ENHANCED TRANSGENE EXPRESSION IN UROTHELIAL CANCER GENE THERAPY WITH HISTONE DEACETYLASE INHIBITOR

0022-5347/05/1742-0747/0 THE JOURNAL OF UROLOGY® Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 174, 747–752, August 2005 Printed in U.S.A.

DOI: 10.1097/01.ju.0000164723.20555.e6

ENHANCED TRANSGENE EXPRESSION IN UROTHELIAL CANCER GENE THERAPY WITH HISTONE DEACETYLASE INHIBITOR TAKATSUGU OKEGAWA,* KIKUO NUTAHARA, REY-CHEN PONG, EIJI HIGASHIHARA AND JER-TSONG HSIEH From the Departments of Urology, University of Kyorin, Tokyo, Japan, and University of Texas Southwestern Medical Center, Dallas, Texas

ABSTRACT

Purpose: Efficient adenoviral infection requires the presence of the coxsackievirus and adenovirus receptor (CAR). We determined whether the histone deacetylase inhibitor FR901228 (Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) increases the efficiency of adenoviral gene therapy in bladder cancer in vivo and in vitro. Materials and Methods: Cytotoxicity studies were performed to determine a minimally cytotoxic FR901228 concentration for bladder cancer cells. The level of CAR expression was determined by fluorescence activated cell scanning and/or reverse transcriptase-polymerase chain reaction analysis in FR901228 treated bladder cell lines. The in vivo effect on adenoviral gene expression was investigated in athymic mice. Results: The concentration of FR901228 showing no or minimal cytotoxicity that was selected for these studies was 0.5 ng/ml for bladder cancer cells. Treatment of cancer cells with 0.5 ng/ml histone deacetylase inhibitor increased CAR RNA levels and acetylated histone H3. This increase was associated with a 5 to 10-fold increase in adenoviral infection, as evidenced by increased transgene expression from a ␤-galactosidase containing adenoviral vector. Intravenous administration of FR901228 enhanced CAR expression in athymic mice. The combination of p53 adenovirus and histone deacetylase inhibitor resulted in significant tumor inhibition in vitro and in vivo. Conclusions: Nontoxic doses of the histone deacetylase inhibitor FR901228 increased CAR RNA levels and resulted in the marked enhancement of transgene expression after adenoviral infections. FR901228 pretreatment may increase the sensitivity of tumor cells to adenoviral gene therapy vectors. KEY WORDS: bladder, bladder neoplasms, adenovirus receptor, histone deacetylases

Urothelial cancer is the second most common urological malignancy and it has a poor prognosis. The variable morphology, natural history and prognosis demonstrate that bladder cancer is not a single disease entity but it occurs in 3 distinct forms, of which each has distinct features.1 According to Mostofi et al more than 90% of bladder cancers are transitional cell carcinomas (TCCs) derived from the urothelium, about 6% to 8% are squamous carcinomas and 2% are adenocarcinomas.2 Using genetic and molecular biological techniques researchers have delineated many critical genes involved in TCC development. Data from numerous research laboratories indicate that exogenous delivery of these genes into TCC can alter the malignant phenotype of the tumor and/or specifically eradicate it.1, 3 Thus, gene therapy has become an attractive regimen, in addition to conventional therapy. Because of the post-mitotic infectivity of adenovirus 5, this virus is commonly used for the adenoviral delivery system of gene therapy for urothelial cancer in clinical trials of phase I gene therapy. Adenoviruses are thought to enter the host cell cytoplasm through specific receptor mediated endocytosis and entry into target cells is the rate limiting step of gene delivery.4 A 46 kDa transmembrane protein with a typical cell adhesion molecule and an Ig-like structure was identified as a coxsackievirus and adenovirus type 5 receptor (CAR).5

