Delivery of 5-Aza-2¶-Deoxycytidine to Cells Using Oligodeoxynucleotides 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. Jones 1,2,3 Departments of 1 Biochemistry and Molecular Biology and 2 Urology and 3 Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California and 4 SuperGen, Inc., Pleasanton, California Abstract The major goal of epigenetic therapy is to reverse aberrant promoter hypermethylation and restore normal function of tumor suppressor genes by the use of chromatin-modifying drugs. Decitabine, or 5-aza-2¶-deoxycytidine (5-aza-CdR), is a well-characterized drug that is now Food and Drug Adminis- tration approved for the treatment of myelodysplastic syndrome. Although 5-aza-CdR is an extremely potent inhibitor of DNA methylation, it is subject to degradation by hydrolytic cleavage and deamination by cytidine deaminase. We show that short oligonucleotides containing a 5-aza-CdR can also inhibit DNA methylation in cancer cells at concen- trations comparable with 5-aza-CdR. Detailed studies with S110, a dinucleotide, showed that it works via a mechanism similar to that of 5-aza-CdR after incorporation of its aza- moiety into DNA. Stability of the triazine ring in aqueous solution was not improved in the S110 dinucleotide; however, deamination by cytidine deaminase was dramatically de- creased. This is the first demonstration of the use of short oligonucleotides to provide effective delivery and cellular uptake of a nucleotide drug and protection from enzymatic degradation. This approach may pave the way for more stable and potent inhibitors of DNA methylation as well as provide means for improving existing therapeutics. [Cancer Res 2007;67(13):6400–8] Introduction Aberrations in DNA methylation are frequently observed in various types of cancer (1–3). Several tumor suppressor genes or cancer-related genes acquire de novo DNA methylation in promoter or regulatory regions leading to inactivation, contribut- ing to tumorigenesis (4–8). DNA hypermethylation can be reversed by demethylating agents and crucial cellular functions reestab- lished in the cells. In recent years, the use of DNA methylation inhibitors has become a promising alternative to patients with myelodysplastic syndrome and hematologic malignancies (9, 10). The most widely known examples of DNA methylation inhibitors are 5-azacytidine (5-aza-CR) and 5-aza-2¶-deoxycytidine (5-aza- CdR); both drugs were initially synthesized as anticancer agents and were later shown to inhibit DNA methylation (11, 12). The clinical use of nucleotides rather than nucleosides is essentially impossible due to the negative charge on the phosphate group that prevents effective cellular uptake. Thus, nucleoside analogues, which are taken up intracellularly and phosphorylated to their respective monophosphates, diphosphates, and triphosphates before incorporation into replicating DNA, leading to covalent trapping of DNA methyltransferases (DNMT), are used (13). 5-Aza- CR and 5-aza-CdR are powerful demethylating agents; nevertheless, they have a number of drawbacks. The aza pyrimidine ring is unstable in aqueous solution, making it difficult to administer, and is quite toxic both in vitro and in vivo (14). Furthermore, the drugs have transient effects, and DNA is gradually remethylated after removal of the drug (15). Yet, another problem arises due to cytidine deaminase, which renders the drugs inactive by converting them into 5-azauridine compounds. In our attempts to synthesize more stable and potent inhibitors of DNA methylation, we found that short oligonucleotides containing an azapyrimidine effectively inhibit DNA methylation in living cells. Here, we focus on S110, a 5¶-AzapG-3¶ dinucleotide, whose aqueous stability and toxicity are quite similar to that of 5-aza-CdR but is protected from deamination by cytidine deaminase. The demethylating activity seems to require incorpo- ration of the azapyrimidine into DNA presumably after degradation of the oligonucleotide by phosphodiesterases and is not limited to a dinucleotide but is seen in trinucleotides and tetranucleotides as well, showing that short oligonucleotides are effective prodrugs for delivery of inhibitors of DNA methylation. The utilization of short oligonucleotides as nucleoside drug delivery vehicles that provides protection against enzymatic degradation might have application for delivery of other nucleoside drugs to cells. Materials and Methods Synthesis of oligonucleotides containing 5-aza-CdR. Dinucleotides, trinucleotides, and tetranucleotides containing 5-aza-CdR were synthesized by standard procedures with modifications to increase coupling times, different oxidizing agents, and use of phenoxyacetyl decitabine phosphor- amidite, instead of phenoxyacetyl cytidine phosphoramidite. A polystyrene- based solid support with loading of 240 Amol/g dG(pac) or D(pac) was used (16, 17). Briefly, synthesis of S53, 5¶-GpAza-3¶ dinucleotide, is described here. Amersham A ¨ KTA Oligopilot 10 system was loaded with a protected decitabine-linked CpG solid support (phenoxyacetyl protection of amino function) and coupled with 2 to 2.5 equivalents of tert -butyl phenoxyacetyl 2¶-deoxyguanosine phosphoramidite in the presence of 60% of 0.3 mol/L benzylthiotetrazole activator in acetonitrile for 2.5 min. The CpG solid support containing protected S53 was treated with 20 mL of 50 mmol/L K 2 CO 3 in methanol for 1 h and 20 min. The coupled product was oxidized with 2 mol/L tert -butylhydroperoxide in dry acetonitrile prepared by dissolving tert -butylhydroperoxide in 80% tert -butylperoxide for 5 min. The dimethoxy trityl protective group was removed with 3% dichloroacetic acid in toluene. The CpG solid support was washed with dry methanol; the filtrate was neutralized by addition of 2 mL of 1 mol/L acetic acid in methanol. The solution was concentrated by rotary evaporation; the Requests for reprints: Peter A. Jones, University of Southern California/Norris Comprehensive Cancer Center and Hospital, 1441 Eastlake Avenue, Room 8302L, Mail Stop 83, Los Angeles, CA 90033-9181. Phone: 323-865-0816; Fax: 323-865-0102; E-mail: jones_p@ccnt.hsc.usc.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-0251 Cancer Res 2007; 67: (13). 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