Elevated serum calcium concentrations in mammary tumor bearing C3H/Fg mice without bony metastases

Sera from eight adult C3H/Fg mice bearing spontaneous mammary gland tumors were analyzed for calcium content in two successive weekly samples. The sera from six mice were fount to have significantly elevated serum calcium concentrations while sera from two were normocalcemic. Intramuscular implantation of mammary gland tumors into 12, 3-month-old female C3H/Fg mice resulted in significant and progressive increases in serum calcium concentrations over a 4-week period. Subcutaneous implants had a similar effect, but gave less consistent results. These data supported the hypothesis that mammary gland tumors are capable of inducing elevated serum calcium concentrations in C3H/Fg mice. It was concluded that low grade elevation of serum calcium occasionally seen in adult female C3H/Fg mice may be caused by incipient mammary gland tumors.     

Characterization of spontaneous mammary gland carcinomas in female baboons

Spontaneous mammary gland carcinomas occurred in five baboons during a 13-year period at Southwest Foundation for Biomedical Research. The affected baboons ranged in age from 21 to 33 years. Menopause in the baboon occurs at approximately 26 years of age. All five animals had typical invasive ductal carcinoma. Morphologically, the tumors were characterized by neoplastic cells arranged from pseudopapillary and cribiform to more poorly differentiated solid cellular growth patterns. Additional features included lack of tubule formation (4/5), marked nuclear pleomorphism (5/5), a high mitotic rate (4/5) and tumor necrosis (4/5). Applying a grading system used for breast cancer in women, four tumors were graded as poorly differentiated carcinomas and one was graded as moderately differentiated. Co-existent ductal carcinoma in situ (DCIS) was observed in three of the mammary tumors. Metastases to the regional lymph nodes were confirmed in two animals, both with histological evidence of lymphovascular invasion in the primary tumor. Distant metastases were observed in only one animal. Immunohistochemical staining for human therapeutic markers revealed 2/5 tumors strongly positive for estrogen receptor, 1/5 strongly positive for progesterone receptor and 4/4 negative for HER2 expression. Although the incidence appears to be low, these five cases of mammary carcinoma in female baboons suggest that when present baboon mammary carcinoma is usually of ductal origin and behaves similar to a human breast carcinoma.     

Multiple cystic sweat gland tumors in transgenic mice

Here we describe gross and microscopic sweat gland tumors found in a transgenic mouse model of breast cancer, which had transforming growth factor α under the control of mouse mammary tumor virus promoter (MMTV-TGFα). Initially, 20% of the mice in the colony were affected. Cystic lesions formed on the phalanges, palmar surfaces of the metacarpals, and plantar surfaces of the metatarsals. The lesions were multifocal and nonulcerated with straw-colored fluid, ranging in size from 1 to 30 mm at the largest dimension. The colony was monitored for 6 mo; during that time, the prevalence of lesions increased to 52% of the mice. Histologically, in most cases the cyst walls were lined by 1 or 2 layers of normal-appearing epithelial cells that resembled basal cells, indicating adenoma. However, 2 cysts from 2 different mice had papillary proliferative projections and extensive disorganized glandular structures that protruded into the cyst cavities, indicating adenocarcinoma. In these 2 cases, the neoplastic cells revealed architectural and cytologic atypia with rare mitoses. Similar findings have previously been observed in sweat gland tumors; however, multiple sweat-gland tumors have not been reported in mice.     

Abnormal ornithine decarboxylase activity in transgenic mice increases tumor formation and infertility

A transgenic mouse line carrying ornithine decarboxylase cDNA as the transgene under the control of a mouse mammary tumor virus long terminal repeat (MMTV LTR) promoter was generated in order to study whether ornithine decarboxylase transgene expression will have any physiological or pathological effect during the entire life of a transgenic mouse. The high frequency of infertile animals and the loss of pups made the breeding of homozygous mice unsuccessful. However, a colony of heterozygous transgenic mice was followed for 2 years. In adult heterozygous transgenic mice, ornithine decarboxylase activity was significantly increased in the testis, seminal vesicle and preputial gland when compared to non-transgenic controls. In contrast, ornithine decarboxylase activity was decreased in the kidney and prostate of transgenic mice. No significant changes in ornithine decarboxylase activity were found in the ovary and mammary gland and only moderate changes in ornithine decarboxylase activity were detected in the heart, brain, pancreas and lung. The most common abnormalities found in adult animals (12 males and 20 females) of the transgenic line were inflammatory processes, including pancreatitis, hepatitis, sialoadenitis and pyelonephritis. Spontaneous tumors were observed in eight animals, including two benign tumors (one dermatofibroma, one liver hemangioma) and six malignant tumors (one lymphoma, one intestinal and three mammary adenocarcinomas and one adenocarcinoma in the lung). No significant pathological changes were found in 17 nontransgenic controls.     

