(Circulation. 1997;96:1899-1905.)
© 1997 American Heart Association, Inc.
Articles |
Adenoviral Constructs Encoding Phosphorylation-Competent Full-length and Truncated Forms of the Human Retinoblastoma Protein Inhibit Myocyte Proliferation and Neointima Formation
From the Division of Cardiovascular Research, Department of Medicine, Tufts University School of Medicine, St. Elizabeth's Medical Center (R.C.S., H.P., L.N.T., K.K., K.W.), Boston, Mass; CANJI, Inc (K.N.W., D.A.), San Diego, Calif; and the Program in Cell, Molecular and Developmental Biology, Sackler School of Biomedical Sciences, Tufts University (H.P., K.W.), Boston, Mass.
Correspondence to Kenneth Walsh, Division of Cardiovascular Research, St Elizabeth's Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail kwalsh{at}opal.tufts.edu
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Abstract |
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Background The retinoblastoma (Rb) protein is a key cell-cycle regulator that controls entry into the S phase by modulating the activity of the E2F transcription factor. We analyzed the effects of full-length phosphorylation-competent and a mutant truncated form of human Rb for their effects on vascular smooth muscle cell (VSMC) proliferation and neointima formation.
Methods and Results A number of mutant forms, both phosphorylation competent and incompetent, of human Rb protein were evaluated for their ability to inhibit E2F activity. The results of these assays indicated that a phosphorylation competent, amino-terminal–truncated Rb protein (Rb56) was a particularly potent inhibitor of E2F-mediated transcription relative to the full-length Rb construct (Rb110). Adenoviral constructs containing Rb56 or Rb110 expressed their respective Rb forms in VSMCs, as determined by Western immunoblot analysis, and were similar in their abilities to arrest the cell cycle, as determined by assays of 3H-thymidine incorporation and by flow cytometric analyses. When examined for their effect on neointima formation after balloon injury of the rat carotid artery, both full-length and truncated forms of Rb inhibited formation of this VSMC-derived lesion.
Conclusions These analyses revealed that the maintenance of high levels of phosphorylation-competent human Rb, either full-length or truncated forms, in VSMCs is an effective method of modulating the extent of intimal hyperplasia that occurs after balloon-induced vascular injury.
Key Words: angioplasty • genes • carotid arteries • muscle, smooth • stenosis
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Introduction |
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The Rb protein is a key modulator of cell-cycle transit because of its ability to control the G1/S-phase transition (for a review, see Goodrich and Lee1 ). This activity appears to be dependent on the ability of Rb to modulate the activity of the E2F family of transcriptional regulators that are required for S-phase progression.2 In the presence of proliferative signals, the Rb protein is phosphorylated at multiple sites by cyclin/cdk complexes to generate hyperphosphorylated Rb, which is unable to repress E2F-dependent transcription.3 4 Rb kinase activity is regulated by a number of cyclin-dependent kinase inhibitors that act to prevent Rb hyperphosphorylation and thereby maintain cells in the G1 phase.5 The proper regulation of Rb is required for the establishment of the postmitotic state in differentiated tissues, and the lack of Rb function can result in tumorigenesis. In myocytes that terminally differentiate, such as skeletal muscle, Rb is maintained in a hypophosphorylated state as a result of high levels of cyclin kinase inhibitor expression.6 7 8 Homozygous deletion of the Rb gene occurs in a number of neoplastic diseases, indicating that the regulation of Rb activity is a key checkpoint preventing uncontrolled proliferation. Regulation of Rb phosphorylation may also be a source of malignant progression, and the loss of cdk inhibitors p16 and/or p15 has been shown to occur in a number of malignancies.9 10 11 12 13
Recently, Chang et al14 showed that a nonphosphorylatable, constitutively active form of murine Rb inhibited VSMC proliferation and reduced neointima formation in the injured rat carotid artery and porcine femoral artery models, suggesting that similar regulatory networks may underlie carcinogenesis and proliferative vessel-wall disorders. In the study of Chang et al,14 the effects of wild-type Rb were not analyzed. In the present study, we evaluate the ability of human Rb, both the phosphorylation-competent full-length form and a truncated form, to inhibit E2F transcriptional activity, VSMC proliferation, and neointima formation in the rat balloon-injury model. The results presented herein suggest that adenovirus-mediated Rb inhibition of VSMC proliferation is, to a large extent, dependent on the quantity of exogenous protein produced.
