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Tumor Biology |
Department of Urology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390 [T. O., R-C. P., Y. L., A. I. S., J-T. H.], and Division of Immunologic and Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104 [J. M. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In our laboratory, we are evaluating the efficacy of recombinant adenovirus on therapy for human bladder cancer. We observed that a wide spectrum of viral sensitivity among human bladder cancer cell lines correlates with their CAR levels (5) . We also found that CAR expression is often down-regulated in cancer specimens but not in adjacent normal tissue. This suggests that altered CAR expression may be involved in the progression of bladder cancer and that decreased CAR levels may result in the poor viral transfection rate in bladder cancer. Therefore, we decided to examine the role of CAR and its structural functional relationship in the biology of bladder cancer and to explore its potential impact on therapy for bladder cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue Samples.
Forty-five patients (36 men and 9 women) received radical cystectomy at
the University of Texas Southwestern Medical Center. Mean patient age
was 67.3 ± 4.2 years (range, 48–81 years). Cancer
stage was assigned according to the Tumor-Node-Metastasis system.
Pathological stages of the 45 patients with bladder cancer were: pTis,
n = 4; pT1,
n = 4; pT2,
n = 9; pT3,
n = 13; pT4,
n = 3; pTxN+,
n = 12. Normal and cancer tissues derived
from the same patient were immediately immersed in liquid nitrogen
within 10 min after removal. A portion of each sample was taken for
histopathological examination, and the remaining tissue was frozen in
liquid nitrogen and stored at -80°C until use. Total cellular RNA
was then prepared and subjected to semiquantitative RT-PCR assay.
Sample Collection and Semiquantitative RT-PCR Analysis.
For semiquantitative RT-PCR, the assay condition was carried out as
described previously (5)
, which is under linear
relationship between PCR product and cDNA levels. Briefly, 2 µg of
total cellular from each tissue were reverse transcribed into the
first-strand cDNA. One-eighth of the cDNA was subjected to a 20-µl
PCR [30 cycles of 92°C (15 s), 55°C (30 s), and 72°C (2 min)
using both the CAR primer set (5'-GCCTTCAGGTGCGAGATGTGTTA-3' and
5'-GAACACGGAGAGCACAGATGAGAC-3'; 0.5 ng/µl) and the GAPDH primer set
(5'-TCGTGGAAGGACTCATGACC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'; 0.5
ng/µl)]. The final PCR products (10 µl) were electrophoresed in a
2% NuSieve agarose gel (3:1 FMC Bioproducts, Rockland, ME) and
quantified with BioMax 1D image analysis software (Eastman Kodak,
Rochester, NY). The relative level of CAR mRNA from each sample was
normalized to GAPDH transcript from the same reaction. We used
densitometric analysis to quantify each PCR transcript level. Then, the
CAR ratio of tumor versus normal from each patient was
calculated as CAR (tumor):GAPDH (tumor):CAR (normal):GAPDH (normal).
Plasmid Construction and Transfection into Bladder Cancer Cells.
CAR cDNA containing the complete open reading frame was cloned by
RT-PCR (5)
. Two CAR mutants (GPI and Tailless) used in
this study were described previously (6)
. To make another
mutant with a complete deletion of the intracellular domain of CAR, the
QuikChange Site-Directed Mutagenesis kit was used. Site-directed
mutagenesis was performed by PCR according to the manufacturer
(Stratagene, La Jolla, CA). The oligonucleotide used for generating two
stop codons immediately after the transmembrane domain of Tailless cDNA
was
5'-CATTGGTCTTATCATCTTTTGATGATAGTCTAGA
GGGCCCG-3'. Briefly, the denatured Tailless plasmid was reannealed with
oligonucleotide primer and subjected to PfuTurbo DNA PCR. After PCR,
the methylated and nonmutated parental DNA template was digested with
DpnI. The XL-1 Blue cells were then transformed with
DpnI-treated DNA for selecting the mutated DNA. Mutants
(i.e., Tailless-m) were verified by DNA sequencing. A
mammalian expression vector (pcDNA3.1) was used as a plasmid control
(Invitrogen, Carlsbad, CA).
To generate an antisense CAR expression vector, pTOPOCAR was digested with BamHI and filled-in. After phenol-chloroform purification, this CAR insert was isolated after EcoRI digestion. The insert was then subcloned into both SmaI and EcoRI sites of pCI-neo (Promega, Madison, WI).
