Procedures for the quantitative protein determination of urokinase and its inhibitor, PAI-1, in human breast cancer tissue extracts by ELISA.
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.
Clinical Research Unit, Department of Obstetrics and Gynaecology, Technical University of Munich, Germany.
Manfred
M
Schmitt
Alexandra S
AS
Sturmheit
Anita
A
Welk
Christel
C
Schnelldorfer
Nadia
N
Harbeck
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D018572:Disease-Free Survival; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D005260:Female; D016591:Histocytological Preparation Techniques; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011933:Reagent Kits, Diagnostic; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D009363:Neoplasm Proteins; D017395:Plasminogen Activator Inhibitor 1; D011933:Reagent Kits, Diagnostic; C051321:SERPINE1 protein, human; D014568:Urokinase-Type Plasminogen Activator