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

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The isoform composition of cathepsin D has been investigated by isoelectric focusing of breast tissue supernatant fluids from patients with breast cancer, benign breast disease and from normal controls. The results 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 metalloproteases. These three isoforms, and not the more acidic isoforms, contained a 31 kD protein band which was recognized by polyclonal antibodies against cathepsin D, suggesting that these isoforms are cathepsin D. Further evidence that these isoforms are cathepsin D came from studies in which the pepstatin-inhibitable protease activity and not the pepstatin-uninhibitable protease activity, bound to and was elutable from, an immunoaffinity resin made by coupling anticathepsin D polyclonal antibodies to agarose. The mean relative percentage of the total breast tissue 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 normal control breast profiles (32+/-5%). An analysis of the three cathepsin D isoforms between pIs 6 to 7 indicated a trend of increased relative 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 cathepsin D isoforms are related to the more neutral isoforms by sialylation 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 apparent increased relative amounts of the most acidic cathepsin D isoform in malignant breast tissue, coupled with the neuraminidase treatment results, provide further evidence that malignant breast tissue cathepsin D is abnormally glycosylated.