Both ErbB1 and ErbB2 are overexpressed or amplified in breast tumours. To examine the effects of activating ErbB receptors in a context that mimics polarized epithelial cells in vivo, we activated ErbB1 and ErbB2 homodimers in preformed, growth-arrested mammary acini cultured in threedimensional basement membrane gels. Activation of ErbB2, but not that of ErbB1, led to a reinitiation of cell proliferation and altered the properties of mammary acinar structures. These altered structures share several properties with early-stage tumours, including a loss of proliferative suppression, an absence of lumen, retention of the basement membrane and a lack of invasive properties. ErbB2 activation also disrupted tight junctions and the cell polarity of polarized epithelia, whereas ErbB1 activation did not have any effect. Our results indicate that ErbB receptors differ in their ability to induce early stages of mammary carcinogenesis in vitro and this three-dimensional model system can reveal biological activities of oncogenes that cannot be examined in vitro in standard transformation assays.The mammary epithelium of an adult breast is organized into ducts and lobules. The ducts end in a highly branched structure referred to as the terminal ductal lobular unit (TDLU). A TDLU is comprised of multiple individual units referred to as mammary acini. Each acinus has a central lumen, a single layer of polarized luminal epithelial cells surrounded by myoepithelial cells, and a basement membrane.We have previously shown that human mammary epithelial cells (MECs) form acini-like structures containing a single layer of polarized, growth-arrested cells when grown within a matrix rich in laminin and collagen IV (Matrigel, derived from the Englebreth-Holm Swarm (EHS) tumour) 1,2 . The epithelial cells within acini in vivo and in culture have an apico-basal distribution of polarity markers such as ZO-1, E-cadherin and α 6 β 4 integrins. They also deposit collagen IV and secrete sialomucin in their basal and apical surfaces, respectively 1,2 , indicating that the acinar structures formed in culture closely mimic the acini in an adult breast.Early stages of breast cancer (hyperplasia and ductal carcinoma in situ (DCIS)) are characterized by an increased proliferation of epithelial cells, a loss of acinar organization and § Correspondence and requests for materials should be addressed to J.S.B. joan_brugge@hms.harvard.edu. † Present addresses: Cold Spring Harbor Laboratories, Cold Spring Harbor, New York 11724, USA (S.K.M.); Department of Basic Medical Sciences, Purdue University, West Lafayette, Indiana 47907, USA (S.L.)Supplementary information is available on Nature Cell Biology's website (http://cellbio.nature.com) or as paper copy from the London editorial office of Nature Cell Biology. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 October 11. filling of the luminal space 3 . However, a lack of acinar organization and the acquisition of invasive behaviour are later events involved...

Tumor cells can evade chemotherapy by acquiring resistance to apoptosis. We investigated the molecular mechanism whereby malignant and nonmalignant mammary epithelial cells become insensitive to apoptosis. We show that regardless of growth status, formation of polarized, three-dimensional structures driven by basement membrane confers protection to apoptosis in both nonmalignant and malignant mammary epithelial cells. By contrast, irrespective of their malignant status, nonpolarized structures are sensitive to induction of apoptosis. Resistance to apoptosis requires ligation of beta4 integrins, which regulates tissue polarity, hemidesmosome formation, and NFkappaB activation. Expression of beta4 integrin that lacks the hemidesmosome targeting domain interferes with tissue polarity and NFkappaB activation and permits apoptosis. These results indicate that integrin-induced polarity may drive tumor cell resistance to apoptosis-inducing agents via effects on NFkappaB.

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively ref lecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.The cell nucleus is organized by a nonchromatin internal structure referred to as the nuclear matrix (NM; refs. 1-3). Identified NM components include coiled-coil proteins (4), cell cycle regulators (5), tissue-specific transcription factors (6, 7), and RNA splicing factors (for review see ref.2). Although splicing factors have been shown to redistribute during cellular differentiation (8, 9) and following the induction of gene expression (10), spatial distribution of nuclear components are thought to be the consequence of changes in gene expression (8, 10, 11). However, whether NM composition and structure may themselves affect gene expression and cellular function has not been examined.To systematically study the effect of cell growth and tissue differentiation on nuclear organization, we used a reconstituted basement membrane (rBM)-directed model of mammary gland morphogenesis (12). The HMT-3522 human mammary epithelial cells (HMECs) were isolated from reduction mammoplasty and became immortalized in culture (13). When embedded within a rBM, these cells arrest growth, organize an endogenous BM, and form polarized acinus-like structures with vectorial secretion of sialomucin into a central lumen (12). We used this model to compare the nuclear organization of HMECs cultured on a plastic surface [two-dimensional (2D) monolayer] vs. a three-dimensional (3D) rBM. Nuclear organization was assessed by examini...

