Clinical Study DNA Methylation Pyrosequencing Assay Is ...downloads.hindawi.com/journals/bmri/2013/486072.pdf · and L of bisul te treated template DNA in a total volume of L. PCRconditionswereasfollows:initialdenaturingat

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 486072 10 pageshttpdxdoiorg1011552013486072

Clinical StudyDNA Methylation Pyrosequencing Assay Is Applicablefor the Assessment of Epigenetic Active Environmentalor Clinical Relevant Chemicals

Ana-Maria Florea

Environmental Toxicology University of Trier FBVI Universitatsring 15 54296 Trier Germany

Correspondence should be addressed to Ana-Maria Florea anamfloreagmailcom

Received 3 April 2013 Revised 19 July 2013 Accepted 22 July 2013

Academic Editor Antoni Camins

Copyright copy 2013 Ana-Maria Florea This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Exposure of cells and organisms to stressors might result in epigenetic changes Here it is shown that investigation of DNAmethylation using pyrosequencing is an alternative for in vitro and in vivo toxicological testing of epigenetic effects inducedby chemicals and drugs An in vitro evaluation of global and CpG site specific DNA methylation upon treatment of cells withchemicalsdrugs is shown Bisulfite genomic sequencing of methylation controls showed highmethylation of LINE1 in methylationpositive control and low methylation in the negative controls The CpG sites within the LINE1 element are methylated at differentlevels In vitro cell cultures show a methylation level ranging from 56 to 49 Cultures of drug resistant tumor cells showsignificant hypomethylation as compared with the originating nonresistant tumor cells The in vitro testing of epigeneticallyactive chemicals (5-methyl-2rsquo-deoxycytidine and trichostatin A) revealed a significant change of LINE1 methylation status upontreatment while specific CpG sites were more prone to demethylation than others (focal methylation) In conclusion DNAmethylation using pyrosequencing might be used not only for testing epigenetic toxinsdrugs but also in risk assessment of drugsfood and environmental relevant pollutants

1 Introduction

Epigenetic factors such as DNAmethylation histone modifi-cations and microRNAs are able to regulate gene expressionand genomic stability [1ndash3] CG dinucleotides have unicfeature in the mammalian genome they can associate in CGscluster regions termed CpG islands [4] DNA methylationoccurs in CpG islands that could be related to transcriptionalsilencing of genes Thus active gens show low methyla-tion in the gene promoter-associated CpG islands whilein downstream-transcribed gene regions and in repetitiveregions such as LINEs and SINEs hypermethylation is found[4ndash8] DNA methylation pattern is maintained due to enzy-matic activity DNA methyltransferase (DNMT) family con-trols de novo and maintenance DNA methylation Thereforeit is possible to modify the DNA methylation status of genesusing agents that hinder the function of DNA methylationenzymes [9]

Retrotransposons are mobile elements with the propertyof moving within a gene via a copy and paste mechanismThus they are responsible for creating genetic variation butalso might be the reason for creation disease-causing muta-tions within the human genome (ie insertional mutagen-esis recombination retrotransposition-mediated and geneconversion-mediated deletion and 31015840 transduction) Activeretrotransposable elements include long interspersed ele-ments (LINEs) and short interspersed elements (SINEs)while approximately 027 of all human disease mutationsare attributable to retrotransposable elements [10] LINE1retrotransposons are present in the human genome (esti-mated 500000 copiesgenome) they disseminate throughRNA sequences in DNA sequences after reverse transcriptionand are then integrated into new genomic loci such asnearby genes Thus they are able to epigenetically disruptthe transcriptome and contribute to processes related totumorigenesis [11]

2 BioMed Research International

A large body of toxicological evidence shows that manysubstances could have an impact on humanrsquos health withoutclear signs of acute or immediate toxicity In chronic exposureto damaging chemicals (long-term toxicity) it is often foundthat the chemicals induce changes in the DNA sequence thusresulting in DNA damage and mutagenesis Neverthelessepigenetic research shows that toxic chemicals could alsomodify the DNA function without changing the sequencedue to for example changing the state of DNA methylationthus inducing deregulated gene expression and genomicinstability [1ndash3 9] Thus DNA methylation might play anintegral role in toxicity through the hypomethylation ofrepetitive DNA elements that impacts chromosomal andtranscriptional stability of the genome and also as a result offocal hypermethylation thus resulting in epigenetic silencingof gene expression (DNA methylation and gene expressionare inversely correlated) [12 13]

Long-term toxicological damage could be caused withoutchanging the DNA sequence thus having important impli-cations on the safety assessment for chemicals drugs andfood [9] Epigenetic alterations may be early indicators ofgenotoxic and nongenotoxic carcinogenic exposure and thusused as biomarkers in the assessment of the carcinogenicpotential of environmental chemical and physical agents andmight be used in cancer risk assessment [14] For exampleenvironmental relevant chemicals such as arsenic cadmiumand bisphenol A have epigenetic modulation property ([3 1516]) but also mixtures of benzene hydroquinone styrenecarbon tetrachloride and trichloroethylene [17]

All major human cancers have a large number ofgenetic alterations and epigenetic abnormalities that mightbe used as biomarkers for the molecular diagnosis of cancerThe genome-wide loss of methylation at CpG sites of thepromoter is a common epigenetic event in malignanciesand may play a role in the process of carcinogenesis [14]while changes in promoter DNA methylation and asso-ciated gene silencing might be used for cancer therapy(eg decitabine and azacitidine) [18] Thus investigation ofchemical induced changes in DNA methylation status couldrepresent an important indicator for chemical induced riskassessment

In this paper it is shown that pyrosequencing is applicablein toxicity tests to investigate the modification in DNAmethylation upon exposure to chemicals in vitro Pyrose-quencing could be adapted to provide accurate quantifiabledata and detailed profiles of DNA methylation patternsunderlying for example cell cycle regulation differentialgene expression and epigenetic effects

2 Material and Methods

Pyrosequencing is a quantitative sequence-based detectiontechnology adaptable for exploratory and testing work intoxicology and also in pharmacological approaches Manyready designed assays (PyroMark CpG Assays) are availablefor pyrosequencing analysis for example more than 84000assays are available for gene-specific CpG islands includingthe mouse and rat genomes (Qiagen Germany EpigenDX

