Delivery of 5-Aza-2¶-Deoxycytidine to Cells Using Oligodeoxynucleotides

Research Article

Christine B. Yoo,1 Shinwu Jeong,3 Gerda Egger,3 Gangning Liang,2,3 Pasit Phiasivongsa,4 Chunlin Tang,4 Sanjeev Redkar,4 and Peter A. Jones1,2,3

Departments of 1Biochemistry and Molecular Biology and 2Urology and 3Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California and 4SuperGen, Inc., Pleasanton, California

Abstract prevents effective cellular uptake. Thus, nucleoside analogues, which are taken up intracellularly and phosphorylated to their The major goal of epigenetic therapy is to reverse aberrant promoter hypermethylation and restore normal function of respective monophosphates, diphosphates, and triphosphates tumor suppressor genes by the use of chromatin-modifying before incorporation into replicating DNA, leading to covalent drugs. Decitabine, or 5-aza-2¶-deoxycytidine (5-aza-CdR), is a trapping of DNA methyltransferases (DNMT), are used (13). 5-Aza- well-characterized drug that is now Food and Drug Adminis- CR and 5-aza-CdR are powerful demethylating agents; nevertheless, tration approved for the treatment of myelodysplastic they have a number of drawbacks. The aza pyrimidine ring is syndrome. Although 5-aza-CdR is an extremely potent unstable in aqueous solution, making it difficult to administer, and inhibitor of DNA methylation, it is subject to degradation by is quite toxic both in vitro and in vivo (14). Furthermore, the drugs hydrolytic cleavage and deamination by cytidine deaminase. have transient effects, and DNA is gradually remethylated after We show that short oligonucleotides containing a 5-aza-CdR removal of the drug (15). Yet, another problem arises due to cytidine deaminase, which renders the drugs inactive by converting can also inhibit DNA methylation in cancer cells at concentrations comparable with 5-aza-CdR. Detailed studies with them into 5-azauridine compounds. S110, a dinucleotide, showed that it works via a mechanism In our attempts to synthesize more stable and potent inhibitors similar to that of 5-aza-CdR after incorporation of its aza- of DNA methylation, we found that short oligonucleotides containing an azapyrimidine effectively inhibit DNA methylation moiety into DNA. Stability of the triazine ring in aqueous ¶ ¶ solution was not improved in the S110 dinucleotide; however, in living cells. Here, we focus on S110, a 5 -AzapG-3 dinucleotide, deamination by cytidine deaminase was dramatically de- whose aqueous stability and toxicity are quite similar to that of creased. This is the first demonstration of the use of short 5-aza-CdR but is protected from deamination by cytidine oligonucleotides to provide effective delivery and cellular deaminase. The demethylating activity seems to require incorpo- uptake of a nucleotide drug and protection from enzymatic ration of the azapyrimidine into DNA presumably after degradation degradation. This approach may pave the way for more stable of the oligonucleotide by phosphodiesterases and is not limited to and potent inhibitors of DNA methylation as well as provide a dinucleotide but is seen in trinucleotides and tetranucleotides as well, showing that short oligonucleotides are effective prodrugs for means for improving existing therapeutics. [Cancer Res delivery of inhibitors of DNA methylation. The utilization of short 2007;67(13):6400–8] oligonucleotides as nucleoside drug delivery vehicles that provides protection against enzymatic degradation might have application Introduction for delivery of other nucleoside drugs to cells. Aberrations in DNA methylation are frequently observed in various types of cancer (1–3). Several tumor suppressor genes or Materials and Methods cancer-related genes acquire de novo DNA methylation in promoter or regulatory regions leading to inactivation, contribut- Synthesis of oligonucleotides containing 5-aza-CdR. Dinucleotides, ing to tumorigenesis (4–8). DNA hypermethylation can be reversed trinucleotides, and tetranucleotides containing 5-aza-CdR were synthesized by demethylating agents and crucial cellular functions reestab- by standard procedures with modifications to increase coupling times, different oxidizing agents, and use of phenoxyacetyl decitabine phosphor- lished in the cells. In recent years, the use of DNA methylation amidite, instead of phenoxyacetyl cytidine phosphoramidite. A polystyrene- inhibitors has become a promising alternative to patients with based solid support with loading of 240 Amol/g dG(pac) or D(pac) was used myelodysplastic syndrome and hematologic malignancies (9, 10). (16, 17). The most widely known examples of DNA methylation inhibitors Briefly, synthesis of S53, 5¶-GpAza-3¶ dinucleotide, is described here. are 5-azacytidine (5-aza-CR) and 5-aza-2¶-deoxycytidine (5-aza- Amersham A¨KTA Oligopilot 10 system was loaded with a protected CdR); both drugs were initially synthesized as anticancer agents decitabine-linked CpG solid support (phenoxyacetyl protection of amino and were later shown to inhibit DNA methylation (11, 12). The function) and coupled with 2 to 2.5 equivalents of tert-butyl phenoxyacetyl clinical use of nucleotides rather than nucleosides is essentially 2¶-deoxyguanosine phosphoramidite in the presence of 60% of 0.3 mol/L impossible due to the negative charge on the phosphate group that benzylthiotetrazole activator in acetonitrile for 2.5 min. The CpG solid support containing protected S53 was treated with 20 mL of 50 mmol/L

