- Email: [email protected]
Endogenous DNA methylation and epimutagenesis
Mutation Research 422 Ž1998. 97–100
Endogenous DNA methylation and epimutagenesis Robin Holliday
)
CSIRO DiÕision of Molecular Science Sydney Laboratory, P.O. Box 184, North Ryde, NSW 1670, Australia
Keywords: DNA methylation; Epimutation; 5-methyldCMP deaminase; Epimutator; Gene silencing; Tumor suppressor
1. Introduction It is now well established that genes can be silenced by the methylation of CpG doublets in their promoter regions. This methylation often occurs in CpG islands during tumour progression, or during the growth of established mammalian cell lines. Although no direct evidence exists, it is normally assumed that such methylation is due to the activity of a de novo DNA methyl transferase, which methylates non-methylated CpG doublets at a low frequency. This initial methylation is subsequently maintained by the same enzyme or another maintenance methyl transferase. We previously showed that genes can be silenced by the incorporation of 5methyldCTP into the DNA of cells permeabilised by electroporation w1,2x. The question therefore arises whether 5-methyldCMP, produced by the repair or turnover of DNA, might be phosphorylated in the cell to 5-methyldCTP and then incorporated into DNA. If this happened, it would provide a different mechanism for de novo DNA methylation, which is not due to DNA methyl transferase activity. It has been argued that this never happens in cells because Ž1. 5-methyldCMP is rapidly deaminated to
) Tel.: q61-2-94905000; Fax: q61-2-94905010; E-mail: [email protected]
thymidine monophosphate, which is then incorporated into DNA, and Ž2. that the kinases which convert monophosphate deoxynucleotides to diphosphate nucleotides do not act on 5-methyldCMP w3,4x. wNote that the subsequent phosphorylation step, from the diphosphate nucleotides to the corresponding triphosphates is non-specific w5,6xx. It is true that an endogenous pathway for DNA methylation would be disastrous for normal cells, in which DNA methylation is tightly regulated. However, it has been known for a long time that transformed cells have irregularities in enzyme metabolism, including the metabolism of pyrimidine nucleosides and nucleotides w7x. It is therefore possible that such cells are not able to fully prevent the conversion of 5-methyldCMP to 5-methyldCTP and its subsequent incorporation into DNA.
2. Results and discussion We have investigated this possibility in an established line of Chinese hamster ovary cells ŽCHO., designated K1. Aminopterin blocks the endogenous synthesis of dTMP, and also purines. The addition of thymidine and hypoxanthine allows growth in the presence of aminopterin ŽHAT medium.. 5-Methyldeoxycytidine Ž5-methyldC. can be substituted for thymidine provided a cellular deaminase is present to convert 5-methyldC to thymidine, or 5-methyldCMP
0027-5107r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 1 9 3 - 6
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R. Hollidayr Mutation Research 422 (1998) 97–100
to dTMP Žsee Fig. 1.. This is known as HAM medium w8x. A concentration of 5-methyldC in HAM medium equivalent to thymidine in HAT medium, allows only slow growth of CHO K1 cells, suggesting that the deaminaseŽs. is not very active. It was found that treatment of K1 cells with the DNA demethylating agent 5-aza-C produced derivatives which grew much more rapidly in HAM medium, and these were designated HAMq. This result suggests that the slow growth of K1 cells, designated HAM sl , is due to methylation and the silencing or partial silencing of a deaminase gene. A single clone of HAMq cells was isolated and treated with the mutagen EMS to induce BrdU resistant TK- derivatives. These isolates cannot grow on HAT medium, but they still grow on HAM medium. This shows that the active deaminase is operating at the nucleotide level, that is, the conversion of 5methyldCMP to dTMP rather than the conversion of 5-methyldC to thymidine Žsee Fig. 1.. Amongst the HAMq BrdU R isolates, one was found that was unable to grow in HAM medium containing 5 mgrml 5-methyldC, although like K1 HAM s1 it did respond to higher concentrations of 5-methyldC in HAM medium Žsee footnote to Table 1.. This BrdU R
Fig. 1. Pathways of uptake into DNA and interconversion of 5-methylcytosine and thymine nucleosides and nucleotides. The enzymes involved are: ŽA. kinases; ŽB. deaminases; ŽC. thymidylate synthetase, and ŽD. DNA polymerase. Any repair process involving excision of a DNA tract will produce thymidine monophosphate and 5-methylcytidine monophosphate. Aminopterin inhibits thymidylate synthetase, and strains resistant to BrdU lack thymidine kinase.