USA) These assays are created using algorithms that providespecific quantification of CpG methylation

Using pyrosequencing for the investigation of DNAmethylation has been proofed as highly reproducible andhighly sensitive in praxis Companies are offering to usersthe possibility of buying ready to use kits for pyrosequencingespecially diagnostic relevant biomarkers such as KRASMGMT p16 LINE1 MLH1 and BRAF (Qiagen GermanyEpigenDX USA) Although the number of available pre-designed ready to buy DNA methylation assays (eg Pyro-Mark CpG Assays) has considerably increased in the marketthe researcher today might be put in the position that thereare no assays available for specific biomarkers Thus it ispossible to create self-designed assays using commercialsoftware (PSQ assay design) or freely available software(Primer3)

21 Cell Culture Normal human dermal fibroblasts (NHDF)were provided from PromoCell GermanyThe cells were iso-lated from normal human foreskin from a 10-year old donorand cryopreserved at passage 2 (P2) using serum-free freez-ingmedium Cryo-SFM (PromoCell Germany)The cultureswere maintained in the incubator at 37∘C 5 CO

2 The

cells were split once they have reached 80ndash95 confluenceby using Trypsin-EDTA (0025) (PAA Germany) Aftercollecting the detached cells in Trypsin inhibitor (PromoCellGermany) the cells were maintained in cell culture dishes(NUC Germany) The fibroblast cultures were maintainedin serum-free fibroblast culture medium without phenolred supplemented with ldquoSupplement Mixrdquo (basic fibroblastgrowth factor 1 ngmL and insulin 5 ngmL) (PromoCellGermany) as well as antibiotics penicillinstreptomycin(PAA Germany)

HACAT cells (DKFZ Germany) are in vitro sponta-neously transformed keratinocytes derived from histologicalnormal skin that grows in DMEM medium (high glu-cose) supplemented with 2mM L-glutamine (PAA) and 10fetal calf serum (PAA Germany) as well as antibioticspenicillinstreptomycin (PAA Germany) The cultures weremaintained at 37∘C with 5 CO

2and splitted as necessary if

they reached 80ndash95 confluence [19]

22 5-Methyl-21015840-deoxycytidine and Trichostatin A Treatment(A + T2) of HACAT Cells HACAT cells were plated in10 cm2 culture dishes at 50 confluence and were left overnight for attachment They were treated (119899 = 6) with thedemethylating agent 5-aza-21015840-deoxycytidine (5-Aza) and thehistone deacetylase inhibitor trichostatin A (TSA) (SigmaGermany) The treatment conditions were done with eitherthe demethylating agent 5-aza-21015840-deoxycytidine (1 120583M for72 h) or the histone deacetylase inhibitor trichostatin A (1 120583Mfor 36 h)

23 Glioma Cell Lines LNT-229 LN-308 and LN-18 Allglioblastoma cell lines were grown under standard conditionand as elsewhere described [20] Dulbeccorsquos modified Eaglersquosmedium (DMEM)was used as basal medium 10 (vv) heat-inactivated fetal bovine serum (FCS) and 1 (vv) penicillin

BioMed Research International 3

Gstreptomycin were added to the medium and cells wereincubated at 37∘C in a humidified atmosphere containing 5CO2 The cell culture of glioma cell lines and temozolomide

(TMZ) resistant cells were elsewhere described [20] Cellpellets from drug sensitive and drug resistant cell lines wereprovided for nucleic acid extraction andpyrosequencing [20]

24 Kidney Carcinoma CKA-CA6 Cells and Human Embry-onic Kidney HEK293 Cells The DNA was kindly providedby PD Dr Csaba Mahotka (Pathology HH UniversityDusseldorf)The DNA extraction was done using the QiagenDNA extraction kit and the company provided protocol Thecells were grown using standard cell culture procedures

25 Nucleic Acids Extraction and Bisulfite Conversion ofthe Genomic DNA Nucleic acid extraction was performedas reported elsewhere [21] using ultracentrifugation andcaesium chloridemethod followed by the phenolchloroformpurification or using the DNA extraction kit from QiagenGermany using the providers protocol One microgramof genomic DNA was further subject of bisulphite trans-formation using the EZ DNA Methylation Gold Kit (HissDiagnostics Germany) in order to be used in pyrosequencingreactions

26 Primer Design Long interspersed nuclear element 1(LINE1 or L1) sequences are highly repeated human retro-transposon sequences and constitute about 17 of the humangenome DNA methylation within the promoter region ofhuman LINE1 elements is important for maintaining tran-scriptional inactivation and for inhibiting transposition [22]Genome-wide losses of DNA methylation within the pro-moter region of human LINE1 elements have been regardedas a common epigenetic event in malignancies and mayplay crucial roles in carcinogenesis [11]This methylationassay amplifies a region of the LINE1 element and servesas a marker for global methylation PyroMark LINE1 fromQiagen Germany has been used following the companyrecommendations and elsewhere described [20] CpG sitesare located in positions 331 to 305 of LINE1 (GenBankaccession number X58075)

Self-designed assays were obtained using the PSQ assaydesign The sequence of LINE1 element was obtained usingRefSeq (GenBank accession number X58075) The locationof each primer combination is shown in Figure 1 With thehelp ofMicrosoftWord (MicrosoftUSA) the LINE1 sequencewas in silico bisulfite converted (all CG =gt YG all C =gt T)and pasted in the primer design software The result of theprimer design is shown in Tables 1 and 2 The LINE1 CpG1-6 is located at the beginning of the sequence (X58075) andis spanned over 6CpG sites while LINE1 CpG7-14 is locatedafter LINE1 CpG1-6 and is spanned over 8 CpG sites

27 Pyrosequencing For pyrosequencing the PyroMarkLINE1 kit (Qiagen Germany) was used following thecompany recommendations In brief each PCR mix con-tained 1times PCR buffer 15mmolL of MgCl

2(final con-

centration) (Qiagen) 02molL of each dNTP (Biobudget

(1)

(2)

(3)

Location of the sequences analyzed for total methylation (LINE1 times58075)PSQ assay design LINE1 repetitive element

(1) LINE1 CpG1-6 (self-designed)(2) LINE1 CpG7-14 (self-designed)(3) LINE1 Qiagen (provided from Qiagen)

