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[CANCER RESEARCH 60, 5031–5036, September 15, 2000]
Advances in Brief
The Dual Impact of Coxsackie and Adenovirus Receptor Expression on Human
Prostate Cancer Gene Therapy
1
Takatsugu Okegawa,
2
Yingming Li,
2
Rey-Chen Pong, Jeffrey M. Bergelson, Jian Zhou, and Jer-Tsong Hsieh
3
Department of Urology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 [T. O., Y. L., R. C. P., J. Z., J. T. H.], and Division of Immunologic and
Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19100 [J. M. B.]
Abstract
In a recent paper, we reported a significant difference in coxsackie and
adenovirus receptor (CAR) from several human bladder cancer cell lines
that correlated with their sensitivities to adenoviral infection (Y. Li, R-C.
Pong, J. M. Bergelson, M, C. Hall, A. I. Sagalowsky, C-P. Tseng, Z. Wang,
and J. T. Hsieh, Cancer Res., 59: 325–330, 1999). In human prostate
cancer, CAR protein is down-regulated in the highly tumorigenic PC3 cell
line, which suggests that, in addition to its function as a viral receptor,
CAR may have a pathophysiological role in prostate cancer progression.
In this paper, we document that CAR does not merely enhance the viral
sensitivity of prostate cancer cells but also acts as a tumor inhibitor for
androgen-independent prostate cancer cells. Our data indicate that CAR
is a potential therapeutic agent for increasing the efficacy of prostate
cancer therapy.
Introduction
Adenovirus is a nonenveloped DNA virus thought to enter the host
cell cytoplasm through a specific receptor-mediated endocytosis. Vi-
rus adsorption occurs when viral fibers, elongated proteins that project
radically from each of 12 vertices of an icosahedral capsid, bind to
specific cellular receptors on a target cell membrane (1). Recently,
two groups (2, 3) reported cloning a unique transmembrane protein for
both coxsackie and CAR.
4
Adenovirus type 5 is frequently used as a
vector for gene therapy. Sequence analysis indicates that this CAR
cDNA encodes a typical immunoglobulin-like membrane protein with
two immunoglobulin domains that interact with adenovirus fiber
protein (2, 3). In addition to the extracellular domain, CAR cDNA
contains a 22-amino acid transmembrane domain and a 107-amino
acid intracellular domain that has a putative tyrosine phosphorylation
site. According to its protein structure, CAR may function not only as
the receptor for adenovirus but also as a cell adhesion molecule.
However, the physiological function of CAR is virtually unknown.
In the past 5 years, many studies (4–6) have explored adenovirus-
based gene therapy on prostate cancer treatment. However, some key
issues related to the efficiency of virus uptake or possible side effects
have not been addressed. For example, recombinant adenoviruses
have proved to be relatively inefficient in airway epithelia because
they bind more poorly to the differentiated ciliated airway epithelia
than to immature airway cells (7). On the other hand, viral proteins are
good immunogens. High dosages of adenovirus may impose a poten-
tial host immune rejection. Obviously, increasing the susceptibility of
target cells to viral infection increases the efficacy of gene therapy.
In our laboratory, we use replication-deficient adenovirus to eval-
uate the efficacy of gene therapy for prostate cancer. Previously, we
reported that CAR expression varies among human bladder cancer
cells, and we demonstrated that increased levels of CAR significantly
enhance the uptake of adenovirus (8). In this study, we document that
the levels of CAR protein expression among three human prostate
cancer cell lines correlate with their in vivo tumorigenic potentials.
This prompts us to examine the effect of increased CAR expression on
the efficacy of prostate cancer therapy. Furthermore, because CAR is
down-regulated in prostate cancer cells, we decided to examine the
functional role of CAR in the growth of prostate cancer. The signif-
icance of these finding is discussed.
Materials and Methods
The PC3 cell lines used in this study were obtained from American Type
Culture Collection (Manassas, VA) and were grown in T medium (9) contain-
ing 5% FBS. A mammalian expression vector, pcDNA3.1/V5/His-TOPO, was
purchased from Invitrogen (Carlsbad, CA). Two replication-deficient recom-
binant viruses, AdCMV-

-gal and AdCMV-p21, were generated as described
previously (2, 10).
