Deregulation of eIF4E: 4E-BP1 in differentiated human papillomavirus-containing cells leads to high levels of expression of the E7 oncoprotein.
ABSTRACT Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers. HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7 mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation, higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-containing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molecular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high level of E7 protein in differentiated cells.
- SourceAvailable from: Vijay Kumar
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ABSTRACT: Cervical cancer is the third most common cancer worldwide, and the development of new diagnosis, prognostic, and treatment strategies is a major interest for public health. Cisplatin, in combination with external beam irradiation for locally advanced disease, or as monotherapy for recurrent/metastatic disease, has been the cornerstone of treatment for more than two decades. Other investigated cytotoxic therapies include paclitaxel, ifosfamide and topotecan, as single agents or in combination, revealing unsatisfactory results. In recent years, much effort has been made towards evaluating new drugs and developing innovative therapies to treat cervical cancer. Among the most investigated molecular targets are epidermal growth factor receptor and vascular endothelial growth factor (VEGF) signaling pathways, both playing a critical role in cervical cancer development. Studies with bevacizumab or VEGF receptor tyrosine kinase have given encouraging results in terms of clinical efficacy, without adding significant toxicity. A great number of other molecular agents targeting critical pathways in cervical malignant transformation are being evaluated in preclinical and clinical trials, reporting preliminary promising data. In the current review, we discuss novel therapeutic strategies which are being investigated for the treatment of advanced cervical cancer.Journal of Cancer. 01/2014; 5(2):86-97.
JOURNAL OF VIROLOGY, July 2006, p. 7079–7088
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 14
Deregulation of eIF4E: 4E-BP1 in Differentiated Human
Papillomavirus-Containing Cells Leads to High Levels of
Expression of the E7 Oncoprotein
Kwang-Jin Oh,1Anna Kalinina,1No-Hee Park,2and Srilata Bagchi1*
Center for Molecular Biology of Oral Diseases, College of Dentistry (M/C 860), University of Illinois at Chicago,
801 South Paulina Street, Chicago, Illinois 60612,1and School of Dentistry, University of California at
Los Angeles, 53-038 CHS, 10833 Le Conte Ave., Los Angeles, California 900952
Received 11 November 2005/Accepted 2 May 2006
Infections with high-risk human papillomaviruses (HPVs) are linked to more than 95% of cervical cancers.
HPVs replicate exclusively in differentiated cells and the function of the HPV E7 oncoprotein is essential for
viral replication. In this study, we investigated the mechanism that regulates E7 expression in differentiated
cells. The level of E7 protein was strongly induced in HPV-containing Caski, HOK-16B, and BaP-T cells during
growth in methylcellulose-containing medium, a condition that induces differentiation. Enhanced expression of
E7 was observed between 4 and 8 h of culturing in methylcellulose and was maintained for up to 24 h. The
increase was not due to altered stability of the E7 protein or an increase in the steady-state level of the E7
mRNA. Instead, the translation of the E7 mRNA was enhanced during differentiation. More than 70 to 80% of
the E7 mRNA was found in the polysome fractions in the differentiated cells. Consistent with this observation,
higher levels of the phosphorylated translator inhibitor 4E-BP1 were observed in differentiated HPV-contain-
ing cells but not in differentiated non-HPV tumor cells or primary keratinocytes. The mTOR kinase inhibitor
rapamycin blocked phosphorylation of 4E-BP1 and significantly decreased the level of E7 protein in Caski
cells, suggesting that phosphorylation of 4E-BP1 is linked to E7 expression. Prevailing models for the molec-
ular mechanisms underlying E7 expression have focused largely on transcriptional regulation. The results
presented in this study demonstrate a significant role of the cellular translation machinery to maintain a high
level of E7 protein in differentiated cells.
The high-risk types of human papillomavirus are the main
etiological factors for cervical cancers (reviewed in references
27, 30, and 45). Furthermore, epidemiological studies have
shown that a significant percentage (30 to 40%) of oral, head,
and neck cancers, as well as other anogenital cancer lesions,
contain these high-risk human papillomaviruses (HPVs) (13).
Cervical cancer alone accounts for almost 12% of all cancers in
women (45). Therefore, elucidation of the mechanism that
contributes to the induction of the HPV-associated cancers is
of major importance. The HPVs infect proliferating epithelial
cells, but the viral DNA replication and structural viral gene
expression are restricted to only the differentiated layers of
epidermis or mucosa (27, 30). All HPV-transformed cancer
tissues express two HPV-encoded oncoproteins, E6 and E7.
Both E6 and E7 possess immortalizing activity. Moreover, con-
tinued expression of the E6 and E7 genes is necessary for the
maintenance of the transformed phenotype (30). Past studies
showed that knocking down the expression of the E7 gene by
RNA interference induced senescence in cervical cancer cells
(18, 22). Therefore, the expression of E7 is directly linked to
the growth and survival of the HPV-associated cancer cells.
The transforming activity of E7 is associated with its ability
to interact with the retinoblastoma tumor suppressor protein
Rb and its ability to induce proteolysis of Rb through the 26S
proteasome (3, 5, 16, 31, 43). One of the major biochemical
functions of Rb is to form repressor complexes with the E2F
family transcription factors and to repress expression of the
replication and cell division genes (reviewed in references 9
and 19). E7 converts the repressor form of E2F (Rb/E2F) to
the activator form (E2F). The E7-mediated conversion of E2F
to the activator form stimulates the expression of DNA repli-
cation enzyme genes, which allows E7 to reactivate cellular
DNA replication in differentiated epithelial cells (reviewed in
references 27 and 30).
