New epigenetic tools for cancer diagnostics

Gonchar D.A., Kuznetsov V.V., Akishev A.G., Abdurashitov M.A.,

Degtyarev S.Kh.

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DNA methylation in mammalians genomes is mostly DNA methylation of CG dinucleotides with formation of 5-methylcytosine (5mC) in both DNA strands.Mammalian DNA-methyltransferases DNMT1, DNMT3a and DNMT3b catalyze a reaction of DNA methylation.DNMT1 maintains DNA methylation pattern in vivo modifying a new strand after replication.DNMT3a and DNMT3b are responsible for DNA methylation de novo. This modification in regulation region (promotor and first exon) of gene results in the gene silencing.

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At present time 5mC is determined mostly by a chemical treatment of DNA with sodium bisulphite, which results in cytosine transformation into uracil, whereas 5mC is resistant against this modification.A subsequent analysis of modified and native DNA allows to locate positions of methylated cytosines instudied DNA.Method of bisulphite conversion is quitesophisticated and often results in obtaining falsepositive data.

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There is another approach — enzymatic methods of determination of DNA methylation. Among enzymatic methods of 5mC determination, so called methyl-sensitive PCR assay (MS PCR) isthe most popular. Determination of DNA methylation by MS PCR proceeds in two steps:DNA hydrolysis with site-specific DNA endonuclease (e.g., restriction enzyme) followedby PCR with primers located upsteam and downstream DNA region of interest.

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This method is based on inability of restriction enzymes, which contain CG dinucleotide in the recognition site, to cut this site if 5mC is present in the dinucleotide.A subsequent PCR from primers, which are located around a chosen recognition site, produces a corresponding DNA fragment if there is a methylated CG-dinucleotide within this site. On the contrary,DNA fragment is not produced in PCR if there isno methylated CG-dinucleotide in a recognition sequence of restriction enzyme.

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HpaII (recognition site CCGG) cleaves DNA sequence CCGG, but doesn't cut C(5mC)GG.Singer-Sam et.al.(Mol. Cell Biol. (1990) Vol.10, 4987-4989) called a method of methyl-sensitive PCR with HpaII as HpaII-PCR assay.

HpaII-PCR assay includes DNA hydrolysis with HpaII followed by PCR with primers located upsteam and downstream DNA region of interest.

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Application of methyl-sensitive PCR assays similar to HpaII-PCR assay is limited by a very short list of recognition sequences of corresponding restriction endonucleases.

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Study of DNMT3a and DNMT3b substrate specificity has shown that both enzymes methylate CG-dinucleotide mostly in DNA sequence PuCGPy. This is a reason why restriction enzymes with recognition sites ACGT and GCGC (MaeII and HhaI, respectively) are widely used in methyl-sensitive PCR study of de novo DNA methylation.

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• DNMT3 is the main enzyme responsible for de novo cytosine modification and epigenetic regulation of human and mammalian genes activity.

• DNMT3 recognizes and methylates a tetranucleotide RCGY in DNA as follows:

5’- Pu C G Py -3’ 5’- Pu(5mC) G Py -3’3’- Py G C Pu -5’ 3’- Py G(5mC) Pu -5’

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New enzymes

BlsI and GlaI

belong to a new type of 5-methylcytosine-directed site-specific DNA endonucleases that cleave only methylated DNA.

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GlaI

Cleaves only methylated DNA and has a recognition site:

5’-Pu(5mC)↓GPy-3’3’-PyG↑(5mC)Pu-5’

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Substrate specificity of DNMT3a, DNMT3b and GlaI

PuCGPy Pu( )GPyPyGCPu PyG( )Pu

5mC5mC

DNMTAdoMet

Pu( )GPy Pu( ) G PyPyG( )Pu Py G ( )Pu

↓↑

5mC 5mC5mC 5mC

GlaI

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BlsICleaves a recognition site

5’-PuPyN↓PuPy-3’3’-PyPu↑NPyPu-5’

carrying at least one 5-methylcytosine (N is not considering) in each DNA strand.

Two sites methylated by Dnmt3 and separated by N form BlsI cleavage site

5’ - Pu (5mC) G Py N Pu (5mC) G Py - 3’3’ - Py G (5mC) Pu N Py G (5mC) Pu - 5’

BlsI recognition site

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BLSI- AND GLAI- PCR ASSAY

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BlsI- и GlaI-PCR assays include DNA hydrolysis with BlsI or GlaI, respectively, followed by PCR with primers located upsteam and downstream DNA region of interest.