Plasmid Construction and Transfection into Prostate Cancer Cells. We
performed RT-PCR to obtain CAR cDNA, with total cellular RNA isolated
from both 253J and RT4 cell lines, and CAR cDNA was assembled as
described previously (9). Two additional CAR mutants (Tailess and GPI) were
used in this study. Tailess CAR is a mutant with a deletion of the cytoplasmic
domain of CAR cDNA (11). GPI cDNA, containing only the extracellular
domain of CAR, was constructed by deleting both transmembrane and cyto-
plasmic domains and then adding a glycolipid anchor domain for membrane
attachment as described previously (11). PC3 cells (2 ⫻10
5
per p-35 plate)
were transfected with 2
g of each plasmid using Lipofectamine transfection
reagent. For selection of stable sublines, 48 h after transfection, cells were split
and selected for neomycin-resistant clones with G-418 (600
g/ml). Resistant
colonies were either pooled or cloned by ring isolation after 2 weeks of
selection.
Determination of CAR Levels by FACS. Cytometric analysis was used to
determine CAR levels for each cell. Briefly, membrane fluorescence staining
was performed on a single-cell suspension with the use of RmcB monoclonal
antibody (11) and FITC-conjugated secondary antibodies as described previ-
ously (12). FACS was performed with a dual-laser Vantage flow cytometer
(Becton Dickinson, Mountain View, CA) which delivered 50 mW at 488 nm
with an Enterprise air-cooled laser. Analysis was performed using LYSYS II
software (Becton Dickinson). The positive population of cells was determined
by gating the right-hand tail of the distribution of the negative control sample
for each individual cell line at 1%. This setting was then used to determine the
percentage of positive cells for each of the above markers for each individual
cell line.
Detection of Virus-mediated Gene Delivery. To determine the viral sen-
sitivity of human prostate cancer cells, 5 ⫻10
5
cells were infected with
different concentrations of AdCMV-

-gal at 37°C in a 5% CO-humidified
incubator. Twenty-four hours after infection, the

-galactosidase activity (13)
Received 1/8/00; accepted 8/2/00.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by Grants CA 73017 (J. T.H.), AI35667, and HL
54734 and an Established Investigator Award from the American Heart Association
(J. M. B.).
2
T. O. and Y. L. contributed equally in this project; both are considered as the first
author.
3
To whom requests for reprints should be addressed, at Department of Urology,
University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas,
TX 75390-9110. Phone: 214-648-3988; Fax: 214-648-8786; E-mail: jhsieh@mednet.
swmed.edu.
4
The abbreviations used are: CAR, coxsackie and adenovirus receptor; FACS, fluo-
rescent-activated cell scanning; m.o.i, multiplicity of infection; EGFR, epidermal growth
factor receptor.
5031
was measured in a 200
l of cell lysate and normalized to the protein
concentration of each sample.
Effect of P21 Adenovirus on the Growth Rate of CAR-expressing
Prostate Cancer Cells in Vitro. The p21 adenovirus was used to determine
the efficacy of gene therapy on the CAR-transfected PC3 sublines. Cells were
plated at a density of 5000 cells in 48-well plates using T medium containing
0.2% FBS and infected with AdCMV-p21 at 0, 1, 10, and 100 m.o.i. At the
indicated time, cells were harvested, and relative cell number was determined
by crystal violet assay (14).
Western Analysis of p21 and pRb Expression. To examine the levels of
p21 and Rb expression in PC3 cells after their infection with the p21 virus, we
conducted a Western assay as described previously (10). The cell lysate was
made by adding 20% SDS containing 1 mMphenylmethylsulfonyl fluoride.
