Selective inhibition of MEK1/2 reveals a differential requirement for ERK1/2 signalling in the regulation of HIF-1 in response to hypoxia and IGF-1.

The transcription factor hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tumour growth and progression, and HIF-1 is regulated through a number of signalling pathways. Here, we investigated the involvement of the mitogen-activated protein kinase (MAPK) signalling pathway in HIF-1 regulation. We found that overexpression of wild-type (WT) extracellular signal regulated protein kinase 1 (ERK1) greatly potentiated HIF-1 activation in hypoxia and HIF-1alpha induced in response to insulin growth-like factor 1 (IGF-1). Conversely, treatment of tumour cells with the MEK1/2 inhibitors PD98059 or U0216, or expression of a dominant-negative form of ERK1 blocked HIF-1 activation in hypoxia without affecting HIF-1alpha induction, localization or binding of HIF-1beta. Interestingly however, the highly selective MEK1/2 inhibitor PD184352 did not inhibit HIF-1 activity or vascular endothelial growth factor (VEGF) induced in response to hypoxia but blocked HIF-1alpha protein and HIF-1 activity induced by IGF-1 stimulation without affecting HIF-1alpha mRNA levels. Finally, we found that ERK5 phosphorylation status was not significantly affected by hypoxia in the presence or absence of PD184352. Taken together, our data suggest that although ERK1/2 signalling is important for HIF-1alpha induction and HIF-1 activity in response to IGF-1, it is dispensable for the induction of HIF-1alpha and activation of HIF-1 in response to hypoxia.

An immunocytochemical and ultrastructural study of heterogeneity in the human breast carcinoma cell line PMC42.

A recently established human breast carcinoma cell line has been reported to exhibit a number of morphological cell types in monolayer cultures as defined by phase-contrast microscopy. The cell line also produces cords of viable cells floating within the culture medium. Cultures of this cell line, grown in monolayer, on collagen gels, and as floating cords of cells, were studied by transmission electron microscopy and immunocytochemistry. A detailed analysis of the staining pattern obtained with a series of monoclonal antibodies with well-defined human breast epithelial cell specificities and a polyclonal antikeratin antibody showed PMC42 to resemble cultures of both normal human breast and other human breast carcinoma cell lines. The pleomorphic nature of PMC42 cultures was confirmed at an ultrastructural level, and of the eight cell types observed by phase-contrast appearance, seven ultrastructural counterparts were observed. In addition, the presence of intracytoplasmic lumina and overt epithelial differentiation confirm a breast epithelial origin for this cell line.

Characterisation of chondroitin/dermatan sulphate proteoglycans synthesised by rat mammary myoepithelial and fibroblastic cell lines.

Chondroitin sulphate proteoglycans were isolated from the culture medium of rat mammary gland fibroblast (Rama 27) and myoepithelial (Rama 401) cell lines which had been labelled with [35S]sulphate. Chromatography on Sepharose CL-4B indicated that the Rama 401 proteoglycan was larger than the Rama 27 proteoglycan (Kav values 0.47 and 0.56, respectively). Treatment of the proteoglycans with alkaline NaBH4 yielded chondroitin sulphate chains with average M(r) values of 37,000 (Rama 401) and 21,000 (Rama 27). Structural analysis of the glycosaminoglycan chains indicated that both were co-polymers of chondroitin and dermatan sulphate although there were differences in the amounts and distribution of the disaccharide repeating units. The M(r) values of the core proteins, determined by immunoblotting, were about 43,000 and 46,000 (Rama 27) and 44,500 (Rama 401). Using an antibody to chondroitin sulphate proteoglycan in immunofluorescence experiments, the proteoglycan was demonstrated on the surface of both cell lines. Rama 27 cells additionally possessed an extensive fibrous extracellular matrix which also stained with the antibody. Staining of sections of lactating mammary gland suggested that the proteoglycan was present in the basement membrane as well as the stromal connective tissue. The presence of chondroitin sulphate proteoglycan in the basement membrane was confirmed by ultrastructural immunolocalisation.

