Intranuclear localization of human papillomavirus 16 E7 during transformation and preferential binding of E7 to the Rb family member p130.
ABSTRACT To study intracellular pathways by which the human papillomavirus 16 oncogene E7 participates in carcinogenesis, we expressed an inducible chimera of E7 by fusion to the hormone-binding domain of the estrogen receptor. The chimeric protein (E7ER) transformed rodent fibroblast cell lines and induced DNA synthesis on addition of estradiol. In coimmunoprecipitation experiments, E7ER preferentially bound p130 when compared to p107 and pRb. After estradiol addition, E7ER localization changed to a more intense intranuclear staining. Induction of E7 function was not correlated with binding to p130 or pRb but rather with intranuclear localization and modest induction of binding to p107.
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ABSTRACT: The p53 tumor suppressor protein, one of the most extensively studied proteins, plays a pivotal role in cellular checkpoints that respond to DNA damage to prevent tumorigenesis. However, the transcriptional control of the p53 gene has not been fully characterized. We report that the transcription factor E2F1 binds only to the E2F1 distal site of the p53 promoter in the human papillomavirus positive carcinoma HeLa cell line. Moreover, we showed that etoposide, a DNA damaging agent, activates p53 transcription through the E2F1 pathway. This increase correlates with apoptosis induction as disruption of this pathway led to reduced apoptosis stimulation by the DNA damaging agent.FEBS letters 08/2013; · 3.54 Impact Factor
Chapter: Tumor Viruses and p53[Show abstract] [Hide abstract]
ABSTRACT: Viruses are potent oncogenic agents and are considered to link to approximately one-fifth of all human malignancies. The study of tumor viruses has provided fundamental bases of how normal cells become cancer cells. A number of virus-encoded oncogenes have been identified and their functions were characterized. One of the striking functional similarities of such virus oncogenes, especially the ones encoded by DNA tumor viruses, is that they bind to and inactivate tumor suppressor genes, such as p53 and pRb. In this chapter, I am going to summarize the function of virus oncogenes focusing on their interactions with p53.01/1970: pages 160-177;
Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 6999–7004, June 1999
Intranuclear localization of human papillomavirus 16 E7 during
transformation and preferential binding of E7 to the Rb family
(retinoblastoma gene product?p107)
K. SMITH-MCCUNE*†‡§, D. KALMAN¶?, C. ROBBINS*, S. SHIVAKUMAR*, L. YUSCHENKOFF*, AND J. MICHAEL BISHOP¶?
*Department of Obstetrics, Gynecology, and Reproductive Sciences,†Cancer Research Institute, and‡Reproductive Endocrinology Center, and¶G. W. Hooper
Foundation and?Department of Microbiology and Immunology, University of California, San Francisco CA 94115
Contributed by J. Michael Bishop, March 26, 1999
human papillomavirus 16 oncogene E7 participates in carci-
nogenesis, we expressed an inducible chimera of E7 by fusion
to the hormone-binding domain of the estrogen receptor. The
chimeric protein (E7ER) transformed rodent fibroblast cell
lines and induced DNA synthesis on addition of estradiol. In
coimmunoprecipitation experiments, E7ER preferentially
bound p130 when compared to p107 and pRb. After estradiol
addition, E7ER localization changed to a more intense in-
tranuclear staining. Induction of E7 function was not corre-
lated with binding to p130 or pRb but rather with intranuclear
localization and modest induction of binding to p107.
To study intracellular pathways by which the
Human papillomavirus (HPV) infection of genital tract epi-
thelium is associated with dysplastic changes in epithelial
growth and is an important prerequisite for the development
of cervical cancer. Two viral genes, E6 and E7, mediate
transformation by HPV (1–4). The majority of cervical carci-
nomas contain truncated forms of HPV, which retains E6 and
E7, integrated into the cellular genome (5–9). HPV16 E7
induces growth of cells in soft agar (1), stimulates DNA
synthesis (10, 11), cooperates with ras to transform primary
cells (12, 13), and overcomes G1 arrest induced by serum
deprivation, actinomycin D, or overexpression of p21 (14–17).