The lysate was sonicated for 30 s on ice, followed by centrifugation for 5 min
at 4°C. From each sample, 20
g of total protein were electrophoresed on a
10% SDS-polyacrylamide gel and electrotransferred to a nitrocellulose mem-
brane. After the membrane was blocked with PBS containing 5% powdered
milk, it was incubated with anti-p21 (6B6; PharMingen, San Diego, CA) or
anti-Rb (G3-245; PharMingen) antibody for 1 h, followed by incubation with
antimouse IgG. After extensive washing, the protein was visualized with an
ECL chemiluminescence detection kit (Amersham, Arlington Heights, IL).
Determination of Both in Vitro and in Vivo Growth Rate of CAR-
transfected PC3 Sublines. To examine the effect of CAR on the growth rate
of cells, we measured the in vitro growth rate of CAR-transfected PC3 cells.
Cells were plated at density of 5,000 cells in 48-well plates with T medium
containing 0.2% FBS. Relative cell numbers were determined by crystal violet
assay at the indicated time.
To determine the in vivo tumor growth of each transfected clone, we
injected 1 ⫻10
6
cells/site at 6 sites s.c. in the flanks of 8- to 10-week-old male
athymic mice. After 2 weeks inoculation, when tumors became palpable,
growth of s.c. tumors was measured weekly with a caliper, and tumor volume
was calculated (volume ⫽length ⫻width⫻height ⫻0.5236) (14).
Statistical Analysis. All data were evaluated by Student’s test. Probabili-
ties ⬍0.05 were considered significant.
Results
Correlation of CAR Levels and Viral Sensitivity of Human
Prostate Cancer. Cytometric analysis of the immunofluorescence
staining of three human prostate cancer cell lines (LNCaP, DU145,
and PC3) indicates that DU145 contains the highest number of CAR-
positive cells (82%). LNCaP contains 63% of CAR-positive cells, and
PC3 contains the lowest numbers of CAR-positive cells (35%). The
viral sensitivity of each cell line, determined by AdCMV-

-gal cor-
related with the percentages of CAR-positive cells (Table 1), suggests
that CAR levels may determine the viral sensitivity of each cell line.
To examine whether the CAR protein is responsible for adenoviral
infection in human prostate cancer cells, we constructed a mammalian
CAR expression vector and transfected it into PC3 cells. After G-418
selection, four independent clones of CAR (PC3-CAR2, -7, -11,
and -14), one vector-transfected clone (PC3-vector), and four inde-
pendent clones of CAR mutants (PC3-Tailess4, PC3-Tailess5, PC3-
GPI4, and PC3-GPI12) were selected, based on the different plasmid
DNA integration pattern (Fig. 2A).
Immunofluorescence staining of the transfected PC3 cells with
CAR monoclonal antibody was performed 1 month after G418 selec-
tion. Results (Table 1) show that variable CAR-positive cells, ranging
from 45 to 86%, were detected among six different PC3 subclones. In
contrast, the CAR-positive cells in PC3-vector were ⬃10%. Cytomet-
ric analysis revealed that, overall, the CAR-positive cell population
was enriched ⬃3–8 times greater than PC3-vector cells. Furthermore,
results obtained from

-galactosidase activity (after infecting each
transfected PC3 with AdCMV-

-gal) indicate that

-galactosidase
activity is proportional to the CAR levels from each clone. In partic-
ular,

-galactosidase activity in PC3-CAR2 cells infected with
100 m.o.i. AdCMV-

-gal was ⬃15 times higher than that in PC3-
vector cells. For two CAR mutants (GPI and Tailess sublines), they
were still sensitive to adenovirus; the

-galactosidase correlated with
the levels of membrane CAR determined by cytometric analysis
(Table 1), suggesting that CAR appears to be a more efficient recep-
tor. Taken together, these data indicate that viral sensitivities of
prostate cancer cell lines correlate with their CAR levels.
Efficacy of Gene Therapy in CAR-expressing Prostate Cancer
Cells. Demonstrating the presence of CAR is one of key determinants
of the efficacy of gene therapy. To do this, a recombinant adenovirus
carrying p21 cDNA, a cyclic kinase inhibitor (10), was used. As
shown in Fig. 1, the PC3-parental, vector and CAR2 cells did not
show growth inhibition at day 6 in the presence of 1 m.o.i. AdCMV-
p21. However, AdCMV-p21 at 10 m.o.i. can exhibit an 84% growth
inhibition rate in PC3-CAR2 cells at day 6 (Fig. 1C), whereas the
same concentration of AdCMV-p21 achieved only a 22% growth
inhibition rate in PC3-parental cells at day 6 (Fig. 1A). More signif-
icantly, as shown in Fig. 1C, the growth inhibition elicited by
AdCMV-p21 at both 10 and 100 m.o.i. was almost identical. This
indicates that the increased expression of CAR protein in PC3 cells
can reduce the concentration of p21 virus by as much as 10 times to
achieve maximal growth inhibition. Western blot analyses (Fig. 1D)
provided direct evidence that p21 protein can be detected in PC3-
CAR2 cells 24 h after infecting with AdCMV-p21 at 10 m.o.i. and that
the elevated p21 protein levels exhibited in a dose- and time-depen-
dent manner. Similarly, the steady-state level of Rb protein, a key
indicator for p21-induced growth inhibition, was reduced, and the
majority of Rb protein was hypophosphorylated in PC3-CAR2 cells
after infecting with p21 virus (Fig. 1E).
Furthermore, the PC3-vector cells with 10% CAR-positive cells
(Table 1) exhibited a strong resistance to p21-induced growth inhibi-
tion (Fig. 1B) because p21 virus failed to infect this clone (Fig. 1D).
This was evidenced by the presence of hyperphosphorylated Rb
protein levels in PC3-vector cells (Fig. 1E). Therefore, CAR protein
appears a critical rate-limiting factor in determining the outcome of
gene therapy.
In Vitro Growth Characteristics of CAR-transfected Prostate
Cancer Cells. Structurally, CAR belongs to the immunoglobulin
superfamily. It shares similar structure with a cell adhesion molecule
such as C-CAM, a potent tumor suppressor in prostate cancer (2, 12).
Also, CAR levels in the three human prostate cancer cell lines seem
to correlate with their in vivo tumorigenicity (15). Therefore, we
decided to examine the effect of CAR on the in vitro growth rate of
PC3 cells. The growth rates of both PC3-CAR2 and -11 cells were
⬃40% that of PC3-vector cells at day 6 (Fig. 2B). The growth rate of
both PC3-CAR7 and 14 cells were ⬃75 and 85% that of PC3-vector
Table 1 Determination of CAR levels by FACS and the efficiency of virus-mediated
gene delivery in CAR-, GPI-, or Tailess-transfected PC3 cells
Cell line FACS (%)
a

-Galactosidase activity (A
405 nm
/
g)
b
Control 10 m.o.i. 100 m.o.i.
DU145 82 0.98 ⫾0.36 35.68 ⫾3.54 225.67 ⫾4.52
LNCaP 63 1.11 ⫾0.87 22.24 ⫾2.87 145.87 ⫾5.61
PC3-parental 35 1.21 ⫾0.32 10.74 ⫾1.36 48.09 ⫾2.35
PC3-vector 11 1.40 ⫾0.54 3.06 ⫾0.99 16.20 ⫾0.88
PC3-CAR2 86 1.25 ⫾0.24 35.96 ⫾0.98 239.39 ⫾8.02
PC3-CAR7 45 1.37 ⫾0.59 19.22 ⫾2.73 77.12 ⫾0.25
PC3-CAR11 63 1.08 ⫾0.49 23.42 ⫾0.99 120.87 ⫾1.90
PC3-CAR14 46 1.17 ⫾0.54 13.25 ⫾0.46 89.46 ⫾4.70
PC3-GPI4 54 1.45 ⫾0.12 23.40 ⫾3.04 117.17 ⫾8.67
PC3-GPI12 61 1.52 ⫾0.28 27.62 ⫾1.21 126.86 ⫾2.21
PC3-Tailess4 80 1.67 ⫾0.92 39.21 ⫾3.98 264.67 ⫾4.85
PC3-Tailess5 62 1.14 ⫾0.83 30.88 ⫾0.87 173.82 ⫾5.32
a
Cells were incubated with RmcB (CAR) before the addition of FITC-conjugated
antimouse IgG secondary antibody. Data are calculated as described in “Materials and
Methods” and presented as the percentage of cells gated positive.
b
Each value was determined in triplicate from two separate experiments.