Rapid modulation of gap junction expression in mouse mammary gland during pregnancy, lactation, and involution.

We investigated the expression of gap junctions in virgin, pregnant, lactating, and involuting mouse mammary gland epithelium with a panel of sequence-specific antibodies to connexins 26, 32, 40 and 43. Indirect immunofluorescence labeling of frozen sections of mammary gland showed that connexin26 was the major connexin in mammary epithelium. Connexins 43, 40, and 32 were not detected. Connexin26 was not detected in the mammary epithelium of virgin mice but was increasingly expressed during pregnancy. At Day 4 of pregnancy, when the mammary gland was composed almost exclusively of ducts, low levels of labeling were detected in the duct epithelium. As pregnancy progressed, the level of labeling with antibodies to connexin26 increased in quantity and intensity. At Day 12, when developing lobules were present, immunolabeling for connexin26 was detected surrounding the developing lumina, which on Day 19 were distended with milk. Labeling of mammary gland reached a maximum on Day 24 (5 days' lactation) but within 24 hr of removal of the litter on Day 28, connexin26 labeling was greatly diminished. No further change in labeling intensity with the antibodies to connexins was detected throughout involution. Double immunofluorescence labeling of 5-day lactating mammary gland with antibodies to connexin26 and anti-keratin 14 or -keratin 19 indicated that the majority of gap junctions detected by this analysis were within the luminal cell population. Western blot analysis of a lactating mammary gland (Day 24) confirmed the absence or low level of expression of connexins 32 and 43, as seen in the immunofluorescence studies, and showed that connexin26 was a dominant antigen expressed in lactating mammary gland epithelium.

Differentiation of separated mouse mammary luminal epithelial and myoepithelial cells cultured on EHS matrix analyzed by indirect immunofluorescence of cytoskeletal antigens.

We have previously demonstrated that purified virgin mouse mammary luminal epithelial and myoepithelial cells promiscuously express cell type-specific cytokeratins when they are cloned in vitro. Changes in cytokeratin expression may be indicators of the loss or change of the differentiated identity of a cell. To investigate the factors that may be responsible for the maintenance of differentiated cellular identity, specifically cell-cell and cell-matrix interactions, we cloned flow-sorted mouse mammary epithelial cells on the extracellular matrix (ECM) derived from the Engelbreth-Holm-Swarm murine sarcoma (EHS matrix). Changes in cell differentiation on EHS, compared with culture on glass, were analyzed by comparing patterns of cytokeratin expression. The results indicate that ECM is responsible for maintenance of the differentiated identity of basal/myoepithelial cells and prevents the inappropriate expression of luminal antigens seen on glass or plastic. Luminal cell identity in the form of retention of luminal markers and absence of basal/myoepithelial antigens, on the contrary, appears to depend on homotypic cell-cell contacts and interactions. The results also show that luminal cells (or a subpopulation of them) can generate a cell layer that expresses only basal cytokeratin markers (and no luminal cytokeratin markers) and may form a pluripotent compartment. (J Histochem Cytochem 47:1513-1524, 1999)

High-pressure freezing for immunocytochemistry.

