Methylation of individual CpG sites Methylation of individual CpG sites within within p16p16 CpG islands CpG islands

in various human gastric lesions in various human gastric lesions

Zheming Lu( 陆哲明） Dajun Deng （邓大君）

Beijing University School of OncologyBeijing University School of Oncology

Beijing Institute for Cancer Research,Beijing Cancer HospitalBeijing Institute for Cancer Research,Beijing Cancer Hospital

R Beroukhim et al. Nature 463, 899-905 (2010)

Identification of significant focal SCNAs across Identification of significant focal SCNAs across 3131 human cancer specimens3131 human cancer specimens

p16p16 is inactivated by genetic alterations (10%) and is inactivated by genetic alterations (10%) and DNA methylation (30%) in many human tumorsDNA methylation (30%) in many human tumors

(Esteller, Oncogene 2002)

• Methylation status of individual CpG sites within Methylation status of individual CpG sites within p16p16 CpG islands CpG islands in vivoin vivo is not well characterized. is not well characterized.

p16p16 is fully methylated in many cancer cell lines is fully methylated in many cancer cell lines..

• The mechanisms lead to fate diversities of The mechanisms lead to fate diversities of p16p16 methylation in different gastric lesions are unknown. methylation in different gastric lesions are unknown.

p16 p16 methylation is stable in cancer cells, but unstable in methylation is stable in cancer cells, but unstable in gastritis lesions.gastritis lesions.

(Wong et al., 1999 MCB)

(Human mammary epithelial cell strains, immortalized with HPV16 E6/E7 transfection)

(Luo et al., 2006 Lab Invest)

M-p16

U-p16 Proportion of M-p16(35 CpG sites)

T12: 0%

T14: 7.89%

T74: 28.91%

T67: 39.14%

(Human gastric carcinoma tissues)

Analyzed fragment of Analyzed fragment of p16p16 CpG island by partial-MSP CpG island by partial-MSP(-355nt~+233nt, including 45+3 CpG sites)(-355nt~+233nt, including 45+3 CpG sites)

partial-MSP primer setpartial-MSP primer set

to enrich to enrich p16p16 CpG islands CpG islands with methylated cytosineswith methylated cytosines

126bp-MSP 151bp-MSP 234bp-MSP83bp-MSP

392bp-DHPLC

More More p16p16 partial-MSP products was detected in partial-MSP products was detected in gastric carcinomas than in normal/gastritisgastric carcinomas than in normal/gastritis

36/40 (90.0)Gastriccarcinoma

<0.01

19/45 (42.2)

7/13 (53.8)

Partial-MSPpositive rate (%)

Chronicgastritis

Normal

Pathologicalchanges

P-value(two-sides)

M 4644 3210 5115 4505 3107 5025 5014 3150 F0310 F0342 F0350 F0414 F0438 F0144 F0566 F0020

578bp

(H.pylori-specific 23S rDNA by PCR assay

[Liu et al., 2008 BMC Microb])F0437 F0815 F1023 F1069 F0973 F0985 F0747 F0867 F1141 F1143 F1011 F0749 F0597 F0773

289bp

More More p16p16 partial-MSP products were detected partial-MSP products were detected in the in the HpHp-positive samples than -negative ones-positive samples than -negative ones

144negative

1530positive

Hp

negativepositive

Partial-MSP

(p<0.001)

Methylation status of individual CpG sites within Methylation status of individual CpG sites within p16p16 CpG island in human gastric mucosa CpG island in human gastric mucosa ((nn=51)=51)

Ca(n=31)

Gastritis(n=14)

Normal(n=6)

Methylation status of individual CpG sites within Methylation status of individual CpG sites within p16p16 CpG island in human gastric mucosa CpG island in human gastric mucosa

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

20%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Pro

po

rtio

n o

f m

eth

yla

tio

n

CpG location

Carcinoma Gastritis Normal

* *

G/A

( p<0.05)

0%

5%

10%

15%

20%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Hp(-)/GC Hp(+)/GC

* *

0%

5%

10%

15%

20%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

Hp(-)/nonGC Hp(+)/nonGC *

** * * *

*

H. pyloriH. pylori infection infection and and p16p16 methylation methylation

Normal or gastritis tissues: nHp(+)=13, nHp(-)= 7

Gastric carcinomas: nHp(+)=17, nHp(-)= 9

(*: p<0.05)

(*: p=0.06)