Several groups, including ours, have documented a wide spectrum of CAR expression among different cancer cell lines and tumor specimens.6 – 8 Previously we have reported that CAR and not integrin (␣v␤3 and ␣v␤5) is the key determinant for viral sensitivity among bladder cancer cell lines and decreased CAR mRNA levels are often seen at the more advanced stages of urothelial cancer.9 These results indicated that CAR levels in patients with urothelial cancer certainly affect the outcome of gene therapy. Some investigators have indicated that histone deacetylase (HDAC) inhibitors enhance adenoviral transgene expression in several different cancer cell lines.10⫺16 We investigated whether the HDAC inhibitor FR901228 could potentially turn on endogenous CAR gene expression in urothelial cell lines. MATERIALS AND METHODS

FR901228 and recombinant viruses. FR901228, a depepsipeptide fermentation product from Chromobacterium violaceum, was used. The replication deficient recombinant viruses AdCMV-␤gal and AdCMV-p53 were generated, as described previously.4, 17 Cell culture. T24, TCC and WH cell lines (American Type Culture Collection, Rockville, Maryland) were grown in T medium containing 5% fetal bovine serum.18 These cells were routinely cultured in a humidified incubator at 37C with 5% CO2. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. For quantitative RT-PCR analysis 2 ␮g total cellular RNA per tissue were used to synthesize first strand cDNA, as described previously.4 An eighth of the cDNA

Submitted for publication October 15, 2004. Supported by grant CA95730. * Correspondence and requests for reprints: Department of Urology, Kyorin University School of Medicine, 6 –20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan (telephone: 0422– 47-5511; FAX: 0422– 42-8431; e-mail: [email protected]). 747

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was subjected to PCR (30 cycles at 92C for 15 seconds, at 55C for 30 seconds and at 72C for 2 minutes) in a 20 ␮l reaction mixture using the CAR primer set (5⬘-GCCTTCAGGTGCGAGATGTGTTA-3⬘ and 5⬘-GAACACGGAGAGCACAGATGAGAC-3⬘ (0.5 ng/␮l) or the glyceraldehyde-3-dehydrogenase (GAPDH) primer set (5⬘-TCGTGGAAGGACTCATGACC-3⬘ and 5⬘-TCCACCACCCTGTTGCTGTA-3⬘ (0.5 ng/␮l). The final PCR products (10 ␮l) were analyzed on 2% NuSieve® agarose gel (3:1). The density of each transcript was determined using BioMax 1D image analysis software (Eastman Kodak, Rochester, New York). Determination of CAR levels by fluorescence activated cell scanning (FACS). Cytometric analysis was done to determine CAR levels for each cell. Briefly, membrane fluorescence staining was performed in a single cell suspension using RmcB monoclonal antibody and fluorescent isothiocyanate conjugated secondary antibodies, as described previously.17, 18 FACS was performed with a dual laser Vantage flow cytometer (Becton Dickinson, Mountain View, California), which delivered 50 mW at 488 nm with an air cooled laser. Analysis was performed using LYSYS II software (Becton Dickinson). The positive population of cells was determined by gating the tail on the right side of the distribution of the negative control sample for each individual cell line at 1%. This setting was then used to determine the percent of positive cells for each described marker for each individual cell line. Western analysis of histone H3 expression. Cell lysate was made by adding 20% sodium dodecyl sulfate containing 1 mM phenylmethylsulfonyl fluoride. The lysate was sonicated for 30 seconds on ice and then centrifuged for 5 minutes at 4C. Total protein (20 ␮g) per sample was subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred to nitrocellulose the membrane. After blocking with phosphate buffered saline (PBS) containing 5% powdered milk the membrane was incubated with rabbit polyclonal antibody against acetylated histone H3 and rabbit polyclonal antibody against histone H3, diluted 1:2,000 in 5% milk (Upstate Biotechnology, Lake Placid, New York) for 1 hour, followed by incubation with antirabbit IgG, diluted 1:10,000. After extensive washing the protein was visualized with an ECL chemiluminescence detection kit (Amersham, Arlington Heights, Illinois). Detection of virus mediated gene delivery. We examined whether the increase in CAR levels could enhance adenoviral infectivity, as determined by transgene expression after infection. We determined virus medicated gene delivery using 2 approaches, namely ␤-galactosidase staining and activity assay. Three bladder cancer cells (150,000 cells per p-60 plate) were incubated with or without 0.5 ng/ml HDAC inhibitor. At the end of the 24-hour incubation period HDAC inhibitor was removed and cells were infected with AdCMV-␤gal. Infected cells were washed with PBS, fixed and stained for ␤-galactosidase activity.4 Adenovirus infected cells were counted microscopically according to the number of positive cells. In the second approach ␤-galactosidase activity was determined. Infected cells were trypsinized and washed once with PBS. The protein concentration of each sample was then determined by the Bradford dye binding procedure (BioRad Laboratories, Hercules, California). ␤-Galactosidase activity was measured in 200 ␮l cell lysate and normalized to the protein concentration of each sample.4 Effect of Ad-CMV-p53 plus HDAC on tumor growth in vitro. The p53 adenovirus was used to determine the efficacy of gene therapy in the bladder cell line incubated with or without 0.5 ng/ml HDAC inhibitor. Cells were plated at a density of 5,000 cells in 48-well plates using T medium containing 0.2% fetal bovine serum and infected with AdCMV-p53 at an MOI of 1. At the indicated time points cells were harvested

