Stromal and epithelial caveolin-1 both confer a protective effect against mammary hyperplasia and tumorigenesis: Caveolin-1 antagonizes cyclin D1 function in mammary epithelial cells

Here, we investigate the role of caveolin-1 (Cav-1) in breast cancer onset and progression, with a focus on epithelial-stromal interactions, ie, the tumor microenvironment. Cav-1 is highly expressed in adipocytes and is abundant in mammary fat pads (stroma), but it remains unknown whether loss of Cav-1 within mammary stromal cells affects the differentiated state of mammary epithelia via paracrine signaling. To address this issue, we characterized the development of the mammary ductal system in Cav-1-/- mice and performed a series of mammary transplant studies, using both wild-type and Cav-1-/- mammary fat pads. Cav-1-/- mammary epithelia were hyperproliferative in vivo, with dramatic increases in terminal end bud area and mammary ductal thickness as well as increases in bromodeoxyuridine incorporation, extracellular signal-regulated kinase-1/2 hyperactivation, and up-regulation of STAT5a and cyclin D1. Consistent with these findings, loss of Cav-1 dramatically exacerbated mammary lobulo-alveolar hyperplasia in cyclin D1 Tg mice, whereas overexpression of Cav-1 caused reversion of this phenotype. Most importantly, Cav-1-/- mammary stromal cells (fat pads) promoted the growth of both normal mammary ductal epithelia and mammary tumor cells. Thus, Cav-1 expression in both epithelial and stromal cells provides a protective effect against mammary hyperplasia as well as mammary tumorigenesis.     

Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status

A Japanese study reported that up to 16% of breast cancer samples harbor a sporadic mutation within the human Cav-1 gene, namely P132L. To date, however, no studies have examined the United States' population. Here, we developed a novel allele-specific real-time PCR assay to detect the Cav-1 P132L mutation in mammary tumor cells isolated by laser capture microdissection from formalin-fixed paraffin-embedded breast cancer samples. We report that the Cav-1 P132L mutation is present in approximately 19% of estrogen receptor alpha (ERalpha)-positive breast cancers but not in ERalpha-negative breast cancers. This is the first demonstration that the P132L mutation is exclusively associated with ERalpha-positive mammary tumors. We also identified six novel Cav-1 mutations associated with ERalpha-positive breast cancers (W128Stop, Y118H, S136R, I141T, Y148H, and Y148S). Thus, the overall incidence of Cav-1 mutations in ERalpha-positive breast cancers approaches 35% (greater than one-third). To mechanistically dissect the functional relationship between Cav-1 gene inactivation and ERalpha expression, we isolated primary mammary epithelial cells from wild-type and Cav-1-/- mice and cultured them in a three-dimensional system, allowing them to form mammary acinar-like structures. Under conditions of growth factor deprivation, Cav-1-deficient mammary acini displayed increased ERalpha levels and enhanced sensitivity toward estrogen-stimulated growth, with specific up-regulation of cyclin D1. Finally, we discuss the possibility that sporadic Cav-1 mutations may act as an initiating event in human breast cancer pathogenesis.     

Quantitative Analysis of Three-Dimensional Human Mammary Epithelial Tissue Architecture Reveals a Role for Tenascin-C in Regulating c-Met Function