K2CO3 in methanol for 1 h and 20 min. The coupled product was oxidized with 2 mol/L tert-butylhydroperoxide in dry acetonitrile prepared by Requests for reprints: Peter A. Jones, University of Southern California/Norris dissolving tert-butylhydroperoxide in 80% tert-butylperoxide for 5 min. The Comprehensive Cancer Center and Hospital, 1441 Eastlake Avenue, Room 8302L, Mail dimethoxy trityl protective group was removed with 3% dichloroacetic acid Stop 83, Los Angeles, CA 90033-9181. Phone: 323-865-0816; Fax: 323-865-0102; E-mail: in toluene. The CpG solid support was washed with dry methanol; the [email protected]. I2007 American Association for Cancer Research. filtrate was neutralized by addition of 2 mL of 1 mol/L acetic acid in doi:10.1158/0008-5472.CAN-07-0251 methanol. The solution was concentrated by rotary evaporation; the

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Short Oligonucleotide DNA Methylation Inhibitors residue was taken up in 200 mmol/L triethylammonium acetate (pH 6.9), on a Ready Gel Tris-HCl Gel, 4% to 15% linear gradient (Bio-Rad washed with 500 AL 50% aqueous acetonitrile, and filtered through a Laboratories) and transferred to a polyvinylidene difluoride membrane syringe filter. S53 was subsequently purified to 95% purity by the A¨KTA using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Explorer 100 high-performance liquid chromatography (HPLC) with a Laboratories). The membrane was hybridized with antibodies against Gemini C18 preparative column (Phenomenex), 250 Â 21.2 mm, 10 Am human DNMT1 (H-300; 1:500; Santa Cruz Biotechnology), p16 (N-20; 1:500; with guard column (Phenomenex), 50 Â 21.2 mm, 10 Am, with 50 mmol/L Santa Cruz Biotechnology), and proliferating cell nuclear antigen (PCNA; triethylammonium acetate (pH 7) in MilliQ water (Mobile Phase A) and PC10; 1:500; Santa Cruz Biotechnology) in TBS-Tween (TBS-T) buffer 80% acetonitrile in MilliQ water (Mobile Phase B), with 2% to 20%/25% (0.01 mol/L Tris, 0.15 mol/L NaCl, and 0.1% Tween 20) with 5% nonfat dry Mobile Phase B in column volumes. The electrospray-mass spectrometry milk overnight at 4jC. The membranes were washed four times with TBS-T À (+) of triethylammonium salt of S53 exhibited m/z 556.1 [M-H] and buffer at room temperature and incubated with secondary antibodies for À 1,113.1 for [2M-H] , and the calculated exact mass for the neutral 1 h at room temperature. Secondary antibodies used were anti-rabbit-IgG- compound C18H24N9O10P is 557.14. horseradish peroxidase (HRP) for DNMT1 and p16 (1:7,500; Santa Cruz Cytidine deaminase assay. Recombinant human cytidine deaminase Biotechnology) and anti-mouse-IgG-HRP for PCNA (1:7,500; Santa Cruz was kindly provided by Alberto Vita (University of Camerino, Italy) and Biotechnology). The membranes were washed six times with TBS-T at room prepared as described in Vincenzetti et al. (18). Purified cytidine deaminase temperature. Proteins were detected with the ECL Western Blotting (0.09–0.1 unit) was incubated with 0.2 mmol/L 5-aza-CdR or S110 in 3 mL of Detection Reagents (GE Healthcare Biosciences Corp.) and by exposure to 0.1 mol/L Tris-HCl (pH 7.5) at 38jC. Percent substrate remaining was Kodak X-OMAT AR film. measured after 45 and 90 min of incubation by a Waters 2695 HPLC system Quantitation of DNA methylation. Genomic DNA (4 Ag) was treated with a 996 photodiode array detector (Waters Corp.). with sodium bisulfite as previously described (19). Methylation analysis Determination of nucleic acid stability. The absorbance of 5-aza-CdR was done using the methylation-sensitive single-nucleotide primer and S110 was measured with a Beckman DU Series 600 UV/visible extension (Ms-SNuPE) assay for p16 5¶-region as previously described Spectrophotometer (Beckman Coulter, Inc.). The wavelengths of maximum (21). The PCR primers used were sense 5¶-TTTGAGGGATAGGGT-3¶ and absorbance of 5-aza-CdR and S110 were 241 and 245 nm, respectively. The antisense 5¶-TCTAATAACCAACCAACCCCTCC-3¶. An initial denaturation initial absorbance was measured immediately after 5-aza-CdR and S110 at 94jC for 3 min was followed by 94jC for 45 s, 62jC for 45 s, and 72jC were dissolved in PBS, and both compounds were incubated at 37jC except for 45 s for 40 cycles. Primers used for Ms-SNuPE analysis were when absorbance reading was taken. 5¶-TTTTTTTGTTTGGAAAGATAT-3¶,5¶-TTTTAGGGGTGTTATATT-3¶, and Cell lines and drug treatment. T24 bladder carcinoma cells and 5¶-GTAGAGTTTAGTT-3¶. Conditions for primer extension were 94jC for HCT116 colon carcinoma cells were obtained from the American Type 1 min, 50jC for 30 s, and 72jC for 20 s. Methylation analysis of X58075 Culture Collection and cultured in McCoy’s 5A medium supplemented with LINE-1 element was carried out with the following primer set. The PCR 10% heat-inactivated FCS, 100 units/mL penicillin, and 100 Ag/mL primers were sense 5¶-TTTTTTGAGTTAGGTGTGGG-3¶ and antisense streptomycin (Invitrogen) in a humidified incubator at 37jCin5%CO2. 