Notchic-ER chimeras display hormone-dependent ... - Nature

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Oncogene (2000) 19, 3914 ± 3924 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc

Notchic-ER chimeras display hormone-dependent transformation, nuclear accumulation, phosphorylation and CBF1 activation Chiara Ronchini1 and Anthony J Capobianco*,1 1

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio, OH 45267-0524, USA

Notch genes encode a family of evolutionarily conserved transmembrane receptors that are involved in many distinct cellular processes such as dierentiation, proliferation and apoptosis. Notch function has been shown to be required both during development and in adult life. Moreover, several studies on spontaneous human tumors and in experimental models demonstrate that three of the four mammalian Notch genes can act as oncogenes. The mechanism by which Notch proteins induce neoplastic transformation is not known. In order to determine the early signaling events mediated by Notch during cellular transformation we constructed several inducible alleles of Notchic by fusing portions of Nic to the hormone-binding domain of the estrogen receptor. Here we show that Notchic-ER chimeras are conditionally activated by 4Hydroxytamoxifen (OHT) in a dose-dependent manner. Clonal RKE cell lines expressing Notchic-ER chimeras display hormone-dependent transformation in vitro. Transformation mediated by Notchic-ER is reversible and chronic stimulation is necessary for the maintenance of the transformed phenotype. In response to hormone activation Notchic-ER chimeras become hyperphosphorylated and accumulate in the nucleus of the cell; indicating that both phosphorylation and nuclear localization are required for Notch transforming activity. Oncogene (2000) 19, 3914 ± 3924. Keywords: notch; hormone inducible activity; phosphorylation; CBF-1; nuclear localization Introduction Notch genes encode a family of evolutionarily conserved transmembrane receptors that are involved in cell fate determination during development. In general, it is believed that Notch signaling can in¯uence many distinct cellular processes such as dierentiation, proliferation and apoptosis (ArtavanisTsakonas et al., 1999; Miele and Osborne, 1999; Milner and Bigas, 1999). The mechanism of Notch signaling remains poorly understood. The prevailing model is that the ligands are presented to Notch via cell to cell contact (Artavanis-Tsakonas et al., 1995; Fortini et al., 1993; Jarriault et al., 1995; Kopan et al., 1994; Nye et al., 1994; Struhl and Adachi, 1998; Struhl et al., 1993). The engagement of ligand somehow induces proteolytic

*Correspondence: AJ Capobianco, Department of Molecular Genetics, University of Cincinnati, College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio, OH 45267-0524, USA Received 12 April 2000; revised 26 May 2000; accepted 1 June 2000

processing that releases the intracellular portion of the receptor (Nic) from the plasma membrane. Once free from the membrane, it is thought that Nic translocates to the nucleus where it interacts with eector molecules to alter gene expression (Ahmad et al., 1995; Fehon et al., 1991; Fortini et al., 1993; Jariault et al., 1995; Kidd et al., 1998; Kopan et al., 1996; Lieber et al., 1993; Schroeter et al., 1998; Struhl and Adachi, 1998; Zagouras et al., 1995). One such eector molecule is CBF1, a transcriptional regulatory protein that functions as a repressor in the absence of activated Notch. Upon activation of Notch and subsequent translocation, it is proposed that Nic associates with CBF1 and in turn activates genes containing CBF1 binding sites in their promoters (Fortini and Artavanis-Tsakonas, 1994; Hsieh et al., 1996; Jarriault et al., 1995; Kao et al., 1998; Oswald et al., 1998; Stifani et al., 1992; Tamura et al., 1995). Although certain aspects of this model are compelling, there is considerable debate on the physiological role of nuclear Notch proteins. Most of the data providing evidence for nuclear function comes from ectopic expression of engineered Nic proteins in both vertebrate and invertebrate cell culture systems (Capobianco et al., 1997; Fortini et al., 1993; Kidd et al., 1998; Kopan et al., 1996; Lecourtois and Schweisguth, 1998; Lieber et al., 1993; Rebay et al., 1993; Schroeter et al., 1998; Struhl and Adachi, 1998). However, a strict correlation of ligand-induced nuclear localization and Notch function remains elusive. There is now signi®cant evidence that Notch function is involved in tumorigenesis. A recurrent theme encountered among Notch proteins involved in tumorigenesis is that Notch proteins are truncated in a very similar fashion. The truncated proteins consist primarily of the intracellular portion of the protein and are not tethered to the plasma membrane. These forms of Notch are thought to be constitutively active. To date the only known example of genetic alterations in the Notch locus in a human cancer is a translocation in the T-cell receptor locus in T-cell acute lymphoblastic leukemia (T-ALL) (Ellisen et al., 1991). However, inappropriate expression of Notch1 and Notch2 has been observed in numerous human cancers of dierent origins (Aster et al., 1994; Daniel et al., 1997; Ellisen et al., 1991; Zagouras et al., 1995). Moreover, truncated forms of Notch1, Notch2 and Notch4/Int3 have been demonstrated to have transforming activity in several dierent systems (Dievart et al., 1999; Gallahan et al., 1987; Girard et al., 1996; Jhappan et al., 1992; Pear et al., 1996; Robbins et al., 1992; Rohn et al., 1996; Smith et al., 1995). Previously, we demonstrated that constitutive forms of Notch1 (N1ic) and Notch2 (N2ic) could transform an E1A immortalized baby rat kidney cell line (RKE) in

Inducible Notch activity C Ronchini and AJ Capobianco

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vitro. In addition, we demonstrated that Nic proteins could cooperate with the adenoviral protein E1A to transform primary baby rat kidney cells (BRK). Furthermore, cells transformed by Nic proteins form colonies in semi-solid media and are tumorigenic in nude mice (Capobianco et al., 1997). In order to study the initial events mediated by Notch in transformation we created conditional alleles of Notchic by constructing chimeras with the human estrogen receptor hormone binding domain (ER) and analysed their conditional activation in this cellular system. We demonstrate that Notchic-ER proteins display hormone-induced transformation that is dose-dependent in vitro. Moreover, we show that, following hormone induction, Notchic-ER accumulates in the nucleus and that the active proteins are phosphorylated. Activation, but not binding, of CBF1 by Notchic-ER is dose dependent. Finally, we report evidence that the transformed phenotype induced by Notch activation is reversible and that chronic stimulation of Notchic-ER molecules is required for maintenance of transformation.

