10.1016@S0022 53470380045 9

184
THE JOURNAL OF UROLOGY
METHODS: The video highlights our technique of laparoscopic radical

cystectomy and orthotopic diversion in a 61 year-old lady with muscle-invasive
transitional cell carcinoma of the bladder (T2G3). An extended bilateral pelvic
lymph node dissection was also achieved laparoscopically. A 6-port transperitoneal
approach was used. The principal operative steps of laparoscopic cystectomy
included: (I) peritoneotomies to include the urachus, (2) transection of the uterine
broad ligaments, (3) clip ligation of both ureters as low as possible, (4) incision of
the endopelvic fascia, (5) control of lateral and posterior vesicle pedicles using
sequential firing of the reticulating Endo-GIA stapler, (6) division of the urethra,
(7) resection of the anterior vaginal wall, (8) intact specimen extraction through the
vaginal vault, and (9) reconstruction of the vagina. An extended bilateral pelvic
lymph node dissection was then performed. Urinary diversion with a Studer pouch
was performed in 5 steps: (I) isolation of distal ileum and stapled ileal-ileal
anastomosis, (2) detubularization and creation of posterior ileal plate, (3)
urethroileal anastomosis using continuous suturing, (4) completion of the anterior
pouch, (5) stented uretero-ileal anastomosis. A 24 Fr. Foley urethral catheter and
2 Jackson-Pratt drains were left in situ.
RESULTS: The procedure was completed laparoscopically within 9.5 hours.
The estimated blood loss was 400 cc. There were no intraoperative complications,
however the patient developed a vesico-vaginal fistula.
CONCLUSIONS: Laparoscopic radical cystectomy and intracorporeally
constructed neobladder for the female patient is feasible. Vaginal extraction of the
specimen eliminates the need for an extraciton incision. Extended bilateral pelvic
lymph node dissection is safe and feasible.
Source of Funding: None.
V710
LAPAROSCOPIC DETRUSORORRHAPHY IN CHILDREN
WITH VESICO-URETERAL REFLUX Nasser Simforoosh, Pejman
Shadpour", Tehran, Iran
INTRODUCTION AND OBJECTIVE: Numerous reports have attested to the
efficacy of antirellux surgery by laparoscopic approach in animal models. Very few
clinical studies have also appeared in the literature. Herein, we present our
encouraging results with due follow up.
METHODS: 24 children (aged 1.5 to 12 years), were treated for symptomatic
anatomical rellux by Lich-Gregoir detrusororrhaphy, via trans peritoneal
laparoscopy. 31 relluxing units were treated altogether. A tunnel averaging 2.5 em
long was created by free-hand suturing, using 2-0 absorbable material. No catheter
or drain was placed in any but 6 cases. The patients have been followed for 3-24
months (mean 5.7) with sonography, urine culture, and cystography.
RESULTS: 96.7% success was gained in primary rellux. Operative and
hospitalization times averaged 113 minutes, and 1.8 days respectively. All patients
have so far maintained sterile urine off antibiotics after the documented resolution
of rellux. Irritative symptoms and hematuria did not occur. No patient experienced
any intra operative complication. One patient reported hesitancy & intermittency
but voids to completion with normal 1I0w in follow-up studies. Two omental
protrusions upon drain removal were treated locally. The procedure was equally
effective in those failing previous antirellux treatment.
CONCLUSIONS: The merits of extravesical laparoscopic antirellux surgery
are obvious in terms of cosmesis, shorter hospital stay, and less morbidity. Surgical
results are durable, and the possibility for minimizing damage to bladder
innervation by limited retrovesical dissection is borne out by this less invasive
method. Clearly, we have not encountered secondary voiding dysfunction in our
patients thus far.
V711
NOVEL "PULL THROUGH" TECHNIQUE OF LAPAROSCOPIC
URETEROINTESTINAL ANASTOMOSIS Sidney C Abreu*,
Inderbir S Gill, Ramani Anup, Amgad Farouk, Andrew Steinberg, Jihad
Kaouk, Gyung Tak Sung, Cleveland, OH
INTRODUCTION AND OBJECTIVE: Laparoscopic radical cystectomy and
ileal conduit urinary diversion have been performed completely intracorporeally
using free hand suturing and in situ knot-tying techniques exclusively. A
challenging and time-consuming surgical step is performing the ureterointestinal
anastomosis. Herein, we present a novel technique of laparoscopic ureterointestinal
anastomosis while has the potential to be technically simpler, quicker, and
non-relluxing.
METHODS: Laparoscopic cystectomy and ileal conduit urinary diversion was
performed completely intracorporeally in 8 farm pigs (2 acute and 6 chronic). Our
novel technique of ureteral reimplantation comprises the following steps: I) The
distal ureter was spatulated for approximately 3cm, and folded over itself with a
single stitch of 4-0 vicryl for approximately 1.5 em. 2) A 7 Fr single-J stent was
delivered through the conduit into the abdominal cavity, then passed into the ureter
up to the renal pelvis. 3) A 2-0 absorbable suture double-armed with CT-I needles
was placed around the stent and passed through the ureteral edge. 5) Each CT-I
Presenting author.
Vol. 169, No.4, Supplement, Monday, April 28, 2003
needle was introduced through the ileotomy into the ileal conduit and then
delivered through the ileal wall 2 em away from the ileotomy. 6) Traction was
applied to the double armed suture as well as to the J stent, telescoping the ureter
into the conduit. 7) The 2 stitches were tied together; thus, anchoring the ureter to
ileal wall. 8) Three to four serosal stitches were placed circumferentially between
the ureter and the ileal conduit.
RESULTS: Mean operative time was 300 minutes. Blood loss was minimal.
One death occurred at 6 weeks postoperatively due to a fistula between the cecum
and the conduit. The pigs were sacrificed at various time intervals (I week - 2
animals, I month - 2 animals, 3 months - I animal). At autopsy, Whitaker
pressure-llow study, ureteral calibration by gentle passage of bougies revealed a
widely patent anastomosis. IVP documented prompt washout of contrast.
Furthermore retrograde loop gram demonstrated a non-reftuxing ureteral
reimplantation.
CONCLUSIONS: This video demonstrates a novel technique of laparoscopic
ureterointestinal anastomosis. Over a 3 month follow-up, this technique proved to
be effective in achieving a widely patent, non-relluxing ureterointestinal
anastomosis. Furthermore, its inherent simplicity allows this delicate reconstructive
part of the operation to be performed in time sensitive manner.
Bladder Cancer: Basic Research (II)

Discussed Poster
Monday, April 28, 2003
1:00-5:00 PM
712
TRANSITIONAL CELL CARCINOMA AFTER CONDITIONAL
TARGETING OF RB AND P53 IN THE BLADDER
UROTHELIUM Axel Bex", Henk Gerrit Van der Poel, Simon Horenblas,
Amsterdam, Netherlands
INTRODUCTION AND OBJECTIVE: Clinical advances in bladder cancer
will require the development of novel animal model systems closely mimicking the
human disease. We developed a system of conditional gene targeting using the
Cre/loxP system that permits temporally controlled mutation of bladder cancer
relevant tumor suppressor genes Rb and p53 in the bladder urothelium.
METHODS: R26cre-ERT-mice expressing Cre-ERT, a fusion between Crerecombinase and a mutated hormone binding domain of the human estrogen
receptor (ERT) under the transcriptional control of the ubiquitiously expressed
ROSA26 locus, permit temporally and spatially controlled Cre mediated
recombination in vivo upon topical application of 4-hydroxy-tamoxifen (OHT).
R26cre-ERT-mice were crossbred with conditional mice carrying homozygote and
heterozygote floxed alleles of Rb and p53. After a lower midline incision R26creERT-mice carrying Rblox/,ox/p53Iox/'ox_, Rblox/w'/p53Ioxf'ox_ and Rb'oxfw'/p53Iox/w,_
combinations received 5 mg OHT intravesically and were observed (wt = wild
type). Controls of the combinations received a pellet without OHT.
RESULTS: Three months after OHT implantation into the bladder R26cre-ERTRb'ox/lo'/p53IOX"OX_ and Rb'ox/w'/p53'Ox/lOx_nrice developed carcinomain situ and muscle
invasive bladder cancer. Control mice and mice carrying Rblox/w'/p531ox/w,_
combinationsdid not develop tumors. Immunohistochemical staining was positive for
cytokeratineand negative for smooth muscle actin, vimentin and desmin. Lymphnode
metastasis or distant metastasis was not observed at that time interval.
CONCLUSIONS: The R26cre-ERT mouse can be used to induce multifocal
somatic mutagenesis in the bladder urothelium in a promotor-independent and
time-controlled manner. After targeting of Rb and p53, carcinoma in situ and
muscle invasive bladder cancer developed as observed in man. By crossbreeding
with mice carrying flexed alleles for Rb, p53, pl6INK4a and E-cadherin, singular
or in combination, we will use the model to investigate genotype-phenotype
relations of transitional cell carcinoma.
713
EFFECTIVE SUICIDE GENE TRANSFER STRATEGY BY A
CHIMERIC ADENOVIRUS VECTOR TO BLADDER CANCER
Kazumasa Matsumoto*, Christian T Freund, Weiguo Jian, Patricia Yotnda,
Elizabeth A Olmsted-Davis, Alan R Davis, Seth P Lerner, Houston, TX
INTRODUCTION AND OBJECTIVE: Binding of adenovirus (Ad) to a human
cell is mediated by the coxsackie and adenovirus (CAR) receptor. A target cell
needs to express sufficient levels of CAR in order to be susceptible to Ad infection.
Down regulation of CAR has been observed in various types of cancers including
high grade and advanced bladder cancer resulting in the potential for decreased
transduction efficacy using standard Ad5 vectors. Other serotypes including Ad35
have been shown to utilize cell entry mechanisms that are independent of CAR. In
this study, we evaluated the efficacy of a novel chimeric vector that combines the
Ad35 CAR independent ceU entry mechanism with a traditional first generation
AdS backbone for bladder cancer gene therapy.
METHODS: A replication deficient chimeric Ad vector named Ad51F35
expressing either GFP or HSV-tk was created by overlap recombination and the
Ad35 fiber gene was incorporated into the AdS backbone. Both Ad5 and Ad51F35
vectors were characterized in human bladder cancer ceUlines T24 (CAR-deficient)
and 5637 (CAR-positive). GFP gene expression was determined by both
fluorescence microscopy and quantitated by Western blot. Suicide gene therapy
efficacy was evaluated by exposing cancer cells to viral doses of 1 to 300 MOl of
Ad5HSV-tk or Ad51F35HSV-tk plus ganciclovir. CeU viability was assessed with
the MIT assay.
RESULTS; Western blot confirmed target gene expression and differential
transduction efficiencies of both vectors in CAR positive and CAR deficient cells
lines. With both Ad vectors, cell viability was suppressed in a dose dependent
manner. Compared to AdS, Ad51F35-HSV-tk was 30-fold more effective in terms
of achieving cell death in CAR negative T24 cells and 3-fold more effective in
CAR positive 5637 cells.
CONCLUSIONS; Our results clearly demonstrate that the chimeric Ad51F3S
vector incorporating the Ad3S fiber protein can greatly augment suicide gene
therapy in CAR down regulated bladder cancer ceUs while maintaining efficacy
comparable to traditional AdS vectors in CAR positive cancer ceUs, providing a
new gene transfer strategy for bladder cancer gene therapy.
Source of Funding: National Cancer Institue.
714
THE PROTEIN PHOSPHATASE ACTIVITY OF PTEN IS
SUFFICIENT TO INHIBIT CELL MIGRATION AND ORTHOTOPIC INVASION BUT NOT TUMORIGENICITY IN BLADDER
CANCER Dan Theodorescu ", John Gildea, Michael Harding,
Charlottesville, VA
INTRODUCTION AND OBJECTIVE: Recent studies have found a higher
frequency of the PTEN tumor suppressor gene alterations in invasive bladder
carcinoma than in superficial disease, suggesting that PTEN is a player in this
process. A role of PTEN in bladder cancer invasion is further suggested by the fact
that PTEN is a regulator of ceU motility, a necessary component of tumor invasion.
However it is unknown whether PTEN is mechanistically involved in tumor
invasion or merely an epiphenomenon and if the former is true, whether this
process is dependent on its protein or lipid phosphatase activities.
METHODS; To address theses issues, we stably transfected human bladder
ceUlines with known invasive phenotypes with either wild type PTEN constructs
or those deficient in the lipid phosphatase (G129E) or both protein and lipid
phosphatase (GI29R) activities. Here we show that chemotaxis was inhibited by
both the wild type and G129E mutant ofPTEN but not by GI29R transfected ceUs.
Using a novel organotypic in vitro invasion assay, we evaluated the impact of wild
type and mutant PTEN transgene expression on the invasive ability of T24T, a
human bladder cancer ceU line with a known invasive phenotype in orthotopic in
vivo models.
RESULTS; Results indicate that the GI29E mutant blocks invasion as
efficiently as wild type PTEN transfection. In contrast to the wild type gene, this
mutant has no effect on cell clonogenicity in agar. To further establish the role of
PTEN in tumor invasion we evaluated vector and PTEN transfected T24T cells in
an orthotopic in vivo assay that faithfuUy reproduces human disease. Microscopic
examination of murine bladders at the completion of this experiment parallels the
results obtained with the organotypic assay. In contrast, subcutaneous
tumorigenicity is not affected by cells expressing the G129E mutant while being
suppressed by those expressing the wild type PTEN transgene.
CONCLUSIONS; In conclusion, our results are the first 'demonstration that the
inhibitory effects of PTEN on ceU motility translate into suppression of in vivo
invasion. These results are also first to show that the protein phosphatase activity
of PTEN does playa critical role in regulating the invasive phenotype. Finally,
these studies validate the use of organotypic in vitro approaches as surrogates of in
vivo invasion aUowing rapid dissection of molecular processes leading to this
phenotype while reducing the number of animals used in such research.
Source of Funding: NCI.
715
DNA MICROARRAY ANALYSIS OF STAGE AND GRADE IN
BLADDER CANCER - TIG3 TUMOUR SIMILARITY TO
MUSCLE INVASIVE DISEASE Edward H Streeter", Oxford, UK;
Tracy Chaplin, London, UK; David W Cranston, Adrian L Harris,
Oxford, UK
INTRODUCTION AND OBJECTIVE; Patients with high grade (G3) TI
bladder cancer have a high risk of developing muscle invasive disease. It is not
clear whether this is due to understaging or early detection of invasive disease, or
185
biological progression from superficial to invasive phenotype. This work aimed to

investigate the genetic expression profiles of various bladder cancers, to determine
if there is evidence for divergent developmental pathways between low and high
risk superficial tumours.
METHODS: RNA from 39 bladder tumours (8 Ta, 18 TI (8 G2, 10 G3), 10 T2
and 3 T3) was hybridised against a reference panel of cell line RNA on dual colour
arrays containing 9,932 genes (HverI.2.1 - Sanger Centre, UK). AdditionaUy,
pooled RNA from 5 tumours within each of 4 risk groups (Ta, T1GII2, T1G3,
Muscle Invasive) were hybridised onto Affymetrix HU-133A arrays, containing
22,000 genes. GeneSpring software was used to determine genes showing a
significant variation by stage, and to perform heirarchical clustering of aU tumours.
Findings were verified using reverse transcriptase-polymerase chain reaction (RTPCR), and ELISA.
RESULTS: Using data from the Sanger arrays, cluster analysis of the 39
individual tumours was performed based on the 75 genes showing most significant
stage variation. This showed a strong association between TlG3 and muscle
invasive tumours. AdditionaUy, analysis of Affymetrix data demonstrated that
genes selected on the basis of their differential expression between Ta and muscle
invasive groups also showed differential expression between low and high grade
TI tumours. Thus gene expression profiles for low grade T1 tumours are similar to
Ta tumours, whilst high grade TI disease shows striking similarity in a large
number of cases with muscle invasive disease. These results were verified
successfully using RT-PCR for 10 selected genes. Additional verification using
ELISA on whole tumour protein extracts from a set of independently chosen
tumours verified one of the selected genes, osteopontin, as being upregulated 6
times in the progression from Ta to muscle invasive disease (p=0.023).
CONCLUSIONS: Biological staging of bladder cancer may more accurately
predict future clinical behaviour than standard histopathological staging alone.
Here evidence is presented to show the similarity in gene expression profiles
between high grade Tl tumours and muscle invasive disease. This supports the
early aggressive treatment of T1G3 tumours, which in many cases may represent
understaged muscle invasive disease.
Source of Funding: Royal CoUege of Surgeons of England; Cancer Research
UK; Martin Charitable Trust.
716
RENDERING BLADDER CANCER SUITABLE FOR ADENOVIRAL GENE THERAPY Markus D Sachs", Berlin, Germany; Michael
Cohen, Wasim Chowdhury, Devon Lacey, Baltimore, MD; Stefan A Loening,
Berlin, Germany; Ronald Rodriguez, Baltimore, MD
INTRODUCTION AND OBJECTIVE: The coxsackie adenovirus receptor
(CAR), which is necessary for adenoviral cell attachment and therefore
adenoviral gene therapy, is down-regulated in clinical bladder cancer. It has
therefore been argued, that bladder tumors are not a good target for adenoviral
gene therapy. Recently it has been shown, that this type of gene inactivation can
be overcome by Histone deacetylase inhibitors (HDACI), resulting in a higher
gene expression, cell differentiation and apoptosis. Valproic acid (VPA) is a
well established drug in the long-term treatment of epilepsy, which was
recently shown to exhibit strong HDACI-activity. Here we show, that VPA can
up-regulate CAR at clinically applicable doses, rendering bladder cancer
suitable for gene therapy.
METHODS: T24 bladder cancer cells were treated for 72 h with VPA at
various doses, cell cycle synchronized and examined for CAR expression by
quantitative rt-PCR, flow cytometry and functional studies by transfection of an
adenovirus that carries the luciferase reporter gene. The results were compared to
CAR-positive 293 cells and CAR-negative L929 cells.
RESULTS; The results are summarized in table I. VPA increased the CARcopy number up to 13.5-fold, which resulted in 66.5% of ceUs expressing CAR on
the cell surface, compared to only 3% of untreated cells. The re-expressed receptor
was functioning as shown by a 38-fold increase in transgene expression.
CONCLUSIONS; Low CAR expression can be overcome at both RNA and
protein levels using Valproic acid (VPA). This drug has been used for the treatment
of seizure disorders for decades, it has been shown to rarely cause mild adverse
effects and can be given on a long-term basis. The significant increase in CAR
expression after VPA treatment could overcome low gene transfer and render
bladder cancer a suitable target for adenoviral cancer gene therapy.
Table 1. Summary of results
0.0
mM VPAtreatnlent
copynumber (foldIn10
crease)
flowcytometry shift (% of
2.9
cells)
luciferase expression (fold
1.0
increase)
0.6
1.2
2.4
5.0
2.0
39
9.2
13.5
61.0
0.0
43
14.8
42.0
66.5
99.8
0.0
4.3
11.5
17.4
38.0
n/a
nla
293 cells 1929 cells
Source of Funding: Max Kade Foundation; Flight Attendant Medical

