Skip to main content
  • Non-peer-reviewed research
  • Published:

Acid Phosphatase Activity in Human Breast Tumors

Abstract

We examined lysosomal participation in the degradation of tumor cells from human breast biopsies, utilizing the histochemical activity and localization of acid phosphatase (AP). Enzyme activities in benign and malignant lesions were compared. AP was faintly detected in normal mammary epithelia and was marked in malignant cells. The histochemical patterns of AP distribution in the breast tissues showed differences between normal and neoplastic cells. AP staining was more intense in ductal carcinoma in situ (DCIS) associated with invasive ductal carcinoma (IDC) cases compared to when IDC was present alone. These results suggest that non-invasive breast cancers, such as DCIS, may be subjected to more lysosomal cellular lysis than is observed in invasive breast cancers, like IDC.

Introduction

It has been shown that lysosomes, acting presumably via their acid hydrolases, are involved in a variety of cytoplasmic degradative changes during physiological processes [1, 2] and regression of mammary tumors [3, 4]. Acid phosphatase (AP) has also been used to monitor cell death and cell lysis [5,6,7,8]. Activities of lysosomal hydrolases were demonstrated to be more marked in cancer cells than in homologous normal tissue [9, 10]. Ductal carcinoma in situ (DCIS), also known as intraductal carcinoma, is characterized by proliferation of presumably malignant epithelial cells within the mammary ductal-lobular system, without light microscopic evidence of invasion into surrounding stroma [11]. An increasing number of women have been diagnosed with DCIS as more sophisticated methods for detecting breast cancer are being used. Autopsies performed on women who died from all kinds of causes showed that 6-16 % of them had DCIS [12, 13]. Over 14 % of breast cancers diagnosed in the United States annually are DCIS [14]. Some DCIS lesions, if left untreated, may progress to invasive breast carcinoma [15]. Invasive ductal carcinoma (IDC) often metastasizes to the axillary lymph nodes and is associated with a relatively poor prognosis compared with other types of breast cancer, such as medullary or tubular carcinoma [16]. Other common sites of metastases for IDC include bone and intraparenchymal sites within the lung, liver and brain [16].

In this report, AP, the marker enzyme for lysosomes [17], was used to visualize these organelles in normal and neoplastic breast tissues. We detected higher AP activity and histochemistry in DCIS associated with IDC compared to IDC alone.

Materials & methods

Tissue Collection & Processing

Normal and malignant breast tissues used in this study were obtained from the Cooperative Human Tissue Network (Philadelphia, PA). Two cases of IDC associated with DCIS and one case of IDC were studied. The patients were 58, 67, and 73 years old at the time of the biopsies. Normal breast tissue from each patient was also examined and considered as controls. Tissues were fixed overnight in 4 % paraformaldehyde at 4°C, placed in Tissue Tek OCT (Miles, Elkhart, IN), frozen in liquid N2, cut as 5 μm sections onto poly-L-lysine coated slides (Sigma, St. Louis, MO), and stored at -20°C until they were stained.

Lysosomal Localization

Lysosomes were localized from slides of frozen sections using a histochemical assay for AP (Sigma) as previously described [18]. Briefly, prior to fixing the slides, 0.6 mL of sodium nitrite solution were added to 0.6 mL of fast garnet GBC solution, mixed by inversion; allowed to stand for 2-4 min; and added to 23 mL of dH2O. Three mL of acetate solution and 3 mL of naphthol AS-BI phosphoric acid solution (provides naphthol AS-BI phosphate, the substrate) were added to the previous solution. Slides were fixed in citrate-acetone-formaldehyde solution at room temperature, in Coplin jars, for 30 sec and rinsed with dH2O for 1 min. Slides were then incubated for 1 h at 37°C in naphthol AS-BI phosphate and fast garnet stain in acetate buffer. Slides were rinsed with tap H2O for 2 min, dried for 15 min, counterstained with methylene blue for 1 min, rinsed with dH2O, and mounted in CrystalMount (Biomeda Corp., Foster City, CA). The presence of acid phosphatase was indicated by distinct red-violet focal precipitates, which were resolved by light microscopy.

Results and discussion

Normal myoepithelial cells showed a moderate amount of lysosomes (Figs. 1,2,3, panels a and b). The increase in lysosomes was apparent in the tumor cells. The acid phosphatase preparations revealed conglomerates (dark granules) in tumor cells from patients with DCIS associated with IDC (Figs. 2 and 3, panels c and d). However, less AP activity was detected in the case of IDC alone (Fig. 1, panels c and d). The tumor cells displayed histochemical, degenerative changes (Figs. 2 and 3, panels c and d). The concentration of AP particles, which characterize lysosomes, was increased in breast tumor cells. Cords of tumor cells were strongly positive for AP, while the stromal tissue was unstained (Figs. 2 and 3, panels c and d). The positive granules are much larger in the breast tumor cells than those in the normal breast cells (Figs. 1,2,3).