Remodeling of the stromal extracellular matrix and elevated expression of specific proto-oncogenes within the adjacent epithelium represent cardinal features of breast cancer, yet how these events become integrated is not fully understood. To address this question, we focused on tenascin-C (TN-C), a stromal extracellular matrix glycoprotein whose expression increases with disease severity. Initially, nonmalignant human mammary epithelial cells (MCF-10A) were cultured within a reconstituted basement membrane (BM) where they formed three-dimensional (3-D) polarized, growth-attenuated, multicellular acini, enveloped by a continuous endogenous BM. In the presence of TN-C, however, acini failed to generate a normal BM, and net epithelial cell proliferation increased. To quantify how TN-C alters 3-D tissue architecture and function, we developed a computational image analysis algorithm, which showed that although TN-C disrupted acinar surface structure, it had no effect on their volume. Thus, TN-C promoted epithelial cell proliferation leading to luminal filling, a process that we hypothesized involved c-met, a proto-oncogene amplified in breast tumors that promotes intraluminal filling. Indeed, TN-C increased epithelial c-met expression and promoted luminal filling, whereas blockade of c-met function reversed this phenotype, resulting in normal BM deposition, proper lumen formation, and decreased cell proliferation. Collectively, these studies, combining a novel quantitative image analysis tool with 3-D organotypic cultures, demonstrate that stromal changes associated with breast cancer can control proto-oncogene function.Most contemporary experimental and clinical breast cancer research studies have focused on the gain or loss of function of specific oncogenes or tumor suppressor genes, respectively, as primary transforming events within the mammary epithelium.¹ Equally compelling evidence, however, demonstrates that the biochemical and -physical nature of the stromal extracellular matrix (ECM) microenvironment surrounding the epithelium also contributes to breast homeostasis and tumorigenesis.^2,3,4 For example, when cultured within a laminin-enriched compliant ECM, normal breast epithelial cells produce an endogenous basement membrane (BM), which directs the formation of polarized, spherical, multicellular acini, each of which contains a single centrally-located lumen.⁵ On the other hand, blocking exuberrant β1 integrin signaling between malignant human breast cancer cells and their BM microenvironment induces phenotypic reversion and functional normalization of mammary acini.⁶ In addition, changes within the mammary ECM, such as those associated with postlactational involution, lead to altered signals to normal and malignant epithelium, failing to support duct development in the former while promoting metastasis in the latter.⁷ Increases in the biophysical stiffness of the normal mammary stromal ECM have also been shown to result in malignant behavior.³ Collectively, these and other studies reinforce the notion that tissue phenotype, specified by the ECM, can exert a dominant effect over gene expression in adjacent epithelial cells.Three-dimensional (3-D) laminin-based cultures have allowed investigators to elucidate the effects of specific oncogenes on mammary epithelial tissue form and function in an appropriate in vivo–like context.⁸ For instance, mammary epithelial cells overexpressing either cyclin D1, or the HPV-16 E7 oncogene, form individual acini that possess normal lumens, but that are of a greater size.⁹ However, overexpression of ErbB2, or coexpression of cyclin D1 and the antiapoptotic protein Bcl-2 in normal mammary epithelial cells, generates a multiacinar hyperproliferative phenotype characterized by luminal filling.^9,10 Thus, it appears that different oncogenes exert distinct site-specific effects on 3-D mammary acinar architecture and behavior. Given this, determining how tumor-associated stromal ECM components alter 3-D tissue structure represents a key step in linking changes within the ECM to the activation or repression of specific genes and signaling pathways operating within the epithelium. One approach to this question is to develop computational tools that can accurately measure changes in mammary epithelial tissue structure as a response to alterations in ECM within a pathobiologically-relevant 3-D microenvironment.Herein, we developed a novel computational model to objectively measure how tenascin-C (TN-C), a stromal ECM glycoprotein induced in experimental and clinical breast cancer,¹¹ affects normal human mammary epithelial 3-D tissue form and function. To accomplish this, we devised an algorithm to quantify acinar surface topography and volume in 3-D cultures of nonmalignant human mammary epithelial (MCF-10A) cells interacting with TN-C. In this way, we discovered that in contrast to control cultures, TN-C–treated cultures were dysmorphic, possessed disrupted endogenous BMs, and acinar interiors filled with proliferating epithelial cells. Because increased expression of the c-met proto-oncogene leads to intraluminal filling and cell proliferation,¹² we hypothesized and thereafter demonstrated that c-met contributes to TN-C–dependent loss of normal mammary epithelial tissue architecture by affecting BM integrity, epithelial cell proliferation, and lumen formation. Thus, the quantitative analyses of mammary epithelial tissue architecture presented herein provide a framework for understanding how the tumor stroma controls the function of a proto-oncogene that is amplified in human breast cancer.     

