Abstract. Functional differentiation in mammary epithelia requires specific hormones and local environmental signals . The latter are provided both by extracellular matrix and by communication with adjacent cells, their action being intricately connected in what appears to be a cascade of events leading to milk production . To distinguish between the influence of basement membrane and that of cell-cell contact in this process, we developed a novel suspension culture assay in which mammary epithelial cells were embedded inside physiological substrata . Single cells, separated from each other, were able to assimilate information from a laminin-rich basement membrane substratum and were induced to express 0-casein . In contrast, a stromal environment of collagen I was not sufficient to REGULATION of differentiation in complex tissues is determined not only by growth factors and hormones, but also by intercellular communication and by inter actions between cells and their extracellular matrix (Stoker et al., 1990) . In epithelial tissue, part ofthe extracellular matrix (ECM) I occurs in the form of a basement membrane, which provides positional information for cells and cues for organizing intracellular structure, as well as signals that regulate cellular behaviour. An ideal model for studying how ECM signals are transduced to control tissue-specific function is the simple epithelium of mammary gland . In adult animals, the cells undergo developmental changes during pregnancy and become highly secretory at lactation . The great advantage of this system is that in culture, mammary epithelial cells regain their differentiated phenotype only under suitable hormonal and substratum conditions . The model can therefore be used to understand the mechanism by which tissue-specific genes are expressed (Bissell and Hall, 1987;Streuli and Bissell, 1991) .Considerable evidence now indicates that basement membrane plays a significant role in regulating mammary phenotype. In vivo, the alveolar epithelium of rodent mammary 1 . Abbreviations used in this paper: DAPI, 4,6-diamidino-2-phenylindole ; ECM, extracellular matrix; EHS, Engelbreth-Holm-Swarm .® The Rockefeller University Press, 0021-9525/91/12/1383/13 $2 .00

Abstract. Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating H-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the/~-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing /3-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the/3-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the cd chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

Red meat is associated with increased risk of colorectal cancer and increases the endogenous formation of N-nitrosocompounds (NOC). To investigate the genotoxic effects of NOC arising from red meat consumption, human volunteers were fed high (420 g) red meat, vegetarian, and high red meat, highfiber diets for 15 days in a randomized crossover design while living in a volunteer suite, where food was carefully controlled and all specimens were collected. In 21 volunteers, there was a consistent and significant (P < 0.0001) increase in endogenous formation of NOC with the red meat diet compared with the vegetarian diet as measured by apparent total NOC (ATNC) in feces. In colonic exfoliated cells, the percentage staining positive for the NOC-specific DNA adduct, O 6 -carboxymethyl guanine (O 6 CMG) was significantly (P < 0.001) higher on the high red meat diet. In 13 volunteers, levels were intermediate on the high-fiber, high red meat diet. Fecal ATNC were positively correlated with the percentage of cells staining positive for O 6 CMG (r 2 = 0.56, P = 0.011). The presence of O 6 CMG was also shown in intact small intestine from rats treated with the N-nitrosopeptide N-acetyl-NV-prolyl-NV-nitrosoglycine and in HT-29 cells treated with diazoacetate. This study has shown that fecal NOC arising from red meat include direct acting diazopeptides or N-nitrosopeptides able to form alkylating DNA adducts in the colon. As these O 6 CMG adducts are not repaired, and if other related adducts are formed and not repaired, this may explain the association of red meat with colorectal cancer. (Cancer Res 2006; 66(3): 1859-65)

Cell exfoliation in the gut is an important cell renewal mechanism. To approach its investigation we applied a novel immunomagnetic technique for isolation of exfoliated cells from human stool. Exfoliated colonocytes were isolated from 168 stool samples. The cells were assessed microscopically using conventional stains and immunohistochemistry. The technique allowed us to obtain well-preserved colonocytes displaying characteristic features of well-differentiated colonic epithelium and positive immunostaining for cytokeratin 5/8. No mucin-producing cells were found. Exfoliated cells did not produce inducible nitric oxide synthase, albeit cultured colon carcinoma cells HT-29 analysed in parallel showed strong immunostaining. Analysis of exfoliated cell numbers in consecutive stool samples from the same subjects revealed considerable interindividual variation. Overall exfoliated colonocyte numbers were relatively low, isolation being unaffected by addition during the procedure of excessive amounts of HT-29 cells. Apoptosis was extremely rare among exfoliated colonocytes. Well-preserved exfoliated colonocytes can be consistently isolated from human faeces using a simple procedure. Our findings suggest that the actual process of cell exfoliation in the human colon may be much less intense than is generally accepted. Exfoliated cell isolation from human stool constitutes a convenient non-invasive approach that can be used for diagnostic and research purposes.

