Azathioprine-induced carcinogenesis in mice according to Msh2 genotype

Abstract

Background: The thiopurine prodrug azathioprine is used extensively in cancer therapy. Exposure to this drug results in the selection of DNA mismatch repair-deficient cell clones in vitro. It has also been suggested that thiopurine drugs might constitute a risk factor for the emergence of human neoplasms displaying microsatellite instability (MSI) because of deficient DNA mismatch repair.

Methods: Azathioprine was administered via drinking water (6-20 mg/kg body weight per day) to mice that were null (Msh2⁻(/)⁻; n = 27), heterozygous (Msh2(+/)⁻; n = 22), or wild type (Msh2(WT); n = 18) for the DNA mismatch repair gene Msh2. Control mice (45 Msh2⁻(/)⁻, 38 Msh2(+/)⁻, and 12 Msh2(WT)) received drinking water lacking azathioprine. The effect of azathioprine on tumorigenesis and survival of the mice was evaluated by Kaplan-Meier curves using log-rank and Gehan-Breslow-Wilcoxon tests. Mouse tumor samples were characterized by histology and immunophenotyping, and their MSI status was determined by polymerase chain reaction analysis of three noncoding microsatellite markers and by immunohistochemistry. Msh2 status of tumor samples was assessed by loss of heterozygosity analyses and sequencing after reverse transcription-polymerase chain reaction of the entire Msh2 coding sequence. All statistical tests were two-sided.

Results: Most untreated Msh2(WT) and Msh2(+/)⁻ mice remained asymptomatic and alive at 250 days of age, whereas azathioprine-treated Msh2(WT) and Msh2(+/)⁻ mice developed lymphomas and died prematurely (median survival of 71 and 165 days of age, respectively). Azathioprine-treated Msh2(+/)⁻ mice developed diffuse lymphomas lacking Msh2 expression and displaying MSI due to somatic inactivation of the functional Msh2 allele by loss of heterozygosity or mutation. By contrast, azathioprine-treated Msh2(WT) mice displayed no obvious tumor phenotype, but histological examination showed microscopic splenic foci of neoplastic lymphoid cells that retained Msh2 expression and did not display MSI. Both untreated and azathioprine-treated Msh2⁻(/)⁻ mice had a reduced lifespan compared with untreated Msh2(WT) mice (median survival of 127 and 107 days of age, respectively) and developed lymphomas with MSI.

Conclusion: Azathioprine-induced carcinogenesis in mice depends on the number of functional copies of the Msh2 gene.