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An aspartic proteinase present in cyst fluid from women with gross cystic breast disease was purified by a procedure involving affinity chromatography on pepstatin-agarose and size-exclusion high performance liquid chromatography. The amino-terminal sequence of the purified breast proteinase was identical to that corresponding to gastric pepsinogen C. Additional data on cleavage specificity, pH optimum, and immunological properties supported the close relationship between both molecules. Northern blot analysis and polymerase chain reaction amplification studies performed on RNAs obtained from normal and pathological breast tissues demonstrated that the protein is produced by mammary carcinomas and cysts, but not by the normal resting mammary gland. Immunohistochemical staining of paraffin-embedded tissue sections confirmed the existence of a subset of tumors that have the ability to synthesize and secrete this pepsin zymogen. On the basis of these results, we suggest that pepsinogen C expression by human mammary epithelium may be involved in the development of breast diseases, being also of potential interest as a biochemical marker of the hormonal imbalance underlying these pathologies.

Cholesterol and apolipoprotein D (apo D) concentrations were measured in cyst fluids and sera from 66 women with gross cystic disease of the breast. Intracystic cholesterol concentrations are about twofold greater than those found in serum, whereas apo D, the major cyst fluid protein, is present in concentrations as much as 1000-fold greater than found in serum. We classified the cyst fluids into two groups by K+/Na+ ratio and albumin concentration and measured intracystic cholesterol and apo D concentrations in each group. Type I cysts had an average content of cholesterol (12.7 mmol/L) moderately lower than that in Type II cysts (17.6 mmol/L). By contrast, the average concentration of apo D in Type I cyst fluids (15.1 g/L) was slightly higher than that in Type II cysts (13.7 g/L). The absence of a correlation between cholesterol concentration and apo D concentration and the results from previous binding analysis suggest that this protein is not involved in the accumulation of cholesterol in breast-cyst fluid.

Cyst fluids from 55 premenopausal women with gross cystic breast disease were classified by K+/Na+ ratio: 19 with ratio over 1 (type I) and 36 with ratio less than 1 (type II). Immunoradiometric assay of cathepsin D in both types of cyst fluids revealed the presence of large amounts of this proteinase. The average concentration of cathepsin D in type I cyst fluids was 63.3 nmol/l, which was significantly higher than that corresponding to type II cyst fluids (35.1 nmol/l). Immunoprecipitation analysis of intracystic cathepsin D demonstrated that this protein was present as the 52 kD non-processed precursor form of the molecule. Since procathepsin D is a useful prognostic marker in breast carcinoma, we suggest that cyst fluid quantification of cathepsin D could aid to detect patients affecting of gross cystic disease with higher risk for developing breast cancer.

We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to study cyst fluids from women with breast gross cystic disease. The subjects could be classified into two categories according to the concentrations of protein GCDFP-70 in the cyst fluid: those with Type I cysts had a very low content of this protein; those with Type II cysts had very high concentrations. Analysis of the amino acid sequence of GCDFP-70 from both cyst fluid types confirmed that this protein is human plasma albumin. The average concentration of albumin found in Type I cyst fluids was 0.32 g/L and that corresponding to Type II was 10.16 g/L. Thus, albumin quantification from cyst fluids or analysis by either polyacrylamide or agarose gel electrophoresis provides a simple procedure for classifying these fluids, yielding results that correlate well with previous classifications based on other measurements such as sodium, potassium, and chloride concentrations. This albumin-based quantification method may improve the classification of breast cysts and might be useful in further studies on functional changes in the cysts and their relationship to breast cancer.

GCDFP(gross-cystic-disease-fluid protein)-24, a progesterone-binding protein present in large amounts in cyst fluid from human breast gross cystic disease, was purified in a one-step procedure by size-exclusion h.p.l.c. Peptide fragments obtained by trypsin digestion of the intact protein were purified by reverse-phase h.p.l.c. and analysed for their amino acid composition and subjected to automated Edman degradation. A search of the National Biomedical Research Foundation Data Bank revealed that all the sequenced tryptic peptides from protein GCDFP-24 matched perfectly with regions present in the amino acid sequence determined for human apolipoprotein D. Additional data on N-terminal sequence of the unblocked proteins, carbohydrate-attachment sites, amino acid composition and molecular-mass estimations supported the identity between both molecules. On the basis of this identity a possible role of apolipoprotein D in progesterone transport is proposed.