5¶-CATCTCACTAAAAAATACCAAACAA-3¶, and the conditions were 94jC Cells were plated (1.5 Â 105 per 60-mm dish) and treated 24 h later with for 3 min followed by 94jC for 45 s, 52jC for 45 s, and 72jC for 45 s for 5-aza-CdR (Sigma-Aldrich) or S110 continuously for 6 days. Each compound 39 cycles. Primers for Ms-SNuPE analysis were 5¶-GGGTGGGAGTGATT-3¶, was dissolved in PBS. The medium was changed 3 days after the initial 5¶-GAAAGGGAATTTTTTGATTTTTTG-3¶,and5¶-TTTTTTAGGTGAGG- treatment and supplemented with a fresh dose of 5-aza-CdR or S110. TAATGTTT-3¶. Conditions for primer extension were 94jC for 1 min, Determination of cytotoxicity. A colony formation assay was used to 50jC for 30 s, and 72jC for 20 s. compare cytotoxicity of 5-aza-CdR and S110 as previously described (19). In vitro hemimethylation assay. An intronic region of p16 was Briefly, T24 cells were plated at a low density (100 per 60-mm dish) and amplified using human genomic DNA as a substrate and a primer set with treated with varying concentrations of 5-aza-CdR and S110. Colonies were a methylated CpG to yield a hemimethylated CpG site. The amplicon (1 ng) allowed to form for 10 to 14 days, fixed with methanol, and stained with 10% was treated with M.Sss1 or human DNMT1 (New England Biolab) in 50 AL Giemsa. The number of colonies from an untreated control plate was used of reaction buffers, as recommended by the manufacturer, for 30 min at 37jC, to calculate the plating efficiency in percent at each concentration. in the presence of S110 of various concentrations. The reaction was stopped Triplicate dishes were used, and error bars are represented by 1 SD of the by heat inactivation of enzymes. Bisulfite-converted DNA was amplified by mean. 5¶-CTCTTACCATCCTCTT-3¶ and 5¶-GAGTTATATTTATGTGATTATTTT-3¶, Nucleic acid isolation. RNA was collected from T24 and HCT116 cells and Ms-SNuPE assay was done using 5¶-TTTTAAAATTTTGTTAATAGTTT- with the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. GAATT-3¶ as a primer. DNA was collected using the DNeasy Tissue kit (Qiagen) according to the Sucrose density gradient ultracentrifugation. Nuclei were prepared manufacturer’s protocol. according to the procedure described in Gal-Yam et al. (22). Briefly, cells Quantitative reverse transcription-PCR analysis. Total RNA (5 Ag) were trypsinized and washed once with PBS. The cells were then was reverse transcribed with Moloney murine leukemia virus reverse resuspended in ice-cold RSB buffer [10 mmol/L Tris-HCl (pH 7.4), transcriptase (Invitrogen) and random primers (Invitrogen). The reverse 10 mmol/L NaCl, 3 mmol/L MgCl2] containing Complete Mini proteinase transcription was carried out in a total volume of 50 AL as previously inhibitors (Roche) and kept on ice for 10 min before dounce homogeni- described (4). The quantitation of mRNA levels was carried out by a real- zation with 0.5% NP40 to break up cell membranes. Nuclei were washed time fluorescence detection method as described previously (15, 20). All twice with RSB plus proteinase inhibitors without the detergent. Purified samples were normalized to the reference gene GAPDH. The primer and nuclei (1 Â 108) were resuspended in 1 mL of RSB containing 0.25 mol/L ¶ ¶ probe sequences are as follows: for p16, sense 5 -CTGCCCAACGCACCGA-3 , sucrose, 3 mmol/L CaCl2, and 100 Amol/L phenylmethylsulfonyl fluoride probe 5¶-6-FAM-TGGATCGGCCTCCGACCGTAACT-BHQ-1 3¶, and antisense and digested with MNase (Worthington Biochemical Corp.) for 15 min at 5¶-CGCTGCCCATCATCATGAC-3¶;forGAPDH,sense5¶-TGAAGGTCG- 37jC, and then the reaction was stopped with EDTA/EGTA (up to GAGTCAACGG-3¶, probe 5¶-6-FAM-TTTGGTCGTATTGGGCGCCTGG-BHQ- 10 mmol/L). After microcentrifugation at 5,000 rpm for 5 min, the nuclear 13¶, and antisense 5¶-AGAGTTAAAAGCAGCCCTGGTG-3¶. The conditions pellet was resuspended in 0.3 mL of the buffer [10 mmol/L Tris-HCl for real-time reverse transcription-PCR are 94jC for 9 min followed by (pH 7.4), 10 mmol/L NaCl] containing 5 mmol/L EDTA/EGTA, gently 45 cycles at 94jC for 15 s and 60jC for 1 min. rocked for 1 h at 4jC, and followed by microcentrifugation to obtain soluble Western blot analysis. Cell pellets were lysed in radioimmunoprecipi- nucleosomes, which were then fractionated through a sucrose density tation buffer containing 0.1% SDS, 0.5% NP40, and 0.5% sodium gradient solution [5-25% sucrose, 10 mmol/L Tris-HCl (pH 7.4), 0.25 mmol/L deoxycholate in PBSand incubated on ice for 30 min. The lysates were EDTA, 300 mmol/L NaCl] at 30,000 rpm for 16 h at 4jC. Fractions were centrifuged at 4jC for 30 min at 14,000 rpm. The supernatant was collected taken from the top of the centrifuge tube into 16 aliquots and subjected and stored at À80jC. Approximately 60 Ag of protein was electrophoresed to Western blot analysis. www.aacrjournals.org 6401 Cancer Res 2007; 67: (13). July 1, 2007

