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Epigenetic Gene Regulation and Cancer Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
Received May 11, 2004; accepted August 27, 2004
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Abstract |
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). DNA intercalators share common structural motifs—the presence of planar polyaromatic systems that bind by insertion between DNA base-pairs. Doxorubicin (Adriamycin), an anthracycline-based DNA intercalator, is one of the most typical anticancer drugs commonly employed in the clinic. It is well accepted that the antitumor activity of doxorubicin is caused by the formation of a cleavable complex of topoisomerase II, resulting in apoptosis (Hickman, 1992; Kiechle and Zhang, 2002). Doxorubicin is indicated in the treatment of a broad spectrum of solid tumors (e.g., breast, bladder, endometrium, thyroid, lung, ovary, stomach, and sarcomas of the bone) and in the treatment of lymphoma, as well as acute lymphoblastic and myeloblastic leukemias (Carter, 1975). One of the most important and clinically relevant side effects of doxorubicin is the induction of cardiomyopathy (Lenaz and Page, 1976). A number of mechanisms have been proposed to explain this effect of doxorubicin, including oxidative stress (Myers et al., 1977), the induction of mitochondrial damage (Wallace, 2003), and changes in gene expression in cardiac myocytes and muscle cells in general (Kurabayashi et al., 1994; Boucek et al., 1999). Therefore, doxorubicin probably affects the activity of other important proteins or pathways; these need to be elucidated to better understand and to take advantage of its antitumor activity.
DNA methyltransferase 1 (DNMT1) is the primary enzyme responsible for maintenance of DNA methylation on genomic DNA (Bestor et al., 1988; Yoder et al., 1996; Pradhan et al., 1999; Yokochi and Robertson, 2002). Disruption of DNMT1 function causes chromosome instability and dysregulation of transcription, and ultimately leads to apoptotic cell death (Robertson, 2001; Ehrlich, 2002; Jones and Baylin, 2002). Cre-mediated deletion of the Dnmt1 gene resulted in demethylation of genomic DNA in cultured murine fibroblasts and led to p53-dependent cell death (Jackson-Grusby et al., 2001), suggesting that loss of DNMT1 induces apoptosis. However, other studies have shown that deletion of the DNMT1 gene is compatible with cell viability in the HCT116 cell line (Rhee et al., 2000, 2002). The DNMT1-/- HCT116 cells exhibited normal morphology except for a slightly slower growth rate compared with the parental or DNMT1+/- cells (Rhee et al., 2000), indicating that loss of DNMT1 did not induce apoptosis in this cell line. Therefore, it was of great interest to determine whether DNMT1 is associated with apoptosis mediated by doxorubicin. Previous work has suggested that DNA methyltransferase activity in crude cell extracts could be inhibited by DNA intercalators (Adams and Rinaldi, 1987); however, the molecular mechanism of intercalator-dependent inhibition of DNMT1 functions in vitro has not been reported.
In this report, we first investigated the effect of the inhibition of DNMT1 activity by DNA intercalating drugs including doxorubicin in an in vitro assay system. Enzymatic studies demonstrate that doxorubicin is a potent inhibitor of DNMT1 activity via DNA intercalation. We also examined doxorubicin-induced cytotoxicity using the HCT116 human colorectal cancer cell line as a model system. Our data show that doxorubicin treatment of HCT116 cells results in massive cell killing by apoptosis at only a particular drug dose (referred to as conditional apoptosis). Lower and, remarkably, higher doses of doxorubicin do not result in significant apoptosis induction. This conditional apoptosis is absent in cells lacking the major DNA methyltransferase DNMT1. However, DNMT1-/- HCT116 cells can be made apoptosisinducible by reintroduction of DNMT1. These results suggest for the first time that DNMT1 is one of the important targets for doxorubicin and that interaction of DNMT1 and doxorubicin may contribute to the induction of apoptosis in cancer cells.
