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 therapyinduced 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 0 overhangs. As measured by g-H2AX induction and foci formation, 6TG induced DNA damage in Rad51d-proficient and Rad51d-deficient cells. However, the extent of g-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.
Thiopurines are a class of chemotherapy drugs used as immunosuppressants in organ transplant patients and for the treatment of childhood cancers. However, approximately 40% of patients develop life‐threatening problems later in life as a direct result of treatment. 6‐thioguanine (6TG) is a thiopurine directly incorporated into the DNA. Processing of the damage by the mismatch DNA repair pathway leads to the formation of double‐stranded breaks repaired by the homologous recombination (HR) machinery. RAD51D is an HR protein, and absence of RAD51D leads to the formation of large multinucleated cells in response to 6TG treatment. Using live cell imaging, I am attempting to determine how these multinucleated cells form. Treated and untreated mouse embryo fibroblasts (MEFs) were cultured in a 37°C chamber containing 5% CO2 and visualized using a Leica ASMDW microscope. Images were captured every three minutes and translated into individual videos. Cell division times and outcomes were determined by manually following each cell and daughter cells. The data suggest multinucleated cells form after aberrant mitotic events and subsequent fusion of the daughter cells during the second cell cycle following treatment. Current data also indicate that multinucleation is more prevalent at lower doses while interphase arrest is more common at a higher dose of 6TG. These studies have the potential to determine the mechanisms of thiopurine‐induced secondary cancers and may possibly be used to identify individuals who may benefit from alternative treatments.
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