Interleukin-1alpha is a paracrine inducer of FGF7, a key epithelial growth factor in benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is an extremely common disease of older men in which there is benign overgrowth of the prostatic transition zone, leading to obstruction of urine outflow. FGF7, a potent growth factor for prostatic epithelial cells, is increased by threefold in BPH and is correlated with increased epithelial proliferation in this condition. Immunohistochemistry of normal and hyperplastic prostate revealed that FGF7-expressing fibroblastic cells were present in higher numbers near the epithelial acini, implying that epithelial cells may express a factor that induces FGF7 expression by stromal cells. Conditioned medium (CM) from primary cultures of prostatic epithelial cells was capable of inducing a two- to sixfold increase in the expression of FGF7 by primary stromal cultures. Blocking experiments with neutralizing anti-interleukin-1alpha (Il-1alpha) antibodies and IL-1Ra, an Il-1alpha receptor antagonist, show that this inducing activity was due to the presence of Il-1alpha in the epithelial CM. Analysis of normal prostatic peripheral zone and BPH tissue by enzyme-linked immunoabsorption assay reveal that Il-1alpha is present at increased levels in hyperplastic prostate and that levels of Il-1alpha correlate strongly with tissue FGF7 concentration in BPH. Therefore Il-1alpha is produced by prostatic epithelial cells and can induce FGF7, a potent epithelial growth factor, which can in turn lead to further epithelial growth and increased Il-1alpha secretion, thus establishing a double paracrine loop that is functionally equivalent to an autocrine growth loop. This double paracrine loop may play a key role in the abnormal proliferation of the transition zone, which is critical to the pathogenesis of BPH.     

MMTV promoter-regulated caveolin-1 overexpression yields defective parenchymal epithelia in multiple exocrine organs of transgenic mice

Caveolin-1 (Cav-1) is a major structural protein of caveolae, specialized plasma membrane invaginations that are involved in a cell-specific fashion in diverse cell activities such as molecular transport, cell adhesion, and signal transduction. In normal adult mammals, Cav-1 expression is abundant in mesenchyme-derived cells but relatively low in epithelial parenchyma. However, epithelial Cav-1 overexpression is associated with development and/or progression of many carcinomas. In this study, we generated and characterized a transgenic mouse model of Cav-1 overexpression under the control of a mouse mammary tumor virus (MMTV) long terminal-repeat promoter, which is predominantly expressed in specific epithelial cells. The MMTVcav-1⁺ transgenic mice were fertile, and females bore litters of normal size with no obvious developmental abnormalities. However, by age 11 months, the MMTVcav-1⁺ mice demonstrated overtly different phenotypes in multiple exocrine organs when compared with their nontransgenic MMTVcav-1⁻ littermates. Cav-1 overexpression in MMTVcav-1⁺ mice produced organ-specific abnormalities, including hypotrophy of mammary glandular epithelia, bronchiolar epithelial hyperplasia and atypia, mucous-cell hyperplasia in salivary glands, elongated hair follicles and dermal thickening in the skin, and reduced accumulation of enzymogen granules in pancreatic acinar cells. In addition, the MMTVcav-1⁺ transgenic mice tended to have a greater incidence of malignant tumors, including lung and liver carcinomas and lymphoma, than their MMTVcav-1⁻ littermates. Our results indicate that Cav-1 overexpression causes organ-specific, age-related epithelial disorders and suggest the potential for increased susceptibility to carcinogenesis.     

Regulation of Cripto-1 signaling and biological activity by caveolin-1 in mammary epithelial cells

Human and mouse Cripto-1 (CR-1/Cr-1) proteins play an important role in mammary gland development and tumorigenesis. In this study, we examined the relationship between Cripto-1 and caveolin-1 (Cav-1), a membrane protein that acts as a tumor suppressor in the mammary gland. Cripto-1 was found to interact with Cav-1 in COS7 cells and mammary epithelial cells. Using EpH4 mouse mammary epithelial cells expressing Cr-1 (EpH4 Cr-1) or Cr-1 and Cav-1 (EpH4 Cr-1/Cav-1), we demonstrate that Cav-1 expression markedly reduced the ability of Cr-1 to enhance migration, invasion, and formation of branching structures in EpH4 Cr-1/Cav-1 cells as compared to EpH4 Cr-1 cells. Furthermore, coexpression of Cav-1 together with Cr-1 in EpH4 Cr-1/Cav-1 cells inhibited Cr-1-mediated activation of c-src and mitogen-activated protein kinase signaling pathways. Conversely, primary mammary epithelial cells isolated from Cav-1 null(-/-)/mouse mammary tumor virus-CR-1 transgenic animals showed enhanced motility and activation of mitogen-activated protein kinase and c-src as compared to Cav-1(+/-)/CR-1 mammary cells. Finally, mammary tumors derived from mouse mammary tumor virus-CR-1 mice showed a dramatic reduction of Cav-1 expression as compared to mammary tissue from normal FVB/N mice, suggesting that in vivo Cav-1 is down-regulated during the process of CR-1-mediated mammary tumorigenesis.     