Research Institute.
186
717
719
THE INHIBITION OF CYCLOOXYGENASE-2 (COX-2) EXPRESSION BY COXSACKIE AND ADENOVIRUS RECEPTOR

(CAR) IN BLADDER CANCER Jer-Tsong Hsieh*, Linya Yang, Rey-
GENE EXPRESSION PROFILES ANALYSIS OF PATIENTS

WITH BLADDER CANCER TREATED WITH BCG Fabien Saint*,
Chen Pong, Hossein Saboorian, Arthur I Sagalowsky, Dallas, TX

INTRODUCTION AND OBJECTIVE: CAR, a high affinity receptor for
Adenovirus type 5, is considered a rate-limiting step of successful adenovirusbased gene therapy. We and other group demonstrated that CAR expressed
heterogeneously among various transitional carcinoma cell (TCC) lines and
specimens. We further showed that CAR could act a tumor inhibitor in TCC
cells. This study is to elucidate the underlying mechanism of CAR action in
TCC cells.
METHODS: Stable transfectants were cloned by transfecting plasmid into
TCC cells with G418 selection. Western blot analysis was used for determining the
steady-state levels of CAR or CDX-2 levels in TCC cell lines. We employed
imrnunostaining to detect both CAR and COX-2 expression in TCC specimens
from 51 patients with bladder cancer. The pathological stages of these specimens
were: pTa (n =25); p'Tis (n=6); pTl (n=4); pT2 (n=6); pT3 (n=8); pT4 (n=2).
For CAR, a new polyclonal antibody against the extracellular domain of CAR was
used. For COX-2, antibody was purchased from Oxford Inc. The tissue
procurement protocol was approved by the institutional IRB committee.
RESULTS: Increased CAR expression in TCC cells could lead to their growth
inhibition. Western blot analysis indicated that increased CAR expression resulted
in reduced expression of COX-2, a protein related with carcinogenesis of several
neoplasms, in CAR-negative TCC cells. In contrast, downregulation of endogenous
CAR in CAR-positive cell by transfecting an antisense CAR vector resulted in
elevating of COX-2 protein levels. By deleting intracellular domain of CAR, this
mutant CAR lost its growth inhibitory activity and failed to suppress COX-2
protein expression. Using irnmunostaining, we found that detectable levels of CAR
was associated with normal transition cell and low-grade TCC, however, decreased
CAR levels were often found in high-grade tumor specimens. In contrast, COX-2
was not detected in normal and low-grade tumor, however, it presented in
high-grade tumor. Most importantly, we observed a reciprocal expression pattern
of CAR and COX-2 in the same specimen. Taken together, CAR appears to be a
potent inhibitor for modulating COX-2 protein expression in TCC cells.
CONCLUSIONS: CAR is a transmembrane protein receptor that could elicit
endogenous signal transduction to suppress cell growth. The underlying
mechanism of CAR in inhibiting TCC cell growth could be due to the suppression
of COX-2 protein expression. This study provides a new insight of relationship
between both CAR and CDX-2.
Source of Funding: NIHINCI.
Marta Sanchez-Carbayo, New York, NY; Sixtina Gil Die: de Medina, Cretei/,
France; Juan Jose Lozano, New York, NY; Hui Zhao, Nicholas Socci, Agnes
Viale, New York, NY; Angel Ortiz, New York, NY; Dominique Chopin, Creteil,
France; Carlos Cordon-Cardo, New York, NY
INTRODUCTION AND OBJECTIVE: Bacillus Calmette-Guerin (BCG) is a
standard treatment for reducing tumor recurrence and delaying progression of
high-risk and intermediate-risk superficial bladder tumors. However, it is not clear
yet which patients are more susceptible to be responders to BCG. The aim of this
study was to evaluate the clinical utility of gene expression profile analysis to
discriminate patients with different response to BCG.
METHODS: We analyzed the expression profiles of 20 superficial bladder
tumors (2 pTa, 18 pTl) of patients receiving their first cycle of BCG, The median
follow-up was 52 months (range: 16 to 88), No evidence of disease was observed
in 13 (65%) of these patients while recurrence was detected in 7 (35%) of them.
Gene expression profiles were performed using the human U133A oligonucleotide
microarrays (Affyrnetrix). Data analysis was performed using the MAS 5.0 and
Genespring software. Bootstrapping techniques were also applied to evaluate the
robustness of the hierarchical clustering analysis.
RESULTS:Genespringsoftwarewas applied to identifygenes that could segregate
patients that recurred from those that showed no evidence of disease during follow-up.
Using t-test and consideringsignificanta p value equal or lower than 0.05, we observed
that 37 genes were able to distinguish responders from non-responders. These genes
revealed the relevance of interferon-inducedproteins, and intracellularsignaling such
as TGF-beta pathway in BCG response. The application of bootstrapping on
hierarchicalclustering analysiscould separate among respondersthose patientswith no
evidence of disease during at least 4 years of follow-up.
CONCLUSIONS: Gene expression profiles analysis has been shown to be a
useful means to further characterize the molecular events associated with BCG
response. Moreover, we have identified novel genes with clinical predictor utility
to select patients more likely to response to BCG therapy.
720
ANTISENSE AGENTS TARGETING THE TYPE 1 INSULINLIKE GROWTH FACTOR RECEPTOR SENSITIZE HUMAN
BLADDER CANCER TO PACLITAXEL AND CISPLATIN
MEDIATED CYTOTOXICITY Giles 0 Hellawell*, London, UK;
Simon Brewster, Val Macaulay, Oxford, UK
718
DIFFERENCES IN THE PROTEIN PROFILE OF INVASIVE AND
NON-INVASIVE BLADDER TUMORS Sigrun Langbein*, Axel
Schaaf. Mannheim, Germany; Markus Meyer, Goettingen, Germany; Maurice
S Michel, Michael Siegsmund, Peter Aiken, Mannheim, Germany
INTRODUCTION AND OBJECTIVE: Bladder carcinoma is an increasing
tumorentity. The initial differentiation between stable (no progression) and
unstable (progression) bladder tumor is not yet possible. No biochemical- or
genetic correlation has been found to objectively prognosticate the risk of
progression. Proteome analysis and profiling might be a useful tool to detect
differences between superficial and invasive bladder carcinomas, and to find
differentially expressed proteins which could lead to an early detection of
potentially aggressive superficial tumors.
METHODS: We analysed 14 primary bladder tumors (9 Ta and 5 2: T3) by
surface enhanced laser desorption / ionisation mass spectrometry (SELDI-TOF,
Ciphergen Biosysterns Inc. Fremont) on different ProteinChip Arrays. The tumor
material was obtained by transurethral resection, imrnediatly shock frozen and
stored at -80 C. We tested non-invasive versus invasive tumors with WCX (weak
cationic exchange) and SAX (strong anionic exchange) ProteinChip Arrays. The
peaks were registered in a range of 1.5 to 98 kD by the ProteinChip Reader
according to an automated data collection protocol.
RESULTS: The non-invasive tumors showed a strong peak at 6.7 and 10.1
kD (WCX-array), whereas the invasive tumors presented only a weak signal.
The peak at 10.1 kD presented much stronger in non-invasive tumors also on
the SAX-array. The invasive tumors showed upregulation at 7.4,9.5, 14.5, 15.1
and 15.9 kD.
CONCLUSIONS: Protein profiling of bladder tumors by the SELDI-TOF
technology is a reliable, sensitive and quick method for detecting differences
between invasive and non-invasive carcinomas. Our data show first results in
differential protein expression in both groups. Further studies with larger patient
groups have to confirm these results. Identifying the proteins may lead to
distinguish between potentially aggressive and non-aggressive superficial bladder
carcinomas.
0
*Presenting author.
INTRODUCTION AND OBJECTIVE: Chemotherapy resistance presents a

significant obstacle to the treatment of advanced bladder cancer. The type I
insulin-like growth factor receptor (IGFlR) plays an important role in progression,
invasion and metastasis of bladder cancer cells. The objective of this study was to
determine whether antisense mediated IGFI R downregulation could enhance the
chemosensitivity of bladder cancer cells in vitro.
METHODS: MGHU-J bladder cancer cells were transfected with IGFIR
antisense oligonucleotides (ASOs) or antisense RNA. Transfected cultures were
treated with paclitaxel, cisplatin or vehicle control, and survival was measured by
colony formation in agar and in sparsely seeded monolayer.
RESULTS: IGFIR ASOs caused dose-dependent inhibition of IGFIR
expression to levels of 40% of those in control-treated cells. This was accompanied
by marked reduction in MGHU-l growth measured by MTS assay, and clonogenic
survival was reduced by up to 70%. Furthermore, IGFIR ASOs enhanced
chemosensitivity to paclitaxel and cisplatin with a 2 to 2.5-fold reduction in IC-50.
Ultrastructural analysis of clones treated with cisplatin confirmed that the ASIGFIR cells were more susceptible to apoptosis.
CONCLUSIONS: IGFIR downregulation enhances chemosensitivity in part
via enhanced susceptibility to apoptosis. In a clinical setting, targeted suppression
of the IGFIR using ASO technology may enhance the effects of conventional
chemotherapy. In vivo studies should be undertaken to assess whether intra-vesical
antisense treatment in combination with chemotherapy may represent a novel
therapy for bladder cancer.
Source of Funding: PPP Foundation; Cancer Research UK.
721
INTRODUCTION OF MIDKINE GENE INTO HUMAN
BLADDER CANCER CELLS ENHANCES THEIR MALIGNANT
PROGRESSION THROUGH ITS ANGIOGENIC ACTIVITY
Muramaki Mototsugu*, Kobe, Japan; Hideaki Miyake, Akashi, Japan; [sao
Hara, Hiroshi Okada, Sadao Kamidono, Kobe, Japan
INTRODUCTION AND OBJECTIVE: Midkine (MK) is a member of a family
of heparin-binding growth factors, which are reported to have an important role in
angiogenesis. MK is overexpressed in a wide range of human carcinomas including
bladder cancer and is considered to be associated with disease progression. In this

study, we introduced MK cDNA into human bladder cell line (UM-UC-3), and
evaluated the effects of the overexpression of MK on the malignant potential of
UM-UC-3 cells both in vitro and in vivo.
METHODS: We introduced the MK cDNA into UM-UC-3 cells which do
not secrete detectable level of MK protein, and generated the MK
overexpressing cell line, UM-UC-3/MK, and the vector-only-transfected cell
line, UM-UC-3/C. The expression of MK from UM-UC-3/MK conditioned
media was confirmed by Western blotting analysis and human umbilical vein
endothelial cell (HUVEC) proliferation assay. We then evaluated the tumor
progression of UM-UC-3 sublines after subcutaneous and orthotopic injection
into athymic nude mice. Established tumors were analyzed by
immunohistochemical staining of blood vessel endothelium using anti-CD3l
and micro vessel density (MVD) was quantified.
RESULTS: Western blotting analysis revealed that high expression of MK
was achieved in heparin affinity-purified conditioned media of UM-UC-3/MK
cells, not in those of parental UM-UC-3 and UM-UC-3/C cells. The expression
of biological active MK was confirmed by HUVEC proliferation assay.
Although no significant differences were found concerning in vitro growth rate
among these sublines, UM-UC-3fMK cells demonstrated significantly
increased tumor growth after subcutaneous as well as orthotopic injection and
enhanced lymph node metastasis from orthotopic injection compared with
parental UM-UC-3 and UM-UC-3fC cells. Moreover, tumor-induced
angiogenesis, as determined by the MVD of the established tumors, was
significantly increased in UM-UC-3/MK tumors.
CONCLUSIONS: These findings suggest that the overexpression of MK leads
to release of an endothelial growth-stimulating activity from transfected cells,
resulting in increase of tumor growth and vascular density in vivo.
722
CYTOGENETIC ANALYSIS OF GENETIC INSTABILITY IN
SUPERFICIAL BLADDER CANCER AND IN NORMAL
APPEARING BLADDER MUCOSA Costantino Leonardo, Annamaria
Cianciulli, Francesco Iori, Franco Giorgio", De Nunzio Cosimo, Peris
Filippo, Cesare Laurenti, Roma, Italy
INTRODUCTION AND OBJECTIVE: To determine wheter genetic
aberrations found in superficial TCC were also present in normal appearing
mucosa.
METHODS: We investigated chromosome 1,7,9,17 aneusomy in 25
patients with TCC and in tissue samples taken from sites of macroscopically
uninvolved urothelium surrounding the tumors (ST), as from distant sites (DS),
using the fluorescence in situ hybridization (FISH) technique on touch imprints
of the resected material. Chromosomal probes were pericentromeric
fluorescent-labeled for use in the FISH assay (Vysis, Inc.) specific for the
centromeric region of chromosome (Chr) I (DI25), 7 (D721), 9 (D925) and 17
(D1721). T-test and Spearman correlation coefficient were used for statistical
analisys.
RESULTS: For 7 DS, I ST of the 25 cases, the FISH assay was not
valuable. Histopathological report of tumor (T) samples showed 12 TaGI-G2
TCC, 12 TlG2-3 and I patient presented papillary hyperplasia. Ten (40%) T
specimens showed aberrations of all chromosomes analyzed and fifteen (60%)
were aneuploid for at least one chromosome. Numerical aberrations of
examined chromosomes with similar patterns were seen in tumor and all
analyzed specimens. Differences were observed in aberration frequencies, Chr
I 88% (T), 50% (ST) and 21% (DS); Chr 7 72% (T), 65% (ST), 42% (DS); Chr
9 76% (T); 54% (ST), 52% (DS); Chr 9: 76% (T), 54% (ST), 52% (DS); Chr
17: 84% (T), 46% (ST), 21% (DS). Statistycal differences were observed
between T vs ST (p=0.002) and T vs DS (p=0.002). Moreover, on the basis of
the histological report, we divided the ST and DS in neoplastic, normal or
pathological-non neoplastic mucosa and compared the mean percentage of
aneusomic cells for chromosome 1,7,9 and 17. Between T and normal mucosa
as ST or DS the chromosomal aberrations (Chr1: 40%, 25%, 15% respectively;
Chr7: 40%; 25%, 15%; Chr9: 50%, 30%, 20%; Chrl7: 45%, 25%, 15%)
showed significative difference (p<0.002). Between T and pathological nonneoplastic ST or DS we observed statistical differences in Chrl (T: 55%, ST:
20%, DS: 20%) and 17 (T: 60%, ST: 25%, DS: 30%), while for Chr 7 (T: 25%,
ST: 40%, DS: 40%) and 9 (T: 50%, ST: 40%, DS: 45%) statistical evaluation
did not reach significance. A strong correlation between frequency of
chromosomal aberrations and histopathological diagnosis (p=O.OOO, r=0.6)
was also observed.
CONCLUSIONS: A population of cells in morphologically normal epithelium
presented genetic aberrations common to those in bladder cancer. These findings
might provide a ground for multiple tumorigenesis.
187
723
ALTERED EXPRESSION OF SPECIFIC GENES IN THE HOX
NETWORK IS ASSOCIATED WITH HUMAN BLADDER
TRANSITIONAL CELL CARCINOMAS Luca Cindolo*, Avellino,
Italy; Monica Cantile, Riccardo Autorino, Giuseppe Di Lorenzo, Yincenzo
Altieri, Clemente Cillo, Naples, Italy
INTRODUCTION AND OBJECTIVE: HOX genes are a network of transcription
factors controlling embryonal development and crucial function of adult eukariotic
cells. The molecularorganizationof this network of 39 genes is unique in the genome
and probably acts regulating phenotypic cell identity. HOX genes are involved in
human congenital, somatic, metabolic and neoplastic alterations. In the present study
we have detected the expression of the complete HOX gene network in human normal
bladder and in transitional cell carcinomas (TCC).
METHODS: We have analysed, through duplex RT-PC, HOX gene expression
in human normal (10) and neoplastic bladder (15). Clinical and pathologic features
have been compared to experimental data to search for specific correlation.
RESULTS: 28/39 genes of the HOX network are expressed in normal bladder.
In contrast, 35/39 genes of the HOX network are active in bladder cancers. A block
of three HOX genes at 3 end of locus C (on chromosome 12) are silent in normal
bladder and actively expressed in almost the totality of bladder cancer tested.
Furthermore paralogous group 11 HOX genes (All, Cll and D11), involved in
reno-uro-genital tract embryonal development, are dramatically altered in their
expression when comparing normal and neoplastic bladder.
CONCLUSIONS: Altered expression of of HOX C4, C5 and C6 and of
paralogous group II HOX genes are associated with human bladder transitional
cell carcinomas. These results strongly suggest the involvement of the HOX gene
network in urothelial oncogenesis and support the potential possibility to target
HOX genes for future cancer therapies.
724
HYPERMETHYLATION OF A E-CADHERIN (CDHl) PROMOTER REGION IN HIGH GRADE TRANSITIONAL CELL
CARCINOMA OF THE BLADDER COMPRISING CARCINOMA
IN SITU (CIS) Yohei Horikawa", Akita, Japan; Kokichi Sugano,
Utsunomiya-shi, Tochigi, Japan; Masanori Shigyo, Chuo-ku, sapporo, Japan;
Yae Kanai, Tadao Kakizoe, Hiroyuki Fujimoto, Chuo-ku, Tokyo, Japan;
Tetsuro Kato, Akita, Japan
INTRODUCTION AND OBJECTIVE: To elucidate the role of methylation in
the promoter region of the CDHI gene in bladder carcinogenesis, particularly in
those comprising carcinoma in situ (CIS).
METHODS: Forty-nine cases of transitional cell carcinoma (TCC) of the
bladder obtained from transurethral resection (TUR) were examined. Methylation
status of the CDHI promoter region was analyzed by a methylation-specific PCR
(MSP) from chemically modified DNA after Na-bisulfite treatment. An LOH on
16q was examined by a blunt-end single-strand DNA conformation polymorphism
(SSCP) analysis using four tetranucleotide repeat microsatellite markers assigned
on 16q13-22.1. E-cadherin expression was evaluated by immunostaining on
formalin-fixed paraffin-embedded tissue sections using anti-E-cadherin murine
monoclonal antibody, HECDI and standard avidin-biotin immunoperoxidase
complex technique.
RESULTS: In the analysis of the 49 bladder TCC samples, CDHI promoter
methylationwas found in 23 (47%). Methylation of the CDHI gene was not correlated
with tumor stage (p=0.2097), but with high-grade TCC (p=0.0416). CDHI promoter
methylation was observed at a significantly higher frequency in the CIS (+) group
compared with the CIS (-) group (89% (16/18) vs. 23% (7f31), p<O.OOOI) and showed
strong correlation with abnormal E-cadherin expression (p<0.0001). We found
16qLOH in 16 (34%) of 47 cases, that was correlated with higher histological grade
(p=0.0069), but not with the presence of the CIS component (p=0.1235).
CONCLUSIONS: This study showed that CDHI gene promoter methylation is
strongly associated with bladder TCC comprising CIS.
725
IDENTIFICATION OF TWO MAGE-A8-ENCODED TUMOR
ASSOCIATED ANTIGEN PEPTIDES IN TRANSITIONAL CELL
CARCINOMA OF THE BLADDER, BASED ON CDNA
MICROARRAY DATA Erez Bar-Haim; Rehovot, Israel; Adrian Paz*,
Shmuel Cytron, petah tikva, Israel; Arthur Machlenkin, Boa; Tirosh, Rehovot,
Israel; Avi Stein, Haifa, Israel; Ezra Yadai, Esther Tzehoval, Rehovot, Israel;
Francois A Lemonnier, Paris, France; Lea Eisenbach, Rehovot, Israel
INTRODUCTION AND OBJECTIVE: Bladder carcinoma is the forth most
common cancer in men and the eighth most common cancer among women. Our
study intended to characterize tumor-associated antigens (TAA) peptides of
transitional cell carcinoma of the bladder (TCC).
188
METHODS: A DNA micro array-based differential display analysis of 10,000

