5-Hydroxymethylcytosine Is Strongly Depleted in Human ... · LC/MS-MS Genomic DNA (1–2 mg) was incubated with 5 units of DNA Degradase Plus (Zymo Research) at 37 C for at least

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5-Hydroxymethylcytosine Is Strongly Depleted in HumanCancers but Its Levels Do Not Correlate with IDH1Mutations

Seung-Gi Jin1, Yong Jiang1, Runxiang Qiu2, Tibor A. Rauch4, Yinsheng Wang3, Gabriele Schackert5,Dietmar Krex5, Qiang Lu2, and Gerd P. Pfeifer1

AbstractThe base 5-hydroxymethylcytosine (5hmC) was recently identified as an oxidation product of 5-methylcytosine

in mammalian DNA. Here, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-20-deoxycytidine (5hmdC) and 5-methyl-20-deoxycytidine (5mdC) in genomicDNA,we investigatedwhether levels of5hmC can distinguish normal tissue from tumor tissue. In squamous cell lung cancers, levels of 5hmdC weredepleted substantially with up to 5-fold reduction compared with normal lung tissue. In brain tumors, 5hmdCshowed an evenmore drastic reductionwith levels up tomore than 30-fold lower than in normal brain, but 5hmdClevels were independent of mutations in isocitrate dehydrogenase-1. Furthermore, immunohistochemicalanalysis indicated that 5hmC is remarkably depleted in many types of human cancer. Importantly, an inverserelationship between 5hmC levels and cell proliferation was observed with lack of 5hmC in proliferating cells. Thedata therefore suggest that 5hmdC is strongly depleted in human malignant tumors, a finding that adds anotherlayer of complexity to the aberrant epigenome found in cancer tissue. In addition, a lack of 5hmC may become auseful biomarker for cancer diagnosis. Cancer Res; 71(24); 7360–5. �2011 AACR.

Introduction

Recently, 5-hydroxymethylcytosine (5hmC) has been iden-tified as an oxidation product of 5-methylcytosine (5mC) inmammalian DNA (1, 2) generated by the a-ketoglutarate–dependent Tet dioxygenases (2, 3). Levels of 5hmC are tissuedependent and the highest levels have been found in thecentral nervous system (4, 5). The biological function of 5hmCis currently unknown. 5hmC may be an intermediate in DNAdemethylation processes that accomplishes the conversion of5mC to cytosine (6). The distribution of 5hmC in embryonicstem cells (7–12), mouse cerebellum (13), and human prefron-tal cortex (14) has beenmapped by array- or sequencing-basedassays. 5hmC was enriched at promoters and within genebodies.

Several hematologic malignancies carry mutations in one ofthe TET genes, TET2 (15). TET2 mutations were linked toaberrant levels of 5hmC and 5mC in these cancer genomes(16, 17). Also, mutations in isocitrate dehydrogenase-1 (IDH1)have been linked to abnormal DNA methylation patterns (18).One attractive proposal is that mutated IDH1 produces a newmetabolite, 2-hydroxyglutarate (2HG; ref. 19), which can inhibitTET proteins potentially leading to altered levels of 5hmC and5mC in tumors (20).

Systematic studies on levels of 5hmC in human cancers arelacking. Here, we have used liquid chromatography/tandemmass spectrometry (LC/MS-MS), to assess the levels of 5-hydroxymethyl-20-deoxycytidine (5hmdC) and 5-methyl-20-deoxycytidine (5mdC) in human lung carcinomas and in braintumor DNA. We also used immunofluorescence staining toassess 5hmC in a series of normal and malignant tissuesections.

Materials and Methods

DNA samplesStage-I lung squamous cell carcinoma (SCC) and adenocar-

cinoma samples and matched normal tissues were obtainedfrom the frozen tumor bank of the City of HopeMedical Centerunder an Institutional Review Board approved protocol. Sam-ples were obtained from tumors without laser-capture micro-dissection. DNA from primary small cell lung cancers andmatched normal lung was obtained from Asterand, BioChain,and Cureline. Normal human brain tissue DNAs of the pre-frontal cortex were obtained from Capital Biosciences andBioChain. DNA from neurons and astrocytes of fetal (24 weeksof gestation) human brain was obtained from ScienCell.