The protein encoded by E7 has a region of shared sequence
homology with both adenovirus E1A and SV40 large T
antigen, which mediates binding to the retinoblastoma gene
product pRb (13, 18). By binding to pRb, E7 alters the
interaction of pRb with the transcription factor E2F-1, result-
ing in activation of E2F-responsive genes (19). E7 protein from
high-risk HPV types, HPV16 and HPV18, binds with a higher
affinity to pRb than E7 protein from low-risk HPV types such
as HPV6 and HPV11 (20). Therefore, the prevailing model is
that E7 protein, by binding to pRb, promotes progression
through the cell cycle. The role of E7 binding to other
Rb-family members such as p107 and p130 has not been
entirely elucidated. However, it is known that induction of
B-myb transcription by E2F-1 is the result of E7 binding to
p107 rather than to pRb (21).
To assess the relationship between transformation and E7
binding to Rb-family members, we have constructed an induc-
ible chimeric molecule consisting of HPV16 E7 fused in-frame
an approach used to render other oncogenes steroid-
dependent (22–27). By using this inducible chimera of E7, we
demonstrate that HPV16 E7 activity is correlated with in-
tranuclear localization of the chimera. In addition, E7 binds to
greater proportions of intracellular p130 and p107 than pRb in
MATERIALS AND METHODS
Construction of E7ER. The HPV16 viral genome (from J.
Palefsky, Univ. of California, San Francisco) was used as the
template to make E7 that lacked a stop codon by using PCR.
of the estrogen receptor [HE14, from P. Chambon, Institute of
Genetics and Molecular and Cellular Biology, Strasborg,
France (28)]. E7 mutants were obtained from K. Vousden
(National Cancer Institute, Frederick, MD) (29, 30), and were
similarly cloned in-frame with ER to create mutant E7ER
chimeras. A replication-defective murine retrovirus vector
containing a neomycin-resistance gene under the control of
the SV40 promoter [pMXE7ER (31)] was used to express the
chimera (Fig. 1A). Viral stocks were generated by transfection
into the packaging cell line ?2 (32) and were used to infect
cultures of 3T3?C7 cells (a derivative of NIH?3T3 cells). The
ER fragment was also subcloned alone into a mammalian
expression vector LNCX (33); LNCX-ER was transfected
directly into 3T3?C7 cells by using calcium phosphate precip-
itation and a clonal cell line selected with a high level of ER
plating cells from transformed foci at low density and isolating
individual colonies. Cells were grown in DMEM lacking
phenol red with 10% stripped calf serum (complete medium).
Serum was stripped of endogenous steroids by incubation with
activated charcoal (1 g?50 ml of serum) for 20 minutes at 4°C.
Soft Agar Assays. Cells were plated in 0.375% low melting
agarose (FMC) in complete medium with 1 ?M E2or 0.1%
ethanol and fed weekly with appropriate medium.
Stimulation of DNA Synthesis. Cells were plated at a density
of 105cells per well into a six-well plate; after the cells reached
confluence, the medium was changed to phenol red-free
DMEM with 0.5% BSA and 5 ?g?ml transferrin (Sigma),
cultured for 36–48 hours, and treated with 1 ?M E2, 0.1%
ethanol, or 10% calf serum for 24 hours. [3H]Thymidine (1
?Ci, Amersham Pharmacia; 1 Ci ? 37 GBq) was added for 45
minutes, and incorporation into DNA was measured. For the
dose–response curve, (Fig. 1C), all media contained a con-
centration of ethanol equivalent to that delivered with the
highest dose of E2.
Immunoprecipitation and Western Blotting. For the West-
ern blot in Fig. 1B, cell pellets were lysed directly in sample
buffer. For immunoprecipitation, cell pellets were lysed in 2 ml
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
PNAS is available online at www.pnas.org.
Abbreviations: E2, estradiol; HPV, human papillomavirus; ER, estro-
gen receptor; CAT, chloramphenicol acetyltransferase.
