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and Its Ligands in Non-Neoplastic and Neoplastic Human Urothelial Cells
From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR) is a
member of the nuclear receptor superfamily of ligand-activated
transcription factors and is expressed in several types of tissue.
Although PPAR reportedly is expressed in normal urothelium,
its function is unknown. We examined the expression of PPAR in
normal urothelium and bladder cancer in an attempt to assess its
functional role. Immunohistochemical staining revealed normal
urothelium to express PPAR uniformly. All low-grade carcinomas were
positive either diffusely or focally, whereas staining was
primarily focal or absent in high-grade carcinomas. A nonneoplastic
urothelial cell line (1T-1), a low-grade (RT4) carcinoma cell
line, and two high-grade (T24 and 253J) carcinoma cell lines in
culture expressed PPAR mRNA and protein. Luciferase assay indicated
that PPAR was functional. PPAR ligands
(15-deoxy-
12,14-prostaglandin
J2, troglitazone and pioglitazone) suppressed the
growth of nonneoplastic and neoplastic urothelial cells in a
dose-dependent manner. However, neoplastic cells were more
resistant than were nonneoplastic cells. Failure to express PPAR or
ineffective transcriptional activity may be some of the mechanisms
responsible for resistance to the inhibitory action of PPAR
ligands.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR) is a
member of the nuclear receptor superfamily of ligand-activated
transcription factors and functions as a regulator of adipocyte
differentiation and lipid metabolism.1,2
PPAR is
expressed in several types of tissue including the kidney, spleen,
colon, breast, and urinary tract as well as adipose
tissue.3-6
Recent studies indicate that this receptor can
induce differentiation in liposarcoma cells,7
monocytes/macrophages,8
and breast cancer cells in
vitro.9
Furthermore, it has been shown that the
growth of colon cancer cells and androgen-independent prostate cancer
cells is inhibited by treatment with PPAR agonists in
vitro and in vivo.10,11
Although these
studies suggest that PPAR may be a potential target for cancer
treatment, Lefebvre and colleagues12
and Saez and
colleagues13
demonstrated that PPAR agonists promoted
the development of colorectal tumors in mice.
We are interested in elucidating the role
of PPAR and its ligands in bladder cancer. Despite its rich
expression in normal urothelium, the role of PPAR in the urothelium
is unknown.5,6
One of the natural ligands for PPAR,
prostaglandin D metabolite
15-deoxy-12,14-prostaglandin
J2
(15d-PGJ2),14,15
is present in urine
abundantly.16
In this study, we examined the expression of
PPAR protein in normal human urothelium and bladder carcinoma tissue
by immunohistochemistry, and tested the effect of two classes of
ligands for PPAR, 15d-PGJ2, and troglitazone
(TRO) and pioglitazone (PIO) (thiazolidinedione
derivatives)10
on the growth of nonneoplastic and
neoplastic human urothelial cells in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Portions of normal ureter derived from 3 nephrectomy specimens
removed for renal cell carcinoma, 2 normal bladder mucosal biopsies,
and 48 bladder carcinoma specimens removed transurethrally were used.
Excised specimens were fixed immediately in cold 4% paraformaldehyde
(EM Science, Gibbstown, NJ) solution. After overnight fixation in the
refrigerator, they were processed by the routine procedure and embedded
in paraffin. Before staining, sections mounted on
poly-L-lysine-coated slides were deparaffinized with
xylene, and rehydrated in graded ethanol. For the purpose of antigen
retrieval, sections were incubated in Target retrieval solution (DAKO,
Carpinteria, CA) at 95°C for 20 minutes and cooled at room
temperature for 20 minutes. After blocking with 3% horse serum in
phosphate-buffered saline, samples were incubated at room temperature
for 3 hours with the monoclonal mouse anti-PPAR antibody (E-8, lot
no.H218; Santa Cruz Biotechnology, Santa Cruz, CA) or the polyclonal
rabbit anti-PPAR1, 2 antibody (Calbiochem, San Diego, CA) diluted to
1:50 or 1:2000 with the blocking solution, and for the subsequent steps
the avidin-biotin-peroxidase complex method with a Vectastain ABC kit
(Vector, Burlingame, CA) was used. Carcinomas were graded according to
the World Health Organization classification.17
Cells and Cell Culture
The three human bladder carcinoma cell lines used were RT4, T24, and 253J.18 The cells were grown in Ham’s F12 (RT4 and T24) or RPMI 1640 (253J) medium supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD) in a humidified atmosphere of 95% air and 5% CO2 at 37°C.
