Susceptibility of lactating rat mammary glands to gamma-ray-irradiation-induced tumorigenesis

Lactating rats of the Wistar-MS strain were irradiated with 260 cGy of gamma rays 21 days after parturition (day 21). Diethylstilbestrol (DES) pellets were implanted one month after termination of nursing and were allowed to remain for one year. A significantly higher incidence (96.4%) of mammary tumors was observed in these rats irradiated during late lactation than in virgin irradiated animals (30.4%). A control group of lactating animals irradiated during late lactation but not treated with DES was also observed for one year; the final incidence of mammary tumors in this group was 35.3%. The latency period was shortest in the DES-treated group irradiated during late lactation. Histological examination showed that the mammary glands of lactating rats were highly developed, with alveoli filled with milk. Five days after weaning, there was degeneration of alveolar tissue, concomitant with a marked decrease in the concentration of estrogen and prolactin receptors. A considerable amount of epithelial tissue remained in the mammary glands during the process of atrophy. When the rats were irradiated 5 days after weaning, and then were treated with DES for one year, the incidence of mammary tumors was 73.3%, significantly higher than that in virgin irradiated rats. However, this incidence was not significantly different from that in animals irradiated during late lactation. These results suggested that the induction of mammary tumors by gamma irradiation before or after weaning was more dependent upon the stage of differentiation in mammary glands than upon the proliferative activity of epithelial cells, and that DES is essential as a promoter for radiation-induced mammary tumorigenesis.     

Protein-coding capacities of polyadenylated RNAs from normal and neoplastic rat mammary tissues

Patterns of gene expression in normal and neoplastic rat mammary tissues were compared by cell-free translation of their total polyadenylated RNAs and by dot blot hybridization of the RNA to cloned complementary DNA probes for six of the major milk proteins, including: Mr 42,000 and 25,000 caseins, X-casein, whey phosphoproteins, kappa-proteins, and alpha-lactalbumin. Distinct but overlapping messenger RNA populations were evident from the translation patterns of normal virgin, pregnant, and lactating mammary glands. Dot blot analysis showed that each milk protein RNA had a different characteristic accumulation profile during pregnancy and lactation. The MTW9 and MCCLX mammary tumor lines, which are transplantable, prolactin dependent for growth, and produce alpha-lactalbumin, both showed high alpha-lactalbumin and Mr 42,000 casein messenger RNA activity. The tumors also had other milk protein RNA sequences, although in different proportions than at any stage during functional differentiation of normal adult mammary gland. Our results indicate that normal pregnant mammary gland expresses all of the abundant milk protein genes prior to detectable milk secretion. The patterns of gene expression in the two mammary tumors do not appear to correspond to any particular stage of functional differentiation of the normal mammary gland.     

Effect of dietary fat or tamoxifen on the expansion of cells harboring Ha-ras oncogenes in mammary glands from methylnitrosourea-treated rats

Diets containing high levels of fat enhance the formation ofmethylnitrosourea (MNU)-induced mammary gland adenocarcinomasin rats, while administration of the antiestrogen tamoxifendecreases the incidence of these tumors. It is not known, however,at what stage during tumor development the fat or tamoxifenexert their effects. Here we have used a PCR/liquid hybridizationand gel retardation assay to determine the effects of dietaryfat and tamoxifen on the growth rate of cells harboring an Ha-rasoncogene in the mammary glands of rats at various times followingMNU administration. Glands from animals on a high-fat diet hadsignificantly higher mutant cell fractions than those on a low-fatdiet at both 30 and 75 days following MNU treatment. In contrast,there was no difference between the mutant cell fractions oftamoxifen-treated animals and controls at either 30 or 70 days.These results suggest that dietary fat promotes tumor formationearly in carcinogenesis by stimulating the growth of cells harboringHa-ras mutations, while tamoxifen delays the appearance of tumorseither by acting as a tumoristatic or tumoricidal agent, orby acting to eliminate or retard the growth of preneoplasticcells just prior to the emergence of tumors.     

