Secreted transforming growth factor ?2 activates
NF-?B, blocks apoptosis, and is essential for the
survival of some tumor cells
Tao Lu*†, Lyudmila G. Burdelya*†, Shannon M. Swiatkowski*, Alexander D. Boiko*, Philip H. Howe‡, George R. Stark*§,
and Andrei V. Gudkov*§
Departments of *Molecular Biology and‡Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195
Contributed by George R. Stark, March 24, 2004
The basis of constitutive activation of NF-?B, essential for survival
and resistance to apoptosis in many tumors, is not well under-
stood. We find that transforming growth factor ?2 (TGF?2), pre-
dominantly in its latent form, is secreted by several different types
of tumor cell lines that exhibit constitutively active NF-?B and that
TGF?2 potently stimulates the activation of NF-?B in reporter cells.
Suppression of TGF?2 expression by small interfering RNA kills
prostate cancer PC3 cells, indicating that the TGF?2–NF-?B path-
way is important for their viability. These findings identify TGF?2
as a potential target for therapeutic strategies to inhibit the
growth of tumor cells that depend on constitutively active NF-?B,
or to sensitize them to treatment with cytotoxic drugs.
prostate cancer ? small interfering RNA ? ELISA ? Smad
tance to apoptotic stimuli, one of the most important aspects of
how the interaction of normal cells with their tissue-specific
environment is regulated. Resistance to apoptosis can be
achieved by means of different genetic alterations, including the
loss of proapoptotic mechanisms (i.e., loss of p53-dependent
signaling in response to stress), and induction or up-regulation
of antiapoptotic mechanisms (i.e., expression of Bcl-2) (1).
Consistently, many tumors acquire constitutive activation of the
normally inducible antiapoptotic transcription factor nuclear
processes, including responses to cytokines and various stresses
(4). Constitutively active NF-?B provides tumor cells with
important selective advantages because it helps to determine
their resistance to both natural [i.e., tumor necrosis factor
(TNF), Fas, or TRAIL] and pharmacological (chemotherapeu-
tic drugs) death stimuli. Factors mediating the constitutive
activation of NF-?B are likely to be targets for anticancer
treatments, making their identification an important clinical
Transforming growth factor ?2 (TGF?2), identified here as a
determinant of the constitutive activation of NF-?B in PC3
tumor cells, belongs to a superfamily of more than 40 factors,
found in vertebrates, insects, and nematodes (5). Three isoforms
of TGF? are known in mammals (6). The TGF?s and their
receptors are expressed ubiquitously in normal tissues and in
most cell lines (7). All TGF?s are secreted as latent precursors
containing active TGF? and latency-associated peptide. In most
cells, latency-associated peptide is linked to an additional pro-
tein, latent TGF? binding protein, forming the large latent
complex (8, 9). Latent TGF?s must be activated to the mature
forms to activate the receptors that mediate Smad-dependent
signaling (10). The activation of TGF? is a complex process
involving conformational changes of latent TGF?, induced
either by the cleavage of latency-associated peptide by proteases
through the actions of endoglycosylases, or by the binding of
latency-associated peptide to proteins such as integrin ?v?5 or
uring progression, tumor cells become increasingly inde-
pendent of negative regulatory controls, including resis-
thrombospondin-1 (11). Members of the TGF? superfamily play
essential roles in early embryonic development, cell mobility,
growth, differentiation, apoptosis, and tumorigenesis (10). De-
spite the name ‘‘transforming growth factor,’’ the role of TGF?
family members in tumorigenesis is complex. Depending on the
cell type, these factors can promote either tumor suppression or
oncogenesis (7). In general, the TGF?s are potent inhibitors of
the growth of various cell types, including epithelial, endothelial,
and hematopoietic, but act as mitogens for fibroblasts.
Here, we have analyzed the mechanism of constitutive NF-?B
activation in the prostate cancer cell line PC3, which is known to
be resistant to TNF-induced apoptosis due to NF-?B activation
(12), and in several other tumor cell lines. A potent NF-?B-
activating factor has been newly identified as TGF?2. The
viability of PC3 cells depends on the production of TGF?2,
presumably because of their addiction to the constitutive acti-
vation of NF-?B.
