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Procedures for the Quantitative Protein Determination of Urokinase and Its Inhibitor, PAI-1, in Human Breast Cancer Tissue Extracts by ELISA

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Breast Cancer Research Protocols

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 120))

Summary

The determination of the protein content of urokinase-type plasminogen activator (uPA) and its inhibitor, PAI-1, in breast cancer tissue extracts is used clinically to identify patients at risk to experience disease recurrence (metastasis) or early death. The serine protease uPA, in concert with its inhibitor PAI-1, promotes tumor cell adhesion, migration, and proliferation, as well as extracellular matrix degradation and, thus, facilitates tumor cell invasion and metastasis. The various technical steps to recover uPA and PAI-1 protein from archived breast cancer tissues and to quantitatively determine uPA and PAI-1 protein content in tumor tissue extracts by enzyme-linked immunosorbent assay (ELISA) are described in detail. The technical steps involved require fresh-frozen breast cancer tissue, a dismembrator machine (ball mill) to pulverize the tissue in the frozen state, detergent (Triton X-100) containing Tris-buffered saline to extract uPA and PAI-1 from the pulverized breast cancer tissue, an ultracentrifuge to separate the detergent fraction from cellular debris, uPA and PAI-1 ELISA kits, protein determination reagents, and a 96-well spectrophotometer (ELISA reader) to assess uPA, PAI-1, and total protein in the detergent extract. The uPA/PAI-1 ELISAs and the protein determination format described are robust and highly sensitive. In addition to the macromethod of tissue disintegration, we present a simple but sensitive micro-extraction procedure using cryostat sections or core biopsies as the source of breast cancer tissue. Such a technique allows rapid and quantitative determination of uPA and PAI-1, even in small breast cancer specimens.

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References

  1. Schmitt, M., Wilhelm, O. G., Reuning, U., et al. (2000) The urokinase plasminogen activator system as a novel target for tumour therapy. Fibrinol. Proteol. 14, 114–132.

    Article  CAS  Google Scholar 

  2. Andreasen., P. A., Kjöller, L., Christensen, L., and Duffy, M. J. (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72, 1–22.

    Article  PubMed  CAS  Google Scholar 

  3. Duffy, M. J. (1996) Proteases as prognostic markers in cancer. Clin. Cancer Res. 2, 613–618.

    PubMed  CAS  Google Scholar 

  4. Schmitt, M., Jänicke, F., and Graeff, H. (1992) Tumor-associated proteases. Fibrinolysis 6, 3–26.

    Article  CAS  Google Scholar 

  5. Schmitt, M., Harbeck, N., Thomssen, C., et al. (1997) Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy. Thromb. Haemost. 78, 285–296.

    PubMed  CAS  Google Scholar 

  6. Reuning, U., Magdolen, V., Wilhelm, O., et al. (1998) Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis. Int. J. Oncology 13, 893–906.

    CAS  Google Scholar 

  7. Jänicke, F., Schmitt, M., Ulm, K., Gössner, W., and Graeff, H. (1989) Urokinasetype plasminogen activator antigen and early relapse in breast cancer. Lancet 2, 1049.

    Article  PubMed  Google Scholar 

  8. Jänicke, F., Schmitt, M., Hafter, R., et al. (1990) The urokinase-type plasminogen activator (u-PA) is a potent predictor of early relapse in breast cancer. Fibrinolysis 4, 69–78.

    Google Scholar 

  9. Jänicke, F., Schmitt, M., and Graeff, H. (1991) Clinical relevance of the urokinase-type and tissue-type plasminogen activators and of their type 1 inhibitor in breast cancer. Sem. Thromb. Hemost. 17, 303–312.

    Article  Google Scholar 

  10. Jänicke, F., Pache, L., Schmitt, M., et al. (1994) Both the cytosols and detergent extracts of breast cancer tissues are suited to evaluate the prognostic impact of the urokinase-type plasminogen activator and its inhibitor, plasminogen activator inhibitor type 1. Cancer Res. 54, 2527–2530.