Distribution of epidermal growth-factor receptors in normal and neoplastic mammary tissues

Epidermal growth factor (EGF) is considered to be mitogenic for proliferation of mammary glands in animals. The action of EGF is mediated by specific EGF receptors (EFG-R). In the present study, we investigated distribution of EGF receptors during various physiological stages of mammary glands, N-methyl-N-nitrosourea (MNU)-induced mammary tumors in rats and human breast cancer samples. EGF receptor concentrations were determined by Scatchard analyses in the membrane fraction of the tissues. Results showed increased EGF receptor levels in the structurally differentiated mammary tissues from pregnant rats; whereas lower concentrations were observed in the functionally differentiated glands from lactating rats. EGF receptors were absent in the majority of the tumors induced by MNU. The loss of EGF receptor was not observed during the first 20 days post carcinogen treatment, but appeared to be correlated with the onset of the tumor. Consistent with the literature, the majority of the steroid receptor positive human breast cancer samples were EGF receptor negative, whereas steroid receptor negative samples contained EGF receptors. These results suggest that the loss of EGF receptors in ovarian hormone dependent mammary tumors does not occur gradually during carcinogenesis but appears to be a characteristic of hormone dependent mammary tumor cells.     

Estrogen receptor binding to nuclei from normal and neoplastic rat mammary tissues in vitro

To investigate the role of hormones in regulating growth of neoplastic mammary cells, we established a heterologous assay for studying interactions of partially purified calf uterine [3H]estradiol-charged estrogen receptor ([3H]ER) with rat tumor nuclei in vitro. This system displays saturable high affinity binding of [3H]ER which is time and salt dependent. Optimal assay conditions required for the heterologous system were identical to those we reported for the homologous calf nuclear binding system. Specificity of [3H]ER binding was demonstrated; 10-fold excess unlabeled estrogen-charged ER (EcR) competed for greater than 90% of the [3H]ER binding sites and binding of [3H]estradiol (not complexed with ER) was less than 1% of [3H]ER binding. Binding of [3H]ER displayed tissue specificity in decreasing order: R3230AC mammary tumor greater than lactating mammary gland = liver greater than kidney greater than lung. Scatchard analysis of saturation data provided estimates of binding affinity to nuclei from R3230AC mammary tumors [Kd, 2.0 +/- 0.3 (SE) nM); the number of binding sites per nucleus for R3230AC tumors was 95,000 +/- 13,800. [3H]ER binding to nuclei isolated from R3230AC rat mammary tumors grown in intact rats was 40% higher than that observed in tumors from ovariectomized animals. Results of administration of individual pharmacological doses of either progesterone or an estrogen to ovariectomized rats did not restore nuclear ER binding levels in R3230AC tumors to those detected in tumors from intact rats. These results suggest that the physiological levels of endogenous hormones produced by the ovaries are important in regulating the number of ER binding sites in nuclei from these mammary tumors.     

Methylation and expression of rat kappa-casein gene in normal and neoplastic rat mammary gland

The kappa-casein mRNA was evaluated in the rat mammary gland during functional differentiation and neoplastic growth. Using a dot-blot assay, the mRNA was barely detectable in the virgin gland; it steadily increased from the onset of gestation and leveled off during lactation. In rat mammary tumors, either primary (7,12-dimethylbenz(a)anthracene-induced) or transplanted (MTW9), the level of kappa-casein mRNA was about 2.5-fold lower than in the lactating gland, but an extensive variation among individual tumors was observed. There was no detectable kappa-casein mRNA in rat liver. In the mammary gland of virgin, 10-day pregnant, and nonlactating females, the DNA sequences within and/or around the kappa-casein gene were found to be hypermethylated at the HpaII-MspI sites as compared to 10-day lactating females. In the two tumors studied, the kappa-casein gene was partially methylated at the same sites. Prolactin treatment induced kappa-casein gene expression in the virgin rat mammary gland but did not result in a change of the methylation status at the HpaII-MspI sites. Under similar conditions of prolactin treatment, however, the methylation of the Sau96I sites was reduced, and an inverse correlation between the onset of kappa-casein gene methylation and kappa-casein gene expression was evident in both the virgin gland and the tumors. Thus, the expression of kappa-casein was found to be inversely correlated with the extent of methylation of the kappa-casein gene, except in the case of the prolactin-stimulated virgin gland.     

Binding of peanut lectin to breast epithelium, human carcinomas, and a cultured rat mammary stem cell: use of the lectin as a marker of mammary differentiation.