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Methods |
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Cell Culture
Rat VSMCs were prepared according to the method of Mader,15 and human VSMCs were cultured by explant outgrowth from unused portions of saphenous veins from coronary bypass surgery (St Elizabeth's Medical Center, Boston, Mass) according to the method described for internal mammary arteries by Pickering et al.16 Cultures were used at or below 10 passages and were grown in DMEM containing 15% FBS (GIBCO BRL). Quiescence was induced by incubation for 72 hours in low-mitogen (0.5% FBS) medium.
Construction of Rb and E2F Expression Plasmids
A partial cDNA (amino acids 95 through 437) of the
E2F gene was obtained from pGEX2T-E2F-XH917 via
PCR amplification. The amplified fragment was digested with
HindIII and subcloned into pCMV to generate pCMV-E2F. The
full-length Rb110 cDNA was obtained from pA9-RB18 by
digestion with Sac I/HindIII and subcloned into
pCMV to generate pCMV-RB110. The RB110-H209 mutant contains the change
TGT (Cys) to GCA (Phe) at position 706 as described
previously.19 The serine-to-alanine mutations in Rb110
788/807/811 were generated by subcloning an EcoRI fragment
of the cDNA into M13mp18 and performing site-directed mutagenesis as
described previously.20 Codon changes used were AGC to GCC
(788), TCA to GCA (807), and AGT to GCT (811). The Rb56 construct was
generated by PCR amplification of the pA9-RB plasmid with the use of a
5 primer (GTCGACATGAACACTATC) containing a Sal I
restriction site and start codon and a 3 primer containing a
HindIII restriction site. The resulting PCR product was
subcloned into pCMV to obtain pCMV-RB56. The H209 and 788/807/811 Rb56
constructs were generated from the corresponding Rb110 constructs.
Construction of Adenovirus Vectors and Their Amplification in
Culture
The recombinant adenoviruses used in the present studies are
based on serotype 5 human adenovirus from which the viral early region
1–encoding E1a, E1b, and pIX proteins have been deleted. This
adenovirus is limited to propagation in 293 cells that produce the Ad5
E1 gene products required for replication. Transfer plasmids
encoding either full-length or truncated Rb were generated from
pACN21 and were in turn used to construct the recombinant
adenoviruses. Either a full-length Rb cDNA (amnio acids 1 through 928),
subcloned as a 2.8-kilobase Xba I, BamHI fragment
from the plasmid pETRbc,22 or a truncated version of Rb
(amino acids 381 through 928), subcloned as a 1.7-kilobase
Xba I, BamHI cDNA fragment, was placed downstream
from the CMV promoter/enhancer and the Ad2 tripartite leader cDNA of
the plasmid pACN. These plasmids were subsequently linearized with
EcoRI and cotransfected (CaPO4; Stratagene) with
either the isolated Cla I–digested large fragment of
H5ilE423 to make Ad-Rb56 (ACN56) containing a partial E4
deletion or with the large fragment from a hybrid virus of
dl32724 and H5ilE4 to create Ad-Rb110 (ACNRb), which
contains deletions in both the E3 and E4 regions of the vector. The
control virus, Ad-ß-Gal (ACBGL), contains the bacterial
ß-galactosidase gene downstream from the CMV
promoter/enhancer.25 Isolation and initial purification of
the viruses were performed by standard methods,26 and
purified virus was prepared according to the method of Huyghe et
al.27
Measurement of the Effects of Rb Variants on an E2F Expression
System in Transiently Transfected Cells
A10 cells were plated at 50% confluence on 10-cm dishes in
growth medium. Transfections used a total of 5 µg of DNA with 90 µg
of DOTAP (Boehringer Mannheim) in Opti-MEM. The DNA-lipid mixture was
incubated for 20 minutes at room temperature in serum-free Opti-MEM,
after which cells were transfected for 5 hours at 37°C. After
transfection, growth medium was added and the cells were incubated for
an additional 48 hours before being harvested.
Cells were cotransfected with 2.4 µg of the test plasmid and 2 µg of an E2F-luciferase reporter plasmid, (E2F)x4-E1bTATA-Luc, containing the PvuII/Sac I fragment from the promoter region of the (E2F)x4-E1bTATA-CAT plasmid28 29 cloned into the Sma I/Sac I site of the pGL2-Basic plasmid (Promega) and positioned to drive the expression of the luciferase gene. Included in the transfection mixture was 0.1 µg of a plasmid (pE2F) containing the E2F1 structural gene under control of the CMV promoter/enhancer, which was necessary to provide expression of sufficient E2F protein to drive the assay. The transfection mixture also contained 0.6 µg of pSV2-AP30 containing the bacterial alkaline phosphatase gene under control of the SV40 promoter/enhancer as an internal control for transfection frequency. The Rb expression plasmids have the various Rb gene constructs downstream from the CMV promoter/enhancer.
