Received for publication, November 20, 2000, and in revised form, March 23, 2001
Keratinocytes are the natural target cells for
infection by human papillomaviruses (HPVs), most of which cause benign
epithelial hyperplasias (warts). However, a subset of papillomaviruses,
the "high risk" HPVs, cause lesions that can progress to
carcinomas. Inflammatory mediators such as tumor necrosis factor-
(TNF-
) and TNF-related apoptosis-inducing ligand (TRAIL) are
produced by cells in response to a viral infection. To determine the
effects of TNF-
and TRAIL on keratinocytes expressing the high risk
HPV-16 oncoprotein E7, human foreskin keratinocytes stably expressing E7 were treated with TNF-
and TRAIL. Treatment with TNF-
alone, but not TRAIL, induced growth arrest and differentiation in
keratinocytes that was almost completely overcome by expression of
HPV-16 E7. Both cytokines induced apoptosis when administered in
combination with the protein synthesis inhibitor cycloheximide, but the
apoptotic response to TRAIL was significantly more rapid and efficient
compared with the response seen after TNF-
treatment. HPV-16
E7-expressing keratinocytes were more prone to both TNF-
- and
TRAIL-mediated apoptosis compared with vector-infected controls.
 |
INTRODUCTION |
The human papillomaviruses
(HPVs)1 are a family of small
DNA viruses with a pronounced tropism for epithelial cells (1). Infection with a member of this large family of viruses usually causes
papillomatous hyperplasia (warts) but lesions caused by a subset of
these viruses, the "high risk" HPVs (such as HPV-16, -18, -31, or
-33) have a propensity for malignant progression. Although the link
between HPVs, cervical cancer, and other anogenital tract carcinomas is
most firmly established (2), there is also evidence linking HPV
infection to carcinomas of oral and respiratory mucosa (3).
During malignant progression, the HPV genome frequently integrates into
the host DNA resulting in the expression of only two viral proteins, E6
and E7, that bind and induce the degradation of the p53 and pRB tumor
suppressor proteins, respectively (4-8). An important function of pRB
is to modulate the activity of members of the E2F family of
transcription factors that play important roles in regulating
the G1/S-phase transition (9, 10). Degradation of
pRB by E7 abrogates this regulatory circuit and relaxes
G1/S checkpoint control (11). E6 promotes the proteasomal
degradation of the tumor suppressor p53, rendering the cell unable to
efficiently execute a program of growth arrest and apoptosis under
conditions of cellular stress, DNA damage, or aberrant growth signals
(10, 12). The loss of cellular surveillance function permits
replication of damaged DNA, resulting in the accumulation of cells with
genomic aberrations and increasing the likelihood of neoplastic changes (13).
Viral infection of an immunocompetent host induces the production and
release of cytokines, powerful mediators of inflammation produced by
fibroblasts, macrophages, lymphocytes, and keratinocytes themselves
(14). TNF-
is one of the main mediators of inflammation in the skin
and mucosa, the first barrier encountered by an epitheliotropic virus
(14). TNF-
and its related cytokines bind to specific members of the
TNF receptor superfamily initiating various signaling pathways that
lead to growth arrest, proliferation, or cell death (15). The cellular
response to a cytokine depends upon the specific ligand-receptor
interaction, the cell type, and the immediate cellular microenvironment
(16).
TNF-R1 contains four cysteine-rich extracellular domains that become
trimerized upon TNF-
binding (16). Ligation of this receptor
approximates and cross-links intracellular death domains that then bind
the death-domain-containing adapter proteins TRADD and FADD.
Oligomerization of these proteins results in caspase activation and
apoptosis (17, 18). In addition, TNF-R1 can simultaneously activate
protective responses via NF-
B-dependent and -independent
gene transcription and protein synthesis (19, 20).
A recently identified member of the TNF family of cytokines, TRAIL has
not been as extensively studied, and its effects on keratinocytes are
largely unknown. Found in a wide range of tissues, TRAIL (also known as
Apo-2 ligand) is a type II transmembrane protein closely homologous to
Fas ligand, TNF-
, and lymphotoxin-
(21-23). Four major receptors
for TRAIL have been characterized. TRAIL-R1 (DR4) and TRAIL-R2 (DR5)
are type I transmembrane proteins with cytoplasmic death domains
homologous to those found in TNF-R1 and Fas (17, 21, 23-25). TRAIL-R3
(also called TRID or DcR1) lacks a cytoplasmic domain and instead is
anchored directly to the cell membrane via a
glycosylphosphatidylinositol link (26-30). TRAIL-R4 (DcR2) contains an
incomplete cytoplasmic death domain (31). TRAIL receptors 3 and 4 cannot initiate the caspase cascade and thereby may confer resistance
to TRAIL-induced apoptosis by competing for ligand. A fifth and most
recently described cell-type-specific receptor, osteoprotegerin,
can be stimulated by TRAIL and is involved in the regulation of bone
resorption (32, 33).
Like other members of the TNF receptor superfamily, the cytoplasmic
domains of TRAIL receptors 1 and 2 are believed to interact with the
death-domain-containing adapter molecules TRADD and FADD to transmit a
death signal. To date, there is conflicting evidence as to which
adapters are actually required, the order of their assembly, and
whether or not there exists as yet unidentified adapter proteins unique
to the TRAIL system (34-38).
