Susceptibility of the mammary gland to carcinogenesis: I Differentiation of the mammary gland as determinant of tumor incidence and type of lesion.

The influence of age and mammary gland differentiation on the incidence of tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) was studied by correlating the development of the mammary glands of 20-180-day-old, virgin Sprague-Dawley rats with the number and type of tumors induced by DMBA administered at those various ages. The number of terminal end buds (TEBs), terminal ducts (TDs), and alveolar buds (ABs)/sq mm and their DNA-labeling indices (DNA-LI) were determined. Highest density of TEB occurred when the rats were 20 days old, decreasing thereafter. DNA-LI ranged between 25.2 and 29 in TEB of rats aged 30-55 days, which was coincident with the highest incidence of carcinomas. With aging, the number of TEBs and their DNA-LI decreased and the number of TDs and ABs increased, although with a low DNA-LI, which correlated with a lower incidence of carcinomas and higher incidence of benign lesions.     

Susceptibility of the mammary gland to carcinogenesis. II. Pregnancy interruption as a risk factor in tumor incidence.

In the rat, pregnancy and lactation prior to carcinogen administration protect the mammary gland from developing carcinomas and benign lesions. In this study, the influence of pregnancy interruption versus full pregnancy and pregnancy plus lactation on the incidence of carcinomas and benign lesions was studied in the mammary glands of rats treated with 7,12-dimethylbenz(a)anthracene (DMBA). Fifty-nine Sprague-Dawley rats were separated into 5 groups: I) rats that had had one pregnancy and one lactation; II) rats that had had one pregnancy without lactation; III) rats that had had pregnancy interrupted at the 12th day of gestation; IV) age-matched virgin rats as a control Group I; and V) age-matched virgin rats as a control for groups II and III. The 5 groups received a single intragastric dose of DMBA (10 mg/100 g body weight), with the exception of 2 animals per group, which were killed 1 hour after an intraperitoneal injection of 2.5 mu Ci 3H-thymidine/g body weight. The number of labeled nuclei per 100 cells (DNA labeling index, LI) was counted in terminal end buds (TEBs), terminal ducts (TDs), and alveolar buds (ABs) of the glands. The number of structures and the DNA-LI were correlated with the incidence of tumors at 22 weeks after DMBA. Pregnancy, with or without lactation, resulted in elimination of TEBs and reduction in the DNA-LI of TDs and ABs. These groups did not develop carcinomas. After the interruption of pregnancy the mammary gland contained numerous TEBs, with a high DNA-LI; 77% of these animals developed carcinomas, and all of them developed benign lesions. Therefore, while pregnancy and lactation protected the mammary gland from developing carcinomas and benign lesions by induction of full differentiation, pregnancy interruption did not elicit sufficient differentiation in the gland to be protective, and these animals were at the same risk as virgin animals treated with the carcinogen.     

Basis of cellular autonomy in the susceptibility to carcinogenesis

This paper summarizes our contributions in the basic understanding of the different susceptibility of the mammary gland to carcinogenesis according to age and parity history. Mammary carcinomas induced by the administration of 7,12-dimethylbenz-(DMBA) to young virgin rats arise from undifferentiated terminal ductal structures called terminal end buds (TEBs). TEBs, that normally differentiate into alveolar buds (ABs) and lobules, under the influence of DMBA develop intraductal proliferations which progress to carcinoma. The high susceptibility of the young virgin rat TEBs to neoplastic transformation is due to its large proliferative compartment, with cells cycling every 10 hours, and to a higher 3H-DMBA uptake. Progressive differentiation of TEBs into ABs and lobules or their regression to terminal ducts (TDs) is seen with aging. Complete differentiation of the gland is attained through pregnancy and lactation or through exogenous administration of chorionic gonadotrophin. The greater differentiation of the gland is manifested as permanent structural changes, consisting in the disappearance of TEBs and in a diminution of the number of TDs due to their differentiation into ABs and lobules. This greater differentiation results in a diminished or total refractoriness of the gland to the carcinogen because ABs and lobules have a lower proliferative compartment, and a longer cell cycle than TEBs and TDs. Cells of parous rats have both in vivo and in vitro lower DMBA-DNA binding capacity, lower DNA synthesis and greater ability to repair DMBA-damaged DNA than cells of young virgin rats. The more efficient DNA repair capacity of the parous rat mammary gland is demonstrated by the induction of unscheduled DNA synthesis and a removal of DMBA-DNA adducts.     

