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.