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DNA Methylation and DNA Methylation and CancerCancer
O
NH2
N
N
Peter Jones
Structure of B-DNA
5'-CG5mCGAATT5mCGCG-3'NDB ID: BDLB72
Structure 1
Replication
De novo
DNMT 3a/b
Maintenance
DNMT 1
Ara-C
Replication
De novo
DNMT 3a/b
MaintenanceDecitabine
DNMT 1
Decitabine
Eukaryotic Methylation
CpG CpG
CH3
CH3CH3
CH3
CpG
CpG
CpG
CpG
Eukaryotic Methylationon Inactive X- Chromosome
CpG CpG
CpG
CpG
CpG
CpG
CH3
CH3 CH3
CH3
Nature 429 457- 463 (2004)
Eukaryotic Methylation
CpG CpG
CH3
CH3CH3
CH3
CpG
CpG
CpG
CpG
Eukaryotic Methylationin Cancer
CpG CpG
CpG
CpG
CpG
CpG
CH3
CH3 CH3
CH3
Exon1 Exon2LINE
Chromosome in Normal Cell
De NovoMethylation
Gene Silencing
TransposonDemethylation
ActivatedTransposon?
5mC TMutations
Mutated Gene
Satellite Demethylation
ChromosInstability
ome
Exon1
Chromosome in Cancer Cell
XLINE Exon2
Epigenetic Therapy
PromoterPromoterMethylationMethylation
DemethylateDemethylate
Vidaza Decitabine
N
NH2
N
O
N
N
NH2
N
O
N
DeoxyriboseRibose
5-Aza-CR 5-Aza-CdR
5Aza-dC
Metabolism of 5Metabolism of 5--azacytidine azacytidine (Vidaza)(Vidaza) and 5and 5--azaaza--2’2’--
deoxycytidine deoxycytidine (Decitabine)(Decitabine)
Phosphatase
DNARNA
5-aza-CTP
5-aza-CDP
5-aza-CMP
5-aza-CR
Uridine – CytidineKinase
5-aza-dCTP
5-aza-dCDP
5-aza-dCMP
5-aza-CdR
DeoxycytidineKinase
Phosphatase
RibonucleotideReductase
(Decitabine)(Vidaza)
Nature 429 457- 463 (2004)
CDK4/6
p14Arf
MDM2
p53pRb
INK4A/ARF
p16INK4a
Treatment of T24 bladder cancer cells with Decitabine causes a reduction of DNMT protein levels and activation of p16
DNMT1
DNMT3b3
0 1 2 3 4Decitabine
p16
kDa
195
130
16
Days
% m
eth
ylat
ion
Days
0 2 440
60
100
80
0 2 40
4
6
2#
po
pu
lat i
on
do
ub
l ing
s Treated
Untreated
Days
INK4b
p15
INK4b
p15
CDK4/6
pRb
p16INK4a p14Arf
MDM2
p53
INK4A/ARF
CDK4/6
pRb
Abnl. Metaph. 20/20 18/18
Genes Induced > 4 Fold by 5Genes Induced > 4 Fold by 5--AzaAza--CdRCdR
Gene Class LD419 (Fibroblast) T24 (Tumor) OverlapInterferon induced 1 10
Cytokine 2 7 SAA1Membrane proteins 2 6
MAGEs 1 5 MAGE4Growth factors 1 4Cytokeratins 4 1 KRT17
Histones 3 3 H2AFL, H2BFHTranscription factors 1 3 STAT1Protease inhibitors 0 3
Proteases 3 2Complement component 1 3 C3
Imprinted gene 1 1 H19Apoptosis 0 2
Miscellaneous 13 12
Total induced genes 34/6,600 (0.5%) 61/6,600 (0.9%)
Genes inducible by interferon 10/34 (29%) 33/61 (54%)
Problems with Epigenetic Therapy
•Remethylation•Labile Drugs•Selectivity
Re-silencing of p16 Gene after 5-Aza-CdR Treatment
10 14 22 300 3 8
5-Aza-CdR (10-6 M)
p16
GAPDH
Days
TFNormal Cells
Pro
gres
sive
Ch
ange
s
Cell division
TF
Cell division
TF
Cell division
Tumor Cells
Problems with Epigenetic Therapy
•Remethylation•Labile Drugs•Selectivity
Chemical Structures
N
NH2
N
O1
2
34
5
6
Ribose Ribose
55--AzacytidineAzacytidine(5(5--AzaAza--CR)CR)
N
NH2
N
O
N3
21
4
5
6
N
N
O1
2
34
5
6
Ribose
CytidineCytidine ZebularineZebularine((ZebZeb))
DNARNA
Zebularine
Covalent binding of DNMTs to zebularine-substitued DNA
DNA
N
N
O
DNA
N
N
ODNMT
DNMT
H
H
HH
H
David Hornby
Kinetics of p16 gene expression afterzebularine treatment in T24 cells
Days After Treatment (500 µM
p1
GAPD
0 1 2 3 4 5 7 9 1 1
Days After
p16
Pro
mot
er
Met
hyl
atio
n
10
9
8
7
6
0 1 2 3 4 5 7 9 1 15
Continuous Treatment of T24 cells with
Zebularine (10 -4 M 4 x10-4 M)
0 5 7 14 21 28 33 4050
60
70
80
90
100
Days of Continuous Treatment
p16
Pro
mo
ter
Met
hyl
atio
n (
%)
Zebularine (10-4 M)
5 7 14 21 28 33 40 5 7 14 21 28 33 400
p16
GAPDH
Zebularine (10-4 M) (10-4 M 4 x 10-4 M)Zebularine
0 5 7 14 21 28 33 40 5 7 14 21 28 33 40
Zebularine (10-4 M 4x10-4M)
Reactivation of Reactivation of p16p16 in EJ6 Tumor Cells in EJ6 Tumor Cells Subcutaneously Subcutaneously Implanted in MiceImplanted in Mice
0
0.5
1
1.5
2
2.5
3
3.5
Control 500 mg/kgoral
1000 mg/kgoral
500 mg/kgi.p.