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Materials and Methods |
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). All experiments in a given panel in Fig. 1 were performed with the same enzyme preparations. DNA methyltransferase activity of DNMT1 was measured using the DNMT-magnetic beads assay (Yokochi and Robertson, 2002). A typical methylation reaction (40 µl) contained 125 nM DNA oligonucleotides [hemimethylated DNA conjugated with a biotin molecule, 34 base pairs (Yokochi and Robertson, 2002)], 30 nM DNMT1, and 600 nM tritium-labeled AdoMet (1 mCi/ml; Amersham Biosciences) in reaction buffer (50 mM Tris, pH 8.0, 5 mM EDTA, 10% glycerol, 10 mM 2-mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride). Experiments were done independently at least three times, and the mean values are shown. Radioactive materials were purchased from Amersham Biosciences. General chemicals were purchased from Sigma (St. Louis, MO), Invitrogen (Carlsbad, CA), and Roche (Indianapolis, IN).
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Cell Culture, Drug Treatment, and Transient Transfection. HCT116 and DNMT1-/- HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. Trypsinized cells were plated onto 100-(10 ml of medium) and 35-mm (2 ml of medium) diameter dishes and standard 96-well flat-bottomed plates (100 µl of medium) and incubated for 24 h before drug treatment. Because growth rates of HCT116 cells and its knockout derivative differ slightly (Rhee et al., 2000), initial cell numbers were adjusted so that the cell concentrations were typically 1 x 105 cells/ml at the time of drug addition. DNMT1-/- HCT116 cells were transfected with GFP or GFP-DNMT1 expression vectors using TransIT-LT1 transfection reagent (Mirus), according to the manufacturer's instructions. Each experiment was repeated three times independently; at least 10 cells expressing green fluorescence were examined and were confirmed to be consistent in terms of doxorubicin sensitivity.
Cell Viability Assay, Trypan Blue Staining, and Apoptosis-Related Assays. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay and Caspase-Glo 3/7 assay (Promega, Rockford, IL) were performed in accordance with the manufacturer's instructions. A fixed concentration of cells (1 x 105 cells/ml at the time of drug addition) was treated with various concentrations of drugs, and the cell viability or caspase activity were measured after 48 h. Parallel reactions were carried out without cells, and this value was subtracted from the experimental values. Results were expressed as the mean of four independent experiments, and error bars represent the S.D. DNA fragmentation in cells treated with doxorubicin was detected as described previously (Yeung, 2002). For Trypan Blue staining, cells in 2 ml of medium were treated with various concentrations of doxorubicin for 48 h, trypsinized, and resuspended in fresh medium; and an equal volume of Trypan Blue stain was then added. The number of live and dead cells was counted on pictures taken with a microscopy system with a digital camera (Nikon, Tokyo, Japan). Results were expressed as the mean of four independent experiments.
Western Blotting and Methyl Acceptance Assay. The DNMT1 antipeptide antibody has been described previously (Yokochi and Robertson, 2002). The PCNA and 
-actin antibodies were purchased from Santa Cruz Biotechnology. Genomic DNA was extracted from HCT116 cells that were treated with various concentrations of doxorubicin (0, 1 x 10-10, 1 x 10-9, 1 x 10-8, 1 x 10-7, 2 x 10-7, 5 x 10-7, 1 x 10-6, 2 x 10-6, 5 x 10-6, 1 x 10-5, 2 x 10-5, 5 x 10-5, and 1 x 10-4 M) for 48 h. Methylation acceptance capability of genomic DNA was measured by in vitro methylation reaction using bacterial CpG methylase SssI (New England Biolabs, Beverly, MA). A reaction solution contained genomic DNA (2 µg), SssI (1 µl), and 900 nM tritium-labeled AdoMet in reaction buffer (50 mM Tris, pH 8.0, 5 mM EDTA, 10% glycerol, 10 mM 2-mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride). After ethanol precipitation, tritium incorporation into genomic DNA was measured by a liquid scintillation counter. Results were presented as the mean of three independent experiments. S.D. was shown as error bars.