Authors' Affiliations: Departments of 1Cancer Biology and 2Neuros-ciences, Beckman Research Institute of the City of Hope, Duarte; 3Depart-ment of Chemistry, University of California Riverside, Riverside, California;4Department of Orthopedic Surgery, Section of Molecular Medicine, RushUniversity Medical Center, Chicago, Illinois; and 5Klinik und Poliklinik f€urNeurochirurgie, Universit€atsklinikum Carl Gustav Carus, Technische Uni-versit€at Dresden, Dresden, Germany

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S.-G. Jin, Y. Jiang, and R. Qiu contributed equally to the work.

Corresponding Author: Gerd P. Pfeifer, Department of Cancer Bio-logy, Beckman Research Institute, City of Hope, 1500 E. DuarteRoad, Duarte, CA 91010. Phone: 626-301-8853; Fax: 626-358-7703;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-2023

�2011 American Association for Cancer Research.

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Twenty-seven astrocytomas (World Health Organization,grade II–III) were obtained on Institutional Review Boardapproved protocols at the Department of Neurosurgery at theUniversity Hospital in Dresden. DNA was isolated by standardprocedures with phenol-chloroform extraction and ethanolprecipitation. Eight additional brain tumor DNAs wereobtained from Asterand. Genomic DNAs from tissues and celllines were isolated by the help of the DNeasy Tissue Kit(QIAGEN).

IDH mutationsFor sequencing of IDH1 and IDH2 exon 4, 40 ng of genomic

DNA was used for PCR amplification using the followingprimers: for IDH1, forward 50-TGCCACCAACGACCAAGTCAand reverse 50-CATGCAAAATCACATATTTGCC; for IDH2, for-ward 50-TGAAAGATGGCGGCTGCAGT and reverse 50-GGGGTGAAGACCATTTTGAA.

Simultaneous quantification of 5mdC and 5hmdC byLC/MS-MSGenomic DNA (1–2 mg) was incubated with 5 units of DNA

Degradase Plus (Zymo Research) at 37�C for at least 2 hours.The stable isotope labeled 5hmdC (21, 22) and labeled 20-deoxyguanosine (Cambridge Isotope Laboratories) wereadded as internal standards. Aliquots of the mixture weresubjected directly to LC/MS-MS analysis. LC/MS-MS wascarried out with a Thermo Accela 600 HPLC pump interfacedwith a TSQ Vantage triple stage quadruple mass spectrometer(Thermo Fisher Scientific). A 2.1 � 50 mm Kinetex XB-C18

column (2.6 mm in particle size and 100 A�in pore size;

Phenomenex) was used for separation at a flow rate of 400mL/min. The TSQmass spectrometer was optimized and set up

in selected reaction monitoring scan mode for monitoring the[MþH]þ ions of 5hmdC (m/z 258.1! 142.1), 5mdC (m/z 242.1! 126.1), dG (m/z 268.1! 152.1), labeled 5hmdC (m/z 261.1!144.1) and labeled dG (m/z 273.1 ! 157.1). Thermo Xcalibursoftware (version 2.1) was used to conduct data analysis.

Immunodot blot analysis for 5hmC was conducted asdescribed previously (14).

ImmunohistochemistryFrozen tissue arrays were from Biochain (catalog no.

T6235700-5 and lot no. B403109). They contain normal braintissue and cranionpharyngioma, normal breast and invasiveductal carcinoma, normal colon and adenocarcinoma, normalskeletal muscle and rhabdomyosarcoma, normal kidney andrenal cell carcinoma, normal liver and hepatocellular carcino-ma, normal lung and SCC, normal pancreas and adenocarci-noma, normal prostate and adenocarcinoma, normal skin andmalignant melanoma, normal small intestine and malignantmesenchymoma, normal stomach and adenocarcinoma, nor-mal uterus and adenocarcinoma, and normal ovary and cysta-denocarcinoma. The tissue sections were boiled in 10 mmol/Lsodium citrate for antigen retrieval followed by blocking with10% goat serum, 0.1% Triton X-100 in PBS for 1 hour at roomtemperature (RT). Sections were incubated with primary anti-5hmCpolyclonal antibody (dilution 1:1,000; ActiveMotif) in 5%goat serum, 0.01% Triton X-100 in PBS at 4�C, overnight. Afterwashing with PBS at RT, sections were incubated with RhodRed-X-AffiniPure conjugated goat anti-rabbit secondary anti-body (dilution 1:200; Jackson ImmnunoResearch) for 1 hour atRT, then washed with PBS and water, and mounted withfluoromount-G solution (SouthernBiotech). Ki67 staining wascarried out with Ki67 antibody (BD Pharmingen; catalog

