overexpressed by infecting cells 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 induction of'apoptosis, we examined effect of SP600126 (JNK inhibitor) on Ad-REIC-induced apoptosis, We determined the protein levelsand phosphorylation state ofJNK and other apoptosis-related proteins by Western blot analysis NCCIT cells were subcutaneously injected into the right flankofnude mice Three weeks after injection of the cells, Ad-REIC or Ad-LaeZ in a 100 JlI buffer was infected intratumorally, The size of tumors was measured every or days over 30 days after the infection (Results) Expression ofREIC/Dkk-3 was reduced in all the human seminomaand non-seminomatous germ 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 abrogated by a JNK inhibitor A single intratumoral injectionofAd-REICmarkedlyinhibitedtumorigenicgrowthofNCCIT cells in nude mice (Fig I) (Conclusion) These results indicate that Ad-REIC may lead to developing less insulting non-genotoxic therapeutic measures against human testicular cancer 2400 PBS o Ad-LacZ o Ad-REIC 2000 rf) ~ 1600 '-'" Ci '0 1200 E ::J e E :g ::J 800 195 Conditionally-Replicative Adenovaral Vectors with Bioluminescence-Based Replication Monitoring Capability for Lung Cancer Julia Davydova, Eric J Brown, Masato Yamamoto '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 novel Cyclooxygenase-Z (Cox-Z) promoter-controlled replieativc agent with bioluminescence-based replication monitoring capability and applied it to treat lung cancer Withexception of'adenoviral death 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-controlledE1 expression cassette and designed six differentstructures such as configurationswith and withoutADP and vectors equipped with different fibers to optimize therapeutic and monitoring vector features The presence or absence of a polyadenylation 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 in Cox-Z-negative A431 lung cancer cells in vitro, supporting the principle that the oncolytic effect of these CRAds depends on the Cox-2 promoter activity.The detected gene expression closely correlated with the viral DNAquantity resulting from viral replication No expression or cytocidal effect was observed in mouse hepatoma BNL-ING-A.2 cells in which human adenoviruses not productively 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 (A43 I) the same Cox2-based vector showed no antitumor effect while its signal was barely detectable and comparable to the negative-control group treated with a non-replicative luciferase vector.Notably, live imagingallowed dynamic visualizationofviral replicationand viral spread in tumors, showing close correlation to therapeutic effect, These data demonstrate that our noninvasive CRAd replication monitoringsystem is applicable for furtherdevelopmentofoncolytic therapeutic agents for lung cancer 196 Adenoviral Delivery of Secretable 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 Moonsup Jeong,' Yong-Sam Kwon,' Soon-I·lye Park,I Min-Tae 400 Park; Chae-Young Kim,' Kung-Won Son.! Yong Ko,2 Paul D o Weeks after vector injection Robbins,' Dai-Wu SeoV Byong-Moon Kim.1 'Research Laboratories, Dong-A Pharm Co., Ltd., Yongln, Kyunggi, Republic ofKorea; 2Neurosurgery; Hanyang University Medical Center; Sungdong, Seoul Republic ofKorea; 'Molecutar Genetics and Biochemistry, University ofPittsburgh, Pittsburgh, PA; "Surgery; University of Pittsburgh, Pittsburgh PA Treatment of malignant gliomas is stilI a significant clinical challenge The conventional therapies for malignant gliomas yield a median survival of only 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 apopS74 Molecular The 4lpy Volume 15 Supplement tã ã\ br 2007 Co pyright â "111(: American Soctc;ty o f Gene Thcrapj- 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)-lnitrosourea (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 effect induced in other sensitive gliomas To examine the effects 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 intratumoral 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 suppressed Through MR Imaging, Ad-stTRAIL alone or combination with BCNU induced significant suppression oftumor mass as compared 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 regression 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, I Carlos Rached,' Daniel Pearson , Manish Shanker, I Sunil Chada.? Rajagopal Rarncsh.' 'Thoracic and Cardiovascular Surgery; M D Anderson Cancer Center; Houston, TX; 2Research and Clinical Development, lntrogen Therapeutics Inc.• Houston, TX Cisplatin (CDDP) is the mainstay chemodrug for cancer treatment 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 Cisplat in (CDDP)-sensitive (2008 ; IC50 = 30 IlM; CDOP-S) and resistant (2008/C 13; IC50 =125 IlM; CDDP-R) ovarian tumor cells with an 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 activation 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, aV~3 or aV~5) expression compared to CDDP-S cells A similar 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 for 2008/C 13) resulted in a significant growth inhibition compared to cells that were treated with Ad-mda7 alone or COOP alone (P Molecular Therapy Volume 15.Supplement I ~by Cop yright © The Americ m Society o r Gene Therapy 2007 < 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;