Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells.

Abstract

Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumor-suppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-kappaB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASSF1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.

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ORIGINAL ARTICLE
Latent membrane protein 1 suppresses RASSF1A expression, disrupts
microtubule structures and induces chromosomal aberrations in human
epithelial cells
C Man1, J Rosa6, LTO Lee4, VHY Lee4, BKC Chow4, KW Lo5, S Doxsey6, ZG Wu7, YL Kwong2,
DY Jin3, ALM Cheung1and SW Tsao1
1Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, People’s
Republic of China;2Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
SAR, People’s Republic of China;3Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong,
Pokfulam, Hong Kong SAR, People’s Republic of China;4Department of Zoology, Faculty of Science, The University of Hong Kong,
Pokfulam, Hong Kong SAR, People’s Republic of China;5Department of Cellular and Anatomical Pathology, Chinese University of
Hong Kong, Shatin, Hong Kong SAR, People’s Republic of China;6Department of Molecular Medicine, University of Massachusetts
Medical School, Worcester, MA, USA and7Department of Biochemistry, Hong Kong University of Science and Technology, Clear
Water Bay, Hong Kong SAR, People’s Republic of China
Epstein–Barr virus (EBV) infection is closely associated
with nasopharyngeal carcinoma (NPC) and can be
detected in early premalignant lesions of nasopharyngeal
epithelium. The latent membrane protein 1 (LMP1) is an
oncoprotein encoded by the EBV and is believed to play a
role in transforming premalignant nasopharyngeal epithe-
lial cells into cancer cells. RASSF1A is a tumor-
suppressor gene commonly inactivated in many types of
human cancer including NPC. In this study, we report a
novel function of LMP1, in down-regulating RASSF1A
expression in human epithelial cells. Downregulation of
RASSF1A expression by LMP1 is dependent on the
activation of intracellular signaling of NF-jB involving
the C-terminal activating regions (CTARs) of LMP1.
LMP1 expression also suppresses the transcriptional
activity of the RASSF1A core promoter. RASSF1A
stabilizes microtubules and regulates mitotic events.
Aberrant mitotic spindles and chromosome aberrations
are reported phenotypes in RASSF1A inactivated cells. In
this study, we observed that LMP1 expression in human
epithelial cells could induce aberrant mitotic spindles,
disorganized interphase microtubules and aneuploidy.
LMP1 expression could also suppress microtubule dy-
namics as exemplified by tracking movements of the
growing tips of microtubules in live cells by transfecting
EGFP-tagged EB1 into cells. The aberrant mitotic
spindles and interphase microtubule organization induced
by LMP1 could be rescued by transfecting RASSF1A
expression plasmid into cells. Downregulation of RASS-
F1A expression by LMP1 may facilitate its role in
transformation of premalignant nasopharyngeal epithelial
cells into cancer cells.
Oncogene (2007) 26, 3069–3080. doi:10.1038/sj.onc.1210106;
published online 13 November 2006
Keywords: Epstein–Barr virus; LMP1; microtubules;
RASSF1A; NF-kB; nasopharyngeal epithelial cells
Introduction
Nasopharyngeal carcinoma (NPC) is a common cancer
in Southern China. The incidence rate among ethnic
Cantonese living in this region is approximately 30-fold
higher compared to incidence rate worldwide. The
histopathological type of NPC in Southern China is
mainly of the undifferentiated type, and is closely
associated with Epstein–Barr virus (EBV) infection.
Ethnical clustering of NPC strongly suggests the inter-
play of genetic susceptibility and environmental factors
in its development (Lo and Huang, 2002). Genetic
predisposition to NPC among Southern Chinese may
confer cellular susceptibility to transformation actions
of EBV encoded oncogenes.
Latent infection of EBV in premalignant nasopharyn-
geal lesions was shown to be an early event in the
development of NPC (Pathmanathan et al., 1995; Lo
et al., 2004b). The EBV encoded latent membrane
protein 1 (LMP1) has been shown to be capable of
transforming in vitro rodent fibroblasts into tumorigenic
cells (Wang et al., 1985). Two domains in the cytoplas-
mic carboxyl tail of LMP1 (CTAR1 and CTAR2), are
essential for activation of multiple cellular signaling
pathways, notably the NF-kB pathway (Eliopoulos and
Young, 2001; Tsao et al., 2002a), which mediates many
downstream pathogenic properties of LMP1.
The p53 and Rb tumor-suppressor genes are crucial in
cell cycle regulation checkpoints and are common targets
Received 18 April 2006; revised 21 September 2006; accepted 29
September 2006; published online 13 November 2006
Correspondence: Professor SW Tsao, Department of Anatomy, The
University of Hong Kong, L01-53, Laboratory Block, Li Ka Shing
Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong
Kong SAR, People’s Republic of China.
E-mail: gswtsao@hkucc.hku.hk
Oncogene (2007) 26, 3069–3080
& 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00
www.nature.com/onc

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of inactivation by viral oncogenes, including large T
antigen of SV40 virus (De Luca et al., 1997) and E6 and
E7 of human papilloma virus (Balsitis et al., 2005).
