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

Parker, Belinda S.; Cutts, Suzanne M.; Nudelman, Abraham; Rephaeli, Ada; Phillips, Don R.; Sukumar, Saraswati

doi:10.4161/cbt.2.3.364

Cited by 26 publications

(18 citation statements)

References 15 publications

(24 reference statements)

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“…Both agents induce global and localized CpG island-demethylation and re-expression of the associated genes in a cellular environment (55,57) and these effects may contribute towards their growth inhibitory properties. Similarly, formaldehyde-activated mitoxantrone, a close structural analogue of pixantrone, can also mediate localized demethylation of CpG islands in several cancer-associated genes (58). A functional consequence of this demethylation was the reactivation of the associated gene.…”

Section: Discussionmentioning

confidence: 99%

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity

Evison

Bilardi

Chiu

et al. 2009

Nucleic Acids Research

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DNA methylation is an epigenetic modification of the mammalian genome that occurs predominantly at cytosine residues of the CpG dinucleotide. Following formaldehyde activation, pixantrone alkylates DNA and particularly favours the CpG motif. Aberrations in CpG methylation patterns are a feature of most cancer types, a characteristic that may determine their susceptibility to specific drug treatments. Given their common target, DNA methylation may modulate the DNA damage induced by formaldehyde-activated pixantrone. In vitro transcription, mass spectrometry and oligonucleotide band shift assays were utilized to establish that pixantrone–DNA adduct formation was consistently enhanced 2–5-fold at discrete methylated CpG doublets. The methylation-mediated enhancement was exquisitely sensitive to the position of the methyl substituent since methylation at neighboring cytosine residues failed to confer an increase in pixantrone–DNA alkylation. Covalent modification of DNA by formaldehyde-activated doxorubicin, but not cisplatin, was augmented by neighbouring CpG methylation, indicating that modulation of binding by CpG methylation is not a general feature of all alkylators. HCT116 colon cancer cells vastly deficient in CpG methylation were 12- and 10-fold more resistant to pixantrone and doxorubicin relative to the wild-type line, suggesting that these drugs may selectively recognize the aberrant CpG methylation profiles characteristic of most tumour types.

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Section: Discussionmentioning

confidence: 99%

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity

Evison

Bilardi

Chiu

et al. 2009

Nucleic Acids Research

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“…19; summarized in Table II). To establish how cytosine methylation caused such a dramatic enhancement of adduct formation together with a corresponding increase of biological activity (19,20) we determined the relative free energy of binding of mitoxantrone to each se- quence (with intercalation of the drug chromophore between the C and G residues) with the drug intercalated in either the major or minor groove, and these energies are summarized in Table II. Binding via the major groove was much more favorable than the minor groove for the non-methylated sequence, and this is consistent with previous NMR studies which revealed that the drug side chains were located in the major groove (29).…”

Section: Fig 4 Effect Of Guanine Modifications On Mitoxantrone-dna mentioning

confidence: 99%

“…Recently, we have shown that in tumor cells in culture the activity of mitoxantrone depends on the methylation status of the cell (20). Mitoxantrone-DNA adduct levels decreased when breast cancer cells were pretreated with the DNA de-methylating agent 5-aza-2-deoxycytidine, 1 and DK08 cells in which the DNA methyltransferase activity had been knocked out (resulting in a 50% decrease of methylation levels compared with the parental HCT116 cells) were also less sensitive to mitoxantrone (20).…”

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confidence: 99%

“…Mitoxantrone-DNA adduct levels decreased when breast cancer cells were pretreated with the DNA de-methylating agent 5-aza-2-deoxycytidine, 1 and DK08 cells in which the DNA methyltransferase activity had been knocked out (resulting in a 50% decrease of methylation levels compared with the parental HCT116 cells) were also less sensitive to mitoxantrone (20). Both of these results are consistent with the view that some of the mechanism of action of mitoxantrone may be due to formaldehyde-mediated formation of mitoxantrone-DNA adducts at methylated CpG sequences.…”

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A Molecular Understanding of Mitoxantrone-DNA Adduct Formation

