Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064 and
2 Cancer Research Laboratory, University of California, Berkeley, CA 94720, USA
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Abstract |
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Abbreviations: AMV, age-matched virgin; BrdU, 5-bromo-2-deoxyuridine; E2, 17ß-estradiol; GH, growth hormone; IGF-I, insulin-like growth factor-I; -lac,
-lactalbumin; MNU, N-methyl-N-nitrosourea; P4, progesterone; PRL, prolactin; RIA, radioimmunoassay; T4, thyroxine.
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Introduction |
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The developmental stage of the mammary gland at the time of exposure to carcinogen has an important effect on the susceptibility of the gland to carcinogenesis (12,13). However, the effects of hormones on mammary carcinogenesis may be even more prevalent for the promotional period of cancer development. It has, for example, been shown that although the incidence of palpable mammary tumors is very low in parous rats after exposure to MNU, the incidence of latent carcinomas is quite high and approaching that of AMV rats (14). This finding supports our hypothesis in that although tumor initiation takes place in the mammary gland of parous rats upon MNU exposure, their hormonal environment appears to be sufficiently altered to prevent the development of frank tumors. However, it has never been directly determined if a defined hormonal treatment will counteract the protective effects parity provides against mammary carcinogenesis.
In the present study, we continue to examine the role hormones play in the parity-associated protection against breast cancer. The objectives were to investigate whether treatments of parous rats with hormones known to affect normal mammary gland development would alter the susceptibility of these animals to MNU-induced cancers.
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Materials and methods |
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Treatments with 17ß-estradiol (E2), progesterone (P4) and thyroxine (T4)
Animals were treated with E2 (Sigma, St Louis, MO), P4 (Sigma), T4 (Sigma), E2 plus P4 (E2 + P4), and E2 plus T4 (E2 + P4 + T4). The steroids were administered by subcutaneous implants of silastic capsules (Helix Medical Inc, Carpinteria, CA). The capsules (2 cm long, 1.98 mm ID/ 3.18 mm OD) contained 20 µg E2 mixed with cellulose, 20 mg P4 or 20 µg E2 mixed with 20 mg P4, when both steroids were administered simultaneously. T4 was administered in the drinking water (3 µg/ml). Controls, both parous and AMV rats received silastic capsules containing cellulose. Treatment commenced when the animals were ~114 days of age and was continued for 1 week when half of the animals in each group were killed for the assessment of circulating hormonal concentrations and mammary gland development at the time of carcinogen injection. The remaining animals in each group that had received identical hormonal treatment to those killed after 1 week of hormonal exposure were injected with MNU (Ash, Stevens, Detroit, MI) and these animals continued to receive hormonal treatment for the next 7 months, when all animals were killed.
To control for the possible effects of the hormonal treatment, per se, on mammary tumorigenesis, an additional group of animals received the same hormonal treatment for the same period of time as the MNU-treated rats but these animals were not injected with the carcinogen (Figure 1).
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Implantation of capsules
Animals were anesthetized with a ketamine (Aveco, Fort Dodge, IO) and xylazine (Mobay, Shawnee, KA) mixture (30 mg ketamine + 6 mg xylazine/kg body wt). Approximately 1.5 cm dorsal incision was made through the skin. A small subcutaneous pocket was made with a pair of forceps where the capsule was implanted and the incision closed with wound clips. Every 2 months the capsules were replaced with freshly packed capsules.
Tumor induction, detection and removal
The carcinogen was administered as described previously (15). Briefly, MNU was dissolved in 0.9% saline, pH 5.0 and heated to 5060°C. The animals were anesthetized as described above and then each received a single intraperitoneal injection of MNU (50 mg/kg body wt). Animals were palpated for detection of mammary tumors, commencing 1 month after the MNU injection. When the tumors had grown to 1.52.0 cm in diameter, rats were anesthetized as described above and the tumors surgically removed and processed for histology. At the end of each experiment, ~33 weeks after MNU injection, all animals were killed by CO2 inhalation, followed by decapitation and trunk blood collection. The blood was centrifuged at 1000 g for 20 min, serum was harvested and stored at 80°C until the hormonal concentrations of various hormones were measured. Mammary tissues were collected and either stored frozen for protein and DNA extraction or fixed in either Tellyesniczky's fixative for whole mounts and regular histological preparations, or in 4% paraformaldehyde for 3 h for immunocytochemistry.
