WESTERN BLOTTING AND ENZYMATIC-ACTIVITY ANALYSIS OF CATHEPSIN-D IN BREAST-TISSUE AND SERA OF PATIENTS WITH BREAST-CANCER AND BENIGN BREAST DISEASE AND OF NORMAL CONTROLS

Increased total antigen amounts of cathepsin D in breast tissue have b een reported to be associated with increased disease recurrence, more frequent metastasis, and increased mortality in breast cancer patients . In the present study, Western blotting analysis has been used for th e first time to determine the relative amounts of precursor and proces sed forms of cathepsin D in sera and breast tissue of patients with br east cancer, benign breast disease, and normal controls. Sera gave sim ilar blots for breast cancer patients and controls with two major form s of cathepsin D (M(r) 52,000 and 27,000). Malignant breast tissue con tained the two forms of cathepsin D found in sera and an additional M( r) 31,000 form which was found in significantly increased (P < 0.001) relative amounts in breast tissue from 43 breast cancer patients [24 /- 12% (SD)] when compared to 51 benign breast disease patients (13 +/ - 8.9%) and 23 normal controls (1.8 +/- 4.4%). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.41) in relative amounts of the M(r) 31,000 form of c athepsin D between proliferative-type and nonproliferative-type fibroc ystic breast disease. A cathepsin D assay has been optimized for human breast tissue and used to demonstrate for the first time significantl y increased (P < 0.001) amounts of pepstatin-inhibitable, cathepsin D- specific activity in breast tissue from 36 breast cancer patients (2.2 +/- 1.4 units/mg of protein) when compared to 47 benign breast diseas e patients (0.63 +/- 0.43) and 23 normal controls (0.24 +/- 0.21). Pre liminary analysis of subgroups of benign breast disease patients sugge sted no significant difference (P = 0.21) in pepstatin-inhibitable, ca thepsin D-specific activity between proliferative-type and nonprolifer ative-type fibrocystic breast disease. The positive correlation (r = 0 .82) of increased amounts of the M(r) 31,000 form of cathepsin D and i ncreased pepstatin-inhibitable, cathepsin D enzymatic activity in mali gnant breast tissue suggests that the M(r) 31,000 form is the proteoly tically active form of the enzyme which may be involved in the develop ment and/or metastatic spread of breast cancer.