Methylation and expression of p16INK4 tumor suppressor gene in primary colorectal cancer tissues.
It is known that p16(INK4) tumor suppressor gene expression in colon cancer cells is repressed by methylation at the CpG island of promoter, but in vivo silencing of p16 gene is not fully understood. Some studies showed that primary colorectal cancer (CRC) tissues often overexpress the p16 protein, while others showed the high incidence of p16 methylation. The aim of this study was to clarify p16 gene regulation in vivo. We used real-time methylation-specific PCR (MSP) to examine density of p16 methylation, and immunohistochemistry, Western blot analysis to determine p16 protein expression. Methylation was detected in 5 CRC cell lines tested and 9 of 21 (42.9%) CRCs. Four of 5 CRC cell lines did not express p16 mRNA, but 6 of 9 CRCs did express p16 mRNA even with methylation. Real-time MSP showed that CRC tissues had a wide variety in methylation density (methylation index: 0.28-0.91) and that highly methylated CRC tissues displayed significantly lower p16 mRNA expression than those with no-methylation or low-methylation. Immunohistochemistry showed that the majority of CRCs (53 of 55: 96.4%) overexpressed the p16 protein. Low p16 expression was associated with lymph node metastasis (p=0.003) and large tumor size (p=0.048). Western blot in a subset of non-tumor and tumor samples showed a consistent overexpression of the p16 protein. These results showed that CRC tissues displayed variable methylation density, which may be characteristics of p16 gene methylation in vivo. Our data suggest that a low p16 expression due to methylation may contribute to tumor enlargement and expansion of CRC.
Kim
Byung No
BN
Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan.
Yamamoto
Hirofumi
H
Ikeda
Kimimasa
K
Damdinsuren
Bazarragchaa
B
Sugita
Yurika
Y
Ngan
Chew Yee
CY
Fujie
Yujiro
Y
Ogawa
Minoru
M
Hata
Taishi
T
Ikeda
Masataka
M
Ohue
Masayuki
M
Sekimoto
Mitsugu
M
Monden
Takushi
T
Matsuura
Nariaki
N
Monden
Morito
M
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000473:pathology; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis* / Q000378:metabolism*; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D007150:Immunohistochemistry; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D019941:Cyclin-Dependent Kinase Inhibitor p16