FHIT alterations in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats

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Abstract
Alteration of the FHIT gene was investigated in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl) amine (BHP) in male Wistar rats. Animals at 6 weeks of age were given 2000 p.p.m. of BHP in drinking water for 12 weeks, then maintained without further treatment until killed at the end of week 25. A total of 25 lung adenocarcinomas were obtained and total RNAs were extracted from each for assessment of aberrant transcription of the FHIT gene by reverse transcription (RT)–polymerase chain reaction (PCR) analysis. Aberrant transcripts were detected in 15 adenocarcinomas (60%) as absence in the regions of nucleotides (nt) −9 to 279, −98 to 279, −98 to 348 or −98 to 447. Genomic DNAs were also extracted from all 25 adenocarcinomas and exons 5–9 were examined for mutations, using PCR–single strand conformation polymorphism (SSCP) analysis and sequencing. A mutation was detected in only one adenocarcinoma (4%), an ACC to ATC (Thr to IIe) transition at codon 76. Southern blot analysis of eight tumors did not show any evidence of gross rearrangement or deletion of the FHIT gene. Western blot analysis revealed reduced expression of Fhit protein in six out of 10 adenocarcinomas (60%). These results suggest that alteration of the FHIT gene may be involved in the development of lung adenocarcinomas induced by BHP in rats.