To determine the effects of the inflammatory cytokines TNF-
and
TRAIL on the natural host cell of human papillomaviruses, the genital
keratinocyte, retroviruses expressing the HPV-16 E6 and E7 oncoprotein
were used to infect HFKs. These cells were treated with cytokines with
and without the protein synthesis inhibitor cycloheximide. TNF-
alone induced growth arrest and differentiation in control HFKs but not
in HFKs expressing HPV-16 E7. HFKs expressing E6 or dominant negative
p53 also arrested in the presence of TNF-
, indicating that
TNF-
-induced cytostasis is p53-independent and can be overcome by
E7. In contrast, TRAIL alone did not inhibit proliferation, suggesting
a basic difference in the way these cytokines and their corresponding
receptors transmit intracellular signals. As in many other cell types,
apoptosis could be induced in cytokine-treated HFKs only with the
concurrent administration of the protein synthesis inhibitor
cycloheximide. TRAIL treatment induced apoptosis more rapidly and
efficiently than TNF-
and expression of the HPV-16 E7 protein
potentiated the cytotoxicity of both TNF-
and TRAIL.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Primary HFKs were prepared from a pool of
several neonatal foreskins obtained after routine circumcision,
following the protocol of Rheinwald and Beckett (39). The cells were
maintained in serum-free keratinocyte growth medium (Keratinocyte-SFM,
Life Technologies, Inc.). IMR-90 fibroblasts were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
Retroviral Infections--
LXSN-based retroviral vectors for
expressing HPV-16 E6 and E7 (40), dominant negative p53 (41), and SV40
large T antigen (42) were prepared using the protocols of Miller and
Rosman (43). Cells were infected with viral supernatants for 4 h
at 37 °C in the presence of 4 µg/ml Polybrene (hexadimethrine
bromide, Sigma Chemical Co.). The viral supernatant was removed, and
the plates were washed twice with phosphate-buffered saline. HFKs were
maintained in normal keratinocyte growth medium for the first 24 h after infection and then selected for 2 days in keratinocyte growth medium containing 200 µg/ml G418 (Calbiochem).
Cytokine Treatment--
Subconfluent plates of HFKs were treated
with the indicated concentrations of TNF-
(R&D Systems), TRAIL
(Alexis), or agonistic Fas antibody (Clone DX 2.1, R&D Systems) for the
indicated periods of time. Fresh media containing the appropriate
cytokine was added to the cultures for each day where the treatment
extended past 24 h.
Reverse Transcriptase Polymerase Chain Reaction--
Total RNA
was extracted from a subconfluent plate of HFKs using the RNeasy Mini
Kit (Qiagen). A 5-µg aliquot was reverse-transcribed with murine
leukemia virus reverse transcriptase (New England BioLabs), and the
resulting cDNA was combined with 5 pM of each primer
pair and PCR Supermix (Life Technologies, Inc.) in a final reaction
volume of 50 µl. A polymerase chain reaction was carried out for 35 cycles (95 °C melting temperature for 1 min; 55 °C annealing temperature for 1 min; 72 °C extension temperature for 1 min). The nucleotide sequences for the PCR primers (Life Technologies, Inc.) have been described previously (44).
Immunological Methods--
Cells were lysed in EBC lysis buffer
(50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40)
supplemented with protease inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 1 µl/ml aprotinin and leupeptin) and
phosphatase inhibitors (2 mM NaF and 0.5 mM
sodium orthovanadate) for 30 min at 4 °C. After centrifugation,
protein concentrations were measured using the Bio-Rad assay. 100 µg
of protein from each sample was subjected to SDS-polyacrylamide gel
electrophoresis and transferred onto a polyvinylidene difluoride
membrane (Immobilon P, Millipore Corp.). The membranes were then
incubated with the appropriate antibodies. The antibodies used were as
follows: TRAIL-R1 (Alexis, 210-730-C100); TRAIL-R2 (Imgenex, IMG-120);
TRAIL-R3 (Alexis, 210-744-R100); TRAIL-R4 (Imgenex, IMG-121); p53
(Ab-6, Oncogene Science); p21 (Ab-1, Oncogene Research Products);
transglutaminase (Ab-II, Neomarkers); actin (Ab-1, Oncogene Research
Products); phospho-Akt (Thr-308, New England BioLabs). Proteins were
detected using the ECL chemiluminescence system (Amersham Pharmacia
Biotech), and quantitation was performed with National Institutes of
Health IMAGE software.
Detection of Cell Proliferation--
Equal numbers of HFKs, as
determined by a cell counter (Coulter), were aliquoted into 35-mm
plates and treated with the appropriate cytokines. At each time point,
the cells were trypsinized in equal volumes of trypsin/EDTA and quantified.
Cell Cycle Analysis--
Cells were trypsinized and stained with
propidium iodide (Sigma), as described previously (45). The samples
were analyzed by fluorescence-activated cell sorting (FACS) using Cell
Quest software (Becton Dickinson).
Detection of Cell Death--
Keratinocytes growing on 35-mm
tissue culture plates were treated with 10 ng/ml TNF-
or TRAIL and
30 µg/ml cycloheximide (Sigma). The cells were stained with 1 µg/ml
bisbenzimide (Hoechst 33258, Sigma) and apoptotic nuclei were
visualized by fluorescence microscopy as described previously (41). 500 cells were counted per sample.
 |
RESULTS |
TNF-
, but Not TRAIL, Induces Growth Arrest in
Keratinocytes--
Although it has been previously reported that
TNF-
treatment causes a cytostatic effect in HFKs (46-49), little
is known about the effects of TRAIL on HFK growth. Therefore, HFKs were
grown with or without 10, 25, or 50 ng/ml TNF-
or TRAIL, and the
cells were counted at regular intervals. As previously reported,
TNF-
treatment of control HFKs had a strong cytostatic effect (Fig. 1a). FACS analysis revealed
that TNF-
-treated cells were mostly arrested in
G0/G1 (Fig. 1a). In contrast,
treatment with 10, 25, or 50 ng/ml TRAIL (Fig. 1a) or 1 or
10 µg/ml agonistic Fas antibody (data not shown) did not affect HFK
growth. To eliminate the possibility that the lack of growth arrest in
response to TRAIL treatment was due to the failure of TRAIL receptor
signaling, an immunoblot for the phosphorylated, active form of c-Akt
was performed. c-Akt is a cell survival factor that is phosphorylated
by the phosphatidylinositol 3'-OH kinase in response to
ligation of surface receptors such as those for platelet-derived growth
factor, insulin-like growth factor, and TNF-
(50-53). HFKs
incubated with 10 ng/ml TNF-
or TRAIL showed strong c-Akt
phosphorylation 2 min after addition of the cytokine to the media,
indicating that TRAIL signaling is intact (Fig. 1b). To
determine whether the cytostatic effect is not only specific for
TNF-
but also keratinocyte-specific, IMR-90 normal human diploid
lung fibroblasts were treated with 10 or 25 ng/ml TNF-
. No growth
inhibition was observed in these cells (Fig. 1c).

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Fig. 1.
a, TNF- causes growth arrest in LXSN
HFKs whereas TRAIL has no effect on keratinocyte growth. LX HFKs were
grown in normal media or media containing 10, 25, or 50 ng/ml TNF-
or TRAIL and were harvested and counted at the indicated time points.