A Table 1. Short oligonucleotide DNA methylation inhibitors CdR and S110 after 0.1 and 1 mol/L treatment (Fig. 2A, top). At 10 Amol/L concentrations, only a small decrease in methylation Compound name Structure Dose (Amol/L) p16 was noted, probably due to side effects of high drug concentrations A induction as we observed previously (12). In fact, 10 mol/L treatment may be too cytotoxic for effective demethylation to take place as the Decitabine 5-Aza-CdR 1 +++ plating efficiency of T24 cells indicates. It is well established that its S110 AzapG 1 +++ cytotoxic dose is not ideal for optimal epigenetic therapy because S53 GpAza 1 +++ these drugs inhibit DNA methylation best at low doses in cell lines S54 GpAzapG 1 +++ as well as in the clinic (25, 26). S55 AzapGpAzapG 1 +++ We then analyzed the DNA methylation status of exon 1 of the S56 pGpAzapAzapG 1 +++ p16 gene in these cells after 6-day treatment by quantitative Ms- S52R AzapsG 100 + SNuPE. In untreated T24 cells, the three CpG sites under analysis S112 HEGpAzapG 100 ++ are located in the first exon of the gene and are almost always fully methylated. The methylation level decreased with increasing NOTE: List of short oligonucleotides that inhibit DNA methylation and concentrations of 5-aza-CdR or S110 in T24 cells (Fig. 2A, middle induce p16 expression in T24 cells after 6 d of continuous treatment. left) and HCT116 cells (Fig. 2A, middle right) with the greatest Dose indicates the lowest concentration at which the induction of p16 demethylation seen at 1 Amol/L 5-aza-CdR. expression is observed. Next, we measured the expression of p16 in both cancer cell Abbreviations: Aza, 5-aza-2¶-deoxycytidine.; HEG, hexaethylene glycol lines. Untreated T24 bladder carcinoma cells do not express p16 phosphate linker. and dose-dependent increases in p16 expression were observed after 6 days of continuous treatment with 5-aza-CdR or S110 (Fig. 2A, bottom left). After HCT116 colorectal carcinoma cells were treated for 6 days, a dose-dependent increase in p16 expression was Results observed with S110, whereas the highest p16 expression was seen Reexpression of p16 in T24 bladder cancer cells by short at 1 Amol/L dose of 5-aza-CdR (Fig. 2A, bottom right). In addition, oligonucleotides. We first treated T24 cells with varying concen- T24 and HCT116 cells treated with either agent for 3 days also trations of oligonucleotides containing the 5-azapyrimidine ring for showed a dose-dependent increase in the level of p16 protein 6 days to test their abilities to induce the expression of the (Fig. 2B), showing the competence of S110 to inhibit DNA methylation-silenced p16 gene in T24 bladder carcinoma cells methylation and induce p16 at both mRNA and protein levels as (Table 1). Induction of p16 is an indirect, yet straightforward, well as 5-aza-CdR. Thus, S110 is able to inhibit DNA methylation at method to detect inhibition of DNA methylation because 5¶-region and induce the expression of the p16 gene in T24 and demethylation of the 5¶ CpG island of p16 results in reexpression HCT116 cells at concentrations comparable to 5-aza-CdR, and the of the gene (4, 23). All short-chain nucleotides tested, regardless of induction of p16 expression by both agents correlated with the their chain lengths, were able to induce robust p16 expression with demethylation at the 5¶-end region of the gene in both cell lines. the exception of S52R (Fig. 1B) and S112 (Fig. 1E), which induced Depletion of DNMT1 levels by S110. To provide more clues to expression to lesser extents (Table 1). S52R, a phosphorothioate the mechanism of action of S110, DNMT1 protein levels in T24 and derivative of S110 (Fig. 1B), is less prone to cleavage by HCT116 cells treated with the compound were analyzed by phosphodiesterases and caused a small induction of p16 at high Western blot analysis (Fig. 2B). A number of studies have provided concentration, suggesting that the cleavage of the oligonucleotides evidence that incorporation of the azapyrimidine ring into DNA is into nucleotides and nucleosides is a determining factor in necessary for the drug to inhibit DNA methylation (13, 27, 28). inducing gene expression. S112 is an S110 derivative with a Work by Hurd et al. (29) showed a covalent bond formation hexaethylene glycol phosphate linker at the 5¶-end (Fig. 1E), which between a DNMT and zebularine-incorporated oligodeoxynucleo- is subject to cleavage in cells. This extra cleavage requirement tide, strengthening the notion that DNA incorporation is an probably slows down the ability of S112 to induce the p16 gene and essential step toward inhibition of methylation by mechanism- thus explains the weaker activity of the compound. Demethylation based inhibitors. It is probable then that S110 works via a similar of the 5¶-end of the p16 gene was observed in cells treated with the mechanism and causes a depletion of extractable DNMT1 in cells. short oligonucleotides, showing that indeed, these compounds are Indeed, partial and complete depletion of DNMT1 protein was bona fide inhibitors of DNA methylation (data not shown). From observed in T24 and HCT116 cells treated with 5-aza-CdR for here on, we focused our detailed characterization on S110 (Fig. 1D) 3 days (Fig. 2B). Similarly, in S110-treated T24 and HCT116 cells, as an example of short oligonucleotide demethylating agents. partial depletion of the enzyme was seen at the 1 Amol/L dose, and Effects of S110 on DNA methylation and p16 gene expression a trace of DNMT1 remained after 10 Amol/L treatment. Our results in T24 and HCT116 cells. We extended on the initial screen done suggest that S110 causes depletion of extractable DNMT1 in cells in Table 1 to determine the dose dependency of S110 using T24 and may work by a similar mechanism as that of 5-aza-CdR in bladder and HCT116 colon cancer cells. First, we assessed global inhibiting DNA methylation. methylation status by determining the methylation level of long Inhibition of DNMT1 in vitro by S110 and fractionation of interspersed nucleotide element-1 (LINE-1) sequences. Repetitive DNMT1 in nuclear extracts using sucrose density gradient DNA elements, such as LINE-1 retrotransposable elements, serve as ultracentrifugation. To validate the notion that S110 works via a useful marker of genome-wide methylation changes and have DNA incorporation after phosphodiesterase cleavage, we next previously been shown to be demethylated upon treatment with determined whether the dinucleotide could inhibit DNMTs in a 5-aza-CdR (24). In both T24 and HCT116 cells, the decrease in the cell-free hemimethylation assay. We tested the ability of S110 to level of methylation was dose dependent and comparable for 5-aza- inhibit a bacterial methylase, M.Sss1, or human DNMT1 activity to

