Liew C T, Li H M, Lo K W, Leow C K, Chan J Y, Hin L Y, Lau W Y, Lai P B, Lim B K, Huang J, Leung W T, Wu S, Lee J C
Department of Anatomical and Cellular Pathology, Faculty of Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin.
Oncogene. 1999 Jan 21;18(3):789-95. doi: 10.1038/sj.onc.1202359.
The tumor suppressor gene p16 (CDKN2/MTS-1/INK4A) is an important component of the cell cycle and inactivation of the gene has been found in a variety of human cancers. In order to investigate the role of p16 gene in the tumorigenesis of hepatocellular carcinoma (HCC), 48 cases of HCC were analysed for p16 alterations by: methylation-specific PCR (MSP) to determine the methylation status of the p16 promoter region; comparative multiplex PCR to detect homozygous deletion; PCR-SSCP and DNA sequencing analysis to identify mutation of the p16 gene. We found high frequency of hypermethylation of the 5' CpG island of the p16 gene in 30 of 48 cases (62.5%) of HCC tumors. Moreover, homozygous deletion at p16 region were present in five of 48 cases (10.4%); and missense mutation were detected in three of 48 cases (6.3%). The overall frequency of p16 alterations, including homozygous deletion, mutation and hypermethylation, in HCC tumors was 70.8% (34 of 48 cases). These findings suggest that: (a) the inactivation of the p16 is a frequent event in HCC; (b) the p16 gene is inactivated by multiple mechanisms including homozygous deletion, promoter hypermethylation and point mutation; (c) the most common somatic alteration of the p16 gene in HCC is de novo hypermethylation of the 5' CpG island; and (d) in contrast to other studies, high frequency of genomic alterations are not uncommon in the 9p21 of the p16 gene. Our results strongly suggest that the p16 gene plays an important role in the pathogenesis of HCC.
肿瘤抑制基因p16（CDKN2/MTS-1/INK4A）是细胞周期的重要组成部分，该基因的失活已在多种人类癌症中被发现。为了研究p16基因在肝细胞癌（HCC）发生中的作用，通过以下方法分析了48例HCC患者的p16改变情况：采用甲基化特异性PCR（MSP）来确定p16启动子区域的甲基化状态；采用比较多重PCR检测纯合缺失；采用PCR-SSCP和DNA测序分析来鉴定p16基因的突变。我们发现，在48例HCC肿瘤患者中有30例（62.5%）的p16基因5' CpG岛出现高频率的甲基化。此外，48例中有5例（10.4%）在p16区域存在纯合缺失；48例中有3例（6.3%）检测到错义突变。HCC肿瘤中p16改变（包括纯合缺失、突变和甲基化）的总体频率为70.8%（48例中的34例）。这些发现表明：（a）p16失活在HCC中是常见事件；（b）p16基因通过多种机制失活，包括纯合缺失、启动子高甲基化和点突变；（c）HCC中p16基因最常见的体细胞改变是5' CpG岛的从头高甲基化；（d）与其他研究不同，p16基因9p21区域的高频率基因组改变并不罕见。我们的结果强烈表明p16基因在HCC的发病机制中起重要作用。
