Cytosine Methylation Enhances Mitoxantrone-DNA Adduct Formation at CpG Dinucleotides
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m009216200
ISSN1083-351X
AutoresBelinda S. Parker, Suzanne M. Cutts, Don R. Phillips,
Tópico(s)Genomics and Chromatin Dynamics
ResumoRecently, 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 majority of CpG dinucleotides in the mammalian genome are methylated and hypermethylation in subsets of genes is associated with various neoplasms. Upon methylation of a 512-base pair DNA fragment (containing the lac UV5 promoter) using HpaII methylase, three CCGG sites downstream of the promoter were methylated at C5 of the internal cytosine residue. In vitrotranscription studies of mitoxantrone-reacted DNA revealed a 3-fold enhancement in transcriptional blockage (and hence adduct formation) exclusively at these methylated sites. In vitrocross-linking assays also revealed that methylation enhanced mitoxantrone adduct formation by 2–3-fold, and methylation of cytosine at a single potential drug binding site on a duplex oligonucleotide also enhanced adduct levels by 3-fold. Collectively, these results indicate preferential adduct formation at methylated CpG sites. However, adducts at these methylated sites exhibited the same stability as nonmethylated sites, suggesting that cytosine methylation increases drug accessibility to DNA rather than being involved in kinetic stabilization of the adduct. 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 majority of CpG dinucleotides in the mammalian genome are methylated and hypermethylation in subsets of genes is associated with various neoplasms. Upon methylation of a 512-base pair DNA fragment (containing the lac UV5 promoter) using HpaII methylase, three CCGG sites downstream of the promoter were methylated at C5 of the internal cytosine residue. In vitrotranscription studies of mitoxantrone-reacted DNA revealed a 3-fold enhancement in transcriptional blockage (and hence adduct formation) exclusively at these methylated sites. In vitrocross-linking assays also revealed that methylation enhanced mitoxantrone adduct formation by 2–3-fold, and methylation of cytosine at a single potential drug binding site on a duplex oligonucleotide also enhanced adduct levels by 3-fold. Collectively, these results indicate preferential adduct formation at methylated CpG sites. However, adducts at these methylated sites exhibited the same stability as nonmethylated sites, suggesting that cytosine methylation increases drug accessibility to DNA rather than being involved in kinetic stabilization of the adduct. N,N,N′,N′-tetramethylethylenediamine base pair(s) Mitoxantrone is a synthetic anticancer agent that is a member of the anthracenedione class of compounds and was originally designed as a simplified analogue of the anthraquinone-containing anthracyclines (1De Vita V.T. Hellman S. Rosenberg S.A. De Vita V.T. Cancer: Principles and Practice of Oncology. J. B. Lippincott Co., Philadelphia1993Google Scholar,2Feofanov A. Sharonov S. Fleury F. Kudelina I. Nabiev I. Biophys. J. 1997; 73: 3328-3336Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Because of the similar cytotoxicity to the anthracyclines yet lack of associated cardiotoxicity (3Myers C.E. Mimnaugh E.G. Yeh G.C. Sinha B.K. Lown J.W. Anthracycline and Anthracenedione-based Anti-cancer Agents. Elsevier, Amsterdam1988: 527-569Google Scholar, 4Pratt W.B. Ruddon R.W. Ensminger W.D. Maybaum S. The Anticancer Drugs. 2nd Ed. Oxford University Press, New York1994Google Scholar), mitoxantrone is an important anticancer agent and has good activity against solid tumors and myeloid cancers (5Cornbleet M.A. Stuart-Harris R.C. Smith I.E. Coleman R.E. Rubens R.D. McDonald M. Mouridsen H.T. Rainer H. Van Oosterom A.T. Smyth J.F. Eur. J. Cancer Oncol. 