ABSTRACT
Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for immortalization of human B cells by EBV. EBNA2 and activated Notch transactivate genes by interacting with the cellular transcription factor RBP-Jκ/CBF1. Therefore, EBNA2 can be regarded as a functional homologue of activated Notch. We have shown previously that the intracellular domain of Notch1 (Notch1-IC) is able to transactivate EBNA2-regulated viral promoters and to induce phenotypic changes in B cells similar to those caused by EBNA2. Here we investigated whether Notch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. Using an EBV-immortalized lymphoblastoid cell line in which EBNA2 function can be regulated by estrogen, we demonstrate that murine Notch1-IC, in the absence of functional EBNA2, is unable to maintain LMP1 expression and to maintain cell proliferation. However, in a lymphoblastoid cell line expressing LMP1 independently of EBNA2, murine Notch1-IC can transiently maintain proliferation after EBNA2 inactivation. After 4 days, cell numbers do not increase further, and cells in the G2 phase of the cell cycle start to die. In contrast to EBNA2, murine Notch1-IC is unable to upregulate the expression of the c-myc gene in these cells.
Epstein-Barr virus (EBV), a lymphotropic human gammaherpesvirus, is associated with several human malignancies. It infects primary resting B cells and induces unlimited proliferation of virus-infected cells in vitro (so called EBV-immortalized lymphoblastoid cell lines). In these cell lines, only a subset of viral genes that code for six EBV nuclear proteins (EBNA1, -2, -3A, -3B, -3C, and -LP) and three latent membrane protein antigens (LMP1, -2A, and -2B) are expressed. Five of these, EBNA1, -2, -3A, and -3C and LMP1, appear to be absolutely required for B-cell immortalization (33).
EBNA2 and LMP1 most likely contribute to B-cell immortalization by activation of cellular pathways physiologically dependent on ligand-receptor interactions. LMP1 mimics a constitutively activated receptor of the tumor necrosis factor receptor family (11, 14, 47) and shares functional characteristics with CD40 (35, 64, 70). Both molecules induce NF-κB (5, 16, 37), stress-activated protein kinases (4, 11, 34), and the JAK/STAT pathway (13, 18, 30). Through activation of these pathways, LMP1 induces B-cell activation markers and cell adhesion molecules (33) and enhances the viability of nonproliferating B cells (70).
EBNA2 is essential for initiation and maintenance of B-cell immortalization (7, 17, 31). Acting as a transcriptional activator, it modulates the transcription of different cellular genes, including the B-cell activation markers CD21 and CD23 and the proto-oncogenes c-fgr (33) and c-myc(28). In addition, it activates transcription of the viral RNAs coding for the various EBV nuclear and membrane proteins (33). EBNA2 does not bind to DNA directly (41, 68), but is recruited to EBNA2-responsive elements by interacting with the transcription factors RBP-Jκ (also called CBF1 and KBF2) (15, 21, 66, 69) and PU.1/Spi-1 (27, 38). RBP-Jκ binding sites are present in all EBNA2-regulated promoters discovered so far. Binding of RBP-Jκ is essential but not sufficient to confer EBNA2 responsiveness on EBNA2-regulated promoters (43).
RBP-Jκ is highly conserved in evolution and ubiquitously expressed. The RBP-Jκ homologue in Drosophila melanogaster is the neurogenic protein Suppressor of Hairless [Su(H)]. In insects as well as in mammals, Su(H)/RBP-Jκ acts downstream of the receptor Notch (3, 39). Four vertebrate homologues of the Notch genes Notch 1 (Tan1), Notch2, Notch3, and Notch4 (int3) have been cloned (54). In mammalian cells, RBP-Jκ usually acts as a transcriptional repressor (10, 23, 65). Activation of the transmembrane receptor Notch by binding of one of its ligands, such as Delta or Jagged, leads to proteolytic cleavage of Notch, resulting in translocation of the intracellular (IC) part (Notch-IC or activated Notch) to the nucleus, where it transactivates genes previously repressed by RBP-Jκ (32, 40, 57, 61).