    PubMed  Google Scholar 

  11. Stack, M. S. and Johnson, D., A. (1994) Human mast cell tryptase activates singlechain urinary-type plasminogen activator (pro-urokinase). J. Biol. Chem. 269, 9416–9419.

    PubMed  CAS  Google Scholar 

  12. Dano, K., Andreasen, P. A., Grondahl-Hansen, J., Kristensen, P., Nielsen, L.S., and Skriver, L. (1985) Plasminogen activators, tissue degradation, and cancer. Adv. Cancer Res. 44, 139–266.

    Article  PubMed  CAS  Google Scholar 

  13. Jänicke, F., Prechtl, A., Thomssen, C., et al. (2001) Randomized adjuvant chemotherapy trial in high-risk, lymph node-negative breast cancer patients identified by urokinase-type plasminogen activator and plasminogen activator inhibitor type 1. J. Natl. Cancer Inst. 93, 913–920.

    Article  PubMed  Google Scholar 

  14. Harbeck, N., Kates, R. E., Look, M. P., et al. (2002) Enhanced benefit from adjuvant chemotherapy in breast cancer patients classified high-risk according to urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (n = 3424). Cancer Res. 62, 4617–4622.

    PubMed  CAS  Google Scholar 

  15. Grøndahl-Hansen, J., Christensen, I. J., Rosenquist, C., et al. (1993) High levels of urokinase-type plasminogen activator and its inhibitor PAI-1 in cytosolic extracts of breast carcinomas are associated with poor prognosis. Cancer Res. 53, 2513–2521.

    PubMed  Google Scholar 

  16. Foekens, J. A., Schmitt, M., van Putten, W. L. J., et al. (1994) Plasminogen activator inhibitor-1 and prognosis in primary breast cancer. J. Clin. Oncol. 12, 1648–1658.

    PubMed  CAS  Google Scholar 

  17. Fernö, M., Bendahl, P. O., Borg, Å., et al. (1996) Urokinase plasminogen activator, a strong independent prognostic factor in breast cancer, analysed in steroid receptor cytosols with a luminometric immunoassay. Eur. J. Cancer 32A, 793–801.

    Article  PubMed  Google Scholar 

  18. Harbeck, N., Dettmar, P., Thomssen, C., et al. (1999) Risk-group discrimination in node-negative breast cancer using invasion and proliferation markers: six-year median follow-up. Br. J. Cancer 80, 419–426.

    Article  PubMed  CAS  Google Scholar 

  19. Harbeck, N., Thomssen, C., Berger, U., et al. (1999) Invasion marker PAI-1 remains a strong prognostic factor after long-term follow-up both for primary breast cancer and following first relapse. Breast Cancer Res. Treat. 54, 147–157.

    Article  PubMed  CAS  Google Scholar 

  20. Duffy, M. J., O’Grady, P., Devaney, D., O’Sioran, L., Fennelly, J. J., Lijnen, H. (1988) Urokinase-plasminogen activator, a marker for aggressive breast carcinomas. Cancer 62, 531–533.

    Article  PubMed  CAS  Google Scholar 

  21. Bouchet, C, Hacene, K., Martin, P. M., et al. (1999) Dissemination risk index based on plasminogen activator system components in primary breast cancer. J. Clin. Oncol. 17, 3048–3057.

    PubMed  CAS  Google Scholar 

  22. Jänicke, F., Thomssen, C., Pache, L., Schmitt, M., and Graeff, H. (1994) Urokinase (uPA) and PAI-1 as selection criteria for adjuvant chemotherapy in axillary node-negative breast cancer patients, in Prospects in Diagnosis and Treatment of Cancer (Schmitt, M., Graeff, H., Jänicke, F., eds.). Elsevier Science, Amsterdam,, pp. 207–218.

    Google Scholar 

  23. Foekens, J. A., Look, M. P., Peters, H. A., van Putten, W. L. J., Portengen, H., and Klijn, J. G. M. (1995) Urokinase-type plasminogen activator and its inhibitor PAI-1: Predictors of poor response to tamoxifen therapy in recurrent breast cancer. J. Natl. Cancer Inst. 87, 751–756.

