Cytosine Methylation Enhances Mitoxantrone-DNA Adduct Formation at CpG Dinucleotides

Parker, Belinda S.; Cutts, Suzanne M.; Phillips, Don R.

doi:10.1074/jbc.m009216200

Cited by 25 publications

(26 citation statements)

References 17 publications

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“…8), and in a cellular environment, this may endow these adducts with the capacity to alter the expression of the genes in which they occur; this is presently under examination. Indeed, other CpG-specific alkylators, including formaldehyde-activated mitoxantrone, mitomycin C, and the carcinogen benzo(a)pyrene diolepoxide, are also enhanced by CpG methylation (Li et al, 2000;Parker et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Formaldehyde-Activated Pixantrone Is a Monofunctional DNA Alkylator That Binds Selectively to CpG and CpA Doublets

Evison¹,

Chiu²,

Pezzoni³

et al. 2008

Molecular Pharmacology

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The topoisomerase II poison mitoxantrone is important in the clinical management of human malignancies. Pixantrone, a novel aza-anthracenedione developed to improve the therapeutic profile of mitoxantrone, can efficiently alkylate DNA after formaldehyde activation. In vitro transcriptional analysis has now established that formaldehyde-activated pixantrone generates covalent adducts selectively at discrete CpG or CpA dinucleotides, suggesting that the activated complex binds to guanine or cytosine (or both) bases. The stability of pixantrone adduct-induced transcriptional blockages varied considerably, reflecting a mixture of distinct pixantrone adduct types that may include relatively labile monoadducts and more stable interstrand cross-links. 6,9-Bis-[[2-(dimethylamino)ethyl]amino]benzo[g]isoquinoline-5,10-dione (BBR 2378), the dimethyl N-substituted analog of pixantrone, could not form adducts, suggesting that pixantrone alkylates DNA through the primary amino functions located in each side chain of the drug. Pixantrone generated DNA adducts only when guanine was present in substrates and exhibited a lack of adduct formation with inosine-containing polynucleotides, confirming that the N2 amino group of guanine is the site for covalent attachment of the drug. Mass spectrometric analysis of oligonucleotide-drug complexes confirmed that formation of covalent pixantrone-DNA adducts is mediated by a single methylene linkage provided by formaldehyde and that this occurs only with guanine-containing double stranded oligonucleotide substrates. CpG methylation, an epigenetic modification of the mammalian genome, significantly enhanced the generation of pixantrone-DNA adducts within a methylated DNA substrate, indicating that the methylated dinucleotide may be a favored target in a cellular environment.

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Section: Discussionmentioning

confidence: 99%

Formaldehyde-Activated Pixantrone Is a Monofunctional DNA Alkylator That Binds Selectively to CpG and CpA Doublets

Evison¹,

Chiu²,

Pezzoni³

et al. 2008

Molecular Pharmacology

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“…It is known that mitoxantrone binds covalently to DNA specifically and preferentially at methylated CpG sites. 7 We predicted that higher the levels of methylated DNA in cells, the greater the mitoxantrone cytotoxicity. Conversely, reducing the level of methylation in cells would result in a lowered cytotoxicity of the drug.…”

Section: Reduced Levels Of Methylation In Cells Reduces Cytotoxicity Ofmentioning

confidence: 99%

“…4 In addition, mitoxantrone can also be activated by formaldehyde in cell-free systems to bind covalently to DNA, 5 specifically at CpG and CpA sequences in DNA, producing labile drug-DNA adducts which can be detected as 'virtual' interstrand crosslinks. 6 Further work revealed that drug binding was enhanced 3-fold when CpG sites in DNA were methylated 7 and that the cytotoxic effects of mitoxantrone are augmented by formaldehyde. AN-9 (pivaloyloxymethyl butyrate), a butyric acid prodrug, upon hydrolysis releases butyric acid, pivalic acid and formaldehyde.…”

Section: Introductionmentioning

confidence: 99%

Mitoxantrone Mediates Demethylation and Re-Expression of Cyclin D2, Estrogen Receptor 14.3.3 Sigma In Breast Cancer Cells

