Study of the cell-extracellular matrix (ECM) interactions. Human colon adenocarcinoma cells as a model system for the analysis of the induction of a malignant phenotype.

Methodologies and Techniques:
Cell cultures. Optical and electron microscopy. Flow cytometry. Northern and Western blot analyses. Determination of enzyme activities. In vivo and in vitro tumorigenicity and metastasis assays. Biocompatibility assays. Biomaterials implantation. Protein purification and characterization. Spectroscopical analysis. Cloning and expression of recombinant proteins. Mutagenesis and chimeric protein design and production.

ECM and Malignant Phenotype in Human Colon Adenocarcinoma Cells

BCS-TC2 cells were established from a human colon adenocarcinoma. They present a low differentiation degree  and almost no tumorigenicity. Coinjection of these cells with specific ECM components in nude mice has allowed the selection of stable cell sublines with different both tumorigenic and metastatic capabilities. Several differences have been detected in the expression of ECM-protein receptors and matrix-specific degrading enxymes.

Key words: colon adenocarcinoma; cell lines; tumorigenicity; metastasis; extracellular matrix; integrins; matrix metalloproteinases.

Image: Cellular responses to the interaction of ECM components with cell surface receptors.

Cell Differentiation and Apoptosis

Differentiation of BCS-TC2 cells can be induced by treatment with short-chain fatty acids, such as butyric acid. Morphological alterations and changes on the expression of specific differentiation markers are gnerated: alkaline phosphatase, carcinoembryonic antigen, ecto-5'-nucleotidase, etc. Terminal differentiation induced by treatment with butyric acid leads to cellular death by apoptosis.

Key words: short-chain fatty acids; butyrate; cell differentiation; apoptosis.

Image: Electron micrography revealing the presence of ecto-5'-nucleotidase.

Annexins

These proteins are characterized by their ability to reversibly interact with acidic phospholipid membranes in the presence of calcium. Annexin V (anchorin CII) interacts with collagen and exhibits a voltage-dependent calcium channel activity, which is increased upon its interaction with collagen, fostering in this way cartilage and bone calcification. Chimeric and mutant forms of anexin V are being used to study the interaction with collagen and lipid vesicles. The relationship between annexins expression and differentiation and cell proliferation, and induction of a malignant phenotype is currently under study.

Key words: anexin; calcification; collagen; liposomes; vesicle aggregation.

Image: Three-dimensional structure of annexin V.

Biomaterials

Collagen-based materials, either as a part of a tissue (i.e. pericardium) or interacting with inorganic supports (i.e. sepiolite) are being studied. Several methods in order to evaluate the biocompatibility of materials, the modification of their mechannical properties and resistance to biodegradation have been developped.

Key words: biomaterials; glutaraldehyde; collagen; sepiolite; calcification; collagenases.

Image: Model proposed for the sepiolite-collagen complex.