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Introduction
Coxsackie and adenovirus receptor (CAR) has primarily
been studied for its role as the initial cell surface attachment
receptor for coxsackie and group C adenoviruses. CAR is a
46 kDa class I membrane glycoprotein with a
carboxy-terminal cytoplasmic domain, a transmembrane domain, and an
extracellular region consisting of 2 immunoglobulin-like
domains, namely, an amino-terminal immunoglobulin (Ig)
variable-related domain (D1), which is distal to the cell
surface, and a proximal IgC2 domain
(D2)[1]. There is evidence for the evolutionary conservation of CAR, with
homologues of the human receptor, hCAR, present in several
mammalian species, including mice[2], rats, dogs, and
pigs[3]. The presence of this protein significantly enhances the
efficiency of adenoviral vector-mediated gene
transfer[4]. However, the normal physiological function of this
membrane protein is still not completely understood.
Recent studies suggest that CAR mediates homotypic
intercellular adhesion as part of the tight and/or adherent
junction[5]. CAR plays a role in tumorigenesis. In this respect,
CAR expression has been correlated with both tumor
suppression[6] and malignant
transformation[7]. More recently, it has been shown that the expression of CAR in highly
tumorigenic CAR-deficient human prostate
cancer[8] and malignant glioma
cells[6] leads to growth inhibition. Given that
CAR is well positioned to participate in these roles, we
investigated the effect of its overexpression on the human
bladder cancer cell in vitro and in
vivo.
Materials and methods
Cell culture and reagents Retroviral packaging cell line
PT67[9], NIH3T3 fibroblasts, and human bladder cancer cell
line T24 were obtained from the American Type Culture
Collection (Manassas, VA, USA). The T24 cell line was
established in 1970 from a urinary bladder
carcinoma[10], which was identified as CAR-negative in the previous
study[11]. The cells were cultured in high-glucose Dulbecco's
modified Eagle's medium (DMEM) (Gibco, NY, USA) at 37 °C
supplemented with 10% fetal bovine serum (FBS). Retroviral
vector pLXSN was purchased from Clontech Co ( BD
Biosciences Clontech, Palo Alto, CA, USA). The plasmid
pTOPOCAR[4], including full length CAR cDNA, was
obtained from Dr Jer-tsong HSIEH (Department of Urology,
University of Texas Southwestern Medical Center, Dallas,
TX, USA).
Construction of CAR-encoding retroviral vector
An EcoR I/BamH I fragment encoding human CAR was obtained
from pTOPOCAR and subcloned into a pLXSN backbone to
generate the retroviral vector, named pLXSN-CAR.
Generation of viral particles and infection of target cells
pLXSN and pLXSN-CAR were transfected into the PT67
packaging cells by Lipofectamine 2000 (Invitrogen, Carlsbad,
CA, USA). PT67 packaging cells were split and grown in the
selective medium with 500 mg/L G418. After 2 weeks, the
G418-resistant colonies were picked and expanded in
medium with 300 mg/L G418, and the supernatant viral particles
were collected. The titer of viral stocks was determined by
infection of the NIH3T3 cells[12]. The titer of pLXSN-CAR
and pLXSN retrovirus was 1.6×109 colony-forming units/L
and 2.0×109 colony-forming units/L, respectively.
For pLXSN-CAR retroviral transduction, the T24 cells
were cultured and allowed to grow to subconfluence. The
medium was then replaced with a 1:1 precipitated mixture of
CAR retroviral supernatant and fresh complete medium.
