Brain tumors, gliomas and astrocytomas, are devastating malignancies that account for 2.3 percent of all US cancer deaths and represent the second most common solid tumor of children. Malignant brain tumors respond poorly to current therapies with a mean survival rate of less than one year despite treatment, Due to the invasive nature of gliomas, particularly glioblastoma multiformes (GBM), localized anti-cancer strategies, such as surgical removal, also fail to adequately halt the disease. Gliomas select for genetic inactivation of multiple tumor suppressor genes, including p53 (>60 percent), PTEN (>75 percent), p16/p I4ARF (50 percent), pRB (30 percent), and epigenetic down-regulation of the p27 Cdk inhibitor. A central hypothesis of anti-cancer therapies holds that replacement of tumor suppressor gene functions in malignant cells will result in specific death or apoptosis of the cancer cell while sparing the surrounding normal tissue. Indeed, tumor cells are undergoing continuous DNA damage and therefore, adenovirus expression of wild type p53 in gliomas by results in specific apoptosis to the glioma tumor cells. We propose to test this hypothesis by generating transducible tumor suppressor proteins. My laboratory has further developed the methodology of protein transduction. Recombinant, bacterially expressed fusion proteins containing an N' terminal protein transduction domain from HIV TAT rapidly transduce into 100 percent of cells. Using this methodology, we have generated and transduced over 60 TAT-fusion proteins from 15-120 kDa. Recently, we have demonstrated the ability of TAT-B-gal protein to transduce into most, if not all, cells and tissues of mouse models in vivo, including across the blood-brain barrier. Thus, in principle and practice, all mammalian cell types are susceptible to protein transduction. We propose to test the anti-cancer effectiveness and specificity of killing glioma tumors in mouse models by transducible tumor suppressor proteins, namely TAT-ARF and TAT-p53, and by a transducible pro-apoptotic viral protein, TAT-Apoptin. TAT-fusion proteins will be analyzed and optimized in vitro and then tested against xenograft intracranial glioma tumors in nude mice and in de novo derived astrocytomas in B8 transgenic ras about2"""""""" mice. In addition, to quantify protein transduction potential in mouse models, we intend to analyze the transduction and refolding rates of TAT-reporter fusion proteins, including TAT-B-gal and TAT-TK. TAT-TK activity will also be monitored in vivo by microPET imaging using '8F-fluoroganciclovir as a positron emitter.
