Patent Application: US-73863403-A

Abstract:
the present invention relates to a dna demethylase inhibitor which comprises s - adenosylmethionine , a metabolite of s - adenosylmethionine or a pharmaceutically acceptable salt thereof .

Description:
s - adenosylmethionine ( adomet ) is the methyl donor of numerous methylation reactions . exogenous administration of adomet was shown to cause hypermethylation of dna and to protect against liver disease and liver cancer in rodent models . in addition , epidemiological data suggest a correlation between the methyl content of diets and colorectal cancer . the current model is that an increased concentration of adomet stimulates dna methyltransferase ( dnmt ) reactions , triggering hypermethylation and protecting the genome against global hypomethylation , a hallmark of cancer . using an assay of active demethylation in hek 293 cells , we show that adomet inhibits active demethylation and expression of an ectopically methylated cmv - gfp plasmid in a dose dependent manner . the inhibition of gfp expression is specific to methylated gfp ; adomet does not inhibit an identical but unmethylated cmv - gfp plasmid . s - adenosylhomocysteine ( adohcy ), an analogue of adomet that differs by a single methyl residue , does not inhibit demethylation or expression of cmv - gfp . adomet but not adohcy inhibits methylated dna binding protein 2 / dna demethylase ( mbd2 / dmtase ) in vitro , suggesting that adomet directly inhibits mbd2 / dmtase , and that the methyl residue on adomet is required for its interaction with mbd2 / dmtase . we also demonstrate that adomet inhibits anchorage independent growth , an indicator of tumorigenesis , of hek 293 and a549 cells . taken together , our data support a new mechanism of action for adomet as an inhibitor of intracellular demethylase activity , which results in hypermethylation of dna . the antitumorigenic effects of adomet are consistent with the hypothesis that demethylase activity is required for tumorigenesis . in vitro methylation of substrates — cmv - gfp ( pegfp - c1 from clontech ; genbank accession # u55763 ) was methylated in vitro by incubating 10 μg of plasmid dna with 12 units of sssi cpg methyltransferase ( new england biolabs ) in the recommended buffer containing 800 μm adomet for 3 hours at 37 ° c . twelve units of sssi and 0 . 16 μmol of adomet were then added and the reaction was further incubated for 3 additional hours . the methylated plasmid was recovered by phenol / chloroform extraction and ethanol precipitation , and complete methylation was confirmed by observing full protection from hpaii digestion . cell culture and transient transfections — human embryonal kidney hek 293 cells ( american type culture collection , under atcc accession # crl 1573 ) were plated at a density of 7 . 5 × 10 4 / well in a 6 well dish and transiently transfected with 80 ng of cmv - gfp ( methylated or mock methylated ) using the calcium phosphate precipitation method as described previously ( 17 ). 0 . 3 μm trichostatin a ( tsa ) was added 24 hours post transfection . after an additional 24 h , cells were treated with or without various concentrations of adomet or adohcy ( 2 - 8 mm ). cells were harvested 72 hours post transfection . each experiment was performed in triplicate , and experiments were performed several times using different cultures of hek 293 cells . western blot analysis — whole cell extracts were prepared using radio immuno precipitation assay buffer according to the santa cruz biotechnology protocol , and protein concentrations were determined using the bradford reagent ( bio rad ). 2 . 5 μg of protein were resolved on a 12 . 5 % sds - polyacrylamide gel and then transferred to pvdf membrane ( amersham pharmacia biotech .). after blocking the nonspecific binding with 5 % skim milk , gfp protein was detected using rabbit polyclonal igg ( santa cruz , sc - 8334 ) at 1 : 500 dilution , followed by peroxidase - conjugated anti rabbit igg ( sigma ) at 1 : 5000 , and enhanced chemiluminescence detection kit ( amersham pharmacia biotech ). blots were quantifed using nih image 1 . 62 software . southern blot analysis — dna was extracted from hek 293 cells using the dneasy tissue kit ( quiagen ). dna was first digested with 50 units of ecor1 , followed by digestion with 20 units of either hpaii or mspi restriction enzymes . samples were subjected to electrophoresis on a 1 . 5 % agarose gel and then transferred to hybond - n + membrane ( amersham ). blots were probed with a 32 p - labeled cmv - gfp cdna probe ( avaii - cfr101 fragment ) synthesized using a random priming labeling kit ( roche ). membranes were hybridized at 68 ° c . for 4 - 6 hours in a buffer containing 0 . 