To generate stable transfectants, both T24 and 253J cells (2 x 105 per p-35 plate) were transfected with 2 µg of cDNA using Lipofectamine transfection reagent (Life Technologies, Rockville, MD). For RT4 cells, 2.5 x 106 (per p-100 plate) cells were transfected with 10 µg of cDNA using electroporation. Cells were selected for neomycin-resistant clones with G-418 (600 µg/ml) 48 h after transfection. Resistant colonies were cloned by ring isolation 2 weeks after selection.
Determination of CAR Levels by FACS.
Cytometric analysis was used to determine CAR levels for each clone.
Briefly, membrane fluorescence staining was performed on a single-cell
suspension using RmcB monoclonal antibody and FITC-conjugated secondary
antibodies (5
, 7)
. Fluorescence-activated cell scanning
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, Mountain View, CA). 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% as described previously (5
, 7)
. 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 bladder cancer cells,
5 x 105 cells were infected with
different concentrations of AdCMV-ß-gal at 37°C (8)
.
Twenty-four h after infection, the ß-gal activity was measured in a
200-µl cell lysate and normalized to the protein concentration of
each sample.
Determination of in Vitro Growth Rate of
CAR-transfected Bladder Cancer Cell Line.
To examine the effect of CAR on the cell growth, cells were plated at a
density of 5000 cells in 48-well plates with T-medium containing 0.2%
fetal bovine serum. Relative cell numbers were determined by crystal
violet assay (9
, 10)
at the indicated time.
Western Analysis of p21 and pRb Expression.
To examine the p21 and Rb protein levels in each transfectant, we
performed Western blot analysis (11)
. The cell lysate was
prepared 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. Twenty µg of
total protein from each sample were subjected to a 10%
SDS-polyacrylamide gel and electrotransferred to a nitrocellulose
membrane. After blocking with PBS containing 5% powdered milk, the
membrane was incubated with either 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).
Cell Adhesion Assay.
Cells were trypsinized into a signal cell suspension at a concentration
of 1 x 106 cells/ml. Cell
suspension (1 ml) in a 1.5-ml Eppendorf tube was mixed gently at room
temperature to allow cell aggregates to form. Samples were taken over a
5-h period, and the number of single cells was determined using a
hemacytometer. The degree of cell adhesion was represented as a
decrease in the percentage of single cells (12)
.
Statistical Analysis.
All data were evaluated using Student’s t test. Probability
values less than 0.05 or 0.01 were considered significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, using semiquantitative RT-PCR assay, a wide spectrum of
CAR mRNA expression was detected. However, we observed that all of the
normal tissue expressed detectable levels of CAR mRNA. As shown in Fig. 1B
, the CAR levels in specimens obtained from
pT1 (0.96 ± 0.11) and
pT2 (0.85 ± 0.26) are similar.
However, based on the pathological feature of each specimen, the mean
values of T:N ratio of CAR in pT3,
pT4, or pTxN+ were
significantly lower than values in pT1
(P < 0.05). Furthermore, the T:N ratio of
CAR in specimens with grade 3 tumor is significant lower than grade 2
tumor (Fig. 1C)
. These data indicate that decreased
expression of CAR is often associated with high grade and/or stage of
human bladder cancer.
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as determined by AdCMV-ß-gal.
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, CAR appears to be a typical immunoglobulin-like protein
containing extracellular, transmembrane, and intracellular domains. To
determine which domain is crucial for adenoviral infection in human
bladder cancer cells, we transfected a series of CAR expression vectors
into T24 cells, including GPI, Tailless, and Tailless-m (Fig. 2A)
. GPI cDNA contains only the extracellular domain of CAR
with an additional glycolipid anchor domain for membrane attachment.
Tailless cDNA contains the extracellular domain, transmembrane domain,
and the first two amino acids from the intracellular domain of CAR.
Tailless-m cDNA contains both the extracellular and transmembrane
domains. For T24 cells, two independent CAR clones (T24-CAR3 and
T24-CAR4), one plasmid control (T24-vector) and six independent clones
of CAR mutants (T24-GPI13, T24-GPI24, T24-Tailless1, T24-Tailless7,
T24-Tailless-m1, and T24-Tailless-m2) were selected according to
different plasmid DNA integration patterns (data not shown).
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Cytometric analysis of the transfected bladder cancer cells using CAR
monoclonal antibody (RmcB) was performed 1 month after G-418 selection.