The requirement for a differentiated layer has made it dif-
ficult to study the productive life cycle of HPVs. The prolifer-
ating keratinocytes are located in the basal layer in contact
with the extracellular matrix glycoproteins in the basement
membrane. Keratinocytes leave the basal layer to undergo a
series of biochemical and phenotypic changes that constitute
the differentiation program. Since the movement away from
the basement membrane is associated with the initiation of the
differentiation process, historically, suspension of keratino-
cytes has been used as a method of triggering differentiation
(17). The HPV-containing epithelial cells express the early
differentiation markers, involucrin, and different cytokeratins
within 24 h of growth in methylcellulose-containing culture
(12, 35). Many events of the HPV life cycles, including expres-
sion of differentiation-specific viral promoters, differentiation-
dependent viral-genome amplifications, and viral DNA repli-
cation, could be efficiently achieved within 24 to 48 h of growth
in methylcellulose-containing medium (6, 11, 12, 21, 35, 39,
40). However, the final steps of virus life cycles, including the
* Corresponding author. Mailing address: Center for Molecular Bi-
ology of Oral Diseases, College of Dentistry (M/C 860), University of
Illinois at Chicago, 801 South Paulina Street, Chicago, IL 60612.
Phone: (312) 413-0683. Fax: (312) 413-1604. E-mail: email@example.com.
production of infectious viral particles, could not be achieved
in the methylcellulose culture system (35). The organotypic
raft culture system is the only in vitro system that allows late-
stage differentiation for production of limited amounts of in-
fectious viral particles from the HPV-infected cells. Therefore,
differentiation in methylcellulose-containing (1.6%) semisolid
medium has been extensively used for analysis of HPV pro-
moters and enhancers, HPV DNA replication, gene expres-
sion, and late protein synthesis.
Previous studies in our laboratory and by other research
groups showed that the HPV16 E7 protein has a short half-life
and the level of E7 in tumor cells is regulated primarily
through proteolysis by the ubiquitin proteasome (16, 34, 43).
E7 plays a critical role in the replication of HPVs in differen-
tiated cells. However, how E7 expression is regulated during
differentiation is currently unknown. In this study, we observed
a sustained induction of E7 protein during differentiation in
semisolid methylcellulose-containing medium. We show that
the increased expression of E7 is due to enhanced translation
of the E7 mRNA in differentiated cells.
MATERIALS AND METHODS
Cells and culture conditions. Caski cells, C33A cells, HaCaT cells, and SCC-25
cells were cultured in Dulbecco’s modified Eagle medium supplemented with
10% fetal bovine serum. Primary human oral keratinocytes, primary human
foreskin keratinocytes, and HPV16-immortalized HOK-16B cells were grown in
keratinocyte growth medium from Cambrex as previously described (32).
HPV16-transformed BaP-T cells were grown in Dulbecco’s modified Eagle me-
dium supplemented with 10% fetal bovine serum and 4 ?g/ml hydrocortisone
(24). Caski, HOK-16B, BaP-T, HaCaT, C33A, and SCC-25 cells and primary oral
and foreskin keratinocytes were induced to differentiate by being cultured in
medium containing 1.6% methylcellulose as previously described (35). For
CaCl2-induced differentiation, primary human oral keratinocytes, primary hu-
man foreskin keratinocytes, Caski cells, and BaP-T cells were grown in medium
containing 1.5 mM CaCl2.
Antibodies and chemicals. [35S]methionine-cysteine was from ICN. m7GTP-
Sepharose, rapamycin, and MG132 were from EMD Bioscience Calbiochem.
The following antisera were from Cell Signaling Technology: eIF4E, phospho-
eIF4E (Ser209), 4E-BP1, and phospho-4E-BP1 (Ser65). The E7 antibody
(ED17), p27 antibody (C-19), and pan-cytokeratin (C11) antibody were from
Santa Cruz Biotech. Unless otherwise stated, all chemicals were from Sigma.
RNase protection assay. Total RNA was isolated from Caski, BaP-T, and
HOK-16B cells with an RNeasy kit (QIAGEN, Valencia, Calif.). The entire
HPV16 E7 open reading frame (ORF) was subcloned in pGEM 4. For genera-
tion of the antisense E7 probe, pGEM-E7 plasmid was linearized with EcoRI
and transcribed in vitro with SP6 polymerase and32P-labeled UTP. For gener-
ating the antisense Skp2 probe, a BamHI-KPN1-digested Skp2 cDNA fragment
was subcloned into pcDNA3, linearized with XbaI, and transcribed in vitro with
T7 polymerase. The cyclophilin-human antisense control template was obtained
from Ambion, Inc. and was transcribed with SP6 RNA polymerase. Five to 10
micrograms of total RNA was hybridized to the32P-labeled antisense-HPV16 E7
probe,32P-labeled antisense human Skp2 probe, or32P-labeled antisense-human
cyclophilin probe at 60°C for 16 h. The hybridization mixture was digested with
RNase A and T1. The products were then separated on a denaturing sequencing
gel (4% acrylamide) and visualized by autoradiography. The percentages of E7
mRNA were quantified by phosphorimaging (Molecular Dynamics).