Figure 1 Long interspersed nuclear element 1 (LINE1 or L1)sequences are highly repeated human retrotransposon sequencesHere the LINE1 GenBank accession number X58075 is shownafter in silico bisulphite conversion the location of the investigatedprimers are shown

Germany) 1120583L of forward and reverse primer (10 pmolL ofeach PCR primer) 2U of HotStar Taq Polymerase (Qiagen)and 2 120583L of bisulfite treated template DNA in a total volumeof 50120583L

PCR conditions were as follows initial denaturing at 95∘Cfor 15 minutes 45 cycles of 95∘C for 20 seconds 50∘C for 20seconds and 72∘C for 20 seconds and final extension at 72∘Cfor 5 minutes giving an amplicon length of 146 bp The PCRproduct was checked by 1 agarose gel electrophoresis (datanot shown) The biotinylated PCR product was purified tosingle-stranded DNA to be the template in a pyrosequencingreaction as recommended by the manufacturer using thePyrosequencing Vacuum Prep Tool (Qiagen Germany)

Forty microliters of biotinylated PCR product wereimmobilized on streptavidin coated sepharose beads There-after the sepharose beads containing the immobilized PCRproduct were purified washed denatured using a 02MNaOH solution and washed again Then 03120583M pyrose-quencing primerwas annealed to the purified single-strandedPCR product and pyrosequencing was performed by Pyro-Mark Q24 (Qiagen Germany) The sequence to analyze wasTTYGTGGTGYGTYGTTTwhile the dispensation orderwasGCTCGTGTAGTCAGTCG [20]

Similarly it was proceeded with the self-designed primersLINE1 CpG1-6 and LINE1 CpG7-14 Primers were orderedat Eurofins (Germany) and reassembled as recommendedby the provider For PCR reaction the HotStar Taq Poly-merase from Qiagen was used PCR conditions 95∘C-5min(1 cycle) 95∘C-1min 57∘C-1min 72∘C-1min (50 cycles)72∘C-5min (1 cycle) 4∘Cinfin The assays were proofed using


Table 1 Self-designed pyrosequencing assays using PSQ assay design are shown Primer score represents the probable quality of the primercombinations (min 0-gt max 100 color score blue gt yellow gt orange gt red) F1 forward primer R1 reverse primer (biotinylated) S1sequencing primer (LINE1 CG1-6)

(a)

Primer set 1 Score 76General warnings

(b)

F1 GGGAGGAGTTAAGATGGT= R1 ATAAACCCCATACCTCAAArarr S1 GGGAGGAGTTAAGATGGT

(c)

PCR product Forward PCR primer F1 Reverse PCR primer R1 Sequencing primer S1Length bp 109 18 19 18Position 51015840-31015840 3-20 111-93 3-20WarningsTm ∘C 602 609 531 GC 330 500 368 500Sequence toanalyze YGAATAGGAA TAGTTTYGGT TTATAGTTTT TAGYGTGAGY GAYGTAGAAG AYGGGTGATT TTTGTATTTT TATTTGA

Table 2 Self-designed pyrosequencing assays using PSQ assay design are shown Primer score represents the probable quality of the primercombinations (min 0-gtmax 100 color score blue gt yellow gt orange gt red) F1 forward primer R1 reverse primer (biotinylated) S1sequencing primer (LINE1 CG7-14)

(a)


(b)

F1 GGTTTATTTTATTAGGGAGTGTTA= R1 AAAAAAAAACTCCCTAACCrarr S1 AGGGAGTGTTAGATAGTGG

(c)

PCR product Forward PCR primer F1 Reverse PCR primer R1 Sequencing primer S1Length bp 127 24 19 19Position51015840-31015840 105-128 231-213 118-136

WarningsTm ∘C 619 593 508 GC 370 292 316 474Sequence toanalyze GYGTAGGTTA TTGTGTGYGY GTATYGTGYG YGAGTYGAAG TAGGGYGAGG TATTGTTTTA TTTGGGAAG

human methylation positive and negative controls as seen inFigure 2 (ordered at EpigenDX USA) The pyrosequencingwas done as recommended by the provider using the Pyro-Gold Reagents 5 times 24 (Qiagen Germany) and the PyroMarkQ24 Qiagen (Germany) and as described in detail above

28 Statistics Statistical analysis was done comparing con-trol versus each condition while Studentrsquos t-test was appliedwhere lowast119875 lt 005

3 Results

31 The Setup and the Quality Control of a DNA MethylationPyrosequencing Assay To make sure that a specific DNAmethylation assay functions in a given laboratory conditionthe methylation PCR reaction followed by pyrosequencingshould be tested Known methylation status DNA samplessuch as positive and negative methylation controls are usedThey are available for purchase at many providers (eg


86 71 77

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060

B1 TTYGTGGTGYGTYGTTTMethylation positive control (EpigenDX) LINE1 Qiagen kit

(a)

E S G C T C G5

T G T A G10

T C A G T15

C G

Methylation negative control (EpigenDX)

2 2 2

0102030405060

minus10

B2 TTYGTGGTGYGTYGTTT

(b)

NA NA NA

024

minus2minus4minus6

A8 TTYGTGGTGYGTYGTTTPCR negative control

E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

Figure 2 Human methylation positive and negative controls (EpigenDX USA) and PyroMark LINE1 kit (Qiagen Germany)

EpigenDX USA Millipore Germany) while mouse and ratmethylation controls are available as well For this paperare shown the results obtained with the human methylationpositive and negative controls bought at EpigenDX USA(Figure 2) and PyroMark LINE1 kit (Qiagen Germany)

As described in the Material and Methods for each testreaction was used 200 ng bisulphite transformed DNA for(Figure 2 from (a) to (c)) (1) methylation positive control (2)methylation negative control (3) PCR negative control Theestablishment of the PCR negative control is very importantin order to check for (i) the contamination of the PCRreaction (ii) specificity of the primers and (iii) size of thePCR product The PCR reaction was assembled as describedat the Material and Methods After quality check on 1-2agarose gel (showing one clear PCR band) the PCR productis purified with the Pyrosequencing Vacuum Prep Tool(Qiagen Germany) and proceeded with the pyrosequencing

