Affiliations of authors: J. C. Cheng, F. A. Gonzales, P. A. Jones, University of Southern California (USC)/Norris Comprehensive Cancer Center and Hospital, Departments of Biochemistry and Molecular Biology and Urology, USC Keck School of Medicine, Los Angeles; C. B. Matsen, E. U. Selker, Institute of Molecular Biology, University of Oregon, Eugene; W. Ye, USC/Norris Comprehensive Cancer Center and Hospital, Department of Preventive Medicine, and USC Keck School of Medicine, Los Angeles; S. Greer, Department of Microbiology and Immunology, Biochemistry and Molecular Biology and Radiation Oncology, University of Miami School of Medicine, Sylvester Cancer Center, Miami, FL; V. E. Marquez, Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD.
Correspondence to: Peter A. Jones, Ph.D., D.Sc., University of Southern California (USC)/Norris Comprehensive Cancer Center and Hospital, Departments of Biochemistry and Molecular Biology and Urology, USC Keck School of Medicine, 1441 Eastlake Ave., Los Angeles, CA 90089-9181 (e-mail: jones_p{at}ccnt.hsc.usc.edu).
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ABSTRACT |
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INTRODUCTION |
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The most well characterized and widely used drugs to inhibit DNA cytosine methylation and reactivate silenced genes are several nucleoside analogs, including 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR) (12,13), and several non-nucleoside drugs, including procainamide (14,15). Both 5-Aza-CR and 5-Aza-CdR have a nitrogen in place of a carbon at position 5 of the pyrimidine ring (Fig. 1). These nucleoside analogs were originally developed as cancer chemotherapeutic agents (16) and are powerful inducers of genes silenced by DNA methylation (17). Although 5-Aza-CR and 5-Aza-CdR are both being tested in international clinical trials, especially for the treatment of acute myeloid leukemia and myelodysplastic syndrome (1820), their instability in neutral solution has complicated their clinical use. 5-Aza-CR has a short half-life [approximately 90 minutes at 50 °C in phosphate-buffered saline (PBS) at pH 7.4 (21)] and is unstable in neutral aqueous solutions, and the hydrolysis products have been well characterized (22). This chemical instability encouraged the development of other analogs, such as 5,6-dihydro-5-azacytidine (23) and pseudoisocytidine (24), which possess more stable ring systems but have not been clinically useful. 5-Fluoro-2'-deoxycytidine also inhibits DNA methylation and reactivates silenced genes when incorporated into DNA (12) but generates 5-fluorodeoxyuridine and its metabolites, which may be toxic (25). Thus, there is still a need for an effective, stable, and minimally toxic inhibitor of DNA methylation.
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MATERIALS AND METHODS |
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N. crassa strains N644 (matA am132 [(am/hph/am)ec42 pJI2]RIP77 inl), N242 (al-2 matA), and N613 (al-2 matA; dim-2) were grown in liquid or solidified medium using standard procedures, as previously described (34). Approximately 2000 conidia (asexual spores) of the indicated strains, grown on minimal medium supplemented with alanine, were used for each plate. Drugs (trichostatin A [TSA], 5-Aza-CR, and zebularine) in varying concentrations were administered from a 4-mm diameter no. 1 paper disk (Whatman International Ltd., Maidstone, U.K.) placed in the middle of each plate shortly after plating the conidia. To test for resistance to hygromycin, 5 mg of hygromycin B (Calbiochem, San Diego, CA) was added in 5 mL of solidified medium after the plate was incubated 24 hours at 32 °C. The plates were then incubated for 2 days at 32 °C and photographed. To test the effect of TSA, 5-Aza-CR, or zebularine on DNA methylation, DNA was isolated from cultures grown to saturation (23 days) in the continuous presence of the drugs and processed as described (35). TSA (Wako Chemicals USA, Inc., Richmond, VA) was dissolved in dimethyl sulfoxide (DMSO), and 5-Aza-CR (Sigma Chemical Co., St. Louis, MO) and zebularine, synthesized as previously described (35), were dissolved in PBS.