Table 1 Growth phenotypes of HAM strains and their BrdU R derivatives Strain
Growth in supplemented MEM sl
K1 HAM K1 HAM sl BrdU R HAMq HAMq BrdU R HAMy HAMy BrdU R
HAT
HAM
BrdC
BrdU
q y q y q y
slow slow q q y y
q q y y q q
y q y q y q
HAM medium contains 5 mgrml 5-methyldC and 10% dialysed serum. BrdC is used at 1 mgrml, and BrdU at 200 mgrml. Hypoxanthine, aminopterin and thymidine are standard concentrations w1x. Both HAM sl and HAMy strains respond to higher concentrations of 5-methyldC in HAM medium Že.g., 100 mgrml.. It is not known whether this is due to residual 5-methyldCMP deaminase activity or traces of contaminating thymidine.
derivative was designated HAMy. It was reactivated with 5-aza-C to produce a TKq strain w9x, and this was found to retain the HAMy phenotype. This result indicates that it has lost the 5-methyldCMP deaminase activity. The derivation of these strains is shown below and their phenotypes are summarised in Table 1.
Table 1 shows that HAMy and HAM sl cells are resistant to BrdC Ž5-bromodC., whereas HAMq cells are inhibited by this analogue. Deoxycytidine kinase acts on 5-methyldC and BrdC, so the resistance of HAM sl and HAMy to BrdC is presumably due to the failure to deaminate BrdCMP to BrdUMP, which kills cells when incorporated into DNA. If 5-methyldCMP deaminase is important to prevent the incorporation of the nucleotide, via the diand triphosphates, into DNA, then the HAMy strain would be expected to have a high frequency of gene silencing. This was found to be the case. It was previously known that BrdU R isolates arise spontaneously in K1 cultures at a rate of 6 = 10y5 , and that
R. Hollidayr Mutation Research 422 (1998) 97–100 Table 2 Spontaneous resistance to BrdU ŽTKy . and TG ŽHPRTy . in different HAM strains Phenotype Kl HAM sl HAMq HAMy
Resistance to BrdU
TG
6.0=10y5 y10y6 1.2=10y2
;10y6a (10y6a 4.6=10y3
The values are rates per cell determined by fluctuation tests based on 10 populations Žmedian value, see Ref. w9x.. a The TGR colonies in K1 are mainly mutations w1x. It is assumed a comparable frequency would occur in HAMq, but this has not been determined. BrdU R isolates are reactivable by 5-aza-C w9x and 23r23 TG R isolates from HAMy were also reactivable.
these isolates are always reactivable by 5-aza-C w9x. Additional evidence was obtained that the TK gene is hemizygous in CHO K1 cells, with one active TKq and one inactive methylated copy. Thus, 6 = 10y5 is the rate of epimutation by de novo methylation in a single gene. Fluctuation tests on the HAMq and HAMy strains demonstrated that the former is ; 50 times more stable than HAM sl , and the latter is about 200 times less stable, with BrdU R colonies arising at a rate of ; 10y2 . Thus the presumed activity of 5-methyldCMP deaminase is inÕersely related to the frequency of epimutation, as would be expected if the enzyme prevents uptake of 5-methyldCMP into DNA. There is one active X linked copy of HPRT Žhypoxanthine phosphoribosyl transferase. in CHO Kl cells, and thioguanine resistant ŽTG R . isolates arise with low frequency Ž; 10y6 .. Most of these are probably gene mutations, as they are not reactivated by 5-aza-C w1x. However, HPRTy isolates obtained by uptake of 5-methyldCTP in permeabilised cells are reactivable by 5-aza-C, which shows that silencing of this gene by methylation can occur. HAMy cells produce TG R derivatives at high frequency, in comparison to HAM sl and HAMq cultures, which are very stable. Twenty-three HAMy TG R isolates were treated with 5-aza-C and all were reactivated to HPRTq, as judged by their ability to grow in HAT medium. The frequencies of epimutation in the three strains with different HAM phenotypes are shown in Table 2. It is evident that the HAMy strain is an epimutator.
