Mcl-1, an early-induction molecule, modulates activin A-induced apoptosis and dierentiation of CML cells Yumi Fukuchi 1 , Masahiro Kizaki* ,1 , Kenji Yamato 2 , Chiharu Kawamura 1 , Akihiro Umezawa 1 , Jun-ichi Hata 1 , Tatsuji Nishihara 3 and Yasuo Ikeda 1 1 Division of Hematology, Department of Internal Medicine and Pathology, Keio University School of Medicine, Tokyo 160-8582, Japan; 2 Section of Molecular Cellular Oncology/Microbiology, Department of Oral Function, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo 113-8519, Japan; 3 Department of Oral Microbiology, Kyushu Dental College, Kitakyushu 803-8580, Japan Activin A, one member of the transforming growth factor (TGF)-b superfamily, is known to be a commitment factor for cell death and dierentiation. In the present study, we demonstrate that human chronic myeloid leukemia (CML) cell lines, KU812 and K562 cells, either induced apoptosis or dierentiation, respectively, by treatment with activin A. During these cell fate decisive events caused by activin A, rapid and transient up-regulation of Mcl-1 was observed in both cell lines. In activin A-induced apoptosis of KU812 cells, continuous up-regulation of Bax was observed. After the decrease in Mcl-1 expression had occurred, activation of caspase-9 and caspase-3 and cleavage of DFF45 were shown to take place in KU812 cells, resulting in the fragmentation of the genomic DNA of the cells. In contrast, the down-regulation of Mcl-1 without up-regulation of Bax caused accumulation of hemoglobin (Hb) contents in activin A-treated K562 cells. Interestingly, erythropoietin (EPO) prevented activin A- induced apoptosis with continuous expression of Mcl-1 and caused KU812 cells to undergo erythroid dierentia- tion. To address the role of Mcl-1 in activin A-treated CML cells, KU812 and K562 cells were stably transfected with cDNA encoding Mcl-1 (designated as KU812/mcl and K562/mcl cells). As in combined eect of activin A and EPO on the parental KU812 cells, activin A induced dierentiation, but not apoptosis, of KU812/mcl cells without modulating Bax levels. Activin A-treated K562/ mcl cells, as well as parental cells, were only dierentiated to erythroid cells. These results suggest that Mcl-1 is an early inducible gene activated by the activin A signaling pathway for both cellular dierentiation and apoptosis, and continuous expression of Mcl-1 may be contributed to dierentiation signals to the erythroid lineage in CML cells. Oncogene (2001) 20, 704 – 713. Keywords: activin A; CML; apoptosis; dierentiation; Mcl-1 Introduction Activin A is highly homologous with transforming growth factor (TGF)-b and consists of two bA subunits (Vale et al., 1986; Ling et al., 1986). It was initially recognized as a gonadal protein that stimulates the release of follicular-stimulating hormone (FSH) from the pituitary gland (Vale et al., 1986; Ling et al., 1986). Several studies have shown that activin A carries out a variety of functions including regulation of hematopoiesis (Eto et al., 1987; Yu et al., 1989; Yamashita et al., 1991). Activin A induces dierentia- tion of murine Friend leukemia cell line and human leukemia cell line K562 cells into hemoglobin (Hb)- containing cells (Murata et al., 1988; Miyamoto et al., 1990); it also modulates dierentiation and prolifera- tion of immature erythroid progenitor cells (Broxmeyer et al., 1988; Shao et al., 1992; Mizuguchi et al., 1993). We have recently isolated an inhibitory factor for mouse B cell hybridoma cells from the culture media of activated mouse macrophages and identified this molecule as being identical to activin A (Nishihara et al., 1993). In addition, we have found that activin A induces cell cycle arrest during the G1 phase. More- over, it induces apoptosis in mouse plasmacytic cells and human myeloma cells (Koseki et al., 1995; Yamato et al., 1996, 1997). It has been shown that activin A might act as a commitment factor for inducing cellular apoptosis and dierentiation as a manner of induction or suppression of gene expression (Shiozaki et al., 1998). However, the mechanism of growth inhibitory eects of activin A in hematopoietic cells has not been elucidated. The Bcl-2 family of proteins are key regulators of apoptosis in many cell types. This family of proteins consists of pro-apoptotic (Bax, Bad, and Bak) and anti-apoptotic (Bcl-2, Bcl-X L , and Mcl-1) subfamilies (Yang and Korsmeyer, 1996; Kroemer, 1997; Gross et al., 1999). Pro- and anti-apoptotic family members can heterodimerize with each other and the balance of these members may act as a rheostar for the suicide program that determines the activities of cell fate (Peters et al., 1998; Moulding et al., 1998; Shimizu et al., 2000; Kroemer and Reed, 2000). Mcl-1, one of the Oncogene (2001) 20, 704 – 713 ª 2001 Nature Publishing Group All rights reserved 0950 – 9232/01 $15.00 www.nature.com/onc *Correspondence: M Kizaki, Division of Hematology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Received 31 July 2000; revised 3 October 2000; accepted 29 November 2000