We investigated the binding of fluorescence-labeled peanut agglutinin (PNA) to breast epithelium. Specific binding of PNA to the mammary glands of female Sprague-Dawley rats increased as the gland matured. Sexually immature rats showed relatively little fluorescence, but this increased in mature and pregnant animals. A maximum was reached in lactating rats in which significant labeling of material within the lumen was observed. PNA was bound exclusively to the epithelial and not the myoepithelial or mesenchymal cells. In tissue culture, a rat mammary epithelial stem cell line, which can be stimulated to differentiate to alveolus-like secretory or myoepithelial cells, showed evidence of PNA binding only on the secretory cells and not on unstimulated or myoepithelial cells. Fibroblast cultures also failed to show significant binding of PNA. Receptor sites on the secretory cells were masked mainly by sialic acid. Human breast sections, like those of the rat, showed fluorescent labeling at the apical region of the epithelial cells; this labeling increased if the tissue had prior treatment with neuraminidase. Breast carcinomas that were morphologically differentiated showed more labeling with PNA than did undifferentiated tumors, which often had weak or sometimes negative labeling. When significant fluorescence was observed, it was localized mainly in the cytoplasm. By contrast, labeling was restricted to the cell periphery in differentiated carcinomas. The use of PNA as a marker for breast epithelial cell differentiation is therefore proposed.     

Suppression of mouse mammary tumorigenesis by long-term tamoxifen therapy

A sustained release of tamoxifen, which produced decreasing serum levels of this drug (24 to 4 ng/mL) over 6 months, suppressed mammary tumorigenesis in virgin or once pregnant C3H/OUJ female mice. Tamoxifen was consistently more effective than early ovariectomy, which only delayed tumorigenesis. Tamoxifen prevented the stimulatory action of cyclical (alternate-month) progesterone administration on mouse mammary tumorigenesis. However, when tamoxifen treatment (12 months) was stopped, progesterone treatment initiated tumorigenesis. In contrast, when long-term tamoxifen treatment was stopped in mice that had not undergone ovariectomy, and estrous cycle returned, the majority of these mice remained tumor free. A comparison of different durations (3, 6, and 12 months) of tamoxifen treatment of virgin mice, starting at approximately 4 months of age, showed an equivalent effect on mammary tumorigenesis. All virgin mice developed tumors by 18 months of age, whereas 80% of the tamoxifen-treated mice were tumor free. Nevertheless, cyclical progesterone administration caused rapid development of tumors after 3 months of tamoxifen treatment; only 15% of these mice were tumor free at 18 months. Cyclical progesterone administration caused an increase in tumorigenesis after 6 months of tamoxifen treatment; 50% of these mice were tumor free at 18 months of age. These data demonstrate the efficacy of tamoxifen to suppress mouse mammary tumorigenesis and demonstrate that continuous tamoxifen therapy is necessary to prevent the development of tumors by progesterone, a stimulatory hormone.     

Growth factor binding to 7,12-dimethylbenz(a)anthracene-induced mammary tumors from rats subject to chronic caloric restriction

Caloric restriction (CR) inhibits tumorigenesis in rodents. To understand the basis for this effect the binding of insulin, insulin-like growth factor I/somatomedin C (IGF-I/Sm-C), insulin-like growth factor II/multiplication stimulating activity (IGF-II/MSA), and epidermal growth factor were examined to membrane preparations of 7,12-dimethylbenz(a)anthracene-induced mammary adenocarcinomas and several normal tissues from female Sprague-Dawley rats. Animals were fed ad libitum (AL) or 25% and 40% calorically restricted diets. Large, palpable (LP) and small, less than or equal to 100 mg, nonpalpable (SNP) tumors were evaluated. Growth factor binding to tumors was differentially affected by CR. IGF-I/Sm-C binding was comparable for AL-LP, AL-SNP, and 25% CR-LP tumors, but elevated in 25% CR-SNP tumors. Scatchard analysis revealed high and low affinity IGF-I/Sm-C binding sites, with AL-SNP and 25% CR-SNP tumors exhibiting similar levels of high affinity sites and at a greater concentration than AL-LP and 25% CR-LP tumors. Insulin binding to mammary tumors was low, i.e., 8- to 13-fold lower than IGF-I/Sm-C binding. The 25% CR-LP and SNP tumors bound 2- to 5-fold more insulin than corresponding AL-LP and SNP tumors. Binding of IGF-II/MSA to these tumor preparations was high, approximately 11- to 25-fold greater than insulin binding, and was unaffected by CR or tumor size. The binding of epidermal growth factor was not detected in any tumor preparations. Receptor binding studies were confirmed with covalent cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Normal tissues exhibited tissue- and growth factor-specific alterations in binding with host CR. Thus, alterations in growth factor binding were not tumor specific, but were less pronounced than in mammary tumors. These findings suggest alterations in IGF-I/Sm-C and insulin binding properties to tumors in relation to CR and tumor size may contribute, in part, to the inhibitory effects of CR on tumorigenesis.     

