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Advances in Brief |
B Activation Is Involved in LMP1-mediated Transformation and Tumorigenesis of Rat-1 Fibroblasts1
Department of Microbiology, The University of Hong Kong, Hong Kong, SAR, China
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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B activation and 100% defective in
activator protein-1 activation. When introduced into Rat-1 cells
through retrovirus, the Rat-1-LMP1TRADD cells showed a
significant reduction of focus formation and decreased tumor growth in
nude mice as compared with Rat-1-LMP1 cells, suggesting that NF-B or
activator protein-1 activation may be important for LMP1-induced cell
transformation. To further delineate the determinants of LMP1-mediated
cellular transformation, a retrovirus with a dominant-negative
IB
was introduced into the transformed Rat-1-LMP1
cells. The presence of IB significantly suppressed both focus
formation and tumorigenicity of Rat-1-LMP1 cells. Our results suggest
that the activation of NF-B may play an important role in
LMP1-mediated cell transformation and tumorigenesis. |
Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Structural analysis of LMP1 reveals that the protein contains at least
three parts: a cytoplasmic NH2 terminus of 24
amino acid residues, a six-transmembrane segment of 162 amino acid
residues, and a cytoplasmic COOH terminus of 200 amino acid residues
(2)
. The six-transmembrane segment was shown to be
required to form cytoplasmic membrane patches. Such oligomerization of
LMP1 in the cytoplasmic membrane may mimic that induced by
ligand-receptor interactions, resulting in a constitutive activation of
receptor molecules (6)
. The cytoplasmic COOH terminus
interacts with two families of proteins, tumor necrosis factor
receptor-associated factors and TRADD protein through two distinct
domains (7
, 8) . These two domains participate in the
activation of NF-B (9
, 10)
and AP-1 (11
, 12)
. The TRADD binding domain of LMP1 is largely responsible for
NF-B activation and is absolutely required for AP-1 activation
(9, 10, 11, 12)
. Interestingly, a series of studies on recombinant
EBV-mediated B-cell growth transformation revealed that this TRADD
binding domain of LMP1 is important for the efficient long-term
outgrowth of EBV-containing lymphoblastoid cell lines (8
, 13
, 14)
. However, the roles of NF-B and AP-1 activation in
LMP1-mediated cellular transformation and tumorigenesis remain to be
elucidated. In this report, we show that the TRADD domain of LMP1, in
addition to being required for NF-B and AP-1 activation, is
important for LMP1-mediated transformation and tumorigenesis of Rat-1
rodent fibroblasts. Furthermore, inhibition of the NF-B pathway by a
dominant-mutant IB results in a significant reduction of LMP1
transforming activity.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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B luciferase reporter
plasmid (ELAM-Luc) was a gift from David Goeddel (Tularik, South San
Francisco, CA), and AP-1 luciferase reporter with four AP-1 sites was a
gift from Zhigang Dong (University of Minnesota, Austin, MN).
Plasmid pSVK3IB expressing a dominant-mutant IB was a gift
from John Hiscott (16)
. An EcoRI fragment with
IB mutant gene was isolated and subcloned into the
EcoRI site of retroviral plasmid pBabe (15)
to
give pBabe-IB. The Rat-1 rat fibroblast cell line was provided by
Dr. Friedrich A. Grasser (Institut fur Medizinische Mikrobiologie und
Hygiene, Hamburg, Germany). PA317 and Bosc23 (American Type Culture
Collection) are amphotropic and ecotropic retrovirus packaging cell
lines, respectively. Cells were cultured in DMEM with 10% serum (Life
Technologies, Inc.).
Retrovirus-mediated Gene Transfer and Detection of the Expressed
Proteins.
To obtain the retroviruses, the retroviral plasmids were transfected
into the ecotropic retrovirus packaging cell line Bosc23 to generate
ecotropic viruses, followed by their infection into the amphotrophic
viral packaging cell line PA317 to produce amphotrophic retroviruses
that are used in all subsequent experiments, as described previously
(15)
. Rat-1 cells were infected with Rv-LMP1,
Rv-LMP1TRADD, or Rv-LNSX (vector control)
retroviruses. At 48 h after infection, the cells were selected
with geneticin (800 µg/ml) for 2 weeks, and thousands of resistant
clones were pooled for subsequent analysis. Similarly, the Rat-1-LMP1
cells were infected with Rv-IB or Rv-Babe (vector control)
retroviruses, selected with puromycin (2 µg/ml), and pooled together
to avoid clonal artifact. The expression of LMP1 was demonstrated by
immunofluorescence and immunoblotting with an LMP1 antibody S12
(15)
, and IB expression was demonstrated by
immunoblotting with antibody SC-203 (Santa Cruz) at 1:200 dilution with
100 µg of cell lysates. A lysate of B95–8 (20 µg), an EBV-positive
B-cell line, was used as the positive control for LMP1.
