Elisei R, Shiohara M, Koeffler H P, Fagin J A
Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Ohio 45267, USA.
Cancer. 1998 Nov 15;83(10):2185-93.
D-type cyclins, in association with the cyclin-dependent kinases CDK4 and CDK6, promote progression through the G1 phase of the cell cycle. CDK activity is modulated by inhibitors such as p15INK4b and p16INK4a. Loss of function of p15INK4b and p16INK4a (multiple tumor suppressor-I and CDK4 inhibitor) determines impairment in the control of the cell cycle and contributes to the transformation of several cell types.
The authors examined 20 thyroid neoplasms (12 papillary carcinomas and 8 follicular adenomas) and 4 human thyroid carcinoma cell lines for gene mutations and epigenetic modifications of the p15INK4b and p16INK4a genes by Southern blot analysis, single strand conformation polymorphism, and a polymerase chain reaction-based methylation assay.
Abnormalities of p16 were found in the four cell lines studied. In follicular carcinoma (WRO) cells, both the p15 and p16 genes were homozygously deleted. Undifferentiated carcinoma (FRO) cells had a nonsense point mutation at codon 72 (CGA-TGA, Arg-Stop) of p16, whereas the poorly differentiated papillary carcinoma (NPA) line harbored a point mutation at the exon 1-intron 1 boundary that altered the donor splicing site and caused an aberrantly spliced form of p16INK4a. Furthermore, p16 allelic loss was evident in the DNA of both FRO and NPA cells. Finally, p16 expression was absent in the ARO cell line, likely due to a de novo methylation of exon 1 of p16INK4a. Regarding the primary thyroid tumors, a missense point mutation at codon 91 was found in 1 of 12 papillary thyroid carcinomas (GCC-GTC, Ala-Val). No mutations were found in follicular adenomas. However, in 6 of 20 primary tumors there was hypermethylation at exon 1 of p16.
The high prevalence of p15 and p16 mutations in the cell lines described suggests involvement of these genes in immortalization in vitro. The p16 defects may have preexisted in a small subclone of the primary tumor that were selected for in vitro. Alternatively, p16 mutations may have arisen de novo during cell culture. Mutations of p15INK4b and p16INK4a do not appear to be critical events in the development of follicular adenomas or papillary carcinomas. However, de novo methylation of the 5' CpG island of p16 is common in primary tumors, indicating that the function of this gene may be lost as an epigenetic event during disease progression.
D型细胞周期蛋白与细胞周期蛋白依赖性激酶CDK4和CDK6结合，促进细胞周期G1期的进程。CDK活性受p15INK4b和p16INK4a等抑制剂调节。p15INK4b和p16INK4a（多肿瘤抑制因子-1和CDK4抑制剂）功能丧失决定了细胞周期控制受损，并促成多种细胞类型的转化。
作者通过Southern印迹分析、单链构象多态性和基于聚合酶链反应的甲基化检测，研究了20个甲状腺肿瘤（12个乳头状癌和8个滤泡性腺瘤）以及4个人类甲状腺癌细胞系中p15INK4b和p16INK4a基因的基因突变和表观遗传修饰。
在所研究的4个细胞系中发现了p16异常。在滤泡癌（WRO）细胞中，p15和p16基因均纯合缺失。未分化癌（FRO）细胞在p16的第72密码子（CGA-TGA，精氨酸-终止密码子）处存在无义点突变，而低分化乳头状癌（NPA）细胞系在外显子1-内含子1边界处存在点突变，改变了供体剪接位点，导致p16INK4a异常剪接形式。此外，FRO和NPA细胞的DNA中均明显存在p16等位基因缺失。最后，ARO细胞系中未检测到p16表达，可能是由于p16INK4a外显子1的从头甲基化。关于原发性甲状腺肿瘤，在12个甲状腺乳头状癌中的1个中发现第91密码子处存在错义点突变（GCC-GTC，丙氨酸-缬氨酸）。滤泡性腺瘤中未发现突变。然而，在20个原发性肿瘤中的6个中，p16外显子1存在高甲基化。
所述细胞系中p15和p16突变的高发生率表明这些基因参与了体外永生化过程。p16缺陷可能在原发性肿瘤的一个小亚克隆中预先存在，并在体外被选择出来。或者，p16突变可能在细胞培养过程中从头产生。p15INK4b和p16INK4a突变似乎不是滤泡性腺瘤或乳头状癌发生中的关键事件。然而，p16的5'CpG岛从头甲基化在原发性肿瘤中很常见，表明该基因的功能可能在疾病进展过程中作为一种表观遗传事件而丧失。
