637. Suppression of VEGF-Mediated Tumor Angiogenesis by Cellular Relocalization of Its Transcriptional Activator HIF-1α

Growth Factors, Protein Delivery, & Animal Models therapy clinical trial development. In this study, we compare two different xenograft models with the goal of investigating the longterm impact of amplifying alkylator-resistant human hematopoietic stem and progenitor cells (HSC) in vivo. The reconstitution efficiency of in vivo-selected human cells in NOD/SCID versus NOD/SCID/ γchainnull mice was compared in parallel transplantation experiments. We have previously demonstrated in NOD/SCID mice significant selection of human SCID-repopulating cells (SRC) and their progeny by expression of MGMTP140K via an oncoretroviral vector when O6benzylguanine and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) were administered in vivo (Cai et al. Molecular Therapy 2006). In our current study, both mouse strains (n = 6-8 per group) were transplanted with sufficient numbers of MGMTP140K-transduced CD34+ cells to obtain high levels of human cell chimerism. In primary recipient NOD/SCID and NOD/SCID/γchainnull mice, high levels of human cell engraftment (70%-85% CD45+ cells), clonogenic cells, and multi-lineage differentiation were present in the bone marrow at 4-months post-transplantation. In both mouse models, a significant enhancement in the total number and percentage of MGMTP140Ktransduced cells in vehicle- versus drug-treated mice was observed (% transduced: NOD/SCID mice, 29% +/- 11% for vehicle control vs. 82% +/- 4% for drug-treated mice; NOD/SCIDγchainnull, 33% +/- 11% for vehicle control vs. 88% +/- 1% for drug-treated mice). For secondary reconstitution experiments, bone marrow samples were stimulated for 40 hours in the presence of interleukin-6 and stem cell factor and equal numbers of human cells derived from NOD/SCID or NOD/SCID/γchainnull mice were transplanted into NOD/SCID or NOD/SCIDγchainnull mice respectively. At 8 weeks post-transplant, low levels of engraftment were observed in secondary NOD/SCID mice receiving human cells from vehicle-treated primary NOD/SCID mice and ranged from 2%-4% human CD45+ cells and only 1%-4% transduced cells. In contrast, in vivo-selected transduced human cells did not reconstitute secondary recipient NOD/SCID mice. In NOD/ SCID/γchainnull mice, high levels of human-cell engraftment were detected. Engraftment of mice receiving bone marrow from vehicletreated mice was 30%-55% human CD45+ with 15-25% of the cells transduced. Engraftment of secondary mice receiving bone marrow from drug-treated mice was 15%-20% human CD45+ with >75% of the remaining human cells transduced. These data indicate that long-lived SRC can be transduced with oncoretroviral vectors and selected in vivo. In contrast to NOD/SCID mice, transplantation of ex vivo manipulated HSC into NOD/SCID/γchainnull mice represents a feasible model in which to test and validate novel strategies that focus on therapeutic manipulation of long-term repopulating cells from human stem-cell sources.

Growth Factors, Protein Delivery, & Animal Models 636. SOD3 Gene Transfer Enhances Tissue Healing by Regulating Endogenous Gene Expression

Juha P. Laurila,1,2 Maria D. Castellone,3 Lilja Laatikainen,1 Merja Haaparanta-Solin,4 Massimo Santoro,3 Mikko O. Laukkanen.1 1 Medicity Research Laboratory, University of Turku, Turku, Finland; 2Turku Graduate School of Biomedical Sciences, University of Turku, Turku, Finland; 3Department of Cellular and Molecular Pathology, University Federico II of Naples, Naples, Italy; 4Turku PET Centre, University of Turku, Turku, Finland. Extracellular superoxide dismutase (SOD3) is an antioxidative enzyme converting superoxide anions into less hazardous hydrogen peroxide. Previous studies have shown adenovirus mediated SOD3 gene transfer to attenuate liver damages, ischemia reperfusion injuries and restenosis. However, the cellular mechanism responsible for delivering SOD3 mediated therapeutic effect is not well understood. Our aim was to clarify the mechanism by using quantitative RT-PCR, S238

immunohistochemistry, western blotting and PET imaging to study the effect of SOD3 after transient transfection in vitro, and adenovirus mediated gene transfer in vivo. Adenovirus SOD3 (AdSOD3) mediated gene transfer into rat hind limb ischemia led to significantly reduced inflammation as evaluated by immunohistochemistry. Further analysis by quantitative RT-PCR showed AdSOD3 gene transfer to reduce expression of inflammatory cytokines and adhesion molecules, and to increase expression of endogenous SOD3 mRNA and VEGF-A mRNA, though this did not entail enhanced angiogenesis. However, SOD3 and VEGF-A may have cooperatively increased AP1 and CRE activity that resulted in increased proliferation in the damaged tissue. Lower inflammation and increased proliferation was also supported by PET imaging, which showed reduced accumulation of F-18-fluorodeoxyglucose in injured tissue after SOD3 gene transfer. Western blotting revealed increased phosphorylation of Mek1/2 and Erk1/2 in vivo. In vitro studies confirmed phosphorylation of Mek1/2 and Erk1/2, and indicated increased activation of Ras. In conclusion, the present work describes a molecular mechanism mediating the therapeutic response of SOD3. Manipulation of the Ras-Erk1/2 signalling pathway offers an explanation to improved tissue recovery in response to SOD3 gene transfer. Thus, the current data emphasizes the significance of SOD3 in the healing process and suggests regulation of a signalling network as a new physiological role for SOD3.

637. Suppression of VEGF-Mediated Tumor Angiogenesis by Cellular Relocalization of Its Transcriptional Activator HIF-1α

Jun Zhang,1 Andrew Lu,1 Yi Lu.1 University of Tennessee Health Science Center, Memphis, TN.

1

Metastasis is the major cause of death in breast cancer patients. It is, therefore, important to block the key step of tumor cell metastasis, such as angiogenesis, in order to effectively treat breast cancer. Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis. In a study of clinical specimens by immunohistochemical staining, we found that breast cancer tissue expressed much higher levels of VEGF than its adjacent normal breast tissue, on the other hand, normal breast tissue expressed high levels of p16 whereas its cancerous counterpart expressed low level (or none) of p16. Restoring of p16 by adenoviral-mediated p16 gene transfer downregulated VEGF gene expression in breast cancer MDAMB-231 and JygMC(A) cells, inhibited angiogenic ability of MDAMB-231 cells both in vitro (HUVEC tubular network formation) and in vivo (dorsal air sac) angiogenic assays, and suppressed metastatic ability of JygMC(A) cells in a spontaneous metastasis animal model. To examine whether p16 modulates VEGF expression by cellular relocalization of HIF-1α, the transcriptional regulator of VEGF gene, an adenoviral vector containing the HIF-1α-HT2 reporter fusion construct (Ad-HIF-1α/HT2) has been generated. By tracing the fluorescence of the reporter gene in the presence or absence of p16 expression, our results indicate that p16 alters cellular localization of HIF-1α in the breast cancer cells, suggesting a potential mechanism for how p16 modulates VEGF expression. Taken together, these results demonstrate that adenoviral-mediated p16 expression downregulated VEGF gene expression in breast cancer cells by modulating cellular localization of HIF-1α protein, inhibited breast cancer-induced angiogenesis, and suppressed breast tumor metastasis. This study suggests that blocking tumor angiogenesis may be an effective approach to suppress tumor metastasis, and p16 gene therapy may have clinical potentials to treat breast cancer.

Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy