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184 THE JOURNAL OF UROLOGY Vol. 169, No 4 Supplement, Monday, April 28, 2003

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 i leum and stapled ileal- ileal

anastomosis, (2) detubularizat ion and creat ion of posterior i leal 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.

V7

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 rel lux 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 1.8days 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 1I0win 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.

Source of Funding: None.

V7

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 technical ly 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, thenpassed 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.

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 sacrif iced 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 loopgram 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-rel luxing ureterointest inal

anastomosis. Furthermore, its inherent simplicity allows this delicate reconstructive

part of the operation to be performed in time sensitive manner.

Source of Funding: None.

Bladder Cancer: Basic Research II

Discussed Poster

Monday, April 28, 2003 1:00-5:00 PM

7

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 Cre

recombinase 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 media ted

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 R26cre

ERT-mice carrying Rblox/,ox/p53Iox/ ox_, Rblox/w /p53Ioxf ox_ and Rb oxfw /p53Iox/w,_

combinations received 5 mg OHT intravesically and were observed w wild

type). Controls of the combinations received a pellet without OHT.

RESULTS:Three months after OHT implantationinto the bladder R26cre-ERT

Rb ox/lo /p53IOX OX_

and Rb

'

/w /p53 Ox/lOx_nrice

developedcarcinomain situand muscle

invasive bladder cancer. Control mice and mice carrying Rblox/w /p531ox/w,_

combinationsdid not develop tumors. Immunohistochemical stainingwas positive for

cytokeratineand negativefor smoothmuscle actin, vimentinand desmin.Lymphnode

metastasis or distant metastasiswas 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-control led manner. After target ing 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.

Source of Funding: None.

7 3

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 sufficientlevels of CAR in order to be susceptible toAd 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

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THE JOURNAL OF UROLOGY

185

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. CeUviability 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-tkwas 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 Ad5 F S

vector incorporating the Ad3S fiber protein can greatly augment suicide gene

therapy in CAR down regulated bladder cancer ceUswhile 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.

7

THE PROTEIN PHOSPHATASE ACTIVITY OF PTEN IS

SUFFICIENT TO INHIBIT CELL MIGRATION AND ORTHO

TOPIC INVASIONBUT NOT TUMORIGENICITY IN BLADDER

CANCER Dan heodorescu John Gildea ichael 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 ceUmotility, a necessary component of tumor invasion.

However i t is unknown whether PTEN is mechanistical ly 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 byGI29R 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 faithfuUyreproduces human disease. Microscopic

examination of murine bladders at the completion of this experiment parallels the

r esult s obt ai ned with the o rganotyp ic assay. In con tr as t, s ubcu taneous

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.

7 5

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

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 (8Ta, 18TI (8G2, 10G3), 10T2

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 aUtumours.

Findings were verified using reverse transcriptase-polymerase chain reaction (RT

PCR), 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 str iking 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 similari ty in gene expression profi les

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 CoUegeof Surgeons of England; Cancer Research

UK; Martin Charitable Trust.

7 6

RENDERING BLADDER CANCER SUITABLE FOR ADENO

VIRAL 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 neces sary for adenoviral cell atta chmen t and therefore

adenoviral gene therapy, is down-regulated in clinical bladder cancer. I t has

therefore been argued, that bladder tumors are not a good target for adenoviral

gene therapy. Recently ithas 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 es tab lished drug in the long-t erm treatment of ep ilepsy, which was

recently shown to exhibit strong HDACI-activity. Here we show, that VPA can

up-regula te CAR at c linica lly applicable doses, rendering bladder cancer

suitable for gene therapy.

METHODS: T24 bladder cancer cells were treated for 72 h with VPA at

various doses, cel l 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 CAR

copy number up to 13.5-fold, which resulted in 66.5 of ceUsexpressing 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

mM

VPAtreatnlent

0.0

0.6

1 2

2 4 5 293 cells

1929 cells

copy

number

foldIn-

10 2.0

39 9.2 13 5 61 0.0

crease

flowcytometry shift

of

2.9

43

14 8

42.0

66.5

99 8

0.0

cells

luciferase

expression fold

1.0 4.3

11 5 17 4 38

n nla

increase

Source

of

Funding:

Max Kade Foundation;

Flight Attendant

Medical

Research Institute.

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THE JOURNAL OF UROLOGY Vol. 169, No.4 Supplement, Monday, April 28, 2003

THE INHIBITION OF CYCLOOXYGENASE-2 (COX-2) EX

PRESSION BY COXSACKIE AND ADENOVIRUS RECEPTOR

(CAR) IN BLADDER CANCER Jer-Tsong Hsieh Linya Yang Rey

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 adenovirus

based gene therapy. We and other group demonst ra ted 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 e lucida te the under lying 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 i ts 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 t ransduct ion to suppress cell growth. The under lying

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.