However, functional interactions of EBV oncogenes with
tumor-suppressor genes during NPC development are
unclear. We have previously identified multiple potential
loci of NPC, for tumor-suppressor genes using high-
resolution allelotyping and array comparative genomic
hybridization. The highest frequencies of allelic losses were
identified in chromosomes 3p (96.3%) and 9p (85.2%) (Lo
et al., 2004b). RASSF1A is a potential tumor-suppressor
gene residing on chromosome 3p21, which is commonly
inactivated in NPC by allelic deletion and promoter
methylation (Chow et al., 2004, 2006). Allelic loss of
RASSF1A locus could be detected in microdissected
tissues from low- and high-grade dysplastic nasopharyn-
geal epithelial lesions (Lo and Huang, 2002; Chow et al.,
2004). RASSF1A is also commonly inactivated in other
human cancers including gastric cancer (Kang et al.,
2002), hepatocellular carcinoma (Yu et al., 2002) and
small-cell lung cancer (Dammann et al., 2001); suggesting
its common involvement in human carcinogenesis. Re-
constitution of RASSF1A expression in a NPC cell line,
C666-1, induced loss of viability, growth suppression,
diminishedinvasivenessand
growth properties (Chow et al., 2004). A lower expression
level of RASSF1A was detected in an immortalized
nasopharyngeal epithelial cell line expressing LMP1 (Tsao
et al., 2002b), which prompted us to investigate if LMP1
may regulate RASSF1A expression in cells.
An important function of RASSF1A is regulation of
microtubule functions (Liu et al., 2003). RASSF1A
colocalizes with cytoplasmic microtubules in interphase
cells. As cells enter prophase during mitosis, RASSF1A
is relocated to the centrosomes, then to the mitotic
spindles at metaphase and spindle poles at anaphase. At
cytokinesis, the protein is relocated to the mid-body
(Song et al., 2004). RASSF1A also stabilizes micro-
tubules (Rong et al., 2004), its expression protects cells
from the action of tubulin depolymerizing drug such as
nocodazole (Vos et al., 2004). Mouse embryonic
fibroblasts generated from RASSF1A null mice showed
increased sensitivity to nocodazole (Liu et al., 2003).
Inactivation of RASSF1A is associated with abnormal
mitotic spindle formation and chromosome instability
(Song et al., 2004; Vos et al., 2004)
In this study, we showed that LMP1 could downregulate
RASSF1A expression, in multiple epithelial cell types. We
have also examined the involvement of potential cellular
signaling pathways in the suppression of RASSF1A. Our
study reveals that downregulation of RASSF1A expression
by LMP1 induces abnormal microtubule function, result-
ing in aberrant mitotic spindles and chromosome instabi-
lity, hence facilitate tumorigenesis.
anchorage-independence
Results
LMP1 downregulates RASSF1A expression
The effect of LMP1 on RASSF1A expression was first
observed in an immortalized nasopharyngeal epithelial
cell line (NP69), which has been previously shown to be
highly sensitive to the transformation action of LMP1
(Tsao et al., 2002b). Western blotting analysis showed
that RASSF1A expression was downregulated in NP69
cells stably expressing LMP1 when compared with the
parental cell line (NP69) (Figure 1a). To confirm that
downregulation of RASSF1A expression in NP69LMP1
is not a result of selective growth of NP69LMP1 clones
with low RASSF1A expression, we next examined the
effectiveness of LMP1 to suppress RASSF1A expression
by transient transfection of LMP1 expression plasmid.
As the transfection efficiency of the immortalized NP69
cells is low, HeLa, HaCaT and SCC1F cells were chosen
as recipient cells because of their higher rates of
transient transfection (50–80%). The prevalent 2117-
LMP1 variant isolated from NPC patients in Hong
Kong (Cheung et al., 1998) was used in this part of the
study. The functional properties of this LMP1 variant
have been previously characterized (Lo et al., 2003,
2004a). Downregulation of RASSF1A could be readily
demonstrated in all the epithelial cell types transiently
transfectedwith the LMP1
(Figure 1b). Furthermore, downregulation of RASSF1A
by transient transfection of LMP1 was shown to be
dose-dependent in HeLa cells, confirming that LMP1 is
directly involved in the downregulation of RASSF1A
expression (Figure 1c). The concentrations of LMP1
plasmids used for transfection were 0.3–1.2mg/106cells,
which is within the normal range of LMP1 concentra-
tions used in cell signaling studies (Eliopoulos et al.,
1999b; Li et al., 2004). Suppression of RASSF1A
expression in LMP1 expressing cells could also be
demonstrated at the transcription level by semiquanti-
tative reverse transcription-polymerase chain reaction
(RT–PCR) (Figure 1d). Consistent with our previous
observations (Chow et al., 2004), RASSF1A mRNA was
undetectable in an EBV positive cell line, C666-1; but
high in HK1, an EBV-negative NPC cell line established
from a well-differentiated squamous carcinoma of NPC
specimen. RASSF1A mRNA could be detected in NP69,
SCC12F and HaCaT cells. The RASSF1A mRNA level
is highly reduced in NP69 cells stably expressing LMP1;
and in SCC12F and HaCaT cells after transient
expression of LMP1. Hence, downregulation of RASS-
F1A expression by LMP1 could be demonstrated at
both transcription and protein expression levels.