5032
ADENOVIRAL RECEPTOR IN PROSTATE CANCER
cells at day 6, respectively. PC3-CAR2 and 11 cells grow more slowly
than PC3-CAR7 and -14 cells because of the higher CAR levels in
both PC3-CAR2 and -11. To rule out this result from the artifact of
stable transfection, we also examined the growth rate of PC3 cells
from transient expression of CAR. As shown in Fig. 3, Aand B, the
growth-inhibitory activity of CAR exhibited in a dose-dependent
manner. This is not due to the cytotoxicity caused by the presence of
the large amount of DNA, because transfecting high concentrations of
both cDNA constructs containing oncogenic protein such as EGFR
and p120
ras
resulted in more cells than that of CAR cDNA (Fig. 3C).
These findings indicate that CAR is a potent tumor inhibitor for
prostate cancer.
With respect to the dual function of the CAR molecule, we further
examined the structural functional relationship between viral binding
and tumor inhibition. The extracellular domain is critical for viral
uptake (11). With the same construct (11) containing only the extra-
cellular domain with a glycolipid anchor for membrane attachment,
two PC3 sublines (GPI4 and -12) were generated. As shown in Table
1, increased CAR expression was detected, and cells were sensitive to
viruses. However, neither subline exhibited growth inhibition under
the same experimental condition (Fig. 2C). These cells appeared to be
tumorigenic in vivo (83%). On the other hand, both Tailess CAR
cDNA-transfected cells (i.e., Tailess 4 and Tailess 5) appeared to be
a potent inhibitor in vitro (Fig. 2C) and in vivo (3%). Therefore, we
believe that CAR is a tumor inhibitor; both the extracellular and
transmembrane domains of CAR are required.
Suppression of in Vivo Tumorigenicity Of PC3 Cells by Increas-
ing CAR Expression. PC3 cells have been shown to be highly
tumorigenic when injected into nude mice (12). To test whether
increased expression of CAR may affect the tumorigenicity of PC3
cells, cells from each clone, including PC3-parental, PC3-vector, and
four clones of PC3-CAR (2, 7, 11, and 14) were injected s.c. into the
flanks of male athymic nude mice, and the incidence of tumor for-
mation and the volumes of the tumors were monitored weekly when
tumors become palpable. As shown in Table 2, the tumor incidence
elicited by PC3-CAR2 showed a significant decrease compared with
that by both PC3-parental and PC3-vector. Overall, the decreased
tumor incidence elicited by four CAR-transfected PC3 cells (Table 2)
correlated with the CAR levels in each clone (Table 1). Table 2 shows
the tumor growth results obtained from three independent experi-
ments. Four weeks after injection, four CAR-transfected PC3 clones
demonstrated a significant (P⬍0.05) tumor growth inhibition com-
pared with PC3-vector. Furthermore, 8 weeks after injection, the
tumor volume induced by four CAR-transfected PC3 clones was
smaller than that induced by both PC3-parental and -vector cells. As
determined by cytometric analysis, the tumor volume has an inverse
correlation with the CAR levels in each clone (Table 1), indicating
that CAR has a dosage effect in suppressing tumor growth. We also
noticed that a few tumors induced by PC3-CAR2 are quite large,
which suggests that these tumors may be an outgrowth of cells
expressing a low level of CAR protein. Using FACS analysis, we
measured CAR levels in the cells derived from these tumors (3%,
Fig. 1. Increased the efficacy of gene therapy in CAR-expressing prostate cancer. PC3-parental cells, PC3-vector cells, and PC3-CAR2 cells were infected with p21 adenovirus at
0, 1, 10, 100 m.o.i. At the indicated time, total cell number was determined by the crystal violet assay. Protein extracts were analyzed by Western blot analysis with p21- or Rb-specific
antibodies. A, growth rate of PC3-parental cells infected with p21 virus; B, growth rate of PC3-vector cells infected with p21 virus. C, growth rate of PC3-CAR2 cells infected with
p21 virus; D, expression of exogenous p21 proteins in PC3 cells infected by p21 adenovirus; E, phosphated Rb levels in p21 virus-infected PC3 clones. ppRb, hyperphosphorylated
form of Rb; pRb, hypophosphorylated form. E, control; F, 1 m.o.i.; 䡺, 10 m.o.i.; f, 100 m.o.i.
