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1650
Human Tumor Growth Is Inhibited by a Vaccinia Virus
Carrying the E2 Gene of Bovine Papillomavirus
Viviana Valadez Graham, Biol. 1
Gerd Sutter, D.V.M., Ph.D.2
Marco V. José, Ph.D.3
Alejandro Garcı́a-Carranca, Ph.D.1
Volker Erfle, D.V.M., Ph.D.2
Norma Moreno Mendoza, Ph.D.4
Horacio Merchant, Ph.D.4
Ricardo Rosales, Ph.D.1
1
Department of Molecular Biology, Instituto de
Investigaciones Biomédicas, Universidad Nacional
Autónoma de México, México City, México
2
GSF-Center for Enviromental and Health Research, Institute of Molecular Virology, Neuherberg-Muenchen, Germany.
3
Department of Biophysics and Biomathematics,
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México City,
México.
4
Department of Cell Biology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México City, México.
BACKGROUND. Papillomavirus is the etiologic agent associated with cervical carcinoma. The papilloma E2 protein is able to regulate negatively the expression of E6
and E7 papilloma oncoproteins. Therefore, a new, highly attenuated vaccinia virus
known as modified vaccinia virus Ankara (MVA), which carries the papillomavirus
E2 gene, was used for the treatment of tumors associated with human papillomavirus.
METHODS. Analysis of expression of the E2 gene from the recombinant vaccinia
virus was performed by reverse transcription–polymerase chain reaction of RNA
isolated from infected cells. Detection of the E2 protein was done by immunoprecipitation from proteins labeled with [35S]-methionine, isolated from infected cells.
The therapeutic effect of the MVA E2 recombinant virus over human tumors was
tested in nude mice bearing tumors generated by inoculation of HeLa cells. Series
of 10 nude mice with tumors of different sizes were injected with MVA, MVA E2, or
phosphate-buffered saline. Tumor size was monitored every week to assess growth.
RESULTS. The MVA E2 recombinant virus efficiently expressed the E2 protein in
BS-C-1 cells. This protein was able to repress, in vivo, the papillomavirus P105
promoter, which controls the expression of the E6 and E7 oncoproteins. In nude
mice the MVA E2 virus reduced tumor growth very efficiently. In contrast, tumors
continued to grow in mice treated with MVA or PBS. The life expectancy of MVA
E2-treated mice was also increased three- to fourfold compared with that of
animals that received MVA or PBS.
CONCLUSIONS. The growth of human tumors was efficiently inhibited by the MVA
E2 recombinant vaccinia virus. The absence of side effects in treated animals
suggested that the MVA E2 virus is a safe biologic agent that could in the future be
used in humans for the treatment of cervical carcinoma. Cancer 2000;88:1650 – 62.
© 2000 American Cancer Society.
KEYWORDS: papillomavirus, vaccinia virus, MVA strain, cervical carcinoma.
Supported in part by grant 3080N (from CONACyT,
Mexico) and by grant IN211394 (from DGAPA–
UNAM, México City, México).
The authors thank Dr. Peter Howley for providing
the pC59 plasmid, Dr. Jaime Berumen for providing the BPV-1 E2 antiserum, and Dr. Carlos Rosales for reading and commenting on the manuscript. They also thank Miriam Guido and Rosa
Maria Dominguez for technical assistance.
Address for reprints: Ricardo Rosales, Ph.D., Department of Molecular Biology, Instituto de Investigaciones Biomédicas–UNAM, Apto. Postal 70228,
Cd. Universitaria, México, D.F.– 04510, Mexico.
Received June 30, 1999; revision received December 1, 1999; accepted December 1, 1999.
© 2000 American Cancer Society
I
n humans, neoplasic transformation has been linked to the presence of human papillomaviruses (HPVs). These viruses can induce
diseases, from warts up to condylomas, and lesions that can progress
to malignant neoplasia. Approximately 1 million people are infected
with HPV every year. It is well known that cervical carcinoma correlates
with the presence of HPVs, particularly types 16, 18, 31, 33, and 35.1–11
In contrast, HPV types 6, 11, 42, and 43 are found only in the anogenital tract and are not associated with cervical carcinoma.1,3,12,13
Cervical carcinoma is a serious health problem. In developing countries, 50,000 women die annually of this malignancy.
Normally, HPVs infect and replicate themselves in the form of an
unintegrated circular episome in keratinocytes of genital mucosa and
perigenital skin. The papillomavirus E1 and E2 gene products regulate
viral DNA replication. The E2 gene product can also activate or
Papilloma E2 Gene in Tumor Therapy/Valadez et al.
repress transcription of different HPV promoters.14,15
In particular, the papillomavirus protein E2 is known
to down-regulate the P105 promoter from HPV type
18, which controls transcription of the E6 and E7 oncogenes. These oncoproteins are expressed in a variety of cervical human tumors.16 The best-studied
HPVs, types 16 and 18, persist extrachromosomally in
precancerous lesions, but they are often integrated
into the cellular genome, causing transformation of
the cell.17 Integration of HPV into the cell genome
leads to a disruption and inactivation of the papillomavirus E2 gene. This event then results in derepression of the E6 and E7 oncogenes. Expression of these
oncogenes appears to be a critical step in the maintenance of the transformed stage and progression to
invasive carcinoma. This mechanism, however, is not
necessarily the only one operating to induce the malignant stage, as suggested by recent studies in which
HPV integration was not detected in at least 30% of the
analyzed tumors.13,18
Due to the strong dependence of cervical carcinoma on infection by HPVs, it is thought that the
induction of a protective stage against these viruses
could help prevent the appearance of cervical tumors.