genes was carried-out, aimed to identify uniquely expressed genes of the tumor that
may serve as potential TAA. One of the overexpressed genes in TCC tumors
compared to normal mucosa was MAGE-A8 gene, which is known to be expressed
also in other tumors and is not expressed in normal tissue. The MAGE-A8 was
screened for HLA-A2.1 binding motifs, six potential peptides were chosen and
synthesized, and peptides binding to HLA-A2.1 was assured. Immunogenicity and
antigenicity of the MAGE-A8 peptides was examined in the HHD system, of
murine class I MHC knock-out mice, transgenic for HLA-A2.1. The
immunogenicity of the MAGE-A8 peptides was examined in three modes of
vaccination; delivered intranasally with cholera toxin as a mucosal adjuvant,
injected into the tail base with complete Freund adjuvant (CFA), or presented
directly as loaded onto cell surface HLA-A2.1 molecules.
RESULTS: The MAGE-A8 gene was expressed in bladder transitional cell
carcinoma, and not in the normal bladder mucosa. High occurrence of MAGE-A8
expression was observed in fresh tumor samples (17 of 23 samples) and TCC lines
(four of eight examined). Two peptides, 8.1 and 8.3 were found to induce CTL that
killed specifically the T24 TCC line in vitro. These two peptides were also able to
prime human peptide specific CTL lymphocyte response of healthy donors.
CONCLUSIONS: These results demonstrate the potential use of the MAGE-A8
peptides for specific immunotherapy of transitional cell carcinoma of the bladder.
METHODS: Ninety-two (92) urothelial specimens including non-malignant

and various grades and stages of urothelial carcinoma obtained by transurethral
resection were analyzed by immunohistochemical (IHC) staining. An affinity
purified polyclonal antibody to the Cables protein was used to detect Cables
expression in formalin-fixed, paraffin-embedded tissue. All specimens were
analyzed for positive staining, partial loss, or total loss of Cables staining of
malignant and non-malignant urothelial cells.
RESULTS: All specimens of non-malignant urothelium (14/14) and low-grade
non-invasive papillary transitional cell carcinoma (12/12) demonstrated uniformly
positive staining for Cables. High-grade non-invasive papillary transitional cell
carcinoma showed 43% (8119) positive, 47% (9/19) partial loss and 10% (2/19)
total loss of Cables expression. Of carcinoma in situ cases, 33% (4/12)
demonstrated positive staining, 50% (6112)partial loss and 17% (2112) total loss of
expression. In lamina propria invasive TCC 9% (1/12) were positive for Cables
expression, 33% (4/12) showed partial loss and 58% (7/12) exhibited total loss.
Muscularis propria invasive TCC demonstrated positive expression in 4% (1/23) of
all specimens, partial loss in 44% (10/23) and total loss of Cables expression in
52% (12/23). Please see table below.
CONCLUSIONS: Cables is a cell-cycle regulatory protein which is expressed
uniformly in non-malignant and low-grade non-invasive TCC of the bladder. The
expression of Cables is lost in the majority of high grade and invasive urothelial
carcinoma, suggesting that inactivation of Cables may play a role in the
pathogenesis of invasive TCC of the bladder.
726
Immunohistochemical Staining for theCables Protein In Urothelial Specimens
THERAPY OF MURINE BLADDER CANCER WITH TUMORSPECIFIC SUICIDE GENE EXPRESSION DRIVEN BY HUMAN
TELOMERASE REVERSE TRANSCRIPTASE PROMOTER IN
SYNGENEIC MOUSE TUMOR MODEL Gia-Shing Shieh, Tainan,
Specimen
Non-Malignant
LowGrade Non-Invasive TCC
High Grade Non-Invasive TCC
Carcinoma InSitu
Lamina Propria Invasive TCC
Muscularis Propria Invasive TCC
Taiwan
INTRODUCTION AND OBJECTIVE: Human telomerase reverse
transcriptase (hTERT) is the key subunit of telomerase, which is highly active in
immortalized cells and over 85% of human cancers but inactive in somatic cells.
Due to the high homology between human and mouse TERT core promoter, the
transcriptional activity of hTERT promoter in syngeneic mouse bladder tumors and
the potential toxicity of hTERT promoter-driven suicide gene in murine somatic
cells are unknown. Thus,we constructed a replication-deficient adenovirus driving
cytosine deaminase (CD) gene driven by hTERT promoter and investigated its
antitumor effect on mouse bladder cancer.
METHODS: MBT-2, immortalized murine embryo fibroblast (NIH-3T3) and
mature fibroblast derived from mouse skin were used. Transient transfection of
EGFP plasmid driven by hTERT promoter was performed by lipofectamine to
assess the activity of hTERT promoter in above cells. The transcriptional activity
of hTERT promoter was quantified using transient co-transfection of phTERTLuciferase and a LacZ reporter plasmid by lipofectamine. We constructed AdhTERT-CD adenovirus as the therapeutic vector. In various concentrations of
adenovirus and 5-ftuorocytosine (5-FC), the cell viability induced via the CD/5-FC
system was analyzed with the WST-I assay. We also tested the anti-tumor effect
of Ad-hTERT-CD in syngeneic mouse tumor models.
RESULTS: The significant expression of EGFP gene driven by hTERT
promoter was demonstrated in MBT-2 and NIH-3T3, but not in mature fibroblast.
In quantification assay of hTERT promoter, the luciferase expression was highest
in MBT-2, moderate in NIH-3T3, and lowest in mature fibroblast. In transfection
study, the Ad-hTERT-CD vector conferred 5-FC sensitivity to MBT-2 and NIH3T3 cells, whereas mature fibroblast remained unaffected. The degree of
cytotoxicity was correlated with the transcriptional activity of hTERT in murine
cell lines. The hTERT promoter-driven CD gene therapy followed by systemic
administration of 5-FC suppressed MBT -2 tumor growth in syngeneic,
immunocompetent mice. The long-term survival was prolonged in mice treated
with Ad-hTERT-CD vector followed by 5-FC therapy.
CONCLUSIONS: By restricting CD expression to MBT-2 cell, the hTERT
promoter allowed the use of suicide genes for cancer therapy without detrimental
effects on murine mature cell. In syngeneic mouse models, the tumor-specific
expression of suicide genes driven by hTERT promoter may be a novel cancer therapy.
Source of Funding: The study is supported by National Science Council,
Taiwan (NSC 90-2314-B-006-162).
727
EXPRESSION OF CABLES,A CELL CYCLE REGULATORY GENE
IS LOST IN INVASIVE TRANSmONAL CELL CARCINOMA OF
THE BLADDER Adam S Feldman", Zoe Tang, Sandra Kirley, Lawrence R
Zukerberg, W Scott McDougal, Chin-Lee Wu, Boston, MA
INTRODUCTION AND OBJECTIVE: We have recently cloned and studied a
novel cellular protein, Cables, whose genetic locus is located at chromosome
18qll-12. Loss of chromosome 18q is frequently observed in urothelial carcinoma.
We investigated the possible involvement of Cables in the pathogenesis of
transitional cell carcinoma (TCC) of the bladder in human tumor specimens.
"Presenting author.
Cables Present
100% (14/14)
100% (12112)
43% (8119)
33% (4/12)
9%(1/12)
4%(1/23)
Partial Loss
Total Loss
47% (9/19)
50% (6112)
33% (4112)
44% (10/23)
10% (2119)
17% (2112)
58% (7/12)
52% (12123)
Source of Funding: Institutional Funding.
728
MOLECULAR DIAGNOSIS AND OUTCOME PREDICTION IN
BLADDER TUMORS BY GENE PROFILING USING CDNA
MICROARRAYS Marta Sanchez-Carbayo*, New York, NY; Nicholas D
Socci, Bronx, NY; Juan Jose Lozano, New York, NY; Wentian Li, Manhasset,
, NY; Thomas Belbin, Michael Prystowsky, Bronx, NY; Angel Ortiz, New
York, NY; Geoffrey Childs, Bronx, NY; Carlos Cordon-Cardo, New York, NY
INTRODUCTION AND OBJECTIVE: The present study was conducted in
order to further characterize the molecular profiles of bladder tumor and validate
new targets involved in bladder cancer progression using a combination of cDNA
and tissue microarray technologies.
METHODS: The transcriptome of IS bladder tumors was compared against a
pool containing equal RNA quantities of four bladder cancer cell lines using cDNA
microarrays containing 17,842 known genes and expressed sequence tags (ESTs).
The potential clinical significance of the selected targets identified by cDNA
microarrays was validated at the microanatomical level using
immunohistochemistry on tissue microarrays containing a cohort of wellcharacterized superficial and invasive bladder carcinomas (n = 173).
RESULTS: Two main clusters segregating superficial from invasive
transitional carcinomas were identified and provided prognostic information.
Cytokeratin 20, neuropilin 2, p21 and p33ING I were selected among the top
ranked molecular targets differentially expressed between superficial and invasive
tumors and validated by immunohistochemistry with tissue microarrays. Their
expression patterns were associated with pathological stage, tumor grade, and
altered RB expression. Moreover, p331NGI expression levels were related to
overall survival. Generation of a support vector machine algorithm revealed the
relevance of WNT signaling and mitotic checkpoint alteration during bladder
cancer progression.
CONCLUSIONS: Gene profiling successfully classified bladder tumors based
on their histopathogenesis and clinical outcome, and identified molecular
biomarkers of potential clinical significance.
729
MULTIFOCAL TRANSITIONAL CELL CARCINOMA OF THE
BLADDER AND UPPER URINARY TRACT: MOLECULAR
SCREENING OF CLONAL ORIGIN USING CD44 ALTERNATIVE SPLICING PATTERNS Hideaki Miyake*, Akashi, Japan;
Isao Hara, Sadao Kamidono, Kobe, Japan; Hiroshi Eto, Akashi, Japan
INTRODUCTION AND OBJECTIVE: CD44 is a widely expressed cell
surface adhesion molecule in which various isoforms arise from alternative RNA
splicing mechanism at the step of cancer initiation. The objective of this study was
to assess whether multifocal transitional cell carcinoma (TCC) of the urothelium is

due to field defect, intraluminal seeding and implantation, or both.
METHODS: Using a series of 19 cases of synchronous multiple urothelial
cancers, we performed RT-PCR analysis using the set of primers that are capable
of amplyfying all CD44 splice variant isoforms. After the PCR products were
electrophoresed, band intensities with areas corresponding to the major isoforms
(i.e., CD44s, CD44vlO and CD44v8-1O) were quantified, and CD44vlO/CD44s,
CD44v8-IO/CD44s and CD44v8-IO/CD44vI0 ratios were calculated. Moreover,
p53 gene mutations in exon lesions of 4 to I I were screened by direct DNA
sequencing.
RESULTS: Among 19 cases, 14 exhibited almost similar CD44vlO/CD44s,
CD44v8-IO/CD44s and CD44v8-IO/CD44vI0 ratios between multiple urothelial
cancers in each case. However, in the remaining 5 cases, these ratios were quite
different between multiple cancer lesions. Furthermore, different types of p53
mutation between multiple cancer lesions were detected in only 3 of 19 cases,
which also showed different values of CD44vI0/CD44s, CD44v8-IO/CD44s and
CD44v8-IO/CD44vlO ratios.
CONCLUSIONS: These findings suggest that at least some of synchronous
multiple TCC of the urothelium seem to be of independent origin based on the
analysis of alternative RNA splicing of CD44. Moreover, these hypothesis was
supported by the evaluation of p53 gene mutations.
730
DOWNSTREAM
SIGNALING
PATHWAYS
AND
ANTIANGIOGENIC EFFECTS OF THE PAN-ERBB TYROSINE
KINASE INHIBITOR CII033 IN A HUMAN BLADDER CANCER
CELL LINE Carlos E Bermejo*, Colin Dinney, Ashish M Kamat, Daniel
Kedar, Maribelis Ruiz, David McConkey, Houston, TX; William L Elliot, Ann
Arbor, MI; Menashe Bar Eli, Houston, TX
INTRODUCTION AND OBJECTIVE: The erb-B receptor family plays an
important role in the pathogenesis of transitional cell carcinoma (TCC) of the
bladder. CI- 1033 is active against all four members of the erb-B receptor tyrosine
kinase family. Previously we determined that epidermal growth factor receptor
(EGFR)-directed therapy inhibited the growth of human TCe. The purpose of this
study was to determine the mechanisms by which therapy with CI-1033 inhibited
the expression of MMP-9 and IL-8 and the growth of human TCC growing within
the bladder of athymic nude mice.
METHODS: 253J B-V TCC cells were treated in vitro with CI-1033 with or
without EGF stimulation; MIT assay confirmed the antiproliferative effects of
CI-1033. Western blot analysis for EGFR, MAPK, JNK, p38, and AKT (total and
phosphorylated) was performed. MMP-9 activity was determined by zymography
and IL-8 production by ELISA. MMP-9 and IL-8 promoter activity was evaluated
following transfection with both promoters within a luciferase reporting construct.
In vivo, 253J B-V cells were injected into the bladder wall of athymic nude mice,
and the mice were treated with saline or CI-1033 (10 mg/kg twice weekly for 3
weeks). Tumors were harvested and weighed at the end of therapy, and IL-8,
MMP-9 and phosphorylated EGFR, MAPK, JNK, p38, and AKT were determined
by immunohistochemical(lHC) analysis of paraffin-embedded tumor.
RESULTS: Western blot analysis demonstrated that CI-1033 inhibited the
phosphorylation of EGFR, MAPK, JNK and AKT while p38 phosphorylation
remained unchanged. Both IL-8 and MMP-9 promoter activity were downregulated
by CI- 1033, and IL-8 expression and MMP-9 activity were suppressed following
treatment with CI-1033. Specific inhibitors of MAPK, JNK, and AKT inhibited
IL-8 expression but to a lesser degree than did CI-1033. In vivo, CI- 1033
significantly suppressed the growth of 253J B-V, compared with results for saline
controls (p<0.05). IHC analysis demonstrated inhibition of IL-8 and MMP-9
expression and decreased phosphorylation ofEGFR, MAPK, JNK, and AKT, while
p38 pbosphorylation remained unchanged.
CONCLUSIONS: CI-I033 mediates a significant antitumor effect against the
highly metastatic 253J B-V cells, which is accompanied by downregulation of the
expressionofIL-8 and activityofMMP-9. This effectis mediatedby JNK, MAPK, and
AKT pathways, although no pathway is dominant. Our data suggest that CI-1033
mediates greater antitumor activity than do more specific downstream inhibitors.
Source of Funding: Supported by National Cancer Institute Grant and by
American Foundation for Urologic Disease Grant (NIH Grant#: ONEOI04).
731
A NOVEL BLADDER CELL PROGRESSION MODEL AND
GENE EXPRESSION PROFILE: THE FL SERIES Brian E
Nicholson", Charlottesville, VA; Jabed Seraj, New Brunswick, NJ; Mark R
Conaway, Dan Theodorescu, Charlottesville, VA
INTRODUCTION AND OBJECTIVE: Animal models to study bladder cancer
metastasis are currently lacking. Thus, we sought to develop an animal model
which recapitulates the same pattern of metastatic disease as seen in patients and
serve as a platform for defining the genes associated with this process.
189
METHODS: Invasive but very poorly metastatic, T24T human urothelial