Alkaline phosphatase activity of transfected cells was measured with the use of CSPD chemiluminescent substrate (Tropix), and luciferase activity was measured with the Luciferase Assay System from Promega. Activity was reported as relative light units based on the ratio of luciferase to alkaline phosphatase activity.
Measurement of the Effects of Adenovirus Infection on Cellular
Proliferation by the 3H-Thymidine–Incorporation
Assay
Quiescent cultures were infected in low-mitogen medium (0.5%
FBS) for 24 hours. After infection, the cultures were transferred to
growth medium and incubated for 14 hours (24 hours for human VSMCs)
to allow entry into the S phase. Cell proliferation was determined by
thymidine incorporation in medium containing 3 µCi/mL
[methyl-3H]-thymidine (6.7 Ci/mmol, NET-027; DuPont NEN)
for 4 hours (12 hours for human VSMCs). Incorporated label was
determined as acid-precipitable material (10% trichloroacetic acid) by
liquid scintillation counting in Scintiverse II (Fisher) with a Beckman
LS 5000TD scintillation counter. All determinations were done in
triplicate.
Flow Cytometric Analysis of Cell-Cycle Progression in
Adenovirus-Infected Cultures
Quiescent rat primary VSMCs were infected for 12 hours at 750
MOI (IU/cell) in low-mitogen medium. After infection, the adenovirus
solution was removed, and the cultures were incubated for 12 hours in
low-mitogen medium before being transferred to growth medium (10% FBS)
for 14 hours to stimulate cell-cycle progression. The cultures were
harvested by trypsinization and fixed in 70% ethanol for 30 minutes at
-20°C. The cells were then stained for DNA content (PBS, 0.1%
Triton X-100, 0.1 mmol/L EDTA, 0.05 µg/mL RNase A,
and 50 µg/mL propidium iodide), and the cell-cycle profile was
determined by flow cytometric analysis with the use of a Becton
Dickinson Vantage flow cytometer and Lysis II cell-cycle
analysis software. Flow cytometry was conducted at the Core
Flow Cytometry Facility of the Dana-Farber Cancer Institute, Boston,
Mass.
Immunoblot Analysis of Rb Expression
in Adenovirus-Infected Cultures
Quiescent primary human VSMC cultures were infected with virus
for 24 hours in low-mitogen medium. After infection, the cultures were
transferred to growth medium (15% FBS) for 24 hours. Whole-cell
extracts were prepared from these cultures,31 and protein
concentrations were determined from the optical densities at 280 and
260 nm. Ten micrograms of each extract was analyzed by SDS-PAGE
on a 7.5% polyacrylamide gel under reducing conditions
(ß-mercaptoethanol). Proteins were transferred by the semidry method
to a polyvinylidene fluoride membrane (Immobilon-P, Millipore) and the
membrane was probed with an anti-human Rb mouse monoclonal antibody
(3C8; 1.7 µg/mL)32 in 2% milk/TBS-T (10
mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, and 0.05%
Tween-20). The secondary antibody (horseradish-peroxidase–conjugated
anti-mouse; Amersham) was used at 1:4000 dilution, and complexes were
visualized with Enhanced Chemiluminescence (ECL) reagent (Amersham).
Exposures were converted to a digital image by use of an Eagle Eye II
video imaging system with Eagle Sight software (version 2.0;
Stratagene), and band intensities were determined by use of One-Dscan
software (version 1.0; Scanalytics).
Measurement of Adenovirus Effects on
Balloon-Catheter–Induced Injury in the Rat Carotid Artery Model
of Restenosis
This model of balloon injury was based on that described by
Clowes et al.33 34 Briefly, the left common carotid artery
of a male Sprague-Dawley rat weighing 400 to 500 g was subjected
to a distending, deendothelializing injury by abrasion
with an inflated 2F embolectomy catheter inserted via the external
carotid artery. The injured segment of the artery was then incubated
with adenovirus (1x109 IU) diluted to 100 µL with 15%
(wt/vol) Poloxamer 407 (BASF)35 for 20 minutes, after
which the viral infusion was withdrawn and the external carotid artery
was ligated. This experimental protocol was approved by the
Institutional Animal Care and Use Committee and complied with the
Guide for the Care and Use of Laboratory Animals (NIH
publication No. 86-23, revised 1985).
Rats were killed 14 days after treatment. The injured segment of the left common carotid artery was dissected free from the surrounding tissue and fixed in 100% methanol until embedded in paraffin. Several 4-µm sections were cut from each specimen. Sections were stained with either hematoxylin and eosin or Richardson's combination elastic-trichrome stain for conventional light microscopic analysis.
