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J Virol, February 1998, p. 1131-1137, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human Papillomavirus Oncoproteins E6 and E7
Independently Abrogate the Mitotic Spindle Checkpoint
Jennifer T.
Thomas and
Laimonis A.
Laimins*
Department of Microbiology-Immunology,
Northwestern University Medical School, Chicago, Illinois 60611
Received 25 July 1997/Accepted 10 November 1997
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ABSTRACT |
The E6 and E7 genes of the high-risk human papillomavirus (HPV)
types encode oncoproteins, and both act by interfering with the
activity of cellular tumor suppressor proteins. E7 proteins act by
associating with members of the retinoblastoma family, while E6
increases the turnover of p53. p53 has been implicated as a regulator
of both the G1/S cell cycle checkpoint and the mitotic
spindle checkpoint. When fibroblasts from p53 knockout mice are treated
with the spindle inhibitor nocodazole, a rereplication of DNA occurs
without transit through mitosis. We investigated whether E6 or E7 could
induce a similar loss of mitotic checkpoint activity in human
keratinocytes. Recombinant retroviruses expressing high-risk E6 alone,
E7 alone, and E6 in combination with E7 were used to infect normal
human foreskin keratinocytes (HFKs). Established cell lines were
treated with nocodazole, stained with propidium iodide, and analyzed
for DNA content by flow cytometry. Cells infected with high-risk E6
were found to continue to replicate DNA and accumulated an octaploid
(8N) population. Surprisingly, expression of E7 alone was also able to
bypass this checkpoint. Cells expressing E7 alone exhibited increased
levels of p53, while those expressing E6 had significantly reduced
levels. The p53 present in the E7 cells was active, as increased levels
of p21 were observed. This suggested that E7 bypassed the mitotic
checkpoint by a p53-independent mechanism. The levels of MDM2, a
cellular oncoprotein also implicated in control of the mitotic
checkpoint, were significantly elevated in the E7 cells compared to the
normal HFKs. In E6-expressing cells, the levels of MDM2 were
undetectable. It is possible that abrogation of Rb function by E7 or
increased expression of MDM2 contributes to the loss of mitotic spindle checkpoint control in the E7 cells. These findings suggest mechanisms by which both HPV oncoproteins contribute to genomic instability at the
mitotic checkpoint.
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INTRODUCTION |
Human papillomaviruses (HPVs) are
small double-stranded DNA viruses that induce hyperproliferative
lesions of cutaneous and mucosal epithelia. Half of the more than 70 identified types of HPVs specifically infect the genital epithelium.
These genital papillomaviruses can be divided into low-risk types,
which induce only benign lesions, and high-risk types, which are
associated with the development of malignant lesions (28).
More than 90% of cervical cancers contain HPV DNA of the high-risk
types (11, 34, 41, 58, 69). The two transforming proteins
encoded by the high-risk HPVs, E6 and E7, function through their
associations with the tumor suppressor proteins, p53 and Rb,
respectively (reviewed in reference 60). E6
facilitates the degradation of p53 through its association with an
accessory protein, E6-AP, a component of the ubiquitin proteolytic
pathway (29, 55, 57, 63). E7 proteins of the high-risk types
bind to Rb (14, 50), as well as to other pocket proteins,
such as p107 and p130 (7, 13), leading to the altered
activities of these cell cycle regulators. The E6 proteins from the
low-risk viruses fail to abrogate p53 functions, while the low-risk E7
proteins bind Rb with substantially reduced affinities (reviewed in
references 28 and 56). These differences are likely responsible for the lack of association of the
low-risk types with malignancy.
p53 is a site-specific DNA binding protein which activates expression
of genes involved in cell cycle control such as the cyclin kinase
inhibitor p21 (15). In response to DNA-damaging agents, p53
levels increase by a posttranscriptional mechanism resulting in arrest
via inhibition of cyclin-associated kinase activity at the
G1/S interface of the cell cycle (reviewed in reference
32). Loss of p53 or expression of mutant p53 results in a failure to arrest in G1, and p53-negative cells
exhibit gene amplification, a marker of genomic instability (40,
68). p53 is commonly mutated in human cancers, many of which
contain amplifications and aneuploid chromosomes, consistent with its
role as a "guardian of the genome" (reviewed in references
36 and 39). In this capacity, p53
has been postulated to play a role in maintaining genomic integrity.
All of these functions of p53 are inhibited by HPV E6 of the high-risk
types (18, 20, 27, 31, 37, 38, 46).
The product of the retinoblastoma gene, Rb, in addition to p53, plays a
significant role in the regulation of the cell cycle and its
checkpoints. Prior to S phase, Rb, in complex with the transcription
factor E2F, is hyperphosphorylated, leading to the release of E2F,
which binds to promoters of numerous genes required for DNA synthesis
(reviewed in reference 62). The binding of E7 to Rb
inhibits the association of Rb with E2F, resulting in constitutive
activation of E2F and expression of these genes (3). Furthermore, Rb can bind p107 and p130, which also negatively regulate
E2F transcription (7, 14). In this way, E7 can modulate the
cell cycle by inappropriately inducing S-phase progression. Additional
studies with E7 have demonstrated the ability of E7 to bypass
p21-mediated G1 arrest following DNA damage (8, 9, 25,
59). The mechanism for abrogation of this checkpoint is independent of p53 and likely acts through deregulation of E2F activity
(26, 48, 54).
In addition to directing the G1/S checkpoint, p53 also
functions in the mitotic spindle checkpoint at G2/M.
Whereas the treatment of wild-type cells with the mitotic spindle
inhibitor nocodazole results in arrest at G2/M, mouse
embryo fibroblasts from p53 knockout mice treated with nocodazole
continue to replicate their DNA, resulting in a significant polyploid
population (6). The absence of p53 results in aberrant
chromosomal replication, implicating it in control of this mitotic
checkpoint. The loss of p53 function in cervical cancer, through the
action of E6, could, by this mechanism, result in the acquisition of
numerous genetic alterations and contribute to the multistep progress
of this disease. In this study, we have examined the ability of the HPV
oncoproteins E6 and E7 to inhibit the mitotic spindle checkpoint.
Expression of either E6 or E7 was found to independently bypass the
mitotic checkpoint. We believe that E6 is able to inhibit this function via degradation of p53. E7 appears to bypass this checkpoint in the
presence of high levels of p53, possibly through the loss of Rb and/or
the increased expression of the cellular oncoprotein MDM2.
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MATERIALS AND METHODS |
Cell culture.