Mammalian Cell Lines and Drug Treatments
All cultures were grown in a humidified incubator at 37 °C in 5% CO2. Stock cultures of the mouse embryonic cell line C3H 10T1/2 Cl8 (10T1/2) (American Type Culture Collection, Manassas, VA) between passages 7 and 15 were grown in 75-cm2 plastic flasks (BD Biosciences Discovery Labware, Billerica, MA) in Eagles basal medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco/Life Technologies, Inc., Palo Alto, CA).
The T24 human bladder carcinoma cell line was obtained from the American Type Culture Collection, and the EJ6 bladder carcinoma cell line was provided by Dr. Eric J. Stanbridge (University of California at Irvine). Both cell lines were cultured in McCoys 5A medium supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin.
To observe the myogenic phenotype, we plated 10T1/2 cells (2500 cells/60-mm dish) and treated the cells 24 hours later with the various concentrations of 5-Aza-CdR, 5-Aza-CR, or zebularine. Control cells were left untreated. The medium was changed 24 hours after the initial drug treatment and every 3 days thereafter until the myogenic phenotype was observed, which was 910 days after initial drug treatment. For methylation analysis, 10T1/2 cells and T24 cells (3 x 105 cells/100-mm dish) were plated and treated 24 hours later with 5-Aza-CdR, 5-Aza-CR, or zebularine at the indicated concentrations. Control cells were not treated with any drugs. For 10T1/2 cells, the medium was changed 24 hours after the initial drug treatment, whereas for T24 cells, the medium was changed 24 hours or 48 hours after the initial drug treatment, as indicated. DNA and RNA were harvested from 10T1/2 cells 72 hours after initial drug treatment and from T24 cells 96 hours after initial drug treatment. The methylation status of the indicated DNA regions was measured in two separate and independent experiments, both of which were done in duplicate.
Nucleic Acid Isolation
DNA was isolated from N. crassa as previously described (36), and DNA and RNA from cultured cells and tumor cells were isolated using the NucleoBond RNA/DNA Midi Kit (Clontech Laboratories, Inc., Palo Alto, CA), according to the manufacturers recommended protocol.
Southern Blot Analysis
Liquid cultures of N. crassa strain N644 inoculated at a concentration of 7 x 104 conidia/mL were grown in the absence or presence of TSA (0.33 and 3.3 µM), 5-Aza-CR (12 and 24 µM), or zebularine (20310 µM) for 4 days at 32 °C with shaking. Because of the instability of 5-Aza-CR, the indicated doses of this drug were added at the beginning of the experiment and after 1 and 2 days of growth. DNA was isolated, and Southern hybridizations were performed on 1-µg DNA samples that had been digested with DpnII or Sau3AI, fractionated by gel electrophoresis, transferred to a nylon membrane, and probed for amRIP or 63 sequences. A control hybridization indicated that the digestions were complete (data not shown).
Reverse TranscriptionPolymerase Chain Reaction Analysis
Reverse transcriptionpolymerase chain reaction (RTPCR) analysis was used to detect p16 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. The protocol, including the primer sequences, probe sequences, and PCR conditions, has been previously described (10).
Quantification of DNA Methylation by Methylation-Sensitive Single Nucleotide Primer Extension
The average methylation at defined CpG sites was quantified using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay as described (37). Briefly, genomic DNA (4 µg) was digested overnight with 4 units of EcoRI or with 4 units of RsaI to cut outside the p16 promoter or the endogenous retroviral sequence class II-d (CII-d), respectively. After digestion, the DNA was denatured for 20 minutes at 95 °C and then treated with 3 M NaOH for 20 minutes at 45 °C. This solution was then treated with 3.6 M sodium bisulfite and 0.1 M hydroquinone for 16 hours at 55 °C in the dark. Treatment of DNA with bisulfite converts unmethylated cytosine residues to uracil, which is then converted to thymine after the primary bisulfite-specific PCR, leaving methylated cytosines unchanged. Bisulfite-converted DNA was purified with the Wizard Plus Minipreps DNA Purification System (Promega Corp., Madison, WI), desulfonated by the addition of 3 M NaOH for 15 minutes at 40 °C, and ethanol precipitated. The sequences of the primers used for bisulfite-treated DNA PCR amplification were as follows: p16 promoter sense, 5'-GTAGGTGGGGAGGAGTTTAG TT-3', and antisense, 5'-TCTAATAACCAACCAACCCCTC CT-3'; CII-d sense, 5'-GTTTATAGGTTTAGAGGTTTT-3', and antisense, 5'-AACACATAAACCTATTTTAAACTTA-3'.
