Evaluation of potential oncogene alterations in the ENU1564 rat mammary tumor model.

The ENU1564 tumor line originated from a rat mammary tumor induced by N-ethyl-N-nitrosourea (ENU), an alkylating chemical carcinogen which induces genetic point mutations. The oncogene abnormalities in rat mammary tumors induced by ENU have not been characterized. In this study, two highly metastatic clones (Br7-C5 and FP2-All) derived from the adenocarcinoma cell line ENU1564, were evaluated for the presence of mutational activation involving the c-Ha-ras, c-neu, and p53 oncogenes. These oncogenes were chosen for investigation based upon their involvement in other ENU-induced rat tumors (c-neu in malignant schwannomas and p53 in nephro-blastomas) or in methylnitrosourea (MNU)-induced rat mammary tumors (c-Ha-ras). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequence-specific oligonucleotide hybridization analyses indicated that no c-Ha-ras codon 12 mutation was present in these tumor cells. PCR-RFLP and single-stranded conformational polymorphism (PCR-SSCP) analyses showed that no sequence changes were present over a 138 base-pair gene fragment spanning codon 664 of the c-neu protooncogene (the site of point mutation in ENU-induced rat nerve tumors). Immunoprecipitation and Western immunoblotting indicated that the p53 protein is neither over-expressed nor mutated in the tumor cells. The results failed to identify specific oncogene alterations in the ENU1564 rat mammary tumor line but ruled out mutational activation of c-Ha-ras (codon 12), c-neu (codon 664), and the p53 genes.