Xing E P, Nie Y, Wang L D, Yang G Y, Yang C S
Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, NJ 08854-8020, USA.
Carcinogenesis. 1999 Jan;20(1):77-84. doi: 10.1093/carcin/20.1.77.
p16INK4a and p15INK4b genes, which encode two functionally related CDK inhibitors, recently emerged as candidate tumor suppressor genes since they were both localized to 9p21, which frequently undergoes hemizygous and homozygous deletion in a variety of tumor types. To determine the mode of inactivation of these two genes in human esophageal squamous cell carcinoma (ESCC), we performed multiple molecular analyses in 60 ESCC specimens from Linxian, China using DNA methylation assay, LOH analysis, deletion screening and SSCP-sequencing. We observed that p16INK4a inactivation was predominantly associated with aberrant methylation in the CpG island of its promoter region, whereas p15INK4b frequently had homozygous deletions. Compared with aberrant methylation, which occurred in 17 of 34 cases, homozygous deletion of p16INK4a and LOH at its nearby D9S942 microsatellite marker were observed at a much lower frequency (17%). Intragenic mutation in p16INK4a gene was rare. In contrast, homozygous deletion in p15INK4b and LOH at the nearby D9S171 marker were observed at frequencies of 35 and 47%, respectively, and the two events were significantly associated with each other. On the other hand, aberrant methylation of p15INK4b was relatively infrequent (6/34) and occurred concomitantly with p16INK4a methylation. Among the 60 cases, only four contained a continuous homozygous deletion spanning both p15INK4b and p16INK4a. Six cases were exclusively deleted at p16INK4a and 17 exclusively deleted at p15INK4b. LOH at D9S942 and D9S171 was also found to be mutually exclusive. Our results suggest that the alteration mode at 9p21 was not uniform, and the two genes were inactivated by distinct mechanisms. Altogether, 68% of the samples harbor at least one type of alteration in p16INK4a gene and 50% of the samples were altered in p15INK4b gene, indicating that they are the frequent inactivating targets during ESCC development.
p16INK4a和p15INK4b基因编码两种功能相关的细胞周期蛋白依赖性激酶（CDK）抑制剂，由于它们都定位于9p21，而9p21在多种肿瘤类型中经常发生半合子和纯合子缺失，因此最近它们作为候选肿瘤抑制基因出现。为了确定这两个基因在人食管鳞状细胞癌（ESCC）中的失活模式，我们使用DNA甲基化检测、杂合性缺失（LOH）分析、缺失筛选和单链构象多态性-测序（SSCP-测序），对来自中国林县的60例ESCC标本进行了多项分子分析。我们观察到，p16INK4a失活主要与其启动子区域CpG岛的异常甲基化有关，而p15INK4b经常发生纯合子缺失。与34例中有17例发生的异常甲基化相比，p16INK4a的纯合子缺失及其附近D9S942微卫星标记处的LOH发生率要低得多（17%）。p16INK4a基因的基因内突变很少见。相反，p15INK4b的纯合子缺失和其附近D9S171标记处的LOH发生率分别为35%和47%，且这两个事件显著相关。另一方面，p15INK4b的异常甲基化相对少见（6/34），且与p16INK4a甲基化同时发生。在这60例病例中，只有4例包含跨越p15INK4b和p16INK4a的连续纯合子缺失。6例仅在p16INK4a处缺失，17例仅在p15INK4b处缺失。在D9S942和D9S171处的LOH也被发现是相互排斥的。我们的结果表明，9p21处的改变模式并不一致，这两个基因通过不同的机制失活。总之，68%的样本在p16INK4a基因中至少有一种类型的改变，50%的样本在p15INK4b基因中发生改变，这表明它们是ESCC发生过程中常见的失活靶点。