Human foreskin keratinocytes (HFKs) were
derived from neonatal human foreskin epithelium as previously described
(22) and were maintained in serum-free keratinocyte growth
medium (KGM; Clonetics). Retrovirally infected cells were grown in
serum-containing medium (44) with mitomycin C (Boehringer
Mannheim)-treated J2 3T3 fibroblast feeders (45) kindly
provided by the Howard Green laboratory.
Infection of HFKs and cell lines.
The retrovirus vector and
construction of the LXSN plasmids have been described previously
(21). PA317 packaging cell lines generating HPV-16 E6, -16 E7, and -16 E6E7 recombinant retroviruses were provided by Denise
Galloway. For infection of HFKs, 1.5 ml of the amphotropic viral
supernatant was combined with 5 ml of KGM containing Polybrene (Sigma)
(9 µg/ml) and added to subconfluent HFKs in a 100-mm dish. After 6 to
8 h, 10 ml of fresh KGM was added to the dish and cells were
allowed to incubate for an additional 12 to 18 h. Cells were then
split 1:3 and replated in serum-containing medium in the presence of
fibroblast feeders. Selection with G418 sulfate (Gibco) (100 to 200 µg/ml) began 2 days postinfection and continued for 8 days. The LKP31
cell line was generated by transfection of HFKs with HPV-31 DNA as
previously described (19).
Flow cytometry analyses.
For cell cycle analyses, cells were
either untreated or treated with nocodazole (Sigma) (50 ng/ml) at
various time points and harvested following trypsinization. Cells grown
with fibroblast feeders were treated with EDTA prior to harvesting to
remove feeders as described elsewhere (45). Harvested cells
(1.5 × 106) were washed with phosphate-buffered
saline and centrifuged, and the cell pellet was resuspended in 0.5 ml
of stain solution (0.1 mg of propidium iodide [PI] per ml, 0.5 mg of
RNase A per ml, 1% PBA-Triton [1 mg of bovine serum albumin per ml
in phosphate-buffered saline-10% Triton X-100], 3.34 mM sodium
citrate, 30 mg of polyethylene glycol per ml). Cells were then passed
through an 18-gauge needle six times and incubated at 37°C for 20 min
with shaking. An equal volume of hypertonic solution (0.1 mg of PI per
ml, 1% PBA-Triton, 0.356 M NaCl, 30 mg of polyethylene glycol per ml)
was added, the cell extracts were sheared again, and samples were
stored at 4°C for at least 6 h. Stained nuclei were analyzed on
a Becton-Dickinson (Mountain View, Calif.) FACScan with Lysis II
software.
Antibodies and Western blot analyses.
Antibodies directed
against p53 protein (Ab-2) and MDM2 (Ab-1) were obtained from Oncogene
Science, Inc. Antibodies directed against p21 protein (clone 6B6) and
cyclin B1 (clone GNS-1) were obtained from PharMingen. Antibodies
against cdc2 p34 (17) were obtained from Santa Cruz
Biotechnology, and horseradish peroxidase-linked sheep anti-mouse
secondary antibody was obtained from Amersham. Nocodazole-treated and
untreated cells were harvested by centrifugation (1,100 rpm; Beckman
TJ6) following trypsinization and lysed with 0.5% Nonidet P-40 lysis
buffer. HFK-16E7 cells were harvested after 96 h of nocodazole
treatment; all other cell populations were harvested after 48 h.
Protein concentrations were determined by Bradford assays, and 100 µg
of whole-cell extract from each cell type was analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then
transferred to a polyvinylidene difluoride membrane (Millipore) and
incubated with 5% milk solution to block nonspecific binding. Primary
and secondary antibodies were incubated with the membrane for 1 h
and 30 min, respectively, followed by chemiluminescence detection as
described by the manufacturer (ECL; Amersham).
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RESULTS |
Abrogation of mitotic checkpoint by E6.
p53 has been shown to
play a role in regulation of the mitotic spindle checkpoint at the end
of S phase in mouse embryo fibroblasts (6). In order to
investigate whether this function of p53 can be inhibited by E6
proteins from high-risk HPV types, we examined the effects of E6
expression on cell cycle progression of nocodazole-treated HFKs. HFKs
were infected with recombinant retroviruses which express the HPV-16 E6
gene or the -16 E6 and E7 genes together. Following selection for
neomycin resistance, colonies were pooled, expanded, and treated with
nocodazole. Flow cytometry analysis of DNA content was performed on
uninfected and HPV oncoprotein-infected HFKs to determine their ability
to arrest at G2/M after exposure to nocodazole (Fig.
1). Normal HFKs were found to accumulate
with a 4N (tetraploid) DNA content after exposure to nocodazole (Fig. 1A and B), and very few cells continued to replicate to an 8N (octaploid) population. Untreated HFK-16E6 cells exhibited a cell cycle
profile identical to that of normal HFKs (Fig. 1C) but failed to arrest
DNA synthesis after nocodazole treatment. Instead, the cells remained
in S phase, resulting in the generation of a substantial 8N population
(Fig. 1D). Moreover, HFK-16E6E7 cells (Fig. 1E and F), as well as HFKs
transfected with the HPV-31 genome (LKP31 cells) (Fig. 1G and H),
exhibited a similar loss of G2/M arrest upon nocodazole
treatment. Infections, treatments, and analyses were repeated at least
three times for normal keratinocytes and E6- and E7-expressing cell
lines.
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FIG. 1.
DNA content flow cytometry analysis of normal and HPV
oncoprotein-expressing HFKs after nocodazole treatment. Cells were
treated with nocodazole for 48 h and harvested, and nuclei were
isolated and stained with PI. (A, C, E, and G) Untreated cells showing
comparable DNA content profiles. 2N represents the DNA content of those
cells in the G0/G1 phase of the cell cycle,
while 4N indicates increased DNA content and is representative of cells
in G2/M. (B) HFKs treated with nocodazole showing an
accumulation of cells at a DNA content of 4N. (D, F, and H)
Nocodazole-treated cells demonstrating a loss of the G2/M
checkpoint and subsequent accumulation at 8N.
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In order to monitor the progression of cell populations through the
cell cycle after nocodazole treatment, we repeated the
above analysis
at successive time points up to 48 h of nocodazole
treatment. As
shown in Fig.
2, HFK-16E6 cells
demonstrated a gradual
change in DNA content from 2N into 4N and 8N
populations. Analysis
of HFK-16E6E7 as well as LKP31 cells showed that
the two cell
lines exhibited similar effects, whereas low-risk E6-alone
cells
did not bypass the checkpoint (data not shown). These data
indicate
that normal human keratinocytes contain a mitotic checkpoint
that
can be abrogated by high-risk E6 protein alone, E6 in combination
with E7, or E6 in the context of the entire HPV-31 genome.