Parker

Buley

Evison

et al. 2004

Journal of Biological Chemistry

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When mitoxantrone is activated by formaldehyde it can form adducts with DNA. These occur preferentially at CpG and CpA sequences and are enhanced 2-3-fold at methylated CpG sequences compared with non-methylated sites. We sought to understand the molecular factors involved in enhanced adduct formation at these methylated sites. This required, first, clarification of factors that contributed to the formation of adducts at CpG sites. For this purpose mass spectrometry of an oligonucleotide duplex (containing a single CpG adduct site) was used to confirm the presence of an additional carbon atom (derived from formaldehyde) on the drug-DNA complex. The effect of 3-flanking sequences was revealed by electrophoretic analysis of oligonucleotidedrug adducts, and the preferred adduct-forming site was identified as 5-CGG-3. Radiolabeled studies of drug-DNA adducts confirmed that the site of attachment involved the exocyclic amino of guanine. Molecular modeling analysis of the relative stability of the intercalated form of mitoxantrone was consistent with observed adduct-forming potential of CG sites with varying flanking sequences. The known preference for adduct formation at methylated CG sites was confirmed by energetics calculations and shown to be due to a shift of equilibrium of the intercalated form of the drug from the major groove (at CG sites) to the minor groove (at methylated CG sites). This increases the relative amount of drug that is located adjacent to the N-2 exocyclic amino of guanine in the minor groove, where covalent linkage is facilitated. These results account for the enhanced covalent binding of mitoxantrone to methylated CG sequences and provide a molecular model of the interactions.

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“…Since hypermethylated genes may be involved in the etiology of the disease and may be early events in neoplastic progression, these genes make attractive targets for therapeutics and disease detection/monitoring (Bovenzi et al, 1999;Evron et al, 2001a;Umbricht et al, 2001;Muller et al, 2003;Oshiro et al, 2003;Parker et al, 2003). Agents that reactivate gene expression silenced by methylation have favorable therapeutic indices and are now entering clinical trials for a variety of cancers (Baylin, 2004;Dowell and Minna, 2004;Egger et al, 2004;Zelent et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

Analysis of methylation-sensitive transcriptome identifies GADD45a as a frequently methylated gene in breast cancer

et al. 2005

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Treatment of the breast cancer cell line, MDAMB468 with the DNA methylation inhibitor, 5-azacytidine (5-AzaC) results in growth arrest, whereas the growth of the normal breast epithelial line DU99 (telomerase immortalized) is relatively unaffected. Comparing gene expression profiles of these two lines after 5-AzaC treatment, we identified 36 genes that had relatively low basal levels in MDAMB468 cells compared to the DU99 line and were induced in the cancer cell line but not in the normal breast epithelial line. Of these genes, 33 have associated CpG islands greater than 300 bp in length but only three have been previously described as targets for aberrant methylation in human cancer. Northern blotting for five of these genes (a-Catenin, DTR, FYN, GADD45a, and Zyxin) verified the array results. Further analysis of one of these genes, GADD45a, showed that 5-AzaC induced expression in five additional breast cancer cell lines with little or no induction in three additional lines derived from normal breast epithelial cells. The CpG island associated with GADD45a was analysed by bisulfite sequencing, sampling over 100 CpG dinucleotides. We found that four CpG's, located approximately 700 bp upstream of the transcriptional start site are methylated in the majority of breast cancer cell lines and primary tumors but not in DNA from normal breast epithelia or matched lymphocytes from cancer patients. Therefore, this simple method of dynamic transcriptional profiling yielded a series of novel methylation-sensitive genes in breast cancer including the BRCA1 and p53 responsive gene, GADD45a.

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

Cited by 26 publications

References 15 publications

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity

CpG methylation potentiates pixantrone and doxorubicin-induced DNA damage and is a marker of drug sensitivity

A Molecular Understanding of Mitoxantrone-DNA Adduct Formation

Analysis of methylation-sensitive transcriptome identifies GADD45a as a frequently methylated gene in breast cancer

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