The results are averages from three plates. Cell cycle analysis was
performed on TNF- - treated populations by FACS analysis of propidium
iodide-stained cells to determine DNA content. Cells in
G0/G1, S, and G2/M at 72 h of
growth in 10 ng/ml TNF- are expressed as a percentage of the total
cells pooled from three 35-mm plates. b, TRAIL signaling is
intact in keratinocytes. SDS-PAGE and immunoblot analysis for threonine
308 phosphorylated Akt was performed on untreated keratinocytes and
keratinocytes exposed to 10 ng/ml TNF- or TRAIL for 2, 7.5, 11.5, and 21.5 min. c, TNF- has no effect on proliferation of
IMR-90 normal human diploid fibroblasts. IMR-90 cells were treated as
above and counted at the indicated time points. The results are
averages from three experiments.
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|
TNF-
-induced Cytostatic Effect Is Overcome by HPV-16
E7--
HPV-16 E7 can interact with pRB and accelerate its
degradation, an effect closely correlated with transformation (5, 41). We wanted to determine if HPV 16 E7 expression could interfere with
TNF-
-mediated growth arrest. Therefore, HPV 16 E7-expressing HFKs
were grown with or without 10, 25, or 50 ng/ml TNF-
or TRAIL, and
the cells were counted at regular intervals. The growth of HPV-16
E7-expressing HFKs was not significantly inhibited at any concentration
of TNF-
(Fig. 2a). HFKs
stably expressing the transformation-defective HPV-16 E7 mutants 16E7
P6-E10, which can bind but not degrade pRB,
and 16E7
D21-C24, which can neither bind nor
degrade pRB (41), were unable to overcome TNF-
-induced growth arrest
(Fig. 2b). To further test whether inactivation of pRB is
linked to abrogation of TNF-
-mediated growth arrest, we tested SV40
large tumor antigen, which can also interact with and inactivate pRB (54). Like HPV-16 E7-expressing cells, HFKs stably expressing SV40
large tumor antigen were resistant to TNF-
-mediated growth arrest at
all concentrations of TNF-
(Fig. 2c, left
panel). In contrast, HFKs expressing a non-transforming pRB
binding-deficient SV40 large tumor antigen mutant (T107) remained
sensitive to TNF-
-mediated growth arrest (Fig. 2c,
right panel). Hence, the ability of a viral oncoprotein to
overcome the TNF-
-mediated growth arrest correlates with pRB
inactivation and cellular transformation.

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Fig. 2.
a, E7 overcomes TNF- -mediated growth
arrest. HFKs infected with E7 were grown in normal media or media
containing 10, 25, or 50 ng/ml TNF- or TRAIL and were harvested and
counted at the indicated time points. Cell cycle analysis was performed
on TNF- -treated populations by FACS analysis of propidium
iodide-stained cells. Cells in G0/G1, S, and
G2/M at 72 h of growth in 10 ng/ml TNF- are
expressed as a percentage of the total cells pooled from three 35-mm
plates. b, the ability of E7 to overcome the TNF-
cytostatic effect correlates with transformation. HFKs expressing the
transformation-deficient E7 mutants HPV-16E7 P6
-E10, which can bind but not degrade pRB (left
panel), and HPV-16E7 D21-C24, which can
neither bind nor degrade pRB (right panel), were incubated
in 10 ng/ml TNF- for the times indicated. c, HFKs
expressing pRB binding competent SV40 large T antigen can overcome the
cytostatic effect. HFKs expressing SV40 large T antigen (left
panel) or the pRB binding-deficient mutant SV40 large T107
(right panel) were grown in normal media or media containing
10 or 50 ng/ml TNF- or 50 ng/ml TRAIL and were harvested and counted
at the indicated time points.
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|
TNF-
-induced Cytostatic Effect Is Independent of p53--
In
HPV-infected cells the E7 protein is co-expressed with E6, which
targets p53, an important mediator of growth arrest, for proteasomal
degradation (7). To determine if p53 mediates the TNF-
cytostatic
effect, HFKs were infected with a retrovirus expressing either the
HPV-16 E6 oncoprotein or a dominant negative form of p53 (55). p53
protein levels were then analyzed by an immunoblot. Cells expressing
HPV-16 E6 contained very low p53 levels, presumably due to E6-mediated
protein degradation (Fig. 3a).
In contrast, p53 levels were increased in cells expressing the dominant
negative mutant, which binds and stabilizes endogenous p53 (Fig.
3a). Cells expressing either E6 or dominant negative p53
underwent growth arrest when incubated with TNF-
(Fig.
3b). This indicates that the TNF-
-induced cytostatic
effect is not mediated by p53.

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Fig. 3.
a, HPV-16 E6 and dominant negative p53
alter levels of endogenous p53 in HFKs. Stable HFK populations
expressing the HPV-16 E6 or a dominant negative version of p53 were
generated by retroviral gene transfer. Steady-state levels of p53 were
determined by immunoblot analysis. b, HPV-16 E6 and DN-p53
expressing cells are sensitive to TNF- -mediated growth arrest. Cells
were incubated in 10 ng/ml TNF- and counted at the times indicated.
The results are averages from three experiments.
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Effects of TNF-
and TRAIL Treatment on Differentiation of LX and
E7 HFKs--
The TNF-
cytostatic effect in keratinocytes may be
related to differentiation (48, 49). We therefore examined HFKs treated with TNF-
for transglutaminase, a late marker of keratinocyte differentiation (56, 57). Transglutaminase expression increased in
response to TNF-
in LXSN- and E6-expressing cells but not in
E7-expressing cells (Fig. 4a).
This demonstrates that the growth inhibitory activity of TNF-
correlates with induction of differentiation in human keratinocytes and
can be resisted by cells expressing HPV-16 E7.

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Fig. 4.
a, TNF- induces markers
of keratinocyte differentiation in LXSN- and E6-infected cells but not
in E7-infected cells. Cells were treated with 10 ng/ml TNF- for the
indicated periods of time. Steady-state levels of transglutaminase and
p21Cip1/Waf1 (upper panels) were determined by
SDS-PAGE and immunoblot analysis. Quantitation of signals are shown
underneath by the bar graphs. Actin was used as a
loading control (lower panel). b, TRAIL does not
cause an increase in markers of keratinocyte differentiation.