Transcription Activity Analysis.
For each transfection, Rat-1 cells were seeded into six-well dishes at
1 x 105 cells/well (Nunc). The
cells were then cotransfected with NF-B reporter plasmid (100
ng/well), pRL-SV40 (3 ng/well), and AP-1-Luc (50 ng/well) and the
indicated amounts of pLNSX-LMP1, pLNSX-LMP1TRADD,
and/or pBabe-IB plasmids. Vector pLNSX or pBabe was supplemented
to a total amount of 1 µg of DNA per transfection. Transfection was
done with the Lipofectamine system (Life Technologies, Inc.). Cell
lysates were made 48 h later and examined by using the dual
luciferase reporter assay system (Promega) to obtain the relative
promoter activities. All experiments were done in triplicate, and a
total of three independent experiments were performed.
Cell Culture Transformation Assay.
For contact inhibition assay (5)
, Rat-1 infectants
(2 x 105 cells/well) were plated
out directly onto six-well dishes. After 7 days, the number of
colonies/well was recorded. For soft agar focus formation assay, Rat-1
infectants (5 x 104 cells/well)
were seeded in soft agar on six-well dishes as described previously
(15)
. The number of foci/fixed area was recorded on the
10th day. The transformation experiments were done in triplicate to
determine mean and SD.
Nude Mice Tumorigenicity Assay.
Athymic nude mice, 4–6 weeks of age, were inoculated s.c. with
different Rat-1 infectants (5 x 106 cells/mouse) in 100 µl of serum-free DMEM
medium. After 3–4 weeks, the dimensions of tumor were measured, and
the representative animals were photographed. Two independent
experiments were carrier out with two sets of independently infected
cultures, each of five five-mouse groups.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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B and AP-1 primarily through the last few amino
acids of LMP1 that interact with TRADD. To investigate the roles of
this TRADD binding site in LMP1-mediated transformation of rodent
fibroblasts, an LMP1TRADD mutant was made,
replacing the amino acid residues required for TRADD interaction YYD at
position 384–386 with ID, as described previously (8)
.
The LMP1TRADD was cloned into a retroviral
plasmid vector (pLNSX), and its sequence was confirmed through DNA
sequencing. Cotransfection studies with NF-B luciferase reporter
indicate that whereas wild-type LMP1 activates NF-B by 8-fold (Fig. 1A
), the LMP1TRADD mutant only activates
NF-B by 2-fold (Fig. 1A
). Similar results were also
obtained from REF52 rat embryonic fibroblasts or 293 human embryonic
kidney cells (data not shown). The result is consistent with other
previous studies of similar LMP1 mutants defective in TRADD binding
(9
, 10) . In parallel, whereas wild-type LMP1 activates
AP-1 by 4 fold in Rat-1 cells, LMP1TRADD shows no
activation of AP-1 (Fig. 1B
), correlating with those of
previous publications (11
, 12)
. Thus, this
LMP1TRADD mutant is 
75% defective in NF-B
activation and 100% defective in AP-1 activation. Retrovirus
Rv-LMP1TRADD was constructed to effectively
introduce LMP1TRADD into Rat-1 cells. Rat-1
fibroblasts were then infected with vector control Rv-LNSX, wild-type
Rv-LMP1, or mutant Rv-LMP1TRADD. Two days after
infections, immunofluorescence analysis was performed, and the results
revealed that 30–50% of the LMP1 retrovirus-infected cells
expressed LMP1 (data not shown). The cells were reseeded and selected
with geneticin. The Rv-LMP1TRADD viral stock had
a slightly higher titer than that of Rv-LMP1 and thus gave 50% more
geneticin-resistant colonies. Thousands of resistant clones for each of
the three infected cultures were pooled together for further passage
under the selection of geneticin. All three drug-selected populations
of retrovirus-infected cells showed no noticeable difference in the
growth rate because the cells were continuously passaged at 1:10
whenever they reached confluence over a period of 5 weeks. The
immunoblot for LMP1 with the cell lysates indicates the presence of
LMP1 and LMP1TRADD in the respectively infected
cells after selection that is absent in the vector-infected cells (Fig. 1C
).