5033
ADENOVIRAL RECEPTOR IN PROSTATE CANCER
38%, 41%, 54%, 57%) and found a strong inverse correlation between
tumor volume (364 mm
3
, 260 mm
3
, 197 mm
3
,94mm
3
,33mm
3
) and
the CAR levels in each clone. Taken together, we believe that CAR is
a potent tumor inhibitor for human prostate cancer.
Discussion
Gene therapy, through either replacing defective or suppressing
gene overexpression, is an innovative approach to the treatment of
malignant and benign disorders. Cytotoxic genes such as HSV-TK
(16) and cytokines for boosting host immunity are good candidates for
cancer gene therapy. Several practical and theoretical (17) consider-
ations make recombinant adenovirus an attractive vector for cancer
gene therapy. For example, adenoviral infection results in episome
DNA replication without the chromosomal integration that may cause
potential genotoxicity. A recombinant adenovirus can carry a large of
transgene (17, 18); these recombinant adenoviruses are structurally
stable, and no genome rearrangement is detected after extensive
amplification (19). Also, adenovirus can infect virtually all of the
epithelial cells, regardless of their cell cycle stage. Importantly, ad-
enoviral infection appears to be linked only to mild disease, such as
acute respiratory disease. However, because viral proteins are cyto-
toxic and immunogenic, repeated administration of adenovirus may
elicit cell-mediated immunity, i.e., infiltration of CD8
⫹
T cell (20).
Therefore, an agent that can increase the infectivity of recombinant
adenovirus at a lower viral dosage may potentially avoid such adverse
side effect while increasing the efficiently of gene delivery.
In recent studies, we reported that a wide spectrum of CAR levels
exists among several human bladder cancer lines (8). Using both virus
binding and virus infectivity assay, we found that the levels of CAR
correlate with viral sensitivity determined. Moreover, we observed
loss or reduced expression of CAR levels in several human bladder
cancer lines. Using Northern blot and quantitative RT-PCR analyses,
we documented that a significant difference in viral receptor levels is
caused by down-regulation of the CAR gene in several resistant
cancer cell lines. Similarly, in other cancer types such as melanoma
and glioma, variable expression of CAR gene is also documented (21,
22). Southern blot analysis indicated that there is no large gene
alteration or rearrangement in the CAR gene between the CAR-
positive and CAR-negative cells (8). This suggests that transcriptional
regulation of the CAR gene is critical for its steady-state levels.
In this study, derived from a patient with a bony metastasis, we
demonstrated that CAR protein levels are down-regulated in an an-
drogen-independent human prostate cancer line (PC3). It appears that
PC3 cells are resistant to adenoviral infection. To revert this viral
resistance, we genetically engineered PC3 cells by increasing 35% of
CAR-positive cells to 86% CAR-positive cells, and we were able to
detect that transgene activity in CAR-positive cells, such as

-galac-
tosidase, increased about 5-fold compared with CAR-negative cells.