8

DIFFERENCES INTHE PROTEIN PROFILEOF INVASIVEAND

NON-INVASIVE BLADDER TUMORS

Sigrun Langbein

x l

Schaaf. Mannheim Germany; MarkusMeyer Goettingen Germany; Maurice

S

Michel Michael Siegsmund Peter Aiken Mannheim Germany

INTRODUCTION AND OBJECTIVE: Bladder carcinoma is an increasing

tumorenti ty. The init ial 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 profil ing 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

0

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.

Source of Funding: None.

*Presenting author.

9

GENE EXPRESSION PROFILES ANALYSIS OF PATIENTS

WITH BLADDER CANCER TREATED WITH BCG Fabien Saint

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: 16to 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 appliedto identifygenesthat couldsegregate

patientsthat recurred from thosethat showedno evidenceof disease duringfollow-up.

Usingt-testandconsideringsignificanta p valueequal or lowerthan0.05, we observed

that 37 genes were able to distinguishresponders from non-responders. These genes

revealedthe relevanceof interferon-inducedproteins, and intracellularsignalingsuch

as TGF-beta pathway in BCG response. The application of bootstrapping on

hierarchicalclusteringanalysiscouldseparateamongrespondersthosepatientswithno

evidenceof 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.

Source of Funding: None.

7

ANTISENSE AGENTS TARGETING THE TYPE 1 INSULIN

LIKE GROWTH FACTOR RECEPTOR SENSITIZE HUMAN

BLADDER CANCER TO PACLITAXEL AND CISPLATIN

MEDIATED CYTOTOXICITY

Giles

0

Hellawell London UK;

Simon Brewster Val Macaulay Oxford UK

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 cel ls 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 inhibit ion of IGFIR

expression to levels of 40 of those in control-treated cells. This was accompanied

by marked reduction in MGHU-l growth measured byMTS 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 AS

IGFIR 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.

7

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

INTRODUCTIONANDOBJECTIVE: 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

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THE JOURNAL OF UROLOGY 187

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

l ine, 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 cel l 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 ana lyzed by

immunohistochemical staining of blood vessel endothelium using anti-CD3l

and microvessel 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 pro liferation assay.

Although no significant differences were found concerning in vitro growth rate

among the se sub li ne s,

UM UC fMK

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 fC cells. Moreover, tumor-induced

angiogenesis, as determined by the MVD of the e stablished 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.

Source of Funding: None.

7

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 det ermine whe te r geneti c

aberrations found in superficial TCC were also present in normal appearing

mucosa.

METHODS: We invest igated chromosome 1,7,9,17 aneusomy in 25

patients with TCC and in tissue samples taken from sites of macroscopical ly

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

f luorescent- 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 papil lary hyperplasia. Ten 40 ) T

specimens showed aberrations of all chromosomes analyzed and fifteen 60 )

were aneup loid for at least one chromosome. Numerical aberra tions of

examined chromosomes with sim ilar 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 his to logica l report, we divided the ST and DS in neoplas ti c, 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

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7 9

MULTIFOCAL TRANSITIONAL CELL CARCINOMA OF THE

BLADDER AND UPPER URINARY TRACT: MOLECULAR

SCREENING OF CLONAL ORIGIN USING CD44 ALTER

NATIVE 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

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.

Immunohistochemical Staining

forthe

Cables

Protein In

Urothelial Specimens

7 8

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 RNAquantities 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 well

characterized superficial and invasive bladder carcinomas (n

=

173).

RESULTS: Two main clusters segregating superf ic ia l from invas ive

transitional carcinomas were identified and provided prognostic information.

Cytokerat in 20, neuropilin 2, p21 and p33INGI 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.

Source of Funding: None.

Source of Funding: Institutional Funding.

Total Loss

10

2119

17

2112

58

7/12)

52 12123

47 9/19)

50 6112

33 4112

44 10/23)

Partial Loss

100

14/14)

100

12112

43 8119

33

4/12)

9 1/12)

1/23)

Cables Presentpecimen

Non-Malignant

Low

Grade

Non-Invasive TCC

High

Grade

Non-Invasive TCC

Carcinoma InSitu

Lamina

Propria Invasive

TCC

Muscularis Propria Invasive

TCC

7

THERAPY OF MURINE BLADDER CANCER WITH TUMOR

SPECIFIC SUICIDE GENE EXPRESSION DRIVEN BY HUMAN

TELOMERASE REVERSE TRANSCRIPTASE PROMOTER IN

SYNGENEIC MOUSE TUMOR MODEL

Gia-Shing Shieh, Tainan,

Taiwan

INTRODUCTION AND OBJECTIVE : Human t el omerase r ever se

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 phTERT

Luciferase and a LacZ reporter plasmid by lipofectamine. We constructed Ad

hTERT-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 NIH

3T3 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

admin is tra tion of 5-FC suppre ss ed MBT-2 tumor g rowth in syngene ic,

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

expressionof suicidegenesdrivenby hTERT promotermaybe a novelcancer therapy.

