Summary
The levels of uPA, its inhibitors PAI-1 and PAI-2, and the uPA receptor (uPAR) have prognostic value in breast cancer. However, different extraction methods and assays kits are used in different laboratories and may directly influence the levels observed. To define a buffer suitable for both PAI-2 and uPAR extraction from breast cancer tissue compatible with hormone receptors and other cytosolic prognosticator assays, we compared PAI-2 and uPAR values obtained by immunoenzymatic assays (American Diagnostica, Green-which, USA) in several extraction conditions: 1) cytosol obtained with the standard hormone receptor buffer; 2) solubilized pellets obtained by Triton X100 extraction of the pelleted membranes obtained with standard hormone receptor buffer; 3) cytosol obtained by direct extraction in the buffer (containing Triton X100) recommended by the manufacturer, after 2 hours or 12 hours of incubation. Cytosol extracts prepared using the standard procedure recommended for hormone receptors gave the highest PAI-2 values. The highest uPAR values were obtained in the subsequent detergent extraction of the pelleted membranes. PAI-2 levels obtained with the kit manufacturer's method after 12 hours of incubation were lower than those obtained after 2 hours of incubation, whereas uPAR levels were similar. We conclude that the most suitable extraction protocol employs standard hormone receptor extraction buffer to obtain a supernatant cytosol fraction for PAI-2 assay, and subsequent detergent extraction of the pelleted membranes to obtain an extract suitable for uPAR.
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References
Duffy MJ, O'Grady P, Devaney D et al.: Urokinase plasminogen activator, a marker for aggressive breast carcinomas, preliminary report. Cancer 62: 531–533, 1988
Jänicke F, Schmitt M, Hafter R, Hollrieder A, Babic R, Ulm K, Gössner W, Graeff H: Urokinase-type plasminogen activator (uPA) antigen is a predictor of early relapse in breast cancer. Fibrinolysis 4: 69–78, 1990
Jänicke F, Schmitt M, Graeff H: 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, 1991
Jänicke F, Schmitt M, Pache L, Ulm K, Harbeck N, Hofler H, Graeff H: Urokinase (uPA) and its inhibitor PAI-1 are strong and independent prognostic factors in node-negative breast cancers. Breast Cancer Res Treat 24: 195–208, 1993
Spyratos F, Martin PM, Hacène K, Romain S, Andrieu C, Ferrero-Poüs M, Deytieux S, Le Doussal V, Tubiana-Hulin M, Brunet M: Multiparametric prognostic evaluation of biological factors in primary breast cancer. J Natl Cancer Inst 84: 1266–1272, 1992
Sumiyoshi K, Serizawa K, Urano T, Takada A, Baba S: Plasminogen activator system in human breast cancer. Int J Cancer 50: 345–348, 1992
Grøndahl-Hansen J, Christensen IJ, Rosenquist C, Brünner N, Mouridsen HT, Danø K, Blichert-Toft M: 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, 1993
Bouchet C, Spyratos F, Martin PM, Hacène K, Gentile A, Oglobine J: Prognostic value of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in breast carcinomas. Br J Cancer 69: 398–405, 1994
Foëkens JA, Schmitt M, van Putten WLJ, Peters HA, Kramer MD, Jänicke F, Klijn GM: Plasminogen activator inhibitor-1 and prognosis in primary breast cancer. J Clin Oncol 12: 1648–1658, 1994
Hollas W, Blasi F, Boyd D: Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer. Cancer Res 51: 3690–3695, 1991
Foëkens JA, Buessecker F, Peters HA, Krainick U, van Putten WLJ, Look MP, Klijn GM, Kramer MD: Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer. Cancer Res 55: 1423–1427, 1995
Rønne E, Hoyer-Hansen N, Pedersen H, Rank F, Osborne CK, Clark GM, Danø K, Grøndahl-Hansen J: Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies. Breast Cancer Res Treat 33: 199–207, 1995
Bianchi E, Cohen RL, Thor AT, Todd RE, Mizukami IF, Lawrence DA, Ljung BM, Schuman MA, Smith HS: The urokinase receptor is expressed in invasive breast cancer but not in normal breast tissue. Cancer Res 54: 861–866, 1994
Duggan C, Maguire T, McDermott E, O'Higgins N, Fennelly JJ, Dully MJ; Urokinase plasminogen activator and urokinase plasminogen activator receptor in breast cancer. Int J Cancer 61: 597–600, 1995
Wiechelman K, Braun R, Fitzpatrick J: Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal Biochem 193: 265–275, 1988
Pyke C, Kristensen P, Ralfkiaer E, Grøndahl-Hansen J, Enksen J, Blasi F, Danø K: Urokinase-type plasminogen activator is expressed in stromal cells and its receptor on cancer cells at invasive foci in human colon adenocarcinomas. Am J Pathol 138: 1059–1067, 1991
Romain S, Spyratos F, Lainé-Bidron C, Bouchet C, Guirou O, Martin PM, Oglobine J, Magdelénat H: Comparative study of four extraction procedures for urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in breast cancer tissues. Eur J Clin Chem Clin Biochem 33: 603–608, 1995
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Bouchet-Bernet, C., Spyratos, F., Andrieu, C. et al. Influence of the extraction procedure on plasminogen activator inhibitor-2 (PAI-2) and urokinase receptor (uPAR) assays in breast cancer tissues. Breast Cancer Res Tr 41, 141–146 (1996). https://doi.org/10.1007/BF01807159
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DOI: https://doi.org/10.1007/BF01807159