Typical good functioning pyrosequencing assay is shownin Figure 2 (PyroMark LINE1 kit (Qiagen Germany)) Inthis case the LINE1 sequence that was assessed by pyrose-quencing has three CpG sites (see location in Figure 1) Overeach of the CpG site the found methylation level and thequality of the obtained result (red = bad quality yellow = passquality and blue = good quality) are shown The pyrogramshows in addition the well analyzed and the sequence toanalyze Furthermore on the 119909-axis the nucleotides injectedby the pyrosequencer are shown while on the 119910-axis theluminescence detected by the LCD camera is shown

In brief in the shown pyrograms (Figure 2) the ldquoErdquorepresents the moment when the enzyme was add to thereaction followed by the substrate ldquoSrdquo that is documentedby a small spike The pyrosequencer added a ldquoGrdquo that is notpresent in the sequence (as in build reaction control) thusno spike was observed Then the nucleotide ldquoCrdquo is added

to the reaction that represents the bisulphite transformationcontrol in this case the bisulphite transformation is completebecause no signal was detected by the pyrosequencer Theactual sequence to analyze starts with a TT (that is double ashigh as the single T) followed by ourmethylated cytosine heremarked with YThe percentages formethylation are the resultof the calculated ratio thymine versus methylated cytosineThe pyrosequencing continues with adding one by one thenucleotides (here G T GG etc) if the nucleotide fits thesequence a light signal is detected if not no light signal isdetected

In the methylation positive control it is shown that eachof the CpG sites has a high level of methylation for exampleCG1 = 86 CG2 = 71 CG3 = 77 In the methylationnegative control it is shown that each of the CpG sites hasa low level of methylation for example CG1 = 2 CG2= 2 CG3 = 2 If one compares in the pyrogram thespike present for the detection of the methylated cytosine inthe methylation negative control does not practically existFurthermore the PCR negative controls show no specificspikes whatsoever of the nucleotides are injected in thepyrosequencing reaction thus this pyrosequencing assay isspecific

32 Different Cell Lines Show Different Methylation Status ofthe LINE1 Element Well-established cell lines were inves-tigated for their intrinsic methylation level of the LINE1element In Figure 3 from (a) to (d) the pyrosequencing ofLINE1 element (Qiagen Germany) a kidney carcinoma cellline is shown (CKA-CA6) followed by human embryonickidney cells (HEK293) spontaneously immortalized humankeratinocytes (HACAT) and primary in vitro cultures ofnormal human dermal fibroblasts (NHDF) The three inves-tigated CpG sites show different levels of methylation in each


47 52 49

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060

minus10

A7 TTYGTGGTGYGTYGTTTCKA-CA6LINE1 Qiagen kit

(a)

E S G C T C G5

T G T A G10

T C A G T15

C G

50 56 51

0102030405060

A8 TTYGTGGTGYGTYGTTTHEK 293

(b)

53 64 65

01020304050

A7 TTYGTGGTGYGTYGTTTHACAT

E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

66 59 59

0255075

100125150

minus25

A1 TTYGTGGTGYGTYGTTTNHDF

E S G C T C G5

T G T A G10

T C A G T15

C G

(d)

Figure 3 Established tumor nontumor and primary cell lines investigated for their intrinsic methylation level of the LINE1 element (QiagenGermany) CKA-CA6 kidney carcinoma HEK293 human embryonic kidney cells spontaneously immortalized human keratinocytes(HACAT) and primary in vitro cultures of normal human dermal fibroblasts (NHDF)

Glo

bal m

ethy

latio

n (

)

LINE1

5649 51 53

79

30

102030405060708090

NH

DF

HAC

AT

CKA-

CA

HEK

293

M+

Mminus

Figure 4 Quantitation of intrinsic methylation level of the LINE1element (Qiagen Germany) in established tumor nontumor andprimary cell lines using several measurement replicates (119899 = 3ndash6)and the correspondent methylation controls (Studentrsquos t-test lowast119875 lt005)

cell line investigated Also the methylation level was slightlydifferent in each cell line (Figure 4) The highest level ofmethylation was found for NHDF that was about 56 (119899 = 5)followed by 53 for HEK293 (119899 = 3) 51 for CKA-CA(119899 = 3) and 49 for HACAT cells (119899 = 5) The measuredmethylation value of methylation positive control was 79while for methylation negative control was 3

33 Drug Sensitive and Drug Resistant Glioma Cell Lines ShowDifferent Methylation Status of the LINE1 Element Three

glioma cell lines were investigated for the methylation levelof LINE1 element as shown in Figure 5 from (a) to (d) forLNT 229 LN 18 and LNT 308 The LINE1 methylation levels(Qiagen Germany) varied dramatically between the threecell lines (Figure 6) the highest methylation was observedin LNT 308 cells followed by LNT 229 and LN 18 In thetemozolomide resistant strains of same cell lines that wereobtained with treatment of increasing concentrations of thedrug a decrease in the global methylation was observed asexemplarily depicted for LNT 308 P (drug sensitive) and LNT308R (drug resistant) (Figure 5(d)) Overall the decrease inglobalmethylationwas significant for LN 18 R and LNT 308Rbut not for LNT 229 P (Studentrsquos t-test lowast119875 lt 005) This is anindication not only that genes responsible for drug resistancemight be activated but also that LINE1 element might playan important role in the temozolomide drug resistance ofglioblastoma [20]

34 The Use of Self-Designed Pyrosequencing Assays inthe Evaluation the Modifications in DNA Methylation ofEpigenetic Regulating ChemicalsDrugs Here the similaritybetween the commercial available LINE1 element kit (QiagenGermany) and the self-designed assays (PSQ primer designQiagen Germany) was investigated (Figure 1 and Tables1 and 2) HACAT cells were treated in vitro with DNAdemethylating agent 5-aza-21015840-deoxycytidine (A) (1120583M for72 h) and the histone deacetylase inhibitor trichostatinA (T2)(1 120583M for 36 h) as shown in the Material and Methods Theresults obtained in the pyrosequencing of LINE1 element kit(Qiagen Germany) are shown in Figure 7

Observe that in the HACAT control cultures the methy-lation level of each CpG site was 53 59 and 63