5 m sodium phosphate ph 6 . 8 , 1 mm edta , 7 % sds , and 0 . 2 mg / ml herring sperm dna . following hybridization , the membranes were washed twice for 10 min in a 5 % sds , 0 . 04 m sodium phosphate ph 6 . 8 , 1 mm edta solution , and then four times for 10 min in the same solution containing 1 % sds . the results of 3 independent experiments were quantified by densitometry ( nih image 1 . 62 ). the demethylated , hpaii digested 529 bp fragment ( u ), and the methylated undigested dna ( m ) were quantified for each sample , and the percent methylation was determined as [[ m /( u + m )]* 100 . adomet preparations for in vitro studies — adomet was prepared as a 50 mm solution in distilled water either by freeze drying a 32 mm adomet solution , which contained 10 % ethanol and 5 mm sulfuric acid ( new england biolabs ) immediately before use or by dissolving lyophilized powder ( sigma ) in distilled water . to control for possible nonspecific effects of remnants of the adomet storage buffer we freeze dried a solution of 5 mm sulfuric acid and 10 % ethanol under the same conditions as done with the adomet solution and added distilled water after lyophilizing . adohcy was purchased from sigma and dissolved in distilled water at a 50 mm concentration . purification of recombinant mbd2 / dmtase from sf9 cells — a fragment containing human mbd2 / dmtase was excised from pcr2 . 1 - dmtase ( 10 ) with bamhi and xhoi and transferred to the baculovirus expression transfer vector pbluebachis2 c ( invitrogen ). pbluebachis2 c - mbd2 / dmtase and bac - n - blue viral dna were co - transfected into the sf9 insect cell line , and recombinant viruses were isolated , identified and amplified according to the manufacturer &# 39 ; s protocol ( invitrogen ) with no modifications . high titer p3 viral stocks were used for infections . insect sf9 cells were cultured in spinner flasks to a density of 2 . 5 × 10 6 cells / ml in grace &# 39 ; s insect cell culture medium supplemented ( 1 ×) from life technologies . for infection , 5 × 10 6 cells were plated in 10 cm tissue culture plates ( sarstedt ) and allowed to settle and attach for 30 minutes . the culture medium was removed and was replaced with 10 ml of the same medium containing mbd2 / dmtase virus at a multiplicity of infection of 10 . the cells were cultured with the virus for 5 days at 27 ° c . and were then harvested by scraping in cold phosphate buffered saline . cell pellets from 10 plates were frozen and kept at − 70 ° c . until they were used for enzyme purification . frozen pellets were thawed in 5 ml of lysis buffer ( 10 mm tris - hcl ph 8 . 0 , 5 mm mgcl 2 , 500 mm nacl , 0 . 05 % tween 20 , 10 % glycerol and 10 mm imidazole ) containing 1 μg / ml of the following protease inhibitors : aprotinin , leupeptin and pefablock . protease inhibitors were added to all the solutions used in the purification . the homogenates were subjected to two cycles of freezing and thawing ( 5 minutes per step ). dna in the homogenate was sheered by passing through a 18 . 5 gauge needle 10 times . the extracts were then subjected to 15 cycles of sonication ( 10 seconds burst , 10 seconds gap per cycle at 20 % of maximal output ). the extracts were centrifuged at 10 , 000 × g for 35 minutes . the supernatant was transferred into a fresh tube and was recentrifuged for additional 25 minutes at 15 , 000 × g . the extract was filtered through a 5 micron filter to remove any particulate matter and the buffer was exchanged on a pd - 10 buffer exchange column ( amersham pharmacia ) with buffer l ( 10 mm tris - hcl ph 8 . 0 , 10 mm mgcl 2 ) containing 50 mm nacl . recombinant mbd2 / dmtase was partially purified by q - sepharose ( pharmacia ) ion exchange chromatography . q - sepharose beads ( 1 ml of swollen beads ) were washed extensively and preequilibrated with buffer l containing 50 mm nacl and divided into 3 equal aliquots . the cell extracts were sequentially bound three times to the 3 aliquots of q - sepharose beads in batch in 15 ml tubes by shaking gently on a nutator for 45 minutes at 4 ° c . following each binding step , the bound beads and unbound supernatant were separated by centrifugation for 2 minutes at 1000 × g and the supernatant was transferred into a new tube and bound with new preequilibrated beads . the bound beads from the three binding steps were joined and resuspended in lysis buffer . the beads were washed in batch 4 times with 5 ml buffer l + 50 mm nacl . for each washing step , the beads were incubated with the wash solution for 15 minutes and were then separated from the wash supernatant by centrifugation for 2 minutes at 1000 × g . following washing , the proteins were eluted in batch ( 30 minutes per step ) with a stepwise nacl gradient in buffer l . each elution step was analyzed for in vitro demethylase activity and for the presence of the recombinant his - tagged mbd2 / dmtase by a western blot analysis using the anti xpress antibody from invitrogen as previously described . mbd2 / dmtase peak elution is at the 0 . 