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¹ USC/Norris Comprehensive Cancer Center, Department of Urology, Biochemistry, and Molecular Biology and ² Department of Medicine, Division of Hematology, Keck School of Medicine, University of Southern California, Los Angeles, California

Requests for reprints: Peter A. Jones, USC/Norris Comprehensive Cancer Center, Department of Urology, Biochemistry, and Molecular Biology, 1441 Eastlake Avenue, Los Angeles, CA 90089. E-mail: jones_p{at}ccnt.hsc.usc.edu

	Abstract

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DNA cytosine methylation plays a considerable role in normal development, gene regulation, and carcinogenesis. Hypermethylation of the promoters of some tumor suppressor genes and the associated silencing of these genes often occur in certain cancer types. The reversal of this process by DNA methylation inhibitors is a promising new strategy for cancer therapy. In addition to the four well-characterized nucleoside analogue methylation inhibitors, 5-azacytidine, 5-aza-2'-deoxycytidine (5-Aza-CdR), 5-fluoro-2'-deoxycytidine, and zebularine, there is a growing list of non-nucleoside inhibitors. However, a systemic study comparing these potential demethylating agents has not been done. In this study, we examined three non-nucleoside demethylating agents, (–)-epigallocatechin-3-gallate, hydralazine, and procainamide, and compared their effects and potencies with 5-Aza-CdR, the most potent DNA methylation inhibitor. We found that 5-Aza-CdR is far more effective in DNA methylation inhibition as well as in reactivating genes, compared with non-nucleoside inhibitors.

	Introduction

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The relationship between epigenetic alterations such as DNA methylation and human carcinogenesis has become increasingly evident (1–3). DNA cytosine methylation is employed in normal cells as a mechanism to silence gene expression, such as in genomic imprinting and X chromosome inactivation (4–6). During cancer development, cells can undergo abnormal hypermethylation of CpG islands in the promoters of tumor suppressor genes, which leads to the silencing of these genes (1–3, 7). Thus, reactivation of tumor suppressor genes by demethylating agents has become a potential and promising area of cancer therapy (8–11). There is a growing list of DNA methylation inhibitors in addition to 5-azacytidine and 5-aza-2'-deoxycytidine (5-Aza-CdR; ref. 12), the first demethylating agents with well-characterized mechanisms of action. The list includes, but is not limited to, 5-fluoro-2'-deoxycytidine, zebularine, antisense oligodeoxynucleotides, mitoxantrone, psammaplin A, procaine, N-acetylprocainamide, procainamide, hydralazine, and (–)-epigallocatechin-3-gallate (EGCG; refs. 11, 13–27).

Hydralazine and procainamide were first reported to have DNA methylation–inhibition properties in 1988 (15). Hydralazine is a vasodilator and is used clinically as an antihypertensive drug. It has been found to decrease the expression of DNA methyltransferases (DNMT1 and DNMT3A), and induces autoimmunity (16). Procainamide is used clinically as an antiarrhythmic, and previous studies have shown that it inhibits DNA methyltransferase activity, thus leading to DNA hypomethylation (19, 20). Recently, EGCG, the major polyphenol in green tea that has been reported to have chemopreventive activity (28, 29), has been reported to directly inhibit the DNA methyltransferase enzyme and reactivate methylation-silenced genes such as RARß and p16 (17).

Despite the identification of an increasing number of DNA methylation inhibitors, there has been no systemic study comparing the DNA-demethylating effects and potencies of these agents. In this study, we compare several potential non-nucleoside DNA methylation inhibitors—EGCG, hydralazine, and procainamide—to the nucleoside analogue methylation inhibitor 5-Aza-CdR (see Fig. 1). We found that 5-Aza-CdR is far more effective both in its DNA methylation inhibition activity and in its ability to reactivate methylation-silenced genes in cancer cells.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Comparison of chemical structures of DNA methylation inhibitors 5-Aza-CdR (A), EGCG (B), hydralazine (C), and procainamide (D).

	Materials and Methods

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Cell Treatments
Cells were seeded at 2 x 10⁵ cells per 100 mm dish 24 hours prior to treatments. Cells were treated with 1 µmol/L 5-Aza-CdR (Sigma-Aldrich Chemical Company, St. Louis, MO), 20 and 30 µmol/L of EGCG, 10 and 20 µmol/L of hydralazine (Sigma-Aldrich Chemical Company), and 100 and 200 µmol/L of procainamide (Sigma-Aldrich Chemical Company). The EGCG sample was a generous gift from Dr. Chung S. Yang (from Unilever Bestfoods; ref. 17), and a separate sample was obtained from Sigma-Aldrich Chemical Company. 5-Aza-CdR was prepared in PBS and was removed after 24 hours, whereas the other treatments were continuous. EGCG was prepared in DMSO and replaced every 2 days. Hydralazine and procainamide were prepared fresh in PBS and replaced daily with new medium. All treatment regimens have been shown to be effective in inhibiting DNA methylation in previous studies (16, 17, 21). Cells were collected after 6 days of treatment. Genomic DNA and total RNA were extracted for subsequent methylation and expression studies using standard methods.