Caveolin-1 mutations (P132L and null) and the pathogenesis of breast cancer: caveolin-1 (P132L) behaves in a dominant-negative manner and caveolin-1 (-/-) null mice show mammary epithelial cell hyperplasia

Caveolin-1 (Cav-1) is the principal structural protein of caveolae membranes that are found in most cells types, including mammary epithelial cells. Recently, we mapped the human CAV1 gene to a suspected tumor suppressor locus (7q31.1/D7S522) that is deleted in a variety of human cancers, as well as mammary tumors. In addition, the CAV1 gene is mutated (P132L) in up to approximately 16% of human breast cancers. The mechanism by which deletion or mutation of the Cav-1 gene contributes to mammary tumorigenesis remains unknown. To understand the role of the Cav-1 (P132L) mutation in the pathogenesis of human breast cancers, we generated the same mutation in wild-type (WT) Cav-1 and studied its behavior in cultured cells. Interestingly, the P132L mutation leads to formation of misfolded Cav-1 oligomers that are retained within the Golgi complex and are not targeted to caveolae or the plasma membrane. To examine whether the Cav-1 (P132L) mutant behaves in a dominant-negative manner, we next co-transfected cells with Cav-1 (P132L) and WT Cav-1, and evaluated their caveolar targeting. Our results indicate that Cav-1 (P132L) behaves in a dominant-negative manner, causing the mislocalization and intracellular retention of WT Cav-1. Virtually identical results were obtained when Cav-1 (P132L) was stably expressed at physiological levels in a nontransformed human mammary epithelial cell line (hTERT-HME1). These data provide a molecular explanation for why only a single mutated CAV1 allele is found in patients with breast cancer. Thus, we next investigated if functional inactivation of Cav-1 gene expression leads to mammary tumorigenesis in vivo. For this purpose, we performed mammary gland analysis on Cav-1-deficient mice (-/-) that harbor a targeted disruption of the Cav-1 gene (a null mutation). Interestingly, we show that inactivation of Cav-1 gene expression leads to mammary epithelial cell hyperplasia, even in 6-week-old virgin female mice. These data clearly implicate loss of functional Cav-1 in the pathogenesis of mammary epithelial cell hyperplasia, and suggest that Cav-1-null mice represent a novel animal model to study premalignant mammary disease.     

Primary culture of human oral epithelial cells. Growth requirements and expression of differentiated characteristics

Epithelial cell cultures were initiated from explants of normal human oral mucosa. Growth parameters, cell type, and degree of maturation/cytodifferentiation were assessed by morphological and surface topographical criteria (light and scanning electron microscopy) together with immunofluorescence studies with a panel of antibodies to cytokeratins and extracellular matrix components. The effects of different media formulations were compared. Whereas stromal cell over-growth soon became apparent in media containing 10% serum, in low serum (0.5%) media containing insulin, hydrocortisone, epidermal growth factor (EGF), and/or cholera toxin (CT), epithelial growth was maintained with minimal or absent stromal cell contamination. Cell proliferation, maturation, and differentiation were modulated by EGF and CT: cultures maintained on EGF showed optimal growth but cells typically displayed only limited differentiation. By contrast, CT promoted considerably more cytodifferentiation but at the expense of proliferative capacity. Both factors together were complementary, resulting in maintenance of cells of a more mature phenotype of high proliferative capacity. Cytokeratins of normal oral epithelium in situ demonstrated characteristic changes in patterns of expression associated with differentiation. In culture, proliferative epithelial cells expressed keratins typical of the basal layer, whereas the most differentiated cells were identified by their strong reactivity with antibodies to epidermal keratins. Less mature cells showed expression of keratins associated with nonstratified epithelia. In cultures maintained with CT but no EGF, there was a tendency for weaker expression of basal type keratins, further suggesting that these cells were maintaining a more differentiated phenotype. Extracellular matrix components (fibronectin, laminin, collagen type IV) were not expressed by any epithelial cells in culture. Irrespective of medium composition, cultures did not survive beyond 100 days (5 or 6 subcultivations) before undergoing an irreversible 'crisis' of growth arrest and onset of degenerative changes.     

