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Nasopharyngeal carcinoma is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded LMP1 has cell transformation property. It suppresses cellular senescence and enhances cell survival in various cell types. Many of the downstream events of LMP1 expression are mediated through its ability to activate NF-kappaB. In this study, we report a novel function of LMP1 to induce Id1 expression in nasopharyngeal epithelial cells (NP69) and human embryonal kidney cells (HEK293). The Id1 is a basic helix-loop-helix (bHLH) protein and a negative transcriptional regulator of p16(INK4a). Expression of Id1 facilitates cellular immortalization and stimulates cell proliferation. With the combination of both specific chemical inhibitors and genetic inhibitors of cell signaling, we showed that induction of Id1 by LMP1 was dependent on its NF-kappaB activation domain at the carboxy-terminal region, CTAR1 and CTAR2. Induction of Id1 by LMP1 may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may promote their malignant transformation.
Oncogene (2004) 23, 44884494
& 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00www.nature.com/oncSHORT REPORTEpsteinBarr virus latent membrane protein 1 (LMP1) upregulates Id1
expression in nasopharyngeal epithelial cellsHM Li1, ZH Zhuang1, Q Wang1, JCS Pang3, XH Wang1, HL Wong1, HC Feng1, DY Jin2,
MT Ling1, YC Wong1, AG Eliopoulos4, LS Young4, DP Huang3 and SW Tsao*,11Department of Anatomy, The University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, China; 2Department of Biochemistry,
Laboratory Block, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, China;3Department ofAnatomical and Cellular Pathology and Institute of Molecular Oncology, The Chinese University of Hong Kong, Prince of Wales
Hospital, Shatin, New Territories, Hong Kong SAR, China;4Institute for Cancer Studies, University of Birmingham, Edgbaston,
Birmingham B15 2TT, UKNasopharyngeal carcinoma is closely associated with
EpsteinBarr virus (EBV) infection. The EBV-encoded
LMP1 has cell transformation property. It suppresses
cellular senescence and enhances cell survival in various
cell types. Many of the downstream events of LMP1
expression are mediated through its ability to activate NF-
jB. In this study, we report a novel function of LMP1 to
induce Id1 expression in nasopharyngeal epithelial cells
(NP69) and human embryonal kidney cells (HEK293).
The Id1 is a basic helixloophelix (bHLH) protein and a
negative transcriptional regulator of p16INK4a. Expression
of Id1 facilitates cellular immortalization and stimulates
cell proliferation. With the combination of both specic
chemical inhibitors and genetic inhibitors of cell signaling,
we showed that induction of Id1 by LMP1 was dependent
on its NF-jB activation domain at the carboxy-terminal
region, CTAR1 and CTAR2. Induction of Id1 by LMP1
may facilitate clonal expansion of premalignant nasopharyngeal epithelial cells infected with EBV and may
promote their malignant transformation.Oncogene (2004) 23, 44884494. doi:10.1038/sj.onc.1207580
Published online 5 April 2004Keywords: EpsteinBarr virus; nasopharyngeal carcinoma; LMP1; Id1; nuclear factor kBNasopharyngeal carcinoma (NPC) is a common cancer
among Southern Chinese. The incidence rate of NPC in
Hong Kong is 25 times higher than other parts of the
world (Jeannel et al., 1999). NPC is characterized by a
high incidence of lymph node metastasis (Sham et al.,
1990) and is closely associated with EpsteinBarr virus
(EBV) infection (Raab-Traub, 2002). The EBV-encoded
latent membrane protein-1 (LMP1) is essential for EBV-
induced immortalization of B lymphocyte (Kaye et al.,1993) Expression of LMP1 transforms Rat-1 broblasts
(Wang et al., 1985) and induces various phenotypic
changes in epithelial cells (Dawson et al., 1990)
including loss of contact inhibition, anchorage-independent growth in soft agar and the ability to grow in
reduced serum concentrations. LMP1 expression is
common in EBV-positive NPC and may play a role in
its development (Fahraeus et al., 1988; reviewed by Tsao
et al., 2002a). LMP1 is an integral membrane protein
consisting of a short N-terminal cytoplasmic domain, a
six-transmembrane spanning domain and a 200-residue-
long carboxy-terminal (C-terminal) cytoplasmic domain
(reviewed by Eliopoulos and Young, 2001). Aggregation
or oligomerization of LMP1 in the membrane is
essential for the activation of cell signaling. LMP1
functions as a constitutively active tumor necrosis factor
receptor (TNFR), activating a number of signaling
pathways in a ligand-independent manner (reviewed by
Hatzivassiliou and Mosialos, 2002). Two distinct functional domains have been identied within the C-
terminal regions: C-terminal activation regions 1 and 2
(CTAR 1 and CTAR2). CTAR1 (residue 194231) has
been shown to initiate cell proliferation and CTAR2
(residue 351386) is essential for permanent lymphoblastoid cell lines outgrowth. Several major signaling
pathways from the C-terminal regions of LMP1 have
been identied including nuclear factor kB (NF-kB); the
mitogen-activated protein kinase (MAP kinase) family
(extracellular signal-regulated kinases (ERK), p38, c-
Jun N-terminal kinases (JNK)), the Janus-activated
kinase (JAK)/signal transducer and activation of
transcription (STAT) and recently the phosphatidylinositol 3-kinase (Dawson et al., 2003).LMP1 is a potent activator of the NF-kB signaling
pathway in cells (Hatzivassiliou and Mosialos, 2002).