4 m nacl step . no demethylase activity was observed in the same fractions prepared in a similar manner from uninfected sf9 cells . preparation of substrate dna for in vitro demethylation assay — a . methylation of substrate — typically , 25 μg of dna from micrococcus lysodeikticus ( sigma , type xi , highly polymerized ) were methylated with m . sssi ( 60 u , new england biolabs , neb ) and adomet ( 3 . 2 mm , neb ) in methylation buffer ( nebuffer2 , neb ) in the presence of 50 mm edta for 3 - 4 hours at 37 ° c . fresh adomet ( 3 . 2mm ) and enzyme ( 40 u ) were then added before incubating at 37 ° c . for additional 3 - 4 hours . to achieve complete methylation , methylation is repeated after adohcy , which is a product of the methylation reaction and inhibits dna methylation , is removed using a microcon 10 concentrator ( millipore ). the degree of methylation was verified by methylation sensitive restriction enzyme analysis ( msp i - hpaii digestion ) on aliquots of the reaction mixture . the dna was purified by phenol - chloroform extraction ( one part of either phenol or chloroform per three parts reaction mixture ). unincorporated nucleotides were removed by nap5 gel filtration chromatography ( amersham pharmacia ) column . the nap5 column was equilibrated with demethylation buffer ( 10 mm tris - hcl , 5 mm mgcl 2 , ph 7 . 0 ). dna containing fractions were combined , concentrated on a microcon 10 , and subjected to a second nap5 desalting column . dna containing fractions were again concentrated as described above . [ α - 32 p ] dgtp labeling of dna — we then prepared either methylated or unmethylated dna that is 32 p labeled at g , the 3 ′ neighbor of the methylated c , as previously described ( 18 ) with the following modifications . 5 μg of either methylated or unmethylated dna in 35 μl double distilled water were denatured and annealed to a hexanucleotide primer by boiling for 10 minutes in the presence of 3 μl random hexanucleotide mix ( 0 . 2 a 260 ) ( roche ). the primed dna was then subjected to template directed extension with the klenow fragment of dna polymerase i in the presence of labeled [ α - 32 p ]- dgtp , either methyl dctp ( for methylated dna ) or dctp for unmethylated dna , dttp and datp . the 3 ′ phosphate of all the 5 ′ neighbors of g including either c or methy c is labeled by this procedure . the labeling was performed in polymerase buffer ( 50 mm nacl , 6 . 6 mm tris - hcl , 6 . 6 mm mgcl 2 , 1 mm dtt , ph 7 . 4 ) with klenow fragment i ( 10 u , roche ), methyl dctp ( 1 mm ) and dctp ( 1 mm ), respectively , datp , dttp ( 1 mm each ) and [ α - 32 p ] dgtp ( 50 μci , perkin elmer life sciences ) for 3 hours at 37 ° c . the reaction mixture was extracted with phenol and chloroform ( one part of each per three parts reaction mixture ). tca precipitation ( 2 ml 10 % trichloroacetic acid , 20 μg herring sperm dna ) of an aliquot showed typically 80 - 95 % labeling efficiency ( 260 , 000 - 320 , 000 cpm per μl , total of 150 μl ). the dna was purified by eluting it twice from a nap5 column and concentrating on a microcon 10 as described above with the following variation : distilled water was used for prewashing and elution in the second column . the final concentration was 5 ng / μl and the specific activity was typically 8 . 0 - 8 . 8 × 10 6 cpm per μg . in vitro demethylation assay — a typical reaction mixture ( 50 μl ) consisted of 25 ng of 32 p - prelabeled dna ( prepared as described above ) incubated in demethylation buffer ( 10 mm tris - hcl , 5 mm mgcl 2 , ph 7 . 0 ) with the purified mbd2 / dmtase ( 5 μl , ˜ 5 ng ) for 24 hours at 37 ° c . in either the absence or presence of adomet and adohcy , respectively . the dna was extracted from the enzyme by incubation in two volumes of dna extraction buffer ( 10 mm tris - hcl , 0 . 5 m nacl , 1 % sds ) containing 0 . 