Abe Masanobu, Okochi Eriko, Kuramoto Takashi, Kaneda Atsushi, Takato Tsuyoshi, Sugimura Takashi, Ushijima Toshikazu
Carcinogenesis Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Jpn J Cancer Res. 2002 Oct;93(10):1100-6. doi: 10.1111/j.1349-7006.2002.tb01211.x.
Hypermethylation of the 5' upstream region (5' region) of the human p16(CDKN2A) (p16) gene is known to cause silencing, which is involved in a wide range of human cancers. For the rat p16 gene, its 5' region has not been cloned, and it is uncertain whether surrogate use of exon 1 alpha is adequate for analysis of p16 silencing. In this study, we observed that methylation analysis of exon 1 alpha gave false positive results in three samples of normal rat mammary epithelia and in two of six primary mammary carcinomas. Therefore, we determined the nucleotide sequence of the 5' region of the rat p16 gene. To confirm that methylation status of the 5' region is correlated with p16 expression, the methylation status was analyzed by bisulfite sequencing and methylation-specific PCR in three samples of normal mammary glands, six samples of mammary carcinomas and four cell lines. The 5' region was demethylated in all of the three normal and six carcinoma samples that fully expressed p16. On the other hand, the 5' region was highly methylated in the 3Y1 cell line, which lacked p16 expression, but without deletion. These results showed that the methylation status of the 5' region was more closely correlated with p16 expression than that of the exon 1 alpha and analysis of the methylation status is useful in examining p16 silencing in various rat tumors.
已知人类p16（CDKN2A）（p16）基因5'上游区域（5'区域）的高甲基化会导致基因沉默，这与多种人类癌症有关。对于大鼠p16基因，其5'区域尚未克隆，并且外显子1α的替代使用是否足以分析p16沉默尚不确定。在本研究中，我们观察到外显子1α的甲基化分析在三个正常大鼠乳腺上皮样品和六个原发性乳腺癌中的两个中给出了假阳性结果。因此，我们确定了大鼠p16基因5'区域的核苷酸序列。为了证实5'区域的甲基化状态与p16表达相关，通过亚硫酸氢盐测序和甲基化特异性PCR分析了三个正常乳腺样品、六个乳腺癌样品和四个细胞系中的甲基化状态。在所有完全表达p16的三个正常样品和六个癌样品中，5'区域均发生了去甲基化。另一方面，在缺乏p16表达但无缺失的3Y1细胞系中，5'区域高度甲基化。这些结果表明，5'区域的甲基化状态与p16表达的相关性比外显子1α更密切，并且甲基化状态分析有助于检查各种大鼠肿瘤中的p16沉默。