99
At present, experiments are in progress to document Ž1. the levels of 5-methyldCMP deaminase in HAM sl , HAMq and HAMy strains and Ž2. the uptake of 3 H labelled 5-methyldCMP into DNA in HAMy strains. It is hoped that these experiments will provide more definitive evidence that de novo methylation and gene silencing can be due to the conversion of 5-methyldCMP to 5-methyldCTP and its incorporation into DNA. It is well known that the genome is destabilised in transformed cells Žreviewed in Ref. w10x.. Not only is the karyotype usually abnormal, but also gene amplification can occur, as well as de novo DNA methylation and gene silencing. In contrast, normal somatic cells have a stable karyotype, no gene amplification and DNA methylation is tightly regulated. Therefore, one of the crucial steps in tumour progression is the loss of genome regulation. It may well be that this is an early step, triggered by mutation in a tumour suppressor gene or oncogene. However, once destabilisation has occurred it is likely that many other types of events follow and that these are important components of the whole process of tumour progression. It is suggested here that abnormalities in pyrimidine metabolism, documented many years ago Žreviewed in Ref. w7x., may have very important consequences. In particular, the inability to get rid of 5-methyldCMP, arising from the breakdown of DNA during the repair of spontaneous damage, may lead to a high level of endogenous de novo methylation and gene silencing. If this is so then the gene coding for 5-methyldCMP deaminase can be regarded as a tumour suppressor gene, since its inactivation—by whatever means—will lead to epimutation, which is one important component of gene destabilisation during tumour progression.
References w1x R. Holliday, T. Ho, Gene silencing in mammalian cells by X uptake of 5-methyl deoxycytidine 5 phosphate, Somatic Cell Mol. Genet. 17 Ž1991. 537–542. w2x J. Nyce, Gene silencing in mammalian cells by direct incorX X poration of electroporated 5-methyl-2 deoxycytidine 5 phosphate, Somatic Cell Mol. Genet. 17 Ž1991. 543–550. w3x J.A. Vilpo, L.M. Vilpo, Nucleoside monophosphate kinase may be a key enzyme preventing salvage of DNA 5-methyl cytosine, Mutat. Res. 286 Ž1993. 217–220.
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R. Hollidayr Mutation Research 422 (1998) 97–100
w4x J.A. Vilpo, L.M. Vilpo, Prevention of DNA 5-methyl cytosine reutilization in human cells, Somatic Cell Mol. Genet. 21 Ž1995. 285–288. w5x M.P. Roisin, A. Kepes, Nucleoside diphosphate kinase of Escherichia coli, a periplasmic enzyme, Biochem. Biophys. Acta 526 Ž1978. 418–428. w6x J. Munoz-Dorado, S. Inouye, M. Inouye, Nucleoside diphosphate kinase from Myxococcus xanthus, J. Biol. Chem. 265 Ž1990. 2707–2712. w7x G. Weber, Biochemical strategy of cancer cells and the
design of chemotherapy, Cancer Research 43 Ž1983. 3466– 3492. w8x T. Chan, C. Long, H. Green, A human-mouse somatic hybrid line selected for human deoxycytidine deaminase, Somatic Cell Genet. 1 Ž1975. 81–90. w9x R. Holliday, T. Ho, Evidence for allelic exclusion in Chinese hamster ovary cells, New Biol. 2 Ž1990. 719–726. w10x T. Lindahl ŽEd.., Genetic Instability in Cancer in Cancer Surveys, 28, Cold Spring Harbor Laboratory Press, New York, 1996.
