41 A novel conditionally oncolytic adenovirus for nasopharyngeal cancer gene therapy

S ]0 25-27 October 2002

CARO 2002 -Innovative Technology in Radiation Medicine

LNCaP:wtp53; DU-145:mtp53; PC-3: null-p53). These studies may shed light on the therapeutic ratio for irradiated prostate tissues and contribute to our understanding of prostate epithelial carcinogenesis. For each cell line, we have characterized radiation (IR)-induced apoptosis, radiosensitivity (ie. both clonogenic and MTT assays) and molecular data pertaining to cell-cycle checkpoints (ie. p53,p21,pRB expression). Results from these studies supported the conclusion that clonogenic survival is related to permanent cell cycle arrest, and not apoptosis. Of interest, DNA repair assays using the comet assay have shown that the malignant cell lines have a decreased capacity for DNA-dsb, and DNA base-excision, repair when compared to normal prostate cultures. Subsequent experiments suggest that malignant prostate cells have altered expression of genes involved in the DNA-dsb homologous recombination pathway (ie. XRCC3/rad50/51), which may partially explain their relative resistance, and genetic instability. Spontaneous and IR-induced oxidative damage are also defective in malignant cells. This is the first study to investigate the relative radiosensitivity and DNA repair capacity of prostate tissues. These data may provide novel targets for prostate radiotherapy radiosensitization based on defective molecular DNA repair. 41 A NOVEL CONDITIONALLY ONCOLYTIC ADENOVIRUS NASOPHARYNGEAL CANCER GENE THERAPY

There was a dose-dependent (0-50 Gy) reduction in endothelial cell density at 24 h after XRT, and this was associated with a similar dose-dependent disruption in blood-spinal cord barrier (BSCB) as demonstrated by albumin immunohistochemistry. After a single dose of 50 Gy to the cervical spinal cord of ASMase +/+ mice, there was a 47.7% reduction in endothelial cell density at 24 h compared to non-irradiated controls (33.8/mm2 vs 17.6/mm2, p = 0.03). No decrease of endothelial cell density was observed in ASMase -/- mice. After 50 Gy, albumin immunoreactivity and Evans blue dye staining around microvessels was observed at 24 h in the spinal cord of ASMase +/+ mice, but not in non-irradiated controls or ASMase -/- mice. Using radiolabelled DTPA as a vascular tracer, DTPA spinal cord/blood ratio was increased in ASMase +/+ but not in +/+ or 4- mice after a single dose of 50 Gy. We conclude that acute BSCB disruption following XRT is ASMase-dependent. Targeting the ASMase pathway may represent a neuro-protective strategy against XRT-induced BBB disruption. 43 CHANGES IN OLIGODENDROGLIAL POPULATIONS AND MYELIN GENE EXPRESSION IN THE CNS AFTER RADIATION

Atkinson. S.. Li, Y., Wong, S. Sunnybrook & Women's College Health Sciences Centre, University of Toronto, Toronto, ON, Canada

FOR

Chia. M. 1, Li, J. 1, Strathdee, C. 1, Huang, D.2, K/amut, H. 1, Liu, F-F. 1 1princess Margaret Hospital, Toronto, ON, Canada; 2Chinese University of Hong Kong, Hong Kong Rationale: Critical to the success of cancer gene therapy is the ability to achieve tumor-specific cytotoxicity and efficient in vivo delivery. We have succeeded in regulating gene expression in nasopharyngeal carcinoma (NPC) by exploiting the exclusive presence of the Epstein Barr virus (EBV) in cancer cells. When EBNA1 binds to a family of repeats (FR) sequence, located in the oriP (oriP-FR), transcription of downstream genes is activated by 1000-fold (Cancer Res 62:171, 2002). We are now poised to construct a conditionally replicating adenovirus, which can spread beyond the initially infected tumour cells. Methods and Materials: The E1A sequence required for adenoviral replication was cloned downstream of oriP-FR, resulting in a novel adv.oriP.E1A. Protein expression was determined using Western blot, cytopathic effects were evaluated in EBV-positive NPC C666-1 cells using the MTT assay. Tumorigenicity and therapeutic experiments were conducted using scid mice. Results: Western blotting for both E1A and fibre protein demonstrated a time-dependent increase in expression in the C666-1 cells, in contrast to minimal expression in EBV-negative NPC cells. Extensive cytopathic effects were observed in the C666-1 cells 3 days after infection with 5 or 10 pfu/cell of adv.oriP.E1A in contrast to no effect observed in a panel of other human cells (normal or malignant). Infection of adv.oriP.E1A in C666-1 cells (15 pfu/cell) prevented tumour formation. Intra-tumoural injection of adv.oriP.E1A (1 x 109 pfu) plus XRT (4 Gy) caused regression of established EBV-positive NPC tumours (C666-1 & C15). Conclusion: We have successfully constructed a novel selectively oncolytic virus for EBV-positive NPC cells, which now allows us to explore additional therapeutic combinatorial strategies that can potentially improve outcome for patients with NPC. 42 GENETIC REGULATION OF ACUTE DISRUPTION OF THE BLOODBRAIN BARRIER FOLLOWING IONIZING RADIATION