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FIG. 2.
DNA content flow cytometry time course analysis of
HFK-16E6 cells after nocodazole treatment. Cells were either left
untreated (UT) or treated with nocodazole for the indicated times. DNA
content profiles demonstrate a gradual, but not complete, loss of cells
in the G0/G1 phase and gain of an 8N DNA
content population upon nocodazole treatment.
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Abrogation of mitotic checkpoint by E7.
To examine whether
E7 also affected the mitotic spindle checkpoint, normal human
keratinocytes were infected with retroviruses expressing HPV-16 E7 and
neomycin-resistant colonies were pooled and expanded. We first observed
that HFK-16E7 cells proliferated at a rate much lower than that of
normal HFKs and HFK-16E6 cells. By measuring growth rates in tissue
culture, we determined that HFK-16E7 cells exhibit approximately a
twofold increase in doubling time compared to normal keratinocytes and
other keratinocytes expressing HPV oncoproteins, which have comparable
doubling times (data not shown). In order to compare effects in
HFK-16E7 cells with those in HFK-16E6 cells after nocodazole treatment,
it was important to examine them at similar points relative to their cycling times, since the accumulation in 8N for the E6-expressing cells
occurred only after an additional cell cycle (Fig. 2). From the above
data, we determined that nocodazole treatment of E7 cells for 96 h
would be equivalent to 48-h treatment for the other cell lines. DNA
content flow cytometry analysis was therefore performed on HFK-16E7
cells at progressive time points up to 96 h (Fig.
3). Unexpectedly, we observed that, in
the presence of nocodazole, HFK-16E7 cells were able to overcome the
mitotic spindle checkpoint equally as well as were HFK-16E6 cells. The
accumulation of cells with an 8N DNA content can be observed in
HFK-16E7 cells as early as 48 h after nocodazole treatment.
Low-risk E7 cells did not overcome the spindle checkpoint following
nocodazole treatment (data not shown). Furthermore, normal HFKs treated
with nocodazole for 96 h still did not bypass the checkpoint and
accumulated with a 4N DNA content (data not shown). This indicates that
the effect of E7 on the mitotic spindle checkpoint was not due to the
extended length of treatment. These data indicate a role for high-risk E7 in abrogation of the mitotic spindle checkpoint.
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FIG. 3.
DNA content flow cytometry time course analysis of
HFK-16E7 cells after nocodazole treatment. Cells were either untreated
(UT) or treated with nocodazole at the indicated times. HFK-16E7 cells
exhibit a loss of the G2/M checkpoint with no cells in
G0/G1 and a gain of cells at 8N after
nocodazole treatment.
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A summary of the cell cycle distribution of all cells analyzed
expressed as population percentages with and without nocodazole
treatment is shown in Table
1. All cells
expressing HPV oncoproteins
exhibit obvious increases in the percentage
of cells reaching
a DNA content of 8N, ranging from 29% for HFK-16E7
to 46% for
LKP31. Normal HFKs have less than 5% of cells falling in
this
population after nocodazole treatment. These data further confirm
that E6 and E7 can independently abrogate the mitotic spindle
checkpoint.
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TABLE 1.
Summary of cell cycle distribution of normal and HPV
oncoprotein-expressing HFKs after nocodazole treatment
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Levels of p53 and p21 in nocodazole-treated cells.
The
observation that E6 can abrogate the mitotic spindle checkpoint was not
unexpected, given the presumed role of p53 and the ability of E6 to
facilitate its degradation. However, it was less clear how E7 could
alter this checkpoint. Either E7 could target p53, or alternatively, it
could act through other, unknown components of the checkpoint. In order
to investigate the mechanism by which E7 can overcome G2/M
arrest, we first analyzed the levels of p53 protein before and after
nocodazole treatment of both E6- and E7-expressing keratinocytes. One
possibility was that nocodazole-treated E7 cells had dramatically
reduced levels of p53. As shown in Fig. 4A, normal HFKs contain significant
levels of p53, which are increased twofold after nocodazole treatment
(lanes 1 and 2). Cells expressing E6, whether alone, in combination
with E7, or in the context of the entire HPV-31 genome, have decreased
levels of p53 due to the ability of E6 to facilitate its degradation
(lanes 3, 4, and 7 to 10). Consistent with previous reports, HFK-16E7
cells contain higher levels of p53 compared to normal HFKs (lanes 5 and
6) (10, 54). Following nocodazole treatment, cells
expressing E6 alone or in combination with E7 did not demonstrate
significant changes in levels of p53 (lanes 4 and 8). However, cells
expressing the entire HPV-31 genome exhibited elevated, though still
low, levels of p53 after nocodazole treatment (lane 10). This is
similar to the increase in p53 levels seen following treatment with
actinomycin D, which arrests cells at G1/S; however, the
molecular basis for it is unclear.
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FIG. 4.
Western blot analysis of normal and HPV
oncoprotein-expressing keratinocytes before and after nocodazole
treatment for cyclin B and cdc2. One hundred micrograms of whole-cell
extract was separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis transferred to a polyvinylidene difluoride membrane,
and Western blot analysis was performed. Odd-numbered lanes represent
untreated samples, and even-numbered lanes show nocodazole-treated
samples. (A) p53 protein levels. (B) p21 protein levels.
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To examine whether the p53 present in the oncoprotein-expressing cells
was functional, we carried out Western blotting analyses
for the kinase
inhibitor p21. p21 is transcriptionally regulated
by p53, and the
levels of p21 have been shown to parallel increases
in p53 (reviewed in
reference
32). As shown in Fig.
4B, the
p21 protein
levels directly correlate with the levels of p53,
indicating that the
p53 present is functional, at least in its
role as a transcriptional
transactivator. Most importantly, the
levels of p21 increased following
nocodazole treatment of E7-expressing
cells. The ability of
E7-containing cells to overcome this checkpoint
appears to be
independent of p53, since both levels and activity
remain high. This
finding is reminiscent of the ability of E7
to bypass the
G
1/S checkpoint after DNA damage. While both checkpoints
are thought to be controlled by p53, E7 does not diminish the
activity
or levels of p53.
MDM2 protein levels in nocodazole-treated cells.
We wanted to
further examine the mechanism by which HFK-16E7 cells were able to
bypass the mitotic checkpoint. Recently, Lundgren et al.