As shown in Table 1
, the amount of CAR-positive cells in T24-CAR2 or
CAR3 was 61 and 45%, respectively. In contrast, the amount of
CAR-positive cells in the T24-vector clone was 
4%. Cytometric
analysis revealed that the CAR-positive cell population was enriched
approximately 10- to 15-fold compared with T24-vector cells. In
contrast, the CAR- positive cells in antisense CAR-transfected 253J and
RT4 clones were decreased (ranging from 50 to 85%) compared with
either parental or vector cells. Results obtained from ß-gal activity
elicited by AdCMV-ß-gal indicated that the ß-gal activity
paralleled CAR levels from each clone.
However, three different CAR mutants (i.e., GPI, Tailless,
and Tailless-m) were still sensitive to adenovirus, which indicates
that the extracellular domain of the CAR protein is critical for virus
binding (Table 1)
. However, we observed a slight decrease of ß-gal in
both T24-GPI10 and T24-GPI13 cells without transmembrane domain of CAR,
suggesting that the transmembrane domain may also contribute to virus
internalization. Nevertheless, these data indicate that viral
sensitivities of bladder cancer cell lines correlate with their CAR
levels.
Cell Adhesion Activity of the CAR Protein.
Because CAR is structurally similar to the immunoglobulin-superfamily
CAM, we thought it important to determine whether CAR behaves like an
adhesion molecule. To do this, the time course of aggregate formation
for all of the transfectants was examined. As shown in Fig. 2B
, T24-parental and T24-vector cells, both with low levels
of CAR, formed few aggregates during the 5-h incubation period.
Generally, all of the CAR-transfected T24 clones formed aggregates in a
time-dependent manner. The extent of aggregation appeared to correlate
well with the level of CAR expression on each subline. Conversely, the
antisense CAR-transfected 253J and RT4 cells with lower levels of CAR
formed few aggregates compared with their parental and plasmid control
cells (Fig. 2, C and D)
. We also noticed that
T24-GPI13 and T24-GPI14 formed less aggregate than other sublines,
suggesting that the transmembrane form of CAR molecule has the
better ability to stabilize the cell-cell interaction between
bladder cells.
In Vitro Growth Characteristics of CAR-transfected
Bladder Cancer Cells.
Cell adhesion molecules such as C-CAM1 play an important role in
suppressing the progression of both prostate and bladder cancers
(12, 13, 14, 15)
. Also, the increased CAR expression can inhibit
both in vitro and in vivo growth of human
prostate cancer cell lines (PC-3; Ref. 7
). Therefore, we
decided to examine the effect of CAR on the in vitro growth
rate of bladder cell line cells. Compared with either T24-parental or
T24-vector cells, the growth rate of T24-CAR2 and T24-CAR3 cells
decreased approximately 43 and 30%, respectively, at day 6 (Fig. 3A)
. Because our data indicated that the extracellular domain
of CAR is critical for viral uptake (Table 1)
, we further examined the
structural functional relationship between the CAR domains and its
growth-inhibitory activity. Two Tailless sublines (i.e.,
T24-Tailless1 and Tailless7) grew even slower than T24-CAR2 or T24-CAR3
cells in vitro (Fig. 3, A and B)
. This
higher degree of growth inhibition elicited by the Tailless cDNA may be
attributable to the higher levels of CAR expression from each subline
(Table 1
and Fig. 3A
). For example, the growth rate of
T24-Tailless1 and T24-Tailless cells decreased approximately 64 and
55% compared with T24-vector cells, respectively, at day 6 (Fig. 3A)
. In contrast, two GPI sublines (i.e.,
T24-GPI10 and GPI13) did not exhibit any growth inhibition (Fig. 3A)
, although these sublines expressed similar CAR levels as
T24-CAR2 cells. Furthermore, both T24-Tailless-m1 and T24-Tailless-m2
cells did not exhibit any growth inhibition (Fig. 3A)
,
although both sublines have a high percentage of CAR-positive cells,
which suggests that the first two amino acids in the intracellular
domains of CAR are critical for its tumor-inhibitory activity. These
data indicate that CAR can act as a membrane receptor to elicit the
growth-inhibitory signal in bladder cancer cells.