Notch signaling plays an essential role in cell fate decisions in all species, including vertebrates (1). The Notch signal transduction pathway has an essential function during embryogenesis and is involved in the differentiation of neuronal precursors, myoblasts, and Malpighian tubules. Notch signaling also appears to play a role in lineage decisions during hematopoesis. It has been reported that Notch signalling is involved in the renewal and differentiation of hematopoietic cells (6, 45), in granulocyte differentiation (44, 56), and in differentiation and maturation of thymocytes (9, 52, 53). Until now, it has been unknown whether Notch is also involved in the differentiation of B cells.
Since both Notch-IC and EBNA2 transactivate genes by interacting with RBP-Jκ, EBNA2 may be regarded as a functional homologue of Notch-IC. Previously, we have shown that EBNA2 can functionally replace Notch1-IC in suppressing differentiation of C2C12 myoblast progenitor cells (55). In Burkitt's lymphoma cells, Notch1-IC is able to transactivate the viral EBNA2-regulated promoters in transient-transfection assays, although to a lesser extent than EBNA2 (22). Stably introduced into Burkitt's lymphoma cell lines, Notch1-IC and EBNA2 upregulate CD21 and LMP2A expression and downregulate immunoglobulin M (IgM) expression to similar extents, but Notch1-IC does not induce CD23 expression and only marginally induces LMP1 transcription (60).
In this study, we addressed the question of whether activated Notch1-IC is able to substitute for EBNA2 in the maintenance of B-cell proliferation. An expression vector coding for a tetracycline (TET)-regulated murine Notch1-IC (mNotch1-IC) was stably introduced into the EREB2-5 cell line. EREB2-5 is an EBV-immortalized cell line established by infection of primary B cells with the EBNA2-deficient P3HR1 virus strain and complementation of the EBNA2 defect by an EBNA2-estrogen receptor fusion protein that renders the function of EBNA2 dependent on the presence of estrogen (31). This system allowed us to investigate whether mNotch1-IC can maintain B-cell proliferation in the absence of functional EBNA2.
MATERIALS AND METHODS
Cell lines and culture conditions.EREB2-5 is a lymphoblastoid cell line generated by infection of CD19-enriched human B cells with an EBV mutant conditional for EBNA2 function (31). The cell line 1194 was established by packaging the mini-EBV plasmid p1480.40, which codes for an estrogen-dependent EBNA2 and a constitutive LMP1 gene, into an EBV coat and infecting primary B cells (70). The cell clone Cl4/p1480.40 was used and designated 1194.
The cell lines were grown in RPMI 1640 cell culture medium supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate, penicillin (100 U/ml), streptomycin (100 μg/ml), and 1 μM β-estradiol.
Plasmids.The pHEBo vector has been described (62). The mNotch1-IC expression plasmid pLS710-12 was cloned by digesting the construct pSG5mNotch1-IC (24) withStuI and XbaI and treating the 2.5-kb fragment with Klenow polymerase. The expression construct BC364A, carrying a tetracycline-regulated promoter, the EBV oriP, and a gene for hygromycin resistance (57a), was digested withBglII, and the 9.7-kb fragment was treated with Klenow polymerase. Ligation of the 2.5- and 9.7-kb fragments resulted in plasmid pLS710-12.
The luciferase reporter construct pGa981-16, carrying a multimerized RBP-Jκ site, has been described (46).
Transfections.Cells were transfected by electroporation using a Bio-Rad gene pulser at 960 μF and 230 V as described previously (22). After stable transfection of the EREB2-5 and 1194 cell clones, the cells were seeded in 96-well flat-bottomed plates and selected in RPMI 1640 medium supplemented with β-estradiol and hygromycin. Tetracycline was added to the cell lines transfected with LS710-12.
Luciferase assays.Cells were harvested and lysed as described previously (43). First, 10 μl of each probe was mixed with 150 μl of test buffer (25 mM glycylglycine [pH 7.8], 5 mM ATP, 15 mM MgSO4) on a 96-well plate. After addition of 100 μl of 11 mM luciferin in 0.5 M Tris-HCl (pH 7.8) to each reaction, the bioluminescence in relative light units was measured with a Micro-Lumat LB 96 P (Berthold, Wildbach, Germany).