Polysome fractionation and RNA analysis. The cells were grown either as a
monolayer or in methylcellulose-containing medium for 14 h. Isolation of poly-
somal RNA by sucrose gradient (15 to 40%) fractionation was performed fol-
lowing a previously described procedure (23). Briefly, cells were incubated at 4°C
for 15 min in buffer A containing 10 mM Tris-HCl (pH 8.0), 140 mM NaCl, 1.5
mM MgCl2, 0.5% NP-40, 20 mM dithiothreitol, 150 ?g/ml cycloheximide, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 500 units/ml of RNasin. The cells
were then homogenized with 15 strokes of a Dounce homogenizer and the nuclei
were removed by spinning at 1,000 ? g for 10 min. The supernatant was sup-
plemented with 665 ?g/ml of heparin and centrifuged at 12,000 ? g for 5 min to
remove mitochondria. A sucrose gradient (15 to 40%) was prepared in buffer A.
Postmitochondrial extract (200 ?l) was layered on the top of a 4-ml gradient and
centrifuged in an SW60 Ti rotor at 40,000 rpm for 90 min. Nine fractions (450 ?l)
were collected and digested with proteinase K (80 ?g) in 1% sodium dodecyl
sulfate (SDS) for 15 min at 37°C. RNA was isolated from individual fractions by
phenol-chloroform extraction. The RNA was analyzed by electrophoresis on a
1.2% formaldehyde agarose gel and stained with ethidium bromide to visualize
the distribution of subpolysomal and polysomal RNA. Fractions 1 to 4 contained
mostly subpolysomal RNA, and fractions 5 to 9 contained predominantly poly-
somal RNA. RNA from each fraction was analyzed for E7 mRNA and Skp2
mRNA by RNase protection assay as described above. The percentages of the E7
mRNA bound to polysomes were quantified by phosphorimaging (Molecular
Pulse-chase analysis using [35S]methionine-cysteine. The Caski cells were
grown as a monolayer or in methylcellulose-containing medium for 14 h. The
cells were labeled with [35S]methionine-cysteine (ICN) for 2 h in methionine-free
medium and then chased in medium containing 200 mg/ml of cold methionine.
Cells harvested at different times were lysed in radioimmunoprecipitation assay
(RIPA) buffer (50 mM Tris-HCl [pH 7.8], 150 mM NaCl, 1% NP-40, 0.1% SDS,
and 0.5% Na-deoxycholate) supplemented with protease inhibitor cocktail, 100
mM NaF, and 2 mM PMSF. Cell lysates measuring equal counts per minute were
immunoprecipitated with the E7 antibody. The E7 immunoprecipitates were
separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and developed by autoradiography. The level of35S-E7 was quan-
tified using a Molecular Dynamics PhosphorImager. The average decay rate of
E7 from two independent experiments is presented.
E7 decay rate analysis. The Caski cells were grown as a monolayer or in
methylcellulose-containing medium for 6 h, 12 h, or 18 h and were treated with
50 ?g/ml of cycloheximide for different time periods (between 1 and 4 h). The
cells were lysed in RIPA buffer containing protease inhibitors and phosphatase
inhibitors; 200 ?g of cell extracts was separated by 12% SDS-PAGE and blotted
to a nitrocellulose membrane, which was probed with E7 antibody.
Western blot analysis. Western blot analysis was performed using a previously
described procedure (3, 43). Cell lysates (50 to 300 ?g) were resolved on SDS-
PAGE, transferred to a nitrocellulose membrane, and probed with primary and
horseradish peroxidase-conjugated secondary antibodies, and signals were de-
tected by enhanced chemiluminescence (ECL; Amersham).
Flow cytometry. For analysis of distribution, 1 ? 106cells were fixed in 70%
ethanol in phosphate-buffered saline, treated with 100 ?g/ml RNase, and stained
with 20 ?g/ml propidium iodide. For each sample, 10,000 cells were analyzed for
DNA content using a Coulter EPICS 753 flow cytometer. The percentages of
cells in G1, S, and G2/M were determined using the EASY2 computer system
m7GTP affinity chromatography. eIF4E and 4E-BP1 were isolated by m7GTP-
Sepharose chromatography as previously described (41). Briefly, cells were lysed
in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton
X-100, 1.5 mm EDTA, 10% glycerol, 20 mM ?-glycerophosphate, 50 mM NaF,
200 ?M NaVO4, 2 mM PMSF, 1.5 ?g aprotinin/ml, and 5 ?g leupeptin/ml. Cell
lysates (500 ?g) were incubated with 20 ?l of m7GTP-Sepharose for 4 h at 4°C
and washed extensively with lysis buffer followed by 1 mM GTP-containing
buffer, and the beads were suspended in SDS sample buffer and boiled for 5 min.
The bound proteins were separated by SDS-PAGE and analyzed by Western blot
assay as described above.