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. Similarly, the growth rate of two antisense
CAR-transfected RT4 cells also increased 2.5-fold compared with both
RT4-parental and RT4-vector cells at day 7 (Fig. 3C)
. These
finding indicate clearly that CAR is a potent growth inhibitor for
bladder cancer cells.
Moreover, Western blot analyses (Fig. 4, A and B)
provided direct evidence that p21
levels increased in T24-CAR2, T24-Tailless1, and T24-Tailless7 cells in
a time-dependent manner, whereas the growing cells (such as
T24-parental, T24-vector control, T24–GPI, and T24-Tailless-m cells)
had very low p21 proteins. Similarly, the steady-state levels of Rb
protein, a key effector for p21-induced grow inhibition, were
substantially reduced in T24-CAR2, T24-Tailless1, and T24-Tailless7.
Also, the majority of Rb protein was hypophosphorylated in these three
sublines (Fig. 4A)
. In contrast, hyperphosphorylated Rb
protein was associated with the growing cells (such as T24-parental,
T24-vector control, T24–GPI, and T24-Tailless-m cells; Fig. 4, A and B
).
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and C, antagonizing
endogenous CAR levels in either 253J or RT4 cells led to a higher
in vitro growth rate. This was evidenced by decreased p21
levels and increased hypophosphorylated Rb levels in these sublines.
These data suggest that CAR is able to elicit the growth-inhibitory
signals, such as p21 and Rb, to modulate cell cycle regulators inside
the nucleus of bladder cancer cells.
Effect of Adhesion Activity of CAR on Growth Inhibition of Bladder
Cancer Cells.
To examine the specificity of the intercellular adhesion mediated
through the CAR molecule, we tested whether RmcB monoclonal antibody
(2)
can block intercellular adhesion. Aggregates formed by
T24-CAR2, T24-GPI, T24-Tailless, and T24-Tailless-m sublines can be
inhibited in the presence of the RmcB monoclonal antibody (Fig. 5, B–H)
. However, T24-parental cells were not inhibited under
the same experimental condition because this line did not form cell
aggregates (Fig. 5A)
. Moreover, aggregates formed by
253J-parental and RT4-parental cells can be partially blocked by RmcB
monoclonal antibody (Fig. 5, I and J)
.
Noticeably, this inhibition proceeded in a dose-dependent
manner. For example, RmcB with 1:10 dilution had greater inhibitory
activity than two other dilutions (1:100 or 1:2500). We used an
antibody (Chemicon, Inc., Temecula, CA) against integrin
vß5 as the control
antibody because vß5
levels in T24, 253J, and RT4 cells were 85.3, 88.9, and 1.7%,
respectively. Nevertheless, the presence of control antibody failed to
impede cell aggregation. These results indicate that the aggregation of
CAR-positive cells can be mediated through CAR proteins.
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. In the presence of RmcB antibody, the growth rate of T24-parental,
T24-GPI13, T24-GPI24 cells, T24-Tailless-m1, and T24-Tailless-m2 did
not change (Fig. 6, A, C, D, G, and H
),
suggesting that the intracellular domain of CAR is required for its
growth suppression. However, the growth rate of T24-CAR2,
T24-Tailless1, and T24-Tailless7 cells increased in the presence of
RmcB antibody (Fig. 6, B, E, and F)
.
Similarly, the RmcB antibody was able to enhance the growth rate of
253J- and RT4-parental cells (Fig. 6, I and J)
.
Moreover, as shown in Table 2
, the neutralizing effect of RmcB antibody on the in vitro
growth inhibition of CAR-positive cells proceeded in a dose-dependent
manner. In all experiments, the control antibody (anti-integrin
vß5) did not have any
effect on CAR-transfected cells. These data indicate that the growth
inhibition of bladder cancer cells can be mediated by the cell adhesion
activity of CAR proteins.
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. Apparently, treatment of RmcB antibody can stimulate the
growth rate of T24-CAR2 and T24-Tailless1 cells that were accompanied
with the decreased p21 levels and increased hyperphosphorylated Rb
levels in these cells. RmcB did not completely suppress the p21 and Rb
levels in T24-Tailless1, which may be attributable to the very higher
CAR expression in these sublines. Also, RmcB antibody failed to block
the cell growth of T24-Tailless-m sublines. This was evidenced by no
elevation of either p21 or hypophosphorylated Rb levels (Fig. 6, G
and H, and Fig. 7A
). Furthermore,
RmcB antibody can restore the growth rate of 253J- and RT4-parental
cells, which was reflected by the decreased p21 levels and the
increased hyperphosphorylated Rb levels in these cells (Fig. 7B)
. These data indicate that interruption of CAR
interaction is critical for blocking the transduction of the
growth-inhibitory signal into the nucleus of bladder cancer cells.