The PCR conditions were as follows: for the p16 promoter, there was an initial denaturation step at 94 °C for 3 minutes, followed by 40 cycles of amplification, with each cycle consisting of 94 °C for 45 seconds, 67 °C for 45 seconds, and 72 °C for 45 seconds, and a final extension step at 72 °C for 10 minutes. For CII-d, there was an initial denaturation step at 95 °C for 2 minutes, followed by 40 cycles of amplification, with each cycle consisting of 95 °C for 1 minute, 50 °C for 50 seconds, and 72 °C for 1 minute, and a final extension at 72 °C for 10 minutes.
The PCR products (templates) were separated by electrophoresis through a 2% agarose gel and purified from the gel using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Each template was eluted in 30 µL of H2O. Duplicates of 4 µL of template were then added to 6 µL of a reaction mixture consisting of 1x PCR buffer, 1 µM SNuPE primers, 1 µCi [32P]dCTP or [32P]dTTP, and 1 unit 1 : 1 Taq/TaqStart antibody (Clontech Laboratories). The single nucleotide extensions involved sequentially incubating the mixture at 95 °C for 1 minute, 50 °C for 2 minutes, and finally 72 °C for 1 minute (p16 promoter), or 95 °C for 1 minute, 46 °C for 30 seconds, and finally 72 °C for 20 seconds (CII-d). The sequences for SNuPE primers were as follows: for p16 promoter, 5'-TTTGAGGGATAGGGT-3', 5'-TTTTAGGGGTGTTATATT-3', and 5'-TTTTTTTGTTTG GAAAGATAT-3'; for CII-d, 5'-GGTATAGTTTGAGTAT-3', 5'-TTTTATTTATTGTTATTATGG-3', and 5'-TATTTTTTA ATAGTATTATTTTTTAT-3'.
The reaction mixtures were combined with 4 µL of stop solution (95% formamide, 20 mM EDTA [pH 8.0], 0.05% bromophenol blue, and 0.05% xylene cyanol) before being denatured at 95 °C for 5 minutes and loaded onto a 15% denaturing polyacrylamide gel (1x TBE [TrisBorateEDTA] buffer, 14.25% acrylamide, 0.75% bis-acrylamide, 7 M urea). Methylation levels were quantified on a PhosphoImager analysis system (Molecular Dynamics, Sunnyvale, CA). The reported methylation status of the p16 promoter and CII-d represents the average methylation levels of three CpG sites from duplicate samples from two independent experiments. Methylation levels, defined as the percentage of DNA that is methylated, were measured by Ms-SNuPE as described previously (37).
In Vivo Experiments
EJ6 cells (5 x 105/injection) suspended in PBS were inoculated subcutaneously into the right and left flanks (along the midaxillary lines) of 4- to 6-week-old male BALB/c nu/nu mice (Harlan, San Diego, CA). Mice (n = 30) were randomly divided into six groups (intraperitoneal control, oral control, intraperitoneal zebularine at 500 mg/kg, oral zebularine at 500 mg/kg, intraperitoneal zebularine at 1000 mg/kg, and oral zebularine at 1000 mg/kg). Each group consisted of five mice (at least six tumors per group; one or two mice per group were randomly killed at earlier time points to establish a time course of expression). After 23 weeks and after macroscopic tumors (50200 mm3) had formed, zebularine or control treatments were initiated. Zebularine, at doses of 500 mg/kg or 1000 mg/kg, was administered daily by intraperitoneal injection or oral gavage in a solution of 0.45% saline over a period of 18 days. Two control groups were used, one mock-treated with 0.45% saline administered by intraperitoneal injection, and the other group mock-treated with 0.45% saline administered by oral gavage over the same period of 18 days. Tumors were measured with calipers, and tumor volumes (TVs) were calculated with the following formula: TV = LD2/2 (where L is the longest diameter and D is the shortest diameter). The fold differences in tumor growth among the various mice groups were calculated using relative TVs (RTVs), which are calculated as follows: RTV = TVn/TV0, where TVn is the tumor volume in mm3 at a given day n and TV0 is the tumor volume in mm3 at day 0 (initial treatment).
