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.