Materials and Methods
Cell Culture and Preparation of Conditioned Media. Human prostate
cancer PC3, DU145, and LNCaP, breast cancer MCF7, 293C6,
and the derived mutant line C6P1Z12 (13), normal fibroblast
WI38, fibrosarcoma HT1080, glioma T98G, mink lung epithelial
CCL64, mouse fibroblast BALB?c-3T3 (American Type Culture
Collection), and human melanoma Mel-29 cells (14) were cul-
collected from cells at 90% confluency, filtered, and stored at
Plasmids, Transfections, and Luciferase Assays. To construct the
small interfering RNA (siRNA)-TGF?2 vector, a DNA frag-
ment containing an inverted repeat of the target sequence
GAAATGTGCAGGATAATTG, homologous to the 932–950
region of human TGF?2 mRNA, spaced by the 9-nt sequence
TTCAAGAGA and a poly(T) stretch as a stop codon for RNA
polymerase III, was synthesized and cloned under control of the
H4 promoter (15) into the 3?LTR of the retroviral vector pLPCX
(16). Colonies of transfected cells were counted 10 days after
puromycin selection. Mixed cell populations were propagated
and tested for TGF?2 secretion. The ?B-luciferase construct
the IP10 gene) or the Smad binding element-luciferase construct
was transfected transiently into the indicated cells. All transfec-
tions were carried out by using the Lipofectamine Plus reagent
Abbreviations: CHX, cycloheximide; EMSA, electrophoretic mobility gel shift assay; siRNA,
tumor necrosis factor.
†T.L. and L.G.B. contributed equally to this work.
§To whom correspondence may be addressed at: Department of Molecular Biology, NC20,
Lerner Research Institute, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleve-
land, OH 44195. E-mail: email@example.com or firstname.lastname@example.org.
© 2004 by The National Academy of Sciences of the USA
May 4, 2004 ?
vol. 101 ?
(Invitrogen Life Technologies, Carlsbad, CA). Efficiencies of
transfections were normalized to ?-galactosidase activity, ex-
pressed from a pCMVLacZ ?-galactosidase reporter plasmid
added to each DNA sample. Luciferase assay was performed
24 h after the cells were treated with TGF?2 (R & D Systems)
or conditioned medium, following the protocol provided by
Promega. Relative luminescence was normalized to total pro-
tein, assayed with the Bio-Rad Protein Assay reagent.
Cytotoxicity Assays. Mouse fibroblast BALB?c-3T3 cells were
treated with conditioned medium or with TGF?2. To determine
TGF? activity, conditioned medium from PC3 cells was pre-
treated with polyclonal anti-TGF?2 or anti-TGF?1 (R & D
Systems) for 1 h at room temperature before being added to
BALB?c-3T3 cells. After incubation, TNF? (0.2 ng?ml, Pepro-
Tech, Rocky Hill, NJ) and cycloheximide (CHX; 0.4 ?g?ml,
Sigma) were added. Control cells were treated with CHX alone.
After incubation overnight, the cells were washed with 1? PBS,
and the number of cells was estimated by using a methylene blue
Electrophoretic Mobility Gel Shift Assay (EMSA). The oligomer used
for an NF-?B binding site (Santa Cruz Biotechnology), was
5?-AGTTGAGGGGACTTTCCCAGGC-3?, labeled with
[?-32P]ATP by the polynucleotide kinase method, following the
protocol provided by Promega. Treated cells were washed,
collected in 1? PBS and pelleted at 3,000 ? g at 4°C for 4 min.
Cytoplasmic extracts were prepared in binding buffer (13). The
binding reaction was carried out at room temperature for 20 min
in a total volume of 20 ?l. Samples were loaded into 6%
polyacrylamide gels in 0.25? Tris borate buffer, pH 8.0. After
electrophoresis, the gels were dried and analyzed by autoradiog-
raphy at ?80°C.
Northern Analysis. A human IL-8 cDNA fragment was labeled
following the protocol provided by Amersham Biosciences.