An immortalized nonneoplastic human urothelial cell line (1T-1) was
established in our laboratory.19
It originated from Hu1
cells20
that were derived from the ureter of a 71-year-old
male undergoing nephrectomy for renal cell carcinoma. The benign nature
of the starting material was confirmed by histological examination of
the remaining portion of the ureter. Seven days after plating when
epithelial outgrowth from explants reached 
2 cm in diameter,
infection with an amphotrophic retrovirus vector LXSN16E6E7, containing
E6 and E7 genes of human papilloma virus type 16 (kindly provided by
Dr. D. Galloway, Fred Hutchinson Cancer Center, Seattle, WA) was
performed overnight in 4 ml of keratinocyte serum-free medium (K-SFM;
Life Technologies) in the presence of 4 µg/ml of polybrene (Sigma,
St. Louis, MO). G418 (Life Technologies) at 200 µg/ml was added at 48
hours for selection of immortalized cells. Cells at passage 16 were
subjected to soft agar assay19
and several colonies were
picked up, expanded, and designated as 1T-1, 1T-2, and 1T-3. One
of the clones (1T-1) was used. 1T-1 cells were maintained in K-SFM
supplemented with 50 µg/ml bovine pituitary extract, 5 ng/ml
epidermal growth factor, 100 U/ml penicillin, and 100 µg/ml
streptomycin (Life Technologies) in a humidified atmosphere of 95% air
and 5% CO2 at 37°C.
Isolation of RNA for Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Cytoplasmic RNA from cultured cells was extracted as follows.
Cells grown in monolayers were harvested at an early confluency. RNA
was prepared by lysing cells in hypotonic buffer containing Nonidet
P-40 (Sigma), followed by removal of nuclei. Cytoplasmic RNA was
reverse-transcribed by Moloney murine leukemia virus reverse
transcriptase (Life Technologies) at 42°C for 60 minutes with the use
of random primers (5 µmol/L; Life Technologies). Subsequently, 1 µl
of the product was subjected to PCR amplification. PCR was performed as
follows. The final concentration of deoxynucleotide triphosphates and
primers in the reaction mixture was 200 µmol/L and 1 µmol/L,
respectively. Taq DNA polymerase (Cetus Perkin-Elmer,
Norwalk, CT) was added to the mixture at a final concentration of 0.05
U/ml, and the reaction was performed in a DNA thermal cycler (Cetus
Perkin-Elmer). To amplify PPAR1 (790 bp) and PPAR2 (877 bp), the
nucleotide bases used were 5'-CCG CTC GAG CGG GCC GCC GTG GCC GCA
GAA-3' as an upstream primer for human PPAR1, 5'-CCG CTC GAG CGG AAA
CCC CTA TTC CAT GC-3' as an upstream primer for human PPAR2, and
5'-AGG AAT TCA TGT CAT AGA TAA CG-3' as a downstream primer for both
PPAR1 and 2 and 5'-GAA ATC CCA TCA CCA TCT TCC AGG-3' as an upstream
primer and 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3' as a downstream primer
for glyceraldehyde-3-phosphate dehydrogenase.21
Western Blotting
Cells grown in monolayers were harvested at subconfluency and
lysed with a lysing buffer [62.5 mmol/L Tris (pH 6.8), 2% sodium
dodecyl sulfate, 10% glycerol, 5% ß-mercaptoethanol, and 7 mol/L
urea] (Sigma). The sample was boiled for 10 minutes and was then
forcefully passaged five times through a 25-gauge needle. The samples
were centrifuged at 12,000 x g for 10 minutes and the
precipitates were discarded. Fifty-µg protein samples of the
supernatant were electrophoresed on 10% sodium dodecyl
sulfate-polyacrylamide gel. Protein was transferred to polyvinylidene
difluoride membrane (Bio-Rad, Hercules, CA), and the membrane was
incubated with an anti-PPAR antibody (E-8; Santa Cruz Biotechnology
or Calbiochem), anti-PPAR binding protein (PBP)
antibody,22
or anti-retinoid X receptor 
(RXR)
antibody (D-20; Santa Cruz Biotechnology) and treated with an enhanced
chemiluminescence kit (Amersham, Arlington Heights, IL). Densitometric
analysis was done with an NIH Image 1.59.