Enhanced expression of E7 protein during growth in methyl-
cellulose-containing medium. HPVs replicate exclusively in
differentiated epithelial cells (reviewed in references 27, 30,
and 45). The E7 oncoprotein encoded by HPV plays a critical
role in differentiated keratinocytes, allowing replication of the
viral DNA (8). However, the regulation of E7 expression dur-
ing differentiation has not been studied. Previous studies show
that culturing of HPV-containing keratinocytes in semisolid
medium containing methylcellulose induces differentiation
that supports HPV DNA amplification and expression of viral
late genes (6, 11, 12, 21, 35, 39, 40). Therefore, to analyze E7
expression during differentiation, the HPV16-containing Caski
cervical carcinoma cells were induced to differentiate by cul-
turing them in medium containing 1.6% methylcellulose, and
the cell lysates were analyzed for the E7 protein using an
7080OH ET AL.J. VIROL.
immunoblot assay. A significant increase (four- to fivefold) in
E7 was observed in Caski cells during growth in methylcellu-
lose (Fig. 1A). The increase in the E7 protein was noticed
between 4 and 8 h, reached maximum level around 14 h, and
remained elevated for up to 20 h of culturing in methylcellu-
lose-containing medium (Fig. 1A). The increase of E7 was not
restricted to the malignant Caski cells but was also observed in
HPV16-immortalized HOK-16B oral epithelial cells and
HPV16-transformed BaP-T cells. Both HOK-16B and BaP-T
cells expressed low levels of E7 protein in actively dividing
monolayer cultures that were barely detectable using Western
blot assays (Fig. 1C and D). However, more than six- to eight-
fold increases in E7 protein were observed when these cells
were allowed to grow in semisolid culture containing methyl-
cellulose (Fig. 1C and D). As in the case of Caski cells, the
increase in E7 protein in HOK-16B and BaP-T cells was rapid,
reached its maximum level at around 14 h, and was maintained
at a comparable level for up to 24 h.
Growth in methylcellulose induces differentiation of epithe-
lial cells, and therefore we analyzed the expression of differ-
entiation-associated genes. An induction of cytokeratins was
observed after 20 h of differentiation in Caski cells (Fig. 1A).
Exit from the cell cycle is a prerequisite for differentiation. A
robust increase in the cyclin-dependent kinase inhibitor p27
expression began at 8 h and continued for the duration of the
time course. In contrast, the downregulation of growth-pro-
moting c-Myc protein occurred rapidly within 2 h of suspension
(data not shown). Flow cytometry analysis of cell cycle distri-
bution revealed that after 16 h of growth in methylcellulose-
containing medium, more than 40% of the cells were arrested
in G2with 4 N DNA content (Fig. 1B). In HOK-16B cells and
BaP-T cells, the induction of differentiation was similar to that
in Caski cells; an induction of the differentiation marker pro-
tein involucrin was detected after 20 h of growth in methylcel-
lulose-containing medium. Interestingly, the increase in the
level of E7 was observed long before the induced expression of
involucrin and keratins. This result suggests that the induction
of E7 is not an effect of differentiation; rather, E7 is induced
prior to the onset of differentiation.
To further confirm that the induced expression of E7 during
growth in methylcellulose is linked to differentiation, the Caski
and BaP-T cells were allowed to differentiate in medium con-
taining 1.5 mM CaCl2. Up to two- to fourfold induction of E7
protein was observed in both Caski and BaP-T cells after CaCl2
treatment (Fig. 1E and F). To determine the level of differen-
tiation during CaCl2treatment, the expression levels of the
differentiation marker involucrin in BaP-T cells and cytokera-
tins in Caski cells were analyzed. A marginal increase in in-
volucrin was observed in BaP-T cells after 36 h of CaCl2treat-
ment (Fig. 1F). Similarly, only a marginal increase in cytokeratins
was observed in Caski cells (data not shown). These results sug-
gest that in HPV-containing cells, CaCl2induces only limited
differentiation in comparison to growth in methylcellulose-con-
taining medium. However, the level of E7 protein was induced
during both methods of differentiation.
FIG. 1. Induction of the E7 protein during growth in methylcellulose-containing medium. (A) Caski cells were grown in methylcellulose-
containing medium and were harvested at the indicated times. Cell lysates (200 ?g) were separated by SDS-PAGE and analyzed by Western blot
assay using E7 antibody and p27 antibody. Cell lysates (25 ?g) were probed with pan-cytokeratin antibody and tubulin antibody. (B) Exponentially
growing Caski cells as adherent culture and Caski cells growing in methylcellulose-containing medium for 14 h were analyzed by flow cytometry.
HOK-16B cells (C) and BaP-T cells (D) grown in methylcellulose-containing medium were harvested at the indicated time. Cell lysates—200 ?g
for E7, 100 ?g for involucrin, and 25 ?g for tubulin—were analyzed using an immunoblot assay. Caski cells (E) and BaP-T cells (F) cultured in
medium containing 1.5 mM CaCl2were harvested at the indicated times. Cell lysates (200 ?g for E7, 50 ?g for tubulin, and 250 ?g for involucrin)
were analyzed using an immunoblot assay.
VOL. 80, 2006E7 EXPRESSION IN DIFFERENTIATING CELLS 7081
Half-life of the E7 protein did not change significantly dur-
ing differentiation in methylcellulose-containing medium. E7
protein has a short half-life (?1 to 2 h), and the steady-state
level of E7 in tumor cells is maintained primarily through
proteolysis by the 26S proteasome (16, 34, 43). To determine
whether the increase in E7 in differentiating cells is due to
increased stability, we analyzed the half-life of E7 using two
different assays. First, we analyzed the half-life of E7 by a
pulse-chase assay using [35S]methionine-cysteine. Caski cells
grown in attached or methylcellulose cultures (14 h) were
labeled with [35S]methionine-cysteine for 2 h and were chased
for 1 to 6 h with unlabeled methionine. Interestingly, signifi-
cantly more35S-labeled E7 was obtained by pulse-labeling of
differentiated Caski cells than by actively dividing cells. How-
ever, the half-life of E7 protein did not change during differ-
entiation (Fig. 2A). The half-life of E7 was between 1 and 2 h
both in actively dividing and in differentiating Caski cells (Fig.
