Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels

Mutation Research 415 Ž1998. 213–218

Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels Yong-Woo Lee 1, Limor Broday, Max Costa

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Institute of EnÕironmental Medicine and Kaplan ComprehensiÕe Cancer Center, New York UniÕersity Medical Center, 550 First AÕenue, New York, NY 10016, USA Received 19 March 1998; revised 14 May 1998; accepted 20 May 1998

Abstract Methylation of DNA plays an important role in organizing the genome into transcriptionally active and inactive zones. Nickel compounds cause chromatin condensation and DNA methylation in the transgenic gptq Chinese hamster cell line ŽG12.. Here we show that nickel is an inhibitor of cytosine 5-methyltransferase activity in vivo and in vitro. In living cells, this inhibition is transient and following a recovery period after nickel treatment, Mtase activity slightly rebounds. Genomic DNA methylation levels are also somewhat decreased following nickel treatment, but with time, there is an elevation of total DNA methylation above basal levels and before any rebound of methyltransferase activity. These results suggest that nickel exposure can elevate total genomic DNA methylation levels even when DNA methyltransferase activity is depressed. These findings may explain the hypermethylation of senescence and tumor suppressor genes found during nickel carcinogenesis and support the model of a direct effect of Ni 2q on chromatin leading to de novo DNA methylation. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Epigenetic; Carcinogenesis; Nickel

1. Introduction In eukaryotic cells, DNA methylation and DNA– protein interactions together, organize the genome into transcriptionally active and inactive zones. DNA methylation in mammals is involved in imprinting, regulation of transcription, and development w1–3x. Changes in methylation patterns during development and differentiation involve de novo methylation, ) Corresponding author. Tel.: q1-914-351-2368; Fax: q1-914351-2118 1 Present address: Inje University, 607 Obang-dong, Kimhae, Kyongsangnamdo, South Korea 621-749.

maintenance methylation, and demethylation. Various diseases, including cancer and the fragile X syndrome, are associated with abnormal DNA methylation w4x. Only a single DNA methyltransferase Ž5-cytosine methyltransferase ŽMtase. EC 2.1.1.37. has been characterized in mammals. This enzyme has a strong preference for hemi-methylated substrate DNA w5x. The enzyme has conserved amino-terminal domain and removal of this domain greatly stimulates the de novo DNA-methyltransferase activity of the enzyme, suggesting that, besides a maintenance methylation activity, this enzyme might also be responsible for de novo methylation w6x. A gene for de novo DNA

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 7 8 - 3

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methylation has recently been characterized in Ascobolus w7x. Developments in the field of cancer research over the past few years have led to an increased understanding of the role DNA methylation may play in tumorigenesis. There are two major mechanisms by which DNA methylation may lead to aberrant cell cycle control. The first one is through the generation of transition mutations via deamination of 5-mCyt to T, resulting in point mutations that inactivate tumor suppressor genes such as p53 w8x. The second mechanism is by altering levels of gene expression through epigenetic effects at CpG islands w4x. This may cause hypomethylation of proto-oncogenes or hypermethylation of tumor suppressor genes with respective increased or decreased gene expression w4x. Methylation errors introduced during development, aging, various carcinogens, some chemotherapeutic agents, and methyl-donor deficient diet, have been shown to result in changes in methylation levels of genomic DNA leading to genetic and epigenetic alterations w4,9x. Oxidative DNA damage was also shown to cause the loss of DNA methylation w10x. Certain nickel compounds including crystalline nickel sulfide ŽNiS. and subsulfide ŽNi 3 S 2 . are potent human and animal carcinogens w11x. The mechanisms of carcinogenic action of these nickel compounds have been difficult to define, particularly since they were generally weak mutagens in most of the traditional test systems w11x. We have previously reported that Ni 3 S 2 and NiS induced the inactivation of the gpt reporter gene in the transgenic gptq Chinese hamster cell line ŽG12. w12x. Analysis of the NiS or Ni 3 S 2 induced 6TG resistant G12 variants, showed that most of the mutants lacked gpt transcription without mutagenesis or deletion of the transgene. The reversion rates of these variants were very high Ž10y4 to 10y2 ., and were enhanced following treatment with the demethylating agent 5azacytidine w12x. The involvement of chromatin condensation and DNA methylation in the inactivation of this transgene by NiS was confirmed w12x. The high incidence of 6TG resistance induced by these nickel compounds in the transgenic G12 cell line, but not in another transgenic cell line, G10, suggested that the location of the transgene in G12 cells near a large and dense heterochromatic region of the genome was a preferential site for nickel’s action. Thus, the