In situ lineage tracking of human prostatic epithelial stem cell fate reveals a common clonal origin for basal and luminal cells

Stem cells accumulate mitochondrial DNA (mtDNA) mutations resulting in an observable respiratory chain defect in their progeny, allowing the mapping of stem cell fate. There is considerable uncertainty in prostate epithelial biology where both basal and luminal stem cells have been described, and in this study the clonal relationships within the human prostate epithelial cell layers were explored by tracing stem cell fate. Fresh-frozen and formalin-fixed histologically-benign prostate samples from 35 patients were studied using sequential cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) enzyme histochemistry and COX subunit I immunofluorescence to identify areas of respiratory chain deficiency; mtDNA mutations were identified by whole mitochondrial genome sequencing of laser-captured areas. We demonstrated that cells with respiratory chain defects due to somatic mtDNA point mutations were present in prostate epithelia and clonally expand in acini. Lineage tracing revealed distinct patterning of stem cell fate with mtDNA mutations spreading throughout the whole acinus or, more commonly, present as mosaic acinar defects. This suggests that individual acini are typically generated from multiple stem cells, and the presence of whole COX-deficient acini suggests that a single stem cell can also generate an entire branching acinar subunit of the gland. Significantly, a common clonal origin for basal, luminal and neuroendocrine cells is demonstrated, helping to resolve a key area of debate in human prostate stem cell biology.     

Immunohistochemical study of expression of Caveolin-1 in canine malignant mammary tumour

Caveolin-1 (Cav-1) is thought to have an important impact on both signal transduction and the mediation of intracellular processes and may contribute to certain steps of carcinogenesis in various types of cancer. Cav-1 immunohistochemical expression was studied in 37 canine malignant mammary tumours to investigate the relationship of histopathological type and histopathologic grade with the expression of Cav-1. Cav-1 was expressed by epithelial cells in 33 of 37 (89.2 %) malignant mammary tumours. There was a significant relationship between epithelial expression of Cav-1 and histological grade of tumours (P?<?0.05); however, no significant relationship was observed between the expression of Cav-1 in myoepithelial and mesenchymal cells and histological grade (P?<?0.05). This study showed that Cav-1 can be used as a prognostic factor and its expression is associated with more malignant canine mammary tumours.     

A temporal requirement for Hippo signaling in mammary gland differentiation,growth, and tumorigenesis

Despite recent progress, the physiological role of Hippo signaling in mammary gland development and tumorigenesis remains poorly understood. Here we show that the Hippo pathway is functionally dispensable in virgin mammary glands but specifically required during pregnancy. In contrast to many other tissues, hyperactivation of YAP in mammary epithelia does not induce hyperplasia but leads to defects in terminal differentiation. Interestingly, loss of YAP causes no obvious defects in virgin mammary glands but potently suppresses oncogene-induced mammary tumors. The selective requirement for YAP in oncogenic growth highlights the potential of YAP inhibitors as molecular targeted therapies against breast cancers.     

Modulation of epithelial differentiation of cultured rat bladder carcinoma

Epithelium-to-mesenchyme transformation plays a key role in tissue remodelling in embryos since it allows cells from the primitive epithelia to migrate to other sites where they participate in the formation of new structures. A similar phenomenon may be involved in the detachment of malignant cells from neighboring primary tumor cells, which is a prerequisite to the invasion of neighboring tissues or the development of metastases. To test this hypothesis, an in vitro model using a rat bladder carcinoma cell line was developed. Cells exhibited epithelial features under standard culture conditions. After exposure to a soluble inducer (acidic FGF) or the specific extracellular matrix components (collagens), the cells acquired a fibroblastic phenotype, separated from one another, and started to move freely on the substrate. Inducers were found to act synergistically on the fibroblastic transformation of carcinoma cells and to promote the penetration of these cells into collagen gels.     

