N-[4-(5-nitro-2-furyl)-2-thiazolyl]
formamide (
FANFT) administration to rats followed by
sodium saccharin results in
transitional cell carcinomas of the bladder, of which 24% harbor an activated H-ras gene. Since
2-amino-4-(5-nitro-2-furyl)thiazole (
ANFT) is the mutagenic and carcinogenic metabolite of
FANFT in vivo, we wished to examine ras activation in in vitro
ANFT-transformed rat bladder epithelial cells as well as four cell lines established in culture from in vivo
FANFT-induced rat
bladder tumors. Screening by Western blotting revealed no enhanced levels of p21ras in
ANFT-transformed cells nor in cells established in culture from
FANFT-induced rat bladder
carcinomas. Further investigations using immunohistochemical staining with a different pan-reactive p21
monoclonal antibody (Cetus Corporation) specific for this method, however, showed two groups of cells from
FANFT-induced rat
bladder tumors had enhanced immunoreactivity. Apart from this, p21ras expression of most of the cells groups varied little from the controls. We examined the reported hot spots (exons 1 and 2) of each of the ras genes (H-, K- and N-ras) by direct sequencing of amplified
DNA. No mutations were present. We conclude, therefore, that
ANFT transformation of primary rat bladder epithelial cells in vitro may not in this case be mediated by ras activation, although this is difficult to determine since others have observed that optimal culture conditions can select for certain populations of cells without ras activation.