Cox-2 CRAds with the imaging cassette demonstrated increasing reporter expression and subsequent oncolytic effect in A549 lung cancer cells but not inCox-Z-negativeA431 lung cancer cells
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Monitoring Capability for Lung Cancer
JuliaDavydova,EricJ Brown,MasatoYamamoto
'Surgery University ofMinnesota, Minneapolis MN.
The employment of Conditionally-replicative adenoviruses
(CRAds) constitutes promising tools for cancer gene therapy However, to maintain safety and efficacy in clinical application,
a noninvasive detection system of viral replication is required
In this study, we developed a novelCyclooxygenase-Z(Cox-Z)
promoter-controlled replieativc agent with bioluminescence-based replication monitoring capability and applied it to treat lung cancer Withexceptionof'adenoviraldeath protein(ADP), most E3genes in our reporter vectors were deleted or mutated and replaced with the firefly luciferase gene We combined the E3-modified vector with the Cox-Zpromoter-controlledE1expression cassetteand designed six differentstructures such as configurationswith and withoutADP and vectors equipped with differentfiberstooptimizetherapeutic and monitoring vector features The presence or absence ofa poly-adenylation signal following the reporter gene has been examined for effect on stability oftransgene expression Cox-2 CRAds with the imaging cassette demonstrated increasing reporter expression and subsequent oncolytic effect in A549 lung cancer cells but not
inCox-Z-negativeA431 lung cancer cells in vitro, supporting the
principle that the oncolytic effect of these CRAds depends on the Cox-2 promoter activity.Thedetectedgene expression closely cor-related with the viral DNAquantity resulting from viral replication
No expression or cytocidal effect was observed in mouse hepatoma BNL-ING-A.2cells in which human adenoviruses do not produc-tively replicate, indicating the dependence of reporter expression
on productive replication In vivo therapeutic data revealed that
the Cox2CRAd_dE3_ADP_Luc vector significantly suppressed the tumor growth of established A549 Cox-2-positive xenografts and its therapeutic effect was as high as that of the fully replicative wild type virus In contrast, the Cox-2-negative xenografts (A43I) the same Cox2-based vector showed no antitumor effect while its signal was barely detectableand comparable to the negative-control group treated with a non-replicative luciferase vector.Notably, live imagingalloweddynamic visualizationofviral replicationand viral spread in tumors,showing close correlation to therapeuticeffect,
These data demonstrate that our noninvasive CRAd replication monitoringsystem is applicablefor furtherdevelopmentofoncolytic therapeutic agents for lung cancer
Trimeric TRAIL in Combination with Conventional Chemotherapy for Brain Tumors Induces
Synergistic Tumor Suppression and Improves Survival in an Intracranial Human Malignant Glioma Xenograft Mouse Model
MoonsupJeong ,'Yong-SamKwon ,'Soon-I·lyePark,IMin-Tae
Park; Chae-Young Kim,' Kung-Won Son.! Yong Ko,2 Paul D
Robbins,' Dai-Wu SeoV Byong-Moon Kim.1
Genetics and Biochemistry, University ofPittsburgh, Pittsburgh, PA; "Surgery; Univer sity of Pittsburgh, Pittsburgh PA
Treatment of malignant gliomas is stilI a significant clinical challenge The conventional therapies for malignant gliomas yield
a median survivalof only 9 months TRAIL is a novel therapeutic candidate for treating a wide variety of cancers In particular, a secreted, soluble trimeric TRAIL (stTRAIL) easily disseminates to nearby cancer cells and induces more extensive induction of
