556. Proteasome Inhibitors Increase Cationic-Lipid Mediated Gene Transfer in A549 Cells In Vitro

Abstract

It has previously been described that proteasome inhibitors improve transduction efficiency of adeno-associated virus in vitro. More recently it was shown that proteasome-inhibitors increase peptide-based non-viral gene transfer in certain cell lines. We have assessed if proteasome inhibitors doxorubicin and LLnL increase Lipofectamine 2000-mediated gene expression in A549 (type II pneumocyte-like) and CFTE cells (cystic fibrosis tracheal epithelium). Cells were grown in 96-well plates and transfected (0.05 mg pLux/well complexed to 0.25 mg Lipofectamine) over-night. During transfection, and for 24 hrs after, cells were exposed to doxorubicin (0.5 mM to 20 mM and 0.5 mM to 8 mM for A549 and CFTE cells, respectively, n = 8 wells/condition, at least 2 independent experiments). 48 hrs after transfection luciferase expression was measured. The effect of doxorubicin on transfection was dose-dependent and peaked at 15 mM (no dox: 6.8|[times]|104|[plusmn]|1.2|[times]|104, dox: 1.3|[times]|10|[plusmn]|1.9|[times]|10 RLU/mg, p<0.05) for A549 cells. In contrast exposure of CFTE cells to 1-4 mM doxorubicin decreased gene expression (p<0.05) compared to no drug control. In parallel experiments, cells were exposed to LLnL (10 mM to 100 mM and 10 mM to 60 mM for A549 and CFTE cells, respectively, n=8 wells/condition, at least 2 independent experiments). Transfection efficiency was dose-dependent in A549 cells and all concentrations of LLnL lead to a significant increase in gene expression (6-18 fold, p<0.05). In CFTE cells LLnL did not significantly increase gene expression. Although, the intracellular processes that lead to gene expression after transfection are not well understood and the mode of action of proteasome inhibitors in the context of lipid-mediated gene transfer is unclear, the data presented here suggest follow up in in vivo studies is warranted.