LXSN-infected HFKs were treated with 10 ng/ml TNF- or TRAIL for the
indicated periods of time. Transglutaminase levels were determined by
SDS-PAGE and immunoblot analysis as in a.
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Because the cdk inhibitor p21Cip1/Waf1 plays an important
role in coupling growth arrest and differentiation in human and murine
keratinocytes in response to Ca2+ (40, 58, 59), we probed
the same blot for p21Cip1/Waf1. There was an increase of
p21Cip1/Waf1 protein in LXSN-, E7-, and E6-infected HFKs
(Fig. 4a). Although p21Cip1/Waf1 levels
increased in E7 populations, this may be due to the ability of E7 to
bind and stabilize p21Cip1/Waf1 (60), which may contribute
to the ability of E7-expressing cells to overcome arrest (40, 61).
To further examine whether growth arrest and differentiation were
specific for TNF-
, we treated HFKs with TRAIL. As expected, TRAIL
treatment, which does not cause growth arrest, did not increase transglutaminase levels (Fig. 4b).
Effects of TNF-
and TRAIL Treatment on the Apoptotic Response of
LX and E7 HFKs--
Because TNF-
and TRAIL differentially affected
keratinocyte proliferation, we wanted to investigate the effects of
these cytokines on apoptosis. In many cell types, TNF-
or TRAIL
induces an apoptotic response only when new protein synthesis is
suppressed by treatment with cycloheximide. HFKs were therefore treated
with 10 ng/ml TNF-
or TRAIL, along with 30 µg/ml cycloheximide.
Under these conditions, TRAIL caused a rapid and strong apoptotic
response whereas the apoptotic response to TNF-
treatment of
keratinocytes was less pronounced (Fig.
5a). Importantly, however,
expression of E7 enhanced HFK apoptosis 2-fold for TNF-
treatment
and almost 4-fold for TRAIL treatment (Fig. 5b).

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Fig. 5.
a, TRAIL causes rapid and efficient
apoptosis in HFKs upon inhibition of new protein synthesis. Normal HFKs
were treated with 30 µg/ml cycloheximide alone or 30 µg/ml
cycloheximide and 10 ng/ml TNF- or TRAIL for the indicated periods
of time. The cells were then fixed in methanol and stained with Hoechst
33258. Apoptotic nuclei were counted as a percentage of total nuclei,
with the data points representing the averages of three experiments.
b, expression of HPV 16 E7 enhances TNF- - and
TRAIL-mediated apoptosis in keratinocytes. LXSN and E7 keratinocytes
were treated with 10 ng/ml TNF- or TRAIL along with 30 µg/ml
cycloheximide for 8 h. The cells were fixed in methanol, and the
nuclei were visualized by Hoechst 33258. Apoptotic nuclei were
counted as a percentage of total nuclei, with the data representing the
averages of 12 experiments.
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Expression of TRAIL and TNF-R1 Receptors in Primary Human
Keratinocytes--
It has been reported that the relative abundance of
TRAIL receptors, in particular the anti-apoptotic decoy receptors
TRAIL-R3 and TRAIL-R4, may contribute to the sensitivity of cells to
TRAIL-mediated apoptosis (26, 27, 29, 31). To determine if TRAIL
receptors R1 through R4 are present in HFKs, reverse
transcriptase-PCR was performed using keratinocyte mRNA as a
template. Signals for TRAIL receptors R1, R2, R3, and R4 were detected
in HFKs (Fig. 6a). Next, we
performed immunoblots on LXSN and E7 HFK extracts. These results
corroborated the results of the reverse transcriptase-PCR experiments
showing expression of these four receptors and demonstrated that they
are present at similar levels in both LX- and E7-expressing cells (Fig.
6b). Similarly, levels of TNF-R1 were also unchanged in
cells expressing E7 (Fig. 6b). Therefore, the observed
differences do not reflect alterations in receptor expression.

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Fig. 6.
Expression of TRAIL and TNF receptors in
HFKs. a, reverse transcriptase-PCR analysis of TRAIL
receptor mRNA expression in primary human keratinocytes.
b, control (LXSN) and HPV-16 E7-expressing HFKs were lysed,
and steady-state levels of TRAIL-R1, -R2, -R3, and -R4 were determined
by SDS-PAGE and immunoblot analysis.
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 |
DISCUSSION |
Upon viral infection, the host initiates a cell-mediated immune
response (62). Inflammatory cytokines are produced that modulate gene
expression through receptor-mediated induction of phosphorylation and
protein interaction cascades (63). Previous studies on the effects of
the inflammatory cytokine TNF-
have shown that it induces growth
arrest in keratinocytes, possibly through differentiation (46-49).
Less is known of the effects of the cytokine TRAIL in the skin and mucosa.
Growth arrest is an effective defense against viral infection, because
HPVs can reproduce only if the host cell's DNA synthesis machinery is
active. The human papillomaviruses overcome this defense by producing
oncoproteins that abrogate the function of p53 and pRB, proteins that
regulate progression through the cell cycle (11). Histologically, this
is seen as cell division within the upper levels of stratified
epithelium normally reserved for quiescent, differentiating cells (64,
65).
Here we show that TNF-
treatment of normal HFKs causes growth arrest
predominantly in the G0/G1 phase of the cell
cycle (Fig. 1a). This effect is not observed even following
treatment with high concentrations of TRAIL or incubation with an
agonistic Fas antibody (Fig. 1a and data not shown,
respectively). Because deficient receptor signaling in keratinocytes
could explain the lack of a cytostatic effect in TRAIL treatment, an
immunoblot was performed for phosphorylated, activated Akt. It is known
that many extracellular stimuli such as growth factors and TNF-
can
influence cell proliferation and survival through receptor-mediated
activation of the mitogen-activate protein kinase and
phosphatidylinositol 3'-OH-kinase/Akt pathways (50-53). Here we
show that TRAIL signaling is intact and functional in keratinocytes by
demonstrating the ability of TRAIL to induce phosphorylation of Akt at
levels equal to that seen in TNF-
treatment (Fig. 1b).
