5-Methylcytosine residues in the DNA (DNA methylation) are formed fromthe transfer of the methyl group from S-adenosylmethionine to the C-5 position of cytosine by the DNA-(cytosine-5) methyltransferases (DNMTs). Although regional hypermethylation and global hypomethylation of the genome are commonly observed in neoplastic cells, how these aberrant methylation patterns occur remains unestablished. We report here that sulfonate-derived methylating agents, unlike N-methylnitrosourea or iodomethane, are potent in depleting DNMT1 proteins in human cells, in addition to their DNA-damaging properties. Their effects on cellular DNMT1 are time and dosage dependent but independent of cell type. Unlike γ-irradiation, these agents apparently do not activate the p53/p21WAF1 DNA damage response pathway to deplete the DNMT1 proteins because cells with wild-type, mutated, or inactivated p53 behave similarly. However, cell cycle analysis and protease assay studies strongly suggest that methylmethanesulfonate may activate a cellular protease to degrade DNMT1. These results explain why reported observations on the effect of alkylating agents on DNMT1 activities in human cells vary significantly and provide a crucial link to understand the mechanism behind genomic hypomethylation.

Although the 5-MeC3 residues constitute only 2–3% of the mammalian genome, they serve important roles in gene regulation such as imprinting, long-term silencing of tissue-specific genes, and X-chromosome inactivation. 5-MeC (DNA methylation)-mediated silencing may be accomplished by methylated cytosine binding proteins (i.e., MeCP2), which target histone deacetylase to condense the region of DNA containing 5-MeC (1), or the inability of transcription factors to bind methylated promoter sites (2, 3). Aberrant methylation, in the forms of regional hypermethylation (4) and global hypomethylation (5) of the genome, are commonly observed in neoplastic cells. They are nonrandom and tumor type-specific (6), suggesting that improper methylation contributes to cancer initiation or progression. Two causative roles for methylation in tumor progression have been observed: (a) silencing of tumor suppressor genes, such as p21WAF1, p16INK4a, or Rb, correlate with hypermethylation of their promoter regions in certain tumors (7); and (b) increased deletion mutation rates leading to genomic instability are associated with global hypomethylation of the genome (8). The observation that a DNMT gene is mutated in the ICF (immunodeficiency, centromeric instability, facial abnormalities) syndrome, which is characterized by hypomethylation and chromosome breakage (9, 10, 11, 12), strengthens the role of DNA methylation in human diseases.

How aberrant methylation occurs in tumor cells remains unestablished. A likely candidate for study is the DNA-(cytosine-5) methyltransferase DNMT1, which constitutes >90% of methylation activities in the mammalian cells (13, 14). It processes the maintenance methylase activities necessary for normal cell growth, as well as unusual de novo methylase activities (15). Therefore, any alterations in the cellular level of DNMT1 protein may potentially lead to changes in DNA methylation patterns. In this study, we addressed the question of whether mutagens can affect the DNMT1 protein by studying the levels of DNMT1 in cells exposed to γ-irradiation and alkylating mutagens. We report here that sulfonate-derived methylating agents, such as MMS and DMS, are potent in depleting DNMT1 proteins in the human cells, a novel addition to their abilities in producing alkylation lesions, such as apurinic sites and single- and double-strand breakage in the DNA (16). This hitherto unknown property of sulfonate-derived methylating agents suggests the multiplicity of these agents in tumor-causing actions.

Although DNMT1 and p21WAF1 proteins are shown to share an inverse relationship throughout the cell cycle and after exposure to γ-irradiation, this relationship is not observed during MMS treatment, which induces DNMT1 depletion in cells with Wt, mutated, or inactivated p53. Results from cell cycle analysis and protease assay suggest that the effects of MMS may be direct or indirect, possibly via the modification of DNMT1 or activation of a cellular protease to degrade DNMT1 proteins. These results may explain why reported observations on the effect of alkylating agents on DNMT1 activities in the human cells vary significantly (17) because MMS and DMS have a distinctive effect on the cellular DNMT1 proteins in a time- and dosage-dependent manner as compared with NMU and MeI.

Treatment of Cells with Mutagens.

All cell lines were obtained from American Type Cell Culture and cultured as specified by American Type Cell Culture. MMS, MeI, DMS, and NMU were purchased from Sigma Chemical Co.-Aldrich. The cells were grown to 50% confluence before commencing drug treatment. Stock solutions of all drugs were made in serum-free media and added to the cells for the indicated times. γ-Irradiation was done using a 60Co source at room temperature.

Cell Lysis and Western Analysis.