Source of Funding: The study is supported by National Science Council,

Taiwan (NSC 90-2314-B-006-162).

7 7

EXPRESSIONOF CABLES,A CELL CYCLE REGULATORYGENE

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 18qis 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.

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

genes wascarried-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 t issue. 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

mu rine c las s I MHC knock -out mice, tra ns ge nic 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 potentialuse of the MAGE-A8

peptides for specificimmunotherapyof transitionalcell carcinomaof the bladder.

Source of Funding: None.

Presenting author.

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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 mult iple 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

mult iple 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.

Source of

Funding: None.

73

DOWNSTREAM

SIGNALING

PATHWAYS

AND

ANTIANGIOGENIC EFFECTS OF THE PAN ERBB TYROSINE

KINASE INHIBITOR CII 33 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 andMMP-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

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METHODS : Cytotoxicity studies were performed to determinea minimally

cytotoxic FR901228 concentration for bladder cancer cells. The levels of CAR

expression weredetermined by FACSand/or RT-PCR analysisof CAR, alpha V

integrin,

and acetylated histoneH3 in controland FR901228-treated bladder cell

lines.The efficiency of adenovirus

mediated

transgeneexpressionwasalsostudied.

RESULTS : The drug concentration showing no or minimal cytotoxicity

selected for these studies was 1 nglml FR901228 for bladder cancer cells.

Treatment of cancer

cells

with a low concentration (I

ng/ml

of the histone

deacetyase inhibitor FR901228increasedCARand alphaV integrinRNAlevels

andacetylated histoneH3.This increase wasassociated witha 10- 50 foldincrease

in adenovirus infection as evidenced by increased

transgene

expression from a

beta-galactosdase containing adenoviralvector.

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 integrinin bladdercancer

cells. This increase mediates enhanced

transgene

expression after adenovirus

infection. Thesestudiessuggesta simple,clinicallypracticalmethod for increasing

the sensitivity of tumorcells to adenoviral gene therapyvectors.

Source of Funding: None.

7

GENECHIP, RAPID

PS3

SEQUENCE

ANALYSIS Sarel Halachmi ,

Steven A Ahrendt, John T Chow, Li Wu, Naomi

l chm

i, Stephen

C

YMg,

Scott Wehage, David

Sidransky,

Baltimore, MD

INTRODUCTION ANDOBJECTIVE: The p53 tumor-suppressor gene is the

mostfrequently mutatedgene in humancancer,includingurological malignancies.

P53

mutation

is associated with poor prognosis, and low responsiveness to

adjuvant

therapy.

Despitetheimportanceofp53as a prognostic factor, it s usefor

clinical practice is limited because immunohistochemical staining has not been

standardized yet,anddirect sequencing is beyondcapabilityof mostclinical labs.

Rapidmutationanalysisof the pS3 gene sequencehas recently been developed

utilizingan oligonucleotide probe array. Our aimwas to compared the mutation

analysis of the same DNA by the pS3 GeneChip and conventional

dideoxynucleotide

sequencing.

METIIODS:

Hundred tumors

were

collected

frompatients undergoing resection of

lung and bladder cancer DNAwas

extracted

withphenol/chloroform.

Manual

p53

Sequencing: Exons5-9of thep53genewereamplified fromtumorDNAusingPCR.

PeR

products

weresequenced by cyclesequencing, separated by electrophoresis on a

po

lyacrylamide

gel, and read by two independent observers. GeneChip pS3 Assay:

DNAfromall patients was also

sequenced

by usingtheGeneChip pS3assay. Exons

2-11of thepS3genewere

amplified

as10separate

amplicons

ina singlePCR

reaction.

Amplified

tumorand reference DNAwere

fragmented

, 3 end

fluoresceinated

labeled

and transfered to the pS3probearrayfor 30min at45C. The probearray wasthen

scanned by

laser.

The emitted lightintenstywas proportional to boundtumorDNA.

Mutations weredetected basedon thedifferences in hybridization intensities between

the reference and unknown sample.