Endogenous DNA methylation and epimutagenesis Robin Holliday
)
CSIRO DiÕision of Molecular Science Sydney Laboratory, P.O. Box 184, North Ryde, NSW 1670, Australia
Keywords: DNA methylation; Epimutation; 5-methyldCMP deaminase; Epimutator; Gene silencing; Tumor suppressor
1. Introduction It is now well established that genes can be silenced by the methylation of CpG doublets in their promoter regions. This methylation often occurs in CpG islands during tumour progression, or during the growth of established mammalian cell lines. Although no direct evidence exists, it is normally assumed that such methylation is due to the activity of a de novo DNA methyl transferase, which methylates non-methylated CpG doublets at a low frequency. This initial methylation is subsequently maintained by the same enzyme or another maintenance methyl transferase. We previously showed that genes can be silenced by the incorporation of 5methyldCTP into the DNA of cells permeabilised by electroporation w1,2x. The question therefore arises whether 5-methyldCMP, produced by the repair or turnover of DNA, might be phosphorylated in the cell to 5-methyldCTP and then incorporated into DNA. If this happened, it would provide a different mechanism for de novo DNA methylation, which is not due to DNA methyl transferase activity. It has been argued that this never happens in cells because Ž1. 5-methyldCMP is rapidly deaminated to
) Tel.: q61-2-94905000; Fax: q61-2-94905010; E-mail: [email protected]
thymidine monophosphate, which is then incorporated into DNA, and Ž2. that the kinases which convert monophosphate deoxynucleotides to diphosphate nucleotides do not act on 5-methyldCMP w3,4x. wNote that the subsequent phosphorylation step, from the diphosphate nucleotides to the corresponding triphosphates is non-specific w5,6xx. It is true that an endogenous pathway for DNA methylation would be disastrous for normal cells, in which DNA methylation is tightly regulated. However, it has been known for a long time that transformed cells have irregularities in enzyme metabolism, including the metabolism of pyrimidine nucleosides and nucleotides w7x. It is therefore possible that such cells are not able to fully prevent the conversion of 5-methyldCMP to 5-methyldCTP and its subsequent incorporation into DNA.
2. Results and discussion We have investigated this possibility in an established line of Chinese hamster ovary cells ŽCHO., designated K1. Aminopterin blocks the endogenous synthesis of dTMP, and also purines. The addition of thymidine and hypoxanthine allows growth in the presence of aminopterin ŽHAT medium.. 5-Methyldeoxycytidine Ž5-methyldC. can be substituted for thymidine provided a cellular deaminase is present to convert 5-methyldC to thymidine, or 5-methyldCMP
0027-5107r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 1 9 3 - 6
98
R. Hollidayr Mutation Research 422 (1998) 97–100
to dTMP Žsee Fig. 1.. This is known as HAM medium w8x. A concentration of 5-methyldC in HAM medium equivalent to thymidine in HAT medium, allows only slow growth of CHO K1 cells, suggesting that the deaminaseŽs. is not very active. It was found that treatment of K1 cells with the DNA demethylating agent 5-aza-C produced derivatives which grew much more rapidly in HAM medium, and these were designated HAMq. This result suggests that the slow growth of K1 cells, designated HAM sl , is due to methylation and the silencing or partial silencing of a deaminase gene. A single clone of HAMq cells was isolated and treated with the mutagen EMS to induce BrdU resistant TK- derivatives. These isolates cannot grow on HAT medium, but they still grow on HAM medium. This shows that the active deaminase is operating at the nucleotide level, that is, the conversion of 5methyldCMP to dTMP rather than the conversion of 5-methyldC to thymidine Žsee Fig. 1.. Amongst the HAMq BrdU R isolates, one was found that was unable to grow in HAM medium containing 5 mgrml 5-methyldC, although like K1 HAM s1 it did respond to higher concentrations of 5-methyldC in HAM medium Žsee footnote to Table 1.. This BrdU R
Fig. 1. Pathways of uptake into DNA and interconversion of 5-methylcytosine and thymine nucleosides and nucleotides. The enzymes involved are: ŽA. kinases; ŽB. deaminases; ŽC. thymidylate synthetase, and ŽD. DNA polymerase. Any repair process involving excision of a DNA tract will produce thymidine monophosphate and 5-methylcytidine monophosphate. Aminopterin inhibits thymidylate synthetase, and strains resistant to BrdU lack thymidine kinase.