Cell pellets were lysed in a modified RIPA buffer [50 mm Tris (pH 8.0), 0.3 m NaCl, 10 mm EDTA, 50 mm NaF, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, and 0.2 mm phenylmethylsulfonyl fluoride] by sonication. The lysates were then centrifuged at 14,000 rpm for 20 min at 4°C (Eppendorf), and the supernatant was recovered. Protein concentrations were determined by Lowry assay. Cell lysates (100 μg) were resolved on 6.9 and 12% SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad). Western analyses were performed as described (18). Monoclonal antibody Mab.2E10 for DNMT1 was described previously (18). Mab.D01 for p53 and Mab.PC10 for PCNA detections were from Santa Cruz Biotechnology. Mabs to actin (clone C4) and p21WAF1 (clone 70) were from Boehringer Mannheim and Transduction Laboratories, respectively. Antimouse immunoglobulin horseradish peroxidase conjugate was from DAKO.

FC.

A Becton and Dickinson FACScan machine was used to acquire and analyze 21,000 events (using Cellquest and Modfit). DNA analysis was by propidium iodide staining (50 μg/ml at 37°C for 30 min) of ethanol (80%, 12 h at −20°C)-fixed cells digested with RNase (20 μg/ml for 30 min) as described previously (19).

Protease V8 Assay.

The assay conditions were described previously (20). Briefly, cells were treated with 0.5 mm MMS and harvested at the described times. The cell pellets were then sonicated in the lysis buffer [50 mm Tris·HCl (pH 7.5), 0.02% NP40, 50 mm NaF, 1 mm EDTA, 1 mm DTT, 0.3 m NaCl, 3% glycerol, and 0.2 mm phenylmethylsulfonyl fluoride]. The supernatant was recovered by centrifugation at 50,000 rpm for 20 min at 4°C and frozen at −80°C. The cell lysates (200 μg) were subjected to 50 ng of protease V8 (Roche Molecular Biochemicals) in the digestion buffer [50 mm Tris·HCl (pH 8), 0.3 m NaCl, 1 mm EDTA, and 5 mm DTT] for 10 min at 37°C. The control experiment was conducted without the protease. The reaction (20 μl) was terminated by the addition of 4× Laemmli buffer (10 μl) and boiled at 100°C for 5 min. The samples were then resolved on a 8% SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blot analysis.

DNMT1 Protein Levels in Normal Fibroblasts (MRC5) Are Reduced after Treatments with γ-Irradiation and Sulfonate-derived Methylating Agents.

To understand how carcinogens may affect DNMT1, we investigated the levels of DNMT1 in MRC5-NT (human fibroblast) cells after exposure to the SN1 methylating agent (NMU), SN2 methylating agents (MeI, MMS, and DMS) or γ-irradiation (for comparison). Cell lysates were prepared at various times after the respective treatments and analyzed by Western blot using 2E10, a Mab specific for DNMT1. Interestingly, although the SN1 alkylating mutagen NMU did not alter DNMT1 levels (Fig. 1, compare a and b), treatments with SN2 alkylating agents affect DNMT1 levels differently. Although these chemicals belong to the same family of methylating agents that react with an Ingold’s SN2 mechanism (21), MMS and DMS, but not MeI (see Fig. 1c), effectively reduced the DNMT1 proteins (Fig. 1, d and e). Thus, the S-O-CH3 group of MMS and DMS may be the critical moiety responsible for DNMT1 depletion (see the chemical structure in Fig. 1). Nevertheless, exposure to γ-irradiation does result in the reduction of DNMT1.

p53/p21WAF1-dependent Regulation of DNMT1 Levels upon γ-Irradiation.