Protein immunoblots.For Western blot analysis, cellular extracts were prepared by sonification in H8 lysis buffer (20 mM Tris [pH 7.0], 2 mM EGTA, 2 mM EDTA, 6 mM β-mercaptoethanol, 50 mM NaF, 100 mM NaCl, 1% sodium dodecyl sulfate [SDS]). The protein concentration was determined, and equal amounts of protein were separated on a Laemmli 10% polyacrylamide–SDS gel. Proteins were transferred onto nitrocellulose filters (Amersham Hybond ECL), and protein expression was analyzed with monoclonal antibodies directed against mNotch1-IC (anti-Flag antibody M2 [Eastman Kodak]), LMP1 (S12; kindly provided by David Thorley-Lawson), and c-Myc (mouse anti-Myc, 9E10; Oncogene Science Inc.). Immunoreactive proteins were detected by peroxidase-coupled secondary antibodies and enhanced chemiluminescence (ECL System; Amersham).
Proliferation kinetics.Cells were grown in the absence or presence of tetracycline for at least 1 week. Cells were washed three times with phosphate-buffered saline (PBS) to remove estrogen and seeded with the same number of viable cells in 10-ml cell culture flasks or 96-well plates in the presence and absence of tetracycline. The number of living cells was counted at three specific time points. Dead cells were excluded by trypan blue dye staining.
MTT assay.Cells were treated as described above before seeding. Samples of 3 × 104 cells were seeded in 100 μl of cell culture medium in triplicate in the absence or presence of estrogen for the indicated time. After incubation with MTT [3-(4,5-dimethylthiazol-2-yl)2 2,5-diphenyl tetrazolium bromide; 0.5 mg/ml] for 4 h, MTT conversion, which correlates with the number of living cells in the assay, was determined in an enzyme-linked immunosorbent assay reader as described (48). Each experiment was repeated at least twice. To compare different experiments, the optical density (OD) on day 0 was set to 1, and normalized OD values are given for the following days.
Cell cycle analysis.Cell cycle analysis was performed by propidium iodide staining. Cells were washed twice in PBS and fixed with 70% ethanol. Cells were incubated for 30 min in PBS–2% fetal calf serum containing RNase A (0.5 mg/m) and propidium iodide (50 μg/ml). All samples were passed through 70-μm mesh prior to fluorescence-activated cell sorting (FACS) analysis.
RESULTS
Stable transfection of mNotch1-IC in EREB2-5 cells.The intracellular part of Notch1 (Notch1-IC) functions as a constitutively active Notch receptor (36). To test whether Notch1-IC can replace EBNA2 in the maintenance of B-cell proliferation, the DNA coding for mNotch1-IC was stably introduced into EREB2-5 cells, a conditionally immortalized lymphoblastoid cell line growing only in the presence of estrogen. Estrogen withdrawal leads to the inactivation of EBNA2, followed by cell cycle arrest (31). A cDNA coding for mNotch1-IC with a Flag tag at the N terminus was cloned behind a tetracycline-regulatable promoter on a pHEBo vector (pLS710-12). This construct was stably introduced into EREB2-5 cells. Hygromycin-resistant single-cell clones were analyzed for mNotch1-IC protein expression in the presence and absence of estrogen and tetracycline by Western blotting. The anti-Flag monoclonal antibody detected a molecule with the expected molecular mass of about 70 kDa. In Fig. 1A, two clones (cl.1 and cl.4) are shown expressing mNotch1-IC in the absence but not in the presence of tetracycline. The presence of estrogen did not influence the expression of mNotch1-IC.
Tetracycline-regulated mNotch1-IC expression in EREB2-5 cells. (A) Extracts from two hygromycin-resistant single-cell clones derived after transfection of EREB2-5 cells with a tetracycline-regulated, Flag-tagged mNotch1-IC (EREB-Notch cl.1 and cl.4) were Western blotted and analyzed by immunostaining with the anti-Flag monoclonal antibody M2. Extracts were prepared in the presence and absence of estrogen and tetracycline. (B) EREB2-5 cells and cells from EREB-Notch clones 1 and 4 were transiently transfected with a promoter-reporter construct containing a multimerized RBP-Jκ binding site. As negative control, a promoter-reporter gene construct without an RBP-Jκ binding site was used. Cells were grown in the presence of estrogen and presence or absence of tetracycline. Four hours before transfection, cells were washed to withdraw estrogen. After transfection, the cells were cultivated for 2 days in the presence (+E) or absence (−E) of estrogen to regulate the function of EBNA2 and in the presence (+N) or absence (−N) of tetracycline to regulate Notch1-IC expression. Luciferase activities were determined as relative light units (RLUs). Mean values and standard deviations of two independent experiments are indicated.
