Adenoviral modification of mouse brain derived endothelial cells, bEnd3, to induce apoptosis by vascular endothelial growth factor.
A second generation genetically-engineered cell-based drug delivery system, referred to as apoptotic-induced drug delivery (AIDD), was developed using endothelial cells (ECs) that undergo apoptosis upon binding of vascular endothelial growth factor (VEGF) to a Flk-1:Fas fusion protein (FF). This new AIDD was redesigned using mouse brain derived ECs, bEnd3 cells, and an adenovirus vector in order to enhance and control the expression of FF. The FF was tagged with a HA epitope (FFHA) and designed to be coexpressed with green fluorescence protein (GFP) by the regulation of cytomegalovirus promoters in the adenovirus vector. bEnd3 cells showed favorable coexpression of FFHA and GFP consistent with the multiplicity of infection of the adenovirus. Immunofluorescence analysis demonstrated that FFHA was localized at the plasma membrane, whereas GFP was predominantly located in the cytoplasm of ECs. Cell death was induced by VEGF, but not by platelet derived growth factor or fibroblast growth factor in a dose-dependent manner (range 2-20 ng/ml), and revealed caspase-dependent apoptotic profiles. The FFHA expressing bEnd3 cells underwent apoptosis when cocultured with a glioma cell (SF188V+) line able to overexpress VEGF. The combined data indicated that the FFHA adenovirus system can induce apoptotic signaling in ECs in response to VEGF, and thus, is an instrumental modification to the development of AIDD.
Mitsuuchi
Y
Y
Department of Pharmacology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Powell
D R
DR
Gallo
J M
JM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D001921:Brain / Q000166:cytology*; D002478:Cells, Cultured; D016543:Central Nervous System Neoplasms / Q000188:drug therapy / Q000473:pathology; D018920:Coculture Techniques; D016503:Drug Delivery Systems / Q000379:methods*; D042783:Endothelial Cells / Q000821:virology; D000939:Epitopes / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D005910:Glioma / Q000188:drug therapy / Q000473:pathology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism; D040262:Receptors, Vascular Endothelial Growth Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D019014:fas Receptor
D000939:Epitopes; C491899:Fas protein, mouse; D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D019014:fas Receptor; D040262:Receptors, Vascular Endothelial Growth Factor