Altered expression and localization of PKC eta in human breast tumors.

Protein kinase C (PKC) eta is a PKC isoform whose upregulation is associated with differentiation in many epithelial tissues, including the rat mammary gland. The purpose of this study was to examine whether PKC eta is altered, in expression or localization, in human breast cancer. Paraffin sections of 49 in situ breast lesions, 29 invasive breast tumors, and nine normal breast biopsies were examined for PKC eta expression by immunohistochemistry. Adjacent regions of normal epithelium, and in situ lesions that were present adjacent to invasive lesions were also analyzed. In normal epithelium, regardless of the presence of adjacent in situ or invasive lesions, PKC eta was present in the cytoplasm of the luminal epithelium, and increased in areas of normal lobular development, similar to normal rat mammary gland. PKC eta staining intensity was homogeneous in normal lobules, but heterogeneous in in situ and invasive lesions, being focally increased in cells with aberrant nuclear morphology. In situ lesions were similar to adjacent normal epithelium in average staining intensity, regardless of whether invasion was also present. However, the invasive lesions themselves were significantly decreased in staining intensity compared to adjacent in situ lesions. In addition, 75% of invasive breast cancer lesions showed decreased staining relative to adjacent normal epithelium, compared to 37% of in situ lesions. The invasive tumors which possessed high PKC eta staining were associated with positive lymph node status. These results demonstrate that quantitative and qualitative alterations in PKC eta occur in human breast cancers.

Expression, tissue distribution, and cellular localization of the antiapoptotic TIP-B1 protein.

TIP-B1 is a novel 27-kDa protein isolated from the cytosol of tumor necrosis factor (TNF)-stimulated cells. Cells preincubated with TIP-B1 are protected from TNF-induced apoptosis. This study showed that, as with normal fibroblasts and U937 histiocytic lymphoma, human MCF7 mammary adenocarcinoma cells were protected from TNF in a concentration-dependent manner by pretreatment with either TNF or purified TIP-B1. Immunoblot and immunohistochemical analyses indicated expression of both TIP-B1 mRNA and protein in MCF7 cells and heart, kidney, brain, liver, ovary, uterus, thymus, spleen, lymph node, and mammary gland cells throughout their development. Expression of TIP-B1 was heterogeneous, with staining of specific cell types within tissues. Based on the ability of TIP-B1 to protect both normal and tumor cells from TNF-induced apoptosis and its broad tissue distribution, with expression only in select cells within those tissues, a role for TIP-B1 in the regulation of TNF-induced effects is strongly indicated.

A developmental atlas of rat mammary gland histology.

The mammary gland is a dynamic tissue that undergoes epithelial expansion and invasion during puberty and cycles of branching and lobular morphogenesis, secretory differentiation, and regression during pregnancy, lactation, and involution. The alteration in the mammary gland epithelium during its postnatal differentiation is accompanied by changes in the multiple stromal cell types present in this complex tissue. The postnatal plasticity of the epithelium, endothelium, and stromal cells of the mammary gland may contribute to its susceptibility to carcinogenesis. The purpose of this review is to assist researchers in recognizing histological changes in the epithelium and stroma of the rat mammary gland throughout development.

Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation.

Protein kinase C (PKC) is involved in signaling that modulates the proliferation and differentiation of many cell types, including mammary epithelial cells. In addition, changes in PKC expression or activity have been observed during mammary carcinogenesis. In order to examine the involvement of specific PKC isoforms during normal mammary gland development, the expression and localization of PKCs alpha, delta, epsilon and zeta were examined during puberty, pregnancy, lactation, and involution. By immunoblot analysis, expression of PKC alpha, delta, epsilon and zeta proteins was increased in mammary epithelial organoids during the transition from puberty to pregnancy. In mammary gland frozen sections, PKCs alpha, delta, epsilon and zeta were stained in the luminal epithelium and myoepithelium, in varying isoform-and developmental stage-specific locations. PKC alpha was found in a punctate apical localization in the luminal epithelium during pregnancy. During lactation, PKC epsilon was present in the nucleus, and PKC zeta was concentrated in the subapical region of the luminal epithelium. Additionally, marked staining for PKCs alpha, delta, epsilon, and zeta was observed in the myoepithelial cells at the base of ducts and alveoli. This basal ductal and alveolar staining differed in intensity in a developmentally-specific fashion. During most time points (virgin, pregnant, lactating, and early involution), myoepithelial cells of the duct were more intensely stained than those lining the alveoli for PKCs alpha, delta, epsilon and zeta. During late involution (days 9-12), the preferential staining of ducts was lost or reversed, and the myoepithelial cells lining the regressing alveolar structures stained equally (PKCs epsilon and zeta) or more intensely (PKCs alpha and delta), coincident with the thickening of the myoepithelial cells surrounding the regressing alveoli. The increased PKC isoform staining at the base of alveoli during involution suggests that alveolar regression may be influenced by alterations in signaling in the alveolar myoepithelium.

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.

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.

Multiple differentiation pathways of rat mammary stromal cells in vitro: acquisition of a fibroblast, adipocyte or endothelial phenotype is dependent on hormonal and extracellular matrix stimulation.

It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblast-like phenotype or differentiate in vitro into mature adipocytes in a hormone-dependent manner. This paper reports that rat mammary stromal cells can also differentiate into capillary-like structures in vitro when cultured on a reconstituted basement membrane (RBM). The differentiation potential of mammary stromal cells was compared with that of human umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a fibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. When plated on reconstituted basement membrane, all three cell types began to migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple structures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular endothelial growth factor (VEGF) and was inhibited by the anti-angiogenic drug suramin. Western blotting analysis demonstrated the presence of the endothelial-specific marker flk-1, as well as the presence of the tight-junction-associated protein ZO-1. Mammary stromal cell differentiation into capillary structures was not a terminal state, since these cells were still able to differentiate into adipocytes when exposed to adipogenic medium. These findings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent manner, and may explain the dramatic changes in stromal composition during both normal mammary gland development and tumorigenesis.

Protein kinase C eta upregulation and secretion during postnatal rat mammary gland differentiation.

The mammary gland has the ability to undergo repeated cycles of tightly regulated postnatal proliferation, differentiation, and apoptosis-mediated regression, providing a model to investigate potential regulators of mammary epithelial growth and differentiation. Protein kinase C eta is a candidate regulator of mammary epithelial differentiation, as increased expression of PKC eta is often observed during the terminal differentiation of many epithelial tissues. In this study, PKC eta expression and localization were characterized during puberty, pregnancy, lactation and involution in isolated rat mammary epithelial cells (MEC), as well as in paraffin-embedded and frozen rat mammary gland sections. By Western blot analysis of whole cell lysates from purified MEC, PKC eta protein expression increased during the shift from resting to a pregnant state. This increased PKC eta protein expression during pregnancy was associated with alveolar rather than ductal development, as immunohistochemical staining for PKC eta was increased in differentiating secretory alveoli, but not ducts. By immunofluorescent staining, PKC eta was stained intensely in an intracellular reticular meshwork throughout the cytosol of alveolar epithelial cells from pregnant mammary gland. During lactation, PKC eta was abundant in apocrine bodies budding from the alveolar epithelium, in the lumen of alveoli, and was present in milk, in association with casein, while being decreased in the cytoplasm of the luminal alveolar epithelium. Staining intensity of alveoli for PKC eta decreased further during involution. Western blotting of subcellular fractions from isolated mammary epithelial cells demonstrated that PKC eta remained associated with the membrane and particulate fractions throughout development. The upregulation of PKC eta in alveolar but not ductal epithelium during pregnancy suggests an association with functional secretory differentiation.