Mammary Epithelial Proliferation Research Articles

Establishment and characterization of a bovine mammary epithelial cell line with unique properties.

Clonal cell lines (BME-UV) were established from primary epithelial cells by stable transfection with a plasmid, carrying the sequence of the simian virus 40 early region mutant tsA58, encoding the thermolabile large T antigen. The BME-UV cells have undergone more than 300 population doublings and produce intranuclear large T antigen. At low confluency, growing islands of cells are apparent exhibiting the characteristic cobblestone morphology of epithelial cells. The BME-UV cells expressed functional markers such as microvilli and desmosomes and biochemical markers of mammary epithelial cells such as a repertoire of cytokeratins. The BME-UV cells are capable of synthesizing low levels of alpha-lactalbumin and alpha s1-casein (50 ng/ml of medium/24 h). One of the cell lines, BME-UV1 showed enhanced proliferation in the presence of epidermal growth factor (EGF) and insulinlike growth factor I (IGF-I). The BME-UV1 cell line is the only known bovine mammary epithelial cell line responsive to EGF. The BME-UV cells grown on collagen at low confluency are capable of developing very long projections that most likely allow for communication between cells at a distance from each other. The BME-UV cells may become a valid model system to examine bovine mammary epithelial proliferation and differentiation and cell-to-cell communication.

New insights in the molecular mechanism of progestin-induced proliferation of mammary epithelium: Induction of the local biosynthesis of growth hormone (GH) in the mammary gland of dogs, cats and humans

In contrast to the protective, anti-proliferative, action of progestins on the development of endometrium cancer, progestins may have local stimulatory and inhibitory effects on the proliferation of mammary epithelium. Until now there was no final molecular explanation of this discrepancy. Prolonged treatment of dogs with depot medroxyprogesterone acetate (DPMA) or with proligestone (PROL) results in enhanced plasma concentrations of growth hormone (GH), insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins, together with the development of benign mammary tumours. The stimulated plasma GH levels do not have the typical pulsatile secretion pattern, and are not sensitive to stimulation with GHRH or to inhibition with somatostatin. The autonomous secretion can be inhibited by the anti-progestin RUU-486. The source of progestin-induced plasma GH levels has been demonstrated to be the canine mammary gland where progestins induce the expression of the gene encoding GH. The expression of the GH gene is restricted to focal areas of hyperplastic epithelium as shown by immunohistochemistry, and is predominantly located in single positive epithelial cells with an intermediate position between luminal- and myo-epithelium. Progestin-induced fibroadenomatous changes in the mammary gland of cats are also associated with locally enhanced GH expression. In both normal, benign and malignant mammary tumours of humans GH mRNA expression has been demonstrated by RT-PCR. The presence of GH mRNA is associated with the presence of immunoreactive GH as shown by immunohistochemistry. Sequence analysis revealed 100% homology to the pituitary expressed GH gene. In malignant mammary tumours of humans and dogs GH expression is also found in specimens negative for progesterone receptors as measured by ligand binding. It is concluded that the gene encoding GH is expressed in the mammary gland of a variety of species, including man. This appears to represent a contribution to the molecular explanation of the action of progestins on proliferation of mammary epithelium. It needs, however, to be proven whether this local biosynthesis of GH in the mammary gland is the cause of the local stimulatory effect of progestins on the proliferation of mammary epithelium.

Identification of mammary-derived growth inhibitor in sheep mammary tissue

This study was conducted to evaluate the presence of mammary-derived growth inhibitor (MDGI) in mammary tissue obtained from 11 lactating or pregnant Dorset ewes. MDGI is a protein involved in regulation of mammary epithelial proliferation and differentiation. Results revealed the presence of a 13 kDa MDGI protein in all samples analyzed. The majority (90%) of MDGI was found in the cytoplasmic fraction and the remaining (10%) in the microsomal fraction of sheep mammary tissue. Results indicated that MDGI protein concentrations in mammary tissue obtained from 110 or 140 days in pregnancy were equal or higher than in mammary tissue obtained from lactating animals. High levels of MDGI in sheep mammary tissue at times of intense growth and proliferation are consistent with a role of MDGI as a differentiation rather than anti-proliferative factor. Treatments that increase MDGI production would favor mammary epithelial cell differentiation and could increase milk production. This is a first report on sheep MDGI.