Based on this idea, different strategies to develop a
safe vaccine or immunotherapeutic agent against cervical carcinoma have been assessed.
Other methods, such as radiotherapy and chemotherapy, have, of course, been used to reduce papillomas and cancer tumors. However, these methods
work efficiently only during the first stages of tumor
development. Later, it becomes very difficult to treat
cervical tumors due to large tumor sizes and the negative side effects that anticancer drugs may have.
Being aware of these difficulties in treating cervical carcinoma, we decided to use a viral vector based
on an attenuated vaccinia virus known as modified
vaccinia virus Ankara (MVA). This virus was developed
and tested as a safe smallpox vaccine.19 It was also
found to be avirulent for normal or immunosuppressed individuals and not to have negative side effects in 120,000 humans inoculated for priming vaccination.20 –22 One reason for the safety of MVA is that
this virus has its viral expression and recombination
mechanisms impaired. Moreover, MVA is capable of
infecting most, if not all, the human cell lines tested
up to now. It has also been demonstrated that MVA is
an excellent vector for expressing foreign genes, such
as the Escherichia coli Lac Z or the phage T7 polymerase,23,24 under nonpermissive conditions in infected
cells. Because of these characteristics, the most successful current strategy for vaccine development involves the use of vaccinia virus vectors.
The approach of expressing a foreign protein via
1651
vaccinia virus vectors has already been used to protect
animals from other virus infections.25,26 Treatment of
rats with live recombinant vaccinia viruses expressing
tumor specific antigens from polyoma virus could prevent cognate tumor development and in some cases
could also induce regression of preexisting tumors.25
In addition, an MVA recombinant virus expressing the
hemagglutinin and the nucleoprotein genes of influenza virus was found to protect mice fully against a
lethal influenza virus challenge.26
Based on this strategy, we decided to investigate
the potential use of a recombinant MVA virus for the
treatment of cervical carcinoma. In order to do this,
we constructed an MVA recombinant virus by inserting the E2 gene from bovine papillomavirus (BPV-1)
into the MVA genome. The E2 gene was chosen because the E2 protein represses expression of E6 and E7
proteins and can also induce p53-mediated apoptosis
in HeLa cells. This protein is also able to arrest cell
growth independently of the transcriptional repression of the endogenous viral E6 and E7 genes.15,27,28
The new recombinant virus was named MVA E2
and its effects over human tumor growth in immunosuppressed animals were investigated. We found that
treatment with this recombinant MVA E2 virus resulted in human tumor growth arrest in nude mice. In
contrast, only a slight inhibition of tumor growth was
observed in animals inoculated with the parental MVA
virus. In control animals just inoculated with phosphate-buffered saline (PBS), tumors continued to
grow indefinitely. These data indicated that the recombinant MVA E2 virus has the capacity of inducing
elimination of papilloma tumors from animals, and
therefore it becomes a potential new therapeutic
agent for cervical carcinoma.
METHODS
Mice
Nude male mice (Mus musculus) age 8 weeks were
purchased from Taconic Laboratory (New York, NY)
and kept under “sterile” conditions in isolated cages.
Cells and Viruses
Monkey kidney (BS-C-1), human carcinoma (HeLa),
and chicken embryo fibroblast (CEF) cells were grown
in Dulbecco modified Eagle medium (DMEM) (Gibco
BRL, Gaithersburg, MD) supplemented with 10% fetal
calf serum (FCS) (Gibco BRL, Gaithersburg, MD). Cells
were maintained in a humidified air-5% CO2 atmosphere at 37 °C. 3T3 F4 cells, which contain integrated
copies of the ␤-galactosidase gene under the control of
the P105 promoter of HPV type 18, were also grown in
DMEM with 10% FCS.
Vaccinia virus strain MVA and MVA E2 recombi-
1652
CANCER April 1, 2000 / Volume 88 / Number 7
FIGURE 1. Schematic map of the MVA
genome and the plasmid pIIIgpt dsP E2
used for insertion of the bovine papilomavirus E2 gene. Hind III restriction endonuclease sites within the genome of MVA are
indicated at the top (kb: kilobase pairs).
Letters at the top represent the MVA fragments generated by Hind III digestion. DNA
sequences (flank 1 and flank 2) adjacent
to deletion III within the A fragment of the
vaccinia genome were cloned into the
plasmid PIIIgptdsP to allow recombination
into the MVA genome. A cassette with
back-to-back copies of a strong synthetic
vaccinia virus early/late promoter (dsP) is
shown. P 7.5 refers to well-characterized
vaccinia virus early/late promoter. A cassette with the E2 gene was inserted in
front of one of the vaccinia virus early/late
promoters. The E. coli guanine phosphoribosyl transferase (gpt) gene under control
of the P 7.5 promoter is also shown.
nant viruses were routinely propagated and titrated by
endpoint dilution in CEF cells to obtain the 50% tissue
culture infectious dose (TCID50).