Results Structure of Notchic-ER chimeric molecules and generation of cell lines expressing the fusion proteins In order to study the mechanism by which Notch transforms cells and to identify its role in early events of transformation, we created chimeric proteins that can be conditionally activated by estrogen-hormones (Figure 1a). These molecules were obtained by fusing amino acids 282 ± 595 of the human estrogen receptor, encoding the hormone binding domain (ER), with dierent alleles of the intracellular portion of the human Notch protein (Nic, amino acids 1759 ± 2556). ER-Nic encodes a chimeric protein with the ER hormone binding domain fused to the N-terminus of Nic. Nic-ER chimera encodes the ER sequences fused to the C-terminus of full-length Nic. Both of these constructs are estimated to express a protein of approximately 122 kDa. ER-NicD2202 encodes a chimera of approximately 83 kDa in which ER sequences are fused at the Nterminus of a Notchic deletion mutant, NicD2202. The Nic sequences expressed in this chimera are deleted of the C-terminal 354 amino acids, from amino acids 2202 to amino acids 2556 (Jeries and Capobianco, 2000). ERNic and ER-NicD2202 encode a C-terminal myc epitope tag to allow detection with 9E10. Constructs are all expressed from the viral LTR in the retroviral expression vector pBabe-puro. In addition, we created a vector that directs the expression of a C-terminal-myc tagged ER hormone binding domain under the control of the LTR (pBP283ER). This construct is used as a control for all experiments. Retroviral particles were produced for each plasmid by transfection into a retroviral packaging cell line. In order to obtain stable cell lines expressing Notchic-ER chimeras, RKE were infected with supernatants containing retroviral particles and selected in puromycin. Isolated puromycin-resistant colonies were picked and propagated in culture. Expression of the fusion proteins was determined by Western blot analysis after immunoprecipitation (IP)

Figure 1 Structure and expression of Notchic-ER chimeric proteins. (a) Amino acids 282 ± 595 of the human estrogen receptor encoding the hormone binding domain (ER) are fused N-terminally to Nic protein (ER-Nic) and to the deletion mutant NotchicD2202 (ER-D2202). ER-Nic and ER-D2202 encode a Cterminal myc epitope tag (m). Nic-ER encodes a chimeric protein in which the ER domain is C-terminal to Nic and it does not encode a myc epitope tag. The predicted size of the chimeric constructs is indicated in kDa on the right. RAM, CBF1 binding domain; NLS, nuclear localization signal; ANK, ankyrin-like repeats; OPA, glutamine-rich region; PEST, region rich in proline, glutamate, serine and threonine. (b) Expression of Notchic-ER chimeras. Lysates from RKE clonal lines were analysed by Western blotting following immunoprecipitation of Notchic-ER proteins. Proteins of the expected size were detected in samples immunoprecipitated with the polyclonal anti-Notch antiserum 925 (IP) (Nic-ER and ER-D2202) or with anti-myc antibody 9E10 (ER-Nic), but not in control samples incubated with pre-immune serum (C). ER-Nic and Nic-ER proteins were detected with antiTAN1 antibody 15A, ER-D2202 was detected with anti-myc antibody 9E10

of the chimeric proteins with polyclonal anti-Notch 925 antiserum (Nic-ER and ER-NicD2202) or anti-myc antibody 9E10 (ER-Nic). IP of the proteins was necessary because the level of expression of our constructs was too low to allow detection from a whole cell lysate of the cells. As shown in Figure 1b, all constructs expressed proteins of the predicted size: a band of about 122 kDa was consistently detected for ER-Nic and Nic-ER proteins and a band of about 83 kDa for ER-NicD2202. The level of expression was similar for every clone and the IP was speci®c, as demonstrated by the failure to detect Notchic-ER proteins from lysates IP with the pre-immune serum as a control. Cell transformation by Notchic-ER chimeras in vitro is hormone-dependent Previously, we demonstrated that Nic proteins could transform RKE cells in culture. RKE cells transformed by Nic were able to form colonies in semisolid media and were tumorigenic in nude mice (Capobianco et al., 1997). To determine if clonal Oncogene


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RKE cell lines expressing Notchic-ER chimeras were transformed we tested them for the ability to grow in semi-solid media. In order to demonstrate that this transforming activity was hormone-dependent, this assay was performed in the presence and absence of 4-hydroxytamoxifen (OHT). Clonal RKE lines expressing ER-Nic and Nic-ER chimeric molecules readily formed colonies in the presence of OHT (Figure 2a). Approximately 5% of ER-Nic and 4% of Nic-ER expressing RKE cells seeded into soft agar were able to form colonies (Figure 2a). In contrast, no colonies were formed in the absence of hormone (Figure 2a,b). Clonal lines expressing the deletion mutant ER-NicD2202 displayed a decreased ability to form colonies in semi-solid media, resulting in an eciency of colony formation of approximately 1%. Moreover, the colonies formed by ER-NicD2202 were much smaller than those obtained from clones expressing ER-Nic or Nic-ER (Figure 2b). No colonies were formed by cell lines expressing the ER hormone binding domain either in presence or in absence of OHT (Figure 2a,b). This result demonstrates that the clonal RKE cell lines expressing Notchic-ER chimeras are transforming and that transformation is speci®cally induced in a hormone-dependent manner. Transformation by Notchic-ER proteins is dose dependent In order to determine if Notchic-ER transformation activity was dependent on hormone dose, we seeded cells into semi solid media containing OHT at concentrations ranging from 1 nM to 1 mM. Nic-ER expressing cells seeded in 1 mM OHT displayed growth characteristics as shown in Figure 2, resulting in approximately 3% of the cells forming colonies in soft agar (Figure 3a). In contrast, when the concentration of OHT is decreased to 0.1 and 0.01 mM OHT there is a decrease in the eciency of colony formation (1.9 and 0.7% respectively, Figure 3a). No colonies were formed either in the presence of 0.001 mM OHT or in the absence of the hormone. Concomitant with a decrease in the eciency of colony formation, the size of the colonies was also much smaller with lower doses of OHT (Figure 3b). ER-NicD2202 cells displayed a weaker transforming activity. However, this activity was still dose-dependent. The percentage of colonies formed in 1 mM OHT was approximately 1.1%. This is consistent to the experiment shown in Figure 2a. In lower concentrations of OHT ER-NicD2202 cells showed a similar decrease in the eciency of colony formation, approximately 0.7% in 0.1 mM and 0.4% in 0.01 mM OHT. No colonies were observed in plates of cells treated with 0.001 mM OHT or in the absence of hormone (data not shown). Colony size was also eected by the dose of OHT. In concentrations of OHT above 0.01 mM colonies were detectable and stained positive for the Thiazolyl blue dye (MTT). Staining with MTT indicates that cells are living. In contrast, there were no MTT stained cells in concentrations below 0.01 mM (Figure 3b). These results demonstrated that Notchic-ER clonal lines are transforming in vitro following speci®c activation. This activation is proportional to the dose of hormone used for induction of the cells.

