Hypermethylation of the Dal‐1 gene in lung adenocarcinomas induced by N‐nitrosobis(2‐hydroxypropyl)amine in rats

Abstract

DAL-1 (differentially expressed in adenocarcinoma of the lung) is an actin-binding protein that has been shown to suppress growth in lung cancer cells. Recently, inactivation of the gene encoding DAL-1 due to hypermethylation has been found in several human malignancies, including lung cancers. To assess the involvement of the Dal-1 gene in rat lung carcinogenesis, we investigated the expression of Dal1 and its methylation status in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week old male Wistar rats (n = 11) were given 2,000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. Total RNA was extracted from 11 lung adenocarcinomas, one from each BHP treated rat, and Dal-1 gene expression was analyzed using real-time quantitative reverse transcription-polymerase chain reaction. Dal-1 expression was significantly reduced in the lung adenocarcinomas compared with three normal lung tissues (P < 0.05). For methylation analysis, bisulfite sequencing was performed using normal lung tissue and tissue from 4 tumors, all of which showed reduced expression of Dal-1. The 5' upstream region was highly methylated in all four adenocarcinomas, whereas this region was unmethylated in normal lung tissue. These results suggest that aberrant methylation of the Dal-1 gene might be involved in the development of lung adenocarcinomas induced in rats by BHP.