Effects of neonatally administered diethylstilbestrol on induction of mammary carcinomas induced by 7, 12-dimethylbenz(a)anthracene in female rats

Various doses of diethylstilbestrol (DES) were administered to rats once at birth. Thereafter, at 50 days after birth, the rats in all groups were given 10 mg 7, 12-dimethylbenz[a]anthracene (DMBA) and undergone necropsy at 300 days after birth. The incidence of mammary carcinomas (MCs) were 50, 54, 91, 39, 19% at 175 days after birth, and 77, 87, 100, 85, 75% at necropsy in the 0, 0.1, 1, 10, 100 microg groups, respectively. The incidence of rats without corpus luteum were 0, 0, 0, 30, 100% at 50 days after birth, and 0, 40, 53, 93, 100% at necropsy in the 0, 0.1, 1, 10, 100 microg groups, respectively. Observation of the whole mount specimens showed a higher number of terminal end buds (TEBs) in the 1 microg group and a lower number in the 100 microg group compared with the control at 50 days after birth. It suggested that the administration of a relatively low dose (1 microg) of DES during neonatal period may increase TEBs, thus resulting in a stimulatory effect on the initiation of MCs.     

Cell size and shape changes in the myoepithelium of the mammary gland during differentiation

We have studied changes in myoepithelial cell size and shape during different stages of mouse mammary gland differentiation by using the fluorescent probe for actin NBD-phallacidin. Pieces of mammary tissue were fixed, mounted on slides, permeabilized with cold acetone (-20 degrees C), and then treated with nitrobenzoxadiazole-phallacidin. Myoepithelial cells lining ducts of glands at all stages of development are spindle-shaped structures oriented parallel to the long axis of the duct at the base of the luminal epithelium. In virgin animals, myoepithelial cells also occur as linear tracts oriented parallel to the long axis of small projections along the sides of ducts and terminal end buds. In early pregnancy, small stellate-shaped cells begin to appear around presumptive secretory units. By late pregnancy, larger star-shaped units of intense fluorescence appear at the base of alveoli. During lactation, both cell bodies and cell processes further enlarge as these interlacing stellate-shaped cells encompass the expanded alveoli. In regressing glands, cell size decreases and the processes appear to retract. Although alveoli are virtually absent in the multipartate resting gland, myoepithelial cells remain around lateral buds of ducts. These myoepithelial cells have two distinct shapes: small star-shaped cells capping the buds and spindle-shaped cells oriented parallel to the long axis of the buds. A comparison of myoepithelial cell shape in virgin mice and nulliparous women indicates a more developed cell in the human gland at this stage of development. Intact segments of mammary gland combined with NBD-phallacidin as a probe for actin provide an ideal system for future studies of the control of myoepithelial cell size and shape and their influence on cell functions.     

The effect of hydroxylamine on the morphology of the rat mammary gland and on the induction of mammary tumors by 7,12-dimethylbenz[a]anthracene.

The effect of hydroxylamine (HA) on the morphology of the rat mammary gland and on the formation of mammory tumors induced by 7,12-dimethylbenz(a)anthracene (DMBA) was studied. Hydroxylamine caused excessive growth and secretory activity in the mammary gland. When it was given after DMBA administration it decreased the number and size of tumors per tumor bearing animal, but increased the median latency period. When HA was given before carcinogen administration it did not affect the incidence of tumors or the number or weight of tumors per tumor bearing animal, but it protected to some extent the normal lobular structure of the gland against the DMBA induced destruction which was evident a few weeks after carcinogen administration. A delayed effect of DMBA treatment was the formation of dark-staining irregular structures which originated either from the larger ducts or from the duct terminals. These formations were morphologically different from the hyperplastic alveolar nodules (HAN) which showed distended ductules of regular shapes and were common in animals treated with HA before DMBA administration.     

The effects of neonatal androgenization on mammary gland mitotic rate and susceptibility of carcinogen-induced mammary dysplastigenesis and tumorigenesis in LEW/Mai rats.

