DNA Methyltransferase Inhibition Induces the Transcription of the Tumor Suppressor p21

Previous lines of evidence have shown that inhibition of DNA methyltransferase (MeTase) can arrest tumor cell growth; however, the mechanisms involved were not clear. In this manuscript we show that out of 16 known tumor suppressors and cell cycle regulators, the cyclin-dependent kinase inhibitor p21 is the only tumor suppressor induced in the human lung cancer cell line, A549, following inhibition of DNA MeTase by a novel DNA MeTase antagonist or antisense oligonucleotides. The rapid induction of p21 expression points to a mechanism that does not involve demethylation of p21 promoter. Consistent with this hypothesis, we show that part of the CpG island upstream of the endogenous p21 gene is unmethylated and that the expression of unmethylated p21 promoter luciferase reporter constructs is induced following inhibition of DNA MeTase. These results are consistent with the hypothesis that the level of DNA MeTase in a cell can control the expression of a nodal tumor suppressor by a mechanism that does not involve DNA methylation. Previous lines of evidence have shown that inhibition of DNA methyltransferase (MeTase) can arrest tumor cell growth; however, the mechanisms involved were not clear. In this manuscript we show that out of 16 known tumor suppressors and cell cycle regulators, the cyclin-dependent kinase inhibitor p21 is the only tumor suppressor induced in the human lung cancer cell line, A549, following inhibition of DNA MeTase by a novel DNA MeTase antagonist or antisense oligonucleotides. The rapid induction of p21 expression points to a mechanism that does not involve demethylation of p21 promoter. Consistent with this hypothesis, we show that part of the CpG island upstream of the endogenous p21 gene is unmethylated and that the expression of unmethylated p21 promoter luciferase reporter constructs is induced following inhibition of DNA MeTase. These results are consistent with the hypothesis that the level of DNA MeTase in a cell can control the expression of a nodal tumor suppressor by a mechanism that does not involve DNA methylation. methyltransferase DNA methyltransferase 1 polymerase chain reaction DNA methylation of cytosine residues located at the dinucleotide sequence CpG can suppress expression of genes either by directly interfering with the binding of transcription factors (1.Becker P.B. Ruppert S. Schutz G. Cell. 1987; 51: 435-443Abstract Full Text PDF PubMed Scopus (196) Google Scholar) or by attracting methylated-DNA binding proteins such as MeCP2 (2.Nan X. Campoy F.J. Bird A. Cell. 1997; 88: 471-481Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar, 3.Nan X. Ng H., H. Jonson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2788) Google Scholar, 4.Jones P.L. Veenstra G.J.C. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2243) Google Scholar). To ascertain that the epigenetic information encoded in the methylation pattern is faithfully maintained, the expression of the maintenance DNA MeTase1 enzyme DNMT1, which catalyzes the transfer of a methyl group fromS-adenosyl-methionine to the 5′ position on cytosines residing at the dinucleotide CpG (5.Gruenbaum Y. Cedar H. Razin A. Nature. 1982; 295: 620-622Crossref PubMed Scopus (309) Google Scholar), is tightly coordinated with DNA replication (6.Araujo F.D. Knox J.D. Szyf M. Price G. Zannis-Hadjopoulos M. Mol. Cell. Biol. 1998; 18: 3475-3482Crossref PubMed Scopus (74) Google Scholar) and the state of growth of the cell (7.Szyf M. Bozovic V. Tanigawa G. J. Biol. Chem. 1991; 266: 10027-10030Abstract Full Text PDF PubMed Google Scholar). Different protooncogenic pathways can up-regulate dnmt1 expression (8.Rouleau J. MacLeod A.R. Szyf M. J. Biol. Chem. 1995; 270: 1595-1601Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and high levels of dnmt1 mRNA have been observed in many cancer cells (9.Kautiainen T.L. Jones P.A. J. Biol. Chem. 1986; 261: 1594-1598Abstract Full Text PDF PubMed Google Scholar, 10.El-Deiry W.S. Nelkin B.D. Celano P. Yen R.W. Falco J.P. Hamilton S.R. Baylin S.