Plasmid Construction
A cDNA copy of the bovine papillomavirus E2 gene
flanked by Bam HI and AscI sites was amplified by the
polymerase chain reaction (PCR) using DNA from
plasmid pC59 (donated by Dr. Peter Howley from the
National Institutes of Health, Bethesda, MD)29 as a
template. This plasmid contains an open reading
frame that covers part of the E1 and E2 papilloma
genes. A set of primers, GS78-CAGCAGGGATCCAGGATGGAGACAGCATGCGAACGT and GS79-CAGCAGGCGCGCCCATCATTGGTGGTGCGCCTT, were used to
amplify the E2 gene from the pC59 plasmid. The PCR
product was cloned into the unique BamHI and AscI
sites of the pIIIgpt dsP plasmid, which carries two
copies of the strong synthetic vaccinia virus early/late
promoter23 (Fig. 1). The resulting plasmid (pIII gpt dsp
E2) was purified by cesium chloride gradients and
used to construct the MVA E2 recombinant virus.
Construction of Recombinant Vaccinia Virus
To generate a recombinant MVA virus, monolayers of
nearly confluent CEF cells in 6-well plates were infected with MVA at a multiplicity of 0.05 tissue culture
infectious dose (TCID50) of MVA per cell. Ninety minutes after infection, cells were transfected with 10 ␮g
of plasmid DNA (pIIIgpt dsP E2) using Lipofectin re-
agent (Gibco BRL, Gaithersburg, MD) as recommended by the manufacturer. At 48 hours after infection, cells were harvested and processed as previously
described.23 Recombinant MVA virus expressing the
E2 protein was selected by 6 consecutive rounds of
plaque purification on CEF cells in the presence of
mycophenolic acid (25␮g/mL).19 Subsequent virus
stocks were also prepared in CEF cells.
Analysis of Viral DNA
Viral DNA was isolated by digestion of virus-infected
cells with proteinase K (50 ␮g/mL) in TE buffer (10
mM Tris HCl, pH ⫽ 8.0, 1 mM ethylenediaminetetraacetic acid), during 8 hours at 37 °C followed by
extraction with phenol/chloroform. DNA was precipitated with ethanol and resuspended in water. Viral
DNA (3 ␮g) was PCR-amplified using 40 cycles of 1
minute at 94 °C, 2 minutes at 45 °C, and 2 minutes at
72 °C. The primers used for amplification were GS82
(5⬘-GGT TGT TGA TGG ATC TGT GAT GCA TGC GAT
AGC TGA-3⬘) and GS83 (5⬘-GAA TGC ACA TAC ATA
AGT ACC GGC ATC TCT AGC AGT-3⬘). These primers
are complementary to sequences located within the
flank 1 and flank 2 regions adjacent to deletion III
within the Hind III A fragment of the vaccinia genome,
respectively (Fig. 1). PCR products were cut with the
restriction enzymes BamHI and AscI, and analyzed by
electrophoresis on 1% agarose gels.
Papilloma E2 Gene in Tumor Therapy/Valadez et al.
Reverse Transcription-PCR Analysis of RNA
Total RNA (3 ␮g) from infected BS-C-1 cells (for the E2
gene) or from infected 3T3 F4 cells (for the ␤-galactosidase gene) was isolated as described30 and transcribed with 20 units of avian myeloma virus reverse
transcriptase (Boehringer Mannheim, Indianapolis,
IN), using 0.5 ␮g of oligo(dt)12–18 in a total volume of
50 ␮L of buffer (50 mM Tris-HCl, pH 8; 70 mM KCl; 10
mM MgCl2; 4 mM dithiothreitol; and 1 mM each of the
4 deoxyribonucleoside triphosphates) at 42 °C for 90
minutes. The cDNA product was purified by phenolchloroform extraction, precipitated with ethanol, and
resuspended in 50 ␮L of water. One ␮L of cDNA was
amplified by 40 cycles of PCR as described above.
Primers used for amplification of the E2 gene were
GS82 and GS83, and for amplification of a 294 bp
within the ␤-galactosidase gene were BGR-10 (5⬘- TCG
CGG TGA TGG TGC TGC GTT GG-3⬘) and BGR-11
(5⬘-GTT ACG ATG CGC CCA TCT ACA CCA A-3⬘).
Detection of the Recombinant Papilloma E2 Protein
BS-C-1 cells grown in 12-well plates were infected with
the MVA or the MVA E2 recombinant virus at a multiplicity of 15 plaque formation unit (pfu) per cell.
Virus were adsorbed on the cells for 1 hour at 37 °C in
DMEM with 2% FCS. Cultures were then supplemented with DMEM containing 10% FCS and incubated at 37 °C in a 5% CO2 atmosphere. Six hours after
infection, the medium was removed, and the cells
were washed once with 1 mL of methionine free
DMEM. To each well, 0.2 mL of methionine free
DMEM supplemented with 50 ␮Ci of [35S]-methionine
(Dupont, Boston, MA) was added and incubated for 30
minutes at 37 °C. Cytoplasmic extracts of infected cells
were then prepared, after removal of the medium, by
incubating the cells in each well with 0.2 mL of lysis
buffer (0.5% Nonidet P-40; 10 mM Tris-HCl, pH 8; 1
mM EDTA) for 10 minutes at 37 °C. Cell extracts were
incubated with an anti-E2 polyclonal serum for 1
hour, and protein-antibody complexes were immunoprecipitated with 50 ␮L of Protein A-Sepharose (Pharmacia Biotech., Uppsala, Sweden). Immunoprecipitates were then washed with PBS, resuspended in 50
␮L of Laemmli sample buffer, and separated by 10%
sodium dodecyl sulfate (SDS)–polyacrilamide gel electrophoresis (PAGE). Resolved proteins were analyzed
by autoradiography.
1653
sidase specific activity32 were determined as previously described. Maximum activity of ␤-galactosidase
(100%) was taken as 104 units/mg of protein from
uninfected cells.