The influence of neonatal testosterone propionate treatment (androgenization) on mammary gland mitotic rate (MR) and susceptibility to 7,12-dimethylbenz(alpha)anthracene (DMBA) carcinogenesis was studied in female LEW/Mai rats. Mammary gland MR in androgenized rats was significantly lower than MR in normal rats at all ages studied. Treatment of androgenized rats with DMBA resulted in a significant increase in mammary gland MR in comparison with untreated androgenized rats. MR in DMBA-treated androgenized rats was similar to MR in DMBA-treated normal rats at most intervals after the introduction of the carcinogen. Although mammary epithelial MR in androgenized rats was significantly lower than that of normal rats of comparable age, no evidence of a decrease in susceptibility of mammary epithelium in androgenized rats to DMBA carcinogenesis was found. Instead, androgenized rats had a higher incidence of DMBA-induced mammary dysplasias, with no change in their morphologic or histologic features, than did normal rats; and there was no change in the incidence, latency, or histopathologic appearance of DMBA-induced mammary tumors in androgenized versus normal rats.     

Effects of progesterone on mammary carcinogenesis when various doses of DMBA were applied directly to rat mammae

To investigate the suggestion that progesterone acts directly on the mammary gland to inhibit carcinogenesis, doses of 500, 100 or 30 microgram of DMBA were applied directly to inguinal mammary glands of 50 d old rats which either received no other treatment or were injected with progesterone (3 mg/d) s.c. for 18 d before dusting the carcinogen. Progesterone pretreatment did not inhibit mammary carcinogenesis in rats dusted with 500 microgram or DMBA. When smaller doses of DMBA (100 microgram or less) were applied, hormone pretreatment markedly reduced carcinogenesis, the inhibitory effect being statistically significant in the group dusted with the smallest dose of carcinogen. As the dusting technique eliminated any observable systemic effects of the carcinogen, it is concluded that the results support the suggestion that progesterone acts directly on the mammary gland to inhibit carcinogenesis and that this effect can be over-ridden if a large enough dose of DMBA (somewhere between 100 and 500 micrograms) is applied. The importance of carcinogen dose to resulting tumour yield was clearly shown by the significant descending gradation in tumour incidences and gradual lengthening of average tumour latent periods in the 3 control groups with decreasing DMBA dose, as well as in the 3 hormone-pretreated groups.     

Cellular composition and organization of ductal buds in developing rat mammary glands: Evidence for morphological intermediates between epithelial and myoepithelial cells

In the developing rat mammary gland, terminal end buds (TEBs), lateral buds and alveolar buds represent the major sites of morphogenetic activity and cellular differentiation. The morphology and cellular composition of these buds from 20-to 22-day-old rats and cycling rats have been studied by immunocytochemical and electron microscopic techniques. The mammary buds are composed of a heterogeneous collection of cells including epithelial and myoepithelial cells, irregular loosely adherent cells, and occasional large clear cells. The irregular, loosely packed cells or cap cells are mainly situated around the periphery of the TEBs and lateral buds. “Chains” of irregularly shaped cells also extend from the peripheral cap cell layer to the center of the TEB; and, where they converge on lumina, they display microvilli and junctional complexes. At the tips of the end buds, the cap cells are of undifferentiated appearance; however, similar cells situated toward the subtending mammary ducts show a gradation in ultrastructure to that of myoepithelial cells. This change is accompanied by an increase in the amounts of immunoreactive myosin and keratin seen within the cells and a 200-fold increase in the thickness of the basement membrane. In contrast, the peripheral cells of the alveolar buds are more closely packed, contain a greater number of myofilaments, and show increased staining with antisera to myosin. We suggest that the undifferentiated cap cells do not represent a discrete cell type, since they show transitional forms to myoepithelial cells within the subtending mammary ducts, and that the tendency toward the myoepithelial phenotype is predominant in the more differentiated structures, the alveolar buds.     

Vanadate disrupts mammary gland development in whole organ culture.

