Aberrant methylation patterns of the Rassf1a gene in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine.
To clarify the involvement of the Rassf1a gene in lung carcinogenesis, we investigated the methylation status in the 5' upstream region of the RAS-association domain family 1, isoform A (Rassf1a) gene in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). Six-week-old male Wistar rats were given 2000 ppm BHP in their drinking water for 12 wk and maintained without further treatment until they were sacrificed at 25 wk. A total of 15 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of expression of the Rassf1a gene by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. To measure the methylation status of the Rassf1a gene, five adenocarcinomas with a marked reduction of the Rassf1a expression and two normal lung tissues were used for a bisulfite sequencing analysis. While the normal lung tissue was unmethylated, all five adenocarcinomas were highly methylated in the 5' upstream region. Genomic DNAs were also extracted from 15 adenocarcinomas, and mutation analysis of the Rassf1a was performed with PCR-single-strand conformation polymorphism (SSCP) analysis. No mutations were detected throughout exons 1-6. The present results suggest that the aberrant methylation may be involved in the inactivation of the Rassf1a gene in the development of lung adenocarcinomas induced by BHP in rats.
Shimizu
Kyoko
K
Laboratory of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, Kowakae, Higashiosaka, Osaka, Japan.
Kato
Ayumi
A
Shigemura
Mayumi
M
Fujii
Hiromasa
H
Honoki
Kanya
K
Tsujiuchi
Toshifumi
T
D000230:Adenocarcinoma / Q000139:chemically induced / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D019175:DNA Methylation; D008168:Lung / Q000502:physiology; D008175:Lung Neoplasms / Q000139:chemically induced / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009602:Nitrosamines / Q000633:toxicity; D051381:Rats; D012016:Reference Values; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D009602:Nitrosamines; C496882:RASSF1 protein, mouse; D025521:Tumor Suppressor Proteins; C012457:diisopropanolnitrosamine