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Figure 2 Hormone dependent transformation by Notchic-ER proteins. (a) Clonal RKE cell lines seeded into soft agar were maintained in culture in presence of 1 mM OHT (+OHT) or in absence of hormone (7OHT) for 4 weeks. Plates were stained with MTT and scanned with UMAX Power Look II scanner. ER, clonal RKE cell line expressing the ER hormone binding domain; Nic-ER, clonal RKE cell line expressing the Nic-ER chimeric protein; ER-Nic, clonal RKE cell line expressing ER-Nic chimera; ER-D2202, clonal RKE cell line expressing ER-NicD2202 fusion protein. (b) Photomicrographs of colonies shown in (a) before staining with MTT. Photographs were taken at 1006 with a Zeiss IM35 inverted microscope. Clonal RKE cell lines and treatment with hormone are as described for (a)


Figure 3 Dose dependent transformation by Notchic-ER chimeric proteins. (a) Clonal RKE cell line expressing the Nic-ER chimera seeded in soft agar in presence of dierent concentrations of OHT, indicated under each plate. Plates were stained with MTT after 3 weeks and scanned by UMAX Power Look II scanner. (b) Photographs of colonies grown in soft agar at dierent concentrations of OHT ([OHT]), after staining with MTT. Photomicrographs were taken as described for Figure 2b. Nic-ER, as for (a); ER-D2202, clonal RKE cell line expressing ER-NicD2202

Activation but not binding of CBF1 by Notchic-ER chimeric proteins is hormone-dependent To determine if Notchic-ER chimeras can induce CBF1 activity in a hormone dependent manner we performed luciferase reporter assays in Hela cells. Hela cells were cotransfected with a luciferase reporter plasmid containing eight CBF1 binding sites upstream of a minimal

SV40 promoter (86CBF1) and various LTR-driven expression plasmids. After transfection the cells were incubated for 48 h in presence or absence of 1 mM OHT. RL-Tk was included in each transfection as control for transfection eciency. In presence of the hormone, transfection of the plasmid expressing ERNic chimera resulted in a 31-fold increase in luciferase activity compared to the background activity observed for control vector pBP283ER (Figure 4a). In contrast, the luciferase activity was only fourfold higher than that observed with ER alone in the absence of OHT. In cells transfected with a plasmid encoding Nic luciferase activity was not hormone dependent. In the absence of hormone, luciferase activity was induced approximately 90-fold, whereas in the absence of OHT the activity was approximately 75-fold compared to the activity detected for pBP283ER vector (Figure 4a). The result of this luciferase assay demonstrates that CBF1 is activated by Notchic-ER chimeras in a hormone dependent manner and that this hormone dependence is a speci®c and intrinsic characteristic of our chimeric constructs. We demonstrated by growth in soft agar that transformation mediated by Notchic-ER molecules is dependent on hormone dose. Therefore, the level of Notchic activity can be modulated in Notchic-ER chimeras by changing the concentration of hormone. To determine if CBF1 activation by Notchic-ER chimeric proteins can be modulated in a dosedependent manner, we performed luciferase assays in Hela cells treated for 48 h with various concentrations of OHT ranging from 0.001 mM to 1.0 mM (Figure 4b). Expression of ER-Nic and Nic-ER resulted in similar levels of luciferase activity from the CBF1 reporter at each concentration of OHT. At the highest concentration of OHT tested (1.0 mM) we observed that Nic-ER proteins induce CBF1 activity approximately 62.5-fold better than the ER domain alone. Expression of ERNicD2202 resulted in a lower level of CBF1 activation, approximately 32-fold better than ER at 1.0 mM (Figure 4b). Luciferase activities for all of the Notchic-ER chimeras displayed a dose dependence down to 0.001 mM OHT. At the lowest concentration of OHT tested (0.001 mM), Nic-ER and ER-Nic activated the CBF1 reporter 9.8- and sevenfold respectively. In contrast, at 0.001 mM OHT ER-NicD2202 failed to activate the CBF1 reporter above background level (absence of OHT). The luciferase activity determined for ER-NicD2202 at each concentration was about twofold lower than that observed for the two constructs expressing chimeric proteins containing Nic. These results demonstrate a speci®c dose dependent activation of CBF1 by Notchic-ER chimeras. Since the mechanism of gene transcription mediated by Nic protein through CBF1 appears to require a direct binding of Nic to CBF1, we tested the ability of Notchic-ER chimeras to interact with CBF1 by GST pull-down assays. Whole cell lysates were prepared from RKE cell lines expressing ER-Nic proteins after incubation overnight in the presence or absence of 1 mM OHT. Cell lysates were split into three equal aliquots. From one of the aliquots we immunoprecipitated Notchic-ER proteins with the anti-Notch antisera 925. The other two aliquots were incubated with either GST or GST-CBF1. Immunoprecipitates were collected by incubation with protein A-sepharose and

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GST complexes were collected by reduced glutathioneagarose. Samples were then separated by PAGE and transferred to nitrocellulose. Notch proteins were detected by immunoblotting with 9E10. ER-Nic protein was speci®cally detected only in the samples incubated with GST-CBF1 or IP with anti-Notch 925, no Notchic-ER proteins were detected in samples incubated with GST (Figure 4c). We observed that the amount of ER-Nic protein detected in samples of cells treated with hormone was higher than in cells treated with ETOH vehicle (Figure 4c). This dierence could be due to a stabilization of ER-Nic induced by OHT. However, ER-Nic bound to GST-CBF1 in the presence and absence of OHT and the binding was proportional to the amount of protein present in the whole cell lysates. This result indicates that the ability of NotchicER to bind CBF1 is independent of hormone activation. Accumulation of Notchic-ER chimeras in the nucleus is hormone-dependent

C

Figure 4 Hormone dependent activation of CBF1 by NotchicER chimeras. (a) CBF1 activation was determined by Luciferase reporter assays in Hela cells. Hela cells were transfected with the indicated expression plasmid plus a luciferase reporter plasmid containing eight copies of a CBF1 consensus sequence upstream of a minimal SV40 promoter. Expression plasmids: pBP283ER expressing ER hormone binding domain (ER), vector expressing ER-Nic chimeric protein (ER-Nic) or vector expressing Notchic (Nic). A reporter vector containing the herpes simplex virus thymidine kinase promoter region upstream of Renilla luciferase cDNA was also included as an internal control for normalization. Luciferase expression was evaluated after 48 h of induction of cells with (+OHT) or without (7OHT) 1 mM OHT. Results are expressed as relative light units as described in Materials and methods. Data are average of four dierent replicates. (b) Activation of CBF1 is OHT dose dependent. Luciferase assays were conducted as described for (a). Luciferase activity was assayed after 48 h of treatment with indicated concentrations of OHT ([4-Hydroxytamoxifen]). ER, control vector pBP283ER; ER-D2202, vector expressing chimeric protein ER-NicD2202; ER-Nic, chimeric construct expressing ERNic fusion protein; Nic-ER, vector expressing Nic-ER chimera. Results are expressed as for (a). (c) CBF1 is able to bind ERNic in the presence or absence of OHT. ER-Nic protein was incubated with either GST-(GST) or GST-CBF1-(CBF) bound Glutathione agarose beads or immunoprecipitated with antiNotch 925 (IP) from a total cell lysate after incubation overnight with (+OHT) or without (7OHT) 1 mM OHT. ERNic protein was detected by Western blot analysis with 9E10 antibody Oncogene