Protein tyrosine kinases and phosphatases are signaling molecules involved in all aspects of development, including proliferation, differentiation, and apoptosis. How disruption of protein tyrosine phosphatase affects mammary gland development is not entirely clear. We examined the effects of sodium vanadate, which is known to primarily inhibit tyrosine phosphatases, in mouse mammary gland development in whole organ culture. Mammary epithelial differentiation was effectively inhibited by vanadate in a dose-dependent manner as indicated by lack of epithelial alveoli compared to the contralateral non-treated gland controls. Mammary glands in the differentiation medium after four days in the presence of vanadate did not differentiate into alveoli. Instead, they exhibited prominent terminal end buds and lost the distinctive epithelial structures. The inhibitory effect of vanadate on mammary epithelial cell differentiation was irreversible after one day of treatment. Immunohistochemical staining for PCNA (Proliferating Cell Nuclear Antigen) showed that vanadate-treated glands exhibited elevated proliferation signals in the differentiation medium. Expression of beta-casein protein in the vanadate-treated glands decreased dramatically and progressively. Short-term exposure (up to 72 hours) of mammary glands to vanadate resulted in an increase in mammary epithelial cell density and loss of organization of the mammary structures. TUNEL assay of mammary glands with prolonged exposure to vanadate revealed widespread apoptosis. Furthermore, some cells were still proliferating or expressing beta-casein after prolonged exposure to vanadate. Taken together, these data indicate that vanadate treatment blocks mammary epithelial cell differentiation and promotes abnormal proliferation and apoptosis, likely through the inhibition of protein tyrosine phosphatase-mediated signaling.     

Oncogenic signaling pathways activated in DMBA-induced mouse mammary tumors

Only about 5% of human breast cancers can be attributed to inheritance of breast cancer susceptibility genes, while the balance are considered to be sporadic in origin. Breast cancer incidence varies with diet and other environmental influences, including carcinogen exposure. However, the effects of environmental carcinogens on cell growth control pathways are poorly understood. Here we have examined oncogenic signaling pathways that are activated in mammary tumors in mice treated with the prototypical polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA). In female FVB mice given 6 doses of 1 mg of DMBA by weekly gavage beginning at 5 weeks of age, all of the mice developed tumors by 34 weeks of age (median 20 weeks after beginning DMBA); 75% of the mice had mammary tumors. DMBA-induced mammary tumors exhibited elevated expression of the aryl hydrocarbon receptor (AhR), c-myc, cyclin D1, and hyperphosphorylated retinoblastoma (Rb) protein. Because of this, the activation of upstream regulatory pathways was assessed, and elements of the Wnt signaling pathway, the NF-kappa B pathway, and the prolyl isomerase Pin-1 were found to be frequently up-regulated in the tumors when compared to normal mammary gland controls. These data suggest that environmental carcinogens can produce long-lasting alterations in growth and anti-apoptotic pathways, leading to mammary tumorigenesis.     

The Relationship of Terminal Duct Hyperplasia to Mammary Carcinoma in 7,12-Dimethylbenz(α)anthracene—Treated LEW/Mai Rats

The evolution of dysplasias and carcinomas in the inguinal mammary glands of LEW/Mai rats given 7,12-dimethylbenz(α)anthracene (DMBA) by gastric gavage was studied with the use of stained whole mounts. Two major dysplasias, hyperplastic terminal end buds (HEBs) and hyperplastic alveolar nodules (HANs), developed prior to mammary carcinomas. HEBs were present in the mammary glands of ~70% of the rats within 1 week following DMBA. The percentage of rats with these lesions and the incidence of HEBs in the mammary gland decreased prior to the appearance of palpable and microscopic tumors. During the time when tumors first became evident (40-80 days), the percentage of rats with HEBs (11%) paralleled the percentage of rats with mammary tumors (12%). The initial percentage of rats with HEBs (~70%) paralleled the final tumor incidence (71%) observed in DMBA-treated rats that were allowed to live until they developed tumors. The histologic features of HEBs resembled those of the carcinomas, and HEBs were present in the immediate vicinity of some of the microscopic and palpable tumors. With only one exception, the location of microscopic tumors in the mammary gland was consistent with their derivation from small terminal ducts. These data are compatible with a developmental relationship between HEBs and mammary carcinoma. HANs, on the other hand, developed relatively late (ie, 30 days) following DMBA administration and became more numerous with the passage of time. Over the period of time when mammary carcinomas first became evident, the percentage of rats with HANs (73%) was inconsistent with a developmental relationship between HANs and mammary carcinoma. This conclusion was supported by the absence of HANs in the vicinity of microscopic tumors, by the dissimilarity between the histologic features of HANs and mammary carcinomas, and by their absence from the mammary gland during the time when at least some of the mammary tumors must have arisen. The results implicate terminal duct hyperplasia in the histopathogenesis of rat mammary carcinomas.     