All mice were killed 24 hours after the last treatment. At this time, tumors were removed and each tumor was divided into two separate portions. One portion was immediately fixed with neutral buffered formalin, embedded in OCT compound, frozen, and then sectioned. The frozen sections were stained with hematoxylin and eosin. All histologic examinations were carried out by light microscopy using a Leica DM LB microscope (Leica Microsystems, Inc., Bannockburn, IL). The other portion of each tumor was used for isolating DNA and total RNA for analysis of the methylation status of p16 promoter by Ms-SNuPE and of gene expression by RTPCR, respectively. All experimental protocols were approved by the Institutional Animal Care and Use Committee, in compliance with the Guide for the Care and Use of Laboratory Animals, University of Southern California.
Determination of Cytotoxicity
Cytotoxicity was assessed using a colony formation assay. Both 10T1/2 cells (250 cells/60-mm dish) and T24 cells (100 cells/60-mm dish) were plated in triplicate and treated after 24 hours with 5-Aza-CdR, 5-Aza-CR, or zebularine at the indicated concentrations. Control plates were not treated with any drugs. After cell colonies were visible (1214 days after treatment), cells were fixed in 100% methanol and stained with 10% Giemsa stain. The total number of colonies was counted, and the percentage of average plating efficiency was determined by dividing the mean colony number on the treated plates or the control plates by the number of cells seeded in the dishes times 100. In this cytotoxicity assay, a decrease in the size of the colonies, as determined by microscopy, is an indication of growth inhibition, and a decrease in the number of the colonies is an indication of cytotoxicity. Two separate and independent experiments were done.
Statistical Analysis
For 10T1/2 cells and T24 cells, analyses of variance (ANOVAs) were performed to test differences in DNA methylation and average plating efficiency among the treatment groups, using experiment-to-experiment variability as the error term. The least significant difference (LSD) method was used for the pair-wise comparisons in contexts where the overall F test was statistically significant at the .05 level (38). The P values from the overall F test of the treatment effect were .001 for DNA methylation, <.001 for average plating efficiency in 10T1/2 cells (Table 1), and <.001 for both DNA methylation and average plating efficiency in T24 cells. The Pearson correlation coefficient was calculated to test the association between p16 promoter methylation status and p16/GAPDH ratio for T24 cells. For the in vivo experiment, tumor volumes of mice were determined for two tumors from each mouse. Prior to analysis, the logarithms of the ratios of tumor volumes at day 18, day 14, day 8, and day 4 divided by the tumor volume at day 0 were determined. For each mouse, the rate of the change in log ratio by day (slope) was estimated using a linear regression model, with tumor and day as the covariates. A lack-of-fit test and visual inspection of the data indicated that the linear pattern fit very well for animals receiving the control and 1000 mg/kg zebularine, and reasonably well for the mice receiving 500 mg/kg zebularine. Then, ANOVA was done using the calculated slope as the dependant variable to investigate the effects of route of administration and dose of zebularine and their interaction on the daily change in (log) ratio of tumor volume (relative to day 0). Pair-wise comparison of the slopes was performed using the LSD method, in cases where the overall F test was statistically significant at the .05 level (37).