§To whom reprint requests should be addressed at: Box 0128, Cancer
Research Institute, 2340 Sutter Street, Room S331, University of
California, San Francisco, CA 94143. e-mail: email@example.com.
of lysis buffer (25 mM Tris?HCl, pH 7.4?150 mM NaCl?2%
NP40?0.5% Na deoxycholate?0.2% Na dodecylsulfate) con-
taining 1 mM Pefabloc, 0.1 mg?ml aprotinin, and 0.1 mg?ml
leupeptin. Five microliters of rabbit polyclonal anti-ER anti-
body (from S. Robbins, University of Calgary) was added per
cell pellet. Antigen–antibody complexes were isolated on
Protein A-Sepharose beads (Sigma) and washed three times in
wash buffer (25 mM Tris, pH 7.4?50 mM NaCl?0.5% Na
deoxycholate?0.2% NP40). Proteins were resolved on 10%
(Fig. 3) or 8% (Fig. 4) polyacrylamide gels, transferred to
nitrocellulose (Schleicher & Schuell), and incubated with 5
?g?ml monoclonal anti-Rb antibody (clone G3–245, PharM-
ingen) or 1 ?g?ml rabbit anti-Rb IgG (Santa Cruz Biotech-
nology), anti-p107 IgG, or anti-p130 IgG (Santa Cruz Bio-
technology), or 1:1,000-diluted anti-ER rabbit antiserum
(from S. Robbins) in Tris-buffered saline. Detection was
macia) using horseradish peroxidase-conjugated sheep anti-
mouse or donkey anti-rabbit Ig (Amersham Pharmacia) or
horseradish peroxidase conjugated-Protein A (Amersham
Pharmacia) for detecting anti-ER antibodies. When sequential
immunoprecipitations were performed, antibody (5 ?g of
mouse or rabbit IgG) was added (for 1–18 hours) to the
supernatant collected from the first immunoprecipitation. For
densitometry, films of Western blots were scanned with Adobe
PHOTOSHOP, and the intensities of bands representing pRb,
p107, and p130 were quantitated by using IMAGEQUANT (Mo-
E7–Rb Interactions in a Mammalian Two-Hybrid System. A
mammalian two-hybrid system was constructed (based on ref.
34). cDNA encoding the retinoblastoma gene product (amino
acids 379–792) was amplifed from a wild-type Rb allele (from
W.-H. Lee, University of Texas Health Science Center, San
Antonio) by using PCR and cloned in-frame into pGal0 (34)
to create g4Rbp. A mutant Rb allele of the pRb pocket domain
(Cys-to-Phe change at amino acid 706, from J. Horowitz, Duke
University Medical Center Durham, NC) was similarly cloned
in-frame into pGal1/0 to create g4Rbpm. Gal4–p107 was
obtained from C. Dang, John Hopkins University, Baltimore.
A Gal4?VP16-dependent luciferase reporter vector
(g5E1Blux) was constructed by cloning five concatemerized
Gal4-binding domains into a luciferase-reporter plasmid PXP1
(35). CV1 cells or PVU1 cells (CV1 cells containing a large
tumor antigen allele with a deletion of the pRb-binding
pocket) were transfected by using the calcium phosphate
expressing chloramphenicol acetyltransferase (CAT) under
control of the SV40 promoter (pSV40CAT) to quantitate
transfection efficiency. In transfections designed to test for
interactions between two proteins, the reporter and
expressing the two fusion proteins (e.g., g4Rbp and E7ER). E2,
tamoxifen, or ethanol were added 1 day after transfection, and
the cells were harvested 48–72 hours later. Luciferase assays
were performed by using Promega reagents. Liquid CAT
assays were performed to quantitate transfection efficiency as
described (36). To obtain relative transfection efficiencies,
results of CAT assays from each plate were divided by results
from a plate transfected with only the SV40CAT and the Gal4
luciferase reporter plasmids. Luciferase values from each plate
were normalized for transfection efficiency by dividing by the
corresponding relative transfection efficiency ratio. Transfec-
luciferase values were averaged. Normalized luciferase values
generally varied by ?5% between plates transfected with the
same set of plasmids.
