Matrilysin 1 [matrix metalloproteinase 7 (MMP7)] is one of the most important metalloproteinases expressed in human tissues. This enzyme is generally not expressed by normal differentiated epithelial colon cells, but has been shown to be up-regulated in human colon adenomas and adenocarcinomas. Little is known about the role of MMP7 in cell invasion and its involvement in proteolytic processes. By searching the ligands of MMP7 in the colonic carcinoma cells HT29, we identified laminin-5/laminin-332 (LN5) as a specific target for MMP7 enzymatic activity. LN5, composed of A3, B3, and ;2 chains, is an important component of epithelial basement membranes where it induces firm adhesion and hemidesmosome formation. In this study, we show that LN5 and MMP7 are coexpressed in HT29 cells as well as in HT29 xenograft tumors and human colorectal adenocarcinomas. We provide evidence that human LN5 is a ligand for MMP7 and that a specific cleavage occurs in its B3 chain, giving rise to a carboxyl-terminal B3 chain fragment of 90 kDa. We have identified the MMP7 cleavage site at position Ala 515 -Ile 516 in the B3 chain. Videomicroscopic analysis of HT29 cells plated on LN5 substrates reveals that the MMP7-processed LN5 significantly enhances cell motility. Moreover, the delayed migration of HT29 cells obtained after specific inhibition of MMP7 reinforces the hypothesis supporting its involvement in cell migration. Altogether, our results show that MMP7 is likely to play a crucial role in the regulation of carcinoma cell migration by targeting specific proteolytic processing of the LN5 B3 chain. (Cancer Res 2006; 66(23): 11228-37)

When cultured in polystyrene dishes subjected to previous treatment and supplied with a serum-containing medium, hog thyroid cells form monolayers displaying dome-like arrangements after three to four days. Cells involved in formation of "domes" are morphologically polarized; the apical microvilli of these cells point toward the culture medium. When the tissue is cultured in untreated polystyrene dishes, thyroid cells remain in suspension; their aggregates swell progressively and form hollow spheres encompassed by a single layer of cells. The polarity of the cells forming such spheres is inverse in comparison to the condition characteristic of the intact thyroid gland. When culture medium is supplemented with TSH, PGE1, PGE2 or dBC, structures resembling true follicles are formed in both types of cultures. Gelatin, added to suspension cultures, is also capable of promoting follicle formation. Cultured thyroid cells regularly form an epithelial layer as a result of the interaction of cellular processes. However, the polarization of this layer depends on culture conditions. Thus, structures with either a normal follicle-like polarization of their cells or showing an inverted type of polarization can be obtained.

Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of ␣1, ␤1 and ␥1 chains) in intestinal cell differentiation or tumorigenesis, an ␣1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin ␣1 chain is not expressed. Upon transfection of the ␣1 chain, the ␣1␤1␥1 trimer was found secreted in the media along with free Key words: laminins; basement membrane; colonic cancer cells; tumor growth; differentiationIt is well established that in multicellular organisms, extracellular matrix molecules control various physiologic activities such as cell growth, migration, apoptosis and differentiation. The extracellular network includes the interstitial matrix and basement membranes. The basement membranes that separate the connective tissue from epithelia, muscle fibers, blood vessels and nerves are formed by a complex set of collagens and of noncollagenous glycoproteins such as laminins, nidogen and of proteoglycans, which have a unique composition in each organ. Laminins are multifunctional molecules forming a family of proteins that display organ, site and developmental specificity. 1,2 The first characterized laminin isoform, laminin-1 (composed of ␣1, ␤1 and ␥1 chains), is a crosslike-shaped molecule assembled into a triplestranded coiled-coil structure designated as the long arm. 3 As for the other extracellular matrix molecules, laminin-1 is made up of specific structural domains that allow self-assembly, permit interaction with other basement membrane molecules and encode information for the cells via specific transmembrane receptors, mostly of the integrin family. 4 Recent studies performed on various organs during development show that laminin-1 has a rather limited expression. 5 Despite such restricted expression, crucial roles for laminin-1 in organ development and in cell differentiation have been demonstrated by cell-coating experiments, blocking antibody and antisense strategies. Several major functions have been assigned to the constitutive ␣1 chain of laminin-1, among which are the polarization of developing kidney tubules, 6,7 lung alveolar formation, 8 development of submandibular glands 9 and polarization and functional differentiation of mammary and intestinal cells. 10,11 Conversely, alterations in cell-matrix and cell-cell interactions have been shown in a number of pathologic situations, such as inflammatory processes, wound healing and malignant transformation. In colorectal cancer, perturbations in the production, deposition and degradation of matrix molecules, among which are laminins, have been observed. [12][13][14][15][16] The role of laminin-1 in tumor growth and metastasis has been evaluated in several cell systems. In particular, it was shown that coinjection of puri...