To analyze whether the stably introduced mNotch1-IC gene is functionally active, a promoter reporter gene construct carrying a multimerized RBP-Jκ binding site (pGa981-16) was transiently transfected into the mNotch1-IC-expressing cell clones (EREB-Notch cl.1 and cl.4) and into the parental EREB2-5 line. Luciferase activity was measured in the presence and absence of estrogen and tetracycline. As shown in Fig. 1B, the reporter gene was transactivated by mNotch1-IC in the absence of EBNA2 in the EREB-Notch cl.1 and cl.4 cells. In the absence of Notch1-IC and EBNA2, luciferase activities were nearly at background levels. The transactivation efficiencies with EBNA2 and mNotch1-IC were comparable. Activation by EBNA2 and mNotch1-IC was dependent on the presence of the RBP-Jκ binding site on the reporter construct. This indicates that mNotch1-IC is functionally active in EREB2-5 cells and can be efficiently regulated by tetracycline.
mNotch1-IC cannot substitute for EBNA2 in the maintenance of proliferation of EREB2-5 cells.To analyze whether mNotch1-IC can maintain the proliferation of EREB2-5 cells in the absence of functional EBNA2, we determined the amount of viable EREB2-5 and EREB-Notch cells after withdrawal of estrogen in the presence and absence of tetracycline. As a positive control, the number of viable EREB2-5 cells in the presence of estrogen was determined. The same number of viable cells were seeded in 96-well plates and stained with MTT reagent after 1, 4, 5, and 7 days. As shown in Fig.2, the OD values, reflecting the number of living cells, decreased after estrogen withdrawal in the parental cell line EREB2-5 and in the Notch1-IC-transfected cell clones. Viability decreased less rapidly in the Notch1-IC-transfected cell clones, but without significant differences in the presence and absence of tetracycline. The MTT assay results were confirmed by cell counting (data not shown). We conclude that mNotch1-IC alone is not sufficient to substitute for EBNA2 in the maintenance of B-cell proliferation.
mNotch1-IC stably expressed in EREB2-5 cells cannot maintain B-cell proliferation. After estrogen withdrawal, the same number of viable EREB and EREB-Notch clone 1 and clone 4 cells in the presence or absence of tetracycline (N), leading to the absence (−N) or presence (+N) of Notch1-IC were seeded in 96-well plates, and the number of viable cells was determined by staining with MTT reagent on the days indicated after estrogen withdrawal. As a positive control, the OD values of the parental line EREB in the presence of estrogen (+E) are shown. At day 5, fresh medium was added to all cells. To compare different MTT assays, OD values at day 1 were normalized to 1. Mean values and standard deviations are shown for triplicates.
mNotch1-IC does not upregulate LMP1 in EREB2-5 cells.The viral protein LMP1 is essential for the proliferation of EBV-immortalized B cells and is known to be activated by EBNA2. To test whether the stably introduced mNotch1-IC gene is able to induce expression of the endogenous LMP1 gene in the absence of functional EBNA2, we performed Western blots. Protein extracts of EREB-Notch clones 1 and 4 grown in the presence and absence of estrogen and tetracycline were analyzed. As shown in Fig.3, LMP1 expression decreased after inactivation of EBNA2 and was not induced by switching on mNotch1-IC expression. We conclude that mNotch1-IC is not able to induce expression of the endogenous LMP1 gene in EREB2-5 cells.
mNotch1-IC stably expressed in EREB2-5 cells cannot induce LMP1 expression. Protein extracts were prepared from EREB-Notch clones 1 and 4 in the presence and absence of estrogen and tetracycline. The protein extracts were Western blotted and analyzed by immunostaining with monoclonal anti-LMP1 antibody S12.
