The CCAAT/enhancer binding proteins (C/EBPs) are differentially expressed throughout mammary gland development and interact with binding sites within the promoter of a milk protein gene, ␤-casein. The specific roles of C/EBP␤ and C/EBP␣ in mouse mammary gland development and differentiation have been investigated in mice that carry targeted deletions of these genes. C/EBP␤ −/− virgin mice exhibited cystic, enlarged mammary ducts with decreased secondary branching. Transplantation of C/EBP␤ −/− mammary epithelium into the cleared mammary fat pads of nude mice confirmed that this defect in ductal morphogenesis was intrinsic to the epithelium. When treated with estrogen/progesterone (E+P) to simulate pregnancy, C/EBP␤ −/− mammary glands displayed only limited lobuloalveolar development and ductal side branching. Primary mammary epithelial cells obtained from E+P-treated C/EBP␤ −/− mice that were cultured on extracellular matrix gels did not functionally differentiate in response to lactogenic hormones despite their organization into three-dimensional structures. Expression of ␤-casein protein was inhibited 85%-100% and whey acidic protein (WAP) was undetectable. In contrast, no detectable alterations in mammary development or ␤-casein expression were observed in mammary outgrowths derived from newborn C/EBP␣ −/− mammary epithelium transplanted into the cleared mammary fat pads of syngeneic hosts. These results demonstrate that C/EBP␤, but not C/EBP␣, is required for ductal morphogenesis, lobuloalveolar development, and functional differentiation of mammary epithelial cells.

One of the most consistent results in the epidemiology of human breast cancer is the inverse relationship of risk and early full-term parity. The goal of this study was to investigate the molecular mechanisms through which early full-term pregnancy protects the breast from cancer development. We used Wistar-Furth (WF) rats as our experimental system and mimicked pregnancy using estrogen and progesterone (E/P). Sexually mature female rats were treated with steroid hormones for 21 days and after 28 days of gland involution, the rats were administered MNU. Rats that received a high dose of 20 microg E and 20 mg P exhibited an 82% reduction in the incidence of mammary adenocarcinomas as compared to the rats receiving only blank pellets. Decreasing doses of E/P were partially protective suggesting that complete differentiation of the gland was not required for refractoriness. We measured the RNA expression levels of several target genes involved in the regulation of mammary cell proliferation and/or differentiation including estrogen receptor (ER) and progesterone receptor (PR), cyclins D1 and D2, the cell cycle inhibitors p16, p21 and p27, and the tumor suppressor p53. At the time of MNU treatment we found no significant differences in the expression of these genes, with the possible exception of p21, indicating that hormone treatment did not result in constitutive changes in expression levels. The numbers of apoptotic cells were low and comparable in the hormone exposed and age-matched virgin gland (AMV) at the time of carcinogen challenge and remained low for 8 days after MNU treatment. The number of BrdU-labeled cells at the time of carcinogen challenge were also low in both the AMV (1.8%) and hormone exposed (0.8%) animals. In contrast, cell proliferation in the AMV (5.7%) was significantly different from both the parous involuted (1.2%) and the E/P-treated involuted (1.5%) animals 8 days after MNU treatment. We interpret these data to indicate that hormone treatment results in mammary epithelial cells that have persistent alterations in intracellular pathways governing proliferation responses to carcinogens.

Ductal morphogenesis in the rodent mammary gland is characterized by the rapid penetration of the stromal fat pad by the highly proliferative terminal endbud and subsequent formation of an arborized pattern of ducts. The role of apoptosis in ductal morphogenesis of the murine mammary gland and its potential regulatory mechanisms was investigated in this study. Significant apoptosis was observed in the body cells of the terminal endbud during the early stage of mammary ductal development. Apoptosis occurred predominately in defined zones of the terminal endbud; 14.5% of the cells within three cell layers of the lumen were undergoing apoptosis compared to 7.9% outside this boundary. Interestingly, DNA synthesis in the terminal endbud demonstrated a reciprocal pattern; 21.1% outside three cell layers and 13.8% within. Apoptosis was very low in the highly proliferative cap cell laver and in regions of active proliferation within the terminal endbud. In comparison to other stages of murine mammary gland development, the terminal endbud possesses the highest level of programmed cell death observed to date. These data suggest that apoptosis is an important mechanism in ductal morphogenesis. In p53-deficient mice, the level of apoptosis was reduced, but did not manifest a detectable change in ductal morphology, suggesting that p53-dependent apoptosis is not primarily involved in formation of the duct. Immunohistochemical examination of the expression of the apoptotic checkpoint proteins, Bcl-x, Bax and Bcl-2, demonstrated that they are expressed in the terminal endbud. Bcl-x and Bcl-2 expression is highest in the body cells and lowest in the nonapoptotic cap cells, implying that their expression is associated with increased apoptotic potential. Bax expression was distributed throughout the terminal endbud independent of the observed pattern of apoptosis. A functional role for Bcl-2 family members in regulating endbud apoptosis was demonstrated by the significantly reduced level of apoptosis observed in WAP-Bcl-2 transgenic mice. The pattern of apoptosis and ductal structure of endbuds in these mice was also disrupted. These data demonstrate that p53-independent apoptosis may play a critical role in the early development of the mammary gland.

Estrogen regulates proliferation and morphogenesis of mammary ductal epithelium by interacting with a specific intracellular estrogen receptor (ER) that acts as a hormone-dependent transcriptional regulator of gene expression. The mechanisms by which ER regulates transcription in response to estrogen have been analyzed extensively in tissue culture and in cell-free systems. These studies have demonstrated that the transcriptional activity of ER is strongly influenced by cellular context and highlight the need to address ER transcriptional activity in an appropriate cellular background. Thus, to gain insight into the mechanistic role of ER in mammary epithelial morphogenesis, we have used an adenoviral gene delivery strategy to introduce an estrogen-responsive reporter gene into the mammary epithelium and to monitor the activity of endogenous ERs in their natural environment where cellular context including stromal-epithelial interactions can be taken into account. Using this approach, we first demonstrated highly efficient adenoviral delivery throughout the mammary epithelium using a beta-galactosidase (betagal) reporter gene under the control of the constitutively active cytomegalovirus (CMV) promoter. Next, we constructed an adenoviral vector by substituting the CMV promoter with an estrogen-dependent promoter fragment-linked betagal (Ad-ERE-tk-betagal). This adenoviral reporter system provides evidence that ER positive mammary epithelial cells display a differential sensitivity in a region-specific manner toward estrogen induction. Our data suggest that the availability of factor(s) other than ER is necessary for ER-mediated gene activation and may be important in modulating the differential responses of mammary epithelial cells to estrogen.