Ultrastructurally, the intracellular lumen appears as a more or less spherical cavity with osmiophilic substance, bordered with microvilli and surrounded by a filamentous network. Epithelial cancer cells, both healthy cultured and hormonally stimulated in vivo, often present such structures, which probably result from a dysfunction of the Golgi apparatus and cytoskeleton. This characterizes an abortive secretory process, which may be the consequence of a great hormonal sensitivity of the cells or of a loss of differentiation criteria when they are cancerous or isolated for culture.

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Little is known about the functional interactions between digestive neuroendocrine tumor cells and their stromal microenvironment. The focus of our study is whether mesenchymal cells modulate peptide expression, cell proliferation, and invasiveness in digestive neuroendocrine tumor cells. We designed an experimental in vivo and in vitro study using the mouse enteroendocrine cell line STC-1. In vivo, STC-1 cells were injected subcutaneously in 18 immunosuppressed newborn rats. At day 21, all animals presented poorly differentiated neuroendocrine tumors with lung metastases. Subcutaneous tumors were usually limited by a capsule containing basement membrane components and myofibroblasts that presented a low mitotic index. Lung tumors were devoid of capsule and poor in myofibroblasts, and their mitotic index was high. The profile of peptide expression in STC-1 tumors was different from that of cultured STC-1 cells. In vitro, STC-1 cells were cultured with fibroblasts of different origins, including dermis, lung, digestive tract, and liver. Based on their origin, myofibroblasts differentially modulated hormone synthesis, proliferation, spreading, and adhesion of STC-1 cells. In conclusion, our results show that site-specific functional interactions between mesenchymal and neuroendocrine cells may contribute to modulating the behavior of digestive neuroendocrine tumors, depending on their growth site.

Epithelial-mesenchymal interactions play a pivotal role in colon cancer invasion and metastasis. We aimed at elucidating the impact of long-term cultivation on the phenotypic and functional characteristics of primary fibroblasts and their interaction with the human colon adenocarcinoma cell line LoVoC5. We used fibroblasts from human colon tumor tissue, normal human colon mucosa, rat normal colon and 2 rat colon-derived myofibroblast cell lines, MIC316 and MG. The following parameters were studied: cell shape and size, growth curve, intermediate filament expression and extracellular matrix synthesis. Coculture models with or without cell contacts were used to test the effects on LoVoC5 cell proliferation, spreading and adhesion. Irrespective of their origin, fibroblastic cells in primary cultures presented marked phenotypic and functional changes with time. Before passage 5, they presented as large, slow-growing cells expressing vimentin and ␣-smooth muscle actin; synthesizing laminin-1, fibronectin and collagens I and IV; and inducing LoVoC5 proliferation, spreading and adhesion. After passage 15, they presented as small, fast-growing cells inconstantly expressing ␣-smooth muscle actin and synthesizing mainly type I collagen. In coculture with or without cell contacts, they inhibited LoVoC5 proliferation and allowed only limited cell spreading and adhesion. Myofibroblastic cell lines presented as large, fast-growing cells expressing vimentin and ␣-smooth muscle actin and synthesizing mainly type I collagen. They had no significant effects on LoVoC5 proliferation, spreading and adhesion. Our results underline the importance of age-dependent variations in colon mesenchymal cells in culture and for the in vitro study of epithelial-mesenchymal interactions in colon cancer.

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