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Short Oligonucleotide DNA Methylation Inhibitors methylate a double-stranded DNA containing a hemimethylated (Fig. 3C). After 5-aza-CdR or S110 treatment, the distribution of CpG site. Figure 3A shows that both M.Sss1 and DNMT1 were able to DNMT1 changed dramatically, and although the majority of the methylate DNA even in the presence of high concentrations of enzyme was present in the low molecular portion of the gradient S110. This result provided important clues to the mechanism of S110 (fractions 4 and 5), it was also found in fractions 6 to 16 along with by showing that the dinucleotide cannot inhibit DNMT1 directly. high molecular weight DNA (Fig. 3C). We interpret this result to Although Fig. 3A showed that the dinucleotide cannot directly show that the incorporation of 5-aza-CdR or the hydrolyzed inhibit DNA methylation, we still lacked direct evidence that S110 product of S110 caused the DNMT1 enzyme to bind very strongly metabolite had to be incorporated into DNA after cleavage. We to the DNA in chromatin. Our experiment strongly suggests that therefore extracted nuclei from 5-aza-CdR or S110-treated HCT116 S110 works through a similar mechanism as 5-aza-CdR after its cells, collected soluble nucleosomes, and size fractionated the cleavage by phosphodiesterases, and that both 5-aza-CdR and S110 nucleosomes by sucrose density gradient centrifugation to byproducts are incorporated into DNA where they covalently determine whether the drugs would trap DNMT1 on chromatin interact with DNMTs. It is most probable that the S110 (Fig. 3B and C). The absorbance showed that DNA is concentrated dinucleotide is cleaved into nucleotides and nucleosides because in fractions 5 to 16 (Fig. 3B) as are the nucleosomes (Fig. 3C). incorporation of a dinucleotide or oligonucleotide is not known to Under the salt concentrations used (300 mmol/L NaCl), DNMT1 occur during DNA synthesis. was not associated with nucleosomes, and the majority of DNMT1 Reverse dinucleotide and phosphorothioate analogues of protein in untreated HCT116 cells was not bound to chromatin S110. To gain further insight as to how the S110 dinucleotide