Stable transfection of mNotch1-IC in conditionally immortalised cell lines that constitutively express LMP1.As LMP1 is known to be essential for the maintenance of proliferation of EBV-immortalized B cells (35), we next asked whether mNotch1-IC is able to maintain B-cell proliferation in the absence of functional EBNA2 if LMP1 is constitutively expressed. To this end, we stably transfected the mNotch1-IC expression construct pLS710-12 into cell line 1194. Cell line 1194 is a primary human B-cell line immortalized by the mini-EBV construct p1480.40, carrying an estrogen-regulated EBNA2 and a constitutively expressed LMP1 gene (70). The 1194 cell system allowed us to compare the function of EBNA2 and mNotch1-IC in context with an LMP1 gene that is expressed independently of EBNA2. The 1194 cells were transfected with the Notch1-IC expression vector pLS710–12 and the pHEBO control vector in parallel and selected for hygromycin resistance. Single-cell clones were analyzed for mNotch1-IC protein expression in the presence and absence of tetracycline. As shown in Fig. 4A, expression of mNotch1-IC was induced by removal of tetracycline in two clones (1194-Notch cl.1 and cl.2). An extract of the 1194 cell line stably transfected with the pHEBo vector (1194-pHEBo) served as a negative control.
Tetracycline-regulated mNotch1-IC expression in 1194 cells. (A) Extracts from two hygromycin-resistant single-cell clones derived after transfection of 1194 cells with a tetracycline-regulated, Flag-tagged mNotch1-IC (1194-Notch clones 1 and 2) were Western blotted and analyzed by immunostaining with the anti-Flag monoclonal antibody M2. Extracts were prepared in the presence and absence of tetracycline. The antibody detected a protein with the expected molecular mass of about 70 kDa, whereas an unspecific signal was visible in all lanes at about 80 kDa. (B) Extracts from the controls EREB-pHEBo and 1194-pHEBo and from the 1194-Notch clone 1 in the presence and absence of estrogen and tetracycline were analyzed by Western blots using monoclonal anti-LMP1 antibody S12.
To investigate whether the stably introduced mNotch1-IC gene is functionally active, we transiently transfected the reporter construct carrying the multimerized RBP-Jκ site (pGa981-16) into the 1194-Notch and 1194-pHEBo clones and measured luciferase activity in the presence and absence of estrogen and tetracycline. pGa981-16 could be transactivated by mNotch1-IC in the absence of EBNA2 (data not shown), indicating that mNotch1-IC is functionally active in 1194 cells.
To verify that LMP1 is expressed independently of EBNA2 in the transfected cell lines 1194-Notch and 1194-pHEBo, we performed Western blot analysis. Protein extracts of the 1194-Notch clone 1 and of the 1194-pHEBo control cells were prepared in the presence and absence of estrogen and tetracycline. The anti-LMP1 antibody detected the strongly expressed LMP1 protein regardless of whether estrogen or tetracycline was present (Fig. 4B), whereas in the pHEBo-transfected EREB2-5 cell line, LMP1 was downregulated after removal of estrogen. This indicates that the expression of LMP1 remained independent of EBNA2 in the transfected single-cell clones of 1194 cells and was not influenced by addition of tetracycline.
After inactivation of EBNA2, mNotch1-IC can transiently maintain proliferation in cooperation with LMP1.To test whether mNotch1-IC and LMP1 together can maintain the proliferation of 1194 cells, we performed cell proliferation assays with 1194-pHEBo and the 1194-Notch clone 1. After estrogen withdrawal, 104 cells were seeded in 96-well plates in the presence and absence of estrogen and tetracycline and stained with MTT after 2, 4, 5, and 7 days (Fig.5A). In the absence of functional EBNA2, OD values for the mNotch1-IC-expressing cells increased for 4 days with the same kinetics as the EBNA2-positive controls but started to decrease at day 5. This indicates that in the presence of mNotch1-IC and absence of EBNA2, the cells proliferate for this period of time. The OD values for the LMP1-expressing cells in the absence of Notch1-IC and EBNA2 remained constant for 4 days as well as those of the 1194-pHEBo control cell line (data not shown), confirming that LMP1, in the absence of functional EBNA2, confers a survival advantage on the cells (70). The results were confirmed by cell counting. For the mNotch1-IC-expressing cells, cell numbers doubled after 4 to 5 days and then declined slowly. In contrast, when mNotch1-IC expression was switched off by the addition of tetracycline, cell numbers remained constant for 4 days and gradually decreased afterwards (Fig. 5B). In the presence of functional EBNA2, the 1194-Notch cells grew continuously. Cell viability remained virtually constant (between 89 and 93%) for 4 to 5 days after estrogen withdrawal in the cell culture without tetracycline and estrogen, indistinguishable from that of the EBNA2-expressing control cells, and started to decrease at day 7 and more substantially at day 9 after estrogen withdrawal (Fig. 5C). In contrast, cell viability started to decrease 2 days after estrogen withdrawal in the absence of functional EBNA2 and Notch-IC.
