Adenoviral transgene expression enhanced by cotreatment with etoposide in cultured cells

The use of adenovirus as a means of DNA transfer into cells has become a common technique in cell culture systems (1,2). While adenoviral-mediated gene transfer is more effective than traditional transfection techniques (e.g., liposome formation or calcium phosphate precipitation), infection of some cell types remains inefficient (3). Previously, induction of DNA synthesis by treatment with DNA-damaging agents such as UV light, γ-irradiation, cis-platinum, and tritiated thymidine has been shown to increase adenoassociated virus (AAV)-mediated transduction efficiency in epithelial cells (4). Other studies revealed that topoisomerase inhibitors, such as etoposide, amscarine, and novobiocin, could increase AAV-mediated gene transduction in cell culture models (5). These authors suggested that use of topoisomerase inhibitors could be used as a mechanism to enhance gene expression in clinical trials or in cell culture (5), and it has been shown that topoisomerases are involved in the packaging, replication, and transcription of the adenoviral genome (6). Hong et al. (7) observed that use of the topoisomerase inhibitors etoposide and camptothecin in combination with gene therapy in a mouse liver tumor model increased AAV-mediated gene expression and effectively controlled tumor growth. Additionally, it has been noted that treatment with DNA-damaging agents in cultured cells can enhance transgene expression in multiple cell types (8–10) and in mouse models (11). In many of these studies, etoposide was used as a means to increase cell death in combination with the overexpression of a proapoptotic protein; therefore, very high concentrations of etoposide were used. In this study, we explored the use of low doses of etoposide as a means to increase adenoviral gene expression without increasing cytotoxicity or apoptosis in cultured cells. Our results indicated that low doses of etoposide, below toxic levels, can be used to increase adenoviral-mediated gene transduction without inducing apoptosis.

UMR106 osteosarcoma cells, which have proven to be resistant to adenoviral infection in our hands in comparison to other cell types (data not shown), were infected with equal amounts of an α-actinin-green fluorescent protein (GFP) adenovirus along with increasing concentrations of etoposide (10–10⁴ nM). Infections were carried out by first incubating cells with virus and etoposide or vehicle (dimethyl sulfoxide; DMSO) in a small volume (700 µL/well of 6-well plate) of tissue culture media supplemented with 10% fetal calf serum for 4 h at 37°C. After the initial 4-h incubation, 2 mL tissue culture media plus etoposide or vehicle were added per well, and the cells were incubated at 37°C for 24 h. After 24 h, cells were washed three times with media and incubated an additional 24 h at 37°C. At 48 h postinfection, media samples were taken, and cells were harvested in sodium dodecyl sulfate (SDS) sample buffer. These experiments showed that as little as 100 nM etoposide effectively increased adenoviral gene expression (Figure 1A). To determine the effect of etoposide treatment on cytotoxicity, a colorimetric assay for lactate dehydrogenase release was used according to the manufacturer's instructions (Roche, Indianapolis, IN, USA). Only doses greater than 500 nM showed increased cytotoxicity compared with vehicle-treated controls (Figure 1B). Therefore, we chose to use 100 nM etoposide in subsequent studies.

To verify that incubation with etoposide did not induce apoptosis, cells were infected with or without a GFP-expressing adenovirus in the presence or absence of 100 nM etoposide. The use of etoposide increased GFP expression approximately 3-fold compared to vehicle-treated controls in UMR106 cells (Figure 2A). Neither etoposide alone nor in combination with adenovirus increased phosphorylation of histone H2A.X(ph-H2A.X), a marker of DNA damage and apoptosis (Figure 2A). Cells treated with 10 ng/mL murine tumor necrosis factor-α and 10 µg/mL cycloheximide were used as a positive control for apoptotic cells. To further test the ability of etoposide to enhance adenoviral gene expression, similar experiments were carried out in rat embryo fibroblast (REF52) cells. Cotreatment with etoposide increased GFP expression approximately 2.5-fold in REF52 cells (Figure 2, D and E). Western blot analysis of poly-ADP-ribose polymerase (PARP) cleavage, a marker for apoptosis, showed that neither etoposide alone nor in combination with adenovirus induced apoptosis in these cells (Figure 2, D and F). Additionally, fluorescence microscopy of GFP expression confirmed that the increase in transgene expression correlated with both an increase in the number of cells expressing the transgene and an increase in the expression level per cell (Figure 2G). Additionally, these effects appear to be specific to viral-mediated transgene expression, since etoposide cotreatment of lipid-mediated transfected (with FuGENE® Transfection Reagent; Roche) cells with etoposide did not increase transgene expression (data not shown).