Endocrinologic milieu and susceptibility of the rat mammary gland to carcinogenesis.

The incidence of DMBA-induced mammary carcinomas in Sprague-Dawley rats depends upon their previous reproductive histories. Young virgin rats (YV) are highly susceptible to the carcinogen, while old virgin rats (OV) are less susceptible, and parous rats (P) are resistant. The authors performed endocrinologic studies in these three groups of rats in order to determine whether the different susceptibility to carcinogenesis, according to the reproductive history, is or is not related to the hormonal milieu. The pituitary, the ovaries, the adrenals, and the mammary glands were processed for light microscopy. Pituitary prolactin (PRL), follicle-stimulating hormone, and luteinizing hormone cells were immunostained by peroxidase-antiperoxidase and quantitated with an image analyzer. Radioimmunoassays of serum and pituitary PRL and serum estradiol were also done. The results showed no differences in the hormonal milieu of YV, OV, and P rats at the time of carcinogen treatment. Several changes were observed after DMBA administration, the most conspicuous being 1) hyperplasia of pituitary PRL cells, 2) high serum PRL levels, 3) nodular hyperplasia of the adrenal cortex, 4) high serum estradiol levels, and 5) lack of adrenal necrosis in P rats and some OV rats. These modifications did not correlate with the degree of susceptibility of YV, OV, and P rats to carcinogenesis, supporting the concept of the importance of the mammary gland differentiation at the moment of carcinogen administration. (Am J Pathol 1982, 109:47-56).     

Influence of strain and sex on the local development of mammary tumors induced by direct application of DMBA powder to rat mammary glands

In order to determine the influence of strain and sex on local carcinogenesis in rat mammary tissue, 1 mg of 7,12-dimethylbenz(a)anthracene (DMBA) was dusted directly onto the exposed mammary gland of 30-day-old Long-Evans (L-E) rats and Sprague-Dawley (S-D) rats. The experiment was terminated 28 weeks after application of the carcinogen. Tumors measuring between 1 and 2 cm in diameter were harvested from female L-E rats with high frequency (85%) and long latency (mean: 23.7 weeks after DMBA dusting), and from female S-D rats with extremely high frequency (98%) and short latency (16.7 weeks). Male rats of both strains were almost identically much less susceptible to DMBA (L-E; 55%, 25.0 weeks, S-D; 53%, 23.9 weeks). Ovariectomized S-D (47%, 24.9 weeks) and orchiectomized S-D (30%, 24.8 weeks) rats, which were gonadectomized at 30 days of age, respectively, were also much less susceptible. A variety of histologies, mostly malignant epithelial, mesenchymal or mixed tumors, were noted in each group. The carcinomatous response in the mammary tissue was much higher in female S-D (96%) than in female L-E (50%) rats, and very low in male and gonadectomized rats (10-20%). In contrast, the sarcomatous response in the mammary tissue was moderate in female and male L-E and male S-D (43-50%) rats, and low in the other groups (15-29%).     

A proposed selective cell carcinogenesis in mammary tumors.

The present study was done to ascertain whether a specific carcinogenic agent has a causal effect on the initial proliferation of only one cell type or whether it acts indiscriminately on all cells in the breast secretory unit. Enzymes histochemistry and electron microscopy were performed on DMBA-induced mammary tumors in female Sprague-Dawley rats and on virus-associated spontaneous mammary tumors in C3H/HEJ mice. The results showed that the chemical carcinogen DMBA affects initial myoepithelial cell proliferation, while virus-associated mammary carcinoma originated from ductular epithelial cell proliferation. To determine whether a specific tumor is composed of a single cell type, tumors were grown in tissue culture. The monolayer was fixed in the usual manner for electron microscopy while in Falcon tissue culture plates. The plates were dissolved in xylene and the monolayer was cut into small pieces and embedded in the plastic media. Electron microscopy performed on the tissue culture and the original tissue from the virus-induced tumors showed the presence of viruses in large numbers. It also suggested the differentiation of basal membrane to form basal lamina and apical plasma membrane into microvilli. This study strongly suggests the presence of selective cell carcinogenesis in the mammary gland.     