    Article  PubMed  CAS  Google Scholar 

  24. Nekarda, H., Schmitt, M., Ulm, K., et al. (1994) Prognostic impact of urokinasetype plasminogen activator and its inhibitor PAI-1 in completely resected gastric cancer. Cancer Res. 54, 2900–2907.

    PubMed  CAS  Google Scholar 

  25. Heiss, M. M., Babic, R., Allgayer, H., et al. (1995) Tumor-associated proteolysis and prognosis, new functional risk factors in gastric cancer defined by the urokinase-type plasminogen activator system. J. Clin. Oncol. 8, 2084–2093.

    Google Scholar 

  26. Ganesh, S., Sier, C. F. M., Griffioen, G., et al. (1994) Prognostic relevance of plasminogen activators and their inhibitors in colorectal cancer. Cancer Res. 54, 4065–4071.

    PubMed  CAS  Google Scholar 

  27. Sato, T., Nishimura, G., Yonemura, Y., et al. (1995) Association of immunohistochemical detection of urokinase-type plasminogen activator with metastasis and prognosis in colorectal cancer. Oncology 52, 347–352.

    Article  PubMed  CAS  Google Scholar 

  28. Ganesh, S., Sier, C. F. M., Heerding, M. M., et al. (1996) Prognostic value of the plasminogen activation system in patients with gastric carcinoma. Cancer 77, 1035–1043.

    Article  PubMed  CAS  Google Scholar 

  29. Verspaget, H. W., Sier, C. F., Ganesh, S., Griffioen, G., and Lamers, C. B. (1995) Prognostic value of plasminogen activators and their inhibitors in colorectal cancer. Eur. J. Cancer 31A, 1105–1109.

    Article  PubMed  CAS  Google Scholar 

  30. Nekarda, H., Schlegel, P., Schmitt, M., et al. (1998) Strong prognostic impact of tumor-associated urokinase-type plasminogen activator in completely resected adenocarcinoma of the esophagus. Clin. Cancer Res. 4, 1755–1763.

    PubMed  CAS  Google Scholar 

  31. Kuhn, W., Pache, L., Schmalfeldt, B., et al. (1994) Urokinase (uPA) and PAI-1 predict survival in advanced ovarian cancer patients (FIGO III) after radical surgery and platinum-based chemotherapy. Gynecol. Oncol. 55, 401–409.

    Article  PubMed  CAS  Google Scholar 

  32. Kuhn, W., Schmalfeldt, B., Reuning, U., et al. (1999) Prognostic significance of urokinase (uPA) and its inhibitor PAI-1 for survival in advanced ovarian carcinoma stage FIGO IIIc. Br. J. Cancer. 79, 1746–1751.

    Article  PubMed  CAS  Google Scholar 

  33. Kobayashi, H., Fujishiro, S., and Terao, T. (1994) Impact of urokinase-type plasminogen activator and its inhibitor type 1 on prognosis in cervical cancer of the uterus. Cancer Res. 54, 6539–6548.

    PubMed  CAS  Google Scholar 

  34. Hofmann, R., Lehmer, A., Buresch, M., Hartung, R., and Ulm, K. (1996) Clinical relevance of urokinase plasminogen activator, its receptor, and its inhibitor in patients with renal cell carcinoma. Cancer 78, 487–492.

    Article  PubMed  CAS  Google Scholar 

  35. Miyake, H., Hara, I., Yamanaka, K., Arakawa, S., and Kamidono, S. (1999) Elevation of urokinase-type plasminogen activator and its receptor densities as new predictors of disease progression and prognosis in men with prostate cancer. Int. J. Oncol. 14, 535–541.

    PubMed  CAS  Google Scholar 

  36. Pedersen, H., Brünner, N., Francis, D., et al. (1994) Prognostic impact of urokinase, urokinase receptor and type 1 plasminogen activator inhibitor in squamous and large cell lung cancer tissue. Cancer Res. 54, 4671–4675.