1000 mg/kgi.p.
Rat
io o
f p1
6/G
AP
DH
(N=5)
Con
trol
500 1000No
cDN
A
p16
GAPDH
Oral (mg/kg)Oral (mg/kg)
500 1000
I.P. (mg/kg)I.P. (mg/kg)
Problems with Epigenetic Therapy
•Remethylation•Labile Drugs•Selectivity
0
100
200
300
400
500
600
700
800
LD419 LD98 T-1 CCD-1070Sk
T24 Calu-1 HCT15 CFPAC-1 PC3 SW48 HT29
CPM/10,000 cells/24 hrs
U/C k activity (pmol/min/mg protein)
INCORPORATION OF 14C-ZEBULARINE IN DNA AND ACTIVITY OF URIDINE/CYTIDINE KINASE
Depletion of DNMT proteins after continuoustreatment with zebularine
0 1 2 3 8
LD419
0 1 2 3 8
T24
40Days
DNMT1
DNMT3b3
PCNA
DNMT3a
Preferential response of cancer cells to DNA methylation inhibitors
FibroblastsFibroblasts Cancer CellsCancer Cells
⇑⇑ ⇓⇓ ⇑⇑ ⇓⇓55--AzaAza--CdR (≥4 fold)CdR (≥4 fold) 34/6,60034/6,600 11/6,60011/6,600 61/6,60061/6,600 2/6,6002/6,600
Zebularine (≥2 fold)Zebularine (≥2 fold) 1/13,0001/13,000 1/13,0001/13,000 12/13,00012/13,000 6/13,0006/13,000
Microarray Analysis of Fibroblasts and Cancer Cells After Continuous Zebularine Treatment
Decrease ≥ 2 -fold Decrease ≥ 2 -fold
Increase ≥ 2 -fold 1 0 Increase ≥ 2 -fold 121 0 6
Fibroblasts [4] Cancer Cells [3]Overlap
Gene Fold Change
MAGEA1 6.0MAGEB2 3.2GAGE3 5.7GAGE6 8.0GAGE7 9.6
GAGE7B 12.4XAGE1 21.2
SPANXA1 258.3
Methylation and Expression of XAGE1 after Continuous Zebularine Treatment
100 200 300 400 500 600 700 800 900 1000
0
20
40
60
80
100
LD98 LD419 T-1 CCD-1070Sk
T24 Cf-Pac-1 HCT15
% M
ET
HY
LA
TIO
N
Untreated
100uM Zebularine
Fold change 0 0 0 0 8 8 49
p16 p16 ↑↑
MLH1 MLH1 ↑↑
TransposonsTransposons
TumorTumorAntigenAntigen
EE--CadherinCadherin↑↑
↓↓ GrowthGrowth
↑↑ ChemosensitivityChemosensitivity
↑ Adhesion
↑ IFN Response
↑ Immunogenicity
PromoterPromoterMethylationMethylation
DemethylateDemethylate
AcknowledgementsUSCAna AparicioChristina BenderJonathan ChengGerda EggerFelicidad GonzalesMark GonzalgoGangning LiangJoy LinCarvell NguyenMihaela VelicescuDan WeisenbergerChristine YooUniversity of MiamiShelly Greer
University of OregonCindy MatsenEric SelkerUniversity of FreiburgMichael LuebbertMichael DaskalakisNCIVictor MarquezDenmarkTorben F. OrntoftThomas ThykjaerDisclosureEpigenomicsSuperGen
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