Morphometric analysis of AgNORs in tubular and papillary parts of canine mammary gland tumors

OBJECTIVE: To test the value of the silver staining nucleolar organizer regions (AgNORs) technique on canine mammary gland tumors using image analysis and to estimate differences in AgNOR parameters in structurally different parts of canine mammary gland tumors. STUDY DESIGN: Analysis was performed on 13 complex type and 10 simple type malignant canine mammary gland tumors containing tubular and/or papillary structures. Ten normal mammary glands were used as controls. Morphometric analysis was done by a computer-assisted image analysis system and consisted of evaluation of nuclear area, number and area of AgNORs per nuclear area, ratio of nuclei with five or more AgNORs, nuclear perimeter, area fraction between nuclear area and area of AgNORs, and area, equivalent diameter, volume equivalent sphere, perimeter and circularity of a singular AgNOR. RESULTS: Distinct differences were detected between normal and malignant mammary gland tissue for all measured parameters. There were no significant differences between the tubular and papillary parts of the same tumor or between the tubular and papillary parts of complex and simple type tumors. CONCLUSION: Despite the fact that no significant differences were found for AgNOR parameters between papillary and tubular structures of mammary gland tumors, the results of grouping tumors by the number of AgNORs indicate that this might help with classification of canine mammary gland tumors.     

Breast cancer protein PS2 synthesis in mammary gland of transgenic mice and secretion into milk

PS2, a small estrogen-inducible secretory polypeptide with structural analogies to a growth factor, is produced by approximately 50% of human breast tumors. The function of PS2 is, however, unknown. To determine whether PS2 may play an autocrine role in the development of mammary tumors we constructed transgenic mice bearing fusion constructs designed to direct the expression of human PS2 in the lactating mammary gland under the control of the whey acidic protein (WAP) promoter. Mouse lines bearing the genomic PS2 gene under the control of the WAP promoter region (WAP-PS2-2) failed to express the transgene. However, mice harboring the fusion construct WAP-PS2-1, in which the PS2 coding sequence is inserted into the 5' untranslated region of the complete WAP gene, were observed to express the transgene. Expression was restricted to the secretory epithelium of the mammary gland during lactation, and PS2 protein was secreted into the milk. Nevertheless, no mammary gland dysplasia was observed, and PS2 expression had no discernable effect upon the physiology and/or development of the suckling young or the transgenic mother.     

Melatonin, experimental basis for a possible application in breast cancer prevention and treatment

The role of the pineal as an oncostatic gland has been studied in animal models of tumorigenesis, especially on those concerning the mammary gland. The general conclusion is that experimental manipulations activating pineal gland, or the administration of melatonin, reduce the incidence and growth rate of chemically-induced murine mammary tumors, while pinealectomy or situations which implicate a reduction of melatonin production usually stimulate mammary carcinogenesis. The direct actions of melatonin on mammary tumors have been suggested because of its ability to inhibit, at physiological doses (1nM), the in vitro proliferation of MCF-7 human breast cancer cells. In this article we review the outstanding findings related to melatonin actions on mammary which, taken together, support a possible usefulness of this indoleamine in the prevention and treatment of mammary gland malignancy.     

Expression pattern of four membrane-type matrix metalloproteinases in the normal and diseased mouse mammary gland

Both mammary gland development and mammary carcinogenesis involve extensive remodeling of the mammary gland extracellular matrix. The expression of four membrane-type matrix metalloproteinases (MT-MMPs) with matrix remodeling potential in development and tumorigenesis was evaluated by in-situ hybridization on mouse mammary gland sections. MT1-MMP and MT3-MMP were found in the mammary stroma mainly around epithelial structures in both developing and mature mammary gland. In contrast, MT2-MMP was found exclusively in the mammary epithelium. Lactating gland expressed none of the examined MT-MMPs. Mammary gland tumors expressed MT1-MMP, MT2-MMP, and MT3-MMP while MT4-MMP was not expressed in any developmental or cancerous stage analyzed here. Our results suggest that MT1-MMP, MT2-MMP, and MT3-MMP may be involved in remodeling of both the normal and diseased mammary gland either directly or indirectly by activation of other MMPs.     