expressionplasmid
Downregulation of RASSF1A expression by LMP1
involves NF-kB activation
As aforementioned, LMP1 could activate multiple cell
signaling pathways in cells including MAPK, JNK, PI3K
and NF-kB. To explore the potential involvement of
these signaling pathways in mediating LMP1-induced
downregulation of RASSF1A, the effects of specific
inhibitors on RASSF1A expression were examined. The
concentrations and treatment times of these inhibitors
for these signaling pathways have been optimized in
previous studies (Li et al., 2004; Wu et al., 2006). The
effectiveness of each inhibitor in suppressing the kinase
activities of MAPK, JNK and PI3K was confirmed by
examining the phosphorylation status of their respective
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downstream targets (Figure 2A, a-c). Inhibition of NF-kB
activation was achieved by co-transfecting LMP1
with a genetic inhibitor of NF-kB (pUSE-IkBa S32/
S36). The IkBa S32/S36 mutant has two mutated
phosphorylation sites (S32 and S36), which resists
phosphorylation degradation and prevents activation
of NF-kB in LMP1 expressing cells. The effective
inhibition of NF-kB was confirmed by examining the
phosphorylation status of IkB (Figure 2B, a). Inhibition
of MAPK, JNK and PI3K pathways was not effective in
restoring LMP1-induced downregulation of RASSF1A
expression (Figure 2A, a-c). In contrary, co-transfection
of LMP1 with the NF-kB inhibitor (IkBa S32/S36)
effectively abolished the LMP1-induced downregulation
of RASSF1A in HeLa and HaCaT cells (Figure 2B, a).
In addition, activating NF-kB signaling by over-
expressing the IkB kinase alpha subunit (IKKa) led
to downregulation of RASSF1A expression, confir-
ming the involvement of NF-kB signaling pathway
(Figure 2B, b).
The C-terminal activation regions of LMP1 are required
to downregulate RASSF1A expression
The C-terminal activation regions 1 and 2 are regions of
LMP1 involved in the activation of NF-kB. We then
examined the ability of LMP1 variants with mutated
CTAR1 and/or CTAR2 regions to downregulate RASS-
F1A expression. The wild-type (wt) and mutant LMP1
plasmids used in this study were kindly provided by
Professor Lawrence Young, University of Birmingham,
UK; which include the pSG5 vector, pSG5B95.8 LMP1,
the pSG5-B95.8LMP1 AxAxA (mutated CTAR1),
pSG5-B95.8LMP1 378STOP (deleted in CTAR2) and
pSG5-B95.8LMP1 AxAxA/378STOP (mutated CTAR1
and deleted CTAR2). This set of mutants have been
used previously in defining the intracellular signaling of
LMP1 (Eliopoulos and Young, 2001; Li et al., 2004).
The experiments were performed in both HeLa and
HaCaT cells. In both cell types, mutation in CTAR1
and deletion in CTAR2 greatly diminished the suppres-
sion effect on RASSF1A downregulation (Figure 2C).
Figure 1
nasopharyngeal epithelial cell line (NP69) and NP69 stably transfected with LMP1 (NP69LMP1). (b) The prevalent 2117-LMP1
variant (pcDNA3.1-2117LMP1) was transiently transfected into HeLa, SCC12F and HaCaT cells. Immunoblot analysis was
performed after 48h of transfection. (c) pcDNA3.1-2117LMP1 expressing plasmid was transfected into HeLa cells with increasing
doses. Effect of LMP1-induced RASSF1A downregulation was determined by immunoblotting. Empty vector (pcDNA3.1) was added
to normalize the total amount of DNA across all transfections. (d) Detection of RASSF1A transcripts using semiquantitative
RT–PCR. An EBV negative cell line (HK1) and an EBV-positive cell line (C666-1) was included. b-actin was used as loading control.
(a–d) Results represent the means7s.d. of three independent experiments.
LMP1 downregulates RASSF1A expression. (a) RASSF1A expression was analysed by immunoblotting in an immortalized
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In HeLa cells, LMP1 mutants deficient in CTAR1 or
CTAR2 region still retained their abilities to down-
regulate RASSF1A (Figure 2C, left panel). In HaCaT
cells, CTAR1 mutant (pSG5-B95.8LMP1 AxAxA)
retained its ability to suppress RASSF1A expression.
However, theCTAR2mutant
378STOP) could not completely retain its ability to
suppress RASSF1A expression (Figure 2C, right panel);
which may be attributed to the stronger ability of
(pSG5-B95.8LMP1
CTAR2 to activate NF-kB signaling (Eliopoulos et al.,
1999b). The difference in response to CTAR1 and
CTAR2 in different cell types is not fully understood,
but may be related to the difference in endogenous
RASSF1A level in different cell types. The HaCaT cell
line has a higher endogenous level of RASSF1A
expression compared to HeLa cells (Figures 1b and
2c). These results conform to our previous observations
that LMP1 suppression of RASSF1A expression involve
Figure 2
LY294002) were used to inhibit ERK1/2, JNK and the Akt pathways in LMP1 expressing HeLa cells. The effects of inhibition were
determined by immunoblot analysis. Treatment conditions were previously optimized (Materials and method section). (B)(a) NF-kB
activation was perturbed by co-transfecting the genetic inhibitor of NF-kB (pUSE-IkBa S32/S36) and pcDNA3.1-2117LMP1 into
HeLa /HaCaT cells. The effect of NF-kB suppression on RASSF1A expression was detected by immunoblotting. (B)(b) NF-kB was
activated by overexpressing IKKa. The plasmid pRc-bactin-IKK plasmid was transfected into HeLa cells and the effect of NF-kB
activation on RASSF1A expression was shown by immunoblotting. (C) Effects of LMP1 variants with mutated CTAR1 and/or
CTAR2 regions on their abilities to downregulate RASSF1A expression. The empty vector pSG5, wt pSG5-B95.8LMP1, pSG5-
B95.8LMP1 AxAxA (mutated CTAR1), pSG5-B95.8LMP1 378STOP (deleted CTAR2) and the pSG5-B95.8LMP1 AxAxA/378STOP
(defective at both CTAR1 and CTAR2 regions) expression plasmids were transfected into HeLa/HaCaT cells. (A–C) Results represent
the means7s.d. of three independent experiments.