Table 1 Growth phenotypes of HAM strains and their BrdU R derivatives Strain
Growth in supplemented MEM sl
K1 HAM K1 HAM sl BrdU R HAMq HAMq BrdU R HAMy HAMy BrdU R
HAT
HAM
BrdC
BrdU
q y q y q y
slow slow q q y y
q q y y q q
y q y q y q
HAM medium contains 5 mgrml 5-methyldC and 10% dialysed serum. BrdC is used at 1 mgrml, and BrdU at 200 mgrml. Hypoxanthine, aminopterin and thymidine are standard concentrations w1x. Both HAM sl and HAMy strains respond to higher concentrations of 5-methyldC in HAM medium Že.g., 100 mgrml.. It is not known whether this is due to residual 5-methyldCMP deaminase activity or traces of contaminating thymidine.
derivative was designated HAMy. It was reactivated with 5-aza-C to produce a TKq strain w9x, and this was found to retain the HAMy phenotype. This result indicates that it has lost the 5-methyldCMP deaminase activity. The derivation of these strains is shown below and their phenotypes are summarised in Table 1.
Table 1 shows that HAMy and HAM sl cells are resistant to BrdC Ž5-bromodC., whereas HAMq cells are inhibited by this analogue. Deoxycytidine kinase acts on 5-methyldC and BrdC, so the resistance of HAM sl and HAMy to BrdC is presumably due to the failure to deaminate BrdCMP to BrdUMP, which kills cells when incorporated into DNA. If 5-methyldCMP deaminase is important to prevent the incorporation of the nucleotide, via the diand triphosphates, into DNA, then the HAMy strain would be expected to have a high frequency of gene silencing. This was found to be the case. It was previously known that BrdU R isolates arise spontaneously in K1 cultures at a rate of 6 = 10y5 , and that
R. Hollidayr Mutation Research 422 (1998) 97–100 Table 2 Spontaneous resistance to BrdU ŽTKy . and TG ŽHPRTy . in different HAM strains Phenotype Kl HAM sl HAMq HAMy
Resistance to BrdU
TG
6.0=10y5 y10y6 1.2=10y2
;10y6a (10y6a 4.6=10y3
The values are rates per cell determined by fluctuation tests based on 10 populations Žmedian value, see Ref. w9x.. a The TGR colonies in K1 are mainly mutations w1x. It is assumed a comparable frequency would occur in HAMq, but this has not been determined. BrdU R isolates are reactivable by 5-aza-C w9x and 23r23 TG R isolates from HAMy were also reactivable.
these isolates are always reactivable by 5-aza-C w9x. Additional evidence was obtained that the TK gene is hemizygous in CHO K1 cells, with one active TKq and one inactive methylated copy. Thus, 6 = 10y5 is the rate of epimutation by de novo methylation in a single gene. Fluctuation tests on the HAMq and HAMy strains demonstrated that the former is ; 50 times more stable than HAM sl , and the latter is about 200 times less stable, with BrdU R colonies arising at a rate of ; 10y2 . Thus the presumed activity of 5-methyldCMP deaminase is inÕersely related to the frequency of epimutation, as would be expected if the enzyme prevents uptake of 5-methyldCMP into DNA. There is one active X linked copy of HPRT Žhypoxanthine phosphoribosyl transferase. in CHO Kl cells, and thioguanine resistant ŽTG R . isolates arise with low frequency Ž; 10y6 .. Most of these are probably gene mutations, as they are not reactivated by 5-aza-C w1x. However, HPRTy isolates obtained by uptake of 5-methyldCTP in permeabilised cells are reactivable by 5-aza-C, which shows that silencing of this gene by methylation can occur. HAMy cells produce TG R derivatives at high frequency, in comparison to HAM sl and HAMq cultures, which are very stable. Twenty-three HAMy TG R isolates were treated with 5-aza-C and all were reactivated to HPRTq, as judged by their ability to grow in HAT medium. The frequencies of epimutation in the three strains with different HAM phenotypes are shown in Table 2. It is evident that the HAMy strain is an epimutator.
