Reconstitution of IL6-Inducible Differentiation of a Myeloid Leukemia Cell Line by Activated Stat Factors Roland Piekorz,* Beate Schlierf,* Renate Burger,† and Gertrud M. Hocke* ,1 *Institute for Microbiology, Biochemistry, and Genetics; and †Department of Medicine III, Division of Hematology/Oncology, University of Erlangen-Nuernberg, Staudtstrasse 5, D-91058 Erlangen, Germany Received August 6, 1998 Differentiation of the myeloid leukemia cell line M1 by treatment with IL6-type cytokines depends on ac- tivation of the Jak/Stat (Janus kinase/signal trans- ducer and activator of transcription) pathway. Defects in this cascade are correlated with an impaired cytokine-inducible differentiation of various other myeloid cell lines. Although treatment with IL-6 in- creased the amount of activated transcription factor Stat3 in the myeloid leukemia line C, differentiation was not induced. However, after cotransfection with expression constructs for the tyrosine kinase Jak2 and Stat factors 3 or 5a, treatment of the cells with IL-6 caused a decrease in the number of viable cells. In parallel, an increase in the percentage of differenti- ated cells occurred. These findings are consistent with the hypothesis that the Jak/Stat signaling cascade plays an important role in cytokine-induced differen- tiation of myeloid leukemia cells. © 1998 Academic Press Myeloid leukemia cells are blocked at an early stage during differentiation and proliferate aberrantly. In culture, some of these cell lines undergo terminal dif- ferentiation by treatment with various agents includ- ing retinoic acid, dimethyl sulfoxide, lipopolysaccha- ride (LPS), phorbol ester (TPA) or cytokines (1, 2). Some of these cytokines are involved in normal hema- topoiesis, e.g. members of the interleukin-6 (IL-6)-type family. Cytokines of this family, leukemia inhibitory factor (LIF) and IL-6, induce differentiation of the mu- rine myeloid leukemia line M1. This effect is mediated by the Jak (Janus kinase)/Stat (signal transducer and activator of transcription) signaling cascade and is de- pendent on the activated transcription factor Stat3 (3, 4). The Jak/Stat signaling cascade is triggered by bind- ing of the cytokine to its specific receptor ligand- binding chain. This leads to the association of the re- ceptor cytokine complex with the common signal transducer chain gp130. Thereby associated Jak ki- nases are activated and phosphorylate Stat factors at a conserved tyrosine residue. Subsequently, Stat factors dimerize, translocate to the nucleus, bind to specific sequences in the promoter region of target genes and modulate their transcriptional activity (5, 6). Treat- ment of M1 cells with LIF or IL-6 causes activation of Stat1 and Stat3 (7) and, additionally, of Stat5a (8-10). Whereas the importance of Stat3 in induction of differ- entiation was clearly demonstrated and an involve- ment of Stat1 was excluded (3, 4), a role for Stat5a has not been demonstrated so far. Stat5a, sharing a 96% homology with Stat5b, was originally identified as mammary gland factor (MGF) (11). Besides its regulation by prolactin, Stat5a is ac- tivated by multiple cytokines including IL-2, IL-3, IL-5, IL-6, erythropoietin, granulocyte-macrophage colony stimulating factor and thrombopoietin. Activation of Stat5 in various cell types is linked with processes of both, differentiation and proliferation (12). Recent findings implicate that in addition to Stat3 activation of Stat5a might be necessary to induce differentiation of myeloid leukemia cells (10). A study, in which various myeloid leukemia lines was investigated, revealed that none of these lines responded with induction of differentiation to treat- ment with IL-6 or LIF. This was due to multiple defects in the Jak/Stat signaling cascade (10). If the differen- tiation potential of myeloid leukemia cells depends on an intact Jak/Stat signaling cascade, reconstitution of the missing parts should render the cells responsive to cytokine-inducible differentiation. One of the cytokine- unresponsive cell lines, the murine myeloblastic leuke- mia line C, was chosen to restore the cytokine re- 1 Corresponding author. Fax: +49-9131-85 85 26. E-mail: ghocke@ biologie.uni-erlangen.de. Abbreviations:  2 M,  2 -macroglobulin; HA, hemagglutinin epitope; HPRT, hypoxanthine phosphoribosyl transferase; IL-6, interleu- kin-6; Jak, Janus kinase; LIF, leukemia inhibitory factor; LPS, li- popolysacharide; MGF, mammary gland factor; RE, response ele- ment; RT-PCR, reverse transcriptase-polymerase chain reaction; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; Stat, signal transducer and activator of transcription; TPA, 12-0-tetradecanoyl- phorbol 13-acetate. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 250, 436 – 443 (1998) ARTICLE NO. RC989335 436 0006-291X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.