To determine if incubation of cells with etoposide affected the ability of exogenously expressed proteins to function in cells, UMR106 cells were infected with increasing concentrations of a myocardin-expressing adenovirus in the presence or absence of etoposide. Myocardin is a serum response factor-associated transcription factor that acts as a master regulator of many smooth muscle-specific genes (12). Myocardin expression was increased in cells infected in the presence of etoposide. And, importantly, expression of the myocardin target genes smooth muscle α-actin, telokin, and myosin light chain kinase were also increased, indicating that etoposide did not inhibit the normal function of myocardin to induce smooth muscle-specific genes (Figure 3). These data also clearly demonstrate that equivalent levels of transgene expression can be achieved with significantly smaller volumes of adenovirus in the presence of etoposide (Figure 3, compare myocardin bands in DMSO 100 versus ETOP 10).

Previous studies have shown that the use of DNA damaging agents, such as etoposide, can enhance the expression of both adenoassociated viral (5) and adenoviral transgenes (11). Many of these studies used high concentrations of drug with the intention in some cases of inducing cell death or decreasing tumor growth. Here we have investigated the possibility of using low doses of the topoisomerase inhibitor etoposide to increase adenoviral transgene expression in cultured cells. We show that incubation of target cells with as little as 100 nM etoposide during the first 24 h of infection results in a significant increase in transgene expression (>2.5-fold). Moreover, this effect was observed for multiple adenoviral vectors (α-actinin-GFP, GFP, myocardin) and in multiple cell types (UMR106 and REF52 cells). Additionally, incubation of cells with etoposide did not induce cytotoxicity, as measured by lactose dehydrogenase (LDH) release (Figure 1B), nor apoptosis, as measured by Western blot analysis of histone H2A.X phosphorylation and PARP cleavage (Figure 2, A, C, D, and F). Interestingly, fluorescence microscopy revealed that the increase in transgene expression appears to be due to both an increased number of cells expressing the transgene and increased expression in each infected cell (Figure 2G). Finally, etoposide did not interfere with the function of an exogenously expressed transgene, as evidenced by myocardin's ability to induce the expression of its smooth muscle-specific target genes (Figure 3).

While the data presented here clearly demonstrate the ability of etoposide to enhance adenoviral transgene expression, the mechanism by which this occurs remains unclear. Other studies suggested that similar effects could be due to the induction of DNA repair synthesis in cell culture (5,13) or suppression of the immune response in animal models (11). While we did not assess the induction of DNA repair mechanisms in this study, the fact that we observed an increase in both the level of transgene expression and the number of cells expressing the transgene suggest that this may contribute to increased transgene expression. Additionally, since adenoviral-mediated gene transfer is receptor-mediated, an etoposide-induced increase in surface expression of the coxsackie adenovirus receptor might be a mechanism contributing to enhanced expression. However, we did not see an additional increase in transgene expression in cells pretreated with etoposide compared with cotreated cells (data not shown), and treatment of cells with etoposide after virus has been washed away enhances transgene expression (data not shown). These observations appear to argue against an increase in receptor expression as a mechanism by which etoposide enhances transgene expression.

In conclusion, these results suggest that the use of etoposide to increase adenoviral-mediated gene expression could be of benefit in cell culture systems, potentially in systems using cell types that are traditionally difficult to infect, such as the UMR106 cells used in this study. Additionally, etoposide could be used to reduce the total amount of adenovirus used, conferring a longer life to adenoviral stocks and a reduction in the risk of adenoviral-mediated cellular toxicity. We propose that use of etoposide during adenoviral infection is an effective means to increase transgene expression with minimal effects on normal cell physiology and may be a valuable tool for researchers to use as a low-cost step to enhance protein expression in cultured cells.