reporter sequence in the G12 cell line was susceptible to heterochromatin spreading and a PEV Žposition-effect variegation.-like phenomenon w12x. Here we have examined the influence of carcinogenic nickel compounds on DNA methyltransferase activity and methylation levels in the mouse 3T3 cell line and also in the B200 nickel resistant cell line w13,14x. We have shown that Ni 2q inhibited DNA Mtase in vivo and in vitro, and also diminished the overall methylation levels in cells. However, following a recovery period after nickel treatment, Mtase activity rebounded slightly but there was also an overall increase in genomic DNA methylation that preceded any change in Mtase activity. These changes may account in part for the hypermethylation of senescence and tumor suppressor genes associated with nickel carcinogenesis w15x.

2. Materials and methods 2.1. Cell culture and nickel treatment BALBrc-3T3 mouse fibroblasts obtained from the American Type Culture Collection were grown in monolayer in DMEM containing 10% fetal bovine serum, 2 mM L-glutamine, 100 unitsrml penicillin, and 100 mgrml streptomycin. The cultures were maintained at 378C in a humidified atmosphere of 95% air: 5% CO 2 . The cells were grown for 24 h and then exposed to 0.3 mgrcm2 Ni 3 S 2 . Following the indicated treatment period, the cells were rinsed twice with saline A and incubated further for the indicated recovery periods. The nickel-resistant cells, B200, were grown as described above, in a medium containing 200 mM NiCl 2 , and harvested after the indicated periods. The B200 cell line was derived from the mouse 3T3 cell line by exposure to 200 mM of NiCl 2 w13x. The B200 cell line is resistant to growth inhibition by 200 mM NiCl 2 and exhibits a transformed phenotype not found in our parental 3T3 cells Ži.e., ability to proliferate in soft agar w13x. This level of NiCl 2 Ž200 mM. inhibits the growth of wild-type 3T3 cells and is cytotoxic, but B200 cells are resistant to these effects w13x. During the exposure, the cells were grown to 60 to 80% confluency, trypsinized, and replated at a 1:4 dilution in a fresh medium containing 200 mM NiCl 2 .

Y.-W. Lee et al.r Mutation Research 415 (1998) 213–218

2.2. DNA Mtase enzyme assay A modification of the assay developed by Adams et al. w16x was used to determine DNA Mtase activity. A total of 1 = 10 7 cells were scraped from plates, pooled into ice-cold PBS and collected by centrifugation. The cells were suspended in 500 ml lysis buffer w50 mM Tris–HCl, pH 7.8, 1 mM DTT, 0.01% NaN3 , 1 mM EDTA, 0.35 mM PMSF, 10% Glycerol, 1% Tween 80, 100 mgrml RNaseAx, and then lysed by four cycles of freezing at y708C and thawing at 378C. Protein concentration was determined by the Bradford assay. Cell lysates containing 5 mg protein were mixed with 0.5 mg poly dI-dC and 1.5 mM S-adenosyl-L-wmethyl-3 Hxmethionine Ž80–85 Cirmmol; Amersham, TRK 581. in a total volume of 20 ml and incubated at 378C for 2 h. The reactions were terminated by adding 300 ml of a stop solution Ž1% SDS, 2 mM EDTA, 5% 2-propanol, 125 mM NaCl, 1 mgrml Proteinase K, 0.25 mgrml carrier DNA. for 1 h at 378C. The DNA was extracted with phenol–chloroform and ethanol precipitated. The recovered DNA was resuspended in 30 ml of 0.3 M NaOH, and incubated for 30 min at 378C. DNA was spotted on GFrC Whatman filter discs, dried, then washed five times with 5% Žwrv. trichloroacetic acid followed by 70% Žvrv. ethanol. Filters were placed in scintillation vials and incubated for 1 h at 608C with 500 ml of 0.5 M perchloric acid. Then 5 ml of scintillation cocktail was added and radioactive 3 H incorporation into DNA was assessed using a Beckman liquid scintillation counter. 2.3. Direct effects of NiCl 2 on DNA Mtase enzyme actiÕity The procedure was the same as above, except that the reaction mixture also contained 0, 100, 200 or 400 mM NiCl 2 . In order to examine the influence of nickel on M.SssI CpG methylase, four units of the enzyme, substituted for cell lysate, were used in the same reaction conditions. 2.4. Genomic DNA methylation leÕel

was incubated with 4 units of SssI, HpaII or HhaI methylases ŽNew England Biolabs., in the presence of 1.5 mM S-adenosyl-L-wmethyl-3 Hxmethionine and 1.5 mM nonradioactive S-adenosylmethionine. The reaction mixtures Ž20 ml., in the manufacturer’s provided buffer containing 0.1 mg RNaseA, were incubated at 378C for 4 h. The reactions were terminated and read as described above for the DNA Mtase enzyme activity assay. Higher levels of w3 Hxmethionine incorporation into DNA indicated lower levels of genomic DNA methylation, while less w3 Hxmethionine incorporation are indicative of higher levels of genomic DNA methylation w17x.