Photoreactive (caged) cyclic AMP analogs induce DNA synthesis in mammary epithelial cells

Dimethoxy-2-nitrobenzyl adenosine-3′,5′ cyclic monophosphate (DMNB) is a metabolically active, photolabile cyclic AMP analog that yields free cyclic AMP upon UV hydrolysis. The analog is useful in that it permits short term, transient elevations of intracellular cyclic AMP. Addition of DMNB (1-10 μM) to mouse mammary epithelial cells, followed by UV irradiation of cells, caused a significant increase in DNA synthesis over that observed with controls, UV irradiation alone or DMNB alone. In subsequent studies, DMNB exhibited a modest, but statistically significant, interaction with epidermal growth factor in promoting DNA synthesis. Effects of DMNB were observed if DNA synthesis was measured as either 3H-thymidine incorporation into DNA or as percent S-phase cells. These results indicate that previously observed effects of agents such as cholera toxin and phosphodiesterase resistant cyclic AMP analogs on mammary epithelial proliferation can be mimicked, at least in part, by a short term pulse of cyclic AMP.

Estrogen and Progesterone Receptors in Benign Breast Epithelium

: Estrogen and progesterone are necessary for lobulo-alveolar development of the human breast, and there is an abundance of epidemiologic literature implicating estrogen and possibly progesterone exposure as promoters of breast malignancy. The investigation of estrogen receptor (ER) and progesterone receptor (PgR) distribution in normal and benign breast tissue may be a measure of susceptibility of the tissue to these hormones. Earlier data from radioligand binding assays showed that benign breast tissue expressed little or no ER; newer immunohistochemical (IHC) methods have led to the investigation of benign breast tissue from at least 1243 women in 12 studies in the last 9 years. These show that the level of expression of ER and PgR in normal breast epithelium is significantly lower than in receptor positive carcinomas. Immunostaining patterns for both receptors show a great deal of heterogeneity. There is general agreement that stromal and myoepithelial cells are negative for both ER and PgR. A growing body of evidence suggests that PgR is the dominant sex steroid receptor in the normal breast. Mean values for PgR positive cells in breast epithelium range from 24% to 29%; ER is positive by IHC in 3% to 15.6% of normal breast epithelial cells. No firm conclusions are possible as yet regarding overexpression in proliferative epithelium. There is agreement that the proportion of ER positive cells declines in the second half of the menstrual cycle, but there is no clear cut relationship between PgR positivity with the menstrual cycle. Oral contraceptive use appears to decrease the proportion of ER positive cells, and increase mammary epithelial proliferation. A recent case-control analysis of epithelial ER and PgR status reports an association of ER positive benign epithelium with the presence of cancer in the breast. Future research should include a systematic quantitative analysis of receptor expression in epithelial proliferative lesions, and the longitudinal follow-up of women who have had receptor testing on benign breast tissue.

Cell interactions in initiation of mammary epithelial proliferation by oestradiol and progesterone in prepubertal heifers.

The acute temporal effects of exogenous oestradiol (0.1 mg/kg per day), progesterone (0.25 mg/kg per day) or both together on the proliferative response of epithelial cells, fibroblasts, adipocytes and endothelial cells in the mammary tissue of prepubertal cross-bred heifers were determined. Mammary biopsies were taken immediately before, then 24, 48 and 96 h after the initiation of daily administration of hormones to three heifers per treatment group. Incorporation of [3H]thymidine into explants prepared from biopsies was evaluated after a 1-h incubation by measuring trichloroacetic acid (TCA)-insoluble radioactivity in explant homogenates as well as by quantitative histoautoradiography. Incorporation expressed as d.p.m./mg tissue or d.p.m./microgram DNA was increased (P < or = 0.05) approximately 11-fold by 96 h in oestradiol-treated heifers. Progesterone-treated animals were unresponsive and heifers treated with both hormones were intermediate in response compared with oestradiol-treated heifers. Autoradiographic data for ductal or terminal duct epithelial cells showed similar dramatic increases in labelling by 24 h with further increased (P < 0.01) labelling by 96 h (5.1% vs 0.1%) in heifers given oestradiol. As with incorporation, tissue from progesterone-treated heifers showed no time or treatment response compared with pretreatment biopsies and tissue from heifers given both showed intermediate responses, i.e. significantly increased labelling by 96 h compared with pretreatment (P < or = 0.05) but less labelling (P < 0.05) than oestradiol-treated heifers. A proliferative response of epithelial cells in oestradiol-treated heifers occurred prior to the response of fibroblasts adjacent to epithelial cells (< or = 50 microns).(ABSTRACT TRUNCATED AT 250 WORDS)

