Mutagenic and Cytotoxic Properties of 6-Thioguanine, S-Methylthioguanine, and Guanine-S6-sulfonic Acid

Abstract

Thiopurine drugs, including 6-thioguanine ( S G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of S G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects.S G in DNA can be methylated by S-adenosyl-L-methionine to give S 6 -methylthioguanine (S 6 mG) and oxidized by UVA light to render guanine-S 6 -sulfonic acid ( SO3H G). Here, we constructed single-stranded M13 shuttle vectors carrying a S G, S 6 mG, or SO3H G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerasedeficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S 6 mG and SO3H G were highly mutagenic, which resulted in G3 A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G3 A transition occurring at a frequency of ϳ10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

Effects of 6-Thioguanine and S6-Methylthioguanine on Transcription in Vitro and in Human Cells

“…The resulting dephosphory-lated DNA was incubated in a 15-l solution containing 1ϫ NEB buffer 4, ATP (50 pmol of cold, premixed with 1.66 pmol of [␥-32 P]ATP), and 10 units of T4 polynucleotide kinase. The mixture was then incubated at 37°C for 30 min and at 65°C for 20 min, after which 10 units of FspI was added and incubated at 37°C for 1 h. The resulting 32 P-labeled restriction fragments were resolved by using 30% polyacrylamide gel (acrylamide:bisacrylamide ϭ 19:1) with 8 M urea and quantified by phosphorimaging analysis (21,22). The relative mutation frequency (RMF) was determined by the ratio of the amount of detectable mutant restriction product to the total amounts of restriction fragments arising from the lesion-bearing genome.…”

“…The resulting solution was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v), and the aqueous portion was dried with a SpeedVac, desalted with HPLC, and dissolved in water. The resultant ODN mixture was subjected to LC-MS/MS analysis following previously described conditions (21,22,35). Briefly, a 0.5 ϫ 150-mm Zorbax SB-C18 column (Agilent Technologies) was used.…”

“…The relative mutation frequency (RMF) was determined by the ratio of the amount of detectable mutant restriction product to the total amounts of restriction fragments arising from the lesion-bearing genome. The relative bypass efficiency (RBE) was calculated using the following formula, RBE ϭ (lesion signal/competitor signal)/(nonlesion control signal/competitor signal) (22,35,38).…”

“…Our recent studies further revealed that S 6 mG is capable of inducing G 3 A mutations at frequencies of 94 and 39% in Escherichia coli and mammalian cells, respectively (8,(21)(22)(23). Additionally, replicative bypass of S G is also mildly mutagenic in these cells, and the resulting S G:T base pairs can be efficiently recognized by MMR proteins; thus, S G may also trigger the postreplicative MMR system and contribute to thiopurine toxicity (21,22,24,25).…”

Glioma: A Tale of Corticosteroids, Thiopurines and Proposed Novel AntiGlioma Small Molecules

“…Indeed, the first step to address the question of whether TPMT plays any role in brain cancer risk after thiopurine treatment is to determine whether TPMT phenotypes in the brain are similar to those from other tissues. Thiopurine drug-associated genotoxicity has been linked to mutagenesis and transformative events [151][152][153]. We hypothesize that TPMT deficiency can lead to greater genotoxicity (i.e.…”