Merrill E. Goldsmith

Inhibition of glioma cells in vitro and in vivo using a recombinant adenoviral vector containing an astrocyte-specific promoter

Gene therapy using the herpes simplex virus thymidine kinase (HSV-TK) gene in combination with the drug ganciclovir (GCV) is a promising approach for the treatment of cancer-inducing gliomas, a tumor with a poor prognosis. In an attempt to limit the toxic effects on normal tissues, we constructed a recombinant adenoviral vector, Adgfa2TK, in which the HSV-TK gene is driven by the promoter for the gene encoding glial fibrillary acidic protein, an intermediate filament protein expressed primarily in astrocytes. Infection by Adgfa2TK of a glial cell line (C6) and a non-glial cell line (MDA-MB-231) revealed markedly increased expression of HSV-TK in glial cells as determined by Western blot. In comparison, high HSV-TK protein levels were produced in both cell lines after infection with a control virus, AdCMVTK, in which the constitutive cytomegalovirus viral promoter was used to direct HSV-TK expression. Infection of two glial cell lines (C6, U251) and two non-glial cell lines (HepG2, MDA-MB-231) with Adgfa2TK followed by GCV treatment revealed high toxicity in glial cell lines (50% growth inhibitory concentration: <2 μg/mL of GCV) with little or no toxicity (50% growth inhibitory concentration: >75 μg/mL) in the non-glial cell lines. In vivo, injection of Adgfa2TK into C6 tumors grown in nude mice followed by intraperitoneal GCV treatment significantly repressed tumor growth compared with the controls. Adgfa2TK may be useful for directing expression of the HSV-TK gene to gliomas.

Recombinant adenovirus-mediated p14ARF overexpression sensitizes human breast cancer cells to cisplatin

p14(ARF), the alternative product from the human INK4a/ARF locus, is one of the major targets for alterations in the development of human cancers. Overexpression of p14(ARF) results in cell cycle arrest and apoptosis. To examine the potential therapeutic role of re-expressing p14(ARF) gene product in human breast cancer, a recombinant adenovirus expressing the human p14(ARF) cDNA (Adp14(ARF)) was constructed and used to infect breast cancer cells. Five days after infection, Adp14(ARF) had considerable cytotoxicity on p53-wild-type MCF-7 cells. A time-course study showed that Adp14(ARF) infection of MCF-7 cells at 100pfu/cell increased the number of cells in G0/G1 phase and decreased that in S and G2/M phases. The presence of apoptotic cells was confirmed using the TUNEL assay. Adp14(ARF)-mediated expression of p14(ARF) also resulted in a considerable increase in the amounts of p53 and its target proteins, p21(WAF1) and MDM2. Furthermore, the combination treatment of MCF-7 cells with Adp14(ARF) and cisplatin resulted in a significantly greater cell death. Together, we conclude that p14(ARF) plays an important role in the induction of cell cycle arrest and apoptosis in breast cancer cells and recombinant adenovirus-mediated p14(ARF) expression greatly increases the sensitivity of these cells to cisplatin. These results demonstrate that the proper combination of Adp14(ARF) with conventional chemotherapeutic drug(s) could have potential benefits in treating breast cancer that carries wild-type p53 gene.

The histone deacetylase inhibitor FK228 given prior to adenovirus infection can boost infection in melanoma xenograft model systems

A major limitation of adenovirus type 5–mediated cancer gene therapy is the inefficient infection of many cancer cells. Previously, we showed that treatment with low doses of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide) increased coxsackie adenovirus receptor (CAR) levels, histone H3 acetylation, and adenovirus infection efficiencies as measured by viral transgene expression in cancer cell lines but not in cultured normal cells. To evaluate FK228 in vivo, the effects of FK228 therapy in athymic mice bearing LOX IMVI or UACC-62 human melanoma xenografts were examined. Groups of mice were treated with FK228 using several dosing schedules and the differences between treated and control animals were determined. In mice with LOX IMVI xenografts (n = 6), maximum CAR induction was observed 24 h following a single FK228 dose of 3.6 mg/kg with a 13.6 ± 4.3-fold (mean ± SD) increase in human CAR mRNA as determined by semiquantitative reverse transcription-PCR analysis. By comparison, mouse CAR levels in liver, kidney, and lung from the same animals showed little to no change. Maximum CAR protein induction of 9.2 ± 4.8-fold was achieved with these treatment conditions and was associated with increased histone H3 acetylation. Adenovirus carrying a green fluorescent protein (GFP) transgene (2 × 109 viral particles) was injected into the xenografts and GFP mRNA levels were determined. A 7.4 ± 5.2-fold increase in GFP mRNA was found 24 h following adenovirus injection into optimally FK228-treated mice (n = 10). A 4-fold increase in GFP protein–positive cells was found following FK228 treatment. These studies suggest that FK228 treatment prior to adenovirus infection could increase the efficiency of adenovirus gene therapy in xenograft model systems. [Mol Cancer Ther 2007;6(2):496–505]

453. The Histone Deacetylase Inhibitor FK228 Can Increase Adenovirus Transgene Protein Expression in Human LOX IMVI Melanoma Xenografts