(42) examined the role of the MDM2 cellular oncoprotein in
the cell cycle and in tumorigenesis in a p53 null background. Their
data indicate that overexpression of MDM2 in mammary cells resulted in
multiple rounds of S phase without intervening mitosis. The phenotype
is similar in p53 wild-type and in p53 null backgrounds. Because it
appeared that HFK-16E7 cells were bypassing the spindle checkpoint
independently of p53, we examined the levels of MDM2 before and after
nocodazole treatment to see if expression of this protein could be
contributing to aneuploidy (Fig. 5). The levels of MDM2 in HFK-16E7 cells (lanes 5 and 6) were found to be
significantly increased above that seen either in HFKs (lanes 1 and 2)
or in E6-expressing cells (lanes 3 and 4), whose levels were very low
or undetectable. Interestingly, HFK-16E6E7 and LKP31 cells also have
undetectable levels of MDM2 (lanes 7, 8, 9, and 10), indicating that
expression of E7 alone is not sufficient to induce overexpression of
MDM2. It is likely that E7 activates factors which function
synergistically with p53 to increase MDM2 levels. These studies were
repeated three times with similar results. Given the previously
documented role of MDM2 in mediating an abrogation of the spindle
checkpoint, it is possible that its elevated expression may contribute
to E7's ability to act in this process.
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FIG. 5.
Western blot analysis comparing levels of MDM2 protein
in normal and oncoprotein-expressing keratinocytes after nocodazole
treatment. Samples were prepared as described in Materials and Methods.
Odd-numbered lanes represent untreated samples, and even-numbered lanes
represent nocodazole-treated samples.
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Cyclin B and cdc2 levels in nocodazole-treated cells.
Additional cell cycle regulators of the G2/M phase of the
cell cycle may also be affected by the expression of the HPV
oncoproteins and consequently contribute to the ability of these cells
to bypass the mitotic spindle checkpoint. Therefore, we examined the
levels of cyclin B and the cdc2 kinase in nocodazole-treated cells,
which are required for entrance into mitosis. As shown in Fig.
6A, cyclin B levels are reduced in
nocodazole-treated normal HFKs compared to untreated cells (lanes 1 and
2). All other cells tested which express the HPV oncoproteins (lanes 3 to 10) show little or no decrease in the levels of cyclin B after
nocodazole treatment. Furthermore, cdc2 levels (Fig. 6B) are also
decreased in normal HFKs after nocodazole treatment (lanes 1 and 2) but
remain high for all other cell lines tested (lanes 3 to 10). The
presence of a noncycling, quiescent population in the E6-expressing
cells (see 2N population [Fig. 1D]) may account for the lower levels of cyclin B and cdc2. However, the levels of cyclins and cdc2 remain
unchanged before and after nocodazole treatment (lanes 3 and 4). These
data suggest that expression of the HPV oncoproteins alone, or in
combination, does not lead to decreased expression of cyclin B and cdc2
following nocodazole treatment.
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FIG. 6.
Western blot analysis of normal and HPV
oncoprotein-expressing keratinocytes before and after nocodazole
treatment for cyclin B and cdc2. Samples were prepared as described in
Materials and Methods. Odd-numbered lanes represent untreated samples,
and even-numbered lanes show nocodazole-treated samples. (A) Cyclin B
protein levels. (B) cdc2 protein levels.
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DISCUSSION |
The present study demonstrates the ability of both E6 and E7
oncoproteins of the HPV high-risk types to bypass the mitotic spindle
checkpoint in human keratinocytes. p53 has been previously shown to be
a component of this checkpoint, and in its absence, cells
inappropriately replicate their DNA without intervening mitoses when
treated with a mitotic spindle inhibitor (6). E6-expressing
cells most likely overcome this checkpoint through their ability to
facilitate the degradation of p53, since the levels of p53 are
dramatically reduced in these cells. This would be consistent with our
observation that E6 proteins from low-risk types, which fail to induce
p53 degradation, cannot overcome the mitotic checkpoint.
Cells expressing E7 alone can also overcome the mitotic checkpoint.
Levels and activity of p53 are high in E7 cells, indicating a
p53-independent mechanism. E7 functions in transformation through binding and inhibiting the activities of Rb, another tumor suppressor protein (reviewed in reference 49). We observed that
cells expressing low-risk E7, which binds Rb with significantly reduced
affinity compared to high-risk E7, are unable to bypass the mitotic
checkpoint. This suggests that deregulation of Rb function may
contribute to loss of this checkpoint. During preparation of this
manuscript, studies by Wahl and coworkers (12) were
published which show that loss of Rb function in Rb
/
fibroblasts resulted in loss of the mitotic spindle checkpoint. This is
consistent with our findings and points to one mechanism by which E7
cells can bypass the checkpoint.
An additional mechanism which may contribute to the ability of
E7-expressing cells to overcome the spindle checkpoint stems from our
observation that the levels of MDM2 are elevated only in E7 cells.
Previous studies have shown that overexpression of MDM2, a cellular
oncogene, can lead to multiple rounds of S-phase replication without
intervening mitosis in mammary epithelial cells (42). Most
importantly, this phenotype is similar in p53 wild-type and p53 null
backgrounds. In the E7-alone-expressing cells, high levels of
functional p53 are present, as demonstrated by inducible p21
expression. Despite these high levels, E7 can overcome this checkpoint,
suggesting that its action is independent of p53. It is possible that
both loss of Rb and overexpression of MDM2 can act cooperatively to
result in loss of the mitotic spindle checkpoint.
MDM2 was originally identified as the product of the murine double
minute 2 gene from a spontaneously transformed BALB/c 3T3 cell line
(2). Overexpression of MDM2 has been shown to increase the
tumorigenic potential of cells in culture (16) and has been found to be amplified in human sarcomas (51). The ability of MDM2 to promote tumorigenesis is a result of its interactions with
tumor suppressor proteins such as p53. The product of the mdm2 oncogene forms a complex with p53 and inhibits
p53-mediated transactivation (47, 52), growth suppression
(17), G1 arrest following DNA damage, and
apoptosis (4, 5, 23). Interestingly, MDM2 expression is
transcriptionally activated by p53 (1, 30, 65), indicating
the presence of an autoregulatory loop between the two proteins. Other
interactions of MDM2 include its association with Rb (66).
This interaction results in the deregulation of E2F-DP1 activity,
normally negatively controlled by Rb, leading to stimulation of S phase
entry. Furthermore, MDM2 stimulates the transcriptional activity of two
cooperating transcription factors, E2F and DP1, which enhances the
stimulation of S phase (43).