Effects of Enriched Environment on COX-2, Leptin and Eicosanoids in a Mouse Model of Breast Cancer

Cyclooxygenase-2 (COX-2) and adipokines have been implicated in breast cancer. This study investigated a possible link between COX-2 and adipokines in the development of mammary tumors. A model of environmental enrichment (EE), known to reduce tumor growth was used for a syngeneic murine model of mammary carcinoma. 3-week-old, female C57BL/6 mice were housed in standard environment (SE) or EE cages for 9 weeks and transplanted orthotopically with syngeneic EO771 adenocarcinoma cells into the right inguinal mammary fat pad. EE housing influenced mammary gland development with a decrease in COX-2 expressing cells and enhanced side-branching and advanced development of alveolar structures of the mammary gland. Tumor volume and weight were decreased in EE housed mice and were associated with a reduction in COX-2 and Ki67 levels, and an increase in caspase-3 levels. In tumors of SE mice, high COX-2 expression correlated with enhanced leptin detection. Non-tumor-bearing EE mice showed a significant increase in adiponectin levels but no change in those of leptin, F₂-isoprostanes, PGF_2α, IL-6, TNF-α, PAI-1, and MCP-1 levels. Both tumor-bearing groups (SE and EE housing) had increased resistin, IL-6, TNF-α, PAI-1 and MCP-1 levels irrespective of the different housing environment demonstrating higher inflammatory response due to the presence of the tumor. This study demonstrates that EE housing influenced normal mammary gland development and inhibited mammary tumor growth resulting in a marked decrease in intratumoral COX-2 activity and an increase in the plasma ratio of adiponectin/leptin levels.     

Chronic administration of dehydroepiandrosterone (DHEA) to female monkey and rat has no effect on mammary gland histology

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, has been shown to exert chemopreventive effect on the development of carcinogen-induced rat mammary tumors. Since little is known on the effect of DHEA administration on mammary gland physiology and histology, we have studied the effect of long-term administration of DHEA to normal female monkey and rat on mammary gland histology as well as on serum DHEA, DHEA sulphate (DHEA-S), testosterone and estradiol levels. In monkeys, DHEA treatment (2 or 10 mg/(kg b.w.day)) induced a dose-related increase in serum DHEA and DHEA-S (above 20-fold) levels. At the highest dose of DHEA, serum testosterone levels were significantly increased (three- to fourfold), while serum estradiol concentration was not modified. DHEA treatment did not modify the histological characteristics of monkey mammary glands. In the rat, following DHEA administration (10 or 100 mg/(kg b.w.day)), a dose-related marked increase in serum DHEA and DHEA-S was observed. Serum testosterone was also increased in DHEA-treated animals, while no significant changes in serum estradiol levels were detected. As in the monkey, the histology of the female rat mammary gland remained unchanged following long-term treatment with any of the two doses of DHEA.     

Integrin-interacting protein Kindlin-2 induces mammary tumors in transgenic mice

Kindlin-2, an integrin-interacting protein, regulates breast cancer progression. However, currently, no animal model to study the role of Kindlin-2 in the carcinogenesis of mammary gland is available. We established a Kindlin-2 transgenic mouse model using a mammary gland-specific promoter, mammary tumor virus (MMTV) long terminal repeat (LTR). Kindlin-2 was overexpressed in the epithelial cells of the transgenic mice. The mammary gland ductal trees were found to grow faster in MMTV-Kindlin-2 transgenic mice than in control mice during puberty. Kindlin-2 promoted mammary gland growth as indicated by more numerous duct branches and larger lumens, and more alveoli were formed in the mammary glands during pregnancy under Kindlin-2 overexpression. Importantly, mammary gland-specific expression of Kindlin-2 induced tumor formation at the age of 55 weeks on average. Additionally, the levels of estrogen receptor and progesterone receptor were decreased, whereas human epidermal growth factor receptor 2 and β-catenin were upregulated in the Kindlin-2-induced mammary tumors. These findings demonstrated that Kindlin-2 induces mammary tumor formation via activation of the Wnt signaling pathway.