Cell Growth Assay
Cells were seeded on a flat-bottom 96-well plate (Falcon, Becton
Dickinson, Franklin Lakes, NJ) at the density of 2 x
103
(1T-1, T24, and 253J) or 5 x
103
(RT4) cells per well in the respective
appropriate growth medium. Twenty-four hours later, cells were grown in
the same medium containing 15d-PGJ2 (0 to 10
µmol/L; Cayman Chemical, Ann Arbor, MI), TRO (0 to 50 µmol/L; a
gift from Parke-Davis, Ann Arbor, MI), or PIO (0 to 50 µmol/L; a gift
from Takeda Chemical Industries, Ltd., Tokyo, Japan). After 24 hours of
incubation, cell proliferation was assessed by Cell proliferation
enzyme-linked immunosorbent assay, BrdU (Roche Molecular Biochemicals,
Mannheim, Germany). We also assessed cell number by manual counting;
cells were seeded on a flat-bottom 6-well plate (Falcon, Becton
Dickinson) at the density of 5 x 104
cells
per well. Twenty-four hours later, cells were treated with above
PPAR ligands. After 3 days, cells were recovered by treatment with
0.05% trypsin-0.53 mmol/L ethylenediaminetetraacetic acid (Life
Technologies) and counted with a hemocytometer.
Transfections and Luciferase Assay
Cells were seeded on a flat-bottom 6-well plate (Falcon, Becton
Dickinson) at the density of 3 x 105
cells
per well in the respective appropriate growth medium. Twenty-four hours
later, transfection was done by using the Effectene transfection
reagent (Qiagen, Valencia, CA) mix with a reporter plasmid PPRE (PPAR
response elements)-TK-LUC produced in our laboratory.23
The PPRE-TK-LUC was constructed by inserting three copies of PPRE
(AGGACAAAGGTCA) into
HindIII/SalI site of TK-LUC. The transfection mix
was replaced with the complete medium with or without PPAR ligands
(15d-PGJ2 and TRO) and was further incubated for
24 hours. The cells were lysed with cell culture lysis reagent
(Promega, Madison, WI). Luciferase activity was measured with the use
of a luciferase assay reagent (Promega) in a scintillation counter
(Aloka, Tokyo, Japan).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein in Normal
Urothelium and Bladder Cancer Tissue
PPAR protein was uniformly expressed in the urothelium of three
normal ureters and two normal bladder mucosal biopsies and was
localized to nuclei (Figure 1A)
. The
staining reaction was more intense in the superficial and intermediate
cells than in the basal cells. Localization and staining intensity were
similar when the results with the two antibodies were compared.
Expression of PPAR protein in bladder carcinoma cells is summarized
in Table 1
. All cases of transitional
cell carcinomas of low grades (grade 1 and 2) demonstrated an intense
nuclear staining either in all cells or focally up to 90% of tumor
cells (Figure 1B)
. In contrast, staining was focal to absent in
high-grade carcinomas (Figure 1, C and D)
. Statistically significant
loss of PPAR expression was evident in grade 3 carcinomas as
compared to the expression in combined grade 1 and 2 carcinomas
(P = 0.0007, Fisher’s exact test).
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in Nonneoplastic and
Neoplastic Urothelial Cells
First, we examined the expression of PPAR mRNA in immortalized
nonneoplastic and neoplastic urothelial cells. PPAR1 mRNA was
detected by RT-PCR in all cell types whereas PPAR2 mRNA was
expressed only in RT4 cells (Figure 2A)
.