cancer cells were injected via the tail vein in nude mice. Incubation was allowed
until the mice showed clinical signs of disease. The mice were then euthanized and
metastases in the lungs were counted and placed in culture for growth. After the
minimal number of passages sufficient to have enough cells for subsequent
injection, cells were injected into another population of nude mice. This protocol
was repeated until three derivatives with progressive lung metastatic potential were
obtained (FLI, FL2, and FL3). RNA was isolated from the lineage during the
logarithmic growth phase, checked for degradation, and applied to the Affymetrix
Human Genome U133A GeneChip array. Gene array analysis on 22,284 gene
probe sets was performed with both the Affymetrix analysis software, as well as the
dCHIP analysis package. Northern blotting and/or RT-PCR were performed on
several genes with the largest changes in expression, both up and down.
RESULTS: The FLI line developed as a single lung metastasis at four months
post-injection,the FL2 line had multiple metastases bilaterally at two months, and the
FL3 line lungs were nearly consumed with metastasesat 3 weeks. 78 genes were found
to be up-regulated2-fold or greater and progressivefrom T24T to FL-3, while 51 genes
were down-regulated 2-fold or greater and progressive. The most up-regulated genes
includedinsulin-likegrowth factor binding protein 4 (45-foldincrease) and chondroitin
sulfate proteoglycan 2 (23-fold increase). The most down-regulated genes included
cyclin D2 (13-folddecrease) and vanin I (6-fold decrease).Northern blot and RT-PCR
analysis confirm the validity of these data.
CONCLUSIONS: The spectrum of this progression model is from the poorly
metastatic T24T through the highly metastatic FL3. Continued characterization of
this model including genetic reconstitution of down-regulated genes or inhibition
of up-regulated genes in metastatic cells will yield new insights into the biology of
bladder cancer metastasis. These insights should yield new therapeutic protocols
directed at distinct molecular targets.
Source of Funding: NIH; NIDDK.
732
GENE EXPRESSION PROFILING OF MUSCLE-INVASIVE
UROTHELIAL CARCINOMA OF THE BLADDER David A
Elkins", Stephen N Thibodeau, Michael L Blute, Michael M Lieber, Horst
Zincke, Stephen J Itturia, John C Cheville, Rochester, MN
INTRODUCTION AND OBJECTIVE: Yearly, over 50,000 new cases of
urothelial carcinoma of the bladder are diagnosed in the United States. Knowledge
of altered gene expression patterns in this cancer would improve our diagnostic and
therapeutic capabilities. To this end, we performed gene expression profiling of
muscle-invasive urothelial carcinoma of the bladder.
METHODS: As our microarray platform, we used the Affymetrix U95A
GeneChip. RNA was extracted from radical cystectomy specimens (n = 15),
normal adjacent urothelium (n = 9) from the same specimens, and four specimens
with carcinoma-in-situ (CIS). RNA labeling, array hybridization, washing and
detection were carried out according to Affymetrix protocol.
RESULTS: We identify many significant differences in gene expression
between normal urothelium and invasive carcinoma. Of particular interest is
osteopontin, a gene upregulated in numerous invasive cancers. Osteopontin is
highly overexpressed in invasive bladder cancer, but not in the preinvasive lesion
CIS. It is the only gene on the array which is highly overexpressed in every
invasive tumor profiled. Other overexpressed genes include cartilage glycoprotein
39, cytokeratin 20, interleukin 8, and ubiquitin-conjugating enzyme E2C, all of
which are known to be overexpressed in cancer. Many of the other overexpressed
and underexpressed genes have not yet been associated with cancer.
CONCLUSIONS: We found overexpression of numerous genes known to be
upregulated in cancer, lending credence to our results. Chief among these is
osteopontin, a gene reported to be overexpressed in other invasive cancers but not yet
associated with bladder cancer. Other alterations of gene expression seen in our
experiments are novel. We believe many of these differences in gene expression offer
diagnostic and therapeutic possibilities for urothelial carcinoma of the bladder.
Source of Funding: Departmentof Laboratory Medicineand Pathology, Mayo Clinic.
733
THE HISTONE DEACETYLASE INHIBITOR FR901228
PERFERENTIALLY ENHANCES ADENOVIRUS TRANSGENE
EXPRESSION IN BLADDER CANCER Takatsugu Okegawa*, Shigeo
Horie, Kikuo Nutahara, Eiji Higashihara, TOKYO, Japan; Jer-Tsong Hsieh,
Dallas, TX
INTRODUCTION AND OBJECTIVE: Efficient adenovirus infection requires
the presence of coxsackie and adenovirus receptor (CAR) and alpha V integrin.
FR901228 is a histone deacetylase inhibitor. The addition of the histone
deacetylase inhibitors after adenovirus infection is known to increase the
expression of viral proteins and transgene expression. In this study, we investigated
whether the histone deacetylase inhibitor FR901228 added before adenovirus
infection increase the efficiency of CAR and alpha V integrin and is associated with
adenoviral transgene expression after infection.
190
THE JOURNAL OF UROLOGY@
METHODS: Cytotoxicity studies were performed to determine a minimally

cytotoxic FR901228 concentration for bladder cancer cells. The levels of CAR
expression were determined by FACS and/or RT-PCR analysis of CAR, alpha V
integrin, and acetylated histone H3 in control and FR901228-treated bladder cell
lines.The efficiency of adenovirus mediated transgene expression was alsostudied.
RESULTS: The drug concentration showing no or minimal cytotoxicity
selected for these studies was 1 nglml FR901228 for bladder cancer cells.
Treatment of cancer cell s with a low concentration (I ng/ml) of the histone
deacetylase inhibitor FR901228increased CAR and alpha V integrin RNA levels
andacetylated histoneH3. This increase was associated with a 10- 50 fold increase
in adenovirus infection as evidenced by increased transgene expression from a
beta-galactosidase containing adenoviral vector.
CONCLUSIONS: We demonstrated that nontoxic doses of the histone
deacetylase inhibitor FR901228, a histone deacetylase inhibitor. can result in
marked increases in expression of CAR and alpha V integrin in bladder cancer
cells. This increase mediates enhanced transgene expression after adenovirus
infection. Thesestudies suggesta simple,clinicallypracticalmethod for increasing
the sensitivity of tumorcells to adenoviral gene therapy vectors.
METHODS: Transgenic mice were generatedthat expressTVA undercontrol