Quantitative DNA Methylation Analysis by Methylation-Sensitive Single-Nucleotide Primer Extension
Genomic DNA was extracted from cells with the Qiagen DNeasy tissue kit (Valencia, CA). Two micrograms of each DNA sample was converted with sodium bisulfite as previously described (30), and each region of interest was amplified by PCR. The PCR conditions for MAGE-A1 were as follows: 94°C for 4 minutes, followed by 40 cycles of denaturation at 94°C for 1 minute, annealing at 53°C for 1 minute, and extension at 72°C for 1 minute, and a final extension at 72°C for 1 minute. The PCR conditions for LINE elements were as follows: 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 1 minute, annealing at 51°C, and extension at 72°C for 1 minute, and a final extension at 72°C for 10 minutes. The PCR conditions for p16 were as follows: 95°C for 3 minutes, followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 62°C for 1 minute, and extension at 72°C for 1 minute, and a final extension at 72°C for 10 minutes. The bisulfite-specific PCR primer sequences are as follows: MAGE-A1 sense, 5'-GTTTATTTTTATTTTTATTTAGGTAGGATT-3', MAGE-A1 antisense, 5'-TTACCTCCTCACAAAACCTAAA-3'; LINE sense, 5'-TTTTTTGAGTTAGGTGTGGG-3', LINE antisense, 5'-CATCTCACTAAAAAATACCAAACAA-3'; p16 sense, 5'-GTAGGTGGGGAGGAGTTTAGTT-3', p16 antisense, 5'-TCTAATAACCAACCAACCCCTCCT-3'. The methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) conditions for MAGE-A1 and p16 were as follows: 95°C for 2 minutes, 50°C for 2 minutes, and 72°C for 1 minute. The Ms-SNuPE conditions for LINE elements were as follows: 95°C for 1 minute, 50°C for 1 minute, and 72°C for 1 minute. The MAGE-A1 SNuPE primers are as follows: 5'-TTTTATTTTTATTTAGGTAGGATT-3', 5'-TGGGGTAGAGAGAAG-3', and 5'-AGGTTTTTATTTTGAGGGA-3'. The LINE SNuPE primers are as follows: 5'-GGGTGGGAGTGATT-3', 5'-GAAAGGGAATTTTTTGATTTTTTG-3', and 5'-TTTTTTAGGTGAGGTAATGTTT-3'. The p16 SNuPE primers are as follows: 5'-TTTTAGGGGTGTTATATT-3', 5'-TTTTTTTGTTTGGAAAGATAT-3', and 5'-TTTGAGGGATAGGGT-3'.

The PCR amplicons were extracted with the Qiagen gel extraction kit, and Ms-SNuPE analysis was done to examine the methylation level changes as previously described (31).

Pyrosequencing
Bisulfite-converted DNA was used for pyrosequencing analysis as previously described (32). Pyrosequencing was done for LINE elements, Alu elements, and MAGE-A1 gene. The primers used are listed as follows: LINE elements sense, 5'-TTTTTTGAGTTAGGTGTGGG-3'; LINE elements antisense, 5'-biotin-TCTCACTAAAAAATACCAAACAA-3'; LINE elements sequencing, 5'-GGGTGGGAGTGAT-3'; Alu elements sense, 5'-biotin-TTTTTATTAAAAATATAAAAATT-3'; Alu elements antisense, 5'-CCCAAACTAAAATACAATAA-3'; Alu elements sequencing, 5'-AATAACTAAAATTACAAAC-3'; MAGE-A1 sense, 5'-biotin-TATTGTGGGGTAGAGAGAAG-3'; MAGE-A1 antisense, 5'-AAATCCTCAATCCTCCCTCAA-3'; MAGE-A1 sequencing, 5'-AACCTAAATCAAATTCCTT-3'.