Parker¹,

Cutts²,

Nudelman³

et al. 2003

Cancer Biology & Therapy

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In addition to its action as a topoisomerase II poison, mitoxantrone is activated by formaldehyde to bind DNA, forming DNA-adducts specifically at 5'CpG and CpA sequences, with an enhancement of adducts at methylated CpG sites. The butyric acid prodrug, AN-9 (pivaloyloxymethyl butyrate), releases formaldehyde upon cellular hydrolysis and our previous studies have shown that mitoxantrone acts synergistically with AN-9 in cytotoxicity assays. In this paper, we investigated the impact of methylation levels in the cell on mitoxantrone-induced cytotoxicity using the colon cancer cell line HCT116 and its derived DNA methyltransferase (DNMT) 1 and DNMT 3a knockout (DKO8) cell line. We found that decreased methylation levels in the DNMT-null cells led to at least a 2-fold reduction in mitoxantrone-induced cytotoxicity. Next, we studied the impact of mitoxantrone alone, and in combination with AN-9, on hypermethylated genes and their mRNA expression in breast cancer cells. Using methylation-specific PCR and RT-PCR, we found that mitoxantrone treatment of breast cancer cell lines resulted in demethylation of the 14.3.3σ, Cyclin D2 and ERα genes, followed by re-expression of their mRNA. The effect of mitoxantrone on re-expression of key genes involved in cell cycle regulation, and ensuing death of the cells may be an additional, previously undiscovered mechanism of action of mitoxantrone.

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“…22 The cytosine residue in CpG sequences (found disproportionately in regulatory promoter regions) is often methylated, with methylation typically occurring after DNA replication. A bis-amino mitoxantrone analogue, named herein WEHI-150 (see Figure 1), was synthesised in an attempt to develop anthracenedione analogues that are more reactive with DNA than mitoxantrone and pixantrone, but are also more likely to form covalent cross-links between the two DNA strands, through the formation of an adduct with a covalent bond to a guanine residue on each strand of the duplex.…”

Section: Introductionmentioning

confidence: 99%

Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA

et al. 2016

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Collins, J. (2016). Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA. Organic and Biomolecular Chemistry, 14 (20), 4728-4738. Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA AbstractThe ability of a bis-amino mitoxantrone anticancer drug (named WEHI-150) to form covalent adducts with DNA, after activation by formaldehyde, has been studied by electrospray ionisation mass spectrometry and HPLC. Mass spectrometry results showed that WEHI-150 could form covalent adducts with d(ACGCGCGT)2 that contained one, two or three covalent links to the octanucleotide, whereas the control drugs (daunorubicin and the anthracenediones mitoxantrone and pixantrone) only formed adducts with one covalent link to the octanucleotide. HPLC was used to examine the extent of covalent bond formation of WEHI-150 with d(CGCGCG)2 and d(CG5MeCGCG)2. Incubation of WEHI-150 with d(CG5MeCGCG)2 in the presence of formaldehyde resulted in the formation of significantly greater amounts of covalent adducts than was observed with d(CGCGCG)2. In order to understand the observed increase of covalent adducts with d(CG5MeCGCG)2, an NMR study of the reversible interaction of WEHI-150 at both CpG and 5MeCpG sites was undertaken. Intermolecular NOEs were observed in the NOESY spectra of d(ACGGCCGT)2 with added WEHI-150 that indicated that the drug selectively intercalated at the CpG sites and from the major groove. In particular, NOEs were observed from the WEHI-150 H2,3 protons to the H1′ protons of G3 and G7 and from the H6,7 protons to the H5 protons of C2 and C6. By contrast, intermolecular NOEs were observed between the WEHI-150 H2,3 protons to the H2′′ proton of the 5MeC3 in d(CG5MeCGCG)2, and between the drug aliphatic protons and the H1′ proton of G4. This demonstrated that WEHI-150 preferentially intercalates at 5MeCpG sites, compared to CpG sequences, and predominantly via the minor groove at the 5MeCpG site. The results of this study demonstrate that WEHI-150 is likely to form interstrand DNA cross-links, upon activation by formaldehyde, and consequently exhibit greater cytotoxicity than other current anthracenedione drugs. Disciplines Medicine and Health Sciences | Social and Behavioral Sciences Publication DetailsKonda, S., Kelso, C., Pumuye, P. P., Medan, J., Sleebs, B. E., Cutts, S. M., Phillips, D. R. & Collins, J. (2016

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Cytosine Methylation Enhances Mitoxantrone-DNA Adduct Formation at CpG Dinucleotides

Cited by 25 publications

References 17 publications

Formaldehyde-Activated Pixantrone Is a Monofunctional DNA Alkylator That Binds Selectively to CpG and CpA Doublets

Formaldehyde-Activated Pixantrone Is a Monofunctional DNA Alkylator That Binds Selectively to CpG and CpA Doublets

Mitoxantrone Mediates Demethylation and Re-Expression of Cyclin D2, Estrogen Receptor 14.3.3 Sigma In Breast Cancer Cells

Reversible and formaldehyde-mediated covalent binding of a bis-amino mitoxantrone analogue to DNA

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