Because we have reported previously that p21WAF1 and DNMT1 protein levels share an inverse co-relationship in the normal human fibroblast and its SV40-transformed counterpart (18), we investigated whether the observed γ-irradiation-induced depletion of DNMT1 proteins in MRC5-NT fibroblasts is a consequence of the p53/p21WAF1 DNA damage response pathway. Two cell lines with Wt p53 status, MRC5-NT and MCF7, were compared with the SV40-transformed MRC5 cells (MRC5-SV). When these cells were subjected to γ-irradiation, the p53 and p21WAF1 proteins were rapidly induced in the Wt p53 cells but not in the SV40-transformed cells, as shown in Fig. 2,A. Interestingly, whenever high levels of p21WAF1 proteins are observed, the DNMT1 proteins are significantly reduced in the p53-proficient MRC5-NT and MCF7 cells (Fig. 2, A.1 and A.2). However, no alterations in the p53 and DNMT1 levels were observed in the SV40-transformed cells under identical conditions (Fig. 2, A.3). These results are consistent with the functional status of the p53 proteins in these cells; whereas the MRC5-NT and MCF7 cells contain functional p53, the p53 proteins in the MRC5-SV are inactivated by the large T antigen. We then studied the cell cycle profiles of these γ-irradiated cells by FC. Fig. 2 shows that after 24 h of γ-irradiation, the MRC5-NT cells underwent G1 and G2 arrest as contrasted with the MRC5-SV cells, which are predominantly at S and G2-M (A.4a and A.4b). The MRC5-NT cells, which have a high level of p21WAF1 and low level of DNMT1 proteins (Fig. 2, A.1), are characterized by significantly lower populations of S-phase cells (Fig. 2, A.4c) than the MRC5-SV cells (Fig. 2, A.4d) that exhibited less variation in their DNMT1 protein levels (Fig. 2, A.3). Two interesting conclusions can be drawn from these results: (a) the induced p53/p21WAF1 proteins (through the DNA damage response pathway) may have a critical role in reducing DNMT1 levels upon γ-irradiation because the relationship between the two proteins can also be demonstrated in an alternative experiment where reduction of DNMT1 proteins by antisense oligonucleotides leads to elevation of the p21WAF1 proteins (22); and (b) there is a close relationship between the status of the cells at the S-phase and their DNMT1 levels, which is consistent with the report that inhibition of DNMT1 negatively affects DNA replication (23).

p53/p21WAF1-independent Regulation of DNMT1 Levels upon MMS Treatment.

Interestingly, when the above cells were treated with MMS, the DNMT1 proteins were reduced in all of the cells as shown in Fig. 2,B. However, in the Wt p53-containing MRC5-NT and MCF7 cells, the observed reduction of DNMT1 proteins is not accompanied by significant changes in the p53 and p21WAF1 protein levels as compared with the γ-irradiated cells (Fig. 2 A). Although the DNMT1 proteins in the MRC5-SV cells are resistant to changes upon γ-irradiation, their levels are significantly reduced upon MMS treatment. These observations are a likely consequence of the direct effect of MMS on the cells and not its breakdown product, because pretreatment of MMS in the medium for 24 h before cell treatment did not have any appreciable effect on cellular DNMT1 levels (data not shown). Therefore, these results strongly suggest that MMS, unlike γ-irradiation, can reduce cellular DNMT1 proteins via a pathway that is independent of p53/p21WAF1.

MMS Reduction of DNMT1 Is Dosage and Time Dependent.

To ascertain the effect of MMS in the depletion of cellular DNMT1 proteins, we studied the time course and dosage effect of MMS on the MRC5 and MRC5-SV cells. The Western blot analysis in Fig. 3 shows that, although actin and PCNA protein levels remain relatively unchanged, significant depletion of DNMT1 proteins were observed 24 h after treatment at a dosage of 0.1 mm MMS for MRC5-NT cells and 0.5 mm MMS for MRC5-SV cells (Fig. 3, A.b for MRC5-NT and B.b for MRC5-SV). Interestingly, FC examination of these cells 24 h after MMS treatment in A.c and B.c show that at doses <0.5 mm, the MRC5-SV cells, unlike the MRC5-NT cells, are able to proceed through the G1 to S-phase transition before arresting at the S-phase. It is notable that treatment of cells with 1–1.5 mm MMS appears to be cytostatic (“freezing”) because no increase in cell numbers are observed (data not shown), nor was the cell population profiles much altered (Fig. 3, A.c and B.c). Thus, in contrast to γ-irradiation, MMS can deplete DNMT1 proteins in the cells irrespective of the functional status of their p53 proteins, abiding mainly to the differences in dosage of MMS. Together, these results suggest that the effect of MMS on DNMT1 may be nonrandom.

Down-Regulation of DNMT1 by MMS Is Independent of Cell Type.

The above observations led us to examine whether the effect of MMS in reducing DNMT1 proteins is a general phenomenon. We treated various cell lines exhibiting various status of p53 with MMS (0.5 mm; see table in Fig. 4,b). Interestingly, a consistent reduction of DNMT1 levels was also observed in all these cells (Fig. 4,a), irrespective of their p53 status (Fig. 4 b). Thus, in contrast with γ-irradiation, MMS induced depletion of DNMT1 protein is unlikely to be attributed to the p53 damage response pathway. Collectively, these data suggest that sulfonate-derived methylating agents can specifically deplete DNMT1 proteins in human cells.

Variation of DNMT1 and p21WAF1 Proteins during the Cell Cycle.