NF-kB activation mediates many downstream events of
LMP1 expression including upregulation of antiapoptotic gene products (Bcl-2, B-1, A20 and cIAP);
proinammatory cytokines (IL6 and IL8); cell surface
antigens (CD40, CD54 and CD95); and angiogenesis
factors (VEGF and COX2). Both CTAR1 and CTAR2
domains of LMP1 are responsible for the activation of
NF-kB. Suppression of NF-kB activation results in*Correspondence: SW Tsao, Department of Anatomy, University of
Hong Kong, Room L01-53, Laboratory Block, Faculty of Medicine
Building, 21 Sassoon Road, Hong Kong;E-mail: [email protected] 30 August 2003; revised 20 January 2004; accepted 28 January
2004; Published online 5 April 2004Latent membrane protein 1 (LMP1) upregulates Id1
HM Li et al4489spontaneous apoptosis of EBV-transformed lymphoblastoid cell lines (Cahir-Mcfarland et al., 2000).NPC is closely associated with the EBV infection. The
EBV infection may facilitate the transformation of
premalignant nasopharyngeal epithelial cells into invasive cancer cells (Pathmanathan et al., 1995). We have
previously shown that LMP1 stimulated proliferation
and induced anchorage-independent growth of an
immortalized nasopharyngeal epithelial cell line
(NP69) (Tsao et al., 2002a; Lo et al., 2003). LMP1-
expressing NP69 cells have a longer survival time upon
withdrawal of growth factors (Lo et al., 2003). In this
study, we have investigated some of the downstream
events mediating the cell proliferation and survival
ability of LMP1-expressing NP69 cells. We have
previously shown that overexpression of Id1 stimulated
proliferation in NPC cells (Wang et al., 2002). There are
many similarities in the biological properties between
LMP1 and Id1, notably in the enhancement of cell
survival and suppression of senescence. We hypothesize
that Id1 expression may be induced by LMP1 in
immortalized nasopharyngeal epithelial cells. Indeed,
Western blotting analysis conrmed that the Id1 protein
was upregulated by 34 folds in the NP69 cells stably
expressing LMP1 (NP69 pLNSX LMP1) compared with
the parental NP69 cells or NP69 cells infected with the
empty vector (NP69 pLNSX) (Figure 1a and b). A
reduction of p16INK4a expression level was also observed
in LMP1-expressing NP69 cells. We have previously
reported that Id1 expression in NPC cells downregulated the Rb/p16INK4a pathway (Wang et al., 2002). This
is in agreement to our previous study in rodent
broblasts, which showed that inactivation of the
CDK4/p16INK4a regulatory pathway was a downstream
event of LMP1 (Yang et al., 2000). The p16INK4a protein
regulates cell cycle G1/S entry and is largely responsible
for the onset of replicative senescence in human
epithelial cells (Ohtani et al., 2001). Induction of Id1
expression by LMP1 may be involved in the suppression
of p16INK4a expression in the nasopharyngeal epithelial
cells. A recent report showed that LMP1 blocks theFigure 1 Induction of Id1 expression by LMP1 in NP69 cells and
HEK293 cells. NP69 is an immortalized nasopharyngeal epithelial cell
line. It was established by transfection of a SV40T expression plasmid
into primary epithelial outgrowth of nonmalignant nasopharyngeal
tissue (Tsao et al., 2002b). NP69 is nontumorigenic in athymic nude
mice and retains many differentiated properties of normal nasopharyngeal epithelial cells. The NP69 cells were propagated in the Dened
Keratinocyte-SFM supplemented with growth factors (Gibco, Grand
Island, NY, USA) and maintained at 371C with 5% CO2 in air.