Therefore, the cytostatic effect appears to be specific for TNF-
. In
addition, this effect is likely specific for keratinocytes, because
treatment of normal human fibroblasts with TNF-
has no effect on
their growth (Fig. 1c).
It has been previously reported that HPV-immortalized cell lines are
less sensitive to TNF-
-induced growth arrest (66). Our experiments
show that cells expressing the HPV-16 E7 oncoprotein overcome the
growth inhibitory effects of TNF-
, even at concentrations of 50 ng/ml (Fig. 2a). This correlates with the ability of HPV-16 E7 to bind and degrade pRB and induce cellular transformation, because
mutants that cannot degrade pRB undergo growth arrest in response to
TNF-
(Fig. 2b). The importance of pRB inactivation and
cellular transformation in overcoming the TNF-
cytostatic effect is
further emphasized by the ability of keratinocytes expressing the SV40
large T antigen to continue to grow in high concentrations of TNF-
(Fig. 2c, left panel). Similar to E7, HFKs
expressing a transformation-deficient SV40 large T antigen that does
not interact with pRB remain sensitive to TNF-
-mediated growth
arrest (Fig. 2c, right panel). Interestingly,
although pRB status appears critical in the mechanism of growth
arrest, TNF-
-induced cytostasis is not mediated by p53, because HFKs
expressing E6 or dominant negative p53 were still arrested by TNF-
(Fig. 3b).
Our results suggest that E7 attenuates both cell cycle arrest and
differentiation in TNF-
-treated HFKs. p21Cip1/Waf1 is a
cyclin-dependent kinase inhibitor that regulates
progression into S-phase by preventing cyclin E/cdk 2 from
phosphorylating and inactivating pRB or by directly inhibiting E2F-1
itself (67). Growth arrest and differentiation caused by
Ca2+ treatment of keratinocytes demonstrates a
corresponding increase in p21Cip1/Waf1 protein levels (40,
58, 59). In our experiments, similar increases in
p21Cip1/Waf1 levels are seen following TNF-
treatment in
HFKs (Fig. 4a). Significantly, a gradual increase in
p21Cip1/Waf1 levels is also observed in TNF-
-treated
E6-expressing keratinocytes, again suggesting p53-independent
p21Cip1/Waf1 regulation during cytokine-mediated
keratinocyte growth arrest and differentiation.
E7 has been shown to bind and stabilize
p21Cip1/Waf1 and abrogate its function during
Ca2+-induced differentiation (40, 60, 61). This could
explain why levels of p21Cip1/Waf1 are generally higher in
E7-expressing cells throughout the course of TNF-
treatment (Fig.
4a). E7-expressing cells may therefore be able to resist
TNF-
-mediated growth arrest not only through the ability of HPV-16
E7 to degrade pRB but also through direct inhibition of
p21Cip1/Waf1. Inhibition of p21Cip1/Waf1 by
antisense mRNA or intracellular injection of
-p21Cip1/Waf1 antibodies has in fact been shown to force
senesced or differentiated cells back into the cell cycle (68, 69).
Levels of transglutaminase, a marker of keratinocyte differentiation,
increase in LXSN and E6 cells grown in TNF-
but not in identically
treated E7 HFKs (Fig. 4a). These findings are consistent with previous studies of E7- and E6/E7-expressing HFKs that have shown
delayed differentiation and continued expression of cyclin A, an
S-phase cyclin, when grown in semisolid media or Ca2+,
conditions that normally favor differentiation (40, 70, 71). Treatment
of LXSN-infected HFKs with TRAIL alone, which has no effect on the
growth of keratinocytes, does not cause an increase in levels of
transglutaminase (Fig. 4b). Therefore, unlike TNF-
, TRAIL
cannot activate a differentiation program.
Apoptotic signaling in response to TRAIL has not been well
characterized. There is conflicting evidence regarding the recruitment of adapter proteins to the receptor and the mechanism of caspase activation. In our study, treatment of keratinocytes with cycloheximide and TNF-
causes relatively low levels of apoptosis, whereas
treatment with TRAIL and cycloheximide causes more rapid and efficient
apoptosis (Fig. 5a).
HFKs expressing the E7 oncoprotein are more prone to both TNF-
- and
TRAIL-mediated apoptosis (Fig. 5b). This is not due to up-regulation of expression of TNF or TRAIL receptors by E7, because receptors R1 through R4 are present in both cell types in equal amounts
(Fig. 6b). Instead, the ability of E7 to enhance E2F
transcriptional activity through degradation of pRB is likely
responsible for this effect. Increased E2F-1 activity has been shown in
other cell types to promote not only progression through S-phase but also apoptosis (72, 73). Previous studies have shown that E7-expressing
keratinocytes exhibited not only enhanced growth, but also spontaneous
apoptosis (74). In cytokine-treated HFKs, free E2F-1 may further
enhance cell death by directly inhibiting anti-apoptotic signaling from
TNF receptor-associated factor 2 (TRAF2) at the level of the
receptor (75).
E7-mediated p21Cip1/Waf1 inactivation, which helps overcome
TNF-
-mediated growth arrest, may also enhance apoptosis. In general,
cyclin-dependent kinase inhibitor expression associated
with senescence or differentiation has been shown to confer resistance
to cell death (76, 77). TNF-
-induced increases in
p21Cip1/Waf1 delay cell death in MCF-7 cells (78). Indeed,
it has been suggested that p21Cip1/Waf1 may need to be
degraded before a cell can even commence apoptosis (79). One possible
mechanism for this could be through the ability of
p21Cip1/Waf1 to directly inhibit caspase 3 (80).
Here we report that HPV-16 E7 can modulate the responses of its natural
host cell to the closely related cytokines TNF-
and TRAIL. Future
studies will try to decipher the nature of TRAIL signaling in
keratinocytes. Dissection and analysis of cytokine signaling pathways
and understanding how this virus disrupts intracellular signaling and
cell cycle control could lead to a better understanding of how HFKs
infected with HPV-16 avoid the immune system and progress to malignancy.
We thank Denise Galloway and Moshe Oren for
providing retroviral packaging cell lines and Lily Yeh and David Alan
Thompson for initial help with keratinocyte cultures. Special thanks to Alexandra Eichten, Sonia Gonzalez, Tammy Piboonniyom, and
Miranda Grace for help and critical comments.