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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), using highly purified recombinant DNMT1 to investigate the effects of DNA intercalators on DNMT1 catalytic activity. Doxorubicin, as well as other DNA inter-calating agents, significantly inhibited DNMT1 activity in vitro (Fig. 1A). We next compared dose-response plots of DNMT1 activity against the DNA substrate in the presence of a fixed concentration of doxorubicin, S-adenosyl-L-homocysteine (AdoHcy), or methylated DNA as enzymatic inhibitors. Both AdoHcy and methylated DNA are product inhibitors of DNMT1, and they require direct binding to the enzyme to inhibit its catalytic activity. Consistent with the velocity equations for an enzymatic reaction under the effect of an inhibitor (Segel, 1993; Copeland, 2000), DNMT1 activity yielded hyperbolic curves in the presence of either AdoHcy or methylated DNA (Fig. 1B). In contrast, DNMT1 inhibition yielded a sigmoidal curve under the effect of doxorubicin inhibition, suggesting that the inhibitory mechanism by doxorubicin might not be explained by a simple "inhibitor-enzyme" interaction model (Copeland, 2000). To identify which factor (DNMT1, DNA, or AdoMet) interacts with doxorubicin to inhibit the DNMT1 catalytic reaction, we employed conditions in which one of the three factors was varied to examine whether it would alter the doxorubicin-dose dependence of DNMT1 activity on the inhibition plots (Fig. 1C). The slope in the relative comparison plot of the inhibition curves was significantly affected only when the amount of DNA was varied (Fig. 1C, left, inset), indicating that the inhibitory effect of doxorubicin is dose-dependent with respect to DNA. These results suggest that the formation of a drug-DNA complex is important for the inhibition of DNMT1 by doxorubicin and exclude the possibility that doxorubicin binds directly to DNMT1 to cause inhibition. Thus, we conclude that doxorubicin inhibits DNMT1 activity via DNA intercalation.
Doxorubicin Demonstrates a Specific Cytotoxicity in the Presence of DNMT1. We next sought to examine the possible consequence of the inhibition of DNMT1 by doxorubicin in human cancer cells. As a reference, we used a different type of anticancer drug, etoposide (VP-16-213). Etoposide is a non-DNA-intercalating drug that inhibits topoisomerase II specifically resulting in p53-dependent apoptosis (Ross et al., 1984) but does not inhibit DNMT1 in vitro (Fig. 1A). A fixed number of HCT116 human colorectal carcinoma cells (DNMT1+/+ and DNMT1-/-) were treated with various concentrations of anticancer agents (doxorubicin and etoposide) for 48 h, and the cell viability was measured by MTS assay. A particular dose (1 x 10-6 M) of doxorubicin specifically decreased cell viability (Fig. 2A), whereas etoposide yielded a simple dose-dependent decrease in cell viability (Fig. 2B). It is noteworthy that the DNMT1-/- HCT116 cells (Rhee et al., 2000) did not demonstrate the specific cytotoxicity after doxorubicin treatment (Fig. 2C). Similar specific cytotoxicity was observed with other DNA intercalators, such as actinomycin D and actinomycin V in DNMT1+/+ HCT116 cells, and in other cell lines, such as HeLa and 293, with doxorubicin (data not shown). The large peak of caspase-3/7 activation at 1 x 10-6 M doxorubicin (Fig. 2D, etoposide control in Fig. 2E) is consistent with the decrease in cell viability (Fig. 2A), suggesting that the cytotoxicity at 1 x 10-6 M doxorubicin is caused by apoptotic cell death. A slight activation of caspase-3/7 was observed (
2-fold) in a broad but nonspecific range of doxorubicin doses between 1 x 10-7 and 1 x 10-5 Min DNMT1-/- cells (Fig. 2F). This is consistent with previous findings that the other major targets of doxorubicin are DNA polymerases and topoisomerases and that the inhibition of these DNA-binding proteins with doxorubicin leads to apoptosis (Hickman, 1992; Kiechle and Zhang, 2002).
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Apoptosis Is Induced by Only a Particular Concentration of Doxorubicin. To further confirm that apoptosis is associated with the decreasing cell viability induced by doxorubicin in DNMT1+/+ HCT116 cells, the numbers of live and dead cells were counted after Trypan Blue staining (Fig. 3A). A large fraction of cells died under the treatment with 1 x 10-6 M doxorubicin, suggesting that the decrease in cell viability is related to massive cell killing. Ten-fold higher (1 x 10-5 M) or 10-fold lower (1 x 10-7 M) doses of doxorubicin, however, did not lead to significant cell death. DNA laddering analysis also supports this notion (Fig. 3B). The improved DNA laddering assay described by Yeung (2002) clearly demonstrated nucleosomal fragmentation, which is a well documented characteristic of apoptosis, at the 1 x 10-6 M doxorubicin dose, whereas random genomic DNA digestion occurred at higher doses (1 x 10-5 M and above). Cells treated with 1 x 10-6 M doxorubicin appeared shrunken and developed blebs on cell surfaces, typical morphological hallmarks of apoptosis (Budihardjo et al., 1999) (Fig. 3C). Our results therefore suggest that apoptosis induced by doxorubicin is "conditional", meaning that only a particular concentration of drug specifically induces apoptosis in DNMT1+/+ HCT116 cells but higher or lower concentrations do not. It should be noted that, at the highest dose (1 x 10-4 M) of doxorubicin, cells blackened, swelled, and burst (Fig. 3C), and showed neither caspase-3/7 activation (Fig. 2F) nor DNA laddering (Fig. 3B). These characteristics are consistent with cell death caused by necrosis (Kroemer et al., 1998).
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Conditional Apoptosis Induced by Doxorubicin Depends on DNMT1 in HCT116 Cells. Consistent with the results of cell viability (Fig. 2C) and caspase activity (Fig. 2F), DNMT1-/- HCT116 cells yielded a simple dose-dependence with regard to doxorubicin concentration in the Trypan Blue staining assay (Fig. 4A). Because DNMT1-/- HCT116 cells grow more slowly than DNMT1+/+ HCT116 cells, differences between these two cell lines may be explained by the difference of cell growth rates. To rule out this possibility, the cell cycle of DNMT1+/+ HCT116 cells was first arrested by other growth inhibitors [e.g., Colcemid (Fig. 4B), nocodazole, hydroxyurea, and aphidicolin (data not shown)]. Doxorubicin-mediated conditional apoptosis was induced even in these growth-arrested cells (Fig. 4B). This result suggests that the difference of doxorubicin sensitivity between DNMT1+/+ and DNMT1-/- cells is not related to cell growth rates and that cell proliferation and DNA replication are not essential for conditional apoptosis. To further confirm the direct contribution of DNMT1 to conditional apoptosis, transient transfection of DNMT1-/- HCT116 cells was performed with either empty green fluorescent protein (GFP) expression vector or GFP-tagged DNMT1 expression vector. After 48 h of transfection, cells were treated with 1 x 10-6 M doxorubicin (the dose inducing apoptosis in DNMT1+/+ cells but not in DNMT1-/- cells) for an additional 48 h. Expression of GFP-DNMT1 caused apoptosis in DNMT1-/- HCT116 cells after treatment with 1 x 10-6 M doxorubicin (Fig. 4C, top), whereas expression of GFP alone did not (Fig. 4C, bottom). Total protein levels of DNMT1 in DNMT1+/+ HCT116 cells were greatly reduced after treatment with 1 x 10-6 M doxorubicin (Fig. 4D), further supporting the notion that DNMT1 in cells is related to conditional apoptosis induced by doxorubicin. Taken together, we conclude that DNMT1 contributes to the conditional apoptosis in HCT116 cells.