Polybrene (Sigma, St Louis, MO, USA) was added to the
culture medium at a final concentration of 8 mg/L to enhance
the binding of the virus to the host cells. The transduction
was repeated the following day. Twenty-four hours post
final transduction, the cells were harvested, resuspended,
and maintained in selective medium containing 0.6 g/L
G418 for up to 14 d. The G418-resistant cell clones were
pooled, expanded, and tested for CAR expression. The
control pLXSN retroviral transduction was performed as
that of the pLXSN-CAR retrovirus. At last, 6 clones of
CAR-positive cells and 8 clones of pLXSN cells were
selected and expanded, respectively. Six CAR clones
continuously expressed high levels of the CAR protein, so all
the 6 CAR clones and 1 of the 8 pLXSN cell clones were
applied for further study. The T24 cells successfully
infected with the pLXSN-CAR retrovirus and the control
pLXSN retrovirus were designated as T24/pLXSN-CAR and
T24/pLXSN, respectively.
Western blotting and immunofluorescence staining
Western blotting and immunofluorescence staining for the
CAR protein in T24 cells infected with the CAR retrovirus
and control virus were carried out as described in a previous
report[13]. Briefly, a total protein extract (30 µg) isolated from
harvested cells was then separated by 12% SDS-PAGE.
Subsequently, the separated protein was transferred to
nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and
the membrane was blocked with 5% non-fat milk in TBS for
1.5 h. The membranes were then incubated for 1.5 h with the
primary antibody against CAR at a dilution of 1:600 (Santa
Cruz, CA, USA), and then hybridized for 1 h with the
secondary antibody at a dilution of 1:5000. Immunodetection
was performed using the ECL detection system (Pierce,
Rockford, IL, USA). Protein loading equivalence was
assessed by the expression of GAPDH.
To confirm the CAR expression, bladder cancer cells were
fixed in 4% paraformaldehyde for 10 min and blocked with
goat serum for 30 min, respectively. The cells were then
incubated at 37 °C for 1 h with mouse anti-human CAR
monoclonal antibody (Upstate Inc, Lake Placid, NY, USA) at a
dilution of 1:150. After washing 3 times with PBS, the
cells were incubated with tetramethylrhodamine
isothio-cyanate (TRITC)-conjugated goat anti-mouse antibody at
37 °C for 1 h. The fluorescence staining intensity and
intercellular location were then examined by fluorescence
inverted microscope (Olympus IX 50, Tokyo, Japan).
Cell proliferation assay Cell growth was evaluated by
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) (Sigma, San Louis, MO, USA) assay. The bladder
cancer cells (1×104/well) were plated in 96-well tissue culture
plates in DMEM containing 10% FBS in a final volume of
0.2 mL. After incubation for 1, 2, 3, and 4 d, the cells were
incubated with 20 µL MTT (at a final concentration of 0.5
mg/mL) at 37 ºC for 4 h. The medium was removed and the
precipitated formazan was dissolved by adding 200 µL DMSO
(Sigma, USA). After shaking for 10 min, the samples were
lysed and the absorbance at 570 nm was detected using a
Bio-Rad Technologies Microplate Reader (USA).
In vitro soft agar colony-formation assay
T24/pLXSN-CAR1, T24/pLXSN-CAR2, T24/pLXSN, and parental T24
cells were collected and dispersed into a suspension of single
cells in growth medium. One thousand cells were
resuspended in 2 mL growth medium containing 0.3% low melting
temperature agarose (Promega, Madison, WI, USA) and were
overlaid in triplicate on 2 mL solidified 0.6% low melting
temperature agarose in growth medium in a 6-well dish. The
dishes were incubated for 2_3 weeks at 37 °C in a 5%
CO2 atmosphere until colonies were formed. Colonies larger than
50 cells were then counted. Descriptive statistics (mean±SD)
on colony sizes were calculated.
In vivo tumorigenicity assay T24/pLXSN-CAR1,
T24/pLXSN, and parental T24 cells were harvested, washed, and
resuspended in serum-free DMEM at the concentration of
5×107 cells/mL, and
1×106 cells were injected subcutaneously
into 6_8-week-old BALB/c-nu/nu mice (n=5 mice per group,
Shanghai Experimental Animal Center, Shanghai, China).