Histology
Mammary gland samples obtained posterior to the lymph node of the right fourth mammary gland and mammary tumors were fixed, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) for inspection and classification of tumors. Whole mount preparations were made of the right first, second and third mammary glands. The glands were removed, fixed and stained with iron hematoxylin (16).
Immunocytochemistry
Paraformaldehyde-fixed mammary tissues were paraffin embedded and sectioned (5 µm). The sections were deparaffinized and rehydrated and then aldehyde groups and endogenous peroxidase activity blocked by incubation with 2% glycine and 0.3% hydrogen peroxide, respectively. Non-specific protein binding was blocked first with 2% dried milk followed by 5% goat serum both diluted in PBS. The tissue sections were then incubated overnight in primary antiserum to cyclin D (Upstate Biotechnology, Lake Placid, NY) diluted to 1:100. After thorough rinsing, the slides were incubated with the secondary antibody (biotinylated goat anti-rabbit IgG; Vector Laboratories, Burlingame, CA) diluted 1:200, followed by incubation with ABC reagent and diaminobenzidine (Vector Laboratories). After addition of a coverslip, the percentage cyclin D labeled mammary epithelial cells for all experimental and control groups was determined by counting immunostained and unstained cells. The cyclin D labeling was assessed at three to four different locations in two epithelial structures of the right fourth mammary gland pair. Therefore, the percentage cyclin D labeling in the mammary gland of each animal was the average of six to eight independent cell counts from different locations in the mammary gland, counting 635.8 ± 15.7 (mean ± SEM) cells at each location. Immunostaining with cyclin D to assess the percentage proliferating cells in the mouse mammary gland has been validated and the method was found to correlate very closely to that for BrdU labeling (17). Precaution was taken to dissect histological samples from each mammary gland at as similar a location as possible, using the lymph node as a landmark.
Circulating concentrations of hormones
Concentrations in serum of E2, P4, T4, prolactin (PRL), growth hormone (GH) and insulin-like growth factor-I (IGF-I) were measured by radioimmunoassays (RIAs). Assay kits from Diagnostic Products (Los Angeles, CA) were used to measure E2, P4 and T4, modified for use with rat serum by diluting the reference standards in charcoal-stripped rat serum. PRL and GH were measured using reagents from NIDDK and IGF-I with an RIA kit from Diagnostic Systems Laboratories (Webster, TX).
Mammary gland differentiation
The -lactalbumin (
-lac) content of the mammary gland was measured for the assessment of mammary gland differentiation at the time of MNU injection. The mammary tissues were finely ground in liquid nitrogen with mortar and pestle and then homogenized using a Polytron homogenizer in 2 vol (w/v) buffer (50 mM TrisHCl, 5 mM MgCl2, pH 7.5) containing 1 mM Pefabloc (Boehringer Mannheim, Indianapolis, IN) and 1 µM pepstatin A (Sigma). After homogenization, a 20 µl sample was collected for measuring total DNA content and the remainder of the preparation extracted for 1 h, followed by centrifugation at 20 000 g for 30 min. The supernatant was collected and assayed for total protein and
-lac concentrations. For measuring the
-lac concentration, an RIA was developed using antiserum generated against rat
-lac (generous gift from Dr Kurt E.Ebner, University of Kansas Medical Center). Rat
-lac was purified from rat milk using a previously developed procedure (18). This purified preparation of
-lac was used as a reference standard and as iodinated tracer. The within and between coefficient variation of the assay was 3.0 and 18.4%, respectively.
Total protein was measured with the BCA protein assay (Pierce, Rockford, IL) using BSA (Fraction V, Sigma) as a standard, and total DNA content of each mammary gland homogenate was measured with fluorometric assay (18) using calf thymus DNA (Sigma) as a standard.
Statistics
The effects of the various hormonal treatments on mammary cancer incidence was analyzed using 2x2 contingency tables and 2-test. The effects of the treatments on tumor load, concentrations of the different hormones in serum, percentage cyclin D labeled cells, DNA content and
-lac concentration in the mammary gland at the time of MNU injection were all analyzed by one-way ANOVA using Fisher's protected least significant difference test. Differences between groups were considered significant for values of P < 0.05.
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Results |
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The number of animals carrying only non-malignant mammary tumors, mostly fibroadenomas, was ~1020% and this did not differ significantly between groups.