1 u proteinase k ( roche ) at 50 ° c . for 2 hours . subsequent phenol - chloroform extraction ( one part of either phenol or chloroform per three parts of reaction volume ) in the presence of trna ( 50 μg ) as a carrier and ethanol precipitation with salt and 95 % ethanol resulted in almost quantitative recovery of the input dna . the dna pellets were resuspended in distilled water ( 8 μl ) and digested with microccocal nuclease to 32 p labeled 3 ′ mononucleotides as described elsewhere ( 27 , 33 ). the labeled mononucleotides were separated by thin layer chromatography and visualized by autoradiography on a phosphorimager plate . the extent of demethylation was quantified from the image with the mcid - m4 software ( imaging research inc .). soft agar assay — hek 293 and a549 cells were plated at a density of 7 . 5 × 10 4 in a 6 well dish , and were treated the following day with either 4 mm adomet or adohcy for an additional 24 hours . for analysis of growth in soft agar , 3 × 10 3 cells were seeded in triplicate onto a six well dish in 4 ml of complete medium containing 0 . 33 % agar solution at 37 ° c . ( 34 ). cells were fed with 2 ml of medium twice weekly . the assay was performed in triplicate , and colonies were counted 21 days after plating for a549 cells , and 7 days after plating for hek 293 cells . high throughput screening of demethylase inhibitors . the assay described here for demonstrating that adomet is a demethylase inhibitor in living cells is used for high - throughput screening of demethylase inhibitors . 5 , 000 hek 293 cells are plated in a 96 well dish and are transfected with methylated cmv - gfp as described above . 24 hours following transfection all the wells are treated with 300 μm tsa . increasing concentrations ( 10 nm - 10 mm ) of representative chemicals from libraries of chemical compounds are added to the wells . following 96 hours the 96 wells plates are read in a fluorescent 96 well reader . control wells and wells with inactive compounds show high fluorescence activity resulting from demethylation and expression of cmv - gfp . wells that were treated with a putative inhibitor show dose dependent inhibition of fluorescence . the wells with hits are scanned for overt toxicity by live microscopy and trypan blue dye exclusion . dna is extracted from the well as described here and the dna is subjected to hpaii digestion as described above to validate that the putative compounds inhibited demethylation . once this is validated the lead compounds is tested in in vitro demethylase assays as described above . adometinhibits tsa induced active demethylation of ectopically methylated and transiently transfected cmv - gfp in a dose dependent manner — there have been several reports demonstrating that exogenous administration of adomet leads to dna hypermethylation ( 6 , 8 , 19 ). similarly , other studies have shown that a decrease in dietary folate , or a depletion of intracellular adomet , results in dna hypomethylation ( 2 ). however , it is not known whether adomet &# 39 ; s effects on methylation are due to changes in dnmt or dna demethylase activities . we utilized a previously described transient transfection based assay system ( fig1 and ( 15 )) to study the effects of adomet on active demethylation of ectopically methylated dna . in prior studies we have shown that in vitro methylated cmv - gfp reporter plasmid is actively demethylated 72 hours following transfection into hek 293 cells when histone hyperacetylation is induced with tsa ( 15 ). since cmv - gfp does not replicate nor is it de novo methylated in hek 293 cells ( 15 ), this assay specifically measures active demethylation in a living cell . we first determined the effects of increasing doses of adomet , or its analogue adohcy , on the demethylation of methylated cmv - gfp ( fig2 ). dna was isolated from hek 293 cells transfected with methylated cmv - gfp dna and treated with either tsa and adomet , or tsa and adohcy . dna was first linearized with the eco ri restriction enzyme , followed by digestion with mspi ( which cleaves the sequence ccgg ) or hpaii ( which cleaves the sequence ccgg only when it is not methylated ). as can be seen in fig2 a and d , the addition of tsa results in nearly complete demethylation of cmv - gfp by endogenous demethylase activity , as indicated by the complete hpaii digestion of cmv - gfp to the 529 bp fragment ( u ). upon the addition of increasing concentrations of adomet ( fig2 b and e ), the percentage of methylated gfp remaining increases in a dose dependent manner , illustrated by the decrease in the ratio of the 529 bp hpaii fragment ( u ) to the undigested dna ( m ). adohcy has an insignificant effect on the demethylation of cmv - gfp , indicating that the methyl moiety of adomet is required for inhibition of demethylation . we then determined whether adomet stimulates de novo methylation of an identical unmethylated cmv - gfp substrate . fig2 c illustrates that unmethylated cmv - gfp , transfected under identical conditions , does not get de novo methylated , even in the presence of 8 mm adomet . this indicates that adomet does not cause an increase in dnmt activity on ectopic cmv - gfp . thus , the likely mechanism by which adomet causes hypermethylation of cmv - gfp in comparison with the tsa treated control is by inhibiting its active demethylation by resident demethylases . adomet reduces tsa induced expression of methylated cmv - gfp in a dose dependent manner , but has no effect on unmethylated cmv - gfp — a number of studies have shown that an increase in adomet inhibits gene expression ( 7 , 8 ), however it is not clear whether adomet specifically affects genes whose methylation state it alters exclusively , or whether it has a non specific effect on gene expression . we took advantage of the cmv - gfp system described above to address this question . we determined whether adomet influences the expression of either methylated cmv - gfp , whose methylation state is affected by adomet , or unmethylated cmv - gfp , whose methylation state is not affected by adomet . hek 293 cells were transiently transfected and treated with tsa and either adomet or adohcy , as described in the previous section . extracts were then prepared and subjected to a western blot analysis using an antibody directed against gfp protein . fig3 a and c illustrate that methylated cmv - gfp is completely repressed in untreated hek 293 cells . this is as expected , since it is well documented that dna methylation leads to gene silencing . the addition of tsa leads to a dramatic induction of gfp expression as expected from the complete demethylation following tsa treatment . upon the addition of increasing amounts of adomet , gfp expression is decreased in a dose dependent fashion . adohcy has no significant effect on the expression of methylated gfp , consistent with its lack of effect of dna demethylation . since our system measures expression and demethylation that is dependent on histone hyperacetylation , there are two possible mechanisms whereby adomet exerts its effects on demethylation ( fig1 ). adomet could directly inhibit a demethylase activity , or it could inhibit histone acetylation , which we have previously shown leads to an inhibition of demethylation ( 15 ). if the latter were true , then adomet should also inhibit the tsa induced expression of non methylated gfp , whose expression is induced by histone acetylation as well . fig3 b and d indicate that this is not the case , since adomet has no significant effect on the induction of non - methylated gfp by tsa . the fact that adomet specifically affects the expression of a methylated copy of cmv - gfp , and not an unmethylated copy , supports the model that adomet inhibits gene expression by directly inhibiting the active demethylation of methylated cmv - gfp . adometinhibits the tumorigenic properties of two different transformed cells lines , a549 human lung carcinoma cells and hek 293 human embryonal kidney cells — it is well documented that a correlation exists between reduced intracellular adomet ( either as a consequence of decreased folate intake or pharmacological intervention ) and an increase in cell proliferation and tumorigenesis ( 2 , 19 ). however , there is no direct demonstration that adomet treatment of a cancer cell can revert its state of transformation . we therefore decided to test whether treatment with adomet could inhibit the transformation potential of hek 293 cells , which were shown here to be responsive to adomet inhibition of demethylase . we also tested adomet effects on the transformed state of a549 , a human lung non - small cell carcinoma cell line , to exclude the possibility that adomet effects are an idiosyncrasy of hek 293 cells . cells were treated for 24 hours with either adomet or adohcy at a concentration of 4 mm . an equal number of living cells were seeded onto soft agar to test their anchorage independent growth , an indicator of tumorigenesis in vitro . as shown in fig4 , both untreated hek293 and a549 cells formed colonies in soft agar , which is characteristic of cancer cell lines . remarkably , after only a single 24 hour treatment with adomet , no colonies are formed with a549 cells , and only a minimal number of colonies are observed with hek 293 cells . in contrast , adohcy does not inhibit anchorage independent growth with either cell line , and even has a slight stimulatory effect . since we have shown here that 4 mm adomet inhibits cellular demethylase activity in hek 293 cells whereas adohcy has no effect at the same concentration , the inhibition of cell transformation is consistent with the hypothesis that hypomethylation is critical for tumorigenesis . adomet &# 39 ; s chemoprotective effects against colorectal cancer might therefore be due to its inhibition of active demethylation . adomet but not adohcy inhibits demethylation activity in vitro — in order to further confirm that the observed effect of adomet and adohcy is due to inhibition of active demethylation and not an indirect effect , in vitro studies with a recombinant mbd2 / dmtase were performed ( 10 ). although it is not certain whether mbd2 / dmtase is responsible for the demethylation seen in hek 293 cells , it is the only demethylase characterized thus far , and the in vitro experiment should test whether adomet can inhibit this demethylase activity . his tagged mbd2 / dmtase was partially purified by chromatography on q - sepharose from nuclear extracts of sf9 cells infected with the recombinant mbd2 / dmtase construct as described in materials and methods . fractions were eluted with a stepwise gradient of nacl and assayed for demethylation activity with a 32 p prelabeled methylated dna from micrococcus lysodeikticus ( fig5 a ). conversion of methyl dcmp to dcmp was almost exclusively detected with the 0 . 4 m nacl fraction , although the 0 . 2 m fraction exhibited some low activity . this correlates with the peak presence of the his tagged recombinant mbd2 / dmtase protein in this fraction as demonstrated by a western blot analysis ( fig5 b ) using an anti - xpress antibody . next , we determined whether adomet inhibits the demethylation activity of mbd2 / dmtase . the aforementioned dna was incubated with mbd2 / dmtase in the presence of increasing adomet concentrations . fig6 a presents the autoradiography of one representative experiment . conversion of methy dcmp to cmp was greatly reduced at 2 mm adomet and abolished completely at concentrations higher than 4 mm . the dose curve in fig6 b is an average of quantitative autoradiography of 3 independent experiments . in order to exclude the possibility that traces of adomet storage buffer components ( ethanol , sulfuric acid ) inhibit demethylation , adomet storage buffer was freeze dried and added to control demethylation reactions in the same volumes as adomet . inhibition of demethylation by this buffer is insignificant ( less than 10 %) as shown by the straight line in fig6 b in comparison to the 100 % inhibition attained with adomet . therefore , traces of buffer components do not significantly contribute to adomet &# 39 ; s inhibition of mbd2 / dmtase activity . moreover , experiments with buffer free adomet showed an identical inhibitory effect ( fig6 b , the curve is an average of experiments performed with both types of adomet preparations ). in contrast to adomet , no inhibition of demethylation occurred in the presence of increasing concentrations of adohcy ( fig6 c ). the curve in fig6 d is based on the quantitative autoradiography of the experiment in fig6 c . these results indicate that the small differences in the chemical structure ( methyl group and positive charge on the sulfur ) between adomet and adohcy are responsible for their different interaction with the mbd2 / dmtase . adomet and adohcy compete for binding to the catalytic site on dnmts . it was therefore proposed that the ratio of adohcy to adomet determines dnmt activity as discussed in the introduction . adohcy inhibits dnmts whereas increased adomet offsets this inhibition . we therefore determined whether a similar relationship applies to mbd2 / dmtase . a competition experiment between adomet and adohcy is presented in fig6 e . increasing concentrations of adohcy were added in the presence of an inhibitory concentration of adomet ( 10 mm ), in a series of demethylation reactions . the results of this experiment illustrate that even a tenfold concentration excess of adohcy to adomet does not diminish inhibition of the demethylase reaction by adomet . this is consistent with the hypothesis that adomet has a higher affinity for mbd2 / dmtase as compared with adohcy . further studies are necessary to elucidate the mode of inhibition : whether adomet is a competitive inhibitor with the substrate dna or an allosteric inhibitor as was demonstrated for methylene