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• a promoter region of CEPBD (CCAAT/enhancer binding protein, delta);

• a promoter region of DAPK1 (death-associated protein kinase 1);

• a promoter and first exon region of RASSF1A (Ras association domain family 1A);

• a promoter and first exon region of SEPT9b (septin 9b);

• a promoter and first exon region of MGMT (O6-methylguanine DNA methyltransferase);

• a promoter and first exon region of RARB (retinoic acid receptor, beta);

• a promoter and first exon region of IGFBP3 (insulin-like growth factor binding protein 3).

Studied DNА regions of human genome

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Structure of regulation region of the studied tumor suppressor genes

SEPT9b (939 bp)

GC GCAGCGC GCGCCGC GCC GC GCCGCGC

d1d2

d3r1r2

r3

GC GCTGCGC

r3d1d2 r1

RASSF1A (804 bp)

CEPBD (341 bp)

GC GCAGCGC

d1 r1

RARB

(853 bp)

d1 r1r2

DAPK1

(357 bp)

GCGCCGCGC

d1 r1d2 r2

d1 r1d2

MGMT

(678 bp)

r1

IGFBP3

(817 bp)

d1

GC GCTGCGT17

DNA preparations from five human cell lines: L-68 (control, lung fibroblast), HeLa (cerbix adenocarcinoma), Raji (Burkitt’s lymphoma), U-937 (histiocystic lymphoma) and Jurkat (acute T-cell leukemia) have been treated separately with:

1) Restriction enzyme with recognition site in studied region (HaeIII for CEPBD, RASSF1A and SEPT9b; FatI for RARB), positive control; 2) GlaI (recognizes 5'-Pu(5mC)GPy-3' [2]); 3) BlsI (recognizes 5'-GCNGC-3' if at least two 5-methylcytosines (N isn't considering) are present in both DNA strands [3]);4) no added enzyme, negative control.

After incubation 4 reaction mixtures have been used as a DNA template for PCR. DNA from Drosophila melanogaster at the same concentration has been used as a negative PCR control.

Protocol of BlsI- and GlaI- PCR assay

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RASSF1A (804 bp)

1, 5, 12, 16, 20 – HaeIII2, 6, 13, 17, 21 – GlaI3, 4, 7, 8, 14, 15, 18, 19, 22, 23 – Negative control

10, 11, 25 – 100 bp DNA ladder9, 24 – control DNA (Drosophila melanogaster)

GCGCTGCGC

r1d1d2 r2

GCGCTGCGC

r1

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GCGCAGCGC

d1 r1

CEPBD (341 bp)

CEBPD 341 bp (d1-r1) L68 HeLa Raji D

1 2 3 4 5 6 7 8 9 10 11 12 13 14

U937 Jurkat D

15 16 17 18 19 20 21 22 23 24

1, 5, 9, 15, 19 – HaeIII2, 3, 6, 7, 10, 11, 16, 17, 20, 21 – GlaI

14, 24 – 100 bp DNA ladder13, 23 – control DNA (Drosophila melanogaster)

GCGCAGCGC

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d1 r1

2, 6, 10, 16, 20 – HaeIII3, 7, 11, 17, 21 – GlaI4, 5, 8, 9, 12, 13, 18, 19, 22, 23 –Negative control

1, 15, 25 – 100 bp DNA ladder14, 24 – control DNA (Drosophila melanogaster)

RARB (853 bp)

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MGMT (356 bp)

U937 Jurkat

M HaeIII GlaI BlsI TaqI HaeIII GlaI BlsI TaqI

L68 HeLa Raji

M HaeIII GlaI BlsI TaqI HaeIII GlaI BlsI TaqI HaeIII GlaI BlsI TaqI

r1d2

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SEPT9b

A 399 bp (d1-r1) U937 Jurkat D

1 2 3 4 5 6 7 8 9 10 11 L68 HeLa Raji D

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Б 197 bp (d2-r2) U937 Jurkat D

1 2 3 4 5 6 7 8 9 10 11 L68 HeLa Raji D

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

GCGCAGCGC GCGCCGCGC GCGCCGCGC

d1d2

d3r1r2

r3

2, 6, 13, 17, 21 – HaeIII3, 7, 14, 18, 22 – GlaI4, 8, 15, 19, 23 – BlsI

1, 11, 12, 26 – 100 bp DNA ladder10, 25 – control DNA (Drosophila melanogaster)

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U937 Jurkat

1 2 3 4 5 6 7 8 9 10 11

L68 HeLa Raji

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

2, 6, 13, 17, 21 – HaeIII3, 7, 14, 18, 22 – GlaI4, 8, 15, 19, 23 – BlsI

5, 9, 16, 20, 24 – Tru9I10, 25 – Negative control 1, 11, 12, 26 – 100 bp DNA ladder

IGFBP3 (817 bp)

r1d1

GCGCTGCGT

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Fig. . BlsI- and GlaI- PCR assay of promoter region (fragment d2-r2, 173 bp in length).