and the relative cell number was determined by crystal violet assay.4 Effect of Ad-CMV-p53 plus HDAC inhibitor on tumor growth in vivo. T24 cells (1 ⫻ 106) mixed with 100 ␮l MatrigelTM were injected subcutaneously into athymic mice. When tumors grew to about 160 mm,3 a single injection of 3.2 ⫻ 108 pfu p53 intratumorally and/or 0.1 mg/kg HDAC inhibitor intravenously was given for 3 consecutive days. Tumor volume was measured weekly for 3 weeks and calculated, as described previously.4 Statistical analysis. All data were evaluated using Student’s t test with probability values less than 0.05 or 0.01 considered significant. RESULTS

Growth inhibition by FR901228. Cytotoxicity studies were performed to determine the minimally cytotoxic concentration of FR901228 for bladder cancer cells. The drug concentration showing no or minimal cytotoxicity that was selected for these studies was 0.5 ng/ml (fig. 1). Determination of CAR levels by FACS and RT-PCR analysis of CAR in control and HDAC treated cell lines. Cytometric analysis of the immunofluorescence staining of 3 human bladder cancer cell lines indicated that WH, TCC and T24 contained low numbers of CAR positive cells. WH contained 34% CAR positive cells, TCC contains 23% CAR positive cells and T24 contained the lowest number of CAR positive cells (14%). Table 1 shows CAR levels, as determined by FACS before and after the administration of HDAC inhibitor. CAR expression was increased approximately 10-fold after incubation with HDAC inhibitor for 72 hours in the T24 cell line. In treated WH and TCC cells CAR expression achieved levels approximately 2 to 3 times those in untreated cells. Furthermore, our results indicated that CAR expression increased in a time dependent manner (table 1). Similar results were obtained when CAR RNA levels after incubation in FR901228 for 72 hours were determined by RT-PCR analysis (fig. 2). Thus, in T24, WH and TCC cells HDAC inhibitor induced CAR expression significantly. Effect of HDAC inhibitors on bladder cell lines. We examined whether HDAC inhibitor might be responsible for this

FIG. 1. Growth inhibition effect on 5,000 human bladder cancer cells of various concentrations of FR901228 for 72 hours with total cell number determined by crystal violet assay. Percent inhibition was calculated as treated-to-control cell ratio. All experiments were repeated at least 3 times.

