205 Knockdown of Lifeguard (LFG/FAIM2) in Glioblastoma Cells Increases Sensitivity to FasL and Doxorubicin drugsuicidesystemalone and incombinationwithgcmcitabine(Eli Lillyand Company)(i p ), Prolifer[.]
drug suicide systemalone and in combinationwith gcmcitabine(Eli Lillyand Company) (i p.), Proliferation in vitro and tumorgrowth in vivowereexamined Results: The combination ofAd5F35-TKlGCV with gemcitabine resulted in higher inhibitory effect compared to treatment withAd5F35-TKlGCValone or gemcitabinealone Combine index (CI) was < I and indicated synergistic cytotoxicity for TCC-SUP CI was I and suggesting additive cytotoxicity for 5637 After 24 hours of treatment, flow cytometry analysis showed that gemcitabine increased the GO/G I in 5637 and TCC-SUP cell lines, compared to Ad5F35-TKlGCV alone After exposure to solvent or gemcitabine for 24 hours, cells transduced with Ad5GFP showed GFP expression was not significantlyincreased in cells treated with gemcitabine as compared with solvent by flow cytometry analysis in 5637 and TCC-SUPcell lines.Tumor volumes were significantly reducedin the mice treatedwith a combinationofAd5F35-TKlGCV and gcrncitabinc compared with treatment with Ad5F35-TKlGCV aloneor gemcitabine alone.Conclusions:A combination ofAd5F35TK/GCV and gemcitabine may be effective in the treatment of bladderTCC producing additive and potentially synergistic effects via differential cell cycle alterations 203 Systemic Stable Radioprotection by Intravenous Administration of Manganese Superoxide Dismutase-Plasmid Liposomes Joel S Greenberger; Tracy Smith,' James J Schlesselman.? Michael W Epperly} I Department ofRadiation Oncology, University ojPittsburgh Cancer Institute, Pittsburgh, PA; ZDepartment ojBiostatistics, University ofPittsburgh, Pittsburgh, PA Organ specificlocalizedadministrationof manganesesuperoxide dismutaseplasmid/liposome complexes(MnSOD-PL) conferssingle fractionand fractionated ionizingirradiationprotectionof the rodent lung, esophagus, oral cavity, intestine and bladder by a mechanism dependentuponmitochondrial localizedantioxidanteffects.lntravenous injectionofMnSOD-PL significantly increasedsurvivalof both maleand femaleC57BL/6NI-lsd mice fromthe LD30/50wholebody irradiation dose of 9.5 Gy To determine if systemic MnSOD-PL mediated improvedsurvival at the expense ofa deleteriousdelayed increase in development of cancer, life shortening, or ncurodegcnerative disease, 50 male and female C57BL/6NHsdmice per group were injectedintravenously with MnSOD-PL(100 ug plasmidDNA in 0.1 ml) and irradiated with control mice to 0, I or 9.5 Gy whole body irradiation twenty-four hours later Moribund or dying mice were sacrificed, and examined for tumors, bone marrow isolated, stainedwith WrightGeirnsa,and examinedforabnormalhemogram Small intestine was fixed in formalin, sectioned and examined for intestinalchanges Femalemice pretreatedwith MnSOD-PLpriorto 9.5 Gy (LD 50130) had an increased survival over the first 30 days which is the time frame for death due to hematopoieticor intestinal damage with 90% survival compared to 58% for the control mice (p < 0.000 I) Between 30 and 330 days there was no significant change in survival rate between the MnSOD-PL and control mice in either 9.5 Gy or 1.0 Gy irradiated groups Male mice pretreated with MnSOD-PLand irradiatedto 9.5 Gy had 75% survival after 30 days compared to 25% for the control mice (p < 0.000I) Between 30 and 170 days there was no significant change in survival rate between9.5 or 1.0Gy groups There was no detectabledifferencein the incidenceof deaths associated with tumor (thymoma), marrow, or intestinal damage All mice are being followed until death The data indicate,at this time, that systemic MnSOD-PLtreatmentis not deteetably harmful to surviving total body irradiated mice Molecular Therapy Volume 15, Supplement I May2007 Copyright © Th e American Society of G ene Th erapy 204 Gene Therapy of Pancreatic Carcinoma Based on Gemcitabine Chemosensitization Using Phosphorylating Fusion Gene Transfer Fabienne VernejouI,2 Barbara Bournct,' Anny Souquc,' Hubert Lulka,' Gilles Cambois,' D Drocourt,' Lucien Pradayrol,' Gerard Tiraby,' Pierre Cordelier,' Louis Buscail.' 'INSERM U858, I2MR, Toulouse France; zResearcll Department, Cayla/Invivogen, Toulouse, France Background: Pancreaticcancer is one ofthe most aggressiveand devastatinghumanmalignancies Exceptingsurgicalresection,there is no efficient treatment The chemotherapeutic agent gemcitabine improves the patient's clinical status but survival is not prolonged We recently designed a new strategy to render gemcitabine more efficient in the treatment of pancreatic cancer using gene therapy (Mol Tiler 2006; /4: 758-67) Methods and results: Wehavegenerated a fusiongene (DCK::UMK),combiningDeoxyCytidine Kinase (DCK) and Uridylate Monophosphate Kinase (UMK) proteins to convert gemcitabine into its toxic phosphorylated metabolite Antitumor effect of DCK::UMK gene expression was tested in vitro both in hamster(PC1.0) and human (BxPc-3, MiaPaca2)pancreatic cancer-derivedcellsand in vivo in an orthotopictransplantable model of pancreatic cancer established in hamsters, WhileJctPEI scarcely transfectedpancreatic-derived