Amplification of Recombinant Adenoviral Transgene Products Occurs by Inhibition of Histone Deacetylase

Abstract

n-Butyrate (butyrate) has been shown to amplify transgene expression in cells infected with E1-defective adenoviruses. The present studies were undertaken in order to better define the actions of butyrate in the context of adenovirus gene expression, and to attempt to elucidate the mechanism by which butyrate mediates the transgene amplification. It was found that butyrate amplified viral transgene expression over a concentration range of 0.5-5 mM, and that the amplification required an exposure of 12-24 hr for maximal effect. Western blot analysis of representative viral proteins showed that butyrate treatment amplified DNA-binding protein, but not fiber protein. A transient adenoviral replication system suggested that butyrate had a modest inhibitory effect on replication of the E1-defective adenovirus. Use of a specific inhibitor of histone deacetylase, trichostatin A (TSA), reproduced the amplification of the viral transgene product achieved with the butyrate. In contrast, adenoviral transgene expression could not be amplified by TSA treatment in a cell line known to have a TSA-resistant histone deacetylase. Butyrate amplified steady-state gene expression of the viral transgene, but had no detectable effects on either DNA-binding protein or fiber steady-state gene expression. Nuclear run-off experiments showed that both butyrate and TSA caused an increase in the viral transgene transcription. It was concluded that inhibitors of histone deacetylase amplify adenoviral transgene expression at the transcriptional level.

A novel N-acyl phosphatidylethanolamine-containing delivery vehicle for spermine-condensed plasmid DNA

“…In the experiments in Table 1 and Figures 10 and 11, an aliquot of a stock solution was added to adjust the Ca 2+ and Mg 2+ concentrations to 1.2 and 0.8 mm, respectively, just before addition to the wells. After 3 h, the samples were aspirated and medium containing 10% heat inactivated fetal bovine serum and either 5 m trichostatin A (Figures 7 and 8 41,42 We found that these inhibitors enhance the transgene expression of both cationic lipoplexes and the liposomal system in the range of two-to 10-fold at 2 g/ml DNA after 18-22 h, similar to previous results 43 .) At this point the cells were washed with Dulbecco's phosphate-buffered saline (PBS) and photomicrographs were taken (Olympus IMT-2 inverted microscope using the 10 × objective).…”

“…These data are presented in Table 1 relative to the liposomal treatment (all data after incubation in sodium butyrate, a nontoxic activator of transgene expression [41][42][43] and at physiological Ca 2+ and Mg 2+ levels; all data normalized in terms of total intracellular esterase activity). In the table, the cell viability and transfection are taken as 1.0 for the N-C12-DOPE/DOPC liposomes, ie numbers greater than 1.0 Figure 9b) or lipid complexes with equal amounts of pEGFP-C1 plasmid DNA for 3 h in serum-free medium.…”

Histone deacetylase inhibitors upregulate expression of the coxsackie adenovirus receptor (CAR) preferentially in bladder cancer cells

“…7 Treatment with NaPheBt or TSA correlated with increased transduction by adenovirus, as shown in Figure 5a. Previous findings show that TSA and butyrate analogs increase adenoviral transgene expression 30 in HeLa and A549 cells. However, it has only recently been determined that such agents enhance adenoviral transgene expression by increasing expression of CAR.…”

Oxamflatin is a novel antitumor compound that inhibits mammalian histone deacetylase

“…E ects of oxam¯atin on the integrated CMV promoter activity n-Butyrate and TSA are known to modulate transcriptional regulatory elements, including viral elements such as retroviral long terminal repeats (Chen et al, 1997), simian virus (Goldberg et al, 1992;Nakajima et al, 1998) and cytomegalovirus promoters (Dion et al, 1997;Tanaka et al, 1991). To analyse the e ect of oxam¯atin on the activity of modulating the integrated viral promoters, we determined the luciferase reporter activity in NIH3T3 cells stably transfected with pBCLuc.…”