FR901228 ENHANCES ADENOVIRAL INFECTION OF UROTHELIAL CANCER CELLS

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TABLE 1. CAR levels on FACS in control and FR901228 treated cells FR901228 (ng/ml)

Mean % FACS ⫾ SD 24 Hrs

p Value vs Control

48 Hrs

p Value vs Control

72 Hrs

p Value vs Control

WH: 0 31 ⫾ 2 29 ⫾ 1 32 ⫾ 1 0.01 34 ⫾ 3 42 ⫾ 2 ⬍0.05 46 ⫾ 3 ⬍0.01 0.1 49 ⫾ 5 ⬍0.05 64 ⫾ 3 ⬍0.01 72 ⫾ 4 ⬍0.01 0.5 71 ⫾ 4 ⬍0.01 69 ⫾ 3 ⬍0.01 71 ⫾ 5 ⬍0.01 TCC: 0 23 ⫾ 3 22 ⫾ 4 21 ⫾ 3 0.01 25 ⫾ 5 35 ⫾ 6 ⬍0.05 41 ⫾ 5 ⬍0.05 0.1 41 ⫾ 4 ⬍0.01 52 ⫾ 7 ⬍0.01 67 ⫾ 6 ⬍0.01 0.5 75 ⫾ 7 ⬍0.01 78 ⫾ 5 ⬍0.01 76 ⫾ 4 ⬍0.01 T24: 0 6⫾1 6⫾1 7⫾1 0.01 10 ⫾ 1 ⬍0.05 18 ⫾ 3 ⬍0.01 21 ⫾ 3 ⬍0.01 0.1 31 ⫾ 3 ⬍0.01 39 ⫾ 4 ⬍0.01 56 ⫾ 5 ⬍0.01 0.5 57 ⫾ 5 ⬍0.01 63 ⫾ 5 ⬍0.01 65 ⫾ 7 ⬍0.01 Cells were incubated with RmcB (CAR) before adding of fluorescein isothiocyanate conjugated antimouse IgG secondary antibody (data are presented as percent of cells gated positive with each data set repeated in triplicate).

phenomenon. Figure 3 shows that incubation with HDAC inhibitor resulted in a marked increase in histone acetylation, as evidenced by immunoblot performed with an antibody recognizing acetylated histone H3. It did not alter total histone H3 levels. Correlation of CAR levels and viral sensitivity of bladder cancers. Although these results indicated that HDAC inhibitor could increase CAR expression, we examined whether this increase would result in enhanced transgene expression after adenoviral infection. ␤-Galactosidase activity after incubation with 0.5 ng/ml HDAC inhibitor for 24 hours was proportional to the CAR level from each cell line, in particular ␤-galactosidase activity in treated, infected T24 cells (MOI 50) (fig. 4). AdCMV-␤-gal was approximately 10 times higher than that in untreated T24 cells. These studies demonstrated that HDAC inhibitor increased CAR levels and resulted in marked enhancement of transgene expression after adenoviral infections. Enhanced growth inhibition with p53 adenovirus and HDAC inhibitor for bladder cancer. To examine whether there was a combined effect of HDAC inhibitor and adenovirus gene therapy bladder cancer cells were treated with p53 adenovirus (MOI 1) or HDAC (0.5 ng/ml). Growth inhibition of bladder cancer cells was greatly increased by the combination compared with each single treatment (fig. 5). We further used an animal model to examine whether HDAC inhibitor and p53 adenovirus had a synergistic effect on tumor growth in bladder cancer cells. Mice were sacrificed weekly 3 weeks after intravenous injection of HDAC inhibitor. CAR expression was found to be increased on RT-PCR and Western blot analysis (fig. 6). The combination of p53 adenovirus and HDAC inhibitor caused significant inhibition of tumor growth (table 2). Body weight in these animals appeared to be the same, suggesting that these agents did not cause any severe toxicity in the host. We did not find any recurrent tumors in animals that received combination therapy. These results indicate that these 2 therapeutic agents have a synergistic effect on bladder cancer with potential clinical applications.

der cancer cells in vitro and in vivo.9 These observations suggest that advanced cancer may be the cells most difficult to infect with adenovirus. Therefore, if CAR expression on

DISCUSSION

In bladder cancers decreased CAR expression has been reported in cell lines and tissue specimens.7, 9 These finding imply that CAR has physiological functions in bladder cancers other than as an adenoviral receptor. Structurally CAR is a typical cell adhesion molecule with homophilic interaction.9 Our studies further demonstrate that the cell adhesion function of CAR is critical for its growth inhibitory effect on human bladder cancer cells.9 Increased CAR expression in CAR negative cells leads to the growth suppression of blad-

FIG. 2. RT-PCR analysis of CAR RNA in control and FR901228 treated cell lines. Cells were incubated with and without FR901228. RNA was isolated and RT-PCR was performed as described. For quantitation (values below lanes) band density of cancer line incubated with 0.5 ng/ml FR901228 was assigned value of 1.00. Each data set was repeated in triplicate.