    PubMed  CAS  Google Scholar 

  37. Pedersen, H., Grøhndahl-Hansen, J., Francis, D., et al. (1994) Urokinase and plasminogen activator inhibitor type 1 in pulmonary adenocarcinoma. Cancer Res. 54, 120–123.

    PubMed  CAS  Google Scholar 

  38. Rosenberg, S. (2000) Modulators of the urokinase-type plasminogen activation system for cancer. Exp. Opin. Ther. Patents 10, 1843–1852.

    Article  CAS  Google Scholar 

  39. Bürgle, M., Kessler, H., Stürzebecher, J., Magdolen, V., Wilhelm, O., and Schmitt, M. (2002) The urokinase-type plasminogen activator system—a new target for tumor therapy, in Proteinase, Peptidase Inhibition: Recent Potential Targets for Drug Development (Smith H. J., Simons, C., eds.). Harwood Academic, New York, pp. 231–248.

    Google Scholar 

  40. Mühlenweg, B., Sperl, S., Magdolen, V., Schmitt, M. and Harbeck N. (2001) Interference with the urokinase plasminogen activator system: a promising therapy concept for solid tumors. Exp. Opin. Pharmacother. 1, 683–691.

    Google Scholar 

  41. Magdolen, U., Krol, J., Sumito, S., et al. (2002) Natural inhibitors of tumor-associated proteases. Radiol. Oncol. 36, 131–143.

    CAS  Google Scholar 

  42. Setyono-Han, B., Stürzebacher, J., Schmalix, W. A., et al. (2005) Suppression of rat breast cancer metastasis and reduction of primary tumor growth by the small synthetic urokinase inhibitor WX-UK1. Thromb. Haemost. 93, 779–786.

    PubMed  CAS  Google Scholar 

  43. http://www.medicalnewstoday.com/index.php?newsid=8684.

  44. Schmitt, M., Lienert, S., Prechtel, D., et al. (2002) The urokinase protease system as a target for breast cancer prognosis and therapy: technical considerations. J. Clin. Ligand Assay 25, 43–52.

    Google Scholar 

  45. Benraad, T. J., Geurts-Moespot, J., Grøndahl-Hansen, J., et al. (1996) Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): Report of an EORTC/BIOMED-1 workshop. Eur. J. Cancer 32A, 1371–1381.

    Article  PubMed  CAS  Google Scholar 

  46. Sweep, C. G. J., Geurts-Moespot, J., Grebenschikov, N., et al. (1998) External quality assessment of trans-European multicentre antigen determinations (ELISA) of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) in human breast cancer tissue extracts. Br. J. Cancer 78, 1434–1441.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Clinical Research Unit, Department of Obstetrics and Gynaecology, Technical University of Munich, Munich, Germany

    Manfred Schmitt, Alexandra S. Sturmheit, Anita Welk, Christel Schnelldorfer & Nadia Harbeck

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  1. Manfred Schmitt
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  2. Alexandra S. Sturmheit
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  3. Anita Welk
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  4. Christel Schnelldorfer
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Editors and Affiliations

  1. School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford, UK

    Susan A. Brooks

  2. Medical Oncology Unit, The Churchill Hospital, Headington, Oxford, UK

    Adrian Harris

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© 2006 Humana Press Inc.

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Schmitt, M., Sturmheit, A.S., Welk, A., Schnelldorfer, C., Harbeck, N. (2006). Procedures for the Quantitative Protein Determination of Urokinase and Its Inhibitor, PAI-1, in Human Breast Cancer Tissue Extracts by ELISA. In: Brooks, S.A., Harris, A. (eds) Breast Cancer Research Protocols. Methods in Molecular Medicine, vol 120. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-969-9:245

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  • DOI: https://doi.org/10.1385/1-59259-969-9:245

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-191-2

  • Online ISBN: 978-1-59259-969-1

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