Histological images of cross sections were projected onto a digitizing board (Summagraphics), and the intimal, medial, and luminal areas were measured by quantitative morphometric analysis with a computerized sketching program (Macmeasure version 1.9; National Institute of Mental Health).
Immunohistochemical Analysis of Rb Expression in
Adenovirus-Infected Tissue
Adenovirus-treated carotid arteries were harvested from rats 2
days after balloon injury and infection. Tissue was fixed in
phosphate-buffered formalin and embedded in paraffin. Cross sections
(4 µm) were cut and dewaxed through xylene and graded alcohols.
Endogenous peroxidase was quenched with 1% hydrogen
peroxide for 30 minutes. Antigen retrieval was performed in 10
mmol/L sodium citrate buffer, pH 6.0, at 95°C for 10 minutes.
A monoclonal anti-Rb antibody (AB-5; Oncogene Sciences) was applied (10
µg/mL) at 4°C for 24 hours. Secondary antibody was applied
from the Unitect Mouse Immunohistochemistry Kit (Oncogene Sciences)
according to the manufacturer's instructions. Antibody complexes were
visualized by use of 3,3-diaminobenzidine (DAB; Vector
Laboratories), and slides were counterstained with hematoxylin.
Statistical Analysis
Differences between groups were analyzed by use of
either an unpaired two-tailed Student's t test or
single-factor ANOVA. Statistical significance was assumed when the
probability of a type I error was <.05. Statistical tests were
conducted with the use of Statview software from Abacus Concepts, Inc
(Macintosh version 4.1).
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Results |
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Evaluation of Full-length and Mutant Forms of Human Rb as Inhibitors of E2F-Mediated Transcription
To study the effects of the Rb protein on proliferation, a number of mutant forms of Rb were generated by either deletion or site-directed mutagenesis. Fig 1A
describes the structure of Rb and indicates regions that were deleted
and/or mutated. The ability of the various mutants to inhibit E2F
transcriptional activity was measured in an E2F reporter assay. This
assay uses a luciferase reporter construct containing four tandem
E2F-DNA regulatory elements positioned to support transcription of the
luciferase gene. As can be seen in Fig 1B, cotransfection of an E2F
expression plasmid, pE2F, produces a 115-fold increase in relative
luciferase activity compared with the E2F-luciferase construct alone,
and the full-length 110-kD Rb protein decreased this activity by 50%
in the fetal rat VSMC line A10. When the cysteine at position 706 of
the Rb110 protein is converted to a phenylalanine (H209), Rb-binding
proteins are no longer able to associate with Rb.19 In the
E2F reporter assay, this form of Rb does not produce a statistically
significant decrease in luciferase activity, suggesting that
E2F-binding activity is essential for repression. In contrast,
site-directed mutagenesis from serine to alanine residues at
phosphorylation site positions 788, 807, and 811 of the
full-length Rb construct (Rb110 788/807/811) reduced relative E2F
transcriptional activity by 71%. Although this represents a
significant repression of E2F transcriptional activity, the greatest
inhibitory effect was observed with an amino-truncated Rb
(Rb56) protein. As can be seen in Fig 1B, Rb56 produced a 98%
reduction of luciferase activity in the E2F reporter assay. The greater
transcriptional repression by Rb56 was statistically significant
compared with the repression observed with Rb110, as determined by
ANOVA analysis (P<.05). The differential effects of
Rb56 and Rb110 on E2F-mediated transcription do not appear to result
from differences in protein expression levels from the Rb effector
plasmids, as determined by Western blot analysis with an
anti-Rb antibody (D.A., PhD, unpublished data, 1996). The specificity
of Rb56 inhibition was demonstrated by inclusion of the H209 mutation,
which does not significantly inhibit luciferase activity. Conversion of
the phosphorylation sites at amino acids 788, 807, and
811 in Rb56 did not further reduce luciferase activity (Fig 1B and
other data not shown).
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Inhibition of VSMC Proliferation by Adenoviral Constructs of
Full-length or a Truncated Form of Human Rb
To compare the efficacy of Rb110 and Rb56 proteins as
inhibitors of proliferation, we constructed
replication-deficient adenoviral vectors expressing these proteins
under control of the CMV promoter. When these vectors were tested for
the ability to inhibit the mitogen-stimulated proliferation of rat and
human VSMCs (Fig 2), as measured by
3H-thymidine incorporation, we observed a potent inhibition
with both the full-length Rb110 (Ad-Rb110) and the truncated Rb56
(Ad-Rb56) viruses at MOIs >100. The dose-response curves were similar
for both forms of Rb in human and rat VSMCs. Ad-ß-Gal did not inhibit
rat or human VSMC proliferation to a significant extent, as can also be
seen in Fig 2.