We further evaluated the efficacy of gene therapy for PC3-cells using
a recombinant AdCMV-p21virus. The elevated levels of p21 protein
in virus-infected cells result in the accumulation of hypophosphor-
ylated Rb, G
1
arrest, and apoptosis (23, 24). Data from this study
(Table 1 and Fig. 1) show that the CAR-transfected PC3 cells (i.e.,
PC3-CAR2) exhibited about 10 times the viral sensitivity of PC3-
vector cells. With Western analysis (Fig. 1D), increased expression of
p21 protein in both a dosage- and time-dependent manner were
Fig. 2. In vitro characterization of CAR-trans-
fected PC3 cells. A, high-molecular-weight DNA
(20
g) was digested with HindIII and subjected to
Southern blot analysis probed with a neo cDNA
probe. In Band C, cells were plated at a density of
5000 cells/ml in 48-well plates in T medium con-
taining 0.2% FBS. Cells were harvested and
counted at the indicated time. The relative cell
numbers were determined by crystal violet assay.
E, PC3-parental; F, PC3-vector; 䡺, PC3-CAR2;
f, PC3-CAR7; ‚, PC3-CAR11; Œ, PC3-CAR14;
〫, PC3-GPI4; ⽧, PC3-GPI12; ƒ, PC3-Tailess4;
, PC3-Tailess5.
5034
ADENOVIRAL RECEPTOR IN PROSTATE CANCER
detected in PC3-CAR2 cells, but not in PC3-parental or PC3-vector
cells. Concurrently, Rb proteins converted to the hypophosphorylated
form were also detected in PC3-CAR2 cells, but not in PC3-parental
or PC3-vector cells (Fig. 1E). Increased expression of p21 protein in
PC3 cells also resulted in apoptosis (data not shown). Taken together,
AdCMV-p21 adenovirus-mediated growth inhibition in PC3-cells can
be enhanced significantly with an increment of their CAR protein
levels. Because a lower dose of adenovirus delivery for CAR-positive
cells can achieve the same therapeutic outcome as a higher dose of
adenovirus, we suggest that a careful determination of CAR status in
target cells must be evaluated before the treatment. By doing so,
excessive in vivo administration of adenovirus may be avoided.
CAR protein contains two immunoglobulin loops on its extracel-
lular, transmembrane, and intracellular domain (6, 7, 25). Therefore,
this protein belongs to the immunoglobulin superfamily. We also
noticed that CAR-transfected PC3 cells (72% for PC3-CAR2, 35% for
PC3-CAR7) can increase the cell attachment on the plate compared
with either PC3-parental (7%) or PC3-vector cells (4%), which indi-
cates that CAR has cell-adhesive activity. Our laboratory has demon-
strated that C-CAM1, an immunoglobulin-like cell adhesion mole-
cule, can inhibit tumor growth effectively in vitro and in vivo (11, 14,
26). Because CAR levels in three human prostate cancer cell line
(LNCaP, DU145, PC3) correlate with their in vivo tumorigenic po-
tential (11) and CAR is down-regulated in human prostate cancer
specimens (data not shown), we were led to examine the biological
function of CAR in prostate cancer. Clearly, our results indicate that
stable transfection of CAR cDNA could inhibit the in vitro growth of
PC3 cells (Fig. 2) and that transient expression of CAR can also
Fig. 3. The growth inhibition of PC3 cells by
transient expression of CAR cDNA. The effect of
transient expression of CAR on the growth of PC3
cells was determined by the method described by
Yeung et al. (27) with minor modification. Cells
(150,000 cells/p-60 plate) were plated in T-medium
containing 5% FBS and cotransfected with different
amount of CAR cDNA,

-gal cDNA (0.2
g), and
control plasmid to make the same concentration (1
g) in each experiment. Twenty-four hours after
transfection, cells were changed to T medium con-
taining 0.2% FBS. At the indicated time, the levels of
CAR expression were determined by RT-PCR (A)
and total blue cells were counted (B, C). Each data set
was repeated in triplicate. The percentage of control