Immunofluorescence. Cells were plated on two-chamber
culture slides (Lab-Tek). At confluence, the medium was
changed to 0.5% BSA and 5 ?g?ml transferrin in DMEM for
24 hours, at which time 1 ?M E2or 0.1% ethanol was added
neomycin-resistance gene. X, Xho; B, BamHI; E, EcoRI; C, ClaI. (B) Lysates of 3 ? 105cells transfected with the ER construct (C7/ER-3) and
E7ER (C7/E7ER 3-3) were analyzed by using Western blot analysis with anti-ER rabbit antiserum (S. Robbins). Positions of 45- and 29-kDa
molecular mass markers are shown to the right. (C) [3H]Thymidine incorporation was measured in starved E7ER-expressing cells (C7/E7ER 1-1)
or a pool of cells transfected with vector alone (C7/neo) in response to increasing doses of E2. Each point is the average of duplicate wells; duplicate
cpm values varied by ?10%. (D) Induction of DNA synthesis was measured in response to 1 ?M E2in clonal cell populations expressing the ER
domain (C7/ER3), wild-type E7 in the context of the ER chimera (E7ER 1-1 and 3-3), and mutant E7 alleles containing a point mutation in the
Rb-binding pocket (E7*ER 4-1 and 4-3) or a deletion of the Rb-binding pocket (E7*ER 5-4). Bar graphs represent the mean from 4 (C7/ER3),
12 (E7ER 1-1), 19 (E7ER 3-3), 3 (E7*ER 4-1 and 4-3), or 2 (E7*ER 5-4) independent assays. Error bars represent the SEM. (E) Cells (104) were
plated in soft agar, and the total number of colonies greater than 6–8 cells in size was counted after 2 (C7/E7, C7/E7ER 1-1 and 3-3) or 4 (C7/ER3)
weeks. Results are expressed as a ratio of the total number of colonies in the presence as compared with the absence of E2.
Structure and function of the E7ER chimera. (A) pMXE7ER contains the E7ER chimera in a retroviral vector with the
7000Microbiology: Smith-McCune et al.Proc. Natl. Acad. Sci. USA 96 (1999)
for 18 hours. Cells were rinsed, fixed in 3.7% formaldehyde in
PBS, incubated in blocking solution (10% calf serum and 0.1%
Triton X-100 in PBS) for 20–30 minutes, rinsed with PBS, and
incubated for 1–2 hours with polyclonal rabbit anti-ER anti-
body (Santa Cruz Biotechnology; 1 ?g?ml in blocking solu-
tion). As a negative control, parallel sets of slides were
incubated with rabbit IgG at 1 ?g?ml. The slides were then
washed 3 times with PBS, incubated with Cy3-conjugated goat
anti-rabbit IgG (The Jackson Laboratory) at 1:500 dilution for
40 minutes, rinsed, mounted, and viewed by using fluorescence
optics with a Texas red filter. 4?,6-Diamidino-2-phenylindole
(DAPI) (2 ?g?ml) was added to the last wash for detection of
nuclei. For in situ lysis, cells were incubated in hypotonic lysis
buffer (10 mM Hepes, pH 7?10 mM KCl?0.1% Triton X-100?
0.5 mM DTT?1.5 mM MgCl2) for 10 min at 4°C before fixation
in 3.7% formaldehyde?PBS (37, 44).
Inducible Transformation in Cells Expressing E7ER. Ex-
pression of E7ER in G418-selected populations was verified
with Western blotting (Fig. 1B). Cells containing ER or E7ER
constructs expressed proteins detected by anti-ER antibodies
corresponding to the predicted sizes (38 kDa and 52 kDa,
respectively). The E7ER chimera was also recognized by an
anti-E7 mAb (data not shown), confirming that the chimeric
molecule expressed the E7 epitope.
To evaluate whether E7 function is inducible in the context
of E7ER, the amount of DNA synthesis was measured over a
range of E2concentrations and found to be maximal between
500 nM and 1 ?M (Fig. 1C). These relatively high concentra-
tions are consistent with the fact that the ER clone used in
these experiments contains a mutation that results in a lower
affinity for E2than wild-type ER (38). Clonal populations of
cells expressing E7ER reproducibly demonstrated an induc-
tion of incorporation of [3H]thymidine in response to 1 ?M E2
ranging from 2- to 10-fold (Fig. 1D). Cells expressing chimeric
molecules with a point mutation in the pRb-binding pocket
(Gly-24), E7*ER 4-1 and 4-3, or a deletion of the pRb-binding
pocket (amino acids 21–35), E7*ER 5-4, showed no induction
of DNA synthesis in response to E2(Fig. 1D), consistent with
previously reported effects of these mutations.