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FR901228 ENHANCES ADENOVIRAL INFECTION OF UROTHELIAL CANCER CELLS

FIG. 3. Western blot analysis of histone H3 shows effect of HDAC inhibitors on FR901228 treated cells. WH and T24 cells were incubated with and without FR901228 for 72 hours. Levels of histone H3 and acetylated histone H3 were determined by Western blot analysis as described. Each data set was repeated in triplicate.

target cells could be increased, this could potentially yield improved clinical efficacy. HDAC inhibitors (FR901228, CHAP31, sodium butyrate and trichostatin A) are a novel class of chemotherapeutic agents, initially identified by their ability to reverse the malignant phenotype of transformed cells. They can induce apoptosis and differentiation and inhibit cell cycle progression, and they have anti-angiogenic and immune stimulatory properties.19 However, the molecular mechanisms underlying their anticancer effects are poorly understood. A number of HDAC inhibitors are currently being tested in early phase clinical trials against various cancers and promising results are being reported, supporting the development of

these compounds for clinical use. In addition, some investigators have indicated that the addition of HDAC inhibitors after transfection increased transgene expression of transiently transfected DNA.10⫺16 We are particularly interested in enhancing transgene expression using HDAC inhibitors. We first tested the cytotoxicity of the HDAC inhibitor FR901228 in bladder cancer lines with weak CAR expression. The drug concentration showing no or minimal cytotoxicity that was selected for these studies was 0.5 ng/ml for 3 bladder cancer cell lines. We further noted that HDAC inhibitor enhanced transgene expression induced by AdCMV-␤-gal in bladder cancer cells. The magnitude of induction by the inhibitor was 3 to 10-fold greater than in the control (figs. 2 to 4). We observed that the growth inhibition of T24 cells was greatly potentiated in vitro and in vivo by combining HDAC inhibitor and Ad-CMV-p53 at a low dose (fig. 6 and table 2). A nontoxic dose of FR901228 increased CAR levels in vitro and in vivo. Thus, combining HDAC inhibitors and the adenoviral delivery system for gene therapy would synergize the therapeutic efficacy for bladder cancer. Several investigations have indicated that some HDAC inhibitors could potentially turn on endogenous CAR gene expression in cancer cells in vitro, suggesting that downregulation of the CAR gene is due to epigenetic control.10⫺16 Kitazono et al also reported that FR901228 can increase CAR gene expression in several cancer cell lines.10, 11 Lee et al reported that butyrate increased the level of CAR in bladder cancers cell with low levels of expression.12 Hemminki et al noted that FR901228 and trichostatin A can increase adenoviral transgene expression in ovarian cancer cells.13 Sachs et al indicated that CAR expression can be up-regulated using

FIG. 4. A, AdCMV-␤-gal expression in control and FR901228 treated cells. Three bladder cancer cell lines (150,000 cells per p-60 plate) were incubated with or without 0.5 ng/ml FR901228. At end of 24-hour incubation FR901228 was removed and cells were infected with AdCMV-␤-gal. Each value was determined in triplicate in 2 separate experiments. B, quantitation of ␤-galactosidase positive cells after adenovirus infection. Infected cells were washed with PBS, fixed and stained for ␤-galactosidase activity. Adenovirus infected cells were counted microscopically according to number of positive cells, indicated as blue cells. Each value was determined in triplicate in 2 separate experiments. m.o.i, MOI. Asterisk indicates significantly different vs no treatment (p ⬍0.01).