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Changes in Global Methylation Status of Genomic DNA Are Not Associated with Conditional Apoptosis. Results presented in Fig. 3A from the Trypan blue cell counting assay clearly indicated that one of the effects of doxorubicin was to cause cell cycle arrest. This effect occurred over a relatively wider range of drug concentration than the conditional apoptosis effect, beginning at 1 x 10-7 M doxorubicin. Because demethylation caused by inhibition of DNMT1 is believed to occur by a passive mechanism, which requires active cell division to dilute out the methylated parental DNA strands, it was not clear whether doxorubicin treatment and conditional apoptosis would be associated with detectable genomic demethylation. To determine this, global DNA methylation levels in doxorubicin-treated DNMT1+/+ HCT116 were examined using the methyl-acceptance assay. Results of this assay (Fig. 4E) revealed that the cellular DNA methylation status of HCT116 cells was not significantly altered after treatment with 1 x 10-6 M doxorubicin. Although some hypomethylation seemed to be occurring at very high doses of doxorubicin, the significance of such results is difficult to ascertain because cells are undergoing high levels of cell death by necrosis under these conditions.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Furthermore, Cre-lox mediated depletion of Dnmt1 in mouse fibroblasts caused loss of Dnmt1 protein, genomic hypomethylation, and induction of p53-dependent apoptosis (Jackson-Grusby et al., 2001). Because cytosine methylation within the promoter regions of genes can cause transcriptional silencing, demethylation may activate the expression of genes that in turn activate p53. In this report, however, we found that conditional apoptosis was induced only when DNMT1 protein and 1 x 10-6 M doxorubicin coexist. Therefore, the latter mechanism—loss of genomic methylation—may not represent the most plausible cause of conditional apoptosis. An alternative mechanism is that catalytically inactive DNMT1 bound to doxorubicin-treated DNA may be perceived by the cell as a form of DNA damage. The cytotoxic effects of 5-aza-2'-deoxycytidine (5-AdC) are believed to be mediated through the irreversible covalent binding of DNA methyltransferase to 5-AdC-substituted DNA, which is then recognized as DNA damage by the cell, resulting in apoptosis (Juttermann et al., 1994). Indeed, it has been shown that 5-AdC treatment causes p53 activation through a traditional DNA damage response pathway, which is consistent with this model (Karpf et al., 2001). Other studies have shown that the introduction of DNMT1-/- DNMT3B-/- double mutations into HCT116 cells did not cause apoptosis, even though these cells exhibited marked genomic hypomethylation (a roughly 95% reduction in methylated cytidine content) (Rhee et al., 2002). In addition, we demonstrated that doxorubicin could induce conditional apoptosis only in DNMT1+/+ HCT116 cells but not in DNMT1-/- HCT116 (this work) or DNMT1-/- DNMT3B-/- HCT116 (data not shown) cells. These results suggest that DNMT1 itself, rather than the secondary demethylation of genomic DNA, is the primary mediator of drug-induced cytotoxicity. Total soluble DNMT1 protein levels were decreased specifically in DNMT1+/+ HCT116 cells treated with the apoptosisinducible concentration of doxorubicin (Fig. 4D). The reduced DNMT1 protein levels may be caused by the formation of an irreversible DNMT1-DNA-doxorubicin complex such that the DNMT1 becomes trapped in an insoluble chromatin fraction during protein extraction. Although there is no direct evidence at this time, we speculate that doxorubicin may stabilize the covalent link between DNMT1 and DNA. The DNMT1-DNA complex could be recognized as DNA damage resulting in apoptosis. The cellular DNA repair machinery may facilitate the degradation or removal of the complex. Analysis of these, and other possibilities, will be the subject of future work.
The antitumor activity of DNA intercalators, including doxorubicin, was believed to be closely related to DNA cleavage depending on topoisomerase II and inhibition of DNA replication (Hickman, 1992; Brana et al., 2001; Kiechle and Zhang, 2002). Our data indicate that DNMT1 is also required for conditional apoptosis induced by a particular concentration of doxorubicin, suggesting that DNA methyltransferase is one of the targets of doxorubicin for apoptosis induction in cancer cells. We propose that the expression levels of DNMT1 in tumor cells may be important criteria that should be taken into account to evaluate the selective cytotoxicity of the drug (Pratt et al., 1994) and to determine the optimal dose regimen.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; 5-AdC, 5-aza-2'-deoxycytidine; DNMT, DNA methyltransferase; cre, cyclization recombination; lox, locus of x-over P1.
1 Current address: Tomoki Yokochi, Ph.D., Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York. 
Address correspondence to: Dr. Keith D. Robertson, Department of Biochemistry and Molecular Biology, University of Florida, Box 100245, Gainesville, FL 32610-0254. E-mail: keithr{at}ufl.edu
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), actinomycin D (
), and actinomycin V (
). Dose-response plots of AdoHcy (
) and etoposide (
) are also shown. B, dose-response plots of DNMT1 activity as a function of DNA substrate concentration in the absence (
; fully methylated DNA at 0.02 µM, 