We previously established a rapid three-dimensional assay for discrimination of normal and malignant human breast epithelial cells using a laminin-rich reconstituted basement membrane. In this assay, normal epithelial cells differentiate into well-organized acinar structures whereas tumor cells fail to recapitulate this process and produce large, disordered colonies. The data suggest that breast acinar morphogenesis and differentiation is regulated by cell-extra-cellular matrix (ECM) interactions and that these interactions are altered in malignancy. Here, we investigated the role of ECM receptors (integrins) in these processes and report on the expression and function of potential laminin receptors in normal and tumorigenic breast epithelial cells. Immunocytochemical analysis showed that normal and carcinoma cells in a three-dimensional substratum express profiles of integrins similar to normal and malignant breast tissues in situ. Normal cells express alpha 1, alpha 2, alpha 3, alpha 6, beta 1 and beta 4 integrin subunits, whereas breast carcinoma cells show variable losses, disordered expression, or downregulation of these subunits. Function-blocking experiments using inhibitory anti-integrin subunit antibodies showed a &gt; 5-fold inhibition of the formation of acinar structures by normal cells in the presence of either anti-beta 1 or anti-alpha 3 antibodies, whereas anti-alpha 2 or -alpha 6 had little or no effect. In experiments where collagen type I gels were used instead of basement membrane, acinar morphogenesis was blocked by anti-beta 1 and -alpha 2 antibodies but not by anti-alpha 3. These data suggest a specificity of integrin utilization dependent on the ECM ligands encountered by the cell. The interruption of normal acinar morphogenesis by anti-integrin antibodies was associated with an inhibition of cell growth and induction of apoptosis. Function-blocking antibodies had no inhibitory effect on the rate of tumor cell growth, survival or capacity to form colonies. Thus under our culture conditions breast acinar formation is at least a two-step process involving beta 1-integrin-dependent cellular growth followed by polarization of the cells into organized structures. The regulation of this pathway appears to be impaired or lost in the tumor cells, suggesting that tumor colony formation occurs by independent mechanisms and that loss of proper integrin-mediated cell-ECM interaction may be critical to breast tumor formation.

This paper considers the experience of developing and implementing a community‐based multidisciplinary rehabilitation service in the City of Wolverhampton. It outlines the process of defining and agreeing the service parameters, objectives and methods of service delivery and includes the main points from an initial evaluation of the team. The article concludes with key messages for consideration by others setting up a similar service.

Lactoferrin is a secreted iron binding protein which is expressed during normal functional development of mammary epithelium. Murine mammary epithelial cell lines competent for milk protein expression were used to identify microenvironmental factors that regulate lactoferrin expression. While lactoferrin was not expressed in adherent monolayer cultures under standard subconfluent conditions on plastic, lactoferrin mRNA and protein steadily accumulated when the cells aggregated to form spheroids on a reconstituted basement membrane gel. However, unlike other milk proteins such as beta-casein, lactoferrin expression was also induced at high cell density in the absence of exogenously added basement membrane or prolactin. These results led us to examine whether changes in cell growth, cell-cell interactions and/or cell shape were responsible for regulation of lactoferrin gene expression. Rounded, non-proliferating cells in suspension in serum-free medium expressed lactoferrin even as single cells. Conversely, lactoferrin expression could be inhibited in non-proliferative cells in serum-free medium by maintaining them in contact with an air-dried extracellular matrix which caused the cells to retain flat, spread morphologies. These findings indicated that cessation of cell growth was not sufficient, that cell-cell interactions were not required, and that cell culture conditions which minimize cell spreading may be important in maintaining lactoferrin expression. Additional data supporting this latter concept were generated by treating spread cells with cytochalasin D. The resulting disruption of microfilament assembly induced both cell rounding and lactoferrin expression. Shape-dependent regulation of lactoferrin mRNA was both transcriptional and post-transcriptional. Surprisingly, treatment of rounded cells with a transcription inhibitor, actinomycin D, produced a stabilization of lactoferrin mRNA, suggesting that transcription of an unstable factor is required for degradation of lactoferrin mRNA. Importantly, lactoferrin mRNA expression was regulated similarly in early passage normal human mammary epithelial cells. In vivo, the changing extracellular matrix components of the mammary gland during different stages of normal and abnormal growth and differentiation may provide different physical constraints on the configurations of cell surface molecules. These physical constraints may be communicated to the cell interior through mechanical changes in the cytoskeleton. Unlike beta-casein whose expression is upregulated by specific integrin-mediated signals, lactoferrin may be representative of a class of proteins synthesized in the mammary gland using basal transcriptional and translational machinery. The suppression of lactoferrin expression that is observed in monolayer culture and in malignant tissues may reflect inappropriate cell shapes and cytoskeletal structures that are manifested under these conditions.

Transforming growth factor-beta (TGF-beta) affects cellular proliferation and differentiation and has been linked to the development of pathologic extracellular matrix production characteristic of progressive renal disease. TGF-beta 1 is conventionally regarded as having growth-inhibitory activity on T lymphocytes. The growth-regulatory activity of TGF-beta 1 depends on the interaction of TGF-beta 1 with its receptors, especially the type I and II receptors. In this study, we describe a CD4+ nephritogenic T cell clone that, unlike a sister clone with an identical TCR Ag receptor, is not growth inhibited by TGF-beta 1, nor is its nephritogenicity affected by exogenous TGF-beta 1. Although this TGF-beta-resistant T cell clone expresses both type I and II TGF-beta receptors on the cell surface, the affinity of this interaction is markedly diminished compared with that of the sister T cell clone, which does undergo growth arrest in response to extracellular TGF-beta 1. The resistant T cell clone expresses one mutant allele for the type I TGF-beta receptor. This mutation consists of a leucine to glutamine substitution at codon 122, a conserved amino acid in the transmembrane domain. We speculate that this mutation alters the interaction between the type I and II TGF-beta receptors following recognition of ligand by the type II receptor.