Adenovirus gene therapy is frequently inefficient because of low levels of coxsackie adenovirus receptor (CAR) the primary adenovirus receptor. Previously, we showed that treatment of a diverse group of cancer cell lines with the histone deacetylase inhibitor FK228 (FR901228, depsipeptide), a drug in phase II clinical trials for the treatment of peripheral and cutaneous T-cell lymphoma, caused increased CAR RNA levels and was associated with a 5-10 fold increase in adenovirus transgene expression following adenovirus infection of FK228-treated cells. Since treatment of cultured cells with 1 ng/ml FK228 caused increased acetylated histone H3, the mechanism for the FK228 effect in vitro may be through protein acetylation. This potential mechanism was also suggested because treatment with the histone deacetylase inhibitors sodium butyrate or trichostatin A also caused increased levels of CAR. Increases in CAR levels, transgene expression, and histone H3 acetylation were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment in vivo was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI human melanoma xenografts. Treatment efficacy was determined by analyzing CAR RNA levels by semi- quantitative RT-PCR analysis or by analyzing CAR protein by western blot analysis. A maximum human CAR increase of 13.6- fold (|[plusmn]|4.3) compared to untreated xenografts was observed 24 h following i.v. injection of mice with 3.6 mg/kg FK228. By comparison, murine CAR RNA levels in livers, kidneys and lungs from the same animals remained unchanged. Xenograft protein from mice treated with 3.6 mg/kg FK228 and analyzed at 6 h following drug treatment showed no increase in CAR protein levels, however, analysis at 24 h showed a 9.2-fold (|[plusmn]|4.8) increase in CAR levels. Based on these results xenograft-bearing mice treated with 3.6 mg/kg FK288 were given an intra-tumor injection of adenovirus carrying a GFP transgene (2E+9 VP) 24 h following FK228 administration and the xenografts were analyzed 24 h later. Analysis of RNA from the entire xenografts showed a 7.2-fold (|[plusmn]|4.5) increase in expression from the adenovirus GFP transgene in the FK228-treated mice compared to untreated controls. In order to determine the mechanism for this effect, the levels of histone H3 acetylation in treated compared to control xenografts were analyzed. Western blot analysis of xenografts from mice showed that 6 h following treatment with 3.6 mg/kg FK228, there was an average 28.2-fold (|[plusmn]|22.9) increase in histone H3 acetylation that remained at 20.4-fold (|[plusmn]|12.6) 24 h following treatment. These studies suggest that the mechanism by which FK228 increases the efficiency of adenovirus gene therapy in vivo is through protein acetylation.