The long-term goal of this application is to use gene therapy in the treatment of cancer. In this grant period both well-characterized and newly identified mediators of apoptosis will be analyzed and employed in studies of tumor reduction in mice.
The specific aims are listed in order of priority. 1) To test whether delivery of FasL by myoblasts is efficacious in the treatment of solid tumors in mice. This approach capitalizes on the attributes of myoblasts that allow them to be grown to large numbers, genetically engineered, and delivered to tissues in vivo where they stably express transgenes. FasL-expressing primary myoblasts, that are not transformed and are defective in Fas, will be injected and tested for their potential as localized antitumor agents that effectively kill cancer cells by three synergistic mechanisms: (a) directly via FasL/Fas-mediated apoptosis, (b) indirectly via FasL-mediated neutrophil invasion, and (c) via a bystander effect as allogeneic stimulants of T and B lymphocytes and natural killer cells. 2) To determine the mechanism of action of proteins implicated by others in the FasL/Fas mediated apoptotic pathway using (a) constitutive and regulatable retroviral vectors designed in the applicant's laboratory to delivery cytotoxic wild type and mutant proteins in a controlled manner to large populations of cells and (b) a novel method for monitoring protein-protein interactions in diverse intact mammalian cell types based on intracistronic beta-galactosidase (beta-gal) complementation of chimeric proteins. 3) To capitalize on the beta-gal complementation technology developed in the applicant's laboratory to identify previously unrecognized interacting proteins with a function in apoptosis, a potential """"""""mammalian two-hybrid screen."""""""" The ultimate goal is to test a well-characterized component of the Fas-mediated cell killing pathway (FasL) directly as an antitumor agent and to identify and characterize other components of the pathway that may serve as future anticancer agents when delivered by myoblasts or other emerging in vivo gene delivery technologies. In conjunction with traditional treatment modalities, these gene therapy strategies may provide potent adjuncts for the localized treatment of solid tumors that are often difficult to access surgically and who treatment with anticancer agents is hindered by dose-limiting bone marrow toxicity.
|Ozawa, Clare R; Banfi, Andrea; Glazer, Nicole L et al. (2004) Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and aberrant angiogenesis. J Clin Invest 113:516-27|
|von Degenfeld, Georges; Banfi, Andrea; Springer, Matthew L et al. (2003) Myoblast-mediated gene transfer for therapeutic angiogenesis and arteriogenesis. Br J Pharmacol 140:620-6|
|Springer, Matthew L; Ozawa, Clare R; Banfi, Andrea et al. (2003) Localized arteriole formation directly adjacent to the site of VEGF-induced angiogenesis in muscle. Mol Ther 7:441-9|
|Brazelton, Timothy R; Nystrom, Michael; Blau, Helen M (2003) Significant differences among skeletal muscles in the incorporation of bone marrow-derived cells. Dev Biol 262:64-74|
|Wehrman, Tom; Kleaveland, Benjamin; Her, Jeng-Horng et al. (2002) Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments. Proc Natl Acad Sci U S A 99:3469-74|
|Springer, Matthew L; Ozawa, Clare R; Blau, Helen M (2002) Transient production of alpha-smooth muscle actin by skeletal myoblasts during differentiation in culture and following intramuscular implantation. Cell Motil Cytoskeleton 51:177-86|
|Munz, Barbara; Hildt, Eberhard; Springer, Matthew L et al. (2002) RIP2, a checkpoint in myogenic differentiation. Mol Cell Biol 22:5879-86|
|Blanco-Bose, W E; Blau, H M (2001) Laminin-induced change in conformation of preexisting alpha7beta1 integrin signals secondary myofiber formation. Dev Biol 233:148-60|
|Blanco-Bose, W E; Yao, C C; Kramer, R H et al. (2001) Purification of mouse primary myoblasts based on alpha 7 integrin expression. Exp Cell Res 265:212-20|
|Blau, H M; Brazelton, T R; Weimann, J M (2001) The evolving concept of a stem cell: entity or function? Cell 105:829-41|
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