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Flow Cytometric Analysis of Cell-Cycle Progression in
Transduced Rat Primary VSMCs
The ability of the adenoviral constructs to inhibit rat aorta VSMC
proliferation is reflected by their ability to induce cell-cycle
arrest, as shown in the Table. The
quiescent mock-infected culture displayed 70.0% of cells in the
G0/G1 phase of the cell cycle.
Proliferating mock-infected and Ad-ß-Gal–infected cultures had a
similar number of cells in the G0/G1
phase (43.9% and 45.9%, respectively). However, when the cells were
infected with the adenovirus encoding the full-length Rb (Ad-Rb110),
70.5% of the culture was in the G0/G1
phase, and in cultures infected with amino-truncated Rb (Ad-Rb56),
76.7% of the culture was in G0/G1.
Cultures transduced with either Rb vector displayed correspondingly
lower proportions of cells in the S and G2/M phases
compared with mock- or Ad-ß-Gal–infected cells. These data further
demonstrate that both the wild-type Rb110 and the amino-truncated Rb56
are similar in their cell-cycle regulatory properties in VSMCs.
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Western Immunoblot Analysis of Human Rb
Expression by Adenovirus-Infected Cells
Ad-Rb56 and Ad-Rb110 expressed their respective proteins as
indicated by Western immunoblot analysis of
whole-cell extracts from infected human VSMCs, as shown in Fig 3. Infection at MOIs >100 with either
Ad-Rb110 or Ad-Rb56 resulted in substantial overexpression of the
recombinant Rb proteins relative to endogenous Rb. The
hypophosphorylated and
hyperphosphorylated forms of endogenous Rb
could be resolved, and the slower-migrating
hyperphosphorylated form was clearly present in the
uninfected proliferating culture but was absent in the quiescent
(serum-starved) culture. Infection with the Ad-ß-Gal control virus
appeared to decrease the extent of endogenous Rb
phosphorylation at an MOI >100. This can be correlated
with a slight inhibition of human VSMC proliferation at these MOIs (Fig 2
). In Ad-Rb56–infected cultures, both the
hyperphosphorylated and
hypophosphorylated forms of endogenous Rb
decreased as Rb56 expression increased. In Ad-Rb110–infected
cultures, we also observed a marked decrease in
hyperphosphorylated Rb expression with increasing
levels of virally encoded Rb expression. These data demonstrate that
the replication-deficient adenoviral constructs can substantially
overexpress the full-length and truncated forms of Rb relative to the
expression of endogenous Rb in VSMCs. Furthermore, these
data reveal that the level of Rb phosphorylation is
sensitive to overall levels of recombinant Rb expression, either
full-length or truncated, and can also be affected to a lesser extent
by transduction with control virus.
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Effect of Human Rb Adenoviral Constructs on Neointima
Formation in the Rat Carotid Artery Model of Restenosis
The ability of the Rb adenoviral constructs to inhibit VSMC
proliferation in vitro led us to test their ability to inhibit the
hyperproliferation of VSMCs in the rat carotid artery model of
stenosis. Chang et al14 reported that an
adenovirus construct encoding a
phosphorylation-resistant form of murine Rb was
able to inhibit neointima formation relative to an
Ad-ß-Gal construct in this model. In Figs 4 and 5,
the results with both phosphorylation-competent human
Rb adenoviral constructs (Ad-Rb110 and Ad-Rb56) and the Ad-ß-Gal
construct are shown. To observe the extent of adenoviral transduction,
we treated injured arteries with either Ad-Rb110 or Ad-ß-Gal and
killed the animals 2 days after infection to harvest both the treated
carotid artery and the contralateral untreated artery as a control.
Tissues sections were stained for the presence of human Rb with a
human-specific monoclonal anti-Rb antibody, and, as shown in Fig 4, extensive staining of the most luminal layers of smooth muscle cells
was observed. Positive staining for human Rb was not observed in either
the Ad-ß-Gal–treated or contralateral control arteries. Also shown
in Fig 4 are representative hematoxylin-eosin–stained
cross sections from adenovirus-treated arteries 14 days after
treatment. As can be observed, Ad-Rb-110 inhibited
neointima formation in this system compared with the lesion
obtained in an Ad-ß-Gal–treated artery. For the full-length Ad-Rb110
construct, a 39% reduction (P=.015) in the intima/media
ratio was obtained compared with Ad-ß-Gal (Fig 5). Similarly, a 43%
reduction (P=.0025) was observed with the Ad-Rb56 construct.
Neointima formation in vessels infected with the Ad-ß-Gal
construct were equivalent to the values observed for saline treatment
of injured vessels.36
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Discussion |
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By virtue of its central importance in controlling S-phase progression, deletion or inactivation of both Rb alleles appears to be involved in malignant progression in a number of tumors. Consistent with its inactivation during tumorigenesis, Rb reintroduction into both malignant and primary cell lines has been shown to inhibit proliferation.37 38 39 40 41 In light of this broad spectrum of activity, Rb is a strong candidate for use as an antiproliferative agent in a number of disorders that are characterized by inappropriate proliferative responses. The proliferation of VSMCs in response to balloon angioplasty may be a relevant example of such an excessive response, and agents that attenuate the proliferation of VSMCs could potentially provide a prophylactic therapy for prevention of restenosis.
The goal of our study was to analyze the regulatory properties of various forms of human Rb in VSMCs. We initiated this study by examining the effects of mutations in Rb that were anticipated to modulate the E2F binding activity. Full-length phosphorylation-competent Rb (Rb110), full-length Rb with mutations at a subset of phosphorylation sites (Rb110 788/807/811), and an amino-terminal–truncated form of Rb (Rb56) all inhibited E2F-mediated transcription from a luciferase reporter construct. The Rb56 construct appeared most potent in inhibiting E2F-mediated transcription. Rb forms with an inactivating mutation (H209) had no effect on transcription. On the basis of these results, we created adenoviral constructs that expressed Rb protein in either the full-length (Ad-Rb110) or the amino-terminal–deleted (Ad-Rb56) forms and compared their ability to inhibit VSMC proliferation.
When examined for their effect on VSMC proliferation in vitro, the full-length Rb110 and truncated Rb56 forms were surprisingly similar in their growth-inhibitory activities. Both the Rb110 and the Rb56 adenoviral constructs were able to bring about similar levels of G0/G1 arrest in rat VSMCs compared with the Ad-ß-Gal– or mock-infected controls, consistent with the notion that Rb functions as an important regulator of the G1- to S-phase transition in VSMCs. The ability of full-length Ad-Rb110 to inhibit VSMC proliferation indicates that high levels of phosphorylation-competent Rb are sufficient to inhibit cell-cycle progression. The Rb adenoviral constructs were also compared in their abilities to inhibit neointima formation in the rat carotid artery model of stenosis. Both constructs were able to significantly reduce the intima/media ratio. Two weeks after injury, Ad-Rb56 reduced the intima/media ratio by 43%, whereas Ad-Rb110 had a similar reduction of 39%. Our results with human Rb constructs compare favorably with the reduction in intima/media ratio obtained by Chang et al14 (42%) using an adenovirus construct that encodes a constitutively active, nonphosphorylatable form of murine Rb. Thus, whereas others have sought to control the level of Rb activity by delivering a form of Rb protein that is insensitive to hyperphosphorylation, the results presented herein suggest that overexpression of phosphorylation-competent Rb is sufficient for inhibition of neointima formation.
Together, these observations indicate that the full-length phosphorylation-competent human form of Rb can function in a similar manner as a mutant nonphosphorylatable murine Rb in the rat carotid artery model system of restenosis. The use of human Rb in clinical applications should lower the possibility of an immune response that may be anticipated when cross-species gene transfer is conducted. For this reason, we believe that wild-type human Rb represents a conservative and practical choice for preventing the VSMC hyperproliferation that occurs after balloon angioplasty.
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Selected Abbreviations and Acronyms |
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Received December 14, 1996; revision received May 12, 1997; accepted May 16, 1997.
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References |
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|---|
1. Goodrich DW, Lee WH. Molecular characterization of the retinoblastoma susceptibility gene. Biochim Biophys Acta. 1993;1155:43-61.[Medline] [Order article via Infotrieve]
2.
Hiebert SW. Regions of the retinoblastoma gene
product required for its interaction with the E2F transcription
factor are necessary for E2 promoter repression and pRb-mediated growth
suppression. Mol Cell Biol. 1993;13:3384-3391.
3.
Kato J, Matsushime H, Hievert SW, Ewen ME, Sherr
CJ. Direct binding of cyclin D to the retinoblastoma gene
product (pRb) and pRb phosphorylation by the cyclin
D-dependent kinase, CDK4. Genes Dev. 1993;7:331-342.
4. Ewen ME, Sluss HK, Sherr CJ, Matsushime H, Kato JY, Livingston DM. Functional interaction of the retinoblastoma protein with mammalian D-type cyclins. Cell. 1993;7:487-497.