of the PSCApromoter. Mice were injected orthotopically with virus producercells
containing green flourescence protein (GFP), luciferase, and/or SV40 large T
antigen (TAG). At various timepoints, some mice were sacrificed, and bladder
tissue observed for GFP. In mice injected with luciferase, the charged couple
devices(CCD)camerawas used to observe the efficiency of injection, progression
of infection, and tumor development over time.
RESULTS: Immunohistochemistry performed on tissues of transgenic mice
confirmed TVA expression in bladder mucosa. Mice injected orthotopically with
viral vectors containingGFP demonstrated urothelial cell infection. CCD images
showed luminescence from infected bladder cells in TVA-positive mice(see
graphic), whilewild-type mice injectedwith Juciferase virus werenot luminescent.
CONCLUSIONS: The TVA system is a novel and efficient method of
introducing oncogenesinto specific cells andimagingurothelium, thereby allowing
TCC develpoment to be followed non-invasively over time.
734
GENECHIP, RAPID PS3 SEQUENCE ANALYSIS Sarel Halachmi",
Steven A Ahrendt, John T Chow, Li Wu, Naomi Halachmi, Stephen C YMg,
Scott Wehage, David Sidransky, Baltimore, MD
INTRODUCTION AND OBJECTIVE: The p53 tumor-suppressor gene is the
mostfrequently mutatedgene in humancancer,includingurological malignancies.
P53 mutation is associated with poor prognosis, and low responsiveness to
adjuvanttherapy. Despitethe importanceof p53 as a prognostic factor, it's use for
clinical practice is limited because immunohistochemical staining has not been
standardized yet, and direct sequencing is beyondcapability of most clinical labs.
Rapid mutation analysis of the pS3 gene sequencehas recently been developed
utilizingan oligonucleotide probe array. Our aim was to compared the mutation
analysis of the same DNA by the pS3 GeneChip and conventional
dideoxynucleotide sequencing.
METIIODS: Hundred tumors werecollected frompatients undergoing resection of
lung and bladder cancer. DNA was extracted with phenol/chloroform. Manual p53
Sequencing: Exons5-9of the p53 genewereamplified fromtumorDNAusingPCR.
PeR products weresequenced by cyclesequencing, separated by electrophoresis on a
polyacrylamide gel, and read by two independent observers. GeneChip pS3 Assay:
DNAfrom all patients was also sequenced by using the GeneChip pS3assay. Exons
2-11of thepS3genewereamplified as 10separate amplicons in a singlePCRreaction.
Amplified tumorand reference DNA werefragmented, 3'end fluoresceinated labeled
and transfered to the pS3probearray for 30 min at 45C. The probearray was then
scanned by laser. The emitted lightintensity was proportional to boundtumorDNA.
Mutations weredetected basedon the differences in hybridization intensities between
the reference and unknown sample.
RESULTS: Cyclesequencing of the conserved regions of the pS3 genedetected
76%of themutations withinthisregion. The GeneChip pS3assaydetected 81% of all
(exons 2-11) mutations. including 80%of themutations within theconserved regions
of the gene. The GeneChip detected 46/52 missense mutations (88%), but 0/5
frameshift mutations. The specificity of directsequencing and of the pS3 GeneChip
assayat detecting pS3mutations were 100% and 98%,respectively.
CONCLUSIONS: GeneChips provides several advantages over direct
sequencing:accuracy, detection of more mutations, and considerable time saving,
as all samples were analyzed with the by a single investigator in 6 weeks.
Conventional sequencing of these samples consumed almost I year. Neither the
pS3 GeneChip or directsequencing were able to detect all of the p53 mutations in
a groupof 100lung cancers. However, the pS3GeneChip provides a rapid screen,
detecting >80% of the mutations with a very low false-positive rate (2%).
Source of funding: None.
735
DEVELOPMENT OF A NOVEL MOUSE MODEL FOR
INDUCTION AND IMAGING OF BLADDER CANCER Isla P
Garraway, Chou Tran; Robert Reiter, Los Angeles, CA
INTRODUCTION AND OBJECTIVE: Transitional cell carcinoma (TCC) is a
common malignancy of the genitourinary (GU) tract, The heterogeneous nature of
TCC has led to studies to elucidate molecular eventsin tumorigenesis. However, the
paucity of animalmodels is a majordrawback. We havetakenadvantage of the avian
sarcoma leukosis virus(IVA) systemto develop a novel mousemodel forTeC.1n this
model, transgenic miceexpress the TVA receptor undercontrol of the prostate stem
cell antigen (PSCA) promoter. TVA expression is limited to GU tissuein thesemice.
Consequently, genedelivery by retroviral infection occursonlyin cellswhereTVA is
expressed. Multiple rounds of infection enabledifferent genes to be introduced intoa
singlecell, including the imaging gene, luciferase, whichimparts luminescence and
enables the natural history of TCC to be tracked non-invasively.
*Presenting author.
Source of funding: MargaretEarly Trust.
736
INACTIVATION OF THE FHIT GENE FAVORS BLADDER
CANCER DEVELOPMENT Andrea Vecchione, Cinzia Sevignani,
Enrico Giamieri, Nicola Zanesi, Roberta Bichi, Hideeshi Ishii. Rossano
Cesari, Leonard G Gomella, Kay Huebner, Carlo Croce, Raffaele Baffa*,
Philadelphia, PA
INTRODUCTION AND OBJECTIVE: The FHIT (Fragile Hyslidine Triad)
gene has been identified in a region at chromosome 3pI4.2, which is frequently
deletedin many tumors. We have previously reported deletionsat the FHIT locus
in 50% of the transitional cancer cell (TCC) cell lines tested and alterations of
FHIT expression in 61% of the primary TCC of the urinary bladder analyzed by
immunohistochemistry. In this report, we have studied the effects of FHIT
transduction in TCC derived cells and the susceptibility of the FHIT knockout mice
to N-butyl-N-(4-hydroxybutil)-nitrosamine (BBN).
METHODS: The SW780 TCC cells and human fetal kidney 293 (HFK)
cells were obtained from the ATCC. We constructed two adenoviral vectors
(ad-FHIT and ad-GFP) as recommended by the manufacturers. cDNAs were
expressed under control of the cytomegalovirus (CMV5) promoter. Each vector
was transfected into HFK-293 cells with plaque isolation and vector
purification after homologous recombination. Adenoviral infection was
performed at the desired MOl at 37 C for I h. Flow cytometry was performed
by standard protocols. For analysis of the tumorigenicity, cells were inoculated
subcutaneously in both flanks of 2 six weeks old male BALB/c nude mice in
each experimental group (Ad-FHIT; Ad-GFP; and vector alone). 28 FHIT1- and
25 FHIT+I+ mice were treated with 0.1 % BBN in drinking water for 13 weeks
and maintained on tap water for two more weeks. Animals were sacrificed at the
end of the 15h week, necropsy performed, and bladder processed for
histopathology.
RESULTS: Flow cytometry analysis of SW780 cells infected with adFHIT, ad-GFP, and control vector showed a significant increase in the
apoptotic cell population in the FHIT transduced cells. Mice injected with
SW780 cells alone and infected with ad-GFP developed a sizable tumor in both
flanks one month after the injection. The two mice injected with SW780 cells
infected with ad-FHIT are still tumor free 5 months after the injection. Six of
28 (21%) of the FHIT f - vs. 2 of 25 (8%) FHIT+I+ mice treated with BBN
developed invasive carcinoma. The difference between the two groups was
statistically significant.
CONCLUSIONS: Our results indicate that FHIT transduction induces
apoptosis in TCC cells, similarly with that observed in other human tumors.
In addition, restoring FHIT expression in SW780 resulted in abrogation
of tumorigenicity in nude mice. Lastly, FHIT-null mouse urothelium is more
sensitive to chemical carcinogens. These observations suggest that FHIT
based gene therapy should be explored as a therapeutic strategy for bladder
cancer.
737
ASSOCIATION BETWEEN A CIA SINGLE NUCLEOTIDE
POLYMORPHISM OF THE ECADHERIN GENE PROMOTER
AND TRANSITIONAL CELL CARCINOMA OF URINARY
BLADDER Zhang Xu*, Xin Ma, Qing-Guo Zhu, Longcheng u. Zhong
Chen, Wuhan, China
INTRODUCTION AND OBJECTIVE: E-cadherin was the prime mediator
of cell-cell adhesion in all epithelial tissues, reduced E-cadherin expression has
been shown to correlate with various carcinomas, including bladder cancer.
The abnormal expression of E-cadherin was consistent with the abnormal level
of E-cadherin mRNA in bladder transitional cell carcinoma (BTCC), but
the molecular mechanism on the down-regulation of E-cadherin mRNA
expression was unclear, which might be related to the single nucleotide
polymorphism (SNP) of E-cadherin gene. Our goal was to study the
relationship between a CIA single nucleotide polymorphism at position-160
from the transcription start site of E-cadherin gene promoter and cancer
occurrence, pathological grades and clinical stages in bladder transitional cell
carcinoma (BTCC).
METHODS: In 50 patients with BTCC and 50 normal controls, DNA
fragment containing -160 position of E-cadherin gene promoter was obtained
by using PCR from the human genomic DNA extracted from peripheral blood.
The PCR products were digested with both Hph I and Afl III, and the digestion
reactions were fractioned on 4% agarose gel. The individual electrophoresis
results accorded with individual genotypes at -160 position of E-cadherin gene
promoter.
RESULTS: The A allele frequencies at -160 position of E-cadherin gene
promoter were significantly higher in BTCC than in normal controls(P<O.O I),
also significantly higher in invasive BTCC than in superficial
carcinoma(P<0.05). However, there was no statistical difference in the A allele
frequency between high pathological grade and low pathological grade of
BTCC(P >0.05).
CONCLUSIONS: Our results demonstrated that a CIA single nucleotide
polymorphism at position -160 from the transcription start site of E-cadherin
gene promoter was closely related to cancer occurrence and clinical stages in
BTCC. the A allele frequencies at this position in invasive bladder cancer are
significantly higher than in superficial cancer. Whether this SNP is a new
predictor of disease progression in BTCC, it still needd to be confirmed in
long-term follow-up studies.
191
738
CISPLATIN-MEDIATED DNA DAMAGE INCREASED
APOPTOSIS BY XERODERMA PIGMENTO SUM GROUP C
PROTEIN IN BLADDER TRANSITIONAL CELL CARCINOMA
Jin Yang*, Indianapolis, IN; Zhiwen Chen, Chongqing, China
INTRODUCTION AND OBJECTIVE: DNA damage recongnition plays an
important role in DNA repair, mutagenesis and apoptosis. Failure to recognize
DNA damage leads to DNA replication without DNA repair and causes genome
instability. XPC protein is an important DNA damage recognition protein that
binds to DNA damage sites at very early stage during DNA repair. We have
investigated the survival statues of NF (normal) cells, cancer cells and xeroderma
pigmentosum group C cells (XPC protein deficiency) in treatment of cisplatin and
found many cancers and the XPC cells were further resistance to cisplatin than NF
cells. We raise the hypothesis that one of the functions of XPC protein is in keeping
sensing DNA damage and as a critical sensor in subsequently biological response
to DNA damage.Not only defect of XPC could cause cancer cell genetic instability
but also involve in reisistant to chemotherapy.
METHODS: Statues of XPC protein in normal cell (NF) and bladder
transitional cell carcinoma cell lines (HI197, T24) were determined by
western-blot. XPC cDNA and control vector were transfected into HIl97 and
T24 cells and stable expression clone cells were selected in level of mRNA
(RPA assay) and protein (western blot). These cells were administrated by
different concentration of cisplatin, and the cell survival assay was performed
through counting the cell number and analysis of cell clone genesis. In parallel
cell survival assay, cell apoptosis assay was done through detecting the DNA
ladder in agarose gel and expression of P53 and P73 were determined through
western-blot.
RESULTS: Expression ofXPC protein in HU97 and T24 were lower than that
in NF cells. The XPC protein increased apparently after transfection of XPC
cDNA. The HI197 and T24 cell transfected with XPC gene increased sensitivity to
cisplatin-mediated DNA damage and showed more cells were killed by the
pathway of apoptosis.
CONCLUSIONS: DNA damage recongnition function of XPC protein playa
critical role in keeping sensitivity to DNA damage and defects in XPC protein lead
to tolerance to DNA damage and apoptosis, On another hand, it helps us in
understanding the mechanism of resistant to chemotherapy in bladder cancer. Also,
it provided a new potential approach of gene therapy to bladder cancer.
739
ASSOCIATION OF THE POLYMORPHISMS OF THE ANTIOXIDANT ENZYME GENES (GPXl AND MNSOD) WITH
BLADDER CANCER RISK IN A JAPANESE POPULATION
Yasushi lchimura", Akita, Japan; Tomonori Habuchi, Kyoto, Japan; Lizhong
Wang, Kenji Mitsumori, Norihiko Tsuchiya, Kazunari Sato, Akita, Japan;
Hiroyuki Nishiyama, Shin Higashi, Osamu Ogawa, Kyoto, Japan; Tetsuro
Kato, Akita, Japan
INTRODUCTION AND OBJECTIVE: There is much evidence that suggests
reactive oxygen species (ROS) produced during normal cellular oxygen
metabolism are an important source of endogeneous DNA damage and may
contribute to carcinogenesis. Among specific mechanisms which counteract
oxidative DNA damage, the glutathione peroxidase I gene (GPXI) and manganese
superoxide dismutase gene (MnSOD) encode main antioxidant enzymes that
detoxify endogeneous ROS. Recently, the association between genetic
polymorphisms of GPXl (Leucine to Proline at codon 198; Leul98Pro) and
MnSOD (Valine to Alauine at ?9 in the signal sequence; Val-9Ala) and cancer
susceptibility has been proposed. In view of a possible relatiouship between ROS
and bladder carcinogenesis, we iuvestigated the relatiouship between the GPXI
and MuSOD polymorphisms and the incidence and progression of bladder cancer.
METHODS: The GPXI and MnSOD genotypes in 213 bladder cancer patients
and 295 normal controls were determined by the PCR-restriction fragment length
polymorphism technique.
RESULTS: The GPXI genotype frequency was 90.8% for Pro!Leu, 9.2% for
Prol Leu and 0% for Leu/Leu genotype in controls and 77.9%,22.1% and 0% in
cases, respectively. The MnSOD genotype frequency was 76.3% for Va1IVal,
21.7% for Val/Ala and 2.0% for Ala/Ala genotype in controls aud 79.3%, 19.2%
and 1.5% in cases, respectively. There was a significant difference in the GPXI
genotype frequency between the case group and the control group (p<O.OOI); the
adjusted odds ratio (aOR) for bladder cancer was 2.89 for the Pro/Leu genotype
compared with the Pro/Pro genotype (95%CI = 1.60-5.22, p<O.OOI). This
association was more significant when the nonsmoking case group was compared
with the control group (aOR = 4.28, 95%CI = 1.91-9.60, p<O.OOI). Compared
with the ProlPro genotype, the Pro/Leu genotype was significantly associated with
advanced tumor stage (Ta-I vs, T2-4; aOR = 2.58, 95%CI = 1.07-6.18, P =
0.034), but showed no significant association with the tumor grade (grade I vs.
grade 2+3). The analysis for the MnSOD polymorphism provided no significant
192
results. However, the presence of MnSOD Ala allele increased the bladder cancer
risk in men with the ProlLeu GPX genotype.
CONCLUSIONS: The present results suggest that the GPXl ProlLeu genotype
may significantly increase a risk of bladder cancer, and the increased risk may be
modified by the Ala-9Val MnSOD polymorphism.
740
MOLECULAR ALLELOTYPING IDENTIFIES PROGRESSION
MARKERS FOR TRANSITIONAL CELL BLADDER CANCER
Rolf Von Knobloch*, Heidrun Brandt, Christian Gack. Axel Heidenreich,
Rainer Hofmann, Marburg, Germany
INTRODUCTION AND OBJECTIVE: Transitional cell bladder cancers have
a heterogeneous biological behavior. Especially in superficial tumours a precise
estimation of potential for progression is mandatory to choose the correct
therapeutic option. To date there are no reliable progression markers as well as
serological markers for bladder cancer available. We applied the fluorescent
Microsatelliteanalysis (MSA) to perform a molecular allelotyping for the
identification of molecular markers and also used the method to perform a
serological tumor diagnosis.
METHODS: Between 1999 and 2001 58 fresh bladder cancers specimens of all
stages and blood samples were prospectively collected during TUR or cystectomy.
DNA from tumors and blood lymphocytes (normal-DNA) was extracted via
phenol-chloroform method, whereas free serum-DNA was extracted using a
commercially available kit (Midi-Kit, Qiagen). We performed fluorescent MSA
with a total of 32 polymorphic markers for the chromosomal regions 3p, 5q, 8p, 8q,
9p, 9q, 13q, 14q, 17p, 17q and 20q. Fragment analysis of the Cy5-labelled PCR
products was carried out on an automated laser sequencer (ALFexpressII,
Amersham Pharmacia Biotech).
RESULTS: The incidence of tumor-DNA alterations (loss of heterozygosity =
LOH; allelic imbalance = AI) was highest for chromosomal regions 3p, 5q, 8p, 9p,
9q und 20q in 43 to 62% of cases. We identified significant differences in
frequency as well as in composition of genetic alterations between non-invasive
pTa- as compared to invasive pTl-pT4-tumors as well as between low and high
grade tumors. Furthermore, alterations at the chromosomal marker sites 5q, 14q
and 17p were significantly (p<0.05) associated with advanced tumor stage and
nuclear differentiation grade, whereas LOH at 8p was only associated with tumor
stage and LOH at 13q only with tumor grade (p<0.05). In 79.3% of the 58 bladder
cancer patients tumor specific serum-DNA alterations were identified. The
presence of serum-DNA-alterations was significantly associated with tumor grade
(p=0.008) but independent from the underlying local tumor stage.
CONCLUSIONS; MSA of the chromosomal regions 5q, 8p, 13q, 14q and 17q
allows the identification of bladder cancers with an inferior prognosis. The
molecular serum-DNA analysis has the potential for application as a serological
tumor marker after radical therapy for G2 to G3-tumors.
Source of Funding: The study was supported by grant No. 912000 from the
Kempkes-Stiftung, Marburg.
741
EXPRESSION OF THE ETS-l PROTO-ONCOGENE IN HUMAN
BLADDER CANCER: CORRELATION WITH TUMOR
INVASION Han Yong Choi, Eun-Tak Kim*, Kwang-Sung Ahn, Eun Kyung
Bae, seoul, South Korea
INTRODUCTION AND OBJECTIVE: Hypoxia, one of microorgan
environments, activates the expression of Ets-I gene. The Ets-I transcription factor
transactivates VEGF and several genes encoding matrix-degrading proteases and is
thought to be involved in both tumor vascularization and invasion. The aims of this
study were to confirm that hypoxia induces the expression of Ets-I gene and to
evaluate the roles of Ets-l gene in bladder cancer development.
METHODS: We examined the expression levels of Ets-I in bladder cancer
cells growing under hypoxia for 12 hours and performed the promoter analysis
using constructed clones conjugated with reporter gene (luciferase). The expression
pattern of Ets-I was immunohistochemically investigated in 67 bladder cancer
specimens; 48 specimens without progression (29 from Ta stage, 19 from Tl stage)
and 19 specimens with progression (13 from Ta stage, 6 from Tl stage). The
activity of matrix metalloproteinases (MMPs) and VEGF expressioin were
examined in bladder cancer cells (T24-Ets-l) using zymographic analysis and
western analysis, respectively.
RESULTS: The expression level of Ets-l gene in bladder cancer cells was
increased 4 times higher in hypoxic condition than in normal condition and the
luciferase activity was threefold when compared with control. The extent and
intensity of immunoreactive Ets-llEts-2 polypeptides in bladder cancer tissue were
statistically much greater than noncancerous bladder tissues. In addition, Ets-I
expression was higher in high-grade than in low-grade bladder cancer and was
higher in advanced than in early stage cancer. Ets-I and MMP positivity were well
correlated with the invasive phenotype. The activity of gelatinase and the
'Presenting author.
expression of MMPs (MMP-l, -2, -3, -9, -13) in transfectants (T24-Ets-l) were
approximately 4 times higher than those in control.
CONCLUSIONS: Microorgan environments such as hypoxia affect the
expression of Ets-l gene. In bladder mucosal cells, hypoxia induced Ets-I affects
the activity ofMMPs known as one of the factors involved in the process of bladder
cancer invasion beyond the muscularis mucosa. These suggest that the change in
the expression level of Ets-I by hypoxia plays a role in malignant transformation
and progression in bladder cancer.
742
FEZI-DEFICIENT MICE ARE MORE SUSCEPTIBLE TO BBN
CARCINOGENESIS Raffaele Baffa*, Cinzia Sevignani, Hideshi Ishii,
Andrea Vecchione, Leonard G Gomella, Carlo Croce, Philadelphia, PA
INTRODUCTION AND OBJECTIVE: FEZIILZTSI is a putative tumor
suppressor gene at 8p22. FEZI is mutated in solid tumors including prostate,
breast, and esophageal cancers. Fez1 expression is absent or reduced in a sub type
of gastric cancer and in more than 60% of transitional cell carcinoma (TCC) of the
urinary bladder. The FEZ] gene encodes a 67-kDa leucine-zipper protein with a
region of similarity to cAMP-dependent activated protein. To further investigate
the role of FEZ] alterations in development of bladder cancer, we decided to use
heterozygous and nullizygous FEZ]-deficient mice in a chemically induced
carcinogenesis model.
METHODS: We utilized the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN),
a potent chemical carcinogen that induces bladder tumors in mice with a yield of
over 90% and one of the very few chemical carcinogens showing specific
carcinogenicity for mouse urothelium. BBN was obtained from TCI (Tokyo
Chemical Industry) America (Portland, OR). Twenty-five FEZI +f+ (13 males, 12
females), seventeen FEZI +f (9 males, 8 females), and sixteen FEZ] -f- (8 males,
8 females) were treated with 0.1% BBN in their drinking water for 13 weeks and
then maintained without any chemical supplement for two additional weeks. The
mice were weighed and observed daily. In all three groups, animals were sacrificed
at the end of the 15th week, necropsy performed, and bladder processed for
histopathological analysis. Histological urotheliallesions were classified into four
categories, namely: I) simple hyperplasia and mild dysplasia; 2) moderate and
severe dysplasia; 3) in situ carcinoma; and 4) invasive carcinoma.
RESULTS: In the FEZI +f+ group simple hyperplasia and mild dyslpasia were
detected in 20 mice (80%), three mice (12%) showed moderate to severe dysplasia,
only two mice presented neoplastic lesions, one with in situ carcinoma (4%) and
the other with invasive carcinoma (4%). Conversely, 14/17 (82%) FEZ] +f- and
15/16 (94%) FEZ] -f- mice harbored an invasive carcinoma. In most of the cases,
we observed squamous differentiation. Mild to moderate dysplasia was diagnosed
in the remaining FEZl-deficient mice. Chi-squared analysis proved a significant
association between FEZI-null genotype and onset of bladder tumors (p<O.OOOl).
CONCLUSIONS: The difference in the incidence of bladder tumors between
the three groups of mice is an important indication of the need for the urothelial cell
to have both FEZI alleles intact to respond efficiently to the damaging action of a
chemical carcinogen. FEZ] may become an excellent candidate for gene therapy in
advanced bladder carcinoma.
743
ANDROGEN DEPENDENT REGULATION OF BCG INDUCED
INTERLEUKIN-6 (IL-6) EXPRESSION IN HUMAN TRANSITIONAL CARCINOMA CELL (TCC) LINES Fanghong Chen*,
Guangjian Zhang, Peter Langenstroer, Yoshiki Iwamoto, William A See,
Milwaukee, WI
INTRODUCTION AND OBJECTIVE: Autocrine expression of IL-6 by TCC
in response to BCG may play an important role in promoting BCG adherence to
TCC, and consequently BCG treatment efficacy. IL-6 expression in response to
BCG requires NF-KB mediated signal transduction. Reports in other systems
suggest that signaling through the NF-KB pathway is regulated by sex steroids
(androgens and estrogens). This study evaluated the influence of androgens on
BCG induced, NF-KB mediated, IL-6 expression.
METHODS: RT-PCR was used to confirm expression of the androgen receptor
in the human TCC lines 253J and T24. A reporter construct containing an androgen
response element was used to establish the integrity of androgen mediated signal
transduction. Subsequently, the dose dependent effect of dihydro-testosterone
(DHT) on BCG induced IL-6 expression, and NF-KB signaling, was evaluated.
Two pharmacologic androgen receptor blockers were used to determine if receptor
blockade inhibited the effect of DHT on activation of the androgen response
element, NF-KB signaling, and BCG induced IL-6 expression.
RESULTS; Both human TCC lines expressed the androgen receptor and
demonstrated intact androgen stimulated signaling pathways. DHT suppressed
BCG induced, NF-KB mediated signaling and IL-6 expression in a dose-dependent
manner. DHT decreased mRNA levels of IL-6, expression of the full-length IL-6
promoter construct, and expression of an NF-KB specific reporter construct in