The signal transduction pathway of Notch proteins remains uncertain. The prevailing model is that upon ligand binding Notch is released from the plasma membrane by proteolysis. The intracellular portion of Notch then translocated to the nucleus where it is involved in the regulation of gene expression. Previously we have shown that constitutively active Nic proteins are localized primarily in the nucleus of RKE cells (Capobianco et al., 1997). Therefore, we wanted to determine if nuclear localization is regulated by hormone in the Notchic-ER chimeras. Subcellular distribution of Notchic-ER proteins was determined by indirect immuno¯uorescence in clonal RKE cell lines expressing the various chimeric alleles. Cells were seeded onto glass microscope slides and treated overnight with 1 mM OHT. Cells stimulated overnight with ETOH served as the control. Cells were ®xed with paraformaldehyde and Notch proteins were detected with 9E10. In the absence of hormone, both ER-Nic and ER-NicD2202 chimeras display a diuse subcellular localization. In contrast, when cells are stimulated with OHT overnight these chimeric molecules appear to accumulate exclusively in the nucleus of the cell. However, the ER domain expressed alone displays a diuse subcellular distribution regardless of the presence of OHT (Figure 5a), indicating that the Notch sequences are required for the redistribution of the chimeric protein. Cells were also stained with DAPI to show the boundaries of the nucleus. In order to con®rm the results obtained by immuno¯uorescence we performed a Western blot analysis of proteins immunoprecipitated from subcellular fractions. Clonal RKE cell lines expressing ERNic and ER-NicD2202 were incubated overnight with or without 1 mM OHT. Cellular lysates were fractionated as described in Materials and methods. Notchic-ER proteins were IP from cytoplasmic and nuclear fractions with anti-Notch 925 and detected with antimyc 9E10 by immunoblotting. Consistent with the results obtained by immuno¯uorescence analysis, we detected an equal amount of protein in the cytoplasmic and nuclear fractions of cells that were not treated with OHT (Figure 5b). In contrast, the proportion of Notchic-ER protein IP from the nuclear fraction of


cells treated with OHT was signi®cantly higher than the protein IP from the cytoplasmic fraction (Figure 5b). This result was observed for both RKE cells expressing ER-Nic and ER-NicD2202 (Figure 5b). To insure that the antibody was present in excess during IP, supernatants were IP a second time with 925. From this analysis we saw that only nuclear fractions from cells treated with OHT contained any detectable Notchic-ER protein remaining (data not shown). As described for Figure 4c, the amount of protein immunoprecipitated from cells treated with OHT was greater than from cells not treated. Although the treatment with hormone resulted in an increase in protein level, the chimera was not equally distributed between the cytoplasmic and nuclear fractions, but it was exclusively localized into the nucleus of the cell. These results demonstrate that in cells treated with OHT Notchic-ER proteins preferentially accumulate in the nucleus, regardless of the quantity of protein.

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Notchic-ER proteins are phosphorylated following hormone activation Western blot analysis revealed a slower migrating form of ER-Nic chimera from cells that had been treated with OHT (Figure 5b). This mobility shift was detected for all three Notchic-ER chimeras. Our hypothesis was that phosphorylation of Notchic-ER chimeras might occur upon activation by hormone. In order to test this hypothesis, we treated RKE cell lines expressing Notchic-ER chimeras with or without 1 mM OHT overnight. Notchic-ER proteins were IP with antiNotch antiserum 925 from whole cell lysates. Samples were divided into three aliquots of equal volume. Two aliquots were incubated at 378C for 20 min with CIP either in the manufacturer's supplied buer or in Phosphate buered saline (PBS). PBS acts as a competitive inhibitor of CIP and should inactivate the enzyme. The third aliquot was incubated in buer without addition of CIP. After incubation with CIP, proteins were separated by SDS ± PAGE and NotchicER chimeras detected by immunoblotting with 9E10 or anti-Notch 15A. ER-Nic chimera IP from cells treated with OHT displayed a slower migrating band than that observed in cells not treated with hormone. Incubation of the immune complexes with CIP from lysates of cells treated with OHT resulted in the conversion of the slower migrating form into a form similar to that observed for untreated cells (Figure 6a). The treatment of immune complexes with CIP in PBS had no eect on the migration of Notchic-ER chimeras (Figure 6a). The same analysis conducted on ER-NicD2202 gave similar results. However, the extent of the mobility

Figure 5 Hormone dependent nuclear translocation of NotchicER chimeras. (a) Subcellular localization of Notchic-ER chimeras was analysed by indirect immuno¯uorescence. Clonal RKE cell lines were stained with anti-myc antibody 9E10 (CY3) after incubation overnight with (+) or without (7) 1 mM OHT. 1 and

2, clonal RKE cell line expressing ER hormone binding domain; 3 and 4, clonal RKE cell line expressing ER-NicD2202; 5 and 6, clonal RKE cell line expressing ER-Nic. Nuclei were stained with DAPI (DAPI). (b) Localization of Notchic-ER proteins in subcellular fractions by Western blot analysis. RKE clones expressing ER-Nic (ER-Nic) and ER-NicD2202 (ER-D2202) chimeras were incubated overnight with (+) or without (7) 1 mM OHT. Cytoplasmic (cyto) and nuclear (nuc) fractions were collected as described in Materials and methods and Notchic-ER chimeric proteins were immunoprecipitated with anti-Notch 925. Notch proteins were detected with anti-myc 9E10 antibody Oncogene


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Figure 6 Hormone induced phosphorylation of Notchic-ER proteins. (a) Cell lines expressing ER-Nic chimera were treated overnight with (+) or without (7) 1 mM OHT. ER-Nic was IP with anti-Notch antisera 925. Immunocomplexes were incubated with buer (NT), calf intestinal phosphatase (CIP) or in presence of the CIP in PBS buer (CIP/PBS) for 20 min at 378C. Proteins were then analysed by Western blotting with 9E10. Arrows indicate the dierent migrating forms. (b) Notchic was immunoprecipitated with anti-Notch antibody 925 from total lysates of clonal RKE cell lines. Proteins were analysed by Western blotting after treatment with CIP enzyme as described for (a). Blots were probed with anti-myc antibody 9E10. The lower panel is a lighter exposure of the same blot. It is to show the appearance of a faster migrating form following treatment with CIP. Arrows are as for (a)

