Progesterone-induced apoptosis in immortalized normal and malignant human ovarian surface epithelial cells involves enhanced expression of FasL Viqar Syed 1 and Shuk-Mei Ho* ,1,2 1 Department of Surgery, University of Massachusetts Medical School, Worcester, MA, USA; 2 Department of Cell Biology and Physiology, University of Massachusetts Medical School, Worcester, MA, USA Progesterone (P4) has been implicated as a protective factor for epithelial ovarian cancers, yet little is known about its mechanism of action. We previously reported that pregnancy-equivalent doses of P4 inhibited the growth of normal and malignant human ovarian surface epithelial (HOSE) cells. Here, we investigated how P4- induced cell death in two immortalized normal (HOSE 642, HOSE 12-12) and two malignant (OVCA 429, OVCA 432) HOSE cell lines. The exposure of HOSE or OVCA cell cultures to 10 6 m P4 induced time-dependent increasesinearlyandlateapoptoticcellsandactivationof caspase-8 and -3, but not that of caspase-9. A general caspase inhibitor Z-VAD effectively blocked the P4- induced cell death in a dose-dependent manner. Compar- able levels of Fas mRNA and protein were expressed in HOSE and OVCA cell lines, and these levels were unaffected by P4. In contrast, levels of FasL mRNA and protein were higher in OVCA cells than in HOSE cells. Interestingly, the hormone enhanced levels of FasL mRNA and protein in HOSE cells, but lowered their levels in OVCA cells. The exposure of HOSE or OVCA cells to an activating anti-Fas antibody induced cell loss, whereas treatment of cells with a blocking anti-FasL antibody reduced the P4-induced cell loss. Cotreatment of cells with the activating anti-Fas antibody and P4 produced additive effects on cell loss. These results reveal for the first time that P4 induces apoptosis in HOSE and OVCA cells via activation of a caspase-8-initiated Fas/ FasL signaling pathway. They also demonstrate differ- ential P4-regulation of FasL expression between HOSE and OVCA cells. Oncogene (2003) 22, 6883–6890. doi:10.1038/sj.onc.1206828 Keywords: caspases; Fas; Fas ligand; caspase-8; hormo- nal carcinogenesis; ovarian neoplasm Introduction Ovarian cancer (OC), primarily epithelial OC, remains the most deadly gynecologic malignancy in the Western world. The incidence of this disease rises after women reach menopause and may be related to declines in specific reproductive hormones such as progesterone (P4) (Cramer et al., 1983; Riman et al., 1998). Conversely, increased exposure to pregnancy levels of progesterone appears to offer protection, since OC risk is reduced in women with higher numbers of births or a history of twin pregnancies (which is associated with higher P4 levels) (Adami et al., 1994, 1995; Lambe et al., 1999). The protective effect of pregnancy may be attributable to removal of genetically damaged cells from the ovarian surface epithelium (OSE) via P4- induced apoptosis. Consistent with this theory, P4 or other progestins have been shown to inhibit cell growth and/or induce apoptosis in normal and malignant human ovarian surface epithelial (HOSE) cells in vitro and in vivo (Bu et al., 1997; Hu and Deng, 2000; Syed et al., 2001; Yu et al., 2001; Rodriguez et al., 2002). Apoptosis is a well-orchestrated, autonomous mode of cell death that eliminates superfluous, damaged, mutated, or aged cells (Zimmermann et al., 2001). The process is mediated by the action of a class of enzymes known as caspases ( cysteine aspartate-specific prote ases) (Alnemri et al., 1996; Earnshaw et al., 1999; Zou et al., 1999) that may be classified broadly into apoptotic initiators (caspase-2, -8, -9, and -10) and executioners (e.g., caspase-3, -6, and -7). The initiators are respon- sible for activating the executioners, which ultimately dismantle cells by cleaving key cellular and nuclear proteins. Two major apoptotic pathways utilizing different initiator caspases have been identified. One involves perturbation of the mitochondria, release of mitochondrial cytochrome C to the cytosol, formation of the Apaf-1 and caspase-9 complex, and subsequent activation of the enzyme and its downstream execu- tioner caspases (caspase-3, -6, and -7) (Zou et al., 1999). The other is set off by the binding of specific ligands to cell surface ‘death receptors’ of the tumor necrosis factor (TNF)-receptor family (Ashkenazi and Dixit, 1998). Such an interaction results in the conversion of initiator procaspases into active enzymes, primarily caspase-8 and -10, and subsequent activation of executioner caspases. Among the TNF-receptor family, Fas (APO-1/CD95) is Received 14 February 2003; revised 13 May 2003; accepted 30 May 2003 *Correspondence: S-M Ho, Department of Surgery, Rm 504, University of Massachusetts Medical School, Lazare Research Building, 364 Plantation Street, 5th Floor, Worcester, MA 01605, USA; E-mail: Shuk-mei.Ho@umassmed.edu Oncogene (2003) 22, 6883–6890 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc