Cancer Res Treat.  2004 Apr;36(2):151-156.

Site-Specific Mutagenesis in Human Cells by Bulky Exocyclic Amino-Substituted Guanine and Adenine Derivatives

• Moon KY

Abstract

PURPOSE
7-Bromomethylbenz[a]anthracene is a well- known mutagen and carcinogen. The aim of this study is to determine the mutagenic potency of its two major DNA adducts [N2-(benz[a]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[a]a2G) and N6-(benz[a]anthracen-7- ylmethyl)-2'-deoxyadenosine (b[a]a6A)] and the simpler benzylated analogs [N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A)] in Ad293 human cells and to compare to their mutagenicity in human cells and E. coli. MATERIALS AND METHODS: The shuttle vector pGP50 is capable of replicating in E. coli and human cells. Modified nucleotides were positioned in the plasmid pGP50 in a manner similar to pGP10 as described (8). Adenovirus transformed human embryonic kidney cells (line 293) were transfected with a shuttle vector containing an adduct. Two days later, the plasmids were recovered and treated with DpnI to remove unreplicated DNA. DH10B E. coli were transformed with the plasmids. Bacteria were cultured with the media containing X-gal, IPTG and ampicillin. Bacteria transformed by the plasmid with the adduct-induced mutation in the initiation codon of lacZ' form white colonies whereas bacteria transformed by the plasmid without mutation form blue colonies. RESULTS: In the human cell site-specific mutagenesis system, bn2G exhibited weak mutagenicity and bn6A was not mutagenic, although b[a]a2G or b[a]a6A produced 8% and 7% mutant colonies, respectively. At the site of the adduct, b[a]a2G induced the G--> T transversion mutation while b[a]a6A produced the A--> G transition mutation. CONCLUSION: These data indicate that bulkier b[a]a2G and b[a]a6A exhibit significantly greater mutagenicity in human cells than in E. coli.

Keyword

Mutagenesis; Mutagen

MeSH Terms

Adenine*
Adenoviridae
Ampicillin
Bacteria
Codon, Initiator
DNA
DNA Adducts
Genetic Vectors
Guanine*
Humans*
Isopropyl Thiogalactoside
Kidney
Mutagenesis
Mutagenesis, Site-Directed*
Nucleotides
Plasmids
Adenine
Ampicillin
Codon, Initiator
DNA
DNA Adducts
Guanine
Isopropyl Thiogalactoside
Nucleotides

Figure

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  Fig. 1 Chemical structure of four bulky aralkylated nucleosides.

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  Fig. 2 The shuttle vector pGP50 is capable of replicating in E. coli and human cells. SV40 ori makes pGP50 capable of replicating in human cells. T-antigen is required to utilize the SV40 ori. Modified nucleotides were positioned in this plasmid in a manner similar to pGP10 as described (8).

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  Fig. 3 Adenovirus transformed human embryonic kidney cells (line 293) were transfected with the shuttle vector containing an adduct. The recovered shuttle vectors were used to transform E. coli where the adduct-induced mutation was scored by the loss of lacZ' activity producing white colonies.

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  Fig. 4 Colony phenotypes in E. coli, resulting from aralkylated nucleosides in dU-containing plasmids or gapped plasmids. The value represents the mean±SE of three experiments.

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  Fig. 5 Colony phenotypes resulting from aralkylated nucleosides in human cells. The difference in mutagenicities was analyzed by one-way analysis of variance (ANOVA), followed by Duncan's multiple range t-test. A significant difference in mutagenicities between aralkylated nucleosides and control is indicated by *P<0.05. The value represents the mean±SE of three expriments.

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