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Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells
J.-T. Hsieh / Urologic Oncology: Seminars and Original Investigations 24 (2006) 553–567
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Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells. Ben-Kasus T, Ben-Zvi Z, Marquez VE, Kelley JA, Agbaria R, Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Biochem Pharmacol 2005;70:121–33 Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5=-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2=deoxycytidine and requires higher doses for equivalent inhibition of DNMT. Commentary Because DNA hypermethylation is very prevalent in bladder cancer, targeting DNA hypermethylation in cancer becomes a new avenue of cancer therapy. 5-Aza-cytidine is a well-known DNA hypomethylation agent that can inhibit DNA methyltransferase activity and result in gene reactivation. Although 5-aza-cytidine is a very potent agent in vitro, it is not active in vivo because of its chemical instability. From recent studies, Zebularine appears to be a promising agent for clinical application because of oral availability and chemical stability. However, Zebularine is less potent than 5-aza-cytidine in vitro because it needs to be phosphorylated and incorporated into DNA. The goal of this study is to better understand Zebularine by analyzing its metabolites. It appears that 2 major Zebularine metabolites (diphosphocholine [Zeb-DP-Chol] and diphosphoethanolamine) have higher incorporation rates into RNA than DNA, which may explain Zebularine’sweaker cytotoxic effect. This study certainly provides a road map to modify Zebularine to be a more potent agent. doi:10.1016/j.urolonc.2006.09.002 Jer-Tsong Hsieh, Ph.D.
Enhanced transgene expression in urothelial cancer gene therapy with histone deacetylase inhibitor. Okegawa T, Nutahara K, Pong RC, Higashihara E, Hsieh JT. Department of Urology, University of Kyorin, Tokyo, Japan. J Urol 2005;174:747–52 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 beta-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. Commentary Regulation of eukaryotic gene expression is the post-translational modification of nucleosomal histones that convert regions of chromosome to transcriptionally active or inactive. The most well studied post-translational modification of histones is the acetylation of
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Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells. Ben-Kasus T, Ben-Zvi Z, Marquez VE, Kelley JA, Agbaria R, Department of Clinical Pharmacology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel. Biochem Pharmacol 2005;70:121–33 Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5=-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2=deoxycytidine and requires higher doses for equivalent inhibition of DNMT. Commentary Because DNA hypermethylation is very prevalent in bladder cancer, targeting DNA hypermethylation in cancer becomes a new avenue of cancer therapy. 5-Aza-cytidine is a well-known DNA hypomethylation agent that can inhibit DNA methyltransferase activity and result in gene reactivation. Although 5-aza-cytidine is a very potent agent in vitro, it is not active in vivo because of its chemical instability. From recent studies, Zebularine appears to be a promising agent for clinical application because of oral availability and chemical stability. However, Zebularine is less potent than 5-aza-cytidine in vitro because it needs to be phosphorylated and incorporated into DNA. The goal of this study is to better understand Zebularine by analyzing its metabolites. It appears that 2 major Zebularine metabolites (diphosphocholine [Zeb-DP-Chol] and diphosphoethanolamine) have higher incorporation rates into RNA than DNA, which may explain Zebularine’sweaker cytotoxic effect. This study certainly provides a road map to modify Zebularine to be a more potent agent. doi:10.1016/j.urolonc.2006.09.002 Jer-Tsong Hsieh, Ph.D.
Enhanced transgene expression in urothelial cancer gene therapy with histone deacetylase inhibitor. Okegawa T, Nutahara K, Pong RC, Higashihara E, Hsieh JT. Department of Urology, University of Kyorin, Tokyo, Japan. J Urol 2005;174:747–52 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 beta-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. Commentary Regulation of eukaryotic gene expression is the post-translational modification of nucleosomal histones that convert regions of chromosome to transcriptionally active or inactive. The most well studied post-translational modification of histones is the acetylation of