Wona. S. 1, Li, y.1, Jain, V. 1, Reilly, R. 2 Haimovitz-Friedman, A. 3 1Sunnybrook & Women's College Health Sciences Centre, University of Toronto, Toronto, ON, Canada; 2 Toronto General Hospital, Toronto, ON, Canada; 3Memorial Sloan-Kettering Cancer Center, New York, NY, USA Acute disruption of blood-brain barrier (BBB) is well recognised following radiation therapy (XRT) to the central nervous system (CNS). XRT-induced apoptosis in endothelial cells has been shown to be dePendent on the acid sphingomyelinase (ASMase) pathway. In this study, we sought to address the causal relationship between XRTinduced acute vascular endotheiial cell apoptosis and BBB disruption, and whether the latter was mediated by the ASMase pathway. Adult rats were given graded single doses of X-ray to the cervical spinal cord. At different time intervals after XRT, the irradiated spinal cord was processed for histologic and immunohistochemical analysis. In rat spinal cord, there was a dose-dependent apoptotic response during the first 24 h after XRT. Apoptotic cells consisted of endothelial and glial cells.

Demyelination is one of the late histopathological lesions seen following radiation therapy (XRT) to the central nervous system (CNS). The oligodendrocyte is thought to be one of the target cells of XRT-induced demyelination. XRT has been shown to result in early apoptosis of oligodendroglial cells and loss in the reproductive capacity of oligodendrocyte progenitor cells (OPC). The aim of this study was to assess changes in oligodendrocyte and OPC populations, myelin gene expression, and their relationship to demyelination following XRT. Adult rats were given single doses of 8 or 22Gy (ED100 for white matter necrosis) to the cervical spinal cord. At various time points after XRT, changes in myelin gene expression were assessed using RTPCR for the gene of the major myelin protein, pip. Oligodendrocytes were identified using immunohistochemistry (IHC) for APC and in situ hybridization for pip, and OPC using NG2 IHC. Consistent with previous findings, most of the apoptotic cells observed at 8h following XRT were oligodendroglial cells. About 25% of TUNEL positive cells however expressed NG2 immunoreactivity. At 24h after 22Gy, there was a significant decrease in oligodendrocyte but not OPC density. The oligodendroglial population continued to decline thereafter after 22Gy but also after 8Gy. For pip gene expression, a reduction was observed at 4-5 weeks after doses of 22Gy and 8Gy. At later time points, a decrease in pip expression was seen beginning at 18 weeks and after a dose of 22Gy only. Using Luxol fast blue staining, no apparent demyelination could be seen before 20 weeks after 22Gy. It is concluded that oligodendrocytes and OPC in the adult CNS undergo early apoptosis after XRT, and that there is an early decrease in myelin gene expression and oligodendroglial population. These early changes however are unlikely directly related to the late demyelination observed. 44 DOWN-REGULATION OF XIAP ENHANCES TRAIL-INDUCED APOPTOSIS IN CHEMO-RESISTANT HUMAN PRIMARY GLIOBLASTOMA CELLS

Roa, W. 1, Chen, H 1, Petruk, K. 1, Fu/ton. D. 1, Shaw, A. 2, Th'ng, j.2, Moore, R.2 Cross Cancer Institute, University of A/berta, Edmonton, AB, Canada Northwestern Ontario Regiona/ Cancer Centre, Lakehead University, Thunder Bay, ON, Canada Background: Recent evidence has suggested that X chromosome-linked Inhibitor of Apoptosis Protein (XIAP) prevents apoptosis by inhibiting effector caspases, and confers tumor resistance to g-irradiation, chemotherapy and cell kill from activated transmember death-receptors. Objective: To examine the role of XlAP in the signallying pathway of Tumor necoris factor-Related Apoptosis-lnducing Ligand (TRAIL) mediated apoptosis in chemo-resistant fresh isolates and early passages of human glioblastoma cells. Materials & Methods: Down-regulator candidates of XIAP and TRAIL were applied in various combinations to fresh isolates of three glioblastoma cell lines that harboured a quantitative spectrum of XIAP. Resulting cytotoxicity was assessed by XTT for cell viability, Hoechst staining for apoptotic nuclear morphology, and Western blots for the levels of proteins that regulate apoptotic pathways, including XIAP, BcI-XL, p53, caspase-3, 9 activities and PARP cleavage. The involvement of mitochondrial pathway marked by the release of