2A). We also analyzed the half-life of E7 at various times
during differentiation. For this analysis, Caski cells growing in
methylcellulose for 6 h, 12 h, and 18 h were treated with
cycloheximide (50 ?g/ml) for 1 to 4 h, and the cell lysates were
analyzed for E7 protein (Fig. 2B). The half-life of E7 protein
did not change significantly during culturing in methylcellu-
Abundance of E7 mRNA did not change significantly during
growth in methylcellulose-containing medium. To analyze
whether the transcript level of E7 is regulated during differ-
entiation, the steady-state level of the E7 mRNA was mea-
sured by RNase protection assay. Compared to growth as a
monolayer culture, the steady-state level of the E7 mRNA
showed little or no change during growth in methylcellulose
(Fig. 3A and B). A modest 1.2- to 1.5-fold increase in the E7
mRNA was observed in differentiating Caski cells (Fig. 3A) but
not in HOK-16B cells (Fig. 3B). Interestingly, the steady-state
level of the E7 transcript did not change significantly in either
cell type, even after 20 h of growth in methylcellulose (Fig. 3).
In addition, the stability of the E7 mRNA did not change
significantly during growth in methylcellulose-containing me-
dium (data not shown).
Increased polysome association of the E7 mRNA during
growth of Caski and BaP-T cells in methylcellulose-containing
medium. To determine whether the induction of E7 was due to
increased translation, we analyzed the association of the E7
mRNA with polysomes in both Caski and BaP-T cells during
growth in methylcellulose-containing medium. Cytosolic ex-
tracts from cells grown in monolayer culture or in methylcel-
lulose were fractionated by sucrose gradient (15 to 40%) to
separate polysomes from monosomes, as described in Materi-
als and Methods. The gradient fractions were assayed for the
distribution of E7 mRNA by RNase protection assay. The
results show a significant increase in polysome-associated E7
mRNA in both differentiating Caski and BaP-T cells (Fig. 4).
In Caski cells, 40 to 45% of the E7 mRNA was associated with
polysomes during growth as a monolayer culture. After 14 h of
growth in methylcellulose-containing medium, over 80% of the
E7 mRNA was found in the polysome fractions. Similarly, in
BaP-T cells, growth in methylcellulose caused an increase in
polysome-bound E7 mRNA from 25 to 30% to more than
70%, indicating enhanced translation of the E7 mRNA. In
contrast, the level of Skp2 mRNA did not change significantly
FIG. 2. The half-life of E7 did not change significantly during differentiation. (A) The Caski cells grown as a monolayer or in methylcellulose-
containing medium (14 h) were labeled with [35S]methionine-cysteine for 2 h and chased with cold methionine for the indicated periods of time.
The35S-labeled E7 protein was immunoprecipitated and analyzed by SDS-PAGE. The level of35S-E7 was quantified by a phosphorimager and
plotted against the time of chase. The averages of two independent experiments are presented. (B) The Caski cells grown as a monolayer or in
methylcellulose-containing medium for 6 h, 12 h, and 18 h were treated with cycloheximide (50 ?g/ml) for 1 to 4 h. Cell lysates (200 ?g) were
separated by SDS-PAGE and analyzed by Western blot assay using E7 antibody.
7082 OH ET AL.J. VIROL.
in either cell type. The changes in the E7 mRNA distribution
correlated with the induction of the E7 protein in both cell
types. Taken together, these data show that the expression of
E7 is regulated at the level of translation initiation during
growth in methylcellulose-containing medium.
The translation inhibitor 4E-BP1 remains inactivated by phos-
phorylation during differentiation in HPV-containing Caski cells
but not primary keratinocytes or non-HPV HaCaT cells. The
association of mRNA with ribosomes is controlled largely by
cellular translation regulators such as eIF4E and 4E-BP1 (re-
viewed in references 14, 15, and 26). Therefore, to gain insight
into the molecular mechanism regulating translation of the E7
mRNA in differentiating cells, we sought to investigate
whether the cellular translation initiators were deregulated
during differentiation of the HPV-containing cells. The trans-
lation initiation factor eIF4E binds to the 5? Cap structure
(m7GpppN, where N is any nucleotide) found in the majority
of eukaryotic mRNA. A major mechanism for control of eIF4E
function is through its interaction with the translation inhibi-
tory protein 4E-BP1. The inhibitory function of 4E-BP1 is
FIG. 3. The steady-state level of the E7 mRNA did not change significantly during growth in methylcellulose-containing medium. Total RNA
was isolated from Caski cells or HOK-16B cells at the indicated times following growth in methylcellulose-containing medium. RNA (10 ?g) was
hybridized to32P-labeled antisense-E7 and antisense-cyclophilin probes and processed by an RNase protection assay as described in Materials and
Methods. The32P-labeled E7 and cyclophilin probes are shown.