It is interesting that HFK-16E6E7 cells and LKP31 cells do not express
high levels of MDM2 since they, too, express E7. These cells contain
low levels of p53, which may explain the lack of MDM2 protein since
MDM2 is transcriptionally activated by p53. These data suggest that p53
may be required for MDM2 expression; however, our data indicate that
expression of p53 alone is not sufficient to induce high levels of
MDM2. For instance, normal HFKs treated with nocodazole contain levels
of p53 similar to that seen in HFK-16E7 cells and yet do not have
detectable levels of MDM2. Furthermore, expression of E7 alone is not
sufficient to induce expression of MDM2, since HFK-16E6E7 and LKP31
cells do not have detectable levels of MDM2. We conclude that E7 may activate factors which function synergistically with p53 to increase MDM2 levels. Finally, recent work by two different groups has found
that MDM2 is able to promote the degradation of p53 (24, 33)
and that there is an inverse correlation between the levels of MDM2 and
those of p53; high levels of MDM2 were shown to correlate with reduced
levels of p53. We do not see decreased levels of p53 due to
overexpression of MDM2 in the HFK-16E7 cells, demonstrating that this
correlation is not true in all cases.
In addition to p53 and MDM2, p21 has been implicated in control of the
mitotic spindle checkpoint. Work from the Vogelstein laboratory has
shown that cells lacking p21 are able to undergo additional S phases
without intervening mitoses when arrested in a G2-like state
(61). Unlike those authors' findings, our E7-alone-expressing cells have elevated levels of p21 and yet still
undergo rereplication through a mechanism which appears to be
independent of p53-p21 activity. Furthermore, recent reports have
demonstrated that some tumor cell lines contain elevated levels of both
p53 and MDM2 but lack significant levels of p21 (35). It is
possible that p53 is not fully functional in these tumor lines. In our
studies, E7-alone-expressing cells contain elevated levels of p53 and
MDM2, as well as functional p21. E7 cells, therefore, appear to be
unusual in that they express high levels of all three of these cell
cycle regulators.
Previous studies by other groups have examined the role of the HPV
oncoproteins in alteration of genomic stability. White et al.
demonstrated that expression of E6 in human fibroblasts results in a
failure to arrest in G1 and G2 following
exposure to the metabolic inhibitor PALA as well as CAD gene
amplification (64). In contrast, E7-expressing cells display
massive cell death with the rare appearance of PALA-resistant aneuploid
cells, which occurs by a p53-independent mechanism. This indicates that each of the HPV oncoproteins alters distinct pathways, resulting in
different types of genomic instability. The alteration of cyclin-CDK complexes by E6, but not by E7, is implicated in this loss of genomic
stability (67). Likewise, Reznikoff et al. (53)
reported statistically significant differences in genomic stability
between E6- and E7-immortalized human uroepithelial cells. In our
study, we observe a similar loss of cell cycle control for E6 and E7 in
their natural host cells, keratinocytes. Both HPV oncoproteins, through
different mechanisms, are able to overcome the mitotic spindle
checkpoint, leading to altered genomic integrity.
We conclude that both HPV oncoproteins contribute to genomic
instability at the mitotic checkpoint: E6 through degradation of p53, a
component of the checkpoint, and E7 through a p53-independent mechanism. The loss of Rb as well as the increased levels of MDM2 in
the E7 cells may play a role in the loss of this checkpoint by
providing the signal to undergo DNA synthesis but not allowing for a
complete transition through mitosis. Both of these mechanisms afford
the accumulation of genetic alterations, which may contribute to the
progressive nature of cervical cancer.
| |
ACKNOWLEDGMENTS |
We thank Denise Galloway for the LXSN retroviral constructs. We
gratefully acknowledge Margaret Ruesch, Neil Clipstone, Kathy Rundell,
and Mary Hummel for critical review of the manuscript and the members
of the Laimins laboratory for helpful discussions. We also thank
Stephanie Gaillard and Robert Caldwell for technical advice.
This work was supported by the Carcinogenesis Training Grant (5T32
CA09560-12) and a Gramm Fellowship Award from Northwestern University
to J.T.T. and by a grant to L.A.L. from the National Institute of
Allergy and Infectious Diseases (AI31494).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology-Immunology, Northwestern University Medical School, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0648. Fax: (312)
503-0649. E-mail: lal{at}merle.acns.nwu.edu.
| |
REFERENCES |
| 1.
|
Barak, Y.,
T. Juven,
R. Haffner, and M. Oren.
1993.
mdm2 expression is induced by wild type p53 activity.
EMBO J.
12:461-468[Medline].
|
| 2.
|
Cahilly-Snyder, L.,
T. Yang-Feng,
U. Francke, and D. L. George.
1987.
Molecular analysis and chromosomal mapping of amplified genes isolated from a transformed mouse 3T3 cell line.
Somatic Cell Mol. Genet.
13:235-244[Medline].
|
| 3.
|
Chellappan, S.,
S. Hiebert,
M. Mudryj,
J. Horowitz, and J. Nevins.
1991.
The E2F transcription factor is a cellular target for the Rb protein.
Cell
65:1053-1061[Medline].
|
| 4.
|
Chen, C.,
J. D. Oliner,
Q. Zhan,
A. J. Fornace, Jr.,
B. Vogelstein, and M. B. Kastan.
1994.
Interaction between p53 and MDM2 in a mammalian cell cycle checkpoint pathway.
Proc. Natl. Acad. Sci. USA
91:2684-2688[Abstract/Free Full Text].
|
| 5.
|
Chen, J.,
X. Wu,
J. Lin, and A. J. Levine.
1996.
mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein.
Mol. Cell. Biol.
16:2445-2452[Abstract].
|
| 6.
|
Cross, S. M.,
C. A. Sanchez,
C. A. Morgan,
M. K. Schimke,
S. Ramel,
R. L. Idzerda,
W. H. Raskind, and B. J. Reid.
1995.
A p53-dependent mouse spindle checkpoint.
Science
267:1353-1356[Abstract/Free Full Text].
|
| 7.
|
Davies, R.,
R. Hicks,
T. Crook,
J. Morris, and K. Vousden.
1993.
Human papillomavirus type 16 E7 associates with a histone H1 kinase and with p107 through sequences necessary for transformation.
J. Virol.
67:2521-2528[Abstract/Free Full Text].
|
| 8.
|
Demers, G.,
S. Foster,
C. Halbert, and D. Galloway.
1994.
Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7 protein.
Proc. Natl. Acad. Sci. USA
91:4382-4386[Abstract/Free Full Text].
|
| 9.
|
Demers, G. W.,
E. Espling,
J. B. Harry,
B. G. Etscheid, and D. A. Galloway.
1996.
Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.
J. Virol.
70:6862-6869[Abstract/Free Full Text].
|
| 10.
|
Demers, G. W.,
C. L. Halbert, and D. A. Galloway.
1994.
Elevated wild-type p53 protein levels in human epithelial cell lines immortalized by the human papillomavirus type 16 E7 gene.