49 52 50

0255075

100

minus25

A1 TTYGTGGTGYGTYGTTTLNT229

E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

30 35 33

0102030405060A3 TTYGTGGTGYGTYGTTTLN18

E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

60 65 64

01020304050

A5 TTYGTGGTGYGTYGTTTLNT308P

E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

53 62 58

0255075

A6 TTYGTGGTGYGTYGTTTLNT308R

E S G C T C G5

T G T A G10

T C A G T15

C G

(d)

Figure 5 Glioma cell lines were investigated for the methylation level of LINE1 element (Qiagen Germany) for LNT 229 LN 18 and LNT308 Temozolomide resistant strains of same cell lines that were obtained with treatment of increasing concentrations of the drug exemplarilydepicted is LNT 308 P (drug sensitive) and LNT 308R (drug resistant)

20

40

60

80

LNT

229P

LNT

229R

LN 1

8P

LN 1

8R

LNT

308P

LNT

308R

Glo

bal m

ethy

latio

n (

)

Cell lines

LINE1

lowast

lowast

Figure 6 Quantitation of intrinsic methylation level of the LINE1element (Qiagen Germany) in Temozolomide sensitive and resis-tant glioma cell lines using severalmeasurement replicates (119899 = 3ndash6)and the correspondent methylation controls (Studentrsquos t-test lowast119875 lt005)

levels that decreased significantly (Studentrsquos t-test lowast119875 lt005) in HACAT A + T2 treated cells to 36 40 and42 The total methylation of the 3CpG sites decreasedsignificantly (Studentrsquos t-test lowast119875 lt 005) HACAT A +T2 treated cells however the decrease was not as high ascompared to the methylation positive and negative controlsAlso the significant (Studentrsquos t-test lowast119875 lt 005) decreasein methylation HACAT A + T2 treated cells was identifiedin each of the CpG sites of the LINE1 element kit (QiagenGermany)

Similar results were obtained with the self-designedassays (PSQ primer design Qiagen Germany) (Figure 1 and

Tables 1 and 2) The pyrosequencing assay Line1 CpG1-6spanned over 5 CpG sites located at the beginning of theLINE1 element As seen with previous assay the significantdecrease in total methylation (over all 5 CpG sites) wasdocumented in Figure 8(a) however each CpG site showeddecreased methylation level that was at least as strong asobserved for the pyrosequencing with the LINE1 elementkit from Qiagen Germany Nevertheless the decrease inthe LINE1 methylation was different in the CpG site afact that might underline that specific CpG sites could bepreferentially targeted by the epigenetic regulators (chemicalsand drugs)

The pyrosequencing assay Line1 CpG7-14 spanned over8 CpG sites located at the beginning of the LINE1 elementafter Line1 CpG1-6 but before the primers of LINE1 elementkit Qiagen Germany Also here the significant decrease intotal methylation (over all 8 CpG sites) was documented inFigure 8(d) Each CpG site showed decreased methylationlevel that was less strong as observed for the pyrosequencingwith the LINE1 element kit from Qiagen Germany andLine1 CpG1-6 As well the decrease in the LINE1methylationwas different in each CpG site a fact that might underlinethat specific CpG sites could be preferentially targeted by theepigenetic regulators (chemicals and drugs)

4 Discussion

Toxic agents that are not mutagenic could cause stableadverse phenotypic changes if they interfered with epigeneticprocesses DNA methylation in regulatory regions such aspromoters and enhancers could silence gene expression andan inverse correlation between gene expression and DNA


53 59 63

0102030405060

A1 TTYGTGGTGYGTYGTTTHACAT control

E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075

A2 TTYGTGGTGYGTYGTTTHACAT A + T2

E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site

HACAT controlHACAT A + T2

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

Figure 7 Assessment of HACAT cells methylation after in vitro chemical treatment using the self-designed assays (PSQ primer designQiagen Germany) HACAT cells were treated in vitro with DNA demethylating agent 5-aza-21015840-deoxycytidine (A) (1120583M for 72 h) and thehistone deacetylase inhibitor trichostatin A (T2) (1 120583M for 36 h)

methylation in promoters was proposedThe process of DNAmethylation has therapeutic and toxic implications Agentsthat interfere with DNAmethylation processes could be usedto remove deleteriousDNAmethylation therapeutically or onthe other hand could introduce DNAmethylation aberrationwith toxic consequences Nevertheless DNA methylationinhibitors were introduced into clinical practice for anti-cancer treatments [9 20]

41 Assessment of DNA Methylation as Toxicological Evi-dence Adverse health effects induced in humans upon expo-sure to environmental components (particulate matter andozone) and pollutants (arsenic cadmium bisphenol A andparaquat) might cause changes the epigenome [3 15 16 2324]) Thus epigenetics is an important mechanism in theability of environmental chemicals to influence health anddisease [3]

In this paper it is shown that LINE1 element can be usedas indicator not only for epigenetic modifying chemicals forglobal methylation but also for focal methylation Commer-cial available kits and self-designed assays can be used In vitroavailable and established cell lines are suitable for investiga-tion of stressors that affect DNAmethylationwhile epigeneticmodifying drugs are able to change the methylation profiles

in vitro These findings are supported by previous publica-tions such as that of Tabish et al 2012 that shows the globalDNA methylation changes in human lymphoblastoid (TK6)cells (in vitro) in response to 5 direct and 10 indirect-actinggenotoxic agents The authors show the effect of exposureof 5-methyl-21015840-deoxycytidine between control and exposedcultures but also to benzene hydroquinone styrene carbontetrachloride and trichloroethylene and they concluded thatchanges in global DNA methylation are an early event inresponse to agents traditionally considered as genotoxic [17]

42 Assessment of DNA Methylation in Clinical PracticeModification in DNAmethylation could be used as indicatorfor increased occupational risk inworkersHints in this direc-tion was given in a recent study of Godderis et al 2012 whereit was found that global DNA hypermethylation was foundin the lymphocytes of solvent-exposed worker populationcompared with the referents (119875 = 0001 119903 = minus0544) and wasnegatively associated with the exposure [25] FurthermoreDNA methylation could be used as early marker in thedevelopment of serious diseases such as cancer [26] andParkinsonrsquos disease [24] Additionally it could be used inclinical diagnostics emerging research demonstrates thatDNA methylation is responsible for cellular differentiation