Distribution of retinoic acid-binding proteins in normal and neoplastic mammary tissues

Cellular retinoic acid-binding proteins were detected in chemically induced mammary tumors using sucrose density gradient analysis. Unlabeled retinoic acid did not displace nonspecific binding in the 5S region but was, however, a competitive inhibitor for the specifically binding 2S component. Mammary gland cytosol fractions from both 1-methyl-1-nitrosourea-treated and untreated as well as from lactating rats contained low levels of retinoic acid-binding proteins. 1-Methyl-1-nitrosourea treatment did not result in the increased number of binding sites. Thus, the increase in the levels of binding proteins in tumors most probably occurred during tumor development and probably was not a result of the carcinogen per se. Retinoids which have been shown to be effective in the chemoprevention of mammary carcinogenesis only partially competed for the binding sites, indicating that they may be metabolized prior to their action as an active chemopreventive agent.     

Antiestrogenic potency and binding characteristics of the triphenylethylene H1285 in MCF-7 human breast cancer cells

The antiestrogenic character and potency of 4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl)-alpha' -ethylstilbene (H1285) and its binding to estrogen receptor and to estrogen-noncompetible antiestrogen binding sites have been studied in MCF-7 human breast cancer cells. H1285 has an affinity for the estrogen receptor (Kd 0.23 nM) which is comparable to that of estradiol (Kd 0.25 nM), and the binding of these two compounds to estrogen receptor is mutually competitive. On high salt sucrose gradients, the sedimentation profiles of nuclear receptor complexes with H1285 and estradiol are different. While the sedimentation profile of the complex with estradiol varies with the buffer composition, being 4.1S in phosphate:thioglycerol: glycerol and predominantly 5.5S in Tris:EDTA buffered gradients, the H1285 receptor complex shows the same sedimentation (5.5S) regardless of the buffer composition. H1285 also binds to estrogen-noncompetable antiestrogen binding sites that are distinct from the estrogen receptor with a low affinity, only 15% that of the antiestrogen tamoxifen. The biological character and potency of H1285 were examined by determining its effects on cell proliferation, cellular progesterone receptor levels, and plasminogen activator activity. In MCF-7 cells, H1285 was a 30- to 100-fold more potent inhibitor of cell proliferation than was the antiestrogen tamoxifen, and it was approximately equipotent with the higher affinity antiestrogen trans-hydroxytamoxifen. H1285 evoked very minimal increases in cellular progesterone receptor levels, and no increase in plasminogen activator activity over a broad range of concentrations (10(-10)-10(-6)M), and it suppressed plasminogen activator activity stimulated by estradiol. Therefore, by the criteria we have used, we conclude that H1285 is a potent and very effective antiestrogen in MCF-7 cells. The ability of estradiol to reverse the suppression of cell proliferation by H1285, and the high affinity of H1285 for estrogen receptor and its low affinity for estrogen-noncompetible antiestrogen binding sites suggest that H1285 exerts its antiestrogenic effects via interaction with the estrogen receptor of these breast cancer cells.     