Virology
198:169-174[Medline].
|
| 11.
|
de Villiers, E.-M.
1994.
Human pathogenic papillomavirus types: an update.
Curr. Top. Microbiol. Immunol.
186:1-12[Medline].
|
| 12.
|
Di Leonardo, A.,
S. H. Khan,
S. P. Linke,
V. Greco,
G. Seidita, and G. M. Wahl.
1997.
DNA replication in the presence of mitotic spindle inhibitors in human and mouse fibroblasts lacking either p53 or pRb function.
Cancer Res.
57:1013-1019[Abstract/Free Full Text].
|
| 13.
|
Dyson, N.,
P. Guida,
K. Munger, and E. Harlow.
1992.
Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interactions with the same set of cellular proteins.
Science
243:934-936.
|
| 14.
|
Dyson, N.,
P. Howley,
K. Munger, and E. Harlow.
1989.
The human papillomavirus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product.
Science
243:934-937[Abstract/Free Full Text].
|
| 15.
|
El-Diery, W. S.,
T. Tokino,
D. B. Velculescu,
D. B. Levy,
J. M. Parsons,
D. L. Trent,
W. E. Mercer,
K. W. Kinzler, and B. Vogelstein.
1993.
WAF1, a potential mediator of p53 tumor suppression.
Cell
75:817-825[Medline].
|
| 16.
|
Fakharzadeh, S. S.,
S. P. Trusko, and D. L. George.
1991.
Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line.
EMBO J.
10:1565-1569[Medline].
|
| 17.
|
Finlay, C. A.
1993.
The mdm-2 oncogene can overcome wild-type p53 suppression of transformed cell growth.
Mol. Cell. Biol.
13:301-306[Abstract/Free Full Text].
|
| 18.
|
Foster, S. A.,
G. W. Demers,
B. G. Etscheid, and D. A. Galloway.
1994.
The ability of human papillomavirus E6 proteins to target p53 for degradation in vivo correlates with their ability to abrogate actinomycin D-induced growth arrest.
J. Virol.
68:5698-5705[Abstract/Free Full Text].
|
| 19.
|
Frattini, M. G.,
H. B. Lim, and L. A. Laimins.
1996.
In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.
Proc. Natl. Acad. Sci. USA
93:3062-3067[Abstract/Free Full Text].
|
| 20.
|
Gu, Z.,
D. Pim,
S. Labrecque,
L. Banks, and G. Matlashewski.
1994.
DNA damage induced p53 mediated transcription is inhibited by human papillomavirus type 18 E6.
Oncogene
9:629-633[Medline].
|
| 21.
|
Halbert, C. L.,
G. W. Demers, and D. A. Galloway.
1991.
The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells.
J. Virol.
65:473-478[Abstract/Free Full Text].
|
| 22.
|
Halbert, C. L.,
G. W. Demers, and D. A. Galloway.
1992.
The E6 and E7 genes of human papillomavirus type 6 have weak immortalizing activity in human epithelial cells.
J. Virol.
66:2125-2134[Abstract/Free Full Text].
|
| 23.
|
Haupt, Y.,
Y. Barak, and M. Oren.
1996.
Cell type-specific inhibition of p53-mediated apoptosis by mdm2.
EMBO J.
15:1596-1606[Medline].
|
| 24.
|
Haupt, Y.,
R. Maya,
A. Kazaz, and M. Oren.
1997.
Mdm2 promotes the rapid degradation of p53.
Nature
387:296-299[Medline].
|
| 25.
|
Hickman, E.,
S. Picksley, and K. H. Vousden.
1994.
Cells expressing HPV 16 E7 continue cell cycle progression following DNA damage induced p53 activation.
Oncogene
9:2177-2181[Medline].
|
| 26.
|
Hickman, E. S.,
S. Bates, and K. H. Vousden.
1997.
Perturbation of the p53 response by human papillomavirus type 16 E7.
J. Virol.
71:3710-3718[Abstract].
|
| 27.
|
Hoppe-Seyler, F., and K. Butz.
1993.
Repression of endogenous p53 transactivation function in HeLa cervical carcinoma cells by human papillomavirus type 16 E6, human mdm-2, and mutant p53.
J. Virol.
67:3111-3117[Abstract/Free Full Text].
|
| 28.
|
Howley, P. M.
1996.
Papillomavirinae: the viruses and their replication, p. 947-978. In
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fundamental virology, 3rd ed.
Lippincott-Raven, Philadelphia, Pa.
|
| 29.
|
Huibregtse, J. M.,
M. Scheffner, and P. M. Howley.
1991.
A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18.
EMBO J.
10:4126-4135.
|
| 30.
|
Juven, T.,
Y. Barak,
A. Zauberman,
D. George, and M. Oren.
1993.
Wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene.
Oncogene
8:3411-3416[Medline].
|
| 31.
|
Kessis, T. D.,
R. J. Slebos,
W. G. Nelson,
M. B. Kastan,
B. S. Plunkett,
S. M. Han,
A. T. Lorincz,
L. Hedrick, and K. R. Cho.
1993.
Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage.
Proc. Natl. Acad. Sci. USA
90:3988-3992[Abstract/Free Full Text].
|
| 32.
|
Ko, L. J., and C. Prives.
1996.
p53: puzzle and paradigm.
Genes Dev.
10:1054-1072[Free Full Text].
|
| 33.
|
Kubbutat, M. H. G.,
S. N. Jones, and K. H. Vousden.
1997.
Regulation of p53 stability by Mdm2.
Nature
387:299-303[Medline].
|
| 34.
|
Laimins, L. A.
1993.
The biology of human papillomavirus: from warts to cancer.
Infect. Agents Dis.
2:74-86[Medline].
|
| 35.
|
Landers, J. E.,
S. L. Cassel, and D. L. George.
1997.
Translational enhancement of mdm2 oncogene expression in human tumor cells containing a stabilized wild type p53 protein.
Cancer Res.
57:3562-3568[Abstract/Free Full Text].
|
| 36.
|
Lane, D. P.
1992.
p53, guardian of the genome.
Nature
358:15-16[Medline].
|
| 37.
|
Lechner, M. S., and L. A. Laimins.
1994.
Inhibition of p53 DNA binding by human papillomavirus E6 proteins.
J. Virol.
68:4262-4273[Abstract/Free Full Text].
|
| 38.
|
Lechner, M. S.,
D. H. Mack,
A. B. Finicle,
T. Crook,
K. H. Vousden, and L. A. Laimins.
1992.
Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.
EMBO J.
11:3045-3052[Medline].
|
| 39.
|
Levine, A. J.,
J. Momand, and C. A. Finlay.
1991.