1984; 20: 1141-1146Abstract Full Text PDF PubMed Scopus (81) Google Scholar).Recently, we have found that formaldehyde facilitates the formation of mitoxantrone-DNA adducts and that these adducts stabilize duplex DNA sufficiently to prevent strand separation in in vitrocross-linking assays (6Parker B.S. Cullinane C. Phillips D.R. Nucleic Acids Res. 1999; 27: 2918-2923Crossref PubMed Scopus (32) Google Scholar). This structure was unstable and heat-labile, therefore not representing a classical covalent cross-link, and like Adriamycin adducts these structures have been termed “virtual cross-links” (6Parker B.S. Cullinane C. Phillips D.R. Nucleic Acids Res. 1999; 27: 2918-2923Crossref PubMed Scopus (32) Google Scholar). The possible potentiation of mitoxantrone cytotoxicity in the presence of formaldehyde may provide an explanation as to why mitoxantrone is most effective against myeloid cancers, since they are known to have higher levels of formaldehyde (7Thorndike J. Beck W.S. Cancer Res. 1977; 37: 1125-1132PubMed Google Scholar). We have also shown that these formaldehyde-activated mitoxantrone-DNA adducts form predominantly at CpG and CpA sequences (8Parker B.S. Cutts S.M. Cullinane C. Phillips D.R. Nucleic Acids Res. 2000; 28: 982-990Crossref PubMed Scopus (31) Google Scholar).The CpG specificity of adduct formation in vitro suggested that methylation may modulate adduct formation in cells. It is known that CpG dinucleotides are underrepresented in the genome with significant depletion of the doublet in ∼99% of the genome in higher eukaryotes, whereas the remaining 1% contains CpG at the expected frequency (9Millard J.T. Beachy T.M. Biochemistry. 1993; 32: 12850-12856Crossref PubMed Scopus (25) Google Scholar, 10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar). In mammalian species, these CpG dinucleotides are known to be highly methylated with 60–90% methylation at the 5-position of cytosine, whereas only 3–5% of isolated cytosine residues occur as 5-methylcytosine (9Millard J.T. Beachy T.M. Biochemistry. 1993; 32: 12850-12856Crossref PubMed Scopus (25) Google Scholar, 10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar). Control of the methylation status of cytosines plays a central role in regulating expression of genes during mammalian development (11Clark S. Proc. Aust. Soc. Biochem. Mol. Biol. 1999; 30: 9-11Google Scholar). In contrast, tumor cells often have aberrant DNA methylation, and it has been hypothesized that altered methylation is an important component of neoplastic transformation (10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar). Past studies have reported hypomethylation of the genome and of specific genes in human tumors when compared with noncancerous cells (12Baylin S.B. Hoppener J.W.M. Bustros A.D. Steenbergh P.H. Lips C.J.M. Nelkin B.D. Cancer Res. 1986; 46: 2917-2922PubMed Google Scholar, 13Jones P.A. Buckley J.D. Adv. Cancer Res. 1990; 54: 1-23Crossref PubMed Scopus (227) Google Scholar, 14Villain A. Vogt N. Dutrillaux B. Malfoy B. FEBS Lett. 1999; 460: 231-234Crossref PubMed Scopus (37) Google Scholar). The demethylation appears to be random, because specific genes that presumably would not contribute to tumor progression (not involved in cell proliferation and suppression) have been found to be hypomethylated (10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar).However, it has also been reported that there is an increase in methyltransferase activity and regions of hypermethylation in some cancer cell lines. These findings are surprising considering the widespread hypomethylation of DNA in tumors. In some cell lines, DNA methylase activity has been found to be abnormally high, with a 30–50-fold increase in virally transformed cells and a several hundred-fold increase in human cancer cells (15El-Deiry W.S. Nelkin B.D. Celano P. Yen R.