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DISCUSSION |
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With both receptor binding and virus infectivity assays, we found that
the levels of CAR correlate with the viral sensitivity of each cell
line determined. To reverse viral resistance of T24 cells, we
genetically engineered T24 cells by increasing CAR-positive cells to
61% (T24-CAR2) or to 45% (T24-CAR3). Transgene activity in
CAR-positive cells such as ß-gal increased about 10- to 30-fold
compared with the control cells (Table 1)
. Because a lower dose of
adenovirus delivery into CAR-positive cells can achieve the same
therapeutic outcome as a higher dose, careful determination of CAR
status in target tissue must be evaluated prior to gene therapy trials
to avoid undesirable cytotoxicity or immune response caused by
excessive administration of adenovirus.
Recently, we reported that a wide spectrum of CAR levels exists among
several human bladder cancer lines (4)
. Similar reports
confirm this observation in other cancer types such as melanoma and
glioma (19, 20, 21)
. With Northern blot and semiquantitative
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. We also found that
bladder cancer specimens from pT3,
pT4, or pTxN+ had
significantly reduced levels of CAR mRNA compared with those from
either pT1 or pT2 (Fig. 1B)
. Also, bladder cancer with high grade expressed less CAR
mRNA than cancer with low grade (Fig. 1C)
. This finding has
several significant implications regarding the impact of CAR status on
the efficacy of gene therapy trials for human bladder cancer and on the
biological role of CAR in disease progression.
We noticed that CAR-transfected T24 cells showed increased cell
aggregation compared with either the T24-parental or the T24-vector
cells, which indicates that CAR is a typical CAM (Fig. 2)
. We also
observed a similar degree of cell aggregation from T24-GPI,
T24-Tailless, and T24-Tailless-m sublines. In addition, the
CAR-specific antibody is also able to interrupt the cell aggregation of
these sublines (Fig. 5)
. Therefore, these data indicate that the
extracellular domain of CAR has an adhesion activity. We observed
decreased cell aggregation in antisense CAR-transfected 253J and RT4
sublines compared with their parental or vector cells. Honda et
al. (22)
report that CAR has the intercellular
adhesion activity in the developing mouse brain, which suggests that
CAR may mediate cell-cell recognition during brain development. Taken
together, we believe that cell adhesion activity of CAR may play a
potential role in both bladder and brain biology.
To determine which CAR functional domain is responsible for its
tumor-inhibitory activity, CAR deletion mutants in T24 cells were
examined using in vitro growth tumorigenicity assay. CAR
from which all but two cytoplasmic domain residues had been deleted
still retained its tumor-inhibitory activities; CAR lacking the entire
cytoplasmic domain or CAR lacking both transmembrane and cytoplasmic
domains did not (Fig. 3A)
. This indicates that both the
transmembrane domain and the first two amino acids (i.e.,
cysteine-cysteine) in the intracellular domain of CAR are critical for
growth inhibition in bladder cancer cells. The cysteine residues in the
intracellular domain of CAR may be a potential site for
posttranslational lipid modification such as farnesylation,
prenylation, and palmitoylation (23, 24, 25, 26)
. It is known that
posttranslational lipid modification plays a critical role in
protein-protein interaction and protein-membrane association, which is
a key mechanism in modulating signal transduction. Therefore, CAR may
associate with effector protein(s) to elicit the growth-inhibitory
signal cascade.
In this study, we also delineated the underlying mechanism elicited by
CAR as a potent growth inhibitor. We examined the expression of two key
cell cycle regulators (p21 and Rb) that are involved in bladder cancer
progression (11)
. In general, elevated levels of p21
protein result in the accumulation of hypophosphorylated Rb, which
leads to G1 cell cycle arrest and/or apoptosis
(27
, 28) . Cell lines with high CAR levels and slow growth
showed high levels of p21 protein (Fig. 4)
. Conversely, cells with
low CAR levels or Tailless-m and GPI clones without essential parts of
cytoplasmic domain had rapid growth showed low p21 protein. Moreover,
we found that decreasing endogenous CAR levels in both 253J and RT4
cells by antisense strategy led to fast cell growth accompanied with
p21 decline and increased inactive Rb protein (Fig. 4, C and D)
. These data suggest that CAR may act as a key mediator
for the contact inhibition occurring among normal cells but absent in
cancer cells.