HEK293 is an immortalized human embryonal kidney cells. It was
used in the transient expression of the LMP1. The HEK293 cells were
maintained at 371C with 5% CO2 in the DME medium (Sigma, St
Louis, MO, USA) supplemented with 5% FBS. (a) Western blotting
analysis of Id1 and p16 in LMP1-stably expressing NP69 (NP69-
pLNSX-LMP1) cells. NP69 cells are the original parental cells.
Constitutive expression of LMP1 in NP69 cells was achieved by
retroviral expression of an NPC-derived LMP1. The construction of
the LMP1 expression vectors had been previously described (Yang
et al., 2000). NP69 cells infected with the empty vector, pLNSX, were
also established as a negative control. Expression of LMP1 in NP69-
pLNSX-LMP1 cells was conrmed. Detailed experimental procedures
of the Western blotting have been described previously (Wang et al.,
2003). The expression of Id1, p16, LMP1 and actin in this experiment
and hereafter were detected by their respective primary antibodies: Id1
(C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p16 (N-20,
Santa Cruz Biotechnology, Santa Cruz, CA, USA), LMP1 (CS 1-4,
DakoCytomation, Glostrup, Denmark or OT22C, Organon Teknika,
Boxtel, the Netherlands) and actin (C-11, Santa Cruz Biotechnology,
Santa Cruz, CA, USA) for 1 h at room temperature. The secondary
antibody used was 1 : 3000 dilution of horseradish peroxidase-linked
secondary antibodies (Cell Signaling Technology, Beverly, MA, USA),
and the proteins bound by the antibodies were detected by the
chemiluminescence reagent ECL Plus (Amersham, Buckinghamshire,
UK). (b) The levels of Id1 expression in cells were quantitated by a
scanning densitometer. Expression of Id1 was normalized against the
actin level. Control expression of Id1 in NP69 cells was set as 1. (c)
Induction of Id1 expression by transiently expression of 2117-LMP1 in
HEK293 cells incubated in the medium with 1% FBS. Genomic
sequence of the prevalent variant of LMP1 (2117-LMP1) in NPC
tumor in Hong Kong was PCR amplied and cloned into the EcoRI
sites of pcDNA3 vector (Invitrogen, Carlsbad, CA, USA). The entire
LMP1 sequence after cloning was conrmed to be inframe by DNA
sequencing. The expression of LMP1 after transfection was examined
by Western blotting. The efciencies of both LMP1 expression vectors
in activating cell signaling were also conrmed by the luciferase
reporter assay for JNK (data not shown) and NF-kB activation. At
24 h before transfection, the HEK293 cells were plated onto a six-well
plate with 3 104 cells per well. The FuGENE 6 Transfection Reagent
(Roche, Indianapolis, IN, USA) was used for transient transfections
according to the manufacturers protocol. Amounts of 1 mg each of
pcDNA3 and pcDNA3 2117-LMP1 were transiently transfected in
HEK293 cells. After transfection, HEK293 cells were incubated in the
DME medium HEPES Modication (Sigma, St Louis, MO, USA) for
48 h with 1% dialysed FBS unless otherwise stated and analysed for
LMP1 and Id1 expression by Western blotting. (d) Expression of Id1 in
the transfected HEK293 was quantitated by a densitometer. The
means of three separate experiments are shownOncogeneLatent membrane protein 1 (LMP1) upregulates Id1HM Li et al4490p16INK4a pathway in human broblasts through the
CRM1-dependent nuclear export of Ets2 (Ohtani et al.,
2003). Ets2 is an important transcription factor for
p16INK4a gene expression. Nuclear export of Ets2 may
reduce the expression of p16INK4a in cells. In that study,
downregulation of p16INK4a by LMP1 via nuclear export
of Ets2 was, however, not observed in a human
osteosarcoma cell line EH1, suggesting that multiple
regulatory mechanisms are involved in the LMP1-
induced blockage of p16INK4a/Rb pathway.We then investigated if transient expression of LMP1
could also induce Id1 expression. Owing to the low
transfection efciency of NP69 cells (around 5%, data
not shown), we used the human embryonal kidney
HEK293 cells as recipient cells to achieve a higher
transfection efciency of LMP1 cloned in pcDNA
expression vector (Invitrogen). The HEK293 cells have
been used extensively in the study of LMP1 signaling.