3. Results and discussion We first measured the Mtase activities and genomic DNA methylation levels in 3T3 and in the nickel-resistant cell line, B200. As shown in Table 1, the Mtase activity and DNA methylation level of nickel resistant and transformed B200 cells were higher than in 3T3 cells. B200 cells exhibited a transformed phenotype including loss of contact inhibition and growth in soft agar w13x. DNA hypermethylation has been associated with acquisition of rodent transformed phenotypes w15x. Next, we examined the effects of nickel compounds on Mtase activity and methylation levels in both cell lines. 3T3 cells were grown in the presence of 0.3 mgrcm2 Ni 3 S 2 for 0, 10 and 24 h. The cells were harvested following different recovery periods and the Mtase and DNA methylation levels were measured. The Mtase activity was reduced following treatment with Ni 3 S 2 ŽTable 2., but after 2 weeks of recovery, the level of the enzyme activity was higher Table 1 Comparison of Mtase activity and genomic DNA methylation levels in 3T3 and B200 cells Cells

Mtase activity a

Genomic DNA methylation level b

3T3 B200

100"1.4% 147"1.2%

100"0.1% 119"2.0%

a

A modification of the methyl-accepting assay w17x was used to determine the methylation level of DNA isolated from 3T3 and B200 cells. DNA Ž200 ng.

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Mtase activity was measured from triplicate samples and expressed as percent of control. b The methylation levels were measured from triplicate samples by a methyl-accepting assay using CpG methylase Ž SssI..

Y.-W. Lee et al.r Mutation Research 415 (1998) 213–218

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Table 2 Effects of Ni 3 S 2 on Mtase activity and genomic DNA methylation levels in 3T3 cells Time Žtreatmentqrecovery.

Mtase activity a

Genomic DNA Methylation level b

0 hq0 day 10 hq0 day 24 hq0 day 24 hq1 day 24 hq3 days 24 hq2 weeks

100 24 24 20 23 140

100 99"4 102"2 105"3 123"3 115"2

a

Relative Mtase activity Ž%. measured from duplicate samples. Methylation levels of genomic DNA were measured from duplicate samples, by methyl-accepting assay using three different methylases Ž SssI, HpaII and HhaI., and calculated by the expression of w1qŽcontrolytreated.rcontrol4x100 3T3 cells were treated for the times indicated with 0.3 mgrcm2 Ž7.8 mM. of Ni 3 S 2 . b

then in the same cells that received no treatment. The overall methylation level did not change significantly immediately after the treatment, but after 3 days of recovery, it increased as high as 23% above the control level ŽTable 2.. It should be noted that an increase in genomic DNA methylation preceded the increase in Mtase activity ŽTable 2.. Chronic effects of soluble nickel Ž200 mM NiCl 2 . were tested in the nickel-resistant B200 cells. The cells were treated for 1, 2 and 3 weeks with NiCl 2 and the level of Mtase activity was reduced as a result of this treatment in this cell line ŽTable 3.. The methylation levels were reduced to 67% of the control level after 1 week of treatment, but subsequently increased up to 131% of the control level ŽTable 3.. Although the treatments with the water-soluble and water-insoluble nickel were different Žsee Section 2., the results showed the same pattern. Mtase activity did not rise to higher than basal level in the B200 cells but these cells already had 47% higher Mtase activity than wild type ŽTable 1. but there was some recovery of activity with time ŽTable 3.. Again, genomic methylation levels rose before any rebound in Mtase activity ŽTable 3.. The enzyme activity in 3T3 cells after Ni 3 S 2 treatment was reduced to 24% of control ŽTable 2., while the level of the enzyme after NiCl 2 treatment was reduced only to 65% of the control ŽTable 3.. This may be explained by the difference in the uptake process and in the effects on nickel resistant