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The ratio of p16/GAPDH mRNA expression in EJ6 tumors in vivo was measured for each mouse in the six groups as described above. The methylation status of p16 promoter was measured only for mice in the control oral, control intraperitoneal, 1000 mg/kg oral, and 1000 mg/kg intraperitoneal groups, for four random tumors from three mice in each group. ANOVA was performed to test the effects of route of administration and dose of zebularine and their interaction with the p16/GAPDH ratio and methylation status. In the analyses, a mouse was nested within the treatment group, and for the methylation status, a tumor was further nested within the mouse. The mouse-to-mouse variability was used as the error term. The LSD method was used for the pair-wise comparisons in cases where the overall F test was statistically significant at the .05 level (38).
All reported P values were two-sided. All confidence intervals (CIs) were calculated using the pooled estimates of the experiment-to-experiment or mouse-to-mouse variability as calculated by the ANOVA. The SAS software package (version 8.12; SAS Institute, Inc., Cary, NC) was used for all statistical analyses.
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RESULTS |
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To elucidate the mechanism of action of zebularine, we took advantage of an artificial locus in N. crassa that includes a copy of the bacterial hygromycin resistance gene hph that had been silenced by DNA methylation as a result of the operation of repeat-induced point mutation on flanking duplicate copies of the Neurospora am gene (33). We previously used a strain (N644) bearing this construct to demonstrate that the histone deacetylase inhibitor TSA causes selective loss of DNA methylation in Neurospora (34). In the present study, we applied zebularine to a paper disc in the middle of a plate containing approximately 2000 N644 conidia and challenged the cells to grow in the presence of hygromycin. A dilution series showed that as little as 2.5 nmol of zebularine applied to the center of the plate was sufficient to permit growth of nearby cells, whereas 20 nmol of zebularine permitted growth that was similar to that observed in the presence of 5-Aza-CR (Fig. 2, A). Some inhibition of growth near the site of application was observed at higher doses of zebularine (Fig. 2
, A). This observation suggested that zebularine, like the known demethylating agent 5-Aza-CR, had reactivated the silenced hph gene.
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To verify that reactivation of hph by zebularine resulted from the inhibition of DNA methylation, we used methylation-sensitive restriction endonucleases and Southern hybridization to assess methylation in representative methylated chromosomal regions as well as in the overall genome. Liquid cultures of strain N644 were grown in the presence or absence of zebularine, TSA, or 5-Aza-CR. We then examined methylation in the 63 region, which is known to be affected by 5-Aza-CR but not by TSA, and in the am sequences flanking hph, which are known to be affected by both drugs (34). Digestions with the DpnII and Sau3AI endonucleases, which both recognize GATC but differ in that Sau3AI digests only sites where the cytosine is unmethylated, revealed that zebularine treatment reduced methylation dramatically in both the amRIP and
63 regions (Fig. 2
, B). Substantial loss of methylation was observed with the lowest concentration tested (20 µM), and cultures grown with 78310 µM zebularine showed virtually no DNA methylation. This suggested that zebularine was acting as a general methylation inhibitor, similar to 5-Aza-CR, a conclusion that was supported by inspection of the total genomic DNA digested with DpnII or Sau3AI and stained with ethidium bromide. The fragments produced by DpnII or Sau3AI digestions appeared equivalent among DNA samples from cultures grown in the presence of either 5-Aza-CR or zebularine, but differed among DNA samples from cultures grown in the presence of TSA or in the absence of drugs (data not shown). Thus, zebularine is a global inhibitor of DNA methylation, similar to 5-Aza-CR, rather than a selective inhibitor, such as TSA, which inhibits histone deacetylase (34,39).
Induction of the Myogenic Phenotype and Inhibition of DNA Methylation in 10T1/2 Mouse Embryo Cells
We next determined whether zebularine could inhibit DNA methylation in mammalian cells. Cytidine analogs with modifications in the 5 position of the ring are powerful inhibitors of DNA methylation and can induce the formation of striated muscle cells in the non-myogenic 10T1/2 mouse embryo cell line (12,21). We tested the ability of zebularine to induce 10T1/2 cells to undergo the myogenic switch (Fig. 3). Control 10T1/2 cells formed flat, even monolayers and appeared epithelioid (Fig. 3
, A), whereas 10T1/2 cells treated with 5-Aza-CdR (Fig. 3
, B) or with 5-Aza-CR (Fig. 3
, C) formed multinucleated myotubes approximately 910 days after initial drug treatment. We found that zebularine also induced muscle cell formation in 10T1/2 cells (Fig. 3
, D). The extent of muscle cell formation in cultures treated with zebularine was less than that induced by either 5-Aza-CdR or 5-Aza-CR (Table 1
).