1.
|
Howley, P. M.
(1991)
Cancer Res.
51,
5019s-5022s[Abstract]
|
2.
|
zur Hausen, H.
(1996)
Biochim. Biophys. Acta
1288,
F55-F78[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Sugerman, P. B.,
and Shillitoe, E. J.
(1997)
Oral Dis.
3,
130-147[Medline]
[Order article via Infotrieve]
|
4.
|
Dyson, N.,
Howley, P. M.,
Munger, K.,
and Harlow, E.
(1989)
Science
243,
934-937[Medline]
[Order article via Infotrieve]
|
5.
|
Münger, K.,
Werness, B. A.,
Dyson, N.,
Phelps, W. C.,
Harlow, E.,
and Howley, P. M.
(1989)
EMBO J.
8,
4099-4105[Abstract]
|
6.
|
Werness, B. A.,
Levine, A. J.,
and Howley, P. M.
(1990)
Science
248,
76-79[Medline]
[Order article via Infotrieve]
|
7.
|
Scheffner, M.,
Werness, B. A.,
Huibregtse, J. M.,
Levine, A. J.,
and Howley, P. M.
(1990)
Cell
63,
1129-1136[Medline]
[Order article via Infotrieve]
|
8.
|
Boyer, S. N.,
Wazer, D. E.,
and Band, V.
(1996)
Cancer Res.
56,
4620-4624[Abstract]
|
9.
|
Sherr, C. J.
(1994)
Cell
79,
551-555[Medline]
[Order article via Infotrieve]
|
10.
|
Sherr, C. J.
(1996)
Science
274,
1672-1677[Abstract/Free Full Text]
|
11.
|
Jones, D. L.,
and Munger, K.
(1996)
Semin. Cancer Biol.
7,
327-337[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Sherr, C. J.,
and Roberts, J. M.
(1995)
Genes Dev.
9,
1149-1163[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Hartwell, L. H.,
and Kastan, M. B.
(1994)
Science
266,
1821-1828[Medline]
[Order article via Infotrieve]
|
14.
|
Kondo, S.,
and Sauder, D. N.
(1997)
Eur. J. Immunol.
27,
1713-1718[Medline]
[Order article via Infotrieve]
|
15.
|
Nagata, S.
(1997)
Cell
88,
355-365[Medline]
[Order article via Infotrieve]
|
16.
|
Wallach, D.,
Varfolomeev, E. E.,
Malinin, N. L.,
Goltsev, Y. V.,
Kovalenko, A. V.,
and Boldin, M. P.
(1999)
Annu. Rev. Immunol.
17,
331-367[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Pan, G.,
O'Rourke, K.,
Chinnaiyan, A. M.,
Gentz, R.,
Ebner, R.,
Ni, J.,
and Dixit, V. M.
(1997)
Science
276,
111-113[Abstract/Free Full Text]
|
18.
|
Liu, Z. G.,
Hsu, H.,
Goeddel, D. V.,
and Karin, M.
(1996)
Cell
87,
565-576[Medline]
[Order article via Infotrieve]
|
19.
|
Natoli, G.,
Costanzo, A.,
Guido, F.,
Moretti, F.,
Bernardo, A.,
Burgio, V. L.,
Agresti, C.,
and Levrero, M.
(1998)
J. Biol. Chem.
273,
31262-31272[Abstract/Free Full Text]
|
20.
|
Natoli, G.,
Costanzo, A.,
Guido, F.,
Moretti, F.,
and Levrero, M.
(1998)
Biochem. Pharmacol.
56,
915-920[CrossRef][Medline]
[Order article via Infotrieve]
|
21.
|
Wiley, S. R.,
Schooley, K.,
Smolak, P. J.,
Din, W. S.,
Huang, C. P.,
Nicholl, J. K.,
Sutherland, G. R.,
Smith, T. D.,
Rauch, C.,
and Smith, C. A.
(1995)
Immunity
3,
673-682[Medline]
[Order article via Infotrieve]
|
22.
|
Griffith, T. S.,
and Lynch, D. H.
(1998)
Curr. Opin. Immunol.
10,
559-563[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Pitti, R. M.,
Marsters, S. A.,
Ruppert, S.,
Donahue, C. J.,
Moore, A.,
and Ashkenazi, A.
(1996)
J. Biol. Chem.
271,
12687-12690[Abstract/Free Full Text]
|
24.
|
Walczak, H.,
Degli-Esposti, M. A.,
Johnson, R. S.,
Smolak, P. J.,
Waugh, J. Y.,
Boiani, N.,
Timour, M. S.,
Gerhart, M. J.,
Schooley, K. A.,
Smith, C. A.,
Goodwin, R. G.,
and Rauch, C. T.
(1997)
EMBO J.
16,
5386-5397[Abstract/Free Full Text]
|
25.
|
Hymowitz, S. G.,
Christinger, H. W.,
Fuh, G.,
Ultsch, M.,
O'Connell, M.,
Kelley, R. F.,
Ashkenazi, A.,
and de Vos, A. M.
(1999)
Mol. Cell
4,
563-571[Medline]
[Order article via Infotrieve]
|
26.
|
Marsters, S. A.,
Sheridan, J. P.,
Pitti, R. M.,
Huang, A.,
Skubatch, M.,
Baldwin, D.,
Yuan, J.,
Gurney, A.,
Goddard, A. D.,
Godowski, P.,
and Ashkenazi, A.
(1997)
Curr. Biol.
7,
1003-1006[Medline]
[Order article via Infotrieve]
|
27.
|
Pan, G.,
Ni, J.,
Wei, Y. F., Yu, G.,
Gentz, R.,
and Dixit, V. M.
(1997)
Science
277,
815-818[Abstract/Free Full Text]
|
28.
|
Sheridan, J. P.,
Marsters, S. A.,
Pitti, R. M.,
Gurney, A.,
Skubatch, M.,
Baldwin, D.,
Ramakrishnan, L.,
Gray, C. L.,
Baker, K.,
Wood, W. I.,
Goddard, A. D.,
Godowski, P.,
and Ashkenazi, A.
(1997)
Science
277,
818-821[Abstract/Free Full Text]
|
29.
|
MacFarlane, M.,
Ahmad, M.,
Srinivasula, S. M.,
Fernandes-Alnemri, T.,
Cohen, G. M.,
and Alnemri, E. S.
