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Biological evaluation of penetration domain and killing domain peptides
Author(s) -
Jarajapu Y. P. R.,
Baltunis J.,
Knot H. J.,
Sullivan S. M.
Publication year - 2005
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.738
Subject(s) - bystander effect , cell culture , cytotoxic t cell , cancer cell , microbiology and biotechnology , chemistry , cell , antennapedia , biology , biochemistry , biophysics , immunology , gene , cancer , gene expression , in vitro , genetics , homeobox
Background Cancer gene therapy must impact the majority of cells to be effective. Current gene delivery systems are unable to achieve sufficient transfer efficiency to the tumor cells. Cell killing can be dramatically increased through a bystander effect. Modeling the gene product with synthetic peptides can identify key elements for creating cell killing through a bystander effect. Methods Fluorescent labeled peptides were used for uptake kinetic studies and determination of intracellular localization in human glioblastoma cell lines, rat glioma cells lines and pressurized rat cerebral arteries. The degree of cell killing was assayed using propidium iodide coupled with fluorescence‐activated cell sorting (FACS) analysis. Results Peptides derived from HIV Tat and Drosophila antennapedia homeodomain were taken up by all tumor and primary cells. Attachment of an Mdm‐2‐binding domain derived from P14 ARF resulted in cell killing and was independent of domain orientation. Uptake kinetics showed rapid uptake for both tumor and primary cells equilibrating with the external media within 10 min. Intraluminal or extraluminal administration of peptides into pressurized cerebral arteries showed a lack of extravasation across the subbasement lamina. Assay of biological activity following intraluminal administration showed selective suppression of response to vasodilation with no effect on response by smooth muscle cells. Conclusions The results from these studies identified: (1) a cell trafficking domain and a cytotoxic domain for killing brain tumor cells; (2) that cell killing was independent of the domain orientations with regard to the cell trafficking domain being at the C‐terminus or N‐terminus; and (3) that the dual domain peptide can also be taken up by endothelial cells as shown by the cerebral artery studies. Hence, localized expression of the cytotoxic gene has the potential to not only kill brain tumor cells, but also tumor endothelium, thus further increasing the effectiveness of the therapy. Copyright © 2005 John Wiley & Sons, Ltd.