Analysis of Tumor Growth
One hundred fifty nude mice were inoculated subcutaneously with 2 ⫻ 106 HeLa cells in 200 ␮L of PBS.
Two or 3 weeks later, when tumor size was between
0.1 and 0.5 cm2, the animals (50 for each group) were
injected directly into the tumor with 5 ⫻ 107 units of
MVA or MVA E2 once a week for 3 weeks. Control
animals received only PBS. The area of tumor growth
was evaluated every week. The relative tumor growth
was calculated by measuring the tumor size every
week and subtracting the size of the tumor in the
previous week. Because tumor growth in these animals followed a sigmoidal curve, and mathematical
studies have shown that tumor growth in animals can
be described with a logistic differential equation, we
decided to use the logistic differential equation
dG共t兲/dt ⫽ rG关1 ⫺ G/k兴,
which describes the tumor growth in the absence of an
immune response. With this equation we can calculate the behavior of tumor growth when different
amounts of MVA E2 virus are used for the tumor
treatment. We can also determine how the tumor
changes over time with or without an external agent
like the MVA E2 virus. In this equation, r is the intrinsic rate of tumor growth and k is the maximum size of
the tumor.33 The solution for the logistic differential
equation is as follows:
G共t兲 ⫽ Gok/关Go ⫹ 共k ⫺ Go兲exp共 ⫺ rt兲兴,
where G(o) is the initial size of the tumor, r and k have
the same definition as above, and t is time. Fitting of
the experimental data (area of tumor size estimated
every week) to the last equation was made using a
nonlinear least-square estimation following a Marquardt procedure.34
Survival Analysis
Survival data were analyzed by the standard Kaplan–
Meier survival curves, using the computer program
GraphPad Prism.33
Identification of Apoptosis in Tumor Cells
␤-Galactosidase Assay
Confluent 3T3 F4 cells were infected with different
amounts of MVA or MVA E2 viruses and incubated at
37 °C. Twelve hours later, cytoplasmic extracts were
prepared, and protein concentration31 and ␤-galacto-
Human tumors grown in nude mice were injected
with MVA or MVA E2 recombinant vaccinia viruses.
After 24 hours of virus infection, tumors were taken
from the animals, fixed in 4% buffered formaldehyde,
and embedded in paraffin. Sections of 6 ␮m adhered
1654
CANCER April 1, 2000 / Volume 88 / Number 7
to slides treated with 0.05% poly-L-Lysine (300,000
daltons of molecular weight) (Sigma, St. Louis, MO)
were deparaffinized in xylol, dehydrated, and incubated with 20 ␮g/mL proteinase K. After washing in
PBS, the slides were treated according to the instructions of the In Situ Cell Death Detection Kit, AP
(Boehringer Mannheim Co., Indianapolis, IN). Briefly,
sections were incubated with TUNEL reaction mixture
for 1 hour at 37 °C, washed in PBS, and analyzed by
fluorescence microscopy (Nikon Inc., Melville, NY).
Positive and negative controls were included for each
experiment. For inducing DNA strand breaks, sections
were treated with 1 ␮g/mL DNAase (Sigma, St. Louis,
MO) for 30 minutes at 37 °C. For negative controls,
sections were incubated in Label solution without terminal deoxynucleotidyl transferase.
RESULTS
Construction and Isolation of the MVA Recombinant
Virus Expressing the Bovine Papillomavirus E2 Gene
Previous work has shown that MVA can serve as an
efficient expression vector and also as a highly attenuated smallpox vaccine.19,23,26,35,36 This virus was used
to construct a recombinant virus carrying a papillomavirus regulatory gene (the E2 gene). The MVA plasmid pIIIgpt dsP (Fig. 1) contains two copies of the
strong synthetic vaccinia virus early/late promoter
and multiple cloning sites. The bovine papillomavirus
E2 gene was inserted into pIIIgpt dsP, as described in
“Methods,” to form the recombinant plasmid pIIIgpt
dsP E2 (Fig. 1). The plasmid pIIIgpt E2 was transfected
into CEF cells. These cells were then infected with
MVA virus to obtain the new MVA E2 recombinant
virus. Homologous recombination occurs between the
flank 1 and flank 2 sequences of the plasmid with the
same corresponding sequences in the vaccinia virus
genome in order to generate a recombinant vaccinia
virus carrying the E2 gene (Fig. 1). Serial dilutions of
the infected cell lysates were plated on CEF cells in the
presence of mycophenolic acid, to select for recombinants.
Characterization of MVA E2 Recombinant Virus
Infected BS-C-1 cells were used to characterize the
MVA E2 DNA. Integration of the E2 gene into the viral
genome was demonstrated by PCR and restriction enzyme analysis. Viral DNA was isolated from infected
cells and two sets of primers (GS82 and GS83) were
used to amplify a DNA fragment covering the region
between flank 1 and flank 2 of the vaccinia virus
sequences (Fig. 1). The amplified DNA fragment was
cut with the restriction enzymes BamHI and AscI in
order to release the E2 gene. The DNA fragment obtained corresponded in size to the E2 gene, and it was
also inserted with the right orientation into the MVA
genome (data not shown).