response to BCG relative to controls. At the IJ-tMconcentration, DHT completely
inhibited BCG induced activation of IL-6. Competitive, pharmacologic blockade of
the androgen receptor inhibited the effect of DHT on BCG induced signaling in a
dose-dependent fashion. IO,.,.M concentrations of both inhibitors restored BCG
signaling to control levels.
CONCLUSIONS: DHT down-regulates NF-KB mediated IL-6 expression by
human TCC lines in response to BCG. This effect is dependant upon a functional
androgen receptor-signaling pathway and can be blocked by inhibition of
androgen/androgen receptor binding. The effect of sex steroid mediated
modulation of BCG signaling on treatment efficacy and toxicity awaits further
study.
Source of Funding: Department of Veterans Affairs.
744
THERAPEUTIC EFFECT OF BACILLUS CALMETTE-GUERIN
INSTILLATION FOR BLADDER CANCER CAN BE IMPROVED
BY COMBINING WITH SUICIDE GENE THERAPY UTILIZING
CYTOSINE DEAMINASE Toru Nishiyama", Masafumi Oyama, Warren
D Heston, Bryan R Williams, Andrew C Novick, Cleveland, OH; William A
Larchian, Elyria, OH
INTRODUCTION AND OBJECTIVE: The objective of this study is to
evaluate the regional treatment of bladder cancer using combination of BCG
instillation and in situ suicide gene therapy with CD transfection and its effect on
distant disease.
METHODS: Superficial bladder cancer was established by simple
instillation of 5xl0 5 MBT-2 cells in 50,.,.1 of RPMI into the lumen of the bladder
of female C3H/HeJ mouse through a urethral catheter. Ten thousand MBT-2
cells in 50,.,.1 of RPMI were inoculated subcutaneously on the flank of the mice
on the same day. Three days later, the mice received intravesical instillation of
120J-tg of BCG in 50,.,.1 of PBS or PBS alone. Two days after instillation of
BCG, mice were transfected intravesically with either cytosine deaminase or
J3-Gal. In situ gene transfer to bladder tumor was accomplished via intravesical
instillation of plasmid DNA/catinonic liposome complexes. These combination
intravesical therapies were repeated 5 times. Mice were also given
intraperitoneal injection (ip) of 5-fluorocytosine (5-FC) or saline for fourteen
days beginning from seven days after tumor inoculation. The mice were divided
into four groups by the treatments; I) BCG + J3-Gal transfection and 5-FC ip,
2) BCG + CD transfection and 5-FC ip, 3) BCG + CD transfection and saline
ip, 4) PBS + CD transfection and saline ip. Mice were followed for bladder
tumor palpation, sizes of subcutaneous tumor and survival. All the treatment
groups consisted of eight mice.
RESULTS: All the groups treated with BCG instillation showed 50% bladder
tumor free survival rate while group 4 showed only 12.5%. As for the distant
bystander effects of the treatments, group 2 showed 50% tumor rejection while
only 12.5% of the mice in the all other groups survived without subcutaneous
tumor.
CONCLUSIONS: These results show regional therapy of bladder cancer using
BCG could have significant systemic anti-cancer effect when combined with
suicide gene therapy using CD/5-FC system.
745
COMPLETELY AUTOMATED MICROCAPILLARY MICROSATELLITE ANALYSIS FOR BLADDER CANCER DETECTION
Sarel Halachmi*, Michal Cohen, Reymond Shargal, Nadin Cohen, Haifa,
Israel; Mark P Schoenberg, Baltimore, MD; Ofer Nativ, Haifa, Israel
INTRODUCTION AND OBJECTIVE: Superficial transitional cell carcinoma
of the bladder (TCC) is a chronic and recurrent disease and in 70% of the cases
even a complete resection will be followed by tumor recurrence. The main goal in
management of TCC patients is early diagnosis of primary and recurrent tumors.
Microsatellite analysis was proven to be highly specific and sensitive but can be
used today as a research tool only with many limitations. The aim of our study was
to improve microsatellite analysis in order to simplify and facilitate the use of this
sensitive and specific method for the detection of TCC non-invasively in urine
samples.
METHODS: Matched samples of blood urine and tumor were taken from II
patients with proven recurrent and primary TCC of bladder, diagnosed by
cystoscopic direct visualization, confirmed by histopathologic analysis.
Additional blood and urine matched samples were taken from 5 healthy people
without any evidence of genito-urinary disease. DNA was extracted from the
matched samples, and subjected to fluorescent microsatellite analysis with 20
primers already proven elsewhere to be highly informative for TCC. PCR
products were analyzed automatically by the Abi 300 genetic analyzer (Perkin
Elmer, USA). Utilizing capillary electrophoresis and argon laser beam, the size
and the fluorescence intensity of the PCR products was determined. Graphic
193
and numeric analysis results enabled determination of microsatellite alterations

in tumor and urine DNA.
RESULTS: We detected matched identical urine and tumoral microsatellite
alterations in 11/11 patients (100%). We had overall 70% informative reactions,
and 33 matched tumor and urine microsatellite alterations. No alterations were
found among the 5 control samples. The automated system analyzed the samples
automatically after the initial programming. Results were displayed graphically and
numerically.
CONCLUSIONS: Our experience demonstrates that microsatellite analysis
detects primary and recurrent superficial bladder cancer in accord with the
observations previously published by other groups. In addition, we demonstrate
that complete automation of this technically challenging assay is feasible and may
facilitate the translation of microsatellite analysis from the research laboratory to
the clinic. Further study in large groups of patients will be required to document the
ease and economy of this form of in vitro diagnostic for the management of patients
with superficial bladder carcinoma.
746
HSVTK-MEDIATED LOCAL AND DISTANT ANTITUMOR
BYSTANDER EFFECT ON T739 BLADDER TUMOR IN T739
MICE Gang Ye*, Weichi Liu, Ronggui Zhang, ChongQing, China
INTRODUCTION AND OBJECTIVE: The bystander effect, produced by
ganciclovir-mediated killing of cells transduced with a herpes simplex virus
thymidine kinase (HSVtk) gene, defines the cooperative killing of non-HSVtktransduced cells. This approach has been used previously to treat experimental
brain tumors. Bladder cancer is very common in urological practice. Its localized
nature and the accessibility of the bladder space make it a potential target for a
similar type of in vivo gene therapy. In this study, we demonstrated the in vivo
bystander effect of mouse bladder cancer (T739) transduced with HSVtk gene
under the attack of ganciclovir (GCV).
METHODS: In our study, the bystander effect was assessed in vivo using
T739 cells. Mixtures of HSVtk transduced T739 cells (T739tk) and parent T739
cells were implanted into the peritonea of T739 mice. When 0.5 to 0.8 ern
tumor nodule could be obviously palpable in abdomen, the tumor was measured
using ultrasound scan and tumor volumes were calculated. Then the animals
were treated with GCV on a daily basis. Furthermore, similar intraperitoneal
animal model was established with parent T739 cells implanted subcutaneously
in the right flank. When tumors attained a size of 0.5 to 0.8 cm, the same
experiment protocol was performed. To evaluate the effects of this treatment on
survival, the animals were carefully observed and underwent necropsy as soon
as possible after death.
RESULTS: Mixtures of T739tk and T739 cells formed nodules after injected
in the peritoneal cavity. These tumors could be inhibited by administration of GCV,
even when T739tk were as few as 10-30%. Unexpectedly, a distant bystander effect
was observed as tumors in the right flank inoculated with only parent T739 cells
became cytostatic showing little further growth compared to controls and had an
increased mononuclear infiltrates. The distance bystander effect was significant
when more than 50% of T739tk was injected into the peritonea. The median
survival of animals treated with tklGCV system was significantly longer than that
of control animals treated with similar protocols.
CONCLUSIONS: The retrovirus-mediated HSVtk gene combined with GCV
therapy is effective in treating established T739 tumor in an in vivo setting. Local
and distance antitumor bystander effects obtained in this experimental model.
Together with histology of regressing tumors, which showed an infiltration of
lymphoid cells, these results are suggestive of an immune-related antitumor
response that could account for the distant bystander effect.
747
METALLOTHIONEIN: A NEW PROGNOSTIC FACTOR IN
UROTHELIAL BLADDER CANCER? Juergen Pannek", Sonja
Schmidtchen, Theodor Senge, Herne, Germany
INTRODUCTION AND OBJECTIVE: Prognosis and treatment of urothelial
bladder cancer depends on the aggressive behavior of the tumor. Although a variety
of tumor markers have been tested until today, the ideal prognostic factor for
treatment stratification has not been found. Thererfore, we analyzed the clinical
usefulness of metallothionein expression as a prognostic factor in urothelial bladder
cancer.
METHODS: In a retrospective study, immunohistochemical expression of
metallothionein in bladder cancer specimens of 122 patients was evaluated. For
imrnunostaining, a mouse monoclonal anti-metallothionein-antibody was used
(DAKO Corporation, Carpinteria, USA). Mean age of the 103 male and the 19
female patients was 68 years. Tumor staging was pTa in 40 patients, pTis in 18
patients, pTl in 20 patients, pT2 in 21 patients, pT3 in 20 patients, and pT4 in 3
patients. Metallothionein expression was assessed semi-quantitatively.
194
THE JOURNAL OF UROLOGY!!>
RESULTS: With a cutoff point of metallothi onein expression in 50% of the

cancer cell s, 5-yea r tumorspecific survival was significantly less in patients
with a high metallot hionein expre ssion (32 vs. 72%). Sig nificant differences
were found for 5-year -recurrence rate (90 % vs. 58%), and disease progre ssion
(78% vs. 54%).
CONCLUSIONS: We demonstrated a correlation between metallothionein
expression and turnor-specifc survival, progression-free survival and recurrencefree survival. Especially in pTl G3 tumors and carcinoma in situ, tumors with 50%
metallothionein-positive cells had a shorter tumor-specific survival, a higher
recurren ce rate and a higher progression rate. Therefore, metallothionein
expression seems to be a promising tumor marker in urothelial cancer.
748
CURCUMIN PREVENTS AY27 BLADDER TRANSITIONAL
CELL TUMOR GROWTH IN FISHER 344 RATS Saleem S Zafar* ,
Sylvania, OH; James A Hampton, Rick W Keck, Steven H Selman. Toledo, OH
[NTRODUCTION AND OBJECTIVE: Our objective in conducting this study
was to determine the efficacy of curcumin as an intravesical agent in the prevention
of superficial bladder cancer recurrence. Our study demonstrates that curcumin is
cytotoxic and prevents post-implantation growth of transitional cell carcinoma in
Fisher 344 rats.
METHODS: The AY27 rat transitional cell cancer cell line was plated on
culture dishes in culture medium. The cells were allowed 24 hours to attach and
were then exposed to curcumin in 0.1% DMSO for 30 minutes. Concentrations of
curcumin ranged from 0 to 500uM. The curcumin was then removed and replaced
with culture medium and the dishes were then placed in the incubator. The
surviving cell colonies were allowed 3 to 5 days to grow and were then fixed and
stained and counted under the microscope. Fisher 344 rats were used to study the
effects of curcumin on the prevention of bladder cancer growth after diathermic
bladder injury. Group I(n = 17) served as a control. The bladders of these rats were
injured, and the AY27 cells were allowed 30 minutes to attach. Subsequently, these
control rats received intravesical insillation of media without curcumin. Group 2
(n= 33) served as the treatment group. The bladders of these rats were also injured
and the AY27 cells were allowed 30 minutes to attach. Curcumin (500uM in 0.1%
DMSO) was instilled intravesically for thirty minutes. All rats were euthanized 3
weeks following treatment, and the bladders were harvested and analyzed both
grossly and histologically for the presence of tumor.
RESULTS: Curcumin was completely lethal at all doses above 200uM in vitro
(using 1,000,000 cells/dish). Tumor was seen in 82.4% (14 of 17 rats) of control
bladders and in 33.3% (II of 33 rats) of the treated bladders (p = 0.002).
CONCLUS IONS: Curcumin is cytotoxic to the AY27 cell line using the cell
colony survival assay. Furthermore, in vivo intravesical curcumin instilled 30
minutes following implantation prevents subsequent tumor growth in a significant
number of rats. Further studies are warranted to determine the clinical utility of
curcumin as an intravesical agent immediately following superficial bladder tumor
resection.
749
ANT IANGIO GEN IC EFFECTS OF FLAVONOIDS ON RAT
URINARY BLADDER EPITHELIAL CANCER INDUCED BY NKeikichi
BUT YL- N-(4HYDRO XYB UT YL )NITROSAMINE
Takata ", Shunsaku Takei, Tokuhiro Iseda, Masayoshi Yokoyama, Ehime,
Japan
INTRODUCTION AND OBJECT[VE: The level of vascular endothelial
growth factor(VEGF) expression is negative or low in normal bladder tissues but
high in human bladder cancer. Both VEGF and microvessel density(MVD) are
associated with tumor cell nuclear grade and clinical stage, and VEGF is positively
correlated with the occurrence and progression of bladder cancer. Tumor cells may
express high levels of VEGF through angiogenesis. In rat urinary bladder
carcinogenesis induced by chemicals, VEGF is implicated in tumor-associated
microvascular angiogenesis . Flavonoids are polyphenolic compounds and have
potential health benefits as antioxidants, anticarcinogens, anti-inflammatory agents
and inhibitors of platelet aggregation in vivo and in vitro. The present study
inve stig ate s whether the oral flavonoid s, cate chin and quercetin, have
chemopreventive effects during the initiation stages of rat urinary bladder
carcinogenesis in vivo.
METHODS: 96 female Wistar rats were divided into ten groups, of which nine
were given 0.05% N-butyl-N-(4-hydroxybutyl)ni trosamine(BBN) in drinking
water. Eight groups were orally administrated with catechin or quercetin (I, 3, 5,
10 mg/day) for 8 or 16 weeks, respectively. The incidence of bladder epithelial
cancer and angiogenesis estimated by histological findings (HE stain), VEGF
immunohistochemistry and measuring MVD was compared among the groups.
Expression of the VEGF gene in bladder epithelium was investigated by reverse
transcription-polymerase chain reaction (RT-PCR).
Presenting author.
RESULTS: None of the rats developed side effects from drugs used in this
study. The final weight of the body and liver did not significantly differ among the
groups. The tumor incidence and the MVD counts in rats given flavonoids
decreased dose-dependen tly decreased compared with those given BBN alone. The
differences in tumor incidence and MVD counts were statistically significant.
VEGP expression in the rat bladder induced by BBN was inhibited by catechin or
quercetin dose-dependently according to immunohistochemical and molecular
biological findings.
CONCLUSIONS: These findings suggest that catechin and quercetin inhibit
chemically induced bladder carcinogenesis, and that such inhibition is related to the
suppression of epithelial angiogenesis. Catechin and quercetin should be of
considerable benefit as chemopreventive age nts against urinary bladder
carcinogenesis .
750
ARE THERE SIGNIFICANT DIFFERENCES BETWEEN THE
PENETRATION OF NAKED PLASMID DNA AND OLIGO
NUCLEOTIDES INTO BLADDER TISSUE? Axel Schaaf", Sigrun
Langbein, Michael Siegsmund, Peter Aiken. Maurice-Stephan Michel,
Mannheim, Germany
INTRODUCTION AND OBJECTIVE: A lot of new strategies for the
treatment of bladder cancer or genetic disorders deal with plasmid DNA or
antisense oligonucleotides. It is of great interest for future clinical trials, to what
extent both of these methods are capable not only to effect suspended cells or
monolayered cell cultures in vitro but how they take effect on deeper cell layers of
solid organs. For this purpose the penetration in cultured cells and the depth of
penetration into an ex vivo porcine bladder of plasmid DNA and oligonucleotides
were compared in this study.
METHODS: RT 112, HT 1197 and UM-UC 3 human bladder carcinoma cell
lines were treated with the pEGFP-NI plasmid (4.7kb) encoding for the enhanced
green fluorescent protein or with a nonsense FITC-Iabeled oligonucleotide. Porcine
bladders were I hour after removal instillated with plasmid or oligonucleotid
containing solutions with/without transfecting agents (Lipofectamine TM 2000).
After incubation, the bladders were cryo-sectioned. Detection of the effected cells
was performed with the help of fluorescense microscopy.
RESULTS: The oligonucleotide treatment of cell cultures with and without
transfecting agents resulted in transfection rates of almost 100% in every case. The
plasmid transfection of the cell lines without transfecting agents rates effected
significant below I% of the cells. The treatment of the cell lines with lipofectamine
during plasmid transfection resulted in transfection rates from 36.61% (RT 112) to
88.69% (UM-UC 3). With the treatment of the porcine bladder with the pEGFP-NI
plasmid only a transfection of cells of the superficial layer could be achieved. ln
contrast, the treatment with oligonucleotides resulted in a transfection of deeper
cell layers, particularly when transfecting agents were used.
CONCLUSIONS: For future bladder cancer treatment strategies it has to be
considered, that even malignant cells in deeper layers of the tissue have to be
effected. This work points out, that not all of the strategies for future treatments are
capable to fulfil this task: plasmid-DNA in contrast to oligonucleotides is not able
to penetrate deeper cell layers, probably because of its larger size. Currently further
studies are underway to determine the potential therapeutic effect of intravesical
oligonucleotide application.
Source of Funding: Hector-Stiftung, Weinheim, Germany.
751
TE RE I EXPRESSION INHIBITS TUMOR GRO WTH AND
ALTE RS GENE EXPRESSION IN HUMAN BLADDER CANCER
Terence W McGarve y*, Trang B Nguyen, John E Tomaszewski, Stanley B
Malkowicz; Philadelphia, PA
INTRODUCTION AND OBJECTIVE: We have described a novel gene,
TERE I, which demonstrates decreased transcript and protein levels in muscle
invasive bladder cancer. Endogenous expression ofTEREI abrogates proliferation
of TCC cell lines and increases genomic stability. The mechanisms by which
TERE [ inhibits tumor cell proliferation are still unknown and the distribution of
TERE I expression in bladder tumors has not been established. The objective of this
study was to establish the level of expression of TEREI in clinical samples,
demonstrate the effect of TEREI expression on tumor growth, and suggest
mechanisms or pathways for growth suppression.
METHODS: The expression of TEREI in protein in a series of bladder tumors
and lymph nodes (n = 34) was demonstrated by immunohistochemistry using a
TEREI specific polyclonal antibody. Tumor formation was measured in vivo using
transduced J82 tumor cells and blank retrovirus transduced J82 cells. These cells
were also employed in a differential microarray (1.2 K).
RESULTS: Immunohistochemistry demonstrated 45.8% of 24 muscularis
propia invasive TCC of the bladder had less than 2% or less cells staining for
TEREl. In addition, five of ten TI (lamina propria) lesions had 2-10% or less of
Vol. 169, No.4, Supplement, Monda y, April 28, 200 3
the cells stained for the TEREI protein . We also found that there was an 88%
decrease in vivo tumor growth ofTEREI transduced J82 cell s compared to control
transdu ced J82 cell s in 20 nud e mice measured over a thirt y day period
(p < 0.OOO5). In a custom atlas array of 1,200 known gene s, we ident ified 8 1
transcript s that were modified between TERE I transduced J82 cells and the control
J82 cells by 4 fold or more . Modified genes were related to cell cycle control,
tyrosine receptors, adhesion, signal transduction , or apopto sis.
CONCL USIO NS: TEREl is significa ntly under-expressed in nearly 50% of
high stage bladder lesions. Re-expre ssion of TEREI in cells with a (-)TERE I
phen otype results in gro wth suppression. Wh ile not es tablishin g a direct
assoc iation to TEREI , multipl e gene transcripts were up and down regulat ed by
TEREI introduction in a pattern consistent with growth inhib ition. These data
suggest a role for TEREl in the growth regulation of bladder cancer.
IHC Slalnlng olT1, (n=10) >12Lesions ollh. Bladder (n=24) or Lymph Nodes (n=4).
per~~::~ce"a
T21esions
lymphnodemet.
T1leslons
4 + (>50%)
11124
(45.8%)
114 (25%)
4/10 (40%)
3~~:'
2+(2.10%)
1+ (2.10%)
no staining
2124 (8.3%)
1/24 (4.2%) 5/24 (20.8%)
5/24 (20.8%)
0/4(0%)
2110(20%)
0/4(0%)
1110(10%)
214 (50%)
3/10 (30%)
1/4(25%)
0/10(0%)
Source of Funding: Veternas Affairs Merit Review Grant.
195
co ntro l of the UPII promoter and Ad-UPII -T NF carrying tumor necrosis factor
(TNF) under control of the UPII promoter were generated. Ad-UPII-TNF was used
to infect bladder and nonbladd er cell lines. Furthermore, the Ad-UPII-TNF or
Ad-UPII-GFP was instillated into an orthotopic human bladder cancer mod el
intravesically,
RESULTS: We cloned a 2542-bp fragment of DNA that flanks 5 of the
Uroplakinll gene from hum an peripheral blood genome DNA . Tran sfection
experiments showed that the DNA segment resulted in preferential expression in
bladder cancer cells with negligible expression in non-bladder cells. Gel shift assay
demonstrated that the promoter region (a 6OO-bp fragment) could bind to the BIU-87
nuclear protein. ELISA showed TNF was produced by bladder cancer cell lines
efficiently with high specificity in comparison with nonbladder cell lines. MTT assay
showed more bladder cancer cells died with increasing MOl of Ad-UPII-TNF. Culture
medium of bladder cancer cell lines infected with Ad-UPI!-TNF could induce L929
cell lines to die. Intravesical instillation of Ad-UPII-TNF also caused decreased tumor
growth in an orthotopic human bladder cancer model. Furthermore, the tissuespecificity of promoter was confirmed using Ad-UPII-GFP in nude mouse.
CONCLUSIONS: These data suggested that most of the cis elements that confer
the bladder-specific and differentiation-dependent expression of human upn gene
must reside in the 2542-bp sequence and indicated that UPI! promoter had strong
potential for gene therapy aimed at the treatment of human bladder cancer.
Source of Funding: China science funding .
752
754
VALIDATION OF THE TISSUE-ARRAY TECHNIQUE AND