     

Non-invasive methods of separating hedgehog (Erinaceus europaeus) age classes and an investigation into the age structure of road kill

The separation of animals into age classes is often fraught with difficulties, but is essential for the interpretation of survival rates and population structure. This study investigated both live hedgehogs and road kill in order to develop techniques that could be utilised to separate age classes in the field. Body measurements were taken from 24 live hedgehogs caught at a site in County Cork, Ireland over a three year period. Additionally, carcasses from around Ireland were collected and measured. There was a significant correlation between the weight and hind foot length of both live and dead hedgehogs. The weight of individuals was also significantly correlated with the body length and jaw length of hedgehogs. In all of these correlations, the sample clustered clearly into two groups with ‘juveniles’ meeting all of the following criteria: a hind foot length of <3.6 cm, a body length of <?16 cm and a jaw length of below 4.5 cm. It is suggested that these parameters, combined with the presence of growing spines, could provide a way of separating age classes in the field. This would be of particular benefit to short term studies, allowing the population structure to be accurately assessed and the reproductive success and sustainability of a population to be investigated. Road kill were also aged using the dentary bone in order to investigate the age profiles of hedgehogs killed on the road. Amongst those killed, 54 % of hedgehogs were 1-year-old or less. The majority of hedgehogs (87 %) were between 0–3-years-old, the oldest females were nine (n?=?2), and the oldest males were eight years old (n?=?2), respectively. Road kill hedgehogs had an average longevity of 1.94 years, and the mean age of females was higher (2.10 years) than males (1.87 years).     

Prolactin binding to dissociated cells from rat mammary tumors and mammary gland.

Prolactin binding activity was studied in suspensions of cells which had been enzymatically dissociated from R3230AC mammary tumors, 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors and lactating rat mammary glands. Prolactin bound specifically with high affinity (apparent binding affinity = 4.0 X 10(9) M-1) to R3230AC tumor cells. Hormone binding at room temperature was proportional to cell number and increased with time of incubation up to 120-180 min. Prolactin binding to R3230AC tumor cells from diabetic animals was reduced by about 50%. Specific prolactin binding activity was also demonstrated in preparations of cells from DMBA-induced tumors and lactating mammary gland. The levels of hormone binding in both dissociated cells and subcellular particles prepared from these tissues varied as follows: DMBA-induced tumors > lactating mammary gland > R3230AC mammary adenocarcinoma.     

Testosterone and dihydrotestosterone concentrations in elephant serum and temporal gland secretions

Serum and temporal gland secretions (TGS) were obtained from mature wild African (Loxodonta africana) and captive Asian elephants (Elephas maximus). Samples were obtained from five cows and eight bulls culled for management purposes in Kruger National Park, South Africa, and from four females and two males residing at the Washington Park Zoo, Portland, Oregon. Our purpose was to describe the levels of the androgens, testosterone (T), and dihydrotestosterone (DHT), and to correlate these observations with sex, species and behavioral status. Male-female differences in serum T were pronounced in the Asian species, whereas male and female concentrations overlapped in the African elephant serum. Serum T concentrations in African females were greater than in Asian females. Serum DHT reflected T levels, except that the striking elevation of testosterone in Asian bulls during musth was not paralleled by equal increases in DHT levels. A species difference observed among males was higher serum T levels in nonmusth Asian bulls (1.84-5.35 ng/ml) compared to the levels in African bulls (0.38-0.68 ng/ml), except for one dominant African bull (6.64 ng/ml). This single African value was still considerably lower than the serum T values of the Asian males during musth. These musth values were the highest serum androgen concentrations: T was between 19 and 40 ng/ml (average 26.10 ng/ml). The TSG values of T and DHT were much higher than serum levels except in the Asian female. T/DHT ratios in TGS were more similar than in serum. One dominant African bull had a T TGS value of 78 ng/ml, which was much higher than the rest of the African males or females, but considerably lower than as Asian bull in musth (547 ng/ml). It seems apparent that a change in androgen status as reflected in serum and TGS levels of T and DHT precedes or is concomitant with overt alteration in behavior in the Asian male. The temporal gland appears to actively concentrate androgens in both African males and females, but in the Asian male the gland secretes only during musth when the greatest concentration of both T and DHT were observed. The apparent difference in the degree of temporal gland secretory activity between the two species suggests a more specific communicative function within the Asian male.     

Effects of perinatal exposure to a synthetic estrogen and progestin on mammary tumorigenesis in mice

The effects of perinatal exposure to synthetic estrogens and progestins on mammary tumorigenesis were studied in female C3H/HeN/MTV + mice. Mice were treated neonatally with 0.001 microgram/day diethylstilbestrol (DES), with 15 micrograms/day 17 alpha-hydroxyprogesterone caproate (HPC), or with oil on days 1-5 of life (birth = day 1). As adults, neonatally hormone-treated mice received long-term treatment with a synthetic estrogen and progestin combination or vehicle. Animals were palpated weekly for mammary gland tumors. The effect of treatment on the probability of tumor development was examined. Neonatal treatment with a low dose of DES increased the probability of mammary-gland tumor formation, whereas neonatal treatment with HPC had a slightly protective effect on tumorigenesis. Subsequent treatment of adult mice with synthetic steroids did not affect mammary gland tumorigenesis in neonatally DES-treated or oil-treated animals. There was a significant interaction between the effect of neonatal HPC treatment and subsequent steroid treatment on mammary tumorigenesis but examination of the data indicated that this interaction was due to the protective effect of HPC in the absence of subsequent exposure to synthetic steroids and the probability of tumor appearance in mice treated with both HPC and synthetic steroids as adults did not differ from that of neonatally oil-treated controls.     

