COMPARATIVE STUDY ON THE INDUCTION OF CYTOSTASIS AND APOPTOSIS BY ICI 182,780 AND TAMOXIFEN IN AN ESTROGEN RECEPTOR-NEGATIVE OVARIAN CANCER CELL LINE Alfredo ERCOLI 1 , Giovanni SCAMBIA 1 , Andrea FATTOROSSI 1 , Giuseppina RASPAGLIO 1 , Alessandra BATTAGLIA 1 , Lucia CICCHILLITTI 1 , Walter MALORNI 2 , Gabriella RAINALDI 2 , Pierluigi BENEDETTI PANICI 1 and Salvatore MANCUSO 1 * 1 Laboratory of Antineoplastic Pharmacology, Zeneca and Department of Gynecology, Catholic University, Rome, Italy 2 Laboratory of Ultrastructures, Istituto Superiore di Sanita ` , Rome, Italy W e have compared the effects of a broad range of clinically relevant concentrations (0.1 to 10 M) of the steroidal pure anti-estrogen ICI 182,780 and the non-steroidal partial anti- estrogen tamoxifen (T AM) on cell proliferation and induction of apoptosis in the estrogen receptor (ER)-negative ovarian carcinoma cell line A2780. Cell proliferation was assessed by evaluating the number of viable cells, changes in cell-cycle distribution and cell replication rate; while apoptosis induc- tion wasassessed by examining nuclear morphological changes associated with apoptotic death and DNA cleavage into 300 and 50 kbp units (large DN A fragmentation) and into 180 bp units (internucleosomal DN A fragmentation). W e provide evidence that 0.1 to 10 M ICI 182,780 and TAM significantly inhibit the growth of A2780 cellsin a dose-dependent fashion. Cytokinetic analysis revealed that only 10 M TAM caused a significant blockade in G 1 and a diminished replication rate. Conversely, we show that 0.1 to 10 M ICI 182,780 and TAM induce apoptosis in a dose-dependent fashion. T he earliest recognizable apoptotic change induced by treating the cells with these 2 drugs was DNA cleavage into 300 and 50 kbp units. T hisstarted to be visible in adherent cells, implying that apoptosis induction by ICI 182,780 and TAM was not deter- mined by the loss of cell–substrate interaction. A further degradation of 300 and 50 kbp DN A fragments occurred in cells that had lost their adhesion to the culture plate. W e observed the ladder pattern typical of internucleosomal DN A cleavage by treating A2780 cells with the highest dose (10 M) of ICI 182,780 and T AM. Lower concentrationsof these 2 drugs (0.1 to 1 M) did not produce such a pattern of DNA fragmentation. Typical features of apoptotic nuclei were detectable after both drug treatments. H owever, cellsunder- going apoptosis induced by ICI 182,780 showed hyper- aggregation of chromatin, whereasTAM-treated cellsprefer- entially exhibited chromatin clumping. Int. J. Cancer 76:47–54, 1998.  1998 Wiley-Liss, Inc. The non-steroidal drug tamoxifen (TAM) is a partial-agonist anti-estrogen widely used as an anti-cancer agent in the treatment of estrogen-dependent malignancies (Buckley and Goa, 1989) by virtue of its blocking of the estrogen receptor (ER)-mediated proliferative effect. An important additional feature of TAM is its effectiveness in the treatment of estrogen-independent neoplasia, such as ER - breast cancer (Jaiyesimi et al., 1995) and ovarian cancer (Markman et al., 1996), although the mechanisms underly- ing the anti-proliferative action of TAM in these tumors are not completely clarified. The clinical usefulness of TAM, however, is debated mainly because its partial-agonist nature induces both agonistic and antagonistic estrogenic effects, depending on the target tissue and the schedule of exposure (Wakeling, 1993). With this scenario, some new pure anti-estrogens with a higher affinity for the ER were synthesized with the belief that they may offer a practical advantage over partial-agonist anti-estrogens in the treat- ment of estrogen-dependent tumors by providing a more efficient block of the ER-mediated proliferative effect (Wakeling and Bowler, 1988; Wakeling, 1993). The steroidal drug ICI 182,780 was selected as the pure anti-estrogen of choice for clinical trials on the basis of its pharmacodynamic and pharmacokinetic properties (Wakeling et al., 1991). So far, the effectiveness of ICI 182,780 has been investigated in the endocrine therapy of ER + breast cancer with favorable preliminary results (DeFriend et al., 1994; Howell et al., 1996). We have demonstrated that micromolar concentra- tions of ICI 182,780 efficiently inhibit in vitro the growth of ER - MCF-7 ADRr breast cancer cells (De Vincenzo et al., 1996), although no definitive data explaining such an anti-proliferative activity of ICI 182,780 on ER-negative cancer cells were as yet available. In the present work, we compared the effects of a broad range (0.1 to 10 μM) of clinically achievable concentrations of ICI 182,780 (DeFriend et al., 1994) and TAM (Trump et al., 1992) on cell proliferation and induction of apoptosis in an ER - experimen- tal model. An ER - human ovarian cancer cell line, A2780, was selected as a suitable model to use in this investigation since these cells carry wild-type p53 genes (Eliopoulos et al., 1995) and a mutant form would prevent the major physiological roles of this protein, namely, G 1 arrest and/or apoptosis induction (Ko and Prives, 1996). The activity of ICI 182,780 and TAM was examined in time-course experiments. Cell proliferation was assessed by evalu- ating the number of viable cells, changes in cell-cycle distribution and cell replication rate. Apoptosis induction was assessed by examining nuclear morphological changes associated with apop- totic death and well-characterized biochemical markers of apopto- sis such as DNA cleavage into 300 and 50 kbp units (large DNA fragmentation) and into 180 bp units (internucleosomal DNA fragmentation) (Bortner et al., 1995). Because loss of adhesion to the culture dishes of tumoral epithelial cells has been described as an apoptosis-related event (Oberhammer et al., 1993; Desjardins and MacManus, 1995), we sought to further our knowledge on the biochemical and morphological apoptotic changes by examining floating and adherent cells separately. MATERIAL AND METHODS Drugs and chemicals ICI 182,780 and TAM were provided by Zeneca (Milan, Italy) as dry powders. ICI 182,780 and TAM stock solutions were prepared in absolute ethanol. Cell cultures The ER - human ovarian carcinoma cell line A2780 was kindly provided by Dr. R.F. Ozols (NCI, Bethesda, MD). Cells were maintained in RPMI 1640 medium supplemented with 10% FCS (Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/ml antibiotics and 0.3 μg/ml glutamine (complete medium). Cells were incubated at 37°C under 95% air/5% CO 2 in a high-humidity atmosphere. Cell viability was assessed routinely using the Trypan blue exclusion assay. *Correspondence to: Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Largo A. Gemelli, 8. I-00168, Rome, Italy. E-mail: Gemelli@cdq.it Received 5 May 1997; Revised 14 November, 1997 Int. J. Cancer: 76, 47–54 (1998)  1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer