CELL CYCLE ACTIVATION BY c-myc IN A BURKITT LYMPHOMA MODEL CELL LINE Alexander PAJIC 1 , Dimitry SPITKOVSKY 3 , Barbara CHRISTOPH 1 , Bettina KEMPKES 1 , Marino SCHUHMACHER 1 , Martin S. STAEGE 1 , Markus BRIELMEIER 1 , Joachim ELLWART 2 , Franz KOHLHUBER 1 , Georg W. BORNKAMM 1 , Axel POLACK 1 and Dirk EICK 1 * 1 Institute for Clinical Molecular Biology and Tumor Genetics, GSF-Research Center for Environment and Health, Munich, Germany 2 Institute for Experimental Haematology, GSF-Research Center for Environment and Health, Munich, Germany 3 Institute for Applied Tumor Virology, German Center for Cancer Research (DKFZ), Heidelberg, Germany The product of the proto-oncogene c-myc (myc) is a potent activator of cell proliferation. In Burkitt lymphoma (BL), a human B-cell tumor, myc is consistently found to be tran- scriptionally activated by chromosomal translocation. The mechanisms by which myc promotes cell cycle progression in B-cells is not known. As a model for myc activation in BL cells, we have established a human EBV-EBNA1 positive B-cell line, P493-6, in which myc is expressed under the control of a tetracycline regulated promoter. If the expression of myc is switched off, P493-6 cells arrest in G0/G1 in the presence of serum. Re-expression of myc activates the cell cycle without inducing apoptosis. myc triggers the expression of cyclin D2, cyclin E and Cdk4, followed by the activation of cyclin E-as- sociated kinase and hyper-phosphorylation of Rb. The tran- scription factor E2F-1 is expressed in proliferating and ar- rested cells at constant levels. The Cdk inhibitors p16, p21, p27 and p57 are expressed at low or not detectable levels in proliferating cells and are not induced after repression of myc. Ectopic expression of p16 inhibits cell cycle progression. These data suggest that myc triggers proliferation of P493-6 cells by promoting the expression of a set of cell cycle acti- vators but not by inactivating cell cycle inhibitors. Int. J. Can- cer 87:787–793, 2000. © 2000 Wiley-Liss, Inc. The proto-oncogene c-myc (myc) plays a key role in the regu- lation of cell proliferation, differentiation and apoptosis (Henriks- son and Lu ¨scher, 1996). myc is activated in response to mitogenic factors and repressed after exposure to anti-proliferative signals. Expression of myc is sufficient to induce proliferation in quiescent mouse fibroblasts. In the absence of survival factors, myc triggers apoptosis (Evan et al., 1992). Constitutive expression of myc as a consequence of genetic alterations contributes to a number of human and animal neoplasias. In Burkitt lymphoma, a human B-cell tumor, myc is chromosomally translocated to immunoglob- ulin gene enhancers and thus transcriptionally activated (for a review, see Spencer and Groudine, 1991). The c-Myc protein (Myc) is a transcription factor that belongs to the common class of basic domain-helix/loop/helix-leucine zipper proteins. Sequence specific DNA binding and transcriptional activation by Myc is dependent on the heterodimerization with the partner protein Max (for a review, see Amati et al., 1998). Several target genes of Myc have been identified (for a review, see Grandoric and Eisenman, 1997; Cole and McMahon, 1999) that are involved in DNA syn- thesis (ODC, CAD) (Bello-Fernandez et al., 1993; Miltenberger et al., 1995), protein synthesis (eIF4E) (Rosenwald et al., 1993; Jones et al., 1996), glucose metabolism (LDH-A) (Shim et al., 1997), iron regulation (IRP-2) (Wu et al., 1999), telomerase main- tenance (hEST2) (Wang et al., 1998), cell cycle progression (cdc25A and cyclin D2) (Galaktionov et al., 1996; Bouchard et al., 1999) and apoptosis (p53) (Reisman et al., 1993; Hermeking and Eick, 1994). Altogether, these data point to Myc as a key inductor of basal proliferative factors. The functions of Myc in cell cycle activation have been studied mainly in rodent fibroblast cell lines in which myc is expressed either constitutively or as a conditionally active chimeric protein (MycER) that results from a genetic fusion to the hormone binding domain of the estrogen receptor (Eilers et al., 1989). MycER induces cell transformation, gene activation and cell cycle progres- sion dependent on the binding of ER ligands. Taking advantage of these systems, Myc has been demonstrated to activate the cyclin E/Cdk2 kinase complex promoting the G1/S-progression in the cell cycle (Steiner et al., 1995; Berns et al., 1997; Prall et al., 1998; for a review see: Amati et al., 1998). This involves at least 3 distinct mechanisms, namely, the inactivation of the Cdk inhibitors p27 (Steiner et al., 1995; Vlach et al., 1996; Pe ´rez-Roger et al., 1997, 1999; Bouchard et al, 1999) and p21 (Hermeking et al., 1995), the induction of D cyclins of cyclin E (Jansen-Du ¨rr et al., 1993; Pe ´rez-Roger et al., 1997, 1999; Bouchard et al., 1999) and the induction of the phosphatase cdc25A, which dephosphorylates Cdk inhibitory sites (Galaktionov et al., 1996). With respect to cyclin E, different mechanisms are discussed by which the protein levels are increased, among them being direct transcriptional ac- tivation by Myc (Pe ´rez-Roger et al., 1997). It is not fully resolved whether expression of myc renders cell cycle progression in fibro- blasts independent of the functions of Cdk4 (Alevizopoulos et al., 1997; Berns et al., 1997; Haas et al., 1997; Pusch et al., 1997; Prall et al., 1998; for a review, see Obaya et al., 1999). This kinase participates in the phosphorylation of Rb and other pocket pro- teins, and is involved in the sequestration of Cdk inhibitors (for a review, see Morgan, 1997). As a model for cell cycle activation by myc in BL cells, we have established the B-cell line P493-6 carrying a conditional, tetracy- cline-regulated myc. For this cell line we could recently show that Myc induces cell growth (increase of cell mass) in the absence of serum without activating the cell cycle (Schuhmacher et al., 1999). Here we have addressed the question how induction of myc affects cell cycle regulating proteins in the presence of serum. MATERIAL AND METHODS DNA constructs The construction of pmyc-tet (BC266) was carried out as fol- lows: The 1924 bp XhoI-HpaI fragment of plasmid pUHD15-1, which carries the expression cassette for tTa under the control of the cytomegalovirus (CMV) promoter (Gossen and Bujard, 1992), was cloned into the SalI and NaeI sites of vector pHEBOPL (Polack et al., 1991). The resulting vector was termed BC89. TP (or LMP2) is the gene for the terminal protein of EBV. The promoter TP-tetO7 was constructed by replacement of the EBNA2 response element (Zimber-Strobl et al., 1993) in the TP-promoter (NruI-BclI fragment) by tetO7 (SmaI-SmaI fragment of pUHC13-3) (Gossen and Bujard, 1992). The promoter was in- Grant sponsor: Deutsche Forschungsgemeinschaft; Grant sponsor: Deut- sche Krebshilfe; Grant sponsor: Fonds der Chemischen Industrie; Grant sponsor: BMBF. *Correspondence to: Dirk Eick, GSF-Research Center for Environment and Health, Marchioninistrasse 25, D-81377 Munich, Germany. Fax: 49 89 7099500. E-mail: eick@gsf.de Received 17 February 2000; Revised 3 April 2000; Accepted 4 April 2000 Int. J. Cancer: 87, 787–793 (2000) © 2000 Wiley-Liss, Inc. Publication of the International Union Against Cancer