Effects of BRCA1 transgene expression on murine mammary gland development and mutagen-induced mammary neoplasia

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.     

Differential expression of claudin 1, 3, and 4 during normal mammary gland development in the mouse

The claudins are a family of tight junction proteins that display varied tissue distribution and preferential specificity. We recently identified by microarray analysis, members of this family, particularly claudin 1 (cldn1), as highly upregulated genes in the mouse mammary gland during early involution. Gene expression was confirmed by immunohistochemistry and real-time PCR. We then examined gene and protein expression throughout normal mammary gland development. The cldn3 gene showed a steady increase in expression from pregnancy to involution, while cldn1 and cldn4 gene expression increased during pregnancy, but decreased sharply by D10 of lactation, and once again was significantly increased by D1 of involution (P < 0.001 for both genes). The different patterns of gene expression observed between cldn3, and cldn1, and 4 suggest that different family members may be functionally important at different times during mouse mammary gland development. All three genes exhibited a high level of expression at day 1 (D1) of involution, followed by a dramatic decrease in gene expression to day 10 of involution. Immunostaining with the cldn3 antibody showed intense staining of epithelial cells; however, a lesser degree of staining was evident with the cldn1 antibody. In addition to the lateral staining of epithelial cells, basal staining was evident at D1 and D2 of involution and cytoplasmic staining was evident during lactation. Since claudins are known to play a role as tight junction proteins, lateral and basal staining may suggest a role in other functions such as vesicle trafficking or remodeling of tight junctions at different stages of mammary gland development. Cldn1 and 3 antibodies also stained epithelial cells in mouse mammary tumors. In summary, cldn1, 3, and 4 are differentially expressed in the mammary gland during pregnancy, lactation, and involution, suggesting different roles for these proteins at different stages of mammary gland function. In addition, cldn1 and cldn3 are detected in mammary tumors and the wide distribution of cldn3 in particular, suggest specific roles for these proteins in mammary tumorigenesis.     

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.     

Testosterone inhibits estrogen-induced mammary epithelial proliferation and suppresses estrogen receptor expression.

This study investigated the effect of sex steroids and tamoxifen on primate mammary epithelial proliferation and steroid receptor gene expression. Ovariectomized rhesus monkeys were treated with placebo, 17beta estradiol (E2) alone or in combination with progesterone (E2/P) or testosterone (E2/T), or tamoxifen for 3 days. E2 alone increased mammary epithelial proliferation by approximately sixfold (P:<0.0001) and increased mammary epithelial estrogen receptor (ERalpha) mRNA expression by approximately 50% (P:<0.0001; ERbeta mRNA was not detected in the primate mammary gland). Progesterone did not alter E2's proliferative effects, but testosterone reduced E2-induced proliferation by approximately 40% (P:<0.002) and entirely abolished E2-induced augmentation of ERalpha expression. Tamoxifen had a significant agonist effect in the ovariectomized monkey, producing a approximately threefold increase in mammary epithelial proliferation (P:<0.01), but tamoxifen also reduced ERalpha expression below placebo level. Androgen receptor (AR) mRNA was detected in mammary epithelium by in situ hybridization. AR mRNA levels were not altered by E2 alone but were significantly reduced by E2/T and tamoxifen treatment. Because combined E2/T and tamoxifen had similar effects on mammary epithelium, we investigated the regulation of known sex steroid-responsive mRNAs in the primate mammary epithelium. E2 alone had no effect on apolipoprotein D (ApoD) or IGF binding protein 5 (IGFBP5) expression, but E2/T and tamoxifen treatment groups both demonstrated identical alterations in these mRNAs (ApoD was decreased and IGFBP5 was increased). These observations showing androgen-induced down-regulation of mammary epithelial proliferation and ER expression suggest that combined estrogen/androgen hormone replacement therapy might reduce the risk of breast cancer associated with estrogen replacement. In addition, these novel findings on tamoxifen's androgen-like effects on primate mammary epithelial sex steroid receptor expression suggest that tamoxifen's protective action on mammary gland may involve androgenic effects.