293C6 cells were treated with TGF?2 (4 nM) for 4, 10, or 24 h,
then washed with cold 1? PBS. Total RNA was extracted with
the TRIzol reagent at room temperature, following the protocol
provided by Invitrogen Life Technologies. After 15 ?g of total
RNA was loaded, each lane was electrophoresed in an agarose?
formaldehyde gel and transferred to a Hybond-N?membrane
(Amersham Biosciences). After UV crosslinking, the transfers
were hybridized with [?-32P]dCTP-labeled probes and analyzed
by autoradiography at ?80°C.
Western Analysis. Cells treated with TGF?2 for 30 or 60 min were
washed with 1? PBS and pelleted at 3,000 ? g at 4°C for 4 min.
Cell pellets were lysed with radioimmunoprecipitation assay
buffer (13). Cellular debris was removed by centrifugation at
16,000 ? g for 10 min. The amount of protein in the supernatant
solution was determined, and samples were heat-treated in 2?
SDS sample loading buffer (13) at 100°C for 5 min. Equal
amounts of samples were fractioned by SDS?PAGE and trans-
ferred to nitrocellulose membranes. Western analysis was per-
formed with primary antibodies, which were visualized with
horseradish peroxidase-coupled secondary antibodies by using
the ECL Western blotting detection system (PerkinElmer Life
and Analytical Sciences).
ELISA. ELISA (Quantikine-Human TGF?2 or ?1 Immunoassay)
was carried out according to the protocol from R & D Systems.
The amount of TGF?2 or ?1 in conditioned medium was
normalized to cell numbers. Neutralizing anti-TGF?2 or anti-
TGF?1 (R & D Systems) was used to test the specificity of the
Conditioned Media from TNF-Resistant Prostate Cancer Cells Protect
tumor cell lines PC3 and DU145, but not LNCaP, the DNA-
binding activity of NF-?B is constitutively high (12, 17). Con-
sistently, PC3 cells are resistant to treatment with TNF, whereas
LNCaP cells are sensitive. To investigate whether resistance to
TNF is an intrinsic or transmissible trait, cell-free media con-
fibroblasts from TNF-induced cell death through NF-?B activation. (A) Pre-
treatment with medium conditioned by PC3 cells (CM-PC3) protects BALB?c-
3T3 cells from TNF-induced death in a dose-dependent manner. CM-PC3
collected after 24 h was used at different concentrations to treat BALB?c-3T3
cells overnight before adding TNF and CHX. Photographs were taken 24 h
later. Cell protection was assayed as described in Materials and Methods. (B)
Time course of the protection mediated by CM-PC3. BALB?c-3T3 cells were
treated with 50% CM-PC3. Photographs of the cells and the percentages of
BALB?c-3T3 cells protected are shown. (C) Activation of NF-?B in BALB?c-3T3
was assayed 24 h later.
Conditioned medium from PC3 cells protects normal BALB?c-3T3
Lu et al.
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ditioned by PC3 or LNCaP cells were transferred to BALB?c-
3T3 indicator cells. These cells are highly sensitive to treatment
with TNF in the presence of CHX, which prevents the blockade
of apoptosis caused by the TNF-mediated activation of NF-?B.
Treatment with cell-free media conditioned by PC3 but not
LNCaP cells protected BALB?c-3T3 cells from the apoptosis
mediated by TNF in a dose- and time-dependent manner (Fig.
1 A and B). The anti-TNF effect did not require the continued
presence of conditioned medium, suggesting that the mechanism
is unlikely to involve the direct inactivation of TNF, but rather
to require the induction of TNF resistance in the indicator cells,
manifest ?2 h after pretreatment (Fig. 1B).
Resistance of PC3 and many other types of cells to TNF is
known to be determined by the activity of NF-?B (18). There-
fore, we tested whether culture media conditioned by PC3 cells
that are capable of inhibiting TNF-mediated apoptosis could
induce NF-?B. Addition of medium conditioned by PC3 or
LNCaP cells to BALB?c-3T3 cells, after transient transfection
with an NF-?B reporter construct, showed that the NF-?B
activating capacity was characteristic of PC3 but not LNCaP cells
(Fig. 1C). Hence, constitutively active NF-?B in PC3 cells is
accompanied by the secretion of factor(s) capable of inhibiting
apoptosis and activating NF-?B in reporter cells.