5. Sherr CJ. G1 phase progression: cycling on cue. Cell. 1994;79:551-555.[Medline] [Order article via Infotrieve]
6. Wang J, Guo K, Walsh K. Inhibition of Rb phosphorylation by myogenesis-induced changes in the subunit composition of the cdk4 complex. Cell Growth Differ. 1996;7:1471-1478.[Abstract]
7.
Schneider JW, Gu W, Zhu L, Mahdavi V, Nadal-Ginard
B. Reversal of terminal differentiation mediated by p107 in
Rb-/- muscle cells. Science. 1994;264:1467-1471.
8. Guo K, Wang J, Andrés V, Smith RC, Walsh K. MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation. Mol Cell Biol. 1995;15:3823-3829.[Abstract]
9. Otterson GA, Kratzke RA, Coxon A, Kim YW, Kaye FJ. Absence of p16INK4 protein is restricted to the subset of lung cancer lines that retains wildtype RB. Oncogene. 1994;9:3375-3378.[Medline] [Order article via Infotrieve]
10.
Okamoto A, Demetrick DJ, Spillare EA, Hagiwara K,
Hussain SP, Bennett WP, Forrester K, Gerwin B, Serrano M, Beach DH,
Harris CC. Mutations and altered expression of p16INK4 in human
cancer. Proc Natl Acad Sci U S A. 1994;91:11045-11049.
11.
Shapiro GI, Edwards CD, Kobzik L, Godleski J, Richards
W, Sugarbaker DJ, Rollins BJ. Reciprocal Rb inactivation and
p16INK4 expression in primary lung cancers and cell lines.
Cancer Res. 1995;55:505-509.
12. Liu Q, Neuhausen S, McClure M, Frye C, Weaver-Feldhaus J, Gruis NA, Eddington K, Allalunis-Turner MJ, Skolnick MH, Fujimura FK. CDKN2 (MTS1) tumor suppressor gene mutations in human tumor cell lines. Oncogene. 1995;10:1061-1067.[Medline] [Order article via Infotrieve]
13.
Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K,
Tavtigian SV, Stockert E, Day R Sr, Johnson BE, Skolnick MH. A
cell cycle regulator potentially involved in genesis of many tumor
types. Science. 1994;264:436-440.
14.
Chang MW, Barr E, Seltzer J, Jiang Y, Nabel GJ, Nabel
EG, Parmacek MS, Leiden JM. Cytostatic gene therapy for vascular
proliferative disorders with a constitutively active form of the
retinoblastoma gene product. Science. 1995;267:518-522.
15. Mader SL. Influence of animal age on the ß-adrenergic system in cultured rat aortic and mesenteric artery smooth muscle cells. J Gerontol Biol Sci. 1992;47:B32-B36.
16. Pickering JG, Weir L, Rosenfield K, Stetz J, Jekanowski J, Isner JM. Smooth muscle cell outgrowth from human atherosclerotic plaque: implications for the assessment of lesion biology. J Am Coll Cardiol. 1992;20:1430-1439.[Abstract]
17.
Shan B, Zhu X, Chen PL, Durfee T, Yang Y, Sharp D, Lee
WH. Molecular cloning of cellular genes encoding
retinoblastoma-associated proteins: identification of a gene with
properties of the transcription factor E2F. Mol Cell
Biol. 1992;12:5620-5631.
18. Huang S, Wang NP, Tseng BY, Lee WH, Lee EH. Two distinct and frequently mutated regions of retinoblastoma protein are required for binding to SV40 T antigen. EMBO J. 1990;9:1815-1822.[Medline] [Order article via Infotrieve]
19.
Kaye FJ, Kratzke RA, Gerster JL, Horowitz JM. A
single amino acid substitution results in a retinoblastoma protein
defective in phosphorylation and oncoprotein
binding. Proc Natl Acad Sci U S A. 1990;87:6922-6926.
20.
Kunkel TA. Rapid and efficient site-specific
mutagenesis without phenotypic selection. Proc Natl Acad
Sci U S A. 1985;82:488-492.
21. Wills KN, Huang WM, Harris MP, Machemar T, Maneval DC, Gregory RJ. Gene therapy for hepatocellular carcinoma: chemosensitivity conferred by adenovirus-mediated transfer of the HSV-1 thymidine kinase gene. Cancer Gene Ther. 1995;2:191-197.[Medline] [Order article via Infotrieve]
22. Huang S, Lee W-H, Lee EY-H. A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product. Nature. 1991;350:160-162.[Medline] [Order article via Infotrieve]
23.
Hemstrom C, Nordqvist K, Pettersson U, Virtanen
A. Gene product of region E4 of adenovirus type 5 modulates
accumulation of certain viral polypeptides. J
Virol. 1988;62:3258-3264.