To understand the mechanism behind the depletion of cellular DNMT1 proteins by γ-irradiation, we first examined the protein levels under normal conditions of cell cycle progression. The MRC5-NT cells were synchronized by serum depletion for 48 h, followed by the addition of fresh serum to stimulate growth. The cells were harvested for FC analysis (Fig. 5,A) as well as Western blotting for DNMT1, p21WAF1, and PCNA proteins (Fig. 5,B). However, the MRC5-SV cannot be examined under these conditions because these cells are not viable under prolonged serum depletion. Fig. 5,B shows that after 48 h of serum depletion, the DNMT1 proteins decrease significantly, whereas the p21WAF1 proteins are elevated (Fig. 5,B, compare the Ctr and 0 h lanes). This is consistent with growth arrest because the populations of the S-phase cells decrease significantly (Fig. 5,C, see the S-phase bar chart). Because growth resumed after 17.5 h of serum stimulation (Fig. 5,C, see the increase in S-phase), the DNMT1 proteins increase while the p21WAF1 levels decrease. The DNMT1 protein peaks at 24 h when the highest population of S-phase cells was observed. Thus, there is a very distinctive inverse relationship between DNMT1 and p21WAF1 protein levels during G1 and S transition (note that G2-M cell populations did not vary at these time points, as shown in Fig. 5 C). Furthermore, at 24, 26, and 30 h after serum stimulation, although the populations of G1 cells are not significantly different, the populations of S and G2-M cells are inversely proportional to each other. Interestingly, both conditions exhibit high DNMT1 but low p21WAF1 protein levels. This suggests that DNMT1 levels are also high at the G2-M phase, which is consistent with the previous immunostaining studies (24).

Protease V8 Assay: MMS May Be Directly or Indirectly Involved in Modification of DNMT1 for Degradation.

Next, we focused on the mechanism(s) leading to the MMS-induced depletion of DNMT1 by Western blotting analysis of cell extracts lysed under nondenaturing conditions (see “Materials and Methods”). Interestingly, Mab.2E10, which was mapped to amino acids 323–489 of DNMT1, recognizes a Mr ∼80,000 protein (Fig. 6, band *a*) in addition to the DNMT1 protein in the cell extracts after treatment with 0.5 mm MMS (Fig. 6,a, Lane 3). However, this smaller DNMT1 species appears to be unstable as its level decreases beyond 3 h of MMS treatment. Unfortunately, it is not possible to compare the relative amounts of the smaller and full-length DNMT1 proteins on the Western blot because transfer efficiency from gel to membrane filter favors the smaller proteins. Because the DNMT1 protein is not significantly decreased, the observed smaller species should be a minor component, i.e., a transient intermediate during the degradation of DNMT1. This result strongly suggests that DNMT1 undergoes proteolysis upon MMS treatment. Therefore, we attempted to mimic this proteolysis of DNMT1 protein by treatment of cell extracts with a protease before Western blotting. The sensitivity of the DNMT1 protein toward protease V8 cleavage was examined. The control experiments in Fig. 6,b shows that upon protease V8 treatment, only a small fraction of the DNMT1 proteins can be cleaved by protease V8 into slightly smaller DNMT1 proteins (because of cleavage at the NH2 and/or COOH terminus) or a Mr ∼80,000 fragment of DNMT1 (because of cleavage at the central region). However, the majority of DNMT1 proteins were readily cleaved by protease V8 into a prominent Mr ∼80,000 fragment in samples after 3 h of MMS treatment (see the disappearance of the Mr ∼200,000 DNMT1 protein in Fig. 6,b). This result suggests that MMS treatments can render the central region of DNMT1 protein susceptible toward cleavage by protease V8. Interestingly, this prominent Mr ∼80,000 fragment arising from protease V8 cleavage of MMS-treated cell extracts coincides with the appearance of the transient low molecular weight DNMT1 species in MMS-treated cell extracts without protease V8 treatment (Fig. 6, a and b, bands *a*). Together, these results suggest that MMS may be involved in the activation of an endogenous protease that cleaves DNMT1 or modification of DNMT1 because exposure to MMS for 1 to 2 h does not render the DNMT1 proteins sensitive to cleavage by protease V8 (Fig. 6 b, Lanes 1 and 2 h).