Conserved antigen expression in epithelial tumors recognized by monoclonal antibody 4A9 generated against canine mammary carcinoma cells

Hybridoma-derived murine monoclonal antibodies (MoAbs) were generated by fusing P3X63-Ag8.653 myeloma cells with splenic cells from BALB/c mouse which had been immunized with viable canine mammary adenocarcinoma cells, CMT-2. Fifteen MoAbs were shown to react with immunizing cells in indirect immunofluorescence (IFA) and enzyme-linked immunosorbent (ELISA) assays. The reactivity of one IgM MoAb, designated 4A9, was evaluated. The antigen recognized by 4A9 on CMT-2 cells appeared to be localized both in cell membrane and cytoplasm against fixed and unfixed preparations by IFA. The 4A9 MoAb was found to bind with four of five canine mammary carcinoma cell lines while no binding was detected with normal fibroblastic cell lines. In vivo tissue distribution of 4A9 antigen was evaluated by indirect immunoperoxidase (IP) assay against formalin-fixed, paraffin-embedded sections of normal and neoplastic tissues. 4A9 MoAb reacted strongly to moderately with 75% of mammary carcinomas, moderately to weakly with 57% of benign mammary tumors, and strongly with squamous cell and perianal gland carcinomas (100%), interstitial cell tumors (100%), transitional cell carcinomas (43%), lung adenocarcinomas (40%), colon carcinomas (33%), and pancreatic adenocarcinomas (20%). Moderate to weak staining was detected with granulosa cell tumors (25%) and apocrine gland adenocarcinomas (50%). Strong reactivity with perianal gland carcinomas contrasted to no reactivity with perianal gland adenomas. No immunostaining was detected with a large variety and number of normal adult and fetal tissues tested; negligible and very restricted staining was observed in a few adult and fetal tissues. Normal mammary gland was negative. Since the antigen is expressed on the cell surface and in the cytoplasm of most mammary carcinoma cells and a variety of other epithelial tumor cells, the 4A9 antibody may have potential application in diagnosis and management of canine mammary cancer and a variety of other epithelial tumors.     

Environmental carcinogens and p53 tumor-suppressor gene interactions in a transgenic mouse model for mammary carcinogenesis

Mouse mammary tumorigenesis is greatly influenced by a variety of exogenous agents, such as MMTV, chemical carcinogens (i.e., polycyclic aromatic hydrocarbons), and radiation, as well as by endogenous/physiological factors, such as steroid hormones, tumor-suppressor genes (i.e., Brca1/2, p53), and gene products of modifier genes. In the mouse model, the most frequently used chemical carcinogen has been 7,12-dimethylbenz[a]anthracene (DMBA), which activates the Ha-ras gene but does not alter the p53 tumor-suppressor gene. However, on an existing background of p53 gene alteration, low doses of DMBA are strongly cocarcinogenic. Using a transgenic model system, in which the p53 gene was deleted in the mammary gland, we examined the carcinogenic effects of a variety of external agents and internal factors given at either low doses or physiological doses. These agents/factors included DMBA, gamma-radiation, Brca2 heterozygosity, and steroid hormones. All agents/factors increased the tumorigenic response of the p53 null mammary cells, even under conditions where no tumorigenic response was observed in the p53 wildtype mammary cell. The strongest cocarcinogenic effect was observed with the steroid hormone progesterone. The majority of tumors were highly aneuploid and composed of nuclear igh-grade cells. The mechanism for the aneuploidy and secondary events associated with high tumorigenicity were examined using array technology. These results demonstrate that, on a background of underlying genetic instability, very low doses of environmental mutagens and mitogens can produce strong cocarcinogenic effects.