Activating missense mutations in Ha-ras-1 genes in a malignant subset of bladder lesions induced by N-butyl-N-(4-hydroxybutyl)nitrosamine or N-[4-(5-nitro-2-furanyl)-2-thiazolyl]formamide.

Urothelial cell cultures generated from urinary bladders from a series of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)- or N-[4-(5-nitro-2-furanyl)-2-thiazolyl]formamide (FANFT)-treated Fischer 344 rats were examined for activating missense mutations in Ha-ras-1 genes. Our overall objective was to identify oncogene-activating mutations in this system and to determine what altered biological properties correlate with such genetic changes. The urinary bladders from the treated animals showed a spectrum of histopathologies, from simple hyperplasia to transitional cell carcinoma (TCC). Using restriction analysis, oligonucleotide hybridization, and DNA sequencing, we found that approximately 20% (3/14) of the bladder cell cultures had acquired oncogenic single-base substitutions in codon 61 of Ha-ras-1 genes (CAA----AAA or CGA). The donor bladder lesions for these three cultures, which also harbored the same ras-activating mutations, were all classified as stage A or B TCCs. However, four other TCCs also arising in this series were found to have normal Ha-ras genes. Whereas approximately half of the bladder cultures derived from the carcinogen-treated rats were nontumorigenic in athymic mice, the three cultures containing ras oncogenes were all highly tumorigenic (forming tumors within 5 wk of injection into athymic mice). These cultures also displayed a high degree of anchorage-independent growth and NIH 3T3-transforming activity in gene transfer assays. The nontumorigenic cultures were derived from bladder lesions that included three hyperplasias and three stage A TCCs. We conclude that ras-activating missense mutations were present in a malignant subset of bladder lesions induced by BBN or FANFT, but most of the lesions in this system appeared to involve genetic alterations elsewhere. Thus other oncogenes besides activated Ha-ras may apparently be associated with the same bladder histopathologies and transformation markers.