Figure 1. Quantitation of 5hmdCand 5mdC in normal lung and lungSCC DNA. A, 5hmdC. The first 18samples are matched normal lung(LN, blue) and lung tumors (LT,green). The last 6 samples are lungtumors without available normaltissue. B, 5mdC. The asterisks (�)indicate that the levels of 5mdCwere significantly reduced in thetumor compared with normal lung(P < 0.05).

Depletion of 5-Hydroxymethylcytosine in Human Cancers

www.aacrjournals.org Cancer Res; 71(24) December 15, 2011 7361




number 550609; dilution 1:20). The anti-5mC antibody wasfrom Eurogentec (catalog no. BI-MECY-0100; dilution 1:200).Slides were counterstained with Hoechst 33258 dye. All flo-rescent images were taken with an inverted Olympus IX 81fluorescence microscope.

Reverse transcriptase PCRQuantitative reverse transcriptase PCR was carried out as

described (23).

Results and Discussion

To determine the levels of 5hmdC and 5mdC in normal andtumor tissues, we developed a sensitive LC/MS-MS assay withisotope-labeled internal standards (Supplementary Fig. S1A).5mdC was quantitated with reference to the dG standard.Supplementary Fig. S1B shows examples of LC separation andhow mass spectrometric analysis of 5hmdC and 5mdC wasachieved. The method is strictly quantitative as shown bystandard curves (Supplementary Fig. S2). We initially testedits performance bymeasuring 5hmdC and 5mdC in several celland tissue DNA samples (Supplementary Fig. S3). The dataobtained were consistent with values reported in the literature(2, 4) and were also generally in agreement with a less quan-titative immunodot blot assay (Supplementary Fig. S4).

Using the LC/MS-MS assay, we measured 5hmdC in 24stage-I lung SCC DNAs and in matched normal lung DNA(Fig. 1A). The levels of 5hmdC, expressed as percentage of dG,were between 0.078% and 0.182% in normal lung. In every SCC

tumor except one (LT2), we saw a significant reduction of5hmdC level compared with the paired normal lung sample(P< 0.05 for each sample pair; t test; Fig. 1A). 5hmdC levels weregenerally 2- to 5-fold lower in the tumors than in normal lung(P¼ 8.88� 10–7; paired t test). We also quantitated 5mdC (Fig.1B). 5mdC was depleted in most tumor samples with a fewexceptions (tumors 1, 2, 6, 7, 15, and 16). In many cases, 5mdClevels were lower by only approximately 5% to 20% (P¼ 0.023;paired t test). IDH1 or IDH2mutations were not found in theselung tumors. We also analyzed 5hmdC in lung adenocarcino-mas and primary small cell lung cancers (Supplementary Fig.S5). As with SCC, 5hmdC was depleted in most of these tumorsrelative to matched normal tissue.

Next, we analyzed the 2 modified 20-deoxynucleosides in 6normal brain DNA samples and in 33 stage II and III astrocy-tomas (astrocytic gliomas) and in 2 glioblastomas. We foundhigh levels of 5hmdC in normal human brain prefrontal cortexDNA (Fig. 2A), in which 5hmdC was between 0.82% and 1.18%of dG. We also measured levels of 5hmdC and 5mdC inastrocytes and in neurons from human fetal brain. Levels of5hmdC were higher (1.45% 5hmdC/dG) in neurons than inastrocytes (0.23% 5hmdC/dG; Supplementary Fig. S6). In braintumors, 5hmdC was strongly depleted relative to normal brain(Fig. 2A). Some astrocytomas contained only 0.03% to 0.04% of5hmdC, a reduction of more than 30-fold (P ¼ 1.55 � 10–11;unpaired t test). Because astrocytomas initiate in neural stemcells or glial progenitor cells, their decreased level of 5hmdCmay be due to either the malignant state or to the cell of originof these tumors. The varying levels of 5hmdC in tumors did not

