941. Development of Cell Penetration Domain and Cell Killing Domain Genes for Treatment of Cancer

Abstract

Fluorescein labeled peptides derived from HIV-Tat (Tat) and Drosophila antennaepedia homeodomain (ANT) were able to intracellularly label two human glioblastoma cell lines (A172 and T98G) and one rat glioma cell line (RG2). The Tat peptide localized to the cell nuclei whereas the ANT sequence was distributed evenly throughout the cytoplasm. A 14 amino acid sequence derived from P14, a cell cycle suppression protein, was added to the C-terminus of each peptide. Increasing the size of the peptide did not inhibit cell entry and did not change fluorescent staining patterns of cells. It did kill each cell line within 1 hr after adding 10 uM of peptide. Tat, ANT and Exon1 peptides by themselves had no effect on cell viability. A dose response curve yielded an IC50 concentration of 9 uM for the Tat-Exon1. Reversing the domain order reduced the IC50 to 4 uM. Uptake studies showed that addition of Exon1 sequence to the Tat sequence reduced the rate of uptake by half. Reversing the domain order, i.e., Exon-1-Tat, restored the rate of uptake to that observed by Tat alone. To gain a better understanding of the behavior of these peptides in a three dimensional cell system, cell penetration and killing were tested in rat cerebral artery organ cultures. These vessels are maintained under physiological pressure and blood flow. Intraluminal administration of the fluorescent peptides produced cell-staining patterns similar to that observed in the in vitro brain tumor cell lines, with cell staining restricted to the endothelium. Adventitial administration of the Tat and ANT peptides yielded smooth muscle cell labeling of the nucleus and cytoplasm, respectively. No labeling of the endothelium was observed. Addition of the cell-killing domain did not inhibit the ability of the peptides to penetrate into the endothelium; however, response to vasoconstriction was diminished where as smooth muscle cell response was not affected. The Tat-ExonI sequence has been cloned into an expression cassette with an upstream signal sequence and a down stream HA epitope tag. Western blots showed that all three tumor cell lines expressed the peptide. Transfection results further showed a direct correlate between transfection efficiency and cell death. Experiments are directed toward proving the bystander effect mediated by the cell penetration domain in an in vivo rat brain tumor model.