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Two factors that limit the clinical potential of 5-Aza-CdR are its instability and its toxicity. We therefore compared the cytotoxicity of zebularine, 5-Aza-CdR, and 5-Aza-CR by measuring their effects on 10T1/2 cell-plating efficiency (Table 1). A dose of 0.3 µM 5-Aza-CdR reduced plating efficiency from a control level of 22.3% (95% CI = 19.7% to 24.8%) to 5.0% (95% CI = 2.4% to 7.6%; P<.001) and was considerably more cytotoxic than either 3 µM 5-Aza-CR (plating efficiency = 15.5%, 95% CI = 12.9% to 18.1%; P<.001) or 30 µM zebularine (plating efficiency = 18.3%, 95% CI = 15.7% to 20.8%; P<.001).
Induction of p16 Gene Expression and Inhibition of DNA Methylation in T24 Human Bladder Carcinoma Cells
We next tested whether zebularine treatment could induce expression of a human tumor suppressor gene that had been silenced by methylation. The T24 human bladder carcinoma-derived cell line contains a transcriptionally silent, hypermethylated p16 gene promoter (10,42) that can be demethylated and reactivated by 5-Aza-CdR, a deoxyribonucleoside analog (28,42). We examined whether 5-Aza-CR and zebularine, which are ribonucleoside analogs, could also induce p16 expression in T24 cells (Fig. 4, A). Untreated control cells showed no p16 expression, whereas cells treated with 3 µM 5-Aza-CdR showed robust expression of p16. Both 5-Aza-CR and zebularine successfully induced p16 expression in a dose-dependent manner, although not as effectively as 5-Aza-CdR. It is interesting to note that zebularine showed time-dependent induction of p16 expression, in that 24 hours of treatment with 300 µM zebularine was insufficient to induce expression but 48 hours of treatment with 300 µM zebularine was. 5-Aza-CR, 5-Aza-CdR, and zebularine also induced similar levels of p16 protein, as confirmed by western blot analysis (data not shown). Thus, our findings suggest that zebularine inhibited DNA methylation in T24 cells in vitro.
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The cytotoxicity of 5-Aza-CdR, 5-Aza-CR, and zebularine in T24 cells was assessed by measuring the average plating efficiency. A dose of 3 µM 5-Aza-CdR decreased plating efficiency by 55.7% (95% CI = 51.5% to 59.8%). 5-Aza-CR decreased plating efficiency in a dose-dependent manner, with 100 µM producing the highest cytotoxicity (39.7% decrease, 95% CI = 35.5% to 43.8%). The increased cytotoxicity associated with increasing doses of zebularine was greater than the increased cytotoxicity associated with increasing the time of drug treatment. The most cytotoxic treatment regimen observed in T24 cells was 1 mM zebularine for 48 hours, which resulted in a decrease in plating efficiency of 17.0% (95% CI = 12.8% to 21.2%). Zebularine was clearly less toxic than 5-Aza-CR in T24 cells at levels that induced similar levels of p16 promoter demethylation (Fig. 4, B). Both agents decreased methylation by approximately 27%29% at concentrations of either 100 µM 5-Aza-CR or 1 mM zebularine for 48 hours; however, the plating efficiency for cells treated with 5-Aza-CR was lower than that of zebularine (34.3%, 95% CI = 31.4% to 37.3% versus 57.0%, 95% CI = 54.1% to 59.9%, respectively; P<.001). Thus, zebularine was minimally cytotoxic in T24 cells, even at high concentrations.
