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Clinical Chemistry 48: 1288-1295, 2002;
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(Clinical Chemistry. 2002;48:1288-1295.)
© 2002 American Association for Clinical Chemistry, Inc.

Quantitative Real-Time Reverse Transcription-PCR Assay for Urokinase Plasminogen Activator, Plasminogen Activator Inhibitor Type 1, and Tissue Metalloproteinase Inhibitor Type 1 Gene Expressions in Primary Breast Cancer

Remedios Castelló1, Amparo Estellés1a, Carlos Vázquez2, Cristina Falcó1, Francisco España1, Sergio M. Almenar2, Carlos Fuster2 and Justo Aznar1

1 Hospital Universitario La Fe, Centro de Investigación, Avda. Campanar 21, 46009 Valencia, Spain.

2 Instituto Valenciano de Oncología, 46009 Valencia, Spain.

aAuthor for correspondence. Fax 34-96-3868718; e-mail estelles_amp{at}gva.es.

Background: The plasminogen activation system and matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, cancer cell invasion, and metastasis.

Methods: Quantitative real-time reverse-transcription-PCR (RT-PCR) assays were developed to quantify urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue metalloproteinase inhibitor type 1 (TIMP-1) mRNA in 54 breast cancer tissues. Gene fragments were amplified in a LightCycler real-time PCR system using gene-specific primers and SYBR Green I. The results were normalized to ß-actin mRNA. We also quantified antigen and functional concentrations of these components.

Results: The intra- and interassay variabilities for mRNA quantification showed mean SDs for the crossing point of 0.12 and 0.15 cycles, respectively. PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and PAI-1 and uPA functional concentrations increased with tumor severity; the increase was statistically significant for PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and for uPA functional concentrations. Node-positive patients showed significantly higher PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations than those who were node negative.

Conclusions: Quantitative real-time RT-PCR is a highly sensitive, reproducible, and fast method for measuring gene expression of PAI-1, uPA, and TIMP-1 in breast cancer. These components may be involved in breast cancer development, and increased mRNA expression may be associated with a worse prognosis.




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