Search

Background: Human glandular kallikrein (hK2) belongs to the serine protease family of enzymes and has high sequence homology with prostate-specific antigen (PSA). The physiological role of hK2 has not as yet been determined, but there is evidence that it can regulate the proteolytic activity of PSA through processing and activating pro-PSA, an inactive precursor. Thus, it is conceivable that these two secreted proteins may coexist in biological fluids. Currently, hK2 is considered an androgen-regulated and prostate-specific protein. Recently, it has been demonstrated that hK2 is expressed in the breast cancer cell line T-47D after stimulation by steroid hormones, and we reported that hK2 can be detected in a subset of breast tumor extracts. These data suggest that hK2 may be expressed in tissues other than the prostate, such as those in which PSA has already been detected. Because hK2 is a secreted protein, it may be present in various biological fluids., Methods: We analyzed milk samples from lactating women, amniotic fluid from pregnant women, and breast cyst fluid from patients with gross breast cystic disease, using a highly sensitive and specific immunoassay for hK2., Results: hK2 was present in all three biological fluids. We suggest that the female breast may produce hK2 and provide evidence that hK2 may have value as an additional marker for the discrimination between type I and type II breast cysts., Conclusions: The female breast produces hK2 in addition to PSA. More studies are necessary to establish the role of this kallikrein in nondiseased breast, gross breast cystic disease, and breast cancer.

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.

Serum prolactin (PRL) concentrations at baseline and after TRH stimulation were determined in 15 healthy women and in 51 premenopausal patients suffering from Gross Cystic Breast Disease. All women were in the luteal phase of the menstrual cycle and patients were divided into three groups according to cyst type at presentation. Basal hormone levels were within the normal range in the control group and in the three cystic breast disease groups. The maximum PRL response to TRH stimulation was significantly higher (p < 0.001) in patients with type I cysts (low Na+/K+ intracystic ratio and apocrine epithelium) than in patients with type II cysts (high Na+/K+ intracystic ratio and flattened epithelium), type III cysts (intermediate Na+/K+ intracystic ratio and mixed epithelium) and in normal women. Serum PRL concentrations corresponding to samples obtained 60 and 90 minutes after stimulation remained higher in the first group of patients. These results led us to consider the existence of an altered central regulation of PRL secretion in patients with type I cysts at presentation.

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.

Breast cyst fluids were aspirated during the mild luteal phase of the menstrual cycle from 88 patients with gross cystic disease. According to intracystic Na+/K+ ratio three categories of cyst were identified: Type I with low Na+/K+ ratio, low levels of chloride, glucose, and pH, and high concentrations of dehydroepiandrosterone-sulphate (DHAS) and apocrine epithelium; Type II with high Na+/K+ ratio presenting levels of chloride, glucose, pH, and DHAS similar to those found in plasma, and flattened epithelium; Type III with intermediate values for the above parameters. The findings suggest that the cysts could correspond to the different functional stages of the epithelium lining the cysts. Cyst classification was evaluated using a discriminant analysis, which allows breast cyst fluids to be assigned within a category defined by the Na+/K+ ratio. The apparent correct rate for each category was more than 87%. The variables that better discriminate among groups of cysts were chloride, glucose, intracystic prolactin, and pH, respectively. These discriminant functions could be used as criteria for an optimal classification of breast cysts, which might help study of the epidemiology of breast cancer and its relationship to gross cystic disease.