B200 cells compared to wild type. Uptake of nickel ions in solution is poor in comparison to phagocytized crystalline nickel sulfides w18x, therefore, 3T3 cells had higher intracellular Ni concentrations than B200 cells. Both wild-type and nickel-resistant cells phagocytized Ni 3 S 2 particles resulting in higher levels of soluble Ni 2q inside the cell than could be achieved by the uptake of soluble Ni 2q w11x. This process has been termed facultative phagocytosis, a phenomenon exhibited by most cultured cells. Cell survival following either 200 mM NiCl 2 or 0.3 mgrcm2 Ni 3 S 2 depended upon treatment time but it should be noted that a 24 h treatment of B200 cells with 200 mM NiCl 2 did not affect cell survival w13x, while 24 h exposure of 3T3 cells to 0.3 mgrcm2 Ni 3 S 2 reduced cell survival about 40% Žnot shown.. The methylation level after nickel treatment was reduced more strikingly in the B200 cells compared to the effect of Ni 3 S 2 in 3T3 cells. In the B200 resistant cells, the starting methylation levels and enzyme activity were higher compared to 3T3 cells ŽTable 1.. It is possible that a decrease in Mtase activity as a result of the nickel treatment might have more rapidly influenced genomic methylation levels in the B200 cells despite the fact that Ni 3 S 2 was more potent at elevating cellular Ni 2q levels. It should be noted that Table 2 shows levels of exposure to Ni 3 S 2 in mgrcm2 , but in parentheses we

Table 3 Chronic effect of NiCl 2 on Mtase activity and genomic DNA methylation levels in B200 cells Length of treatment

Mtase activity a

Genomic DNA methylation level b

No treatment 1 week 2 weeks 3 weeks 3 weeksy2 days c

100 65 65 59 75

100 67 88 128 131

a

Relative Mtase activity Ž%. measured from duplicate samples. Methylation levels of genomic DNA were measured from duplicate samples, by methyl-accepting assay using three different methylases Ž SssI, HpaII and HhaI., and calculated by the expression of w1qŽcontrolytreated.rcontrol4x100. c NiCl 2 was removed 2 days prior to cell harvest. Nickel-resistant B200 cells were treated with 200 mM NiCl 2 for the times indicated in the table. Mtase activity and levels of DNA methylation were measured as described in Section 2. b

Y.-W. Lee et al.r Mutation Research 415 (1998) 213–218 Table 4 Inhibition of Mtase activity by NiCl 2 in vitro NiCl 2 ŽmM.

CpG methlase Ž SssI. a

3T3 cell lysatea

0 100 200 400

100 65 38 11

100 90 80 66

a

Mtase activity was measured from duplicate samples and expressed as percent of control.

also show levels in mM as well, since Ni 3 S 2 is partially soluble in media w11x. It has previously been reported that interactions of nickel with proteins and other intracellular molecules represented a likely pathway for its effects upon gene expression w11x. It was also shown that nickel disrupted binding of proteins to DNA w19x. In order to check this possibility on the DNA methyltransferase enzyme, we assessed the effect of various NiCl 2 concentrations on DNA Mtase activity in 3T3 cell lysate and on the bacterial CpG methylase Ž SssI.. The enzyme activity in both cases decreased with increasing NiCl 2 concentration ŽTable 4.. This experiment showed that Ni 2q directly inhibited the Mtase enzyme activity, and it may explain the immediate loss in DNA methylation in cells following nickel treatment. However, the level at which nickel ions inhibited the enzyme activity in vitro is low considering the levels that inhibit other enzymes, or considering the levels that are cytotoxic to cells w11,20x. The mechanism of the direct inhibition of Mtase activity has not been studied. There are known inhibitors of DNA methyl-transferase such as the nucleotide analogs Ž5-azacytidine., and analogs or inhibitors of the synthesis of AdoMet and AdoHcy w4x. But in contrast to such stable inhibition mechanisms, the inhibition of the Mtase activity by nickel was only transient, and after a recovery period the enzyme activity recovered slightly, but when Mtase activity was still depressed, much higher levels of genomic DNA methylation were found compared with untreated cells. We have previously shown that nickel induces heterochromatic specific hypermethylation w12x. It is possible that the high levels of genomic DNA methylation that were measured after long recovery periods or chronic treatment, reflect a component of the mecha-

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nism responsible for nickel induced DNA hypermethylation by its ability to enhance chromatin condensation which triggers de novo DNA methylation w12,15x. In the B200 cells, the resistance to NiCl 2 was associated with a high incidence of heterochromatic abnormalities involving fusions at the centromeres w13x. The relationship between the nickelinduced increase in genomic methylation and nickel-induced changes in chromatin structure require further study, but both are likely to play a role in the inactivation of tumor suppressor genes by DNA hypermethylation during nickel carcinogenesis w15,21x.

Acknowledgements This work was supported by grants ES05512 and ES00260 from the National Institute of Environmental Health Sciences and grant CA16037 from the National Cancer Institute.

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