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overexpressed by infectingcells with an adenovirus vector carrying
REICIDkk-3 (Ad-REIC).A vector carrying LacZ (Ad-LacZ) was
used as a negative control Seventy two hours after infection of the
virus vectors at 20 MOl,apoptotie cells were monitored by TUNEL
method To see whether activation of c-Jun terminal kinase (JNK)
is causally linked to the inductionof'apoptosis,we examined effect
of SP600126 (JNK inhibitor) on Ad-REIC-inducedapoptosis, We
determined the protein levelsand phosphorylation state ofJNK and
otherapoptosis-relatedproteins by Western blot analysis NCCIT
cells were subcutaneously injected intothe right flankofnude mice
Three weeks after injection of the cells, Ad-REIC or Ad-LaeZ in
a 100JlI bufferwas infectedintratumorally,The size of tumors
was measured every 3 or 4 days over 30 days after the infection
(Results)Expression ofREIC/Dkk-3 was reduced in all the human
seminomaandnon-seminomatousgerm cell tumortissuesexamined
Overexpression ofREICIDkk-3 using Ad-REIC induced apoptosis
in a testicular germ cell cancer cell line NCCIT but not in normal
human fibroblasts JNK was activatedbyAd-REICand the induction
ofapoptosis was abrogatedby a JNK inhibitor.Asingle intratumoral
injectionofAd-REICmarkedlyinhibitedtumorigenicgrowthofNC-CIT cells in nude mice (Fig.I).(Conclusion) These results indicate
that Ad-REIC may lead to developing less insultingnon-genotoxic
therapeutic measures against human testicular cancer
Co pyright © " 111(: Ameri can S octc;ty o f Gene
Trang 2Thcrapj-tosis than that of full length transmembrane TRAIL In this study,
we compared tumor suppressive activity of adenovirus mediated
delivery of stTRAIL (Ad-stTRAIL) with
1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU) treatment,the conventional adjuvant therapy for
malignant glioma and evaluated the anti-tumor efficacy ofcombining
Ad-stTRAIL and BCNU Although Ad-stTRAIL or BCNU alone
induced tumor-killing effects in vitro ,some gliomas were resistant
to each treatment However,the combination of Ad-stTRAIL and
BCNU reversed their resistance and enhanced tumor-killing
ef-fect induced in other sensitive gliomas To examine the efef-fects of
stTRAIL and BCNU in vivo ,a disease animal model was used where
U87-MG cells were injected into the brain ofathymic mouse The
median survival ofxenografted mice was 38.0± 1.7 days The
in-tratumoral treatment with Ad-stTRAIL alone produced an increased
survival (46.0±3.4 day) as compared with systemically delivered
BCNU (43.0±3.2 day),and the combination of both treatments
caused a longer growth delay and a significant increased survival
(54.0 ± 5.0 day) MR imaging allowed the precise localization of
tumors in brain and showed how the established tumors were
sup-pressed Through MR Imaging, Ad-stTRAIL alone or combination
with BCNU induced significant suppression oftumor mass as
com-pared with BCNU alone.In situ immunohistochemical staining for
TRAIL and Cyelin A and TUNEL staining showed the established
tumors were derived from human malignant gliomas and the
regres-sion ofAd-stTRAIL treated tumor mass was due to the apoptotic cell
death induced by expressed stTRAlL from Ad-stTRAIL Our results
suggest that Ad-stTRAIL treatment together with chemotherapy
may achieve maximal tumor control and is a promising alternative
or cooperative adjuvant therapy for malignant brain tumor
197 mda-7 (INGN-241) Kills Chemoresistant
Cancer Cells and Restores Chemosensitivity to
Cisplatin
Began Gopalan,ICarlos Rached,' Daniel Pearson,1Manish
Shanker,ISunil Chada.? Rajagopal Rarncsh.'