(1997)
J. Biol. Chem.
272,
25417-25420[Abstract/Free Full Text]
|
30.
|
Schneider, P.,
Bodmer, J. L.,
Thome, M.,
Hofmann, K.,
Holler, N.,
and Tschopp, J.
(1997)
FEBS Lett.
416,
329-334[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Degli-Esposti, M. A.,
Dougall, W. C.,
Smolak, P. J.,
Waugh, J. Y.,
Smith, C. A.,
and Goodwin, R. G.
(1997)
Immunity
7,
813-820[Medline]
[Order article via Infotrieve]
|
32.
|
Simonet, W. S.,
Lacey, D. L.,
Dunstan, C. R.,
Kelley, M.,
Chang, M. S.,
Luthy, R.,
Nguyen, H. Q.,
Wooden, S.,
Bennett, L.,
Boone, T.,
Shimamoto, G.,
DeRose, M.,
Elliott, R.,
Colombero, A.,
Tan, H. L.,
Trail, G.,
Sullivan, J.,
Davy, E.,
Bucay, N.,
Renshaw-Gegg, L.,
Hughes, T. M.,
Hill, D.,
Pattison, W.,
Campbell, P.,
and Boyle, W. J.
(1997)
Cell
89,
309-319[Medline]
[Order article via Infotrieve]
|
33.
|
Emery, J. G.,
McDonnell, P.,
Burke, M. B.,
Deen, K. C.,
Lyn, S.,
Silverman, C.,
Dul, E.,
Appelbaum, E. R.,
Eichman, C.,
DiPrinzio, R.,
Dodds, R. A.,
James, I. E.,
Rosenberg, M.,
Lee, J. C.,
and Young, P. R.
(1998)
J. Biol. Chem.
273,
14363-14367[Abstract/Free Full Text]
|
34.
|
Yeh, W. C.,
Pompa, J. L.,
McCurrach, M. E.,
Shu, H. B.,
Elia, A. J.,
Shahinian, A.,
Ng, M.,
Wakeham, A.,
Khoo, W.,
Mitchell, K.,
El-Deiry, W. S.,
Lowe, S. W.,
Goeddel, D. V.,
and Mak, T. W.
(1998)
Science
279,
1954-1958[Abstract/Free Full Text]
|
35.
|
Wajant, H.,
Johannes, F. J.,
Haas, E.,
Siemienski, K.,
Schwenzer, R.,
Schubert, G.,
Weiss, T.,
Grell, M.,
and Scheurich, P.
(1998)
Curr. Biol.
8,
113-116[Medline]
[Order article via Infotrieve]
|
36.
|
Schneider, P.,
Thome, M.,
Burns, K.,
Bodmer, J. L.,
Hofmann, K.,
Kataoka, T.,
Holler, N.,
and Tschopp, J.
(1997)
Immunity
7,
831-836[Medline]
[Order article via Infotrieve]
|
37.
|
Kischkel, F. C.,
Lawrence, D. A.,
Chuntharapai, A.,
Schow, P.,
Kim, K. J.,
and Ashkenazi, A.
(2000)
Immunity
12,
611-620[Medline]
[Order article via Infotrieve]
|
38.
|
Sprick, M. R.,
Weigand, M. A.,
Rieser, E.,
Rauch, C. T.,
Juo, P.,
Blenis, J.,
Krammer, P. H.,
and Walczak, H.
(2000)
Immunity
12,
599-609[Medline]
[Order article via Infotrieve]
|
39.
|
Rheinwald, J. G.,
and Beckett, M. A.
(1981)
Cancer Res.
41,
1657-1663[Abstract]
|
40.
|
Jones, D. L.,
Alani, R. M.,
and Munger, K.
(1997)
Genes Dev.
11,
2101-2111[Abstract/Free Full Text]
|
41.
|
Jones, D. L.,
Thompson, D. A.,
and Munger, K.
(1997)
Virology
239,
97-107[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Chang, T. H.,
Ray, F. A.,
Thompson, D. A.,
and Schlegel, R.
(1997)
Oncogene
14,
2383-2393[CrossRef][Medline]
[Order article via Infotrieve]
|
43.
|
Miller, A. D.,
and Rosman, G. J.
(1989)
BioTechniques
7,
980-982[Medline]
[Order article via Infotrieve]
|
44.
|
Kothny-Wilkes, G.,
Kulms, D.,
Poppelmann, B.,
Luger, T. A.,
Kubin, M.,
and Schwarz, T.
(1998)
J. Biol. Chem.
273,
29247-29253[Abstract/Free Full Text]
|
45.
|
Thompson, D. A.,
Belinsky, G.,
Chang, T. H.-T.,
Jones, D. L.,
Schlegel, R.,
and Münger, K.
(1997)
Oncogene
15,
3025-3036[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Symington, F. W.
(1989)
J. Invest. Dermatol.
92,
798-805[Abstract]
|
47.
|
Detmar, M.,
and Orfanos, C. E.
(1990)
Arch. Dermatol. Res.
282,
238-245[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Pillai, S.,
Bikle, D. D.,
Eessalu, T. E.,
Aggarwal, B. B.,
and Elias, P. M.
(1989)
J. Clin. Invest.
83,
816-21[Medline]
[Order article via Infotrieve]
|
49.
|
Kono, T.,
Tanii, T.,
Furukawa, M.,
Mizuno, N.,
Taniguchi, S.,
Ishii, M.,
and Hamada, T.
(1990)
J. Dermatol.
17,
409-413[Medline]
[Order article via Infotrieve]
|
50.
|
Datta, S. R.,
Brunet, A.,
and Greenberg, M. E.
(1999)
Genes Dev.
13,
2905-2927[Free Full Text]
|
51.
|
Pastorino, J. G.,
Tafani, M.,
and Farber, J. L.
(1999)
J. Biol. Chem.
274,
19411-19416[Abstract/Free Full Text]
|
52.
|
Ozes, O. N.,
Mayo, L. D.,
Gustin, J. A.,
Pfeffer, S. R.,
Pfeffer, L. M.,
and Donner, D. B.
(1999)
Nature
401,
82-85[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Delhase, M.,
Li, N.,
and Karin, M.