LMP1-induced downregulation of RASSF1A involves NF-kB activation. (A) Signaling inhibitors (U0126, JNKII and
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NF-kB activation, and is mediated through the CTAR
regions.
LMP1 suppresses RASSF1A promoter activity through
NF-kB activation
The effect of LMP1 expression on the transcription of
RASSF1A was then examined using a promoter region
of RASSF1A cloned 3.1kb upstream from the ATG site
of RASSF1A. Various deletion mutants of this 3.1kb
RASSF1A promoter region (?3101/?1, ?1190/?1,
?431/?1, ?431/þ15, ?300/?1, ?202/?1) were sub-
cloned into the pGL3-basic vector, upstream of the
luciferase coding sequence. These promoter constructs
were then examined for their usage in HK1 cells, a well-
differentiated squamous NPC (Huang et al., 1980),
which expressed high level of RASSF1A (Chow et al.,
2006). The transfection efficiency of the luciferase
reporter of RASSF1A promoter was normalized by co-
transfecting the cells with the pRL-SV40 as internal
control. The two deleted RASSF1A promoter-reporter
constructs (?431/?1 and ?431/þ15) were shown to
havethehighestpromoter
(Figure 3a), and were selected for co-transfection studies
with the wt LMP1 plasmid and LMP1 mutants. Human
embrogenic kidney (HEK)293 cells were used in this
part of study, as RASSF1A expression is high in this cell
line. In agreement with the immunoblot study, wt B95.8
LMP1 effectivelysuppressed
usage. The suppression of RASSF1A promoter usage
by this prototype (B95.8 LMP1) and the prevalent
variant (2117-LMP1) could be restored by co-transfec-
tion with the NF-kB super suppressor (pUSE-IkBaS32/
S36) (Figure 3b and c). The ability to suppress
RASSF1A promoter usage was lost in the LMP1
mutant (B95.8-LMP1 AxAxA/378STOP mutant), defi-
cient in both CTAR regions. In comparison, the CTAR2
mutant (B95.8-LMP1 378STOP) is less effective com-
pared to the CTAR1 mutant (B95-8-LMP1 AxAxA) in
suppressing RASSF1A transcription; which may be
attributed to the stronger ability of CTAR2 to activate
NF-kB signaling (Eliopoulos et al., 1999b). The promo-
ter experiments confirm CTAR2 as the region respon-
sible for RASSF1A suppression. There is, however,
discrepancy between the immunoblot studies (Figure 2c)
and the promoter analysis in defining the exact region
involved (Figure 3b and c). One explanation is that
HEK293 cells may have a higher endogenous transcrip-
tion rate of RASSF1A compared to HeLa cells;
however, the exact mechanism for the discrepancy
remains to be elucidated.
usagein HK1cells
RASSF1Apromoter
LMP1 induces abnormal interphase microtubules and
mitotic spindles
Loss of RASSF1A function has been previously
reported to induce microtubule defects, mitotic check-
point deregulation and chromosome instability (Song
et al., 2004; Vos et al., 2004). We then examined if
downregulation of RASSF1A expression by LMP1 may
result in similar phenotypes. First of all, we examined
the effect of LMP1 on microtubule phenotypes by
transfecting both the parental cell line (NP69) and
the stable transfectant (NP69LMP1) with pEGFP-a-
tubulin; and observed for alterations of microtubule
morphology using live cell imaging microscopy. In the
parental NP69 cells, typical microtubule networks were
observed (Figure 4a). Defective microtubule poly-
merization was observed in NP69LMP1 cells, which
revealed as punctuated structures in the cytoplasm. To
ensure that these abnormal phenotypes observed were
not artifacts of transfection or cytotoxic effects due to
overexpression of proteins, the cells were examined
under phase contrast microscopy for the presence of
intact membrane structure (Figure 4a, top row). They
were subsequently fixed and immunostained with
DM1a-anti-a-tubulin antibody to confirm that the
distorted structures observed in live cell microscopy
were truly microtubule in origin. Two hundred cells
from the parental NP69 cell line and the NP69LMP1
stable cell line were quantitated for defective micro-
tubules. Seventy five percent of the cells were shown to
be defective in microtubule polymerization compared
with 8% in the parental cells (Figure 4a).
Effect of LMP1 expression on microtubule perturba-
tion was also investigated in other epithelial cell types
(HaCaT, SCC12F and HeLa). For tracking LMP1-
transfected cells, the DsRed expression plasmid was
used as a transfection marker. Cells were co-transfected
with LMP1 and DsRed expression plasmids at a 3:1
ratio. Interphase microtubules and mitotic spindles were
examined only in DsRed-positive cells. Distorted
microtubules and multipolar spindles were detected in
LMP1-transfected cells (Figure 4b, bottom row);
whereas normal interphase microtubules and bipolar
spindles were detected in most of the control cells
transfected with the empty vector (Figure 4b). Two
hundred interphase cells and 50 mitotic cells in random
microscopic fields were quantitated for abnormal
microtubules and spindles in each cell line. Around
30% of the LMP1-transfected cells revealed abnormal
microtubule organization while 50–60% of LMP1-
transfected cells revealed abnormal mitotic spindles
(Figure 4b). The higher percentage of multipolar
spindles observed compared to the lower percentage of
defective interphase microtubules observed in LMP1-
transfected cells, suggest that spindle abnormalities may
be induced by other effects of LMP1 beside microtubule
perturbation.