In order to confirm that the MVA E2 recombinant
virus induced the expression of the E2 gene, BS-C-1
cells were infected with this virus, and the presence of
E2 RNA and E2 protein was then analyzed. First-strand
reverse transcription products, derived from purified
total RNA from MVA E2–infected and uninfected BSC-1 cells, were amplified by PCR with the primers
GS78 and GS79. The PCR product derived from the E2
gene is a 942 bp fragment. Twenty-four hours after
MVA E2 infection, papilloma E2 RNA could be easily
detected in BS-C-1 cells (Fig. 2A). E2 RNA also augmented considerably by 72 hours postinfection (Fig.
2B). This result confirmed that MVA E2 was in fact
directing the expression of the E2 gene in infected
cells and also suggested that there was an accumulation of E2 RNA during infection.
Synthesis of the E2 protein was confirmed by metabolic labeling with [35S]-methionine of BS-C-1 cells
infected with MVA or MVA E2 recombinant virus. In
each case the labeled polypeptides were immunoprecipitated with an anti-E2 polyclonal serum. These antibodies reacted specifically with a 48 kDa protein,
which corresponded in size to the E2 gene product
(Fig. 2C). This result showed that a complete E2 protein was being produced inside MVA E2–infected cells.
MVA E2 Recombinant Virus Represses Specifically the
Human Papillomavirus P105 Promoter
In order to determine the efficacy of the MVA E2
recombinant virus to repress the papillomavirus P105
promoter, which negatively regulates the E6 and E7
oncogenes, a stable 3T3 F4 cell line containing the
␤-galactosidase gene controlled by the P105 promoter
of papillomavirus type 18 was infected with MVA E2.
After infection with either MVA or MVA E2 recombinant viruses, total RNA from these 3T3 F4 –infected
cells was isolated, and the specific RNA for ␤-galactosidase analyzed by reverse transcription–PCR. The
DNA fragment of 295 bp corresponding to an internal
portion of the ␤-galactosidase gene could be easily
detected in these cells. The MVA E2 recombinant virus
reduced the transcription activity from the P105 promoter by about 50% as indicated by a reduction in the
amount of ␤-galactosidase RNA (Fig. 3A). The enzymatic activity of ␤-galactosidase was also reduced in a
dose-dependent manner by MVA E2, up to 60% from
the activity of uninfected cells (Fig 3B). In contrast,
MVA infection did not cause any reduction in ␤-galactosidase activity (Fig. 3B).
Papilloma E2 Gene in Tumor Therapy/Valadez et al.
1655
FIGURE 2. Expression of the E2 gene and production of the E2 protein from
MVA E2. (A) Amplification of E2 RNA. Cellular RNA from BS-C-1 cells infected with
either with MVA (lanes 1–3) or MVA E2 (lanes 4 – 6) was purified 24 hours after
infection. The E2 RNA was amplified by RT-PCR. The quantities, in ␮L, of the PCR
product are indicated above each lane. The band representing the E2 gene
transcript is marked by an arrow. M represents DNA size markers in bp. (B) E2
RNA accumulation. Cellular RNA from BS-C-1 cells was isolated at different times
postinfection and processed as described in (A). Three ␮L of the PCR product
were analyzed at 24, 48, and 72 hours postinfection (hpi) on an agarose gel. (C)
Immunoprecipitation analysis of cytoplasmic extracts from BS-C-1 cells infected
with MVA or MVA E2. At 6 hours after infection, cells were labeled with
[35S]-methionine for 60 or 120 minutes. Cell lysates were prepared and 50 ␮L
were immunoprecipitated with an anti-E2 polyclonal antibody. Immunoprecipitates were resolved by SDS-PAGE and autoradiographed. Numbers on the left
indicate the position and molecular mass in kilodaltons (kDa) of protein standards. The band representing the E2 protein (48 kDa) is marked by an arrowhead.
1656
CANCER April 1, 2000 / Volume 88 / Number 7
FIGURE 3. Repression of human papillomavirus P105 promoter by MVA E2 recombinant virus. (A) RNA from MVA- or MVA E2–infected 3T3-F4 cells was used
to amplify the internal fragment of the ␤-galactosidase gene product (arrow) by RT-PCR. Quantities, in ␮L, of the PCR product are indicated above each lane. (B).
␤-galactosidase activity. 3T3 F4 cells were infected for 12 hours with different amounts (1, 5, or 10 pfu) of MVA or MVA E2 recombinant virus. Cytoplasmic extracts
were prepared, and ␤-galactosidase activity was determined. Maximum activity (100%) is the activity of ␤-galactosidase from noninfected cells. Results are mean ⫾
standard error of two experiments done in triplicate.
MVA E2 Recombinant Virus Reduced Human Tumor
Growth in Nude Mice
Because the E2 protein can repress the expression of
E6 and E7 oncogenes and also promote apoptosis in
some cancer cells,37 we decided to evaluate the effect
of the MVA E2 recombinant virus on human tumors
grown in nude mice. Groups of 50 tumor-bearing
nude mice were injected with MVA or MVA E2 virus
using 5 ⫻ 107 infectious units per injection, or just
with PBS directly into the tumor. This dose of virus has
been shown to be adequate for good infection and
stimulation of the immune system in normal animals.26,35 Mice were treated once a week for 3 weeks.
A significant reduction in tumor size was observed in
MVA E2–treated animals (Fig. 4). In contrast, mice
injected with MVA showed only a very slight reduction
in tumor growth. No untreated or PBS-treated animals
showed any reduction in tumor size (Fig. 4). When
tumors in control mice reached about 4 –5 cm2 in size,
most animals needed to be sacrificed. These results
clearly showed that the MVA E2 virus is capable of
stopping human tumor growth.