cancer cells, DCK::UMKexpression strongly sensitizes pancreatic cancer cells to gemcitabine both in vitro and in vivo Wefoundthat expressingDCK::UMK dramatical1y reduced cel1 proliferation in vitro, and pancreatic tumor growth, in vivo fol1owing treatment with gemcitabine Because of the low transfectionefficiencyof JetPEI in this model, we furtherscrutinized the molecular mechanisms involved in such antitumoral activity Wefound that the antitumoral bystander effect observed fol1owing DCK::UMKgene transferwas mainlydue to cell death byapoptosis oftransfected as wel1 as non-transfected cells Conclusions: Delivering DCK::UMK fusiongene usingJet PEl non viralvectorsensitizes pancreatic cancer cells to gemcitabine treatment Intra-tumorally delivery of DCK::UMK gene in combination to gemcitabine is of high interest for pancreatic cancer management 205 Knockdown of Lifeguard (LFG/FAIM2) in Glioblastoma Cells Increases Sensitivity to FasL and Doxorubicin Bryan C Nikolai, Nikunj V Somia 'Genetics, Cell Biology and Development, University ofMinnesota, Minneapolis, MN Lentiviral vectors are promising vehicles for the delivery of genetic material to target cel1s due in part to their ability to integrate into the genome of non-dividing cel1s The relatively largegenome ofthese virusesallows deliveryof numerouscis-acting elementsfor stablegene expressionincludingpromoter/gene cassettesfacilitating shRNAproductionand subsequentknockdownoftarget genes This type of therapeuticgene ablation is particularly attractive in cancer biology of aggressive tumors where over-expression or ectopic expression of certaingenes contributesto cell proliferationor apoptotic escape In this study we report the knockdown of endogenous Lifeguard (LFG, FAIM2), an inhibitor of Fas-mcdiated cell death, in a glioblastoma cell line Lentiviraldelivery ofshRNA efficiently decreasesthe amount ofLFG and rendersnormallyresistantglioma cells sensitive to membrane-bound Fas ligand Given that some chemotherapeutic drugs act by activating the Fas apoptotic pathway, we examined the chemosensitivityof cells with reduced LPG expression We demonstrate a significant increase in toxicity from a clinically relevant chemotherapeutic agent, Doxorubicin, in the LFG-deficient glioma cells Our data suggest that decreasing LFG S77 expression or small drug inactivation of LFO may be a valuable adjuvant to chemotherapy of gliomas 206 Systemic Osteoprotegerin Gene Therapy Restores Tumor-Induced Bone Loss in a Therapeutic Model of Breast Cancer Bone Metastasis Diptiman Chanda,' Tatyana Isayeva,' Sanjay Kumar,' April F Szafran,I Kurt R Zinn.?Selvarangan Ponnazhagan.' I Pathology, The University ofAlabama at Birmingham, Blrmlngham, AL; "Medicine, The University ofAlabama at Birmingham, Birmingham, AI 9"b -I -1 "to - "b - 23 "" I 3H:.b - Figure 1: Lentiviral hRNA constructs knock down LFG transcript Northern blot 20ug of'total RNA per well from cotransfected 293T cells was probed with hLFG cDNA Each transfection received 20ug lentiviral sh construct and I ug LFG expression plasmid 4-hour exposure; bottom, EtBr stain, ~ :~ : .1 ' - • Fast Toxicity in GUobIu loma Celli 1"',-1 i -'- "I- Figure 2: G'obl.astc.nacels 31'e Breast cancer is the second most leading cause ofcancer death in womcn and metastasizes to bone in greaterthan 80% ofpaticnts with advanceddisease In breastcancer, bone metastasisis predominantly osteolytic, which is a major cause of morbidity, frequently causing severe intractable bone pain, susceptibility to fractures, hypercalcemia and nerve compression syndrome resulting in significant decline in quality of life of these patients Osteoprotegerin (OPO) is a soluble decoy receptor for RANKL and thus prevents binding of RANKL to RANK leading to subsequent inactivation of the osteoclast activity OPO therefore promises tremendous hope for potential clinical use towards the management of osteolytic bone metastasis In the present study we tested the effects of systemic OPO therapy by using rAAVencoding human OPO.Fc in a mouse therapy model of osteolytic breast cancer The rAAV used in this study comprised the ligand binding domain of human OPO fused to the Fe domain of human IgO under the control ofCMV/chicken p actin promoter High level expressionand bioactivityofrAAV-OPO Fe were determined by western blot and osteoclast inhibition assay respectively Bone metastaticbreast cancer model was developed by injecting2x I 01 osteolytic MDA-MB-435breast cancer cells, constitutively expressing luciferase, into the left ventricle of6-week-old female BALB/c-lllI/1l1I mice Seven days after the transplantation ofthe cells skeletal metastases were confirmed by bioluminescence imaging At this time 3xIOII particles of rAAY.OPO-Fc or OFP, was injected in the quadriceps of the hind limbs Narve animals did not receive any treatment Therapeutic benefits were determined weeks after the initiation of therapy and results were analyzed by bioluminescence imaging (for tumor progression), DEXA (for bone densitometry), micro-C'I (for 3D-trabecular bone structure) and histomorphometry Systemic OPO.Fc gene therapy decreased bone lysis associated with MDA-MB-435 bone metastasis as determined by significant increase (p