One of the salient findings in this report is the inverse relationship between DNMT1 and p21WAF1 proteins when p53-proficient cells MRC5-NT and MCF7 cells are exposed to γ-irradiation (Fig. 2, A.1 and A.2). This phenomenon appears to be triggered by the p53-dependent DNA damage response, because it is not observed in MRC-SV cells lacking active p53. In these studies, the FC analysis and Western blotting show that 24 h after irradiation, normal fibroblasts MRC5-NT cells are arrested at both G1 and G2 phases of the cell cycle with major reduction of S-phase cells. This observation is accompanied by decreased DNMT1 but elevated p21WAF1 protein levels. In contrast, the p21WAF1-deficient MRC5-SV cells are accumulated at the S and G2-M phases (Fig. 2, A.4d) with unaltered DNMT1 levels. In summary, the inverse relationship between the p21WAF1 and DNMT1 proteins occurs in the p53-proficient MRC5-NT cells during their cell cycle and exposure to γ-irradiation (or DNA-damaging agents that induce p53). It will be interesting to discover whether the proliferating cell nuclear antigen, which is a target for binding by p21WAF1 or DNMT1 (18), can play a role in mediating this interdependent regulation of the two proteins.

Unlike γ-irradiation, there are two distinctive observations from cells exposed to MMS for 24 h: (a) MMS depletes the DNMT1 proteins (Figs. 2,B and 4) in the cells irrespective of their p53 status (Fig. 4) and without altering their p21WAF1 protein levels (Fig. 2,B); (b) although their DNMT1 proteins are consistently depleted by MMS, the cell cycle profiles of the MMS-treated MRC5-NT and MRC5-SV cells (Fig. 3, A.c and B.c) are significantly different. These observations suggest that the depletion of DNMT1 proteins by MMS is specific, and it follows a mechanism that is different from γ-irradiation.

Aberrant methylation is a common observation in the genome of cancer cells; however, its underlying mechanism is unknown. Although exposure to DNA-damaging agents has been an important etiological factor in human cancer, it remains unclear whether altered methylation patterns occur as a consequence of DNA damage induced by mutagens. Indeed, exposure to NMU, which did not affect DNMT1 protein (Fig. 1 b), can cause hypermethylation in p21WAF1-deficient cells because of de novo methylation of the O6-methylguanine-containing DNA by DNMT1 (18). In this report, however, we show that a class of SN2 alkylators can specifically cause depletion of the cellular DNMT1 proteins. It remains to be established whether exposure to this carcinogen can lead to genome hypomethylation.

Although MMS and DMS are simple molecules (Fig. 1), the mechanism behind their intrinsic ability to specifically deplete cellular DNMT1 proteins appears to be complicated, because no significant changes in other protein levels were detected (Fig. 3). However, some parameters can be precluded: (a) DNA damage may not be involved because MeI does not cause DNMT1 depletion (Fig. 1), although it produces a similar spectrum of DNA lesions in the DNA as MMS (25); and (b) the p53/p21WAF1 DNA damage response pathway does not seem to be critical because MMS can induce DNMT1 depletion in a number of cells with mutated p53 or inactivated p53 (Fig. 4). However, the protease assay provides an important clue. Fig. 6,a shows that a transient and smaller DNMT1 species (band *a*) can be detected in the cells treated with MMS for 3 h and beyond. Interestingly, protease V8 can also cleave DNMT1 in these MMS-treated samples, mimicking the formation of the smaller DNMT1 species (as shown in Fig. 6,b). These results suggest that a protease may be involved in the MMS-induced depletion of DNMT1. Although we are unclear why protease V8 cannot cleave the DNMT1 in cells treated with MMS for 1–2 h (Fig. 6 b, Lanes 1 and 2), the recent observation that MMS can also induce a number of kinase activities (26, 27), such as ATM (ataxia telangiectasia mutated) and hChk 1 and 2 (human homologues of the yeast RAD53 and CDS1 proteins), provide the following possibilities: (a) DNMT1 may require modification by MMS-induced kinase activities before their cleavage; (b) the protease may be a downstream effector of the MMS response pathway; and (c) MMS may directly modify the structure of DNMT1 protein. Because MMS specifically depletes DNMT1, one may consider the possibility that a low dosage of MMS may be as effective as 5-azacytidine analogues in chemotherapy.

Fig. 1.

DNMT1 protein levels in normal fibroblasts (MRC5) are reduced after γ-irradiation or sulfonate-derived methylating agent treatment. Cells were treated with various mutagens, and cell extracts (100 μg) were subjected to Western blot analyses for DNMT1 using monoclonal antibody Mab.2E10. Left panel: a, untreated cells; b, NMU (1.5 mm) treated; c, MeI (1.5 mm) treated; d, MMS (1.5 mm) treated; e, DMS (1.5 mm) treated; f, subjected to γ-irradiation at 5 Gy. Numbers represent hours after treatment. Right panel: chemical structures of the respective methylating agents used on the left panel. The methylating moiety is circled.

Fig. 1.