The p53 tumour suppressor gene.
Nature
351:453-456[Medline].
|
| 40.
|
Livingstone, L. R.,
A. White,
J. Sprouse,
E. Livanos,
T. Jacks, and T. D. Tlsty.
1992.
Altered cell cycle arrest and gene amplification potential accompany loss of wild-type p53.
Cell
70:923-935[Medline].
|
| 41.
|
Lowy, D. R.,
R. Kirnbauer, and J. T. Schiller.
1994.
Genital human papillomavirus infection.
Proc. Natl. Acad. Sci. USA
91:2436-2440[Abstract/Free Full Text].
|
| 42.
|
Lundgren, K.,
R. Montes de Oca Luna,
Y. B. McNeill,
E. P. Emerick,
B. Spencer,
C. R. Barfield,
G. Lozano,
M. P. Rosenberg, and C. A. Finlay.
1997.
Targeted expression of MDM2 uncouples S phase from mitosis and inhibits mammary gland development independent of p53.
Genes Dev.
11:714-725[Abstract/Free Full Text].
|
| 43.
|
Martin, K.,
D. Trouche,
C. Hagemeier,
T. S. Sorenson,
N. B. La Thangue, and T. Kouzarides.
1995.
Stimulation of E2F1/DP1 transcriptional activity by MDM2 oncoprotein.
Nature
375:691-694[Medline].
|
| 44.
|
Meyers, C.,
M. G. Frattini,
J. B. Hudson, and L. A. Laimins.
1992.
Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.
Science
257:971-973[Abstract/Free Full Text].
|
| 45.
|
Meyers, C., and L. A. Laimins.
1994.
In vitro systems for the study and propagation of human papillomaviruses.
Curr. Top. Microbiol. Immunol.
186:199-215[Medline].
|
| 46.
|
Mietz, J. A.,
T. Unger,
J. M. Huibregtse, and P. M. Howley.
1992.
The transcriptional transactivation function of wild-type p53 is inhibited by SV40 large T-antigen and by HPV-16 E6 oncoprotein.
EMBO J.
11:5013-5020[Medline].
|
| 47.
|
Momand, J.,
G. P. Zambetti,
D. C. Olson,
D. George, and A. J. Levine.
1992.
The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.
Cell
69:1237-1245[Medline].
|
| 48.
|
Morozov, A.,
P. Shiyanov,
E. Barr,
J. M. Leiden, and P. Raychaudhuri.
1997.
Accumulation of human papillomavirus type 16 E7 protein bypasses G1 arrest induced by serum deprivation and by the cell cycle inhibitor p21.
J. Virol.
71:3451-3457[Abstract].
|
| 49.
|
Munger, K.,
M. Scheffner,
J. M. Huibregtse, and P. M. Howley.
1992.
Interactions of HPV E6 and E7 oncoproteins with tumour suppressor gene products.
Cancer Surv.
12:197-217[Medline].
|
| 50.
|
Munger, K.,
B. Werness,
N. Dyson,
W. Phelps,
E. Harlow, and P. Howley.
1989.
Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product.
EMBO J.
8:4099-4105[Medline].
|
| 51.
|
Oliner, J. D.,
K. W. Kinzler,
P. S. Meltzer,
D. L. George, and B. Vogelstein.
1992.
Amplification of a gene encoding a p53-associated protein in human sarcomas.
Nature
358:80-83[Medline].
|
| 52.
|
Oliner, J. D.,
J. A. Pietenpol,
S. Thiagalingam,
J. Gyuris,
K. W. Kinzler, and B. Vogelstein.
1993.
Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53.
Nature
362:857-860[Medline].
|
| 53.
|
Reznikoff, C. A.,
C. Belair,
E. Savelieva,
Y. Zhai,
K. Pfeifer,
T. Yeager,
K. J. Thompson,
S. DeVries,
C. Bindley,
M. A. Newton,
G. Sekhon, and F. Waldman.
1994.
Long-term genome stability and minimal genotypic and phenotypic alterations in HPV16 E7-, but not E6-, immortalized human uroepithelial cells.
Genes Dev.
8:2227-2240[Abstract/Free Full Text].
|
| 54.
|
Ruesch, M. N., and L. A. Laimins.
1997.
Initiation of DNA synthesis by human papillomavirus E7 oncoproteins is resistant to p21-mediated inhibition of cyclin E-cdk2 activity.
J. Virol.
71:5570-5578[Abstract].
|
| 55.
|
Scheffner, M.,
J. M. Huibregtse,
R. D. Vierstra, and P. M. Howley.
1993.
The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53.
Cell
75:495-505[Medline].
|
| 56.
|
Scheffner, M.,
H. Romanczuk,
K. Munger,
J. M. Huibregtse,
J. A. Mietz, and P. M. Howley.
1994.
Functions of human papillomavirus proteins.
Curr. Top. Microbiol. Immunol.
186:83-99[Medline].
|
| 57.
|
Scheffner, M.,
B. A. Werness,
J. M. Huibregtse,
A. J. Levine, and P. M. Howley.
1990.
The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53.
Cell
63:1129-1136[Medline].
|
| 58.
|
Schiffman, M. H.
1994.
Epidemiology of cervical human papillomavirus infections.
Curr. Top. Microbiol. Immunol.
186:55-81[Medline].
|
| 59.
|
Slebos, R. J.,
M. H. Lee,
B. S. Plunkett,
T. D. Kessis,
B. O. Williams,
T. Jacks,
L. Hedrick,
M. B. Kastan, and K. R. Cho.
1994.
p53-dependent G1 arrest involves pRb-related proteins and is disrupted by the human papillomavirus 16 E7 oncoprotein.
Proc. Natl. Acad. Sci. USA
91:5320-5324[Free Full Text].
|
| 60.
|
Vousden, K. H.
1994.
Interactions between papillomavirus proteins and tumor suppressor gene products.
Adv. Cancer Res.
64:1-24[Medline].
|
| 61.
|
Waldman, T.,
C. Lengauer,
K. W. Kinzler, and B. Vogelstein.
1996.
Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21.
Nature
381:713-716[Medline].
|
| 62.
|
Weinberg, R. A.
1995.
The retinoblastoma protein and cell cycle control.
Cell
81:323-330[Medline].
|
| 63.
|
Werness, B. A.,
A. J. Levine, and P. M. Howley.
1990.
Association of human papillomavirus types 16 and 18 proteins with p53.
Science
248:76-79[Abstract/Free Full Text].
|
| 64.
|
White, A. E.,
E. M. Livanos, and T. D. Tlsty.
1994.
Differential disruption of genomic integrity and cell cycle regulation in normal human fibroblasts by the HPV oncoproteins.