In order to obtain additional information about
the specific effects of MVA and MVA E2 recombinant
viruses on human tumor growth in nude mice, we
decided to use a logistic differential equation. This
type of mathematical model has been shown to be
useful for describing tumor growth in the absence of
an immune response.33 Fitting the solution of the
logistic model to our experimental data resulted in a
remarkably good match (continuous lines, Fig. 4). This
means that the treatments with MVA or MVA E2 did
not affect the underlying mechanisms of tumor
growth, but rather reduced the maximum size of the
tumor. The intrinsic rate of tumor growth was slightly
reduced when MVA (r ⫽ 0.14) or MVA E2 ( r ⫽ 0.13)
were used in comparison with the value obtained for
PBS (r ⫽ 0.16). However, the maximum size of tumor
in MVA E2–treated animals was much smaller than in
MVA- or PBS-treated mice. These results taken together indicated that the MVA E2 recombinant virus
and, most likely, the E2 protein are the main factors
determining the reduction of the maximum size of the
tumor.
Papilloma E2 Gene in Tumor Therapy/Valadez et al.
1657
creasing the life expectancy of tumor-bearing animals
almost fourfold (Fig. 6). These data indicated that MVA
E2 treatment was indeed the responsible factor for
tumor arrest and that it also helped the animals to live
longer.
DISCUSSION
FIGURE 4. Growth rate analysis of HeLa cell tumors in nude mice after MVA
or MVA E2 treatment. Human tumors grown in nude mice were injected with
MVA, MVA E2, or just phosphate-buffered saline (PBS). Average (from all
treated animals) tumor size per week is shown (symbols). Equations describing
tumor growth for each treatment are as follows: G ⫽ 3.57/(1 ⫹
exp[(3.0624) ⫺ (0.163624)*t]), where k ⫽ 3.57, r ⫽ 0.163624, Go ⫽ 0.2 for
PBS; G ⫽ 2.72/(1 ⫹ exp[(2.27365) ⫺ (0.145432)*t]), where k ⫽ 2.72, r ⫽
0.145432, Go ⫽ 0.2 for MVA; G ⫽ 1.3/(1 ⫹ exp[(2.35396) ⫺ (0.135547)*t]),
where k ⫽ 1.3, r ⫽ 0.135547, Go ⫽ 0.2 for MVA E2. In each case, t ⫽ time
(days), Go ⫽ initial tumor size (cm2), and r ⫽ intrinsic growth rate (cm2/day).
Equations are fitted to data of tumor growth for each treatment. Results of a
nonlinear least-square estimation of the parameters following the procedure of
Maquardt are also shown (continuous lines). Arrows indicate times when virus
injections were made.
MVA E2 Infection Induces Apoptosis in Tumor Cells
In Vivo
Human HeLa tumors in nude mice were infected with
MVA or MVA E2 recombinant virus. After 24 hours of
infection, tumors were excised and processed for detection of apoptotic bodies. MVA E2 induced the formation of clusters of apoptotic cells in various parts of
the tumor. Apoptotic cells were indicated by the presence of high fluorescent nuclei (Fig. 5B and C). In
these regions of the tumor, nuclei appeared to be
surrounded by unstained cytoplasm, and none of the
peripheral cells were stained. These results clearly indicated that apoptotic bodies were present in discrete
tumor regions where the E2 protein was very likely
present. In uninfected or MVA-infected tumors, very
few, if any, apoptotic bodies were detected (Fig. 5).
These results clearly showed that infection with MVA
E2 induced tumor death not by simple necrosis, but by
inducing (most likely through the E2 protein) programmed cell death.
The MVA E2 Recombinant Virus Increased the Life
Expectancy of Mice Bearing Human Tumors
It was observed that mice treated with MVA E2 recombinant virus survived longer than MVA- or PBS-treated
mice. This indicated that MVA E2 is capable of in-
A new recombinant virus carrying the papilloma E2
gene (MVA E2) was constructed and its therapeutic
properties for human tumors analyzed. In this study,
we found that expression of the E2 protein in human
cancer cells through MVA E2 infection was able to stop
tumor growth.
Cervical carcinoma is a very serious health problem affecting thousands of women all over the world.
Papillomaviruses are the main infectious agents that
cause this disease. The papillomavirus proteins E6 and
E7 are the molecules responsible for cell transformation.38 These proteins achieve their effects by interacting with two cellular proteins. E6 binds to the protein
p53 and promotes its degradation. p53 is a tumor
suppressor gene that plays a central role in controlling
the cell cycle. So, when E6 binds to p53, the papilloma
protein eliminates the tumor suppressor activity of
p53. Similarly, E7 binds to the retinoblastoma (Rb)
protein, provoking its inactivation. The Rb gene product forms a complex with the transcription factor E2F,
which is required for the transcription of cellular
genes that drive the cell into the S-phase of the cell
cycle. Inactivation of Rb results in the release of the
E2F transcription and, in turn, stimulation of cellular
DNA replication.
Expression of the viral E6 and E7 oncoproteins is
negatively regulated by the binding of the E2 gene
product to their promoters. This has been clearly
shown for the P105 promoter of HPV type 18 in vivo
and in vitro.16,39 – 41 When the viral DNA gets integrated into the cell genome, the E2 gene is disrupted
or inactivated. The lack of E2 protein then results in
activation of transcription of the E6 and E7
genes.16,28,39,41– 43 This event probably represents the
most critical step in progression to invasive carcinoma.9,16,28 Moreover, the E2 protein has been reported
to be the main factor promoting cell growth arrest and
apoptosis in some cancer cells.27,37 Based on this information, we reasoned that it could be possible to
suppress tumor growth if the E2 protein could be
introduced into tumor cells.