In order to confirm that downregulation of RASSF1A
expression by LMP1 contributed to the abnormal
microtubule phenotype in LMP1 transfected cells, we
performed co-transfection studies in HeLa cells using
LMP1 and RASSF1A expression vectors. Expression of
RASSF1A effectively rescued the abnormal microtubule
phenotypes induced by LMP1 (Figure 4c). We further
confirmed that NF-kB is involved in contributing to the
abnormal microtubule organization in the LMP1-
expressing cells; by co-transfecting the NF-kB inhibitor
(pUSE-IkBa S32/S36) and the LMP1 expression plas-
mid into SCC12F, HaCaT and HeLa cells. The empty
vector, pSG5, was added where appropriate, to equalize
the amount of DNA across all transfections. We
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observed a six to tenfold increase in microtubule defects
in all three cell types transiently transfected with LMP1;
whereas most control cells revealed normal interphase
microtubule structures and bipolar mitotic spindles
(Figure 4d). Consistent with the promoter-reporter
luciferase study, blocking NF-kB activation could
effectively protect the cells from perturbed microtubule
organization in LMP1-expressing cells (Figure 4d).
LMP1 inhibits microtubule dynamics in cells
The distortion of interphase microtubules and increased
in multipolar spindles in LMP1 expressing cells,
suggested that LMP1 may interfere with microtubule
dynamics. Retinal pigment epithelial (RPE) cells, which
are commonly used in the study of microtubule
dynamics was used in this part of study. The RPE cells
used were stably transfected with pEGFP-EB1 expres-
sion plasmid (Rosa et al., 2006). Microtubules’ plus ends
are binding sites for EB1 protein. EB1 is a microtubule-
dynamic regulating protein binding to the tip of growing
microtubules (Karsenti and Vernos, 2001). The pattern
of EGFP-EB1 expression could accurately reflect
the dynamics of the polymerizing microtubules. Images
of the EB1-transfected RPE cells were acquired by
Figure 3
promoter region (?3101/?1, ?1190/?1, ?431/?1, ?431/þ15, ?300/?1, ?202/?1) were co-transfected with pRL-SV40 (internal
control) into HK1 cells. Dual luciferase assays were performed 48h after transfection. Promoter usages of the RASSF1A reporter
constructs were measured as ratios of the firefly luciferase activities to the control renilla activities. Empty vector was added to
normalize the amount of vectors across all transfections. Relative promoter activities were normalized by the luciferase activity of the
vector control (pGL-basic). (b) The RASSF1A promoter construct ?431/?1 was co-transfected in HEK293 cells, with the prototype
and the set of mutants or the HK prevalent variant; in the presence or absence of the NF-kB inhibitor (pUSE-IkBa S32/S36). Promoter
activities were measured as (a). Ratios of the promoter activities between LMP1 expressing cells and the vector control of less than one
represent suppression of RASSF1A promoter activity. (c) Same study performed using the RASSF1A promoter construct –431/þ15.
(a–c) Results represent the means of triplicate readings7s.d. of three independent experiments.
LMP1-dependent suppression of RASSF1A promoter activity involves NF-kB. (a) Deletion mutants of the 3.1kb RASSF1A
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Figure 4
its stable transfectant NP69LMP1 was performed by transient transfection using EGFP-a-tubulin. Live cells with abnormal
microtubule morphology were quantified and imaged by stacked optical sections using confocal microscopy (n¼200). Cells were
subsequently checked under phase contrast for intact membranes. Difference described is statistically significant (t-test; Po0.0001,
indicated by asterisk). (b) HaCaT, SCC12F and HeLa cells were co-transfected with 2117-LMP1 and a transfection marker (pDs-Red)
at a 3:1 ratio. Empty vector (pcDNA3.1) was added to normalize the amount of vectors across all transfections. Interphase
microtubules (n¼200) and mitotic spindles (n¼50) were analysed and quantified, after immunofluorescent staining with DM1a-anti-a-
tubulin. Images were acquired by stacked optical sections using confocal microscopy. Interphase cells which lacked network-like
organization were considered aberrant; and mitotic cells with monopolar or multipolar spindles were considered as abnormal.
Representative images shown are sets of data from HaCaT cells. Differences described between control and LMP1 expressing cells are
statistically significant (t-test; Po0.0005, indicated by single asterisk; Po0.0001, indicated by double asterisks). (c) The role of
RASSF1A in LMP1-induced microtubule abnormalities was studied by co-transfecting B95.8-LMP1, the RASSF1A expression
plasmids and the transfection marker (DsRed) into HeLa cells. Subsequent immunofluorescent analysis, image acquisition and
quantification of microtubule defects, were performed as in (b). The difference described between cells expressing LMP1 alone and
coexpressing LMP1 and RASSF1A, is statistically significant (t-test; Po0.0001, indicated by asterisk). (d) The involvement of NF-kB
in the induction of microtubule defects was revealed by co-transfection of LMP1, pEGFP-a-tubulin and the genetic inhibitor of NF-kB
(pUSE-IkBa S32/S36) in all transient systems previously used. Empty vector (pcDNA3.1) was added to normalize the amount of
vectors across all transfections. Microtubule and spindle defects in LMP1-expressing cells were analysed, quantified and imaged as
in (b, c). The set of data shown were representative images from the SCC12F cell line. Difference described is statistically significant
(t-test; Po0.0005, indicated by single asterisk; Po0.001, indicated by double asterisks). Bars represent 8mm for all panels.