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FR901228 ENHANCES ADENOVIRAL INFECTION OF UROTHELIAL CANCER CELLS

FIG. 5. In vitro effect of HDAC inhibitors on AdCMV-p53. Effect on growth of 5,000 T24 bladder cancer cells of p53 adenovirus at MOI of 1 and/or 0.5 ng/ml FR901228 with mean cell number ⫾ SD determined by crystal violet assay. Single asterisk indicates significantly different vs control (p ⬍0.05). Double asterisks indicate significantly different vs control (p ⬍0.01).

trichostatin A and sodium phenylbutyrate in CAR negative bladder cancer cells.16 Recently Taura et al reported that a novel HDAC inhibitor enhanced adenovirus mediated transgene expression in animal models of colon and gastric cancer.15 Our results in vitro and in vivo are consistent with these previous reports that suggested increases in adenoviral transgene expression and CAR. A number of HDAC inhibitors are currently in phase I and II clinical trials, and encouraging results have been reported for the treatment of hematological malignancies and solid tumors.19 A high dose of chemotherapeutic agent is often associated with undesirable toxicity in nontarget cells. The promise of gene therapy is to deliver tissue and tumor specific gene targeting that increases tumor cell toxicity and death but decreases systemic toxicity in the patient. Using an HDAC inhibitor we were able to determine that the status of histone acetylation associated with the CAR gene signifi-

FIG. 6. A, RT-PCR of CAR in T24 tumors weekly for 3 weeks (Wk.) after initial treatment with FR901228 intravenously in vivo. B, Western blot analysis of CAR in T24 tumors weekly for 3 weeks after initial treatment with FR901228 intravenously in vivo. For quantitation (values below lanes) density of band from tumor line after 3 weeks treatment with FR901228 was assigned value of 1.00.

cantly impacted its gene transcription in cells expressing low levels of CAR. Therefore, it is intriguing to combine these 2 regimens in a way that may not only enhance their therapeutic efficacy, but also decrease undesired side effects. Our recent study investigated the underlying mechanism by which HDAC inhibitor enhanced the expression of a transgene delivered by adenovirus.20 We identified the core promoter sequence from the human CAR gene and found that activation of the CAR gene promoter is modu-

TABLE 2. Synergistic effect of combining FR901228 and ad-CMV-p53 on in vivo growth of T24 bladder cancer cells Tumor No.

Mm3 Tumor Vol (% inhibition vs wk 0) Wk 0

Wk 1

Wk 2

Wk 3

FR901228: 1 176 189 (0) 234 (0) 301 (0) 2 184 211 (0) 268 (0) 321 (0) 3 167 199 (0) 234 (0) 322 (0) 4 152 203 (0) 254 (0) 326 (0) 5 173 227 (0) 283 (0) 347 (0) 6 148 193 (0) 264 (0) 315 (0) Mean ⫾ SD 167 ⫾ 13 203 ⫾ 13 203 ⫾ 13 322 ⫾ 14 ad-CMV-p53: 1 164 183 (0) 145 (12) 122 (26) 2 174 189 (0) 178 (0) 103 (41) 3 156 201 (0) 167 (0) 139 (11) 4 184 199 (0) 178 (0) 128 (30) 5 149 182 (0) 173 (0) 115 (23) 6 172 169 (2) 132 (23) 89 (48) Mean ⫾ SD 167 ⫾ 12 187 ⫾ 11 162 ⫾ 18 116 ⫾ 16* FR901228 ⫹ ad-CMV-p53: 1 185 201 (0) 121 (35) 73 (61) 2 186 189 (0) 115 (38) 82 (56) 3 164 174 (0) 107 (35) 59 (64) 4 178 190 (0) 108 (39) 66 (63) 5 156 184 (0) 128 (18) 89 (43) 6 182 188 (0) 113 (38) 71 (61) Mean ⫾ SD 175 ⫾ 12 187 ⫾ 8 115 ⫾ 7* 73 ⫾ 10* Athymic mice were inoculated subcutaneously with 1 ⫻ 106 cells mixed with 100 ␮l Matrigel subcutaneously in flanks at ages 6 to 8 weeks and tumor size was determined using formula, length ⫻ width ⫻ height ⫻ 0.5236. * Significantly different vs week 1 control (p ⬍0.01).