PROGNOSTIC VALUE OF CYCLIN E, P53, P21 AND P27 IN
MUSCLE INVASIVE BLADDER CANCER Jerome Rigaud, Helene
ABERRANT PROMOTOR METHYLATION PROFILE OF

BLADDER CANCER Choong H Noh *, Woo-Chul Moon, Sam Y Moon,
Hovington, Eva Latulippe, Annie Lessard, Helene Larue, Louis Lacombe,

Yves Fradet, Quebec, QC, Canada
INTRODUCTIO N AND OBJECfIVE: The aim of this stud y was to evaluate
the tissue-array technique for immun ohistochem ical analysis and the prognostic
value of expression of Cyclin E, pS3, p2l and p27 proteins in muscle invasive
bladder cancer.
METH ODS : Tumor tissu e from a total of 236 different muscle invasive
bladder cancers were included in tissue-arra ys by taking a minimum of 4 cores of
0,6 mm of diameter for each of the formalin-fixed paraffin embedded samples .
T umour markers of cell cycle Cycl in E, p53, p21 and p27 were analysed by
immunohistoc hemistry in whole tissue sections and in tissue -arra ys of the same
specimens. The positive or negative expre ssion of tumour markers for each sample
was co mpared between tissue-arrays and whole tissue sections. Furthermore, we
analysed the correl ation between the expression of these tumour mar kers and
clinico pathological variables by using a chi squar e test . Overall survival and
disease free survival according to the expres sion of tumour markers was studied by
Kaplan-Meier method with differences mea sured by log rank test.
RESULTS : For the four tumour markers, a mean of 77.1 % of cores were
interpretable but only 3.2 % of samples were non-informative. Concordance of
expr ession of tumours markers between whole tissue sections and tissue-arrays was
observed in 96.0 %, 94.4 %, 88.2 % and 90.8 % for Cyclin E, p53, p21 and p27
respe cti vely. Over-expression of Cyclin E, p53, p21 and p27 was noted in 46.0 %,
32 .9 %, 54. 1 % and 30 .7 %. Ove r-expres sion of Cyclin E and p53 was correl ated
with grade 3 (p= 0.05 and p=0.03 respectively). None of these four tumour
markers had a statistically signific ant prognostic value on overall survi val and
disease free survival.
CONCLUSIONS : Tissue -array technique is a valuable and accurate method for
immunohistochemica l analysis in muscle invasi ve bladder cancer. Expre ssion of
Cyclin E, p53 , p21 and p27 had no progn ostic value on survival of muscle invasive
bladder cancer patient s in the present cohort.
753
TARGETING GENE THERAPY TO BLADDER CANCER USING
A NOVEL BLADDER-SPECIFIC PROMOTER Zhu Hongjianr, Guo
Yinglu, Zeng Xiangfu, Zhang Zhiwen, Zhang Zhiqing, Beij ing, China
INTRODUCTION AND OBJE CTIVE: Differential expression of the desired
gene product in the target tissue is central to the ge ne therapy. One approach is to
use tissue-specific promoter to dri ve therapeut ic genes. UroplakinIl (UPII) is a
bladder specific membrane protein. The feasibility of tissue-specific gene therapy
for bladder cancer using human UPII promoter was investigeted.
METHODS : Two oligonucleotide primers were synthesized for PCR
amplification using a DNA template isolated from human peripheral blood . DNA
fragment cloned by PCR amplification was trans fected into five different tissue cell
lines, bladder cancer celliines BIU-8 7 and nonbladder cell lines GRC-I (renal
cance r), AS49 (lung cancer), EC (vascular endothelium)and Hs27 (skin fibroblast)
using luciferase as reporter gene. The nuclear extr act was prepared from BIU-87.
DNA-protein interactions were examined by gel shift assay. Two recombinant
ade noviruses : Ad-UPII-GFP carrying green lluorescence protein (GFP ) under
Tae H Uhm, Myung R Oh; Seoul, South Korea

INTRODUCTION AND OBJECTIVE: Promotor meth ylat ion provid es
alternati ve pathway to gene delet ion or mutation for the loss of tumor suppresso r
gene functi ons. Thi s epigenetic change may be a new marker of hum an cance rs.
We herein investigated the aberrant methylation profile of bladder cancers to
identify its role in bladder carcinoge nesis and its diagnostic value for bladder
cancer.
METHODS : Gene promotor meth ylati on was analyz ed in 50 bladd er
transiti onal cell car cinoma tissue s a nd histologi cally normal muc osal tissue s
aro und bladder can cer . In add ition , we a nalyze d 29 nonmalignant bladder
mucosal tissue s. The meth ylation status of 12 gen es were analyz ed by
meth ylati on speci fic pol ymerase ch ain reaction , bisulfite genomic sequenci ng
and methylation genotyping oli go nuc leo tide microarray an al ysi s . Th e
meth ylation frequency and methylati on index of each bladder tissue gro up were
comparativ ely anal yzed.
RESULTS: Bladder cancer tissues commonly showed aberrant promotor
hypermethylation for RASSFlA (62 .0 %), retinoic acid re ceptor(RAR)beta(58.0%), H-cadherin(S4.0%), E-cadherin (38.0%) and pI6(32.0%), whereas ,
meth ylation was not common for de ath as sociated protein kinase(IO.O%),
FHIT(2 .0%), adenomatous polyposis co li(2.0%), MGMT(4.0%), p7 3(4 .0%),
pI5(4.0%) and pl4(4.0%). Benign bladd er mucosa rarely showed abe rrant
methylation except RAR-beta(27.6%). In contrast, normal looking mucosa aroun d
bladder cancer showed aberrant methylation in high frequency similar to bladder
cancer. The methylation index of bladder cancer and normal looking mucosa
around bladder cancer was 0.29 and 0.23, respectively, both of which were
significa ntly higher than that of beni gn bladder mucosa (0.0 2, p<O .O1).
Rem arkably, majority(94%) of the bladd er ca ncer tissue s showe d aberrant
methylation of at least one of 4 genes including RASSFIA, H-cad, E-cad and p16.
CONCLUSIONS: Aberrant prom otor methylation may be a cri tical step in
development of bladd er cancer. Aberrant methylation of RASSFIA, H-cadherin,
E-cadherin and pl6 may be a prom ising new tumor marker for the diagnosis of
bladder canc er. The se aberrant methylation may arise as a field change in
precancerou s stage. Promotor methylation of some genes may develop in bladder
uroth elium secondary to inflammati on and aging pr ocess as well as carcinogenesis.
Source of Funding: No .
755
A REAL-TIME QUANTITATIVE ANALYSIS FOR HTERT AND
HTR MRNA MAYBE A TUMOR MARKER ASSOCIATED WITH
TUMOR STAGE AND GRADE FOR BLADDER CANCER Yoshio
Takihana", Manabu Kamiyama, Yasuhisa Furuya, Takashi Tuchida, Mizuya
Fukasawa, Isao Araki, Nobuaki Tanabe, Masayuki Takeda, Yamanashi,
Japan
INTRODUCTION AND OBJECfIVE: The expression of the telomerase
subunits such as human telomer ase reverse transcriptase (hTERT) and human
telomerase RNA component (hTR) may be associated with tumor development and
progression. The recent study dem onstrated that the telomerase subunits as well as
telomerase activity were almost expre ssed in the tissue of bladder cancer. We
evalu ated the relationship between hTERT and hTR mRNA quantification using
real-time PCR and the tumor stage and grade in bladder cancer.
196
METHODS: We examined the hTERT and hTR mRNA in 29 specimens with

bladder cancer (Grade: Grade I, 10 cases; Grade II, 12 cases and Grade ill, 7 cases.
Stage: pTa, 13 cases; pTl-T4, 16 cases). We immediately froze all of specimens
obtained from TUR-Bt and isolated the total RNA using RNeasy Kit (QIAgen). We
measured the real-time RT-PCR quantification of hTERT, hTR and GAPDH mRNA
expression based on TaqMan technology using Smart Cycler. The normalized hTERT
and hTR values were presented as the hTERT-GAPDH ratio (IOOXhTERT/GAPDH)
and the hTR-GAPDH ratio (IOOXhTRfGAPDH), respectively.
RESULTS: The normalized hTERT values were 0.50.3 in tumor Grade I,
3.7 1.3 in Grade II and 18.68.2 (MeanSE) in Grade III, respectively. The
normalized hTERT values in Grade III were significantly higher than the level in
Grade I and 2. The normalized hTERT in pTa tumor stage (0.60.3) was
significantly lower than pTl-T4 (1O.83.9). The values of normalized hTR in pTa
(20.25.5) were significantly lower than pTl-T4 (46.09.4). The normalized hTR
expression was not correlated with tumor stage. There was no significant difference
in the normalized hTERT and hTR values according to number of tumor.
CONCLUSIONS: Our results demonstrated that the normalized hTERT
mRNA correlated with the progression of stage and increase of grade in bladder
cancer. The quantitative analysis of hTERT and hTR mRNA might be a novel
marker for the progression and therapeutic strategies.
756
THYMIDINE PHOSPHORYLASE AND BLADDER CANCER DIRECT TUMORIGENICITY IN VIVO AND A TARGET FOR
ANTI-ANGIOGENIC THERAPY Edward H Streeter", Gillian
Hutchinson, Adam Jones, Adrian L Harris, Roy Bicknell, Oxford, UK
INTRODUCTION AND OBJECTIVE: Angiogenesis, the development of a
tumour neovasculature, is a critical step towards cancer growth and metastasis. It
is a poor prognostic marker in bladder cancer and other solid tumours. Thymidine
phosphorylase (TP) is an angiogenic enzyme associated with bladder cancer
invasion and progression. Vascular endothelial growth factor (VEGF) is a powerful
angiogenic factor also strongly associated with bladder cancer angiogeneisis. Here
we demonstrate the effect of TP in vivo, and the use of anti-angiogenic therapy to
prevent tumour development.
METHODS: RT1l2 bladder cancer cells transfected with TP (2TlO) or with
empty vector (EVil) were implanted into the bladders of nude rats at laparotomy.
Animals were given either N-acetylcysteine (NAC) in drinking water (IOmg/ml),
intraperitoneal anti-vascular endothelial growth factor antibodies (VG76e, 500mcg
twice weekly), both therapies or neither. Rats were analysed at 6 weeks for bladder
tumours and the presence of metastases. Lectin staining was performed and
microvessel density assessed.
RESULTS: The table details the incidence of bladder tumours in each group of
animals. TP transfection significantly increases tumorigenicity of RTll2 bladder
cancer cells. In untreated rats, 10/20 implanted with 2Tl 0 cells developed bladder
tumours, vs lfl4 EVil implanted rats (p=O.OI, Fisher's exact test). there was no
evidence of metastatic disease. NAC or VG76e alone showed a possible reduction
in the incidence of tumors. Combination therapy eliminated tumour growth
(p=0.03). Therapies were well tolerated with no obvious side effects. Qualitative
differences were noted between treated and untreated tumour vasculature.
CONCLUSIONS: TP transfection greatly enhances RTll2 tumorigenicity.
This is the first demonstration of the direct role of TP in bladder carcinogenesis in
vivo. Additionally, the use of combination anti-angiogenic therapy proved highly
effective in preventing tumor growth, which promises future clinical efficacy. A
further study on the use of oral thymidine phosphorylase inhibitor to prevent
bladder tumour growth is in progress.
TPtransfectlon andantiangigenic agents vstumorgrowth
Cell line
EV11
2T10
2T10
2T10
2T10
Treatment
Bladder tumours (n/total)
Control
Conliol
1114
10120
VG76e
NAC
VG76e + NAC
218
217
017
Source of Funding: Royal College of Surgeons of England; Cancer Research

