Cytosine methylation enhances mitoxantrone-DNA adduct formation at CpG dinucleotides

Recently, we have shown that mitoxantrone can be activated by formaldehyde in vitro to form DNA adducts that are specific for CpG and CpA sites in DNA . The CpG specificity of adduct formation prompted investigations into the effect of cytosine methylation (CpG) on adduct formation, since the majorit y of CpG dinucleotides in the mammalian genome are methylated and hypermeth ylation in subsets of genes is associated with various neoplasms. Upon meth ylation of a 512-base pair DNA fragment (containing the lac UV5 promoter) u sing HpaII methylase, three CCGG sites downstream of the promoter were meth ylated at C5 of the internal cytosine residue. In vitro transcription studi es of mitoxantrone-reacted DNA revealed a 3-fold enhancement in transcripti onal blockage (and hence adduct formation) exclusively at these methylated sites. lit vitro cross-linking assays also revealed that methylation enhanc ed mitoxantrone adduct formation by 2-3-fold, and methylation of cytosine a t a single potential drug binding site on a duplex oligonucleotide also enh anced adduct levels by 3-fold. Collectively, these results indicate prefere ntial adduct formation at methylated CpG sites. However, adducts at these m ethylated sites exhibited the same stability as nonmethylated sites, sugges ting that cytosine methylation increases drug accessibility to DNA rather t han being involved in kinetic stabilization of the adduct.