(2000)
Nature
406,
367-368[CrossRef][Medline]
[Order article via Infotrieve]
|
54.
|
DeCaprio, J. A.,
Ludlow, J. W.,
Figge, J.,
Shew, J. Y.,
Huang, C. M.,
Lee, W. H.,
Marsilio, E.,
Paucha, E.,
and Livingston, D. M.
(1988)
Cell
54,
275-283[Medline]
[Order article via Infotrieve]
|
55.
|
Gottlieb, E.,
Haffner, R.,
von Ruden, T.,
Wagner, E. F.,
and Oren, M.
(1994)
EMBO J.
13,
1368-1374[Abstract]
|
56.
|
George, M. D.,
Vollberg, T. M.,
Floyd, E. E.,
Stein, J. P.,
and Jetten, A. M.
(1990)
J. Biol. Chem.
265,
11098-11104[Abstract/Free Full Text]
|
57.
|
Greenberg, C. S.,
Birckbichler, P. J.,
and Rice, R. H.
(1991)
FASEB J.
5,
3071-3077[Abstract/Free Full Text]
|
58.
|
Missero, C.,
Calautti, E.,
Eckner, R.,
Chin, J.,
Tsai, L. H.,
Livingston, D. M.,
and Dotto, G. P.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5451-5455[Abstract]
|
59.
|
Alani, R. M.,
Hasskarl, J.,
and Munger, K.
(1998)
Mol. Carcinog.
23,
226-233[CrossRef][Medline]
[Order article via Infotrieve]
|
60.
|
Jones, D. L.,
Thompson, D. A.,
Suh-Burgmann, E.,
Grace, M.,
and Munger, K.
(1999)
Virology
258,
406-414[CrossRef][Medline]
[Order article via Infotrieve]
|
61.
|
Funk, J. O.,
Waga, S.,
Harry, J. B.,
Espling, E.,
Stillman, B.,
and Galloway, D. A.
(1997)
Genes Dev.
11,
2090-2100[Abstract/Free Full Text]
|
62.
|
Kyo, S.,
Inoue, M.,
Hayasaka, N.,
Inoue, T.,
Yutsudo, M.,
Tanizawa, O.,
and Hakura, A.
(1994)
Virology
200,
130-139[CrossRef][Medline]
[Order article via Infotrieve]
|
63.
|
Baker, S. J.,
and Reddy, E. P.
(1998)
Oncogene
17,
3261-3270[Medline]
[Order article via Infotrieve]
|
64.
|
Smith-McCune, K.,
Kalman, D.,
Robbins, C.,
Shivakumar, S.,
Yuschenkoff, L.,
and Bishop, J. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
6999-7004[Abstract/Free Full Text]
|
65.
|
Cheng, S.,
Schmidt-Grimminger, D. C.,
Murant, T.,
Broker, T. R.,
and Chow, L. T.
(1995)
Genes Dev.
9,
2335-2349[Abstract]
|
66.
|
Villa, L. L.,
Vieira, K. B.,
Pei, X. F.,
and Schlegel, R.
(1992)
Mol. Carcinog.
6,
5-9[Medline]
[Order article via Infotrieve]
|
67.
|
Delavaine, L.,
and La Thangue, N. B.
(1999)
Oncogene
18,
5381-5392[CrossRef][Medline]
[Order article via Infotrieve]
|
68.
|
Nakanishi, M.,
Adami, G. R.,
Robetorye, R. S.,
Noda, A.,
Venable, S. F.,
Dimitrov, D.,
Pereira-Smith, O. M.,
and Smith, J. R.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
4352-4356[Abstract]
|
69.
|
Ma, Y.,
Prigent, S. A.,
Born, T. L.,
Monell, C. R.,
Feramisco, J. R.,
and Bertolaet, B. L.
(1999)
Cancer Res.
59,
5341-5348[Abstract/Free Full Text]
|
70.
|
Ruesch, M. N.,
and Laimins, L. A.
(1998)
Virology
250,
19-29[CrossRef][Medline]
[Order article via Infotrieve]
|
71.
|
Woodworth, C. D.,
Waggoner, S.,
Barnes, W.,
Stoler, M. H.,
and DiPaolo, J. A.
(1990)
Cancer Res.
50,
3709-3715[Abstract]
|
72.
|
Kowalik, T. F.,
DeGregori, J.,
Schwarz, J. K.,
and Nevins, J. R.
(1995)
J. Virol.
69,
2491-2500[Abstract]
|
73.
|
Shan, B.,
and Lee, W. H.
(1994)
Mol. Cell. Biol.
14,
8166-8173[Abstract]
|
74.
|
Stoppler, H.,
Stoppler, M. C.,
Johnson, E.,
Simbulan-Rosenthal, C. M.,
Smulson, M. E.,
Iyer, S.,
Rosenthal, D. S.,
and Schlegel, R.
(1998)
Oncogene
17,
1207-1214[CrossRef][Medline]
[Order article via Infotrieve]
|
75.
|
Phillips, A. C.,
Ernst, M. K.,
Bates, S.,
Rice, N. R.,
and Vousden, K. H.
(1999)
Mol. Cell
4,
771-781[CrossRef][Medline]
[Order article via Infotrieve]
|
76.
|
Wang, J.,
and Walsh, K.
(1996)
Science
273,
359-361[Abstract]
|
77.
|
Chaturvedi, V.,
Qin, J. Z.,
Denning, M. F.,
Choubey, D.,
Diaz, M. O.,
and Nickoloff, B. J.
(1999)
J. Biol. Chem.
274,
23358-23367[Abstract/Free Full Text]
|
78.
|
Jiang, Y.,
and Porter, A. G.
(1998)
Biochem. Biophys. Res. Commun.
245,
691-697[CrossRef][Medline]
[Order article via Infotrieve]
|
79.
|
Zhang, Y.,
Fujita, N.,
and Tsuruo, T.
(1999)
Oncogene
18,
1131-1138[CrossRef][Medline]
[Order article via Infotrieve]
|
80.
|
Suzuki, A.,
Tsutomi, Y.,
Akahane, K.,
Araki, T.,
and Miura, M.
(1998)
Oncogene
17,
931-939[CrossRef][Medline]
[Order article via Infotrieve]
|