Estrogen receptor cleavage and plasminogen activation by enzymes in human breast tumor cytosol

Estrogen receptors in cytoplasmic extracts of breast tumors from more than 40 patients were separately analyzed by gel filtration and/or ultracentrifugation under diverse conditions. Resultant patterns are presented for specimens from 11 women with infiltrating duct carcinoma and are representative of results obtained in all samples of sufficient size and receptor content (approximately 40 fmol/mg cytosol protein) for accurate determination of hydrodynamic parameters. Estradiol-binding components of intracellular origin were distinguished from the serum contaminant, sex hormone-binding globulin by their high affinity for diethylstilbestrol and negligible affinity for 5 alpha-dihydrotestosterone. The predominant molecular forms of the receptors, but not the steroid specificity, varied dramatically with experimental factors, including the duration of the fractionation procedure, ionic strength, and the presence of protease inhibitors, particularly the bacterial tripeptides N-acetyl- and N-propionyl-L-leucyl-L-leucyl-DL-arginine aldehydes (leupeptin). At least three discrete forms of the intracellular receptors were detected. The smallest labeled complex, the mero-receptor, with a sedimentation coefficient of about 3S and a Stokes radius of about 24 A, was formed during prolonged analysis of control cytosol in hypotonic or hypertonic buffers. Complexes with an intermediate sedimentation coefficient (approximately 5S) and Stokes radius (approximately 34A) were detected when control cytosol was analyzed rapidly in hypotonic buffer or when cytosol containing 50 nM leupeptin was analyzed in hypertonic buffer. The largest receptor form (10.5S, 71A) was predominant in cytosol prepared with 50 mM leupeptin and analyzed in hypotonic buffer. In this small series of patients, there was no obvious correlation between the molecular form of the receptors and the clinical status or eventual responsiveness to endocrine therapy. Preliminary studies of endogenous proteolytic enzymes in breast tumor cytosol that may be involved in mero-receptor formation included assays of plasminogen activators (EC 3.4.21.-) by fibrinolytic and spectrofluorometric techniques. The detection of high concentrations of plasminogen activators in several tumor cytosols and the inhibition of this activity by leupeptin, which stabilizes the large receptor forms in this and other systems, are consistent with a possible role of these enzymes in receptor cleavage.