Figure 2. Quantitation of 5hmdCand5mdC in normal brain DNA and instage II/III astrocytomas. A, 5hmdCquantitation in normal brain (BN,blue) and in brain tumors (BT, greenor orange). Samples BT1–16, BT25,BT27–29, and BT32-36 were stageIII astrocytomas; BT17-24, BT26,BT30, and BT31 were stage IIastrocytomas; and BT37 and BT38were glioblastomas. B, 5mdC in BNand in BTs. Tumors with no IDH1mutation are shown in green;tumors with IDH1 R132H are shownin orange. The sample BT26 had aminor allele frequency of IDH1R132H. Sample BT25 had the raremutation R132G.

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Cancer Res; 71(24) December 15, 2011 Cancer Research7362




correlate with patient age or whether the tumor was stage II orIII or with patient survival (data not shown). Levels of 5mdCshowed only a small reduction in some brain tumors (Fig. 2B; P¼ 0.3; unpaired t test). There was no correlation between levelsof 5hmdC and levels of 5mdC. A substantial fraction of stages IIand III gliomas contain mutations in IDH1 and much morerarely, in IDH2 (24). We determined the mutation status ofIDH1 at codon 132 (Supplementary Fig. S7). We identified 16stage II/III tumorswith the typical codon R132Hmutation, and17 stage II/III tumors without any IDH1 mutation. The R132HIDH1 mutation produces a neomorphic enzyme with thecapacity to generate 2HG (19). We expected that IDH1-mutanttumors would have lower levels of 5hmdC according to thepresumed role of 2HG as an inhibitor of TET oxidases (seeSupplementary Fig. S8). Surprisingly, however, the levels of5hmdC were evenly distributed between the low and highranges, both in IDH1 wild type and in IDH1-mutant tumors(Fig. 2A; P¼ 0.53; t test, nonpaired). This finding is in contrastto a previous report, which observed a significant reduction of5hmC in IDH1-mutant gliomas by immunohistochemistry (20).Similarly, IDH1-mutant and wild-type cases did not showdifferences in levels of 5mdC (Fig. 2B).

To investigate whether loss of 5hmdC is a feature of humancancers in general, we conducted immunohistochemical stain-ing with an anti-5hmC antibody (Fig. 3 and Supplementary Fig.S9). This antibody was verified previously by us and used fordetecting 5hmC in early embryos (14, 23). Normal tissuesections and corresponding tumor were stained with thisantibody. We observed substantial 5hmC staining of almostall cells in most normal tissues. However, staining in corre-sponding tumors was universally decreased with only a fewcells (<10%) staining positive for 5hmC. The only exceptionwasa tumor originating in the colon (Supplementary Fig. S9).Additional lung tumor slides were also analyzed (Supplemen-tary Fig. S10) including tumor and adjacent normal lung(Supplementary Fig. S10A).

We also did parallel staining of several normal and tumorsections for 5mC using an anti-5mC antibody (SupplementaryFig. S11) and did not observe a substantial decrease of 5mCstaining in the tumors. This means that the loss of 5hmC is notsimply due to loss of 5mC in tumors. We then determinedwhether reduced staining of 5hmC in tumors is due toincreased cell proliferation. We used an anti-Ki67 antibody tostain proliferating cells. Sections of normal brain were almost

Figure 3. Immunohistochemicalanalysis of 5hmC on human tissuearrays. Human tissue arrayscontaining samples of malignanttumor and corresponding normaltissue were stained with anti-5hmCantibody. Staining with Hoechst33258 is shown as a control. Themagnification of all panels is10-fold.