Ultrastructural immunocytochemistry requires that minimal damage to antigens is imposed by the processing methods. Immersion fixation in cross-linking fixatives with their potential to damage antigens is not an ideal approach and rapid freezing as an alternative sample-stabilization step has a number of advantages. Rapid freezing at ambient pressure restricts the thickness of well-frozen material obtainable to approximately 15 microm or less. In contrast, high-pressure freezing has been demonstrated to provide ice-crystal-artefact-free freezing of samples up to 200 microm in thickness. There have been few reports of high-pressure freezing for immunocytochemical studies and there is no consensus on the choice of post-freezing sample preparation. A range of freeze-substitution time and temperature protocols were compared with improved tissue architecture as the primary goal, but also to compare ease of resin-embedding, polymerization and immunocytochemical labelling. Freeze-substitution in acetone containing 2% osmium tetroxide followed by epoxy-resin embedding at room temperature gave optimum morphology. Freeze-substitution in methanol was completed within 18 h and in tetrahydrofuran within 48 h but the cellular morphology of the Lowicryl-embedded samples was not as good as when samples were substituted in pure acetone. Acetone freeze-substitution was slow, taking at least 6 days to complete, and gave blocks which were difficult to embed in Lowicryl HM20. Careful handling of frozen samples avoiding rapid temperature changes reduced apparent ice-crystal damage in sections of embedded material. Thus a slow warm-up to freeze-substitution temperature and a long substitution time in acetone gave the best results in terms of freezing quality and cellular morphology. No clear differences emerged between the different freeze-substitution media from immunocytochemical labelling experiments.

Cell proliferation in the human mammary epithelium. Differential contribution by epithelial and myoepithelial cells.

The ductal system of the human breast consists of epithelial, myoepithelial, and basal clear cells. By labeling ducts and alveoli dissected from reduction mammoplasty specimens with 3H-thymidine in vitro and labeling human breast organoids xenografted in nude mice in vivo, it was found that cellular proliferation in the human breast is virtually confined to epithelial and basal clear cells. A pulse label of 3H-thymidine in organ culture explants was followed over a period of time, and it was found that myoepithelial cells originate from a precursor cell population within the mammary epithelium after a number of cell divisions. Myoepithelial cells were not seen to divide when fully mature.

Effects of growth factors on proliferation on basal and luminal cells in human breast epithelial explants in serum-free culture.

A method of culturing human breast epithelium is described in which viable explants can be maintained in protein-free medium while retaining the capacity of responding to added hormones and growth factors for at least 7 days. Culture parameters were chosen to provide maximum sensitivity of detection of proliferative responses by autoradiography. Under basal conditions, the mean thymidine labeling index of the explants was 0.08%. After stimulation with insulin, hydrocortisone, and cholera toxin (I,H,CT), a combination known to stimulate proliferation in human breast epithelium in vitro, the mean labeling index was 15.7%. Stimulation of explants with epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha resulted in mean labeling indices of 6.6 and 10.8%, respectively. Autoradiography at the ultrastructural level demonstrated that in I,H,CT-stimulated explants the majority of the labeled cells were luminal, with only 1.5% being basal cells. In contrast, after EGF and TGF-alpha basal cells accounted for 11.5 and 18.5% of the labeled population. These results indicate that this system provides an in vitro assay of proliferative activity in the normal human breast that enables comparisons to be made between both the luminal and the basal cells in the explants and their counterparts in monolayer culture prepared from flow sorted cells. Thus, growth responses dependent on cell-to-cell interactions or stromal modulation can be identified.

Topographical arrangement of basement membrane proteins in lactating rat mammary gland: comparison of the distribution of type IV collagen, laminin, fibronectin, and Thy-1 at the ultrastructural level.

The topographical distribution of type IV collagen, laminin, fibronectin, and the thymocyte differentiation antigen Thy-1 in the basement membrane of the lactating rat mammary gland was investigated. Small cubes of tissue, which had not been subjected to prior fixation or freezing, were incubated with monospecific or monoclonal antibodies to these proteins, and the antibodies were located by an indirect immunoperoxidase staining technique and observed in the electron microscope. The lamina densa stained uniformly with antibodies to type IV collagen and laminin. In addition, both laminin and type IV collagen were present in semiperiodic clusters that traversed the lamina lucida from the cell surface to the lamina densa. Fibronectin was present only in the semiperiodic clusters and not elsewhere in the basement membrane. These clusters were irregularly spaced along the cell surface and heterogeneous in size. It remains to be determined if these three proteins are present in the same clusters. Thy-1 was largely present on the lamina densa and not on the lamina lucida. The Thy-1 staining of the lamina densa occurred in discrete maxima and minima. These maxima occurred in regions adjacent to Thy-1-bearing stromal cells. Thus, the topographical distribution of proteins within a basement membrane varies in a nonrandom manner, and local factors can modify this distribution. We suggest that this topographical variability may play a role in cell recognition and signalling processes that occur across the basement membrane.