Genes Dev.
8:666-677[Abstract/Free Full Text].
|
| 65.
|
Wu, X.,
J. H. Bayle,
D. Olson, and A. J. Levine.
1993.
The p53-mdm-2 autoregulatory feedback loop.
Genes Dev.
7:1126-1132[Abstract/Free Full Text].
|
| 66.
|
Xiao, Z.,
J. Chen,
A. J. Levine,
N. Modjtahedi,
J. Xing,
W. R. Sellers, and D. M. Livingston.
1995.
Interaction between the retinoblastoma protein and the oncoprotein MDM2.
Nature
375:694-698[Medline].
|
| 67.
|
Xiong, Y.,
D. Kuppuswamy,
Y. Li,
E. M. Livanos,
M. Hixon,
A. White,
D. Beach, and T. D. Tlsty.
1996.
Alteration of cell cycle kinase complexes in human papillomavirus E6- and E7-expressing fibroblasts precedes neoplastic transformation.
J. Virol.
70:999-1008[Abstract].
|
| 68.
|
Yin, Y.,
M. A. Tainsky,
F. Z. Bischoff,
L. C. Strong, and G. M. Wahl.
1992.
Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles.
Cell
70:937-948[Medline].
|
| 69.
|
zur Hausen, H., and E. De Villiers.
1994.
Human papillomaviruses.
Annu. Rev. Microbiol.
48:427-447[Medline].
|
J Virol, February 1998, p. 1131-1137, Vol. 72, No. 2
0022-538X/98/$04.00+0
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Nakahara, T., Nishimura, A., Tanaka, M., Ueno, T., Ishimoto, A., Sakai, H.
(2002). Modulation of the Cell Division Cycle by Human Papillomavirus Type 18 E4. J. Virol.
76: 10914-10920
[Abstract]
[Full Text]
-
Chien, W.-M., Noya, F., Benedict-Hamilton, H. M., Broker, T. R., Chow, L. T.
(2002). Alternative Fates of Keratinocytes Transduced by Human Papillomavirus Type 18 E7 during Squamous Differentiation. J. Virol.
76: 2964-2972
[Abstract]
[Full Text]
-
Gaillard, S., Fahrbach, K. M., Parkati, R., Rundell, K.
(2001). Overexpression of Simian Virus 40 Small-T Antigen Blocks Centrosome Function and Mitotic Progression in Human Fibroblasts. J. Virol.
75: 9799-9807
[Abstract]
[Full Text]
-
Duensing, S., Duensing, A., Flores, E. R., Do, A., Lambert, P. F., Munger, K.
(2001). Centrosome Abnormalities and Genomic Instability by Episomal Expression of Human Papillomavirus Type 16 in Raft Cultures of Human Keratinocytes. J. Virol.
75: 7712-7716
[Abstract]
[Full Text]
-
Oh, S. T., Kyo, S., Laimins, L. A.
(2001). Telomerase Activation by Human Papillomavirus Type 16 E6 Protein: Induction of Human Telomerase Reverse Transcriptase Expression through Myc and GC-Rich Sp1 Binding Sites. J. Virol.
75: 5559-5566
[Abstract]
[Full Text]
-
Duensing, S., Lee, L. Y., Duensing, A., Basile, J., Piboonniyom, S.-o., Gonzalez, S., Crum, C. P., Münger, K.
(2000). The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Proc. Natl. Acad. Sci. USA
10.1073/pnas.170093297v1
[Abstract]
[Full Text]
-
Flores, E. R., Allen-Hoffmann, B. L., Lee, D., Lambert, P. F.
(2000). The Human Papillomavirus Type 16 E7 Oncogene Is Required for the Productive Stage of the Viral Life Cycle. J. Virol.
74: 6622-6631
[Abstract]
[Full Text]
-
Zheng, L., Chen, Y., Riley, D. J., Chen, P.-L., Lee, W.-H.
(2000). Retinoblastoma Protein Enhances the Fidelity of Chromosome Segregation Mediated by hsHec1p. Mol. Cell. Biol.
20: 3529-3537
[Abstract]
[Full Text]
-
Chang, Y. E., Laimins, L. A.
(2000). Microarray Analysis Identifies Interferon-Inducible Genes and Stat-1 as Major Transcriptional Targets of Human Papillomavirus Type 31. J. Virol.
74: 4174-4182
[Abstract]
[Full Text]
-
Crish, J. F., Bone, F., Balasubramanian, S., Zaim, T. M., Wagner, T., Yun, J., Rorke, E. A., Eckert, R. L.
(2000). Suprabasal expression of the human papillomavirus type 16 oncoproteins in mouse epidermis alters expression of cell cycle regulatory proteins. Carcinogenesis
21: 1031-1037
[Abstract]
[Full Text]
-
Ogston, P., Raj, K., Beard, P.
(2000). Productive Replication of Adeno-Associated Virus Can Occur in Human Papillomavirus Type 16 (HPV-16) Episome-Containing Keratinocytes and Is Augmented by the HPV-16 E2 Protein. J. Virol.
74: 3494-3504
[Abstract]
[Full Text]
-
Seavey, S. E., Holubar, M., Saucedo, L. J., Perry, M. E.
(1999). The E7 Oncoprotein of Human Papillomavirus Type 16 Stabilizes p53 through a Mechanism Independent of p19ARF. J. Virol.
73: 7590-7598
[Abstract]
[Full Text]
-
Harrington, E. A., Bruce, J. L., Harlow, E., Dyson, N.
(1998). pRB plays an essential role in cell cycle arrest induced by DNA damage. Proc. Natl. Acad. Sci. USA
95: 11945-11950
[Abstract]
[Full Text]
-
Geraghty, R. J., Krummenacher, C., Cohen, G. H., Eisenberg, R. J., Spear, P. G.
(1998). Entry of Alphaherpesviruses Mediated by Poliovirus Receptor-Related Protein 1 and Poliovirus Receptor. Science
280: 1618-1620
[Abstract]
[Full Text]
-
Liu, Y., Hong, Y., Androphy, E. J., Chen, J. J.
(2000). Rb-independent Induction of Apoptosis by Bovine Papillomavirus Type 1 E7 in Response to Tumor Necrosis Factor alpha. J. Biol. Chem.
275: 30894-30900
[Abstract]
[Full Text]
-
Duensing, S., Lee, L. Y., Duensing, A., Basile, J., Piboonniyom, S.-o., Gonzalez, S., Crum, C. P., Munger, K.
(2000). The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle. Proc. Natl. Acad. Sci. USA
97: 10002-10007
[Abstract]
[Full Text]
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Abstract
Introduction