Figure 1. Chemical structures of 5-aza-CdR( A), S52 (B), S53 (C), S110 (D), and S112 (E). 5-Aza-CdRis a deoxycytidine with an extra nitrogen at the 5-position of the pyrimidine ring. S52 is a phosphorothioate analogue of S110. There are two optical isomers of S52: S52S and S52R. S53 is a dinucleotide with a guanosine at the 5¶-end and 5-aza-CdRat the 3 ¶-end. S110 is a reverse dinucleotide of S53 containing a 5-aza-CdRat the 5 ¶-end followed by a guanosine. S112 is a triethylamine salt of 5¶-AzapG-3¶ dinucleotide with a hexaethylene glycol phosphate at the 5¶-end. www.aacrjournals.org 6403 Cancer Res 2007; 67: (13). July 1, 2007

Figure 2. Re-expression of p16 and inhibition of DNA methylation by S110 in cancer cells. T24 or HCT116 cells were treated with varying concentrations of 5-aza-CdRand S110 continuously for 6 d ( A). Global methylation changes were measured at LINE-1 elements and the 5¶-region of the p16 gene by Ms-SNuPE. Quantitative real time reverse transcription-PCRanalysis of the p16 gene was done with normalization against the GAPDH reference gene. White columns, cells treated with 5-aza-CdR; black columns, S110 treatment. Columns, mean from three independent experiments; bars, SD. T24 or HCT116 cells were treated with 5-aza-CdRor S110 for 3 d ( B). Whole-cell lysates were collected and separated on a polyacrylamide gel and blotted against specific antibodies for human DNMT1, p16, and PCNA.

inhibits DNA methylation, we used S110 analogues (i.e., S53), the stability of S110 with that of 5-aza-CdR, we measured the which is a reverse dinucleotide of S110, and S52R and S52S, which absorbance of 5-aza-CdR and S110 at their respective wavelengths k j are two optical isomers of phosphorothioate analogues of S110 of maximum absorbance ( max) over time at 37 C in PBSsolution (Fig. 1B and C). S53 would allow us to determine whether the at pH 7.4. The absorbance of 5-aza-CdR showed a sharp decay in sequence of the dinucleotide plays a role in inhibiting DNA the first 30 h of incubation with a half-life of about 20 h (Fig. 5A). methylation. In addition, the phosphorothioate analogues would The half-life of S110 was 21 h, with 65% of its initial absorbance help us determine whether a free 5-aza-CdR is released from the remaining for as long as 200 h (Fig. 5A). This residual absorbance dinucleotide, or if S110 directly inhibits DNA methylation. The at 245 nm is attributed to the guanosine ring in the dinucleotide, phosphate backbones of phosphorothioate analogues S52R and the absorbance of which did not decrease during the course of the S52S are not readily cleaved, hence retaining the dinucleotide experiment. In addition, the stability of both drugs was comparable structure for longer period of time. T24 cells were treated contin- when monitored for 90 min at 38jC (pH 7.5) by HPLC (see Fig. 5B). uously for 6 days with these compounds at varying concentrations. Furthermore, additional HPLC analysis of 5-aza-CdR and S110 A dose-dependent induction of p16 and demethylation of p16 stability at room temperature over time showed that both exon 1 were seen in T24 cells treated with S53, whereas the compounds have similar half-lives (data not shown). Therefore, expression and the DNA methylation status at the 5¶ end region whether the 5-azacytosine ring was in a single nucleotide or a of p16 remained unaffected in cells treated with S52R and S52S dinucleotide, the stability of the compound was not affected, and (Fig. 4A and B). Overall, this suggests that the order of bases in the half-lives of these two compounds in aqueous solution at 37jC the dinucleotide does not play a crucial role in inhibiting DNA remained the same. methylation, but the cleavage of the dinucleotide is critical in Enzymatic degradation of 5-aza-CdR and S110 by human rendering the compound active, suggesting once again that the cytidine deaminase. 5-aza-CdR is quickly catabolized into 5- dinucleotide does not work as a whole and has to be cleaved into azadeoxyuridine by cytidine deaminase in vivo (30). We incubated individual nucleotides and nucleosides. either 5-aza-CdR or S110 with human recombinant cytidine Stability of 5-aza-CdR and S110 at 37°Cin neutral aqueous deaminase at 38jC and measured the amount of substrate solution. A drawback of 5-aza-CdR is its short half-life in aqueous remaining over time by HPLC (Fig. 5B). A slow decrease in the solution due to rapid hydrolysis of the azapyrimidine. To compare total amount of 5-aza-CdR in the absence of cytidine deaminase