-W.C. Falco J.P. Hamilton S.R. Baylin S.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3470-3474Crossref PubMed Scopus (293) Google Scholar). However, this is not accompanied by increased global methylation (16Szyf M. Knox D.J. Milutinovic S. Slack A.D. Araujo F.D. Ann. N. Y. Acad. Sci. 2000; 910: 156-174Crossref PubMed Scopus (29) Google Scholar). Baylin et al. (12Baylin S.B. Hoppener J.W.M. Bustros A.D. Steenbergh P.H. Lips C.J.M. Nelkin B.D. Cancer Res. 1986; 46: 2917-2922PubMed Google Scholar) have examined methylation of the calcitonin gene in human lymphoid and acute myeloid malignancies in both cultured and uncultured tumors and detected increased methylation at CCGG sites in the 5′ region of the gene. An increase in methylation was found in 90% of patients with non-Hodgkin's lymphoid neoplasms and in 95% of tumor cell DNA extracted from patients with acute nonlymphocytic leukemia. These unusual methylation patterns in the calcitonin gene were found much less frequently in other tumor types (12Baylin S.B. Hoppener J.W.M. Bustros A.D. Steenbergh P.H. Lips C.J.M. Nelkin B.D. Cancer Res. 1986; 46: 2917-2922PubMed Google Scholar, 13Jones P.A. Buckley J.D. Adv. Cancer Res. 1990; 54: 1-23Crossref PubMed Scopus (227) Google Scholar). More recently, Clark and colleagues (17Melki J.R. Vincent P.C. Clark S.J. Cancer Res. 1999; 59: 3730-3740PubMed Google Scholar) analyzed the methylation pattern of a number of tumor-related genes in leukemia and found that 90% of acute myeloid leukemia patients exhibited an abnormal methylation pattern with hypermethylation in at least one gene, and 75% exhibited an increase in methylation in two or more of the target genes. This was in contrast to the normal control samples, which were essentially unmethylated. Hypermethylation in leukemia is therefore not limited to single genes (17Melki J.R. Vincent P.C. Clark S.J. Cancer Res. 1999; 59: 3730-3740PubMed Google Scholar). In contrast, a recent global analysis of the methylation status of 1200 CpG islands in 98 primary human tumors found that some tumors (including breast and testicular tumors) displayed relatively decreased levels of methylation; however, others (including myeloid leukemias) displayed a higher frequency of methylation (18Costello J.F. Fruhwald M.C. Smiraglia D.J. Rush L.J. Robertson G.P. Gao X. Wright F.A. Feramisco J.D. Peltomaki P. Lang J.C. Schuller D.E., Yu, L. Bloomfield C.D. Caligiuri M.A. Yates A. Nishikawa R. Huang H.-J.S. Petrelli N.J. Zhang X. O'Dorisio M.S. Held W.A. Cavenee W.K. Plass C. Nat. Genet. 2000; 25: 132-138Crossref PubMed Scopus (1156) Google Scholar).Since mitoxantrone-induced DNA adducts have been found to occur preferentially at CG sites, which are the dinucleotide substrates of methylation, and because significant change in methylation status accompanies neoplasia, we aimed to establish whether methylation modulates the ability of mitoxantrone to form adducts with CpG sequences. The ultimate objective is to provide insight into the mechanism of adduct formation at methylated DNA sites in tumor cells and to establish how the extent of methylation of DNA contributes to the sensitivity of cells to mitoxantrone. Mitoxantrone is a synthetic anticancer agent that is a member of the anthracenedione class of compounds and was originally designed as a simplified analogue of the anthraquinone-containing anthracyclines (1De Vita V.T. Hellman S. Rosenberg S.A. De Vita V.T. Cancer: Principles and Practice of Oncology. J. B. Lippincott Co., Philadelphia1993Google Scholar,2Feofanov A. Sharonov S. Fleury F. Kudelina I. Nabiev I. Biophys. J. 1997; 73: 3328-3336Abstract Full Text PDF PubMed Scopus (48) Google Scholar). Because of the similar cytotoxicity to the anthracyclines yet lack of associated cardiotoxicity (3Myers C.E. Mimnaugh E.G. Yeh G.C. Sinha B.K. Lown J.W. Anthracycline and Anthracenedione-based Anti-cancer Agents. Elsevier, Amsterdam1988: 527-569Google Scholar, 4Pratt W.B. Ruddon R.W. Ensminger W.D. Maybaum S. The Anticancer Drugs. 