Because vaccinia viruses are excellent vehicles for
introduction of foreign genes into cells, we used the
highly attenuated vaccinia virus strain MVA to deliver
the E2 protein to tumor cells. We also decided to use
the MVA vaccinia virus as a recombinant vector because it is very efficient in expressing many foreign
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CANCER April 1, 2000 / Volume 88 / Number 7
FIGURE 5. MVA E2–induced apoptosis
in tumor cells. Nude mice bearing tumors were injected with MVA or MVA E2
virus directly into the tumor. Twentyfour hours later, infected tumors were
isolated and processed for detection of
apoptosis. (A) MVA-treated tumor. (B)
MVA E2–treated tumor. (C) MVA E2–
treated tumor at 10-fold higher magnification. (*) indicates large clusters of
apoptotic cells with fluorescent nucleus.
Different stages of programmed cell
death are shown: 1) nucleus with perinuclear chromatin; 2) pignotic nuclei,
and 3) apoptotic bodies. Scale bar is 50
␮m.
genes, leading to the production of large quantities of
protein in infected cells.23,24 In addition, MVA vaccinia
virus is a safe virus because it possesses a high degree
of attenuation due to its loss of 30,000 base pairs of
genetic material, and because its use in humans was
extensively documented during the worldwide eradication of smallpox.19 –21,36 MVA has also a great vaccine potential due to its capacity for stimulating the
immune system.26 In the particular case of papillomavirus tumors, it has already been reported that recombinant vaccinia viruses expressing the E6 and E7 oncoproteins are able to protect 70 – 80% of the
immunized rats from a challenge with tumor cells.
These animals did not present tumor development
after virus treatment.44
In the current study, infection of cells with our
MVA E2 recombinant virus resulted in very good expression of the protein E2 inside these cells, as demonstrated by the presence of both the mRNA and the
actual protein (Fig. 2). Considering that one of the
major functions of E2 is the repression of transcription
from the P105 promoter, it was essential to confirm
that E2 was really functioning inside cells. Repression
of the promoter would result in lower levels of the
protein E7. Direct measurements of this protein are,
however, very difficult to perform. It has been esti-
Papilloma E2 Gene in Tumor Therapy/Valadez et al.
1659
FIGURE 5. (Continued)
mated that in HeLa cells, E7 has a very short life—just
13.5 minutes.45 So detecting changes in synthesis, catabolism, or immunoreactivity of E7, even in response
to external stimuli, turns out to be very hard. Although
an estimation of E7 can be obtained when determinations are made in tissue culture cells, where it is possible to control the number of cells used in each experiment, analyzing changes in the E7 protein
concentration inside the tumor is even more difficult
because it is complicated to determine the number of
cells in the tumor mass. To avoid these difficulties, we
looked at the level of transcription of a reporter gene
expressed by the P105 papillomavirus promoter. Previous work from our group has shown that the E2
protein of bovine papillomavirus is able to repress
different promoters, including the P105 promoter, of
genital and cutaneous HPV by directly binding to HPV
promoters.14
␤-galactosidase gene is under control of the P105
promoter in 3T3 F4 cells. When these cells were infected with MVA E2, a dose-dependent inhibition of
transcription from the P105 promoter was observed
(Fig. 3). These results confirmed that E2 is correctly
expressed in infected cells and that it is a functional
protein with the capacity of repressing transcription
from the P105 promoter. These results are in agreement with previous reports showing that it is possible
to repress 100% of the transcriptional activity of different papillomavirus promoters by expressing the E2
protein of bovine papillomavirus inside cells.16,40,41
Because MVA E2 was successfully inducing the
production of E2 protein in cells, we decided to infect
tumor cells that were growing in an animal, to see
whether the presence of the E2 protein in them could
inhibit tumor formation. Nude mice, which have a
deficient immune system, were inoculated subcutaneously with 2 ⫻ 106 HeLa cells in 200 ␮L of PBS. Two or
3 weeks later, when tumors had grown to about 0.2
cm2, the animals’ tumors were injected directly with
5 ⫻ 107 pfu of MVA or MVA E2. This dose was chosen
because it provides a good response in immunocompetent animals26,35 and because it is the amount normally used in humans.46,47 We also wanted to evaluate
whether this amount of virus would induce negative
side effects in the animals. Infection of human cancer
cells with MVA E2 dramatically inhibited tumor
growth in nude mice (Fig. 4). In contrast, animals
treated with the parental MVA virus or just PBS had
tumors that continued growing. The only difference
between MVA and MVA E2 is the presence of the E2
gene and therefore the expression of this protein in
infected cells. Our observations in nude mice support
the hypothesis that by introducing the E2 protein into
cancer cells, the transcription of the E6 and E7 oncogenes will stop. Nude mice may not reflect what will
be observed with tumors in a human system. However, we wanted to investigate the direct effects of
MVA E2 on tumors, without the participation of a
complete immune system, to understand better the
direct actions of MVA E2 on cancer cells within a
tumor. Currently, experiments are underway to assess
whether the efficacy of MVA E2 against tumors in
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CANCER April 1, 2000 / Volume 88 / Number 7
FIGURE 6. MVA E2 increased the survival of tumor-bearing mice. Nude mice
bearing human tumors of different sizes
were inoculated with phosphate-buffered saline (PBS), control MVA virus, or
MVA E2 virus directly into the tumor,
with 5 ⫻ 107 infectious units per injection once a week for 3 weeks. The
number of living animals in each group
was determined each week for up to 4
months. Inoculation with MVA E2 (open
squares) significantly prolonged the survival of mice compared with MVA or PBS
treatment (solid symbols). This experiment
was done with 50 animals for each treatment using different virus preparations.
nude mice is maintained in immunocompetent animals.
