The Fas antigen is a transmembrane receptor that can trigger apoptosis in a variety of tumor and hematopoietic cells. Ovarian follicular atresia and luteolysis are thought to occur by apoptosis. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, we demonstrated that human granulosa/luteal cells express the Fas antigen. An anti-human Fas antigen monoclonal antibody (Fas mAb; clone CH-11), which induces apoptosis in other cell types by binding to the Fas antigen, induced significant cell death (30%) in cultures pretreated with interferon gamma (IFN gamma). This agrees with studies on tumor cell lines showing that IFN gamma enhances cytotoxic effects of Fas mAb. Granulosa/luteal cells exhibited morphological characteristics typical of apoptosis, including membrane blebbing and condensed chromatin. DNA fragmentation into oligonucleosomal units of approximately 180 bp, typical of apoptosis, was detected at elevated levels in Fas mAb-treated cultures via 3' end-labeling and gel electrophoresis. Examination of cultured cells in situ for apoptotic DNA cleavage by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) indicated that more apoptotic death occurred in Fas mAb-treated cultures than in control cultures. Effects of hCG-induced luteinization of cultures on Fas mAb-induced cytotoxicity was examined: combined pretreatment with IFN gamma and hCG induced a synergistic increase in Fas mAb-induced cytotoxicity (40%) over that obtained with IFN gamma-pretreatment alone (15%). In summary, granulosa/luteal cells express the Fas antigen and are sensitive to Fas mAb-induced apoptosis. Human CG synergized with IFN gamma to increase Fas mAb-induced death.(ABSTRACT TRUNCATED AT 250 WORDS)

The major cause of mortality in breast cancer is the progression of metastatic disease. Although loss of oestrogen receptors (ER) and progesterone receptors is associated with more aggressive disease, 30% of patients with receptor-positive, node-negative tumours develop metastases (Price et al, 1997). A component of the metastatic process is invasion, which is accomplished when the cells can transit the extracellular matrix (ECM) layer. More accurate prognoses might be possible if factors could be identified that either promote invasion or are markers for it, independent of steroid receptor status. Specific treatments to prevent invasion might also be developed.Thrombin is a growth-regulatory protein at nanomolar concentrations. Thrombin is also a serine proteinase that is generally associated with coagulation. Its proenzyme, prothrombin, occurs in the plasma at concentrations up to 3 µM (Shapiro and McCord, 1978). Thrombin and its associated receptor, TR-1, are involved in the activation of platelets (McNicol and Gerrard, 1993;Vittet and Chevillard, 1993), growth stimulation of immature rat uterine stromal cells (Arena et al, 1996), alteration of permeability in vascular endothelial cells (DeMichele and Minnear, 1992;Garcia et al, 1995), DNA synthesis in astrocytoma cells (Aragay et al, 1995) and endothelial cell growth in angiogenesis (Fidler and Ellis, 1994). Thrombin also has been implicated in tumour progression (Wojtukiewicz et al, 1995).The initial step in characterization of thrombinÕs role in breast tumour cell invasion is a description of the presence of TR-1 on invasive breast tumour cell lines and of thrombinÕs influence on in vitro invasion by those cell lines. We report the presence of TR-1 and activity of thrombin in stimulating invasion by the MDA-MB231 breast cancer cell line and its absence in other, less aggressive, breast tumour cell lines. MATERIALS AND METHODS Cell cultureCells were cultured in DulbeccoÕs modified Eagle medium (DMEM) without phenol red, with streptomycin, penicillin, nonessential amino acids, glutamine and 5% serum (Hyclonedefined calf serum for MDA-MB231 and MDA-MB436; Gibco fetal calf serum for MCF-7 cells).Conditioned medium was prepared by incubating confluent 3T3 cultures with bovine serum albumin (BSA) medium (DMEM without phenol red, containing 0.1% BSA and antibiotics) for 24 h. Conditioned medium was stored frozen and was centrifuged immediately before use to remove cells and debris. Invasion assaysInvasion chambers with 8-µm pores were purchased from BectonÐDickinson. Chambers were used without modification for chemotaxis assays. Matrigel-coated chambers were used for invasion assays. Matrigel, purchased from BectonÐDickinson, was diluted in ice-cold DMEM without additions. One hundred microlitres (38 µg) was pipetted into each chamber in a 24-well plate. The plate was incubated at 37°C for 1 h, then medium was removed by aspiration and replaced with 400 µl of DMEM. Chambers were either used immediately or after overnight storage at 4°C.Assays were performed in 24...