25

30

35

40

45

50

HACAT control HACAT A + T2Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45

HACAT control HACAT A + T2

Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)

Figure 8 Assessment of HACAT cells methylation after in vitro chemical treatment using LINE1 element kit (Qiagen Germany) HACATcells were treated in vitro with DNA demethylating agent 5-aza-21015840-deoxycytidine (A) (1120583M for 72 h) and the histone deacetylase inhibitortrichostatin A (T2) (1 120583M for 36 h)

and when measured in whole peripheral blood serves notonly to distinguish cancer cases from controls [26] but alsoto distinguish between the drug sensitive and drug resistantcells

Acknowledgment

The practical work was done during the employment atHeinrich-Heine University of Dusseldorf Moorenstraszlige 540225 Dusseldorf Germany

References

[1] M Berdasco and M Esteller ldquoHot topics in epigenetic mecha-nisms of aging 2011rdquo Aging Cell vol 11 no 2 pp 181ndash186 2012

[2] P Salpea V R Russanova T H Hirai et al ldquoPostnataldevelopment- and age-related changes in DNA-methylationpatterns in the human genomerdquoNucleic Acids Research vol 40no 14 pp 6477ndash6494 2012

[3] S Singh and S S Li ldquoEpigenetic effects of environmentalchemicals bisphenol a and phthalatesrdquo International Journal ofMolecular Sciences vol 13 no 8 pp 10143ndash10153 2012

[4] C Vinson and R Chatterjee ldquoCG methylationrdquo Epigenomicsvol 4 no 6 pp 655ndash663 2012

[5] C C Oakes D J Smiraglia C Plass J M Trasler and BRobaire ldquoAging results in hypermethylation of ribosomal DNAin sperm and liver of male ratsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 100 no4 pp 1775ndash1780 2003

[6] K Swisshelm CM Disteche JThorvalsden A Nelson and DSalk ldquoAge-related increase in methylation of ribosomal genes


and inactivation of chromosome-specific rRNA gene clusters inmouserdquoMutation Research vol 237 no 3-4 pp 131ndash146 1990

[7] S Maegawa G Hinkal H S Kim et al ldquoWidespread andtissue specific age-related DNA methylation changes in micerdquoGenome Research vol 20 no 3 pp 332ndash340 2010

[8] R FThompson G Atzmon C Gheorghe et al ldquoTissue-specificdysregulation of DNA methylation in agingrdquo Aging cell vol 9no 4 pp 506ndash518 2010

[9] M Szyf ldquoThe implications of DNA methylation for toxicologytoward toxicomethylomics the toxicology of DNA methyla-tionrdquo Toxicological Sciences vol 120 no 2 Article ID kfr024pp 235ndash255 2011

[10] P A Callinan and M A Batzer ldquoRetrotransposable elementsand human diseaserdquo Genome Dynamics vol 1 pp 104ndash1152006

[11] N Rodic andKH Burns ldquoLong interspersed element-1 (LINE-1) passenger or driver in human neoplasmsrdquo PLOS Geneticsvol 9 no 3 Article ID e1003402 2013

[12] L Liu T Van Groen I Kadish et al ldquoInsufficient DNAmethylation affects healthy aging and promotes age-relatedhealth problemsrdquo Clinical Epigenetics vol 2 no 2 pp 349ndash3602011

[13] E Gilson and B Horard ldquoComprehensive DNA methylationprofiling of human repetitive DNA elements using an meDIP-on-repArray assayrdquo Methods in Molecular Biology vol 859 pp267ndash291 2012

[14] I Koturbash F A Beland and I P Pogribny ldquoRole of epigeneticevents in chemical carcinogenesis-a justification for incorporat-ing epigenetic evaluations in cancer risk assessmentrdquoToxicologyMechanisms and Methods vol 21 no 4 pp 289ndash297 2011

[15] B Wang Y Li C Shao Y Tan and L Cai ldquoCadmium and itsepigenetic effectsrdquo Current Medicinal Chemistry vol 19 no 16pp 2611ndash2620 2012

[16] TG Rossman andC B Klein ldquoGenetic and epigenetic effects ofenvironmental arsenicalsrdquo Metallomics vol 3 no 11 pp 1135ndash1141 2011

[17] A M Tabish K Poels P Hoet and L Godderis ldquoEpigeneticfactors in cancer risk effect of chemical carcinogens on globalDNAmethylation pattern in human TK6 cellsrdquo PLoS ONE vol7 no 4 Article ID e34674 2012

[18] H Tsai H Li L Van Neste et al ldquoTransient low doses ofDNA-demethylating agents exert durable antitumor effects onhematological and epithelial tumor cellsrdquo Cancer Cell vol 21no 3 pp 430ndash446 2012

[19] P Boukamp R T Petrussevska D Breitkreutz J HornungA Markham and N E Fusenig ldquoNormal keratinization ina spontaneously immortalized aneuploid human keratinocytecell linerdquo Journal of Cell Biology vol 106 no 3 pp 761ndash771 1988

[20] C Happold P Roth W Wick et al ldquoDistinct molecular mech-anisms of acquired resistance to temozolomide in glioblastomacellsrdquo Journal of Neurochemistry vol 122 no 2 pp 444ndash4552012

[21] J van den Boom M Wolter R Kuick et al ldquoCharacterizationof gene expression profiles associated with glioma progressionusing oligonucleotide-based microarray analysis and real-timereverse transcription-polymerase chain reactionrdquo AmericanJournal of Pathology vol 163 no 3 pp 1033ndash1043 2003

[22] A F Burden N C Manley A D Clark S M Gartler CD Laird and R S Hansen ldquoHemimethylation and Non-CpGmethylation levels in a promoter region of human LINE-1 (L1)repeated elementsrdquo Journal of Biological Chemistry vol 280 no15 pp 14413ndash14419 2005

[23] J W Holloway S Savarimuthu Francis K M Fong and I AYang ldquoGenomics and the respiratory effects of air pollutionexposurerdquo Respirology vol 17 no 4 pp 590ndash600 2012

[24] M Kong M Ba H Liang et al ldquo5rsquo-Aza-dC sensitizes paraquattoxic effects on PC12 cellrdquo Neuroscience Letters vol 524 no 1pp 35ndash39 2012