'Thoracic and Cardiovascular Surgery; M D Anderson Cancer
Center; Houston, TX ;2Research and Clinical Development,
lntro-gen Therapeutics Inc • Houston, TX
Cisplatin (CDDP) is the mainstay chemodrug for cancer
treat-ment that inelude cancer of the lung,breast and colon However,
development ofchemotherapy-resistant tumor cells during treatment
is a major problem in cancer therapy Therefore, novel therapeutic
strategies that can restore chemosensitivity and/or effectively kill
drug resistant tumor cells are warranted.In the present study we
have utilized a gene therapy strategy and investigated if the tumor
suppressor/cytokine melanoma differentiation associated gene-7
(mda-7) can kill chemoresistant tumor cells Treatment of Cisplatin
(CDDP)-sensitive (2008;IC50= 30 IlM; CDOP-S) and resistant
adenovirus vector carrying the mda-7 gene (Ad-mda7; INGN-241)
resulted in effective growth inhibition of both cell lines However,
CDDP-R cells were found to be at least two times more sensitive
to Ad-mda7 than CDDP-S cells and demonstrated increased
activa-tion of proapototic markers Molecular analysis for the enhanced
sensitivity of CDDP-R cells to Ad-mda7 revealed no significant
increase in the transduction efficiency or adenovirus receptor (CAR,
enhanced sensitivity to Ad-mda7 was observed in CDDP-R lung
tumor (H1437R) cells compared to CDDP-S (1-11437)cells These
results showed Ad-mda7 can effectively kill CDDP-R cells of both
ovarian and lung origin We next determined if Ad-mda7 can restore
chemosensitivity to CDDP-R cells Treatment of CDDP-R cells
with Ad-mda7 (3000 vp/eell) plus CDDP (25~IM ; IC50=125 IlM
to cells that were treated with Ad-mda7 alone or COOP alone (P
C op yright © T heAmeric m Societyo Gene Therapy
< 0.05; 87% inhibition).Additionally,the growth inhibitory effect
in Ad-mda7 plus CODP-treated CDOP-R cells was synergisitic compared to cells that were untreated or treated with COOP«
5%),Ad-luciferase (Ad-luc; <5%», Ad-mda7 (12%), or Ad-Iue plus CDDP (20%) The underlying mechanism by which Ad-mda7 (INGN-24I ) restores chemosensitivty is not known and is currently under investigation in the laboratory Finally, our studies show that Ad-mda7 (INGN-24I) effectively kills chemoresistant tumor cells when used as monotherapy and in combination with CODP restores chemosensitivity to CDDP.Our findings are of clinical importance and warrant further investigation for clinical translation
198 Engineering Cytosine Deaminase/Uracii Phosphoribosyltransferase Fusion for Improved Cancer Gene Therapy
Andressa Ardiani,'Michi Fuchita,IMargaret E.Black.':'
'School a/Molecular Bioscience Washington State University, Pullman, If 'll; 2Departmenl a/Pharmaceutical Sciences Wash-ington Stale University, Pul/man If'il.
Suicide gene therapy is a particularly attractive cancer therapy because of its ability to localize toxicity to tumor cells We are interested in the bacterial uracil phosphoribosyltransferase (UPRT) and its application in conjunction with the cytosine deaminase (CD)/5-fluorocytosine (5FC) system to enhance tumor ablation Cytosine deaminase (CD) is an enzyme responsible for deaminat-ing cytosine to form uracil It also recognizes 5FC, ananti-fungal drug,and is able to convert 5FC into 5-nuorouracil (5FU),a highly toxic anti-cancerdrug UPRT is an important enzyme involved in the pyrimidine salvage pathway, catalyzing the phosphorylation of uracil to uracil-monophosphate The enzyme also phosphorylates 5FU to form 5FUMP which is then further catalyzed to antimetabo-lites by endogenous enzymes.Because the conversion of 5FC to 5FU by bCD has been shown to be rate limiting, our lab performed regio-specificrandom mutagenesis within the substrate binding site
of bCD From these series of experiments, we have successfully identified 3 bCD variants.Kinetic analyses ofthe 3 variants suggest that substrate preference for 5FC is shifted by a decrease of normal substrate (cytosine) specificity of 100-fold to an increased
speci-ficity for 5FC by approximately 19-fold In vitro cytotoxicity and
bystander effect assays were performed in rat C6 glioma,HCTll6
colorectal and DU 145prostate cancer cells In vitro data revealed that
cells stably transfected with these mutants have 3- to 18-fold lower IC,,,values than wild type transfected cells.Experiments also showed that bCD mutants have significant increase in bystander activities compared to wild type bCD It has been previously demonstrated that the fusion of CD with UPRT (CO/UPRT) along with 5FC im-parts a greater tumor killing activity than CD alone Therefore,to further enhance the production of cytotoxic compounds,we sought
to incorporate improvements previously identified in bCD into new fusion constructs containing both bCD and UPRT activities
We hypothesize that such fusion enzymes will offer more efficient prodrug activation,confer significantly improved sensitivity toward 5FC and therefore,improved tumor ablation The fusion constructs
were evaluated for their cell killing effect and bystander effect in
vitro Mutants with exceptional prod rug converting properties will
be beneficial because they allow administration of lower doses of 5FC, thereby minimizing side effects without the loss of potency
The use ofsuch novel mutant fusion constructs will advance suicide gene therapy treatment for cancer and improve the likelihood or complete tumor ablation
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