Using a GFP expression vector (Clontech) as a
transfection marker, we were able to achieve over 80%
efciency of transfection in HEK293 cells (data not
shown). Upregulation of Id1 expression after transfection with the LMP1-expressing plasmid was consistently
observed in HEK293 cells (Figure 1c and d). The
experiments have been repeated several times to conrm
the ability of LMP1 to induce Id1 expression in
HEK293 cells.We then investigated the possible signaling mechanism involved in the induction of Id1 expression by
LMP1 using specic cell signaling inhibitors. A panel of
chemical inhibitors of cell signaling was used to examine
their effects on the Id1 expression in NP69 cells
constitutively expressing LMP1. The duration of treatment and concentration of these chemical inhibitors had
been previously optimized. The expression of Id1 was
effectively inhibited by BAY 11-7085, which could
inhibit NF-kB activation (Figure 2a and b) but not by
PD098059 and SP600125, inhibitors of MAPkinase and
SAPkinase respectively (Figure 2c and d). On the other
hand, potential transcription factor binding sites in the
50 region of human Id1 sequence were searched with
MatInspector 2.2. based on the TRANSFAC 4.0
transcription factor database (Quandt et al., 1995).
The human Id1 promoter sequence (GenBank Accession
Number U57645) (Nehlin et al., 1997) upstream of the
TATA box (position 130) was used as the input. The
search was limited to the vertebrate transcription factor
binding sites, and only those sequences that scored equal
or higher than the matrix similarity threshold (0.85) and
core similarity threshold (0.75) in the output were
treated as the potential binding sites. Two potential
NF-kB binding sites (Zabel et al., 1991) were found in
the human Id1 promoter at position 1110 and 1947.
The involvement of NF-kB activation in the expression
of Id1 was further conrmed by cotransfecting the
genetic inhibitor of NF-kB, IkB (S32A/S36A), with
LMP1 expression vector into HEK293 cells. Cotransfection of IkB (S32A/S36A) mutant, which specically
inhibits activation of NF-kB, suppressed Id1 expression
induced by LMP1 (Figure 2e). Suppression of Id1
expression was not observed in cotransfection experiments of LMP1 with dominant-negative mutants of
MAP kinase (pcDNA3.1c XP-MEK1) and SAPkinase
(pEBG SEK (KR)). On the other hand, activation of
NF-kB by the IKKa expression in turn upregulated the
expression of Id1 in HEK293 cells (Figure 2f). These
observations suggest that activation of NF-kB is the
major signaling event mediating induction of Id1
expression by LMP1.NF-kB activation is a major cellular signaling pathway of LMP1, which mediates many downstream events
of LMP1. The CTAR1 and CTAR2 are the major
domains of LMP1 involved in NF-kB activation
(Eliopoulos and Young, 2001). We next examined their
involvement in the induction of Id1 expression by
transfecting the wild-type LMP1 and mutant LMP1
decient in CTAR1 and/or CTAR2 expression into
HEK293 cells and examined for their ability to induce
the Id1 expression. The LMP1 mutants used were
pSG5_B95.8-LMP1 AxAxA (decient at CTAR1 region), pSG5_B95.8-LMP1 378 STOP (decient in
CTAR2 region) and pSG5_B95.8-LMP1 AxAxA/378
STOP (decient in both CTAR1 and CTAR2 regions).
Their intracellular signaling abilities have been characterized in a previous study (Eliopoulos et al., 1999).