24.
Ginsberg HS, Lundholm-Beauchamp U, Horswood RL, Pernis
B, Wold WS, Chanock RM, Prince GA. Role of early region 3 (E3)
in pathogenesis of adenovirus disease. Proc Natl Acad Sci
U S A. 1989;86:3823-3827.
25. Wills KN, Maneval DC, Menzel P, Harris MP, Sutjipto S, Vaillancourt MT, Huang WM, Johnson DE, Anderson SC, Wen SF, Bookstein R, Shepard HM, Gregory RJ. Development and characterization of recombinant adenoviruses encoding human p53 for gene therapy of cancer. Hum Gene Ther. 1994;5:1079-1088.[Medline] [Order article via Infotrieve]
26. Graham FL, Prevec L. Adenovirus-based expression vectors and recombinant vaccines. Biotechnology. 1992;20:363-390.[Medline] [Order article via Infotrieve]
27. Huyghe BG, Liu X, Sutjipto S, Sugarman BJ, Horn MT, Shepard HM, Scandella CJ, Shabram P. Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography. Hum Gene Ther. 1995;6:1403-1416.[Medline] [Order article via Infotrieve]
28.
Helin K, Chin-Lee W, Fattaey AR, Lees JA, Dynlacht BD,
Ngwu C, Harlow E. Heterodimerization of the transcription factors E2F-1
and DP-1 leads to cooperative trans-activation. Genes
Dev.. 1993;7:1850-1861.
29.
Zhu L, van den Heuvel S, Helin K, Fattaey A, Ewen M,
Livingston D, Dyson N, Harlow E. Inhibition of cell
proliferation by p107, a relative of the retinoblastoma
protein. Genes Dev. 1993;7:1111-1125.
30. Henthorn P, Zervos P, Raducha M, Harris H, Kadesch T. Expression of human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression. Proc Natl Acad Sci U S A. 1988;87:6922-6926.
31. Andrés V, Fisher S, Wearsch P, Walsh K. Regulation of Gax homeobox gene transcription by a combination of positive factors including MEF2. Mol Cell Biol. 1995;15:4272-4281.[Abstract]
32. Wen SF, Nodelman M, Nared-Hood K, Duncan J, Geradts J, Shepard HM. Retinoblastoma protein monoclonal antibodies with novel characteristics. J Immunol Methods. 1994;169:231-240.[Medline] [Order article via Infotrieve]
33. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle cell growth in the absence of endothelium. Lab Invest. 1983;49:327-333.[Medline] [Order article via Infotrieve]
34. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1983;49:208-215.[Medline] [Order article via Infotrieve]
35. March KL, Madison JE, Trapnell BC. Pharmacokinetics of adenoviral vector-mediated gene delivery to vascular smooth muscle cells: modulation by poloxamer 407 and implications for cardiovascular gene therapy. Hum Gene Ther. 1995;6:41-53.[Medline] [Order article via Infotrieve]
36.
Pastore CJ, Isner JM, Bacha PA, Kearney M, Pickering
JG. Epidermal growth factor receptor–targeted cytotoxin
inhibits neointimal hyperplasia in vivo. Circ
Res. 1995;77:519-529.
37.
Takahashi R, Hashimoto T, Xu HJ, Hu SX, Matsui T, Miki
T, Bigo-Marshall H, Aaronson SA, Benedict WF. The retinoblastoma
gene functions as a growth and tumor suppressor in human bladder
carcinoma cells. Proc Natl Acad Sci U S A. 1991;88:5257-5261.
38.
Huang HJS, Yee JK, Shew JY, Chen PL, Bookstein R,
Friedmann T, Lee EY, Lee WH. Suppression of the neoplastic
phenotype by replacement of the Rb gene in human cancer
cells. Science. 1988;242:1563-1566.
39. Ookawa K, Shiseki M, Takahashi R, Yoshida Y, Terada M, Yokota J. Reconstitution of the RB gene suppresses the growth of small-cell lung carcinoma cells carrying multiple genetic alterations. Oncogene. 1993;8:2175-2181.[Medline] [Order article via Infotrieve]
40. Wang NP, To H, Lee WH, Lee EY. Tumor suppressor activity of RB and p53 genes in human breast carcinoma cells. Oncogene. 1993;8:279-288.[Medline] [Order article via Infotrieve]
41.
Bookstein R, Shew JY, Chen PL, Scully P, Lee WH.
Suppression of tumorigenicity of human prostate carcinoma cells by
replacing a mutated RB gene. Science. 1990;247:712-715.
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