was calculated as the number of blue cells from each
transfection versus either 0
gofCAR(B)or0.8
g
of EGFR (C). CAR: a, 0
g; b, 0.1
g; c, 0.4
g; d,
0.8
g. EGFR: e, 0.8
g. Ras: f, 0.8
g. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
Table 2 Tumor incidence and growth rate of CAR-transfected prostate cancer cells
Clone
Tumor incidence (%)
a
Mean volume (mm
3
⫾SD)
b
Experiment 1 Experiment 2 Experiment 3
28 days 56 days 28 days 56 days 28 days 56 days 28 days 56 days
PC3-parental 44/60 (73) 47/60 (78) 37 ⫾8 345 ⫾95 32 ⫾12 307 ⫾81 49 ⫾6 265 ⫾85
PC3-vector 51/60 (85) 51/60 (85) 46 ⫾8 346 ⫾63 46 ⫾8 374 ⫾64 54 ⫾13 312 ⫾75
PC3-CAR2 24/54 (44) 27/54 (50) 28 ⫾5
c
232 ⫾60
c
18 ⫾4
c
153 ⫾25
c
23 ⫾19
c
168 ⫾63
c
PC3-CAR7 34/54 (63) 34/54 (63) 37 ⫾6
d
302 ⫾50
c
29 ⫾12
d
240 ⫾20
c
41 ⫾13
c
227 ⫾72
c
PC3-CAR11 33/54 (61) 34/54 (63) 29 ⫾7
c
270 ⫾59
c
25 ⫾6
c
214 ⫾95
c
31 ⫾11
c
184 ⫾54
c
PC3-CAR14 35/54 (65) 36/54 (67) 36 ⫾10
d
307 ⫾94
c
22 ⫾4
c
223 ⫾22
c
32 ⫾12
c
211 ⫾60
c
a
Tumor incidence is calculated from three independent experiments.
b
Athymic mice were inoculated s.c. with 1 ⫻10
6
cells/sites at six sites s.c. in the flanks of the 6- to 8-week-old animals at day 0. Tumor size was determined by the formula
length ⫻width ⫻height ⫻0.5236.
c
The CAR-transfected PC3 cells showed significant tumor growth inhibition compared with PC3-parental and PC3-vector cells (P⬍0.05).
d
The CAR-transfected PC3 cells showed significant tumor growth inhibition compared with PC3-vector cells (P⬍0.05).
5035
ADENOVIRAL RECEPTOR IN PROSTATE CANCER
inhibit the cell growth in a dose-dependent manner (Fig. 3). By
injecting the CAR-transfected PC3 cell-induced tumors into athymic
nude mice s.c., we observed a decrease of in vivo tumor incidence and
tumor growth rate (Table 2). A similar growth-inhibitory effect of
CAR is thus observed in human bladder cancer lines (data not shown).
These results demonstrate that CAR expression in prostate cancer
cells could be a potent growth inhibitor from in vitro and in vivo.
However, the mechanism of action of CAR protein in prostate
cancer is still unknown. We have recently shown that the intracellular
domain but not the immunoglobulin domain of C-CAM1 is crucial for
its tumor-inhibitory activity (26). This suggests that the intracellular
domain may be able to elicit a signaling pathway in prostate cancer.
Interestingly, our data indicate that the extracellular domain of CAR
is essential for viral infection (Table 1). In addition to the extracellular
domain of CAR, the transmembrane domain is required for growth
inhibition (Fig. 2C). It is possible that the transmembrane domain of
CAR can interact with other peripheral proteins associated with mem-
brane that leads to signal transduction. The analysis of detailed mech-
anism is under way. Nevertheless, all of the information derived from
further study can be translated into the development of CAR as a new
agent for improving gene therapy.
In conclusion, our findings indicate that increased expression of
CAR protein can inhibit tumors growth in vitro and in vivo. The
tumor-suppressing effect of CAR and its adenoviral receptor nature
indicate that CAR has a dual effect to potentiate prostate cancer gene
therapy. We therefore believe that CAR proteins have significant
biological and therapeutic implications for human prostate cancer.
Further study for up-regulating the endogenous CAR gene in prostate
cancer will make a significant contribution not only to the prostate
cancer therapy but also to the application of gene therapy of other
cancer types.
Acknowledgments
We thank Andrew Webb for editing the manuscript and Dr. John D.
McConnell for the critical reading of the manuscript.
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