LMP1 induces abnormal interphase microtubules and mitotic spindles. (a) Analysis of microtubule structures in NP69 and
LMP1 suppresses RASSF1A expression
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confocal laser scanning microscope at 3s intervals
(Supplementary movies S1–S5). EB1-expressing RPE
cells transfected with the empty vector (pSG5) revealed
sharp and abundant EGFP foci distributed along the
polymerizing microtubules (Figure 5a, Supplementary
S1). When EB1-expressing RPE cells were transfected
with the wt B95.8 LMP1 plasmid, a high percentage
(40%) of cells has complete suppression of microtubule
dynamics revealed in loss of EGFP-EB1 movements.
EB1 movements were abolished as the EGFP tagged
EB1 failed to bind to polymerizing microtubules in the
LMP1-transfected cells. In some LMP1-transfected
cells, the EGFP-EB1 appeared to loop around the
nucleus, indicating aggregation of microtubule struc-
tures (Figure 5a, Supplementary S2–S4). RPE cells
expressing the LMP1 mutants defective at either
CTAR1 or CTAR2 region induced similar abnormal
phenotype; although less number of cells revealed
complete
when the LMP1-expressing cells are defective at
CTAR2; which may due to its potent activation of
NF-kB (Eliopoulos et al., 1999b). LMP1 mutant
defective in both C-terminal activating regions, had
minimal effect on microtubule dynamics (Figure 5a,
Supplementary S5).
suppression ofmicrotubule dynamic
LMP1 overexpression induces chromosomal aberrations
in RPE cells
Microtubule defects commonly lead to loss of cell
polarity and spindle abnormalities (Doxsey, 2001),
resulting in mitotic chaos during chromosome segrega-
tion. We examined if chromosome abnormalities could
be observed in LMP1 expressing cells. We have pre-
viously reported that stable expression of LMP1 could
induce aneuploidy and structural chromosome rear-
rangements in an SV40T immortalized nasopharyngeal
Figure 5
stably expressing EGFP-EB1 were co-transfected with wt LMP1 or the LMP1 mutants or the empty vector pSG5 with the transfection
marker (Ds-Red) to examine the effect of LMP1 on microtubule dynamics. Cells which revealed sharp, abundant EGFP-EB1 foci
distributed along the polymerizing microtubules were quantified as normal. Cells with no EGFP-EB1 tracks of movements were
quantified as abnormal. Bars represent 8mm for all panels. (b) Cytogenetics analyses were performed on the RPE hTert cells and the
RPE hTert cells transfected with the B95.8-LMP1 wt expression plasmid and harvested after 48h without any drug selection. Thirty
cells from each cell line were analysed and karyotyped according to ISCN (1995). RPE hTert cells reveal a cytogenetic profile of
44,X,t(2;X)(p13;q21),?3[20]/45,X,t(2;X)(p13;q21)[10][cp30]. RPE hTert cells transfected with LMP1 reveal a cytogenetic profile of
45,X,t(2;X)(p13;q21)[22]/64,X,der(X)del(q23),þder(X)del(q21)x2,der(1)add(1)(p11),þder(1)del(1q24),þ2,þder(2),t(2;X)(p13;q21),
del(4)(p12),þ5,þ6,þder(6)del(q15),þder(7)add(q35)x2,þ8,þ8,add(9)(p23),þder(10)add(q22),þ12,þ12,?13,þ14,þ17,þ17,
þ17,18,þ19,þ19[8] [cp30]. Arrows represent translocations between chromosomes 2 and X. The difference described is statistically
significant (t-test; Po0.005).
LMP1 inhibits microtubule dynamics and induces chromosomal aberrations in RPE cells. (a) Immortalized RPE hTert cells
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epithelial cells (Zhang et al., 2004). We have chosen the
telomerase-immortalized retinal pigment epithelial cells
(RPE hTert) because of their relatively normal and
diploid karyotype, compared to cancer cells and other
viral-protein immortalized cell types (Boukamp et al.,
1997; Duensing and Munger, 2002). RPE hTert only
harbors one balanced translocation between chromo-
somes 2 and X (Figure 5b, i). RPE hTert cells were
transiently transfected with LMP1 and harvested for
cytogenetic analyses. Transient expression of LMP1
effectively induced chromosomal abnormalities in RPE
hTert cells (Figure 5b, ii); in which 30% of these cells
revealed a hypotriploid karyotype. Detail cytogenetic
alterations are described in the legend of Figure 5. This
is the first demonstration that transient LMP1 expres-
sion could induce chromosomal instability in human
epithelial cells.
Discussion
EBV infection is an early event in the development of
NPC and has been postulated to be involved in the
malignant transformation of premalignant nasopharyn-
geal epithelium into cancer. The EBV encoded LMP1 is
believed to have oncogenic properties and were detected
in premalignant lesions of nasopharyngeal epithelium
(Pathmanathan et al., 1995).