Tumor dimensions were estimated based on bisecting
diameters measured with a caliper, and the tumor volume was
approximated using the formula 0.5 (ab2), where a is the long
measured axis of the tumor, and b is the short measured axis
of the tumor[14]. Tumor volume was measured at least weekly
using the above formula. The mice were sacrificed after 5
weeks to measure tumor weight. All experiments on the
animals complied with the Guidelines of Animal Care of Xi'an
Jiaotong University.
Statistical analysis All data were expressed as mean±SD.
Student's t-test was used. P value <0.05 was considered
statistically significant. All statistical tests were performed
with statistical analysis software (SPSS, Chicago, IL, USA).
Results
Determination of expression of CAR in the
G418-resistant stable T24/pLXSN-CAR transfectant
The pLXSN-CAR vector containing full-length CAR cDNA was successfully
constructed. The expression of the CAR protein was
detected by Western blot analysis and immunofluorescence
staining. We found a high level of 46 kDa CAR expression in
the T24/pLXSN-CAR cell clones. The CAR protein was
undetectable in the parental T24 cells and control T24/pLXSN
cells (Figure 1). Immunofluorescence staining was performed
to confirm whether the retrovirus could infect and express
the CAR protein in T24 cells. The intensity of the
CAR-specific cell membrane immunofluorescence signal
significantly increased in the T24/pLXSN-CAR cells, while in the
T24/pLXSN and parental T24 cells, only a poor signal was
observed (Figure 2).
Effect of CAR overexpression on T24 cell growth
To examine the effect of the CAR protein on the cell growth of
T24 cells, we determined the growth rate of the CAR
transfectant, control, and parental cells. Both T24/pLXSN
and T24 cells exhibited a higher growth rate than
T24/pLXSN-CAR1 and T24/pLXSN-CAR2, which was statistically
significant (P<0.05; Figure 3B). Noticeably, no significant
difference in the cell growth rate between the T24/pLXSN and
T24 cells was detected (P>0.05).
Effect of CAR on anchorage-independent growth
The increased expression of CAR in T24 cells caused a
significant reduction in the number of colonies formed by the
T24/pLXSN-CAR1 cells (71±9) and T24/pLXSN-CAR2 cells
(80±12) compared with the number of colonies formed by
parental T24 or T24/pLXSN cells (216±24 and 202±14,
respectively, P<0.05; Figure 4). In contrast, no significant
difference was detected in the clone number between
parental T24 cells and T24/pLXSN cells (P>0.05). Since
anchorage-independent growth is considered in
vitro tumorigenic assay, these data suggested that the overexpression of CAR
could cause tumor inhibition in vitro.
Reduced tumor growth of T24 cells with elevated CAR
expression Tumor xenografts were established by
subcutaneous injection of 1×106 cells from parental, control and
CAR-transfected cells into the flank of 6_8-week-old
BALB/c-nu/nu mice (n=5 mice per group). The mean tumor volume on d
7, 14, 21, 28, and 35 are shown in Figure 5A.
T24/pLXSN-CAR1 tumors were much smaller than T24/pLXSN and
parental T24 tumors (P<0.05). The volume of the T24 tumors
was not significantly different from that of the T24/pLXSN
tumors (P>0.05). On the thirty-fifth day after implantation,
the average weight of the T24/pLXSN-CAR1 tumors was,
significantly lower (217±30 mg) than that of tumors formed
by the parental T24 cells (651±84 mg) and T24/pLXSN cells
(600±52 mg, P<0.05; Figure 5B). The overexpression of CAR
in T24 cells resulted in a significant inhibitory effect on the
growth of the T24 tumors.
