ELEVATIONS IN CATHEPSIN-B PROTEIN-CONTENT AND ENZYME-ACTIVITY OCCUR INDEPENDENTLY OF GLYCOSYLATION DURING COLORECTAL TUMOR PROGRESSION

Western blots, enzyme assays, protein glycosylation studies, and immun ohistochemical staining were used to characterize cathepsin B expressi on at successive stages of colorectal tumor progression, In normal col on mucosa and premalignant adenomas, cathepsin B expression was predom inantly due to mature two-chain protein detected on Western blots as t he nonglycosylated 27-kDa form, with overexpression of this protein oc curring in only 4 of 18 adenomas, Overexpression increased significant ly in Dukes A and B carcinomas (26 of 37 cases), with cathepsin B prot ein generally detectable in carcinomas as a combination of both 27-kDa nonglycosylated and 28-kDa glycosylated mature two-chain forms, Glyco sylated cathepsin B protein in carcinoma extracts was sensitive to PNG ase F but resistant to Endo H, indicating a pattern consistent with co mplex rather than high mannose type glycosylation, When sorted by adva ncing tumor stage, peak expression of cathepsin B protein occurred in carcinomas involved in local invasion compared with adenomas or metast atic cancers, At all stages, cathepsin B activity correlated significa ntly with the levels of heavy chain mature cathepsin B protein (r = 0. 6682, p < 0.0001) irrespective of glycosylation, Immunohistochemical s taining of cathepsin B protein revealed fine diffuse cytoplasmic stain ing in both adenomas and carcinomas compared with coarse granular cyto plasmic staining (typical of lysosomes) seen in matched normal mucosa, Our results demonstrate several sequential, apparently independent ch anges in cathepsin B expression during colorectal tumor progression in cluding early changes in subcellular localization, up-regulation of ca thepsin B protein and activity in invasive cancers, and altered protei n glycosylation detected in malignant tumors at all stages.