Nuclear thyroid hormone receptors in C3H/HeN mouse mammary glands and spontaneous tumors

Saturable, high-affinity binding sites for 3,5,3'-triiodo-L-thyronine (T3) were identified in isolated nuclei and solubilized chromatin extracts of mammary glands, spontaneous mammary tumors, and liver from C3H/HeN mice. Receptor concentration in whole mammary gland nuclei (254 fmol/mg DNA) was only about one-half that of mouse liver nuclei (536 fmol/mg DNA), but in molecular weight (55,000) and in their affinity for various thyroid hormone analogues, the binding was essentially identical. Saturation analysis of T3 binding in a series of individual spontaneous mammary tumors and pooled lactating mammary glands indicated that the concentrations of T3-binding sites of the mammary gland are conserved in the transition to neoplasms and are somewhat increased in the largest tumors. Thyroxine binding was identical in capacity to T3 binding in mammary gland nuclei and nuclear extract but showed a higher binding capacity than did T3 in the largest tumors. High-performance molecular exclusion chromatography did show a difference between mammary gland and liver in the distribution of competible [125I]T3 binding between two macromolecular forms; the excluded peak (Mr greater than 450,000) comprised 56% of the T3 binding in the liver but only 9% in the mammary gland with the included peak (Mr 55,000) contributing the balance of binding in each case. Spontaneous mammary tumor resembled the mammary gland in the macromolecular distribution of specific T3 binding (16% excluded). Thymidine uptake showed only a modest decrease in the larger tumors (greater than 2.0 g), while nuclear histone acetylase activity was significantly decreased in this group. Neither measurement showed a significant correlation with T3 or thyroxine binding capacity.     

A comparative study of inner membrane enzymes and transport systems in mitochondria from R3230AC mammary tumor and normal rat mammary gland.

Mitochondria from a rat mammary tumor (R3230AC) have been compared with mitochondria from pregnant and lactating rat mammary glands, with particular attention paid to inner membrane enzymes and Transport proteins. In the tumor the mitochondrial adenosine triphosphatase was not activated by 2,4-dinitrophenol, in contrast to the mammary mitochondria from lactating or pregnant rats. Translocation of adenosine diphosphate across the inner membrane was found to be more rapid in the tumor by virtue of lovered Km adenosine diphosphate and raised Vmax. Transport of phosphate and dicarboxylic acids occurred at similar rates in all three types of mitochondria. The inner membrane proteins were also examined directly by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and some differences are noted. These results, although they indicate subtle differences between the inner mitochondrial membranes of tumor as compared with those of pregnant or lactating rat mammary glands, cannot form the basis of an explanation for enhanced glucose utilization and aerobic lactic acid production in this tumor.     

Refractoriness to mammary tumorigenesis in parous rats: is it caused by persistent changes in the hormonal environment or permanent biochemical alterations in the mammary epithelia?

Administration of a single i.v. injection of 50 mg N-methyl-N-nitrosourea(MNU)/kg body wt to 50- to 60-day-old virgin rats, 120-day-oldvirgin rats, and 120-day-old parous rats (Sprague-Dawley; n= 18–37) resulted in a high incidence of mammary carcinomasin the virgin animals (97.3% in 50- to 60-day-old virgin rats;75.0% in 120-day-old virgin rats), but mammary carcinomas didnot develop in the parous rats. The concentrations in serumof various mammotropic hormones were measured in identical groupsof rats at the time of MNU treatment. Growth hormone (GH) concentrationwas significantly reduced in parous rats, as compared with youngor age-matched virgin rats. The concentrations of prolactin,17ß-estradiol, progesterone, corticosterone and thyroxinewere not significantly altered in the parous rats compared tothe two groups of virgin animals. Histological examination ofthe mammary glands from the three groups of rats showed thatthe epithelia of the parous animals were in a stage of regression,whereas the mammae of the young virgin rats showed the highestdegree of lobulo-alveolar development. The levels of estrogenreceptor (ER), epidermal growth factor (EGF) receptor (EGF-R)and GH receptor (GHR) in the mammary glands of the animals werealso measured. We found a reduction in the receptor levels forboth estrogen and EGF in mammary tissues from parous animals.Receptors for GH were present in normal mammary tissues fromboth virgin and parous rats. We hypothesize that the reductionin the circulating concentration of GH caused the reduced susceptibilityof parous rats to mammary carcinogenesis possibly by decreasingthe levels of ER and/or EGF-R in the mammary gland.     