Discussion
CAR is a 46 kDa transmembrane glycoprotein, first
identified as a high affinity receptor for both coxsackie and
adenovirus (types 2 and 5)[1,2]. The presence of this protein
significantly enhances the efficiency of adenoviral
vector-mediated gene transfer[4]. Structurally, CAR is a typical
Ig-like molecule with 2 Ig domains that interact with the
adenovirus fiber protein. Most likely, the structure of this
protein suggests that CAR may have other functions in
addition to being a virus receptor[1]
. Recent reports have indicated that CAR has homophilic interactions and appears
to form a complex with ZO-1 in the tight junction of
polarized epithelial cells[15]. Also, the loss of the CAR expression
is associated with high grade
cancer[16], and hence, it is
involved in cell differentiation.
Using semiquantitative RT-PCR, an apparent down-
regulation of CAR mRNA levels in the invasive tumors
compared with superficial bladder cancer specimens has been
documented[16]. Moreover, the study using
immunohistochemical staining indicates that bladder cancers reveal a
marked downregulation of CAR associated with stage and
grade[17]. This suggests that altered CAR expression would
be involved in the progression of bladder cancer. In
addition to bladder cancer, the downregulation of CAR is also
reported in ovarian cancer[18],
melanoma[19], and head and neck
cancer[20]. It has also been reported that the increased
expression of the CAR gene leads to the inhibition of several
of these tumors. However, the detailed characterization of
the biological function of CAR is still lacking.
In the present study, we have examined the effect of CAR
on bladder cancer cells based on cell growth in
vitro and tumor growth in vivo. Using a highly efficient retroviral
vector, we found that pLXSN-CAR-infected T24 cells grew
much slower than the control retrovirus-infected or parental
T24 cells. Noticeably, elevated CAR in T24 cells caused a
significant reduction in the number of colony formations
when T24 cells were plated on a semisolid agar plate. To
support the in vitro results, animal xenografts have resulted
in the same conclusion: tumors formed by T24 cells infected
with the CAR retrovirus grow significantly slower than those
by T24 cells infected with empty retroviral or parental T24
cells.
Our findings have demonstrated that CAR serves as a
potent tumor suppressor in bladder cancer cells both
in vitro and in vivo. Nevertheless, some reports suggest that the
downregulation of CAR will reduce the volume of lung
cancer in a mouse xenograft model[21], implying that the
biological effect of CAR in different cancer types may vary.
There-fore, further study is needed for elucidating the different
mechanisms of the action of CAR in the development of
different types of cancer.
Acknowledgement
We would like to thank Jer-tsong HSIEH (Department of
Urology, University of Texas Southwestern Medical Center,
5323 Harry Hines Boulevard, Dallas, TX, USA) for criticism
and encouragement.
References
1 Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA,
Krithivas A, Hong JS, et al. Isolation of a common receptor for
Coxsackie B viruses and adenoviruses 2 and 5. Science 1997;
275: 1320_3.
2 Tomko RP, Xu R, Philipson L. HCAR and MCAR: the human
and mouse cellular receptors for subgroup C adenoviruses and
group B coxsackie viruses. Proc Natl Acad Sci USA 1997; 94:
3352_6.
3 Fechner H, Haack A, Wang H, Wang X, Eizema K, Pauschinger
M, et al. Expression of coxsackie adenovirus receptor and
alphav-integrin does not correlate with adenovector targeting
in vivo indicating anatomical vector barriers. Gene Ther 1999; 6:
1520_35.
4 Li Y, Pong RC, Bergelson JM, Hall MC, Sagalowsky AI, Tseng
CP, et al. Loss of adenoviral receptor expression in human
bladder cancer cells: a potential impact on the efficacy of gene
therapy. Cancer Res 1999; 59: 325_30.
5 Coyne CB, Bergelson JM. CAR: a virus receptor within the tight
junction. Adv Drug Deliv Rev 2005; 57: 869_82.
6 Kim M, Sumerel LA, Belousova N, Lyons GR, Carey DE, Krasnykh
V, et al. The coxsackie and adenovirus receptor acts as a tumour
suppressor in malignant glioma cells. Br J Cancer 2003; 88:
1411_6.