Addition of E2to soft agar assays of E7ER-expressing clonal
cell lines resulted in a significant induction of growth of
colonies in soft agar, as shown in Fig. 1E. E2had little effect
on cells carrying either the vector alone or wild-type E7;
cloning efficiencies were either low (?1%) or high (12%),
respectively. The estrogen receptor itself did not confer E2-
dependent induction of DNA synthesis (Fig. 1D) or growth in
soft agar (Fig. 1E), confirming that the observed effects are
caused by the E7 moiety in the chimera.
Interaction of E7ER with pRb and p107 in a Mammalian
Two-Hybrid System. To document that chimeric E7ER mol-
ecules interact with pRb-family members in the intracellular
environment, we constructed a modified mammalian two-
transactivation function that can be measured in the context of
the Gal4 promoter (39); this property of the molecule was
exploited to detect interactions between E7ER and pRb or
p107 expressed as fusion proteins with a Gal4 DNA-binding
domain. Thus, whether complexes form between E7ER and
either Gal4pRb or Gal4p107, the ER transactivation domain
will be brought into sufficient proximity of the transcriptional
machinery by the Gal4 DNA-binding domain to activate
transcription of a luciferase reporter gene under control of the
Gal4 promoter (g5E1BLux). In cells transfected with the
reporter construct alone, the addition of E2did not increase
the basal levels of luciferase activity (Fig. 2A). In cells co-
transfected with the reporter construct, E7ER and the Gal4
fusion vector containing a truncated version of pRb retaining
the Rb-pocket domain (amino acids 379–792) (g4Rbp), high
levels of luciferase activity were induced by E2, whereas only
background levels were found in its absence (Fig. 2A). A single
point mutation in the Rb pocket known to result in loss of
association of pRb with viral oncoproteins resulted in loss of
the induction of luciferase activity (Fig. 2A, g4Rbpm). These
results confirm that the interaction between E7ER and pRb is
occurring through the expected domains in pRb. In addition,
E7ER showed ligand-dependent transactivation of the Gal4
promoter in association with two other forms of Gal4–pRb
fusion proteins, consisting of amino acids 2–928 and amino
acids 379–928 of pRb (data not shown). The Gal4–p107
chimera (g4p107) similarly interacted with E7ER as measured
by luciferase activity (Fig. 2C). However, the ER construct
alone did not exhibit interaction with either pRb or p107 (Fig.
2C), and the E7ER chimera in the absence of E2 failed to
induce luciferase activity (Fig. 2B). HPV16 E7 itself also
contains a transactivation domain (13) which can function in
the two-hybrid system; cotransfection of HPV16 E7 and
Gal4–pRb results in induction of luciferase activity (Fig. 2B).
In the presence of tamoxifen [which binds to the estrogen
the CAT reporter, and various constructs as indicated. Luciferase values were normalized to transfection efficiency as described in the text. Fold
induction refers to the normalized luciferase value of the experimental cells (average of two plates) divided by the normalized luciferase value of
cells transfected with the luciferase reporter and CAT reporter alone. E2(1 ?M), tamoxifen (1 ?M), or ethanol was added as indicated. PVU1
cells were used in A, whereas CV1 cells were used in B and C; the enhanced induction of luciferase activity in A may be because of the fact that
the large tumor antigen expressed by these cells replicates the Gal4-based plasmids that have an SV40 origin. Columns represent the average and
error bars represent the SE of duplicate transfections. See Materials and Methods for descriptions of g5E1BLux, g4Rbp, and g4Rbpm.
