Biomedical subjects

S F Shoemaker

Publications and source records attributed to S F Shoemaker.

7 recordsLinked to original sources

PubMed2001

TNFalpha induces NFkappaB/p50 in association with the growth and morphogenesis of normal and transformed rat mammary epithelial cells.

In contrast to the cytotoxic or cytostatic effect of TNFalpha on many breast cancer cell lines, TNFalpha stimulates growth and morphogenesis of normal rat mammary epithelial cells (MEC). The present studies were carried out to determine whether there are intrinsic differences between normal and malignant MEC which may explain the differing responsiveness to TNFalpha. Freshly isolated rat MEC organoids from normal mammary gland or 1-methyl-1-nitrosourea-induced mammary tumors were treated with TNFalpha for 21 days. Unexpectedly, TNFalpha stimulated growth and morphogenesis of both normal and transformed MEC in primary culture, although in transformed cells its effects were delayed and the majority of the colonies were histologically abnormal, with multiple cell layers and no lumen. Since NFkappaB is a key mediator of TNFalpha action and has been implicated in carcinogenesis, the expression of the p50, p52, p65, and c-rel NFkappaB proteins in normal and transformed MEC was determined. Expression of p52 was significantly reduced in tumor cells, and p50 was absent, although its putative precursor, p105 was abundant. There were no changes in the levels of p65 or c-rel. TNFalpha induced a pronounced and sustained increase of a p50 homodimeric NFkappaB/DNA complex in both normal and transformed MEC. However, in transformed MEC, NFkappaB binding was initially undetectable but then increased in response to TNFalpha. Thus, NFkappaB expression and DNA binding activity are altered during mammary carcinogenesis. In addition, the significant increase in NFkappaB/p50 DNA-binding was temporally coincident with TNFalpha-induced growth and morphogenesis, suggesting that it may play a significant role in both normal development and carcinogenesis.

Adenocarcinoma↗

PubMed2000

Mammary fibroblasts stimulate growth, alveolar morphogenesis, and functional differentiation of normal rat mammary epithelial cells.

Stromal-epithelial interactions play a profound role in regulating normal and tumor development in the mammary gland. The molecular details of these events, however, are incompletely understood. A novel serum-free transwell coculture system was developed to study the natural paracrine interactions between mammary epithelial cells (MEC) and mammary fibroblasts (MFC) isolated from normal rats during puberty. The MEC were cultured within a reconstituted basement membrane (RBM) in transwell inserts with or without MFC in the lower well. The presence of MFC stimulated epithelial cell growth, induced alveolar morphogenesis, and enhanced casein accumulation, a marker of the functional differentiation of MEC, but did not induce ductal morphogenesis. Potent mitogenic, morphogenic, and lactogenic effects were observed when the MFC were cultured either on plastic or within a layer of RBM. Although most MFC maintained on plastic died after 1 wk in serum-free medium, fibroblast survival was enhanced significantly when the MFC were cultured within the RBM. Taken together, this in vitro model effectively reconstitutes a physiologically relevant three-dimensional microenvironment for MEC and MFC, and seems ideal for studying the locally derived factors that regulate the developmental fate of the epithelial and fibroblast compartments of the mammary gland.

Animals↗

PubMed1999

Conjugated linoleic acid inhibits proliferation and induces apoptosis of normal rat mammary epithelial cells in primary culture.

The trace fatty acid conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis when fed prior to carcinogen during pubertal mammary gland development or during the promotion phase of carcinogenesis. The following studies were done to investigate possible mechanisms of these effects. Using a physiological model for growth and differentiation of normal rat mammary epithelial cell organoids (MEO) in primary culture, we found that CLA, but not linoleic acid (LA), inhibited growth of MEO and that this growth inhibition was mediated both by a reduction in DNA synthesis and a stimulation of apoptosis. The effects of CLA did not appear to be mediated by changes in epithelial protein kinase C (PKC) since neither total activity nor expression nor localization of PKC isoenzymes alpha, beta II, delta, epsilon, eta, or zeta were altered in the epithelium of CLA-fed rats. In contrast, PKCs delta, epsilon, and eta were specifically upregulated and associated with a lipid-like, but acetone-insoluble, fibrillar material found exclusively in adipocytes from CLA-fed rats. Taken together, these observations demonstrate that CLA can act directly to inhibit growth and induce apoptosis of normal MEO and may thus prevent breast cancer by its ability to reduce mammary epithelial density and to inhibit the outgrowth of initiated MEO. Moreover, the changes in mammary adipocyte PKC expression and lipid composition suggest that the adipose stroma may play an important in vivo role in mediating the ability of CLA to inhibit mammary carcinogenesis.

Adipocytes↗

PubMed1999

Selective changes in EGF receptor expression and function during the proliferation, differentiation and apoptosis of mammary epithelial cells.