99
At present, experiments are in progress to document Ž1. the levels of 5-methyldCMP deaminase in HAM sl , HAMq and HAMy strains and Ž2. the uptake of 3 H labelled 5-methyldCMP into DNA in HAMy strains. It is hoped that these experiments will provide more definitive evidence that de novo methylation and gene silencing can be due to the conversion of 5-methyldCMP to 5-methyldCTP and its incorporation into DNA. It is well known that the genome is destabilised in transformed cells Žreviewed in Ref. w10x.. Not only is the karyotype usually abnormal, but also gene amplification can occur, as well as de novo DNA methylation and gene silencing. In contrast, normal somatic cells have a stable karyotype, no gene amplification and DNA methylation is tightly regulated. Therefore, one of the crucial steps in tumour progression is the loss of genome regulation. It may well be that this is an early step, triggered by mutation in a tumour suppressor gene or oncogene. However, once destabilisation has occurred it is likely that many other types of events follow and that these are important components of the whole process of tumour progression. It is suggested here that abnormalities in pyrimidine metabolism, documented many years ago Žreviewed in Ref. w7x., may have very important consequences. In particular, the inability to get rid of 5-methyldCMP, arising from the breakdown of DNA during the repair of spontaneous damage, may lead to a high level of endogenous de novo methylation and gene silencing. If this is so then the gene coding for 5-methyldCMP deaminase can be regarded as a tumour suppressor gene, since its inactivation—by whatever means—will lead to epimutation, which is one important component of gene destabilisation during tumour progression.
References w1x R. Holliday, T. Ho, Gene silencing in mammalian cells by X uptake of 5-methyl deoxycytidine 5 phosphate, Somatic Cell Mol. Genet. 17 Ž1991. 537–542. w2x J. Nyce, Gene silencing in mammalian cells by direct incorX X poration of electroporated 5-methyl-2 deoxycytidine 5 phosphate, Somatic Cell Mol. Genet. 17 Ž1991. 543–550. w3x J.A. Vilpo, L.M. Vilpo, Nucleoside monophosphate kinase may be a key enzyme preventing salvage of DNA 5-methyl cytosine, Mutat. Res. 286 Ž1993. 217–220.
100
R. Hollidayr Mutation Research 422 (1998) 97–100
w4x J.A. Vilpo, L.M. Vilpo, Prevention of DNA 5-methyl cytosine reutilization in human cells, Somatic Cell Mol. Genet. 21 Ž1995. 285–288. w5x M.P. Roisin, A. Kepes, Nucleoside diphosphate kinase of Escherichia coli, a periplasmic enzyme, Biochem. Biophys. Acta 526 Ž1978. 418–428. w6x J. Munoz-Dorado, S. Inouye, M. Inouye, Nucleoside diphosphate kinase from Myxococcus xanthus, J. Biol. Chem. 265 Ž1990. 2707–2712. w7x G. Weber, Biochemical strategy of cancer cells and the
design of chemotherapy, Cancer Research 43 Ž1983. 3466– 3492. w8x T. Chan, C. Long, H. Green, A human-mouse somatic hybrid line selected for human deoxycytidine deaminase, Somatic Cell Genet. 1 Ž1975. 81–90. w9x R. Holliday, T. Ho, Evidence for allelic exclusion in Chinese hamster ovary cells, New Biol. 2 Ž1990. 719–726. w10x T. Lindahl ŽEd.., Genetic Instability in Cancer in Cancer Surveys, 28, Cold Spring Harbor Laboratory Press, New York, 1996.