shift for ER-NicD2202 was less than the one observed for clones expressing ER-Nic (data not shown). We demonstrated that Notchic-ER proteins are phosphorylated upon hormone activation. We wanted to determine if this phosphorylation occurs within Notchic sequences or within the ER hormone binding domain. We performed a similar experiment as described above using RKE cell lines expressing Nic. Treatment of Nic with CIP results in a faster migrating form. This form is not usually detected indicating that most of the Nic expressed in the cell is highly phosphorylated. When immune-complexes are treated with CIP in PBS the mobility of the Nic protein is not altered (Figure 6b). Notchic-ER activity is required for maintenance of cell transformation We have demonstrated that transformation of RKE cells in vitro by Notchic-ER chimeric proteins is hormone dependent. Clonal RKE cell lines expressing Notchic-ER proteins readily formed colonies in semisolid media when cultured in the presence of OHT. We wanted to determine if transformation by Notchic-ER proteins was reversible and if Notch activity was required for the maintenance of the transformed state. Clonal RKE cell lines expressing various Notchic-ER proteins were selected by drug resistance in the absence of OHT. These cells were selected for expression of our constructs but not for the acquisition of a transformed phenotype. To determine if transformation by NotchicER proteins was reversible we maintained a culture of RKE cells expressing Notchic-ER in the presence of Oncogene

Figure 7 Notchic-ER activity is required for maintenance of transformation. Clonal RKE cell line expressing Nic-ER chimeric protein was maintained in culture in presence of 1 mM OHT for the indicated time in days (Days). After each indicated day cells were harvested and seeded into soft agar in absence (7OHT) or presence (+OHT) of 1 mM OHT. After 3 weeks, colonies were stained and scanned as described for Figure 2a

1 mM OHT for over 1 month. At various time intervals cells were harvested and seeded into soft agar in the presence or absence of 1 mM OHT. Results for this experiment are shown in Figure 7. Over a period of 30 days with chronic stimulation with OHT RKE cell lines expressing Nic-ER were able to revert to a nontransformed state. Colonies readily formed in semisolid media in the presence of OHT for cells pretreated with hormone for 30 days. In contrast, no colonies were formed if cells were seeded in the absence of OHT (Figure 7). Discussion The use of the hormone binding domain of the estrogen receptor as a way to control the transforming activity of heterologous proteins was ®rst described for MYC-ER (Eilers et al., 1989). This strategy has been successfully employed to investigate the role of many dierent proteins in transformation, gene expression, signal transduction and regulation of the cell cycle (Burk and Klempnauer, 1991; Capobianco and Gilmore, 1993; Eilers et al., 1989; Jackson et al., 1993; Kruse et al., 1997; Rehberger et al., 1997; Samuels et al., 1993; Superti-Furga et al., 1991; Vigo et al., 1999). By fusing the hormone binding domain of the human estrogen receptor with dierent portions of


Notchic protein, we created inducible Notch proteins. This system provides an important tool in order to de®ne the role of Notch in transformation and to study the initial events in the Notch signaling pathway. The involvement of Notch proteins in neoplasia is well documented for three of the mammalian Notch genes. Previously we demonstrated that the intracellular domains of Notch1 and Notch2 transformed baby rat kidney cells immortalized by E1A (Capobianco et al., 1997). Here we show that clonal RKE cell lines expressing Notchic-ER chimeras form colonies in soft agar only in the presence of OHT, demonstrating that the transformation of these cell lines is conditionally activated by hormone. In addition, transformation by Notchic-ER was dosedependent in that the number and size of the colonies increase concomitantly with increasing concentration of OHT. Oncogenesis is often associated with overexpression or misregulation of oncoproteins. For example, overexpression of erbB2, due to ampli®cation of the gene, is associated with an aggressive phenotype of cancers of dierent tissues (Eccles et al., 1994; Lofts and Gullick, 1992). It seems likely that the biological eects of Notch proteins also correlate to the dose of functional protein present in cells. Experiments using mosaic ¯ies indicate that the level of expression of Notch can regulate the fate of dierentiation of cells (Heitzler and Simpson, 1991). We have now demonstrated that cellular transformation requires a certain threshold level of active Notchic using our Notchic-ER chimeras. Oncogenesis is a multistep process that requires accumulation of somatic mutations. It is now widely accepted that genomic instability plays a major role in tumorigenesis and many oncogenes are capable of inducing genomic instability. Neoplastic transformation mediated by Notch might involve the induction of subsequent genetic alterations that could make Notch activity dispensable for maintenance of transformation. If this is the case the transformed phenotype induced by Notch should be irreversible and should not require a chronic activation of the protein. Such a mechanism has been described for MYCER oncogenes. An overexpression of MYC generates multiple genetic abnormalities that result in the acquisition of a proliferative state that can be maintained independently of MYC activation over many days (Chernova et al., 1998; Felsher and Bishop, 1999). In order to address if the mechanism of transformation mediated by Notch can follow this scheme, we cultured the clonal RKE cell lines expressing Notchic-ER chimeras in the presence of OHT over a period of 30 days. At dierent time points we tested these cells for their ability to grow in soft agar in the presence or absence of hormone. After 30 days, we observed formation of colonies only in the presence of hormone activation. We demonstrated that clonal RKE cell lines expressing Notchic-ER chimeras chronically stimulated with hormone acquire a transformed phenotype. The failure of these cells to form colonies in semi-solid media in the absence of OHT indicates that the induced transformation is reversible and a chronic activation of Nic is required for its maintenance. However, we cannot exclude that such a mechanism of transformation is involved in tumorigenesis induced by Notch, it might be that