Organs are made of the organized assembly of different cell types that contribute to the architecture necessary for functional differentiation. In those with exocrine function, such as the breast, cell–cell and cell–extracellular matrix (ECM) interactions establish mechanistic constraints and a complex biochemical signaling network essential for differentiation and homeostasis of the glandular epithelium. Such knowledge has been elegantly acquired for the mammary gland by placing epithelial cells under three-dimensional (3D) culture conditions. Three-dimensional cell culture aims at recapitulating normal and pathological tissue architectures, hence providing physiologically relevant models to study normal development and disease. The specific architecture of the breast epithelium consists of glandular structures (acini) connected to a branched ductal system. A single layer of basoapically polarized luminal cells delineates ductal or acinar lumena at the apical pole. Luminal cells make contact with myoepithelial cells and, in certain areas at the basal pole, also with basement membrane (BM) components. In this chapter, we describe how this exquisite organization as well as stages of disorganization pertaining to cancer progression can be reproduced in 3D cultures. Advantages and limitations of different culture settings are discussed. Technical designs for induction of phenotypic modulations, biochemical analyses, and state-of-the-art imaging are presented. We also explain how signaling is regulated differently in 3D cultures compared to traditional two-dimensional (2D) cultures. We believe that using 3D cultures is an indispensable method to unravel the intricacies of human mammary functions and would best serve the fight against breast cancer.

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 microm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive "water window", where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (lambda = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.

BackgroundBasoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated.ResultsWe demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity.ConclusionThese results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.

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The coiled-coil protein NuMA is an important contributor to mitotic spindle formation and stabilization. A potential role for NuMA in nuclear organization or gene regulation is suggested by the observations that its pattern of nuclear distribution depends upon cell phenotype and that it interacts and/or colocalizes with transcription factors. To date, the precise contribution of NuMA to nuclear function remains unclear. Previously, we observed that antibody-induced alteration of NuMA distribution in growth-arrested and differentiated mammary epithelial structures (acini) in threedimensional culture triggers the loss of acinar differentiation. Here, we show that in mammary epithelial cells, NuMA is present in both the nuclear matrix and chromatin compartments. Expression of a portion of the C terminus of NuMA that shares sequence similarity with the chromatin regulator HPC2 is sufficient to inhibit acinar differentiation and results in the redistribution of NuMA, chromatin markers acetyl-H4 and H4K20m, and regions of deoxyribonuclease I-sensitive chromatin compared with control cells. Short-term alteration of NuMA distribution with anti-NuMA C-terminus antibodies in live acinar cells indicates that changes in NuMA and chromatin organization precede loss of acinar differentiation. These findings suggest that NuMA has a role in mammary epithelial differentiation by influencing the organization of chromatin. INTRODUCTIONThe nuclear mitotic apparatus protein (NuMA) was first described in 1980 (Lydersen and Pettijohn, 1980) and observed to concentrate at the spindle poles during mitosis. Subsequently NuMA, variously named SPN antigen, p240 antigen, centrophilin, 1F1/1H1 antigen, SP-H antigen, and W1 antigen (Compton et al., 1991(Compton et al., , 1992Kallajoki et al., 1991Kallajoki et al., , 1993Maekawa et al., 1991;Tousson et al., 1991;Yang et al., 1992;Maekawa and Kuriyama, 1993;Tang et al., 1993), was shown to be a critical player in the formation and stabilization of the mitotic spindle, notably by associating with the minus end of spindle microtubules and interacting with dynein, dynactin, and LGN (Compton and Cleveland, 1993;Gaglio et al., 1995;Merdes et al., 2000;Du et al., 2001;Gehmlich et al., 2004). In addition to its location at the poles of the mitotic spindle, NuMA has been reported in the nucleus of both cycling and growth-arrested cells, as shown by immunostaining of cells in culture and tissue biopsy sections (Lelièvre et al., 1998;Merdes and Cleveland 1998;Gribbon et al., 2002;Taimen et al., 2004). However, a role for NuMA in interphase remains to be determined. The hypothesis that NuMA might organize nuclear structure (Compton and Cleveland, 1994) is supported by the existence of a long central coiled-coil region in the protein and the prevalence of NuMA in the nucleus upon detergent extraction. Furthermore, NuMA binds DNA matrix attachment regions (MARs) in vitro (Ludérus et al., 1994), and the cleavage of NuMA precedes DNA degradation during apoptosis (Weaver et al., 1996). The likelihood of a link...