Lactogenic hormones increase epidermal growth factor messenger RNA content of mouse mammary glands

Epidermal growth factor (EGF) is known to stimulate mammary epithelial proliferation, has been identified in milk and is expressed in lactating mammary epithelia. This study examined hormonal control of EGF mRNA in mammary glands of mice. Prepro-EGF mRNA (4.7 kb) was detected during lactation (and increased significantly during this period), whereas a smaller EGF-like RNA (.5 kb) was at highest levels in mammary glands of virgin and pregnant mice. The 4.7 kb RNA was polyadenylated, whereas .5 kb RNA was not. In mammary gland organ cultures from steroid-primed mice, the combinations of insulin+hydrocortisone and insulin+prolactin+hydrocortisone increased both prepro-EGF and β-casein mRNA expression. When hydrocortisone was present there was a decrease in mammary gland content of EGF-like RNA (.5 kb band). We conclude that prepro-EGF mRNA expression in mouse mammary tissue is under the control of the lactogenic hormones prolactin and hydrocortisone.

Growth factors in normal and malignant mammary epithelial proliferation

Growth factors in normal and malignant mammary epithelial proliferation

Is EGF a physiological inhibitor of mouse mammary casein synthesis? Unphysiological responses to pharmacological levels of hormones

It has been observed that EGF inhibits the induction of casein synthesis by mouse mammary tissue in vitro in addition to acting as a promoter of mammary epithelial proliferation. However, since the circulating level of EGF increased during lactation, and since functional EGF receptors are retained by the lactating cells, it seemed unlikely that EGF is an inhibitor of mammary differentiation in vivo . The current studies demonstrate, in fact, that EGF inhibits the induction of casein synthesis in vitro only when insulin is present in the culture medium at unphysiologically high concentrations. Other artifactual responses to high levels of hormones are described.

3T3-L1 Adipocytes promote the growth of mammary epithelium

Murine mammary epithelium grows in association with predominantly adipocyte stroma in vivo. To investigate potential growth-promoting effects of adipocytes on mammary epithelium, we developed a co-culture system of mammary epithelium and adipocytes by taking advantage of the 3T3-L1 cell line. These cells undergo adipocyte differentiation when the culture reaches confluence and growth ceases. Mid-pregnant murine mammary epithelium was plated on lethally irradiated feeder layers of 3T3-L1 adipocytes, undifferentiated 3T3-L1 cells, 3T3-C2 fibroblasts (a subclone of 3T3 cells that does not undergo adipocyte differentiation), or tissue culture plastic. Mammary epithelial colony size on adipocyte feeder layers was 2-fold larger than colonies on 3T3-C2 cells and 4-fold larger than colonies on tissue culture plastic. Measurement of tritiated thymidine [ 3H]TdR incorporation and labelling index in mammary cells was significantly higher on adipocytes than on other feeder layers or plastic. There was a 6-fold increase in mammary cell number after 5 days in culture when mammary epithelium was plated on substrate-attached material (‘extracellular matrix’) derived from 3T3-L1 cells and a 4-fold increase in cell number when plated on plastic in conditioned medium derived from 3T3-L1 adipocytes compared with growth on plastic in unconditioned medium. We conclude that interaction of mammary epithelium with adipocytes results in a marked increase in proliferation of mammary epithelium and that extracellular components may mediate this effect.