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3470-3474Crossref PubMed Scopus (295) Google Scholar). It has been proposed that increased DNA MeTase levels are a downstream component of oncogenic programs (11.Szyf M. Trends Pharmacol. Sci. 1994; 7: 233-238Abstract Full Text PDF Scopus (101) Google Scholar) and that they play a causal role in cellular transformation (12.Wu J. Issa J.P. Herman J. Bassett Jr., D.E. Nelkin B.D. Baylin S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8891-8895Crossref PubMed Scopus (254) Google Scholar, 13.MacLeod A.R. Szyf M. J. Biol. Chem. 1995; 270: 8037-8043Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). This hypothesis has been supported by the observation that a reduction in DNA MeTase levels by either 5-aza-deoxycytidine (13.MacLeod A.R. Szyf M. J. Biol. Chem. 1995; 270: 8037-8043Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 14.Laird P.W. Jacksongrusby L. Fazeli A. Dickinson S.L. Jung W.E. Li E. Weinberg R.A. Jaenisch R. Cell. 1995; 81: 197-205Abstract Full Text PDF PubMed Scopus (660) Google Scholar), DNA MeTase antisense mRNA or antisense oligonucleotides can reverse tumor growth in vivo and in vitro (13.MacLeod A.R. Szyf M. J. Biol. Chem. 1995; 270: 8037-8043Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 14.Laird P.W. Jacksongrusby L. Fazeli A. Dickinson S.L. Jung W.E. Li E. Weinberg R.A. Jaenisch R. Cell. 1995; 81: 197-205Abstract Full Text PDF PubMed Scopus (660) Google Scholar, 15.Ramchandani S. MacLeod A.R. Pinard M. von Hofe E. Szyf M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 684-689Crossref PubMed Scopus (168) Google Scholar). The mechanisms responsible for cellular transformation by DNMT1 and the reversal of transformation by DNA MeTase inhibition are unknown. One attractive hypothesis is that high levels of DNA MeTase lead to ectopic methylation and inactivation of tumor suppressor genes such as p16 (16.Merlo A. Herman J.G. Mao L. Lee D.J. Gabrielson E. Burger P.C. Baylin S.B. Sidransky D. Nat. Med. 1995; 1: 686-692Crossref PubMed Scopus (1878) Google Scholar). Similarly, the inhibition of DNA MeTase could result in demethylation and lead to activation of p16 (17.Otterson G.A. Khleif S.N. Chen W. Coxon A, B. Kaye F.J. Oncogene. 1995; 11: 1211-1216PubMed Google Scholar, 18.Gonzalgo M.L. Hayashida T. Bender C.M. Pao M.M. Tsai Y.C. Gonzales F.A. Nguyen H.D. Nguyen T.T. Jones P.A. Cancer Res. 1998; 58: 1245-1252PubMed Google Scholar). However, because both the increase in DNA MeTase levels and its inhibition by pharmacological inhibitors are global processes, it is difficult to understand how they could predictably result in a discrete change of the methylation state of specific sites. A more likely explanation is that ectopic methylation of tumor suppressors in tumor cells is a slow and stochastic process, but the aberrant methylation events are selected because they confer a growth advantage. This hypothesis is supported by the observation that in tumors bearing one mutant and one normal allele of p16, only the normal allele is methylated (19.Myohanen S.K. Baylin S.B. Herman J.G. Cancer Res. 1998; 58: 591-593PubMed Google Scholar). Similarly, partial inhibition of DNA MeTase with pharmacological agents results in a stochastic demethylation of a specific site only in a fraction of the cells at each round of replication. An alternative possibility is that the DNMT1 protein might have a more direct and immediate effect on the state of cellular growth and transformation (20.Szyf M. Cancer Metast. Rev. 1998; 17: 219-231Crossref PubMed Scopus (28) Google Scholar). To investigate this possibility we inhibited DNA MeTase and examined the expression of genes known to control the cell cycle. In the past, 5-aza-deoxycytidine has been used as the standard inhibitor of DNA MeTase (21.Jones P.A. Cell. 1985; 40: 485-486Abstract Full Text PDF PubMed Scopus (298) Google Scholar). Unfortunately, the incorporation of 5-aza-deoxycytidine into DNA and subsequent trapping