9 Real time DAPK1

U-937

JurkatL-68

HeLa

Raji

Pretreated DNA

With GlaI

With BlsI

With HaeIII

Methylation: Raji - >99%; L-68, HeLa, U-937, Jurkat - <1%

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(+) - methylation; (-) – no methylation

Abnormal DNA methylation of regulation regions of tumor suppressor genes

PCR assay BlsI/GlaI-PCR assay GlaI-PCR assay

Tumor suppressorgene

Cell line

DAPKI CEPBD SEPT9b RASSF1A

IGFBP3 MGMT RARB

L-68fibroblast - - - - - - -HeLacervix adenocarcinoma - - + - - - +RajiBurkitt’s lymphoma + + + + + - +U-937histiocytic lymphoma - + + - + + +Jurkatacute T-cell leukemia - - - + + - +

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Comparison of bisulphite conversion and BlsI- and GlaI- PCR assay

A quantity of DNA for analysis:2-5 DNA molecules for BlsI- and GlaI- PCR assay

Fidelity of BlsI- and GlaI- PCR assay – 2%Bisulphite conversion – 15%

BlsI- and GlaI- PCR assay analyzes DNA fragments from 100 to 10000 b.p., while bisulphite conversion only 150-200 b.p.

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GLAD-PCR ASSAY

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Introduction to GLAD-PCR assay

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There is one vital disadvantage of BlsI- and GlaI- PCR assay – it is good for epigenetic typing of cancer cell lines and is hardly ever may be applied in clinical practice because the studied DNA samples include unmethylated DNA from stroma, blood cells, etc.

A new GLAD-PCR assay we have developed recently allows to determine minimal quantities of methylated sites in presence of excess of unmethylated DNA.

GLAD-PCR assay may find a wide application in routine clinical practice

Method

NNNNNRCNNNNNYG

CH3

GYNNNNNCRNNNNNCH3

Double stranded methylated DNA

Universaladapter

GlaILigase

NNNNNNNNNNNN

2. Adapter ligation3. Real-time PCR1. GlaI digestion

Genomeprimer

TaqManprobe

Hybridprimer

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GLAD-PCR assay

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GlaI hydrolysis and Ligation Adapter Dependent PCR (GLAD-PCR) is the novel method to determine R(5mC)GY sites produced by methylation with DNMT3A and DNMT3B. GLAD PCR analysis is performed in one tube and includes 3 steps: DNA hydrolysis with site-specific methyl-directed DNA endonuclease GlaI, universal adapter ligation and Real-time PCR with Taqman probe.

One primer is designed for DNA region of interest, structure of another primer is based on an adapter sequence.

Studied genes

1 — transcription start. H — position of hybrid primer.

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Studied DNA

Malignant cell linesRaji — Burkitt’s lymphoma,Jurkat — acute T-cell leukemia,U-937 — histiocytic lymphoma,HeLa — cervix adenocarcinoma,

ControlL-68 — fibroblast cell line,G — human peripheral blood DNA,Mouse — A/He mouse DNA, negative control

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GLAD PCR analysis of DNA methylation in regulatory region of tumor suppressor genes

Amplification chart of GLAD PCR assay of 15 ng DNA per reaction using Bio-Rad CFX96. We accept Raji DNA methylation to be 100%.

CEBPD RARB

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GLAD PCR analysis of DNA methylation in regulatory region of tumor suppressor genes

Sensitivity determination of the GLAD PCR assay

CEBPD RARB

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Epigenetic typing of human cancer cell lines by GLAD PCR assay

* — gene regulator of cell differentiation

DNAGene . Raji Jurkat U-937 Hela L-68 G Mouse

RASSF1 100 55 — — — — —

CEBPD 100 1,3 1,0 — — — —

TWIST1 100 1,0 0,5 — — — —

EGFR 100 1,0 1,2 — — — —

LIN28 100 90 100 90 — — —

RARB 100 60 90 100 1 1 —

HS3ST2 100 12 13 55 0,5 0,5 —

NANOG* 1,1 0,4 0,4 1,1 55 100 —

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Conclusions

A new method of GLAD PCR assay has been developed to study DNA methylation. Method includes GlaI hydrolysis of studied DNA, the universal adapter ligation and subsequent real-time PCR of the studied RCGY site. Method is performed in one tube, takes about four hours and allows to determine several copies of methylated DNA.GLAD PCR assay has been applied to study aberrant methylation of selected RCGY site in regulatory regions of tumor suppressor genes. GLAD PCR assay has revealed different patterns of RCGY sites methylation in four malignant cell lines. All studied RCGY sites are highly methylated in Raji cells and unmethylated in control fibroblast line.GLAD PCR assay may be used for determination of methylation status of particular RCGY site and for a rapid epigenetic characterization of malignant cells.

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