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(E) over time is indicative of hydrolysis of the 5-azapyrimidine Cytotoxicity of 5-aza-CdR and S110 in T24 cells. Finally, we ring in water. A rapid decrease in 5-aza-CdR was observed in the compared the cytotoxicities of 5-aza-CdR and S110 in T24 bladder first 45 min of incubation with human cytidine deaminase, and only carcinoma cells by measuring effects of the compounds on the about 2% of the total substrate remained after 90 min (Fig. 5B, .). In plating efficiency of T24 cells. 5-Aza-CdR decreased the plating contrast, S110 was subject to hydrolytic cleavage as found earlier efficiency in a dose-dependent manner, with no colonies forming at (Fig. 5A) but was resistant to enzymatic degradation by cytidine 10 Amol/L concentration (Fig. 5C). S110 was slightly less toxic than deaminase; the majority of S110 remained in the solution after 5-aza-CdR at the doses tested up to 1 Amol/L concentration but 90 min of incubation with cytidine deaminase (Fig. 5B, x). Incu- displaying similar toxicity at 10 Amol/L concentration. Similar bation of S110 in human plasma showed similar results, and the results were obtained for HCT116 cells (data not shown). Thus, the dinucleotide persisted for 40 min with very little decrease when cytotoxicity of S110 as measured by plating efficiency is quite measured by HPLC compared with a 33% decrease in the level of similar to 5-aza-CdR in T24 cells. 5-aza-CdR (data not shown). Although the dinucleotide structure cannot prevent the hydrolysis of the 5-azacytosine ring, it may greatly lengthen the half-life of the drug by protecting it from Discussion deamination, potentially increasing the efficacy of the drug in Short oligonucleotides were synthesized to test whether the patients as a result. stability of 5-aza-CdR, a cancer chemotherapeutic agent, could be

Figure 3. Inhibition of DNMT1 in vitro by S110 and fractionation of DNMT1 in nuclear extracts using sucrose density gradient ultracentrifugation. A fragment of double-stranded DNA containing a hemimethylated CpG site (1 ng) was incubated with M.Sss1 or recombinant human DNMT1 in the presence of S110 for 30 min at 37jC(A). Heat inactivation of the enzyme (-Enz) was followed by bisulfite conversion and DNA methylation analysis by quantitative Ms-SNuPE. HCT116 cells were plated and treated with 1 Amol/L 5-aza-CdRor 1 Amol/L S110 for 24 h (B and C). Cells were harvested and lysed for nuclei extraction. All nuclei were subjected to partial micrococcal nuclease digestion, which yielded nucleosomal fragments of various sizes. The nucleosomes diffused from the digested nuclei were separated by sucrose gradient centrifugation, and 16 fractions were collected. Protein from each fraction was analyzed by Western blot analysis using specific antibodies to human DNMT1 and histone H3. Absorbance of each fraction was measured at k = 260 nm before TCA precipitation. x, untreated (Unt) cells; n, cells treated with 5-aza-CdR; E, S110-treated cells. Histone H3 is a loading control for the presence of chromatin.

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the longer-chain oligonucleotides, further improving stability. In practice, this may be very advantageous in terms of broadening the applications of epigenetic therapy to patients with solid tumors. Oligonucleotides have been widely studied as chemotherapeu- tics, and in most cases, the base sequence plays a pivotal role in giving them therapeutic effects. For example, oligonucleotides with CpG motifs activate toll-like receptors and elicit immune responses (31). Antisense morpholino oligonucleotides and small interfering RNAs all use sequence specificities of endogenous molecules to inhibit either transcription or translation of target genes (32, 33). Unlike the existing approaches, the short oligonucleotides we have developed do not require any sequence specificities and simply provide protection against enzymatic degradation before the DNA