LMP1 mutants with deciency in either CTAR1 or
CTAR2 regions were still capable of inducing Id1
expression. However, LMP1 mutants decient in both
CTAR1 and CTAR2 (Figure 3a and b) failed to induce
Id1 expression. These results indicated that deletion of
both regions were required to abolish the LMP1-
mediated Id1 expression and was concordant with
previous studies that both CTAR1 and CTAR2 were
involved in activation of NF-kB (reviewed by Eliopoulos and Young, 2001).Variations in LMP1 sequences are common in EBV-
associated diseases. LMP1 natural sequence variants
differ in their potential to activate cellular signaling
pathways (Fielding et al., 2001). We then compared the
ability of two LMP1 variants, 2117-LMP1 and B95.8-
LMP1, to induce the Id1 expression and activate NF-kB
signaling. The 2117-LMP1 is the prevalent LMP1
variant isolated from NPC of Hong Kong Chinese
(Cheung et al., 1998; GenBank Accession Number
AY519199). It carries a 30 bpdeletion in the C-terminal
and has multiple substitutions at various codons in the
transmembrane domain and signaling domains of C-
terminal. The B95.8-LMP1 (GenBank Accession Number X01995) is the prototypic LMP1 isolated from a case
of infectious mononucleosis. Both the genomic 2117-
LMP1 and B95.8-LMP1 were cloned into identical
expression vector, pcDNA3. The sequences of both
LMP1 expression vectors had been determined to ensure
the correct reading frame in both expression vectors
(data not shown). The 2117-LMP1 was more potent
than the B95-8 LMP1 to activate the Id1 expression.
The enhanced activity of 2117-LMP1 to activate the Id1
expression was closely related to its activity of NF-kB
activation. The optimal dose of LMP1 plasmid used in
the activation of Id1 was correlated with the optimal
dose in the activation of NF-kB detected by a luciferase
reporter system and the phosphorylation status of IkBaOncogeneLatent membrane protein 1 (LMP1) upregulates Id1
HM Li et al4491Figure 2 Effects of cell signaling inhibitors on the expression of Id1 in LMP1-expressing cells. The effects of cell signal inhibition on
the Id1 expression were examined by applying both chemical inhibitors and genetic inhibitors of major cell signaling pathways that can
be activated by LMP1. Inhibition of cell signaling in NP69 cells expressing LMP1 (NP69 pLNSX LMP1) was achieved by specic
chemical inhibitors. The cells were plated at 3 104 cells per well in a six-well plate 24 h before treatment with chemical inhibitors. The
cell signaling inhibitors used were: BAY 11-7085 (Calbiochem, San Diego, CA, USA) (Pierce et al., 1997) for NF-kB inhibition,
PD098059 (Calbiochem, San Diego, CA, USA) for MAPkinase inhibition and SP600125 (Calbiochem, San Diego, CA, USA) for
SAPkinase inhibition. Ascending concentrations of the respective cell signaling inhibitors were used with reference to their optimum
dose and duration of treatment. The effects of the chemical inhibitors on the cells were evaluated with the Western blotting against
phospho-IkBa (5A5, Cell Signaling Technology, Beverly, MA, USA), phospho-MEK1/2 (Cell Signaling Technology, Beverly, MA,
USA) and phospho-c-jun (KM-1, Santa Cruz Biotechnology, Santa Cruz, CA, USA) respectively. Inhibition of cell signals in HEK293
cells was achieved by transfecting the HEK293 cells with genetic inhibitors specic for activation of NF-kB (pUSEamp( )IkB (S32A/
S36A)) (Upstate, Waltham, MA, USA) (Beg et al., 1992), MAPkinase (pcDNA3.1c XP-MEK1) (Cowley et al., 1994) and SAPkinase
(pEBG SEK (KR)) (Sanchez et al., 1994). (a) Different concentrations (0, 5, 10, 20 mm) of BAY 11-7085 were added to inhibit
specically NF-kB in NP69-pLNSX-LMP1 cells. (b) Densitometer scanning of the Id1 expression in NP69 LMP1 cells treated by