By Western blotting, PPAR (58 kd) was detected at varying levels
in all cell types (Figure 2B)
. Expression of PPAR protein was
reduced by 49, 63, and 68%, respectively, in RT4, T24, and 253J cells
as compared to the expression in 1T-1 cells. By immunocytochemical
staining, the protein was uniformly localized to nuclei in all cells in
all cell lines (data not shown).
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We next tested the effect of PPAR ligands on the growth of
immortalized nonneoplastic and neoplastic cells. Treatment with PPAR
ligands suppressed the growth of all tested cells in a dose-dependent
manner (Figure 3)
. Low-dose
15d-PGJ2 (0.5 µmol/L) almost completely
inhibited the growth of 1T-1 cells. In contrast, all carcinoma cell
lines were resistant to its suppressive effect at concentrations up to
1 to 5 µmol/L (Figure 3A)
.
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. Treatment with another synthetic PPAR ligand, PIO, also showed
the similar inhibitory effect on nonneoplastic and neoplastic human
urothelial cell lines except 253J cells (Figure 3C)
.
Ligand-Induced Transcriptional Activity of PPAR
We examined the transcriptional activity of PPAR using a
luciferase reporter plasmid containing a PPAR response element. TRO
ranging from 0.1 µmol/L to 50 µmol/L and
15d-PGJ2 from 0.1 to 10 µmol/L were tested. The
concentrations of TRO shown in Figure 4
are those that resulted in the best transcriptional activity. We could
not examine the activity of RT4 cells because of low transfection
efficiency. Treatment of 1T-1 cells with TRO (0.1 µmol/L) strikingly
increased the transcription of luciferase gene by 5.2-fold. In T24 and
253J cells TRO (10 or 20 µmol/L) activated transcription by twofold
(Figure 4)
. Treatment with 15d-PGJ2 (0.1
µmol/L) increased luciferase activity by twofold in 1T-1 cells.
However, luciferase activity by 15d-PGJ2 (up to
10 µmol/L) was not detected in T24 and 253J cells (data not shown).
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Protein in Nonneoplastic and
Neoplastic Urothelial Cells
We examined the expression of PBP, a PPAR co-activator, and
RXR, a PPAR heterodimeric partner, by Western blotting. PBP
protein (165 kd) was detected only in RT4 cells (Figure 5)
. RXR protein (55 kd) was
expressed in all cell lines. 1T-1 and RT4 cells expressed RXR
protein at a much higher level than did T24 and 253J cells (Figure 5)
.
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ligand,
9-cis-retinoic acid (9-cis-RA) on a PPAR
ligand. In RT4, T24, and 253J cells, 9-cis-RA (5 µmol/L)
enhanced the inhibitory effect of TRO by 1.1-fold, 1.5-fold, and
twofold, respectively. The growth of 1T-1 cells was inhibited
completely by treatment with 9-cis-RA (5 µmol/L) alone
whereas it had no effect on the growth of all carcinoma cell lines
(data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ligands mostly inhibit
the growth of breast,9
prostate,10
and colon
cancer11
cells in vitro and in vivo.
We demonstrated here that PPAR was expressed in nonneoplastic and
neoplastic urothelial cell lines at both mRNA and protein levels.
However, the sensitivity to the inhibitory effects by PPAR ligands
(15d-PGJ2, TRO, and PIO) was variable.
Nonneoplastic urothelial cell line 1T-1 was sensitive to the inhibitory
effects of PPAR ligands. In contrast, carcinoma cell lines were
resistant.
Studies by others using various carcinoma cell lines all indicate that
PPAR ligands reduce growth rate.10,11,24,25
Thus
PPAR ligands have been suggested to be a useful therapeutic agent
for breast, prostate, or colon carcinomas.9-11
The
rationale behind this approach is that whereas normal cells either do
not express PPAR or, if they express, only at a very low level,
whereas carcinoma cells express the receptor abundantly.10
This allows selective action of PPAR ligands on neoplastic cells and
induces cell death or differentiation. However, there have been
recent reports indicating that treatment with a PPAR ligand promotes
the development of colon tumor in Min+/- mice that lack one functional
copy of the APC tumor suppresser gene.12,13
Normal human ureter reportedly expressed PPAR protein.5
In the present study, we confirmed this observation.