Inhibition of radiogenic mammary carcinoma in rats by estriol or tamoxifen

Mammary carcinomas have been induced by 3.5 Gy whole-body gamma radiation administered at age 40 to 50 days to virgin female Sprague-Dawley rats. In 142 irradiated controls carcinoma incidence averaged 7.8% in survivors observed less than 300 days and 38.3% of those surviving longer (P less than 0.001 by t test). Mammary cancer promotion was inhibited by two methods: estriol (E3) 638 micrograms/month (2.2 microns/mo) subcutaneously for natural life span begun 2 weeks after exposure reduced cancer incidence from 76% in controls to 48% after 331 to 449 mean days observation until neoplasia was palpable (P less than 0.02 by chi-square analysis). Uterine weights were similar in control and treated groups, and were 15% to 18% greater than uteri of nonirradiated controls from other simultaneous experiments. Six monthly 638-micrograms doses of 17 alpha ethinyl estriol (EE3) reduced tumors from 88% in controls to 64% (P less than 0.05 by chi-square analysis) and delayed cancer onset (P less than 0.01-0.04 by life table analysis). Ethinyl estradiol (EE2) after 6 months' treatment similarly delayed mammary tumor development reducing incidence to 75% (NS), with a six-fold increase in nonmammary epithelial malignant tumors. Estriol administration begun between 3 days before to 5 days after radiation did not alter mammary cancer incidence in six experiments. Monthly implantation of 2.5 mg tamoxifen (4.44 microns/mo) started 2 weeks after radiation reduced mammary cancer incidence from 83% to 14% after 307 to 314 days' observation (P less than 0.001 by chi-square analysis). Treated rats had atrophic ovaries and uteri consistent with blockade of endogenous estradiol activity. Short-term parenteral E3 or EE3 therapy using 10 to 30 micrograms/kg/day (35-100 microns/kg/day) rapidly differentiated virgin rat mammary glands without impairment of subsequent estrus cycles and offers an alternative to castration or life-long antiestrogen therapy for reduction of risk of radiogenic mammary carcinoma.     

Prevention of age-related spontaneous mammary tumors in outbred rats by late ovariectomy

Background: Breast cancer prevention trials have shown that the antiestrogen tamoxifen inhibits development of estrogen receptor (ER)-positive tumors. In Sprague-Dawley rats, removal of ovarian function in young animals can reduce the incidence of spontaneous age-dependent mammary tumors. However, it is not known whether removal of ovaries late in life, before middle age onset, can still prevent mammary tumor development. Methods: In this study we used Hsd:Sprague-Dawley^® SD^® (Hsd) rats to determine the effect of late ovariectomy on mammary tumor development. Intact, sham-ovariectomized and ovariectomized rats were followed until 110 weeks of age, or over their life span. In some experiments, palpable tumors were surgically removed upon presentation. Results: Removal of ovaries before middle age onset (∼5-7 months) inhibited development of spontaneous mammary tumors by 95%. Only one mammary tumor was observed in 19 late ovariectomized animals while 47 total tumors developed in 42 non-ovariectomized animals. Tumor incidence was reduced from 73.8 to 5.3% (relative risk = 0.05, 95% CI = 0.0072-0.354). The frequency of mammary carcinomas in non-ovariectomized virgin female rats was one in eight rats. Spontaneous rat carcinomas expressed ER and other biomarkers, such as cyclin D1. When palpable tumors were removed by surgical excision, tumor multiplicity increased from 0.76 to 1.61 tumors per rat. Surprisingly, ovariectomy increased the 110-week survival rate and maximum life span of Hsd rats. Conclusion: Late ovariectomy prevents spontaneous mammary tumor development in Hsd rats. This animal model may be useful for evaluating novel interventions in breast cancer prevention.     

