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744 Improvement of the TK/GCV Strategy for the Treatment of Gliomas by 5 Fluorouracil and TK30, a More Potent TK Enzyme Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© ������[.]
ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER isogenic tk vector (TOZ.1) in the presence or absence of GCV Expression of TOIκB was confirmed by Western blotting, and the bioactivity to inhibit the NF-κB translocation to the nucleus was examined by electrophoretic motility shift assay In human glioblastoma U-87MG cell line, dimer of p50/p50 of NF-κB had already been translocated in the nucleus without receptor mediated signals through TNF-α With infection of TOIκB to U-87MG cells, the translocation of NF-κB was significantly inhibited, and the caspase activity was found to be significantly upregulated in TOIκB infected U-87MG cells, when compared with that in TOZ.1 infected cells At an MOI of 0.1, the cytotoxicity against U-87MG human glioblastoma cells were examined TOIκB infection alone killed 85% of U-87MG cells, and in the presence of GCV, all the cells were killed In U-87MG cells that NF-κB had already translocated to the nucleus, cells had become apoptotic by infection of TOIκB, and under the presence of GCV, HSV-tk/GCV system enhanced the cytotoxicity by the bystander effects of HSV-TK/ GCV system 742 Over-Expression of TIMP-3 Causes Apoptosis in Lung Cancer Cells Katherine M Finan,1 Greg Hodge,2 Ann M Reynolds,1 Sandra Hodge,1 Mark D Holmes,1 Andrew H Baker,3 Paul N Reynolds.1 Department of Thoracic Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia; 2Department of Haematology, Women’s and Children’s Hospital, Adelaide, South Australia, Australia; 3Division of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom New options are needed for the treatment of advanced lung cancer Tumour invasion and spread involves a complex interplay between tumour, stromal and inflammatory cells with the extracellular matrix (ECM) The matrix metalloproteinases (MMPs) are a family of enzymes involved in ECM maintenance and tissue inhibitors of matrix metalloproteinases (TIMPs) are important factors in the regulation of the activity of MMPs Gene delivery of TIMP and has been shown to inhibit tumour growth in animal models There is evidence that TIMP may also be therapeutic in this way, and in contrast to TIMP and 2, may directly induce apoptosis in tumour cells, although this has not yet been evaluated in lung cancer Hypothesis: Overexpression of TIMP delivered by adenoviral (Ad) vectors to lung cancer cells causes apoptosis and increased cell death Aims: To compare the effect of TIMP 1, and gene delivery on lung cancer cell lines Methods: A549, 1299, H1466 and 522 cells were plated at a known concentration, then infected with Ad vectors carrying the genes for TIMP 1, and Following infection the cells were examined for changes consistent with apoptosis and cell death Analysis by flow cytometry using Propidium Iodide and Anexin V staining was used to establish the presence of apoptosis Cell counts were used to evaluate any reduction in cell number Results: For all lines maximal reduction in cell number was seen with TIMP overexpression and this was dose dependent Flow cytometry confirmed the induction of apoptosis by TIMP (eg for A549 cells 32.5 ± 6.3% at 72 hours vs 2.2 ± 2.1% for uninfected cells) Rates of apoptosis with TIMP and infected cells were similar to controls Conclusion: TIMP induces a reduction in cell numbers and causes increased apoptosis in lung cancer cell lines Further study using animal models of lung cancer is thus warranted and is currently being pursued Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy 743 Suicide Gene Therapy Using HSVtk/GCV in Combination with Cell Cycle-Altering Drugs Tiina Pasanen,1 Anne Karppinen,1 Leena Alhonen,1 Juhani Jänne,1 Jarmo Wahlfors.1 Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute for Molecular Scieces, Kuopio, Finland Suicide gene therapy with HSV-TK and ganciclovir (GCV) has been widely used against malignant cells and its efficacy has been demonstrated in a variety of different in vitro and in vivo models However this gene therapy regimen needs to be enhanced for a true clinical success We studied whether polyamine biosynthesis inhibition by 2-difluoromethylornithine (DFMO), a clinically tested and well-tolerated chemotherapeutic drug, can increase the cytotoxicity of HSV-TK/GCV in 9L rat glioma cells Polyamines putrescine, spermidine and spermine are cationic molecules that are indispensable to cell growth and differentiation The hypothesis was that prolonged S-phase caused by polyamine biosynthesis inhibition, would lead to accumulation of cells in this particular cell cycle phase When the cells are released from this block, there would be higher proportion of cells in the middle of DNA-synthesis phase Because HSV-TK/GCV cytotoxicity is killing cells only after their DNA has been replicated, the preceding DFMO treatment would make the cell destruction more efficient When the cells were exposed to GCV or days after removal of DFMO from growth medium, the cytotoxicity was increased up to 2.5-fold