Advertisement

Breast Cancer Research and Treatment

, Volume 40, Issue 3, pp 257–263 | Cite as

Quantification of uPA receptor expression in human breast cancer cell lines by cRT-PCR

  • Gerhard Sliutz
  • Helena Eder
  • Heinz Koelbl
  • Clemens Tempfer
  • Leo Auerbach
  • Christian Schneeberger
  • Christian Kainz
  • Robert Zeillinger
Report

Abstract

The conversion of plasminogen to active plasmin is thought to be a crucial step in the process of extracellular matrix degradation associated with metastatic spread. Activation of plasminogen is initiated by urokinase plasminogen activator (uPA). The binding of uPA to the uPA cell surface receptor (uPA-R) accelerates plasmin generation from plasminogen and localizes uPA activity to the cell surface.

We investigated the mRNA-expression of uPA-R in 19 different human breast cancer cell lines. In a competitive reverse transcription polymerase chain reaction (cRT-PCR) we simultaneously co-amplified two different RNA templates bearing the same primer recognition sequences, the cell line RNA and a known amount of anin vitro synthesized uPA-R-RNA internal standard. We analyzed the two PCR products differing 50 bp in size by agarose gel electrophoresis and calculated the initial uPA-R-RNA template concentration from the relative intensities of the bands quantified by video densitometry.

We grouped the investigated cell lines according to theirin vitro invasiveness according to literature. Cell lines with a high potential of invasiveness showed a higher expression of uPA-R compared to those with a low potential of invasiveness (Student's t-test,p 0.04). In addition to that we compared the uPA-R mRNA levels with uPA-R, uPA, and PAI-1 protein levels in culture supernatants and cell lysates. The obtained results in breast cancer cell lines with different invasiveness and in benign epithelial cell lines revealed the complex cooperation of the urokinase type proteolytic pathway.

uPA, uPA-R, and PAI-1 are to be considered as a diagnostic tool rather than assaying a particular molecule alone. Our findings support the hypothesis that the urokinase proteolytic pathway plays a central role in the acquisition of an invasive phenotype and favors its potential use as a prognostic marker in patients with breast cancer.

Key words

breast cancer cRT-PCR fibrinolytic system plasminogen activator uPA-R expression 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Dano K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L: Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res 44: 139–266, 1985PubMedGoogle Scholar
  2. 2.
    Ellis V, Scully MF, Kakkar VV: Plasminogen activation initiated by single-chain urokinase-type plasminogen activator. Potentiation by U937 monocytes. J Biol Chem 264: 2185–2188, 1989PubMedGoogle Scholar
  3. 3.
    Estreicher A, Muhlhauser J, Carpentier JL, Orci L, Vassalli JD: The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes. J Cell Biol 111: 783–792, 1990PubMedGoogle Scholar
  4. 4.
    Roldan AL, Cubellis MV, Masucci MT, Behrendt N, Lund LR, Dano K, Appella E, Blasi F: Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis [published erratum appears in EMBO J 1990 May; 9 (5): 1674]. EMBO J 9: 467–474, 1990PubMedGoogle Scholar
  5. 5.
    Behrendt N, Ronne E, Ploug M, Petri T, Lober D, Nielsen LS, Schleuning WD, Blasi F, Appella E, Dano K: The human receptor for urokinase plasminogen activator. NH-2 terminal amino acid sequence and glycosylation variants. J Biol Chem 265: 6453–6460, 1990PubMedGoogle Scholar
  6. 6.
    Grondahl-Hansen J, Christensen IJ, Rosenquist C, Brunner N, Mouridsen HT, Dano 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, 1993PubMedGoogle Scholar
  7. 7.
    Duffy MJ, Reilly D, O'Sullivan C, O'Higgins N, Fennelly JJ, Andreasen P: Urokinase-plasminogen activator, a new and independent prognostic marker in breast cancer. Cancer Res 50: 6827–6829, 1990PubMedGoogle Scholar
  8. 8.
    Pedersen H, Brunner N, Francis D, Osterlind K, Ronne E, Hansen HH, Dano K, Grondahl-Hansen J: 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, 1994PubMedGoogle Scholar
  9. 9.
    Brunner N, Grondahl-Hansen J, Peters HA, van Putten WL, Look MP, Pappot H, Ronne E, Dano K, Klijn JG, Foekens JA: Prognostic significance of urokinase plasminogen activator receptor in breast cancer (meeting abstract). Proc Am Assoc Cancer Res 36: A597–16X, 1995Google Scholar
  10. 10.
    Kury FD, Schneeberger C, Sliutz G, Kubista E, Salzer H, Medl M, Leodolter S, Swoboda H, Zeillinger R, Spona J: Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation. Oncogene 5: 1403–2408, 1990PubMedGoogle Scholar
  11. 11.
    Bae SN, Arand G, Azzam H, Pavasant P, Torri J, Frandsen TL, Thompson EW: Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-basedin vitro assays. Breast Cancer Res Treat 24: 241–255, 1993PubMedGoogle Scholar
  12. 12.
    Wang AM, Doyle MV, Mark DF: Quantitation of mRNA by the polymerase chain reaction [published erratum appears in Proc Natl Acad Sci USA 1990 Apr; 87 (7): 2865]. Proc Natl Acad Sci USA 86: 9717–9721, 1989PubMedGoogle Scholar
  13. 13.
    Siebert PD, Larrick JW: Competitive PCR. Nature 359: 557–558, 1992PubMedGoogle Scholar
  14. 14.
    Bastholm L, Nielsen MH, De Mey J, Dano K, Brunner N, Hyer-Hansen G, Ronne E, Elling F: Confocal fluorescence microscopy of urokinase plasminogen activator receptor and cathepsin D in human MDA-MB-231 breast cancer cells migrating in reconstituted basement membrane. Biotechnic Histochem 69: 61–67, 1994Google Scholar
  15. 15.
    Caron de Fromentel C, Nardeux PC, Soussi T, Lavialle C, Estrade S, Carloni G, Chandrasekaran K, Cassingena R: Epithelial HBL-100 cell line derived from milk of an apparently healthy woman harbours SV40 genetic information. Exp Cell Res 160: 83–94, 1985PubMedGoogle Scholar
  16. 16.
    Duffy MJ, Reilly D, McDermott E, O'Higgins N, Fennelly JJ, Andreasen PA: Urokinase plasminogen activator as a prognostic marker in different subgroups of patients with breast cancer. Cancer 74: 2276–2280, 1994PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Gerhard Sliutz
    • 1
  • Helena Eder
    • 1
  • Heinz Koelbl
    • 1
  • Clemens Tempfer
    • 1
  • Leo Auerbach
    • 1
  • Christian Schneeberger
    • 1
  • Christian Kainz
    • 1
  • Robert Zeillinger
    • 1
  1. 1.Department of Gynecology and ObstetricsGeneral Hospital Vienna, Medical School, University of ViennaViennaAustria

Personalised recommendations