The efficient way in which MVA E2 infection was
able to reduce tumor growth in mice (Fig. 4) suggested
that the E2 protein, produced inside infected cells,
could also get inside the surrounding cells and then
stop the transcription of the E6 and E7 genes, and
maybe also promote apoptosis in these cells as well.
Ninety days after the MVA E2 recombinant vaccinia
virus treatment was finished, mice again showed tumor growth. It is likely that the E2 protein that appears
inside tumor cells is capable of not only repressing
transcription from the P105 promoter inside tumor
cells, but also inducing apoptosis of all E2-expressing
cells. However, tumor cells that did not acquire the E2
protein would not be eliminated, and they could continue dividing until new tumors appeared again.
Macroscopic observation of tumor lesions indicated that tumor necrosis was present in most animals
treated with the MVA E2 virus, whereas in MVAtreated animals only a small spot of necrosis was visible after 3 weeks of treatment. We reasoned that
dying cells in MVA E2–treated tumors were dying due
to apoptosis triggered by the E2 protein. When tumor
sections were analyzed for immunohistochemical detection of DNA strand breaks, a large number of apoptotic cells were observed in MVA E2–infected tumors.
Very few apoptotic bodies were seen in control tumors
(Fig. 5). These observations also supported the idea
that the MVA E2 recombinant virus is capable of stopping tumor growth by inducing apoptosis, most likely
through expression of the E2 protein.
Mathematical analyses of our results with the formulas describing tumor growth for each treatment
(Fig. 4) suggested that it may be possible not only to
reduce the maximum size of the tumor, but also, given
the logistic pattern of growth, to reduce tumor growth
significantly if there is sufficient E2 protein inside all
tumor cells. This conclusion is in good agreement with
the fact that the E2 protein can induce suppression of
growth and cell cycle arrest leading to apoptosis.15,27,37
In conclusion, it is possible to stop tumor growth by
introducing the E2 protein inside all cancer cells. It is
also probable that a complete tumor disappearance
could be achieved if the E2 protein were present inside
every cancer cell. Tumor growth inhibition by MVA E2
treatment also resulted in a significant increase in life
expectancy for these animals (Fig. 6). Mice survived
three or four times longer than animals treated with
MVA or PBS. This means that although our recombinant virus did not completely eliminate all tumors in
mice, it can efficiently reduce the tumor burden in
these animals and therefore be a very good candidate
for a new therapeutic agent.
Other experiments, similar to ours, underline the
protective properties of other MVA recombinant viruses as tools for anticancer therapy. One of these
viruses, which carries the hemagglutinin (HA) and nucleoprotein (NP) genes of the influenza virus, was able
to protect immunized mice against a lethal influenza
virus challenge.26 Another virus, a trivalent simian immunodeficiency virus (SIV)–recombinant MVA virus
carrying the gap, pol, and env genes, appeared to
affect the extent and pattern of SIV replication following challenge.35 Other types of viruses, such bovine
enteroviruses, have been shown to prolong the lifespans of mice bearing human carcinoma cells.22,48
Related experiments have been performed with recombinant vaccinia virus vectors using the wild-type
virus expressing the large-T (LT), middle-T (MT), and
small-T (ST) antigens of polyoma virus. Immunization
of animals with these recombinant viruses could prevent the proliferation of cognate tumors.22,48 The use
of BPV-1 proteins cloned into vaccinia virus vectors
Papilloma E2 Gene in Tumor Therapy/Valadez et al.
also resulted in reduced proliferation of tumors.49 By
using other types of viruses, such as bovine enterovirus, as immunizing agents, it was possible to prolong
the life-spans of rabbits in which a T cell like leukaemia was induced by injecting F-647a cells (F-647 is an
HTLV-1 transformed T-cell line).22
The E2 protein may have other antitumor properties besides its direct effects on malignant cells, as
suggested by recent reports in which immunization of
rabbits with E1 and E2 proteins (of cottontail rabbit
papillomavirus, or CRPV) could prevent the formation
of a new focus of papillomas. This treatment also
produced strong regression of preexisting papillomas.50 Taken together, these observations strongly
support the idea that by using the E2 protein it is
possible to induce papilloma tumor regression. Therefore, the usefulness of our new recombinant virus for
the treatment of cervical carcinoma is evident.
These reports, all taken together, show that viruses are effective tools for inducing expression of
particular proteins in cells and that they are capable of
inducing, in this manner, a protective response in the
treated animals. The current report shows that the
introduction of the E2 gene product into human cancer cells by means of our recombinant vaccinia virus
(MVA E2) is able to stop human tumor growth. MVA
E2 virus was also able to prolong the life expectancy of
animals harboring tumors without showing any side
effects. The MVA E2 recombinant virus is in fact a very
good therapeutic agent for human papilloma tumors
present in immunosuppressed animals. Our results
suggest, then, that MVA E2 virus is safe biologic agent
and that it is possible to use this new recombinant
vaccinia virus for the treatment of cervical carcinoma.
Experiments are now in progress to explore further the
role of the E2 protein in the arrest of different types of
tumors and also in immunocompetent animals.
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