different dosages of BAY 11-7085 was shown. All results represent three independent experiments and the error bars indicate standard
error of the means. (c) Different concentrations (0, 25, 50, 75 mm) of PD098059 were added to inhibit specically MAPkinase in NP69-
pLNSX-LMP1 cells. (d) Different concentrations (0, 5, 10, 20 mm) of SP600125 were added to inhibit specically SAPkinase in NP69-
pLNSX-LMP1 cells. Actin protein level was detected as a loading control. (e) The effects of inhibition of NF-kB, MAPkinase and
SAPkinase on LMP1-induced Id1 expression were examined by cotransfection of 0.5 mg of pcDNA3 2117-LMP1 together with 0.5 mg
of pUSEamp( )IkB (S32A/S36A), pcDNA3.1c XP-MEK1 and pEBG SEK (KR) respectively into HEK293 cells. The Id1 expression
was determined by Western blotting analysis. The chart shows the relative levels of Id1 protein in the cells by densitometer scanning. (f)
Upregulation of Id1 through activation of NF-kB. In order to study whether activation of NF-kB pathway could upregulate Id1,0.8 mgof IkB kinase a (HA-IKKa) (DiDonato et al., 1997) was transfected into HEK293 cells. Transfection of 0.8 mg pcDNA3 was
applied as a negative control. Western blotting analysis of Id1 in the transfected cells was shown. Actin protein level was detected as a
loading controlOncogeneLatent membrane protein 1 (LMP1) upregulates Id1HM Li et al4492Figure 3 Effects of the expression of LMP1 with mutations at
CTAR1 and CTAR2 regions on the Id1 expression. The prototype
LMP1 sequence (B95.8-LMP1) was originated from infectious
mononucleosis patients, and the construction and properties of
pSG5 B95.8-LMP1 (wt), pSG5 B95.8-LMP1 AxAxA/378 STOP,
pSG5 B95.8-LMP1 AxAxA, pSG5 B95.8-LMP1 378 STOP had
been previously described (Eliopoulos et al., 1999). Amounts of0.1 mg each of the wild type and the mutant B95.8-LMP1 were
transfected into HEK293 cells respectively. The pSG5 empty vector
was also added to make the total amount of DNA in each
transfection as 1 mg. (a) Western blotting analysis of Id1 in the
transfected HEK293 cells was shown. Actin protein level was
detected as a loading control. (b) Densitometer measurement of Id1
expression induced by wild-type LMP1 and mutant LMP1(Figure 4). The lowered ability of LMP1 to induce the
Id1 expression at higher doses (1.5 mg of plasmid) may
be related to the cytotoxicity of LMP1 expression at
high concentrations (Hammerschmidt et al., 1989). The
cytotoxicity of LMP1 at high level can be inferred from
the decrease in the expression of b-Gal gene used as a
control to normalize the variation in transfection
efciencies among each sample (Table 1).All the above results in this report support the
conclusion that induction of Id1 expression by LMP1
is likely to be mediated by the activation of NF-kB. The
signicance of LMP1 induction in nasopharyngeal
epithelial cells is not fully understood at this stage. Id1
has been shown to be upregulated in cells that had
overcome replicative senescence and also in cells with
enhanced survival in growth factor-deprived environment. Id1 expression could extend the lifespan of human
keratinocytes and, in some cases, immortalize them
(Alani et al., 1999). We have also shown that the Id1
expression itself could induce MAPkinase and cell
proliferation in prostatic carcinoma cells (Ling et al.,
2002). Recently, we have also reported that LMP1
expression in an immortalized nasopharyngeal epithelial
cell line (NP69) could stimulate an array of genes
involved in cell survival (Lo et al., 2003). LMP1
expression could be detected in the premalignant
nasopharyngeal lesions (Pathmanathan et al., 1995).