Differences in mediated mutagenesis and poly cyclic aromatic hydrocarbon metabolism in mammary cells from pregnant and virgin rats

The physiological state of pregnancy confers significant resistanceto poly cyclic aromatic hydrocarbon (PAH)-induced mammary tumorigenesis.We have tested the abilities of primary mammary cells from pregnantand virgin rats cultured on collagen-coated plates to metabolizePAHs and convert these carcinogens to mutagenic derivatives.Using a cell-mediated mutagenesis assay, we found that mammaryepithelial cells from pregnant rats produced half the levelsof mutagenic 7,12-dimethylbenz[a]anthracene (DMBA) metabolitesformed by cells from virgin rats. Pregnant-derived mammary cellsalso showed consistently lower levels of metabolism of DMBAand benzo[a]pyrene (B[a]P) than cells from virgin rats. H.p.l.c.analysis of B[a]P metabolism by these cell populations indicatedno significant qualitative differences in the extracellularand intracellular metabolites formed. We have previously shownthat mammary cells from rat strains exhibiting significant differencesin susceptibility to PAH-induced tumors have equivalent qualitativeand quantitative abilities to metabolize PAH carcinogens. Ourcurrent data suggest that modifications in mammary tumor susceptibilityfound in various physiological states, unlike genetically determineddifferences, may be related in part to an altered ability toactivate chemical carcinogens within the mammary gland.     

Tamoxifen inhibition of estrogen receptor-alpha-negative mouse mammary tumorigenesis

Tamoxifen reduces the relative risk of breast cancer developing from specific premalignant lesions. Many breast cancers that arise after tamoxifen treatment are estrogen receptor-alpha (ER-alpha)-negative, although premalignant lesions such as atypical ductal hyperplasia are highly ER-alpha-positive. The p53 null mouse mammary epithelial transplant model is characterized by ER-alpha-positive premalignant lesions that give rise to both ER-alpha-positive and ER-alpha-negative tumors. Given this progression from ER-alpha-positive to ER-alpha-negative lesions, we tested the ability of tamoxifen to block or delay mammary tumorigenesis in several versions of this model. In groups 1 and 2, p53 null normal mammary epithelial transplants were maintained in virgin mice. In groups 3 to 5, the p53 null and mammary transplants were maintained in mice continuously exposed to high levels of progesterone. In groups 6 and 7, transplants of the premalignant outgrowth line PN8a were maintained in virgin mice. Tamoxifen blocked estrogen signaling in these mice as evidenced by decreases in progesterone-induced lateral branching and epithelial proliferation in the mammary epithelium. Tamoxifen did not alter the elevated levels of progesterone in the blood while significantly reducing the circulating level of prolactin. Tamoxifen reduced tumor incidence in p53 null normal mammary epithelial transplants maintained in virgin mice from 55% to 5% and in progesterone-stimulated mice from 81% to 21%. The majority of the resultant tumors were ER-alpha-negative. Tamoxifen also significantly delayed tumorigenesis in the ER-alpha-positive high premalignant line PN8a from 100% to 75%. These results show that tamoxifen delays the emergence of ER-alpha-negative tumors if given early in premalignant progression.     

Prevention of age-related spontaneous mammary tumors in outbred rats by late ovariectomy

Background: Breast cancer prevention trials have shown that the antiestrogen tamoxifen inhibits development of estrogen receptor (ER)-positive tumors. In Sprague–Dawley rats, removal of ovarian function in young animals can reduce the incidence of spontaneous age-dependent mammary tumors. However, it is not known whether removal of ovaries late in life, before middle age onset, can still prevent mammary tumor development. Methods: In this study we used Hsd:Sprague–Dawley^® SD^® (Hsd) rats to determine the effect of late ovariectomy on mammary tumor development. Intact, sham-ovariectomized and ovariectomized rats were followed until 110 weeks of age, or over their life span. In some experiments, palpable tumors were surgically removed upon presentation. Results: Removal of ovaries before middle age onset (∼5–7 months) inhibited development of spontaneous mammary tumors by 95%. Only one mammary tumor was observed in 19 late ovariectomized animals while 47 total tumors developed in 42 non-ovariectomized animals. Tumor incidence was reduced from 73.8 to 5.3% (relative risk = 0.05, 95% CI = 0.0072–0.354). The frequency of mammary carcinomas in non-ovariectomized virgin female rats was one in eight rats. Spontaneous rat carcinomas expressed ER and other biomarkers, such as cyclin D1. When palpable tumors were removed by surgical excision, tumor multiplicity increased from 0.76 to 1.61 tumors per rat. Surprisingly, ovariectomy increased the 110-week survival rate and maximum life span of Hsd rats. Conclusion: Late ovariectomy prevents spontaneous mammary tumor development in Hsd rats. This animal model may be useful for evaluating novel interventions in breast cancer prevention.