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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 Controls¹

Department of Chemistry and Center for Molecular Bioscience and Biotechnology, Lehigh University, Bethlehem, Pennsylvania 18015 [D. C. S., S. B., L. M. W., J. A. A.]; Department of Pathology [S. T., S. L.] and Department of Surgery [M. K. L., T. Z], St. Luke's Hospital, Bethlehem, Pennsylvania 18015; and STC Diagnostics, Bethlehem, Pennsylvania 18015 [S. N.]

² To whom requests for reprints should be addressed, at CMBB, Iacocca Hall, 111 Research Drive, Lehigh University, Bethlehem, PA 18015.

Increased total antigen amounts of cathepsin D in breast tissue have been 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 the first time to determine the relative amounts of precursor and processed forms of cathepsin D in sera and breast tissue of patients with breast cancer, benign breast disease, and normal controls. Sera gave similar blots for breast cancer patients and controls with two major forms of cathepsin D (M_r 52,000 and 27,000). Malignant breast tissue contained 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 cathepsin D between proliferative-type and nonproliferative-type fibrocystic breast disease.

A cathepsin D assay has been optimized for human breast tissue and used to demonstrate for the first time significantly 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 disease patients (0.63 ± 0.43) and 23 normal controls (0.24 ± 0.21). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.21) in pepstatininhibitable, cathepsin D-specific activity between proliferative-type and nonproliferative-type fibrocystic breast disease. The positive correlation (r = 0.82) of increased amounts of the M_r 31,000 form of cathepsin D and increased pepstatin-inhibitable, cathepsin D enzymatic activity in malignant breast tissue suggests that the M_r 31,000 form is the proteolytically active form of the enzyme which may be involved in the development and/or metastatic spread of breast cancer.

¹ This research was supported in part by grants from St. Luke's Hospital and STC Diagnostics. L. M. W. was supported in part by a USPHS training grant (GM 08351).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/15/93. Accepted 10/29/93.

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