiple comparison).
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Fig. 6. Effect of PhIP and DMBA exposure on serum estradiol-17ß levels. Virgin rats received a single dose of PhIP (150 mg/kg, p.o.), or DMBA (25 mg/kg, p.o.) as described in the Materials and methods. The level of serum estradiol-17ß in each rat (n = 6 per group) is shown individually.
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Discussion
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Extensive lobulo-alveolar development and functional differentiation of the mammary gland occurs during pregnancy and lactation under the influence of lactogenic hormones (12). After weaning, involution of the mammary gland involves remodeling of the gland through the process of apoptosis to a state resembling a mature virgin gland (12,18). In the current study, the administration of PhIP during involution of the mammary gland retarded the process of involution apparently by inhibitory effects on normal programmed cell death. Twenty-four hours after the last dose of PhIP, there were significantly fewer apoptotic cells in glands from PhIP-treated rats than from control rats. The apoptotic index in the mammary gland was ~2.5-fold higher with control than with PhIP rats. Consistent with an inhibitory effect of PhIP on apoptosis in the mammary gland, the gland from PhIP-treated rats showed larger alveoli and more epithelial cells.
The expression of the apoptosis-regulating proteins, Bax and Bcl-2 were also affected by PhIP administration. Bax has been shown to be an important regulator of apoptosis in the involuting mammary gland, with involution associated with increased expression of Bax, a pro-apoptotic protein (22). In the current study, the expression of Bax in the involuting mammary gland was reduced by treatment with PhIP. This was observed by both western blotting and by immunohistochemistry. The expression of Bcl-2, a protein that promotes cell survival, was accordingly higher in PhIP-treated rats than in control rats as detected by western blotting and immunohistochemistry. The ratio of expression of Bcl-2:Bax is also a factor in apoptosis (31). In the current study, this ratio was 8.4-fold higher in PhIP-treated rats than in control rats. These findings are consistent with inhibitory effects of PhIP treatment on apoptosis in the involuting mammary gland.
The mechanism of the inhibition of mammary gland involution by PhIP is not fully understood, but it may be partly related to higher serum prolactin levels ensuing from PhIP exposure. Prolactin has been shown to inhibit mammary gland involution (32). Thus, higher serum prolactin levels would be expected to retard apoptosis occurring during involution. The current study showed that PhIP significantly increases serum prolactin levels in female rats. The increase in serum prolactin associated with PhIP exposure may be potentially related to its effects on catecholamine metabolism in the hypothalamus. In vitro studies have indicated that PhIP inhibits the activity of L-DOPA amino acid decarboxylase, an enzyme involved in the production of dopamine in the hypothalamus (33). Lower dopamine levels would be expected to lead to an increase in prolactin level (34). In vitro studies with other HCAs such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) have also raised the possibility that additional enzymes involved in catecholamine metabolism may be potentially affected by PhIP (33). Although effects of PhIP on catecholamine metabolism and dopamine levels have not been demonstrated in vivo, the presence of PhIPDNA adducts in the brains of rats and monkeys (35,36) supports the idea that PhIP transverses the blood brain barrier and, therefore, may potentially reach the hypothalamus to alter catecholamine metabolism.
Although multiple doses of PhIP induce mammary carcinomas in virgin rats (7,9), in the involuting rat mammary gland model described here, PhIP induced a high incidence of fibroadenomas. Just one of 12 rats developed a carcinoma despite the high-fat diet, a strong promoter of carcinomas induced by PhIP (7,9). The low yield of carcinomas is consistent with the inhibitory effects of pregnancy and lactation on chemical carcinogen-induced carcinomas (37,38). In addition, the high yield of fibroadenomas with PhIP is also in accordance with the ideas of Russo and Russo (38,39) that the type of lesion induced is dependent on the target site and the state of differentiation of the mammary gland at the time of carcinogen administration. Fibroadenomas are thought to arise from differentiated alveolar and lobular structures whereas carcinomas arise from the less differentiated epithelial cells of ductal structures such as terminal end buds and terminal ducts (38,39). Although benign, fibroadenomas are nevertheless a health problem in women (40). In the light of the inhibitory effects of PhIP on mammary gland involution, it is plausible that the development of fibroadenomas in this rat model was associated with abnormalities in mammary gland involution.
PhIP has previously been shown to induce mammary carcinomas in virgin rats; the mechanisms for the induction of mammary carcinomas include PhIPDNA adduct formation, inhibition of differentiation and proliferation of terminal end buds (10). The effect of PhIP on serum prolactin levels is a hitherto unrecognized action of this compound that may also partially explain the carcinogenic targeting of PhIP to the virgin mammary gland. Several studies with rats show that a positive correlation exists between the induction of mammary tumors by DMBA and prolactin levels (41). Prolactin is also important for the growth of established carcinogen-induced rat mammary tumors (13,42,43). Studies by Heuson et al. (44) have shown that chemical carcinogen-induced tumors can either be inhibited or stimulated, respectively, by prolactin-decreasing drugs such as 2-bromoergocryptine or prolactin-increasing drugs such as perphenazine. It is likely that the elevation in prolactin levels by PhIP would serve as a promotional factor in the development of mammary gland carcinomas. Although estrogen is essential for the development of mammary carcinomas (12), a surge in serum estradiol-17ß was not associated with PhIP exposure, at least not under the dosage conditions and time during the estrous cycle examined here. In contrast, DMBA increased estradiol-17ß in three of the six rats examined, consistent with previous findings (29,30). Thus, an increase in serum prolactin levels but not estradiol-17ß appears to be associated with exposure of female rats to PhIP.
In summary, the results from this study indicate that PhIP delays involution of the rat mammary gland via an inhibitory effect on apoptosis. Although the mechanisms for the effects of PhIP on involution are not fully understood, speculatively, the inhibition of apoptosis by PhIP may involve an indirect effect of PhIP on serum prolactin levels which would slow involution. The ability of PhIP to increase prolactin levels is also likely to play a role in the mammary gland carcinogenicity of PhIP. Whether PhIP modulates the levels or activities of other hormones or growth factors that regulate involution or has a direct effect on involution, for example by modifying the expression of specific genes in the mammary gland that are involved in growth factor signaling, requires further consideration.
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Acknowledgments
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The authors thank Unnur P.Thorgeirsson (NCI) for diagnoses of tumor histopathology, Barbara K.Vonderhaar (NCI) for providing nb2 cells and information on the prolactin assay, Doug-Young Ryu (NCI) for assistance with animal studies, and Snorri Thorgeirsson (NCI) for helpful suggestions.
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Notes
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1 To whom correspondence should be addressed Email: elizabeth_snyderwine{at}nih.gov 
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Received November 30, 1998;
revised February 16, 1999;
accepted March 25, 1999.