In the absence of estrogen, 1194-Notch cells continue to proliferate for 4 days. (A) 1194-Notch clone 1 cells were kept in the absence of estrogen for up to 7 days, and the number of viable cells was determined by MTT conversion on the days indicated. The rate of MTT conversion at day 0 was set to 1, and OD values were normalized accordingly. (B) After estrogen withdrawal, the same number of viable 1194-Notch clone 1 cells in the presence or absence of estrogen (E) and tetracycline (N), leading to the presence or absence of EBNA2 (+E and −E, respectively) and Notch1-IC (+N and −N, respectively), were seeded in 10-ml cell culture flasks. Cell growth was determined by counting the number of living cells on days 0, 2, 4, and 7 in triplicates. Dead cells were excluded by trypan blue dye staining. (C) The viability of the cells was determined as the ratio of the number of living to total cells. Average values of four experiments are shown. The standard deviations never exceeded 24% of the respective single values.
We next addressed the question of whether, in the absence of EBNA2, the Notch1-IC-expressing 1194 cells arrest in a specific phase of the cell cycle. Cells were stained with propidium iodide 3, 6, 9, and 13 days after estrogen withdrawal to determine the percentage of cells in the G1 and G2 phases by FACS. As shown in Fig.6, the ratio of Notch1-IC-expressing cells in G1 and G2 was nearly constant for 6 days, and then cells in G1 started to accumulate (Fig. 6A). In contrast, the 1194-pHEBo control cells were almost completely arrested in G1 4 days after estrogen withdrawal (Fig. 6B). We conclude from these experiments that B cells expressing both mNotch1-IC and LMP1 are able to maintain proliferation transiently.
1194-Notch cells pass through the cell cycle for 6 days. 1194-Notch (A) and 1194-pHEBo (B) cells were stained with propidium iodide 0, 3, 4, 6, 9, or 13 days after estrogen withdrawal. The relative numbers of cells in the G1 and G2phases of the cell cycle were determined by FACS analysis.
Notch1-IC and LMP1 can transiently maintain proliferation in the absence of c-Myc.We have shown recently that the c-mycgene can be upregulated by EBNA2 and is a direct target of this viral transactivator (28, 31). To examine whether mNotch1-IC can maintain c-myc expression, Western blot analyses were performed using protein extracts of 1194-Notch and 1194-pHEBo cells maintained in the presence of estrogen and for 1 or 2 days in the absence of estrogen as well as tetracycline. As shown for the 1194-Notch clone 1 in Fig. 7, c-Myc could be detected only if EBNA2 was functionally active. We conclude that mNotch1-IC cannot maintain c-Myc expression in 1194 cells, yet B-cell proliferation can be maintained by mNotch1-IC and LMP1 in the absence of c-Myc for at least 4 days.
mNotch1-IC cannot upregulate c-Myc expression in 1194 cells. Extracts from 1194-pHEBo and 1194-Notch cells (clone 1) grown in the presence and absence of tetracycline were prepared 0, 1, and 2 days after estrogen withdrawal. The protein extracts were Western blotted and analyzed by immunostaining with the anti-Myc monoclonal antibody 9E10.
DISCUSSION
Both EBNA2 and activated Notch (Notch-IC) interact with RBP-Jκ and thereby stimulate expression of genes otherwise repressed by this transcription factor. Therefore, these two proteins have been proposed to be functionally equivalent. Recent studies from our laboratory have supported the concept of functional homology between EBNA2 and activated Notch1 but have also revealed obvious differences. We could show that mNotch1-IC is able to transactivate EBNA2-responsive viral promoters (22), although to a lesser extent than EBNA2. After stable transfection into EBNA2-negative Burkitt's lymphoma cells, Notch1-IC, on one hand, upregulates CD21 and LMP2A and downregulates IgM expression, similarly to EBNA2 (60); on the other hand it, does not upregulate CD23 expression and affects LMP1 transcription only marginally.