completely devoid of Ki67 antigen but brain tumors containedmany Ki67-positive cells that lacked 5hmC staining (Supple-mentary Fig. S12). Similarly, there was little Ki67 staining innormal lung, but in adjacent carcinoma tissue, we saw mutu-ally exclusive staining of Ki67 and 5hmC (Fig. 4 and Supple-mentary Fig. S10). The same was found for sections of breast,pancreatic tumors, and uterus tumors (Supplementary Fig.S13). Tissue sections of normal small intestine showed strongKi67 staining for proliferating cells at the bottom of crypts withlack of 5hmC staining, whereas the more differentiated cellscontained high levels of 5hmC and lacked Ki67 staining (Fig. 4).Thus, one straightforward explanation for the loss of 5hmdC intumors is the enhanced rate of cell proliferation in tumors thatcould lead to a passive loss of 5hmdC, which is not a substratefor DNA methyltransferase 1 (25). We noticed that not all cellsthat lack 5hmC staining in the tumors are Ki67 positiveperhaps due to past history of proliferation leading to perma-nent loss of 5hmC.

The inverse link between 5hmC levels and cell proliferationmay also reflect intrinsic differences in cell type, as we arecomparing differentiated cells with undifferentiated cancercells that carry stem cell–like properties or originate fromsomatic stem cells. Another alternative is the possible exis-tence of aberrations in 5hmC production or elimination path-

ways in tumors. Mutations in TET genes have not beenreported in solid tumors. With the exception of TET3 in brain,there was no substantial reduction of TET gene expression inlung and brain tumors relative to normal tissue as confirmedby reverse transcriptase PCR (Supplementary Fig. S14).

The loss of 5hmdC in tumors may have profound effectson DNA methylation patterns. For example, if 5hmC is anintermediate in DNA demethylation, its loss at specificgenomic locations may make these sequences more proneto acquire methylation. It will be essential to understand themechanisms of how 5hmC is lost in tumors. Finally, loss of5hmC could become a useful molecular biomarker forcancer detection and diagnosis, perhaps in conjunction withKi67 staining.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by NIH grants CA084469 and AG036041 to G.P.Pfeifer, NS075393 to Q. Lu, and CA101864 to Y. Wang.

Received June 16, 2011; revised September 20, 2011; accepted October 3, 2011;published OnlineFirst November 3, 2011.

Figure 4. Inverse relationshipbetween 5hmC and Ki67 staining.Sections of normal lung and lungtumor, normal prostate andprostate tumor, and normal smallintestine were stained with anti-5hmC (red) and anti-Ki67antibodies (green) to markproliferating cells. Note themutuallyexclusive staining of 5hmC andKi67 in the tissue sections. Forexample, proliferating cells inintestinal crypts arepositive forKi67but negative for 5hmC.

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Cancer Res; 71(24) December 15, 2011 Cancer Research7364




References1. Kriaucionis S, Heintz N. The nuclear DNA base 5-hydroxymethylcy-

tosine is present in Purkinje neurons and the brain. Science2009;324:929–30.

2. Tahiliani M, Koh KP, Shen Y, Pastor WA, Bandukwala H, Brudno Y,et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosinein mammalian DNA by MLL partner TET1. Science 2009;324:930–5.

3. Ito S, D'Alessio AC, Taranova OV, Hong K, Sowers LC, Zhang Y. Roleof Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal andinner cell mass specification. Nature 2010;466:1129–33.

4. GlobischD,MunzelM,MullerM,Michalakis S,WagnerM,KochS, et al.Tissue distribution of 5-hydroxymethylcytosine and search for activedemethylation intermediates. PLoS One 2010;5:e15367.

5. Szwagierczak A, Bultmann S, Schmidt CS, Spada F, Leonhardt H.Sensitive enzymatic quantification of 5-hydroxymethylcytosine ingenomic DNA. Nucleic Acids Res 2010;38:e181.

6. Wu SC, Zhang Y. Active DNA demethylation: many roads lead toRome. Nat Rev Mol Cell Biol 2010;11:607–20.

7. Ficz G, Branco MR, Seisenberger S, Santos F, Krueger F, Hore TA,et al. Dynamic regulation of 5-hydroxymethylcytosine in mouse EScells and during differentiation. Nature 2011;473:398–402.

8. Koh KP, Yabuuchi A, Rao S, Huang Y, Cunniff K, Nardone J, et al. Tet1and Tet2 regulate 5-hydroxymethylcytosine production and cell line-age specification in mouse embryonic stem cells. Cell Stem Cell2011;8:200–13.

