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

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Abstract
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.