[25] L Godderis K De Raedt A M Tabish et al ldquoEpigeneticchanges in lymphocytes of solvent-exposed individuals rdquo Epige-nomics vol 4 no 3 pp 269ndash277 2012

[26] E A Houseman W P Accomando D C Koestler et alldquoDNA methylation arrays as surrogate measures ofcell mixturedistributionrdquo BMC Bioinformatics vol 13 no 1 p 86 2012

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


A large body of toxicological evidence shows that manysubstances could have an impact on humanrsquos health withoutclear signs of acute or immediate toxicity In chronic exposureto damaging chemicals (long-term toxicity) it is often foundthat the chemicals induce changes in the DNA sequence thusresulting in DNA damage and mutagenesis Neverthelessepigenetic research shows that toxic chemicals could alsomodify the DNA function without changing the sequencedue to for example changing the state of DNA methylationthus inducing deregulated gene expression and genomicinstability [1ndash3 9] Thus DNA methylation might play anintegral role in toxicity through the hypomethylation ofrepetitive DNA elements that impacts chromosomal andtranscriptional stability of the genome and also as a result offocal hypermethylation thus resulting in epigenetic silencingof gene expression (DNA methylation and gene expressionare inversely correlated) [12 13]

Long-term toxicological damage could be caused withoutchanging the DNA sequence thus having important impli-cations on the safety assessment for chemicals drugs andfood [9] Epigenetic alterations may be early indicators ofgenotoxic and nongenotoxic carcinogenic exposure and thusused as biomarkers in the assessment of the carcinogenicpotential of environmental chemical and physical agents andmight be used in cancer risk assessment [14] For exampleenvironmental relevant chemicals such as arsenic cadmiumand bisphenol A have epigenetic modulation property ([3 1516]) but also mixtures of benzene hydroquinone styrenecarbon tetrachloride and trichloroethylene [17]

All major human cancers have a large number ofgenetic alterations and epigenetic abnormalities that mightbe used as biomarkers for the molecular diagnosis of cancerThe genome-wide loss of methylation at CpG sites of thepromoter is a common epigenetic event in malignanciesand may play a role in the process of carcinogenesis [14]while changes in promoter DNA methylation and asso-ciated gene silencing might be used for cancer therapy(eg decitabine and azacitidine) [18] Thus investigation ofchemical induced changes in DNA methylation status couldrepresent an important indicator for chemical induced riskassessment

In this paper it is shown that pyrosequencing is applicablein toxicity tests to investigate the modification in DNAmethylation upon exposure to chemicals in vitro Pyrose-quencing could be adapted to provide accurate quantifiabledata and detailed profiles of DNA methylation patternsunderlying for example cell cycle regulation differentialgene expression and epigenetic effects

2 Material and Methods

Pyrosequencing is a quantitative sequence-based detectiontechnology adaptable for exploratory and testing work intoxicology and also in pharmacological approaches Manyready designed assays (PyroMark CpG Assays) are availablefor pyrosequencing analysis for example more than 84000assays are available for gene-specific CpG islands includingthe mouse and rat genomes (Qiagen Germany EpigenDX

USA) These assays are created using algorithms that providespecific quantification of CpG methylation

Using pyrosequencing for the investigation of DNAmethylation has been proofed as highly reproducible andhighly sensitive in praxis Companies are offering to usersthe possibility of buying ready to use kits for pyrosequencingespecially diagnostic relevant biomarkers such as KRASMGMT p16 LINE1 MLH1 and BRAF (Qiagen GermanyEpigenDX USA) Although the number of available pre-designed ready to buy DNA methylation assays (eg Pyro-Mark CpG Assays) has considerably increased in the marketthe researcher today might be put in the position that thereare no assays available for specific biomarkers Thus it ispossible to create self-designed assays using commercialsoftware (PSQ assay design) or freely available software(Primer3)

21 Cell Culture Normal human dermal fibroblasts (NHDF)were provided from PromoCell GermanyThe cells were iso-lated from normal human foreskin from a 10-year old donorand cryopreserved at passage 2 (P2) using serum-free freez-ingmedium Cryo-SFM (PromoCell Germany)The cultureswere maintained in the incubator at 37∘C 5 CO

2 The

cells were split once they have reached 80ndash95 confluenceby using Trypsin-EDTA (0025) (PAA Germany) Aftercollecting the detached cells in Trypsin inhibitor (PromoCellGermany) the cells were maintained in cell culture dishes(NUC Germany) The fibroblast cultures were maintainedin serum-free fibroblast culture medium without phenolred supplemented with ldquoSupplement Mixrdquo (basic fibroblastgrowth factor 1 ngmL and insulin 5 ngmL) (PromoCellGermany) as well as antibiotics penicillinstreptomycin(PAA Germany)

HACAT cells (DKFZ Germany) are in vitro sponta-neously transformed keratinocytes derived from histologicalnormal skin that grows in DMEM medium (high glu-cose) supplemented with 2mM L-glutamine (PAA) and 10fetal calf serum (PAA Germany) as well as antibioticspenicillinstreptomycin (PAA Germany) The cultures weremaintained at 37∘C with 5 CO

2and splitted as necessary if

they reached 80ndash95 confluence [19]

22 5-Methyl-21015840-deoxycytidine and Trichostatin A Treatment(A + T2) of HACAT Cells HACAT cells were plated in10 cm2 culture dishes at 50 confluence and were left overnight for attachment They were treated (119899 = 6) with thedemethylating agent 5-aza-21015840-deoxycytidine (5-Aza) and thehistone deacetylase inhibitor trichostatin A (TSA) (SigmaGermany) The treatment conditions were done with eitherthe demethylating agent 5-aza-21015840-deoxycytidine (1 120583M for72 h) or the histone deacetylase inhibitor trichostatin A (1 120583Mfor 36 h)

23 Glioma Cell Lines LNT-229 LN-308 and LN-18 Allglioblastoma cell lines were grown under standard conditionand as elsewhere described [20] Dulbeccorsquos modified Eaglersquosmedium (DMEM)was used as basal medium 10 (vv) heat-inactivated fetal bovine serum (FCS) and 1 (vv) penicillin









2(final con-


(1)

(2)

(3)










(a)


(b)