Peripubertal human breast development.

The microanatomical and histological appearance of the human breast has been studied during puberty. The macroscopic architecture of the mammary epithelial tree was identified and correlated with the histological appearance of material excised from defined regions of the breast preparations. Between ages 13 yrs and 15 yrs the human breast shows evidence of ductal elongation and branching, with lobules formed by lateral and dichotomous branching. The majority of ducts are lined by a two-layered epithelium consisting of recognisable myoepithelial and luminal cells. Less-well-defined multilayered regions were also observed in some areas, apparently at the site of lateral branching or early lobular development.

Developmental expression and assembly of connexins into homomeric and heteromeric gap junction hemichannels in the mouse mammary gland.

During the development of the mammary gland, duct-lining epithelial cells progress through a program of expansive proliferation, followed by a terminal differentiation that allows for the biosynthesis and secretion of milk during lactation. The role of gap junction proteins, connexins, in the development and function of this secretory epithelium was investigated. Connexins, Cx26 and Cx32, were differentially expressed throughout pregnancy and lactation in alveolar cells. Cx26 poly-(A)(+) RNA and protein levels increased from early pregnancy, whereas Cx32 was detectable only during lactation. At this time, immunolocalization of connexins by confocal microscopy and immunogold labeling of high-pressure frozen freeze-substituted tissue showed that both connexins colocalized to the same junctional plaque. Analysis of gap junction hemichannels (connexons) isolated from lactating mammary gland plasma membranes by a rate-density centrifugation procedure, followed by immunoprecipitation and by size-exclusion chromatography, showed that Cx26 and Cx32 were organized as homomeric and heteromeric connexons. Structural diversity in the assembly of gap junction hemichannels demonstrated between pregnant and lactating mammary gland may account for differences in ionic and molecular signaling that may physiologically influence the onset and/or maintenance of the secretory phenotype of alveolar epithelial cells.

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin.

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.

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

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.

HGF/SF: a potent cytokine for mammary growth, morphogenesis and development.

The mammary gland is a renewing tissue in which morphogenetic processes and differentiation occur cyclically during the menstrual cycle, pregnancy and lactation. These events have been shown to be dependent upon epithelial-mesenchymal interactions. Studies of the effects of individual factors, their cellular source and their target cell populations in the different developmental stages of the mammary gland are greatly facilitated by the accessibility of this organ and the application of new techniques that allow purification of the major epithelial and stromal components of this tissue. Here we demonstrate that HGF/SF and its cellular receptor, c-met, are expressed and regulated temporally during mouse mammary development and differentiation. We show that human and mouse mammary fibroblasts produce HGF/SF and that HGF/SF is not only mitogenic but morphogenic and motogenic for both human and mouse mammary epithelial cells. We have found that human luminal and myoepithelial cells express c-met differentially and that HGF/SF has different effects on these two mammary epithelial cell populations. HGF/SF is mitogenic for luminal cells but not myoepithelial cells, and morphogenic to myoepithelial cells but not luminal cells. This is discussed in the context of the proliferative compartments in the normal mammary gland and the potential role of the myoepithelial cells to act as the skeleton for ductal development.

Epidermal growth factor receptor expression on human breast luminal and basal cells in vitro.

