Ficorella Corrado, Cannita Katia, Ricevuto Enrico, Toniato Elena, Fusco Carlo, Sinopoli Nuntia Teresa, De Galitiis Federica, Di Rocco Zorika Christiana, Porzio G, Frati Luigi, Gulino Alberto, Martinotti Stefano, Marchetti Paolo
Department of Experimental Medicine, University of L'Aquila, Italy.
Oncol Rep. 2003 Jan-Feb;10(1):169-73.
The objective of this study was to investigate the contribution of p16 inactivation in gastric cancer and to compare it with p53. A cohort of 34 primary GCs were analyzed for p16 mutations and transcriptional silencing of the gene due to hypermethylation of the promoter. SSCP analysis and direct sequencing of exons 1 and 2 of the p16 gene were performed to detect any structural alterations. The methylation specific PCR (MSP) assay was applied to reveal hypermethylation of the 'CpG' island in the regulatory region using specific primer pairs for methylated and unmethylated nucleotides after a chemical reaction converting cytosines into uracile when unmethylated. SSCP and direct sequencing analysis did not detect any p16 mutations. The MSP assay showed 4 MSP(+) variants (11.8%). Three MSP(+) were stage III-IV disease and 1 MSP(+) was detected in an early stage disease (IB). All MSP(+) were diffuse type adenocarcinomas. The MSP(+) samples were different from previously reported samples harboring p53 mutations in the same cohort. These data increase the number of gastric cancers showing alterations of either p53 or p16 to 29.4% (10/34). Functional inactivation by hypermethylation of the p16 locus and p53 mutations could play a significant, complementary role in the pathogenesis of sporadic gastric cancer.
本研究的目的是调查p16失活在胃癌中的作用，并将其与p53进行比较。对34例原发性胃癌患者进行队列分析，检测p16基因的突变情况以及由于启动子高甲基化导致的基因转录沉默。采用单链构象多态性（SSCP）分析和对p16基因外显子1和2进行直接测序，以检测任何结构改变。应用甲基化特异性PCR（MSP）检测法，在将未甲基化的胞嘧啶通过化学反应转化为尿嘧啶后，使用针对甲基化和未甲基化核苷酸的特异性引物对，揭示调控区域中“CpG”岛的高甲基化情况。SSCP和直接测序分析未检测到任何p16突变。MSP检测法显示有4个MSP（+）变异体（11.8%）。3个MSP（+）为III-IV期疾病，1个MSP（+）在早期疾病（IB期）中检测到。所有MSP（+）均为弥漫型腺癌。MSP（+）样本与同一队列中先前报道的携带p53突变的样本不同。这些数据表明，显示p53或p16改变的胃癌数量增加到29.4%（10/34）。p16基因座的高甲基化导致的功能失活和p53突变可能在散发性胃癌的发病机制中发挥重要的互补作用。