E7 and E7ER interactions with pRb in a mammalian two-hybrid system. Cells were transfected with the luciferase reporter (g5E1Blux),
Microbiology: Smith-McCune et al.Proc. Natl. Acad. Sci. USA 96 (1999) 7001
no transactivation of the luciferase reporter was measured in
cells cotransfected with E7ER and Gal4–pRb or Gal4–p107
(Fig. 2C). These results indicate that, in the context of the
E7ER chimera, the endogenous transactivation domain of E7
Intracellular Binding of E7ER to pRb, p107, and p130. By
using polyclonal antisera to the ER domain, intracellular
complexes containing E7ER were immunoprecipitated from
cell lysates and analyzed with Western blotting. Comparison of
Fig. 3A with Fig. 3B shows that pRb is measurable in the cells
but that only a small proportion of the endogenous pRb is
detectable in immunocomplexes with E7ER. In addition,
equivalent proportions of pRb were complexed with E7ER in
the presence or absence of 1 ?M E2 (Fig. 3A and 4E),
indicating that complex formation did not correlate with
activation of E7 function. E7 is known to result in decreased
levels of pRb protein because of proteolysis (40), and indeed
the amount of pRb in E7ER-expressing cells was lower than
the amount in ER-expressing cells (Fig. 3B, E7ER 1-1 and 3-3
of pRb immunoprecipitated with E7ER as a proportion of the
total pRb in the cognate cell lysate. Therefore, the lack of
induction of pRb binding on E7ER activation is independent
of the effect of E7ER on pRb levels.
When the blot in Fig. 3A was reprobed with antibodies to
p107, interaction of E7ER with p107 was readily detected (Fig.
3C). In addition, on treatment with E2, the amount of p107 in
the immune complex with E7ER increased 2.6-fold (? 0.47,
uncomplexed p107 was unchanged. The ER domain alone
showed no detectable interaction with p107 in the presence or
absence of E2 (Fig. 3C). Thus, complex formation between
p107 and E7ER increased when E7 activity was induced
compared with cells in which E7 was present but biologically
inactive. This is in contrast to the binding of E7ER to pRb,
which was difficult to detect and did not vary with the
activation state of E7.
Significantly greater proportions of p130 than pRb or p107
were detected in complexes with E7ER. In Fig. 4A, the
position of pRb migration is marked to the right of the figure,
but no pRb complexed to E7ER was detectable in this
experiment. However, on reprobing the blot in Fig. 4A with
anti-p130 antibodies, p130 was easily detectable (Fig. 4B).
Sequential immunoprecipitation of cell lysates, first with an-
ti-ER antibodies and then with anti-p130 antibodies (Fig. 4C),
revealed that significant proportions of the intracellular p130
(?65%) were associated with E7ER (Fig. 4E).
In Fig. 4D, E7mutER, which contains a point mutation in
the pRb-binding pocket (Gly-24), was also analyzed in coim-
munoprecipitation experiments. This mutant allele was unable
to bind to p130. A similar result was found by using a mutation
of E7 lacking the pRb-binding pocket (amino acids 21–35)
(data not shown). These results indicate that the domain of E7
involved in p130 binding overlaps with that used to bind pRb
Intracellular Localization of E7ER Changes On Activation
of the Construct. It was puzzling that induction of E7 effects
To test the possibility that the E7ER chimera might sequester
Rb-family members within cellular compartment(s), where
they are inactive, we compared the intracellular localization of
E7ER before and after exposure to E2. The immunofluores-
cent staining pattern of E7ER changed to an intense nuclear
signal after addition of E2(Fig. 5B vs. Fig. 5A). This shift of
E7ER localization mirrored the staining pattern of cells ex-
known translocation of ER from the cytoplasm to the nucleus
on ligand binding (27). Immunofluorescent staining with the
anti-ER antibody of cells carrying the vector alone showed
faint cytoplasmic staining that did not change in the presence
of E2(data not shown).
Discrepancies remain in the literature regarding the intra-
cellular localization of E7: it appears to be cytoplasmic when
cells are fractionated but nuclear when studied by using
immunofluorescence (41–43), a paradox that has been ex-
plained by the hypothesis that cell fractionation disrupts the
association of E7 with the nucleus (41). To study E7ER
localization directly, we lysed cells in situ before fixation and
examined nuclei on the substrate by using immunofluores-
cence (37, 44). We found a dramatic increase in the amount of
E7ER associated with the nucleus in the presence as compared
with the absence of E2(Fig. 5 E and F).