Hormonal Effects on Mammary Cytology

This reviews cytological studies of hormonal effects on the mammary gland. Normal lobuloalveolar growth in the intact animal requires the stimulation of estrogen and progesterone. Exogenous estrogen alone brings about limited growth in ovariectomized mice but a combination of exogenous estrogen and progesterone brings about growth similar to early pregnancy. Proliferation of mammary epithelium is under the direct influence of estrogen and progesterone. Alveolar and terminal duct epithelia undergo a marked morphological change during lactogenesis. Ultrastructural differentiation of the epithelium occurs immediately before and after parturition. The need for pituitary and adrenal hormones in addition to ovarian steroids for the progression of lactogenesis has been shown by endocrine ablation studies. Midpregnancy mouse mammary tissue exposed in vitro to the lactogenic hormone combination of glucocorticoid insulin and prolactin develops alveolar cells displaying all the accepted ultrastructural correlates of lactogenesis and lactation. This emphasizes the primary action of these 3 hormones on mammary epithelial structure. Human chorionic somatomammotrophin will substitute for pituitary prolactin in initiating secondary development of mouse and rabbit mammary tissue in vitro. Growth hormone is not needed in culture for lactogenesis unless insulin cortocoid and prolactin are in minimal amounts. Nonsecretory midpregnancy mouse mammary cells treated with insulin divided in vitro into cells ultrastructurally identical to parent cells. These daughter cells when treated with hydrocortisone developed a granular endoplasm reticulum but not milk protein or lactose synthesis. They divide only once and cannot be maintained long in a secretory state. Cytomorphological studies of induced lactogenesis in pseudopregnant rabbits showed that mammary cells do not develop to full synthesizing capacity until 72 hours after prolactin treatment and some hours after organelle development. Steroid hormones may inhibit lactogenesis either alone or in concert with lactogenic hormones. The observation that dairy cows yield significant milk prepartum shows that mammary growth and development are not absolutely parallel with elevated hormones at partuition. Also a gland-specific lactogenic inhibitor mechanism independent of hormones has been shown in cows.

A radioautographic study of cell proliferation in the mammary gland of the pregnant mouse.

AbstractProliferation of the mammary gland epithelium on days 2, 3, 4, 6, 8, 12, 16, and 19 pregnancy has been studied in mice, by means of radioautography. Each animal received an intraperitoneal injection of 25°c of tritiated thymidine and was killed one hour later. The inguinal mammary glands were processed for paraffin sectioning and radioautographs prepared using the dipping technique. The rate of mammary epithelial cell proliferation was quantitated for each animal by determining the percent of labeled epithelial cells in a large sample (&gt; 2000 cells) of cells (labeling index).The data showed that the proliferative activity of the mammary epithelium during pregnancy followed a bimodal distribution. The first evidence of a proliferative response to pregnancy was noted on day 3. By day 4, the labeling index reached a maximum of 25.3%. The labeling index declined on days 6 and 8 to 7.5% and 8.0% respectively. On day 12, the labeling index increased to 12.1%, then fell to 7.9% and 1.3% on day 16 and 19, respectively.Separate labeling indexes were determined for the interlobular epithelium (ducts) on day 12, 16, and 19. The data showed that the labeling indexes of the interlobular and intralobular epithelium during these stages of pregnancy were not significantly different. The correlation of the hormones secreted during pregnancy with the data on mammary epithelial proliferation is discussed.

Cell proliferation in the mammary gland during late pregnancy and lactation

AbstractNulliparous, CFW mice were injected with 25 m̈c of tritiated thymidine on day 19 of pregnancy, and days 1, 2, 3, 5, 7, 10, 15 and 20 of lactation. The animals were killed one hour after injection. The inguinal mammary glands were removed and processed for paraffin sectioning. Radioautographs were prepared, using the dipping technique.Quantitation of mammary epithelial cell proliferation for the intra‐ and interlobular (ducts) epithelium was performed by determining the percent of labeled epithelial cells in a large sample of cells (labeling index). It was concluded that epithelial cell sample sizes of 1,000–2,000 cells were adequate to measure mammary epithelial proliferation. A wave of epithelial proliferation was observed during early lactation. In the intralobular epithelium, a peak labeling index of 11.1% was attained on day two of lactation whereas a peak labeling index of 7.9% was observed on day three of lactation in the interlobular epithelium. Cells of the connective tissue and vascular bed proliferated in response to the growth of the mammary epithelium. Myoepithelial cells were frequently labeled on days two and three of lactation.