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 increases in CAR and |[alpha]|v-integrin RNA levels. Treatment of cells with 1 ng/ml FK228 prior to adenovirus infection was associated with a 5-10 fold increase in adenovirus transgene expression. Increases in transgene expression were not found in cultured normal cells from breast, liver or kidney following similar FK228 treatment. The effect of FK228 treatment was examined in athymic mice bearing advanced-stage subcutaneous LOX IMVI or UACC-62 human melanoma xenografts. The efficacy of treatment was evaluated by analyzing CAR and |[alpha]|v-integrin RNA and protein. Both types of xenografts (n=3) had increased CAR RNA levels as determined by semi-quantitative RT-PCR analysis 6 h following the last of three treatments with 3.6 mg/kg FK228. The LOX IMVI xenografts showed an 8.0-fold (|[plusmn]|0.9) and the UACC-62 xenografts showed a 5.1-fold (|[plusmn]|0.4) increase over untreated mice. The LOX IMVI system was chosen for further study because of greater response to FK228. Mice with LOX IMVI xenografts (n=6) treated with single 3.6 mg/kg FK228 dose and analyzed at 6 h following drug administration showed a 10.7-fold (|[plusmn]|4.9) increase in CAR levels while at 24 h there was a 13.6-fold (|[plusmn]|4.3) increase. By comparison, CAR levels in the livers, kidneys and lungs from the same animals remained unchanged. Xenografts from mice (n=6) treated with 3.6 mg/kg and analyzed by western blot analysis 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 protein. Little change in |[alpha]|v-integrin RNA or protein was observed under any conditions. Based on these results xenograft-bearing mice (n=10) treated with a single dose of 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 following virus injection. 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. Immunohistochemistry of formalin-fixed paraffin-embedded xenografts using an antibody to GFP showed higher levels of GFP protein expression diffusely spread throughout the xenografts in FK228 treated mice 18 h and 24 h following adenovirus injection. Thus, as previously observed in vitro, the FK228 induced increase in adenovirus transgene expression also occurs in vivo. These studies suggest that FK228 treatment can increase the efficiency of adenovirus gene therapy in vivo.

618. Coxsackie-Adenovirus Receptor (CAR) Levels Can Be Increased in Human Xenografts Followed by Treatment with FK228, a Histone Deacetylase Inhibitor

Efficient adenovirus infection requires the presence of coxsackie-adenovirus receptor (CAR) and αv integrin on the surface of cells. Previously, we showed that treatment of several cancer cell lines with a low concentration of the histone deacetylase inhibitor FK228 (FR901228, depsipeptide) (1 ng/ml) caused an increase in the RNA levels of CAR and αv-integrin. FK228 pre-treatment was associated with a 5–10 fold increase in adenoviral transgene expression following adenovirus infection. The levels of CAR and αv integrin RNA were not increased in cultured normal cells from breast, liver or kidney following similar FK228 treatment. These results suggest that FK228, a drug currently in phase II clinical trials for the treatment of patients with peripheral or cutaneous T-cell lymphoma, may result in preferential enhancement of adenoviral transgene expression in cancer cells. To further evaluate this differential sensitivity between normal and cancer cells, we examined the effect of FK228 in athymic mice bearing advanced-stage subcutaneous LOX IMVI and UACC-62 human melanoma xenografts and MDA-MB-231 human breast cancer xenografts. Mice with melanoma xenografts were treated with FK228 iv q4d × 3 (days 1, 5, and 9) with one of three doses- 0.7, 1.6, or 3.6 mg/kg/dose. The highest dose was effective in causing tumor regression. Animals were sacrificed 6 h, 24 h, or 48 h following the last dose of FK228 and tissues were harvested. The levels of CAR and αv integrin RNA were monitored in mouse liver, kidney and lung and in the human xenografts using semi-quantitative RT-PCR analysis. The levels of CAR RNA were increased in the xenografts. The time of maximum increase in the levels of CAR RNA in the xenografts was 6 h after the administration of 3.6 mg/kg/dose FK228. The levels of αv integrin RNA in the xenografts were unchanged as were the levels of both CAR and αv integrin, in the livers, kidneys and lungs from the same animals. In order to define an optimal dose and schedule of FK228 administration for CAR induction, mice with LOX IMVI xenografts were treated with FK228 one, two, or three times. The tissues were then harvested either 6 or 24 hours following each dose and the levels of CAR and αv integrin RNA were determined. The results showed that the optimal dose for CAR induction was 24 hours after a single treatment with 3.6 mg/kg FK228 where CAR RNA levels were increased by 14-fold. Accordingly, CAR protein levels increased by 3–8 fold. In mice with MDA xenografts treated with a single dose of 1.6, 3.6 or 5.4 mg/kg FK228, CAR protein levels increased after 48 hours by 4-fold following the 3.6 mg/kg dose, whereas αv integrin levels remained unchanged. Because we have previously shown a correlation between the level of CAR and the extent of adenovirus infection in vitro, these results suggest that FK228 may preferentially increase the levels of adenovirus infection in cancer cells in vivo as it did in vitro. Experiments are in progress to determine if FK228 pre-treatment can increase the efficiency and selectivity of adenovirus gene therapy in vivo.