DNMT1 protein levels in normal fibroblasts (MRC5) are reduced after γ-irradiation or sulfonate-derived methylating agent treatment. Cells were treated with various mutagens, and cell extracts (100 μg) were subjected to Western blot analyses for DNMT1 using monoclonal antibody Mab.2E10. Left panel: a, untreated cells; b, NMU (1.5 mm) treated; c, MeI (1.5 mm) treated; d, MMS (1.5 mm) treated; e, DMS (1.5 mm) treated; f, subjected to γ-irradiation at 5 Gy. Numbers represent hours after treatment. Right panel: chemical structures of the respective methylating agents used on the left panel. The methylating moiety is circled.

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Fig. 2.

Effect of γ-irradiation and MMS on cellular DNMT1 proteins. Panel A.1–3, MRC5, MCF7, and MRC5.SV were subjected to γ-irradiation (5 Gy) in a time course experiment. Western blots of the cell extracts were performed using Mab.2E10 for DNMT1, Mab.C70 for p21WAF1, and Mab.D01 for p53. 4a-b, DNA profiles of the γ-irradiated cells analyzed by FC. The diagrams represent the gated cell populations (events, Y axis) with different DNA contents stained by propidium iodide (FL2-A on the X axis defines the G1, S, or G2 cells). 4c-d, bar charts used to show the percentage of cells in G1, S, and G2-M populations from FC analysis in a-b. Panel B is similar to panel A.1–3, except MMS (0.5 mm) is used instead of γ-irradiation.

Fig. 2.

Effect of γ-irradiation and MMS on cellular DNMT1 proteins. Panel A.1–3, MRC5, MCF7, and MRC5.SV were subjected to γ-irradiation (5 Gy) in a time course experiment. Western blots of the cell extracts were performed using Mab.2E10 for DNMT1, Mab.C70 for p21WAF1, and Mab.D01 for p53. 4a-b, DNA profiles of the γ-irradiated cells analyzed by FC. The diagrams represent the gated cell populations (events, Y axis) with different DNA contents stained by propidium iodide (FL2-A on the X axis defines the G1, S, or G2 cells). 4c-d, bar charts used to show the percentage of cells in G1, S, and G2-M populations from FC analysis in a-b. Panel B is similar to panel A.1–3, except MMS (0.5 mm) is used instead of γ-irradiation.

Close modal
Fig. 3.

Reduction of DNMT1 protein by MMS is dosage and time dependent. MRC5-NT and MRC5-SV cells were subjected to MMS treatment at different concentrations. A, Wt p53-containing MRC5-NT cells after 6 and 24 h treatment, respectively. a-b, Western blots of the extracts performed with Mab.2E10 for DNMT1, Mab.C4 for actin, and PC10 for PCNA. c, DNA profiles as analyzed by FC of the cells after 24 h treatment with MMS. B is the similar experiment with MRC5.SV cells.

Fig. 3.

Reduction of DNMT1 protein by MMS is dosage and time dependent. MRC5-NT and MRC5-SV cells were subjected to MMS treatment at different concentrations. A, Wt p53-containing MRC5-NT cells after 6 and 24 h treatment, respectively. a-b, Western blots of the extracts performed with Mab.2E10 for DNMT1, Mab.C4 for actin, and PC10 for PCNA. c, DNA profiles as analyzed by FC of the cells after 24 h treatment with MMS. B is the similar experiment with MRC5.SV cells.

Close modal
Fig. 4.

Reduction of cellular DNMT1 proteins by MMS is independent of cell type. Cells were treated with MMS (0.5 mm) for various durations, and Western blot analyses of the lysates were performed using Mab.2E10 for DNMT1. a, Western blots of the lysates from: 1., WI38; 2., U2OS; 3., VA13; 4., MDA-MB231; 5., HL60; and 6., NCI-H1299 cells. Numbers are hours after drug treatment. b, table showing the origins and p53 status of the cell types.

Fig. 4.

Reduction of cellular DNMT1 proteins by MMS is independent of cell type. Cells were treated with MMS (0.5 mm) for various durations, and Western blot analyses of the lysates were performed using Mab.2E10 for DNMT1. a, Western blots of the lysates from: 1., WI38; 2., U2OS; 3., VA13; 4., MDA-MB231; 5., HL60; and 6., NCI-H1299 cells. Numbers are hours after drug treatment. b, table showing the origins and p53 status of the cell types.

Close modal
Fig. 5.

Cell cycle synchronization studies of MRC5-NT fibroblasts. MRC5-NT cells were cultured in medium containing 0.01% FBS serum for 48 h. The arrested G0 (0 hr) cells were then released by the addition of medium containing 10% serum and harvested at the indicated times. A, FC showing the DNA profiles of the cells. Ctr, untreated cells; 0 hr, sample after treatment with 0.01% FBS for 48 h and others are samples harvested at respective hours after the addition of 10% FBS. B, Western blots of DNMT1, p21WAF1, and PCNA proteins. C, percentage of cells at G1, S, and G2-M represented by histograms from the FC data in A.