RESULTS : Cyclesequencing of the conserved regions of thepS3genedetected

76 of themutationswithinthisregionTheGeneChip pS3assaydetected 81 of all

exons

2-11)

mutation

s. 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 thepS3GeneChip

assayat detecting pS3mutations were 100 and98 ,respectively.

CONCLUSIONS: GeneChips provides several advantages over direct

sequencing:accuracy, detectionof more

mutations

, and considerable time saving,

as all samples were analyzed with the by a single investigator in 6 weeks.

Conventional sequencng of these samples

consumed

almost I year. Neither the

pS3GeneChip or directsequencing were able to detectall of thep53 mutationsin

a groupof 100lungcancers. However the pS3GeneChip provides a rapid screen,

detecting >80 of themutations with a very lowfalse-positive rate (2 ).

Source of funding: None.

7 5

DEVELOPMENT OF A NOVEL MOUSE MODEL

FOR

INDUCTION AND IMAGING OF BLADDER CANCER

Isla P

Garraway, Chou Tran;Robert Reiter, Los Angeles, CA

INTRODUCTION ANDOBJECTIVE: Transitional cell carcinoma (TCC)is a

common malignancy

of the genitourinary (GU)

tr ct

The heterogeneous natureof

TCChas ledto studies to elucidate molecular eventsin tumorigenesis. However, the

paucity of animalmodels is a majordrawback. Wehavetakenadvantage of theavian

sarcoma

leukosis virus(IVA) systemtodevelop a

novel

mousemodel forTeC.1n this

model, transgenic miceexpress theTVA receptor undercontrol of the

prostate

stem

cell antigen PSCA promoter. TVA expression is limited toGUtissuein thesemice.

Consequently,

gene

delivery

by retroviral

infection

occursonlyin cellswhereTVAis

expressed. Multiple

rounds

of infection enable

different genes

tobe introduced intoa

singlecell, including the imaging gene, luciferase, which imparts luminescence and

enables the natural history ofTCCto betracked non-invasively.

*Presenting author.

METHODS : Transgenic mice weregeneratedthat expressTVA undercontrol

of thePSCApromoter Micewere injected orthotopically withvirusproducercells

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)camerawasusedto observetheefficiency of injection progression

of infection, and tumordevelopment over time.

RESULTS:

Immunohstochemistryperformed on tissues of transgenic mice

confirmed TVAexpression in bladdermucosa. Mice injected orthotopcally with

viral vectors containingGFPdemonstrated urothelial cell infection. CCD images

showed luminescence from infected bladder cells in TVA-positive mice(see

graphic), whilewild-typemice injectedwith

Juciferase

virus werenot luminescent.

CONCLUSIONS: The TVA system is a novel and efficient method of

introducingoncogenesintospecific cells andimagingurothelium , thereby allowing

TCC develpoment to be

followed

non-invasively over time.

Source of funding: MargaretEarlyTrust.

7 6

INACTIVATION OF THE IT 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 (FragileHyslidineTriad)

gene has been identified in a region at chromosome 3pI4.2, which is frequently

deletedin manytumors. We havepreviously reported deletionsat the

FHIT

locus

in 50 of the transitional cancer cell

TCC

cell lines testedand alterations of

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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

transductionin 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 tran sfec ted into HFK-293 cells with p la qu e isolatio n and

vector

pur if icat ion after homologous recombination. Adenovi ra l infec tion 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 IT

1

-

and

25

HIT I

mice were treated with 0.1 BBN in drinking water for 13weeks

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 infec ted with ad

FHIT, ad-GFP,

and contro l vector showed a significant increase in the

apoptotic cell populat ion in the FHIT t ransduced cells. Mice injected with

SW780 cells alone and infected with ad-GFP developed a sizable tumor in both

flanks one month after the inject ion. The two mice injected with SW780 cells

infected with ad-FHIT are stil l tumor free 5 months after the injection. Six of

28 (21 ) of the

IT

f

vs. 2 of 25 (8 )

HIT 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 express ion in SW780 resul ted in abrogat ion

of tumorigenicity in nude mice. Lastly, FHIT-null mouse urothelium is more

sensi tive to chemical carcinogens. These observations suggest that

FHIT

based gene therapy should be explored as a therapeutic strategy for bladder

cancer.

Source of Funding: None.

ASSOCIATION BETWEEN A

CI

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 transi tional cell carcinoma (BTCC), but

the molecu lar mechan ism on the down- regula tion of E-cadhe ri n 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 CI single nucleotide polymorphism at position-160

from the transcrip tion start site of E-cadherin gene p romote r and cancer

occurrence, pathological grades and clinical stages in bladder transitional cell

carcinoma (BTCC).