of DNA MeTase enzyme unto the replicating DNA (22.Wu J.C. Santi D.V. Prog. Clinic. Biol. Res. 1985; 198: 119-129PubMed Google Scholar) results in pleiotropic effects that confound the interpretation of the results (23.Juttermann R. Li E. Jaenisch R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11797-11801Crossref PubMed Scopus (582) Google Scholar). Therefore, we have developed two other approaches to inhibit DNA MeTase: antisense oligonucleotides (15.Ramchandani S. MacLeod A.R. Pinard M. von Hofe E. Szyf M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 684-689Crossref PubMed Scopus (168) Google Scholar) and oligonucleotide-based DNA MeTase antagonists that form a stable complex with DNA MeTase and inhibit its activity at an EC50 of 60 nm (24.Szyf M. Bigey P. Curr. Res. Mol. Ther. 1998; 1: 93-101Google Scholar, 25.Bigey P Knox J.D. Croteau S. Bhattacharya S.K. Théberge J. Szyf M. J. Biol. Chem. 1999; 274: 4594-4606Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). We have previously shown that inhibition of DNA MeTase by DNA MeTase antagonists results in a rapid inhibition of DNA replication that is inconsistent with a stochastic model (25.Bigey P Knox J.D. Croteau S. Bhattacharya S.K. Théberge J. Szyf M. J. Biol. Chem. 1999; 274: 4594-4606Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In this manuscript we demonstrate that inhibition of DNA MeTase results in the rapid induction of the known tumor suppressor and cell cycle regulator p21 by a mechanism that does not involve DNA methylation of the p21 promoter. A549 cells, a human non-small cell lung carcinoma cell line (26.Giard D.J. Aaronson S.A. Todaro G.J. Arnstein P. Kersey J.H. Dosik H. Parks W.P. J. Natl. Cancer Inst. 1973; 51: 1417-1423Crossref PubMed Scopus (1857) Google Scholar) (ATCC: CCL 185), were grown in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal calf serum and 2 mm glutamine. HEK 293 cells, a human adenovirus type 5 transformed human embryonal kidney cell line (27.Graham F.L. Smiley J. Russel W.C. Nairn R J. Gen. Virol. 1977; 36: 59-74Crossref PubMed Scopus (3507) Google Scholar) (ATCC, CRL 1573), were grown in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum and 2 mm glutamine. For oligonucleotide treatment A549 cells were plated at a concentration of 2.5 × 105 cells/100-mm tissue culture dish 18 h prior to treatment. The DNA MeTase antagonist (3118) used in our study is a phosphorothioate modified hemimethylated hairpin of the following sequence: 5′-CTGAA(methyl)CGGAT(methyl)CGTTTCGATCCGTTCAG-3′. The sequence of the inactive analog used in our study (3188) is identical; it is also phosphorothioate modified but has a 2′-O-methyl modification of the sugar backbone. The oligos 3118 and 3188 have the same structure as the DNA MeTase antagonist 3016 and the inactive analog 3060, which were previously described by us (25.Bigey P Knox J.D. Croteau S. Bhattacharya S.K. Théberge J. Szyf M. J. Biol. Chem. 1999; 274: 4594-4606Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), except that 3118 and 3188 bear a fluorescein tag at the 5′ end. The sequence of the phosphorothioate antisense oligodeoxynucleotide used in this study is: 5′-AAGCATGAGCACCGTTCTCC-3′ (as) and its mismatch control oligonucleotide (mm) has a 3-base pair mismatch. For determining p21 mRNA stability, the 4-h oligonucleotide treatment was followed by treatment for and For binding A549 cells were with for h in following which the medium was by a growth medium for and the cells were For HEK 293 cells were plated 18 h prior to at a concentration of 5 × 105 cells/100-mm tissue culture The medium was h and the cells were h was the cells at the points the treatment standard of the were to an two as by the The for antisense for p16, and the and The for antisense for and The were on a to and by The of the was by and was to the of the control mRNA in each cell were standard and on a for DNA MeTase and for to and the binding with p21 protein was p21 at followed by at and DNMT1 protein was as described previously (15.Ramchandani