|
B activation in LMP1-mediated
cell transformation and tumorigenesis, we used a mutant gene encoding a
nonphosphorylatable IB that resulted in a constitutive
suppression of NF-B despite the presence of the activators
(16)
. A pBabe-IB plasmid was constructed for
generating IB retrovirus (Rv-IB). This pBabe-IB
plasmid, when cotransfected into Rat-1 cells with pLNSX-LMP1,
significantly inhibited LMP1-mediated activation of the NF-B
reporter (Fig. 1D
). At a 1:1 ratio against cotransfecting
pLNSX-LMP1, pBabe-IB inhibited LMP1-mediated NF-B activation
by 75%; and at 3:1 ratio, it almost completely inhibited LMP1-mediated
NF-B activation. To examine the effect of this IB
mutant in LMP1-transformed Rat-1 cells, the Rat-1-LMP1 cells were
infected with either Rv-Babe (vector) or Rv-IB retrovirus. Two
days after infection, the cells were split 1:5 into six-well dishes and
selected with puromycin. Although all of the mock-infected
Rat-1-LMP1 cells died, thousand of colonies emerged from each well and
covered the wells in 4 days for both Rv-Babe- and Rv-IB-infected
cells. They were again seeded into 10-cm dishes with puromycin
selection. Both Rat-1-LMP1/Babe and Rat-1-LMP1/IB were
continuously passaged for 5 weeks together with Rat-1-LMP1 cells.
Again, no obvious difference in cell growth was observable among these
cultures, suggesting that IB did not have a significant effect on
cell growth. The expression of IB in these cells was further
verified by immunoblot with SC-203 antibody against IB (Santa
Cruz) that yielded a specific band at
Mr 38,000, representing
IB protein in the newly established culture (Fig. 1E
).
A nonspecific, slow migrating band indicated a similar amount of
protein loaded in each lane.
Earlier deletion analysis of LMP1 yielded conflicting results of the
sequence requirement for the transformation of rodent fibroblasts in
culture (4
, 5)
. To examine the effect of
LMP1TRADD mutation in transforming Rat-1
fibroblasts and to evaluate the role of NF-B activation in the
LMP1-mediated transformation process, the respectively infected cells
were analyzed for contact inhibition of growth (5)
and
anchorage-independent growth in soft agar (3)
. Cells from
thousands of drug-resistant clones were pooled together for all
transforming assays to avoid the artifacts from a few selected clones.
Reproducible results on transformation assays were obtained from three
experiments with independently infected Rat-1 cells. The results of two
experiments are shown in Table 1
. The data indicated a significant reduction in the number of colonies
and foci in Rat-1-LMP1TRADD cells in both assays
when compared with that in Rat-1-LMP1 cells. It is also apparent that
Rv-LMP1TRADD maintains a residual transforming
activity when compared with the negative control Rv-LNSX in both
assays. Thus, the results underlined the importance of TRADD binding in
LMP1-mediated cell transformation, pointing to the mutation in this
domain as the factor leading to a dramatic reduction of LMP1
transforming potential. Our findings are partially consistent with a
previous report that showed the removal of the last 23 amino acids of
LMP1 resulted in its inability to induce colony formation of Rat-1
cells (5)
. Because LMP1-mediated TRADD binding resulted in
the activation of a number of signaling cascades, particularly the
activation of NF-B, we further investigated the role of NF-B
activation in LMP1-mediated transformation by examining the
transforming properties of Rat-1-LMP1 cells after the superinfection by
Rv-IB. Similarly, the transformation assays were done three times
with Rat-1-LMP1 independently infected with Rv-Babe or Rv-IB that
gave consistent results. The results from two of them showed that the
introduction of this dominant-negative IB into the transformed
Rat-1-LMP1 cells resulted in a significant reduction of colony and
focus numbers in both transformation assays, whereas the control
virus-infected cells (Rat-1-LMP1/Babe) had little effect (Table 1)
.
