ANALYSIS OF BREAST-TISSUE CATHEPSIN-D ISOFORMS FROM PATIENTS WITH BREAST-CANCER, BENIGN BREAST DISEASE AND FROM NORMAL CONTROLS

The isoform composition of cathepsin D has been investigated by isoele ctric focusing of breast tissue supernatant fluids from patients with breast cancer, benign breast disease and from normal controls. The res ults indicated the presence of several acid protease isoforms between pi values of 2 to 8. Three of these isoforms (with approximate pIs of 6.0, 6.4 and 7.0) were pepstatin-inhibitable but not inhibitable by a mixture of protease inhibitors for seryl, cysteinyl and metalloproteas es. These three isoforms, and not the more acidic isoforms, contained a 31 kD protein band which was recognized by polyclonal antibodies aga inst cathepsin D, suggesting that these isoforms are cathepsin D. Furt her evidence that these isoforms are cathepsin D came from studies in which the pepstatin-inhibitable protease activity and not the pepstati n-uninhibitable protease activity, bound to and was elutable from, an immunoaffinity resin made by coupling anticathepsin D polyclonal antib odies to agarose. The mean relative percentage of the total breast tis sue protease activity associated with pepstatin-inhibitable activity ( i.e. cathepsin D) was significantly increased (p<0.01) in five breast cancer isoform profiles (64+/-4%, mean+/-S.D.) when compared to five n ormal control breast profiles (32+/-5%). An analysis of the three cath epsin D isoforms between pIs 6 to 7 indicated a trend of increased rel ative amounts of the most acidic isoform in the breast cancer isoform profiles when compared to isoform profiles from benign breast disease and normal control breast tissues. Evidence that the more acidic cathe psin D isoforms are related to the more neutral isoforms by sialylatio n came from studies in which neuraminidase treatment of breast tissue supernatant fluids led to decreased amounts of the most acidic isoform with a concomitant increase in the more neutral isoforms. The apparen t increased relative amounts of the most acidic cathepsin D isoform in malignant breast tissue, coupled with the neuraminidase treatment res ults, provide further evidence that malignant breast tissue cathepsin D is abnormally glycosylated.