thioguanine-anhydrous and Chromosomal-Instability

Thiopurines are part of a clinical regimen used for the treatment of autoimmune disorders and childhood acute lymphoblastic leukemia. However, despite these successes, there are also unintended consequences such as therapy-induced cancer in long-term survivors. Therefore, a better understanding of cellular responses to thiopurines will lead to improved and personalized treatment strategies. RAD51D is an important component of homologous recombination (HR), and our previous work established that mammalian cells defective for RAD51D are more sensitive to the thiopurine 6-thioguanine (6TG) and have dramatically increased numbers of multinucleated cells and chromosome instability. 6TG is capable of being incorporated into telomeres, and interestingly, RAD51D contributes to telomere maintenance, although the precise function of RAD51D at the telomeres remains unclear. We sought here to investigate: (1) the activity of RAD51D at telomeres, (2) the contribution of RAD51D to protect against 6TG-induced telomere damage, and (3) the fates of Rad51d-deficient cells following 6TG treatment. These results demonstrate that RAD51D is required for maintaining the telomeric 3' overhangs. As measured by γ-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of γ-H2AX telomere localization following 6TG treatment was higher in Rad51d-deficient cells than in Rad51d-proficient cells. Using live-cell imaging of 6TG-treated Rad51d-deficient cells, two predominant forms of mitotic catastrophe were found to contribute to the formation of multinucleated cells, failed division and restitution. Collectively, these findings provide a unique window into the role of the RAD51D HR protein during thiopurine induction of mitotic catastrophe. Environ. Mol. Mutagen. 59:38-48, 2018. © 2017 Wiley Periodicals, Inc.

Topics: Animals; Cell Line; Chromosomal Instability; DNA Damage; DNA-Binding Proteins; Homologous Recombination; Mice; Mitosis; Telomere; Thioguanine

Thiopurines are extensively used as immunosuppressants and in the treatment of childhood cancers, even though there is concern about therapy-induced leukemias and myelodysplastic syndromes resulting from thiopurine use. Following metabolic activation, thiopurines are incorporated into DNA and invoke mismatch repair (MMR). Recognition of 6-thioguanine (6-thioG) in DNA by key MMR proteins results in cell death rather than repair. There are suggestions that homologous recombination (HR) is involved downstream of MMR following thiopurine treatment, but the precise role of HR is poorly understood. In this study, we demonstrate that cells deficient in RAD51D (a RAD51 paralogue) are extremely sensitive to 6-thioG. This sensitivity is almost completely rescued by the deletion of Mlh1, which suggests that HR is involved in the repair of the 6-thioG-induced recombinogenic lesions generated by MMR. Furthermore, 6-thioG induces chromosome aberrations in the Rad51d-deficient cells. Interestingly, Rad51d-deficient cells show a striking increase in the frequency of triradial and quadriradial chromosomes in response to 6-thioG therapy. The presence of these chromatid exchange-type aberrations indicates that the deficiency in RAD51D-dependent HR results in profound chromosomal damage precipitated by the processing of 6-thioG by MMR. The radials are notable as an important source of chromosomal translocations, which are the most common class of mutations found in hematologic malignancies. This study thus suggests that HR insufficiency could be a potential risk factor for the development of secondary cancers that result from long-term use of thiopurines in patients.

Topics: Animals; Cell Line; Chromosomal Instability; DNA; DNA Damage; DNA Mismatch Repair; DNA-Binding Proteins; G2 Phase; Giant Cells; Mice; Recombination, Genetic; Thioguanine

Exonuclease-1 (EXO1) mediates checkpoint induction in response to telomere dysfunction in yeast, but it is unknown whether EXO1 has similar functions in mammalian cells. Here we show that deletion of the nuclease domain of Exo1 reduces accumulation of DNA damage and DNA damage signal induction in telomere-dysfunctional mice. Exo1 deletion improved organ maintenance and lifespan of telomere-dysfunctional mice but did not increase chromosomal instability or cancer formation. Deletion of Exo1 also ameliorated the induction of DNA damage checkpoints in response to gamma-irradiation and conferred cellular resistance to 6-thioguanine-induced DNA damage. Exo1 deletion impaired upstream induction of DNA damage responses by reducing ssDNA formation and the recruitment of Replication Protein A (RPA) and ATR at DNA breaks. Together, these studies provide evidence that EXO1 contributes to DNA damage signal induction in mammalian cells, and deletion of Exo1 can prolong survival in the context of telomere dysfunction.

Topics: Animals; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Proliferation; Chromosomal Instability; DNA Damage; DNA, Single-Stranded; Exodeoxyribonucleases; Gamma Rays; Gene Deletion; Gene Fusion; Genotype; Intestinal Mucosa; Longevity; Mice; Mice, Inbred C57BL; Mice, Knockout; Mutagens; Phenotype; Protein Serine-Threonine Kinases; Replication Protein A; RNA; Signal Transduction; Telomerase; Telomere; Thioguanine