(c)



(a)


(b)


(c)





3 Results



86 71 77

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G


2 2 2

0102030405060

minus10


(b)

NA NA NA

024

minus2minus4minus6


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)










47 52 49

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060

minus10


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G

50 56 51

0102030405060


(b)

53 64 65

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

66 59 59

0255075

100125150

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


Glo

bal m

ethy

latio

n (

)

LINE1

5649 51 53

79

30

102030405060708090

NH

DF

HAC

AT

CKA-

CA

HEK

293

M+

Mminus








49 52 50

0255075

100

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

30 35 33


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

60 65 64

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

53 62 58

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


20

40

60

80

LNT

229P

LNT

229R

LN 1

8P

LN 1

8R

LNT

308P

LNT

308R

Glo

bal m

ethy

latio

n (

)

Cell lines

LINE1

lowast

lowast






4 Discussion



53 59 63

0102030405060


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)








25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of









2(final con-


(1)

(2)

(3)










(a)


(b)


(c)



(a)


(b)


(c)





3 Results



86 71 77

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G


2 2 2

0102030405060

minus10


(b)

NA NA NA

024

minus2minus4minus6


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)










47 52 49

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060

minus10


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G

50 56 51

0102030405060


(b)

53 64 65

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

66 59 59

0255075

100125150

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


Glo

bal m

ethy

latio

n (

)

LINE1

5649 51 53

79

30

102030405060708090

NH

DF

HAC

AT

CKA-

CA

HEK

293

M+

Mminus








49 52 50

0255075

100

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

30 35 33


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

60 65 64

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

53 62 58

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


20

40

60

80

LNT

229P

LNT

229R

LN 1

8P

LN 1

8R

LNT

308P

LNT

308R

Glo

bal m

ethy

latio

n (

)

Cell lines

LINE1

lowast

lowast






4 Discussion



53 59 63

0102030405060


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)








25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



(a)


(b)


(c)



(a)


(b)


(c)





3 Results



86 71 77

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G


2 2 2

0102030405060

minus10


(b)

NA NA NA

024

minus2minus4minus6


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)










47 52 49

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060

minus10


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G

50 56 51

0102030405060


(b)

53 64 65

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

66 59 59

0255075

100125150

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


Glo

bal m

ethy

latio

n (

)

LINE1

5649 51 53

79

30

102030405060708090

NH

DF

HAC

AT

CKA-

CA

HEK

293

M+

Mminus








49 52 50

0255075

100

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

30 35 33


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

60 65 64

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

53 62 58

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


20

40

60

80

LNT

229P

LNT

229R

LN 1

8P

LN 1

8R

LNT

308P

LNT

308R

Glo

bal m

ethy

latio

n (

)

Cell lines

LINE1

lowast

lowast






4 Discussion



53 59 63

0102030405060


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)








25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


86 71 77

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G


2 2 2

0102030405060

minus10


(b)

NA NA NA

024

minus2minus4minus6


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)










47 52 49

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060

minus10


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G

50 56 51

0102030405060


(b)

53 64 65

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

66 59 59

0255075

100125150

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


Glo

bal m

ethy

latio

n (

)

LINE1

5649 51 53

79

30

102030405060708090

NH

DF

HAC

AT

CKA-

CA

HEK

293

M+

Mminus








49 52 50

0255075

100

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

30 35 33


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

60 65 64

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

53 62 58

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


20

40

60

80

LNT

229P

LNT

229R

LN 1

8P

LN 1

8R

LNT

308P

LNT

308R

Glo

bal m

ethy

latio

n (

)

Cell lines

LINE1

lowast

lowast






4 Discussion



53 59 63

0102030405060


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)








25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


47 52 49

E S G C T C G5

T G T A G10

T C A G T15

C G

0102030405060

minus10


(a)

E S G C T C G5

T G T A G10

T C A G T15

C G

50 56 51

0102030405060


(b)

53 64 65

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

66 59 59

0255075

100125150

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


Glo

bal m

ethy

latio

n (

)

LINE1

5649 51 53

79

30

102030405060708090

NH

DF

HAC

AT

CKA-

CA

HEK

293

M+

Mminus








49 52 50

0255075

100

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

30 35 33


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

60 65 64

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

53 62 58

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


20

40

60

80

LNT

229P

LNT

229R

LN 1

8P

LN 1

8R

LNT

308P

LNT

308R

Glo

bal m

ethy

latio

n (

)

Cell lines

LINE1

lowast

lowast






4 Discussion



53 59 63

0102030405060


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)








25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


49 52 50

0255075

100

minus25


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

30 35 33


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

60 65 64

01020304050


E S G C T C G5

T G T A G10

T C A G T15

C G

(c)

53 62 58

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(d)


20

40

60

80

LNT

229P

LNT

229R

LN 1

8P

LN 1

8R

LNT

308P

LNT

308R

Glo

bal m

ethy

latio

n (

)

Cell lines

LINE1

lowast

lowast






4 Discussion



53 59 63

0102030405060


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)








25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


53 59 63

0102030405060


E S G C T C G5

T G T A G10

T C A G T15

C G

(a)

36 40 42

0255075


E S G C T C G5

T G T A G10

T C A G T15

C G

(b)

LINE1 (Qiagen)

25

35

45

55

65

75

HACATcontrol

HACATA + T2

ME+ MEminus

Treatment

Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

(c)

0

10

20

30

40

50

60

70

CG1

CG2

CG3

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)








25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


25

30

35

40

45

50


Glo

bal m

ethy

latio

n (

)

lowastlowastlowast

LINE1 CpG1-6

(a)


20253035404550556065

CG1

CG2

CG3

CG4

CG5

CpG site

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

25

30

35

40

45


Glo

bal m

ethy

latio

n (

)

Treatment

lowastlowastlowast

LINE1 CpG7-14

(c)

0

20

40

60

80

100

120

CG1

CG2

CG3

CG4

CG5

CG6

CG7

CG8

CpG site


lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(d)



Acknowledgment


References

































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of





Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Documents

Clinical Study DNA Methylation Pyrosequencing Assay Is ...downloads.hindawi.com/journals/bmri/2013/486072.pdf · and L of bisul te treated template DNA in a total volume of L. PCRconditionswereasfollows:initialdenaturingat