In this study, a novel function of LMP1 in down-
regulation of RASSF1A expression was identified; which
supports its role in early pathogenesis of NPC. Recent
studies have shown that RASSF1A plays important
roles in regulating events in mitosis and has marked
influence on microtubules’ behavior as well as on
chromosome instability (Mathe, 2004). RASSF1A is a
microtubule-associated protein and could be localized to
the spindle poles in mitotic cells, where its expression
would regulate mitosis (Vos et al., 2004). Cells depleted
in RASSF1A exit mitosis prematurely with severe
chromosome segregation defects (Song et al., 2004).
The tumor-suppressor function of RASSF1A in NPC
has been demonstrated in a RASSF1A-deficient NPC
cell line (C666-1). As mentioned in the introduction
section, RASSF1A could be inactivated by methylation
or deletion in human cancers. Although RASSF1A is
viewed as being indispensable for cell division, given the
catastrophic mitotic defects induced by abrogation of
this protein, there has not been any study of how a viral
oncogene could regulate this protein. In this study, we
showed that LMP1 could modulate the organization
and/or function of microtubules; hence, this viral
oncoprotein may induce multipolar spindles and chro-
mosome instability. These are unreported phenotypes of
LMP1. Nonetheless, these phenotypes are associated
with those in RASSF1A inactivated cells. We further
demonstrated that the effects of LMP1 on microtubules
are via RASSF1A, by rescuing the phenotype in cells
co-transfected with both LMP1 and RASSF1A. In this
study, we have elaborated some of the mechanisms
involved in the downregulation of RASSF1A by LMP1.
The two functional domains (CTAR1 and CTAR2) of
this viral oncogene are known to be involved in NF-kB
activation (Eliopoulos et al., 1999b). The LMP1 mutant,
deficient in both CTAR domains, was incapable of
downregulating RASSF1A expression; suggesting that
NF-kB activation may mediate LMP1-induced down-
regulation of RASSF1A. Based on the RASSF1A
promoter analyses and the microtubule dynamic studies,
we further confirmed the importance of the LMP1
CTAR2 region in suppression of RASSF1A.
We previously revealed that overexpression of survivin
could dramatically reduce microtubule nucleation and
dynamics (Rosa et al., 2006). Interestingly, survivin is
also a downstream target of LMP1 expression and
NFkB activation. The implication of LMP1 expression
in chromosome instability was revealed in another
study, showing that expression of LMP1 increases the
sensitivity of epithelial cells to DNA damage resulting in
micronuclei formation (Liu et al., 2004).
The demonstration that a viral oncogene could
downregulate a tumor-suppressor gene is of direct
relevance to the role of tumorigenic virus in the
pathogenesis of human cancers. The inactivation of
tumor-suppressor gene by tumorigenic virus is well-
documented in human papilloma virus. The expression
of the E6 and E7 oncogenes could inactivate the p53 and
Rb genes, respectively. It was indicated that the E6/E7
oncogene could activate aurora A and induce polyploidy
(Patel et al., 2004). In NPC, a close association with
EBV infection is present. EBV infection is an early and
clonal event in NPC (Pathmanathan et al., 1995). LMP1
could be detected at high level in premalignant lesion
of nasopharyngeal epithelium and low-grade NPC
(Pathmanathan et al., 1995). The findings that LMP1
could downregulate RASSF1A expression and induce
chromosome instability support a role of EBV encoded
oncogene in the pathogenesis of NPC.
Materials and methods
Cell lines
NP69 (Tsao et al., 2002b) and NP69LMP1 (Lo et al., 2003)
cells are immortalized nasopharyngeal epithelial cell lines,
previously established in our laboratory. HK1 is a cancer cell
line established from a well-differentiated NPC (Huang et al.,
1980), and was used in our previous RASSF1A studies (Chow
et al., 2004, 2006). HeLa and HEK293 cells were from ATCC
(Manassas, VA, USA). Both cell types were maintained in
Rosewell’s Park Memorial Institute 1640 medium supplemen-
ted with 10% fetal bovine serum (FBS). HEK293 cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% FBS. HaCaT and SCCF12 cells were
kindly provided by Dr Dawson, University of Birmingham,
UK. They were maintained in DMEM and DMEM: HAMF12
medium in a 3:1 ratio, respectively. The RPE hTert were
obtained from Clontech (CA, USA); and has been used in
previous studies of microtubule function (Rosa et al., 2006).
Transient transfections and plasmids
Transient transfections were achieved using Fugene 6 transfec-
tion reagent (Roche Molecular Biochemicals Inc., IN, USA)
according to the manufacturer’s recommendations. Sources
and references of the expression plasmids used are: prevalent
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LMP1 variant of NPC (2117-LMP1) isolated from an NPC
xenograft (Cheung et al., 1998); prototype LMP1 (B95.8-
LMP1) was cloned from the B95.8 cell line. The prevalent
variant was cloned into the pcDNA3.1 expression vector
(Invitrogen, CA, USA). The wt and mutants prototype LMP1
(pSG5 B95.8-LMP1 wt, pSG5 B95.8-LMP1 AxAxA, pSG5
B95.8-LMP1 378STOP and pSG5 B95.8-LMP1 AxAxA/
378STOP) were provided by Professor Lawrence Young,
University of Birmingham, UK. The cloning and biological
properties of the wt and mutant LMP1 have been previously
described (Eliopoulos et al., 1999a). Genetic suppressor of
NF-kB (pUSE-IkBa S32/S36) and the activator of NF-kB
(pRc-bactin-IKKa) had been described previously (Jin et al.,
1999). The plasmid for imaging
(pEGFP-a-tubulin) and the plasmid for imaging dynamics of
microtubule plus-ends binding protein in live cells (pEGFP-
EB1) had been described (Rosa et al., 2006). Transfection
marker (pDsRed) was obtained from Clonetech laboratories
Inc., CA, USA.