Figure 1
figure 1

Infiltrating Ductal Carcinoma (IDC). a, b) Faint histochemical activity (dark granules) is seen in the epithelial cells from normal breast tissue and is negative in the connective tissue.. c) The florid complex and irregularly shaped epithelial bridges resulted in superimposed micropapillary features. d) Enhanced enzyme activity is evident in the epithelial elements. X 200, a and c; X 400, b and d.

Figure 2
figure 2

IDC Associated with Ductal Carcinoma in situ (DCIS). a, b) Very few AP positive granules (dark granules) are seen in the normal breast. Stromal fibroblasts contain no enzyme activity. c, d) Cords of tumor cells are strongly positive while the stromal tissue is unstained (clear areas). X 200, a and c; X 400, b and d.

Figure 3
figure 3

IDC Associated with DCIS. a, b) Normal breast myoepithelia has is sparsely stained for AP. c) Strong enzyme activity is evident throughout the cytoplasm of the tumor cells. The AP positive granules (dark areas) are much larger than those in the control tissue (a, b). The majority of the tumor cells are AP positive, suggesting that they are undergoing cell death. d) Intense AP activity in the cords of tumor cells. Note that AP staining is absent in the stroma. X 200, a and c; X 400, b and d.

Histochemical reactions for acid phosphatase were demonstrated in all cases of breast cancer that we examined. The reaction was most constant in the epithelial cells and negligible in connective tissues. This observation is in agreement with previous reports [19, 20]. It seems that increased acid phosphatase activity is a constant feature in neoplastic transformation [21], and this was noted in all the cases of breast carcinomas we studied. Acid phosphatase activity has been used to indicate that lysosomes participate in the execution of cell death in a variety of tissues [22, 23] and in the regression of mammary carcinomas [24, 25]. Apoptosis could represent a barrier to the progression of invasive cancer [26, 27]. Our observations support this hypothesis in that we detected more AP activity in DCIS relative to IDC. Moreover, a recent study found that apoptosis comprises only 11 cells per mm2 of tissue in 288 cases of invasive breast cancer [28]. Acid phosphatase has been used as a marker of metastatic bone disease and response to treatment in breast cancer patients [29]. In this light, our findings suggest that the level of AP activity in breast tumors may be correlated to the amount of apoptosis that is going on in the cells.

References

  1. Anton E, Brandes D, Barnard S: Lysosomes in uterine involution: distribution of acid hydrolases in luminal epithelium. Anat Rec. 1969, 164: 231-251.

    Article  CAS  PubMed  Google Scholar 

  2. Frost JL, Brandes D: Nonspecific esterases in rat prostatic epithelial cells. J Histochem Cytochem. 1967, 15: 589-595.

    Article  CAS  PubMed  Google Scholar 

  3. Brandes D, Anton E: The role of lysosomes in cellular lytic processes. 3. Electron histochemical changes in mammary tumors after treatment with cytoxan and vitamin A. Lab Invest. 1966, 15: 987-1006.

    CAS  PubMed  Google Scholar 

  4. Brandes D, Sloan KW, Anton E, Bloedorn F: The effect of x-irradiation on the lysosomes of mouse mammary gland carcinomas. Cancer Res. 1967, 27: 731-746.

    CAS  PubMed  Google Scholar 

  5. Yamamoto S, Sawada K, Shimomura H, Kawamura K, James TN: On the nature of cell death during remodeling of hypertrophied human myocardium. J Mol Cell Cardiol. 2000, 32: 161-175. 10.1006/jmcc.1999.1064.

    Article  CAS  PubMed  Google Scholar 

  6. Heryanto B, Yoshimura Y, Tamura T, Okamoto T: Involvement of apoptosis and lysosomal hydrolase activity in the oviducal regression. during induced molting in chickens: a cytochemical study for end labeling of fragmented DNA and acid phosphatase. Poult Sci J. 1977, 76: 67-72.

    Article  Google Scholar 

  7. Beem EP, Hillebrand MJ, Benckhuijsen C, Overdijk B: Origin of the increased activity of beta-glucuronidase in the soluble fraction of rat mammary tumors during ovariectomy-induced regression. Cancer Res. 1987, 47: 3980-3987.

    CAS  PubMed  Google Scholar 

  8. Sarraf CE, Bowen ID: Kinetic studies on a murine sarcoma and an analysis of apoptosis. Br J Cancer. 1986, 54: 989-998.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Isidoro C, Demoz M, De Stefanis D, Baccino FM, Bonelli G: High levels of proteolytic enzymes in the ascitic fluid and plasma of rats bearing the Yoshida AH-130 hepatoma. Invasion Metastasis. 1995, 15: 116-124.

    CAS  PubMed  Google Scholar 

  10. Sinadinovic J, Cvejic D, Savin S, Micic JV, Jancic-Zguricas M: Enhanced acid protease activity of lysosomes from papillary thyroid carcinoma. Cancer. 1989, 63: 1179-1182.