TGF?2 Is the Major Protective Factor Secreted by PC3 Cells and Is
Essential for Their Survival. The activation of NF-?B that results in
blockade of apoptosis could be mediated by cytokines known to
be produced by prostate cancer cells (19). By using polyclonal
neutralizing antibodies against several NF-?B-inducing cyto-
kines, we found that anti-TGF?2 (but not anti-TGF?1 or anti-
clusterin) almost completely blocked protection (Fig. 2A), indi-
cating that TGF?2 is likely to be primarily responsible for the
antiapoptotic effect of media conditioned by PC3 cells. Consis-
similar to that of conditioned media (data not shown).
The secretion of NF-?B-inducing factors by PC3 cells suggests
that constitutively active NF-?B is maintained in these cells by
autocrine regulation, and TGF?2 seems to be the major factor.
To test this possibility further, we analyzed the consequences of
suppressing TGF?2 production on the phenotype of PC3 cells by
using siRNA. PC3 cells expressing GFP were infected with a
retrovirus (pLPCX-siTGF?2) encoding a hairpin loop siRNA
representing a fragment of TGF?2 mRNA, expressed from an
H4 promoter (15). A construct expressing siRNA against GFP
was used as a control. MCF7 and LNCaP cells, infected with the
same virus, were used as examples of cells that do not produce
TGF?2. Interestingly, the number and sizes of colonies from
pLPCX-siTGF?2-infected PC3 cells were dramatically reduced
in comparison to cells infected with the control virus, and the
not shown) cells.
Rare, slowly growing colonies, formed after transduction with
but not by anti-TGF?1. CM-PC3 was pretreated with polyclonal antibodies for 1 h at room temperature before being added to BALB?c-3T3 cells. (B) Reduction
in colony number and colony size is caused by transduction of siTGF?2 into PC3 cells. The number of colonies per well (average three) was determined 10 days
after puromycin selection. The experiment was repeated three times with similar results. Colony numbers were normalized for transfection efficiencies,
determined by using a ?-galactosidase reporter assay. The CMV-LacZ plasmid was added to each transfection mixture. (C) PC3 cells stably transfected with a
construct expressing siRNA against TGF?2 express reduced amounts of TGF?2. The amount of TGF?2 in 5 ml of culture media conditioned for 24 h by 106PC3
media from PC3-siTGF?2 cells have decreased NF-?B activation ability in 293C6?B indicator cells. Indicator cells were incubated with conditioned media for 24 h.
(E) Conditioned media from PC3-siTGF?2 cells showed decreased ability to activate NF-?B in LNCaP cells, as assayed by EMSA. LNCaP cells were treated with
different conditioned media. To show the specificity, IL-1 was used as a control. The binding of labeled probe was blocked by competition with a 100-fold excess
of unlabeled probe.
TGF?2 mediates the survival of PC3 cells by inducing NF-?B. (A) Protection of BALB?c-3T3 cells from TNF?CHX by CM-PC3 is prevented by anti-TGF?2,
www.pnas.org?cgi?doi?10.1073?pnas.0402048101 Lu et al.
anti-TGF?2 siRNA, were expanded and tested for TGF?2
production in comparison with similar colonies generated after
transfection with control siRNA. Conditioned media from PC3
cells transduced with siRNA against TGF?2 contained about
one-third as much total TGF?2 as did media from PC3 cells
transduced with siRNA against GFP, determined by ELISA
(Fig. 2C). Anti-TGF?2 was used to pretreat the media to show
the specificity of the assay (Fig. 2C). The former media were
proportionally less capable of inducing NF-?B-mediated tran-
scription in 293C6 indicator cells (Fig. 2D) and specific DNA
binding in LNCaP (Fig. 2E) or BALB?c 3T3 cells (data not
shown). TGF?2 production increased gradually during propa-
gation of the siTGF?2-PC3 cell population, up to the level of the
original PC3 cells, suggesting that TGF?2 provides a selective
advantage (data not shown). We generated cells in which the
superrepressor I?B (SR-I?B) suppressed the NF-?B response
(20). These cells were sensitive to TNF even in the absence of
CHX, and the sensitivity to TNF induced by SR-I?B could not
be overcome by preincubating the cells with conditioned media
from PC3 cells or by recombinant TGF?2 (data not shown).