FIG. 4. Increased polysome association of the E7 mRNA during differentiation. Caski cells (left) and BaP-T cells (right) were grown in a
monolayer culture (A) or in methylcellulose-containing medium for 14 h (B). Cytosolic extracts were fractionated on a sucrose gradient (15 to 40%)
as described in Materials and Methods. RNA isolated from each fraction (numbers indicated at the top) was analyzed for the presence of the E7
mRNA and Skp2 mRNA by an RNase protection assay. Staining for rRNA by ethidium bromide in gradient fractions is shown for each sample.
Fractions 1 to 5 represent subpolysomal RNA, and fractions 6 to 9 represent polysome-associated RNA.
VOL. 80, 2006 E7 EXPRESSION IN DIFFERENTIATING CELLS7083
regulated primarily by phosphorylation (2, 4, 10, 14, 15, 26, 33).
The hypophosphorylated form of 4E-BP1 competes with an-
other translation initiation protein, eIF4G, to bind eIF4E and
blocks its function (2, 15, 33). In contrast, the hyperphosphor-
ylated form of 4E-BP1 cannot bind to eIF4E and cannot re-
press translation. The function of eIF4E is also regulated by
phosphorylation. We analyzed the phosphorylation status of
both 4E-BP1 and eIF4E during the differentiation of Caski
cells, using antibodies specific for phospho-eIF4E (Ser209) and
phospho-4E-BP1 (Ser65). Increased phosphorylation of 4E-
BP1 was observed in Caski cells within 4 h of growth in differ-
entiating culture, and most remarkably, the phosphorylation of
4E-BP1 remained high throughout the differentiation phase,
for up to 20 h (Fig. 5). More than 60% of 4E-BP1 was in
phosphorylated form in Caski cells after 14 h of growth in
methylcellulose-containing medium (Fig. 5). As a control, we
analyzed phosphorylation of 4E-BP1 during differentiation of
normal human keratinocytes and non-HPV HaCaT skin kerati-
nocytes. In both primary human keratinocytes and non-HPV
HaCaT skin keratinocytes, 4E-BP1 was dephosphorylated at
between 4 and 8 h of growth in methylcellulose-containing
medium. In these cells, less than 5% of 4E-BP1 remained
phosphorylated after 14 h of growth in methylcellulose-con-
taining medium (Fig. 5). The higher level of dephosphorylated
4E-BP1 in differentiating cells suggests repression of Cap-de-
pendent translation. A decrease in the steady-state level of
4E-BP1 was also observed in differentiating non-HPV cells. In
contrast, the steady-state level or the phosphorylation status of
eIF4E did not change during differentiation of keratinocytes.
Incidentally, the kinetics of induction of phospho-4E-BP1 par-
alleled the induction of E7 in Caski cells. Taken together, these
results suggest that the induced phosphorylation of 4E-BP1 is
linked to increased Cap-dependent translation of E7 mRNA in
differentiating Caski cells (Fig. 5).
Induced phosphorylation of 4E-BP1 was also observed in
differentiating HPV-containing HOK-16B and BaP-T oral ke-
ratinocytes (Fig. 6). In comparison, in primary oral keratino-
cytes, a dramatic decrease in the phospho-4E-BP1 was ob-
served during differentiation. The level of eIF4E or phospho-
eIF4E, on the other hand, did not change significantly in
differentiating normal or HPV-containing oral keratinocytes.
We further analyzed the phosphorylation of 4E-BP1 in SCC-
25, a non-HPV oral cancer cell line, and in C33A, a non-HPV
cervical cancer cell line. Similar to primary keratinocytes, a
significant decrease in phospho-4E-BP1 was observed during
differentiation in methylcellulose-containing medium. The
steady-state level of eIF4E was comparable in all the cell types
studied (Fig. 6). In comparison to primary keratinocytes, a
higher level of phospho-4E-BP1 was also observed in Caski
cells during calcium-induced differentiation (data not shown).
To evaluate the relationship between the increased phos-
phorylation of 4E-BP1 and the initiation of translation, the
interaction of 4E-BP1 with eIF4E was examined by chroma-
tography of cell lysates on 7-methyl GTP-Sepharose (Cap af-
finity column). For this experiment, Caski and HaCaT cells
were grown in monolayer cultures or in methylcellulose-con-
taining medium for 14 h and the cell extracts were allowed to
bind to Cap affinity resins. In Caski cells, there was no detect-
able change in the ratio of 4E-BP1 to eIF4E bound to the
column during differentiation (Fig. 7A). However, there was a
dramatic increase in the ratio of 4E-BP1 to eIF4E bound to the
Cap affinity column when extracts from differentiating non-
HPV cells were analyzed (Fig. 7A). The lack of increase in the
ratio of 4E-BP1 to eIF4E upon differentiation of HPV-con-
taining cells as opposed to that in non-HPV cells during dif-
ferentiation is significant. Phosphorylation of 4E-BP1 disrupts
its interaction with eIF4E and the Cap complex. The enhanced
binding of 4E-BP1 with the 7-methyl GTP-Sepharose is con-
sistent with the acute hypophosphorylation of 4E-BP1 in
HaCaT cells during differentiation.