longer times are necessary for the establishment of new genetic alterations. We demonstrated that Notchic-ER chimeras are phosphorylated following activation by hormone. Phosphatase treatment of Notchic-ER proteins following IP from lysates of cells treated with OHT revealed that the shift in mobility observed in treated cells was due to addition of phosphate. Phosphorylation of Notchic-ER occurs to a high stoichiometry. Usually the transition between the unphosphorylated and the phosphorylated forms is complete: the detectable proteins are present only in the phosphorylated form following treatment with hormone. Moreover, following CIP treatment, it is often possible to distinguish more than one band, this is indicative of the presence of multiple phosphorylation forms and of the presence of multiple phosphorylation sites in Notchic. This is supported also by the observation that ER-NicD2202 displays a lesser shift than Notchic-ER chimeras. Several sites of phosphorylation have been identi®ed in the human estrogen receptor. Therefore, it is possible that the phosphorylation we are detecting in response to hormone is related to the phosphorylation of the ER portion of our chimeras. However, most of the phosphorylation sites are localized in the Nterminal portion of the estrogen receptor that is not present in Notchic-ER. Amino acids 282 ± 595 contain tyrosine (Tyr537) and serine (Ser294). These are described as minor sites of phosphorylation and are constitutively phosphorylated (Arnold et al., 1995; Le Go et al., 1994). Furthermore, we demonstrate using similar criteria that Nic expressed in transformed RKE cells is phosphorylated. Nic from these cell lysates are nearly exclusively phosphorylated as determined by the fact that only one band is detected in Western blot analysis. Upon phosphatase treatment we could detect a faster migrating band. Therefore, we conclude that the phosphorylation induced by hormone activation of Notchic-ER chimeras occurs on the Notchic sequence present in the fusion protein. In support of our data, phosphorylation of Notch has been previously described in Drosophila (Kidd et al., 1989, 1998). We have shown by both subcellular fractionation of RKE cells and by indirect immuno¯uorescence (IF) that Notchic-ER proteins accumulate in the nucleus following hormone activation. In the absence of hormone Notchic-ER molecules are non-transforming and display a diuse whole-cell staining as detected by IF. The fractionation data are in concordance with the IF data in that unstimulated cells have approximately equal amounts of Notchic-ER in the cytoplasm and the nucleus. If nuclear localization is critical for transformation, why are RKE cells expressing Notchic-ER not transformed in the absence of hormone? There are at least two arguments that can be made to answer this question. We have shown that there is a critical threshold level of Notchic-ER activation necessary to elicit cellular transformation. One possibility to explain the localization data is that the amount of Notchic in the nucleus is simply below this threshold level. The other possibility is phosphorylation. We have shown that in the absence of hormone Notchic-ER proteins are not phosphorylated (or at least hypophosphorylated) in both the cytoplasmic and nuclear fractions. Therefore, one could argue that phosphorylation might be critical to Nic activity. We have observed that in the

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presence of OHT there is an increase in the quantity of Notchic-ER chimeric proteins. Currently, we do not know the mechanism of this increase. One possibility is that phosphorylation increases the stability of the chimera. However, we favor a qualitative versus a quantitative model of Notch activation. We have previously shown that the expression level of Notchic does not correlate with the extent of the transformed phenotype (Capobianco et al., 1997). Therefore, we do not believe that this increase in total protein is responsible for the conditional nature of the chimera. CBF1, Su(H), has been shown to be an important downstream eector for Notch function. For all three Notchic-ER chimeras we observed a very tight hormonal regulation of CBF1 activity. The ability of CBF1 to transactivate a CBF1 site containing luciferase reporter gene also displayed a remarkable dose dependence. Our hypothesis is that regulation of this activity primarily lies in the phosphorylation state of Nic and in its subcellular distribution. We showed that GST-CBF1 was able to anity precipitate Notchic-ER proteins from RKE cells that had been treated with either OHT or ETOH, indicating that CBF1 is not excluded from access to Notch in the absence of OHT. Recently we have demonstrated that mutants that lack the RAM domain are incapable of binding CBF1 and still retain the ability to transform RKE cells (Jeries and Capobianco, 2000). Although the Notchic-ER chimeras displayed hormone dependent activation of CBF1 in Hela cells we do not believe this activity is necessary for transformation. In agreement with our hypothesis, Dumond et al. (2000) have reported CBF-1 independent transformation by Notchic. Materials and methods Cells and transformation assay Hela, RKE and Phoenix cells (Grignani et al., 1998) were cultured in Dulbecco's modi®ed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). In order to obtain stable cell lines expressing Notchic-ER chimeras, pBabe-puro retroviral vectors expressing NotchicER constructs were transfected in Phoenix cells by lipofectamine method. 56105 RKE cells were incubated at 378C for 24 h in presence of the retroviral containing supernatants and 8 mg/ml of Polybrene. Notchic-ER clones were selected by picking isolated puromycin-resistant colonies and propagated in culture in DMEM supplemented with 10% FBS and 2 mg/ml of puromycin. Growth in semi-solid medium Approximately 104 cells of clonal RKE lines expressing Notchic-ER chimeric proteins were seeded in presence of OHT at the indicated concentration or in an equivalent volume of ethanol (ETOH) vehicle (absence of hormone) in 3 ml of DMEM without phenol red containing 30% FBS and 0.3% low melting temperature (LMT) agarose. The cells were overlaid onto a base of 4 ml of medium containing 0.5% LMT agarose and fed every 5 days with media containing 2 mM OHT or ETOH vehicle until colonies were visible. The percentage of cells forming colonies was determined by counting the number of colonies in 24 random ®elds at 1006 magni®cation on an inverted microscope. The number of colonies obtained from counting 24 ®elds was averaged and multiplied by 70 to obtain the total number of colonies. We determined that the area of a ®eld of our microscope at this

Oncogene

magni®cation is 1/70 of the total surface of 22.1 cm2 of the 60 mm plates used for the assay. The percentage was then determined by dividing the total number of colonies formed by the total number of cells seeded. Plasmids Plasmids were constructed using standard recombinant DNA protocols. Enzymes were purchased from New England Biolabs (Beverly, MA, USA) and used according to the manufacturer's speci®cations. Bp3hbER is a pBabe-puro retroviral vector containing amino acids 282 ± 595 of the human estrogen receptor (ER) (kindly provided by M McMahon, UCSF). This sequence encodes the hormone binding domain of the receptor without an initiating methionine. Nic-ER chimeric construct was obtained by subcloning a PCR fragment encoding Notchic (Nic) into Bp3hbER vector. The resulting Nic-ER plasmid encodes a Nic-ER chimeric protein in which ER domain is fused C-terminally to Notchic full length. The expression of the protein is under control of LTR promoter. We created a pBabe-puro vector that directs the expression of hormone binding domain of human estrogen receptor (pBP283ER). This construct was obtained introducing an initiator methionine 5 prime to aa 282 of ER by PCR. This fragment was then subcloned into a modi®ed pBabe-puro containing a myc-epitope tag. ER-Nic and ER-NicD2202 chimeric constructs were generated by subcloning the sequences of Nic and NicD2202 into pBP283ER, respectively. ER-Nic and ER-NicD2202 encode for chimeric proteins in which the ER domain is fused Nterminally to the Notchic and a C-terminal myc epitope tag. Analysis of Notchic-ER proteins Clonal RKE cell lines expressing Notchic-ER chimeras were grown until con¯uent and treated with 1 mM OHT or an equal volume of 100% ETOH vehicle overnight. Cells were then harvested and lysed in standard lysis buer (150 mM NaCl, 50 mM HEPES pH 7.4, 1.5 mM EDTA, 10% glycerol, 1% Nonidet P-40) containing protease inhibitors (Leupeptin 5 mg/ml, Aprotin 2 mg/ml, Pefabloc 2 mM) and 0.5 mM dithiothreitol (DTT). Whole cell lysates were centrifugated at 100 000 g for 30 min and Notchic-ER proteins were immunoprecipitated (IP) from cleared supernatants with indicated antibodies for 1 h at 48C. Immunocomplexes were collected by adsorbtion to Protein A immobilized on sepharose CL-4B beads (Sigma) for 1 h at 48C. Proteins were then separated by SDS ± PAGE and transferred to nitrocellulose by standard procedure. Notchic-ER proteins were detected by immunoblotting with anti-myc antibody 9E10 or anti-Notch antibody bTAN15A and visualized by enhanced chemiluminescence Kit (ECL, Amersham, Bucks, UK). For subcellular localization, cellular fractions were collected as follows: clonal RKE cell lines expressing Notchic-ER chimeric proteins were maintained overnight in presence of 1 mM OHT or 100% ETOH. Cells were harvested and lysed in hypolysis buer (40 mM Tris-HCl pH 7.5, 10 mM NaCl, 1 mM EDTA) containing protease inhibitors and 0.5 mM DTT. Cells were disrupted by dounce homogenization in a glass dounce (Kontes Glass Company) with a B type pestle. Whole cell lysates were centrifugated at 1600 g and pellets containing nuclei were washed in 1 ml of hypolysis buer and recentrifugated. Nuclear extracts were prepared by lysis of nuclei in high salt concentration buer (40 mM Tris-HCl pH 7.5, 420 mM NaCl, 1 mM EDTA) containing protease inhibitors and 0.5 mM DTT. Supernatants from the 1600 g speed spin of the whole cell lysates were centrifugated at 100 000 g. The supernatants of this centrifugation were saved as cytoplasmic fractions and the pellets as membrane fractions. Membranes were washed and solubilized in