G/A

Distribution of Distribution of p16p16 clones with different no. clones with different no. of methylated CpG sites in gastric mucosaof methylated CpG sites in gastric mucosa

p16p16 clones with clones with 6 methylated CpG sites 6 methylated CpG sites in gastric mucosain gastric mucosa

Carcinoma[77/460; 16.7%]

Gastritis[18/217; 8.3%]

Normal[5/67; 7.5%]

88bp83bp

M U M U M U M U M U

M.Ctrl U.Ctrl TE buffer Sample-1 Sample-2

83bp-MSP

(+) (-)

partial-

MSP

(+) 33 22

(-) 1 5

83bp-MSP

(+) (-)

Hp(+) 28 15

(-) 5 25

Results of the 83bp-MSP assay confirm Results of the 83bp-MSP assay confirm those of the partial-MSP assaythose of the partial-MSP assay

(p=0.044) (p<0.0001)

Results of the 83bp-MSP assay confirm those of Results of the 83bp-MSP assay confirm those of clone sequencing of the partial-MSP productsclone sequencing of the partial-MSP products

(*: p<0.05)

(n=32) (n=19)

*

*

G/A

(Trend test, p=0.021)

Methylation of Methylation of p16p16 near its TSS region near its TSS region correlated with pathological changes of correlated with pathological changes of gastric mucosa by the new MSP assaygastric mucosa by the new MSP assay

Nucleosome positioning of Nucleosome positioning of p16p16 CpG island by CpG island by a serial of PCR assays a serial of PCR assays

Preparation of mono-nucleosomal DNA fragment by MNase digestion

(Bai H, et al., 2008, Chin J Cancer Res)

(Fatemi et al, 2005 NAR)

Methylation status of individual CpG sites within Methylation status of individual CpG sites within p16p16 CpG island in human gastric mucosa CpG island in human gastric mucosa ((nn=51)=51)

Carcinoma(n=31)

Gastritis(n=14)

Normal(n=6)

(Wong et al., 1999 MCB)

(Human mammary epithelial cell strains, with HPV16 E6/E7 transfection)

• Will the unmethylated Will the unmethylated p16p16 be methylated under be methylated under its repressor pressure?its repressor pressure?

Subclone C9 Subclone E3 MGC803 AGS

DAPI

P16

ExpressionExpression status of status of p16 p16 and and bikuninbikunin in AGS, MGC in AGS, MGC803, and the fused subclones803, and the fused subclones

The modal chromosome number (The modal chromosome number (MedianMedian) of AGS, ) of AGS, MGC803, and a fused subclone cellsMGC803, and a fused subclone cells

0

0. 5

1

1. 5

2

2. 5

3

3. 5

38 42 45 46 47 48 49 50 77 96

Chromosome Chromosome No. No. ((nn=20):=20):

0

0. 5

1

1. 5

2

2. 5

3

3. 5

4

4. 5

50 54 56 57 58 59 60 61 62 63 118 120

48 in AGS cells

58 in MGC803 cells

89 in fused cells0

0. 5

1

1. 5

2

2. 5

3

3. 5

40 80 83 88 89 92 93 94 95 96 97 98

Hemi-methylated CpG islands of Hemi-methylated CpG islands of p16p16 and and bikuninbikunin in th in the fused subclones by bisulfite-clone sequencing e fused subclones by bisulfite-clone sequencing

Methylation of CpG islands of p16 in the fused subMethylation of CpG islands of p16 in the fused subclones by bisulfite-clone sequencingclones by bisulfite-clone sequencing

ConclusionsConclusions

• CpG islands of p16 are partially methylated at the protein coding nucleosomes within its exon-1 in gastritis tissues.

• p16 methylation could be fully extended to its 5-UTR and promoter regions in gastric carcinoma tissues.

• Nucleosome is the basic methylation unit of CpG islands for p16 (and other genes such as MLH1 and MT3).

• p16 methylation may extend from the exon-1 to promoter.

Dr. Zheming Lu: partial-MSP and extensive clone sequencing

Dr. Qiang Li: fused cell subcloning

Dr. Zhaojun Liu: DNA methylation detection by DHPLC; Hp by PCR

Ms. Jing Zhou: 83bp-MSPand new MethyLight

Cooperation withDr. Jiafu JiDr. Kaifeng PanDr. Budong Zhu

Dr. Hua Bai: nucleosome positioning

Ms. Liankun Gu: pathology