The FRAP/mTOR kinase pathway leads to phosphorylation
of 4E-BP1 (2, 15, 33). To correlate the induced phosphoryla-
tion of 4E-BP1 with the expression of E7, we treated Caski
cells with the inhibitor of mTOR kinase, rapamycin. Actively
FIG. 5. Induced and sustained phosphorylation of 4E-BP1 in differentiating HPV-containing Caski cells but not in normal human keratinocytes
or non-HPV HaCaT cells. Cell lysates of normal human keratinocytes (150 ?g), Caski cells (100 ?g), and HaCaT cells (100 ?g) grown in
methylcellulose-containing medium for the indicated times were fractionated by SDS-PAGE and analyzed by Western blot assay using antibodies
against phospho-4E-BP1 (Ser65), 4E-BP1, eIF4E, and phospho-eIF4E (Ser 209). Two hundred micrograms of Caski cell lysates was analyzed for
E7 protein using a Western blot assay.
7084 OH ET AL. J. VIROL.
dividing Caski cells in a monolayer culture or Caski cells grow-
ing in methylcellulose-containing medium were treated with
rapamycin (100 ng/ml) for 12 h. As expected, during rapamycin
treatment, a significant decrease of the phospho-4E-BP1 was
readily observed in both the actively dividing and differentiat-
ing cells (Fig. 7B). Similarly, a significant decrease in E7 pro-
tein level was observed in rapamycin-treated Caski cells (Fig.
7B). Rapamycin treatment did not change the steady-state
level of E7 mRNA (Fig. 7B). Taken together, these results
suggest that the induced and sustained phosphorylation of
4E-BP1 allows enhanced translation of E7 mRNA in differen-
HPVs replicate in differentiated cells, and the viral oncopro-
tein E7 makes the host differentiated cells replication compe-
tent to support virus growth. In this study, we investigated the
mechanism by which E7 is regulated during differentiation.
Due to the lack of a simple cell culture system to grow the
HPVs, studies on the HPV oncogenes are restricted to HPV-
containing cell lines. An important new observation of this
study is the finding that a significantly higher level of E7 is
expressed during differentiation of HPV-containing tumor
cells. Studies on the mechanism of the E7 induction revealed
that the increase was due to induced and sustained translation
of the E7 transcript in the differentiated cells. Concomitantly,
we observed significant changes in the phosphorylation pattern
of a key translation regulatory protein, 4E-BP1, in HPV-con-
taining tumor cells during differentiation. The enhanced phos-
phorylation of 4E-BP1 predicts increased Cap-dependent
translation in differentiating HPV-expressing cells.
Mobilization of the translation machinery, instead of the
transcription machinery, to induce E7 during differentiation of
HPV-containing tumor cells is intriguing. Multiple recent stud-
ies have shown that the deregulation of Cap-dependent trans-
lation by overexpression or induced phosphorylation of key
translational regulators plays a major role in transformation
and tumor formation (reviewed in references 4, 26, 28, and 36).
The translation factor eIF4E promotes tumor formation and
cooperates with c-Myc in lymphomagenesis (37). The activa-
tion of the translation complex has been shown to be essential
for the genesis and maintenance of the malignant phenotype in
human mammary epithelial cells (1). The oncogene Bcr-Abl
kinase modulates the translation regulators ribosomal protein
S6 and 4E-BP1 in chronic myelogenous leukemia cells to in-
duce translation (25). Similarly, in this study we observed en-
hanced translation of the E7 mRNA in differentiated cells,
which is linked to enhanced phosphorylation of 4E-BP1. Ex-
pression of high levels of E7 protein would support the ability
of papillomaviruses to reinitiate the growth cycle in differen-
The translation of most eukaryotic mRNAs involves the Cap
initiation complex eIF4F (15). The eIF4F complex involves
three proteins: the Cap-binding protein eIF4E, the RNA he-
licase eIF4A, and the scaffold protein eIF4G (reviewed in
references 14, 15, and 26). Among them, eIF4E is a limiting
translation factor and a major target for translation regulation.
4E-BP1 regulates the initiation of Cap-dependent translation
by association with the Cap complex through eIF4E. The bind-
ing of 4E-BP1 to the Cap complex depends on phosphorylation
(2, 10, 15, 33). Hypophosphorylated 4E-BP1 competes with
eIF4G to bind eIF4E to block formation of active translation
FIG. 6. Changes in the levels of 4E-BP1 and eIF4E during growth in methylcellulose-containing medium. Lysates of primary oral keratinocytes
(200 ?g), HOK-16B cells (150 ?g), BaP-T cells (100 ?g), Caski cells (50 ?g), SCC-25 cells (100 ?g), and C33A cells (50 ?g) grown in monolayer
cultures or in methylcellulose-containing medium were analyzed by a Western blot assay using 4E-BP1 and eIF4E antibodies.
VOL. 80, 2006E7 EXPRESSION IN DIFFERENTIATING CELLS7085
complex. Hyperphosphorylation of 4E-BP1 inhibits association
of eIF4E to 4E-BP1 and results in stimulation of translation (2).
