We previously demonstrated that H-ras1 oncogene mutations detected in N-nitroso-N-methylurea (NMU)-induced mammary tumors arose as background mutations within rat mammary cells (RMCs) and that NMU promoted the outgrowth of these preexisting mutants. We have now detected a putative DNA structure in the H-ras1 promoter of RMCs in vivo that was absent in NMU-induced mammary tumor cells. Analysis of the promoter in RMCs as a function of time after exposure to carcinogens indicated that NMU, but not 7,12-dimethylbenz(a)anthracene, initiated the loss of this structure with a half-life of 7 days. Although loss of the structure was irreversible in cells that gave rise to tumors, it was restored in normal RMCs by 120 days after exposure and was present in normal RMCs of animals bearing tumors, even 1 year after NMU exposure. The structure was also abrogated in RMCs during pregnancy and restored after lactation was terminated, suggesting that reversible regulation of the structure by hormones contributed to normal RMC growth. Thus, NMU may promote abnormal RMC growth by mimicking the effects of hormones on DNA conformation. We hypothesize that the NMU-induced alterations in promoter conformation irreversibly deregulates H-ras1 expression in initiated cells, thereby increasing the phenotypic penetrance of the conditional H-ras1 mutations.
This study was supported by NIH Program Project Grant ES 05622 (William G. Thilly) awarded to the MIT Center for Environmental Health Sciences and Cancer Center Core Grant CA 15704 (Robert Day) awarded to the Fred Hutchinson Cancer Research Center. B. H. was the recipient of a postdoctoral fellowship from the Fonds de la Recherche en Santé du Québec, Quebec, Canada.