HPV16 E7 Preferentially Binds p130. Previous reports
suggest that the capability of E7 to transform cells arises, at
least in part, from its ability to complex with the nuclear
phosphoprotein pRb (20, 45). E7 also interacts with the
pRb-like protein p107 (21) and with a 130-kDa protein that
resembles the E1A-associated protein p130 (46). In addition,
E7 peptides compete with E1A for binding to p130 (47). Our
results show that E7ER, like E7, complexes with the pRb-
family members in both biochemical and in vivo assays. Sur-
prisingly, the relative proportion of intracellular p130 bound to
E7ER is far greater than the proportions of pRb or p107.
Indeed, ?65% of the p130 in the cell was present in complexes
with E7ER, compared with 5% of pRb and 25% of p107 (Fig.
Recent reports have indicated that the Rb-family members
have different functions in regulating cell growth and differ-
entiation and that p130 is the predominant member present in
quiescent cells (recently reviewed in ref. 48). In addition, p130
has been implicated in the G1–G0 transition (49, 50). The
predominant intracellular E2F activity, E2F-4, is bound pri-
marily to p130 in quiescent cells (51, 52) and switches to
association with p107 and pRb as cells pass through the G1–S
In skin, the Rb-family members are expressed in different
histological compartments, with pRb and p107 localized pre-
dominantly in the basal and parabasal levels and p130 in the
upper, differentiated cell layers (50). When HPV infects
epithelium, productive viral replication occurs in the same
differentiated layers where p130 is expressed. The histology of
cervical dysplasia demonstrates aberrant differentiation and
an increased number of mitotically active cells, consistent with
an inability of cervical keratinocytes to undergo terminal
differentiation. Our work suggests that the effect of HPV16 E7
in human disease may derive in part from interactions with
p130, which may modify keratinocyte differentiation and
thereby facilitate viral replication.
p107 association on E2 treatment. Confluent starved cultures were
treated overnight with E2 (1 ?M) (?) or ethanol (?) and immuno-
precipitated with anti-ER antibodies (A and C) and then sequentially
with anti-pRb antibodies (B). Western blots were probed with anti-Rb
antibodies (A and B) or anti-p107 antibodies (C). Each lane consists
of lysate from 2 ? 107cells expressing the E7ER construct (3-3 and
1-1) or the ER construct (ER-3).
Minimal association of E7ER with pRb and induction of
7002Microbiology: Smith-McCune et al. Proc. Natl. Acad. Sci. USA 96 (1999)
Nuclear Localization Is Important for E7-Induced Trans-
formation. The data presented here indicate that properties of
E7, such as induction of DNA synthesis and growth of cells in
soft agar, are rendered E2-dependent in the context of the
E7ER allele. In a mammalian two-hybrid assay (Fig. 2), the
biochemical readout for interactions between E7ER and pRB
or p107 were dependent on the presence of E2. However,
results from the coimmunoprecipitation experiments sug-
gested that the amounts of endogenous p130 and pRb asso-
ciated with E7ER were unchanged on activation of E7, and the
amount of p107 was modestly induced. It is possible the results
of the immunoprecipitation experiments represent indiscrim-
inate accessibility of Rb family members to E7ER secondary
to lysis of cells, but this seems unlikely given the pronounced
differences in the proportions of Rb-family members bound to
E7ER within the same cell lysate and the observation that
binding to p107 was consistently induced on activation of
Our results suggest two explanations for E2dependence of
E7ER function. One possibility is that the transformation
elicited by E7 in these assays depended on induction of
association with p107 and was independent of association with
pRb and p130. This interpretation is supported by data
showing that the induction of B-myb expression by E7 de-
of C7/E7ER 1-1 cells (A and B) or C7/ER-3 (C and D) were treated overnight with 1 ?M E2(B and D) or ethanol (A and C) and prepared for
immunofluorescence as described. (Bar ? 20 ?m.) (E–J) In situ lysis reveals differential nuclear localization of E7ER on E2 treatment.