The expression of EGF receptors has been studied on luminal and basal cells of human breast in vitro. Primary cultures of normal adult human breast epithelium were prepared as single cell suspensions containing a mixture of luminal and basal cells. The cells were simultaneously immunolabelled with antibodies recognising EMA (luminal epithelial cells), CALLA/CD10 (basal cells) and the epidermal growth factor receptor (EGFR). Flow cytometric analysis of these triple labelled cells detected low levels of EGFR on both cell types, with proportionally more EGFR on basal cells compared with luminal cells. Separated populations of basal and luminal cells were prepared from single cell suspensions by flow sorting or by immunomagnetic methods and cultured with and without EGF. Increased proliferation was detected in both cell types in the presence of EGF. To determine the localisation of the EGF receptor, purified cell populations were immunolabelled with anti-EGFR antibody and an FITC-labelled second antibody for fluorescence light microscopy and colloidal gold-labelled antibody for scanning electron microscopy (SEM). Low levels of EGFR were detected by indirect immunofluorescence on both cell types with higher levels on basal cells compared with luminal cells. The detailed subcellular distribution of the receptor was examined by SEM, with gold-labelling of EGFR detected using a field emission scanning electron microscope with a YAG crystal backscattered electron detector. Both luminal and basal cells expressed EGFR over the upper surface of individual cells when these were growing in isolation, but when cells formed part of a confluent island, levels of EGFR on the upper surface of cells were obviously reduced. Observations made by SEM on cells at the edges of such confluent islands showed that cultured basal cells expressed much higher levels of EGFR on their basal, as compared with their upper surfaces.

Gap junction distribution and connexin expression in human breast.

Expression of the gap junction proteins (connexins) in human breast epithelium was studied in vivo and in vitro. A panel of sequence-specific anti-peptide antibodies was used to examine four connexin (Cx) isoforms by indirect immunofluorescence labeling. Antibodies to Cx43 readily detected gap junctions between the basal cells in major ducts, but less so within lobular/alveolar structures. Cx26 immunoreactivity was less abundant in beast epithelium but was observed between the luminal cells in major ducts and to a lesser extent in lobular/alveolar structures. Ultrastructural studies of normal human breast showed gap junctions between basal cells in ducts and lobules, but not between luminal cells or between luminal and basal cells. Immunomagnetically separated luminal and basal cells were grown in vitro. Basal cells expressed Cx43 at cell-cell attachment points whereas luminal cells showed only small amounts of immunolabeling with the Cx26 antibody which was generally not associated with the cell borders. Microinjection of Lucifer yellow into cultured luminal or basal cells indicated that basal cells have high levels of gap junctional communication, but dye transfer between luminal cells was difficult to detect by transfer of Lucifer yellow. Western blot analysis of purified luminal and basal cells indicated the presence of mainly Cx43 in both cell types. Polymerase chain reaction analysis of breast mRNA identified message for CX43 and to a lesser extent for Cx26; Cx32 was not detected in human breast, although it was present in mouse mammary gland. mRNA extracted from cloned cultures of human luminal and basal cells contained message for Cx43 and Cx26 in both cell types.

Growth factor stimulation of proliferating cell nuclear antigen (PCNA) in human breast epithelium in organ culture.

Normal human breast explants were maintained in serum-free culture for 7 days in the presence of either insulin hydrocortisone and cholera toxin (I/H/CT), epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha). Explants were labelled with [3H] thymidine, fixed in methacarn and processed for autoradiography. Parallel sections were immunolabelled with anti-PCNA antibody and analysed with a CAS 200 image analyser. Thymidine labelling index (TLI) and PCNA expression produced similar results with both indices increased in response to I/H/CT, EGF and TGF-alpha. In sections double labelled for PCNA and autoradiography the majority of labelled cells were positive for both markers.

Evidence for susceptibility genes to familial Wilms tumour in addition to WT1, FWT1 and FWT2.

Three loci have been implicated in familial Wilms tumour: WT1 located on chromosome 11p13, FWT1 on 17q12-q21, and FWT2 on 19q13. Two out of 19 Wilms tumour families evaluated showed strong evidence against linkage at all three loci. Both of these families contained at least three cases of Wilms tumour indicating that they were highly likely to be due to genetic susceptibility and therefore that one or more additional familial Wilms tumour susceptibility genes remain to be found.