Reverse Transcription-PCR and Quantitative Real-time Reverse Transcription-PCR
Total RNA was extracted from cells with the Qiagen RNeasy miniprep kit. Reverse transcription was done with Moloney murine leukemia virus reverse transcriptase and random hexamers from Promega (Madison, WI). Reverse transcription-PCR was done for the p16 gene as previously described (14) using the following primers: p16 sense, 5'-AGCCTTCGGCTGACTGGCTGG-3'; p16 antisense, 5'-CTGCCCATCATCATGACCTGGA-3'. PCR conditions for the p16 gene were as follows: 94°C for 3 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute, annealing at 57°C for 30 seconds, and extension at 72°C for 40 seconds, and a final extension at 72°C for 5 minutes. We also did quantitative real-time reverse transcription-PCR analysis as previously described (33) using DNA Engine Opticon System (MJ Research, Hercules, CA). The primers used are listed below: MAGE-A1 sense, 5'-GAACCTGACCCAGGCTCTGTG-3'; MAGE-A1 antisense, 5'-CCACAGGCAGATCTTCTCCTTG-3'; MAGE-A1 fluorogenic probe, 5'-CAAGGTTTTCAGGGGACAGGCCAAC-3'; MAGE-B2 sense, 5'-CGGCAGTCAAGCCATCATG-3'; MAGE-B2 antisense, 5'-TTGCGGCGTTTCTCACG-3'; MAGE-B2 fluorogenic probe, 5'-TCGTGGTCAGAAGAGTAAGCTCCGTGC-3'; RARß sense, 5'-CCCTTCACTCTGCCAGCTG-3'; RARß antisense, 5'-GCCCAGGTCCAGTCGGA-3'; RARß fluorogenic probe, 5'-AAATACACCACGAATTCCAGTGCTGACCA-3'; p16 sense, 5'-AGCCTTCGGCTGACTGGCTGG-3'; p16 antisense, 5'-CTGCCCATCATCATGACCTGGA-3'; p16 fluorogenic probe, 5'-TGGATCGGCCTCCGACCGTAACT-3'. The real-time reverse transcription-PCR conditions for all four genes were as follows: 95°C for 9 minutes, followed by 45 cycles of denaturation at 95°C for 15 seconds and annealing at 60°C for 1 minute.

	Results

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5-Aza-CdR Is Considerably More Effective in DNA Methylation Inhibition than Non-Nucleoside Agents
The quantitative Ms-SNuPE and pyrosequencing methods were used to compare the methylation status of several loci in the genome before and after treatment with potential inhibitors. Ms-SNuPE analysis was done to examine the methylation levels of the p16 promoter, MAGE-A1, and LINE repetitive elements (Fig. 2). EGCG from Sigma seemed to be more toxic than EGCG from Unilever Bestfoods; the highest doses of EGCG with surviving cells tested are shown in Figs. 2 and 3: for T24 cells, EGCG from Unilever Bestfoods was used at 20 µmol/L. For HT29 cells, 30 µmol/L of EGCG from Unilever Bestfoods and 30 µmol/L of EGCG from Sigma were used. For PC3 cells, 30 µmol/L of EGCG from Unilever Bestfoods and 20 µmol/L of EGCG from Sigma were used.

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Comparison of the methylation inhibition potencies of the various agents by Ms-SNuPE. Ms-SNuPE results of the methylation levels of p16 promoter (A), MAGE-A1 (B), and LINE elements (C). T24, HT29, and PC3 cells were treated with 5-Aza-CdR for 24 h or with hydralazine, procainamide, or EGCG continuously. Cells were collected on day 6. Columns, percentage of methylation of two independent experiments; bars, ± SD. The percentage of methylation is calculated as the average cytosine / (cytosine + thymine) signal ratio of three separate CpG sites for each region examined. Unt, untreated; Aza, 1 µmol/L 5-Aza-CdR; E-20 or 30(S), 20 or 30 µmol/L of EGCG from Sigma; E-20 or 30(UB), 20 or 30 µmol/L of EGCG from Unilever Bestfoods; H-20, 20 µmol/L of hydralazine; P-200, 200 µmol/L of procainamide.

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Comparison of the methylation inhibition potencies of the various agents by pyrosequencing. Pyrosequencing results of the methylation levels of Alu repetitive elements (A), MAGE-A1 (B), and LINE repetitive elements (C). T24, HT29, and PC3 cells were treated with 5-Aza-CdR for 24 h or with hydralazine, procainamide, or EGCG continuously. Cells were collected on day 6. The percentage methylation is calculated as the cytosine / (cytosine + thymine) ratio of the most proximal CpG sites to the primers. Columns, average percentage of methylation of two independent experiments; bars, ± SD. Unt, untreated; Aza, 1 µmol/L 5-Aza-CdR; E-20 or 30(S), 20 or 30 µmol/L of EGCG from Sigma; E-20 or 30(UB), 20 or 30 µmol/L of EGCG from Unilever Bestfoods; H-20, 20 µmol/L of hydralazine; P-200, 200 µmol/L of procainamide.