tetrahydrofolate reductase . furthermore , we do not know how mbd2 / dmtase recognizes adomet on a structural basis . the currently accepted mechanism for the effects of the methyl donor adomet on dna methylation and tumorigenesis is founded on the assumption that the dna methylation reaction is irreversible and defined exclusively by the dnmt . taking advantage of our previously developed assay of demethylase activity in living cells ( fig1 ), we tested an alternative hypothesis : that adomet inhibits demethylase activity . if the steady state methylation status of dna is maintained by an equilibrium of dnmt and demethylase activities ( 14 ), then inhibition of the demethylase side of the equilibrium should result in hypermethylation . therefore , the reported dna hypermethylation effects of exogenous adomet might be caused in part by inhibiting the level of demethylase activity in tumor cells . the main advantage of the system used in this paper is that it studies active demethylation exclusively , without interference from either replication dependent passive demethylation or de novo dnmt activities ( 15 ). we show here that exogenous adomet inhibits tsa stimulated demethylation of ectopically methylated and transiently transfected cmv - gfp dna ( fig2 b , e ). since methylation inhibits the expression of cmv - gfp ( 15 ), inhibition of demethylation of cmv - gfp results in reduction of gfp protein expression ( fig3 a , c ), illustrating that adomet affects both demethylation of dna and gene expression . this association of inhibition of demethylation and silencing of gene expression prompted us to rule out the possibility that adomet has a general , methylation - independent inhibitory effect on gene expression , or a general toxic effect , which also might result in inhibition of expression . it is possible that adomet increases histone methyltransferase activity , resulting in hypermethylation of k9 on h3 histones , which has been shown to correlate with inhibition of acetylation . inhibition of acetylation was shown to inhibit expression and demethylation of cmv - gfp ( 16 ). to address this alternative possibility , we measured in parallel the effects that adomet might have on methylated as well as unmethylated cmv - gfp plasmid , both transfected and treated with exogenous adomet under equivalent conditions . we first show that adomet treatment does not result in de novo methylation of unmethylated cmv - gfp ( fig2 c ). thus , exogenous adomet does not stimulate dna methylation as might be predicted by the current hypothesis of adomet &# 39 ; s mechanism of action . second , we show that exogenous adomet does not inhibit expression of unmethylated cmv - gfp under conditions where a clear inhibition of expression of methylated cmv - gfp is observed ( fig3 b , d ). thus adomet specifically affects the expression of methylated genes . to our knowledge , this is the first demonstration that adomet specifically targets methylated dna . this result also rules out the possibility that adomet exerts a general toxic effect on the cell . our data therefore demonstrate that exogenous adomet specifically affects methylated dna and prevents its expression . this most probably occurs by inhibiting an endogenous demethylase activity , resulting in hypermethylation of cmv - gfp and methylation - dependent repression . we used the unmethylated analogue of adomet , adohcy , as a control . adohcy differs from adomet by a single methyl group . we show that adohcy has no effect on either gene expression ( fig3 a , c ) or demethylation ( fig2 b , e ). taken together , these results indicate that both activities of adomet , inhibition of demethylation and inhibition of gene expression , are tightly associated and that they are both dependent on the methyl moiety in adomet . we also demonstrate that adomet inhibits the tumorigenic potential of both hek 293 and a549 cells at the same concentration that it inhibits demethylation and expression of methylated dna ( fig4 ). this is consistent with epidemiological and clinical data , as well as with animal experiments using liver cancer models ( 2 ). since we show that adomet specifically inhibits the expression of methylated genes , we suggest that part of the anti tumorigenic effects of adomet could be explained by the inhibition of demethylation and expression of genes that are required for anchorage independent growth . further experiments will be required to identify the genes whose expression are inhibited by adomet and are required for anchorage independent growth and tumorigenesis . in addition , we show that adomet directly inhibits recombinant mbd2 / dmtase activity ( fig5 ) in a dose dependent manner using an in vitro assay ( fig6 ). this supports the hypothesis that adomet directly inhibits demethylase activity in living cells . adohcy does not inhibit mbd2 / dmtase at the same concentrations ( fig6 ). since an increase in intracellular adohcy was previously shown to be associated with hypomethylation , we tested the possibility that either adohcy stimulates mbd2 / dmtase activity , or that it competes with adomet binding to mbd2 / dmtase and relieves adomet inhibition . our results suggest that adohcy does not interact with demethylase and that it has no effect on adomet inhibition of this enzyme in vitro . our results support the conclusion that the methyl group in adomet is required for its interaction with mbd2 / dmtase . although our results demonstrate that exogenous adomet inhibits mbd2 / dmtase in vitro and in living cells , there is no evidence that the intact adomet is the inhibitor . it is still possible that a metabolite of adomet is the active compound . further experiments are required to test this possibility . nevertheless , our experiments demonstrate that pharmacological administration of adomet inhibits active demethylation , alters gene expression , and inhibits tumorigenesis . further experiments are also required to demonstrate that the demethylase activity that is inhibited by adomet in hek 293 cells is indeed mbd2 / dmtase . it is possible that other demethylases are responsible for demethylating ectopically methylated dna in hek 293 cells . nevertheless , our results provide a first proof of principle that adomet is an inhibitor of demethylase activity . thus , in addition to its role as a cofactor of transmethylation reactions , adomet can also act as a regulator of dna methylation metabolism by inhibiting demethylase activity . our data further emphasize that the demethylase side of the methylation equilibrium has to be taken into account when dissecting the mechanism of action of drugs that modify the dna methylation pattern . based on our data , we suggest that adomet can alter dna methylation patterns by inhibiting demethylase , which is expressed in some or most cells ( fig7 ). in this case , a reduction in the intracellular levels of adomet by methyl deficient diets removes this inhibition and increases the demethylase tone , resulting in active demethylation of dna which could take place even in postmitotic tissue . interestingly , adomet has recently been shown to inhibit the overall demethylation of a cg site in the 5 ′ of the myogenin gene during c2c12 differentiation ( 8 ). however , this report did not determine whether adomet stimulated dnmt or inhibited dna demethylase . the fact that adomet inhibits anchorage independent growth ( fig4 ) suggests that active demethylation might be especially important for tumorigenic or anchorage independent growth . this is consistent with the well - documented observations of global hypomethylation in cancer cells . there is evidence that adomet &# 39 ; s tumor protective mechanism involves dna methylation since this protection is removed when the animals are co treated with 5 - azacytidine and adomet ( 6 ). in accordance with this hypothesis , we have recently shown that antisense inhibition of mbd2 / demethylase inhibits tumorigenesis ( 20 ). it is tempting to speculate that certain genes that are required for anchorage independent growth might be inhibited by methylation and activated by a demethylase activity . inhibition of the demethylase tone by adomet is proposed to result in silencing of these genes . further experiments are obviously required to identify the genes that are suppressed by adomet and are required for anchorage independent growth . if adomet &# 39 ; s mechanism of action in inhibiting tumorigenesis involves inhibition of demethylation , it would support the hypothesis that demethylation plays a causal role in tumorigenesis , and serve as a warning against using inhibitors of dna methylation as anticancer agents . while the invention has been described in connection with specific embodiments thereof , it will be understood that it is capable of further modifications and this application is intended to cover any variations , uses , or adaptations of the invention following , in general , the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth , and as follows in the scope of the appended claims . 2 . pascale , r ., simile , m ., de , m . m . and feo , f . 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