However, in bladder carcinoma tissues as well as in carcinoma cell
lines, its expression seemed affected by the degree of malignancy: in
low-grade (grades 1 and 2) carcinomas, PPAR protein was uniformly or
diffusely demonstrated by immunohistochemistry, whereas in high-grade
(grade 3) carcinomas the expression of PPAR protein was mostly
heterogeneous or absent.
In the in vitro study we demonstrated that high-grade
carcinoma cell lines T24 and 253J expressed RXR at a lower level
than did the nonneoplastic cell line 1T-1 and a low-grade carcinoma
cell line RT4. Expression of co-factor PBP was almost exclusive to RT4
cells. The luciferase reporter assay indicates that ligand-induced
transcriptional activity of PPAR is most active in 1T-1 in which a
fivefold to sixfold increase was observed at the TRO concentration as
low as 0.1 µmol/L. On the other hand, transcriptional activity of
carcinoma cells was low (up to twofold) requiring ligand concentrations
as high as 10 to 20 µmol/L. Treatment with
15d-PGJ2 did not induce transcriptional activity.
It is possible that mutations in PPAR gene may affect
ligand-dependent transcriptional activity.26,27
In human
colon cancer,26
two missense mutations were detected in
the ligand-binding domain and impaired the function of the protein. One
of mutations showed a normal response to synthetic ligands but
decreased transcription when exposed to natural ligands. Though
15d-PGJ2 significantly inhibited the growth of
neoplastic urothelial cells, it induced no PPRE luciferase activity.
The result suggests that the growth-inhibitory action of
15d-PGJ2 may depend on some other mechanisms. For
example, according to a recent report,28
15d-PGJ2 is a direct inhibitor of IkB kinase
(IKKß) and prevents nuclear factor-kB activation.
Based on these observations we suggest several mechanisms to account
for the differential response of urothelial cells to PPAR ligands:
nonneoplastic cells are highly sensitive to their cytocidal effects
because of their efficient transcriptional activity whereas carcinoma
cells are resistant because of low transcriptional activity or because
of their failure to express PPAR. Our data suggest that intravesical
administration of PPAR ligand in an attempt to treat bladder cancer
may result in severe cytotoxic effects on normal urothelial cells
before therapeutic effects on cancer cells can be demonstrated.
Furthermore some cancer may be totally refractory because of their
mutations or failure to express PPAR. Additional studies are needed
before therapy with PPAR ligands is attempted.
|
Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants CA 14649, CA 33511, and GM 23750; and by the Joseph L. Mayberry Sr. Research fund.
Accepted for publication April 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2, a lipid-activated transcription factor. Cell 1994, 79:1147-1156[Medline]
(PPAR) and its coactivators steroid receptor coactivator-1 and PPAR-binding protein PBP in the brown fat, urinary bladder, colon, and breast of the mouse. Am J Pathol 1998, 153:349-354 and the retinoid X receptor. Proc Natl Acad Sci USA 1997, 94:237-241 promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 1998, 93:241-252[Medline]
. Mol Cell 1998, 1:465-470[Medline]
(Troglitazone) has potent antitumor effect against human prostate cancer both in vitro and in vivo. Cancer Res 1998, 58:3344-3352. Nat Med 1998, 4:1046-1052[Medline]
promotes the development of colon tumors in C57BL/6J-APCmin/+mice. Nat Med 1998, 4:1053-1057[Medline]
enhance colon polyp formation. Nat Med 1998, 4:1058-1061[Medline]
and promotes adipocyte differentiation. Cell 1995, 83:813-819[Medline]
. Cell 1995, 83:803-812[Medline]
9, 12-13, 14-dihydroprostaglandin D2 in human urine. J Biol Chem 1988, 263:16619-16625. Gene Exp 1996, 6:185-195
induces growth arrest and differentiation markers of human colon cancer cells. Jpn J Cancer Res 1999, 90:75-80[Medline]
and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci USA 1998, 95:8806-8811 associated with human colon cancer. Mol Cell 1999, 3:799-804[Medline]
associated with severe insulin resistance, diabetes mellitus and hypertension. Nature 1999, 402:880-883[Medline]
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