Fig. 5.

Cell cycle synchronization studies of MRC5-NT fibroblasts. MRC5-NT cells were cultured in medium containing 0.01% FBS serum for 48 h. The arrested G0 (0 hr) cells were then released by the addition of medium containing 10% serum and harvested at the indicated times. A, FC showing the DNA profiles of the cells. Ctr, untreated cells; 0 hr, sample after treatment with 0.01% FBS for 48 h and others are samples harvested at respective hours after the addition of 10% FBS. B, Western blots of DNMT1, p21WAF1, and PCNA proteins. C, percentage of cells at G1, S, and G2-M represented by histograms from the FC data in A.

Close modal
Fig. 6.

Protease V8 assay for DNMT1 after MMS treatment. MRC5.SV cells were treated with 0.5 mm of MMS and harvested at the indicated times. Cell lysates were partially digested with protease V8 before electrophoresis on an 8% SDS-PAGE. Western blots were done using Mab.2E10. a, Western blot of cell lysates without protease V8 digestion. b, Western blot of cell lysates digested with protease V8 for 10 min at 37°C. Lanes labeled 2C refer to the untreated sample at 2 h, whereas the numbered lanes are samples treated with MMS for the respective hours and Lane V8 for protease V8 alone. *a*, *b*, and *c*, possible species derived from the degradation of DNMT1.

Fig. 6.

Protease V8 assay for DNMT1 after MMS treatment. MRC5.SV cells were treated with 0.5 mm of MMS and harvested at the indicated times. Cell lysates were partially digested with protease V8 before electrophoresis on an 8% SDS-PAGE. Western blots were done using Mab.2E10. a, Western blot of cell lysates without protease V8 digestion. b, Western blot of cell lysates digested with protease V8 for 10 min at 37°C. Lanes labeled 2C refer to the untreated sample at 2 h, whereas the numbered lanes are samples treated with MMS for the respective hours and Lane V8 for protease V8 alone. *a*, *b*, and *c*, possible species derived from the degradation of DNMT1.

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported in part by the National Science and Technology Board of Singapore.

3

The abbreviations used are: 5-MeC, 5-methylcytosine; MMS, methylmethanesulfonate; DMS, dimethylsulfate; NMU, N-methyl-N-nitrosourea; MeI, iodomethane; Mab, monoclonal antibody; FC, flow cytometry; Wt, wild type.

We thank T. W. Koh for assistance with cell synchronization studies and Y. H. Tan for support.