7 Anders M, Hansen R, Ding RX, Rauen KA, Bissell MJ, Korn WM.
Disruption of 3D tissue integrity facilitates adenovirus infection
by deregulating the coxsackievirus and adenovirus receptor. Proc
Natl Acad Sci USA 2003; 100: 1943_8.
8 Okegawa T, Li Y, Pong RC, Bergelson JM, Zhou J, Hsieh JT.
The dual impact of coxsackie and adenovirus receptor
expression on human prostate cancer gene therapy. Cancer Res 2000;
60: 5031_6.
9 Miller AD, Chen F. Retrovirus packaging cells based on 10A1
murine leukemia virus for production of vectors that use
multiple receptors for cell entry. J Virol 1996; 70: 5564_71.
10 Bubenik J, Baresova M, Viklicky V, Jakoubkova J, Sainerova H,
Donner J. Established cell line of urinary bladder carcinoma
(T24) containing tumour-specific antigen. Int J Cancer 1973;
11: 765_73.
11 Lai YJ, Pong RC, McConnell JD, Hsieh JT. Surrogate marker for
predicting the virus binding of urogenital cancer cells during
adenovirus-based gene therapy. Biotechniques 2003; 35: 186_94.
12 Bodine DM, McDonagh KT, Brandt SJ, Ney PA, Agricola B,
Byrne E, et al. Development of a high-titer retrovirus producer
cell line capable of gene transfer into rhesus monkey
hematopoietic stem cells. Proc Natl Acad Sci USA 1990; 87: 3738_42.
13 Li L, He DL. Transfection of promyelocytic leukemia in
retro-virus vector inhibits growth of human bladder cancer cells. Acta
Pharmacol Sin 2005; 26: 610_5.
14 Guo QL, You QD, Wu ZQ, Yuan ST, Zhao L. General gambogic
acids inhibited growth of human hepatoma SMMC-7721 cells
in vitro and in nude mice. Acta Pharmacol Sin 2004; 25: 769_74.
15 Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT, Bergelson
JM. The coxsackievirus and adenovirus receptor is a
transmembrane component of the tight junction. Proc Natl Acad Sci USA
2001; 98: 15 191_6.
16 Okegawa T, Pong RC, Li Y, Bergelson JM, Sagalowsky AI, Hsieh
JT. The mechanism of the growth-inhibitory effect of coxsackie
and adenovirus receptor (CAR) on human bladder cancer: a
functional analysis of car protein structure. Cancer Res 2001; 61:
6592_600.
17 Sachs MD, Rauen KA, Ramamurthy M, Dodson JL, De Marzo
AM, Putzi MJ, et al. Integrin alpha(v) and coxsackie adenovirus
receptor expression in clinical bladder cancer. Urology 2002;
60: 531_6.
18 Dmitriev I, Krasnykh V, Miller CR, Wang M, Kashentseva E,
Mikheeva G, et al. An adenovirus vector with genetically
modified fibers demonstrates expanded tropism via utilization of a
coxsackie and adenovirus receptor-independent cell entry
mechanism. J Virol 1998; 72: 9706_13.
19 Hemmi S, Geertsen R, Mezzacasa A, Peter I, Dummer R. The
presence of human coxsackievirus and adenovirus receptor is
associated with efficient adenovirus-mediated transgene
expression in human melanoma cell cultures. Hum Gene Ther 1998; 9:
2363_73.
20 Li D, Duan L, Freimuth P, O'Malley BW Jr. Variability of
adenovirus receptor density influences gene transfer efficiency and
therapeutic response in head and neck cancer. Clin Cancer Res
1999; 5: 4175_81.
21 Qin M, Escuadro B, Dohadwala M, Sharma S, Batra RK. A novel
role for the coxsackie adenovirus receptor in mediating tumor
formation by lung cancer cells. Cancer Res 2004; 64: 6377_80. |