2nd Ed. Oxford University Press, New York1994Google Scholar), mitoxantrone is an important anticancer agent and has good activity against solid tumors and myeloid cancers (5Cornbleet M.A. Stuart-Harris R.C. Smith I.E. Coleman R.E. Rubens R.D. McDonald M. Mouridsen H.T. Rainer H. Van Oosterom A.T. Smyth J.F. Eur. J. Cancer Oncol. 1984; 20: 1141-1146Abstract Full Text PDF PubMed Scopus (81) Google Scholar). Recently, we have found that formaldehyde facilitates the formation of mitoxantrone-DNA adducts and that these adducts stabilize duplex DNA sufficiently to prevent strand separation in in vitrocross-linking assays (6Parker B.S. Cullinane C. Phillips D.R. Nucleic Acids Res. 1999; 27: 2918-2923Crossref PubMed Scopus (32) Google Scholar). This structure was unstable and heat-labile, therefore not representing a classical covalent cross-link, and like Adriamycin adducts these structures have been termed “virtual cross-links” (6Parker B.S. Cullinane C. Phillips D.R. Nucleic Acids Res. 1999; 27: 2918-2923Crossref PubMed Scopus (32) Google Scholar). The possible potentiation of mitoxantrone cytotoxicity in the presence of formaldehyde may provide an explanation as to why mitoxantrone is most effective against myeloid cancers, since they are known to have higher levels of formaldehyde (7Thorndike J. Beck W.S. Cancer Res. 1977; 37: 1125-1132PubMed Google Scholar). We have also shown that these formaldehyde-activated mitoxantrone-DNA adducts form predominantly at CpG and CpA sequences (8Parker B.S. Cutts S.M. Cullinane C. Phillips D.R. Nucleic Acids Res. 2000; 28: 982-990Crossref PubMed Scopus (31) Google Scholar). The CpG specificity of adduct formation in vitro suggested that methylation may modulate adduct formation in cells. It is known that CpG dinucleotides are underrepresented in the genome with significant depletion of the doublet in ∼99% of the genome in higher eukaryotes, whereas the remaining 1% contains CpG at the expected frequency (9Millard J.T. Beachy T.M. Biochemistry. 1993; 32: 12850-12856Crossref PubMed Scopus (25) Google Scholar, 10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar). In mammalian species, these CpG dinucleotides are known to be highly methylated with 60–90% methylation at the 5-position of cytosine, whereas only 3–5% of isolated cytosine residues occur as 5-methylcytosine (9Millard J.T. Beachy T.M. Biochemistry. 1993; 32: 12850-12856Crossref PubMed Scopus (25) Google Scholar, 10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar). Control of the methylation status of cytosines plays a central role in regulating expression of genes during mammalian development (11Clark S. Proc. Aust. Soc. Biochem. Mol. Biol. 1999; 30: 9-11Google Scholar). In contrast, tumor cells often have aberrant DNA methylation, and it has been hypothesized that altered methylation is an important component of neoplastic transformation (10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar). Past studies have reported hypomethylation of the genome and of specific genes in human tumors when compared with noncancerous cells (12Baylin S.B. Hoppener J.W.M. Bustros A.D. Steenbergh P.H. Lips C.J.M. Nelkin B.D. Cancer Res. 1986; 46: 2917-2922PubMed Google Scholar, 13Jones P.A. Buckley J.D. Adv. Cancer Res. 1990; 54: 1-23Crossref PubMed Scopus (227) Google Scholar, 14Villain A. Vogt N. Dutrillaux B. Malfoy B. FEBS Lett. 1999; 460: 231-234Crossref PubMed Scopus (37) Google Scholar). The demethylation appears to be random, because specific genes that presumably would not contribute to tumor progression (not involved in cell proliferation and suppression) have been found to be