Low Dose Histone Deacetylase Inhibitor, Depsipeptide (FR901228), Promotes Adenoviral Transduction in Human Rhabdomyosarcoma Cell Lines

Purpose. Transduction of rhabdomyosarcoma (RMS) cells with adenoviral vectors for in vivo and in vitro applications has been limited by the low to absent levels of coxackie and adenovirus receptor (CAR). This study investigates the potential use of low doses of a histone deacetylase inhibitor, depsipeptide (FR901228), currently in Phase II human trials, to enhance adenoviral uptake in six rhabdomyosarcoma cell lines. Methods. Differences in adenoviral uptake in the presence and absence of depsipeptide (FR901228) were assessed using an adenoviral construct tagged with green fluorescent protein. Changes in CAR and αv integrin expression RMS in response to pretreatment with depsipeptide (FR901128) was determined using RT-PCR. Results. Pretreatment of five of six RMS cell lines with 0.5 ng/ml of depsipeptide (FR901228) for 72 h resulted in increased viral uptake as assessed by green fluorescent protein expression. RT-PCR analysis for CAR showed that in four of these five cell lines, CAR expression was increased 2.8–8.1-fold in cells treated with depsipeptide (FR901228) as compared to control. αv integrin expression was substantially increased in the one cell line, RH5, which showed increased GFP expression in response to depsipeptide (FR901228) pretreatment but a minimal increase in CAR expression. Conclusions. Depsipeptide (FR901228) can be used as a vehicle to enhance adenoviral transduction in a majority of RMS cells. The mechanism of increased viral uptake appears to mediate via upregulation of CAR.


Introduction
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Novel treatments are required to improve the poor survival rates of patients that present with advanced disease. Gene therapy, using adenoviral constructs, is one potential therapeutic strategy. Adenoviral infections are advantageous because of their ability to infect both dividing and nondividing cells, their large packaging capacity and their ability to propagate high-titer viral stocks. However, the relatively low efficiency of recombinant gene transfer to tumor cells, including RMS cells, has been a disadvantage to the use of adenovirus in gene therapy.
Adenoviral infection of a cell requires the binding of the knob domain of the fiber capsid protein of the virus to the primary cellular receptor, coxackie and adenovirus receptor (CAR). 1 The attachment the virus to CAR may be facilitated by heparin sulfate proteoglycans or sialic acid. 2,3 Subsequent internalization of the virion by receptor-mediated endocytosis is potentiated by the interaction of RGD peptide sequences in the penton base of the virus with secondary host cell receptors, integrins a v b 3 and a v b 5. 4 In addition, adenoviral entry requires the activation of the phosphatidylinositol-3-OH kinase and the Rho family GTPase signaling pathways which target the actin cytoskeleton. 5 How actin facilitates internalization of the virus is unknown.
A number of studies have reported that cancer cells express only low levels of CAR and are therefore poorly infected by adenovirus. [6][7][8] Using flow cytometry and immunohistochemistry, Cripe et al. have shown that RMS cell lines and primary tumor samples, respectively, have low to absent levels of CAR but adequate a v integrin levels. Also, increasing CAR levels through transfection of CAR cDNA into RMS cells rendered them highly susceptible to adenovirus infection. 9 In a number of cell types, methods to achieve CAR-independent gene transfer have been utilized. These include modifications in adenoviral vectors as well as the use of polycations to alter the electrostatic repulsion between the negatively charged cell surface and adenoviral particles. 6,[9][10][11][12][13] An alternative strategy would be to increase the level of CAR in tumor cells.
Depsipeptide (FR901228), is a histone deacetylase inhibitor isolated from Chromobacterium violaceum No. 968. 14,15 By increasing the acetylation of histones and thereby activating transcription, histone deacetylase inhibitors (HDIs) modulate the expression of genes involved in a number of cellular processes including inhibition of cell growth, promotion of differentiation and reversion of a transformed phenotype. 16,17 Previous reports have shown that inhibitors of histone deacetylase, trichostatin A and butyrate, can increase adenoviral transgene expression at the transcriptional level. [18][19][20] Recently, Kitazono et al. demonstrated an increase in CAR, a v integrin and adenovirus-mediated gene expression in several carcinoma cell lines and hematopoetic cells pretreated with depsipeptide (FR901228). 8,21 In the present study, we describe the use of low dose depsipeptide to enhance adenoviral transduction in a panel of six rhabdomyosarcoma cell lines.

Materials and Method
Cell culture and reagents Rh30, Rh5, Rh13 and Rh18 are cell lines cultured from human alveolar rhabdomyosarcoma tumors. 22 RD4a is a subclone of a well established human embryonal rhabdomyosarcoma cell line, RD. 23 CTR (obtained from Dr. Maria Tsokos, National Cancer Institute, Bethesda, MD) is a human embryonal rhabdomyosarcoma cell line. 24 All cell lines were cultured in RPMI 1640 with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine at 37 C and 5-6% CO 2 .
Depsipeptide, FR901228, was obtained from the Pharmaceutical Management Branch, Cancer Therapy Evaluation Program, National Cancer Institute (Bethesda, MD).