LMP1-induced Id1 expression may stimulate cell proliferation, enhancing cell survival and escape from
cellular senescence, so that may confer selective growth
advantage in nasopharyngeal epithelial cells.The relationshipbetween LMP1 and Id1 expression in
NPC is likely to be complex and deserves further study
to fully understand its signicance. Immunocytochemistry staining of LMP1 in NPC biopsies generally
revealed a low and heterogeneous expression level
(reviewed in Tsao et al., 2002a). Multiple cellular
pathways are believed to be involved in the activation
of Id1 in cancer cells. Overexpression of Id1 is
commonly detected in non-EBV-associated malignancies (Sikder et al., 2003). We postulate that advanced
NPC may be less dependent on the Id1 induction by
LMP1 as they commonly acquire additional genetic
alterations, for example, p16 and RASSF1A inactivation, which facilitate survival of cells and their escape
from senescence. LMP1 expression is commonly detected in premalignant lesions of NPC and has been
postulated to play a role in transformation of premalignant nasopharyngeal epithelial cells into invasive
cancer cells (Pathmanathan et al., 1995). LMP1 induction of Id1 expression may have a more signicant role
at the early stage of NPC development. Our ndings
support a role of LMP1 in the transformation of
premalignant epithelial cells into invasive cancer cells
at the early stage of NPC development.In summary, we have identied a novel function
of LMP1 in the induction of Id1 expression in
human nasopharyngeal epithelial cells through
activation of NF-kB, and the CTAR1 and CTAR2
regions of the LMP1 are involved in the induction of
Id1. Our study supports a role of LMP1 in cellular
immortalization and transformation of premalignant
nasopharyngeal epithelial cells at an early stage of
carcinogenesis.AbbreviationsNPC, nasopharyngeal carcinoma; EBV, EpsteinBarr virus;
LMP1, latent membrane protein 1; Id, inhibition of DNAOncogeneLatent membrane protein 1 (LMP1) upregulates Id1
HM Li et al4493Figure 4 Comparison of 2117-LMP1 and B95.8-LMP1 in the induction of Id1 and NF-kB in HEK293 cells. (a) Different
concentrations (0, 0.1, 0.5, 1.5 mg) of pcDNA3 2117-LMP1 plasmids and pcDNA3 B95.8-LMP1 expression plasmids were used in the
transient transfection of HEK293 cells. pcDNA3 was added to equalize the total amount of DNA to 1.5 mg in each transfection.
Western blotting analysis of LMP1, phospho-IkBa (p-IkBa) and Id1 in the transfected HEK293 cells was shown. Actin protein level
was detected as a loading control. (b) Quantitation of LMP1 expression in transfected cells. (c) Quantitation of phospho-IkBa
expression in transfected cells. (d) Quantitation of Id1 expression in transfected cells. (e) Comparison of NF-kB activation by 2117-
LMP1 and B95.8-LMP1. A total of 1 106 HEK293 cells were cotransfected with NF-kB luciferase reporter (3Enh.kBconA-Luc
reporter) and with 0.1, 0.5 and 1.5 mg pcDNA3 2117-LMP1 and pcDNA3 B95.8-LMP1 using a standard calcium phosphate technique
previously described (Blake et al., 2001). The RSV-b-Gal vector was also transfected as control to normalize the transfection efciency.
At 36 h post-transfection, cells were lysed in reporter lysis buffer and subjected to luciferase assay by luminometer. The results
represent the means of three independent experiments relative to b-Gal controlOncogeneLatent membrane protein 1 (LMP1) upregulates Id1HM Li et al4494Table 1 Effects of different concentrations of 2117-LMP1 and B95.8-LMP1 in NF-kB and b-Gal activitiesPlasmid transfected Luciferase mean b-Gal mean Relative normalized NF-kB activitypcDNA3 6018 5202 10.15 mg 2117-LMP1 611430 3694 1430.5 mg 2117-LMP1 813928 2837 2481.5 mg 2117-LMP1 439576 2496 1520.15 mg B95.8-LMP1 339179 3300 890.5 mg B95.8-LMP1 314063 3019 901.5 mg B95.8-LMP1 181087 2891 54The raw mean readings of NF-kB and b-Gal activities affected by different concentrations of 2117-LMP1 and B95.8-LMP1 as shown in Figure 4e
were listed. The s.d. was shown in Figure 4ebinding or differentiation; NF-kB, nuclear factor kB; IkBa,
inhibitor of kB alpha; FBS, fetal bovine serum.AcknowledgementsWe are grateful to Dr Zhenguo Wu at Hong Kong University
of Science and Technology for providing PD098059, Dr
Leonard I. Zon at Harvard Medical School for providing
pEBG SEK (KR), and Dr Michael Karin at University of
California-San Diego for providing HA-IKKa. This project
was supported by the Research Grant Council, Hong Kong
(HKU7356/02M) and a research retreat grant (Faculty of
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