Effects of Zebularine on Human Bladder Carcinoma Cells In Vivo
Because zebularine inhibited DNA methylation in N. crassa and cultured animal cells, we next examined the ability of the drug to reactivate silenced mammalian genes in vivo. We used a tumorigenic derivative of T24 cells, EJ6 cells, which also have a methylated p16 gene promoter. EJ6 bladder cells were inoculated subcutaneously into the right and left flanks of male BALB/c nu/nu mice at 46 weeks of age. When macroscopic tumors were evident (approximately 23 weeks later), the mice were treated with zebularine at concentrations of 500 mg/kg or 1000 mg/kg, administered either by intraperitoneal injection or oral gavage. We decided to examine oral gavage as a possible route of administration, because zebularine is very stable and has a half-life of approximately 44 hours at 37 °C in PBS at pH 1.0 and approximately 508 hours at pH 7.4 (data not shown). Frozen sections of resected tumors from control mice and mice treated with zebularine were stained with hematoxylin and eosin to observe for any morphologic changes. Tumors from control mice had a high ratio of tumor cells to stroma, whereas tumors from mice treated with either 1000 mg/kg orally or 1000 mg/kg intraperitoneally had a much lower ratio of tumor cells to stroma (Fig. 5, A). Moreover, tumor growth appeared inhibited in all treated groups compared with tumor growth in the control group. Compared with the growth of tumors from the control mice, tumor growth was statistically significantly reduced in the groups treated either with 1000 mg/kg orally (P<.001) or intraperitoneally (P<.001) (Fig. 5
, B). The mean fractions of daily change in tumor size were 0.97 (95% CI = 0.96 to 0.99), 0.99 (95% CI = 0.97 to 1.01), and 1.06 (95% CI = 1.04 to 1.08) for 1000 mg/kg intraperitoneal, 1000 mg/kg oral, and the control, respectively. Weight loss was minimal in all control and treated groups. The average maximum weight loss observed in the group treated with 1000 mg/kg zebularine by intraperitoneal administration was approximately 11% (95% CI = 4% to 19%) (Fig. 5
, C), suggesting that zebularine was minimally toxic in these mice. In addition, none of the mice died from any treatment during the course of the experiment (data not shown).
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DISCUSSION |
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The mechanism of action of zebularine as a DNA methylation inhibitor presumably requires incorporation into DNA after phosphorylation of zebularine to the diphosphate level and conversion to a deoxynucleotide. After the conversion to the deoxy-zebularine triphosphate and subsequent incorporation into DNA in place of a cytosine base, the 2-(1H)-pyrimidinone would most likely base pair with guanine, resulting in two WatsonCrick hydrogen bonds instead of three. It is unlikely that zebularine would be incorporated in place of thymine, because this would lead to a single WatsonCrick hydrogen bond, which is very weak and unstable. Replacing cytosine by 2-(1H)-pyrimidinone would result in the formation of a tight complex that can lead to potent inhibition of DNA methylation, as reported by Hurd et al. (32). In fact, this structural formation was recently demonstrated by Zhou et al. (43) in a crystallized complex showing that the 2-(1H)-pyrimidinone ring can be flipped out of the helix to form a covalent bond with the active cysteine group in the DNA methyltransferase. Furthermore, our preliminary experiments have shown a marked decrease in the level of DNA methyltransferase 1 protein (44) in cells treated with zebularine (data not shown). The novelty of our studies is that we demonstrate that zebularine can act as a pro-drug leading to inhibition of DNA methylation and gene activation in fungal and mammalian cells.
Although the potency of zebularine was similar to that of 5-Aza-CR in N. crassa, it was less effective than either 5-Aza-CR or 5-Aza-CdR in the animal cell in vitro systems. Higher doses of zebularine were required for the induction of p16 gene expression and demethylation of its promoter sequence. Because the strength of the covalent complex between DNA methyltransferase and modified DNA containing 5-Aza-CdR (45) or 2-(1H)-pyrimidinone (32) would be expected to be comparable, differences in transport or metabolic activation of zebularine and 5-Aza-CR in mammalian cells could account for the differences in potencies between the two drugs. 5-Aza-CdR (or 5-Aza-CR) must be phosphorylated to its nucleotide form by deoxycytidine kinase (or uridinecytidine kinase) and subsequently incorporated into replicating DNA to inhibit DNA methyltransferase (40,4649). Presumably, zebularine is metabolized in the same way, but perhaps the uridinecytidine kinase has a lower binding affinity for zebularine than for 5-Aza-CR, resulting in less activation and incorporation of zebularine into DNA, thus necessitating higher dosages of the drug.
