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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.]
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ABSTRACT |
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Introduction |
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In the past 5 years, many studies (4, 5, 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 potential 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 evaluate 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 significance of these finding is discussed.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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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 cytoplasmic domains and then adding
a glycolipid anchor domain for membrane attachment as described
previously (11)
. PC3 cells (2 x 105 per p-35 plate) were transfected with
2 µg 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
µg/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 previously
(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 sensitivity of human prostate cancer cells,
5 x 105 cells were infected with
different concentrations of AdCMV-ß-gal at 37°C in a 5%
CO-humidified incubator. Twenty-four hours after infection, the
ß-galactosidase activity (13)
was measured in a 200 µl
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 mM phenylmethylsulfonyl fluoride. The
lysate was sonicated for 30 s on ice, followed by centrifugation
for 5 min at 4°C. From each sample, 20 µg of total protein were
electrophoresed on a 10% SDS-polyacrylamide gel and electrotransferred
to a nitrocellulose membrane. 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 x 106 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 x widthx height x 0.5236) (14) .
Statistical Analysis.
All data were evaluated by Student’s test. Probabilities <0.05 were
considered significant.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, suggests that CAR levels may determine the viral sensitivity of each
cell line.
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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%. Cytometric analysis
revealed that, overall, the CAR-positive cell population was enriched
3–8 times greater than PC3-vector cells. Furthermore, results
obtained from ß-galactosidase activity (after infecting each
transfected PC3 with AdCMV-ß-gal) indicate that ß-galactosidase
activity is proportional to the CAR levels from each clone. In
particular, ß-galactosidase activity in PC3-CAR2 cells infected with
100 m.o.i. AdCMV-ß-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 ß-galactosidase correlated
with the levels of membrane CAR determined by cytometric analysis
(Table 1)
, suggesting that CAR appears to be a more efficient receptor.
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
significantly, 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-dependent 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)
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exhibited a strong resistance to p21-induced growth inhibition (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 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
, A and 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 p120ras
resulted in more cells than that of CAR cDNA (Fig. 3
C).
These findings indicate that CAR is a potent tumor inhibitor for
prostate cancer.
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, 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 Increasing 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 formation 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
experiments. Four weeks after injection, four CAR-transfected PC3
clones demonstrated a significant (P < 0.05)
tumor growth inhibition compared 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%, 38%, 41%,
54%, 57%) and found a strong inverse correlation between tumor volume
(364 mm3, 260 mm3, 197
mm3, 94 mm3, 33
mm3) and the CAR levels in each clone. Taken
together, we believe that CAR is a potent tumor inhibitor for human
prostate cancer.
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Discussion |
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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
androgen-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
ß-galactosidase, 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
hypophosphorylated Rb, G1 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 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 extracellular,
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
indicates that CAR has cell-adhesive activity. Our laboratory has
demonstrated that C-CAM1, an immunoglobulin-like cell adhesion
molecule, 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 potential (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 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 membrane that leads to signal
transduction. The analysis of detailed mechanism 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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{at}mednet.swmed.edu
4 The abbreviations used are: CAR, coxsackie and
adenovirus receptor; FACS, fluorescent-activated cell scanning; m.o.i,
multiplicity of infection; EGFR, epidermal growth factor receptor.
Received 1/ 8/00. Accepted 8/ 2/00.
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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K. A. Rauen, D. Sudilovsky, J. L. Le, K. L. Chew, B. Hann, V. Weinberg, L. D. Schmitt, and F. McCormick Expression of the Coxsackie Adenovirus Receptor in Normal Prostate and in Primary and Metastatic Prostate Carcinoma: Potential Relevance to Gene Therapy Cancer Res., July 1, 2002; 62(13): 3812 - 3818. [Abstract] [Full Text] [PDF]
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 A. Hemminki, K. R. Zinn, B. Liu, T. R. Chaudhuri, R. A. Desmond, B. E. Rogers, M. N. Barnes, R. D. Alvarez, and D. T. Curiel In Vivo Molecular Chemotherapy and Noninvasive Imaging With an Infectivity-Enhanced Adenovirus J Natl Cancer Inst, May 15, 2002; 94(10): 741 - 749. [Abstract] [Full Text] [PDF]
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,
PC3-parental; •, PC3-vector; 
, PC3-CAR2; 
, PC3-CAR7; 
,
PC3-CAR11; 
, PC3-CAR14; 
, PC3-GPI4; 
, PC3-GPI12; 
,
PC3-Tailess4; 
, PC3-Tailess5.