We observed both induced and sustained phosphorylation of
4E-BP1 in differentiating HPV-containing cells. The induction
of 4E-BP1 phosphorylation coincided with the induction of E7
protein in Caski cells, suggesting that the translation inhibitor
4E-BP1 plays a major role in the accumulation of E7. Treat-
ment of Caski cells with the TOR kinase inhibitor rapamycin
blocked both the phosphorylation of 4E-BP1 and the expres-
sion of E7 in Caski cells. The phosphorylation of 4E-BP1 was
sustained for up to 20 h without significant loss during differ-
entiation of multiple HPV-containing cells, including Caski,
HOK-16B, and BaP-T. In contrast, the phosphorylation of
4E-BP1 was reduced in primary keratinocytes and non-HPV
cancer cells during differentiation in methylcellulose-contain-
ing medium. Consistent with this observation, significantly
higher levels of 4E-BP1 were found to be associated with the
7-methyl GTP affinity resins in differentiating HaCaT cells,
suggesting repression of Cap-dependent translation.
The molecular mechanism of sustained phosphorylation of
4E-BP1 during differentiation of HPV-containing cells is not
elucidated in this study. However, these results support the
notion that the formation of the functional translation initia-
tion complex is induced and maintained during differentiation
of HPV-containing cells. A recent study showed that eIF4E,
but not 4E-BP1, is strongly phosphorylated during the onset of
TPO-induced differentiation of UT7-mpl cells (7). Increased
levels of eIF4E were also observed in differentiating lung tu-
mor cell lines (42). The level of eIF4E or phospho-eIF4E did
not change significantly during differentiation of HPV-contain-
ing cells. It is possible that the modifications of translation
regulators during differentiation are cell type specific.
Viruses often use host DNA replication machinery to repli-
cate viral DNAs, cellular transcription machinery to transcribe
viral mRNAs, and cellular translation machinery to ensure
translation of the viral mRNA. Different viruses utilize differ-
ent strategies to use the host translation machinery for en-
hanced synthesis of viral polypeptides (38). However, little is
known about how the human papillomaviruses interact with
the cellular translation machinery. In high-risk oncogenic
HPVs, E7 is expressed from a bicistronic E6/E7 pre-mRNA
(44). The most abundant mRNAs transcribed from oncogenic
HPVs are derived from the p97 promoter. The HPVs contain
a differentiation-specific enhancer sequence that induces ex-
pression of late genes in differentiated cells. The differentia-
tion-dependent promoter is not responsible for expression of
E6/E7 mRNA (20). The E7 ORF is the second ORF after the
E6 ORF in the E6/E7 mRNA. No mRNA that encodes E7 as
the first open reading frame has been identified. In Caski cells,
the most abundant in vivo mRNA is E6*IE7 in which part of
the E6 ORF is spliced out, suggesting that E7 is formed from
this mRNA. Thus, the 5? end of the E7 mRNA contains
uAUGs and an sORF from E6 (29). It is possible that these
uAUGs and the E6 ORF control translation of the E7 ORF.
FIG. 7. (A) Increased level of 4E-BP1 associates with the m7GTP resin in differentiating HaCaT cells but not in Caski cells. Lysates (500 ?g)
of Caski cells or HaCaT cells grown in methylcellulose-containing medium (for 14 h) were incubated with m7GTP-Sepharose beads as described
in Materials and Methods. The proteins bound to the beads were analyzed for 4E-BP1 and eIF4E using a Western blot assay. (B) Rapamycin blocks
expression of E7 protein in Caski cells. Caski cells grown as adherent culture or in methylcellulose-containing medium were treated with rapamycin
(100 ng/ml) for 12 h. (Left) Lysates from attached (350 ?g) or differentiated (200 ?g) cells were analyzed for E7, tubulin (25 ?g), and 4E-BP1 (100
?g) using a Western blot assay. (Right) Total RNA (10 ?g) isolated from dimethyl sulfoxide (DMSO)- or rapamycin-treated cells was analyzed
for E7 mRNA and cyclophilin mRNA using an RNase protection assay.
7086 OH ET AL.J. VIROL.
The results described in this report show that Cap-depen-
dent translation is enhanced in differentiating HPV-containing
cells, in marked contrast to many viruses that impaired such
translation. In herpes simplex virus type 1-infected cells, viral
gene product ICP0 stimulates Cap-dependent translation to
support viral DNA replication in quiescent differentiated cells
(41). ICP0 stimulates phosphorylation of eIF4E and 4E-BP1 to
enhance the assembly of active eIF4E complex, and this pro-
cess is crucial for productive viral growth and efficient reacti-
vation of latent infection (41). Since productive HPV infec-
tions in differentiated cells depend on the E7 function, the
induction of E7 at the onset of differentiation might constitute
a critical step in the HPV life cycle. The HPVs replicate ex-
clusively in the differentiated epithelial cells, and the HPV late
proteins are translated exclusively in the differentiated epithe-
lial cells. Our studies show that Cap-dependent translation is
enhanced in differentiating HPV-containing cells, in contrast
to differentiating non-HPV cells. Parallel to the ICP0 protein
of herpes simplex virus type 1, an HPV protein is likely involved
in enhancing Cap-dependent translation in HPV-containing cells.
A recent study showed that the HPV oncoprotein E6 associates
with GADD34/PP1 to block PKR-initiated translation inhibition
(23a). Future studies will be important in determining the identity
of the HPV protein involved in enhancing Cap-dependent trans-
lation machinery in differentiating cells.
We thank Ruben H. Kim of the UCLA School of Dentistry for
primary oral keratinocytes. We thank Pradip Raychaudhuri of the
Department of Biochemistry and Molecular Genetics at UIC for his
critical comments on the manuscript.
This work was supported by grants DE12506 and AG24138 from the
National Institutes of Health.
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