926. The Combination of P19ARF-HIV Tat Cytotoxic Gene Expression with AAT-1 PDX Increases Cell Killing of Brain Tumor Cells

Abstract

A synthetic cytotoxic gene was developed to kill tumor cells through a bystander effect; the application being to kill not only transfected tumor cells but the cells surrounding the transfected cells. Rather than relying on a cytotoxic ligand/receptor interaction, a combination of an intracellular cytotoxic gene element with a membrane permeability domain was developed. Peptide modeling studies identified a 28 amino acid sequence composed of an HIV Tat derived membrane permeability domain combined with a cytotoxic P19ARFdoamin that could be taken up by brain tumor cells and endothelial cells resulting in their death (Y.P.R. Jarajapu et.al (2005) J. of Gene Med. 7: 908-917). The DNA sequence encoding for the 28 amino acid sequence was cloned into a expression cassette containing an upstream alkaline phosphatase signal sequence and a downstream extrinsic epitope tag derived from influenza hemmaglutinin (HA tag). Western blot analysis confirmed the expression of P19ARF-Tat and Tat-P19ARF. Immunohistochemical staining of transfected cells showed intracellular staining characteristic of the endoplasmic reticulum of viable cells and nuclear staining characteristic of dead cells. In vitro cell killing studies with brain tumor cells varied the DNA dose/well to yield transfection efficiencies ranging from 5 % to 20%. An 8% transfection efficiency yielded a 50% reduction in viable cells, and 18% transfection efficiency yielded a 90% reduction in viable cells, indicative of a bystander cell killing mechanism. Gel electrophoresis analysis of fluorescein labeled Tat-P19ARFpeptide showed proteolysis of intact peptide into discrete bands following a 1 hr incubation with conditioned media from both human and brain tumor cells. Protease inhibitor studies coupled with immune precipitation identified furin as the protease responsible for cleaving the peptide. Addition of recombinant alpha-1 antitrypsin PDX, a selective furin inhibitor with nM binding affinity, increased the cytotoxicity of the expressed P19ARF-Tat in a dose dependent manner with the highest dose of recombinant protein increasing the degree of cell killing three fold.These studies characterize the expression of a cytotoxic gene whose mechanism of cell killing has been engineered to be through a bystander effect. The secretion of furin by tumor cells was inactivating the gene product through proteolysis. Addition of the furin inhibitor increased the amount of P19ARF-Tat thus increasing the cell killing activity of the cytotoxic gene. Coexpression of this inhibitor with the cytotoxic gene product should further improve the cytotoxic activity against the tumors. A synthetic cytotoxic gene was developed to kill tumor cells through a bystander effect; the application being to kill not only transfected tumor cells but the cells surrounding the transfected cells. Rather than relying on a cytotoxic ligand/receptor interaction, a combination of an intracellular cytotoxic gene element with a membrane permeability domain was developed. Peptide modeling studies identified a 28 amino acid sequence composed of an HIV Tat derived membrane permeability domain combined with a cytotoxic P19ARFdoamin that could be taken up by brain tumor cells and endothelial cells resulting in their death (Y.P.R. Jarajapu et.al (2005) J. of Gene Med. 7: 908-917). The DNA sequence encoding for the 28 amino acid sequence was cloned into a expression cassette containing an upstream alkaline phosphatase signal sequence and a downstream extrinsic epitope tag derived from influenza hemmaglutinin (HA tag). Western blot analysis confirmed the expression of P19ARF-Tat and Tat-P19ARF. Immunohistochemical staining of transfected cells showed intracellular staining characteristic of the endoplasmic reticulum of viable cells and nuclear staining characteristic of dead cells. In vitro cell killing studies with brain tumor cells varied the DNA dose/well to yield transfection efficiencies ranging from 5 % to 20%. An 8% transfection efficiency yielded a 50% reduction in viable cells, and 18% transfection efficiency yielded a 90% reduction in viable cells, indicative of a bystander cell killing mechanism. Gel electrophoresis analysis of fluorescein labeled Tat-P19ARFpeptide showed proteolysis of intact peptide into discrete bands following a 1 hr incubation with conditioned media from both human and brain tumor cells. Protease inhibitor studies coupled with immune precipitation identified furin as the protease responsible for cleaving the peptide. Addition of recombinant alpha-1 antitrypsin PDX, a selective furin inhibitor with nM binding affinity, increased the cytotoxicity of the expressed P19ARF-Tat in a dose dependent manner with the highest dose of recombinant protein increasing the degree of cell killing three fold. These studies characterize the expression of a cytotoxic gene whose mechanism of cell killing has been engineered to be through a bystander effect. The secretion of furin by tumor cells was inactivating the gene product through proteolysis. Addition of the furin inhibitor increased the amount of P19ARF-Tat thus increasing the cell killing activity of the cytotoxic gene. Coexpression of this inhibitor with the cytotoxic gene product should further improve the cytotoxic activity against the tumors.