Caveolin-1(-/-)- and caveolin-2(-/-)-deficient mice both display numerous skeletal muscle abnormalities, with tubular aggregate formation

Here, we examine the role of "non-muscle" caveolins (Cav-1 and Cav-2) in skeletal muscle biology. Our results indicate that skeletal muscle fibers from male Cav-1(-/-) and Cav-2(-/-) mice show striking abnormalities, such as tubular aggregates, mitochondrial proliferation/aggregation, and increased numbers of M-cadherin-positive satellite cells. Notably, these skeletal muscle defects were more pronounced with increasing age. Because Cav-2-deficient mice displayed normal expression levels of Cav-1, whereas Cav-1-null mice exhibited an almost complete deficiency in Cav-2, these skeletal muscle abnormalities seem to be due to loss of Cav-2. Thus, Cav-2(-/-) mice represent a novel animal model-and the first genetically well-defined mouse model-that can be used to study the pathogenesis of tubular aggregate formation, which remains a poorly understood age-related skeletal muscle abnormality. Finally, because Cav-1 and Cav-2 were not expressed within mature skeletal myofibers, our results indicate that development of these abnormalities probably originates in stem/precursor cells, such as satellite cells or myoblasts. Consistent with this hypothesis, skeletal muscle isolated from male Cav-3(-/-) mice did not show any of these abnormalities. As such, this is the first study linking stem cells with the genesis of these intriguing muscle defects.     

Maintenance of normal human breast organoids within rat mammary fat pads in organ culture

Summary Normal human breast organoids, derived by collagenase digestion of reduction mammaplasty tissue specimens, have been cultured in vitro for up to 28 days after injection into organ cultures of virgin rat mammary fat pads. The culture medium was serum-free Waymouth's MB 752/ 1 with hormonal additives. The rat mammary tissue responded well to growth-promoting and lactogenic stimuli in the culture medium, in agreement with previous investigations. Using immunohistochemistry casein was identified in rat epithelia exposed to lactogenic medium. Human organoids in culture remained viable but did not show hormone-responsiveness. Electron microscopy confirmed the presence of both luminal epithelial cells and myoepithelial cells.The serum-free culture of normal human breast organoids in a three-dimensional matrix provides a system in which to study factors controlling growth and differentiation.     

Caveolin-1 and accelerated host aging in the breast tumor microenvironment: chemoprevention with rapamycin, an mTOR inhibitor and anti-aging drug

Increasing chronological age is the most significant risk factor for human cancer development. To examine the effects of host aging on mammary tumor growth, we used caveolin (Cav)-1 knockout mice as a bona fide model of accelerated host aging. Mammary tumor cells were orthotopically implanted into these distinct microenvironments (Cav-1(+/+) versus Cav-1(-/-) age-matched young female mice). Mammary tumors grown in a Cav-1-deficient tumor microenvironment have an increased stromal content, with vimentin-positive myofibroblasts (a marker associated with oxidative stress) that are also positive for S6-kinase activation (a marker associated with aging). Mammary tumors grown in a Cav-1-deficient tumor microenvironment were more than fivefold larger than tumors grown in a wild-type microenvironment. Thus, a Cav-1-deficient tumor microenvironment provides a fertile soil for breast cancer tumor growth. Interestingly, the mammary tumor-promoting effects of a Cav-1-deficient microenvironment were estrogen and progesterone independent. In this context, chemoprevention was achieved by using the mammalian target of rapamycin (mTOR) inhibitor and anti-aging drug, rapamycin. Systemic rapamycin treatment of mammary tumors grown in a Cav-1-deficient microenvironment significantly inhibited their tumor growth, decreased their stromal content, and reduced the levels of both vimentin and phospho-S6 in Cav-1-deficient cancer-associated fibroblasts. Since stromal loss of Cav-1 is a marker of a lethal tumor microenvironment in breast tumors, these high-risk patients might benefit from treatment with mTOR inhibitors, such as rapamycin or other rapamycin-related compounds (rapalogues).     