Hence, we have shown that the TGF?2 secreted by PC3 cells is
apoptosis through the activation of NF-?B.
The Rapid and Direct Activation of NF-?B by TGF?2 May Go Through
a Smad-Independent Signaling Pathway. To test the effect of
TGF?2 on NF-?B activation in human cell lines, we established
an indicator assay by stably transfecting 293C6 cells with a
?B-luciferase construct. In contrast to the results of an activity
assay that depends upon Smad activation, in which TGF?2
reached its maximal effect at ?0.4 nM (Fig. 3A), NF-?B was
activated in a dose-dependent manner up to 4.0 nM (Fig. 3B).
EMSA showed that the activation occurred within 5 min and
persisted for at least 4 h (Fig. 3C). Consistently, activation of
IL-8, a typical NF-?B target gene, was induced by treatment with
TGF?2 (Fig. 3D). Because the maximal activation of NF-?B or
Smad occurs at concentrations of TGF?2 that differ by about
10-fold, these two responses are likely to be due to the activation
of distinct signaling pathways.
Some Tumor Cell Lines with Constitutive NF-?B Secrete TGF?2. To
estimate how general the phenomenon of production of TGF?2 by
tumor cells with constitutively active NF-?B might be, we tested its
presence in media from PC3 and eight other cell lines by using a
quantitative ELISA. Low or insignificant levels of active TGF?2
were found in the media conditioned by all cells except PC3.
However, much higher levels of TGF?2 were found in media
conditioned by several cell lines after treatment of the media with
HCl, which converts latent TGF? to the active form (Table 1).
Therefore, several of the cell lines tested produce TGF?2, predom-
inantly in its latent form. Interestingly, all of these cell lines display
constitutive activation of NF-?B, assayed by ELISA (Table 1) or
EMSA (Fig. 4A), including C6P1Z12 (Table 1), a mutant line
derived from 293C6 cells after selection for constitutive activation
of NF-?B (13). Consistent with the predominant presence of latent
activation of Smad2 (Fig. 4B), although the Smad pathway was
capable of responding to active TGF?2 in these cells (Fig. 4 B and
C). These observations suggest that latent TGF?2 production is a
property of many tumor cell lines with constitutively active NF-?B.
Although TGF?1 is also capable of activating NF-?B (data not
shown), the NF-?B activating and antiapoptotic effect of media
as shown by the results with neutralizing antibodies (Fig. 2A). We
do not see effects of TGF?1, probably because low levels of this
A large body of literature suggests that, during the early phase
of epithelial tumorigenesis, TGF?s, especially TGF?1, inhibit
primary tumor development and growth by inducing cell-cycle
arrest and possibly apoptosis, serving as the proapoptotic factors
through Smad-dependent signaling pathways (21, 22). However,
in late stages of progression, as tumor cells evade the growth
inhibition by TGF?s because of inactivation of their signaling
pathways or aberrant regulation of cell-cycle machinery, the role
Dose–response of Smad promoter activation after TGF?2 treatment. 293C6
cells were transfected transiently with a Smad binding element-luciferase
construct for 24 h and then treated with TGF?2 for another 24 h before
Stable 293C6?B indicator cells were treated with TGF?2 for 24 h before
luciferase assay. (C) Time course of activation of NF-?B by TGF?2. Cells were
treated with TGF?2 (2 nM). IL-1-treated cells were used as controls. Note that
the time of exposure of the gel in the experiment was much less than in the
TGF?2 experiment. EMSAs were done as described in Materials and Methods.
(D) TGF?2-induced IL-8 expression in 293C6 cells. Cells were treated with
TGF?2. Northern analysis was done as described in Materials and Methods.
The activation of NF-?B by TGF?2 is rapid and dose-dependent. (A)
Lu et al.
May 4, 2004 ?