5-Aza-CdR Is Considerably More Effective in Reactivating Silenced Genes in Cancer Cells
To examine the ability of 5-Aza-CdR and the non-nucleoside agents to reactivate gene expression, we did reverse transcription-PCR for the p16 gene. Figure 4 shows a representative result of three independent reverse transcription-PCR experiments using p16 as the indicator gene. Only 5-Aza-CdR was able to activate the expression of the p16 gene.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Effects of the various agents on the reactivation of p16. Reverse transcription-PCR analysis of p16 gene expression in T24, HT29, and PC3 cells. The three cell lines were treated with various agents for 6 d. Reaction products were analyzed on ethidium bromide–stained agarose gels. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Unt, untreated; DMSO, DMSO control (same amount added as the EGCG 30 µmol/L sample); Aza, 1 µmol/L 5-Aza-CdR; E-20 and 30(S), 20 and 30 µmol/L of EGCG from Sigma; E-20 and 30(UB), 20 and 30 µmol/L of EGCG from Unilever Bestfoods; H-10 and 20, 10 and 20 µmol/L of hydralazine; P-100 and 200, 100 and 200 µmol/L of procainamide.

	Discussion

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Previous studies have shown that EGCG, hydralazine, and procainamide are able to reduce DNA methylation and reactivate gene expression in cancer cells (15–21). We examined a total of six different genes/repetitive elements in three separate cell lines for their DNA methylation levels using quantitative Ms-SNuPE and pyrosequencing, and their mRNA expression levels by real-time reverse transcription-PCR and reverse transcription-PCR. Both the Ms-SNuPE and pyrosequencing results show that only the nucleoside analogue 5-Aza-CdR can very reliably reduce methylation in all three cell lines. All three non-nucleoside agents have much weaker, if any, demethylating activities, with procainamide being the only agent able to reduce DNA methylation of LINE elements in T24 cells. The slight differences between the Ms-SNuPE and the pyrosequencing results for the LINE elements may be attributed to the fact that the Ms-SNuPE method examined three separate CpG sites, whereas only one CpG site was assayed in the pyrosequencing method. The expression studies with reverse transcription-PCR also show that only 5-Aza-CdR was able to appreciably reactivate MAGE-A1, MAGE-B2, and p16 genes as shown previously in other studies (34–37). The RARß gene may require the simultaneous administration of 5-Aza-CdR along with a histone deacetylase inhibitor such as trichostatin A for its reactivation in these cell lines (38).

At present, we cannot explain the discrepancy between our data and earlier studies. There are many potential reasons for this, however, these other agents seem unlikely to be robust and reliable inhibitors of DNA methylation. The discrepancies could arise from one or more of the following possibilities: the actions of the non-nucleoside agents could be gene-specific or cell line–specific, the treatment methods might have been ineffective to show efficacy, or the methods of analysis were different from previous studies.

We do not believe that the discrepancies were solely due to the set of genes in our study because we examined some of the genes that have been shown to be responsive to these agents in other studies, such as p16 and RARß (17, 21). In addition, we examined global methylation level changes with LINE and Alu repetitive elements and did not observe methylation inhibition from the non-nucleoside agents.

Testing with different cell lines could be another source of discrepancy (15, 16, 19, 20). We examined the effect of DNA methylation inhibitors on T24, HT29, and PC3 cells. From our results, it is apparent that different cell lines have different sensitivities to these agents. Other studies with Jurkat (16) and LnCAP (19) cell lines have shown apparent methylation inhibition activities of hydralazine and procainamide, respectively. Perhaps studies with different cell lines and/or a higher dose regimen will show the demethylating effect of these agents. However, we followed the treatment methods that were reported to be effective in previous studies for the non-nucleoside agents, and therefore we do not believe this to be the cause of the discrepancy (16, 17, 21). Nevertheless, longer treatments with these agents might be able to induce noticeable methylation inhibition (19).

Finally, discrepancies could arise from different methods of study. We used Ms-SNuPE and pyrosequencing analyses, two quantitative and reliable methods, to measure methylation levels. It is possible that the differences between the methods we employed and other methods such as methylation-specific PCR could lead to different results.

Green tea, which contains EGCG, is often consumed habitually. Additionally, hydralazine and procainamide are both used for long-term management. The possible weaker demethylating effects of these agents should not be ignored. Although they are considerably weaker in their DNA methylation inhibition activity compared with 5-Aza-CdR, it is feasible that long-term usage of these agents might have small effects. However, one should consider the potential and feasibility of these non-nucleoside agents in chemotherapy regimens. Plasma levels of procainamide >10 µg/mL (36.8 µmol/L) are associated with toxicity in a patient such as ventricular tachycardia or fibrillation (39). The concentrations we tested in the cell culture were much higher than the toxic plasma level. Taken together, our results do not support the idea that the three non-nucleoside agents tested are likely to be effective as epigenetic therapies with clinical or preventative actions.

	Footnotes

 
Grant support: National Cancer Institute grants CA82422 and CA83867.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/27/05; revised 8/ 8/05; accepted 8/17/05.

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