We also verified whether cell cycle blockage per se could yield similar effect as DFMO Our results from serum deprivation experiments showed that, despite of apparent cell growth and cell cycle phase distribution effects, serum starvation was weaker enhancer of HSV-TK/GCV cytotoxicity than DFMO Finally, the general utility of HSV-TK/ GCV DFMO combination was tested in another tumor cell type, human prostate carcinoma cell line DU-145 DFMO sensitized these cells to HSV-TK/ GCV cytotoxicity, but the effect was less prominent than in 9L cells In conclusion, we have demonstrated that a correctly timed induction of DFMO-mediated polyamine biosynthesis inhibition can enhance the efficiency of HSV-TK/GCV gene therapy in vitro The observed synergistic effect is potentially useful in clinical trials because, as opposed to use of other cell cyclealtering drugs, DFMO has already been tested in the treatment of human tumors and used as chemo preventive regimen with excellent tolerability We are currently testing whether other cell cycle-altering drugs can enhance ganciclovir-mediated cytotoxicity Furthermore, DFMO treatment in combination with HSVtk/GCV therapy will be tested in vivo 744 Improvement of the TK/GCV Strategy for the Treatment of Gliomas by 5-Fluorouracil and TK30, a More Potent TK Enzyme Gaelle V Roullin,1 Emmanuelle F Germain,1 Manuel P Caruso.1 Cancer Research Center, Laval University, Quebec, QC, Canada BACKGROUND: Gene transfer of the Herpes Simplex virus thymidine kinase (TK) associated with gangiclovir (GCV) administration represents a promising approach to treat malignant gliomas TK phosphorylates GCV into GCV-monophosphate, which is further converted into the di- and triphosphate forms by cellular enzymes GCV-triphosphate is incorporated into elongating DNA, which leads to cell death Complete tumor eradication has been obtained in animal experiments with only a small proportion of cells expressing TK (TK cells) This phenomenon, called the bystander effect (BE), is mainly due to the transfer of phosphorylated GCV from TK cells to neighbouring TK negative cells In clinical trials, the low level of gene transfer and inhomogeneous distribution of the vector within the tumor, have been highlighted Moreover, an effective S287 ONCOLYTIC VIRUS AND SUICIDE GENE THERAPY FOR CANCER drug concentration is difficult to obtain in the vicinity of the tumor since GCV does not cross readily the blood brain barrier We have previously reported a major improvement by using TK30, a much more potent mutant of TK: TK30-expressing cells (TK30 cells) were killed more efficiently than TK cells (10- to 100-fold) and the BE was strongly increased too The use of 5-fluorouracil (FU) has been shown by others to enhance the efficacy of the TK/GCV strategy FU is a thymidylate synthase inhibitor which acts by decreasing the pools of thymidine, the major competitor for GCV phosphorylation In this study, we have investigated the potential of FU to interact with GCV on four different human (SKI1, SKMG4, U87 and U251) and three rat (C6, F98 and 9L) TK- or TK30 glioma cells METHODS: The various cell lines were retrovirally infected to stably express the mutant TK30 or the wild-type TK Drug sensitivities were determined by their median inhibitory concentration (IC50) using conventional MTT proliferation assays BE experiments with either TK30 or TK were realized by mixing ratios of infected and non-infected cells The multiple drug-effect analysis of Chou-Talalay was used to qualify the interaction between GCV and FU RESULTS: As calculated by the Chou-Talalay method, at least an additive effect of the drugs was noted in proliferation experiments with all glioma TK30 cells In BE experiments carried out with SKI1 and SKMG4 cells, potentiation and/or synergism were observed and the synergism was stronger in TK- than in TK30 cells Nevertheless, the IC50 (GCV) was still much lower with TK30- vs TK cells in the presence of FU (by a factor of 35 and 126 for SKI1 and SKMG4, respectively) Interestingly, except for U251 cells, a strong synergistic effect was also found when non-infected cells alone were treated with GCV + FU, a phenomenon unreported until now The mechanisms of this antitumor effect as well as in vivo studies are presently under investigation CONCLUSION: Based on these data, the local delivery of GCV + FU associated with TK30 gene transfer should be considered to optimize the TK/GCV strategy by efficiently eliminating both infected and more importantly non-infected glioma cells In vivo experiments will address the antitumoral efficacy of this system and also the possible loco-regional toxic side effects of the drug combination CANCER TARGETED GENE THERAPY II 745 Combination of OncoVEX with Chemotherapy for Cancer Treatment Ziqun Han,1 Jennifer Hu,2 Binlei Liu,1 Michael Robinson,1 Ruth Branston,1 Charles Coombes,2 Robert Coffin.1 BioVex Ltd, Abingdon, Oxon, United Kingdom; 2Cancer Cell Biology, Hammermsith Hospital Campus, Imperial College of Science and Medicine, London, United Kingdom OncoVEX is an oncolytic version of HSV with several improvements as compared to oncolytic versions of HSV previously tested in the clinic These