We examined the relationship between the adhesion activity of CAR and
its capacity to inhibit growth of bladder cancer. A CAR-specific
antibody inhibited aggregation of all CAR-positive cell lines (Fig. 5)
and restored the growth rate in cell lines expressing full-length CAR
as well as CAR containing the "functional" cytoplasmic domain
(Table 2
and Fig. 6
). CAR antibody-alleviated growth suppression
of T24-CAR, T24-Tailless, 253J, and RT4 cells was further evidenced by
the decreased p21 protein and increased Rb phosphorylation levels (Fig. 7)
.
We showed previously that C-CAM1 can still inhibit tumor growth without the first immunoglobulin domain (13 , 29) that is required for its homophilic interaction (30) . In contrast, now it appears that CAR-mediated adhesion may be critical for initiating a growth-inhibitory signal cascade. However, intercellular adhesion elicited by CAR is essential, but not sufficient, for its growth-inhibitory activity because the intracellular domain of CAR is needed for recruiting effector protein(s) that modulates cell cycle regulators inside the nucleus of bladder cancer cells. Therefore, a search for any downstream effector(s) is warranted.
Our findings indicate that increased expression of CAR protein can enhance viral uptake and inhibit tumor growth by increasing intercellular contact. This dual function indicates that CAR has significant biological and therapeutic potential to enhance therapy for human bladder cancer. Moreover, our findings indicate that CAR is not just a viral receptor, and it can elicit a negative signal cascade to modulate cell cycle regulators inside the nucleus of bladder cancer cells. These results can be integrated to formulate a new strategy for bladder cancer therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work is supported in part by Grant
NIHHL54734 (to J. M. B.). 
2 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: JT.Hsieh{at}UTSouthwestern.edu
3 The abbreviations used are: CAR, coxsackie and
adenovirus receptor; RT-PCR, reverse transcription-PCR; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; CAM, cell adhesion molecule;
FACS, fluorescent-activated cell scanning; AdCMV-ß-gal, adenoviral
cytomegalovirus-ß-galactosidase; GPI, glycosylphosphatidylinositol;
Rb, retinoblastoma.
Received 5/ 1/01. Accepted 6/29/01.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R.-C. Pong, R. Roark, J.-Y. Ou, J. Fan, J. Stanfield, E. Frenkel, A. Sagalowsky, and J.-T. Hsieh Mechanism of Increased Coxsackie and Adenovirus Receptor Gene Expression and Adenovirus Uptake by Phytoestrogen and Histone Deacetylase Inhibitor in Human Bladder Cancer Cells and the Potential Clinical Application. Cancer Res., September 1, 2006; 66(17): 8822 - 8828. [Abstract] [Full Text] [PDF]
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J. K. Earel Jr., R. L. VanOosten, and T. S. Griffith Histone Deacetylase Inhibitors Modulate the Sensitivity of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Resistant Bladder Tumor Cells Cancer Res., January 1, 2006; 66(1): 499 - 507. [Abstract] [Full Text] [PDF]
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H. C.A. Graat, P. I.J.M. Wuisman, V. W. van Beusechem, J. E. Carette, W. R. Gerritsen, J. Bras, G. R. Schaap, G. J.L. Kaspers, A. Ogose, W. Gu, et al. Coxsackievirus and Adenovirus Receptor Expression on Primary Osteosarcoma Specimens and Implications for Gene Therapy with Recombinant Adenoviruses Clin. Cancer Res., March 15, 2005; 11(6): 2445 - 2448. [Full Text] [PDF]
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F. Kuhnel, B. Schulte, T. Wirth, N. Woller, S. Schafers, L. Zender, M. Manns, and S. Kubicka Protein Transduction Domains Fused to Virus Receptors Improve Cellular Virus Uptake and Enhance Oncolysis by Tumor-Specific Replicatin |
), T24-vector (
), T24-CAR2 (
),
T24-GPI13 (•), T24-GPI24 (
), T24-Tailless1 (
), T24-Tailless7
(
), T24-Tailless-m1 (
), T24-Tailless-m2 (
). C,
253J-parental (
); T24-GPI10 (x);
T24-GPI13 (•); T24-GPI24 (