    Article  CAS  PubMed  Google Scholar 

  11. Winchester DP, Jeske JM, Goldschmidt RA: The diagnosis and management of ductal carcinoma in-situ of the breast. CA Cancer J Clin. 2000, 50: 184-200.

    Article  CAS  PubMed  Google Scholar 

  12. Alpers CE, Wellings SR: The prevalence of carcinoma in situ in normal and cancer-associated breasts. Hum Pathol. 1985, 16: 796-807.

    Article  CAS  PubMed  Google Scholar 

  13. Nielsen M, Jensen J, Andersen J: Precancerous and cancerous breast lesions during lifetime and at autopsy. A study of 83 women. Cancer. 1984, 54: 612-615.

    Article  CAS  PubMed  Google Scholar 

  14. Ernster VL, Barclay J, Kerlikowske K, Wilkie H, Ballard-Barbash R: Mortality among women with ductal carcinoma in situ of the breast in the population-based surveillance, epidemiology and end results program. Arch Intern Med. 2000, 160: 953-958. 10.1001/archinte.160.7.953.

    Article  CAS  PubMed  Google Scholar 

  15. Silverstein MJ, Cohlan BF, Gierson ED, Furmanski M, Gamagami P, Colburn WJ, Lewinsky BS, Waisman JR: Duct carcinoma in situ: 227 cases without microinvasion. Eur J Cancer. 1992, 28: 630-634.

    Article  CAS  PubMed  Google Scholar 

  16. Harris JR, Lippman ME, Veronesi U, Willett W: Breast cancer (1). N Engl J Med. 1992, 327: 319-328.

    Article  CAS  PubMed  Google Scholar 

  17. Pelletier G, Novikoff AB: Localization of phosphatase activities in the rat anterior pituitary gland. J Histochem Cytochem. 1972, 20: 1-12.

    Article  CAS  PubMed  Google Scholar 

  18. Halaby R, Zakeri Z, Lockshin RA: Metabolic events during programmed cell death in insect labial glands. Biochem Cell Biol. 1994, 72: 597-601.

    Article  CAS  PubMed  Google Scholar 

  19. Fanger H, Barker BE: Histochemistry of breast diseases. I. Phosphatases. Arch Pathol. 1959, 67: 293-305.

    CAS  Google Scholar 

  20. Livni N, Laufer A: Histochemical studies of human breast tumors: Activity of alkaline phosphatase, acid phosphatase and glucose-6-phosphate dehydrogenase. Pathol Microbiol. 1975, 42: 159-170.

    CAS  Google Scholar 

  21. Filmus JE, Podhajcer OL, Mareso E, Guman N, Mordoh J: Acid phosphatase in human breast cancer tissue. Cancer. 1984, 53: 301-305.

    Article  CAS  PubMed  Google Scholar 

  22. Zakeri Z, Quaglino D, Ahuja HS: Apoptotic cell death in the mouse limb and its suppression in the hammertoe mutant. Dev Biol. 1994, 165: 294-297. 10.1006/dbio.1994.1255.

    Article  CAS  PubMed  Google Scholar 

  23. Jones HE, Bowen ID: Acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster. Cell Biol Int. 1993, 17: 305-315. 10.1006/cbir.1993.1066.

    Article  CAS  PubMed  Google Scholar 

  24. Shamberger RJ: Lysosomal enzyme changes in growing and regressing mammary tumours. Biochem J. 1969, 111: 375-383.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lanzerotti RH, Gullino PM: Activities and quantities of lysosomal enzymes during mammary tumor regression. Cancer Res. 1972, 32: 2679-2685.

    CAS  PubMed  Google Scholar 

  26. Bodis S, Siziopikou KP, Schnitt SJ, Harris JR, Fisher DE: Extensive apoptosis in ductal carcinoma in situ of the breast. Cancer. 1996, 77: 1831-1835. 10.1002/(SICI)1097-0142(19960501)77:9<1831::AID-CNCR11>3.0.CO;2-0.

    Article  CAS  PubMed  Google Scholar 

  27. Symonds H, Krall L, Remington L, Saenz-Robles M, Lowe S, Jacks T, Van Dyke T: p53-dependent apoptosis suppresses tumor growth and progression in vivo. Cell. 1994, 78: 703-712.

    Article  CAS  PubMed  Google Scholar 

  28. Lipponen P, Aaltomaa S, Kosma VM, Syrjanen K: Apoptosis in breast cancer as related to histopathological characteristics and prognosis. Eur J Cancer. 1994, 30A: 2068-2073.

    Article  CAS  PubMed  Google Scholar 

  29. Wada N, Ishii S, Ikeda T, Enomoto K, Kitajima M: Serum tartrate resistant acid phosphatase as a potential marker of bone metastasis from breast cancer. Anticancer Res. 1999, 19: 4515-4521.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Reginald Halaby.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Halaby, R., Abdollahi, J. & Martinez, M.L. Acid Phosphatase Activity in Human Breast Tumors . Breast Cancer Res 3, E002 (2001). https://doi.org/10.1186/bcr296

Download citation

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/bcr296

Keywords