MTT assay
Cells were plated in triplicate in 96-well plates at a density of 5 Â 10 3 /well. Twenty-four hours later, media was removed and replaced with media containing various concentrations of depsipeptide (FR901228). Control cells were treated with identical concentrations of DMSO (diluent for depsipeptide). After 72 h, cell number was determined using colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) (Sigma Chemical Co., St. Louis, MO) assay as previously described. 25,26 Absorbencies were measured on a Titertek enzymelinked immunosorbent assay reader using a test wavelength of 570 nm and a reference wavelength of 690 nm. The optical density measurements were converted to cell number using a standard curve.

Adenoviral infection
RMS cells were plated at a density of 2.5 Â 10 5 cells in six-well plates. After 24 h, 0.5 ng/ml of depsipeptide (FR901228) was added to designated wells and DMSO to the control wells. Seventy-two hours later the cells were incubated for 1 h with equal amounts of adenovirus, AdU1, in serum free media. AdU1 is an E1 and E3 deleted replication defective type 5 adenovirus with a GFP cassette driven by a CMV promoter. 27 AdU1 titre of 1 Â 10 11 TCID 50 /ml was determined by the TCID 50 assay as described by Qbiogene (Carlsbad, CA) except that the presence of GFP-positive cells instead of cytopathy was used as an indicator of viral infection.

Results and Discussion
We first determined the effect of various concentrations of depsipeptide (FR901228) on six RMS cell lines since depsipeptide (FR901228) has been shown to have antitumor activity as well as alter the growth of tumor cells. The growth inhibition of RMS cells treated with 0.5 ng/ml depsipeptide (FR901228) for 72 h was less than 20% compared to controls (Fig. 1a). Since our purpose was to use depsipeptide to modulate the efficiency of viral infection in these tumor cells and not to use the drug for its antitumor activity, we conducted the subsequent experiments with 0.5 ng/ml of depsipeptide (FR901228). As shown in Fig. 1b for the CTR cell line, an increase in acetylated histone H3 occurs at this low dose of depsipeptide (FR901228).
In order to study the ability of depsipeptide (FR901228) to enhance adenovirus-mediated gene transfer in RMS cells, we infected six RMS cell lines with an adenoviral vector containing a CMV promoter driven green fluorescent protein (GFP) gene. Cells infected with this virus can be visualized under a fluorescent microscope and GFP can be quantitated by Western analysis. As shown in Fig. 2a, five of the six cell lines pretreated with depsipeptide (FR901228) had increased GFP expression compared to the untreated control cells. The degree of GFP protein expression, quantitated by Western blot analysis, was 8.6-2.6-fold greater in the depsipeptide-treated cells then in the untreated control cells (Fig. 2b). No GFP protein expression was detected in the CTR cell line. Adenoviral infection is mainly dependent on the presence of CAR on the surface of normal as well as malignant cells. Because CAR expression has been reported to be low or sometimes absent in RMS cell lines and tissues, we investigated whether the increased adenoviral expression observed in the RMS cell lines treated with depsipeptide (FR901228) was due to increased CAR expression in these cell lines. Using RT-PCR, we were able to detect transcripts for CAR in five of six cell lines. In addition, four of six RMS cells treated with depsipeptide for 72 h showed a 2.8-8.1-fold increase in CAR expression compared to untreated cells (Fig. 3a)  Densitometry was performed using NIH Image 1.60 software and was used to calculate the fold change in protein expression. a-Tubulin was used to normalize the protein loading.
(FR901228), and showed the greatest increase in adenoviral uptake in response to depsipeptide (FR901228) pretreatment. Unlike Rh30, RH5 had minimally upregulated CAR expression in the presence of depsipeptide (FR901228); however, a 2.8fold increase in adenoviral uptake in response to pretreatment with depsipeptide was observed. In contrast, a v integrin expression was present in all six cell lines, and was modulated by depsipeptide (FR901228) 1.5-6.9-fold in four of the six RMS cell lines, including RH5 (Fig. 3b). The increased adenoviral uptake in RH5 cells in response to depsipeptide (FR901228) may be due to a 6.6-fold increase in a v integrin. Even though we detected a 2fold increase a v integrin expression in the CTR cell line, we observed no viral uptake in this cell line. This is probably due to the lack of CAR expression in both the presence and absence of depsipeptide (FR901228), suggesting that the presence of CAR is critical for adenoviral gene transfer. We conclude that depsipeptide (FR901228) modulates adenovirus transgene expression through upregulation of the viral receptor, CAR and in some cases a v integrin, in RMS cells. The change in gene expression is variable between the different RMS cell lines. These findings are similar to what has been reported for carcinoma cell lines and hematopoetic cells. 8,21 The mechanism of increased gene expression is presumed to be release of transcription repression through inhibition of histone deacetylases. As an efficient means of exogenous gene expression, the addition of low dose depsipeptide (FR901228) to promote adenoviral uptake may be a simple and useful approach for in vitro studies of rhabdomyosarcoma cells. Since depsipeptide (FR901228) is currently in adult Phase II trials and in a pediatric Phase I trial, the use of this agent in vivo is plausible and could be used for the purpose of increasing the efficacy of adenoviral mediated gene therapy.  Fig. 3. Effect of depsipeptide (FR901228) on RNA expression of CAR (a) and a v integrin ( b). RMS cells were treated for 72 h with 0.5 ng/ml of depsipeptide (FR901228) (þ) or DMSO (À) and then harvested for RNA extraction. Levels of gene expression for human CAR, a v integrin, and GAPDH were assessed using semi-quantitative RT-PCR. GAPDH was used to normalize the loading. Densitometry was performed using NIH Image 1.60 software and was used to calculate the fold change in protein expression.