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of TGF?s is often switched from tumor suppression to promo-
tion. For example, among TGF? family members, TGF?1 is
regarded as an important regulator of normal and malignant
prostate tissues. In the nonmalignant prostate, TGF?1 stimu-
lates cell differentiation, inhibits epithelial cell proliferation, and
induces epithelial cell death (23). In contrast, prostate cancer
cells frequently lose their TGF?1 receptors and acquire resis-
Accordingly, high expression of TGF?1 and loss of TGF?
receptor expression have been associated with a particularly bad
prognosis in prostate cancer patients (23). We have found that
TGF?2 secreted by prostate cancer PC3 cells, and not TGF?1,
serves as the major protector against the TNF-mediated death of
BALB?c-3T3 cells through NF-?B activation and is the key
determinant of the survival of PC3 cells. Therefore, quite
differently from the well accepted dominant role of TGF?1 in
prostate cancer development, our study reveals an important
role of another TGF? family member, TGF?2, in this process.
Functional interactions between TGF? and NF-?B in tumor
cells have been addressed in previous publications, which, how-
ever, provide a confusing view, with TGF? playing the role of
either an inhibitor or activator of NF-?B-mediated signaling in
different cell types (24–26). Assayed in stably transfected human
is rapid (Fig. 3C). However, because maximal activation of Smad
(Fig. 3A) or NF-?B (Fig. 3B) occurs at concentrations of TGF?2
that differ by 10-fold or more, these two responses to TGF?2 are
due to the activation of distinct pathways and might even use
somewhat different receptors.
The growth-inhibiting effects of TGF?s on epithelial cells are
mediated by Smad-dependent signaling, explaining why many
tumor cells acquire defects in TGF? receptors or Smad2 (27).
However, this is not the case in PC3 cells, which still retain a
normal response to TGF?2 (Fig. 4C), indicating that the TGF?
receptors and Smads are still functional in these cells. Further-
more, Western analysis showed that Smad2 is not activated
constitutively in PC3 cells or in several other cell lines (Fig. 4B).
ELISA data (Table 1) further confirm that most cancer cell lines
with constitutive NF-?B secrete different levels of TGF?2,
mainly in its latent form. These data strongly suggest that,
although the Smad-dependent signaling pathway is still intact in
PC3 cells, it is not activated in growing cells. Our findings,
together with published information concerning functional in-
teractions between TGF? and NF-?B, allow us to build the
following model: Secretion of TGF? causes a dual effect on
tumor cells of epithelial origin. On the one hand, it suppresses
the growth of these cells through Smad-dependent signaling. On
the other hand, TGF? secretion can be beneficial for tumor cells
by causing the constitutive activation of NF-?B. It is beneficial
to tumors to keep the NF-?B-activating role of TGF? but get rid
of its growth-suppressive effect. To achieve this, tumor cells
acquire mutations in either TGF? receptors or Smads (27). Does
latent TGF?2 activate NF-?B directly to achieve a protective
effect in PC3 cells? Further studies are needed to address this
Suppressing the production of TGF?2 causes PC3 cells to die,
and a similar effect is caused by inactivating NF-?B through the
ectopic expression of the SR-I?B (12), suggesting that PC3 cells
have become addicted to the constitutive autocrine activation of
NF-?B mediated by TGF?2. This observation identifies TGF?2 as
an important potential target for therapeutic suppression designed
and to sensitize them to treatment with cytotoxic drugs.
This work was supported by National Institutes of Health Grants P01
CA62220 (to G.R.S.) and CA88071 (to A.V.G.).
Table 1. ELISA of TGF?2 in conditioned media
Active TGF?2, pg?106cellsTotal TGF?2, pg?106cells
Without antibodyWith antibody Without antibodyWith antibody
Media collected after 24 h were assayed with and without treatment with polyclonal anti-TGF?2. Total TGF?2 was assayed after
exposure to 1 M HCl, to release latency-associated peptide. Active TGF?2 was assayed without exposure to HCl.
NF-?B activity and TGF?2 overexpression. (A) Constitutive NF-?B activation in
several tumor cell lines. Cells were cultured, samples were collected, and
tumor cells. Cells were collected, and Western assays were performed. (C)
Phosphorylation of Smad2 on Ser-465?467 upon TGF?2 treatment of the
tumor cells. Cells with high TGF?2 expression (according to the ELISA) were
treated with TGF?2 for 30 or 60 min, and Western assays were performed.