Since EBNA2 is essential for initiation and maintenance of B-cell immortalization by EBV, the next obvious question was whether mNotch1-IC can substitute for EBNA2 in the maintenance of B-cell proliferation. To address this question, we stably introduced mNotch1-IC, cloned behind a tetracycline-regulatable promoter, into the lymphoblastoid EREB2-5 cell line, in which EBNA2 activation is dependent upon estrogen and can be switched on and off. An important conclusion of this work is that activated Notch1 is unable to maintain proliferation of EREB2-5 cells after EBNA2 has been switched off. A number of arguments support the notion that the failure of Notch1-IC to rescue B-cell proliferation is an inherent property of Notch1-IC and is not due to a weakness of our experimental system. First, after transient transfection of promoter reporter gene constructs containing a multimerized RBP-Jκ site, the stably introduced Notch1-IC could transactivate luciferase reporter constructs to the same extent as EBNA2. Second, we analyzed CD21 expression in the stably transfected EREB2-5 cell clones. Four days after estrogen removal, CD21 expression was high in the presence and low in the absence of Notch1-IC (data not shown), indicating that Notch1-IC is able to upregulate CD21 expression in this cell line in the same way as it does in Burkitt's lymphoma cells (60). Third, in the same experimental system, c-myc expressed from the same tetracycline-regulated promoter is able to rescue proliferation in the absence of estrogen (50a, 57a).
Since the viral LMP1 protein, in concert with EBNA2, plays a crucial role in the induction and maintenance of proliferation, we asked whether mNotch1-IC can induce expression of the endogenous LMP1 gene similarly to EBNA2. In fact, mNotch1-IC proved to be unable to induce LMP1 expression in EREB2-5 cells, providing an explanation for the finding that proliferation of EREB2-5 cells cannot be maintained by mNotch1-IC. The notion that Notch1-IC is unable to maintain LMP1 expression in EREB2-5 cells after estrogen deprivation is in keeping with the finding that induction of LMP1 transcription by a stably transfected Notch1-IC construct in Burkitt's lymphoma cells is very weak and could only be visualised after stabilizing the RNA by the addition of cycloheximide (60). The different responsiveness of some promoters to EBNA2 and Notch1-IC points to a functional association of EBNA2 (and eventually also of Notch1-IC) with additional cellular or viral transcription factors besides RBP-Jκ that contribute to a different EBNA2 versus Notch1 response (27). It has been shown, for instance, that EBNA-LP cooperates with EBNA2 in upregulating the LMP1 promoter (19, 49). Cooperation between EBNA-LP and EBNA2 but not between EBNA-LP and Notch1-IC might thus account for the fact that Notch1-IC is unable to substitute for EBNA2.
Since (i) LMP1 is essential for the proliferation of EBV immortalized cells and (ii) mNotch1-IC cannot induce LMP1 expression in EREB2-5 cells, we next addressed the question of whether activated Notch1 can take over the role of EBNA2 in the maintenance of proliferation if LMP1 is constitutively expressed. To this end, we stably transfected a tetracycline-regulated activated Notch1 gene into the EBV-immortalized cell line 1194. This cell line has been immortalized by a mini-EBV construct that expresses LMP1 constitutively from the simian virus 40 promoter and expresses EBNA2 as a hormone-regulatable estrogen receptor fusion protein (70). This isogenic system, conditional for both EBNA2 and activated Notch1, allowed us to compare the effects of EBNA2 and mNotch1-IC on the background of constitutive LMP1 expression.
Most significantly, EBNA2-deprived cells expressing both mNotch1-IC and LMP1 maintain proliferation for 4 days, resulting in doubling of the cell number. After 4 days, however, the cell numbers do not increase further, and the viability of the cells starts to decline. The ratio of cells in G1 and G2 phases remains nearly constant until day 6 (Fig. 6), suggesting that the cells are passing through the cell cycle during this time. At later time points, the decrease in cell number and viability becomes more clearly apparent, as does the preferential decrease in the number of cells in G2versus G1.