METHODS: In 50 patients with BTCC and 50 normal con trols, 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 AflIII, 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 posit ion of E-cadherin gene

promoter were significantly higher in BTCC than in normal controls(P

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THE JOURNAL OF UROLOGY Vol. 169, No.4 Supplement, Monday, April 28, 2003

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.

Source of Funding: None.

74

MOLECULAR ALLELOTYPING IDENTIFIES PROGRESSION

MARKERS FO R TRANSITIONAL CELL BLADDER CANCER

o l 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

Microsatell iteanalysis (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

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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-KBmediated IL-6 expression by

human TCC lines in response to BCG. This effect is dependant upon a functional

androgen receptor -s ignaling pathway and can be blocked by inhibition of

androgen/androgen receptor binding. The effec t of sex s teroid media ted

modulation of BCG signaling on treatment efficacy and toxicity awaits further

study.

Source of Funding: Department of Veterans Affairs.

7

THERAPEUTIC EFFECT OF BACILLUS CALMETTE-GUERIN

INSTILLATION FOR BLADDER CANCER CANBE IMPROVED

BYCOMBINING 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 es tab li sh ed by simp le

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-tgof BCG in

50 1

of PBS or PBS alone. Two days after ins til la tion 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-Galtransfection and 5-FC ip,

2) BCG

CD transfect ion and 5-FC ip, 3) BCG

CD transfection and saline

ip, 4) PBS CD transfect ion 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.

Source of Funding: None.

7

COMPLETELY AUTOMATED MICROCAPILLARY MICRO

SATELLITE ANALYSISFOR 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

pat ient s with proven recur rent and primary TCC of bladder, d iagnosed 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 informat ive 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 intensi ty of the PCR products was determined. Graphic

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 studyin large groups of patients will berequired to document the

ease and economy of this form of in vitro diagnostic for the management of patients

with superficial bladder carcinoma.

Source of Funding: None.

7 6

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-HSVtk

transduced 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 accessibili ty 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 parentT739

cells were implanted into the pe ritonea 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 intraperi toneal

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 theperitoneal 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.

Source of Funding: None.

7 7

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 thetumor. Although a variety

of tumor markers have been tested unti l 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.

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RESULTS: With a cutoff point of metallothionein expression in 50 of the

cancer cells , 5-year tumorspecific survival was significantly less in patients

with a high metal lothionein expression 32 vs. 72 ). Significant differences

were found for 5-year-recurrence rate 90 vs. 58 ), and disease progression

78 vs. 54 ).

CONCLUSIONS: We demonstrated a correlation between metallothionein

expression and turnor-specifc survival, progression-free survival and recurrence

free survival. Especially in p

Tl

G3 tumors and carcinoma in situ, tumors with 50

metallothionein-positive cells had a shorter tumor-specific survival, a higher

recurrence rate and a higher progression rate. Therefore , meta llothionein

expression seems to be a promising tumor marker in urothelial cancer.

Source of Funding: None.

7 8

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 ANDOBJECTIVE: Our objective in conducting this study

wasto 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 l ine 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).

CONCLUSIONS: 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.

Source of Funding: None.

7 9

ANTIANGIOGENIC

EFFE

CTS OF FLAVONOIDS ON RAT

URINARY BLADDER EPITHELIAL CANCER INDUCED BY N

BUTYL-N-

4 H

YDROXYBUTYL

NITRO

SAMINE K

iki hi

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 angiogenesi s. In ra t 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

investiga te s whe ther the o ra l flavonoids, catechin and que rceti n, have

chemopreventive effects during the init iat ion 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)nitrosamine 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-dependently 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 epithel ial angiogenesis. Catechin and quercetin should be of

cons iderable benefit as chemopreventive agents against urinary bladder

carcinogenesis.

Source of Funding: None.

75

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 lo t of new strategies for the

treatment of bladder cancer or genetic di sorders deal with plasmid DNA or

antisense oligonucleotides.

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 fluorescentprotein 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 t issue have to be

effected. This work points out, that not all of the strategies forfuture 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 Fundin

g: Hector-Stiftung, Weinheim, Germany.

7

TEREI EXPRESS ION INHIBITS TUMOR GROWTH AND

ALTERS GENE EXPRESSION IN HUMAN BLADDER CANCER

Terence W McGarvey , 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 l ines and increases genomic stabili ty. 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 effec t of TEREI expression on tumor growth, and sugges t

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 wasmeasured 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

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Source of Funding: Veternas Affairs Merit Review Grant.

control of the UPII promoter and Ad-UPII-TNF carrying tumor necrosis factor

(TNF) under control of the UPII prom