Serum-starved cultures of C7/E7ER 1-1 cells were treated overnight with 1 ?M E2(F, H, and J) or ethanol alone (E, G, and I). Cells were lysed
in hypotonic buffer before fixation and then prepared for immunofluorescence with anti-ER antibodies (E and F), DAPI (G and H) or rabbit IgG
(I and J). (Bar ? 50 ?m.)
Immunofluorescent localization of E7ER. (A–D) Altered intracellular localization on E2treatment. Serum-starved confluent cultures
C and D, untreated cell lysates were immunoprecipitated first with anti-ER antibodies and then sequentially with anti-p130 antibodies;
(E). E7 mutER is a clonal population expressing a point mutation in the Rb-binding pocket (Gly-24). A second clonal cell line gave identical results.
Data in E represents comparisons by densitometry scanning of the amounts of pRb, p107, and p130 immunoprecipitated with anti-ER antibodies
compared with the sum of the amounts immunoprecipated with anti-ER and subsequently with the relevant Rb-family member antibody. Data from
two (p107) or three (pRb and p130) independent experiments were averaged; error bars represent the SEM.
E7ER preferentially binds to p130. Experiments were performed as in Fig. 3. In A, ? or ? refers to the absence or presence of E2. In
Microbiology: Smith-McCune et al.Proc. Natl. Acad. Sci. USA 96 (1999) 7003
pended on p107 but not pRb binding (21); it is weakened by
our observation that interaction with p107 was only modestly
induced (2.6-fold) on E7ER activation. A second explanation
is that binding of E7 to Rb-family members per se is not as
important for E7 activity as is the intracellular localization of
these complexes. After the addition of E2, E7ER localized to
the nucleus (Fig. 5). Our data further support the hypothesis
that the nucleus is the physiologically important site of E7
action. The E7 sequence has no obvious nuclear localization
signal, and the mechanism by which wild-type E7 is targeted
to the nucleus is unknown. Mutations in E7 that bind pRB but
are nontransforming have been described (53–55). The phe-
notype of these mutants may be caused by their inability to
localize to the correct intracellular compartment. Because
E7ER is localized to the nucleus at least in part because of the
ER domain, it will be interesting to test these mutants in the
of nuclear localization can restore transforming capability.
Our results indicate that the formation of E7ER complexes
with the Rb-family members is not sufficient for transforma-
tion and suggest that intranuclear concentration of the com-
plexes is essential for cell cycle reentry. This interpretation
runs counter to the currently held view that E7 acts by leading
to release of transcriptionally active E2F on binding to the
Rb-family members. Recent data have indicated that the
activity of endogenous E2F4 (the predominant E2F partner of
p130) is regulated by changes in its intracellular localization,
from nuclear in G1?G0cells to cytoplasmic in cycling cells (56).
In addition, pRb-E2F, p107-E2F, and p130-E2F species have
distinct patterns of intracellular localization that vary inde-
pendently during the cell cycle (56). E2F response elements in
B-myb, cyclin A, and cdc2 are occupied in G0(57), suggesting
cell cycle reentry. Our data suggest a model in which E7-Rb
family member complexes play an active, and perhaps direct,
role in E2F transciptional regulation. The experimental system
described in this report will allow us to study the effects of E7
activation on intracellular localization of the Rb and E2F
family members and the transcriptional repression or activa-
tion of E2F sites in response to these changes.
We thank P. Chambon for providing the HE14 ER clone, C. Dang
for the Gal4–p107 construct, J. Horowitz for the mutant Rb allele,
W.-H. Lee for the wild-type Rb clone, M. McMahon for the retroviral
K. Vousden for E7 mutants, and A. Alberts, L. Deiss, M. McMahon,
S. Robbins, E. Shtivelman, and S. Schirm for helpful suggestions. This
research was supported by the G. W. Hooper Research Foundation,
the Irwin Foundation, the University of California Cancer Research
Coordinating Committee, and Public Health Service Grants HD31057
and CA61797. S.S. was supported by a grant from the Cowell Foun-
dation. K.S.-M. acknowledges past support of the Reproductive Sci-
entist Development Program of the National Institute of Child Health
and Human Development and the American Gynecological and
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