Circulating concentration of hormones
The concentrations of E2 in serum of animals treated with E2 were on average ~59 pg/ml. These concentrations were not significantly different from the average circulating concentrations of E2 for the other groups (Table II). However, the circulating concentrations of E2 in treated rats were much more consistent than those in untreated animals, where the E2 concentrations in blood were very erratic and frequently below detectable levels for the assay (0.4 pg/ml). In any event, the physiological effects of the E2 treatment were quite obvious, as the concentrations in serum of other hormones were effected. In particular, the circulating levels of PRL, GH and P4 were all increased significantly in animals treated with E2 as compared with all other groups, except the P4 concentration of P4-treated rats and the GH concentration of T4-treated rats, where these hormonal concentrations were also increased; however, to a lesser extent than in the E2 treatment groups. T4 treatment elevated the circulating concentrations of T4 significantly over all other groups and, interestingly, the parous rats that did not receive any hormonal treatment had significantly higher T4 levels in serum than the parous rats treated with E2, P4 and E2 + P4 and the AMV. The concentration of IGF-I in blood was significantly lower in parous animals treated with E2 alone than in all other groups, except parous rats treated with P4 alone. The serum concentration of GH in parous, untreated rats tended to be lower than in other groups of animals, but in contrast to what we had seen before (8), this trend did not reach a significant level. Also, no reduction was found in the circulating levels of PRL in the parous animals although parity has previously been shown to blunt PRL release in rats (9,10). This discrepancy was probably caused by difference in protocol used for the blood sampling in the present study in that the status of the estrous cycle was not monitored, and insufficient handling of the rats prior to collection of the blood and, therefore, the blood sampling may have caused stress-related release of pituitary hormones.
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Concentration of -lactalbumin in mammary tissues
The concentration of -lac was used to assess the level of differentiation of the mammary tissues (Figure 3C
). Interestingly, the mammary glands of untreated parous rats showed significantly higher
-lac concentrations in the mammary gland than all other groups or 7.7 ± 1.3 µg
-lac/mg DNA (mean ± SEM). The least differentiated mammary tissues were found in animal groups treated with E2 alone or E2 in combination with other hormones. In these groups the content of the mammary gland was 1.67 ± 0.9 µg
-lac/mg DNA in E2-treated rats; 0.68 ± 0.2 µg
-lac/mg DNA in E2 + P4-treated rats and 0.57 ± 0.1 µg
-lac/mg DNA in parous animals treated with E2 in combination with P4 and T4. Parous rats treated with P4 and T4 alone had
-lac concentrations of 3.59 ± 0.8 and 4.27 ± 0.9 µg
-lac/mg DNA, respectively, and AMV had 3.23 ± 0.5 µg
-lac/mg DNA (mean ± SEM).
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Discussion |
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We assessed the proliferation rate (percentage cyclin D-labeled and total DNA content) as well as the levels of differentiation (-lac content) of the mammary glands of parous rats after 7 days of hormonal treatment. The same parameters were assessed in the mammary glands of untreated parous rats and AMV at the same time. Important findings emerged from these assessments. Firstly, the mammary glands of parous rats not receiving any hormonal treatment had the highest level of differentiation as compared with all the other animal groups, but any hormonal treatment of the parous rats reduced the level of differentiation. E2 treatment, either alone or, more dramatically, with P4 was the most effective in reducing mammary differentiation. At the same time, the rate of proliferation of the hormone-treated parous rats was increased. Therefore, it appears that some proportion of the mammary epithelia that survive the involution of the gland after weaning are differentiated. This is in keeping with what Russo and his associates maintain, namely that the mammary gland of the parous rat retains a higher level of differentiation after involution as compared with AMV rats (46). However, with a hormonal environment that stimulates mammary gland development, the gland becomes highly susceptible to carcinogenesis, so much so that it even exceeds the susceptibility in AMV rats. Therefore, the mammary epithelia from the parous animals is capable of rapid proliferation under the appropriate hormonal stimulation. It should be pointed out, in this context, that the whole mount preparations of the parous rats revealed structures associated with mammary gland proliferation (endbuds) even in animals that did not receive any hormonal treatment (Figure 2