Figure 4. Re-expression of p16 and inhibition of DNA methylation by S53 but not by S52 in T24 cancer cells. T24 cells were treated with 5-aza-CdR, S53, a reverse dinucleotide of S110, S52S, and S52R, and the phosphorothioate analogues of S110 at varying concentrations continuously for 6 d and harvested for RNA isolation (A). Quantitative real-time reverse transcription-PCRanalysis of p16 normalized against GAPDH. T24 cells treated with 5-aza-CdR, S53, S52S, and S52Rwere subject to DNA methylation analysis at the 5 ¶-region of the p16 gene by Ms-SnuPE (B). The level of DNA methylation in percent is an average of three CpG sites in exon 1 of the p16 gene. improved without compromising its potency to inhibit DNA methylation. Comparison of the S110 dinucleotide containing 5-aza-CdR showed that S110 works via a similar mechanism of action to the parent compound to inhibit DNA methylation, induce expression of the p16 tumor suppressor gene, and inhibit tumor cell growth (Figs. 2–5). Characterization of S110 showed that the stability in aqueous solution and cytotoxicity is still comparable with that of 5-aza-CdR; however, a major improvement was accomplished in terms of resistance to enzymatic deamination (Fig. 5B). Deamination of 5-aza-CdR by cytidine deaminase rapidly depletes the plasma level of the drug, resulting in low bioavailability that has been frequently pointed out as one of the major drawbacks of 5-aza-CdR (30). S110 dinucleotide is resistant to cytidine deaminase deamination probably due to the substrate Figure 5. Comparison of stability and cytotoxicity of 5-aza-CdRand S110. specificity of the enzyme, which may potentially increase the half- The initial absorbance was measured immediately after the compounds were life of the drug, enhance bioavailability, and make the drug more dissolved in PBS (A). Both compounds were incubated at 37jC, and the efficacious. Lowering the dose requirement may also reduce the absorbance of each compound at its respective wavelength of maximum absorbance was measured over time. The decrease in absorbance was toxicity in patients as well as diminishing common side effects of expressed as percent initial absorbance. Enzymatic degradation of 5-aza-CdR 5-aza-CdR, such as myelosuppression. and S110 by recombinant human CDA was compared (B). Recombinant CDA (0.1 unit) was incubated with 5-aza-CdRor S110 (0.2 mmol/L) at 38 jC, and Trinucleotides and tetranucleotides containing one or more percent substrate remaining was determined by HPLC. x, S110 with CDA; 5-aza-CdR residues in the chain showed demethylating activity as n, S110 alone; ., 5-aza-CdRwith CDA; E, 5-aza-CdRalone. Cytotoxicity was well, showing that the cleavage of these oligonucleotides is extremely compared by measuring the average plating efficiency (C). T24 cells were plated at a low density and treated with 5-aza-CdRor S110 24 h later. Number of colonies efficient (Table 1; data not shown). Additionally, the hydrolytic from a control or untreated dish was expressed as 100% and the treated dishes cleavage of the 5-azacytosine ring may presumably be improved in as percent untreated. Data from triplicate dishes. Columns, mean; bars, SD.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2007 American Association for Cancer Research. Short Oligonucleotide DNA Methylation Inhibitors incorporation of the active moiety following endonuclease and/or likely candidates for the cleavage of oligonucleotides are the 11 phosphodiesterase cleavage. Our findings suggest that once these distinct subfamilies of phosphodiesterases that are abundant in all prodrugs are delivered to the target site and taken up intracellu- cell types (46). Phosphodiesterases catalyze the hydrolysis of larly, they are cleaved and metabolized to induce effects of similar 3¶-ester bonds of cyclic AMP and cyclic GMP into their respective levels to the parent molecule. This concept eliminates the need for monophosphate moeities, leaving a phosphate group at the 5¶-end chemical modifications such as lipid esterification and complex (47, 48). It is probable then that these enzymes, when they process delivery vehicles that are commonly required for prodrugs. the oligonucleotides, also leave the phosphate group on the 5¶-end Although it is most likely that these oligonucleotides are cleaved of the nucleoside in the sequence, hence yielding both nucleotides and incorporated into DNA to inhibit DNA methylation, it is still and nucleosides. However, it is not clear from preliminary data not clear as to whether the cleavage occurs extracellularly or whether the phosphodiesterases are the main player involved in intracellularly. It is not likely that the oligonucleotides are processing these inhibitors, and further work is necessary to assess processed into 5-aza-CdR or its nucleotide by DNases in plasma their role in oligonucleotide metabolism. before entering the cell because there is a little or weak Regardless of the mechanism, our data show that these short exonuclease activity in plasma, and endonucleases are known to oligonucleotides are quite efficient in entering the cell and fragment DNA into larger pieces, thereby protecting the short inhibiting DNA methylation. Dinucleotides, trinucleotides, or oligonucleotides from degradation (34–36). Furthermore, CpG tetranucleotides containing one or more 5-aza-CdR residues are oligodeoxynucleotides must retain the CpG motifs to generate as effective as 5-aza-CdR in inducing p16 expression and immune response, which provides evidence that short oligonucleo- decreasing global methylation levels. These short-chain inhibitors tides are not subject to nuclease degradation extracellularly (37). It may be more resistant to cytidine deaminase degradation and be is probable then that the short oligonucleotide DNA methylation particularly useful for clinical utility. We have therefore intro- inhibitors retain their structure when they enter the cell and are duced a novel drug delivery system in which the oligonucleotides only cleaved in the cells. are not restricted to their sequences for therapeutic potential and Oligonucleotides are thought to internalize themselves into cells provide protection against enzymes involved in the drug through both passive and active mechanisms (31). Much effort has metabolism. This may find numerous applications in delivering been devoted to expedite such uptake with liposomal encapsula- 5¶-phosphates to cells. In addition, other nucleoside drugs that are tion and peptide-mediated delivery of these oligonucleotides subject to enzymatic degradation may benefit by using this (38, 39). Several membrane-bound proteins and receptors were delivery method. The true merits of these short oligonucleotide found to interact with oligonucleotides and facilitate their cellular DNA methylation inhibitors may only be realized in a clinical uptake (40–43). There are also speculations about cell surface setting. channel proteins that conduct nucleic acids and oligonucleotides (44). Other transmembrane proteins are thought to cleave phosphodiester and phosphosulfate bonds at the cell surface (45). Acknowledgments There are therefore many possible mechanisms by which short Received 1/19/2007; revised 3/28/2007; accepted 4/24/2007. oligonucleotides may enter the cell. Grant support: NIH grant R01 CA82422. The costs of publication of this article were defrayed in part by the payment of page Once the oligonucleotides are internalized, they are probably charges. This article must therefore be hereby marked advertisement in accordance cleaved into individual nucleotides and nucleosides. The most with 18 U.S.C. Section 1734 solely to indicate this fact.

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