Open AccessCloning of the 5’Upstream Region of the Rat p16 Gene and Its Role in Silencing
Author(s) -
Abe Masanobu,
Okochi Eriko,
Kuramoto Takashi,
Kaneda Atsushi,
Takato Tsuyoshi,
Sugimura Takashi,
Ushijima Toshikazu
Publication year - 2002
Publication title -
japanese journal of cancer research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.035
H-Index - 141
eISSN - 1349-7006
pISSN - 0910-5050
DOI - 10.1111/j.1349-7006.2002.tb01211.x
Subject(s) - gene silencing , methylation , dna methylation , biology , exon , cdkn2a , bisulfite sequencing , cancer research , microbiology and biotechnology , gene , cpg site , bisulfite , carcinogenesis , gene expression , genetics
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 la is adequate for analysis of p16 silencing. In this study, we observed that methylation analysis of exon la gave false positive results in three samples of normal rat mammary epitheliums 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 1α and analysis of the methylation status is useful in examining p16 silencing in various rat tumors.