improvements are (i) OncoVEX is derived from a clinical isolate of HSV with enhanced anti-tumour potency as compared to previously used laboratory strains (ii) tumour selective replication is enhanced via increased expression of US11 in addition to deletion of ICP34.5 (iii) ICP47, which usually blocks antigen presentation in HSV infected cells, is deleted A version of this virus additionally encoding the gene for GM-CSF is currently in a Phase I clinical trial in a number of tumour types This virus is designed to provide a direct anti-tumour effect by tumour selective virus replication and also to induce an anti-tumour immune response enhanced by the expression of GM-CSF It is anticipated that in routine clinical practice oncolytic viruses are likely to be combined with standard therapies such as chemo- or radiotherapy The work S288 reported here assesses the anti-tumour effects of OncoVEX in combination with a number of first line chemotherapeutic agents (e.g taxol and cisplatin) in in vitro models Work in vivo is underway Results so far demonstrate that OncoVEX treatment of tumour cells either prior to, concurrently with, or after addition of the drug provides enhanced drug sensitivity and that the chemotherapeutic agents tested not reduce the effectiveness of the oncolytic effect Initial conclusions are therefore that combination of OncoVEX with anti-cancer drug therapy may be beneficial for cancer treatment 746 Effective Therapy of Metastatic Ovarian Cancer with an Oncolytic Herpes Simplex Virus Incorporating Two Membrane-Fusion Mechanisms Mikihito Nakamori,1 Xinping Fu,1 Feng Meng,1 Aiwu Jin,1 Lihua Tao,1 Robert C Bast, Jr.,2 Xiaoliu Zhang.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX; 2M.D.Anderson Cancer Center, The University of Texas, Houston, TX Replication-competent herpes simplex virus (oncolytic HSV) holds considerable promise for treating malignant solid tumors including ovarian cancer However, the potency of the virus needs improvement if its full clinical potential is to be realized In our previous studies, we showed that incorporation of membrane fusion capability into an oncolytic HSV, either by screening for a syncytial HSV mutant after random mutagenesis or by inserting a hyperfusogenic glycoprotein from gibbon ape leukemia virus (GALV.fus) into the viral genome, can significantly increase the antitumor capability of the virus We recently constructed a newer version of fusogenic oncolytic HSV by incorporating both membrane fusion mechanisms into a single virus In vitro characterization of this doubly fusogenic oncolytic HSV (Synco-2D) showed that this virus produces a distinctive syncytial phenotype, leading to a significantly increased tumor cell killing ability, compared with that of a nonfusogenic virus When injected directly into the abdominal cavity of mice bearing human ovarian cancer xenografts, Synco-2D eradicated all tumor masses in 75% of the animals, whereas no animals in the conventional oncolytic HSV treated group was tumorfree Our results indicate that this newly generated fusogenic oncolytic HSV is a promising candidate for clinical testing against advanced ovarian cancer 747 Comparaison of K-Ras Antisense Oligonucleotides and siRNAs Strategies in Human Pancreatic Cancer Amor Hajri, Hazare Nouari, Severine Wack, Marc Aprahamian Tumor Biology and Gene Therapy Dept., IRCAD-INSERM U375, Strasbourg, France Human pancreatic cancer is an extremely aggressive and incurable carcinoma because of its poor sensitivity to chemotherapy and/or radiotherapy Point mutations of the K-ras gene are detected in > 90% of human pancreatic cancers and may play an important role in tumorigenesis Antisense therapeutics with phosphorothioate oligonucleotides (PS ODN) and short interfering RNAs (siRNA) targeting specific gene expression represent a promising strategy for pancreatic cancer treatment The aim of the present work was to address a comparative study concerning the antitumor activity of synthetic PS-ODN and siRNA antisense targeting specific k-ras mutation (k-Ras mut12) The siRNA was delivered as a synthetic 19-22 bases and as a recombinant vector for stable expression The antitumor activity was evaluated (i) in vitro, in cell culture with different human pancreatic cancer cell lines (BxPc3, Panc1, Capan1) The endogenous Ras expression was determined by RT-PCR and Western-blotting Antiproliferative and cytotoxic effects were evaluated by [3H] thymidine incorporation and MTT tests (ii) In Molecular Therapy Vol 7, No 5, May 2003, Part of Parts Copyright © The American Society of Gene Therapy ... is a promising candidate for clinical testing against advanced ovarian cancer 747 Comparaison of K-Ras Antisense Oligonucleotides and siRNAs Strategies in Human Pancreatic Cancer Amor Hajri, Hazare... drug-effect analysis of Chou-Talalay was used to qualify the interaction between GCV and FU RESULTS: As calculated by the Chou-Talalay method, at least an additive effect of the drugs was noted... Hazare Nouari, Severine Wack, Marc Aprahamian Tumor Biology and Gene Therapy Dept., IRCAD-INSERM U3 75, Strasbourg, France Human pancreatic cancer is an extremely aggressive and incurable carcinoma