Because 5-Aza-CdR is active at one-tenth the concentration of 5-Aza-CR (12), we investigated the possibility that the deoxyribose form of zebularine, 2'-deoxyzebularine, would be a more effective demethylating agent than its corresponding ribonucleoside form. We found that 2'-deoxyzebularine neither induced the myogenic phenotype nor inhibited DNA methylation of the CII-d locus in 10T1/2 cells or the hph locus in Neurospora (data not shown). Its inactivity in mammalian cells may be a reflection of different specificities or binding affinities of the enzymes for their corresponding substrates. Perhaps deoxycytidine kinase, the most likely enzyme to phosphorylate 2'-deoxyzebularine, does not recognize or bind well to this compound because it lacks the 4-amino group on the pyrimidine ring. Zebularine is probably phosphorylated by uridinecytidine kinase, which may be insensitive to the missing 4-amino group. Because only 2'-deoxyzebularine is expected to be incorporated into DNA, the conversion of zebularine-5'-diphosphate to 2'-deoxyzebularine-5'-diphosphate by ribonucleotide reductase could be an additional rate-limiting step. The metabolic activation of zebularine to 2'-deoxyzebularine-5'-triphosphate is currently under investigation. In addition, cytidine and uridine are known to be strong competitive inhibitors of 5-Aza-CR phosphorylation (50); it is therefore possible that both cytidine and uri-dine can competitively inhibit the phosphorylation of zebularine to a greater degree than that of 5-Aza-CR, thus accounting for the 10-fold difference in potency in animal cells. Other possibilities are that cytidine deaminase may sequester zebularine to a certain extent, thus lowering the effective concentration of the drug, or that cytidine deaminase inhibition may lead to increased levels of deoxycytidine and cytidine levels, thus affecting the anabolism of zebularine. It is also important to consider that zebularine is probably incorporated into RNA as well as into DNA. Indeed, we found that the growth inhibitory effects of zebularine on N. crassa do not depend on the dim-2 DNA methyltransferase (data not shown), which is the only DNA methyltransferase active in vegetative tissues of N. crassa (51). Perhaps the toxicity of zebularine reflects incorporation into RNA.
The finding that zebularine was able to induce p16 expression in a time-dependent manner suggests that this drug may be usefully administered over extended periods. The instability and toxicity associated with 5-Aza-CR and 5-Aza-CdR were shown in a previous study (52), which demonstrated that the administration of T24 cells with 5-Aza-CdR was limited to a single 24-hour treatment. The stability of zebularine in both acidic and neutral solutions is most likely responsible for its more extended inhibitory effect on methylation of the p16 promoter region. This finding suggests new possibilities for the application of zebularine in future in vitro and in vivo studies.
Our studies with BALB/c nu/nu mice demonstrate that silenced genes can be reactivated by zebularine in vivo, as shown previously with 5-Aza-CdR (42). However, what is unique and exciting about zebularine is that this is the first time a methylation inhibitor has been shown to exhibit an in vivo antitumor effect via oral administration. This observation further supports the notion that the drug is acid-stable. Additionally, the apparent decreased tumorigenicity of zebularine-treated EJ6 cells and minimal weight loss observed in these zebularine-treated mice further support the clinical potential of zebularine. These findings raise the possibility that this drug, or a related DNA methylation inhibitor with minimal toxicity, may be clinically useful to reverse aberrant DNA methylation, restoring critical gene function in vivo and thereby treating certain cancers.
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We thank Carvell Nguyen for helpful comments on the manuscript and Mihaela Velicescu, Dan Weisenberger, and Louis Dubeau for their assistance and valuable discussions. We also thank Jiapeng Huang for his help with the microscopic slides.
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Manuscript received July 29, 2002; revised December 3, 2002; accepted January 7, 2003.
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