standard lysis buer. Subcellular fractions were then adjusted to 210 mM NaCl and 1% Nonidet P-40. Notchic-ER proteins were IP from every fraction and analysed as described above. For phosphorylation assays, Notchic-ER chimeras were IP from whole cell lysates of clonal RKE cell lines expressing the molecules as described. Immunocomplexes adsorbed to Protein A sepharose beads were divided into three aliquots of equal volume and incubated at 378C for 20 min. Two aliquots were incubated in manufacturer's supplied buer in presence or absence of 1 ml of calf intestinal alkaline phosphatase (CIP, 10 000 U/ml, New England Biolabs). The third aliquot was incubated with CIP in phosphate buered saline. Samples were then suspended in 26 sample buer and Notchic-ER chimeras analysed by Western immunoblotting. Immunofluorescence Clonal RKE cell lines expressing Notchic-ER proteins were grown on poly-L-lysine treated glass slides in DMEM. Cells were incubated overnight with 1 mM OHT or an equal volume of 100% ETOH. The following day cells were ®xed in 3% paraformaldehyde on ice for 30 min, permeabilized with 0.2% Triton X-100 on ice for 5 min and preincubated for 1 h at RT in PBS buer supplemented with 3% FBS and 0.05% Tween 20. Notchic-ER chimeras were detected by incubation of the cells for 1 h at RT with anti-myc 9E10 antibody, followed by incubation for 30 min at RT with CY3conjugated anti-mouse antibody (Jackson Laboratory). Transactivation assay Hela cells were transiently cotransfected by lipofectamine (Gibco-BRL) (16 mg per 1.6 mg of total DNA) with 0.4 mg of CBF1 luciferase reporter plasmid (86CBF1), 0.4 mg of pRLTK control reporter vector and 0.8 mg of pBabe-puro vectors expressing Nic or Notchic-ER chimeric proteins as indicated. The 86CBF1 luciferase reporter plasmid contains eight copies of a CBF1 consensus binding sequence (GTGGGAA) upstream of a minimal SV40 promoter in pGL2-Promoter DNA vector (kindly provided by PD Ling, (Fuentes-Panana and Ling, 1998)). pRL-TK contains herpes simplex virus thymidine kinase promoter region upstream of a cDNA encoding Renilla luciferase and was used as internal control for normalization of luciferase activity. Transfected cells were incubated for 48 h in presence of 1 mM OHT or of an equal

volume of 100% ETOH vehicle. Lysates were then prepared and luciferase activity analysed using the Dual-Luciferase Reporter Assay System (Promega), following manufacturer's speci®cations. Luciferase and Renilla luciferase luminescence were measured by a single sample luminometer (Femtomaster FB 12, Zylux Corporation). Results are reported as relative light units, calculated for every sample by dividing the value of luminescence of luciferase enzyme by the corresponding value of luminescence of Renilla luciferase.

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GST-fusion protein pull down Clonal RKE cell lines expressing Notchic-ER chimeras were lysed in standard lysis buer. Pre-clearing of lysates was performed by incubation for 1 h at 48C with GST-bound Gluthathione agarose beads (Sigma). Supernatants were collected after centrifugation at 13 000 g and split in three aliquots. Two aliquots were incubated overnight at 48C with GST- or GST-CBF1 adsorbed to Glutathione agarose beads. From the third aliquot Notchic-ER proteins were IP with anti-Notch 925 as described. Recovered proteins were analysed by Western immunoblotting. Antibodies Monoclonal antibody anti-Notch1 bTAN15A was prepared as described elsewhere (Zagouras et al., 1995). Tissue culture supernatant containing this antibody was used to a 1 : 10 dilution. Anti-Notch Rabbit polyclonal antiserum 925 is directed against aa1759 ± 2095 of human Nic protein. Mouse monoclonal antibody 9E10 is directed against a myc epitope. It was used to a 1 : 1000 dilution in immuno¯uorescence and 1 : 5000 dilution in Western blot analysis.

Acknowledgments We thank members of the Capobianco laboratory for support and technical assistance. We thank David Robbins and his laboratory for insightful comments on our work. We also thank Gary Nolan (Stanford), Paul Ling (Baylor) and Martin MacMahon (UCSF) for kindly providing reagents used in this study. This work was funded in part by grants from the American Cancer Society (RPG LBC99465 to AJ Capobianco and the National Cancer Institute (ROI CA 83736 to AJ Capobianco).

References Ahmad I, Zagouras P and Artavanis-Tsakonas S. (1995). Mech. Dev., 53, 73 ± 85. Arnold SF, Obourn JD, Jae H and Notides AC. (1995). Mol. Endocrinol., 9, 24 ± 33. Artavanis-Tsakonas S, Matsuno K and Fortini ME. (1995). Science, 268, 225 ± 232. Artavanis-Tsakonas S, Rand MD and Lake RJ. (1999). Science, 284, 770 ± 776. Aster J, Pear W, Hasserjian R, Erba H, Davi F, Luo B, Scott M, Baltimore D and Sklar J. (1994). Cold Spring Harb. Symp. Quant. Biol., 59, 125 ± 136. Burk O and Klempnauer KH. (1991). EMBO J., 10, 3713 ± 3719. Capobianco AJ and Gilmore TD. (1993). Virology, 193, 160 ± 170. Capobianco AJ, Zagouras P, Blaumueller CM, ArtavanisTsakonas S and Bishop JM. (1997). Mol. Cell. Biol., 17, 6265 ± 6273. Chernova OB, Chernov MV, Ishizaka Y, Agarwal ML and Stark GR. (1998). Mol. Cell. Biol., 18, 536 ± 545. Daniel B, Rangarajan A, Mukherjee G, Vallikad E and Krishna S. (1997). J. Gen. Virol., 78, 1095 ± 1101.