UK.
757
LOSS OF ANGIOGENIC INHIBITOR PIGMENT EPITHELIUMDERIVED FACTOR INDUCES UROTHELIAL HYPERPLASIA
AND HIGH STROMAL VASCULARITY IN A MURINE MODEL
Jennifer A Doll*, Veronica M Stellmach, Mona L Cornwell, Susan E
Crawford, Chicago, IL
INTRODUCTION AND OBJECTIVE: Angiogenesis is required for the
progressive growth and metastasis of all solid tumors. While the role of angiogenic
inducers in bladder carcinoma has received much attention , only a few studies have
*Presenting author.
focused on the role of endogenous angiogenesis inhibitors in bladder development

or disease. We found that a potent inhibitor of angiogenesis, pigment epitheliumderived factor (PEDF), strongly localizes to the human bladder epithelium. We
postulated that PEDF may be an important endogenous inhibitor of the bladder
vasculature and its loss could lead to dysregulated angiogenesis and growth of this
organ.
METHODS: PEDF null mice were generated by homologous recombination
using a PEDF null allele. Gross and microscopic examinations were performed on
age-matched wildtype (n=lO) and null (n=lO) mice at 3 months and more than 6
months of age. The bladder tissue was formalin-fixed and multiple sections
analyzed for epithelial hyperplasia, cytologic atypia, proliferative activity, and
stromal microvascular density using immunohistochemical stains for PCNA and
Factor VIII.
RESULTS: In contrast to the normal, uniform layer of bladder epithelial cells,
the PEDF knockout animals revealed a moderate degree of urothelial hyperplasia
months of age) and a more than 2 fold increase in stromal
at an early onset
vascularity over controls. By six months, the hyperplasia in null animals progressed
to a more severe phenotype with a significant increase in PCNA positive cells and
evidence of cytologic atypia.
CONCLUSIONS: These observations suggest that PEDF is important for
normal murine bladder development by suppressing the growth of both endothelial
and epithelial cells. Therapeutic replacement of this endogenous inhibitor of
angiogenesis may offer a new modality for stabilizing the growth of angiogenicdependent lesions of the bladder.
Source of Funding: NIH grant CA64329 to S.E. Crawford.
758
VASCULAR ENDOTHELIAL GROWTH FACTOR DIRECTLY
MODULATES MATRIX METALLOPROTEINASE-9 EXPRESSION BY HUMAN BLADDER CANCER THROUGH NEURO
PILIN RECEPTOR ACTIVATION Beryl Eve, Paul Sweeney, Houston,
TX; Diane Bielenberg, Boston, MB; Jerald J Killion, Sun-Jin Kim, Michael
Rosenblum, Houston, TX; Johannes Waltenberger, Ulm, Germany; Menashe
Bar-Eli, David McConkey, Colin P Dinney*, Houston, TX
INTRODUCTION AND OBJECTIVE: The purpose of these experiments was
to determine the effects of forced vascular endothelial growth factor expression on
tumorigenicity of human bladder cancer cells growing within the bladder of
athymic nude mice.
METHODS: 253J TCe cells, which are poorly tumorigenic and nonmetastatic
and which expresses low basal levels of VEGF, were stably transfected with the
full-sequence sense VEGF I65 cDNA. VEGF and MMP-9 expressions were
evaluated in vitro by Northern blotting and ELISA. MMP-9 activity was
dertermined by Zymography, and MMP-9 promoter activity within a luciferase
reporter construct. VEGFR expression was determined by RT-PCR and neuropilin
expression by FACS analysis. VEGF binding studies were performed in the
presence of labeled and unlabeled VEGF, 65' The VEGF '65and Neo-transfectants
along with the parental 253J cells were implanted into the bladder of athymic nude
mice, and growth was evaluated after three months.
RESULTS: VEGF and MMP-9 expression, collagenase activity, and MMP-9
promoter activity were all higher in the transfected cells and in the parental 253J
cells exposed to exogenous recombinant human VEGF'65' RT-PCR and VEGFR-l
and -2 in the 253J and its transfectants. Specific binding assays and FACS receptor
analyses confirmed that VEGF 165 interacted directly with neuropilin receptor in
these cells. A role for VEGF-neuropilin receptor interaction in promoting MMP-9
expression was confirmed using neutralizing antibodies to VEGF and neuropilin.
Transfection of the 253J cells with VEGF'65 induce a tumorigenic phenotype in
vivo characterized by expression of VEGF and MMP-9.
CONCLUSIONS: Together, these data reveal a novel, direct role for VEGFneuropilin receptor interaction in promoting the aggressiveness of human bladder
cancer cells.
Source of Funding: NIH.
759
IN VIVO RETROVIRAL MEDIATED GENE TRANSFER INTO
BLADDER UROTHELIUM RESULTS IN PREFERENTIAL
TRANSDUCTION OF TUMORAL CELLS Pierre Mongiat-Artus**,
Nicolas Dumey, Olivier Cussenot, Odile Cohen-Haguenauer, Paris, France
INTRODUCTION AND OBJECTIVE: Superficial bladder tumors frequently
relapse after resection, escape to intra-vesical BCG therapy and may become
invasive. New therapeutics are therefore needed to achieve cure, and gene therapy
is such a new and appealing therapy. We attempted to achieve transduction of
urothelial bladder cancer cells with direct intra-vesical infusion of a retroviral
vector earring a reporter gene.
METHODS: We have evaluated the feasibility of in vivo gene transfer in
directly infusing retroviral supernatant into dog bladder towards transduction of
superficial urothelium. The canine animal was chosen since it can present with
spontaneous bladder carcinomas mimicking human pathology. In a preliminary

study, the effect of urine on retroviral mediated gene transduction was evaluated in
vitro or during in vivo infusion. Here, transduction with an amphotrophic vector
was monitored through expression of marker genes (nlsLacZ and NeoR). Five
human malignant epithelial cell-lines and primary cultures of bladder urothelium
from normal dogs were significantly transduced. We then investigated transduction
of urothelial cells following bladder infusion of normal and spontaneous
transitional cell carcinoma (TCC) bearing dogs.
RESULTS: Transduced cells (blue-nuclei after X-gal staining) were evidenced
in primary cultures of normal epithelium. Analysis of tumor cryosections
evidenced transduction of urothelial cancer cells since a positive PCR signal was
detected and superficial layers stained positive for X-gal with almost no passing
through lamina propria. In vivo transduction varied from I to 15% (mean 5%) and
shown to be preferential for dividing malignant cells. Normal epithelium either
originating from sound dogs or from normal epithelium of tumor-bearing animals
was negative for X-gal staining.
CONCLUSIONS: These results demonstrate for the first time the feasibility of
in vivo retroviral transduction of superficial bladder cancer using clinically relevant
procedure.
increased (2.5x) binding to E-cadherin expressing cell lines (p=O,0003). This

increased binding was abrogated by blocking antibody specific for CDI03. In
normal urolthelium and TCC, CDlO3 was expressed by approximately 50% of T
lymphocytes (CD3) and 70% of cytotoxic T lymphocytes (CTL) (CD8). A few
lymphocytes were infiltrating tumours but most were clustered in the surrounding
stroma. In 25/26 cases of TCC more CDlO3 lymphocytes were present in the
stroma than within the tumour (p=0.OOO6), There was no correlation between
E-cadherin expression and infiltration of tumours by CDI03+ lymphocytes.
CONCLUSIONS: The CD103~E-cadherin interaction is important for
adhesion to and cytotoxicity of bladder cancer cells by CD 103+ lymphocytes. This
population consisted of CD3 + and CD56 + cells, suggesting that loss of Ecadherin during tumour progression may impair natural and T cell-mediated
anti-tumour cytotoxicity, and may adversely effect the response to BCG
immunotherapy. However, the failure of infiltration of tumours by lymphocytes
was not directly related to intensity of E-cadherin expression. Impairment of
lymphocyte migration may be due to factors such as the local cytokine
microenvironment.
Source of Funding: MRC Clinical Training Fellowship,
760
TUMOR SUPPRESSOR ROLE OF KISS-} IN BLADDER
CANCER: LOSS OF KISS-} EXPRESSION IS ASSOCIATED
WITH BLADDER CANCER PROGRESSION AND CLINICAL
OUTCOME Marta Sanchez-Carbayo", Paola Capodieci, Carlos CordonCardo, New York, NY
INTRODUCTION AND OBJECTIVE: The aim of this study was to evaluate
the role of KiSS-l in bladder cancer progression by characterizing its messenger
RNA (mRNA) expression levels in a comprehensive collection of bladder cancer
cell lines, as well as in normal urothelium, and in a large cohort of bladder tumors.
METHODS: The expression profiles of nine bladder cancer cell lines were
compared against a pool containing equal total RNA quantities of each of them
(8,976 genes and ESTs). We also compared 15 primary bladder tumors versus a
pool of four bladder cancer cell lines using also cDNA microarrays (17,842 genes
and ESTs). The expression pattern of KiSS-l transcripts was analyzed using in situ
hybridization in nine bladder cancer cells, paired normal urothelium and bladder
tumor samples (n=25), and tissue microarrays of bladder tumors (n= 173).
RESULTS: Lower expression of KiSS-l was revealed in bladder cancer cells
derived from the most advanced bladder tumors, When comparing 15 primary bladder
tumors versus a pool of four bladder cancer cell lines, lower transcriptlevels of KiSS-l
were observed in the invasive bladder carcinomas as compared to superficialtumors.
KiSS-l expressionratios provided prognostic information,Using in situ hybridization,
we observed complete loss of KiSS-1 in all invasive tumors under study as compared
to their respective normal urotheliurn. The expression of KiSS-1 was found to be
significantly associated with histopathological stage, Patients with lower KiSS-l
expression showed a direct correlation with overall survival in a subset of bladder
tumors whose follow-up was available (n=69). We did not observe any significant
differential KiSS-1 expression along cell cycle by sorting analysis.
CONCLUSIONS: A potential tumor suppressor role in bladder cancer was
revealed for KiSS-I. Moreover, it showed predictive value by identifying patients
with poor outcome.
761
THE ROLE OF E-CADHERIN IN BLADDER CANCER
IMMUNOTHERAPY Joanne Cresswell*, Wai Keong Wong, John Kirby,
David Neal, Newcastle upon Tyne, UK
INTRODUCTION AND OBJECTIVE: This study examines a novel mechanism
of tumour immune escape with important implications for BCG immunotherapy of
bladder cancer, E-cadherin is an epithelial cell adhesion molecule, down-regulatedby
invasive and metastatic cancers, Most intra-epitheliallymphocytes express a specific
counter-receptorfor E-cadherin, AE{37 integrin (CDI03). The role of the CDI03-Ecadherin interaction to adhesion and cytotoxity in vitro, and to tumour infiltrating
lymphocytesin situ in bladder cancer was studied,
METHODS: Expression of CDlO3 was induced on peripheral blood
lymphocytes in a mixed lymphocyte reaction (MLR) supplemented with TGFI3.
A sensitive flow cytometric adhesion assay and a redirected chromium release
assay were used to assess the effect of CD103 upregulation and blockade on
adhesion to and cytotoxicity of bladder cancer cells. Frozen sections of bladder
cancers (n=26) and of normal urothelium (n=4) were dual-stained for CD3,
CD8 or CDlO3 and Evcadherin. Cell counts were performed by light
microscopy and E-cadherin intensity measured by immunofluorescence and
confocal microscopy.
RESULTS: CD103 expression was upregulated to 60% of PBLs. Compared to
control lymphocytes the CDlO3 upregulated population showed significantly
197
Kidney and Ureteral Cancer:

Basic Research (II)
Discussed Poster
Monday, April 28, 2003
1:00-5:00 PM
762
IDENTIFICATION OF PATIENTS AT HIGH RISK OF RENAL
CELL CARCINOMA PROGRESSION AFTER RADICAL
NEPHRECTOMY Nathalie Rioux-Leclercq*, Jean Philippe Moulinoux,
Rennes, France; Jean Yves Bansard, 35033, France; Bernard Lobel,
Francois Guille, Alejandro R Rodriguez. Jean-Jacques Patard, Rennes,
France
INTRODUCTION AND OBJECTIVE: Renal cell carcinoma (RCC) is known
to show a wide variation in biologic behavior and clinical outcome. Markers of cell
differentiation, cell cycle, proliferation, apoptosis, cellular adhesion and growth
factors have been evaluated in association with usual prognostic factors as a
prognostic tool.
METHODS: 73 patients (47 males, 26 females; median age: 64 years)
undergoing radical nephrectomy for RCC, were included in our study, The
expressions of virnentin, Ki-67, p53, Bcl2, Fas, Fas-L, CD44H, VEGF and CD34
were studied by immunohistochemistry. TUNEL assay was used to detect in situ
apoptosis, Immunostaining, tumour stage, nuclear grade and tumor size were
correlated with observed progression cause-specific survival. Multiple
correspondence analysis and ascending hierarchical classification were performed
to determine significant prognostic factors.
RESULTS: 29 patients died, 22 from RCC with a median survival time of 14
months. With regard to cause-specific survival Ki-67 labelling index more than or
equal to 20%, CD44H expression more than or equal to 20%, and VEGF
expression more than or equal to 30%, were significant independent variables both
in patients with metastases (p<0,04, p<0.02, p<O.OOOOI; respectively), and in
patients without metastases (p<0.006; p<O.OOOOI; p<O.OOOOI; respectively),
Multicorrespondence analysis included TNM, nuclear grade, tumour size, Ki67,
CD44H and VEGF expression stratified our patients in 2 groups. Group I patients
are defined by the expression of less than 4 variables among the 6 analyzed
variables. Group 2 patients were characterized by the expression of 4 or more than
4 variables. Median cancer-specific survival for group I patients was 73 months,
compared to 37 months for group 2 patients (p<O.OOOOI).
CONCLUSIONS: We have demonstrated that the patients at high risk of RCC
progression (greater than 50% at 24 months) could be identified by a multivariate
correspondence analysis using Ki-67 labelling index, CD44 and VEGF expression
in association with tumour size, nuclear grade and tumour stage.
Source of Funding: None,
763
INTRACELLULAR 72 KDA HEAT SHOCK PROTEIN (HSP72)A PROGNOSTIC MARKER OF RESPONSE TO IMMUNOTHERAPY IN METASTATIC RENAL CELL CARCINOMA Jan
Roigas*, Stefan Richter, Serdar Deger, Andreas H Wille, Stefan Hauptmann,
Stefan A Loening, Berlin, Germany
INTRODUCTION AND OBJECTIVE: Immunotherapy with biomodulators
such as interleukin-2 (IL-2) and interferon-a2 is currently the main treatment option
of metastatic RCC. Beside other mechanisms, IL-2 acts via activation of NK cells.

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184

THE JOURNAL OF UROLOGY

METHODS: The video highlights our technique of laparoscopic radical

Vol. 169, No.4, Supplement, Monday, April 28, 2003

Bladder Cancer: Basic Research (II)

Vol. 169, No.4, Supplement, Monday, April 28, 2003

THE JOURNAL OF UROLOGY

biological progression from superficial to invasive phenotype. This work aimed to

293 cells 1929 cells

Source of Funding: Max Kade Foundation; Flight Attendant Medical

Vol. 169, No.4, Supplement, Monday, April 28, 2003

THE JOURNAL OF UROLOGY

THE INHIBITION OF CYCLOOXYGENASE-2 (COX-2) EXPRESSION BY COXSACKIE AND ADENOVIRUS RECEPTOR

GENE EXPRESSION PROFILES ANALYSIS OF PATIENTS

Chen Pong, Hossein Saboorian, Arthur I Sagalowsky, Dallas, TX

INTRODUCTION AND OBJECTIVE: Chemotherapy resistance presents a

Vol. 169, No.4, Supplement, Monday, April 28, 2003

bladder cancer and is considered to be associated with disease progression. In this

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THE JOURNAL OF UROLOGY

METHODS: A DNA micro array-based differential display analysis of 10,000

Vol. 169, No.4, Supplement, Monday, April 28, 2003

METHODS: Ninety-two (92) urothelial specimens including non-malignant

Immunohistochemical Staining for theCables Protein In Urothelial Specimens

Source of Funding: Institutional Funding.

THE JOURNAL OF UROLOGY

Vol. 169, No.4, Supplement, Monday, April 28, 2003

to assess whether multifocal transitional cell carcinoma (TCC) of the urothelium is

METHODS: Invasive but very poorly metastatic, T24T human urothelial

THE JOURNAL OF UROLOGY@

METHODS: Cytotoxicity studies were performed to determine a minimally

Vol. 169, No.4, Supplement, Monday, April 28, 2003

METHODS: Transgenic mice were generatedthat expressTVA undercontrol

Source of funding: MargaretEarly Trust.

Vol. 169, No.4, Supplement, Monday, April 28, 2003

THE JOURNAL OF UROLOGY

Vol. 169, No.4, Supplement, Monday, April 28, 2003

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Vol. 169, No.4, Supplement, Monday, April 28, 2003

promoter construct, and expression of an NF-KB specific reporter construct in

THE JOURNAL OF UROLOGY

and numeric analysis results enabled determination of microsatellite alterations

THE JOURNAL OF UROLOGY!!>

RESULTS: With a cutoff point of metallothi onein expression in 50% of the

Vol. 169, No.4, Supplement, Monday, April 28, 2003

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Vol. 169, No.4, Supplement, Monda y, April 28, 200 3

1/24 (4.2%) 5/24 (20.8%)

Source of Funding: Veternas Affairs Merit Review Grant.

VALIDATION OF THE TISSUE-ARRAY TECHNIQUE AND

ABERRANT PROMOTOR METHYLATION PROFILE OF

Hovington, Eva Latulippe, Annie Lessard, Helene Larue, Louis Lacombe,

Tae H Uhm, Myung R Oh; Seoul, South Korea

THE JOURNAL OF UROLOGY

Vol. 169, No.4, Supplement, Monday, April 28, 2003

METHODS: We examined the hTERT and hTR mRNA in 29 specimens with

Bladder tumours (n/total)

Source of Funding: Royal College of Surgeons of England; Cancer Research

focused on the role of endogenous angiogenesis inhibitors in bladder development

Vol. 169, No.4, Supplement, Monday, April 28, 2003

spontaneous bladder carcinomas mimicking human pathology. In a preliminary

THE JOURNAL OF UROLOGY

increased (2.5x) binding to E-cadherin expressing cell lines (p=O,0003). This