To describe what is going on in these cells in more detail, we studied the expression of Myc after estrogen and tetracycline deprivation. We have shown previously that EBNA2 induces expression of the c-myc gene in EREB2-5 cells (31) and that the c-myc gene is a direct target of EBNA2 (28). In contrast, Notch1-IC was unable to maintain the expression of Myc in either EREB2-5 cells (data not shown) or 1194 cells. Myc as a positive regulator of G1-specific cyclin-dependent kinases, plays an essential role in the progression of cells through the G1-to-S-phase transition (50). It was thus unexpected but highly reproducible that cells expressing Notch1-IC and LMP1 and apparently not expressing Myc progress through the cell cycle for several days before they arrest in G1. This indicates that Myc is not absolutely essential for cell cycle progression for at least one or two rounds. It has been shown recently that rat fibroblasts lacking the c-myc gene are still able to proliferate, although substantially more slowly than their Myc-expressing counterparts (42). This suggests that other genes, including Myc target genes, play an essential role in the maintenance of proliferation and can substitute for Myc. The data are compatible with the model that the level of a long-lived Myc target which is essential for proliferation might gradually decline when the c-myc gene is switched off, leading to delayed proliferation arrest after several days.
It is obvious from the comparison of 1194 cells (constitutively expressing LMP1) to Notch1-IC-expressing 1194 cells that Notch1-IC contributes to the phenotype of intermittent induction of proliferation. It is not clear whether Notch1-IC contributes to proliferation or cell survival in this context. In mice, constitutively active Notch1 can render thymocytes resistant to glucocorticoid-induced apoptosis (8) and appears to inhibit apoptosis in erythroleukemia cells (58). Recently, it has been demonstrated that Notch1 interacts with Nur77 and protects T cells from Nur77-dependent apoptosis induced by T-cell receptor cross-linking (26). Therefore, it is tempting to speculate that Notch1-IC also has an antiapoptotic effect in B cells and that the proliferative effect is conferred by LMP1 (35).
In the absence of EBNA2 and mNotch1-IC, the number of LMP1-expressing living cells remained constant for 4 days, at which time the viability of the cells had already started to decrease. This indicates that the actual cell numbers reflect an equilibrium between renewal and cell death. Thus, LMP1 not only promotes cell survival through induction of antiapoptotic genes such as bcl-2 and A20 (12, 20, 37), it also triggers some cells to proliferate while other cells are dying. This is in accordance with the observations that LMP1 can induce DNA synthesis in resting B cells (51), has an impact on cell cycle regulation (cyclin D2) (2), induces increased thymidine incorporation in estrogen-deprived EREB2-5 cells (70), and is required for EBV-induced B-cell proliferation (30a, 35).
In summary, we have shown that activated Notch1 is unable to substitute for EBNA2 in the maintenance of proliferation of EBV-immortalized cells, most likely because it is unable to maintain LMP1 and c-myc expression. Possibly the inactivation of EBNA2 also leads to reduced expression of EBNA3A, -3C, and -LP. EBNA3A and -3C have been shown to be essential for B-cell immortalization (63) and EBNA-LP cooperates with EBNA2 in inducing quiescent cells to progress from G0 to G1(59). It remains to be seen whether other members of the Notch family, notably Notch2, may better substitute for EBNA2 than activated Notch1.
Notch2 was shown to be overexpressed in Hodgkin's lymphoma cells (29). EBV-positive Hodgkin's lymphoma cells express LMP1 and LMP2 in the absence of EBNA2. A synergistic effect of LMP1 and Notch2 may induce proliferation of Hodgkin's lymphoma cells as a crucial step in the development of Hodgkin's disease. In certain B-cell lines (Raji and BCBL1) and in the spleen, Notch2 rather than Notch1 has been found to be expressed (25, 67). It will therefore be interesting to see whether Notch2 has a higher potential to substitute for EBNA2 in EBV-induced B-cell proliferation than Notch1.
ACKNOWLEDGMENTS
We thank David Thorley-Lawson for kindly providing the anti-LMP1 antibody S12 and Bettina Kempkes for providing the cell line EREB2-5. We are grateful to Berit Jungnickel for critically reading the manuscript.
This work was supported by Die Deutsche Forschungsgemeinschaft (Str 461/1-1; Forschergruppe Multiprotein-Komplexe in der Genexpression, and SFB 455) and Fonds der Chemischen Industrie.
FOOTNOTES
- Received 24 August 2000.
- Accepted 1 December 2000.
- Copyright © 2001 American Society for Microbiology