). Also supporting the proliferative capability of mammary epithelial cells from parous animals is the finding that isolated mammary cells and mammary explants from parous mice have only slightly reduced efficiency in filling cleared mammary fat pads of virgin female hosts as compared with virgin donors (19). Another finding worth noticing is that although the mammary glands of all parous rats treated with E2 alone or with E2 + P4 showed a significant increase in cell proliferation, the correlation between cell growth and mammary carcinogenesis was not always obvious when all the animal groups were considered. For example, no difference was found in the percentage cyclin D immunostaining or total DNA content of mammary glands of parous untreated rats and AMV animals. However, the susceptibility of these two groups to mammary tumor development was significantly different. Therefore, it appears that a high cell proliferation rate, per se, is not essential for high susceptibility of the gland to tumorigenesis, rather that the activity of particular genes is the determining factor. These genes would then either be largely non-expressed or show a high level of expression in the parous glands without hormonal stimulation, but the opposite situation would be found in the mammary gland of AMV rats. We have, for example, shown that the expression of both the estrogen receptor and the epidermal growth factor receptor are significantly reduced in mammary glands of parous rats compared with the glands of AMV animals (8). It has also been shown that the expression of BRCA1 and BRCA2 genes is increased in the parous mouse mammary gland compared with age-matched virgin animals (20,21); a finding that may be of great importance as inactivation of these genes is associated with increased risk in developing familial breast cancer (22). Obviously, more work is needed here to determine in detail the differences in gene expression of parous versus virgin mammary glands and how they relate to the changes in gene expression after hormonal treatments that has been shown to increase mammary cancer development.
It is known that treatment with E2 stimulates the release of both PRL (23) and GH (24,25); and PRL, either alone or synergistically with E2, stimulates P4 production (26,27). In this study, we found that all animal groups treated with E2 had elevated concentrations in serum of PRL, GH and P4. This complicates all interpretations of the results, as E2 could be acting directly on the mammary gland to affect development and tumorigenesis, it could be acting by stimulating the release of other hormones that, in turn, stimulate the gland, but most likely both of these events are taking place, as evidence for the importance of all these hormones in mammary gland growth, differentiation and possibly carcinogenesis is numerous (28).
Administration of P4 alone did significantly increase mammary tumorigenesis in the parous rats. Here the effect on the mammary gland may be more direct than those of E2 because the P4 treatment did not obviously affect the circulating concentrations of other hormones. Progesterone treatment has been shown to stimulate chemically induced mammary cancer in rats (29,30), although the effects of P4 or other hormonal treatments on pregnancy-associated protection against mammary cancer were not known before. Accompanying the increase in mammary tumorigenesis of P4-treated rats was a significant increase in the cell proliferation and reduction in -lac content of the mammary gland. However, these changes in growth and differentiation of the mammary gland were not as pronounced as after E2 treatment. Nevertheless, association of mammary carcinogenesis with increase in proliferation and reduction in differentiation is apparent.
Reports on the effects of thyroid hormones on chemically induced mammary cancer in rats are conflicting, with some researchers reporting some stimulatory effects (31) but others very little effects (32) or even reduction with high doses of thyroxine (33). We found that treatment of parous rats with T4 alone caused a slight increase in mammary tumor incidence, but this was not significantly different from untreated parous rats. Here again, a reduction was seen in the -lac content of the mammary gland but no significant effects were seen in cyclin D immunostaining or DNA content of the mammary gland after T4 treatment.
Untreated control animals (carrying cellulose capsules) and animals that received hormonal treatment only, but were not injected with MNU did not, in most cases, develop palpable mammary tumors. The only exceptions were that one animal each in the parous rat groups treated with E2 + P4 and E2 + P4 + T4 developed mammary carcinoma. Although the tumor incidence in these two groups was not significantly different from that of untreated controls, this finding is noteworthy. For example, long-term treatment with high doses of 17ß-estradiol and progesterone has been shown to cause mammary cancer in rats after 67 months of latency (34), and evidence suggests that estradiol or estradiol metabolites may be genotoxic carcinogens in their own right (35), quite aside from the hormonal effects of estradiol to stimulate tumor progression and perhaps cancer initiation (12,28). The results presented here, although not representing a large sample size, support this notion.
In conclusion, parous rats that show almost complete refractoriness to chemically induced mammary carcinogenesis acquire high susceptibility after hormonal treatment. The increase in mammary tumorigenesis was usually associated with a low level of differentiation and high rate of proliferation in the mammary gland, but this correlation was not always apparent, indicating that activation or inactivation of specific genes was required for high mammary cancer incidence.
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Notes |
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Acknowledgments |
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References |
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