S. MacLeod A.R. Pinard M. von Hofe E. Szyf M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 684-689Crossref PubMed Scopus (168) Google Scholar). was reverse with reverse and the in the of to the of reverse of reverse as by the incorporation were to in the of of a DNA that with the same of but a that is by The following and the were p21 mRNA sequence previously described L. T. P. Zannis-Hadjopoulos M. Price G. J. Mol. Biol. 1997; PubMed Scopus Google at at and used to at were as for 1 for 1 and for 1 DNA binding was by sequence as described G. Lee J. 1998; 17: PubMed Scopus Google Scholar), except that the binding reaction 1 mm mm mm mm mm 2 of of cell and of R.A. D.E. Lee Nature. 1998; PubMed Scopus Google Scholar). The were on a was as described previously with J. M. Res. 1994; PubMed Scopus Google Scholar). of A549 DNA were to the of described were used as for subsequent The of the reaction were the by the and the were the The used for the of the p21 at at at and at was by the J. Tanigawa G. Szyf M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). p21 mRNA level was by of and human p21 The levels of expression of p21 mRNA were by and in each to the of as by with 18 oligonucleotide M. J.G. Mol. PubMed Scopus Google Scholar). The was used to HEK 293 cells with either 2 of luciferase reporter or luciferase reporter constructs or of the p21 promoter upstream of the site J.C. W.S. Nature. 1997; PubMed Scopus Google Scholar). The cells were with 5 of either control DNA or a dnmt1 antisense expression in 1 of was with to control for by The luciferase activity was as described previously W.S. T. R. D. W.E. B. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). DNA MeTase activity was of HEK 293 protein as described previously (7.Szyf M. Bozovic V. Tanigawa G. J. Biol. Chem. 1991; 266: 10027-10030Abstract Full Text PDF PubMed Google Scholar). The reaction was out in a of mm 5 mm 1 as a methyl and mm of a hemimethylated oligonucleotide as a methyl as described previously (7.Szyf M. Bozovic V. Tanigawa G. J. Biol. Chem. 1991; 266: 10027-10030Abstract Full Text PDF PubMed Google Scholar). a at the incorporation of methyl into the DNA was by of The methylation of other in the as as endogenous DNA methylation were by the the same in the of the hemimethylated The were the for each The results are as the of To inhibition of DNA MeTase in cancer cells results in induction of the expression of tumor we A549 human lung carcinoma cells with either antisense oligonucleotides (as) dnmt1 mRNA or control or a DNA MeTase antagonist (3118) and its inactive control (3188) for 5 The DNA MeTase antagonist was previously shown to inhibit DNA MeTase activity in control A549 cells of 25.Bigey P Knox J.D. Croteau S. Bhattacharya S.K. Théberge J. Szyf M. J. Biol. Chem. 1999; 274: 4594-4606Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The antisense oligonucleotide was previously shown to the of levels of DNMT1 protein at an 60 nm not of inhibition of DNA MeTase were used to the possibility that the observed a the cells was to an with two of that the level of expression of 16 known cell cycle and tumor in p21 was the only known tumor suppressor that was induced by both p21 protein levels are also induced treatment with both DNA MeTase antisense oligonucleotides and antagonists as by the shown in 2 of DNA MeTase p21 is of cell A549 cells with either antisense oligonucleotides to 1 mRNA antisense mismatch control DNA MeTase antagonist inactive analog of the antagonist or the for or 5 The of p21 protein and DNA MeTase protein are by The of induction of p21 following inhibition of DNA MeTase can an as to the mechanism We to the DNA MeTase antagonist (3118) because it directly with DNA MeTase In the antisense by mRNA which to the reduction of protein and the of DNA MeTase might confound the interpretation of the inhibitor of DNA methylation have its effect at the of DNA replication. Therefore, the fraction of cells that are at a specific of increase with the of replication The level of p21 mRNA following DNA MeTase