Activity and properties of alpha-L-fucosidase are dependent on the state of enterocytic differentiation of HT-29 colon cancer cells.
Previously we have demonstrated an impairment in the activity of alpha-L-fucosidase in colon tumours. In order to establish an in vitro model to study this enzyme in colon cancer, we have determined the activity and properties of the enzyme during the differentiation of HT-29 colon cancer cells. Cultures were committed to differentiate into enterocyte-like cells by placing them in a culture medium without glucose for 18-21 days. The state of differentiation was evaluated by assaying the activity of enterocytic marker enzymes, and the acquisition of enterocyte morphology was assessed by electron microscopy. The alpha-L-fucosidase activity was determined using a fluorometric method. Intracellular levels of alpha-L-fucosidase activity are lower in non-differentiated cells (3.0 +/- 1.01 U/mg) than in differentiated ones (9.2 +/- 4.09 U/mg) (P < 0.001). This variation is not due to a greater secretion of the enzyme to the culture medium, and properties such as pH optimum or the affinity towards substrate are not dependent on differentiation. The enzyme however, is more stable at acidic pH and at high temperatures, and V(max) is higher in differentiated cells. Moreover, in undifferentiated cells the enzyme is mainly in a monomeric form whereas multimeric forms of the enzyme appear only upon differentiation. Most of these changes are very similar to those previously observed between normal colon tissue and colon tumours. Thus, we suggest that differentiation of HT-29 colon cancer cells could be used as a model to study the alterations of the enzyme alpha-L-fucosidase during the progression of the tumoural process.
Departamento de Bioquímica, Area de Bioquímica y Biología Molecular, Universidad de Vigo, E-36280 Vigo, Spain.
Ana
A
Merino-Trigo
Francisco Javier
FJ
Rodríguez-Berrocal
Encarnación
E
de Miguel
María
M
Páez de la Cadena
D015153:Blotting, Western; D002454:Cell Differentiation; D002850:Chromatography, Gel; D003110:Colonic Neoplasms / Q000201:enzymology / Q000473:pathology* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D019073:HT29 Cells; D006801:Humans; D008854:Microscopy, Electron; D005644:alpha-L-Fucosidase / Q000378:metabolism*
D005644:alpha-L-Fucosidase