2-(N-acetoxy-N-acetylamino)fluorene mutagenesis in mammalian cells: sequence-specific hot spot.

Abstract

Mutations induced by 2-(N-acetoxy-N-acetylamino)fluorene were studied using temperature-sensitive simian virus 40 (SV40) mutants as probe in monkey kidney cells. In vitro treatment of the SV40 virions with 2-(N-acetoxy-Nacetylamino)fluorene increased mutagenesis and decreased survival in the viral progeny. A lethal hit of approximately 85 acetylaminofluorene adducts per SV40 genome was calculated.UV irradiation of cells prior to infection did not modify the results. Molecular analysis of independent SV40 revertants showed that 2-(N-acetoxy-N-acetylamino)fluorene induces base substitutions that are located not opposite putative acetylaminofluorene adducts but next to them. Moreover, a hot spot of mutation restoring a true wild-type genotype was observed in 10 of the 16 revertants analyzed. This hot spot, not targeted opposite a major DNA lesion, was not observed using UV light as damaging agent in the same genetic assay, Two models involving the stabilization, by acetylaminofluorene adducts, of the secondary structure of a specific quasipalindromic SV40 sequence are proposed to explain this sequence-specific hot spot.The recent discovery that some human cancers are associated with single point mutation in the ras gene family (1-3) enhanced the interest in the molecular mechanisms by which mutations arise. The most convenient method to approach mutagenesis in mammalian cells is to use exogenous probes (4-8). These probes must be well-defined and entirely dependent on the host cell for replication and for repair. Simian virus 40 (SV40) is the virus that best fits these requirements. Indeed, the SV40 system allowed Bourre and Sarasin (8) to show that UV light gives rise to base substitution opposite putative UV-induced lesions.2-(N-Acetoxy-N-acetylamino)fluorene (AAAF) has been widely used to induce tumors in animals. This carcinogen binds both to proteins and to guanine in nucleic acids. Among the DNA lesions (dG-AAF), the major one, 2-[N-(deoxyguanosin-8-yl)-N-acetylamino]fluorene, gives rise to local modifications in the DNA structure (9). Such adducts are mutagenic in bacterial cells, leading essentially to targeted frameshift mutations (10, 11). Since very little is known about AAAF mutagenesis in mammalian cells, we have determined the mutational specificity of this carcinogen on the SV40 large tumor (T) antigen gene and compared it with the UV-induced mutations determined in the same genetic system-composed of a reversion assay from a temperaturesensitive tsA58 SV40 mutant to wild-type growth at the restrictive temperature.We report here that in vitro treatment of SV40 by AAAF induces both a decrease in progeny survival and an increase in viral mutagenesis. The molecular analysis of AAAFinduced revertants reveals a hot spot of mutation not local- 9556The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Respective roles of pyrimidine dimer and pyrimidine (6-4) pyrimidone photoproducts in UV mutagenesis of simian virus 40 DNA in mammalian cells

“…These sites are included in restriction enzyme-recognizing sequences which could form small hairpin structures. We suggest that these structures may be involved in the observed spontaneous mutation hot spots already reported for some other secondary DNA structures (13,23,27). Indeed, one of the mutations (position 2533) is a silent one and therefore not responsible for the phenotypic reversion.…”

Mutagenic Specificity of (Acetylamino)fluorene-Derived DNA Adducts in Mammalian Cells

“…Among these deletions were a number of -1 frameshifts that occurred in runs of dAs. Gentil et al (14) reported that reversion of a temperaturesensitive mutation in SV40, produced by N-AAAF treatment of the vector followed by replication in African green monkey kidney cells, was mainly due to base substitutions at A:T base pairs. Finally, Klein et al (15) found that mutations were not associated with a site-specifically positioned dG-AAF adduct in a repair-deficient human cell/ shuttle vector system.…”

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

“…The latter hypothesis is supported by recent studies on the specific mutational changes needed to activate specific oncogenes (7,37,44). Therefore, we and others (3,4,8,9,11,12,14,21,22,30,32,40,41) have begun to study the specific kinds of mutations induced by carcinogens in mammalian cells.…”