Lee Y Y, Kang S H, Seo J Y, Jung C W, Lee K U, Choe K J, Kim B K, Kim N K, Koeffler H P, Bang Y J
Department of Internal Medicine, Han Yang University School of Medicine, Seoul, Korea.
Cancer. 1997 Nov 15;80(10):1889-96. doi: 10.1002/(sici)1097-0142(19971115)80:10<1889::aid-cncr3>3.0.co;2-j.
It has been suggested that cyclin-dependent kinase inhibitors (CDKIs), including p16 and p15, are tumor suppressor genes. Alterations of CDKIs have been found in most types of cancer. However, little is known about the status of p16 and p15 genes, including methylation of the promoter region, in gastric carcinoma.
Thirty-six primary gastric tumors and 9 gastric carcinoma cell lines were examined for alterations of the p16 and p15 genes. Deletion of the p16 and p15 genes was assessed by Southern blot analysis, expression by Northern blot analysis, and mutation by polymerase chain reaction-single strand conformation polymorphism followed by direct sequencing. The methylation status of the 5' CpG island of the p16 gene was evaluated using methylation-sensitive restriction enzymes, and reversal of the transcriptional block of the p16 gene was determined by Northern blot analysis after treatment with 5-aza-2'-deoxycytidine.
Homozygous deletions of the p16 and 15 genes from 2 of 9 gastric carcinoma cell lines were found. In contrast, no deletions were detected in 36 primary gastric tumors, and one primary tumor showed rearrangements of the p16 and p15 genes. Two gastric carcinoma cell lines showed a point mutation and an insertional mutation of the p16 gene, respectively; however, no point mutations were noted for the p16 and p15 genes in any of the primary gastric tumors. Constitutive levels of p16 mRNA expression in gastric carcinoma cell lines were quite heterogeneous; four gastric carcinoma cell lines had no detectable p16 mRNA and 6 gastric carcinoma cell lines had negligible expression of p15 mRNA. Of 10 primary gastric tumors, only 1 tumor expressed p16 mRNA. Furthermore, abnormal DNA methylation patterns of the p16 gene were found in 2 gastric carcinoma cell lines through the use of methylation-sensitive restriction enzymes. These cell lines lacked expression of p16 mRNA without deletions of the p16 gene. These transcriptional blocks were reversed by treatment with 5-aza-2'-deoxycytidine.
Deletions or mutations of the p16 and p15 genes are uncommon in primary gastric carcinomas. However, defective mRNA transcription, sometimes by aberrant DNA methylation, might be one of the pathways of inactivation of the p16 gene that leads to the development of gastric carcinoma.
有人提出细胞周期蛋白依赖性激酶抑制剂（CDKIs），包括p16和p15，是肿瘤抑制基因。在大多数类型的癌症中都发现了CDKIs的改变。然而，关于胃癌中p16和p15基因的状态，包括启动子区域的甲基化，人们了解甚少。
对36例原发性胃癌肿瘤和9个胃癌细胞系进行p16和p15基因改变的检测。通过Southern印迹分析评估p16和p15基因的缺失，通过Northern印迹分析评估表达情况，通过聚合酶链反应-单链构象多态性随后直接测序评估突变情况。使用甲基化敏感限制酶评估p16基因5' CpG岛的甲基化状态，并用5-氮杂-2'-脱氧胞苷处理后通过Northern印迹分析确定p16基因转录阻断的逆转情况。
在9个胃癌细胞系中有2个发现p16和15基因的纯合缺失。相比之下，在36例原发性胃癌肿瘤中未检测到缺失，且有1例原发性肿瘤显示p16和p15基因重排。2个胃癌细胞系分别显示p16基因的点突变和插入突变；然而，在任何原发性胃癌肿瘤中p16和p15基因均未发现点突变。胃癌细胞系中p16 mRNA表达的组成水平差异很大；4个胃癌细胞系未检测到p16 mRNA，6个胃癌细胞系p15 mRNA表达可忽略不计。在10例原发性胃癌肿瘤中，只有1例肿瘤表达p16 mRNA。此外，通过使用甲基化敏感限制酶在2个胃癌细胞系中发现了p16基因异常的DNA甲基化模式。这些细胞系缺乏p16 mRNA表达且p16基因无缺失。用5-氮杂-2'-脱氧胞苷处理可逆转这些转录阻断。
p16和p15基因的缺失或突变在原发性胃癌中并不常见。然而，有缺陷的mRNA转录，有时是由异常的DNA甲基化引起的，可能是导致胃癌发生的p16基因失活途径之一。
