WESTERN BLOTTING AND ISOFORM ANALYSIS OF CATHEPSIN-D FROM NORMAL AND MALIGNANT HUMAN BREAST CELL-LINES

Cathepsin D from normal (Hs578Bst) and malignant (MCF7, MDA-MB-231) br east cell lines has been characterized with regard to its kinetic prop erties, activity levels, precursor and processed M(r) forms, and isofo rm composition. Normal cell cathepsin D appears to have a more neutral pH optimum (pH 3.5) than the cancer cell line (pH 3.0-3.2) and greate r activity between pH values of 4.0 to 4.5. The two cancer cell lines have approximately 1.5 to 2.0-fold increased total acid protease activ ity and 2 to 3-fold increased pepstatin-inhibitable protease activity (i.e. cathepsin D) when compared to the normal breast cell line. Weste rn blotting indicates that a major processed form of cathepsin D for a ll three cell lines occurs at 31 kDa. The cancer cell lines contain si gnificant amounts of cathepsin D precursors of 47 and 42 kDa whereas t he normal cell line contains little if any of these precursors. Isoele ctric focusing indicates that the normal cell line contains approximat ely 50% of its total acid protease activity at pIs above 4 whereas the cancer cell lines contain 70-80% of their protease activity at such p Is. In addition, the cancer cell lines contain two to three major isof orms between pIs of 5.5 and 6.3 which were not present in the normal c ell line. The isoforms from pi values of 5.5 to 7.3 for all three cell lines are 100% pepstatin-inhibitable. In addition, Western blot analy sis indicates that these isoforms contain the processed 31 kDa form of cathepsin D. The combined results indicate that the two breast cancer cell lines are similar to biopsied malignant breast tissue in exhibit ing altered acid protease isoform profiles with increased relative amo unts of pepstatin-inhibitable and immunoreactive acid protease activit y (cathepsin D) compared to normal breast tissue or cells.