Smad2 is not constitutively active in tumor cells with constitutive
www.pnas.org?cgi?doi?10.1073?pnas.0402048101 Lu et al.
1. Gurova, K. V. & Gudkov, A. V. (2003) J. Cell. Biochem. 88, 128–137. Download full-text
2. Rayet, B. & Gelinas, C. (1999) Oncogene 18, 6938–6947.
3. Baldwin, A. S. (2001) J. Clin. Invest. 107, 241–246.
4. Karin, M., Cao, Y., Greten, F. R. & Li, Z. W. (2002) Nat. Rev. Cancer 2, 301–310.
5. Massague, J. M. (1998) Annu. Rev. Biochem. 67, 753–791.
6. Govinden, R. & Bhoola, K. D. (2003) Pharmacol. Ther. 98, 257–265.
7. Massague, J. & Chen, Y. G. (2000) Genes Dev. 14, 627–644.
8. Gentry, L. E., Lioubin, M. N., Purchio, A. F. & Marquardt, H. (1988) Mol. Cell.
Biol. 8, 4162–4168.
9. Gray, A. M. & Mason, A. J. (1990) Science 247, 1328–1330.
10. Lawrence, D. A. (1996) Eur. Cytokine Network 7, 363–374.
11. Roberts, A. B. (1998) Miner. Electrolyte Metab. 24, 111–119.
12. Gasparian, A. V., Yao, Y. J., Lu, J., Yemelyanov, A. Y., Lyakh, L. A., Slaga,
T. J. & Budunova, I. V. (2002) Mol. Cancer Ther. 1, 1079–1087.
13. Sathe, S. S., Sizemore, N., Li, X., Vithalani, K., Commane, M., Swiatkowski,
S. M. & Stark, G. R. (2004) Proc. Natl. Acad. Sci. USA 101, 192–197.
14. Kichina, J. V., Rauth, S., Das Gupta, T. K. & Gudkov, A. V. (2003) Oncogene
15. Myslinski, E., Ame, J. C., Krol, A. & Carbon, P. (2001) Nucleic Acids Res. 29,
16. Miller, A. D. & Rosman, G. J. (1989) BioTechniques 7, 980–990.
17. Palayoor, S. T., Youmell, M. Y., Calderwood, S. K., Coleman, C. N. & Price,
B. D. (1999) Oncogene 18, 7389–7394.
18. Muenchen, H. J., Lin, D. L., Walsh, M. A., Keller, E. T. & Pienta, K. J. (2000)
Clin. Cancer Res. 6, 1969–1977.
19. Teicher, B. A., Kakeji, Y., Ara, G., Herbst, R. S. & Northey, D. (1997) In Vivo
20. Miagkov, A. V., Kovalenko, D. V., Brown, C. E., Didsbury, J. R., Cogswell,
J. P., Stimpson, S. A., Baldwin, A. S. & Makarov, S. S. (1998) Proc. Natl. Acad.
Sci. USA 95, 13859–13864.
Liebermann, D. A., Bottinger, E. P. & Roberts, A. B. (2003) J. Biol. Chem. 278,
22. Valderrama-Carvajal, H., Cocolakis, E., Lacerte, A., Lee, E. H., Krystal, G.,
Ali, S. & Lebrun, J. J. (2002) Nat. Cell Biol. 4, 963–969.
23. Wikstrom, P., Damber, J. & Bergh, A. (2001) Microsc. Res. Tech. 52, 411–
24. Han, S. H., Yea, S. S., Jeon, Y. J., Yang, K. H. & Kaminski, N. E. (1998)
J. Pharmacol. Exp. Ther. 287, 1105–1112.
25. Arsura, M., Panta, G. R., Bilyeu, J. D., Cavin, L. G., Sovak, M. A., Oliver, A. A.,
Factor, V., Heuchel, R., Mercurio, F., Thorgeirsson, S. S. & Sonenshein, G. E.
(2003) Oncogene 22, 412–425.
26. Sakurai, H., Chiba, H., Sugita, T. & Toriumi, W. (1999) J. Biol. Chem. 274,
27. Schutte, M. (1999) Ann. Oncol. 10, Suppl. 4, 56–59.
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