1
Nan X., Ng H. H., Johnson C. A., Laherty C. D., Turner B. M., Eisenman R. N., Bird A. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.
Nature (Lond.)
,
393
:
386
-389,  
1998
.
2
Tate P. H., Bird A. P. Effects of DNA methylation on DNA-binding proteins and gene expression.
Curr. Opin. Genet. Dev.
,
3
:
226
-231,  
1993
.
3
Campanero M. R., Armstrong M. I., Flemington E. K. CpG methylation as a mechanism for the regulation of E2F activity.
Proc. Natl. Acad. Sci. USA
,
97
:
6481
-6486,  
2000
.
4
Toyota M., Issa J-P. J. The role of DNA hypermethylation in human neoplasia.
Electrophoresis
,
21
:
329
-333,  
2000
.
5
Soares J., Pinto A. E., Cunha C. V., Andre S., Barao I., Sousa J. M., Cravo M. Global DNA hypomethylation in Breast Carcinoma. Correlation with Prognostic Factors and Tumor Progression.
Cancer (Phila.)
,
85
:
112
-118,  
1999
.
6
Costello J. F., Fruhwald M. C., Smiraglia D. J., Rush L. J., Robertson G. P., Gao X., Wright F. A., Feramisco J. D., Peltomäki P., Lang J. C., Schuller D. E., Yu L., Bloomfield C. D., Caligiuri M. A., Yates A., Nishikawa R., Su Huang H. J., Petrelli N. J., Zhang X., O’Dorisio M. S., Held W. A., Cavenee W. K., Plass C. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns.
Nat. Genet.
,
25
:
132
-138,  
2000
.
7
Baylin S. B., Herman J. G., Graff J. R., Vertino P. M., Issa J-P. Alterations in DNA methylation: a fundamental aspect of neoplasia.
Adv. Cancer Res.
,
72
:
141
-196,  
1998
.
8
Chen R. Z., Pettersson U., Beard C., Jackson-Grusby L., Jaenisch R. DNA hypomethylation leads to elevated mutation rates.
Nature (Lond.)
,
395
:
89
-93,  
1998
.
9
Miniou P., Jeanpierre M., Blanquet V., Sibella V., Bonneau D., Herbelin C., Fischer A., Niveleau A., Viegas-Pequignot E. Abnormal methylation pattern in constitutive and facultative (X inactive chromosome) heterochromatin of ICF patients.
Hum. Mol. Genet.
,
3
:
2093
-2102,  
1994
.
10
Xu G. L., Bestor T. H., Bourc’his D., Hsieh C. L., Tommerup N., Bugge M., Hulten M., Qu X., Russo J. J., Viegas-Pequignot E. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene.
Nature (Lond.)
,
402
:
187
-191,  
1999
.
11
Okano M., Bell D. W., Haber D. A., Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.
Cell
,
99
:
247
-257,  
1999
.
12
Hansen R. S., Wijmenga C., Luo P., Stanek A. M., Canfield T. K., Weemaes C. M., Gartler S. M. The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome.
Proc. Natl. Acad. Sci. USA
,
96
:
14412
-14417,  
1999
.
13
Pradhan S., Bacolla A., Wells R. D., Roberts R. J. Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.
J. Biol. Chem.
,
274
:
33002
-33010,  
1999
.
14
Lei H., Oh S. P., Okano M., Juttermann R., Goss K. A., Jaenisch R., Li E. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells.
Development (Camb.)
,
122
:
3195
-3205,  
1996
.
15
Tan N. W., Li B. F. L. Interaction of oligonucleotides containing 6-O-methylguanine with human DNA (cytosine-5-)-methyltransferase.
Biochemistry
,
29
:
9234
-9240,  
1990
.
16
Schwartz J. L. Monofunctional alkylating agent-induced S-phase-dependent DNA damage.
Mutat. Res.
,
216
:
111
-118,  
1989
.
17
Jones P. A., Buckley J. D. The role of DNA methylation in cancer.
Adv. Cancer Res.
,
54
:
1
-23,  
1990
.
18
Chuang L. S., Ian H. I., Koh T. W., Ng H. H., Xu G., Li B. F. L. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1.
Science (Wash. DC)
,
277
:
996
-1000,  
1997
.
19
Teo A. K. C., Oh H. K., B. Ali R., Li B. F-L. The modified human DNA repair enzyme MGMT is a negative regulator of the estrogen receptor-mediated transcription upon alkylation DNA damage.
Mol. Cell. Biol.
,
21
:
7105
-7114,  
2001
.
20
Oh H. K., Teo A. K. C., B. Ali R., Lim A., Ayi T. C., Yarosh D. B., Li B. F-L. Conformational change in human DNA repair enzyme O6-methylguanine-DNA methyltransferase upon alkylation of its active site by DNA damage-related SN1 and direct-acting SN2 alkylating agents: breaking a “salt-link”?.
Biochemistry
,
35
:
12259
-12266,  
1996
.
21
Boffa L. C., Bolognesi C. Methylating agents: their target amino acids in nuclear proteins.
Carcinogenesis (Lond.)
,
6
:
1399
-1401,  
1985
.
22
Fournel M., Sapieha P., Beaulieu N., Besterman J. M., MacLeod A. R. Down-regulation of human DNA-(cytosine-5) methyltransferase induces cell cycle regulators p16(ink4A) and p21(WAF/Cip1) by distinct mechanisms.
J. Biol. Chem.
,
274
:
24250
-24256,  
1999
.
23
Knox J. D., Araujo F. D., Bigey P., Slack A. D., Price G. B., Zannis-Hadjopoulos M., Szyf M. Inhibition of DNA methyltransferase inhibits DNA replication.
J. Biol. Chem.
,
275
:
17986
-17990,  
2000
.
24
Vogel M. C., Papadopoulos T., Muller-Hermelink H. K., Drahovsky D., Pfeifer G. P. Intracellular distribution of DNA methyltransferase during the cell cycle.
FEBS Lett.
,
236
:
9
-13,  
1988
.
25
Lawley, P. D. Carcinogenesis by alkylating agents. In: C. E. Searle, Chemical Carcinogens (ed.), Vol. 1, Chapter 7, ACS Monograph 182, pp. 325–484. Washington, DC: American Chemical Society, 1984.
26
Shieh S. Y., Ahn J., Tamai K., Taya Y., Prives C. The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.
Genes Dev.
,
14
:
289
-300,  
2000
.
27
Hoeijmakers, Jan H. J. Genome maintenance mechanisms for preventing cancer.
Nature (Lond.)
,
11
:
366
-374,  
2001
.