Therefore, keeping a constitutive NF-B activation appears to be
important for the maintenance of the transforming properties of
Rat-1-LMP1 cells.
|
and Table 2
) suggesting that an intact TRADD domain is also important for inducing
tumor formation of Rat-1 cells in the nude mice model. The tumorigenic
result of the Rv-IB superinfected cells further revealed
information on the requirement of downstream signal for LMP1-induced
tumor growth. Specifically, the presence of IB in Rat-1-LMP1
cells resulted in a significant reduction of tumor size in nude mice
(Fig. 2B
and Table 2
), whereas control Rv-Babe-infected
cells showed little difference when compared with the parental
Rat-1-LMP1 cells. Therefore, the result suggests that a constitutive
activation of NF-B by LMP1 may play an important role in
LMP1-mediated tumorigenesis.
|
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B primarily
mediated through the TRADD binding domain of the protein. This TRADD
domain of LMP1 is important for EBV-mediated B-cell transformation
(8
, 13 , 14)
. In this study, we used two different
approaches, an LMP1TRADD mutant and a
dominant-negative mutant IB, to examine the mechanism of
LMP1-mediate transformation of immortalized rodent fibroblasts. A
common target affected by these two approaches is the reduction of
NF-B activity, and in fact, the residual NF-B activation activity
of the pLNSX-LMP1TRADD mutant is comparable with
that of cotransfecting pLNSX-LMP1 and pBabe-IB at a 1:1 ratio (a
75% reduction when compared with LMP1 alone). The cell lines
established from them resulted in a clear and a similar degree of
reduction in both in vitro transformation and in
vivo tumorigenesis assays. Although LMP1 may still contribute to
cell transformation through other processes, our results positively
established, for the first time, that the LMP1-mediated NF-B
activation is important for its transforming activity. There has been a
number of studies revealing the involvement of members of the NF-B
family in tumorigenesis; in particular, Rv-rel,
Bcl-3, and Lyt10 were all associated with cancers
(17, 18, 19)
. In addition to being involved in the regulation
of cytokines, cell adhesion molecules, and antiapoptotic genes,
emerging evidence also implicates the roles of NF-B in cell
proliferation (20)
and in platelet-derived growth factor
signaling process (21)
. In parallel with our findings, the
constitutive activation of NF-B is essential for the transformation
of Rat-1 cells by Tax protein of human T-cell leukemia virus type 1
(22)
.
In summary, our results indicate that the TRADD binding domain is
important for LMP1-mediated cell transformation, and the introduction
of a dominant-negative IB into the transformed Rat-1-LMP1
significantly inhibits its tumorigenicity. Thus, LMP1-mediated NF-B
activation is important for it to induce the transformation and
tumorigenicity of Rat-1 fibroblasts.
|
Acknowledgments |
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B plasmid, David
Goeddel for NF-B reporter plasmid, Zhigang Dong for AP-1 reporter
plasmid, Friedrich A. Grasser for Rat-1 cells, David Thorley-Lawson for
S12 antibody, and Joanne Zhong and Dongyan Jin for critical reading of
the manuscript.
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FOOTNOTES |
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1 Supported by grants from Research Grant Council,
Committee on Research and Conference Grants of Hong Kong, and
the Croucher Foundation (to L. C.). 
2 To whom requests for reprints should be
addressed, at Department of Microbiology, The University of Hong Kong,
Hong Kong, SAR, China. Phone: 852-2855-4822; Fax: 852-2855-1241;
E-mail: lcao{at}hkucc.hku.hk
3 The abbreviations used are: LMP, latent membrane
protein; TRADD, tumor necrosis factor receptor-associated death domain;
NF, nuclear factor; AP-1, activator protein-1.
Received 11/ 9/99. Accepted 2/17/00.
|
REFERENCES |
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B. Proc. Natl. Acad. Sci. USA, 94: 12592-12597, 1997.B-mediated transcription by mutant derivatives of the latent membrane protein of Epstein-Barr virus. J. Virol., 69: 2968-2976, 1995.[Abstract]
B and cell surface phenotype via two effector regions in its carboxyl-terminal cytoplasmic domain. Oncogene, 10: 549-560, 1995.[Medline]
B block Tat-tumor necrosis factor synergistic activation of human immunodeficiency virus type 1 gene expression and virus multiplication. J. Virol., 70: 5777-5785, 1996.[Abstract]
B p50. Cell, 67: 1075-1087, 1991.[Medline]
B function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition. Mol. Cell. Biol., 19: 2690-2698, 1999.B is a target of AKT in anti-apoptotic PDGF signalling. Nature (Lond.), 401: 86-90, 1999.[Medline]
B is essential for transformation of rat fibroblasts by the human T-cell leukemia virus type I Tax protein. EMBO J., 15: 873-887, 1996.[Medline]
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