Methylation of individual CpG sites within p16 CpG islands in various human gastric lesions Zheming Lu( 陆哲明） Dajun Deng （邓大君） Beijing University School of Oncology Beijing Institute for Cancer Research,Beijing Cancer Hospital

R Beroukhim et al. Nature 463, (2010) Identification of significant focal SCNAs across 3131 human cancer specimens

p16 is inactivated by genetic alterations (10%) and DNA methylation (30%) in many human tumors (Esteller, Oncogene 2002)

Methylation status of individual CpG sites within p16 CpG islands in vivo is not well characterized. p16 is fully methylated in many cancer cell lines.Methylation status of individual CpG sites within p16 CpG islands in vivo is not well characterized. p16 is fully methylated in many cancer cell lines. The mechanisms lead to fate diversities of p16 methylation in different gastric lesions are unknown. p16 methylation is stable in cancer cells, but unstable in gastritis lesions.The mechanisms lead to fate diversities of p16 methylation in different gastric lesions are unknown. p16 methylation is stable in cancer cells, but unstable in 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 p16 CpG island by partial-MSP (-355nt~+233nt, including 45+3 CpG sites) partial-MSP primer set to enrich p16 CpG islands with methylated cytosines 126bp-MSP 151bp-MSP 234bp-MSP83bp-MSP 392bp-DHPLC

More p16 partial-MSP products was detected in gastric carcinomas than in normal/gastritis 36/40 (90.0)Gastric carcinoma < /45 (42.2) 7/13 (53.8) Partial-MSP positive rate (%) Chronic gastritis Normal Pathological changes P-value (two-sides) M F0310 F0342 F0350 F0414 F0438 F0144 F0566 F bp

(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 F bp More p16 partial-MSP products were detected in the Hp-positive samples than -negative ones 144negative 1530positive Hp negativepositive Partial-MSP (p<0.001)

Methylation status of individual CpG sites within p16 CpG island in human gastric mucosa (n=51) Ca (n=31) Gastritis (n=14) Normal (n=6)

Methylation status of individual CpG sites within p16 CpG island in human gastric mucosa * * G/A ( p<0.05)

* * * * * * * * * H. pylori infection and p16 methylation Normal or gastritis tissues: n Hp(+) =13, n Hp(-) = 7 Gastric carcinomas: n Hp(+) =17, n Hp(-) = 9 (*: p<0.05) (*: p=0.06) G/A

Distribution of p16 clones with different no. of methylated CpG sites in gastric mucosa

p16 clones with  6 methylated CpG sites in gastric mucosa Carcinoma [77/460; 16.7%] Gastritis [18/217; 8.3%] Normal [5/67; 7.5%]

88bp 83bp M U M U M U M U M U M.Ctrl U.Ctrl TE buffer Sample-1 Sample-2 83bp-MSP (+)(-) partial- MSP (+)3322 (-)15 83bp-MSP (+)(-) Hp (+)2815 (-)525 Results of the 83bp-MSP assay confirm those of the partial-MSP assay (p=0.044)(p<0.0001)

Results of the 83bp-MSP assay confirm those of clone sequencing of the partial-MSP products (*: p<0.05) (n=32) (n=19) * * G/A

(Trend test, p=0.021) Methylation of p16 near its TSS region correlated with pathological changes of gastric mucosa by the new MSP assay

Nucleosome positioning of p16 CpG island by 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 p16 CpG island in human gastric mucosa (n=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 p16 be methylated under its repressor pressure?Will the unmethylated p16 be methylated under its repressor pressure?

Subclone C9 Subclone E3 MGC803 AGS DAPI P16 Expression status of p16 and bikunin in AGS, MGC803, and the fused subclones

The modal chromosome number (Median) of AGS, MGC803, and a fused subclone cells Chromosome No. (n=20): 48 in AGS cells 58 in MGC803 cells 89 in fused cells

Hemi-methylated CpG islands of p16 and bikunin in the fused subclones by bisulfite-clone sequencing

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

Conclusions 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-MSP and new MethyLight Cooperation with Dr. Jiafu Ji Dr. Kaifeng Pan Dr. Budong Zhu Dr. Hua Bai: nucleosome positioning Ms. Liankun Gu: pathology