9. Pastor WA, Pape UJ, Huang Y, Henderson HR, Lister R, Ko M, et al.Genome-wide mapping of 5-hydroxymethylcytosine in embryonicstem cells. Nature 2011;473:394–7.

10. WuH, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, et al. Genome-wideanalysis of 5-hydroxymethylcytosine distribution reveals its dual func-tion in transcriptional regulation inmouse embryonic stemcells. GenesDev 2011;25:679–84.

11. Williams K, Christensen J, Pedersen MT, Johansen JV, Cloos PA,Rappsilber J, et al. TET1 and hydroxymethylcytosine in transcriptionand DNA methylation fidelity. Nature 2011;473:343–8.

12. Xu Y, Wu F, Tan L, Kong L, Xiong L, Deng J, et al. Genome-wideregulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase inmouse embryonic stem cells. Mol Cell 2011;42:451–64.

13. SongCX, Szulwach KE, Fu Y, Dai Q, Yi C, Li X, et al. Selective chemicallabeling reveals the genome-wide distribution of 5-hydroxymethylcy-tosine. Nat Biotechnol 2011;29:68–72.

14. Jin SG,WuX, Li AX, Pfeifer GP. Genomicmapping of 5-hydroxymethyl-cytosine in the human brain. Nucleic Acids Res 2011;39:5015–24.

15. DelhommeauF,DupontS, Della Valle V, JamesC, TrannoyS,MasseA,et al. Mutation in TET2 in myeloid cancers. N Engl J Med 2009;360:2289–301.

16. KoM,HuangY, JankowskaAM,PapeUJ, TahilianiM,BandukwalaHS,et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancerswith mutant TET2. Nature 2010;468:839–43.

17. Figueroa ME, Abdel-Wahab O, Lu C, Ward PS, Patel J, Shih A, et al.Leukemic IDH1 and IDH2 mutations result in a hypermethylationphenotype, disrupt TET2 function, and impair hematopoietic differen-tiation. Cancer Cell 2010;18:553–67.

18. Noushmehr H, Weisenberger DJ, Diefes K, Phillips HS, Pujara K, Ber-man BP, et al. Identification of a CpG island methylator phenotype thatdefines a distinct subgroup of glioma. Cancer Cell 2010;17:510–22.

19. Dang L, White DW, Gross S, Bennett BD, Bittinger MA, Driggers EM,et al. Cancer-associated IDH1mutations produce 2-hydroxyglutarate.Nature 2009;462:739–44.

20. XuW,YangH, Liu Y, YangY,WangP, KimSH, et al. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases. Cancer Cell 2011;19:17–30.

21. Cao H, Wang Y. Collisionally activated dissociation of protonated 20-deoxycytidine, 20-deoxyuridine, and their oxidatively damaged deri-vatives. J Am Soc Mass Spectrom 2006;17:1335–41.

22. LaFrancoisCJ, Fujimoto J, Sowers LC. Synthesis and characterizationof isotopically enrichedpyrimidine deoxynucleoside oxidationdamageproducts. Chem Res Toxicol 1998;11:75–83.

23. Iqbal K, Jin SG, Pfeifer GP, Szabo PE. Reprogramming of the paternalgenome upon fertilization involves genome-wide oxidation of 5-methylcytosine. Proc Natl Acad Sci U S A 2011;108:3642–7.

24. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, et al.IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009;360:765–73.

25. Valinluck V, Sowers LC. Endogenous cytosine damage products alterthe site selectivity of human DNA maintenance methyltransferaseDNMT1. Cancer Res 2007;67:946–50.






2011;71:7360-7365. Published OnlineFirst November 3, 2011.Cancer Res Seung-Gi Jin, Yong Jiang, Runxiang Qiu, et al.

MutationsIDH1but Its Levels Do Not Correlate with 5-Hydroxymethylcytosine Is Strongly Depleted in Human Cancers

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5-Hydroxymethylcytosine Is Strongly Depleted in Human ... · LC/MS-MS Genomic DNA (1–2 mg) was incubated with 5 units of DNA Degradase Plus (Zymo Research) at 37 C for at least