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FR901228 ENHANCES ADENOVIRAL INFECTION OF UROTHELIAL CANCER CELLS

lated by histone acetylation but not by DNA methylation in bladder and prostate cancer cells. We noted that changes in the chromatin structure of the CAR promoter enhance CAR expression in these cancer cells. The human CAR gene is highly inducible in cancer cells and the promoter sequence identified can be used as a screening system for finding other potential inducers. CONCUSIONS

Increased CAR expression using noncytotoxic doses of the HDAC inhibitor FR901228 in target cells could decrease the dose of virus and further enhance the therapeutic efficacy of gene therapy. In addition, these data could provide a good rationale for the evaluation of the HDAC inhibitor FR901228 as a neoadjuvant to adenoviral gene therapy. REFERENCES

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9. Okegawa, T., Pong, R. C., Li, Y., Bergelson, J. M., Sagalowsky, A. I. and Hsieh, J. T.: The mechanism of growth-inhibitory effect of coxsackie and adenovirus receptor (CAR) on human bladder cancer: a functional analysis of CAR protein structure. Cancer Res, 61: 6592, 2001 10. Kitazono, M., Goldsmith, M. E., Aikou, T., Bates, S. and Fojo, T.: Enhanced adenovirus transgene in malignant cells treated with the histone deacetylase inhibitor FR901228. Cancer Res, 61: 6328, 2001 11. Kitazono, M., Rao, V. K., Robey, R., Aikou, T., Bates, S., Fojo, T. et al: Histone deacetylase inhibitor FR901228 enhances adenovirus infection of hematopoietic cells. Blood, 99: 2248, 2002 12. Lee, C. T., Seol, J. Y., Park, K. H., Yoo, C. G., Kim, Y. W., Ahn, C. et al: Differential effects of adenovirus-p16 on bladder cancer cell lines can be overcome by the addition of butyrate. Clin Cancer Res, 7: 210, 2001 13. Hemminki, A., Kanerva, A., Liu, B., Wang, M., Alvarez, R. D., Siegal, G. P. et al: Modulation of coxsackie-adenovirus receptor expression for increased adenoviral transgene expression. Cancer Res, 63: 847, 2003 14. Goldsmith, M. E., Kitazono, M., Fok, P., Aikou, T., Bates, S. and Fojo, T.: The histone deacetylase inhibitor FK228 preferentially enhances adenovirus transgene expression in malignant cells. Clin Cancer Res, 9: 5394, 2003 15. Taura, K., Yamamoto, Y., Nakajima, A., Hata, K., Uchinami, H., Yonezawa, K. et al: Impact of novel histone deacetylase inhibitors, CHAP31 and FR901228 (FK228), on adenovirusmediated transgene expression. J Gene Med, 6: 526, 2004 16. Sachs, M. D., Ramamurthy, M., Poel, H., Wickham, T. J., Lamfers, M., Gerritsen, W. et al: Histone deacetylase inhibitors upregulate expression of the coxsackie adenovirus receptor (CAR) preferentially in bladder cancer cells. Cancer Gene Ther, 11: 477, 2004 17. Okegawa, T., Li, Y., Pong, R. C., Bergelson, J. M., Zhou, J. and Hsieh, J. T.: The dual impact of coxsackie and adenovirus receptor expression on human prostate cancer gene therapy. Cancer Res, 60: 5031, 2000 18. Camps, J. L., Chang, S. M., Hsu, T. C., Freeman, M. R., Hong, S. J., Zau, H. E. et al: Fibroblast-mediated acceleration of human epithelial tumor growth in vivo. Proc Natl Acad Sci USA, 87: 75, 1990 19. Johnstone, R. W.: Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov, 1: 287, 2002 20. Pong, R. C., Lai, Y. J., Chen, H., Okegawa, T., Frenkel, E., Sagalowsky, A. et al: Epigenetic regulation of coxsackie and adenovirus receptor (CAR) gene promoter in urogenital cancer cells. Cancer Res, 63: 8680, 2003