Epidermal growth factor (EGF) is a multifunctional regulator of mammary epithelial cells (MEC) that transduces its signals through the EGF receptor (EGFR). To clarify the role of the EGFR in the mammary gland, EGFR expression, localization and function were examined during different developmental stages in rats. Immunoblot analysis demonstrated high levels of EGFR during puberty, pregnancy and involution as well as at sexual maturity, and low levels throughout lactation. An immunohistochemical assay was used to show that EGFR was distinctly expressed in a variety of cell types throughout mammary glands from virgin rats and rats during pregnancy and involution, and was down-regulated in all cell types throughout lactation. To examine the relationship between EGFR expression and function, primary MEC were cultured under conditions that induced physiologically relevant growth, morphogenesis and lactogenesis. Cultured MEC expressed an in vivo-like profile of EGFR. EGFR was high in immature MEC, down-regulated in functionally differentiated MEC, and then up-regulated in terminally differentiated and apoptotic MEC. An inhibitor of the tyrosine kinase domain of EGFR was used to demonstrate that EGFR signaling was required for growth and differentiation of immature MEC, and for survival of terminally differentiated MEC, but not for maintaining functional differentiation.

Adenocarcinoma↗

PubMed1995

Prolactin and epidermal growth factor regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01-10 micrograms/ml) and EGF (1-100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland.

Animals↗

PubMed1995

Hydrocortisone and progesterone regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture.

The mechanisms of action of, and resistance to, the steroidal regulators of normal mammary epithelial and breast cancer cell development are only partially understood. A major obstacle to research progress has been the difficulty in supporting physiologically relevant development of normal mammary epithelial cells (MEC) under defined serum-free conditions. A primary culture system was developed in our laboratory that permits nonfunctional rat MEC to undergo extensive proliferation, functional differentiation, as well as multilobular and lobuloductal branching alveolar morphogenesis. In the studies reported here, the contributions of hydrocortisone and progesterone during the coordinate induction of cellular proliferation, organoid morphogenesis, and functional capacity were assessed. Hydrocortisone (0.1-10 microgram/ml) induced alveolar and multilobular branching morphogenesis, suppressed lobuloductal branching morphogenesis, and enhanced casein accumulation. Hydrocortisone also played a role in maintaining alveolar as well as multilobular branching morphogenesis and casein levels. Progesterone (0.01-1 microgram/ml) induced cellular proliferation as well as multilobular and lobuloductal branching morphogenesis, and suppressed casein accumulation. At a supraphysiological concentration (10 micrograms/ml), progesterone inhibited cell growth, alveolar branching morphogenesis, and casein accumulation. MEC cultured without progesterone for up to 1 week retained the ability to respond when subsequently exposed to this steroid. Reversibility studies suggested that progesterone was required for the induction, but not the maintenance of the mitogenic, morphogenic, and lactogenic effects. This physiologically relevant primary culture system can be used to study the factors that regulate steroid responsiveness as well as the cross-talk between steroid and growth factor receptor signaling pathways in normal MEC and breast cancer cells.

Animals↗

PubMed1992

Regulation of rat mammary epithelial cell proliferation and differentiation by tumor necrosis factor-alpha.

The effect of tumor necrosis factor-alpha (TNF alpha) on the growth and differentiation of normal rat mammary epithelial cells was evaluated using a model system in which cells were grown within a reconstituted basement membrane under defined serum-free medium conditions. TNF alpha (5-10,000 U/ml) stimulated mammary epithelial cell proliferation both under conditions previously considered optimal for their growth as well as in medium deficient in epidermal growth factor (EGF). Moreover, TNF alpha could completely substitute for EGF for cell proliferation. Under optimal conditions, TNF alpha had no effect on morphological differentiation, but in medium either lacking or deficient in EGF or when suboptimal reconstituted basement membrane was used, TNF alpha (5-100 U/ml) had a marked stimulatory effect on lobular and ductal morphogenesis. The effect of TNF alpha on functional differentiation, as assessed by casein production, was more complex. In optimal lactogenic medium, TNF alpha (10-10,000 U/ml) inhibited casein production. In the absence of EGF, however, the effect of TNF alpha appeared to follow a bell-shaped curve. Thus, omission of EGF per se resulted in a marked suppression of casein production, possibly secondary to an inhibition of morphological development. At low concentrations (approximately 5 U/ml), TNF alpha stimulated casein production in parallel to its stimulation of morphological differentiation, although not to the same extent as in medium containing optimal levels of EGF. However, once maximal stimulation of morphogenesis had been achieved, further increasing the TNF alpha concentration from 5 to 100 U/ml resulted in a concentration-dependent inhibition of casein production. This suggests that TNF alpha may have a direct inhibitory effect on casein gene expression. In summary, this is the first study to report that the multifunctional cytokine TNF alpha is a potential regulator of the growth and development of the mammary gland.

Animals↗