microtubulestructure
Transient reporter assays
The effect of LMP1 expression on the transcription of
RASSF1A was examined using a 3.1kb promoter region of
RASSF1A upstream from the ATG site of the reported
RASSF1A cDNA sequence. Various deletion fragments of the
3kb RASSF1A promoter region (?3101/?1, ?1190/?1, ?431/
?1, ?431/þ15, ?300/?1, ?202/?1) were subcloned into the
pGL3-basic vector, upstream of the luciferase coding sequence
and examined for the promoter usage in transfected cells. The
suppressing effect of LMP1 on RASSF1A promoter activity
was assayed as previously described (Li et al., 2004). For a
typical transfection, 0.25mg of RASSF1A reporter, 0.01mg of
control reporter (pRL-SV40) and 0.1mg of LMP1 expression
plasmid were co-transfected with Fugene 6 transfection
reagents (Roche Molecular Biochemicals Inc., USA). Readings
were taken using the Dual-Luciferase Reporter Kit (Promega,
WI, USA). All experiments were conducted in triplicates using
independent cultures.
Immunofluorescence analysis
Cells were fixed in ?201C methanol, blocked with 1% bovine
serum albumin, permeabilized with 0.5% Triton X-100 and
stained with antibody against a-tubulin using DM1a-anti-a-
tubulin (Doxsey et al., 1994); followed by Rhodamine-
conjugated anti-mouse antibody (Dako, Glostrup, Denmark).
Microtubule morphology was captured by acquiring stacked
optical sections using multiphoton confocal microscopy (Zeiss
LSM 510, NY, USA), with ?63 objective from eight random
fields. Two hundred interphase cells and 50 mitoses were
scored for microtubule defects and abnormal mitotic spindles.
Live microscopy of GFP-EB1
RPE hTert cells stably expressing EGFP-EB1 were seeded
onto glass bottom microwell dishes (MatTek Corporation,
Ashland, MA, USA) EGFP-EB1. Images at defined zoom
(?63 objective, ?1.5 zoom) and depth (0.2mm) were acquired
every 3s for 3min using a confocal laser scanning microscope
(Zeiss LSM 510) and compacted into time-lapse movies.
Where indicated, cells were co-transfected with various LMP1
expression plasmids and mutants.
Karyotypic analysis
Cytogenetic analyses were performed as described previously
(Li et al., 2006). Briefly, colcemid solution (10mg/ml) (Invitro-
gen, CA, USA) was added for 10–16h, exposed to hypotonic
solution (0.06 M KCl) and fixed in methanol plus acetic acid
(3:1 ratio) for three times. Metaphase spreads were treated by
0.025% trypsin and subsequently stained (Giemsa Gurr R66,
Biomedical specialties, CA, USA). Thirty cells from each cell
line were karyotyped according to the International System for
Human Cytogenetic Nomenclature (ISCN, 1995) using the
Cytovision software (Applied imaging, Hampshire, UK).
RT–PCR
Detection of RASSF1A transcripts were performed by
semiquantitative RT–PCR analysis as previously described
(Chow et al., 2004). Primers used were, forward: 50-
CAGATTGCAAGTTCACCTGCCACTA-30,
GATGAAGCCTGTGTAAGAACCGTCCT-30; which flank
exon 2ab and exon 4 of the gene; and the annealing
temperature was at 651C (28 cycles). Relative levels of b-actin
expression were normalized as loading control.
reverse:50-
Western blotting analysis
Cells were harvested and protein concentrations were deter-
mined as previously described (Li et al., 2004). Protein (30mg)
was resolved on 10% sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE), transferred onto a nitrocel-
lulose membrane (Immobilon-P; Millipore, Bedford, MA,
USA). Primary antibodies used were anti-RASSF1A (1:500,
eBioscience, CA, USA), anti-LMP1 (1:2000, CS1-4, Dakocy-
tomation, Glostrup, Denmark), anti-NF-kB (p65, 1:1000,
Santa Cruz Biotechnology, CA, USA), anti-p-IkBa (1:500,
SantaCruzBiotechnology,
(1:1000, Santa Cruz Biotechnology, CA, USA), antiactin
(1:1000, Santa Cruz Biotechnology, CA, USA). Chemical
inhibitors used were: JNK inhibitor II for the JNK pathway
and U0126 for the ERK1/2 pathway and LY294002 for the Akt
pathway. Concentrations and durations of treatments were
previously optimized (Li et al., 2004). Actin was used as
loading control. Relative protein expression levels were
quantified by gel documentation system (Ultra-Violet Product
Ltd, CA, USA).
CA,USA), antihistone
Statistical method
Student’s t-test was used where applicable. The mean
percentage and standard error of three independent experi-
ments and at least 200 interphase cells or 50 mitotic cells
evaluated per experiment are given.
Acknowledgements
This project was supported by the research Grant council,
Hong Kong (Grant numbers: HKU7356/02M, N_HKU728/
04, HKU7 and CRCG 10205784). We also acknowledged the
support from the Core Imaging Facility of the Li Ka Shing
Faculty of Medicine, University of Hong Kong for the live cell
imaging study.
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