hypomethylated (10Holliday R. Grigg G.W. Mutat. Res. 1993; 285: 61-67Crossref PubMed Scopus (264) Google Scholar). However, it has also been reported that there is an increase in methyltransferase activity and regions of hypermethylation in some cancer cell lines. These findings are surprising considering the widespread hypomethylation of DNA in tumors. In some cell lines, DNA methylase activity has been found to be abnormally high, with a 30–50-fold increase in virally transformed cells and a several hundred-fold increase in human cancer cells (15El-Deiry W.S. Nelkin B.D. Celano P. Yen R.-W.C. Falco J.P. Hamilton S.R. Baylin S.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3470-3474Crossref PubMed Scopus (293) Google Scholar). However, this is not accompanied by increased global methylation (16Szyf M. Knox D.J. Milutinovic S. Slack A.D. Araujo F.D. Ann. N. Y. Acad. Sci. 2000; 910: 156-174Crossref PubMed Scopus (29) Google Scholar). Baylin et al. (12Baylin S.B. Hoppener J.W.M. Bustros A.D. Steenbergh P.H. Lips C.J.M. Nelkin B.D. Cancer Res. 1986; 46: 2917-2922PubMed Google Scholar) have examined methylation of the calcitonin gene in human lymphoid and acute myeloid malignancies in both cultured and uncultured tumors and detected increased methylation at CCGG sites in the 5′ region of the gene. An increase in methylation was found in 90% of patients with non-Hodgkin's lymphoid neoplasms and in 95% of tumor cell DNA extracted from patients with acute nonlymphocytic leukemia. These unusual methylation patterns in the calcitonin gene were found much less frequently in other tumor types (12Baylin S.B. Hoppener J.W.M. Bustros A.D. Steenbergh P.H. Lips C.J.M. Nelkin B.D. Cancer Res. 1986; 46: 2917-2922PubMed Google Scholar, 13Jones P.A. Buckley J.D. Adv. Cancer Res. 1990; 54: 1-23Crossref PubMed Scopus (227) Google Scholar). More recently, Clark and colleagues (17Melki J.R. Vincent P.C. Clark S.J. Cancer Res. 1999; 59: 3730-3740PubMed Google Scholar) analyzed the methylation pattern of a number of tumor-related genes in leukemia and found that 90% of acute myeloid leukemia patients exhibited an abnormal methylation pattern with hypermethylation in at least one gene, and 75% exhibited an increase in methylation in two or more of the target genes. This was in contrast to the normal control samples, which were essentially unmethylated. Hypermethylation in leukemia is therefore not limited to single genes (17Melki J.R. Vincent P.C. Clark S.J. Cancer Res. 1999; 59: 3730-3740PubMed Google Scholar). In contrast, a recent global analysis of the methylation status of 1200 CpG islands in 98 primary human tumors found that some tumors (including breast and testicular tumors) displayed relatively decreased levels of methylation; however, others (including myeloid leukemias) displayed a higher frequency of methylation (18Costello J.F. Fruhwald M.C. Smiraglia D.J. Rush L.J. Robertson G.P. Gao X. Wright F.A. Feramisco J.D. Peltomaki P. Lang J.C. Schuller D.E., Yu, L. Bloomfield C.D. Caligiuri M.A. Yates A. Nishikawa R. Huang H.-J.S. Petrelli N.J. Zhang X. O'Dorisio M.S. Held W.A. Cavenee W.K. Plass C. Nat. Genet. 2000; 25: 132-138Crossref PubMed Scopus (1156) Google Scholar). Since mitoxantrone-induced DNA adducts have been found to occur preferentially at CG sites, which are the dinucleotide substrates of methylation, and because significant change in methylation status accompanies neoplasia, we aimed to establish whether methylation modulates the ability of mitoxantrone to form adducts with CpG sequences. The ultimate objective is to provide insight into the mechanism of adduct formation at methylated DNA sites in tumor cells and to establish how the extent of methylation of DNA contributes to the sensitivity of cells to mitoxantrone.
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