Dievart A, Beaulieu N and Jolicoeur P. (1999). Oncogene, 18, 5973 ± 5981. Dumont E, Fuchs KP, Bommer G, Christoph B, Kremmer E and Kempkes B. (2000). Oncogene, 19, 556 ± 561. Eccles SA, Modjtahedi H, Box G, Court W, Sandle J and Dean CJ. (1994). Invasion Metastasis, 14, 337 ± 348. Eilers M, Picard D, Yamamoto KR and Bishop JM. (1989). Nature, 340, 66 ± 68. Ellisen LW, Bird J, West DC, Soreng AL, Reynolds TC, Smith SD and Sklar J. (1991). Cell, 66, 649 ± 661. Fehon RG, Johansen K, Rebay I and Artavanis-Tsakonas S. (1991). J. Cell. Biol., 113, 657 ± 669. Felsher DW and Bishop JM. (1999). Proc. Natl. Acad. Sci. USA, 96, 3940 ± 3944. Fortini ME and Artavanis-Tsakonas S. (1994). Cell, 79, 273 ± 282. Fortini ME, Rebay I, Caron LA and Artavanis-Tsakonas S. (1993). Nature, 365, 555 ± 557. Fuentes-Panana EM and Ling PD. (1998). J. Virol., 72, 693 ± 700. Gallahan D, Kozak C and Callahan R. (1987). J. Virol., 61, 218 ± 220. Oncogene


3924

Girard L, Hanna Z, Beaulieu N, Hoemann CD, Simard C, Kozak CA and Jolicoeur P. (1996). Genes. Dev., 10, 1930 ± 1944. Grignani F, Kinsella T, Mencarelli A, Valtieri M, Riganelli D, Lanfrancone L, Peschle C, Nolan GP and Pelicci PG. (1998). Cancer Res., 58, 14 ± 19. Heitzler P and Simpson P. (1991). Cell, 64, 1083 ± 1092. Hsieh JJ, Henkel T, Salmon P, Robey E, Peterson MG and Hayward SD. (1996). Mol. Cell. Biol., 16, 952 ± 959. Jackson P, Baltimore D and Picard D. (1993). EMBO J., 12, 2809 ± 2819. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R and Israel A. (1995). Nature, 377, 355 ± 358. Jeries S and Capobianco AJ. (2000). Mol. Cell. Biol., 20, 3928 ± 3941. Jhappan C, Gallahan D, Stahle C, Chu E, Smith GH, Merlino G and Callahan R. (1992). Genes Dev., 6, 345 ± 355. Kao HY, Ordentlich P, Koyano-Nakagawa N, Tang Z, Downes M, Kintner CR, Evans RM and Kadesch T. (1998). Genes Dev., 12, 2269 ± 2277. Kidd S, Baylies MK, Gasic GP and Young MW. (1989). Genes Dev., 3, 1113 ± 1129. Kidd S, Lieber T and Young MW. (1998). Genes Dev., 12, 3728 ± 3740. Kopan R, Nye JS and Weintraub H. (1994). Development, 120, 2385 ± 2396. Kopan R, Schroeter EH, Weintraub H and Nye JS. (1996). Proc. Natl. Acad. Sci. USA, 93, 1683 ± 1688. Kruse U, Iacovoni JS, Goller ME and Vogt PK. (1997). Proc. Natl. Acad. Sci. USA, 94, 12396 ± 12400. Le Go P, Montano MM, Schodin DJ and Katzenellenbogen BS. (1994). J. Biol. Chem., 269, 4458 ± 4466. Lecourtois M and Schweisguth F. (1998). Curr. Biol., 8, 771 ± 774. Lieber T, Kidd S, Alcamo E, Corbin V and Young MW. (1993). Genes Dev., 7, 1949 ± 1965. Lofts FJ and Gullick WJ. (1992). Cancer Treat Res., 61, 161 ± 179. Miele L and Osborne B. (1999). J. Cell Physiol., 181, 393 ± 409.

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Milner LA and Bigas A. (1999). Blood, 93, 2431 ± 2448. Nye JS, Kopan R and Axel R. (1994). Development, 120, 2421 ± 2430. Oswald F, Liptay S, Adler G and Schmid RM. (1998). Mol. Cell. Biol., 18, 2077 ± 2088. Pear WS, Aster JC, Scott ML, Hasserjian RP, Soer B, Sklar J and Baltimore D. (1996). J. Exp. Med., 183, 2283 ± 2291. Rebay I, Fehon RG and Artavanis-Tsakonas S. (1993). Cell, 74, 319 ± 329. Rehberger S, Gounari F, DucDodon M, Chlichlia K, Gazzolo L, Schirrmacher V and Khazaie K. (1997). Exp. Cell. Res., 233, 363 ± 371. Robbins J, Blondel BJ, Gallahan D and Callahan R. (1992). J. Virol., 66, 2594 ± 2599. Rohn JL, Lauring AS, Linenberger ML and Overbaugh J. (1996). J. Virol., 70, 8071 ± 8080. Samuels ML, Weber MJ, Bishop JM and McMahon M. (1993). Mol. Cell. Biol., 13, 6241 ± 6252. Schroeter EH, Kisslinger JA and Kopan R. (1998). Nature, 393, 382 ± 386. Smith GH, Gallahan D, Diella F, Jhappan C, Merlino G and Callahan R. (1995). Cell Growth Dier., 6, 563 ± 577. Stifani S, Blaumueller CM, Redhead NJ, Hill RE and Artavanis-Tsakonas S. (1992). Nat. Genet., 2, 343. Struhl G and Adachi A. (1998). Cell, 93, 649 ± 660. Struhl G, Fitzgerald K and Greenwald I. (1993). Cell, 74, 331 ± 345. Superti-Furga G, Bergers G, Picard D and Busslinger M. (1991). Proc. Natl. Acad. Sci. USA, 88, 5114 ± 5118. Tamura K, Taniguchi Y, Minoguchi S, Sakai T, Tun T, Furukawa T and Hongo T. (1995). Curr. Biol., 5, 1416 ± 1423. Vigo E, Muller H, Prosperini E, Hateboer G, Cartwright P, Moroni MC and Helin K. (1999). Mol. Cell. Biol., 19, 6379 ± 6395. Zagouras P, Stifani S, Blaumueller CM, Carcangiu ML and Artavanis-Tsakonas S. (1995). Proc. Natl. Acad. Sci. USA, 92, 6414 ± 6418.