Cellular localization of aquaporins along the secretory pathway of the lactating bovine mammary gland: An immunohistochemical study

In this study we examined the cellular localization of aquaporins (AQPs) along the secretory pathway of actively lactating bovine mammary glands using immunohistochemistry. Mammary tissues examined included secretory ducts and acini, gland cisterns, teats, stromal and adipose tissues. Aquaporin 1 (AQP1) was localized in capillary endothelia throughout the mammary gland in addition to myoepithelial cells underlying teat duct epithelia. AQP2 and AQP6 were not detected and AQP9 was found only in leukocytes. AQP3 and AQP4 were observed in selected epithelial cells in the teat, cistern and secretory tubuloalveoli. AQP5 immunopositivity was prominent in the cistern. AQP3 and AQP7 were found in smooth muscle bundles in the teat, secretory epithelial cells and duct epithelial cells. These immunohistochemical findings support a functional role for aquaporins in the transport of water and small solutes across endothelial and epithelial barriers in the mammary gland and in the production and secretion of milk.     

Light-microscopic stereology for the study of the ventral and dorsal prostates of the syrian hamster: Strategy,techniques, and applications

A stereological approach to studying the architecture of the ventral and dorsal prostates of the Syrian hamster is described. In this approach, the prostate is considered to consist of acinar and interacinar compartments. The acinar compartment is divided into luminal, epithelial, lamina proprial, and muscular stromal compartments. Volume fractions of acini and interacinar tissue are calculated with reference to the volume of the gland; those of the acinar components are calculated with reference to the acinar volume. Volume fractions are determined from point counts. The surface fraction of the secretory epithelium is determined from intercept counts, with reference to the volume of the epithelium. By assuming that the acini are cylindrical, the acinar length fraction is calculated with reference to the volume of the gland. Absolute values for the volumes of different components, epithelial surface area, and acinar length can be determined from the weight of the gland, its specific gravity, and the volume fraction of that component, or epithelial surface fraction, or acinar length fraction, respectively. Finally, from these values and assuming a cylindrical shape for acini, characteristics of the typical acinus, including its radius, the thicknesses of its epithelium, lamina propria, and muscular stroma, and the amount of mucosal folding can be calculated from simple geometrical formulae. This approach is illustrated from a consideration of the ventral and dorsal prostates of 20-week-old Syrian hamsters.     

Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy-chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non-specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.     

The functional interplay between EGFR overexpression, hTERT activation, and p53 mutation in esophageal epithelial cells with activation of stromal fibroblasts induces tumor development, invasion, and differentiation

Esophageal cancer is a prototypic squamous cell cancer that carries a poor prognosis, primarily due to presentation at advanced stages. We used human esophageal epithelial cells as a platform to recapitulate esophageal squamous cell cancer, thereby providing insights into the molecular pathogenesis of squamous cell cancers in general. This was achieved through the retroviral-mediated transduction into normal, primary human esophageal epithelial cells of epidermal growth factor receptor (EGFR), the catalytic subunit of human telomerase (hTERT), and p53(R175H), genes that are frequently altered in human esophageal squamous cell cancer. These cells demonstrated increased migration and invasion when compared with control cells. When these genetically altered cells were placed within the in vivo-like context of an organotypic three-dimensional (3D) culture system, the cells formed a high-grade dysplastic epithelium with malignant cells invading into the stromal extracellular matrix (ECM). The invasive phenotype was in part modulated by the activation of matrix metalloproteinase-9 (MMP-9). Using pharmacological and genetic approaches to decrease MMP-9, invasion into the underlying ECM could be suppressed partially. In addition, tumor differentiation was influenced by the type of fibroblasts within the stromal ECM. To that end, fetal esophageal fibroblasts fostered a microenvironment conducive to poorly differentiated invading tumor cells, whereas fetal skin fibroblasts supported a well-differentiated tumor as illustrated by keratin "pearl" formation, a hallmark feature of well-differentiated squamous cell cancers. When inducible AKT was introduced into fetal skin esophageal fibroblasts, a more invasive, less-differentiated esophageal cancer phenotype was achieved. Invasion into the stromal ECM was attenuated by genetic knockdown of AKT1 as well as AKT2. Taken together, alterations in key oncogenes and tumor suppressor genes in esophageal epithelial cells, the composition and activation of fibroblasts, and the components of the ECM conspire to regulate the physical and biological properties of the stroma.