antagonist treatment was by which that the induction of p21 mRNA was rapid and that it the h to 5 These results are consistent with a mechanism that does not involve demethylation of specific sites. The also the induction of p21 following treatment with a DNA MeTase antagonist observed in the is a the of induction of p21 mRNA and protein The level of p21 mRNA induction is observed treatment the levels of p21 protein to increase to 5 following treatment 2 Similarly, DNMT1 protein is 1 antisense treatment p21 protein levels to increase to 5 2 A explanation is that the of protein is that of the in of protein following induction of p21 possibility is that inhibition of DNA MeTase p21 protein levels by an mechanism as by M. S. N. MacLeod R.A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). are to this DNA methylation is to play a role in the of p21 gene expression because the p21 gene is in the cells DNA methylation is to an in gene expression J. Szyf M. Razin A. Cedar H. DNA and Scholar). CpG in the promoter have been shown to to in many cancer cells S.B. Herman J.G. Issa J.P. Cancer Res. 1998; PubMed Google Scholar) and are to involved in of To study the state of methylation of p21 we a of the of the p21 promoter that also with the p21 CpG island The to methylated in the cells These results are consistent with the hypothesis that DNA MeTase antagonists not p21 mRNA levels by demethylation of its promoter. p21 a role in the to DNA and has been shown to binding in its promoter and to a downstream of W.S. T. R. D. W.E. B. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). results not the hypothesis that mRNA is induced following treatment with DNA MeTase inhibitors An alternative possibility is that either the of the oligonucleotide inhibitors or the inhibition of DNA methylation are as DNA in an activation of and an increase in DNA binding to G. Lee J. 1998; 17: PubMed Scopus Google Scholar). To this hypothesis, we the shown in of A549 cells with the DNA results in activation of and binding to its This a complex by as previously shown G. Lee J. 1998; 17: PubMed Scopus Google Scholar). In treatment with DNA MeTase inhibitors does not sequence have that the of the p21 mRNA could by such as J. G. P. S. A. J. 1995; PubMed Scopus Google Scholar) and M. E. T. J. 1997; PubMed Scopus Google Scholar). It has also been shown that p21 mRNA a in its that is by the mRNA proteins B. M. H. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). To DNA MeTase antagonists increase the of p21 we A549 cells with either the DNA MeTase antagonist (3118) or the inactive analog the mRNA transcription inhibitor was to the and an and the level of p21 mRNA was by a at points of treatment. observed in DNA MeTase antagonist treatment does not the of p21 that induction of p21 mRNA at the p21 luciferase reporter constructs were used to that the induction of p21 at the in cells, which not bear a CpG are Therefore, directly the hypothesis that induction of p21 transcription by inhibition of DNMT1 by a mechanism that is of methylation of the p21 promoter. HEK 293 cells were used in because they are The results in A show that inhibition of DNMT1 by expression of a antisense results in induction of both p21 promoter luciferase reporter An of DNA MeTase activity that the of the dnmt1 antisense in a reduction of DNA MeTase activity in the of HEK cells To out the possibility that the p21 promoter luciferase reporter constructs are methylated in HEK cells and that antisense treatment this we DNA HEK cells to with which is to methylation at the or which is to this methylation. The DNA was to a the p21 promoter luciferase reporter as a in one of the have in The does not that methylation of the in HEK cells The induction of p21 by DNMT1 inhibition does not the of upstream to the promoter and does not the of This also that the induction of p21 promoter activity by DNA MeTase inhibition is of the methylation of the promoter at other in the of DNA MeTase has been previously shown to arrest the growth of tumor cells (13.MacLeod