6-Thioguanine and S⁶-methylthioguanine are mutagenic in human cells

Abstract

Thiopurines are effective immunosuppressants and anticancer agents. However, the long-term use of thiopurines was found to be associated with a significantly increased risk of various types of cancer. To date, the specific mechanism(s) underlying the carcinogenicity associated with thiopurine treatment remain(s) unclear. Herein, we constructed duplex pTGFP-Hha10 shuttle vectors carrying a 6-thioguanine ((S)G) or S⁶-methylthioguanine (S⁶mG) at a unique site and allowed the vectors to propagate in three different human cell lines. Analysis of the replication products revealed that although neither thionucleoside blocked considerably DNA replication in any of the human cell lines, both (S)G and S⁶mG were mutagenic, resulting in G→A mutation at frequencies of ~8% and ~39%, respectively. Consistent with what was found from our previous study in E. coli cells, our data demonstrated that the mutagenic properties of (S)G and S⁶mG provided significant evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Figure 1. The structures of guanine (Gua), 6-thioguanine (^SG) and S⁶-methylthioguanine (S⁶mG)

Figure 2. Method for the construction of lesion-containing duplex vector

(A) A schematic diagram showing the procedures for the preparation of the lesion-containing plasmid. See Materials and Methods for details. (B) Enzymatic digestion and ligation for the insertion of ^SG- or S⁶mG-carrying ODN into gapped pTGFP-Hha10 vector. ‘X’ represents guanine, ^SG or S⁶mG, and the A:A mismatch site is underlined. The restriction recognition sites are highlighted in bold and cleavage sites are indicated by solid triangles. (C) Agarose (1%) gel electrophoresis for monitoring the processes for the construction of the ^SG-bearing pTGFP-Hha10 vector. (D) Restriction digestion and post-labeling for assessing the integrity of the control and lesion-containing vectors (see text).

Figure 3. Determination of the bypass efficiencies and mutation frequencies of ^SG or S⁶mG

(A) Restriction digestion of PCR products of progeny genome arising from in-vivo replication of ^SG- and S⁶mG-bearing genomes in mammalian cells. The PCR fragments were digested with SacI, and the nascent terminal phosphate groups in the resulting DNA fragments were removed by shrimp alkaline phosphatase. The 5’ termini were subsequently labeled with [γ-³²P]ATP, and the resulting DNA fragments were further digested with FspI and resolved on a 30% denaturing PAGE gel. (B) Denaturing PAGE for assessing the bypass efficiencies and mutation frequencies of ^SG and S⁶mG in 293T, GM00627 and GM04429 cells.

Figure 4. Bypass efficiencies (A) and mutation frequencies (B) of guanine, ^SG and S⁶mG in 293T, GM00627 and GM04429 cells

Black, gray and white columns represent the results for substrates carrying guanine, ^SG and S⁶mG, respectively. The data represent the means and standard deviations of results from three independent transfection experiments.