A.R. Szyf M. J. Biol. Chem. 1995; 270: 8037-8043Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 14.Laird P.W. Jacksongrusby L. Fazeli A. Dickinson S.L. Jung W.E. Li E. Weinberg R.A. Jaenisch R. Cell. 1995; 81: 197-205Abstract Full Text PDF PubMed Scopus (660) Google Scholar, 15.Ramchandani S. MacLeod A.R. Pinard M. von Hofe E. Szyf M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 684-689Crossref PubMed Scopus (168) Google Scholar, 25.Bigey P Knox J.D. Croteau S. Bhattacharya S.K. Théberge J. Szyf M. J. Biol. Chem. 1999; 274: 4594-4606Abstract Full Text Full Text PDF PubMed Scopus (32) Google however, the mechanisms involved are not clear. An attractive hypothesis is that inhibition of DNA MeTase results in the demethylation of tumor suppressors that were previously by methylation. In of this hypothesis it has been shown that the tumor suppressor p16, which is methylated and inactive in many tumor cells (16.Merlo A. Herman J.G. Mao L. Lee D.J. Gabrielson E. Burger P.C. Baylin S.B. Sidransky D. Nat. Med. 1995; 1: 686-692Crossref PubMed Scopus (1878) Google Scholar), could by treatment with an inhibitor of DNA 5-aza-deoxycytidine (16.Merlo A. Herman J.G. Mao L. Lee D.J. Gabrielson E. Burger P.C. Baylin S.B. Sidransky D. Nat. Med. 1995; 1: 686-692Crossref PubMed Scopus (1878) Google Scholar, G.A. Khleif S.N. Chen W. Coxon A, B. Kaye F.J. Oncogene. 1995; 11: 1211-1216PubMed Google Scholar, 18.Gonzalgo M.L. Hayashida T. Bender C.M. Pao M.M. Tsai Y.C. Gonzales F.A. Nguyen H.D. Nguyen T.T. Jones P.A. Cancer Res. 1998; 58: 1245-1252PubMed Google Scholar). In this manuscript we the hypothesis that alternative mechanisms are involved in the arrest of cell growth by DNA MeTase For this A549, a cell line with a of was to this J. H. H. H. T. T. H. A. N. J. N. J. Cancer Res. 1997; 88: PubMed Scopus Google Scholar). novel inhibitors of DNA MeTase that inhibit DNA MeTase by mechanisms were used to increase the that the effects observed were a of DNA MeTase of 16 genes known to the cell only the gene was shown to induced following inhibition of DNA MeTase. The effects of DNA MeTase inhibitors on the growth of A549 cells, which we have previously (25.Bigey P Knox J.D. Croteau S. Bhattacharya S.K. Théberge J. Szyf M. J. Biol. Chem. 1999; 274: 4594-4606Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), can by an induction of The ectopic expression of p21 has been shown to arrest the growth of tumor cells T. Nat. Med. 1995; 1: PubMed Scopus Google Scholar), and its inhibition of p21 is known to into and Y. G.J. H. D. R. D. Nature. 1993; PubMed Scopus Google Scholar, Y. J. J. Nature. 1995; PubMed Scopus Google Scholar). These p21 to play a role in such as by and inhibition J. G.J. D. 1995; PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In p21 can directly arrest DNA replication in to DNA by binding to cell S. G.J. D. B. Nature. 1994; 36: Scopus Google Scholar). is the p16 the induction of p21 expression is rapid induction h the and points to a novel mechanism of DNA methylation. that at a of the p21 promoter is not methylated and demonstrate the of DNA MeTase inhibition to expression of a luciferase reporter gene the control of an unmethylated p21 promoter A that antisense inhibition mRNA in a carcinoma cell line results in an increase of p21 protein levels M. S. N. MacLeod R.A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). results by that the same results are DNA MeTase is directly inhibited by a hairpin phosphorothioate oligos as as inhibited by antisense the induction of p21 in a cell line, evidence that the increase in p21 protein DNA MeTase inhibition is a of p21 mRNA and that p21 induction does not a change in the methylation state of its promoter. It is p21 induction is by a in DNA MeTase activity or DNA MeTase are to this In the of DNA MeTase to p21 as as with p21 for its binding site on cell Ng G. Li 1997; PubMed Scopus Google Scholar), that the increased DNA MeTase expression observed in tumor cells p21 and result in We W. S. for the luciferase reporter constructs and W. Lee for